Engineered artificial antigen presenting cells and lipid nanoparticles for tumor infiltrating lymphocyte expansion

Abstract

In some embodiments, compositions and methods relating to isolated artificial antigen presenting cells (aAPCs) are disclosed, including aAPCs comprising a U937 cell modified to express one or more co-stimulatory molecules including CD64, CD86, 4-1BBL and / or OX40L. Further provided are lipid nanoparticles (LNPs) conjugated to one or more co-stimulatory molecules including CD64, CD86, 4-1BBL and / or OX40L. In some embodiments, methods of expanding tumor infiltrating lymphocytes (TILs) with aAPCs or LNPs and methods of treating cancers using TILs after expansion with aAPCs or LNPs are also disclosed.

Description

Attorney Docket No. 5134-WOENGINEERED ARTIFICIAL ANTIGEN PRESENTING CELLS AND LIPID NANOPARTICLES FOR TUMOR INFILTRATING LYMPHOCYTE EXPANSIONFIELD

[0001] Modified artificial antigen presenting cells (aAPCs) and lipid nanoparticles (LNPs) for expansion of tumor infiltrating lymphocytes are disclosed.BACKGROUND

[0002] Treatment of bulky, refractory cancers using adoptive autologous transfer of tumor infiltrating lymphocytes (TILs) represents a powerful approach to therapy for patients with poor prognoses. Gattinoni, et al. , Nat. Rev. Immunol. 2006, 6, 383-393. A large number of TILs are required for successful immunotherapy, and a robust and reliable process is needed for commercialization. This has been a challenge to achieve because of technical, logistical, and regulatory issues with cell expansion. IL-2-based TIL expansion followed by a ‘'rapid expansion process” (REP) has become a preferred method for TIL expansion because of its speed and efficiency. Dudley, et al. , Science 2002, 298, 850-54; Dudley, et al. , J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, etal., J. Immunother. 2003, 26, 332-42. However, although REP can result in a 1,000-fold expansion of TILs over a 14-day period, it requires a large excess (e.g, 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs), often from multiple donors, as feeder cells, as well as anti-CD3 antibody (OKT-3) and high doses of IL-2. Dudley, et al.. J. Immunother. 2003, 26, 332-42. Despite their high performance, PBMCs have multiple drawbacks, including the large numbers of allogeneic PBMCs required, the need to obtain PBMCs by leukapheresis from multiple healthy donors, the resulting inter-donor variability' in PBMC viability after cryopreservation and variable TIL expansion results, the risk of undetected viral pathogens causing downstream patient infections, and the extensive and costly laboratory testing of each individual donor cell product to confirm sterility and quality (including viral contaminant testing) and to test expansion properties.

[0003] Three primary signals are required for T cell activation: a) engagement between TCR and stimulatory signal pMHC for specific activation (or anti-CD3 for non-specific activation); b) binding between CD28 and co-stimulatory signal CD80 / 86 for proliferation; and c) presence of cytokines for differentiation. Wong, et al.. Engineered Regenration 2021. Artificial APCs provide a number of benefits, such as the ability to produce a master cellAttorney Docket No. 5134-WO bank, the absence of allogeneic cells, and the potential to decrease the duration of the manufacturing process, etc.

[0004] Unfortunately, aAPCs developed for use in the expansion of TILs have suffered from poor performance when compared to PBMCs. including alterations of the phenotypic properties of the input TILs, as well as poor expansion performance and / or high variability in expansion results. Because of the large number of potential cells that might be adapted for use as aAPCs and the unpredictability of identifying suitable candidates, the focus of aAPC development for polyclonal TILs to date has been solely on the well-established K562 cell line. Butler and Hirano, Immunol. Rev. 2014, 257, 191-209. For example, K562 cells modified to express 4-1BBL (CD137L) were tested in pre-REP culture (but not in REP culture) to determine enhancement of TIL expansion from tumor digest, but PBMCs were still required to be used in conjunction with K.562 cells to obtain TIL expansion. Friedman, et al., J. Immunother. 2011, 34, 651-661. Other engineered K562 cells modified to express CD64, CD86, and 4-1BBL were tested and achieved TIL expansion that was at best comparable to PBMCs, and most likely less than PBMCs, and also suffered from skewing of the polyclonal TIL phenotype to a less favorable CD8+ / CD4+T cell ratio. Ye, et al., J. Translat. Med. 2011, 9, 131. Recently, K562 cells modified to express CD86, 4-1BBL (CD137L), high affinity Fc receptor (CD64) and membrane-bound IL-15 have also been shown to propagate TIL (post-REP) at equivalent numbers compared to PBMC feeders, but with the additional complexity of membrane-bound IL-15. Forget, et al., J. Immunother. 2014, 37, 448-60. Other systems developed have lacked critical costimulatory molecules, have led to unfavorable T cell phenotypic skewing, or have required additional interleukins (such as IL-21). Butler and Hirano, Immunol. Rev. 2014, 257, 191-209. Overall, K562 modified aAPCs have not been shown to provide for consistent expansion of TILs with acceptable variability while also performing better than PBMCs in other measures including overall expansion cell counts. Alternative aAPCs besides K562 cells have been successful in other cell expansion methods, but have not achieved the same performance as PBMCs with the unique polyclonal subset of cells that make up TILs. Maus, et al., Nat. Biotechnol. 2002, 20, 143-148; Suhoski, et al., Mol. Ther. 2007, 75, 981-988.SUMMARY

[0005] The U937 promonocytic cell line represents an excellent human monocyte model. The present disclosure provides the unexpected finding that engineered U937 cells, transduced with additional costimulatory molecules, including CD64, CD86 (B7-2), 4-1BBLAttorney Docket No. 5134-WO(CD137L), and / or OX40L (CD134L), provide for superior and highly efficient expansions of TILs in large numbers with minimal variability, reduced cost, and no reliance on human blood samples as a source of PBMCs, with the benefit of using an aAPC which can be produced efficiently from a master cell bank. CD64, CD86 and 4-1BBL are costimulatory molecules that provide costimulatory signals for T cell activation. The U937 cells transduced with additional costimulatory molecules are useful, for example, in the expansion of TILs for use in cancer immunotherapy and other therapies.

[0006] Some embodiments disclosed herein provide an artificial antigen presenting cell (aAPC) comprising a U937 cell expressing one or more co-stimulatory molecules. In some embodiments, the one or more co-stimulatory molecules are at least one of a CD64 protein, a CD86 protein, a 4-1 BBL protein, or an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD86 protein. In some embodiments, the one or more co-stimulatory molecules comprise a 4-1BBL protein. In some embodiments, the one or more co-stimulatory molecules comprise an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and a CD86 protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and a 4- 1BBL protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and a 4-lBBL protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, a 4-1BBL protein, and an OX40L protein. In some embodiments, the aAPC can stimulate and expand a T cell contacted with the aAPC. In some embodiments, the aAPC can stimulate and expand tumor infiltrating lymphocytes (TILs) contacted with the aAPC. In some embodiments, the aAPC expands a population of TILs by at least 50-fold over a period of 7 days in a cell culture medium comprising IL-2 at a concentration of about 3000 lU / mL and OK.T-3 antibody at a concentration of about 30 ng / mL.

[0007] In some embodiments, the CD64 protein comprises SEQ ID NO:4, or a sequence comprising one or more conservative amino acid substitutions thereof. In some embodiments, the CD86 protein comprises SEQ ID NO: 1, or a sequence comprising one or more conservative amino acid substitutions thereof. In some embodiments, the 4-1 BBL proteinAttorney Docket No. 5134-WO comprises SEQ ID NO:2, or a sequence comprising one or more conservative amino acid substitutions thereof. In some embodiments, the OX40L protein comprises SEQ ID NO: 3, or a sequence comprising one or more conservative amino acid substitutions thereof. In some embodiments, the nucleic acid encoding CD64 comprises SEQ ID NO: 12. In some embodiments, the nucleic acid encoding CD86 comprises SEQ ID NO:9. In some embodiments, the nucleic acid encoding 4-1BBL comprises SEQ ID NO: 10. In some embodiments, the nucleic acid encoding OX40L comprises SEQ ID NO: 11.

[0008] Some embodiments disclosed herein provide a lipid nanoparticle (LNP) comprising one or more co-stimulatory molecules. In some embodiments, the one or more co-stimulatory molecules are at least one of a CD64 protein, a CD86 protein, a 4-1 BBL protein, or an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD86 protein. In some embodiments, the one or more co-stimulatory molecules comprise a 4-1 BBL protein. In some embodiments, the one or more co-stimulatory molecules comprise an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and a CD86 protein. In some embodiments, the one or more co- stimulatory molecules comprise a CD64 protein, and a 4-1 BBL protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and a 4-1BBL protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, a 4-1 BBL protein, and an OX40L protein. In some embodiments, the LNP can stimulate and expand a T cell contacted with the LNP. In some embodiments, the LNP can stimulate and expand tumor infiltrating lymphocytes (TILs) contacted with the LNP. In some embodiments, the LNP expands a population of TILs by at least 50-fold over a period of 7 days in a cell culture medium comprising IL-2 at a concentration of about 3000 lU / mL and OKT-3 antibody at a concentration of about 30 ng / mL.

[0009] In some embodiments, the CD64 protein comprises SEQ ID NO:4, or a sequence comprising one or more conservative amino acid substitutions thereof. In some embodiments, the CD86 protein comprises SEQ ID NO: 1, or a sequence comprising one or more conservative amino acid substitutions thereof. In some embodiments, the 4-1BBL protein comprises SEQ ID NO: 2, or a sequence comprising one or more conservative amino acidAttorney Docket No. 5134-WO substitutions thereof. In some embodiments, the OX40L protein comprises SEQ ID NO: 3, or a sequence comprising one or more conservative amino acid substitutions thereof.

[0010] In some embodiments, the LNP comprises: (a) at least one ionizable lipid; (b) at least one neutral lipid; (c) cholesterol and / or a modified derivative thereof; and (d) at least one polymer conjugated lipid and / or a modified derivative thereof. In some embodiments, the one or more co-stimulatory molecules are covalently conjugated to at least one component of the LNP. In some embodiments, the component to which the one or more co-stimulatory molecules are covalently conjugated is the modified derivative of the polymer conjugated lipid. In some embodiments, the covalent conjugation comprises a covalent bond forming reaction selected from the group consisting of a [1,4] -conjugate addition (i.e., Michael addition), [4+2] cycloaddition, [3+2] dipolar cycloaddition, nucleophilic addition, transition metal-catalyzed cross-coupling reaction, carbonyl condensation reaction, and reductive amination. In some embodiments, the covalent conjugation reaction comprises a [1,4]- conjugate addition reaction (i.e., Michael addition). In some embodiments, the [1,4]- conjugate addition occurs between the modified derivative of the polymer conjugated lipid which is further conjugated to a mal eimide moiety and a cysteine thiol of a polypeptide. In some embodiments, the cystine thiol of the polypeptide is derived from a reduced disulfide bridge of the one or more co-stimulatory molecules.

[0011] Some embodiments disclosed herein provide a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising a step of contacting a population of TILs with a population of aAPCs disclosed herein or a population of LNPs disclosed herein in a cell culture medium, wherein the population of TILs is expanded.

[0012] In some embodiments, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 lU / mL and OK.T-3 antibody at an initial concentration of about 30 ng / mL. In some embodiments, the population of TILs is expanded by at least 50-fold over a period of 11 days. In some embodiments, the population of TILs is expanded by at least 200-fold over a period of 11 days. In some embodiments, the population of TILs is expanded by at least 500-fold over a period of 11 days. In some embodiments, the expansion is performed using a gas permeable container. In some embodiments, the ratio of the population of TILs to the population of aAPCs or the population of LNPs is between 1 to 100 and 1 to 500. In some embodiments, the ratio of the population of TILs to the population of aAPCs or the population of LNPs is about 1 to 250.Attorney Docket No. 5134-WO

[0013] Some embodiments disclosed herein provide a method of making a therapeutic population of tumor infiltrating lymphocytes (TILs), wherein the method comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OK.T-3. and a population of artificial antigen presenting cells (aAPCs) modified to express one or more costimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs.

[0014] Some embodiments disclosed herein provide a method of making a therapeutic population of tumor infiltrating lymphocytes (TILs), wherein the method comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OK.T-3, and a population of lipid nanoparticles (LNPs) conjugated to one or more co-stimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs.

[0015] In some embodiments, the method further comprises: (d) harvesting therapeutic population of TILs. In some embodiments, the method further comprises: (e) transferring the harvested therapeutic population of TILs into an infusion bag. In some embodiments, the method further comprises: (1) cry' opreserving the infusion bag comprising the harvestedAttorney Docket No. 5134-WO therapeutic population of TILs using a cryopreservation process. In some embodiments, the first expansion is performed over a period of about 3-11 days. In some embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the second expansion is performed over a period of about 7-11 days. In some embodiments, the second expansion is performed over a period of about 11 days. In some embodiments, the first expansion is performed over a period of about 11 days, and the second expansion is performed over a period of about 11 days. In some embodiments, the first expansion is performed over a period of about 3-12 days. In some embodiments, the first expansion is performed over a period of about 12 days. In some embodiments, the second expansion is performed over a period of about 7-12 days. In some embodiments, the second expansion is performed over a period of about 12 days. In some embodiments, the first expansion is performed over a period of about 12 days, and the second expansion is performed over a period of about 12 days. In some embodiments, step (b) and step (c) are performed in a closed system, wherein the transition from step (b) to step (c) occurs without opening the closed system. In some embodiments, the transition from step (c) to step (d) occurs without opening the closed system. In some embodiments, the transition from step (d) to step (e) occurs without opening the closed system.

[0016] In some embodiments, the one or more co-stimulatory molecules are at least one of a CD64 protein, a CD86 protein, a 4-1 BBL protein, or an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and a CD86 protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and a 4-1BBL protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and a 4-1BBL protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and an OX40L protein. In some embodiments, the one or more co- stimulatory molecules comprise a CD64 protein, a CD86 protein, a 4-lBBL protein, and an OX40L protein.

[0017] Some embodiments disclosed herein provide a method of treating a cancer patient with a therapeutic population of tumor infiltrating lymphocytes (TILs) comprising the steps of:Attorney Docket No. 5134-WO(a) obtaining and / or receiving a first population of TILs from a tumor resected from the patient;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs. wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL -2, OKT-3, and a population of artificial antigen presenting cells (aAPCs) modified to express one or more costimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs; and(d) administering a therapeutically effective dosage of the therapeutic population of TILs to the patient with the cancer.

[0018] Some embodiments disclosed herein provide a method of treating a cancer patient with a therapeutic population of tumor infiltrating lymphocytes (TILs) comprising the steps of:(a) obtaining and / or receiving a first population of TILs from a tumor resected from the patient;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, w herein the first expansion is performed for about 3-14 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of lipid nanoparticles (LNPs) conjugated to one or more co-stimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs; and(d) administering a therapeutically effective dosage of the therapeutic population of TILs to the patient with the cancer.Attorney Docket No. 5134-WO

[0019] In some embodiments, the cancer is anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papilloma virus (HPV), central nervous system associated cancer (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma, and primitive neuroectodermal tumor), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC), hypophary nx cancer, larynx cancer, nasophary nx cancer, oropharynx cancer, and pharynx cancer), kidney cancer, liver cancer, lung cancer (including non-small-cell lung cancer (NSCLC) and small-cell lung cancer), melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), penile cancer, rectal cancer, renal cancer, renal cell carcinoma, sarcoma (including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other bone and soft tissue sarcomas), thyroid cancer (including anaplastic thyroid cancer), uterine cancer, or vaginal cancer.

[0020] In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the second population of TILs 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 five 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 cyclophosphamide is administered with mesna. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting on the day after the administration of the TILs to the patient. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting on the same day as administration of the TILs to the patient. In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 lU / kg of aldesleukin, or a biosimilarAttorney Docket No. 5134-WO or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerance.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The foregoing summary, as well as the following detailed description, will be better understood when read in conjunction with the appended drawings.

[0022] Figure 1: Exemplary Gen 2 (process 2 A) chart providing an overview of Steps A through F.

[0023] Figure 2A-2C: Process flow chart of an embodiment of Gen 2 (process 2A) for TIL manufacturing.

[0024] Figure 3: Exemplary process for generating U937 based aAPCs expressing costimulatory' proteins.

[0025] Figure 4: Map of transfer vector pGenLenti.

[0026] Figure 5: TIL total viable cell (TVC, left) and expansion (right) are non-significant between TIL process using iU937 CD64OECD86OE41BBL0E(iU9373OE) and iPBMC feeder cells.

[0027] Figure 6: iU937 CD64OECD86OE41BBL0E(iU9373OE) alters the ratio of CD8+(left) and CD4+(right) cells compared to iPBMC feeder cells.

[0028] Figure 7: REP'd TILs are mostly TEM and show no significant difference between different feeder cells.

[0029] Figure 8: CD8+CD39’CD69’ population in REP’d TILs using U937 CD64O£CD86OE41BBLOFis significantly higher compared to PBMC feeder cells.

[0030] Figure 9: PD1+, CD27 . and CD127+expressions are significantly different in CD4+REP’d TILs using U937 CD64OECD86OE41BBLOEcompared to PBMC feeder cells.

[0031] Figure 10: PD1+and CD28+expressions are significantly different in CD8+REP’d TILs using U937 CD64O£CD86OE41BBLO£compared to PBMC feeder cells.

[0032] Figure 11: U937-based aAPCs generated higher TIL cell count and viability in comparison to K562-based aAPCs.

[0033] Figure 12: U937-based aAPCs generated higher percentage of CD3+ TILs in comparison to K562-based aAPCs.Attorney Docket No. 5134-WO

[0034] Figure 13: U937-based aAPCs generated similar CD4+ / CD8+ TIL distribution in comparison to K562-based aAPCs.

[0035] Figure 14: CCR7 and CD45RA expression data by REP’d TILs using U937- and K562-based aAPCs.

[0036] Figure 15: U937-based aAPCs generated similar percentage of CD4+ / CD39- / CD69- (left) or CD8+ / CD39- / CD69- (right) TILs in comparison to K562-based aAPCs.

[0037] Figure 16: Expression of activation and exhaustion markers by REP’d TILs using U937-based aAPCs and K562-based aAPCs.

[0038] Figure 17: Expression of EOMES, TbeL TCF1 and Tox by REP’d TILs using U937-based aAPCs and K562-based aAPCs.

[0039] Figure 18: Ml 152 REP TIL re-REP’d with iU937JO£feeder cells achieved comparable or better killing of autologous tumor cell line than iPBMC feeder cells.

[0040] Figures 19A-19C: iU9375C® cells were analyzed by FACS for expression of CD64 (Figure 19A), CD86 (Figure 19B), and 4-1BB-L (Figure 19C). The cells exhibited overexpression of all three markers compared to parental U937 cells.

[0041] Figure 20: TILs REP’d in the presence of iU9373OFhad similar fold expansion compared to TILs REP’d with iPBMCs.

[0042] Figures 21A-21C: TILs REP’d in the presence of iU937,c® had similar total viable cells (TVC, Figure 21A), fold expansion (Figure 21B), and percent viability (Figure 21C) compared to TILs REP’d with PBMCs, regardless of tumor type or donor.

