Methods of making tumor reactive peripheral blood lymphocytes (TRPBL)

Abstract

Provided herein are methods of making tumor reactive peripheral blood lymphocytes (trPBLs) using PBMCs obtained from a patient. The method comprises enriching trPBLs from PBMCs using markers that are expressed or secreted by the trPBLs, such as 4- IBB, CD103, CXCL13, IFN-y, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, CD49a, etc. Further provided are use of the trPBLs or pharmaceutical compositions thereof for the treatment of cancers.

Description

Attorney Docket No.116983-5132-WO METHODS OF MAKING TUMOR REACTIVE PERIPHERAL BLOOD LYMPHOCYTES (trPBL) CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 665,916, filed June 28, 2024, which is herein incorporated by reference in its entirety. BACKGROUND

[0002] Tumor infiltrating lymphocyte (TIL) therapy has shown clinical benefit for patients with solid tumors (Chesney et al., Journal for ImmunoTherapy of Cancer 2022;10:e005755; Schoenfeld et al., SITC Annual Meeting 2021). However, for the next generation TIL therapy, identification of tumor-reactive T cells represents an essential step in improving the clinical specificity and potency of TIL therapy.

[0003] Recent studies have suggested that circulating T cells from peripheral blood of cancer patients may be a source of tumor-reactive T cells with a more stem-like phenotype (Pauken et al., J. Exp. Med.2021, 218, e20200920; Lucca et al., J. Exp. Med.2021, 218, e20200921; Yossef et al., Cancer Cell 2023, 41, 1-12). However, due to the extremely low abundance of the tumor-reactive T cells in peripheral blood, there remains a challenge for their usage as therapy for cancer patients (Cafri et al., Nat. Comun.2019, 10, 449; Malekzadeh et al., Clin. Cancer Res.2020, 26, 1267-1276; Strønen et al, Science 2016, 352, 1337-1341).

[0004] Thus, there remains a need for methods of making tumor reactive peripheral blood lymphocytes (trPBLs) using PBMCs obtained from a patient. Provided herein are methods of enriching and expanding trPBLs from PBMCs using markers that are expressed or secreted by trPBLs. BRIEF SUMMARY

[0005] Some embodiments disclosed herein provide a method for expanding tumor-reactive peripheral blood lymphocytes (trPBLs), comprising: a) obtaining a sample of peripheral blood mononuclear cells (PBMCs) from a subject; b) selecting T cells in the sample of PBMCs expressing one or more markers for trPBL to produce a population of cells enriched DB2 / 650358429.1 1Attorney Docket No.116983-5132-WO with trPBLs; and c) culturing the population of cells enriched with trPBLs in a cell culture medium to produce an expanded population of trPBLs.

[0006] In some embodiments, the one or more markers for trPBL is selected from the group consisting of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD1, CD39, CD38, and CD49a. In some embodiments, the selecting comprises contacting the sample of PBMCs with an antibody to the one or more markers for trPBL. In some embodiments, the selecting further comprises contacting the sample of PBMCs with a secondary antibody capable of binding to the antibody to the one or more markers for trPBL. In some embodiments, the antibody or the secondary antibody is conjugated to a magnetic nanoparticle. In some embodiments, the selecting comprises passing the sample of PBMCs through a magnetic capture zone disposed in a channel of a microfluidic device. In some embodiments, the microfluidic device comprises a plurality of magnetic capture zones, wherein the plurality of magnetic capture zones is disposed to spatially separate cells with different degrees of magnetization.

[0007] In some embodiments, the sample of PBMCs is obtained via a leukopak from the subject. In some embodiments, the sample of PBMCs is obtained via continuous trapping from the subject.

[0008] In some embodiments, the cell culture medium comprises IL-21 at a concentration of about 10 ng / mL, and IL-15 at a concentration of about 10 ng / mL. In some embodiments, the cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine. In some embodiments, the L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. In some embodiments, the NAD+ booster is NAD+. In some embodiments, the NAD+ is present at a concentration of about 10 µM to 100 µM. In some embodiments, the NAD+ is present at a concentration of about 50 µM.

[0009] In some embodiments, the method further comprises transducing the population of cells enriched with trPBLs from step (b) with a recombinant lentiviral particle comprising a nucleic acid sequence encoding tethered IL-12 (TeIL-12) and / or tethered IL-15 (TeIL-15) to produce a population of gene-edited trPBLs. In some embodiments, the method further comprises activating the population of cells enriched with trPBLs from step (b) for 1 day or 2 DB2 / 650358429.1 2Attorney Docket No.116983-5132-WO days before transducing the population of cells with the recombinant lentiviral particle. In some embodiments, the activating step comprises contacting the population of cells enriched with trPBLs from step (b) with a cytokine selected from the group consisting of IL-2, IL-15, IL-21, IL-7, and a combination thereof. In some embodiments, the activating step comprises contacting the population of cells enriched with trPBLs from step (b) with TransAct. In some embodiments, the activating step comprises contacting the population of cells enriched with trPBLs from step (b) with TransAct at a ratio of 1:100. In some embodiments, the transducing step is conducted at a concentration of 105cells / mL. In some embodiments, the transducing step is conducted at a multiplicity of infection (MOI) of about 10 to about 40. In some embodiments, the transducing step is conducted in the presence of RetroNectin or Vectofusin-1. In some embodiments, the transducing step comprises centrifugation. In some embodiments, the transducing step is conducted in the presence of Lentiboost. In some embodiments, the method further comprises resting the population of cells for 2 day or 3 days after the transducing step.

[0010] In some embodiments, the method further comprises administering to the subject an inhibitor of an immune checkpoint gene before obtaining the sample of PBMCs from the subject. In some embodiments, the immune checkpoint gene is selected from the group consisting of PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR.

[0011] In some embodiments, step (c) is performed for a period that lasts about 1-10 days. In some embodiments, step (c) is performed for a period that lasts about 3-5 days.

[0012] Some embodiments disclosed herein provide a population of trPBLs produced by the method disclosed herein.

[0013] Some embodiments disclosed herein provide a gene-edited trPBL expressing an exogenous IL-12 or a variant thereof.

[0014] In some embodiments, the IL-12 is a tethered IL-12 (TeIL-12). In some embodiments, the TeIL-12 comprises a membrane anchor, and a human IL-12 p40 subunit fused to a human DB2 / 650358429.1 3Attorney Docket No.116983-5132-WO IL-12 p35 subunit. In some embodiments, the human IL-12 p35 subunit has the amino acid sequence of SEQ ID NO:60 and the human IL-12 p40 subunit has the amino acid sequence of SEQ ID NO:61. In some embodiments, the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:62. In some embodiments, the gene-edited trPBL further expresses an exogenous IL-15 or a variant thereof. In some embodiments, the IL-15 is a human IL-15. In some embodiments, the human IL-15 has the amino acid sequence of SEQ ID NO:64. In some embodiments, the IL-15 is a tethered IL-15 (TeIL-15). In some embodiments, the TeIL- 15 comprises a membrane anchor, and a human IL-15. In some embodiments, the TeIL-15 has the amino acid sequence of SEQ ID NO:73.

[0015] Some embodiments disclosed herein provide a pharmaceutical composition comprising a population of trPBLs or a population of the gene-edited trPBLs discloses herein.

[0016] Some embodiments disclosed herein provide a method of treating a cancer in a patient in need thereof comprising administering the pharmaceutical composition disclosed herein to the patient.

[0017] In some embodiments, the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma. In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the trPBLs to the patient. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for one day. In some embodiments, the cyclophosphamide is administered with mesna. In some embodiments, the patient is not DB2 / 650358429.1 4Attorney Docket No.116983-5132-WO treated with a non-myeloablative lymphodepletion regimen prior to administering the trPBLs to the patient. In some embodiments, the method further comprises a step of treating the patient with an IL-2 regimen starting on the day after the administration of trPBLs to the patient. In some embodiments, the method further comprises a step of treating the patient with an IL-2 regimen starting on the same day as administration of trPBLs to the patient. In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg of aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerance. In some embodiments, the method comprises no step of treating the patient with an IL-2 regimen. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 depicts an exemplary nucleic acid that allows for expression of a membrane anchored IL-12 (TeIL-12) and PD-1 shRNA in embodiments of the subject PBLs provided herein.

[0019] Figure 2: Shows an exemplary expression vector encoding NFAT driven TeIL-12 and EF1α driven bicistronic tCD19 and Component X.

[0020] Figure 3: Shows a schematic illustration of an exemplary trPBL production process. DETAILED DESCRIPTION I. Introduction

[0021] Provided herein are compositions and methods for the treatment of cancers using tumor reactive peripheral blood lymphocytes (trPBLs), wherein the trPBLs may be selected, enriched, trained, and / or modified to express one or more immunomodulatory agents (e.g., cytokines) on their cell surface. The immunomodulatory agents associated with the trPBLs provide a localized immunostimulatory effect that can advantageously enhance PBL survival and / or anti-tumor activity in a patient recipient. As such, the compositions and methods disclosed herein provide effective cancer therapies. II. Definitions

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention DB2 / 650358429.1 5Attorney Docket No.116983-5132-WO belongs. All patents and publications referred to herein are incorporated by reference in their entireties.

[0023] The terms “co-administration,” “co-administering,” “administered in combination with,” “administering in combination with,” “simultaneous,” and “concurrent,” as used herein, encompass administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the present invention, for example, a plurality of PBLs or TILs) to a subject so that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0024] The term “in vivo” refers to an event that takes place in a subject’s body.

[0025] The term “in vitro” refers to an event that takes places outside of a subject’s body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.

[0026] The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and / or organ which has been removed from a subject’s body. Aptly, the cell, tissue and / or organ may be returned to the subject’s body in a method of surgery or treatment.

[0027] The terms “peripheral blood mononuclear cells” and “PBMCs” refers to a peripheral blood cell having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. Optionally, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs include antigen presenting cells.

[0028] The terms “peripheral blood lymphocytes” and “PBLs” refer to lymphocytes, such as T cells, collected from peripheral blood. In some embodiments, PBLs are separated from whole blood or apheresis product from a donor. In some embodiments, PBLs are separated from whole blood or apheresis product from a donor by positive or negative selection of a T cell phenotype, such as the T cell phenotype of CD3+ CD45+.

[0029] The term “rapid expansion” means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more DB2 / 650358429.1 6Attorney Docket No.116983-5132-WO preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about 100-fold over a period of a week. A number of rapid expansion protocols are described herein.

[0030] By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cell populations can include genetically modified TILs.

[0031] By “population of cells” (including PBLs) herein is meant a number of cells that share common traits. In general, populations generally range from 1 X 106to 10 X 1010in number, with different PBL populations comprising different numbers.

[0032] By “cryopreserved PBLs” herein is meant that PBLs, either primary, bulk, or expanded, are treated and stored in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity, “cryopreserved PBLs” are distinguishable from frozen tissue samples which may be used as a source of primary PBLs.

[0033] By “thawed cryopreserved PBLs” herein is meant a population of PBLs that was previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures wherein PBLs may be administered to a patient.

[0034] TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45RA, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. DB2 / 650358429.1 7Attorney Docket No.116983-5132-WO

[0035] The term “cryopreservation media” or “cryopreservation medium” refers to any medium that can be used for cryopreservation of cells. Such media can include media comprising 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, as well as combinations thereof. The term “CS10” refers to a cryopreservation medium which is obtained from Stemcell Technologies or from Biolife Solutions. The CS10 medium may be referred to by the trade name “CryoStor® CS10”. The CS10 medium is a serum-free, animal component-free medium which comprises DMSO.

[0036] The term “central memory T cell” refers to a subset of T cells that in the human are CD45RO+ and constitutively express CCR7 (CCR7hi) and CD62L (CD62hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secret IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells are predominant in the CD4 compartment in blood, and in the human are proportionally enriched in lymph nodes and tonsils.

[0037] The term “effector memory T cell” refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45RO+, but have lost the constitutive expression of CCR7 (CCR7lo) and are heterogeneous or low for CD62L expression (CD62Llo). Transcription factors for effector memory T cells include BLIMP1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon-γ, IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in blood, and in the human are proportionally enriched in the lung, liver, and gut. CD8+ effector memory T cells carry large amounts of perforin.

[0038] The term “closed system” refers to a system that is closed to the outside environment. Any closed system appropriate for cell culture methods can be employed with the methods of the present invention. Closed systems include, for example, but are not limited to, closed G-containers. Once a tumor segment is added to the closed system, the system is no opened to the outside environment until the PBLs or TILs are ready to be administered to the patient.

[0039] The terms “fragmenting,” “fragment,” and “fragmented,” as used herein to describe processes for disrupting a tumor, includes mechanical fragmentation methods such as crushing, slicing, dividing, and morcellating tumor tissue as well as any other method for disrupting the physical structure of tumor tissue. DB2 / 650358429.1 8Attorney Docket No.116983-5132-WO

[0040] The terms “peripheral blood mononuclear cells” and “PBMCs” refers to a peripheral blood cell having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as an antigen presenting cell (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0041] The term “anti-CD3 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti- CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0042] The term “OKT-3” (also referred to herein as “OKT3”) refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). A hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 is also deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No.86022706. TABLE 1. Amino acid sequences of muromonab (exemplary OKT-3 antibody). Identifier Sequence (One-Letter Amino Acid Symbols)known as interleukin-2, and includes all forms of IL-2 including human and mammalian DB2 / 650358429.1 9Attorney Docket No.116983-5132-WO forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, e.g., in Nelson, J. Immunol.2004, 172, 3983-88 and Malek, Annu. Rev. Immunol.2008, 26, 453-79, the disclosures of which are incorporated by reference herein. The amino acid sequence of recombinant human IL-2 suitable for use in the invention is given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL- 2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the invention is given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug bempegaldesleukin (NKTR-214, pegylated human recombinant IL-2 as in SEQ ID NO:4 in which an average of 6 lysine residues are N6substituted with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H- fluoren-9-yl)methoxy]carbonyl), which is available from Nektar Therapeutics, South San Francisco, CA, USA, or which may be prepared by methods known in the art, such as the methods described in Example 19 of International Patent Application Publication No. WO 2018 / 132496 A1 or the method described in Example 1 of U.S. Patent Application Publication No. US 2019 / 0275133 A1, the disclosures of which are incorporated by reference herein. Bempegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the invention are described in U.S. Patent Application Publication No. US 2014 / 0328791 A1 and International Patent Application Publication No. WO 2012 / 065086 A1, the disclosures of which are incorporated by reference herein. Alternative forms of conjugated IL-2 suitable for use in the invention are described in U.S. Patent Nos.4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated by reference herein. Formulations of IL-2 suitable for use in the invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated by reference herein.

[0044] In some embodiments, an IL-2 form suitable for use in the present invention is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the invention are described in U.S. Patent Application Publication Nos. US 2020 / 0181220 A1 and US 2020 / 0330601 A1, the DB2 / 650358429.1 10Attorney Docket No.116983-5132-WO disclosures of which are incorporated by reference herein. In some embodiments, and IL-2 form suitable for use in the invention is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugating moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is at E62. In some embodiments, the amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, the amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to an unnatural amino acid. In some embodiments, the unnatural amino acid comprises N6-azidoethoxy-L- lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO- lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2- amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p- propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L- phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4- propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3- oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine. In some embodiments, the IL-2 conjugate has a decreased affinity to IL-2 receptor α (IL-2Rα) subunit relative to a wild-type IL-2 polypeptide. In some embodiments, the decreased affinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% decrease in binding affinity to IL-2Rα relative to a wild-type IL-2 DB2 / 650358429.1 11Attorney Docket No.116983-5132-WO polypeptide. In some embodiments, the decreased affinity is about 1-fold, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300- fold, 500-fold, 1000-fold, or more relative to a wild-type IL-2 polypeptide. In some embodiments, the conjugating moiety impairs or blocks the binding of IL-2 with IL-2Rα. In some embodiments, the conjugating moiety comprises a water-soluble polymer. In some embodiments, the additional conjugating moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N- acryloylmorpholine), or a combination thereof. In some embodiments, each of the water- soluble polymers independently comprises PEG. In some embodiments, the PEG is a linear PEG or a branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES). In some embodiments, each of the water-soluble polymers independently comprises a glycan. In some embodiments, each of the water-soluble polymers independently comprises polyamine. In some embodiments, the conjugating moiety comprises a protein. In some embodiments, the additional conjugating moiety comprises a protein. In some embodiments, each of the proteins independently comprises an albumin, a transferrin, or a transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of IgG. In some embodiments, the conjugating moiety comprises a polypeptide. In some embodiments, the additional conjugating moiety comprises a polypeptide. In some embodiments, each of the polypeptides independently comprises a XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugating moiety is directly bound to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugating moiety is indirectly bound to the isolated and purified IL-2 polypeptide through a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker comprises Lomant’s reagent dithiobis (succinimidylpropionate) DSP, 3′3′- DB2 / 650358429.1 12Attorney Docket No.116983-5132-WO dithiobis(sulfosuccinimidyl proprionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3′- dithiobispropionimidate (DTBP), 1,4-di-(3′-(2′-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), such as e.g.1,5- difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′- dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine, benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N′-ethylene-bis(iodoacetamide), or N,N′-hexamethylene- bis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker. In some embodiments, the heterobifunctional linker comprises N-succinimidyl 3-(2- pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chain N-succinimidyl 3-(2-pyridyldithio) propionate (sulfo- LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl- 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB), sulfosuccinimidyl(4- iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ- maleimidobutyryloxy)succinimide ester (GMBs), N-(γ-maleimidobutyryloxy) sulfosuccinimide ester (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl 4- (((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4- iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive cross-linkers such as 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1- carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), N- hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4- azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate DB2 / 650358429.1 13Attorney Docket No.116983-5132-WO (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4- azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4′-azido-2′-nitrophenyl amino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-Nos), sulfosuccinimidyl-2-(m-azido-o- nitrobenzamido)-ethyl-1,3′-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3′- dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sulfo- sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7- azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamido)-4- (iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio) propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoyl hydrazide (ABH), 4- (ρ-azidosalicylamido)butylamine (AsBA), or p-azidophenyl glyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises a maleimide group, optionally comprising maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo- sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyoxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the invention is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form suitable for use in the invention is pegylated as disclosed in U.S. Patent Application Publication No. US 2020 / 0181220 A1 and U.S. Patent Application Publication No. US 2020 / 0330601 A1. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, DB2 / 650358429.1 14Attorney Docket No.116983-5132-WO T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in the invention lacks IL-2R alpha chain engagement but retains normal binding to the intermediate affinity IL-2R beta-gamma signaling complex. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5.

[0045] In some embodiments, an IL-2 form suitable for use in the invention is nemvaleukin alfa, also known as ALKS-4230 (SEQ ID NO:6), which is available from Alkermes, Inc. Nemvaleukin alfa is also known as human interleukin 2 fragment (1-59), variant (Cys125>Ser51), fused via peptidyl linker (60GG61) to human interleukin 2 fragment (62-132), fused via peptidyl linker (133GSGGGS138) to human interleukin 2 receptor α-chain fragment (139-303), produced in Chinese hamster ovary (CHO) cells, glycosylated; human interleukin 2 (IL-2) (75-133)-peptide [Cys125(51)>Ser]-mutant (1-59), fused via a G2peptide linker (60- 61) to human interleukin 2 (IL-2) (4-74)-peptide (62-132) and via a GSG3S peptide linker (133-138) to human interleukin 2 receptor α-chain (IL2R subunit alpha, IL2Rα, IL2RA) (1- 165)-peptide (139-303), produced in Chinese hamster ovary (CHO) cells, glycoform alfa. DB2 / 650358429.1 15Attorney Docket No.116983-5132-WO The amino acid sequence of nemvaleukin alfa is given in SEQ ID NO:6. In some embodiments, nemvaleukin alfa exhibits the following post-translational modifications: disulfide bridges at positions: 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168- 199 or 168-197 (using the numbering in SEQ ID NO:6), and glycosylation sites at positions: N187, N206, T212 using the numbering in SEQ ID NO:6. The preparation and properties of nemvaleukin alfa, as well as additional alternative forms of IL-2 suitable for use in the invention, is described in U.S. Patent Application Publication No. US 2021 / 0038684 A1 and U.S. Patent No.10,183,979, the disclosures of which are incorporated by reference herein. In some embodiments, an IL-2 form suitable for use in the invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, an IL-2 form suitable for use in the invention has the amino acid sequence given in SEQ ID NO:6 or conservative amino acid substitutions thereof. In some embodiments, an IL-2 form suitable for use in the invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or variants, fragments, or derivatives thereof. In some embodiments, an IL-2 form suitable for use in the invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to amino acids 24-452 of SEQ ID NO:7, or variants, fragments, or derivatives thereof. Other IL-2 forms suitable for use in the present invention are described in U.S. Patent No.10,183,979, the disclosures of which are incorporated by reference herein. Optionally, in some embodiments, an IL-2 form suitable for use in the invention is a fusion protein comprising a first fusion partner that is linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having the receptor antagonist activity of IL- 1Rα, and wherein the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, wherein the mucin domain polypeptide linker comprises SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8 and wherein the half-life of the fusion protein is improved as compared to a fusion of the first fusion partner to the second fusion partner in the absence of the mucin domain polypeptide linker. TABLE 2. Amino acid sequences of interleukins. Identifier Sequence (One-Letter Amino Acid Symbols)DB2 / 650358429.1 16Attorney Docket No.116983-5132-WO SEQ ID NO:4 PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE 60 Aldesleukin ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120 ITFSQSIIST LT 132 SEQ ID NO:5 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE 60antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VH or the VL, wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VHor the VL, wherein the IL-2 molecule is a mutein, and wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administration of an antibody described in U.S. Patent Application Publication No. US 2020 / 0270334 A1, the disclosures of which are incorporated by reference herein. In some DB2 / 650358429.1 17Attorney Docket No.116983-5132-WO embodiments, the antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VHor the VL, wherein the IL-2 molecule is a mutein, wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells, and wherein the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of: a IgG class light chain comprising SEQ ID NO:39 and a IgG class heavy chain comprising SEQ ID NO:38; a IgG class light chain comprising SEQ ID NO:37 and a IgG class heavy chain comprising SEQ ID NO:29; a IgG class light chain comprising SEQ ID NO:39 and a IgG class heavy chain comprising SEQ ID NO:29; and a IgG class light chain comprising SEQ ID NO:37 and a IgG class heavy chain comprising SEQ ID NO:38.

[0047] In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR1 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR2 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR3 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR1 of the VL, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR2 of the VL, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR3 of the VL, wherein the IL-2 molecule is a mutein.

[0048] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, in the middle region of the CDR or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine engrafted protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine engrafted protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL-2 sequence replaces all or part of a CDR sequence. The replacement by the IL-2 molecule can be the N-terminal region of the CDR, in the middle region of the CDR or at or near the C-terminal region the CDR. A replacement by the IL-2 molecule can be as few as one or two amino acids of a CDR sequence, or the entire CDR sequences. DB2 / 650358429.1 18Attorney Docket No.116983-5132-WO

[0049] In some embodiments, an IL-2 molecule is engrafted directly into a CDR without a peptide linker, with no additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, an IL-2 molecule is engrafted indirectly into a CDR with a peptide linker, with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0050] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some instances, the IL-2 mutein comprising an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, the IL-2 mutein comprises an amino acid sequence in Table 1 in U.S. Patent Application Publication No. US 2020 / 0270334 A1, the disclosure of which is incorporated by reference herein.