[0043] Figure 22A-22B: TILs REP’d in the presence of iU937’O£had similar percent frequency of CD4+ (Figure 22A) and CD8+ (Figure 22B) TILs compared to TILs REP’d with PBMCs, regardless of tumor type or donor.

[0044] Figures 23A-23D: TILs REP’d in the presence of iU9375C® had similar percent frequency of Tnaive (Figure 23A), TCM (Figure 23B), TEM (Figure 23C), and TEMRA (effector memory cells re-expressing CD45RA; Figure 23D) for CD4+ (top panel of each figure) and CD8+ (bottom panel of each figure) TILs compared to TILs REP’d with PBMCs.

[0045] Figures 24A-24B: TILs REP’d in the presence of IU9375U / ;had similar percent frequency of CD4+CD39-CD69- TILs (Figure 24A) and CD8+CD39-CD69- TILs (Figure 24B) compared to TILs REP’d with PBMCs.Attorney Docket No. 5134-WO

[0046] Figures 25A-25B: TILs REP’d in the presence of iU9373O£had similar percent frequency of CD4+PD1+ TILs (Figure 25A) and CD8+PD1+ TILs (Figure 25B).

[0047] Figure 26: The number of clonotypes (determined by TCR seq) was similar in preREP TILs, TILs that were REP’d with iPBMCs, and TILs that were REP’d with iU9375OE

[0048] Figure 27: Relative abundance of clonotypes with different frequencies was similar between preREP TILs, TILs that were REP’d with iPBMCs, and TILs that were REP’d with iU9373O£.

[0049] Figure 28: A ratio of 1 :50 or 1 : 100 iU9375OAcells resulted in similar fold change as 1:250 PBMCs, regardless of tumor type. The ratio of TILs to iU9373C® cells used in the REP process was varied from 1 :50 (TILs: iU937’O£) to 1:750. iPBMCs were used at 1:250 (TILs:PBMCs).

[0050] Figure 29: A ratio of 1 :50 or 1 : 100 iU9375<J / cells resulted in similar percent viability (Figure 29) as 1:250 PBMCs. regardless of tumor type. The ratio of TILs to iU9373O£cells used in the REP process was varied from 1:50 (TILs: iU937JO£) to 1 :750. iPBMCs were used at 1 :250 (TILs:PBMCs).

[0051] Figure 30: TILs REP’d with U9373O£cells or iPBMCs showed similar activity in a tumoroid killing assay.DETAILED DESCRIPTION

[0052] 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 belongs. All patents and publications referred to herein are incorporated by reference in their entireties.I. Definitions

[0053] 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 to a human subject so that both active pharmaceutical ingredients and / or their metabolites are present in the human 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 pharmaceuticalAttorney Docket No. 5134-WO ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present is also encompassed in the methods described herein.

[0054] The term “in vivo" refers to an event that takes place in a subject’s body.

[0055] 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.

[0056] 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.

[0057] 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 T cell receptor (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.

[0058] 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 affect 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 human subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, etc. which can readily be determined by one of ordinary skill in the art. 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 on the particularAttorney Docket No. 5134-WO compounds or agents chosen, the dosing regimen to be followed, whether the compound or agent is administered in combination with other compounds or agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound or agent is carried.

[0059] A “therapeutic effect” as that term is used herein, encompasses a therapeutic benefit and / or a prophylactic benefit in a human subject. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0060] “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” is 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 the therapeutic compositions described herein is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0061] 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 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.

[0062] 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), Thl and Thl7 CD4+T cells, natural killer cells, dendritic cells and Ml macrophages. TILs include both primary7and secondary7TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to herein as “freshly harvested” or “a first population of TILs”). and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but notAttorney Docket No. 5134-WO limited to bulk TILs and expanded TILs (“REP TILs’' or “post-REP TILs”, or “second population of TILs” or “third population of TILs” where appropriate).

[0063] 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 a|3, 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.

[0064] By “cryopreserved TILs” herein is meant that TILs are treated and stored in the range of about -150°C to -60°C. General methods for cry opreservation are also described elsewhere herein, including in the Examples. For clarity, “cryopreserved TILs” are distinguishable from frozen tissue samples which may be used as a source of primary' TILs.

[0065] By “thawed cryopreserved TILs” herein is meant a population of TILs 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 TILs may be administered to a patient.

[0066] By “population of cells” (including TILs) herein is meant a number of cells that share common traits.

[0067] The term “central memory T cell” (TCM) refers to a subset of T cells that in the human are CD45R0+ and constitutively express CCR7 (CCR7hl) and CD62L (CD62hl). 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.

[0068] The term “effector memory T cell” (TEM) refers to a subset of human or mammalian T cells that, like central memory' T cells, are CD45R0+, but have lost the constitutive expression of CCR7 (CCR710) and are heterogeneous or low for CD62L expression (CD62L10). The surface phenotype of central memory T cells also includes TCR. CD3, CD127 (IL-7R), and IL-15R. Transcription factors for effector memory T cells include BLIMP 1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon- / , IL-4, and IL-5. Effector memory' TAttorney Docket No. 5134-WO 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.

[0069] The terms “sequence identity,” “percent identity,” and “sequence percent identity” 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 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. Govemmenf s National Center for Biotechnology Information BLAST web site. Comparisons betw een 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.

[0070] The term “conservative amino acid substitutions” means amino acid sequence modifications which do not abrogate the binding of an antibody to an antigen or a protein to its ligand. Conservative amino acid substitutions include the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common phy sicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys); Class V (He, Leu, Vai, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gin, or Glu, is a conservative substitution. Thus, a predicted nonessential amino acid residue in a 4-1 BBL or CD86 protein is preferably replaced with another amino acidAttorney Docket No. 5134-WO residue from the same class. Methods of identifying amino acid conservative substitutions which do not eliminate antigen or ligand binding are well-known in the art (see, e.g.. Brummell, et al., Biochemistry 1993, 32, 1180-1187; Kobayashi, et al., Protein Eng. 1999, 12, 879-884 (1999); and Burks, et al., Proc. Natl. Acad. Sci. USA 1997, 94, 412-417).

[0071] The term '‘retrovirus” refers to RNA viruses that utilize reverse transcriptase during their replication cycle, wherein retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase. The double-stranded DNA form is integrated into the chromosome of the infected cell (a “provirus”). The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles. At each end of the provirus are structures called “long terminal repeats” or “LTRs.” The LTR contains numerous regulator ' signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome. Several genera included within the family Retroviridae , including CzVerwrzvzr MS A, Oncovirus A, Oncovirus B, Oncovirus C, Oncovirus D, Lentivirus, Gammaretrovirus, and Spumavirus. Some of the retroviruses are oncogenic (i.e., tumorigenic), while others are not. The oncoviruses induce sarcomas, leukemias, lymphomas, and mammary carcinomas in susceptible species. Retroviruses infect a wide variety of species, and may be transmitted both horizontally and vertically. Because they are integrated into the host DNA, they are capable of transmitting sequences of host DNA from cell to cell. Example gammaretroviral vectors include those derived from the amphotropic Moloney murine leukemia virus (MLV-A), which use cell surface phosphate transporter receptors for entry and then permanently integrate into proliferating cell chromosomes. The amphotropic MLV vector system has been well established and is a popular tool for gene delivery' (See, e.g., Gordon and Anderson, Curr. Op. Biotechnol., 1994, 5, 611-616 and Miller, et al.. Meth. Enzymol., 1993, 217, 581-599, the disclosures of which are incorporated herein by reference.

[0072] The term “lentivirus” refers to a genus that includes HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2), visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritis-encephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly centralAttorney Docket No. 5134-WO nervous system infection in cattle; and simian immunodeficiency virus (SIV), which cause immune deficiency and encephalopathy in sub-human primates. Diseases caused by these viruses 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 (z.e., T cells).

[0073] 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 CD3c. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0074] The term “OKT-3” (also referred to herein as OKT3") refers to a monoclonal antibody 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 Biotec GmbH, Bergisch Gladbach, Germany) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. 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.

[0075] The term “IL-2” (also referred to herein as “IL2”) refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2 including human and mammalian 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. 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 nonglycosylatedAttorney Docket No. 5134-WO human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, CA, USA. NKTR-214 and pegylated IL-2 suitable for use herein is described in U.S. Patent Application Publication No. US 2014 / 0328791 Al and International Patent Application Publication No. WO 2012 / 065086 Al, the disclosures of which are incorporated by reference herein. Alternative forms of conjugated IL-2 suitable for use herein are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261 and 4902,502, the disclosures of which are incorporated by reference herein. Formulations of IL-2 suitable for use herein are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated by reference herein.

[0076] In some embodiments, an IL-2 form suitable for use herein includes an 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 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 VH or 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 Al, the disclosures of which are incorporated by reference herein. 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 VH or the VL, wherein the IL-2 molecule is a mutein, wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulator}’ T cells.

[0077] 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-2Attorney Docket No. 5134-WO 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.

[0078] 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.

[0079] In some embodiments, an IL-2 molecule is engrafted directly into a CDR without a peptide linker, with no additional ammo 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.

[0080] 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 an ammo acid sequence in Table 1 in U.S. Patent Application Publication No. US 2020 / 0270334 Al, the disclosure of which is incorporated by reference herein.

[0081] In some embodiments, the antibody cytokine engrafted protein comprises IgG.IL2F71A.Hl or IgG.IL2R67A.Hl of U.S. Patent Application Publication No. 2020 / 0270334 Al, 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 antibodyAttorney Docket No. 5134-WO 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.

[0082] 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-4 suitable for use herein 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).

[0083] 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 and y 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).

[0084] 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 w ith a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available fromAttorney Docket No. 5134-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).

[0085] The term “myeloid cell” as used herein refers to cells of the myeloid lineage or derived therefrom. The myeloid lineage includes a number of morphologically, phenotypically. and functionally distinct cel] types including different subsets of granulocytes (neutrophils, eosinophils, and basophils), monocytes, macrophages, erythrocytes, megakaryocytes, and mast cells. In certain embodiments, the myeloid cell is a cell derived from a cell line of myeloid lineage.

[0086] As used herein, the term “CD86 protein” may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NO: 1 or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NOT, e.g. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0087] As used herein, the term “4-1BBL” or “CD137L” may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NO:2 or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO:2, e.g. 91%. 92%. 93%. 94%. 95%, 96%, 97%, 98% or 99%.

[0088] As used herein, the term “CD64 protein” may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NO: 4 or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO:4, e.g. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0089] As used herein, the term “OX40L” or “CD134L” may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NOT or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NOT. e.g. 91%. 92%. 93%, 94%. 95%, 96%, 97%, 98% or 99%.

[0090] The term “biosimilar” means a biological product, including a monoclonal antibody or fusion 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 MedicinesAttorney Docket No. 5134-WOAgency. 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 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 clinicalAttorney Docket No. 5134-WO 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 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 regulator}' agencies.

[0091] As used herein, the term “variant” encompasses but is not limited to proteins, antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference protein or 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 protein or antibody. The variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of aAttorney Docket No. 5134-WO reference protein or 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 protein or antibody. The term “variant’’ also includes pegylated antibodies or proteins.

[0092] "Pegylation” refers to a modified antibody, or a fragment thereof, or protein 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, antibody fragment, or protein. Pegylation may, for example, increase the biological (e.g., serum) half life of the antibody or protein. 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-poly ethylene glycol or polyethylene glycol-maleimide. The antibody or protein to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies and proteins described herein, as described for example in European Patent Nos. EP 0154316 and EP 0401384.

[0093] The term “adjuvant” as used herein is defined as any molecule to enhance an antigen-specific adaptive immune response.

[0094] The term “alkenyl” as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms.

[0095] Examples include, but are not limited to vinyl. -CH=C“CCH2, -CH=CH(CH3), - CH=C(CH. 2, -C(CH3)=CH2, -C(CH3>=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl. butadienyl. pentadienyl, and hexadienyl among others.

[0096] The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy , ethoxy , propoxy , butoxy, pentyloxy , hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and theAttorney Docket No. 5134-WO like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

[0097] The term “alkyd” as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or. in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyd groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-penty 1, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-buty l, t-butyl, neopenty l, isopentyd, and 2,2- dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0098] The term “alkyny 1” as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to - CACH, -CAC(CH3), -CAC(CH2CH3), -CH2CACH, -CH2CAC(CH3), and -CH2CAC(CH2CH3) among others.

[0099] The term “alkydene” or “alkydenyl” as used herein refers to a bivalent saturated aliphatic radical (e.g., -CH2-, -CH2CH2-, and -CH2CH2CH2-, inter aha). In certain embodiments, the term may be regarded as a moiety’ derived from an alkene by opening of the double bond or from an alkane by removal of two hydrogen atoms from the same (e.g.. - CH2-) different (e.g., -CH2CH2-) carbon atoms. Similarly, the terms “heteroalky denyl”, “cycloalkyleny 1”, “heterocycloalkylenyl”, and the like, as used herein, refer to a divalent radical of the moiety corresponding to the base group (e.g., heteroalkyd, cycloalkyl, and / or heterocycloalkyl). A divalent radical possesses two open valencies at any position(s) of the group, wherein each radical may be on a carbon atom or heteroatom. Thus, the divalent radical may form a single bond to two distinct atoms or groups, or may form a double bond with one atom.Attorney Docket No. 5134-WO

[0100] The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylgly cerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0101] The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylgly cerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0102] The term “aryl” as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyL triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof.

[0103] The term “monovalent cation” as used herein refers to any positively charged (+ 1) organic or inorganic ion. Non-limiting examples include H , NH41, Li+, Na4, K , Cu‘, Ag4, Cs+. and Au+.

[0104] The term “cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH of about 7.0). It has been found that cationic lipids comprising alkyl chains with multiple sites of unsaturation, e.g., at least two or three sites of unsaturation, are particularly useful for forming lipid particles with increased membrane fluidity. A number of cationic lipids and related analogs, which are also useful in the present disclosure, have been described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Pat. Nos.5,208,036; 5,264,618; 5,279,833; 5,283,185;Attorney Docket No. 5134-WO5,753,613; and 5,785,992; and PCT Publication No. WO 96 / 10390, the disclosures of which are herein incorporated by reference in their entirety for all purposes. Non-limiting examples of cationic lipids are described in detail herein. In some cases, the cationic lipids comprise a protonatable tertiary amine (e.g., pH titratable) head group, Cl 8 alkyl chains, ether linkages between the head group and alky l chains, and 0 to 3 double bonds. Such lipids include, e.g., DSDMA, DLinDMA, DLenDMA, and DODMA.

[0105] The term “conjugated lipid” as used herein refers to a lipid which is conjugated to one or more polymeric groups, which inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, PEG conjugated to ceramides (e.g., U.S. Pat. No.5, 885, 613. the disclosure of which is herein incorporated by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In preferred embodiments, non-ester containing linker moieties are used.

[0106] The term “cycloalkyl” as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbomyl, adamantyl, bornyl, camphenyl. isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbomyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term “cycloalkeny l” alone or in combination denotes a cyclic alkenyl group.

[0107] The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.Attorney Docket No. 5134-WO

[0108] The term “haloalkyl’' group, as used herein, includes mono-halo alkyl groups, polyhalo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1 -di chloroethyl, 1,2-dichloroethyl, l,3-dibromo-3,3- difluoropropyl, perfluorobutyl. and the like.

[0109] The term “helper lipid” as used herein refers to a lipid capable of increasing the effectiveness of delivery of lipid-based particles such as cationic lipid-based particles to a target, preferably into a cell. The helper lipid can be neutral, positively charged, or negatively charged. In certain embodiments, the helper lipid is neutral or negatively charged. Nonlimiting examples of helper lipids include l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl- 2-oleoyl-sn-glycero-3phosphocholin (POPC) and l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0110] The term “heteroalkyl” as used herein by itself or in combination with another term, means, unless otherwise stated, a non-cyclic stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quatemized. The heteroatom(s) (e.g., O. N, P. and S) may be placed at any intenor position of the heteroalkyl group or at either terminal position at which the group is attached to the remainder of the molecule.

[0111] The term “heteroaryl” as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth.

[0112] Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl. tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl,Attomey Docket No. 5134-WO benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl. purinyl, xanthinyl, adeninyl. guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein.

[0113] Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl). N-hydroxytetrazolyl. N- hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyL 3- anthracenyl), thiophenyl (2 -thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydry 1, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4- imidazolyl. 5-imidazolyl), triazolyl (1.2.3-triazol-l-yl, l,2,3-triazol-2-yl 1,2,3 -tri azol-4-yl. l,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2 -thiazolyl, 4- thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4- pyridazinyl, 5 -pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6- quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1 -isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5- isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b] furanyl, 5-benzo[b]furanyl, 6-benzo[b] furanyl, 7- benzo[b] furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2.3- dihydro-benzo[b]furanyl). 4-(2,3-dihydro-benzo[b]furanyl). 5-(2.3-dihydro-benzo[bJfuranyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b] furanyl), benzo[b]thiophenyl (2- benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6- benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3- dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro- benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro- benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2-indolyl, 3- indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl, 4- indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1 -benzimidazolyl, 2- benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8- benzimidazolyl), benzoxazolyl (1 -benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1- benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5 -benzothiazolyl, 6-benzothiazolyl, 7-Attorney Docket No. 5134-WO benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H- dibenz[b.f|azepine (5H-dibenz[b,f] azepin- 1-yl, 5H-dibenz[b,f]azepine-2-yl, 5H- dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f|azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,11- dihydro-5H-dibenz[b,f| azepine (10,1 l-dihydro-5H-dibenz[b,f| azepine- 1-yl, 10,11-dihydro- 5H-dibenz[b,f|azepine-2-yl, 10,1 l-dihydro-5H-dibenz[b,f|azepine-3-yl, 10,11 -dihydro-5H- dibenz[b.f|azepine-4-yl, 10,l l-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like.

[0114] The term “heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturated or fully saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi- and tricyclic ring systems where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. A heterocycloalkyl can include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized and a nitrogen heteroatom optionally can be substituted. Representative heterocycloalkyl groups include, but are not limited, to the following exemplary groups: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0115] The term “heterocyclyl” as used herein refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase “heterocyclyl group” includes fused ring species including those that include fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted asAttorney Docket No. 5134-WO discussed herein. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl. triazolyl. tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl. and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with groups such as those listed herein.

[0116] The term “hy drocarbon” or “hydrocarbyl” as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups.

[0117] The term “ionizable lipid” as used herein refers to a lipid (e.g., a cationic lipid) having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7.

[0118] As used herein, the term “hydrocarbyl” refers to a functional group derived from a straight chain, branched, or cyclic hydrocarbon, and can be alkyl, alkenyl, alkynyl, aryl, cycloalky l, acyl, or any combination thereof. Hydrocarbyl groups can be shown as (Ca- Cb)hydrocarbyl, wherein a and b are integers and mean having any of a to b number of carbon atoms. For example, (Cl-C4)hydrocarbyl means the hydrocarbyl group can be methyl (Cl), ethyl (C2), propyl (C3), or buty l (C4), and (CO-Cb)hydrocarbyl means in certain embodiments there is no hydrocarbyl group.

[0119] The term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simpleAttorney Docket No. 5134-WO lipids,’' which include fats and oils as well as waxes; (2) "compound lipids.” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0120] As used herein, “lipid encapsulated” can refer to a lipid particle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., a protein cargo), with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form an SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle).

[0121] The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e g., 1-1,000 nm) which includes one or more lipids and / or additional agents.

[0122] The term “lipid particle” is used herein to refer to a lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site of interest. In the lipid particle of the disclosure, which is typically formed from a cationic lipid, a non-cationic lipid, and a conjugated lipid that prevents aggregation of the particle, the active agent or therapeutic agent may be encapsulated in the lipid, thereby protecting the agent from enzymatic degradation.

[0123] The term “monovalent” as used herein refers to a substituent connecting via a single bond to a substituted molecule. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond.

[0124] The term “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0125] The term “non-cationic lipid” refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.

[0126] The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegy lated lipids are known in the art and include 1 - (monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s- DMG), DSPE-PEG- DBCO, DOPE-PEG-Azide, DSPE-PEG-Azide, DPPE-PEG-Azide. DSPE-PEG-Carboxy- NHS, DOPE-PEG-Carboxylic Acid, DSPE-PEG-Carboxylic acid and the like.Attorney Docket No. 5134-WO

[0127] The terms "about" and “approximately” mean within a statistically meaningful range of a value. Such a range can be within an order of magnitude, such as within 10%, and preferably within 5% or 1% 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 ordinary7skill 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 quantity7or 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.

[0128] 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 can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”II. Artificial Antigen Presenting Cells

[0129] Provided herein are artificial antigen presenting cells (aAPCs) which can be used to stimulate the proliferation and / or function of an immune cell that is co-cultured with the aAPCs. Using aAPCs provides the following potential benefits: 1) production of a master cell bank; 2) production of a similar or improved quality7cell product, e.g., purity, potency, dose, and viability; and 3) decreasing the manufacturing process duration.Attorney Docket No. 5134-WO

[0130] In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a tumor infiltrating lymphocyte (TIL). In some embodiments, the aAPCs may provide multivalent signaling that is essential for full activation and expansion of the TILs cocultured with the aAPCs. In some embodiments, the aAPCs may stimulate the viability’, phenoty pe, and / or tumor killing function of the TILs co-cultured with the aAPCs.

[0131] Therefore, provided herein are aAPCs engineered to express one or more costimulatory molecules that are essential for T cell activation and expansion. In an embodiment is provided an isolated artificial antigen presenting cell (aAPC) comprising a U937 cell that is genetically modified to express one or more co-stimulatory molecules.A. Co-Stimulatory Molecules

[0132] In some embodiments, the one or more co-stimulatory molecules are at least one of: a CD64 protein, a CD86 protein, a 4-1BBL protein, or an OX40L protein. Example sequences for human CD64, human CD86, human 4-1BBL (CD137L), and human OX40L (CD134L) are given in Table 1.TABLE 1. Amino acid and nucleic acid sequences for co-stimulatory molecules.Attorney Docket No. 5134-WO

[0133] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NO:4, and conservative amino acid substitutions thereof, and wherein the CD64 protein is expressed on the surface of the U937 cell.

[0134] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1 and conservative amino acid substitutions thereof, and wherein the CD86 protein is expressed on the surface of the U937 cell.

[0135] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a 4-1 BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:2, and conservative amino acid substitutions thereof, and wherein the 4-1BBL protein is expressed on the surface of the U937 cell.

[0136] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:3, and conservative amino acid substitutions thereof, and wherein the OX40L protein is expressed on the surface of the U937 cell.

[0137] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NO:4, and conservative amino acid substitutions thereof, and a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1. and conservative aminoAttorney Docket No. 5134-WO acid substitutions thereof, and wherein the CD64 protein and the CD86 protein are expressed on the surface of the U937 cell.

[0138] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NO:4, and conservative amino acid substitutions thereof, and a 4-1 BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:2, and conservative amino acid substitutions thereof, and wherein the CD64 protein and the 4-1 BBL protein are expressed on the surface of the U937 cell.

[0139] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NO:4, and conservative amino acid substitutions thereof, and an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:3, and conservative amino acid substitutions thereof, and wherein the CD64 protein and the OX40L protein are expressed on the surface of the U937 cell.

[0140] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:2, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the U937 cell.

[0141] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1, and conservative amino acid substitutions thereof, and an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:3, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the OX40L protein are expressed on the surface of the U937 cell.

[0142] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO: 2, and conservative amino acid substitutions thereof, and an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:3, and conservative amino acid substitutions thereof, and wherein the 4-1 BBL protein and the OX40L protein are expressed on the surface of the U937 cell.Attorney Docket No. 5134-WO

[0143] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NO:4, and conservative amino acid substitutions thereof, a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1, and conservative amino acid substitutions thereof, and a 4-1 BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:2, and conservative amino acid substitutions thereof, and wherein the CD64 protein, the CD86 protein and the 4-1BBL protein are expressed on the surface of the U937 cell.

[0144] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NO:4, and conservative amino acid substitutions thereof, a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1, and conservative amino acid substitutions thereof, and an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:3, and conservative amino acid substitutions thereof, and wherein the CD64 protein, the CD86 protein and the OX40L protein are expressed on the surface of the U937 cell.

[0145] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NO:4. and conservative amino acid substitutions thereof. a 4-lBBL protein comprising an amino acid sequence as set forth in SEQ ID NO:2, and conservative amino acid substitutions thereof, and an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:3, and conservative amino acid substitutions thereof, and wherein the CD64 protein, the 4-1BBL protein and the OX40L protein are expressed on the surface of the U937 cell.

[0146] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:1, and conservative amino acid substitutions thereof, a 4-lBBL protein comprising an amino acid sequence as set forth in SEQ ID NO:2, and conservative amino acid substitutions thereof, and an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:3, and conservative amino acid substitutions thereof, and wherein the CD86 protein, the 4-1 BBL protein and the OX40L protein are expressed on the surface of the U937 cell.Attorney Docket No. 5134-WO

[0147] In an embodiment, an aAPC as described herein comprises a U937 cell, wherein the cell is modified to express a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NO:4, and conservative amino acid substitutions thereof, a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1, and conservative amino acid substitutions thereof, a 4-lBBL protein comprising an amino acid sequence as set forth in SEQ ID NO:2, and conservative amino acid substitutions thereof, and an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:3. and conservative amino acid substitutions thereof, and wherein the CD64 protein, the CD86 protein, the 4-1BBL protein and the OX40L protein are expressed on the surface of the U937 cell.

[0148] In an embodiment, an aAPC as described herein comprises a U937 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD64, a nucleic acid encoding CD86 and a nucleic acid encoding 4- 1BBL, and wherein the U937 cell expresses CD64, CD86 and 4-1BBL. In an embodiment, the aAPC comprise a U937 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD64, a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the U937 cell expresses CD64, CD86 and 4-1BBL. In an embodiment, a method of preparing any of the foregoing embodiments of aAPCs is provided.

[0149] In an embodiment, an aAPC as described herein comprises a U937 cell modified to express a CD64 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:4, a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO: 1 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:2, wherein the CD64 protein, the CD86 protein and the 4-1 BBL protein are expressed on the surface of the U937 cell. In an embodiment, an aAPC as described herein comprises a U937 cell modified to express a CD64 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:4, a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO: 1 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:2, wherein the CD64 protein, the CD86 protein and the 4-1BBL protein are expressed on the surface of the U937 cell. In an embodiment, the aAPC comprises a U937 cell modified to express a CD64 protein comprising a sequence with greater than 97% identity to an amino acidAttorney Docket No. 5134-WO sequence as set forth in SEQ ID NO:4, a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO: 1 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:2, wherein the CD64 protein, the CD86 protein and the 4-1BBL protein are expressed on the surface of the U937 cell. In an embodiment, the aAPC comprises a U937 cell modified to express a CD64 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:4, a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO: 1 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:2. wherein the CD64 protein, the CD86 protein and the 4-1BBL protein are expressed on the surface of the U937 cell. In an embodiment, the aAPC comprises a U937 cell modified to express a CD64 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:4, a CD86 protein comprising a sequence with greater than 95% identity7to an amino acid sequence as set forth in SEQ ID NO: 1 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:2, wherein the CD64 protein, the CD86 protein and the 4-1BBL protein are expressed on the surface of the U937 cell. In an embodiment, the aAPC comprises a U937 cell modified to express a CD64 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:4, a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO: 1 and a 4- 1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:2, wherein the CD64 protein, the CD86 protein and the 4-1BBL protein are expressed on the surface of the U937 cell. In an embodiment, a method of preparing any of the foregoing embodiments of aAPCs is included.

[0150] In an embodiment is disclosed an aAPC comprising a U937 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO: 7, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6, and conservative amino acid substitutions thereof. In an embodiment, the aAPC comprises a U937 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:7. and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising anAttorney Docket No. 5134-WO amino acid sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 6, and conservative amino acid substitutions thereof. In an embodiment, a method of preparing any of the foregoing embodiments of aAPCs is provided.

[0151] In an embodiment, an aAPC is disclosed comprising a U937 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO: 7 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6. In an embodiment, the aAPC comprises a U937 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO: 7 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO: 6. In an embodiment, the aAPC comprises a U937 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO: 7 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6. In an embodiment, the aAPC comprises a U937 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:7 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6. In an embodiment, the aAPC comprises a U937 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:7 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity' to an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6. In an embodiment, the aAPC comprises a U937 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:7 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity' to an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6. In an embodiment, a method of preparing any of the foregoing embodiments of aAPCs is included.Attorney Docket No. 5134-WO

[0152] In an embodiment, an aAPC is provided comprising a U937 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO: 8, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6, and conservative amino acid substitutions thereof. In an embodiment, the aAPC comprises a U937 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:8. and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 6, and conservative amino acid substitutions thereof. In an embodiment, a method of preparing any of the foregoing embodiments of aAPCs is included.

[0153] In an embodiment, an aAPC is provided comprising a U937 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6. In an embodiment, the aAPC comprises a U937 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO: 8 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6. In an embodiment, the aAPC comprising a U937 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6. In an embodiment, the aAPC comprises a U937 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO: 8 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6. In an embodiment, the aAPC comprises a U937 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO: 8 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence asAttomey Docket No. 5134-WO set forth in SEQ ID NO:5 or SEQ ID NO:6. In an embodiment, the aAPC comprises a U937 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO: 8 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6. In an embodiment, a method of preparing any of the foregoing embodiments of aAPCs is included.

[0154] The sequences for the ligands to which human CD86 binds (CD28 and CTLA-4), the ligand to which human 4-1BBL binds (4-1BB), and the ligand to which human OX40L binds (0X40) are given in Table 2.TABLE 2. Amino acid sequences for human CD28, human CTLA-4, human 4- IBB, and human OX-40.B. Methods of Preparing Artificial Antigen Presenting Cells

[0155] In an embodiment, a method of preparing an aAPC includes the step of stable incorporation of genes for production of one or more co-stimulatory proteins disclosed herein. In an embodiment, a method of preparing an aAPC includes the step of retroviral transduction. In an embodiment, a method of preparing an aAPC includes the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, e.g., in Levine, et al., Proc. Nat ’I Acad. Sci. 2006, 103, 17372-77 ; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., J. Virology 1998, 72, 8463-71, and U.S. Patent No. 6.627,442, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of gamma-retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, e.g., Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which isAttorney Docket No. 5134-WO incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include systems wherein the transposase is provided as DNA expression vector or as an expressible RNA or a protein such that long-term expression of the transposase does not occur in the transgenic cells, for example, a transposase provided as an mRNA (e.g, an mRNA comprising a cap and poly-A tail). Suitable transposon- mediated gene transfer systems, including the salmonid-type Tel-like transposase (SB or Sleeping Beauty transposase), such as SB10, SB1 1, and SBIOOx, and engineered enzymes with increased enzymatic activity, are described in, e.g., Hackett, et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the disclosures of each of which are incorporated by reference herein.

[0156] In an embodiment, a method of preparing an aAPC includes the step of stable incorporation of genes for transient production of one or more co-stimulatory proteins disclosed herein. In an embodiment, a method of preparing an aAPC includes the step of electroporation. Electroporation methods are know n in the art and are described, <?.g., in Tsong, Biophys. J. 1991, 60, 297-306, and U.S. Patent Application Publication No. 2014 / 0227237 Al. the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler. et al., Proc. Natl. Acad. Sci. 1979, 76. 1373-1376; and Chen and Okay area, Mol. Cell. Biol. 1987, 7. 2745-2752; and in U.S. Patent No. 5,593,875, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of liposomal transfection. Liposomal transfection methods, such as methods that employ a 1 : 1 (w / w) liposome formulation of the cationic lipid 7V-[ 1 -(2,3-dioleyloxy)propyl ]-n,n,n- trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Feigner, et al, Proc. Natl. Acad. Sci. USA. 1987, 84. 7413-7417 and in U.S. Patent Nos. 5,279,833; 5,908,635; 6,056,938; 6.110,490; 6,534.484; and 7,687,070, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of transfection using methods described inAttorney Docket No. 5134-WOU.S. Patent Nos. 5.766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated by reference herein.

[0157] In an embodiment, the aAPC is transduced by first using the Gateway cloning method (commercially available from ThermoFisher, Inc.) to prepare vector for lentiviral transduction, followed by lentiviral transduction using the vector and one or more associated helper plasmids, as is also described elsewhere herein. In the Gateway cloning method, a gene is selected (such as a gene encoding one or more co-stimulatory proteins disclosed herein) and is then provided with primers and amplified using PCR technology with the help of an attB tagged primer pair. The PCR fragment is then combined with a donor vector (pDONR, such as pDONR221) that includes attP sites to provide an entry clone, using the BP reaction. An integration reaction between the attB and the attP sites combines the PCR fragment with the donor vector. The resulting entry clone contains the gene of interest flanked by attL sites. The LR reaction is then used to combine the entry clone with a destination vector to produce an expression vector. In the LR reaction, a recombination reaction is used to link the entry clone with the destination vector (such as pLV430G) using the attL and attR sites and a clonase enzyme. The attL sites are already found in the entry clone, while the destination vector includes the attR sites. The LR reaction is carried out to transfer the sequence of interest into one or more destination vectors in simultaneous reactions.

[0158] In embodiments, the gene(s) of interest includes one or more of a gene encoding CD64, CD86, 4-1BBL, OX40L or any combination thereof. In embodiments, the gene of interest encodes CD64. In embodiments, the gene of interest encodes CD86. In embodiments, the gene of interest encodes 4-1 BBL. In embodiments, the gene of interest encodes OX40L. In embodiments, the genes of interest encode CD64 and CD86. In embodiments, the genes of interest encode CD64 and 4-1BBL. In embodiments, the genes of interest encode CD64 and OX40L. In embodiments, the genes of interest encode CD86 and 4-1 BBL. In embodiments, the genes of interest encode CD86 and OX40L. In embodiments, the genes of interest encode 4-1 BBL and OX40L. In embodiments, the genes of interest encode CD64, CD86, and 4- 1BBL. In embodiments, the genes of interest encode CD64, CD86, and OX40L. In embodiments, the genes of interest encode CD64, 4-1 BBL, and OX40L. In embodiments, the genes of interest encode CD86, 4-1 BBL, and OX40L. In embodiments, the genes of interest encode CD64, CD86, 4-1BBL, and OX40L. In embodiments, the genes of interest are clonedAtorney Docket No. 5134-WO into a single vector. In embodiments, the genes of interest are cloned into two or more separate vectors.

[0159] After the vector for lend viral transduction is prepared, virus for transduction of APCs is prepared. In embodiments, the vector for lentiviral transduction is transfected into a packaging cell. In embodiments, a kit, such as the TRANSIT® Lentivirus System (Minis, Madison, WI), Lenti-X™ packaging system (Takara), Lenti-vpak packaging kit (OriGene), IDLV Lenti packaging kit (OriGene), pPACKHl HIV Lend vector Packaging Kit (System Biosciences), or any other available kit or packaging system. In embodiments, the vector for lentiviral transduction is transfected into the packaging cell using any transfection method. In embodiments, the vector for lentiviral transduction is transfected into the packaging cell along with one or more helper plasmids. In embodiments, the one or more helper plasmids encode necessary’ viral proteins for packaging and assembly of the vector particles. For example, necessary viral proteins may include, without limitation, Gag / Pol (structural proteins). Rev (involved in RNA export), and an envelope protein (e.g., VSV-G for broad tropism). In embodiments, the necessary’ viral proteins include one or more of Gag-Pol, Tat, Rev, Nef, Vpr, Vpu, Vif. or vesicular stomatitis virus G (VSV-G) protein, or any combination thereof. In embodiments, the packaging cell is a HEK 293 cell. In embodiments, the packaging cell is a HEK 293T cell, HEK 293T / 17 cell, HEK 293-F cell. The packaging cell may be grown in adherent culture or suspension culture. After transfection, lentivirus particles including the gene(s) (e.g., one or more genes encoding one or more co-stimulatory proteins) may be collected from the supernatant. In embodiments, the lentiviral particles are frozen after harvest. In embodiments, the lentiviral particles are titered.

[0160] Once the lentiviral particles are obtained, APCs may’ be infected (transduced) with the lentiviral particles to produce aAPCs. In embodiments, the aAPCs are assayed for expression of the one or more genes. In embodiments, aAPCs expressing the one or more genes are isolated. In embodiments, aAPCs expressing the one or more genes are cloned, e.g. to produce a clonal aAPC cell line.

[0161] In some embodiments, the aAPCs described herein may be grown and maintained under serum-based media and / or serum free media. According to an exemplary method. aAPCs may be cultured in 24 well plates at a cell density of about I x IO6cells per well for 3 to 5 days. The cells may then be isolated and / or washed by centrifugation and resuspended in media or cryopreserved in an appropriate cryopreservation media (e.g., CRYOSTORIO (BioLife Solutions)) and stored in a -80 °C freezer.Attorney Docket No. 5134-WO

[0162] In some embodiments, the aAPCs described herein may be grown in the presence of serum-based media. In some embodiments, the aAPCs described herein by may be grown in the presence of serum-based media that includes human serum (hSerum) containing media (e.g., cDMEM with 10% hSerum).

[0163] In some embodiments, the aAPCs described herein may be grown in the presence of serum free media. In some embodiments, the serum free media may be selected from the group consisting of CTS OPTMIZER (ThermoFisher), XVIVO-20 (Lonza), Prime T Cell CDM (Irvine), XFSM (MesenCult), and the like.III. Lipid Nanoparticles (LNPs)

[0164] In an embodiment, nanoparticles (LNPs) engineered with one or more costimulatory molecules, e.g., CD64, CD86, 4-1BBL, and OX40L, which can be used as APCs in a method of expanding TILs, are provided.

[0165] In some embodiments, the LNPs are those disclosed in PCT Patent Publication No. WO 2024 / 077232 A2, the content of which is herein incorporated by reference in its entirety.

[0166] In certain embodiments, the LNP comprises at least one ionizable lipid.

[0167] In certain embodiments, the LNP comprises at least one neutral lipid.

[0168] In certain embodiments, the LNP comprises cholesterol and / or a modified derivative thereof.

[0169] In certain embodiments, the LNP comprises at least one polymer conjugated lipid and / or modified derivative thereof, and / or a modified derivative thereof.