[0051] In some embodiments, the antibody cytokine engrafted protein comprises an HCDR1 selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22 and SEQ ID NO:25. In some embodiments, the antibody cytokine engrafted protein comprises an HCDR1 selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13 and SEQ ID NO:16. In some embodiments, the antibody cytokine engrafted protein comprises an HCDR1 selected from the group consisting of HCDR2 selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26. In some embodiments, the antibody cytokine engrafted protein comprises an HCDR3 selected from the group consisting of SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:27. In some embodiments, the antibody cytokine engrafted protein comprises a VH region comprising the amino acid sequence of SEQ ID NO:28. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:29. In some embodiments, the antibody cytokine engrafted protein comprises a VL region comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine engrafted protein comprises a light chain comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted protein comprises a VH region comprising the amino acid sequence of SEQ ID NO:28 and a VLregion comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of DB2 / 650358429.1 19Attorney Docket No.116983-5132-WO SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine engrafted protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334 A1, or variants, derivatives, or fragments thereof, or conservative amino acid substitutions thereof, or proteins with at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody components of the antibody cytokine engrafted protein described herein comprise immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab. In some embodiments, the antibody cytokine engrafted protein described herein has a longer serum half-life than a wild-type IL-2 molecule such as, but not limited to, aldesleukin or a comparable molecule. In some embodiments, the antibody cytokine engrafted protein described herein has a sequence as set forth in Table 3. TABLE 3: Sequences of exemplary palivizumab antibody-IL-2 engrafted proteins Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO:13 MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML 60 IL-2 TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE 120 TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT 153 60 120 60 120 60 120 60 120 60 120 5 60 120Attorney Docket No.116983-5132-WO SEQ ID NO:26 IWWDDKK 7 HCDR2 IMGT SEQ ID NO:27 ARSMITNWYF DV 12 HCDR3 IMGT SEQ ID NO:28 QVTLRESGPA LVKPTQTLTL TCTFSGFSLA PTSSSTKKTQ LQLEHLLLDL QMILNGINNY 60 V KNPKLTAMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV 120 IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG WIRQPPGKAL 180 EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC ARSMITNWYF 240 DVWGAGTTVT VSS 253 SEQ ID NO:29 QMILNGINNY KNPKLTAMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR 60 Heavy chain PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG 120 WIRQPPGKAL EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC 180 ARSMITNWYF DVWGAGTTVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV 240 TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR 300 VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV AVSHEDPEVK 360 FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALAAPIEK 420 TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT 480 PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 533 SEQ ID NO:30 KAQLSVGYMH 10 LCDR1 kabat SEQ ID NO:31 DTSKLAS 7 LCDR2 kabat SEQ ID NO:32 FQGSGYPFT 9 LCDR3 kabat SEQ ID NO:33 QLSVGY 6 LCDR1 chothia SEQ ID NO:34 DTS 3 LCDR2 chothia SEQ ID NO:35 GSGYPF 6 LCDR3 chothia SEQ ID NO:36 DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR 60 V FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIK 106 SEQ ID NO:37 DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR 60 Light chain FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIKRTVA APSVFIFPPS 120 DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 180 SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 213 SEQ ID NO:38 QVTLRESGPA LVKPTQTLTL TCTFSGFSLA PTSSSTKKTQ LQLEHLLLDL QMILNGINNY 60 Light chain KNPKLTRMLT AKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV 120 IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG WIRQPPGKAL 180 EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC ARSMITNWYF 240 DVWGAGTTVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT 300 SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR VEPKSCDKTH 360 TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV AVSHEDPEVK FNWYVDGVEV 420 HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALAAPIEK TISKAKGQPR 480 EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF 540 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 583 SEQ ID NO:39 DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR 60 Light chain FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIKRTVA APSVFIFPPS 120 DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 180 SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 213 The term “IL-4” (also referred to herein as “IL4”) refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naïve helper T cells (Th0 cells) to Th2 T cells. Steinke and Borish, Respir. Res.2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching to IgE and IgG1expression from B cells. Recombinant human IL-4 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human DB2 / 650358429.1 21Attorney Docket No.116983-5132-WO IL-15 recombinant protein, Cat. No. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the invention is given in Table 2 (SEQ ID NO:9).

[0052] The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue- derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell development within the thymus and survival within the periphery. Recombinant human IL-7 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the invention is given in Table 2 (SEQ ID NO:10).

[0053] The term “IL-15” (also referred to herein as “IL15”) refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated by reference herein. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa. Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No.34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the invention is given in Table 2 (SEQ ID NO:11).

[0054] The term “IL-21” (also referred to herein as “IL21”) refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc.2014, 13, 379-95, the disclosure of which is incorporated by reference herein. IL-21 is primarily produced by natural killer T cells and activated human CD4+T cells. Recombinant human IL- 21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from DB2 / 650358429.1 22Attorney Docket No.116983-5132-WO multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, Cat. No.14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the invention is given in Table 2 (SEQ ID NO:12).

[0055] When “an anti-tumor effective amount”, “a tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising PBLs described herein may be administered at a dosage of 104to 1011cells / kg body weight (e.g., 105to 106, 105to 1010, 105to 1011, 106to 1010, 106to 1011,107to 1011, 107to 1010, 108to 1011, 108to 1010, 109to 1011, or 109to 1010cells / kg body weight), including all integer values within those ranges. PBLs (including in some cases, genetically modified PBLs) compositions may also be administered multiple times at these dosages. The PBLs (including, in some cases, genetically engineered PBLs) can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg, et al., New Eng. J. of Med.1988, 319, 1676). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0056] The term “hematological malignancy”, “hematologic malignancy” or terms of correlative meaning refer to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematological malignancies are also referred to as “liquid tumors.” Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin’s lymphoma, and non-Hodgkin’s lymphomas. The term “B cell hematological malignancy” refers to hematological malignancies that affect B cells.

[0057] The term “liquid tumor” refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as marrow infiltrating lymphocytes (MILs). DB2 / 650358429.1 23Attorney Docket No.116983-5132-WO

[0058] The term “microenvironment,” as used herein, may refer to the solid or hematological tumor microenvironment as a whole or to an individual subset of cells within the microenvironment. The tumor microenvironment, as used herein, refers to a complex mixture of “cells, soluble factors, signaling molecules, extracellular matrices, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, foster therapeutic resistance, and provide niches for dominant metastases to thrive,” as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare because of immune suppression by the microenvironment.

[0059] In some embodiments, the invention includes a method of treating a cancer with a population of PBLs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of PBLs according to the invention. In some embodiments, the population of PBLs may be provided wherein a patient is pre-treated with nonmyeloablative chemotherapy prior to an infusion of PBLs according to the present invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / d for 2 days (days 27 and 26 prior to PBL infusion) and fludarabine 25 mg / m2 / d for 5 days (days 27 to 23 prior to PBL infusion). In some embodiments, after non-myeloablative chemotherapy and PBL infusion (at day 0) according to the invention, the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU / kg every 8 hours to physiologic tolerance.

[0060] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (“cytokine sinks”). Accordingly, some embodiments of the invention utilize a lymphodepletion step (sometimes also referred to as “immunosuppressive conditioning”) on the patient prior to the introduction of the PBLs of the invention.

[0061] The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, or the manner of administration. The term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and / or cell migration). The specific dose will vary depending DB2 / 650358429.1 24Attorney Docket No.116983-5132-WO on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

[0062] The terms “treatment”, “treating”, “treat”, and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment”, as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. “Treatment” is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition. For example, “treatment” encompasses delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g., in the case of a vaccine.

[0063] The term “heterologous” when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0064] The terms “sequence identity,” “percent identity,” and “sequence percent identity” (or synonyms thereof, e.g., “99% identical”) in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain DB2 / 650358429.1 25Attorney Docket No.116983-5132-WO alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S. Government’s National Center for Biotechnology Information BLAST web site. Comparisons between two sequences can be carried using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0065] As used herein, the term “variant” encompasses but is not limited to antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference antibody by way of one or more substitutions, deletions and / or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody. The variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody. The term variant also includes pegylated antibodies or proteins.

[0066] By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs, expanded TILs (“REP TILs”) as well as “reREP TILs” as discussed herein. reREP TILs can include for example second expansion TILs or second additional expansion TILs.

[0067] TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45RA, CD95, PD-1, and CD25. Additionally, and DB2 / 650358429.1 26Attorney Docket No.116983-5132-WO alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs may further be characterized by potency – for example, TILs may be considered potent if, for example, interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. TILs may be considered potent if, for example, interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, greater than about 1000 pg / mL.

[0068] The term “deoxyribonucleotide” encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and / or to the linkages between deoxyribonucleotide in the oligonucleotide.

[0069] The term “RNA” defines a molecule comprising at least one ribonucleotide residue. The term “ribonucleotide” defines a nucleotide with a hydroxyl group at the 2' position of a b-D-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Nucleotides of the RNA molecules described herein may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0070] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods. DB2 / 650358429.1 27Attorney Docket No.116983-5132-WO

[0071] The terms “about” and “approximately” mean within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the terms “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. Moreover, as used herein, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.

[0072] The transitional terms “comprising,” “consisting essentially of,” and “consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of” excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinary associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. All compositions, methods, and kits described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”

[0073] The terms “antibody” and its plural form “antibodies” refer to whole immunoglobulins and any antigen-binding fragment (“antigen-binding portion”) or single chains thereof. An “antibody” further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light DB2 / 650358429.1 28Attorney Docket No.116983-5132-WO chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VHand VLregions of an antibody may be further subdivided into regions of hypervariability, which are referred to as complementarity determining regions (CDR) or hypervariable regions (HVR), and which can be interspersed with regions that are more conserved, termed framework regions (FR). Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen epitope or epitopes. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0074] The term “antigen” refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule capable of being bound by an antibody or a TCR if presented by major histocompatibility complex (MHC) molecules. The term “antigen”, as used herein, also encompasses T cell epitopes. An antigen is additionally capable of being recognized by the immune system. In some embodiments, an antigen is capable of inducing a humoral immune response or a cellular immune response leading to the activation of B lymphocytes and / or T lymphocytes. In some cases, this may require that the antigen contains or is linked to a Th cell epitope. An antigen can also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen will preferably react, typically in a highly specific and selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be induced by other antigens.

[0075] The terms “monoclonal antibody,” “mAb,” “monoclonal antibody composition,” or their plural forms refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific to certain receptors can be made using knowledge and skill in the art of injecting test subjects with suitable antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional characteristics. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such DB2 / 650358429.1 29Attorney Docket No.116983-5132-WO as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies will be described in more detail below.

[0076] The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “fragment”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHand CH1 domains; (iv) a Fv fragment consisting of the VLand VHdomains of a single arm of an antibody, (v) a domain antibody (dAb) fragment (Ward, et al., Nature, 1989, 341, 544-546), which may consist of a VHor a VLdomain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VLand VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLand VHregions pair to form monovalent molecules known as single chain Fv (scFv); see, e.g., Bird, et al., Science 1988, 242, 423-426; and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed within the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. In some embodiments, a scFv protein domain comprises a VH portion and a VLportion. A scFv molecule is denoted as either VL-L-VHif the VLdomain is the N-terminal part of the scFv molecule, or as VH-L-VL if the VH domain is the N-terminal part of the scFv molecule. Methods for making scFv molecules and designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No.4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol 9: 132-137 (1991), the disclosures of which are incorporated by reference herein. DB2 / 650358429.1 30Attorney Docket No.116983-5132-WO

[0077] The term “human antibody,” as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0078] The term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0079] The term “recombinant human antibody”, as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (such as a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. DB2 / 650358429.1 31Attorney Docket No.116983-5132-WO

[0080] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.

[0081] The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”

[0082] The term “human antibody derivatives” refers to any modified form of the human antibody, including a conjugate of the antibody and another active pharmaceutical ingredient or antibody. The terms “conjugate,” “antibody-drug conjugate”, “ADC,” or “immunoconjugate” refers to an antibody, or a fragment thereof, conjugated to another therapeutic moiety, which can be conjugated to antibodies described herein using methods available in the art.

[0083] The terms “humanized antibody,” “humanized antibodies,” and “humanized” are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. Humanized forms of non-human (for example, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a 15 hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol.1992, 2, 593-596. The antibodies described herein may also be modified to employ any Fc variant DB2 / 650358429.1 32Attorney Docket No.116983-5132-WO which is known to impart an improvement (e.g., reduction) in effector function and / or FcR binding. The Fc variants may include, for example, any one of the amino acid substitutions disclosed in International Patent Application Publication Nos. WO 1988 / 07089 A1, WO 1996 / 14339 A1, WO 1998 / 05787 A1, WO 1998 / 23289 A1, WO 1999 / 51642 A1, WO 99 / 58572 A1, WO 2000 / 09560 A2, WO 2000 / 32767 A1, WO 2000 / 42072 A2, WO 2002 / 44215 A2, WO 2002 / 060919 A2, WO 2003 / 074569 A2, WO 2004 / 016750 A2, WO 2004 / 029207 A2, WO 2004 / 035752 A2, WO 2004 / 063351 A2, WO 2004 / 074455 A2, WO 2004 / 099249 A2, WO 2005 / 040217 A2, WO 2005 / 070963 A1, WO 2005 / 077981 A2, WO 2005 / 092925 A2, WO 2005 / 123780 A2, WO 2006 / 019447 A1, WO 2006 / 047350 A2, and WO 2006 / 085967 A2; and U.S. Patent Nos.5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784; the disclosures of which are incorporated by reference herein.

[0084] The term “chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0085] A “diabody” is a small antibody fragment with two antigen-binding sites. The fragments comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., European Patent No. EP 404,097, International Patent Publication No. WO 93 / 11161; and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0086] The term “glycosylation” refers to a modified derivative of an antibody. An aglycoslated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Aglycosylation may increase the affinity of the antibody for DB2 / 650358429.1 33Attorney Docket No.116983-5132-WO antigen, as described in U.S. Patent Nos.5,714,350 and 6,350,861. Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8− / − cell lines were created by the targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see e.g. U.S. Patent Publication No.2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example, European Patent No. EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1,6 bond-related enzyme, and also describes cell lines which have a low enzyme activity for adding fucose to the N- acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication WO 03 / 035835 describes a variant CHO cell line, Lec 13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, et al., J. Biol. Chem.2002, 277, 26733-26740. International Patent Publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N- acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech.1999, 17, 176-180). Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino, et al., Biochem.1975, 14, 5516-5523. DB2 / 650358429.1 34Attorney Docket No.116983-5132-WO

[0087] “Pegylation” refers to a modified antibody, or a fragment thereof, that typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Pegylation may, for example, increase the biological (e.g., serum) half life of the antibody. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, as described for example in European Patent Nos. EP 0154316 and EP 0401384 and U.S. Patent No.5,824,778, the disclosures of each of which are incorporated by reference herein.

[0088] The term “biosimilar” means a biological product, including a monoclonal antibody or protein, that is highly similar to a U.S. licensed reference biological product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product. Furthermore, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies. Biological products or biological medicines are medicines that are made by or derived from a biological source, such as a bacterium or yeast. They can consist of relatively small molecules such as human insulin or erythropoietin, or complex molecules such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN), a protein approved by drug regulatory authorities with reference to aldesleukin is a “biosimilar to” aldesleukin or is a “biosimilar thereof” of aldesleukin. In Europe, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological applications in Europe is Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC, as amended and therefore in Europe, the biosimilar may be authorized, approved for authorization or subject of an application for authorization under Article 6 of DB2 / 650358429.1 35Attorney Docket No.116983-5132-WO Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The already authorized original biological medicinal product may be referred to as a “reference medicinal product” in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP Guideline on Similar Biological Medicinal Products. In addition, product specific guidelines, including guidelines relating to monoclonal antibody biosimilars, are provided on a product-by-product basis by the EMA and published on its website. A biosimilar as described herein may be similar to the reference medicinal product by way of quality characteristics, biological activity, mechanism of action, safety profiles and / or efficacy. In addition, the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product. Thus, a biosimilar as described herein may be deemed to have similar or highly similar quality characteristics to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have a similar or highly similar safety profile to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product. As described herein, a biosimilar in Europe is compared to a reference medicinal product which has been authorized by the EMA. However, in some instances, the biosimilar may be compared to a biological medicinal product which has been authorized outside the European Economic Area (a non-EEA authorized “comparator”) in certain studies. Such studies include for example certain clinical and in vivo non-clinical studies. As used herein, the term “biosimilar” also relates to a biological medicinal product which has been or may be compared to a non-EEA authorized comparator. Certain biosimilars are proteins such as antibodies, antibody fragments (for example, antigen binding portions) and fusion proteins. A protein biosimilar may have an amino acid sequence that has minor modifications in the amino acid structure (including for example deletions, additions, and / or substitutions of amino acids) which do not significantly affect the function of the polypeptide. The biosimilar may comprise an amino acid sequence having a sequence identity of 97% or greater to the amino acid sequence of its reference medicinal product, e.g., 97%, 98%, 99% or 100%. The biosimilar may comprise one or more post-translational modifications, for example, although not limited to, glycosylation, oxidation, deamidation, and / or truncation which is / are different to the post-translational modifications of the reference medicinal product, provided that the differences do not result in a change in safety and / or efficacy of the medicinal product. The biosimilar may have an identical or different DB2 / 650358429.1 36Attorney Docket No.116983-5132-WO glycosylation pattern to the reference medicinal product. Particularly, although not exclusively, the biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety concerns associated with the reference medicinal product. Additionally, the biosimilar may deviate from the reference medicinal product in for example its strength, pharmaceutical form, formulation, excipients and / or presentation, providing safety and efficacy of the medicinal product is not compromised. The biosimilar may comprise differences in for example pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles as compared to the reference medicinal product but is still deemed sufficiently similar to the reference medicinal product as to be authorized or considered suitable for authorization. In certain circumstances, the biosimilar exhibits different binding characteristics as compared to the reference medicinal product, wherein the different binding characteristics are considered by a Regulatory Authority such as the EMA not to be a barrier for authorization as a similar biological product. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies.

[0089] The term “recombinant lentiviral RNA molecule” refers to a single stranded RNA genome that may comprise at least a portion of a lentivirus genome, including 5’ and 3’ long terminal repeat (LTR) sequences. In some embodiments, the lentivirus genome may be modified to inhibit replication and limit pathogenicity, while retaining function. For example, the env gene, the gag gene, the pol gene, and the rev gene may be removed from the lentivirus genome that is comprised by the recombinant lentiviral RNA molecule. In some embodiments, the recombinant lentiviral RNA molecule is comprised by a lentiviral vector or a recombinant lentiviral particle as described elsewhere in this disclosure.

[0090] The term “lentiviral particle” or “lentiviral virion” refers to lentivirus, which is a subset of retrovirus. Lentiviruses can deliver significant amounts of genetic information into host cells and integrate it into the cellular genome, making genetically-engineered lentiviruses one of the most efficient tools of gene delivery. These lentiviruses contain a promoter which is used to control the expression of a transgene or shRNA but no virulence genes, making them safe to use in the laboratory.

[0091] The term “recombinant lentiviral particle” or “recombinant lentiviral virion” refers to a lentiviral particle or lentiviral virion produced by gene recombinant technologies. A recombinant lentiviral particle or lentiviral virion can be produced using any suitable method, such as by transducing or transfecting a packaging cell-line with a nucleic acid encoding the viral genome and subsequently isolating newly packaged viral particles. It is understood that DB2 / 650358429.1 37Attorney Docket No.116983-5132-WO the recombinant technologies may be performed at a stage upstream of production of the viral vector itself. For example, recombinant technologies may be used to produce a plasmid, and the plasmid may then be produced at a larger scale, and finally the plasmid may be introduced into a cell line for packaging to produce the viral vector.

[0092] The term “lentiviral vector” refers to a recombinant lentiviral particle comprising a recombinant lentiviral RNA molecule, which contains at least a portion of a lentivirus genome, including 5’ and 3’ LTRs, and a nucleotide sequence encoding one or more genes of interest (GOIs). The lentivirus genome may be modified to inhibit replication and limit pathogenicity, while retaining function. For example, the env gene, the gag gene, the pol gene, and the rev gene may be removed from the lentivirus genome and included in helper plasmids.

[0093] The term “transfer vector” refers to a recombinant DNA plasmid containing a nucleotide sequence encoding the recombinant lentiviral RNA molecule, which contains at least a portion of a lentivirus genome, including 5’ and 3’ LTRs, and a nucleotide sequence encoding one or more genes of interest (GOIs),, while one or more “helper plasmid(s)” or “envelope plasmid(s)” contain(s) genes for proteins that appear on the surface of the lentiviral particle or are essential for the function of the lentiviral particle. The transfer vector can be transfected into a packaging cell line along with the helper plasmid(s) to produce lentiviral vectors comprising the nucleotide sequence encoding the one or more GOIs as part of a recombinant lentiviral RNA molecule.

[0094] The term “Env” refers to the envelope glycoproteins on the surface of the lentiviral particle encoded by the env gene in the lentiviral genome. The Env protein contains a surface subunit and a transmembrane subunit. In some embodiments, the Env protein is selected from the group consisting of baboon retroviral envelope (Ba-EVTR), vesicularstomatitis-virus-G protein (VSV-G), and RD114.

[0095] The term “Gag” refers to the structural protein encoded by the gag gene in the lentiviral genome. The Gag protein is generated as a standalone protein or as a fusion protein with the Pol protein (Gag-Pol).

[0096] The term “Pol” refers to the reverse transcriptase and integrase enzymes encoded by the pol gene in the lentiviral genome. The Pol protein is generated as a fusion protein with the Gag protein (Gag-Pol). DB2 / 650358429.1 38Attorney Docket No.116983-5132-WO

[0097] The term “Rev” refers to the protein encoded by the rev gene in the lentiviral genome. Rev bears a leucine-rich nuclear export signal (NES) and, via association with the Rev response element (RRE), mediates nuclear-to-cytoplasmic transport of the partially spliced and unspliced RNAs, resulting in production of Gag, Gag-Pol, Env, and accessory proteins (Pollard & Malim, Annu. Rev. Microbiol., 1998, 52, 491532).

[0098] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human. III. Nucleic Acid Molecules Encoding Cytokines

[0099] Provided herein are nucleic acid molecules comprising a nucleotide sequence encoding one or more genes of interest (GOIs), e.g., cytokines selected from the group consisting of IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof. In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12). In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a TeIL-12 and a tethered IL-15 (TeIL-15). In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a TeIL- 12 and a tethered IL-2 (TeIL-2).

[0100] In some embodiments, the nucleotide sequences encoding the one or more GOIs, e.g., cytokines selected from the group consisting of IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL- 18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof, are provided by two or more nucleic acid molecules. For example, provided herein are a nucleic acid molecule comprising a nucleotide sequence encoding IL-12 or a variant thereof; and a second nucleic acid molecule comprising a nucleotide sequence encoding IL-15 or a variant thereof. In some embodiments, provided herein are a nucleic acid molecule comprising a nucleotide sequence encoding IL-12 or a variant thereof; and a second nucleic acid molecule comprising a nucleotide sequence encoding IL-2 or a variant thereof.

[0101] In some embodiments, the cytokine is a tethered cytokine. For example, the cytokine is linked to a cell membrane anchor moiety that allows the tethering of the cytokine to the cell surface. Suitable cell membrane anchor moieties include, for example, DB2 / 650358429.1 39Attorney Docket No.116983-5132-WO transmembrane domains of endogenous cell surface proteins and fragments thereof. Exemplary transmembrane domains that can be used include, for example, B7-1, B7-2, and CD8a transmembrane domains and fragments thereof. In some embodiments, the cell membrane anchor moiety further includes a transmembrane and intracellular domain of an endogenous cell surface protein or fragment thereof. In some embodiments, the cell membrane anchor moiety is a B7-1, B7-2 or CD8a transmembrane-intracellular domain or fragment thereof. In certain embodiments, the cell membrane anchor moiety is a CD8a transmembrane domain having the amino acid sequence of IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO:40). In certain embodiments, the cell membrane anchor moiety is a B7-1 transmembrane-intracellular domain having the amino acid sequence of LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO:41).

[0102] In certain embodiments, the cell membrane anchor moiety is a non-peptide cell membrane anchor moiety. In exemplary embodiments, the non-peptide cell membrane anchor moiety is a glycophosphatidylinositol (GPI) anchor. GPI anchors have a structure that includes a phosphoethanolamine linker, glycan core, and phospholipid tail. In some embodiments, the glycan core is modified with one or more side chains. In some embodiments, the glycan core is modified with one or more of the following side chains: a phosphoethanolamine group, mannose, galactose, sialic acid, or other sugars.

[0103] The membrane anchored cytokine may include linkers that allow for the linkage of components of the membrane anchored cytokine (e.g. a cytokine to a cell membrane anchor moiety). Suitable linkers include linkers that are at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues in length. In some embodiments, the linker is 5-10, 10-15, 15-20, 20-25, 25-30, 30- 35, 35-40, 45-50, 50-60 amino acids in length. Suitable linkers include, but are not limited: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker. In some embodiments, the linker is a peptide linker that optionally comprises Gly and Ser. In certain embodiments, the peptide linker utilize a glycine-serine polymer, including for example (GS)n (SEQ ID NO:42), (GSGGS)n (SEQ ID NO:43), (GGGS)n (SEQ ID NO:44), (GGGGS)n (SEQ ID NO:45), (GGGGGS)n (SEQ ID NO:46), and (GGGGGGS)n (SEQ ID NO:47), where n is an integer of at least one (and generally from 3 to 10). Additional linkers that can be used with the present compositions and methods are described in U.S. Patent Publication Nos. US 2006 / 0074008, US 20050238649, and US 2006 / 0024317, each of which is incorporated by reference herein in its DB2 / 650358429.1 40Attorney Docket No.116983-5132-WO entirety, and particularly in pertinent parts related to linkers. In some embodiments, the peptide linker is SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO:48).

[0104] In some embodiments, the linker is a cleavable linker. In exemplary embodiments, the cleavable linker allows for the release of the cytokine into the tumor microenvironment. Cleavable linkers are also useful in embodiments, wherein two membrane anchored cytokines are co-expressed in the same cell. In exemplary embodiments, the linker is a self-cleaving 2A peptide. See, e.g., Liu et al., Sci. Rep.7(1):2193 (2017), which is incorporated by reference in relevant parts relating to 2A peptides. 2A peptides are viral oligopeptides that mediate cleavage of polypeptides during translation in eukaryotic cells. In some embodiments, the 2A peptide includes a C-terminus having the amino acid sequence GDVEXiNPGP (SEQ ID NO:49), wherein Xi is any naturally occurring amino acid residue. In certain embodiments, the 2A peptide is a porcine teschovirus-12A peptide (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO:50). In some embodiments, the 2A peptide is an equine rhinitis A virus 2A peptide (GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO:51). In certain embodiments, the 2A peptide is a foot-and-mouth disease virus 2A peptide: (GSGEGRGSLLTCGDVEENPGP, SEQ ID NO:52). In some embodiments, the cleavable linker includes a furin-cleavable sequence. Exemplary furin-cleavable sequences are described for example, Duckert et al., Protein Engineering, Design & Selection 17(1):107-112 (2004), and US Patent No.8,871,906, each of which is incorporated herein by reference, particularly in relevant parts relating to furin-cleavable sequences. In some embodiments, the linker includes a 2A peptide and a furin-cleavable sequence. In exemplary embodiments, the furin-cleavable 2A peptide includes the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:53).

[0105] In some embodiments, the linker is a degradable linker (e.g., a disulfide linker) such that under physiological conditions, the linker degrades, thereby releasing the cytokine. In some embodiments, the cytokine is reversibly linked to functional groups through a degradable linker such that under physiological conditions, the linker degrades and releases the cytokine. Suitable degradable linkers include, but are not limited to: a protease sensitive linker that is sensitive to one or more enzymes present in biological media such as proteases in a tumor microenvironment such a matrix metalloproteases present in a tumor microenvironment or in inflamed tissue (e.g. matrix metalloproteinase 2 (MMP2) or matrix metalloproteinase 9 (MMP9)). DB2 / 650358429.1 41Attorney Docket No.116983-5132-WO

[0106] In other embodiments, the linker is an enzyme-sensitive linker. Exemplary cleavable linker include those that are recognized by one of the following enzymes: metalloprotease MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. See, e.g., US Patent Nos.8,541,203 and 8,580,244, each of which is incorporated by reference in its entirety and in pertinent parts related to cleavable linkers.

[0107] In certain embodiments, the membrane anchored cytokine includes a signal peptide that facilitates the translocation of the cytokine to the cell membrane. Any suitable signal peptide that facilities the localization of the cytokine to the cell membrane can be used. In some embodiments, the signal peptide does not interfere with the bioactivity of the cytokine. Exemplary signal peptide sequences include, but are not limited to: human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal sequence, human prolactin signal sequence, and human IgE signal sequence. In certain embodiments, the fusion protein includes a human IgE signal sequence. In exemplary embodiments, the human IgE signal sequence has the amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO:54). In some embodiments, the human IgE signal sequence includes the amino acid sequence NIKGSPWKGSLLLLLVSNLLLCQSVAP (SEQ ID NO:55). In some embodiments, the signal peptide sequence is an IL-2 signal sequence having the amino acid sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO:56).

[0108] The nucleic acid molecule can further comprise a native or normative promoter operably linked to the nucleotide sequence encoding one or more cytokines. In some embodiments, the nucleotide sequence encoding each cytokine is operably linked to the same promoter. In some embodiments, the nucleotide sequence encoding each cytokine is operably linked to a different promoter. Preferably, the promoter is functional in T cells. The selection of a promoter, e.g., strong, weak, inducible, tissue-specific and developmental- specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non- viral promoter or a viral promoter, e.g., a nuclear factor of activated T-cells (NFAT) promotor, an EF-1a promoter, a cytomegalovirus (CMV) promoter, a CAG promotor, an MND promoter, or an SSFV promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus. DB2 / 650358429.1 42Attorney Docket No.116983-5132-WO

[0109] “NFAT promoter” as used herein means one or more NFAT responsive elements linked to a minimal promoter of any gene expressed by T-cells. Preferably, the minimal promoter of a gene expressed by T-cells is a minimal human IL-2 promoter. The NFAT responsive elements may comprise, e.g., NFATl, NFAT2, NFAT3, and / or NFAT4 responsive elements. The NFAT promoter (or functional portion or functional variant thereof) may comprise any number of binding motifs, e.g., at least two, at least three, at least four, at least five, or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or up to twelve binding motifs. TABLE 4 – NFAT Promoter Related Sequences. Description Nucleic Acid Sequence T G A T A G C A T GGC T

[0110] In a preferred embodiment, the NFAT promoter comprises six NFAT binding motifs. See, e.g., US Patent No.8,556,882, which is incorporated by reference in its entirety and particularly for pertinent parts relating to NFAT promoters. In some embodiments, the NFAT promoter system controls expression of one or more cytokines. In certain embodiments, the cytokine is selected from the group consisting of IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM- CSF, GCSF, or a variant thereof. In some embodiments, the cytokine is a tethered cytokine. In some embodiments, the NFAT promoter system controls expression of IL-12, or a variant DB2 / 650358429.1 43Attorney Docket No.116983-5132-WO thereof. In some embodiments, the NFAT promoter system controls expression of IL-15, or a variant thereof. In some embodiments, the NFAT promoter system controls expression of IL- 18, or a variant thereof. In some embodiments, the NFAT promoter system controls expression of TeIL-12. In some embodiments, the NFAT promoter system controls expression of TeIL-15. In some embodiments, the NFAT promoter system controls expression of TeIL-18.

[0111] As used herein, “interleukin 12”, “IL-12” and “IL12” all refer to an interleukin that is a heterodimeric cytokine encoded by the IL-12A and IL-12B genes (Genbank Accession numbers: NM_000882 (IL-12A) and NM_002187 (IL-12B)). IL-12 is composed of a bundle of four alpha helices and is involved in the differentiation of native T cells into TH1 cells. It is encoded by two separate genes, IL-12A (p35) and IL-12B (p40). The active heterodimer (referred to as 'p70'), and a homodimer of p40 are formed following protein synthesis. IL-12 binds to the IL-12 receptor, which is a heterodimeric receptor formed by IL- 12R-β1 and IL-12R-β2. IL-12 is known as a T cell-stimulating factor that can stimulate the growth and function of T cells. In particular, IL-12 can stimulate the production of interferon gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) from T cells and natural killer (NK) cells and reduce IL-4 mediated suppression of IFN-γ. IL-12 can further mediate enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. Moreover, IL-12 can also have anti-angiogenic activity by increasing production of interferon gamma, which in turn increases the production of the chemokine inducible protein-10 (IP-10 or CXCL10). IP-10 then mediates this anti-angiogenic effect. Thus, without being bound by any particular theory of operation, it is believed that IL-12 can increase the survivability and / or anti-tumor effects of the PBL compositions provided herein.

[0112] In some embodiments, the IL-12 is a full length IL-12, a fragment or a variant of IL-12. In some embodiments, the IL-12 is a human IL-12 or a variant human IL-12. In exemplary embodiments, the IL-12 is a biological active human IL-12 variant. In some embodiments, the IL-12 includes a 1, 2, 3,4 ,5 ,67, 8, 9, or 10 mutations as compared to a wild-type IL-12.

[0113] In some embodiments, the IL-12 comprises an IL-12 p35 subunit or a variant thereof. In some embodiments, the IL-12 p35 subunit is a human IL-12 p35 subunit. In some embodiments, the IL-12 p35 subunit has the amino acid sequence of SEQ ID NO:60. In certain embodiments, the IL-12 comprises an IL-12 p40 subunit or a variant thereof. In some embodiments, the IL-12 p40 subunit has the amino acid sequence of SEQ ID NO:61. In DB2 / 650358429.1 44Attorney Docket No.116983-5132-WO certain embodiments, the IL-12 is a single chain IL-12 polypeptide comprising an IL-12 p35 subunit attached to an IL-12 p40 subunit. Such IL-12 single chain polypeptides advantageously retain one or more of the biological activities of wildtype IL-12. In some embodiments, the single chain IL-12 polypeptide described herein is according to the formula, from N-terminus to C-terminus, (p40)-(L)-(p35), wherein “p40” is an IL-12 p40 subunit, “p35” is IL-12 p35 subunit and L is a linker. In other embodiments, the single chain IL-12 is according to the formula from N-terminus to C-terminus, (p35)-(L)-(p40). Any suitable linker can be used in the single chain IL-12 polypeptide including those described herein. Suitable linkers can include, for example, linkers having the amino acid sequence (GGGGS)xwherein x is an integer from 1-10. Other suitable linkers include, for example, the amino acid sequence GGGGGGS. Exemplary single chain IL-12 linkers than can be used with the subject single chain IL-12 polypeptides are also described in Lieschke et al., Nature Biotechnology 15: 35-40 (1997), which is incorporated herein in its entirety by reference and particularly for its teaching of IL-12 polypeptide linkers. In an exemplary embodiment, the single chain IL-12 polypeptide is a single chain human IL-12 polypeptide (i.e., it includes a human p35 and p40 IL-12 subunit). DB2 / 650358429.1 45Attorney Docket No.116983-5132-WO TABLE 5 – IL-12 Related Sequences. DB2 / 650358429.1 46Attorney Docket No.116983-5132-WO Description Amino Acid Sequence RNLPVATPDPGMFPCLHHSQNLLRAVSNMLA T L TI D W A P P V D K S K H R P R L P V RDB2 / 650358429.1 47Attorney Docket No.116983-5132-WO TeIL-12-Lr1-Ar2 (nucleotide)ATGGATTGGACCTGGATTCTGTTCCTCGTGGCCGCCGCTACCCGCGTGCACTCCATCTGGGAGCTGAAGAAAGACGTGTACGTGGTGGAATTGGATTGGTACC G C G T G A T C T C T C A G C T C T A A C G T G G G T C A G G G T A C C G A T C G C A CDB2 / 650358429.1 48Attorney Docket No.116983-5132-WO CCAGATGTAGAGAGCGCCGCCGCAACGAGCGCCT GCGTCGTGAGAGCGTGAGGCCTGTGTGA (SEQ ID NO:63) des, an IgE signal sequence peptide; amino acids 529-553: peptide linker; amino acids 554-601: membrane anchor), wherein the nucleic acid is operably linked to an NFAT promoter, an EF- 1a promoter, a CMV promotor, a CAG promotor, an MND promoter, or an SSFV promoter, as described herein. See, e.g., US Patent No.8,556,882, which is incorporated by reference in its entirety and particularly for pertinent parts relating to NFAT promoters for IL-12 expression.

[0115] In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a cytokine selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof. In some embodiments, the cytokine is a tethered cytokine. In some embodiments, the cytokine is under the control of an EF1a promotor, a CMV promotor, a CAG promotor, an MND promotor, or an SSFV promoter.

[0116] As used herein, “interleukin 15”, “IL-15” and “IL15” all refer to an interleukin that binds to and signals through a complex composed of an IL-15 specific receptor alpha chain (IL-15Rα), an IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132) (e.g., Genbank Accession numbers: NM_00000585, NP_000576 and NP_751915 (human); and NM_001254747 and NP_001241676 (mouse)). IL-15 has been shown to stimulate T cell proliferation inside tumors. IL-15 also is able to extend the survivability of effector memory CD8+ T cells and is critical for the development of NK cells. Therefore, without being bound by any particular theory of operation, it is believed that modified PBLs associated with an IL-15s described herein exhibit enhanced survival and / or anti-tumor effects.

[0117] IL-15 has a short half-life of less than 40 minutes in vivo. Modifications to IL-15 monomer can improve its in vivo pharmacokinetics in the treatment of cancers. These modifications have generally centered on improving the trans-presentation of IL-15 with the alpha subunit of IL-15 receptor, IL-15Rα. Such modifications include: 1) pre-association of IL-15 and its soluble receptor a-subunit-Fc fusion to form IL-15: IL-15Rα-Fc complex (see, e.g., Rubinstein et al., Proc Natl Acad Sci U.S.A.103:9166–71 (2006)); 2) expression of the DB2 / 650358429.1 49Attorney Docket No.116983-5132-WO superagonist IL-15-sIL-15Rα-sushi protein (see, e.g., Bessard et al., Molecular cancer therapeutics 8: 2736-45 (2009)); and 3) pre-association of human IL-15 mutant IL-15N72D with IL-15Rα-Fc sushi-Fc fusion complex (see, e.g., Zhu et al., Journal of Immunology 183: 3598-6007 (2009)).

[0118] In some embodiments, the IL-15 is a tethered IL-15 (TeIL-15). In some embodiments, the TeIL-15 comprises the amino acid sequence of SEQ ID NO:73 (amino acids 1-18: human IgE signal sequence peptide; amino acids 132-157: peptide linker; amino acids 158-205: membrane anchor).

[0119] In some embodiments, the IL-15 is a full length IL-15, a fragment or a variant of IL-15. In some embodiments, the IL-15 is a human IL-15 or a variant human IL-15. In exemplary embodiments, the IL-15 is a biological active human IL-15 variant. In some embodiments, the IL-15 includes a 1, 2, 3, 4 ,5, 6, 7, 8, 9, or 10 mutations as compared to a wild-type IL-15. In certain embodiments, the IL-15 includes an N72D mutation relative to a wild type human IL-15. In some embodiments, the variant IL-15 exhibits IL-15Rα binding activity.

[0120] In some embodiments, the IL-15 includes an IL-15 and an extracellular domain of an IL-15Rα. In certain embodiments, the IL-15 includes an IL-15 and an IL-15Rα fused to an Fc domain (IL-15Rα-Fc) TABLE 6 – IL-15 Related Sequences. Description Amino Acid Sequence V K G K F S K P :DB2 / 650358429.1 50Attorney Docket No.116983-5132-WO ITCPPPMSVEHADIWVKSYSLYSRERYICNSG Human IL-15R-alpha-Su (65aa truncated FKRKAGTSSLTECVLNKATNVAHWTTPSLK extracellular domain) CIR (SEQ ID NO: 66) K K L I V Q I V K G K G K P A I G K P A ) Q S L G Y T CDB2 / 650358429.1 51Attorney Docket No.116983-5132-WO CAGTCTATGCACATTGACGCAACACTTTACACCGA GAGCGATGTGCACCCCTCTTGCAAGGTGACTGCC ATGAAATGTTTCTTGCTCGAATTGCAGGTCATCTC C C G T G T T A C Gincludes a complex of human IL-15 and soluble human IL-15Rα. The combination of human IL-15 with soluble human IL-15Rα forms an IL-15 SA complex that possesses greater biological activity than human IL-15 alone. Soluble human IL-15Rα, as well as truncated versions of the extracellular domain, has been described in the art (Wei et al., 2001 J of Immunol.167: 277-282). The amino acid sequence of human IL-15Rα is set forth in SEQ ID NO: 72. In some embodiments, the IL-15SA includes a complex of human IL-15 and soluble human. IL-15Rα comprising all or a portion of the extracellular domain, without the transmembrane or cytoplasmic domain. In some embodiments, the IL-15SA includes a complex of human IL-15 and soluble human IL-15Rα that includes the full extracellular domain or a truncated form of the extracellular domain which retains IL-15 binding activity.

[0122] In some embodiments, the IL-15SA includes a complex of human IL-15 and soluble human IL-15Rα that includes a truncated form of the extracellular domain which retains IL-15 binding activity. In some embodiments, the soluble human IL-15Rα includes amino acids 1-60, 1-61, 1-62, 1-63, 1-64 or 1-65 of human IL-15Rα. In some embodiments, the soluble human IL-15Rα includes amino acids 1-80, 1-81, 1-82, 1-83, 1-84 or 1-85 of human IL-15Rα. In some embodiments, the soluble human IL-15Rα includes amino acids 1- 180, 1-181, or 1-182 of human IL-15Rα.

[0123] In some embodiments, the cytokine is an IL-15SA comprising a complex of human IL-15 and soluble human IL-15Rα comprising a truncated form of the extracellular domain which retains IL-15 binding activity and comprises a Sushi domain. The Sushi domain of IL-15Rα is described in the art as approximately 60 amino acids in length and DB2 / 650358429.1 52Attorney Docket No.116983-5132-WO comprises 4 cysteines. (Wei et al., 2001). Truncated forms of soluble human IL-15Rα which retain IL-15 activity and comprise a Sushi domain are useful in IL-15SA of the present disclosure.

[0124] In some embodiments, the cytokine includes a complex comprising soluble human IL-15Rα expressed as a fusion protein, such as an Fc fusion as described herein (e.g., human IgG1 Fc), with IL-15. In some embodiments, IL-15SA comprises a dimeric human IL- 15RαFc fusion protein (e.g., human IgG1 Fc) complexed with two human IL-15 molecules.

[0125] In some embodiments, the cytokine is an IL-15SA cytokine complex that includes an IL-15 molecule comprising an amino acid sequence set forth in SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO:68, or SEQ ID NO:69. In some embodiments, an IL-15SA cytokine complex comprises a soluble IL-15Rα molecule comprising a sequence of SEQ ID NO:66, SEQ ID NO: 70 or SEQ ID NO:71.

[0126] In some embodiments, the cytokine is an IL-15SA cytokine complex that includes a dimeric IL-15RαFc fusion protein complexed with two IL-15 molecules. In some embodiments, IL-15-SA comprises a dimeric IL-15RαSu (Sushi domain) / Fc (SEQ ID NO:65) and two IL-15N72D (SEQ ID NO:64) molecules (also known as ALT-803), as described in US20140134128, incorporated herein by reference. In some embodiments, the IL-15SA comprises a dimeric IL-15RαSu / Fc molecule (SEQ ID NO: 65) and two IL-15 molecules (SEQ ID NO: 67). In some embodiments, the IL-15SA comprises a dimeric IL-15RαSu / Fc molecule (SEQ ID NO: 65) and two IL-15 molecules (SEQ ID NO:68). In some embodiments, the IL-15SA comprises a dimeric IL-15RαSu / Fc molecule (SEQ ID NO:65) and two IL-15 molecules (SEQ ID NO:69).

[0127] In some embodiments, the IL-15SA includes a dimeric IL-15RαSu / Fc molecule (SEQ ID NO:65) and two IL-15 molecules having amino acid sequences selected from SEQ ID NO: 64, 67, 68, and 69.

[0128] In some embodiments, the IL-15SA includes a soluble IL-15Rα molecule (SEQ ID NO:66) and two IL-15 molecules (SEQ ID NO:64). In some embodiments, the IL- 15SA comprises a soluble IL-15Rα molecule (SEQ ID NO:66) and two IL-15 molecules (SEQ ID NO:67). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO:66) and two IL-15 molecules (SEQ ID NO:68). In some embodiments, the IL- 15SA comprises a soluble IL-15Rα molecule (SEQ ID NO:66) and two IL-15 molecules (SEQ ID NO:69). DB2 / 650358429.1 53Attorney Docket No.116983-5132-WO

[0129] In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO:70) and two IL-15 molecules (SEQ ID NO:64). In some embodiments, the IL- 15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 70) and two IL-15 molecules (SEQ ID NO:67). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 70) and two IL-15 molecules (SEQ ID NO:68). In some embodiments, the IL- 15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 70) and two IL-15 molecules (SEQ ID NO:69).

[0130] In some embodiments, the IL-15SA includes a soluble IL-15Rα molecule (SEQ ID NO:71) and two IL-15 molecules (SEQ ID NO:64). In some embodiments, the IL- 15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 71) and two IL-15 molecules (SEQ ID NO:67). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 265) and two IL-15 molecules (SEQ ID NO:68). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 265) and two IL-15 molecules (SEQ ID NO:69).

[0131] In some embodiments, the IL-15SA comprises a dimeric IL-15RαSu / Fc (SEQ ID NO:65) molecule and two IL-15 molecules (SEQ ID NO:68). In some embodiments, the IL-15SA includes a dimeric IL-15RαSu / Fc (SEQ ID NO:65) molecule and two IL-15 molecules (SEQ ID NO:69).

[0132] In some embodiments, the IL-15SA includes SEQ ID NO:65 and SEQ ID NO:66. In some embodiments IL-15SA comprises SEQ ID NO:67 or SEQ ID NO:68. In some embodiments the IL-15SA comprises SEQ ID NO:67 and SEQ ID NO:65. In some embodiments the IL-15SA comprises SEQ ID NO:68 and SEQ ID NO:65. In some embodiments the IL-15SA comprises SEQ ID NO:69 and SEQ ID NO:65. In some embodiments, the IL-15SA comprises SEQ ID NO:67 and SEQ ID NO:66. In some embodiments the IL-15SA comprises SEQ ID NO:68 and SEQ ID NO:66.

[0133] As used herein, “interleukin 18”, “IL-18,” “IL18,” “IGIF,” “IL-1g,” “interferon-gamma inducing factor,” and “IL1F4,” all refer to an interleukin that is a heterodimeric cytokine encoded by the IL-18 gene (e.g., Genbank Accession numbers: NM_001243211, NM_001562 and NM_001386420). IL-18, structurally similar to IL-1β, is a member of IL-1 superfamily of cytokines. This cytokine, which is expressed by many human lymphoid and nonlymphoid cells, has an important role in inflammatory processes. IL-18 in combination with IL-12 can activate cytotoxic T cells (CTLs), as well as natural DB2 / 650358429.1 54Attorney Docket No.116983-5132-WO killer (NK) cells, to produce IFN-γ and, therefore, contributes to tumor immunity. Thus, without being bound by any particular theory of operation, it is believed that IL-18 can enhance the anti-tumor effects of the PBL compositions provided herein.

[0134] In some embodiments, the IL-18 is a tethered IL-18 (TeIL-18). In some embodiments, the TeIL-18 comprises the amino acid sequence of SEQ ID NO:132 (amino acids 1-18: human IgE signal sequence peptide; amino acids 176-200: peptide linker; amino acids 201-248: membrane anchor). In some embodiments, the TeIL-18 comprises the amino acid sequence of SEQ ID NO:134 (amino acids 1-18: human IgE signal sequence peptide; amino acids 175-199: peptide linker; amino acids 200-247: membrane anchor).

[0135] In some embodiments, the IL-18 is a full length IL-18, a fragment or a variant of IL-18. In some embodiments, the IL-18 is a human IL-18 or a variant human IL-18. In exemplary embodiments, the IL-18 is a biological active human IL-18 variant. In some embodiments, the IL-18 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations as compared to a wild-type IL-18 (SEQ ID NO:75). In some embodiments, the bioactive variant is a decoy resistant IL-18 variant (“DR-IL18,” or “DR-IL-18”) that provides IL-18 signaling activity even in the presence of an inhibitory molecule such as IL-18 binding protein (IL-18BP). Exemplary IL-18 variants that can be included in the subject modified PBLs described herein are shown below in Table 7. Additional IL-18 variants that can be included in the subject modified PBLs are described in WO 2022 / 094473, which is incorporated by reference in its entirety and particular with respect to disclosures relating to variant DR-IL-18.