[0170] In certain embodiments, the LNP comprises a co-stimulatory molecule specific to binding to a surface molecule of a target cell. In embodiments, the co-stimulatory molecule includes one or more of CD64, CD86, 4-1 BBL, OX40L or any combination thereof, or a fragment thereof. In embodiments, the co-stimulatory molecule includes CD64. In embodiments, co-stimulatory molecule includes CD86. In embodiments, the co-stimulatory molecule includes 4-1 BBL. In embodiments, the co-stimulatory molecule includes OX40L. In embodiments, the co-stimulatory molecules include CD64 and CD86. In embodiments, the co-stimulatory molecules include CD64 and 4-1 BBL. In embodiments, the co-stimulatory molecules include CD64 and OX40L. In embodiments, the co-stimulatory molecules include CD86 and 4-1BBL. In embodiments, the co-stimulatory molecules include CD86 and OX40L. In embodiments, the co-stimulatory molecules include 4-1 BBL and OX40L. InAttorney Docket No. 5134-WO embodiments, the co-stimulatory molecules include CD64, CD86, and 4-1BBL. In embodiments, the co-stimulatory molecules include CD64, CD86, and OX40L. In embodiments, the co-stimulatory molecules include CD64, 4-1BBL, and OX40L. In embodiments, the co-stimulatory molecules include CD86, 4-1BBL, and OX40L. In embodiments, the co-stimulatory molecules include CD64, CD86, 4-1BBL, and OX40L. In embodiments, the co-stimulatory molecules are associated with a single LNP. In embodiments, the co-stimulatory molecules are associated with two or more separate LNPs. In embodiments, the co-stimulatory molecules are conjugated to the LNP. In embodiments, the co-stimulatory molecules are covalently linked to the LNP (e.g. via covalent attachment to a lipid or PEG). In embodiments, the co-stimulatory molecules are associated with the LNP. In embodiments, the co-stimulatory molecules are associated with the LNP via one or more hydrophobic domains of the co-stimulatory molecule(s).

[0171] In certain embodiments, the co-stimulatory molecule is covalently conjugated to at least one component of the LNP.

[0172] In certain, non-limiting, exemplary embodiments, the present disclosure provides a LNP. In certain embodiments, the LNP comprises (a) at least one ionizable lipid. In certain embodiments, the LNP comprises (b) at least one neutral lipid. In certain embodiments, the LNP comprises (c) at least one cholesterol compound and / or modified derivative thereof. In certain embodiments, the LNP comprises (d) at least one polymer conjugated lipid and at least one compound of Formula (II), or a salt, solvate, stereoisomer, or isotopologue thereof.

[0173] In certain, non-limiting, exemplary embodiments, the present disclosure provides a LNP. In certain embodiments, the LNP comprises (a) at least one ionizable lipid of Formula (I), or a salt, solvate, stereoisomer, or isotopologue thereof. In certain embodiments, the LNP comprises (b) at least one neutral lipid. In certain embodiments, the LNP comprises (c) at least one cholesterol compound and / or modified derivative thereof. In certain embodiments, the LNP comprises (d) at least one polymer conjugated lipid. In certain embodiments, the LNP comprises (e) at least one co-stimulatory molecule . In certain embodiments, the co- stimulatory molecule is covalently conjugated to at least one component of the LNP.

[0174] In certain, non-limiting, exemplary embodiments, the present disclosure provides a LNP. In certain embodiments, the LNP comprises (a) at least one ionizable lipid of Formula (1). or a salt, solvate, stereoisomer, or isotopologue thereof. In certain embodiments, the LNP comprises (b) at least one neutral lipid. In certain embodiments, the LNP comprises (c) atAttorney Docket No. 5134-WO least one cholesterol compound and / or modified derivative thereof. In certain embodiments, the LNP comprises (d) at least one polymer conjugated lipid and at least one compound of Formula (II), or a salt, solvate, stereoisomer, or isotopologue thereof.

[0175] In certain embodiments, the at least one ionizable lipid comprises an ionizable lipid of Formula (I), or a salt, solvate, stereoisomer, or isotopologue thereof:wherein:R1" and R are each independently ;R2a, Rzb, R2C. R2d, R2e, R2f, R2g, and R2hare each independently selected from the group consisting of H, optionally substituted C1-C12 alkyl, optionally substituted C2- Ci2 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C?-CJ?. alkenyl, optionally substituted C2- C 12 alkynyl, optionally substituted C7-C13 aralkyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C 10 heteroaryl; each occurrence of R3a, Rjb, and Rdcis independently selected from the group consisting of H, -(optionally substituted C1-C6 alkylenyl)-C(=:O)OR4, -(optionally substituted Ci- Co alkylenyl)-C(=O)N(R4)(R3), -(optionally substituted Ci-Ce alkylenyl)-C(=O)R4, - (optionally substituted C1-C6 al kylenyl)-(R4), -C(:=O)OR4, -C(=O)N(R4)(R5), -C(>O)R4, and R4. wherein no more than one of each occurrence of R’a, RdB, and R3cis H;R4is selected from the group consisting of optionally substituted C1-C28 alky l, optionally substituted C2-C28 heteroalkyl, optionally substituted C2-C8 cycloalkyl, optionally substituted C2-Cs heterocycloalkyd, optionally7substituted C2-C28 alkenyl, and optionally7substituted C2-C28 alkynyl;R3is selected from the group consisting of H and optionally substituted Ci-Ce alkyl;Each occurrence of L1is independently7selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, - (optionally substituted C 1-C 12 alkynylenyl)-X~, -(optionally substituted C 1-C 12 heteroalkylenyl)-X-, -X-(optionally substituted C1-C12 alkylenyl)-, -X-(optionalIy substituted C2-C 12 alkenylenyl)-, -X-(optionally substituted Cl -Cl 2 alkynylenyl)-, -X- (optionally substituted C 1-C 12 heteroalkylenyl)-, optionally substituted C3-C8 cycloalkylenyl. and optionally substituted C-2-Cs heterocyloalkylenyl; each occurrence of X, if present, is independently selected from the group consisting of a bond, -NIR7^)-, and -O-;Attorney Docket No. 5134-WO and each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4.

[0176] In certain embodiments, at least one selected from the group consisting of R2\ Rzb, R2c, R2d, R2®, R21, R2g, and R2his H. In certain embodiments, at least two selected from the group consisting of R2a, R2D, R2C. R2d, R2e, R2f, R2g, and R2bare H. In certain embodiments, at least three selected from the group consisting of R23, R2b, R2C, R2d, R2e, R2£, R2g, and R2hare H. In certain embodiments, at least four selected from the group consisting of R2a, R2b, R2C, R2d, R2e, R2f, R2g, and R2!1are H. In certain embodiments, at least five selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R21, R2g, and R2ilare H. In certain embodiments, at least six selected from the group consisting of R23. R21’, R R2ii, R2e, R2f, R2g, and R2hare H. In certain embodiments, at least seven selected from the group consisting of R2a, R2b, R2e, R2d, R2e, R2f. R2« and R2hare H. In certain embodiments, each of R2a, R2b. R2C, R2<!, R \ R2f, R2g, and R2hare H.

[0177] In certain embodiments, Lfis -CH2-. In certain embodiments, L1is -(CH?.)?.-. In certain embodiments, L1is -(CH2)3-. In certain embodiments, L1is -(CH2)10-. In certain embodiments, L1is -(CH2)2O-. In certain embodiments. L1is -(CH2)3O-. In certain embodiments, L!is -CH2CH(OR’)CH2-. In certain embodiments, L;is -(CH2)2NR3c-. In--N N-*'* — f )■ — certain embodiments, L1is2—2. In certain embodiments, L1is ' —fIn certain0 embodiments, L1is For instances of L which are asymmetric (e.g., -(CH2J3O-) it is understood that the present disclosure encompasses both possible orientations (e.g , - (CH2)3O- and -O(CH2)3-).

[0178] In certain embodiments, the ionizable lipid of Formula (I) is:, of Formula (I) is:. In certain embodiments, the ionizable lipid of Formula (I) is:Attorney Docket No. 5134-WOcertain embodiments, the ionizable lipid of Formula (I) is:**»»•

[0179] In certain embodiments, the ionizable lipid of Formula (I) is:certain embodiments, the ionizable lipid of Formula (I) is:4OR3R'"(Formula (I) is:

[0180] In certain embodiments,CH2CH(OH)(optionally substituted C1-C28 alkyl). In certain embodiments, Riais - CH2CH(OH)(optionally substituted C2-C2.8 alkenyl). In certain embodiments, R3ais- CH2CH2C(=O)O(optionaJly substituted C1-C28 alkyl). In certain embodiments, Rjais - CH2CH?.C(=O)NH(optionally substituted CJ-CZH alkyl). In certain embodiments, R’Bis H. In certain embodiments, R3bis -CH2CH(OH)(optionally substituted C1-C28 alkyl). In certain embodiments, R3bis -CH2CH(OH)(optionally substituted C2-C28 alkenyl). In certain embodiments, R3bis -CH2CH2C(=O)O(optional1y substituted C1-C28 alkyl). In certain embodiments, R?bis -CH2CH2C(=O)NH(optionally substituted C1-C28 alkyl). In certain embodiments, R'cis H. In certain embodiments. R^ is -CH2CH(OH)(optionally substituted C1-C28 alkyl). In certain embodiments, R3cis -CH2CH(OH)(optionally substituted C2- C28 alkenyl). In certain embodiments, R3cis -CH2CH2C(=O)O(optionaHy substituted C1-C28Attorney Docket No. 5134-WO alky 1 ). In certain embodiments, R3'- is -CH2CH2C(:=O)NI {(optionally substituted Ci- C28 alkyl).

[0181] In certain embodiments, R‘‘!is -CH2CH(OH)(CH2)9CH3. In certain embodiments, Rais -CH2CH(OH)(CH2)11CH3. In certain embodiments, R3ais - CH2CH(OH)(CH2)13CH3. In certain embodiments, R3bis -CH2CH(OH)(CH2)9CH3. In certain embodiments, R’bis -CH2CH(OH)(CH2)11CH3. In certain embodiments, R’bis - CH2CH(OH)(CH2)13CH3. In certain embodiments, R3cis -CH2CH(OH)(CH2)9CH3. In certain embodiments, R3cis -CH2CH(OH)(CH2)11CH3. In certain embodiments, R3cis - CH2CH(OH)(CH2) 13CH3.

[0182] In certain embodiments, each occurrence of optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted ary], optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is independently optionally substituted with at least one substituent selected from the group consisting of Cl- Ce alkyl, Ca-Cs cycloalkyl, Ci-C* haloalkyl, Ci-C? haloalkoxy, phenoxy, halogen, CN, NO2, OH, N(R’)(R”), C(=O)R’, C(-O)OR’, OC(==O)OR', C(-O)N(R’)(R”), S(===O)2N(R’)(R"), N(R')C(==O)R”, N(R’)S(=O)2R”, C2-CS heieroaryl. and phenyl optionally substituted with at least one halogen, wherein each occurrence of R’ and R” is independently selected from the group consisting of H, Ci-Cr, alkyl, C?-C« cycloalkyl, Ci-Ce haloalkyl, benzyl, and phenyl.

[0183] In certain embodiments, the ionizable lipid of Formula (I) is:1 , r-((2-(2-(4-(2-((2-(2-(bis(2-hy droxytetradecyl)amino)ethoxy )ethyl)(2- hydroxytetradecyl)amino)ethyl)piperazin-l-yl)ethoxy)ethyl)azanediyl)bis(tetradecan-2-oi) (C 14-494).Attorney Docket No. 5134-WO

[0184] In certain embodiments, the at least one ionizable lipid comprises about 1, 2, 3, 4, 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, 30, 31, 32, 33, 34. 35, 36, 37, 38. 39. 40. 41, 42, 43, 44. 45, 46, 47, 48, 49, 50, 51. 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64. 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79.80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91. 92, 93, 94, 95, 96, 97, 98, or about 99 mol% of the LNP.

[0185] In certain embodiments, the at least one ionizable lipid comprises less than about 1. 2, 3, 4, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62. 63, 64. 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87. 88, 89. 90. 91. 92. 93. 94, 95, 96, 97, 98, or about 99 mo]% of the LNP.

[0186] In certain embodiments, the at least one ionizable hpid comprises more than about 1, 2, 3, 4, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39. 40. 41. 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60. 61. 62, 63, 64, 65. 66. 67. 68, 69, 70, 71. 72. 73, 74, 75, 76.77, 78, 79, 80, 81, 82, 83, 84, 85. 86. 87. 88, 89, 90, 91. 92, 93, 94, 95, 96, 97, 98, or about 99 mol% of the LNP.

[0187] In certain embodiments, the at least one ionizable lipid comprises about 10, 11. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. 22, 23. 24. 25. 26. 27. 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42. 43. 44, 45, 46, 47. 48, 49, or about 50 mol% of the LNP.

[0188] In certain embodiments, the at least one ionizable lipid comprises less than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24. 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45. 46. 47. 48. 49, or about 50 mol% of the LNP.

[0189] In certain embodiments, the at least one ionizable hpid comprises more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24. 25, 26, 27, 28, 29, 30, 31, .32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44. 45. 46. 47, 48, 49, or about 50 mol% of the LNP.

[0190] In certain embodiments, the at least one ionizable lipid comprises about 40 mol% of the LNP. In certain embodiments, the at least one ionizable lipid comprises about 41 mol% of the LNP.

[0191] In certain embodiments, the neutral lipid comprises dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylcholine (DSPC). InAttorney Docket No. 5134-WO certain embodiments, the neutral lipid is dioleoylphosphatidylethanolamine (DOPE). In certain embodiments, the neutral lipid is dioleoylphosphatidylethanolamine (DOPE)

[0192] In certain embodiments, the at least one neutral lipid comprises about 5. 6. 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31. 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44. or about 45 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises less than 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. 30, 31, 32, 33, 34, 35, 36, 37. 38, 39, 40, 41, 42, 43, 44, or about 45 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises more than about 5, 6, 7, 8. 9, 10, 1 1, 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30. 31, 32. 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or about 45 mol% of the LNP.

[0193] In certain embodiments, the at least one neutral lipid comprises about 30 mol% of the LNP. In certain embodiments, the LNP comprises about 30 mol% DOPE.

[0194] In certain embodiments, the cholesterol and / or modified derivative thereof comprises 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. 30, 31, 32, 33, 34, 35. 36. 37, 38, 39, 40. 41. 42. 43, 44, 45, 46. 47. 48, 49, or about 50 mol% of the LNP.

[0195] In certain embodiments, the cholesterol and / or modified derivative thereof comprises less than 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, 30, 31, 32, 33, 34. 35, 36. 37. 38. 39. 40. 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mol% of the LNP.

[0196] In certain embodiments, the cholesterol and / or modified derivative thereof comprises more than about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17. 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34. 35, 36. 37. 38. 39. 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mol% of the LNP.

[0197] In certain embodiments, the cholesterol and / or modified derivative thereof is cholesterol In certain embodiments, the chol esterol comprises about 25 mol% of the LNP In certain embodiments, the cholesterol comprises about 25.6 mol% of the LNP.

[0198] In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises about 0.1 , 0.2, 0.3, 0 4, 0.5, 0.6, 0 7, 0.8, 0.9, 1.0, 1.1 ,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5.0, 5.1, 5.2. 5.2,5.4, 5.5, 5.6, 5.7. 5 8, 5.9, 6.0, 6 1. 6.2, 6.2, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0. 7.1, 7.2, 7.2. 7 4,7.5, 7.6. 7.7, 7.8, 7 9, 8.0. 8.1, 8.2, 8.2. 8.4. 8.5, 8.6, 8.7. 8.8, 8.9, 9.0, 9.1. 9.2, 9.2, 9.4. 9.5,Attorney Docket No. 5134-WO9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.2, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 1 1.2,11.2, 11.4, 11.5, 11.6, 1 1.7, 11.8, 11.9, 12 0, 12.1. 12.2. 12.3, 12.4, or about 12.5 mol% of the LNP.

[0199] In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,I.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1. 2.2, 2.3, 2.4, 2.5. 2.6, 2.7, 2.8, 2.9. 3.0,3.1, 3.2, 3.3, 3.4. 3.5, 3.6, 3.7, 3.8. 3.9, 4.0, 4.1, 4.2. 4.3, 4.4, 4.5. 4.6. 4.7, 4.8, 4.9. 5.0. 5.1,5.2, 5 2, 5.4, 5.5, 5.6, 5 7, 5.8, 5.9, 6 0, 6.1, 6.2, 6.2, 6 4. 6.5, 6.6, 6 7, 6.8, 6.9, 7.0, 7 1 , 7.2,7.2, 7.4, 7.5, 7.6, 7 7, 7.8, 7.9, 8.0, 8 1, 8.2, 8.2, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9 2, 9.2,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.2. 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1,I I.2, 11.2, 1 1.4, 1 1.5, 11.6, 11.7, 1 1.8, 11.9, 12.0. 12.1. 12.2. 12.3, 12.4, or about 12.5 mol% of the LNP.

[0200] In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises more than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3. 1.4, 1.5, 1.6. 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4. 2.5, 2.6, 2.7, 2.8. 2.9,3.0. 3.1, 3.2, 3.3. 3 4. 3 5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1. 4 2, 4.3, 4.4, 4 5, 4.6, 4.7. 4.8. 4.9, 5.0,5.1, 5.2, 5.2, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.2, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1.7.2, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.2, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,9.2, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1 , 10.2, 10.2, 10.4, 10 5, 10.6, 10.7, 10.8, 10.9, 1 1.0,11.1, 11.2, 1 1.2, 1 1.4, 11.5, 11.6, 1 1.7, 1 1.8, 11.9. 12.0. 12.1. 12.2, 12.3, 12.4, or about 12.5 mol% of the LNP.

[0201] In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises about 2.5 mol% of the LNP.

[0202] In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises a polyethylene glycol (PEG) conjugated lipid and / or modified derivative thereof. In certain embodiments, the at least one polyethylene glycol (PEG) conjugated lipid and / or modified derivative thereof comprises C14-PEG2000. In certain embodiments, C14-PEG2000 comprises (l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-]methoxy(polyethyiene glycol)-2000|:Attorney Docket No. 5134-WO

[0203] In certain embodiments, the maleimide moiety is covalently linked to the PEG chain of the PEG-conjugated lipid. Anon-limiting example of covalent linkage to PEG chain of the PEG-conjugated lipid includes:. Alternatively, the covalent linkage to the PEG chain may comprise including but not limited to an amide bond.

[0204] In certain embodiments, the surface molecule of a target cell is a surface antigen of a CD4+ T cell. In certain embodiments, the surface molecule of a target cell is a surface antigen of a CD8+ T cell.

[0205] In certain embodiments, the co-stimulatory molecule is at least one selected from the group consisting of an antibody against CD3 (aCD3) find an antibody against. CD28 (aCD28), or a fragment thereof.

[0206] In certain embodiments, the component to which the co-stimulatory molecule is conjugated is the polymer conjugated lipid and / or modified derivati ve thereof and / or modified derivative thereof.

[0207] In certain embodiments, the co-stimulatory molecule is covalently conjugated to the polymer conjugated lipid and / or modified derivative thereof.

[0208] In certain embodiments, the covalent conjugation comprises a covalent bond forming reaction selected from the group consisting of a [l,4]-conjugate addition (i.e., Michael addition), [4+2] cycloaddition, [3+2] dipolar cycloaddition, nucleophilic addition, transition metal-catalyzed cross-coupling reaction, carbonyl condensation reaction, and reductive amination

[0209] In certain embodiments, the covalent conjugation reaction comprises a [1.4]- conjugate addition reaction (i.e., Michael addition).