[0136] In some embodiments, the variant IL-18 includes a stability mutation pair selection from: C38S / C68S, C38S / C68G, C38S / C68A, C38S / C68D, and C38S / C68N [relative to the human wild-type IL-18 - SEQ ID NO: 75]. In some embodiments, the variant IL-18 includes mutations at amino acid positions M51 (e.g., M51E, M51R, M51K, M51T, M51D, or M51N), K53 (e.g., K53G, K53S, K53T, or K53R), Q56 (e g., Q56G, Q56R, Q56L, Q56E, Q56A, Q56V, or Q56K), D110 (e.g., D110S, D110N, D110G, D110K, D110H, D110Q, or D110E) and N111 (e.g., N111G, N111R, N111S, N111D, N111H, or N111Y) in addition to a stabilizing mutation pair selected from: C38S / C68S, C38S / C68G, C38S / C68A, C38S / C68D, and C38S / C68N [relative to the human wild type IL-18 - SEQ ID NO: 75], In some such cases the stabilized IL-18 variant polypeptide additionally includes a mutation at amino acid position S105 (e.g., S105D, S105A, S105N, S105R, S105D, or S105K); and in DB2 / 650358429.1 55Attorney Docket No.116983-5132-WO some cases, further includes mutations at amino acid positions P57 (e.g., P57A, P57L, P57G, or P57K) and M60 (e.g., M60L, M60R, M60K, or M60Q). TABLE 7 – IL-18 Related Sequences. Description Amino Acid Sequence YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEV T D T E T K E L E T K E T K E TDB2 / 650358429.1 56Attorney Docket No.116983-5132-WO Description Amino Acid Sequence ISVKDEKISTLSCENKIISFKEMNPPDNIKDTK E T K E T K E T D V T T D T DDB2 / 650358429.1 57Attorney Docket No.116983-5132-WO Description Amino Acid Sequence YFGKLESQLSVIRNLNDQVLFIDQGNRPLFET D V T A D D T D TIS D I S D I SDB2 / 650358429.1 58Attorney Docket No.116983-5132-WO Description Amino Acid Sequence KERDLFKLILKKEDELGDRSIMFTTQHED D I S D I S T D T D T D D I S D I SDB2 / 650358429.1Attorney Docket No.116983-5132-WO Description Amino Acid Sequence DIIFFKRRVPGHNHKMQFESSSYEGYFLACE D I S T D D I S D I S D I S T D T KDB2 / 650358429.1Attorney Docket No.116983-5132-WO Description Amino Acid Sequence RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDI S T K E V T C V T C V T C T C T C V T CDB2 / 650358429.1Attorney Docket No.116983-5132-WO Description Amino Acid Sequence T K E T D T C T C T K V T C V T C V T CDB2 / 650358429.1 62Attorney Docket No.116983-5132-WO Description Amino Acid Sequence EKERDLFKLILKKEDELGDRSIMFTVQNED V T D T K E T C T D T C T C T C L FIDB2 / 650358429.1 63Attorney Docket No.116983-5132-WO Description Amino Acid Sequence ISMYKDSQPRGMAVTISVKCEKISTLSCENKIISF F G C G C C C G C A G G C G Q L FI E Y Q N C A ADB2 / 650358429.1 64Attorney Docket No.116983-5132-WO Description Amino Acid Sequence CCATCTTTATCATCTCCAAGTACTCCGATTCTC A C G G G G C T DAccession numbers: NM_001207006 and NP_001193935 (human); and NM_0001291041 and NP_001277970 (mouse)) all refer to a member of a cytokine that binds to IL-21 receptor and has potent regulatory effects on cells of the immune system, including natural killer (NK) cells and cytotoxic cells and binds to IL-21 receptor that can destroy virally infected or cancerous cells. Thus, without being bound by any particular theory of operation, it is believed that IL-21 can increase the survivability and / or anti-tumor effects of the PBL compositions provided herein.

[0138] In some embodiments, the IL-21 is a tethered IL-21. In some embodiments, the tethered IL-21 comprises the amino acid sequence of SEQ ID NO: 137 (amino acids 1-18: human IgE signal sequence peptide; amino acids 152-197: peptide linker; amino acids 198- 245: membrane anchor).

[0139] In some embodiments, the IL-21 is a human IL-21 (SEQ ID NO: 136). In some embodiments, the IL-21 associated with the modified PBL is a full length IL-21, a fragment or a variant of IL-21. In some embodiments, the IL-21 is a human IL-21 or a variant human IL-21. In exemplary embodiments, the IL-21 is a biological active human IL- 21 variant. In some embodiments, the IL-21 includes a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations as compared to a wild-type IL-21. DB2 / 650358429.1 65Attorney Docket No.116983-5132-WO TABLE 8 – IL-21 Related Sequences. Description Amino Acid Sequence QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEQS P S G RAccession numbers: NM_000586 and NP_000577 (human) all refer to a member of a cytokine that binds to IL-2 receptor. IL-2 enhances activation-induced cell death (AICD). IL- 2 also promotes the differentiation of T cells into effector T cells and into memory T cells when the initial T cell is also stimulated by an antigen, thus helping the body fight off infections. Together with other polarizing cytokines, IL-2 stimulates naive CD4+ T cell differentiation into Th1 and Th2 lymphocytes and impedes differentiation into Th17 and follicular Th lymphocytes. IL-2 also increases the cell killing activity of both natural killer cells and cytotoxic T cells. Thus, without being bound by any particular theory of operation, it is believed that IL-2 can increase the survivability and / or anti-tumor effects of the PBL compositions provided herein.

[0141] In some embodiments, the IL-2 is a tethered IL-2. In some embodiments, the tethered IL-2 comprises the amino acid sequence of SEQ ID NO: 139 (amino acids 1-20: human IL-2 signal peptide; amino acids 154-178: peptide linker; amino acids 179-226: membrane anchor).

[0142] In some embodiments, the IL-2 is a human IL-2 (SEQ ID NO: 138). In some embodiments, the IL-2 associated with the modified PBL is a full length IL-2, a fragment or a variant of IL-2. In some embodiments, the IL-2 is a human IL-2 or a variant human IL-2. In exemplary embodiments, the IL-2 is a biological active human IL-2 variant. In some DB2 / 650358429.1 66Attorney Docket No.116983-5132-WO embodiments, the IL-2 includes a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations as compared to a wild-type IL-2. TABLE 9 – IL-2 Related Sequences. Description Amino Acid Sequence MYRMQLLSCIALSLALVTNSAPTSSSTKKTQF M Q L L V IS S S T T A G G G A G A C A G C G C C C Ccomprises a nucleotide sequence that encodes a truncated CD-19 (tCD19).

[0144] In some embodiments, the nucleic acid molecule provided herein further comprises a nucleotide sequence that encodes an shRNA. In some embodiments, the shRNA inhibits the expression of an immune checkpoint gene. DB2 / 650358429.1 67Attorney Docket No.116983-5132-WO

[0145] Non-limiting examples of immune checkpoint genes that may be silenced or inhibited by the shRNA include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. For example, immune checkpoint genes that may be silenced or inhibited the shRNA may be selected from the group comprising PD-1, CTLA-4, LAG-3, TIM-3, Cish, CBL-B, TIGIT, TET2, TGFβ, and PKA. BAFF (BR3) is described in Bloom, et al., J. Immunother., 2018, in press. According to another example, immune checkpoint genes that may be silenced or inhibited the shRNA may be selected from the group comprising PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof. Exemplary PD-1 shRNA sequences are provided in the table below. TABLE 10 – PD-1 shRNA sequences Construct DNA sequence PD-1 GGATTTCCAGTGGCGAGAGAACTCGAGTTCTCTCGCCACTGGAAATCC (SEQ D Dfurther comprises long terminal repeat (LTR) sequences, for example, a 5’ LTR sequence and DB2 / 650358429.1 68Attorney Docket No.116983-5132-WO a 3’ LTR sequence (Fig.2). The LTR sequences are essential for viral genome integration into the host cell genome and viral gene expression in the host cell.

[0147] In an embodiment of the invention, the nucleic acid molecule disclosed herein is in the form of a recombinant lentiviral RNA molecule. For example, the recombinant lentiviral RNA molecule may comprise at least a portion of a lentivirus genome, including 5’ and 3’ LTRs. In some embodiments, the lentivirus genome may be modified to inhibit replication and limit pathogenicity, while retaining function. For example, the env gene, the gag gene, the pol gene, and the rev gene may be removed from the lentivirus genome and included in helper plasmids. In some embodiments, the recombinant lentiviral RNA molecule is comprised by a lentiviral vector or a recombinant lentiviral particle as described elsewhere in this disclosure.

[0148] In another embodiment of the invention, the nucleic acid molecule disclosed herein is in the form of a recombinant lentiviral proviral DNA molecule. For example, the recombinant lentiviral proviral DNA molecule is comprised by a host cell, e.g., a gene-edited PBL as disclosed herein. In some embodiments, the recombinant lentiviral proviral DNA molecule is integrated into the genome of the host cell, e.g., a gene-edited PBL as disclosed herein. In some embodiments, recombinant lentiviral proviral DNA molecule is comprised by a packaging cell line as described elsewhere in this disclosure. In some embodiments, the recombinant lentiviral proviral DNA molecule is integrated into the genome of the packaging cell line. TABLE 11 – Exemplary nucleic acid molecule sequences C DNA sequence on G G T A G G TDB2 / 650358429.1 69Attorney Docket No.116983-5132-WO - CCAGGTCAAGGAGTTCGGTGATGCAGGACAGTACACGTGTCACAAGGGCG tC GGGAGGTCTTGTCCCATAGCCTGCTGCTCCTGCACAAGAAGGAAGACGGC D ATCTGGTCTACGGATATCCTTAAAGACCAGAAGGAGCCCAAGAACAAAAC G G A G G T C G G G G C C G A T T G G C T G G A T A A G C C CDB2 / 650358429.1 70Attorney Docket No.116983-5132-WO TTGTCTTTGGAGCTGAAAGATGACCGGCCGGCTCGTGACATGTGGGTGAT GGAGACCGGCCTGCTGCTGCCGCGCGCCACGGCCCAGGATGCGGGGAAGT ACTACTGTCACCGCGGCAACCTGACCATGTCGTTCCACTTGGAGATCACCG A G G T A G G T G C C G G A G G T G G G C C G T T G GDB2 / 650358429.1 71Attorney Docket No.116983-5132-WO GTCGTGAGAGCGTGAGGCCTGTGTGAGAATTCCGCCCCCCCCCCCCCCCG GGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTC GGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAA T G G A A A C C C T G A T C A C G G G G TDB2 / 650358429.1 72Attorney Docket No.116983-5132-WO E TCTGTTCCTCGTGGCCGCCGCTACCCGCGTGCACTCCATCTGGGAGCTGAA F1 GAAAGACGTGTACGTGGTGGAATTGGATTGGTACCCCGACGCCCCAGGCG a- AGATGGTGGTTCTGACTTGCGACACGCCGGAGGAGGACGGCATCACCTGG T G C C G G A G G T C G G G G C C G A T T G G C T G G A T A A G CDB2 / 650358429.1 73Attorney Docket No.116983-5132-WO TGGGCCAAGGACCGCCCTGAGATCTGGGAAGGAGAGCCTCCGTGCGTGCC CCCGCGTGATTCGCTCAACCAGAGCCTGTCCCAGGACCTCACGATGGCAC CCGGTAGCACCCTGTGGTTATCCTGCGGCGTCCCCCCCGACTCCGTGTCCC T G A A T A C T T A G G T A G G T G C C G G A G G TDB2 / 650358429.1 74Attorney Docket No.116983-5132-WO CGCGACTGTCATTTGTCGGAAGAACGCGAGCATCTCCGTACGGGCACAGG ATCGCTATTACTCCAGCAGTTGGTCAGAGTGGGCTTCGGTTCCGTGTTCTG GAGGAGGTGGCGGCGGCTCACGCAACCTGCCCGTGGCGACGCCGGACCCG G C C G A T T G G C T G G A T A A G C C C T G A T A G TDB2 / 650358429.1 75Attorney Docket No.116983-5132-WO G G T A G G T G C C G G A G G T G G G C C G T T G G TDB2 / 650358429.1 76Attorney Docket No.116983-5132-WO ATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCG CCAGAACACAGGCTTGATCACGCGTGCCACCATGGATTGGACCTGGATCC TGTTCCTGGTGGCTGCAGCCACCCGTGTTCATTCCCAGGTCACCGACATCA T G GIV. Recombinant Expression Vectors, Lentiviral Expression Systems, Packaging Cell Line

[0150] In an embodiment of the invention, the inventive nucleic acid molecule disclosed herein is carried in a recombinant expression vector. Accordingly, an embodiment of the invention provides a recombinant expression vector comprising any of the inventive nucleic acid molecules described herein with respect to other aspects of the invention.

[0151] In some embodiments, the nucleotide sequences encoding the one or more GOIs, e.g., cytokines selected from the group consisting of IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL- 18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof, are provided by two or more recombinant expression vectors. For example, provided herein are a recombinant expression vector comprising a nucleotide sequence encoding IL-12 or a variant thereof; and a second recombinant expression vector comprising a nucleotide sequence encoding IL-15 or a variant thereof. In some embodiments, provided herein are a recombinant expression vector comprising a nucleotide sequence encoding IL-12 or a variant thereof; and a second recombinant expression vector comprising a nucleotide sequence encoding IL-2 or a variant thereof.

[0152] For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The recombinant DB2 / 650358429.1 77Attorney Docket No.116983-5132-WO expression vector can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring or non-naturally-occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or intemucleotide linkages do not hinder the transcription or replication of the vector. The vector may contain regulatory nucleic acid sequences which provide for expression of the inventive nucleic acid.

[0153] The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas Life Sciences, Glen Bumie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).

[0154] Bacteriophage vectors, such as λGT10, λGT11, λZap II (Stratagene), λEMBL4, and λNMI 149, also can be used. Examples of plant expression vectors useful in the context of the invention include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the invention include pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0155] In some embodiments, the recombinant expression vector is a viral vector. Suitable viral vectors include, without limitation, lentiviral, retroviral, alphaviral, vaccinial, adenoviral, adenoassociated viral, herpes viral, and fowl pox viral vectors, and preferably have a native or engineered capacity to transform T cells.

[0156] The recombinant expression vectors can be prepared using standard recombinant DNA techniques described in, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, (4th Ed.) Cold Spring Harbor Laboratory Press, New York (2012). Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 µ plasmid, λ, SV40, bovine papilloma virus, and the like.

[0157] The recombinant expression vector can comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the DB2 / 650358429.1 78Attorney Docket No.116983-5132-WO type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate, and taking into consideration whether the vector is DNA- or RNA-based.

[0158] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the recombinant expression vectors include, for instance, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0159] The recombinant expression vector can comprise a native or normative promoter operably linked to the nucleic acid molecule. Preferably, the promoter is functional in T cells. The selection of a promoter, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., an NFAT promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long- terminal repeat of the murine stem cell virus.

[0160] Further provided herein is a lentiviral expression system comprising a transfer vector comprising the nucleic acid molecules disclosed herein which can be transcribed into a recombinant lentiviral RNA molecule for packaging into a lentiviral particle, and one or more helper vectors or helper plasmids encoding an Env protein, a Gag protein, a Pol protein, and a Rev protein. In some embodiments, the Env protein is selected from the group consisting of Ba-EVTR, VSV-G, and RD114.

[0161] In some embodiments, the recombinant lentiviral RNA molecule may comprise at least a portion of a lentivirus genome, including 5’ and 3’ LTRs. In some embodiments, the lentivirus genome may be modified to inhibit replication and limit pathogenicity, while retaining function. For example, the env gene, the gag gene, the pol gene, and the rev gene may be removed from the lentivirus genome and included in helper plasmids. In some embodiments, the recombinant lentiviral RNA molecule is comprised by a lentiviral vector or recombinant lentiviral particle as described elsewhere in this disclosure. DB2 / 650358429.1 79Attorney Docket No.116983-5132-WO

[0162] In some embodiments, two helper vectors or helper plasmids encode an Env protein, a Gag protein, a Pol protein, and a Rev protein. In some embodiments, three helper vectors or helper plasmids encode an Env protein, a Gag protein, a Pol protein, and a Rev protein. In some embodiments, four helper vectors or helper plasmids encode an Env protein, a Gag protein, a Pol protein, and a Rev protein. Any combination of helper vectors or helper plasmids can be used to encode an Env protein, a Gag protein, a Pol protein, and a Rev protein. For example, in some embodiments, one helper vectors or helper plasmids encode an Env protein, a Gag protein, and a Pol protein. In some embodiments, one helper vectors or helper plasmids encode a Gag protein, a Pol protein, and a Rev protein. In some embodiments, one helper vectors or helper plasmids encode a Gag protein, and a Pol protein. In some embodiments, one helper vectors or helper plasmids encode en Env protein, and a Rev protein.

[0163] A packaging cell line can be used to package a nucleic acid, e.g., an RNA encoding a transgene, into a lentiviral vector. Accordingly, the systems and methods described herein may comprise, e.g., a lentiviral packaging cell line comprising at least one plasmid adapted for the production of a lentiviral vector, e.g., a lentiviral vector optionally comprising a transgene. Various lentiviral components useful for the production of a lentiviral vector are known in the art. See for example Zufferey et al., 1997, Nat. Biotechnol. 15:871-875 and Dull et al, 1998, J. Virol.72(11): 8463 -8471. The different functions suitable for the production of a lentiviral vector can be provided to the packaging cells in a lentiviral packaging system comprising one or more nucleic acids (e.g., plasmids), e.g., at least one, two, three, or four plasmids, wherein one plasmid encodes a retroviral envelope protein (Env plasmid), one plasmid encodes one or more retroviral packaging proteins, e.g., Gag and Pol proteins (packaging plasmid or Gag-Pol plasmid), one plasmid encodes a lentiviral Rev protein (Rev plasmid) and one or more plasmids comprising at least one gene of interest (GOI) expression cassette (transfer vector). In some embodiments, the lentiviral packaging system further comprises, or a method described herein comprises use of, at least one, two, three, or four plasmids. In some embodiments, the lentiviral packaging system further comprises, or a method described herein comprises use of, a fifth plasmid. In certain embodiments, a method described herein comprises transfecting five plasmids into the packaging cell, wherein the fifth plasmid does not encode a protein of the lentiviral vector packaging system. In some embodiments, the lentiviral packaging system comprises one or more nucleic acids (e.g., plasmids), e.g., five plasmids, wherein one plasmid encodes an DB2 / 650358429.1 80Attorney Docket No.116983-5132-WO expression vector, one plasmid encodes a Tat (e.g., pcDNATat), one plasmid encodes a Rev protein (e.g., pHCMV-Rev), one plasmid encodes a gagpol (e.g., pHCMV-gagpol), and one plasmid encodes VSV-G (e.g., pVSVG), e.g., as described in Rout-Pitt et al., J Biol. Methods 5(2): 1-9, 2018). In some embodiments, a plasmid may comprise a dual gene expression cassette, e.g., a bicistronic cassette, e.g., a bicistronic construct encoding two transgenes of interest. In some embodiments, the first transgene of interest encodes a first cytokine, e.g., TeIL-12, and the second transgene of interest encodes a second cytokine, e.g., IL-15. In some embodiments the retroviral packaging proteins are derived from a lentivirus, e.g., lentiviral packaging proteins, e.g., lentiviral gag and pol proteins.

[0164] In some embodiments, the lentiviral gag protein is a wild-type lentiviral gag protein, and in other embodiments it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the lentiviral pol protein is a wild-type lentiviral pol protein, and in other embodiments it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the rev protein is a wild-type rev protein, and in other embodiments it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the lentiviral vector packaging system may be a pseudotyped lentiviral vector packaging system, comprising a modified envelope protein, e.g., an envelope protein derived from a different virus or a chimeric envelope protein, e.g., the Env plasmid may encode a Ba-EVTR Env protein.

[0165] In some embodiments, a lentiviral vector is generated using a packaging system comprising pMDLgpRRE, pRSV-Rev and pMD.G plasmids (Dull et al., supra), but using a kanamycin resistance marker, e.g., a marker that confers resistance to both kanamycin and neomycin, e.g., neomycin phosphotransferase II instead of an ampicillin gene.

[0166] Therefore, further provided here is a packaging cell line comprising a recombination expression vector disclosed herein, for example, a transfer vector, comprising the nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL- 12) and optionally a cytokine selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL- 15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof. In some embodiments, the cytokine is a tethered cytokine. In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a TeIL-12 and a tethered IL-15 (TeIL-15). In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a TeIL-12 and a tethered IL-18 (TeIL-18). DB2 / 650358429.1 81Attorney Docket No.116983-5132-WO

[0167] In some embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding an shRNA. In some embodiments, the shRNA inhibits the expression of an immune checkpoint gene. In some embodiments, the shRNA inhibits the expression of PD-1.

[0168] Non-limiting examples of immune checkpoint genes that may be silenced or inhibited by the shRNA include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. For example, immune checkpoint genes that may be silenced or inhibited the shRNA may be selected from the group comprising PD-1, CTLA-4, LAG-3, TIM-3, Cish, CBL-B, TIGIT, TET2, TGFβ, and PKA. BAFF (BR3) is described in Bloom, et al., J. Immunother., 2018, in press. According to another example, immune checkpoint genes that may be silenced or inhibited the shRNA may be selected from the group comprising PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof.

[0169] In some embodiments, the packaging cell line is a human cell line. In some embodiments, the packaging cell line is a 293T cell line.

[0170] In some embodiments, the packaging cell line is a stable packaging cell line produced by the EuLV® system (Shenzhen Eureka Biotechnology Co., Limited, Shenzhen, China) described in Xue et al., Cell & Gene Therapy Insights 2022; 8(2), 199–209, the content of which is incorporated by reference in its entirety.

[0171] Any suitable protocols known in the art may be used in developing the stable packaging cell line. For example, Broussau et al., Mol. Thera., 2008, 16:500-507 describes an inducible packaging cell line, 293SF-PacLV, for production of lentiviral vectors in serum- free culture, the content of which is hereby incorporated by reference in its entirety. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2S-M2) of the tetracycline (Tet) switch, is established first. Clones stably expressing the Gag / Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G), are generated next. Expression of DB2 / 650358429.1 82Attorney Docket No.116983-5132-WO Rev and VSV-G is tightly regulated by the cumate and Tet switches in the 293SF-PacLV cell line. Two approaches are used to generate the packaging cell lines. In the first approach (Two-step), a stable clone expressing the Gag / Pol and Rev genes is first generated and characterized before producing a clone expressing VSV-G and additional Rev. In the second approach (One-shot), all the LV components (Gag / Pol, rev, and VSV-G) are added simultaneously through a single transfection event.

[0172] Two-step approach: A cell line derived from a clone of 293 cells (293SF) adapted to grow in suspension and in serum-free medium that expresses CymR is generated. 293SF were transfected with pMPGBFP / CMV5-CymR / tk-neo and a resistant pool of cells is isolated in the presence of neomycin. Clones are then isolated by limiting dilution of the pool. The clones are maintained for 6 weeks in the absence of selective pressure in order to test their stability. The CymR function is analyzed using an adenoviral vector (AdV) expressing β-galactosidase (β-gal) regulated by the CMV5-CuO promoter. The best β-gal On / Off ratio in the presence and absence of cumate is used in order to select the clone producing the optimal quantity of CymR. Clone G, which showed an On / Off ratio of 14, is selected as a recipient for the rtTA2S-M2 transactivator.293SF-CymR-G cells are transfected with plasmid pUDHrtTA2S-M2.hygro. A pool of hygromycin-resistant cells is generated, and clones are obtained by limiting dilution of the pool in the absence of selection. The functioning of CymR and rtTA2S-M2 produced by the clones is tested using an AdV encoding the green fluorescent protein (GFP) regulated by the TR5 and CuO promoters. The clone with the best On / Off ratio is chosen for the development of the packaging cell lines.

[0173] This is done by transfecting it with pMPG-RSV-Rev / CMV-Gag / polRRE, a plasmid that encodes the Rev, Gag, and pol genes of human immunodeficiency virus-1, as well as the resistance for phleomycin as a fusion protein with GFP. Phleomycin-resistant clones are isolated in 96-well plates. The clones with the best GFP expression level are analyzed for LV production by transient transfection with pCSII-CMV5-GFPq and VSV-G. The titers obtained with the best clones are 10- to 55-fold lower than those obtained when the same clones are transfected with plasmids encoding Rev regulated by Rous sarcoma virus (RSV) (pRSV-Rev). Two clones (#19 and #64) are then subcloned by limiting dilution and analyzed for LV production by transient transfection. As observed for the parent clones, the LV titer obtained is significantly higher in the presence of additional Rev.

[0174] Because the previous results indicate that the quantity of Rev produced by the 293SF-Rev-Gag-Pol cells is not optimal, two of the best subclones (#19-17 and #64-8) are DB2 / 650358429.1 83Attorney Docket No.116983-5132-WO co-transfected with plasmids encoding the puromycin resistance (pPuro), Rev (pkCMV5- CuO-Rev) and VSV-G (pTR5-CuO-VSVg-IRES-GFPq) to generate a cell line expressing VSV-G and more Rev. VSV-G is regulated by the cumate switch and the Tet switch, whereas Rev is regulated by the cumate switch only. The puromycin-resistant clones are first screened for VSV-G expression by measuring GFP (through the IRES-GFP) after induction with Dox and cumate. The LV production is then analyzed by transient transfection with pCSII-CMV5- GFPq. Co-transfection of the best clones with pRSV-Rev increases the LV titer by only twofold to fourfold, which is lower than the earlier level. The three best clones are subcloned in the absence of selective pressure. LV production is analyzed by transient transfection as described earlier. The production from the six best subclones can be improved by only twofold to threefold after the addition of Rev, thereby indicating that the quantity of Rev is nearly optimal. The efficacy of the double switch is investigated by measuring the increase of GFP expression (from the stably integrated VSV-G-IRES-GFPq cassette) after induction of clone #16-22. An induction factor over 2,500 is observed in the presence of cumate and Dox. GFP expression in the presence of the two inducers is much higher than when each inducer is used separately. In the presence of cumate only, the GFP expression level increases by a factor of 4.5, thereby indicating that the cumate switch improves the tightness of the Tet switch by this factor.

[0175] One-shot approach: Because Rev is required for the efficient expression of the protease from the Gag gene, which has been reported to be cytotoxic, even a relatively small amount of Rev could be detrimental to the cells. For this reason, a packaging cell line expressing Rev under very tight regulation is generated. pTR5-CuO-Rev, which contains the Rev coding sequence doubly regulated by the Tet switch and the cumate switch is constructed. The 293SF-CymR-rtTA2S-M2 cell line is co-transfected with plasmids encoding Rev, Gag, Pol, VSV-G, and the resistance for puromycin. Stable clones are selected in the presence of puromycin. Clones having the highest GFP induction factor after addition of Dox and cumate are analyzed for LV production by transfection with transfer vector pCSII- CMV5-GFPq. The two best clones are then subcloned without selection and analyzed for LV production. The transfection of the best subclones with pCSIICMV5-GFPq and pRSV-Rev does not increase the amount of LVs produced, thereby suggesting that the quantity of Rev produced by the clones is not limiting.

[0176] The LV production levels obtained in the best clones when using the Two-step (#16-22) and One-shot (#29-6) approaches is then compared. Titers of 8.6 x 106and 2.6 x 107DB2 / 650358429.1 84Attorney Docket No.116983-5132-WO TU / ml are obtained for #16-22 and #29-6, respectively. The amount of p24 produced after induction is also evaluated by enzyme-linked immunosorbent assay (ELISA). The amounts of p24 produced are 39 and 571 ng / ml by #16-22 and #29-6, respectively. The fact that the specific activity (TU / ng of p24) is 219,000 for #16-22 and 45,000 for #29-6 indicates that the latter clone produced more defective or empty virions.

[0177] Briefly, Ba-EVTR, gag / pol, and rev are stably inserted into 293T cells to obtain packaging cell populations, and the optimal packaging cell line is obtained through monoclonal and titer screening. Then, a transfer vector comprising the nucleic acid molecules disclosed herein, i.e., a transfer vector encoding the GOI, for example, a transfer vector comprising the nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12) and optionally a cytokine selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof, is integrated into the optimal packaging cell line. In some embodiments, the cytokine is a tethered cytokine. In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a TeIL-12 and a tethered IL-15 (TeIL-15). In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a TeIL-12 and a tethered IL-18 (TeIL-18).

[0178] In some embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding an shRNA. In some embodiments, the shRNA inhibits the expression of an immune checkpoint gene. In some embodiments, the shRNA inhibits the expression of PD-1.

[0179] Non-limiting examples of immune checkpoint genes that may be silenced or inhibited by the shRNA include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. For example, immune checkpoint genes that may be silenced or inhibited the shRNA may be selected from the group comprising PD-1, CTLA-4, LAG-3, TIM-3, Cish, CBL-B, TIGIT, TET2, TGFβ, and PKA. BAFF (BR3) is described in Bloom, et al., J. Immunother., 2018, in press. According to another example, immune checkpoint genes that may be silenced or inhibited the shRNA DB2 / 650358429.1 85Attorney Docket No.116983-5132-WO may be selected from the group comprising PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof.

[0180] In some embodiments, the packaging cell line comprises a recombinant DNA encoding an Env protein, a Gag protein, a Pol protein, and a Rev protein. In some embodiments, the recombinant DNA encoding one or more of the Env protein, the Gag protein, the Pol protein, and the Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Env protein, the Gag protein, and the Pol protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Env protein, the Gag protein, and the Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Env protein, the Pol protein, and the Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Gag protein, the Pol protein, and the Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the Env protein is selected from the group consisting of Ba-EVTR, VSV-G, and RD114. In some embodiments, the Env protein is a Ba-EVTR Env protein. In some embodiments, the recombinant DNA encoding one or more of the Env protein, the Gag protein, the Pol protein, and the Ba-EVTR Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Ba-EVTR Env protein, the Gag protein, and the Pol protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Ba-EVTR Env protein, the Gag protein, and the Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Ba-EVTR Env protein, the Pol protein, and the Rev protein is integrated into the genome of the packaging cell line.

[0181] In some embodiments, the recombinant DNA encoding the Env protein, the Gag protein, the Pol protein, and the Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the nucleic acid molecule disclosed herein, e.g., the recombinant lentiviral proviral DNA molecule, is integrated into the genome of the packaging cell line.

[0182] In some embodiments, the different functions for production of a lentiviral vector are provided to a plurality of host cells, e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells (e.g., plurality of Expi293F cells growing in suspension under serum-free conditions) by transfection, e.g., transient or stable transfection, of a lentiviral packaging DB2 / 650358429.1 86Attorney Docket No.116983-5132-WO system adapted for producing lentiviral vectors. In some embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of host cells, e.g., HEK293 cells, e.g., Expi293F cells are transfected. Methods for transfection or infection are well known by those of skill in the art. In some embodiments, at least 0.3pg, at least 0.4pg. at least 0.5pg, at least 0.6pg. at least 0.7pg, at least 0.8pg cells, at least 0.9pg, or at least 1.0 pg of lentiviral packaging system is provided per million cells for transfection. In some embodiments, a transfection reagent is used for transfecting the host cells, e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells. In some embodiments, a transfection reagent is used. Transfection reagents are well known in the art and are available from commercial suppliers. Examples of transfection reagents include but are not limited to, Lipofectamine™ (Invitrogen), Polifectamine, LentiTran (Origene), PEIpro® (Polyplus), FectoVIR® - AAV (Polyplus), and ProFection® (Promega). V. Recombinant Lentiviral Particle And Method Of Making

[0183] This disclosure provides, in some embodiments, method of making a recombinant lentiviral particle. The following general steps may be used. First, culturing a population of packaging cells in a cell culture medium. Once sufficient numbers of packaging cells are obtained, the desired nucleic acids, such as a lentiviral vector comprising a nucleic acid molecule encoding the cytokines disclosed herein, can be introduced into the packaging cells. Additional nucleic acids that may be introduced into the packaging cells include plasmids that promote packaging, e.g., plasmids encoding viral gag, pol, env, and rev. The packaging cells then begin to produce recombinant lentiviral particles. After transfection, a nuclease such as benzonase may be added to the culture media.

[0184] In some embodiments, a stable packaging cell line with the VSV-G, gag / pol, and rev integrated into the genome can be used for the production of the recombinant lentiviral particles. In some embodiments, the stable packaging cell line further comprises the lentiviral vector comprising a nucleic acid molecule encoding the cytokines disclosed herein integrated into the genome.

[0185] Any suitable protocols known in the art may be used for producing the recombinant lentiviral particles using a stable packaging cell line. For example, Broussau et al., supra, describes lentiviral particle production using a stable packaging cell line, the content of which is hereby incorporated by reference in its entirety. DB2 / 650358429.1 87Attorney Docket No.116983-5132-WO

[0186] Neither the preparation of large quantities of plasmid nor the transfection procedure would be necessary if a cell line (a producer) that synthetizes all the essential viral functions including the viral RNA are available. Using such a cell line, LV production can be initiated solely by adding the inducers, such as Dox and cumate. The ability of packaging cells to generate stable producers is evaluated. Clones #29-6 and #16-22 are transduced with a conditional-SIN-LV, produced by transfecting packaging cells with the transfer vector pLVR2-GFP23. Clones are isolated by limiting dilution of the pool of transduced cells. The clones having the highest GFP expression following induction with Dox are expended. The production in serum-free suspension culture of the best clones from parent cells #16-22 (16- 22-22) and #29-6 (#29-6-14) is tested. The medium is changed on a daily basis and the number of infectious particles is determined by flow cytometry.

[0187] The amount of p24 produced is also analyzed by ELISA. The behavior of the two clones is quite similar, except for the absolute amount of LVs produced, which is two to three times higher for clones #29-6-14. The number of infectious particles produced increases every day until it reaches a maximum at day 4. It then decreases progressively. The highest titer (3.4 x 107TU / ml) is obtained at day 4 when #29-6-14 is used. The quantity of p24 produced by the cells at different time points follows the same pattern. On day 1, the specific activity of the viral preparation (TU / ng of p24) is 54,000 and 166,000 for #16-22-22 and #29- 6-14, respectively, and decreases progressively. At the end of the production (day 6 or 7) the specific activity is lower by a factor of 3 to 4, thereby indicating that more defective particles are produced at later time points.

[0188] The relative stability of four different producer clones derived from packaging cells #29-6 is evaluated. The LV production is compared after 4 weeks and 18 weeks of culture in the absence of selective pressure. The titers obtained with these four clones does not decrease significantly after 18 weeks of culture. These data indicate that, using the packaging cells described in this study, it is possible to obtain producers whose long-term stability is more than adequate for a large-scale production process. In addition, tests to detect the presence of replication-competent lentiviruses are carried out by infecting 293 cells using concentrated stock of LVs. No replication-competent lentiviruses are detected by ELISA analysis for P24, and no VSV-G is detected by polymerase chain reaction (PCR).

[0189] Because subcloning and analysis of individual clones is a time-consuming process, whether high levels of LVs can be obtained using a pool of cells is investigated. For this experiment, packaging cells #29-6 are transduced with a conditional-SIN-LV produced DB2 / 650358429.1 88Attorney Docket No.116983-5132-WO by transfecting packaging cells with pTet07-CSII-CMVGFPq. The production efficiency of the pool of producers is then tested in serum-free suspension culture in shake flasks. The medium is changed on a daily basis and the number of infectious particles is determined by flow cytometry. The production pattern of the pool is very similar to that of the two clones, except for the fact that the highest titer (1.9 x 107TU / ml) is obtained at day 3 instead of day 4, and that the duration of useful production is one day shorter.

[0190] Without wishing to be bound by theory, in some embodiments, the cell culture medium is a source of contaminating nucleic acids to the final lentiviral preparation, e.g., the culture medium may contain packaging cell DNA from lysed packaging cells. Accordingly, addition of benzonase to the cell culture medium may degrade the contaminating nucleic acids, allowing for improved purification of the recombinant lentiviral particles.

[0191] Next, recombinant lentiviral particles can be harvested from the packaging cell culture to begin purification of the recombinant lentiviral particles. In some embodiments, harvesting of recombinant lentiviral particles comprises separating the supernatant or cell culture media from the packaging cell. In some embodiments, the packaging cell is not lysed before clarification. In some embodiments, the packaging cells may be lysed, and the lysate may be clarified.

[0192] Naturally occurring lentiviruses are a genus of viruses of the Retroviridae family, characterized by a long incubation period. Lentiviruses can typically deliver a significant amount of genetic information into the DNA of the host cell. Examples of lentiviruses include HIV (human immunodeficiency vims; including HIV type 1, and HIV type 2), the etiologic agent of the human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritis-encephalitis vims, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia vims, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency vims (FIV), which causes immune deficiency in cats; bovine immune deficiency vims (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency vims (SIV), which cause immune deficiency and encephalopathy in sub- human primates. Diseases caused by these vimses are characterized by a long incubation period and protracted course. Usually, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T-cells). DB2 / 650358429.1 89Attorney Docket No.116983-5132-WO

[0193] Further provided herein is a recombinant lentiviral particle comprising the recombinant lentiviral RNA molecule disclosed herein. In some embodiments, the recombinant lentiviral particle is produced by the packaging cell line disclosed herein.

[0194] In some embodiments, the recombinant lentiviral particle comprises the recombinant lentiviral vector disclosed herein. In some embodiments, the recombinant lentiviral vector comprises the recombinant lentiviral RNA molecule disclosed herein. In some embodiments, the recombinant lentiviral particle further comprises a capsid enclosing the recombinant RNA viral genome. In some embodiments, the recombinant lentiviral particle further comprises an Env protein selected from the group consisting of Ba-EVTR, VSV-G, and RD114. In some embodiments, the recombinant lentiviral particle further comprises a reverse transcriptase. VI. Method Of Enriching and Expanding trPBLs

[0195] In some embodiments of the present invention directed to methods for enriching and expanding tumor reactive peripheral blood lymphocytes (trPBLs). In some embodiments, the methods may further comprise one or more steps of gene-editing at least a portion of the trPBLs in order to enhance their therapeutic effect. As used herein, “gene-editing,” “gene editing,” and “genome editing” refer to a type of genetic modification in which DNA is permanently modified in the genome of a cell, e.g., DNA is inserted, deleted, modified or replaced within the cell’s genome. In some embodiments, gene-editing causes the expression of a DNA sequence to be silenced (sometimes referred to as a gene knockout) or inhibited / reduced (sometimes referred to as a gene knockdown). In other embodiments, gene-editing causes the expression of a DNA sequence to be enhanced (e.g., by causing over- expression). In accordance with embodiments of the present invention, gene-editing technology is used to enhance the effectiveness of a therapeutic population of trPBLs.

[0196] In some embodiments of the present invention, provided herein is a method of expanding trPBLs, comprising: a) obtaining a sample of peripheral blood mononuclear cells (PBMCs) from a subject; b) selecting T cells in the sample of PBMCs expressing one or more markers for trPBL to produce a population of cells enriched with trPBLs; and c) culturing the population of cells enriched with trPBLs in a cell culture medium to produce an expanded population of trPBLs. DB2 / 650358429.1 90Attorney Docket No.116983-5132-WO

[0197] In some embodiments of the present invention, provided herein is a method of making a population of gene-edited trPBLs, comprising: a) obtaining a sample of peripheral blood mononuclear cells (PBMCs) from a subject; b) selecting T cells in the sample of PBMCs expressing one or more markers for trPBL to produce a population of cells enriched with trPBLs; and c) culturing the population of cells enriched with trPBLs in a cell culture medium to produce an expanded population of trPBLs, and d) at any time, transducing the population of trPBLs with the recombinant lentiviral particle disclosed herein to produce the population of gene-edited trPBLs, wherein the population of gene-edited PBLs expresses one or more cytokines, such as IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof. A. Obtaining PBLs

[0001] In general, PBLs are initially obtained from peripheral blood mononuclear cells (“PBMCs”) for further manipulation as described herein.

[0002] In some embodiments, PBMCs can be obtained from the circulating blood collected from a patient using any number of techniques known to the skilled artisan, such as Ficoll™ separation.

[0003] In some embodiments, PBMCs can be obtained by apheresis from the circulating blood of a patient. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, PBMCs can be obtained by leukapheresis from the circulating blood of a patient.

[0004] In one embodiment, PBMCs collected by apheresis or leukapheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+ -free, Mg2+ -free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis or leukapheresis sample may be removed and the cells directly resuspended in culture media.

[0005] In some embodiments, PBMCs can be obtained by apheresis or leukapheresis from the circulating blood of a patient, wherein the patient has been administered an inhibitor of an DB2 / 650358429.1 91Attorney Docket No.116983-5132-WO immune checkpoint gene before obtaining the sample of PBMCs from the patient. In some embodiments, the immune checkpoint gene is selected from the group consisting of PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR.

[0006] In some embodiment, PBLs may be isolated from PBMCs by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+ CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. B. Enriching Tumor Reactive PBLs

[0007] Once isolated, the PBMCs and / or the PBLs may be enriched for tumor reactive PBLs (trPBLs). In some embodiments, enriching trPBLs may comprise selecting PBLs that express or secrete one or more trPBL-specific markers.

[0008] As used herein, a “trPBL-specific marker” or “trPBL marker” refers to a cell- membrane protein expressed on trPBLs specifically, or a cytokine or chemokine that is secreted by trPBLs specifically. For example, a trPBL marker can be a cell-membrane protein or a cytokine or chemokine that is expressed / secreted by more than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of trPBLs, and / or expressed / secreted by less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of non-tumor reactive PBLs. In some embodiments, a trPBL marker can be a cell-membrane protein or a cytokine or chemokine that is expressed / secreted by trPBLs at a level that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or at least 100-fold higher than the level by non-tumor reactive PBLs. In some embodiments, trPBL-specific markers include 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD49a, CD38, etc.

[0009] The enriching of trPBLs can be achieved through a variety of methods. According to some embodiments, enriching trPBLs comprises selecting 4-1BB positive (4-1BB+) PBLs. DB2 / 650358429.1 92Attorney Docket No.116983-5132-WO According to some embodiments, enriching trPBLs comprises selecting CD103 positive (CD103+) PBLs. According to some embodiments, enriching trPBLs comprises selecting CXCL13 positive PBLs. According to some embodiments, enriching trPBLs comprises selecting IFN-γ positive PBLs. According to some embodiments, enriching trPBLs comprises selecting CXCR6 positive (CXCR6+) PBLs. According to some embodiments, enriching trPBLs comprises selecting CD200 positive (CD200+) PBLs. According to some embodiments, enriching trPBLs comprises selecting HLA-DR positive (HLA-DR+) PBLs. According to some embodiments, enriching trPBLs comprises selecting CD69 positive (CD69+) PBLs. According to some embodiments, enriching trPBLs comprises selecting PD- 1 positive (PD-1+) PBLs. According to some embodiments, enriching trPBLs comprises selecting CD39 positive (CD39+) PBLs. According to some embodiments, enriching trPBLs comprises selecting CD38 positive (CD38+) PBLs. According to some embodiments, enriching trPBLs comprises selecting CD49a positive (CD49a+) PBLs.

[0010] In some embodiments, selecting 4-1BB positive (4-1BB+) PBLs is performed by staining PBLs with an anti-4-1BB antibody. In some embodiments, the anti-4-1BB antibody is a polyclonal antibody e.g., a mouse anti-human polyclonal antibody, a goat anti-human polyclonal antibody, etc. In some embodiments, the anti-4-1BB antibody is a monoclonal antibody.

[0011] In some embodiments, selecting CD103 positive (CD103+) PBLs is performed by staining PBLs with an anti-CD103 antibody. In some embodiments, the anti-CD103 antibody is a polyclonal antibody e.g., a mouse anti-human polyclonal antibody, a goat anti-human polyclonal antibody, etc. In some embodiments, the anti-CD103 antibody is a monoclonal antibody. In some embodiments, the anti-CD103 antibody is conjugated to a magnetic particle.

[0012] In some embodiments, selecting CXCL13 positive (CXCL13+) PBLs is performed by staining PBLs with an anti-CXCL13 antibody. In some embodiments, the anti-CXCL13 antibody is a polyclonal antibody e.g., a mouse anti-human polyclonal antibody, a goat anti- human polyclonal antibody, etc. In some embodiments, the anti-CXCL13 antibody is a monoclonal antibody. In some embodiments, the anti-CXCL13 antibody is conjugated to a magnetic particle.

[0013] In some embodiments, selecting IFN-γ positive (IFN-γ+) PBLs is performed by staining PBLs with one or more anti-IFN-γ antibodies. In some embodiments, the anti-IFN-γ DB2 / 650358429.1 93Attorney Docket No.116983-5132-WO antibody is conjugated to an anti-CD45 antibody. In some embodiments, the anti-IFN-γ antibody is a monoclonal antibody. In some embodiments, the anti-IFN-γ antibody is conjugated to a magnetic particle. In some embodiments, the IFN-γ positive PBLs are selected using the CliniMACS®Cytokine Capture System (IFN-γ) (Miltenyi Biotec).

[0014] In some embodiments, selecting CXCR6 positive (CXCR6+) PBLs is performed by staining PBLs with an anti-CXCR6 antibody. In some embodiments, the anti-CXCR6 antibody is a polyclonal antibody e.g., a mouse anti-human polyclonal antibody, a goat anti- human polyclonal antibody, etc. In some embodiments, the anti-CXCR6 antibody is a monoclonal antibody. In some embodiments, the anti-CXCR6 antibody is conjugated to a magnetic particle.

[0015] In some embodiments, selecting CD200 positive (CD200+) PBLs is performed by staining PBLs with an anti-CD200 antibody. In some embodiments, the anti-CD200 antibody is a polyclonal antibody e.g., a mouse anti-human polyclonal antibody, a goat anti-human polyclonal antibody, etc. In some embodiments, the anti-CD200 antibody is a monoclonal antibody. In some embodiments, the anti-CD200 antibody is conjugated to a magnetic particle.

[0016] In some embodiments, selecting HLA-DR positive (HLA-DR+) PBLs is performed by staining PBLs with an anti-HLA-DR antibody. In some embodiments, the anti-HLA-DR antibody is a polyclonal antibody e.g., a mouse anti-human polyclonal antibody, a goat anti- human polyclonal antibody, etc. In some embodiments, the anti-HLA-DR antibody is a monoclonal antibody. In some embodiments, the anti-HLA-DR antibody is conjugated to a magnetic particle.

[0017] In some embodiments, selecting CD69 positive (CD69+) PBLs is performed by staining PBLs with an anti-CD69 antibody. In some embodiments, the anti-CD200 antibody is a polyclonal antibody e.g., a mouse anti-human polyclonal antibody, a goat anti-human polyclonal antibody, etc. In some embodiments, the anti-CD69 antibody is a monoclonal antibody. In some embodiments, the anti-CD69 antibody is conjugated to a magnetic particle.

[0018] In some embodiments, selecting PD-1 positive (PD-1+) PBLs is performed by staining PBLs with an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is a polyclonal antibody e.g., a mouse anti-human polyclonal antibody, a goat anti-human polyclonal antibody, etc. In some embodiments, the anti-PD-1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-1 antibody is conjugated to a magnetic particle. DB2 / 650358429.1 94Attorney Docket No.116983-5132-WO

[0019] In some embodiments, selecting CD39 positive (CD39+) PBLs is performed by staining PBLs with an anti-CD39 antibody. In some embodiments, the anti-CD39 antibody is a polyclonal antibody e.g., a mouse anti-human polyclonal antibody, a goat anti-human polyclonal antibody, etc. In some embodiments, the anti-CD39 antibody is a monoclonal antibody. In some embodiments, the anti-CD39 antibody is conjugated to a magnetic particle.

[0020] In some embodiments, selecting CD38 positive (CD38+) PBLs is performed by staining PBLs with an anti-CD38 antibody. In some embodiments, the anti-CD38 antibody is a polyclonal antibody e.g., a mouse anti-human polyclonal antibody, a goat anti-human polyclonal antibody, etc. In some embodiments, the anti-CD38 antibody is a monoclonal antibody. In some embodiments, the anti-CD38 antibody is conjugated to a magnetic particle.

[0021] In some embodiments, selecting CD49a positive (CD49a+) PBLs is performed by staining PBLs with an anti-CD49a antibody. In some embodiments, the anti-CD49a antibody is a polyclonal antibody e.g., a mouse anti-human polyclonal antibody, a goat anti-human polyclonal antibody, etc. In some embodiments, the anti-CD49a antibody is a monoclonal antibody. In some embodiments, the anti-CD49a antibody is conjugated to a magnetic particle.

[0022] In some embodiments, separating from the sample a population of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing PBLs may comprise magnetically separating the population from the PBMCs as described in PCT Publication No. WO2023229979, the content of which is hereby incorporated by reference in its entirety. In some embodiments, magnetically separating from the sample a population of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes may comprise immunomagnetically labeling the population of PBLs followed by magnetic separation within the microfluidic device.