[0210] In certain embodiments, the [1.4] -conjugate addition occurs between a PEG- polyethylene glycol (PEG) conjugated lipid and / or modified derivative thereof which is further conjugated to a maleimide moiety’ and a cysteine thiol of a polypeptide.

[0211] In certain embodiments, the cysteine thiol of the polypeptide is derived from a reduced disulfide bridge of a polypeptide.

[0212] Alternative non-limiting examples of complementary functional groups for conjugation of the lipid-conjugate and co-stimulatory molecule include: (a) a nucleophile and electrophile (e.g., SN1 or SN2 reaction of an hydroxyl and benzyl chloride or an amine and a carboxylic acid or derivative thereof); (b) an azide and an alkyne (i.e., [3+2] cycloaddition orAttorney Docket No. 5134-WO‘‘click" reaction); and (c) a diene and a dienophile (e.g., substituted butadiene and substituted maleimide) via a Diels- Alder [4+2] cycloaddition, inter alia.

[0213] It is appreciated that any of a number covalent bond forming reactions (e.g., SN2, condensation. Diels-Alder reaction (i.e., [4+2] cycloaddition), [3+2] dipolar cycloaddition, and transition metal catalyzed cross-coupling, inter alia) may be employed to prepare the conjugated compositions of the present disclosure. It is understood that, given a particular bond forming reaction (e.g., SN2 reaction), one skilled in the art would readily recognize the requisite functional groups suitable for each component (i.e., conjugated lipid and co-stimulatory molecule) necessary7to achieve conjugation. Additionally, one skilled in the art of organic synthesis would be apprised of the necessary additional reagents and / or catalyst necessary to achieve covalent bond formation.

[0214] In certain embodiments, the LNP has a molar ratio of PEG-polyethylene glycol (PEG) conjugated lipid and / or modified derivative thereof and PEG-polyethylene glycol (PEG) conjugated lipid and / or modified derivative thereof further conjugated to a maleimide moiety selected from the group consisting of about 10: 1 , 9: 1, 8: 1, 7: 1. 6: 1, 5: 1, 4: 1. 3: 1 , 2: 1, 1: 1 , 1 :2. 1 :3, 1 :4. 1 :5, 1:6, 1:7, 1 :8, 1 :9. and 1 : 10.

[0215] In certain embodiments, the LNP has a molar ratio of PEG-polyethylene glycol (PEG) conjugated lipid and / or modified derivative thereof and PEG-polyethylene glycol (PEG) conjugated lipid and / or modified derivative thereof further conjugated to a maleimide moiety' of about 5: 1 .

[0216] In certain embodiments, the LNP has a molar ratio of polymer conjugated lipid and modified derivative of the conj ugated lipid further conj ugated to a maleimide moiety selected from the group consisting of about 10: 1, 9: 1, 8: 1, 7: 1 , 6: 1, 5: 1 , 4: 1, 3: 1, 2: 1, 1: 1. 1 :2, 1 :3, 1 :4. 1 :5, 1:6, 1 :7. 1:8, 1:9, and 1 : 10.

[0217] In certain embodiments, the modified derivative of the polymer conjugated lipid is a compound of Formula (II), or a salt, solvate, stereoisomer, or isotopologue thereof:wherein:Attorney Docket No. 5134-WOR5” and R5bare each independently selected from the group consisting of - C(=O)(optionally substituted C1-C28 alkyl), -C(=O)(optionally substituted C2-C28 alkenyl), -C(=O)(optionally substituted C2-C2.8 alkynyl). optionally substituted Ci- C28 alkyl, optionally substituted C2-C28 alkenyl, and optionally substituted C2-C28 alkynyl;Z is a monovalent cation;L2comprises n units of, o writsunits of9wherein each - — in L is a C-0 or C-N bond;Det is a co-stimulatory molecule, wherein is C-S bond;R6aand R6bare each independently selected from the group consisting of H and C1-C6 alkyl;11, 0, and p are each independently 1, 2, 3, 4, or 5; q is an integer ranging from 1 to 100; and r and s are each independently an integer ranging from 1 to 10.

[0218] In certain embodiments, R5a is C(==O)(C5-C20 alkyl). In certain embodiments, R5a is C(=O)(CH2)16CH3. In certain embodiments, R5b is C(:=:O)(C5-C20 alkyl). In certain embodiments, R5b is C(=O)(CH2)16CH3.

[0219] In certain embodiments,

[0220] In certain embodiments,

[0221] In certain embodiments, the compound of Formula (II) is:Attorney Docket No. 5134-WO

[0222] In certain embodiments, Det comprises at least one of an antibody of CD3 (aCD3) and an antibody of CD28 (aCD28). In certain embodiments, Det comprises an antibody of CD3 (aCD3). In certain embodiments, Det comprises an antibody of CD28 (aCD28). In certain embodiments, Det comprises an antibody of CD3 (aCD3) and an antibody of CD28 (aCD28). In certain embodiments, Det comprises an antibody of CD3 (aCD3) only In certain embodiments, Det composes an antibody of CD28 (aCD28) only. In certain embodiments, the antibody of CD3 and the antibody of CD28 have a ratio ranging from about 100: 1, 90: 10, 80:20, 70:30. 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, or about 1: 100 (aCD3:aCD28).

[0223] In certain embodiments, Det comprises one or more of CD64, CD86, 4-1 BBL, OX40L or any combination thereof, or a fragment thereof. In embodiments Det comprises CD64. In embodiments, Det comprises CD86. In embodiments, Det comprises 4-1BBL. In embodiments, Det comprises OX40L. In embodiments, Det comprises CD64 and CD86. In embodiments, Det comprises CD64 and 4-1BBL. In embodiments, Det comprises CD64 and OX40L. In embodiments, Det comprises CD86 and 4-1BBL. In embodiments, Det comprises CD86 and OX40L. In embodiments, Det comprises 4-1 BBL and OX40L. In embodiments, Det comprises CD64, CD86, and 4-1BBL. In embodiments, Det comprises CD64, CD86, and OX40L. In embodiments, Det comprises CD64, 4-1BBL, and OX40L. In embodiments, Det comprises CD86, 4-1 BBL. and OX40L. In embodiments, t Det comprises CD64, CD86, 4- 1BBL, and OX40L.

[0224] In certain embodiments, (d) comprises the polymer conjugated lipid and the compound of formula (II), wherein the polymer conjugated lipid and the compound of formula (II) have a molar ratio of about 4.9:0.1 , 4.8:0.2, 4.7:0.3, 4.6.0.4. 4.5:0.5, 4.4:0.6, 4.3:0.7. 4.2:0.8, 4.1:0.9, 4.0: 1.0, 3.9: 1.1. 3.8: 1.2, 3.7: 1.3, 3.6: 1.4, 3.5: 1.5. 3.4: 1.6, 3.3: 1.7,3.2: 1.8. 3.1: 1.9, 3.0:2,0, 2.9:2. 1, 2.8:2.2, 2.7:2.3, 2.6:2.4. 2.5:2.5, 2.4:2.6, 2.3:2.7, 2.2:2.8.2.1 :2.9, 2.0:3.0, 1.9:3. 1, 1 8:3 2, 1.7:3.3, 1.6:3.4, 1.5:3.5, 1.4:3.6, 1 .3:3.7. 1.2:3.8, 1. 1 :3.9,1.0:4.0, 0.9:4.1, 0.8:4.2, 0.7:4.3, 0.6:4.4, 0.5:4.5, 0.4:4.6, 0.3:4.7, 0.2:4.8, or about 0. L4.9.

[0225] In certain embodiments, the LNP has a molar ratio of (a):(b):(c):(d) of about 40:30:25:2.5. In certain embodiments, the LNP has a molar ratio of (a):(b):(c):(d) of about 41 :30.8:25.6:2.5. In certain embodiments, (d) comprises the polymer conjugated lipid and the compound of formula (II) having a ratio of about 2. 1:0.4.

[0226] In certain embodiments, the LNP further comprises at least one cargo selected from the group consisting of a nucleic acid molecule and a therapeutic agent.Attorney Docket No. 5134-WO

[0227] In certain embodiments, the therapeutic agent is at least one selected from the group consisting of a small molecule, a protein, and an antibody.

[0228] In certain embodiments, the LNP comprises a nucleic acid molecule.

[0229] In certain embodiments, the nucleic acid molecule is a DNA molecule or anRNA molecule.

[0230] In certain embodiments, the nucleic acid molecule is selected from the group consisting of cDNA. mRNA, miRNA, siRNA, modified RNA, antagomir, antisense molecule, and a targeted nucleic acid, or any combination thereof.

[0231] In certain embodiments, the nucleic acid molecule encodes a chimeric antigen receptor (CAR).

[0232] In certain embodiments, the CAR is specific for binding to a surface antigen of a pathogenic cell or a tumor cell

[0233] In certain embodiments, the surface antigen is selected from the group consisting of CD4, CD8, CD1, CD2, CDS, CD5, CD7, CD16, CD19, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD33, CD38, CD39, CD40L, CD44, CD45. CD62L, CD69, CD73, CD80, CD8.3, CD86, CD95, CD103, CD1I9, CD123, CD126, CD150, CD153, CD154, CD161, CDI83, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18RI, CTLA-4, 0X40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6. CCR7, k light chain. ROR1, ErbB2, ErbB3, ErbB4. EGFR vIII, carcinoembiyonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL13R-a2, MUCL VEGF-A, Tem8, FAP, EphA2, HER2, MUC16, CA9, GD2, GD3, HMW-MAA, CDI71, Lewis Y, G250 / CALX, HLA-AI MAGE Al , HAL-A2 NY-ESO-1 , PSC1, folate receptor-a, 8H9, NC AM, VEGF, 5T4, Fetal AchR, NKG2D ligands, TEM1, and TEM8.

[0234] In certain embodiments, the nucleic acid molecule encodes mRNA. In certain embodiments, the nucleic acid molecule encodes sgRNA. In certain embodiments, the nucleic acid molecule encodes mRNA and sgRNA. In certain embodiments, the mRNA encodes a therapeutic protein. In certain embodiments, the therapeutic protein is a CRISPR-associated protein. In certain embodiments, the CRISPR-associated protein is CRISPR-associated protein 9 (Cas9).

[0235] In certain embodiments, the therapeutic agent is a CRISPR-associated protein. In certain embodiments, the CRISPR-associated protein is CRISPR-associated protein 9 (Cas9).Attorney Docket No. 5134-WOA. Co-Stimulatory Molecules

[0236] In various embodiments, the LNPs are conjugated to one or more costimulatory molecules as disclosed herein.

[0237] In some embodiments, the LNPs are conjugated to one or more co-stimulatory molecules including one or more of a CD64 protein, a CD86 protein, a 4-1 BBL protein, or an 0X40L protein. Example sequences for human CD64, human CD86, human 4-1BBL (CD137L), and human OX40L (CD134L) are given in Table 1.

[0238] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NO:4, and conservative amino acid substitutions thereof, is provided.

[0239] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1 and conservative amino acid substitutions thereof, is provided.

[0240] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a4-lBBL protein comprising an amino acid sequence as set forth in SEQ IDNO:2, and conservative amino acid substitutions thereof, is provided.

[0241] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:3, and conservative amino acid substitutions thereof, is provided.

[0242] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NO:4, and conservative amino acid substitutions thereof, and a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: I, and conservative amino acid substitutions thereof, is provided

[0243] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a CD64 protein comprising an amino acid sequence as set forth m SEQ ID NO:4, and conservative amino acid substitutions thereof, and a 4-1 BBL protein comprising an amino acid sequence as set forth in SEQ ID NO: 2, and conservative amino acid substitutions thereof, is provided.

[0244] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NO:4. and conservative amino acid substitutions thereof, and an OX40L protein comprising an aminoAttorney Docket No. 5134-WO acid sequence as set forth in SEQ ID NO: 3, and conservative amino acid substitutions thereof, is provided.

[0245] In an embodiment, a population of LNPs. wherein the LNPs are conjugated to a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:2, and conservative ammo acid substitutions thereof, is provided.

[0246] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1, and conservative amino acid substitutions thereof, and an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO: 3, and conservative amino acid substitutions thereof, is provided.

[0247] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a 4-lBBL protein comprising an ammo acid sequence as set forth in SEQ ID NO:2, and conservative amino acid substitutions thereof, and an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO: 3, and conservative amino acid substitutions thereof, is provided.

[0248] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NON, and conservative amino acid substitutions thereof, a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1 . and conservative amino acid substitutions thereof, and a 4-1 BBL protein comprising an ammo acid sequence as set forth in SEQ ID NO:2, and conservative amino acid substitutions thereof, is provided.

[0249] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NON, and conservative amino acid substitutions thereof, a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1, and conservative amino acid substitutions thereof, and an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:3, and conservative amino acid substitutions thereof, is provided.

[0250] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a CD64 protein comprising an ammo acid sequence as set forth in SEQ ID NON, and conservative amino acid substitutions thereof, a 4-1 BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:2, and conservative amino acid substitutions thereof, andAttorney Docket No. 5134-WO an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:3, and conservative amino acid substitutions thereof, is provided.

[0251] In an embodiment, a population of LNPs. wherein the LNPs are conjugated to a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1, and conservative amino acid substitutions thereof, a 4*1 BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:2, and conservative amino acid substitutions thereof, and an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:3, and conservative amino acid substitutions thereof, is provided.

[0252] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a CD64 protein comprising an amino acid sequence as set forth in SEQ ID NON, and conservative amino acid substitutions thereof, a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO: 1 , and conservative ammo acid substitutions thereof, a 4- IBBL protein comprising an ammo acid sequence as set forth in SEQ ID NO:2, and conservative amino acid substitutions thereof, and an OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO: 3, and conservative ammo acid substitutions thereof, is provided.

[0253] In an embodiment, a population of LNPs, wherein the LNPs are conjugated to a CD64 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NON, a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO: 1 and a 4-1BBL protein comprising a sequence with greater than 99% identity' to an amino acid sequence as set forth in SEQ ID NON, is provided. In an embodiment, the LNPs are conjugated to a CD64 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NON, a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:1 and a 4-1BBL protein comprising a sequence with greater than 98% identity' to an amino acid sequence as set forth in SEQ ID NON. In an embodiment, the LNPs are conjugated to a CD64 protein comprising a sequence with greater than 97% identity’ to an ammo acid sequence as set forth in SEQ ID NON, a CD86 protein comprising a sequence with greater than 97°% identity to an ammo acid sequence as set forth in SEQ ID NO: 1 and a 4-1BBL protein comprising a sequence with greater than 97% identity7to an amino acid sequence as set forth in SEQ ID NON. In an embodiment, the LNPs are conjugated to a CD64 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NON, a CD86 protein comprising a sequence with greater than 96% identity to an amino acidAttorney Docket No. 5134-WO sequence as set forth in SEQ ID NO: 1 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:2. In an embodiment, the LNPs are conjugated to a CD64 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:4, a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO: I and a 4-1BBL protein comprising a sequence with greater than 95% identity to an ammo acid sequence as set forth in SEQ ID NO:2. In an embodiment, the LNPs are conjugated to a CD64 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:4, a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO: 1 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:2B. Conjugation of Co-Stimulatory Molecules to the LNPs

[0254] In certain embodiments, the one or more co-stimulatory molecules are conjugated to the LNP. Exemplary methods of conjugation can include, but are not limited to, covalent bonds, electrostatic interactions, and hydrophobic (“van der Waals”) interactions. In certain embodiments, the conjugation is a reversible conjugation, such that the delivery' vehicle can be disassociated from the one or more co-stimulatory molecules upon exposure to certain conditions or chemical agents. In some embodiments, the conjugation is an irreversible conjugation, such that under normal conditions the delivery vehicle does not dissociate from the one or more co-stimulatory molecules.

[0255] In some embodiments, the conjugation comprises a covalent bond between an activated polymer conjugated lipid and the one or more co-stimulatory molecules. The term “activated polymer conjugated lipid’’ refers to a molecule comprising a lipid portion and a polymer portion that has been activated via functionalization of a polymer conjugated lipid with a first coupling group. In certain embodiments, the activated polymer conjugated lipid comprises a first coupling group capable of reacting with a second coupling group. In certain embodiments, the activated polymer conjugated lipid is an activated pegylated lipid. In certain embodiments, the first coupling group is bound to the lipid portion of the pegylated lipid. In some embodiments, the first coupling group is bound to the polyethylene glycol portion of the pegylated lipid. In certain embodiments, the second functional group is covalently attached to the one or more co-stimulatory molecules.Attorney Docket No. 5134-WO

[0256] The first coupling group and second coupling group can be any functional groups known to those of skill in the art to together form a covalent bond, for example under mild reaction conditions or physiological conditions. In some embodiments, the first coupling group or second coupling group are selected from the group consisting of maleimides, N- hydroxysuccinimide (NHS) esters, carbodiirnides, hydrazide, pentafluorophenyl (PFP) esters, phosphines, hydroxymethyl phosphines, psoralen, imidoesters, pyridyl disulfide, isocyanates, vinyl sulfones, alpha-haloacefids, and azides, acyl azides, alkyl azides, diazirines, benzophenone, epoxides, carbonates, anhydrides, sulfonyl chlorides, cyclooctyne, aldehydes, and sulfhydryl groups. In some embodiments, the first coupling group or second coupling group is selected from the group consisiting of free amines (-NH2), free sulfhydryl groups (- SH), free hydroxide groups ( -OH). carboxylates, hydrazides, and alkoxyamines. In some embodiments, the first coupling group is a functional group that is reactive toward sulfhydryl groups, such as maleimide, pyridyl disulfide, or ahaloacetyl. In certain embodiments, the first coupling group is a maleimide.

[0257] In certain embodiments, the second coupling group is a sulfhydryl group. The sulfhydryl group can be installed on the one or more co-stimulatory molecules using any method known to those of skill in the art. In certain embodiments, the sulfhydryl group is present on a free cysteine residue. In certain embodiments, the sulfhydryl group is revealed via reduction of a disulfide on the one or more co-stimulatory molecules, such as through reaction with 2 -mercaptoethyl amine. In certain embodiments, the sulfhydryl group is installed via a chemical reaction, such as the reaction between a free amine and 2- iminothilane or N-succinimidyl S-acetylthioacetate (SATA).

[0258] In some embodiments, the polymer conjugated lipid and the one or more co- stimulatory molecules are functionalized with groups used in ‘■‘click’’ chemistry.Bioorthogonal "click” chemistry comprises the reaction between a functional group with a 1.3-dipole, such as an azide, a nitrile oxide, a nitrone, an isocyanide, and the link, with an alkene or an alkyne dipolarophiles. Exemplary' dipolarophiles include any' strained cycloalkenes and cycloalkynes known to those of skill in the art, including, but not limited to, cyclooctynes, dibenzocyclooctynes, monofluorinated cyclcooctynes, difluorinated cyclooctynes, and biarylazacyclooctynone

[0259] In certain embodiments, the one or more co-stimulatory molecules are conjugated to the LNP using maleimide conjugation.Attorney Docket No. 5134-WOIV. Methods of Expanding Tumor Infiltrating Lymphocytes and T Cells

[0260] In an embodiment, a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising a step of contacting a population of TILs with a population of aAPCs or a population of LNPs disclosed herein in a cell culture medium, wherein the population of TILs is expanded, is provided.