[0023] In some embodiments, immunomagnetically labeling a population of PBLs can comprise attaching - directly or indirectly - a magnetic label to a trPBL marker displayed by at least one of the PBLs. In some embodiments, the marker can be 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD49a, CD38, or a combination thereof. In some embodiments, the marker can be 4-1BB. In some embodiments, the marker can be CD103. In some embodiments, the marker can be CXCL13. In some embodiments, the marker can be CD103. In some embodiments, the marker can be IFN-γ. In some DB2 / 650358429.1 95Attorney Docket No.116983-5132-WO embodiments, the marker can be CD200. In some embodiments, the marker can be CXCR6. In some embodiments, the marker can be HLA-DR. In some embodiments, the marker can be CD69. In some embodiments, the marker can be PD-1. In some embodiments, the marker can be CD39. In some embodiments, the marker can be CD38. In some embodiments, the marker can be CD49a. In some embodiments, the PBLs are labeled by an antibody targeting a surface marker of interest (e.g., an anti-CD103 antibody). In some embodiments, the cells are labeled by a multimer targeting a surface marker of interest (e.g., an MHC multimer). In some embodiments, the antibody or multimer is conjugated to a magnetic micro or nano particle (MNP). In some embodiments, the antibody or multimer is labeled by a secondary antibody conjugated with an MNP. In some embodiments, after labeling, the PBLs obtain a level of magnetization as a function of the expression level of the surface marker recognized by the antibody or multimer.

[0024] In some embodiments, the microfluidic device is configured to isolate the population of trPBLs from a population of PBLs based on the levels of magnetization exhibited by the PBLs. For example, the microfluidic device may comprise a sorting chamber comprising several separated zones with varying heights. In some embodiments, in each zone, microstructures are patterned to generate capture pockets that create low-velocity zones for trapping magnetically labeled PBLs. In some embodiments, during operation, the microfluidic device is sandwiched by arrays of magnets that generate constant magnetic fields in the sorting chamber and is connected to a syringe pump for fluidic processing. Without being bound by any theory, when the cells are added to the device, they experience two major forces – the magnetic force generated by the interaction between MNPs and constant magnetic field, and a fluidic drag force which is defined by the fluidic velocity in a specific zone. When the magnetic force overcomes the drag force, a cell acquires enough trapping force to stay in a specific zone. Otherwise, a cell would be flushed into the next zone with a lower drag force, and eventually into the syringe if it cannot be captured by any zone. After sorting, captured cells at each zone are recovered by removing the external magnets.

[0025] In some embodiments, the microfluidic device exhibits about 20 % to about 98 % capture efficiency when capturing a population of rare cells (e.g., a population of trPBLs). In some embodiments, the microfluidic device exhibits about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 20 % to about 92 %, about 20 % to about 94 %, about 20 % to about 96 %, about 20 % to about 98 %, about 30 % to about DB2 / 650358429.1 96Attorney Docket No.116983-5132-WO 40 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 30 % to about 92 %, about 30 % to about 94 %, about 30 % to about 96 %, about 30 % to about 98 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 40 % to about 92 %, about 40 % to about 94 %, about 40 % to about 96 %, about 40 % to about 98 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 50 % to about 92 %, about 50 % to about 94 %, about 50 % to about 96 %, about 50 % to about 98 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 60 % to about 92 %, about 60 % to about 94 %, about 60 % to about 96 %, about 60 % to about 98 %, about 70 % to about 80 %, about 70 % to about 90 %, about 70 % to about 92 %, about 70 % to about 94 %, about 70 % to about 96 %, about 70 % to about 98 %, about 80 % to about 90 %, about 80 % to about 92 %, about 80 % to about 94 %, about 80 % to about 96 %, about 80 % to about 98 %, about 90 % to about 92 %, about 90 % to about 94 %, about 90 % to about 96 %, about 90 % to about 98 %, about 92 % to about 94 %, about 92 % to about 96 %, about 92 % to about 98 %, about 94 % to about 96 %, about 94 % to about 98 %, or about 96 % to about 98 % capture efficiency when capturing a population of rare cells (e.g., a population of trPBLs). In some embodiments, the microfluidic device exhibits about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, about 92 %, about 94 %, about 96 %, or about 98 % capture efficiency when capturing a population of rare cells (e.g., a population of trPBLs). In some embodiments, the microfluidic device exhibits at least about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, about 92 %, about 94 %, or about 96 % capture efficiency when capturing a population of rare cells (e.g., a population of trPBLs). In some embodiments, the microfluidic device exhibits at most about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, about 92 %, about 94 %, about 96 %, or about 98 % capture efficiency when capturing a population of rare cells (e.g., a population of trPBLs).

[0026] In some embodiments, the methods of isolating a population of trPBLs described herein achieves a higher cell recovery than a standard cell-sorting approach (e.g., fluorescence activated cell sorting, or MACS) performed on the same sample. In some embodiments, the microfluidic approach to cell sorting described herein achieves a higher cell recovery than a standard cell sorting approach, while retaining similar purity. DB2 / 650358429.1 97Attorney Docket No.116983-5132-WO

[0027] In some embodiments, the microfluidic approach to cell sorting described herein achieves a higher cell recovery by at least about 0.5 fold to about 50 fold as compared to a standard cell-sorting approach. In some embodiments, the microfluidic approach to cell sorting described herein achieves a higher cell recovery by about 0.5 fold to about 1 fold, about 0.5 fold to about 2 fold, about 0.5 fold to about 2.5 fold, about 0.5 fold to about 5 fold, about 0.5 fold to about 7.5 fold, about 0.5 fold to about 10 fold, about 0.5 fold to about 20 fold, about 0.5 fold to about 30 fold, about 0.5 fold to about 40 fold, about 0.5 fold to about 50 fold, about 1 fold to about 2 fold, about 1 fold to about 2.5 fold, about 1 fold to about 5 fold, about 1 fold to about 7.5 fold, about 1 fold to about 10 fold, about 1 fold to about 20 fold, about 1 fold to about 30 fold, about 1 fold to about 40 fold, about 1 fold to about 50 fold, about 2 fold to about 2.5 fold, about 2 fold to about 5 fold, about 2 fold to about 7.5 fold, about 2 fold to about 10 fold, about 2 fold to about 20 fold, about 2 fold to about 30 fold, about 2 fold to about 40 fold, about 2 fold to about 50 fold, about 2.5 fold to about 5 fold, about 2.5 fold to about 7.5 fold, about 2.5 fold to about 10 fold, about 2.5 fold to about 20 fold, about 2.5 fold to about 30 fold, about 2.5 fold to about 40 fold, about 2.5 fold to about 50 fold, about 5 fold to about 7.5 fold, about 5 fold to about 10 fold, about 5 fold to about 20 fold, about 5 fold to about 30 fold, about 5 fold to about 40 fold, about 5 fold to about 50 fold, about 7.5 fold to about 10 fold, about 7.5 fold to about 20 fold, about 7.5 fold to about 30 fold, about 7.5 fold to about 40 fold, about 7.5 fold to about 50 fold, about 10 fold to about 20 fold, about 10 fold to about 30 fold, about 10 fold to about 40 fold, about 10 fold to about 50 fold, about 20 fold to about 30 fold, about 20 fold to about 40 fold, about 20 fold to about 50 fold, about 30 fold to about 40 fold, about 30 fold to about 50 fold, or about 40 fold to about 50 fold as compared to a standard cell-sorting approach. In some embodiments, the microfluidic approach to cell sorting described herein achieves a higher cell recovery by about 0.5 fold, about 1 fold, about 2 fold, about 2.5 fold, about 5 fold, about 7.5 fold, about 10 fold, about 20 fold, about 30 fold, about 40 fold, or about 50 fold as compared to a standard cell-sorting approach. In some embodiments, the microfluidic approach to cell sorting described herein achieves a higher cell recovery by at least about 0.5 fold, about 1 fold, about 2 fold, about 2.5 fold, about 5 fold, about 7.5 fold, about 10 fold, about 20 fold, about 30 fold, or about 40 fold as compared to a standard cell-sorting approach. In some embodiments, the microfluidic approach to cell sorting described herein achieves a higher cell recovery by at most about 1 fold, about 2 fold, about 2.5 fold, about 5 fold, about 7.5 fold, about 10 fold, about 20 fold, about 30 fold, about 40 fold, or about 50 fold as compared to a standard cell-sorting approach. DB2 / 650358429.1 98Attorney Docket No.116983-5132-WO

[0028] In some embodiments, any of the microfluidic devices disclosed in PCT Publication No. WO 2014 / 166000, the contents of which are incorporated by reference herein, can be used to separate the trPBLs from the peripheral blood sample. In some embodiments, once the trPBLs have been separated, the trPBLs may be eluted from the microfluidic device by removing an attractant acting on the trPBLs (e.g., via removal of a magnetic field). In some embodiments, the captured trPBLs can then be expanded, enhanced, or a combination thereof, by any of the methods disclosed herein.

[0029] Microfluidic devices disclosed herein can be configured to magnetically sort a population of cells. In some embodiments, the population of cells may comprise a population of PBLs. In some embodiments, the population of PBLs may be labeled with magnetic nanoparticles. In some embodiments, each of the magnetic nanoparticles can be about 0-50 nm in diameter, 51- 100 nm in diameter, 100-150 nm in diameter, or 150 – 200 nm in diameter. In some embodiments, each of the magnetic nanoparticles can be about 50 nm in diameter. In some embodiments, compared with conventional microbeads, magnetic nanoparticles can have improved colloidal stability, which may be useful for processing larger samples. In some embodiments, cells labeled with magnetic nanoparticles may be difficult to capture because their orders-of-magnitude lower magnetic susceptibilities, compared to microbeads, result in lower capture efficiencies. Therefore, in some embodiments, the microfluidic devices disclosed herein can include flow rate-reducing structures that give rise to localized regions of lower flow rate, as a sample comprising the cells is flowed through the device. In some embodiments, the presence of such low flow velocity regions can enable capture of the magnetically labeled cells.

[0030] In some embodiments, a microfluidic device disclosed herein can comprise a microfluidic chip. In some embodiments, the microfluidic the chip can comprise a sorting chamber. In some embodiments, the sorting chamber can be etched or molded into the chip. In some embodiments, the sorting chamber can be in communication with a flow inlet and a flow outlet. In some embodiments, the flow inlet can be configured to receive a sample, e.g., a peripheral blood sample comprising a population of trPBLs suspended in a fluid medium, and the outlet can be configured for delivering the fluid medium depleted of said trPBLs. In some instance tubing can be connected to the inlet such that the fluid medium can be delivered into the inlet through the tubing. In some embodiments, tubing can be connected to the outlet such that the fluid medium can be received from the outlet through the tubing. In some embodiments, the tubing can be silicone tubing. In some embodiments, the microfluidic DB2 / 650358429.1 99Attorney Docket No.116983-5132-WO device can comprise a syringe pump capable of controlling the flow rate of fluid medium at the inlet.

[0031] In some embodiments, the sorting chamber can comprise at least one magnetic capture zone. In some embodiments, the sorting chamber can comprise a plurality of magnetic capture zones. In some embodiments, the sorting chamber can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 magnetic capture zones. In some embodiments, the sorting chamber can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 magnetic capture zones. In some embodiments, at least two of the magnetic capture zones can vary in height. In some embodiments, the magnetic capture zones can range from 50 – 800 pm in height. In some embodiments, sorting chamber can comprise three magnetic capture zones, one about 100 pm in height, one about 200 pm in height, and one about 400 pm in height.

[0032] In some embodiments, the microfluidic device can comprise at least one array of magnets positioned on the outer surface of the microfluidic chip, disposed above or below the sorting chamber, such that a magnetic field can be created in the magnetic capture zones by the at least one array of magnets. In some embodiments, the microfluidic device can comprise two arrays of magnetics positioned on two outer surfaces of the microfluidic chip above and below the sorting chamber such that a magnetic field is created in the magnetic capture zones by the two arrays of magnets. In some embodiments, magnets can be positioned in two arrays, with alternating polarities on opposing sides of the sorting chamber. In some embodiments, the at least one array of magnets can produce a magnetic field strength between .1 - .5 T, .5 - 1 T, or 1 - 1.5T in the magnetic capture zones. In some embodiments, the at least one array of magnets can produce a magnetic field strength between .5 - 1 T in the magnetic capture zones. In some embodiments, the magnets can comprise neodymium magnets. In some embodiments, the magnets can comprise N52 Nd FeB magnets.

[0033] In some embodiments, a magnetic capture zone can comprise a plurality of microstructures. In some embodiments, the microstructures can be flow rate-reducing structures configured to improve capture cells labeled with magnetic nanoparticles in the flow. In some embodiments, the microstructures can produce localized regions of lower flow rate, which may allow for capture of the particles (e.g., the reduced flow rate may allow the magnetic force to overcome the drag force on the particles). In some embodiments, the structures can be designed to avoid trapping of non-target particles. For example, despite being lower in flow rate, the regions of lower flow rate may still have enough flow velocity DB2 / 650358429.1 100Attorney Docket No.116983-5132-WO (that is, the flow rate may be at least nonzero) for non-target particles to be washed from the device, while target particles may be trapped in the low flow rate region. In some embodiments, the microstructures are X-shaped.

[0034] In some embodiments, the device can comprise a plurality of magnetic capture zones, with a first zone comprising the inlet of the sorting chamber, a final magnetic capture zone comprising the outlet of the sorting chamber, and a plurality of magnetic capture zones disposed between the first magnetic capture zone and the final magnetic capture zone. In some embodiments, the size or pattern of the microstructures can vary among the capture zones. In some embodiments, the heights can vary among the magnetic capture zones. In some embodiments, the first zone can exhibit the highest linear velocity and thus can retain cells with high magnetic content because the retaining magnetic force can overcome the drag force exerted by the locally high flow velocity. In some embodiments, the other magnetic capture zones can exhibit gradually reduced linear velocities, with the final magnetic capture zone exhibiting the lowest velocity. This design can allow cells with high levels of magnetization to be captured in the first zone of the device, whereas cells with lower magnetization can become sorted in later zones according to level of magnetization.

[0035] In some methods disclosed herein, a population of PBLs suspended in fluid can be propelled through the inlet, across the magnetic capture zones and through the outlet. In some embodiments, magnetically labelled cells can be captured in the capture zones if the magnetic force exerted on the cells is sufficient to overcome the drag force compelling the cell to flow through the capture zone. Otherwise, a cell would be flushed into the next zone with a lower drag force, and eventually into the syringe if it cannot be captured by any zone. After sorting, captured cells at each zone can be recovered by removing the external magnets.

[0036] In some methods disclosed herein, a population of cells can be loaded into the micro fluidic device, through the inlet, at a flow rate of at least 1 milliliters (mL) per hour, 3 mL / h, 6 mL / h, 9 mL / h, 12 mL / h, 15 mL / h, 18 mL / h, 21 mL / h, 24 mL / h, 27 mL / h, 30 mL / h, 35 mL / h, 40 mL / h, 45 mL / h, or 50 mL / h. The magnetic force exerted on the cells can be determined by, for example, the size of the magnetic nanoparticle the number of magnetic nanoparticles attached to the cell, the size of the cell, and the strength of the applied magnetic field. In some embodiments, the relationship between drag force and linear flow velocity in a microfluidic device capable of magnetically capturing particles by leveraging flow rate DB2 / 650358429.1 101Attorney Docket No.116983-5132-WO reducing microstructures can be disclosed in PCT Publication No. WO 2014 / 166000, the content of which is incorporated by reference herein in its entirety.

[0037] In some embodiments, the microfluidic approaches to cell sorting disclosed herein can yield a population of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes about 2 fold to about 20 fold higher than a population of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA- DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes separated from a peripheral blood sample from the subject using fluorescence activated cell sorting (FACS). In some embodiments, the microfluidic approaches to cell sorting described herein can yield a population of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes about 2 fold to about 4 fold, about 2 fold to about 7 fold, about 2 fold to about 8 fold, about 2 fold to about 10 fold, about 2 fold to about 12 fold, about 2 fold to about 14 fold, about 2 fold to about 16 fold, about 2 fold to about 17 fold, about 2 fold to about 18 fold, about 2 fold to about 19 fold, about 2 fold to about 20 fold, about 4 fold to about 7 fold, about 4 fold to about 8 fold, about 4 fold to about 10 fold, about 4 fold to about 12 fold, about 4 fold to about 14 fold, about 4 fold to about 16 fold, about 4 fold to about 17 fold, about 4 fold to about 18 fold, about 4 fold to about 19 fold, about 4 fold to about 20 fold, about 7 fold to about 8 fold, about 7 fold to about 10 fold, about 7 fold to about 12 fold, about 7 fold to about 14 fold, about 7 fold to about 16 fold, about 7 fold to about 17 fold, about 7 fold to about 18 fold, about 7 fold to about 19 fold, about 7 fold to about 20 fold, about 8 fold to about 10 fold, about 8 fold to about 12 fold, about 8 fold to about 14 fold, about 8 fold to about 16 fold, about 8 fold to about 17 fold, about 8 fold to about 18 fold, about 8 fold to about 19 fold, about 8 fold to about 20 fold, about 10 fold to about 12 fold, about 10 fold to about 14 fold, about 10 fold to about 16 fold, about 10 fold to about 17 fold, about 10 fold to about 18 fold, about 10 fold to about 19 fold, about 10 fold to about 20 fold, about 12 fold to about 14 fold, about 12 fold to about 16 fold, about 12 fold to about 17 fold, about 12 fold to about 18 fold, about 12 fold to about 19 fold, about 12 fold to about 20 fold, about 14 fold to about 16 fold, about 14 fold to about 17 fold, about 14 fold to about 18 fold, about 14 fold to about 19 fold, about 14 fold to about 20 fold, about 16 fold to about 17 fold, about 16 fold to about 18 fold, about 16 fold to about 19 fold, about 16 fold to about 20 fold, about 17 fold to about 18 fold, about 17 fold to about 19 fold, about 17 fold to about 20 fold, about 18 fold to about 19 fold, about 18 fold to about 20 fold, or about 19 fold to about 20 fold higher than a population of 4-1BB, CD103, CXCL13, IFN- DB2 / 650358429.1 102Attorney Docket No.116983-5132-WO γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes separated from a peripheral blood sample from the subject using fluorescence activated cell sorting. In some embodiments, the microfluidic approaches to cell sorting described herein can yield a population of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes about 2 fold, about 4 fold, about 7 fold, about 8 fold, about 10 fold, about 12 fold, about 14 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold, or about 20 fold higher than a population of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes separated from a peripheral blood sample from the subject using fluorescence activated cell sorting. In some embodiments, the microfluidic approaches to cell sorting described herein can yield a population of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes at least about 2 fold, about 4 fold, about 7 fold, about 8 fold, about 10 fold, about 12 fold, about 14 fold, about 16 fold, about 17 fold, about 18 fold, or about 19 fold higher than a population of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA- DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes separated from a peripheral blood sample from the subject using fluorescence activated cell sorting. In some embodiments, the microfluidic approaches to cell sorting described herein can yield a population of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes at most about 4 fold, about 7 fold, about 8 fold, about 10 fold, about 12 fold, about 14 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold, or about 20 fold higher than a population of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes separated from a peripheral blood sample from the subject using fluorescence activated cell sorting.

[0038] In some embodiments, the microfluidic approaches to cell-sorting disclosed herein can achieve a percent recovery about 40 % to about 99 % of the 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes. In some embodiments, the microfluidic approaches to cell-sorting disclosed herein can achieve a percent recovery about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 85 %, about 40 % to about 90 %, about 40 % to about 95 %, about 40 % to about 96 %, about 40 % to about 97 %, about 40 % to about 98 %, about 40 % to about 99 %, about 50 % to about 60 %, about 50 DB2 / 650358429.1 103Attorney Docket No.116983-5132-WO % to about 70 %, about 50 % to about 80 %, about 50 % to about 85 %, about 50 % to about 90 %, about 50 % to about 95 %, about 50 % to about 96 %, about 50 % to about 97 %, about 50 % to about 98 %, about 50 % to about 99 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 85 %, about 60 % to about 90 %, about 60 % to about 95 %, about 60 % to about 96 %, about 60 % to about 97 %, about 60 % to about 98 %, about 60 % to about 99 %, about 70 % to about 80 %, about 70 % to about 85 %, about 70 % to about 90 %, about 70 % to about 95 %, about 70 % to about 96 %, about 70 % to about 97 %, about 70 % to about 98 %, about 70 % to about 99 %, about 80 % to about 85 %, about 80 % to about 90 %, about 80 % to about 95 %, about 80 % to about 96 %, about 80 % to about 97 %, about 80 % to about 98 %, about 80 % to about 99 %, about 85 % to about 90 %, about 85 % to about 95 %, about 85 % to about 96 %, about 85 % to about 97 %, about 85 % to about 98 %, about 85 % to about 99 %, about 90 % to about 95 %, about 90 % to about 96 %, about 90 % to about 97 %, about 90 % to about 98 %, about 90 % to about 99 %, about 95 % to about 96 %, about 95 % to about 97 %, about 95 % to about 98 %, about 95 % to about 99 %, about 96 % to about 97 %, about 96 % to about 98 %, about 96 % to about 99 %, about 97 % to about 98 %, about 97 % to about 99 %, or about 98 % to about 99 % of the 103+ lymphocytes. In some embodiments, the microfluidic approaches to cell-sorting disclosed herein can achieve a percent recovery about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, or about 99 % of the 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes. In some embodiments, the microfluidic approaches to cell- sorting disclosed herein can achieve a percent recovery at least about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, or about 98 % of the 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes. In some embodiments, the microfluidic approaches to cell-sorting disclosed herein can achieve a percent recovery at most about 50 %, about 60 %, about 70 %, about 80 %, about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, or about 99 % of the 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD-1, CD39, CD38, and / or CD49a expressing lymphocytes.

[0039] In some embodiments, enriching of trPBLs is performed using a cell sorting method. In some embodiments, the cell sorting method is a flow cytometry method, e.g., flow activated cell sorting (FACS). In some embodiments, the intensity of the fluorophore in both DB2 / 650358429.1 104Attorney Docket No.116983-5132-WO the first population and the population of PBMCs is used to set up FACS gates for establishing levels of intensity that correspond to PBLs negative for one of the markers, and PBLs positive for one of the markers, respectively. In some embodiments, the cell sorting method is performed such that the gates are set at high, medium (also referred to as intermediate), and low (also referred to as negative) using the PBMC, the FMO control, and the sample itself to distinguish the marker-negative or marker-positive populations. In some embodiments, the PBMC is used as the gating control. In some embodiments, the negatives are gated based upon the FMO. In some embodiments, the gating is set up for each sort. In some embodiments, the gating is set-up for each sample of PBMCs. In some embodiments, the gating template is set-up from PBMC’s every 10 days, 20 days, 30 days, 40 days, 50 days, or 60 days. In some embodiments, the gating template is set-up from PBMC’s every 60 days. In some embodiments, the gating template is set-up for each sample of PBMC’s every 10 days, 20 days, 30 days, 40 days, 50 days, or 60 days. In some embodiments, the gating template is set-up for each sample of PBMC’s every 60 days. C. First Expansion

[0040] In some embodiments, the tumor reactive PBLs resulting from the enrichment step are cultured in a first cell culture medium. In some embodiments, no first expansion of the tumor reactive PBLs is performed.

[0041] In some embodiments, the tumor reactive PBLs resulting from the enrichment step are cultured in serum containing IL-2 under conditions that favor the growth of PBLs over tumor and other cells. In some embodiments, the tumor reactive PBLs resulting from the enrichment step are incubated in 2 mL wells in media comprising inactivated human AB serum with 6000 IU / mL of IL-2. In some embodiments, the tumor reactive PBLs are cultured for a period of days, generally from 1 to 14 days. In some embodiments, the tumor reactive PBLs are cultured for a period of 1 to 4 days. In some embodiments, the tumor reactive PBLs are cultured for a period of 1 to 3 days. In some embodiments, the tumor reactive PBLs are cultured for a period of 5 to 11 days. In some embodiments, the tumor reactive PBLs are cultured for a period of 7 to 10 days. In some embodiments, the tumor reactive PBLs are cultured for a period of about 9 days.

[0042] In some embodiments, the first cell culture medium is referred to as “CM”, an abbreviation for culture media. In some embodiments, CM for the first expansion consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and DB2 / 650358429.1 105Attorney Docket No.116983-5132-WO 10 mg / mL gentamicin. In embodiments where cultures are initiated in gas-permeable flasks with a 40 mL capacity and a 10 cm2gas-permeable silicon bottom (for example, G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN, each flask may be loaded with 10–40 × 106viable tumor digest cells or 5–30 tumor fragments in 10–40 mL of CM with IL-2. Both the G- Rex10 and 24-well plates may be incubated in a humidified incubator at 37°C in 5% CO2 and 5 days after culture initiation, half the media may be removed and replaced with fresh CM and IL-2 and after day 5, half the media may be changed every 2–3 days.

[0043] In some embodiments, the culture medium used in the expansion processes disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and / or a serum replacement. In some embodiments, the serum-free or defined medium is used to prevent and / or decrease experimental variation due in part to the lot-to-lot variation of serum- containing media.

[0044] In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and / or serum replacement. In some embodiments, the basal cell medium includes, but is not limited to CTS™ OpTmizer™ T-cell Expansion Basal Medium , CTS™ OpTmizer™ T-Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0045] In some embodiments, the serum supplement or serum replacement includes, but is not limited to one or more of CTS™ OpTmizer T-Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more ingredients selected from the group consisting of glycine, L- histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L- hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturated transferrin, insulin, and compounds containing the trace element moieties Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+and Zr4+. In some embodiments, the DB2 / 650358429.1 106Attorney Docket No.116983-5132-WO defined medium further comprises L-glutamine, sodium bicarbonate and / or 2- mercaptoethanol.

[0046] In some embodiments, the CTS™OpTmizer™ T-cell Immune Cell Serum Replacement is used with conventional growth media, including but not limited to CTS™ OpTmizer™ T-cell Expansion Basal Medium, CTS™ OpTmizer™ T-cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0047] In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined medium is from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the total serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

[0048] In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T- cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1L CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together prior to use. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), along with 2- mercaptoethanol at 55mM. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the media is 55µM. DB2 / 650358429.1 107Attorney Docket No.116983-5132-WO

[0049] In some embodiments, the defined medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1L CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together prior to use. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), along with 2- mercaptoethanol at 55mM. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2- mercaptoethanol, and 2mM of L-glutamine, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further comprises about 3000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L- glutamine, and further comprises about 6000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2- mercaptoethanol, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 3000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 1000 IU / mL to about 6000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher DB2 / 650358429.1 108Attorney Docket No.116983-5132-WO Scientific) and about 2mM glutamine, and further comprises about 3000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 6000 IU / mL of IL-2. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the media is 55µM.

[0050] In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of from about 0.1mM to about 10mM, 0.5mM to about 9mM, 1mM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to about 5 mM. In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2mM.

[0051] In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of from about 5mM to about 150mM, 10mM to about 140mM, 15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about 100mM, 35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about 80mM, 55mM to about 75mM, 60mM to about 70mM, or about 65mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55mM. In some embodiments, the final concentration of 2-mercaptoethanol in the media is 55µM.

[0052] In some embodiments, the defined media described in International PCT Publication No. WO / 1998 / 030679, which is herein incorporated by reference, are useful in the present invention. In that publication, serum-free eukaryotic cell culture media are described. The serum-free, eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting the growth of cells in serum- free culture. The serum-free eukaryotic cell culture medium supplement comprises or is obtained by combining one or more ingredients selected from the group consisting of one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the defined medium further comprises L- glutamine, sodium bicarbonate and / or beta-mercaptoethanol. In some embodiments, the defined medium comprises an albumin or an albumin substitute and one or more ingredients DB2 / 650358429.1 109Attorney Docket No.116983-5132-WO selected from group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more ingredients selected from the group consisting of glycine, L- histidine, L-isoleucine, L-methionine, L- phenylalanine, L-proline, L- hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturated transferrin, insulin, and compounds containing the trace element moieties Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+and Zr4+. In some embodiments, the basal cell media is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0053] In some embodiments, the concentration of glycine in the defined medium is in the range of from about 5-200 mg / L, the concentration of L- histidine is about 5-250 mg / L, the concentration of L-isoleucine is about 5-300 mg / L, the concentration of L-methionine is about 5-200 mg / L, the concentration of L-phenylalanine is about 5-400 mg / L, the concentration of L-proline is about 1-1000 mg / L, the concentration of L- hydroxyproline is about 1-45 mg / L, the concentration of L-serine is about 1-250 mg / L, the concentration of L- threonine is about 10-500 mg / L, the concentration of L-tryptophan is about 2-110 mg / L, the concentration of L-tyrosine is about 3-175 mg / L, the concentration of L-valine is about 5-500 mg / L, the concentration of thiamine is about 1-20 mg / L, the concentration of reduced glutathione is about 1-20 mg / L, the concentration of L-ascorbic acid-2-phosphate is about 1- 200 mg / L, the concentration of iron saturated transferrin is about 1-50 mg / L, the concentration of insulin is about 1-100 mg / L, the concentration of sodium selenite is about 0.000001-0.0001 mg / L, and the concentration of albumin (e.g., AlbuMAX® I) is about 5000- 50,000 mg / L.

[0054] In some embodiments, the non-trace element moiety ingredients in the defined medium are present in the concentration ranges listed in the column under the heading “Concentration Range in 1X Medium” in Table 12 below. In other embodiments, the non- trace element moiety ingredients in the defined medium are present in the final concentrations listed in the column under the heading “A Preferred Embodiment of the 1X DB2 / 650358429.1 110Attorney Docket No.116983-5132-WO Medium” in Table 12. In other embodiments, the defined medium is a basal cell medium comprising a serum free supplement. In some of these embodiments, the serum free supplement comprises non-trace moiety ingredients of the type and in the concentrations listed in the column under the heading “A Preferred Embodiment in Supplement” in Table 12 below. TABLE 12: Concentrations of Non-Trace Element Moiety Ingredients Ingredient A preferred Concentration range A preferred embodiment in in 1X medium embodiment in 1X

[0055] In some embodiments, the osmolarity of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmolarity is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g / L, or about 2.2 g / L sodium bicarbonate. The defined medium can be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100 μM), 2-mercaptoethanol (final concentration of about 100 μM). DB2 / 650358429.1 111Attorney Docket No.116983-5132-WO

[0056] In some embodiments, the defined media described in Smith, et al., Clin Transl Immunology, 4(1) 2015 (doi: 10.1038 / cti.2014.31) are useful in the present invention. Briefly, RPMI or CTS™ OpTmizer™ was used as the basal cell medium, and supplemented with either 0, 2%, 5%, or 10% CTS™ Immune Cell Serum Replacement.

[0057] In some embodiments, the cell medium in the first and / or second gas permeable container is unfiltered. The use of unfiltered cell medium may simplify the procedures necessary to expand the number of cells. In some embodiments, the cell medium in the first and / or second gas permeable container lacks beta-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0058] In some embodiments, the growth media during the first expansion comprises IL-2 or a variant thereof. In some embodiments, the IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments the IL-2 stock solution has a specific activity of 20-30×106IU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 20×106IU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 25×106IU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 30×106IU / mg for a 1 mg vial. In some embodiments, the IL- 2 stock solution has a final concentration of 4-8×106IU / mg of IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of 5-7×106IU / mg of IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of 6×106IU / mg of IL-2. In some embodiments, the first expansion culture media comprises about 10,000 IU / mL of IL-2, about 9,000 IU / mL of IL-2, about 8,000 IU / mL of IL-2, about 7,000 IU / mL of IL-2, about 6000 IU / mL of IL-2 or about 5,000 IU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 9,000 IU / mL of IL-2 to about 5,000 IU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 8,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 7,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 6,000 IU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, DB2 / 650358429.1 112Attorney Docket No.116983-5132-WO about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000 IU / mL, between 2000 and 3000 IU / mL, between 3000 and 4000 IU / mL, between 4000 and 5000 IU / mL, between 5000 and 6000 IU / mL, between 6000 and 7000 IU / mL, between 7000 and 8000 IU / mL, between 1000 and 5000 IU / mL, or about 8000 IU / mL of IL-2.

[0059] In some embodiments, the first expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first expansion can proceed for 1 day to 14 days. In some embodiments, the first expansion can proceed for 2 days to 14 days. In some embodiments, the first expansion can proceed for 3 days to 14 days. In some embodiments, the first expansion can proceed for 4 days to 14 days. In some embodiments, the first expansion can proceed for 5 days to 14 days. In some embodiments, the first expansion can proceed for 6 days to 14 days. In some embodiments, the first expansion can proceed for 7 days to 14 days. In some embodiments, the first expansion can proceed for 8 days to 14 days. In some embodiments, the first expansion can proceed for 9 days to 14 days. In some embodiments, the first expansion can proceed for 10 days to 14 days. In some embodiments, the first expansion can proceed for 11 days to 14 days. In some embodiments, the first expansion can proceed for 12 days to 14 days. In some embodiments, the first expansion can proceed for 13 days to 14 days. In some embodiments, the first expansion can proceed for 14 days. In some embodiments, the first expansion can proceed for 1 day to 11 days. In some embodiments, the first expansion can proceed for 2 days to 11 days. In some embodiments, the first expansion can proceed for 3 days to 11 days. In some embodiments, the first expansion can proceed for 4 days to 11 days. In some embodiments, the first expansion can proceed for 5 days to 11 days. In some embodiments, the first expansion can proceed for 6 days to 11 days. In some embodiments, the first expansion can proceed for 7 days to 11 days. In some embodiments, the first expansion can proceed for 8 days to 11 days. In some embodiments, the first expansion can proceed for 9 days to 11 days. In some embodiments, the first expansion can proceed for 10 days to 11 days. In some embodiments, the first expansion can proceed for 11 days. In some embodiments, the first expansion can proceed for 5 days to 7 days. In some embodiments, the first expansion can proceed for 6 days to 7 days. In some embodiments, the first expansion can proceed for 7 days to 12 days. In some embodiments, the first expansion can proceed for 8 days to 12 days. In some embodiments, the first expansion can proceed for 9 days to 12 days. In some embodiments, the first expansion can proceed for 10 days to 12 DB2 / 650358429.1 113Attorney Docket No.116983-5132-WO days. In some embodiments, the first expansion can proceed for 7 days. In some embodiments, the first expansion can proceed for 9 days.

[0060] In some embodiments, the first expansion is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the PBL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX -10 or a G-REX -100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0061] In some embodiments, the first cell culture medium comprises 6000 IU / mL of IL-2. In some embodiments, the first cell culture medium comprises 3000 IU / mL of IL-2. In some embodiments, the first cell culture medium comprises 2000 IU / mL of IL-2. In some embodiments, the first cell culture medium comprises 1000 IU / mL of IL-2. D. Activation

[0062] In some embodiments, after the first expansion (pre-REP) step the PBLs are activated by adding anti-CD3 agonist and anti-CD28 agonist, such as TransAct, to the culture medium and culturing for about 1 to 3 days, wherein the PBLs will be transduced with the recombinant lentiviral particle disclosed herein to produce the population of gene-edited PBLs.

[0063] In some embodiments, the step of activating the trPBLs after the enrichment step or the second population of PBLs obtained from the first expansion or pre-REP step can be performed for a period that is, is about, is less than, is more than, 1 day, 2 days, 3 days, or a range that is between any of the above values. For example, in some embodiments, the step of activating the trPBLs after the enrichment step or the second population of PBLs is performed for about 1 day. In some embodiments, the step of activating the trPBLs after the enrichment step or the second population of PBLs is performed for about 2 days. In some embodiments, the step of activating the trPBLs after the enrichment step or the second population of PBLs is performed for about 3 days.

[0064] In some embodiments, the step of activating the trPBLs after the enrichment step or the second population of PBLs (obtained from the first expansion or pre-REP step) is performed using anti-CD3 agonist and anti-CD28 agonist, such as TransAct. In some embodiments, the step of activating the trPBLs after the enrichment step or the second population of PBLs is performed using TransAct at 1:10 dilution, at 1:17.5 dilution, at 1:20 DB2 / 650358429.1 114Attorney Docket No.116983-5132-WO dilution, at 1:25 dilution, at 1:30 dilution, at 1:40 dilution, at 1:50 dilution, at 1:60 dilution, at 1:70 dilution, at 1:80 dilution, at 1:90 dilution, or at 1:100 dilution.

[0065] In some embodiments, the step of activating the trPBLs after the enrichment step or the second population of PBLs (obtained from the first expansion or pre-REP step) can be performed by adding the anti-CD3 agonist and anti-CD28 agonist, such as TransAct, to the first cell culture medium. In some embodiments, the step of activating the trPBLs after the enrichment step or the second population of PBLs can be performed by replacing the first cell culture medium with a cell culture medium comprising the anti-CD3 agonist and anti-CD28 agonist, such as TransAct.

[0198] In some embodiments, the method may comprise activating the population of PBLs for 1 day, 2 days, 3 days, or 4 days before transducing the population of PBLs with the recombinant lentiviral particle. In some embodiments, the activating step comprises contacting the population of PBLs with a cytokine selected from the group consisting of IL-2, IL-15, IL-21, IL-7, and a combination thereof.

[0199] In some embodiments, the activating step comprises contacting the population of PBLs with 20ng / mL IL-15. In some embodiments, the activating step comprises contacting the population of PBLs with 10 ng / mL IL-7. In some embodiments, the activating step comprises contacting the population of PBLs with TransAct. In some embodiments, the activating step comprises contacting the population of PBLs with TransAct at a ratio of 1:100. In some embodiments, the activating step is conducted after the first expansion step and before the second expansion step. In some embodiments, the activating step is conducted after the enrichment step and before the second expansion step without a first expansion step. E. Lentiviral Particle Production

[0200] In some embodiments, recombinant lentiviral particles are produced using a recombinant expression vector, one or more helper plasmids encoding viral gag, pol, env, and rev, and / or a packaging cell line disclosed herein.

[0201] The following general steps may be used. First, culturing a population of packaging cells in a cell culture medium. Once sufficient numbers of packaging cells are obtained, the desired nucleic acids, such as a transfer vector comprising a nucleic acid molecule encoding the cytokines disclosed herein, can be introduced into the packaging cells. Additional nucleic acids that may be introduced into the packaging cells include plasmids DB2 / 650358429.1 115Attorney Docket No.116983-5132-WO that promote packaging, e.g., plasmids encoding viral gag, pol, env, and rev. The packaging cells then begin to produce recombinant lentiviral particles.

[0202] In some embodiments, HEK 293T cells are resuspended in virus culture medium (10%FBS, DMEM (high glucose, Glutamine), sodium pyruvate (1% w / v), sodium bicarbonate (0.075%) or HEPES, no Pen / strep).

[0203] On the next day, a Gag / Pol helper plasmid, a Rev helper plasmid, a BaEVTR or VSV-G helper plasmid, and a transfer vector are mixed at 1 µg / µL concentration at a 1:1 molar ratio. Following mixing, 30 µL of TransIT®-Lenti reagent (Mirus Bio) is added and incubated at room temperature for 10 min in 1mL Opti-MEM™ media per HEK 293T dish. HEK 293T cells are then incubated at 37°C, 5% CO2for 2 days.

[0204] Culture supernatant is removed from dishes and fresh media is added to each dish, centrifuged at 200 g for 5 min to remove cellular debris, and then filtered using a .45 µM filter. Viral supernatant is then concentrated via ultracentrifugation for 2 hrs at 60,000 g. Following ultracentrifugation, supernatant is removed and viral pellet is redissolved in Opti- MEM™ media. The viral particle harvesting step may be repeated to optimize viral production. F. Transduction

[0205] In some embodiments, after the activation step, the PBLs are transduced with the recombinant lentiviral particle disclosed herein.

[0206] In some embodiments, the transduction step is performed at a PBL concentration of about 104– 106cells / mL. In some embodiments, the transduction step is performed at a PBL concentration of about 104cells / mL. In some embodiments, the transduction step is performed at a PBL concentration of about 105cells / mL. In some embodiments, the transduction step is performed at a PBL concentration of about 106cells / mL. In some embodiments, the transducing step is performed at a multiplicity of infection (MOI) of about 10 to about 40. In some embodiments, the transducing step is performed at an MOI of about 10. In some embodiments, the transducing step is performed at an MOI of about 20. In some embodiments, the transducing step is performed at an MOI of about 30. In some embodiments, the transducing step is performed at an MOI of about 40.

[0207] In some embodiments, the transducing step is conducted in the presence of RetroNectin or Vectofusin-1. In some embodiments, the transducing step comprises DB2 / 650358429.1 116Attorney Docket No.116983-5132-WO centrifugation. In some embodiments, the transducing step is conducted in the presence of Lentiboost. In some embodiments, the transducing step is conducted in the presence of Lentiboost at a ratio of 1:50.

[0208] In some embodiments, the transducing step is conducted after the first expansion step and before the second expansion step. In some embodiments, the transducing step is conducted after the enrichment step and before the second expansion step without a first expansion step.

[0209] In some embodiments, the method further comprises resting the population of PBLs for 1 day, 2 days, 3 days, or 4 days after the transducing step. G. Second Expansion

[0066] In some embodiments, the PBL cell population is expanded in number after the enrichment step, the first expansion step, and / or the transduction step. This further expansion is referred to herein as the second expansion, which can include expansion processes generally referred to in the art as a rapid expansion process (REP). In some embodiments, the second expansion step is performed without the first expansion step, for example, after the enrichment step and / or the transduction step. The second expansion is generally accomplished using a culture media comprising a number of components, including feeder cells, a cytokine source, and an anti-CD3 agonist antibody, in a gas-permeable container.

[0067] In some embodiments, the second expansion (which can include expansions sometimes referred to as REP) of PBL can be performed using any flasks or containers known by those of skill in the art. In some embodiments, the second expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second expansion can proceed for about 3 days to about 11 days. In some embodiments, the second expansion can proceed for about 1 day to about 10 days. In some embodiments, the second expansion can proceed for about 1 day to about 9 days. In some embodiments, the second expansion can proceed for about 1 day to about 7 days. In some embodiments, the second expansion can proceed for about 1 day to about 5 days. In some embodiments, the second expansion can proceed for about 1 day. In some embodiments, the second expansion can proceed for about 2 days. In some embodiments, the second expansion can proceed for about 3 days. In some embodiments, the second expansion can proceed for about 4 days. In some embodiments, the second expansion can proceed for about 5 days. In some embodiments, the second expansion can proceed for DB2 / 650358429.1 117Attorney Docket No.116983-5132-WO about 6 days. In some embodiments, the second expansion can proceed for about 7 days. In some embodiments, the second expansion can proceed for about 8 days. In some embodiments, the second expansion can proceed for about 9 days. In some embodiments, the second expansion can proceed for about 10 days.

[0068] In some embodiments, the second expansion can be performed in a gas permeable container using the methods of the present disclosure (including for example, expansions referred to as REP). For example, PBLs can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). The non-specific T-cell receptor stimulus can include, for example, an anti-CD3 agonist antibody, such as about 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). PBLs can be expanded to induce further stimulation of the PBLs in vitro by including one or more antigens during the second expansion, including antigenic portions thereof, such as epitope(s), of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 μΜ MART-1 :26-35 (27 L) or gpl 00:209-217 (210M), optionally in the presence of a T-cell growth factor, such as 300 IU / mL IL-2 or IL-15. Other suitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. PBLs may also be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2- expressing antigen-presenting cells. Alternatively, the PBLs can be further re-stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the re-stimulation occurs as part of the second expansion. In some embodiments, the second expansion occurs in the presence of irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0069] In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000 IU / mL, between DB2 / 650358429.1 118Attorney Docket No.116983-5132-WO 2000 and 3000 IU / mL, between 3000 and 4000 IU / mL, between 4000 and 5000 IU / mL, between 5000 and 6000 IU / mL, between 6000 and 7000 IU / mL, between 7000 and 8000 IU / mL, or between 8000 IU / mL of IL-2.

[0070] In some embodiments, the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 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, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 µg / mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0071] In some embodiments the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of PBLs to PBMCs and / or antigen-presenting cells in the rapid expansion and / or the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In some embodiments, the ratio of PBLs to PBMCs in the rapid expansion and / or the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of PBLs to PBMCs in the rapid expansion and / or the second expansion is between 1 to 100 and 1 to 200.

[0072] In some embodiments the APCs are ENLIST cells (Alloplex Biotherapeutics). ENLIST cells are derived from a common tumor cell line to express an array of immunomodulatory ligands designed to specifically engage and activate receptors on various subsets of PBMC in a coordinated fashion. In some embodiments, the ratio of PBLs to ENLIST cells in the rapid expansion and / or the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In some embodiments, the ratio of PBLs to P ENLIST DB2 / 650358429.1 119Attorney Docket No.116983-5132-WO cells in the rapid expansion and / or the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of PBLs to ENLIST cells in the rapid expansion and / or the second expansion is between 1 to 100 and 1 to 200.

[0073] In some embodiments the APCs are artificial APCs (aAPCs). In some embodiments, the ratio of PBLs to aAPCs and / or antigen-presenting cells in the rapid expansion and / or the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In some embodiments, the ratio of PBLs to aAPCs in the rapid expansion and / or the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of PBLs to aAPCs in the rapid expansion and / or the second expansion is between 1 to 100 and 1 to 200.

[0074] In some embodiments, REP and / or the second expansion is performed in flasks with the bulk PBLs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody and 3000 IU / mL IL-2 in 150 ml media. Media replacement is done (generally 2 / 3 media replacement via respiration with fresh media) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REX flasks and gas permeable containers as more fully discussed below.

[0075] In some embodiments, the second expansion (which can include processes referred to as the REP process) is shortened to 1-5 days, wherein the PBLs expanded by such a second expansion have been transduced with the recombinant lentiviral particle disclosed herein to produce the population of gene-edited PBLs. In some embodiments, the second expansion is shortened to 3 days.

[0076] In some embodiments, REP and / or the second expansion may be performed using T-175 flasks and gas permeable bags as previously described (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother.2003, 26, 332-42) or gas permeable cultureware (G-Rex flasks). In some embodiments, the second expansion (including expansions referred to as rapid expansions) is performed in T-175 flasks, and about 1 x 106PBLs suspended in 150 mL of media may be added to each T-175 flask. The PBLs may be cultured in a 1 to 1 mixture of CM and AIM-V medium, supplemented with 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3. The T-175 flasks may be incubated at 37° C in 5% CO2. Half the media may be exchanged on day 5 using 50 / 50 medium with 3000 IU per mL of IL-2. In some embodiments, on day 7 cells from two T-175 flasks may be combined in a 3 DB2 / 650358429.1 120Attorney Docket No.116983-5132-WO L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU per mL of IL-2 was added to the 300 ml of PBL suspension. The number of cells in each bag was counted every day or two and fresh media was added to keep the cell count between 0.5 and 2.0 x 106cells / mL.

[0077] In some embodiments, the second expansion (which can include expansions referred to as REP) may be performed in 500 mL capacity gas permeable flasks with 100 cm gas- permeable silicon bottoms (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5 × 106or 10 × 106PBLs may be cultured with PBMCs in 400 mL of 50 / 50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3 (OKT3). The G-Rex 100 flasks may be incubated at 37°C in 5% CO2. On day 5, 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 × g) for 10 minutes. The PBL pellets may be re-suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL of IL-2, and added back to the original G-Rex 100 flasks. When PBLs are expanded serially in G-Rex 100 flasks, on day 7 the PBL in each G-Rex 100 may be suspended in the 300 mL of media present in each flask and the cell suspension may be divided into 3100 mL aliquots that may be used to seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may be added to each flask. The G-Rex 100 flasks may be incubated at 37° C in 5% CO2and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 may be added to each G-REX 100 flask. The cells may be harvested on day 14 of culture.

[0078] In some embodiments, the second expansion (which can include expansions referred to as REP) may be performed in 500 mL capacity gas permeable flasks with 100 cm gas- permeable silicon bottoms (G-REX-100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5 × 106or 10 × 106PBLs may be cultured with PBMCs in 400 mL of 50 / 50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per mL of anti-CD3 (OKT3). The G-REX-100 (or G- REX100M) flasks may be incubated at 37°C in 5% CO2. On day 5, 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 × g) for 10 minutes. The PBLs pellets may be re-suspended with 150 mL of fresh medium with 5% human AB serum, 6000 IU per mL of IL-2, and added back to the original GREX-100 flasks. When PBLs are expanded serially in GREX-100 flasks, on day 10 or 11 the PBLs can be moved to a larger flask, such as a GREX-500 (or G-REX500M). The cells may be harvested DB2 / 650358429.1 121Attorney Docket No.116983-5132-WO on day 14 of culture. The cells may be harvested on day 15 of culture. The cells may be harvested on day 16 of culture. In some embodiments, media replacement is done until the cells are transferred to an alternative growth chamber. In some embodiments, 2 / 3 of the media is replaced by aspiration of spent media and replacement with an equal volume of fresh media. In some embodiments, alternative growth chambers include GREX flasks and gas permeable containers as more fully discussed below. In some embodiments, the process employed varying centrifugation speeds (400g, 300g, 200g for 5 minutes) and varying numbers of repetitions.