[0261] In an embodiment, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 lU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL. In some embodiments, the population of TILs is expanded by at least 50- fold over a period of 1 1 days In some embodiments, the population of TILs is expanded by at least 200-fold over a period of 1 1 days. In some embodiments, the population of TILs is expanded by at least 500-fold over a period of 1 1 days. In some embodiments, the expansion is performed using a gas permeable container. In some embodiments, the ratio of the population of TIL s to the population of a APCs or the population of LNPs is between I to 100 and 1 to 500. In some embodiments, the ratio of the population of TILs to the population of aAPCs or the population of LNPs is about 1 to 250.

[0262] In some embodiments, a method of expanding a therapeutic population of tumor infiltrating lymphocytes (TILs) comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of artificial antigen presenting cells (APCs) modified to express one or more co-stimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs.

[0263] In some embodiments, a method of expanding a therapeutic population of tumor infiltrating lymphocytes (TILs) comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) performing a first expansion by culturing the first population of TILs in a cell cultureAttorney Docket No. 5134-WO medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; and (c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of lipid nanoparticles (LNPs) conjugated to one or more co-stimulatory molecules, to produce a third population of TILs. wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs. wherein the third population of TILs comprises the therapeutic population of TILs.

[0264] In some embodiments, a method of expanding a therapeutic population of tumor infiltrating lymphocytes (TILs) comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of artificial antigen presenting cells (APCs) modified to express one or more co-stimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs. wherein the third population of TILs comprises the therapeutic population of TILs.

[0265] In some embodiments, a method of expanding a therapeutic population of tumor infiltrating lymphocytes (TILs) comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of lipid nanoparticles (LNPs) conjugated to one or more co-stimulatoiy molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs.Attorney Docket No. 5134-WO

[0266] In some embodiments, a method of expanding a therapeutic population of tumor infiltrating lymphocytes (TILs) comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of artificial antigen presenting cells (APCs) modified to express one or more co-stimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-12 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs.

[0267] In some embodiments, a method of expanding a therapeutic population of tumor infiltrating lymphocytes (TILs) comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of lipid nanoparticles (LNPs) conjugated to one or more co-stimulatory molecules, to produce a third population of TILs. wherein the second expansion is performed for about 7-12 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs.

[0268] In some embodiments, a method of expanding a therapeutic population of tumor infiltrating lymphocytes (TILs) comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-12 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of theAttorney Docket No. 5134-WO second population of TILs with additional IL-2, OKT-3, and a population of artificial antigen presenting cells (APCs) modified to express one or more co-stimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-12 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs.

[0269] In some embodiments, a method of expanding a therapeutic population of tumor infiltrating lymphocytes (TILs) comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-12 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of lipid nanoparticles (LNPs) conjugated to one or more co-stimulatory molecules, to produce a third population of TILs. wherein the second expansion is performed for about 7-12 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs.

[0270] In some embodiments, a method of expanding a therapeutic population of tumor infiltrating lymphocytes (TILs) comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) adding the first population of TILs into a closed system;(c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-11 days (or 3-12 days) to obtain the second population of TILs, and wherein the transition from step (b) to step (c) occurs without opening the system; and(d) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of artificial antigen presenting cells (APCs) modified to express one or more co-stimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-11 (or 7-12 days) days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs, andAttorney Docket No. 5134-WO wherein the transition from step (c) to step (d) occurs without opening the system.

[0271] In some embodiments, a method of expanding a therapeutic population of tumor infiltrating lymphocytes (TILs) comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) adding the first population of TILs into a closed system;(c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-11 days (or 3-12 days) to obtain the second population of TILs, and wherein the transition from step (b) to step (c) occurs without opening the system; and(d) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of lipid nanoparticles (LNPs) conjugated to one or more co-stimulatory molecules, to produce a third population of TILs. wherein the second expansion is performed for about 7-11 days (or 7-12 days) to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs, and wherein the transition from step (c) to step (d) occurs without opening the system.

[0272] In some embodiments, a method of expanding a therapeutic population of tumor infiltrating lymphocytes (TILs) comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) adding the first population of TILs into a closed system;(c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-11 days (or 3-12 days) to obtain the second population of TILs, and wherein the transition from step (b) to step (c) occurs without opening the system;(d) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of artificial antigen presenting cells (APCs) modified to express one or more co-stimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days (or 7-12 days) to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs, andAttorney Docket No. 5134-WO wherein the transition from step (c) to step (d) occurs without opening the system;(e) harvesting therapeutic population of TILs, wherein the transition from step (d) to step(e) occurs without opening the system;(f) transferring the harvested therapeutic population of TILs into an infusion bag, wherein the transition from step (e) to step (f) occurs without opening the system; and(g) cry opreserving the infusion bag comprising the harvested therapeutic population of TILs using a cryopreservation process.

[0273] In some embodiments, a method of expanding a therapeutic population of tumor infiltrating lymphocytes (TILs) comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) adding the first population of TILs into a closed system;(c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-11 (or 3-12 days) days to obtain the second population of TILs, and wherein the transition from step (b) to step (c) occurs without opening the system; and(d) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of lipid nanoparticles (LNPs) conjugated to one or more co-stimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-1 1 days (or 7-12 days) to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs, and wherein the transition from step (c) to step (d) occurs without opening the system;(e) harvesting therapeutic population of TILs, wherein the transition from step (d) to step(e) occurs without opening the system;(f) transferring the harvested therapeutic population of TILs into an infusion bag, wherein the transition from step (e) to step (I) occurs without opening the system; and(g) cry opreserving the infusion bag comprising the harvested therapeutic population of TILs using a cryopreservation process.

[0274] In an embodiment, a method of expanding a population of tumor infiltrating lymphocytes (TILs) using a population of the aAPCs or LNPs of the present disclosure comprises TIL expansion process called the Gen 2 Process as disclosed herein.Attorney Docket No. 5134-WOV. Gen 2 TIL Manufacturing Processes

[0274] An exemplary family of TIL processes known as Gen 2 (also known as process 2A) containing some of these features is depicted in Figures 1 and 2A-2C. An embodiment of Gen 2 is shown in Figures 2A-2C.

[0275] In some embodiments, the TILs may be cryopreserved. Once thawed, they may also be restimulated to increase their metabolism prior to infusion into a patient.

[0276] In some embodiments, the first expansion (including processes referred to as the pre-REP as well as processes shown in Figure 1 as Step A) is shortened to 3 to 14 days and the second expansion (including processes referred to as the REP as well as processes shown in Figure 1 as Step B) is shortened to 7 to 14 days, as discussed in detail below as well as in the examples and figures. In some embodiments, the first expansion (for example, an expansion described as Step B in Figure I) is shortened to 11 days and the second expansion (for example, an expansion as described in Step D in Figure 1) is shortened to 11 days. In some embodiments, the combination of the first expansion and second expansion (for example, expansions described as Step B and Step D in Figure 1) is shortened to 22 days, as discussed in detail below and in the examples and figures.

[0277] The “Step” Designations A, B, C, etc., below are in reference to Figure 1 and in reference to certain embodiments described herein. The ordering of the Steps below and in Figure 1 is exemplary and any combination or order of steps, as well as additional steps, repetition of steps, and / or omission of steps is contemplated by the present application and the methods disclosed herein.A. STEP A: Obtain Patient Tumor Sample

[0278] In general, TILs are initially obtained from a patient tumor sample and then expanded into a larger population for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein and optionally evaluated for phenotype and metabolic parameters as an indication of TIL health.

[0279] A patient tumor sample may be obtained using methods known in the art, generally via surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells. In some embodiments, multilesional sampling is used. In some embodiments, surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cellsAttorney Docket No. 5134-WO includes multilesional sampling (i.e., obtaining samples from one or more tumor sites and / or locations in the patient, as well as one or more tumors in the same location or in close proximity). In general, the tumor sample may be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid tumor may be of lung tissue. In some embodiments, useful TILs are obtained from non-small cell lung carcinoma (NSCLC). The solid tumor may be of skin tissue. In some embodiments, useful TILs are obtained from a melanoma.

[0280] Once obtained, the tumor sample is generally fragmented using sharp dissection into small pieces of between 1 to about 8 mm3, with from about 2-3 mm3being particularly useful. In some embodiments, the TILs are cultured from these fragments using enzy matic tumor digests. Such tumor digests may be produced by incubation in enzymatic media (e.g, Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicine, 30 units / mL of DNase and 1.0 mg / mL of collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests may be produced by placing the tumor in enzy matic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37 °C in 5% CO2. followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density7gradient separation using FICOLL branched hydrophilic polysaccharide may be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133 Al, the disclosure of which is incorporated by reference herein. Any of the foregoing methods may be used in any of the embodiments described herein for methods of expanding TILs or methods treating a cancer.

[0281] Tumor dissociating enzyme mixtures can include one or more dissociating (digesting) enzymes such as. but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotry psin, chymopapain, trypsin, caseinase, elastase, papain, protease type XIV (pronase), deoxyribonuclease I (DNase), try psin inhibitor, any other dissociating or proteolytic enzyme, and any combination thereof.Attorney Docket No. 5134-WO

[0282] In some embodiments, the dissociating enzymes are reconstituted from lyophilized enzymes. In some embodiments, lyophilized enzymes are reconstituted in an amount of sterile buffer such as HBSS.

[0283] In some instances, collagenase (such as animal free- type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 2892 PZ U / vial. In some embodiments, collagenase is reconstituted in 5 mL to 15 mL buffer. In some embodiment, after reconstitution the collagenase stock ranges from about 100 PZ U / mL-about 400 PZ U / mL, e.g., about 100 PZ U / mL-about 400 PZ U / mL, about 100 PZ U / mL-about 350 PZ U / mL, about 100 PZ U / mL-about 300 PZ U / mL, about 150 PZ U / mL-about 400 PZ U / mL, about 100 PZ U / mL, about 150 PZ U / mL, about 200 PZ U / mL, about 210 PZ U / mL, about 220 PZ U / mL, about 230 PZ U / mL, about 240 PZ U / mL, about 250 PZ U / mL, about 260 PZ U / mL, about 270 PZ U / mL, about 280 PZ U / mL, about 289.2 PZ U / mL, about 300 PZ U / mL, about 350 PZ U / mL, or about 400 PZ U / mL.

[0284] In some embodiments, neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzy me may be at a concentration of 175 DMC U / vial. In some embodiments, after reconstitution the neutral protease stock ranges from about 100 DMC / mL-about 400 DMC / mL, e.g.. about 100 DMC / mL-about 400 DMC / mL, about 100 DMC / mL-about 350 DMC / mL, about 100 DMC / mL-about 300 DMC / mL. about 150 DMC / mL-about 400 DMC / mL, about 100 DMC / mL, about 1 10 DMC / mL, about 120 DMC / mL, about 130 DMC / mL, about 140 DMC / mL, about 150 DMC / mL, about 160 DMC / mL, about 170 DMC / mL, about 175 DMC / mL, about 180 DMC / mL, about 190 DMC / mL, about 200 DMC / mL, about 250 DMC / mL. about 300 DMC / mL, about 350 DMC / mL, or about 400 DMC / mL.

[0285] In some embodiments, DNAse I is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme was at a concentration of 4 KU / vial. In some embodiments, after reconstitution the DNase I stock ranges from about 1 KU / mL-10 KU / mL, e.g., about 1 KU / mL, about 2 KU / mL. about 3 KU / mL, about 4 KU / mL, about 5 KU / mL, about 6 KU / mL, about 7 KU / mL, about 8 KU / mL, about 9 KU / mL, or about 10 KU / mL.

[0286] In some embodiments, the stock of enzymes is variable and the concentrations may need to be determined. In some embodiments, the concentration of the lyophilized stock canAttorney Docket No. 5134-WO be verified. In some embodiments, the final amount of enzyme added to the digest cocktail is adjusted based on the determined stock concentration.

[0287] In some embodiment, the enzyme mixture includes about 10.2-ul of neutral protease (0.36 DMC U / mL), 21.3 pL of collagenase (1.2 PZ / mL) and 250-ul of DNAse I (200 U / mL) in about 4.7 mL of sterile HBSS.

[0288] As indicated above, in some embodiments, the TILs are derived from solid tumors. In some embodiments, the solid tumors are not fragmented. In some embodiments, the solid tumors are not fragmented and are subjected to enzymatic digestion as whole tumors. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C, 5% CO2. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase. DNase, and hyaluronidase for 1-2 hours at 37°C, 5% CO2 with rotation. In some embodiments, the tumors are digested overnight with constant rotation. In some embodiments, the tumors are digested overnight at 37°C, 5% CO2 with constant rotation. In some embodiments, the whole tumor is combined with the enzymes to form a tumor digest reaction mixture.

[0289] In some embodiments, the tumor is reconstituted with the lyophilized enzy mes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0290] In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock for the collagenase is a 100 mg / mL 10X working stock.

[0291] In some embodiments, the enzyme mixture comprises DNAse. In some embodiments, the working stock for the DNAse is a 10,000 lU / rnL 10X working stock.

[0292] In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock for the hyaluronidase is a 10 mg / mL 10X working stock.

[0293] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 1000 lU / mL DNAse, and 1 mg / mL hyaluronidase.

[0294] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 500 lU / mL DNAse, and 1 mg / mL hyaluronidase.Attorney Docket No. 5134-WO

[0295] In general, the harvested cell suspension is called a "primary cell population” or a “freshly harvested” cell population.

[0296] In some embodiments, fragmentation includes physical fragmentation, including for example, dissection as well as digestion. In some embodiments, the fragmentation is physical fragmentation. In some embodiments, the fragmentation is dissection. In some embodiments, the fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from digesting or fragmenting a tumor sample obtained from a patient.

[0297] In some embodiments, where the tumor is a solid tumor, the tumor undergoes physical fragmentation after the tumor sample is obtained in, for example, Step A (as provided in Figure 1). In some embodiments, the fragmentation occurs before cryopreservation. In some embodiments, the fragmentation occurs after cryopreservation. In some embodiments, the fragmentation occurs after obtaining the tumor and in the absence of any cry opreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the multiple fragments comprise about 4 to about 50 fragments, wherein each fragment has a volume of about 27 mm3. In some embodiments, the multiple fragments comprise about 30 to about 60 fragments with a total volume of about 1300 mm3to about 1500 mm3. In some embodiments, the multiple fragments comprise about 50 fragments with a total volume of about 1350 mm3. In some embodiments, the multiple fragments comprise about 50 fragments with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the multiple fragments comprise about 4 fragments.

[0298] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragment is obtained by sharp dissection. In some embodiments, the tumor fragment is between about 1 mm3and 10 mm3. In some embodiments, the tumor fragment is between about 1 mm3and 8 mm3. In some embodiments, the tumor fragment is about 1 mm3. In some embodiments, the tumor fragment is about 2 mm3. In some embodiments, the tumor fragment is about 3 mm3. In some embodiments, the tumor fragment is about 4 mm3. In some embodiments, the tumor fragment is about 5 mm3. In some embodiments, the tumor fragment is about 6 mm3. In some embodiments, the tumor fragmentAttorney Docket No. 5134-WO is about 7 mm3. In some embodiments, the tumor fragment is about 8 mm3. In some embodiments, the tumor fragment is about 9 mm3. In some embodiments, the tumor fragment is about 10 mm3. In some embodiments, the tumors are 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumors are 1 mm x 1 mm x 1 mm. In some embodiments, the tumors are 2 mm x 2 mm x 2 mm. In some embodiments, the tumors are 3 mm x 3 mm x 3 mm. In some embodiments, the tumors are 4 mm x 4 mm x 4 mm.

[0299] In some embodiments, the tumors are resected in order to minimize the amount of hemorrhagic, necrotic, and / or fatty tissues on each piece. In some embodiments, the tumors are resected in order to minimize the amount of hemorrhagic tissue on each piece. In some embodiments, the tumors are resected in order to minimize the amount of necrotic tissue on each piece. In some embodiments, the tumors are resected in order to minimize the amount of fatty tissue on each piece.

[0300] In some embodiments, the tumor fragmentation is performed in order to maintain the tumor internal structure. In some embodiments, the tumor fragmentation is performed without performing a sawing motion with a scalpel. In some embodiments, the TILs are obtained from tumor digests. In some embodiments, tumor digests were generated by incubation in enzyme media, for example but not limited to RPMI 1640, 2 mM GLUTAMAX, 10 mg / mL gentamicin. 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS™. Miltenyi Biotec. Auburn, CA). After placing the tumor in enzyme media, the tumor can be mechanically dissociated for approximately 1 minute. The solution can then be incubated for 30 minutes at 37 °C in 5% CO2 and it then mechanically disrupted again for approximately 1 minute. After being incubated again for 30 minutes at 37 °C in 5% CO2. the tumor can be mechanically disrupted a third time for approximately 1 minute. In some embodiments, after the third mechanical disruption if large pieces of tissue were present, 1 or 2 additional mechanical dissociations were applied to the sample, with or without 30 additional minutes of incubation at 37 °C in 5% CO2. In some embodiments, at the end of the final incubation if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using Ficoll can be performed to remove these cells.

[0301] In some embodiments, the harvested cell suspension prior to the first expansion step is called a “primary cell population” or a “freshly harvested” cell population.Attorney Docket No. 5134-WO

[0302] In some embodiments, cells can be optionally frozen after sample harvest and stored frozen prior to entry into the expansion described in Step B, which is described in further detail below, as well as exemplified in Figure 1.1. Pleural effusion T-cells and TILs

[0303] In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of the T-cells or TILs for expansion according to the processes described herein is a pleural fluid sample. In some embodiments, the sample is a pleural effusion derived sample. In some embodiments, the source of the T-cells or TILs for expansion according to the processes described herein is a pleural effusion derived sample. See, for example, methods described in U.S. Patent Publication US 2014 / 0295426, incorporated herein by reference in its entirety for all purposes.

[0304] In some embodiments, any pleural fluid or pleural effusion suspected of and / or containing TILs can be employed. Such a sample may be derived from a primary or metastatic lung cancer, such as NSCLC or SCLC. In some embodiments, the sample may be derived from secondary metastatic cancer cells which originated from another organ, e.g., breast, ovary', colon or prostate. In some embodiments, the sample for use in the expansion methods described herein is a pleural exudate. In some embodiments, the sample for use in the expansion methods described herein is a pleural transudate. Other biological samples may include other serous fluids containing TILs, including, e.g., ascites fluid from the abdomen or pancreatic cyst fluid. Ascites fluid and pleural fluids involve very' similar chemical systems; both the abdomen and lung have mesothelial lines and fluid forms in the pleural space and abdominal spaces in the same matter in malignancies and such fluids in some embodiments contain TILs. In some embodiments, wherein the disclosed methods utilize pleural fluid, the same methods may be performed with similar results using ascites or other cyst fluids containing TILs.

[0305] In some embodiments, the pleural fluid is in unprocessed form, directly as removed from the patient. In some embodiments, the unprocessed pleural fluid is placed in a standard blood collection tube, such as an EDTA or Heparin tube, prior to further processing steps. In some embodiments, the unprocessed pleural fluid is placed in a standard CellSave® tube (Veridex) prior to further processing steps. In some embodiments, the sample is placed in the CellSave® tube immediately after collection from the patient to avoid a decrease in the number of viable TILs. The number of viable TILs can decrease to a significant extent withinAttorney Docket No. 5134-WO24 hours, if left in the untreated pleural fluid, even at 4°C. In some embodiments, the sample is placed in the appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, the sample is placed in the appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.