[0079] In some embodiments, the second expansion (including expansions referred to as REP) is performed in flasks with the bulk PBLs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody and 3000 IU / mL IL-2 in 150 ml media. In some embodiments, media replacement is done until the cells are transferred to an alternative growth chamber. In some embodiments, 2 / 3 of the media is replaced by aspiration of spent media followed by infusion with fresh media. In some embodiments, alternative growth chambers include G-REX flasks and gas permeable containers as more fully discussed below.

[0080] In some embodiments, the second expansion culture medium (e.g., sometimes referred to as CM2 or the second cell culture medium), comprises IL-2, OKT-3, as well as the antigen-presenting feeder cells (APCs), as discussed in more detail below.

[0081] In some embodiments, the culture medium used in the expansion processes disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and / or a serum replacement. In some embodiments, the serum-free or defined medium is used to prevent and / or decrease experimental variation due in part to the lot-to-lot variation of serum- containing media.

[0082] In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and / or serum replacement. In some embodiments, the basal cell medium includes, but is not limited to CTS™ OpTmizer™ T-cell Expansion Basal Medium , CTS™ OpTmizer™ T-Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), DB2 / 650358429.1 122Attorney Docket No.116983-5132-WO RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0083] In some embodiments, the serum supplement or serum replacement includes, but is not limited to one or more of CTS™ OpTmizer T-Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more ingredients selected from the group consisting of glycine, L- histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L- hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturated transferrin, insulin, and compounds containing the trace element moieties Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+and Zr4+. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate and / or 2- mercaptoethanol.

[0084] In some embodiments, the CTS™OpTmizer™ T-cell Immune Cell Serum Replacement is used with conventional growth media, including but not limited to CTS™ OpTmizer™ T-cell Expansion Basal Medium, CTS™ OpTmizer™ T-cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0085] In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined medium is from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the total serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium. DB2 / 650358429.1 123Attorney Docket No.116983-5132-WO

[0086] In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T- cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1L CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together prior to use. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), along with 2- mercaptoethanol at 55mM. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the media is 55µM.

[0087] In some embodiments, the defined medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1L CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together prior to use. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), along with 2- mercaptoethanol at 55mM. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2- mercaptoethanol, and 2mM of L-glutamine, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further comprises about 3000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L- glutamine, and further comprises about 6000 IU / mL of IL-2. In some embodiments, the DB2 / 650358429.1 124Attorney Docket No.116983-5132-WO CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2- mercaptoethanol, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 3000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 1000 IU / mL to about 6000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 3000 IU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 6000 IU / mL of IL-2. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the media is 55µM.

[0088] In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of from about 0.1mM to about 10mM, 0.5mM to about 9mM, 1mM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to about 5 mM. In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2mM.

[0089] In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of from about 5mM to about 150mM, 10mM to about 140mM, 15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about 100mM, 35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about 80mM, 55mM to about 75mM, 60mM to about 70mM, or about 65mM. In some embodiments, the serum-free medium or defined medium is supplemented DB2 / 650358429.1 125Attorney Docket No.116983-5132-WO with 2-mercaptoethanol at a concentration of about 55mM. In some embodiments, the final concentration of 2-mercaptoethanol in the media is 55µM.

[0090] In some embodiments, the defined media described in International PCT Publication No. WO / 1998 / 030679, which is herein incorporated by reference, are useful in the present invention. In that publication, serum-free eukaryotic cell culture media are described. The serum-free, eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting the growth of cells in serum- free culture. The serum-free eukaryotic cell culture medium supplement comprises or is obtained by combining one or more ingredients selected from the group consisting of one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the defined medium further comprises L- glutamine, sodium bicarbonate and / or beta-mercaptoethanol. In some embodiments, the defined medium comprises an albumin or an albumin substitute and one or more ingredients selected from group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more ingredients selected from the group consisting of glycine, L- histidine, L-isoleucine, L-methionine, L- phenylalanine, L-proline, L- hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturated transferrin, insulin, and compounds containing the trace element moieties Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+and Zr4+. In some embodiments, the basal cell media is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0091] In some embodiments, the concentration of glycine in the defined medium is in the range of from about 5-200 mg / L, the concentration of L- histidine is about 5-250 mg / L, the concentration of L-isoleucine is about 5-300 mg / L, the concentration of L-methionine is about 5-200 mg / L, the concentration of L-phenylalanine is about 5-400 mg / L, the DB2 / 650358429.1 126Attorney Docket No.116983-5132-WO concentration of L-proline is about 1-1000 mg / L, the concentration of L- hydroxyproline is about 1-45 mg / L, the concentration of L-serine is about 1-250 mg / L, the concentration of L- threonine is about 10-500 mg / L, the concentration of L-tryptophan is about 2-110 mg / L, the concentration of L-tyrosine is about 3-175 mg / L, the concentration of L-valine is about 5-500 mg / L, the concentration of thiamine is about 1-20 mg / L, the concentration of reduced glutathione is about 1-20 mg / L, the concentration of L-ascorbic acid-2-phosphate is about 1- 200 mg / L, the concentration of iron saturated transferrin is about 1-50 mg / L, the concentration of insulin is about 1-100 mg / L, the concentration of sodium selenite is about 0.000001-0.0001 mg / L, and the concentration of albumin (e.g., AlbuMAX® I) is about 5000- 50,000 mg / L.

[0092] In some embodiments, the non-trace element moiety ingredients in the defined medium are present in the concentration ranges listed in the column under the heading “Concentration Range in 1X Medium” in Table 13 below. In other embodiments, the non- trace element moiety ingredients in the defined medium are present in the final concentrations listed in the column under the heading “A Preferred Embodiment of the 1X Medium” in Table 13. In other embodiments, the defined medium is a basal cell medium comprising a serum free supplement. In some of these embodiments, the serum free supplement comprises non-trace moiety ingredients of the type and in the concentrations listed in the column under the heading “A Preferred Embodiment in Supplement” in Table 13 below. TABLE 13: Concentrations of Non-Trace Element Moiety Ingredients Ingredient A preferred Concentration range A preferred embodiment in in 1X medium embodiment in 1XDB2 / 650358429.1 127Attorney Docket No.116983-5132-WO Reduced Glutathione 10 1-20 1.5 Ascorbic Acid-2- 330 1-200 50 PO (M S lt)so e e o e s, e os oa y o e e e e u s e ee a ou and 350 mOsmol. In some embodiments, the osmolarity is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g / L, or about 2.2 g / L sodium bicarbonate. The defined medium can be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100 μM), 2-mercaptoethanol (final concentration of about 100 μM).

[0094] In some embodiments, the defined media described in Smith, et al., Clin Transl Immunology, 4(1) 2015 (doi: 10.1038 / cti.2014.31) are useful in the present invention. Briefly, RPMI or CTS™ OpTmizer™ was used as the basal cell medium, and supplemented with either 0, 2%, 5%, or 10% CTS™ Immune Cell Serum Replacement.

[0095] In some embodiments, the cell medium in the first and / or second gas permeable container is unfiltered. The use of unfiltered cell medium may simplify the procedures necessary to expand the number of cells. In some embodiments, the cell medium in the first and / or second gas permeable container lacks beta-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0096] In some embodiments, the second expansion is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the PBL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX -10 or a G-REX -100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0097] In some embodiments, the steps of the method are completed within a period of about 1-11 days. In some embodiments, the steps of the method are completed within a period of about 1 day. In some embodiments, the steps of the method are completed within a period of about 2 days. In some embodiments, the steps of the method are completed within a period of about 3 days. In some embodiments, the steps of the method are completed within a period of DB2 / 650358429.1 128Attorney Docket No.116983-5132-WO about 4 days. In some embodiments, the steps of the method are completed within a period of about 5 days. In some embodiments, the steps of the method are completed within a period of about 6 days. In some embodiments, the steps of the method are completed within a period of about 7 days. In some embodiments, the steps of the method are completed within a period of about 8 days. In some embodiments, the steps of the method are completed within a period of about 9 days. In some embodiments, the steps of the method are completed within a period of about 10 days. In some embodiments, the steps of the method are completed within a period of about 11 days. In some embodiments, the steps of the method are completed within a period of about 12 days. In some embodiments, the steps of the method are completed within a period of about 13 days. In some embodiments, the steps of the method are completed within a period of about 14 days. In some embodiments, the steps of the method are completed within a period of about 15 days. In some embodiments, the steps of the method are completed within a period of about 16 days. In some embodiments, the steps of the method are completed within a period of about 17 days. In some embodiments, the steps of the method are completed within a period of about 18 days. In some embodiments, the steps of the method are completed within a period of about 19 days. In some embodiments, the steps of the method are completed within a period of about 20 days. In some embodiments, the steps of the method are completed within a period of about 21 days. In some embodiments, the steps of the method are completed within a period of about 22 days.

[0098] In some embodiments, the antigen presenting cells (APCs) are PBMCs. According to some embodiments, the PBMCs are irradiated. According to some embodiments, the PBMCs are allogeneic. According to some embodiments, the PBMCs are irradiated and allogeneic. According to some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0099] In some embodiments, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 IU / mL and 6000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2000 IU / mL and 6000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2500 IU / mL and 6000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3000 IU / mL and 6000 IU / mL in the cell culture medium in the first expansion. In some DB2 / 650358429.1 129Attorney Docket No.116983-5132-WO embodiments, the IL-2 is present at an initial concentration of between 3500 IU / mL and 6000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 4000 IU / mL and 6000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 4500 IU / mL and 6000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 5000 IU / mL and 6000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 5500 IU / mL and 6000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1000 IU / mL and 5000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 IU / mL and 5000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2000 IU / mL and 5000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2500 IU / mL and 5000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3000 IU / mL and 5000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3500 IU / mL and 5000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 4000 IU / mL and 5000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 4500 IU / mL and 5000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1000 IU / mL and 4000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 IU / mL and 4000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2000 IU / mL and 4000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2500 IU / mL and 4000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3000 IU / mL and 4000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3500 IU / mL and 4000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between DB2 / 650358429.1 130Attorney Docket No.116983-5132-WO 1000 IU / mL and 3000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 IU / mL and 3000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2000 IU / mL and 3000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2500 IU / mL and 3000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1000 IU / mL and 2000 IU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 IU / mL and 2000 IU / mL in the cell culture medium in the first expansion.

[0100] In some embodiments, the second expansion step, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

[0101] In some embodiments, the first expansion is performed using a gas permeable container. In some embodiments, the second expansion is performed using a gas permeable container.

[0102] In some embodiments, the first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. In some embodiments, the second cell culture medium and / or third culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0103] In some embodiments, the second cell culture medium is exchanged after a period of time, for example, 4 days, 5 days, 6 days, 7 days, etc. In some embodiments, the second cell culture medium is exchanged after 4 days. In some embodiments, the second cell culture medium is exchanged after 5 days. In some embodiments, the second cell culture medium is exchanged after 6 days. In some embodiments, the second cell culture medium is exchanged after 7 days. 1. Feeder Cells and Antigen Presenting Cells

[0104] In some embodiments, the second expansion procedures described herein require an excess of feeder cells during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units DB2 / 650358429.1 131Attorney Docket No.116983-5132-WO from healthy blood donors. The PBMCs are obtained using standard methods such as Ficoll- Paque gradient separation.

[0105] In general, the allogeneic PBMCs are inactivated, either via irradiation or heat treatment, and used in the REP procedures, as described in the examples, which provides an exemplary protocol for evaluating the replication incompetence of irradiate allogeneic PBMCs.

[0106] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the PBL expansion procedures described herein if the total number of viable cells on day 14 is less than the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion).

[0107] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the PBL expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion). In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL OKT3 antibody and 3000 IU / mL IL-2.

[0108] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the PBL expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion). In some embodiments, the PBMCs are cultured in the presence of 5-60 ng / mL OKT3 antibody and 1000-6000 IU / mL IL-2. In some embodiments, the PBMCs are cultured in the presence of 10-50 ng / mL OKT3 antibody and 2000-5000 IU / mL IL-2. In some embodiments, the PBMCs are cultured in the presence of 20-40 ng / mL OKT3 antibody and 2000-4000 IU / mL IL-2. In some embodiments, the PBMCs are cultured in the presence of 25-35 ng / mL OKT3 antibody and 2500-3500 IU / mL IL-2.

[0109] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are ENLIST cells (Alloplex Biotherapeutics). In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of PBLs to antigen- presenting feeder cells in the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, DB2 / 650358429.1 132Attorney Docket No.116983-5132-WO about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In some embodiments, the ratio of PBLs to antigen-presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of PBLs to antigen-presenting feeder cells in the second expansion is between 1 to 100 and 1 to 200.

[0110] In some embodiments, the second expansion procedures described herein require a ratio of about 2.5x109feeder cells to about 100x106PBLs. In other embodiments, the second expansion procedures described herein require a ratio of about 2.5x109feeder cells to about 50x106PBLs. In yet other embodiments, the second expansion procedures described herein require about 2.5x109feeder cells to about 25x106PBLs.

[0111] In some embodiments, the second expansion procedures described herein require an excess of feeder cells during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. The PBMCs are obtained using standard methods such as Ficoll- Paque gradient separation. In some embodiments, artificial antigen-presenting (aAPC) cells are used in place of PBMCs.

[0112] In some embodiments, artificial antigen presenting cells are used in the second expansion as a replacement for, or in combination with, PBMCs. 2. Cytokines and Other Additives

[0113] The expansion methods described herein generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.

[0114] Alternatively, using combinations of cytokines for the rapid expansion and or second expansion of PBLs is possible, with combinations of two or more of IL-2, IL-15 and IL-21 as is described in U.S. Patent Application Publication No. US 2017 / 0107490 A1, the disclosure of which is incorporated by reference herein. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, IL-15 and IL-21, with the latter finding particular use in many embodiments. The use of combinations of cytokines specifically favors the generation of lymphocytes, and in particular T-cells as described therein.

[0210] In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-2. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL DB2 / 650358429.1 133Attorney Docket No.116983-5132-WO or lower. In some embodiments, the first cell culture medium or the second cell culture medium contains no added IL-2. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and / or IL-21 at a concentration of about 1 ng / mL to about 100 ng / mL. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and / or IL-21 at a concentration of about 10 ng / mL. In some embodiments, the first cell culture medium comprises IL-2 and IL-21. In some embodiments, the first cell culture medium comprises IL-2 at 3000 IU / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and a protein kinase B (AKT) inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Oridonin, Herbacetin, Tehranolide, Isoliquiritigenin, Scutellarin, and Honokiol. 3. Modifiers of T Cell Metabolism

[0211] In vitro expansion alters PBL cellular state, which correlates with ACT efficacy (Chiffelle, J., et al., bioRxiv 2023, the content of which is hereby incorporated by reference in its entirety). Rapidly expanding PBLs have bioenergetic and biosynthetic needs. Increasing the availability of essential cofactors such as L-arginine and NAD+ may support synthesis of major biomass components. To functionally reinvigorate cells during in vitro expansion may produce PBLs with renewed effector features.

[0212] Therefore, provided herein are modifiers of T cell metabolism that increase availability of essential cofactors L-arginine and NAD+ to support synthesis of major biomass components in PBLs, which may be added to the second expansion (REP) of PBLs.

[0213] L-arginine, a building block for protein synthesis, improves T cell function via metabolic modification (Geiger R., et al., Cell 2016: 167; 829-842; Fultang, L., Blood 2020; 136: 1155-60; the contents of which are hereby incorporated by reference in their entireties).

[0214] NAD+ (nicotinamide adenine dinucleotide), an electron acceptor, is heavily consumed during biomass synthesis, and supplementation improves T cell function (Canto C, et al., Cell Metabolism 2015; 22:31-53; Wang Y., et al., Cell Reports 2021; 36:1-12; the contents of which are hereby incorporated by reference in their entireties). DB2 / 650358429.1 134Attorney Docket No.116983-5132-WO

[0215] NAD+ (Nicotinamide adenine dinucleotide) is a central coenzyme in cellular metabolism, particularly in redox reactions where it cycles between oxidized (NAD+) and reduced (NADH) states. In the context of TILs, NAD+ plays a critical role in modulating their metabolic fitness and function. PBLs operate in the tumor microenvironment, which is often characterized by nutrient scarcity and hypoxia. In this challenging microenvironment, PBLs must adapt their metabolism to sustain their anti-tumor activity.

[0216] NAD+ enhances the metabolic fitness of T cells in several ways:

[0217] Glycolys...

Claims

1. Attorney Docket No.116983-5132-WO WHAT IS CLAIMED IS:

1. A method for expanding tumor-reactive peripheral blood lymphocytes (trPBLs), comprising: a) obtaining a sample of peripheral blood mononuclear cells (PBMCs) from a subject; b) selecting T cells in the sample of PBMCs expressing one or more markers for trPBL to produce a population of cells enriched with trPBLs; and c) culturing the population of cells enriched with trPBLs in a cell culture medium to produce an expanded population of trPBLs.

2. The method of claim 1, wherein the one or more markers for trPBL is selected from the group consisting of 4-1BB, CD103, CXCL13, IFN-γ, CXCR6, CD200, HLA-DR, CD69, PD1, CD39, CD38, and CD49a.

3. The method of claim 1 or 2, wherein the selecting comprises contacting the sample of PBMCs with an antibody to the one or more markers for trPBL.

4. The method of claim 3, further comprising contacting the sample of PBMCs with a secondary antibody capable of binding to the antibody to the one or more markers for trPBL.

5. The method of claim 4, wherein the antibody or the secondary antibody is conjugated to a magnetic nanoparticle.

6. The method of claim 5, wherein the selecting comprises passing the sample of PBMCs through a magnetic capture zone disposed in a channel of a microfluidic device.

7. The method of claim 6, wherein the microfluidic device comprises a plurality of magnetic capture zones, wherein the plurality of magnetic capture zones is disposed to spatially separate cells with different degrees of magnetization.

8. The method of any one of claims 1-7, wherein the sample of PBMCs is obtained via a leukopak from the subject.

9. The method of any one of claims 1-7, wherein the sample of PBMCs is obtained via continuous trapping from the subject.

10. The method of any one of claims 1-9, wherein the cell culture medium comprises IL-21 at a concentration of about 10 ng / mL, and IL-15 at a concentration of about 10 ng / mL. DB2 / 650358429.1 188 Attorney Docket No.116983-5132-WO 11. The method of any one of claims 1-10, wherein the cell culture medium comprises OKT- 3, antigen presenting cells (APCs), and L-arginine.

12. The method of claim 11, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM.

13. The method of claim 11, wherein the L-arginine is present at a concentration of about 5 mM.

14. The method of any one of claims 1-13, wherein the cell culture medium comprises an NAD+ booster.

15. The method of claim 14, wherein the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator.

16. The method of claim 14, wherein the NAD+ booster is NAD+.

17. The method of claim 16, wherein the NAD+ is present at a concentration of about 10 µM to 100 µM.

18. The method of claim 16, wherein the NAD+ is present at a concentration of about 50 µM.

19. The method of any one of claims 1-18, further comprising transducing the population of cells enriched with trPBLs from step (b) with a recombinant lentiviral particle comprising a nucleic acid sequence encoding tethered IL-12 (TeIL-12) and / or tethered IL-15 (TeIL- 15) to produce a population of gene-edited trPBLs.

20. The method of claim 19, further comprising activating the population of cells enriched with trPBLs from step (b) for 1 day or 2 days before transducing the population of cells with the recombinant lentiviral particle.

21. The method of claim 20, wherein the activating step comprises contacting the population of cells enriched with trPBLs from step (b) with a cytokine selected from the group consisting of IL-2, IL-15, IL-21, IL-7, and a combination thereof.

22. The method of claim 20, wherein the activating step comprises contacting the population of cells enriched with trPBLs from step (b) with TransAct.

23. The method of claim 20, wherein the activating step comprises contacting the population of cells enriched with trPBLs from step (b) with TransAct at a ratio of 1:

100.

24. The method of any one of claims 19-23, wherein the transducing step is conducted at a concentration of 105cells / mL. DB2 / 650358429.1 189 Attorney Docket No.116983-5132-WO 25. The method of any one of claims 19-23, wherein the transducing step is conducted at a multiplicity of infection (MOI) of about 10 to about 40.

26. The method of any one of claims 19-25, wherein the transducing step is conducted in the presence of RetroNectin or Vectofusin-1.

27. The method of any one of claims 19-26, wherein the transducing step comprises centrifugation.

28. The method of any one of claims 19-27, wherein the transducing step is conducted in the presence of Lentiboost.

29. The method of any one of claims 19-28, further comprising resting the population of cells for 2 day or 3 days after the transducing step.

30. The method of any one of claims 1-29, further comprising administering to the subject an inhibitor of an immune checkpoint gene before obtaining the sample of PBMCs from the subject.

31. The method of claim 20, wherein the immune checkpoint gene is selected from the group consisting of PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR.

32. The method of any one of claims 1-21, wherein step (c) is performed for a period that lasts about 1-10 days.

33. The method of any one of claims 1-21, wherein step (c) is performed for a period that lasts about 3-5 days.

34. A population of trPBLs produced by the method of any one of claims 1-33.

35. A gene-edited trPBL expressing an exogenous IL-12 or a variant thereof.

36. The gene-edited trPBL of claim 35, wherein the IL-12 is a tethered IL-12 (TeIL-12).

37. The gene-edited trPBL of claim 36, wherein the TeIL-12 comprises a membrane anchor, and a human IL-12 p40 subunit fused to a human IL-12 p35 subunit. DB2 / 650358429.1 190 Attorney Docket No.116983-5132-WO 38. The gene-edited trPBL of claim 37, wherein the human IL-12 p35 subunit has the amino acid sequence of SEQ ID NO:60 and the human IL-12 p40 subunit has the amino acid sequence of SEQ ID NO:

61.

39. The gene-edited trPBL of claim 36, wherein the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:

62.

40. The gene-edited trPBL of any one of claims 35-39, further expressing an exogenous IL- 15 or a variant thereof.

41. The gene-edited trPBL of claim 40, wherein the IL-15 is a human IL-15.

42. The gene-edited trPBL of claim 41, wherein the human IL-15 has the amino acid sequence of SEQ ID NO:

64.

43. The gene-edited trPBL of any one of claims 40-42, wherein the IL-15 is a tethered IL-15 (TeIL-15).

44. The gene-edited trPBL of claim 43, wherein the TeIL-15 comprises a membrane anchor, and a human IL-15.

45. The gene-edited trPBL of claim 43, wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:

73.

46. A pharmaceutical composition comprising the population of trPBLs according to claim 34 or a population of the gene-edited trPBLs according to any one of claims 35-45.

47. A method of treating a cancer in a patient in need thereof comprising administering the pharmaceutical composition according to claim 46 to the patient.

48. The method of claim 47, wherein the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

49. The method of claim 47 or 48, further comprising the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the trPBLs to the patient. DB2 / 650358429.1 191 Attorney Docket No.116983-5132-WO 50. The method of claim 49, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days.

51. The method of claim 49, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days.

52. The method of claim 49, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for one day.

53. The method of any one of claims 50-52, wherein the cyclophosphamide is administered with mesna.

54. The method of claim 47 or 48, wherein the patient is not treated with a non-myeloablative lymphodepletion regimen prior to administering the trPBLs to the patient.

55. The method of any one of claims 47-54, further comprising a step of treating the patient with an IL-2 regimen starting on the day after the administration of trPBLs to the patient.

56. The method of any one of claims 47-55, further comprising a step of treating the patient with an IL-2 regimen starting on the same day as administration of trPBLs to the patient.

57. The method of claim 55 or 56, wherein the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg of aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerance.

58. The method of any one of claims 47-54, comprising no step of treating the patient with an IL-2 regimen. DB2 / 650358429.1 192

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