[0306] In some embodiments, the pleural fluid sample from the chosen subject may be diluted. In some embodiments, the dilution is 1: 10 pleural fluid to diluent. In other embodiments, the dilution is 1:9 pleural fluid to diluent. In other embodiments, the dilution is 1 :8 pleural fluid to diluent. In other embodiments, the dilution is 1:5 pleural fluid to diluent. In other embodiments, the dilution is 1 :2 pleural fluid to diluent. In other embodiments, the dilution is 1: 1 pleural fluid to diluent. In some embodiments, diluents include saline, phosphate buffered saline, another buffer or a physiologically acceptable diluent. In some embodiments, the sample is placed in the CellSave® tube immediately after collection from the patient and dilution to avoid a decrease in the viable TILs, which may occur to a significant extent within 24-48 hours, if left in the untreated pleural fluid, even at 4°C. In some embodiments, the pleural fluid sample is placed in the appropriate collection tube within 1 hour, 5 hours. 10 hours. 15 hours, 24 hours, 36 hours, up to 48 hours after removal from the patient, and dilution. In some embodiments, the pleural fluid sample is placed in the appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal from the patient, and dilution at 4°C.

[0307] In still other embodiments, pleural fluid samples are concentrated by conventional means prior to further processing steps. In some embodiments, this pre-treatment of the pleural fluid is preferable in circumstances in which the pleural fluid must be cryopreserved for shipment to a laboratory performing the method or for later analysis (e.g., later than 24-48 hours post-collection). In some embodiments, the pleural fluid sample is prepared by centrifuging the pleural fluid sample after its withdrawal from the subject and resuspending the centrifugate or pellet in buffer. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspensions, before it is cryopreserved for transport or later analysis and / or processing.

[0308] In some embodiments, pleural fluid samples are concentrated prior to further processing steps by using a filtration method. In some embodiments, the pleural fluid sample used in further processing is prepared by filtering the fluid through a filter containing a known and essentially uniform pore size that allows for passage of the pleural fluid throughAttorney Docket No. 5134-WO the membrane but retains the tumor cells. In some embodiments, the diameter of the pores in the membrane may be at least 4 LIM. In other embodiments the pore diameter may be 5 pM or more, and in other embodiment, any of 6, 7, 8, 9, or 10 pM. After filtration, the cells, including TILs, retained by the membrane may be rinsed off the membrane into a suitable physiologically acceptable buffer. Cells, including TILs, concentrated in this way may then be used in the further processing steps of the method.

[0309] In some embodiments, pleural fluid sample (including, for example, the untreated pleural fluid), diluted pleural fluid, or the resuspended cell pellet, is contacted with a lytic reagent that differentially lyses non-nucleated red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps in circumstances in which the pleural fluid contains substantial numbers of RBCs. Suitable lysing reagents include a single lytic reagent or a lytic reagent and a quench reagent, or a lytic agent, a quench reagent and a fixation reagent. Suitable lytic systems are marketed commercially and include the BD Pharm Lyse™ system (Becton Dickenson). Other lytic systems include the Versalyse™ system, the FACSlyse™ system (Becton Dickenson), the Immunoprep™ system or Erythrolyse II system (Beckman Coulter, Inc ), or an ammonium chloride system. In some embodiments, the lytic reagent can vary with the primary requirements being efficient lysis of the red blood cells, and the conservation of the TILs and phenotypic properties of the TILs in the pleural fluid. In addition to employing a single reagent for lysis, the lytic systems useful in methods described herein can include a second reagent, e.g., one that quenches or retards the effect of the lytic reagent during the remaining steps of the method, e.g., Stabilyse™ reagent (Beckman Coulter, Inc.). A conventional fixation reagent may also be employed depending upon the choice of lytic reagents or the preferred implementation of the method.

[0310] In some embodiments, the pleural fluid sample, unprocessed, diluted or multiply centrifuged or processed as described herein above is cryopreserved at a temperature of about -140°C prior to being further processed and / or expanded as provided herein.B. STEP B: First Expansion

[0311] In some embodiments, the present methods provide for obtaining young TILs, which are capable of increased replication cycles upon administration to a subject / patient and as such may provide additional therapeutic benefits over older TILs (i.e., TILs which have further undergone more rounds of replication prior to administration to a subject / patient). Features of young TILs have been described in the literature, for example in Donia, et al.,Attorney Docket No. 5134-WOScand. J. Immunol. 2012, 75, 157-167; Dudley, et al., Clin. Cancer Res. 2010, 16, 6122- 6131; Huang, et al., J. Immunother. 2005, 28, 258-267; Besser, et al., Clin. Cancer Res. 2013, 19, 0F1-0F9; Besser, et al., J. Immunother. 2009, 32:415-423; Robbins, et al.. J. Immunol. 2004, 173, 7125-7130; Shen, et al., J. Immunother., 2007, 30, 123-129; Zhou, et al., J. Immunother. 2005, 28, 53-62; and Tran, et al., J. Immunother., 2008, 31, 742-751, each of which is incorporated herein by reference.

[0312] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited, but large number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant), determine the binding specificity and downstream applications of immunoglobulins and T-cell receptors (TCRs). Provided herein is a method for generating TILs which exhibit and increase the T-cell repertoire diversity. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity as compared to freshly harvested TILs and / or TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity as compared to freshly harvested TILs and / or TILs prepared using methods referred to as process 1C. In some embodiments, the TILs obtained in the first expansion exhibit an increase in the T-cell repertoire diversity. In some embodiments, the increase in diversity is an increase in the immunoglobulin diversity and / or the T-cell receptor diversity. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin heavy chain. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin light chain. In some embodiments, the diversity is in the T-cell receptor. In some embodiments, the diversity is in one of the T-cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha and / or beta. In some embodiments, there is an increase in the expression of T- cell receptor (TCR) alpha. In some embodiments, there is an increase in the expression of T- cell receptor (TCR) beta. In some embodiments, there is an increase in the expression of TCRab (i.e., TCRa / 0).

[0313] After dissection or digestion of tumor fragments, for example such as described in Step A of Figure 1, the resulting cells are cultured in serum containing IL-2 under conditions that favor the grow th of TILs over tumor and other cells. In some embodiments, the tumorAttorney Docket No. 5134-WO digests are incubated in 2 mL wells in media comprising inactivated human AB serum with 6000 lU / mL of IL-2. This primary cell population is cultured for a period of days, generally from 3 to 14 days, resulting in a bulk TIL population, generally about 1 x 108bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of 7 to 14 days, resulting in a bulk TIL population, generally about 1 x 108bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of 10 to 14 days, resulting in a bulk TIL population, generally about 1 x io8bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a bulk TIL population, generally about 1 x io8bulk TIL cells.

[0314] In some embodiments, expansion of TILs may be performed using an initial bulk TIL expansion step (for example such as those described in Step B of Figure 1, which can include processes referred to as pre-REP) as described below and herein, followed by a second expansion (Step D. including processes referred to as rapid expansion protocol (REP) steps) as described below under Step D and herein, followed by optional cryopreservation, and followed by a second Step D (including processes referred to as restimulation REP steps) as described below and herein. The TILs obtained from this process may be optionally characterized for phenotypic characteristics and metabolic parameters as described herein.

[0315] In embodiments where TIL cultures are initiated in 24-well plates, for example, using Costar® 24-well cell culture cluster, flat bottom (Coming Incorporated, Coming, NY. each well can be seeded with 1 x io6tumor digest cells or one tumor fragment in 2 mL of complete medium (CM) with IL-2 (6000 lU / mL; Chiron Corp., Emeryville, CA). In some embodiments, the tumor fragment is between about 1 mm3and 10 mm3.

[0316] In some embodiments, the first expansion culture medium is referred to as “CM”, an abbreviation for culture media. In some embodiments, CM for Step B consists of RPMI 1640 with GLUTAMAX, supplemented with 10% human AB serum. 25 mM HEPES. and 10 mg / mL gentamicin. In embodiments where cultures are initiated in gas-permeable flasks with a 40 mL capacity7and a 10 cm2gas-permeable silicon bottom (for example, G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask was loaded with 10-40 x 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 were incubated in a humidified incubator at 37°C in 5% CO2 and 5 days after culture initiation, half the media was removed and replaced with fresh CM and IL-2 and after day 5, half the media was changed every 2-3 days.Attorney Docket No. 5134-WO

[0317] 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 serumcontaining media.

[0318] 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 (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0319] 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+, A13+, 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.

[0320] 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 MediumAttorney Docket No. 5134-WO(MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0321] 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.

[0322] In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T- cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ may be used. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of IL 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 55pM.

[0323] In some embodiments, the defined medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ may be used. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of IL 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)Attorney Docket No. 5134-WO(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 lU / rnL to about 8000 lU / 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 lU / 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 lU / 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 lU / mL to about 8000 lU / 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 lU / 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 lU / mL to about 6000 lU / 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 lU / mL to about 8000 lU / 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 lU / 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 lU / 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 55pM.Attorney Docket No. 5134-WO

[0324] In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of from about 0. ImM to about lOmM, 0.5mM to about 9mM, ImM 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.

[0325] 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, lOmM to about 140mM, 15mM to about 130mM, 20mM to about 120mM, 25mM to about 1 lOmM, 30mM to about lOOmM, 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 55 pM.

[0326] In some embodiments, the defined media described in International PCT Publication No. WO / 1998 / 030679, which is herein incorporated by reference, may be used. 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 saturatedAttorney Docket No. 5134-WO transferrin, insulin, and compounds containing the trace element moieties Ag+. A13+, Ba2+, Cd2+, Co2+. Cr3+, Ge4+, Se4+. Br. T, Mn2+, P, Si4+, V5+, M06+, 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 (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0327] 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.

[0328] 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 IX Medium” in Table 3 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 IX Medium” in Table 3. 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 3 below.Attorney Docket No. 5134-WOTABLE 3: Concentrations of Non-Trace Element Moiety Ingredients

[0329] 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 310Attorney Docket No. 5134-WO 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 pM), 2-mercaptoethanol (final concentration of about 100 pM).

[0330] In some embodiments, the defined media described in Smith, et al., Clin Transl Immunology, 4(1) 2015 (doi: 10. 1038 / cti.2014.31) may be used. 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.

[0331] 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 |3ME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0332] After preparation of the tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, the tumor digests are incubated in 2 mL wells in media comprising inactivated human AB serum (or, in some cases, as outlined herein, in the presence of an aAPC cell population) with 6000 lU / mL of IL-2. This primary cell population is cultured for a period of days, generally from 10 to 14 days, resulting in a bulk TIL population, generally about 1 x 108bulk TIL cells. In some embodiments, the growth media during the first expansion comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments the IL-2 stock solution has a specific activity of 20-30 x 106lU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 20xl06lU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 25* 106lU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 30xl06lU / mg for a 1 mg vial. In some embodiments, the IL- 2 stock solution has a final concentration of 4-8 xlO6lU / mg of IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of 5-7x 106lU / mg of IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of 6* 106lU / mg of IL-2. In some embodiments, the IL-2 stock solution is prepare as described in Example 5. In some embodiments, the first expansion culture media comprises about 10,000 lU / mL of IL-2, about 9,000 lU / mL of IL-2, about 8.000 lU / mL of IL-2, about 7,000Attorney Docket No. 5134-WO lU / mL of IL-2, about 6000 lU / mL of IL-2 or about 5,000 lU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 9,000 lU / mL of IL-2 to about 5,000 lU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 8,000 lU / mL of IL-2 to about 6,000 lU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 7,000 lU / mL of IL-2 to about 6,000 lU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 6,000 lU / 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 lU / 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 lU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 lU / mL, about 1500 lU / mL, about 2000 lU / mL, about 2500 lU / mL, about 3000 lU / mL, about 3500 lU / mL, about 4000 lU / mL, about 4500 lU / mL, about 5000 lU / mL, about 5500 lU / mL, about 6000 lU / mL, about 6500 lU / mL, about 7000 lU / mL, about 7500 lU / mL, or about 8000 lU / mL of IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000 lU / mL, between 2000 and 3000 lU / mL, between 3000 and 4000 lU / mL. between 4000 and 5000 lU / mL. between 5000 and 6000 lU / mL, between 6000 and 7000 lU / mL, between 7000 and 8000 lU / mL, or about 8000 lU / mL of IL- 2.

[0333] In some embodiments, first expansion culture media comprises about 500 lU / mL of IL-15, about 400 lU / mL of IL-15, about 300 lU / mL of IL-15, about 200 lU / mL of IL-15, about 180 lU / mL of IL-15, about 160 lU / mL of IL-15, about 140 lU / mL of IL-15, about 120 lU / mL of IL- 15. or about 100 lU / mL of IL- 15. In some embodiments, the first expansion culture media comprises about 500 lU / mL of IL-15 to about 100 lU / mL of IL-15. In some embodiments, the first expansion culture media comprises about 400 lU / mL of IL-15 to about 100 lU / mL of IL-15. In some embodiments, the first expansion culture media comprises about 300 lU / mL of IL- 15 to about 100 lU / mL of IL- 15. In some embodiments, the first expansion culture media comprises about 200 lU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 lU / mL of IL-15. In some embodiments, the cell culture medium further comprises IL- 15. In some embodiments, the cell culture medium comprises about 180 lU / mL of IL-15.

[0334] In some embodiments, first expansion culture media comprises about 20 lU / mL of IL-21, about 15 lU / mL of IL-21, about 12 lU / mL of IL-21, about 10 lU / mL of IL-21, about 5 lU / mL of IL-21, about 4 lU / mL of IL-21, about 3 lU / mL of IL-21, about 2 lU / mL of IL-21,Attorney Docket No. 5134-WO about 1 lU / mL of IL-21, or about 0.5 lU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 20 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 15 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 12 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 10 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 5 lU / mL of IL-21 to about 1 lU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 2 lU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 lU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 lU / mL of IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 lU / mL of IL-21.

[0335] In some embodiments, the cell culture medium comprises an anti-CD3 agonist antibody, e.g. 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, and about 1 pg / 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.

[0336] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in a cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4- 1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is urelumab, utomilumab, EU-101, a fusion protein, or fragments, derivatives, variants, biosimilars, or combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 0.1 pg / mL and 100 pg / mL. In some embodiments, the TNFRSF agonist is added at aAttorney Docket No. 5134-WO concentration sufficient to achieve a concentration in the cell culture medium of between 20 pg / mL and 40 pg / mL.

[0337] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 lU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and wherein the one or more TNFRSF agonists comprises a 4-lBB agonist.

[0338] In some embodiments, the first expansion culture medium is referred to as “CM”, an abbreviation for culture media. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 with GLUTAMAX, supplemented with 10% human AB serum, 25 mM HEPES, and 10 mg / mL gentamicin. In embodiments where cultures are initiated in gas-permeable flasks with a 40 mL capacity and a 10cm2gas-permeable silicon bottom (for example, G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask was loaded with 10-40xl06viable tumor digest cells or 5-30 tumor fragments in 10-40mL of CM with IL-2. Both the G-REX10 and 24-well plates were incubated in a humidified incubator at 37°C in 5% CO2 and 5 days after culture initiation, half the media was removed and replaced with fresh CM and IL-2 and after day 5, half the media was changed every72-3 days. In some embodiments, the CM is the CM1 described in the Examples, see, Example 1. In some embodiments, the first expansion occurs in an initial cell culture medium or a first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium comprises IL-2.

[0339] In some embodiments, the first expansion (including processes such as for example those described in Step B of Figure 1, which can include those sometimes referred to as the pre-REP) process is shortened to 3-14 days, as discussed in the examples and figures. In some embodiments, the first expansion (including processes such as for example those described in Step B of Figure 1, which can include those sometimes referred to as the pre- REP) is shortened to 7 to 14 days, as discussed in the Examples, as well as including for example, an expansion as described in Step B of Figure 1. In some embodiments, the first expansion of Step B is shortened to 10-14 days. In some embodiments, the first expansion is shortened to 11 days, as discussed in, for example, an expansion as described in Step B of Figure 1.

[0340] In some embodiments, the first TIL 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.Attorney Docket No. 5134-WOIn some embodiments, the first TIL expansion can proceed for 1 day to 14 days. In some embodiments, the first TIL expansion can proceed for 2 days to 14 days. In some embodiments, the first TIL expansion can proceed for 3 days to 14 days. In some embodiments, the first TIL expansion can proceed for 4 days to 14 days. In some embodiments, the first TIL expansion can proceed for 5 days to 14 days. In some embodiments, the first TIL expansion can proceed for 6 days to 14 days. In some embodiments, the first TIL expansion can proceed for 7 days to 14 days. In some embodiments, the first TIL expansion can proceed for 8 days to 14 days. In some embodiments, the first TIL expansion can proceed for 9 days to 14 days. In some embodiments, the first TIL expansion can proceed for 10 days to 14 days. In some embodiments, the first TIL expansion can proceed for 11 days to 14 days. In some embodiments, the first TIL expansion can proceed for 12 days to 14 days. In some embodiments, the first TIL expansion can proceed for 13 days to 14 days. In some embodiments, the first TIL expansion can proceed for 14 days. In some embodiments, the first TIL expansion can proceed for 1 day to 11 days. In some embodiments, the first TIL expansion can proceed for 2 days to 11 days. In some embodiments, the first TIL expansion can proceed for 3 days to 11 days. In some embodiments, the first TIL expansion can proceed for 4 days to 11 days. In some embodiments, the first TIL expansion can proceed for 5 days to 11 days. In some embodiments, the first TIL expansion can proceed for 6 days to 11 days. In some embodiments, the first TIL expansion can proceed for 7 days to 11 days. In some embodiments, the first TIL expansion can proceed for 8 days to 1 1 days. In some embodiments, the first TIL expansion can proceed for 9 days to 11 days. In some embodiments, the first TIL expansion can proceed for 10 days to 11 days. In some embodiments, the first TIL expansion can proceed for 11 days.

[0341] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 are employed as a combination during the first expansion. In some embodiments, IL-2, IL-7, IL- 15, and / or IL-21 as well as any combinations thereof can be included during the first expansion, including for example during a Step B processes according to Figure 1, as well as described herein. In some embodiments, a combination of IL-2. IL-15, and IL-21 are employed as a combination during the first expansion. In some embodiments, IL-2, IL- 15, and IL-21 as well as any combinations thereof can be included during Step B processes according to Figure 1 and as described herein.Attorney Docket No. 5134-WO

[0342] In some embodiments, the first expansion (including processes referred to as the pre-REP; for example. Step B according to Figure 1) process is shortened to 3 to 14 days, as discussed in the examples and figures. In some embodiments, the first expansion of Step B is shortened to 7 to 14 days. In some embodiments, the first expansion of Step B is shortened to 10 to 14 days. In some embodiments, the first expansion is shortened to 11 days.

[0343] In some embodiments, the first expansion, for example, Step B according to Figure 1, is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the TIL 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.1. Cytokines and Other Additives

[0344] 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.

[0345] Alternatively, using combinations of cytokines for the rapid expansion and or second expansion of TILs is additionally 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 Al, 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, or 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.

[0346] In some embodiments, Step B may also include the addition of OKT-3 antibody or muromonab to the culture media, as described elsewhere herein. In some embodiments, Step B may also include the addition of a 4- IBB agonist to the culture media, as described elsewhere herein. In some embodiments. Step B may also include the addition of an OX-40 agonist to the culture media, as described elsewhere herein. In other embodiments, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists such as a thiazolidinedione compound, may be used in the culture media during Step B, as described in U.S. Patent Application Publication No. US 2019 / 0307796 Al, the disclosure of which is incorporated by reference herein.Attorney Docket No. 5134-WOC. STEP C: First Expansion to Second Expansion Transition

[0347] In some cases, the bulk TIL population obtained from the first expansion, including for example the TIL population obtained from for example, Step B as indicated in Figure 1, can be cryopreserved immediately, using the protocols discussed herein below. Alternatively, the TIL population obtained from the first expansion, referred to as the second TIL population, can be subjected to a second expansion (which can include expansions sometimes referred to as REP) and then cryopreserved as discussed below. Similarly, in the case where genetically modified TILs will be used in therapy, the first TIL population (sometimes referred to as the bulk TIL population) or the second TIL population (which can in some embodiments include populations referred to as the REP TIL populations) can be subjected to genetic modifications for suitable treatments prior to expansion or after the first expansion and prior to the second expansion.

[0348] In some embodiments, the TILs obtained from the first expansion (for example, from Step B as indicated in Figure 1) are stored until phenotyped for selection. In some embodiments, the TILs obtained from the first expansion (for example, from Step B as indicated in Figure 1) are not stored and proceed directly to the second expansion. In some embodiments, the TILs obtained from the first expansion are not cryopreserved after the first expansion and prior to the second expansion. In some embodiments, the transition from the first expansion to the second expansion occurs at about 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 from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 3 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 4 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 4 days to 10 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 7 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 14 days from when fragmentation occurs.

[0349] In some embodiments, the transition from the first expansion to the second expansion occurs at 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 from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day toAttorney Docket No. 5134-WO14 days from when fragmentation occurs. In some embodiments, the first TIL expansion can proceed for 2 days to 14 days. In some embodiments, the transition from the first expansion to the second expansion occurs 3 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs6 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 12 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 13 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 2 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 3 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs7 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to theAttorney Docket No. 5134-WO second expansion occurs 9 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days from when fragmentation occurs.

[0350] In some embodiments, the TILs are not stored after the first expansion and prior to the second expansion, and the TILs proceed directly to the second expansion (for example, in some embodiments, there is no storage during the transition from Step B to Step D as shown in Figure 1). In some embodiments, the transition occurs in closed system, as described herein. In some embodiments, the TILs from the first expansion, the second population of TILs, proceeds directly into the second expansion with no transition period.

[0351] In some embodiments, the transition from the first expansion to the second expansion, for example, Step C according to Figure 1, is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the TIL 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 bioreactor. In some embodiments, the closed system bioreactor is a single bioreactor.D. STEP D: Second Expansion

[0352] In some embodiments, the TIL cell population is expanded in number after harvest and initial bulk processing for example, after Step A and Step B, and the transition referred to as Step C, as indicated in Figure 1). 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); as well as processes as indicated in Step D of Figure 1. The second expansion is generally accomplished using a culture media comprising a number of components, including feeder cells (e.g. the aAPCs described herein) and / or LNPs as described herein, a cytokine source, and an anti-CD3 antibody, in a gas-permeable container.

[0353] In some embodiments, the second expansion or second TIL expansion (which can include expansions sometimes referred to as REP; as well as processes as indicated in Step D of Figure 1) of TIL can be performed using any TIL flasks or containers know n by those of skill in the art. In some embodiments, the second TIL expansion can proceed for 7 days, 8 days. 9 days. 10 days, 11 days, 12 days. 13 days, or 14 days. In some embodiments, the second TIL expansion can proceed for about 7 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 8 days to about 14 days. In someAttorney Docket No. 5134-WO embodiments, the second TIL expansion can proceed for about 9 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 10 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 11 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 12 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 13 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 14 days.

[0354] 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; as well as processes as indicated in Step D of Figure 1). For example, TILs 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 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). TILs can be expanded to induce further stimulation of the TILs 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 pM MART-1 :26-35 (27 L) or gpl 00:209-217 (210M), optionally in the presence of a T-cell grow th factor, such as 300 lU / mL IL-2 or IL-15. Other suitable antigens may include, e.g., NY-ESO-1, TRP-L TRP-2. tyrosinase cancer antigen. MAGE-A3, SSX-2, and VEGFR2. or antigenic portions thereof. TIL 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 TILs 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.

[0355] In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 lU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 lU / mL. about 1500 lU / mL. about 2000 lU / mL, about 2500 lU / mL, about 3000 lU / mL, about 3500 lU / mL, about 4000Atorney Docket No. 5134-WO lU / mL, about 4500 lU / mL, about 5000 lU / mL, about 5500 lU / mL, about 6000 lU / mL, about 6500 lU / mL. about 7000 lU / mL, about 7500 lU / mL, or about 8000 lU / mL of IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000 lU / mL, between 2000 and 3000 lU / mL, between 3000 and 4000 lU / mL, between 4000 and 5000 lU / mL, between 5000 and 6000 lU / mL, between 6000 and 7000 lU / mL, between 7000 and 8000 lU / mL. or between 8000 lU / mL of IL-2.

[0356] 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, and about 1 pg / 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.

[0357] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in a cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4- 1BB agonist. In some embodiments, the TNFRSF agonist is a 4- IBB agonist, and the 4- IBB agonist is urelumab, utomilumab, EU-101, a fusion protein, or fragments, derivatives, variants, biosimilars, or combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 0.1 pg / mL and 100 pg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 pg / mL and 40 pg / mL.

[0358] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 HJ / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and wherein the one or more TNFRSF agonists comprises a 4-lBB agonist.Attorney Docket No. 5134-WO

[0359] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 are employed as a combination during the second expansion. In some embodiments, IL-2, IL-7, IL- 15, and / or IL-21 as well as any combinations thereof can be included during the second expansion, including for example during a Step D processes according to Figure 1, as well as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 are employed as a combination during the second expansion. In some embodiments, IL-2, IL- 15, and IL-21 as well as any combinations thereof can be included during Step D processes according to Figure 1 and as described herein.

[0360] In some embodiments, the second expansion can be conducted in a supplemented cell culture medium comprising IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2. OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen-presenting cells (APCs; also referred to as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e. , antigen presenting cells).

[0361] In some embodiments, the second expansion culture media comprises about 500 lU / mL of IL-15, about 400 IU / mL of IL-15, about 300 IU / mL of IL-15, about 200 IU / mL of IL-15, about 180 IU / mL of IL-15, about 160 IU / mL of IL-15, about 140 IU / mL of IL-15, about 120 IU / mL of IL- 15, or about 100 IU / mL of IL-15. In some embodiments, the second expansion culture media comprises about 500 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture media comprises about 400 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture media comprises about 300 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture media comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15.

[0362] In some embodiments, the second expansion culture media comprises about 20 IU / mL of IL-21, about 15 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL- 21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, theAttorney Docket No. 5134-WO second expansion culture media comprises about 20 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 15 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 12 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 10 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 5 lU / mL of IL-21 to about 1 lU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 2 lU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 lU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 lU / mL of IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 lU / mL of IL-21.

[0363] In some embodiments, the aAPCs as described herein are used. In some embodiments, LNPs as described herein are used. In some embodiments, the antigen- presenting feeder cells (aAPCs) are PBMCs. In some embodiments, the ratio of TILs to aAPCs 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 TILs 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 TILs to aAPCs in the rapid expansion and / or the second expansion is between 1 to 100 and 1 to 200.

[0364] In some embodiments, REP and / or the second expansion is performed in flasks with the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder cells or aAPCs or LNPs as described herein, 30 mg / mL OKT3 anti-CD3 antibody and 3000 lU / 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.

[0365] In some embodiments, the second expansion (which can include processes referred to as the REP process) is shortened to 7-14 days, as discussed in the examples and figures. In some embodiments, the second expansion is shortened to 11 days.Attorney Docket No. 5134-WO

[0366] 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 106TILs suspended in 150 mL of media may be added to each T-175 flask. The TILs 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 L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU per mL of IL-2 is added to the 300 mL of TIL suspension. The number of cells in each bag is counted every day or two and fresh media added to keep the cell count between 0.5 and 2.0 x 106cells / mL.

[0367] In some embodiments, the second expansion (which can include expansions referred to as REP, as w ell as those referred to in Step D of Figure 1) 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 x 106or 10 x 106TIL may be cultured with aAPCs or LNPs 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 x g) for 10 minutes. The TIL 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 TIL are expanded serially in G-REX-100 flasks, on day 7 the TIL 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 3 100 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% CO2 and 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.

[0368] In some embodiments, the second expansion (including expansions referred to as REP) is performed in flasks with the bulk TILs being mixed with a 100- or 200-fold excess ofAttorney Docket No. 5134-WO inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody and 3000 lU / mL IL-2 in 150 mL media. In some embodiments, media replacement is done until the cells are transferred to an alternative grow th chamber. In some embodiments, 2 / 3 of the media is replaced by respiration with fresh media. In some embodiments, alternative growth chambers include G- REX flasks and gas permeable containers as more fully discussed below7.

[0369] In some embodiments, the second expansion (including expansions referred to as REP) is performed and further comprises a step wherein TILs are selected for superior tumor reactivity. Any selection method known in the art may be used. For example, the methods described in U.S. Patent Application Publication No. 2016 / 0010058 Al, the disclosures of which are incorporated herein by reference, may be used for selection of TILs for superior tumor reactivity7.

[0370] Optionally, a cell viability7assay can be performed after the second expansion (including expansions referred to as the REP expansion), using standard assays known in the art. For example, a trypan blue exclusion assay can be done on a sample of the bulk TILs, which selectively labels dead cells and allows a viability assessment. In some embodiments, TIL samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to the standard Cellometer K2 Image Cytometer Automatic Cell Counter protocol.

[0371] In some embodiments, the second expansion (including expansions referred to as REP) of TIL can be performed using T-175 flasks and gas-permeable bags as previously described (Tran, et al., 2008, J Immunother., 31, 742-751, and Dudley, et al. 2003, J Immunother., 26, 332-342) or gas-permeable G-REX flasks. In some embodiments, the second expansion is performed using flasks. In some embodiments, the second expansion is performed using gas-permeable G-REX flasks. In some embodiments, the second expansion is performed in T-175 flasks, and about 1 x 106TIL are suspended in about 150 mL of media and this is added to each T-175 flask. The TIL may be cultured with irradiated (50 Gy) allogeneic PBMC as “feeder” cells, aAPCs, or LNPs, at a ratio of 1 to 100 (or any ratio, such as between 1:25 and 1 :500) and the cells cultured in a 1 to 1 mixture of CM and AIM-V medium (50 / 50 medium), supplemented with 3000 lU / mL of IL-2 and 30 ng / mL of anti-CD3. The T-175 flasks are incubated at 37°C in 5% CO2. In some embodiments, half the media is changed on day 5 using 50 / 50 medium with 3000 lU / mL of IL-2. In some embodiments, on day 7, cells from 2 T-175 flasks are combined in a 3 L bag and 300 mL of AIM-V with 5%Attorney Docket No. 5134-WO human AB serum and 3000 lU / rnL of IL-2 is added to the 300 mL of TIL suspension. The number of cells in each bag can be counted every day or two and fresh media can be added to keep the cell count between about 0.5 and about 2.0 x io6cells / mL.

[0372] In some embodiments, the second expansion (including expansions referred to as REP) are performed in 500 mL capacity flasks with 1 0 cm2gas-permeable silicon bottoms (G-REX-100, Wilson Wolf) about 5 x 106or 10 x 106TIL are cultured with irradiated allogeneic PBMC at a ratio of 1 to 100 in 400 mL of 50 / 50 medium, supplemented with 3000 lU / mL of IL-2 and 30 ng / mL of anti-CD3. The G-REX-100 flasks are incubated at 37°C in 5% CO2. In some embodiments, on day 5, 250mL of supernatant is removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellets can then be resuspended with 150 mL of fresh 50 / 50 medium with 3000 IU / mL of IL-2 and added back to the original G-REX-100 flasks. In embodiments where TILs are expanded serially in G-REX-100 flasks, on day 7 the TIL in each G-REX-100 are suspended in the 300 mL of media present in each flask and the cell suspension was divided into three 100 mL aliquots that are used to seed 3 G-REX-100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 lU / mL of IL-2 is added to each flask. The G-REX-100 flasks are incubated at 37°C in 5% CO2 and after 4 days 150 mL of AIM-V with 3000 lU / mL of IL-2 is added to each G-REX-100 flask. The cells are harvested on day 14 of culture.

[0373] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited, but large number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant), determine the binding specificity and downstream applications of immunoglobulins and T-cell receptors (TCRs). Without being bound by theory, the methods described herein may generate TILs which exhibit and increase the T-cell repertoire diversity. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity. In some embodiments, the TILs obtained in the second expansion exhibit an increase in the T-cell repertoire diversity . In some embodiments, the increase in diversity is an increase in the immunoglobulin diversity and / or the T-cell receptor diversity'. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin heavy chain. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin light chain. In some embodiments, the diversity is in the T-cell receptor. In some embodiments, the diversity is in one of the T-cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alphaAttorney Docket No. 5134-WO and / or beta. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) beta. In some embodiments, there is an increase in the expression of TCRab (i.e., TCRa / p).

[0374] 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 artificial antigen-presenting feeder cells (aAPCs) or LNPs, as discussed in more detail below.

[0375] 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 serumcontaining media.

[0376] 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 (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0377] 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, insuhn, and compounds containing the trace element moieties Ag+, A13+, Ba2+, Cd2+, Co2+, Cr3+,Attorney Docket No. 5134-WOGe4+, Se4+, Br, T. Mn2+, P, Si4+, V5+, M06+, Ni2+, Rb+, Sn2+ and Zr4+. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate and / or 2-mercaptoethanol .

[0378] 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 (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0379] 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.

[0380] In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T- cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ may be used. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of IL CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 rnL 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 w ith 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 55pM.Attorney Docket No. 5134-WO

[0381] In some embodiments, the defined medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ may be used. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of IL CTS™ OpTmizer™ T- cell Expansion Basal Medium and 26 rnL 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 lU / rnL to about 8000 lU / 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 lU / 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 lU / 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 lU / mL to about 8000 lU / 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 lU / 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 lU / mL to about 6000 lU / 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 lU / mL to about 8000 lU / rnL 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) (ThermoFisherAttorney Docket No. 5134-WOScientific) and about 2mM glutamine, and further comprises about 3000 lU / 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 lU / 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 55pM.

[0382] In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of from about O. lmM to about lOmM, 0.5mM to about 9mM, ImM 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.

[0383] 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, lOrnM to about 140mM, 15mM to about 130mM, 20mM to about I20mM, 25mM to about I lOmM, 30mM to about lOOmM, 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 55 pM.

[0384] In some embodiments, the defined media described in International PCT Publication No. WO / 1998 / 030679, which is herein incorporated by reference, may be used. 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 fromAttorney Docket No. 5134-WO 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+, A13+, Ba2+, Cd2+, Co2+, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, M06+, N12+, 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 (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0385] 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.

[0386] 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 IX Medium'’ in Table 3. 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 IX Medium” inAttomey Docket No. 5134-WOTable 3. 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 3.

[0387] 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 pM), 2-mercaptoethanol (final concentration of about 100 pM).

[0388] In some embodiments, the defined media described in Smith, et al., Clin Transl Immunology, 4(1) 2015 (doi: 10. 1038 / cti.2014.31) may be used. 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.

[0389] 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 |3ME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0390] In some embodiments, the second expansion, for example, Step D according to Figure 1. is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the TIL 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.

[0391] In some embodiments, the step of rapid or second expansion is split into a plurality of steps to achieve a scaling up of the culture by: (a) performing the rapid or second expansion by culturing TILs in a small scale culture in a first container, e.g., a G-REX-100 MCS container, for a period of about 3 to 7 days, and then (b) effecting the transfer of the TILs in the small scale culture to a second container larger than the first container, e.g., a G-Attorney Docket No. 5134-WOREX-500-MCS container, and culturing the TILs from the small scale culture in a larger scale culture in the second container for a period of about 4 to 7 days.

[0392] In some embodiments, the step of rapid or second expansion is split into a plurality of steps to achieve a scaling out of the culture by: (a) performing the rapid or second expansion by culturing TILs in a first small scale culture in a first container, e.g., a G-REX- 100 MCS container, for a period of about 3 to 7 days, and then (b) effecting the transfer and apportioning of the TILs from the first small scale culture into and amongst at least 2, 3, 4, 5, 6, 7, 8, 9. 10. 11. 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers that are equal in size to the first container, wherein in each second container the portion of the TILs from first small scale culture transferred to such second container is cultured in a second small scale culture for a period of about 4 to 7 days.

[0393] In some embodiments, the first small scale TIL culture is apportioned into a plurality of about 2 to 5 subpopulations of TILs.

[0394] In some embodiments, the step of rapid or second expansion is split into a plurality of steps to achieve a scaling out and scaling up of the culture by: (a) performing the rapid or second expansion by culturing TILs in a small scale culture in a first container, e.g., a G- REX-100 MCS container, for a period of about 3 to 7 days, and then (b) effecting the transfer and apportioning of the TILs from the small scale culture into and amongst at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers that are larger in size than the first container, e.g., G-REX-500MCS containers, wherein in each second container the portion of the TILs from the small scale culture transferred to such second container is cultured in a larger scale culture for a period of about 4 to 7 days.

[0395] In some embodiments, the step of rapid or second expansion is split into a plurality of steps to achieve a scaling out and scaling up of the culture by: (a) performing the rapid or second expansion by culturing TILs in a small scale culture in a first container, e.g., a G- REX-100 MCS container, for a period of about 5 days, and then (b) effecting the transfer and apportioning of the TILs from the small scale culture into and amongst 2, 3 or 4 second containers that are larger in size than the first container, e.g., G-REX-500 MCS containers, wherein in each second container the portion of the TILs from the small scale culture transferred to such second container is cultured in a larger scale culture for a period of about 6 days.Attorney Docket No. 5134-WO

[0396] In some embodiments, upon the splitting of the rapid or second expansion, each second container comprises at least 108TILs. In some embodiments, upon the splitting of the rapid or second expansion, each second container comprises at least 108TILs, at least 109TILs, or at least IO10TILs. In one exemplary embodiment, each second container comprises at least IO10TILs.

[0397] In some embodiments, the first small scale TIL culture is apportioned into a plurality of subpopulations. In some embodiments, the first small scale TIL culture is apportioned into a plurality of about 2 to 5 subpopulations. In some embodiments, the first small scale TIL culture is apportioned into a plurality of about 2, 3, 4, or 5 subpopulations.

[0398] In some embodiments, after the completion of the rapid or second expansion, the plurality of subpopulations comprises a therapeutically effective amount of TILs. In some embodiments, after the completion of the rapid or second expansion, one or more subpopulations of TILs are pooled together to produce a therapeutically effective amount of TILs. In some embodiments, after the completion of the rapid expansion, each subpopulation of TILs comprises a therapeutically effective amount of TILs.

[0399] In some embodiments, the rapid or second expansion is performed for a period of about 3 to 7 days before being split into a plurality of steps. In some embodiments, the splitting of the rapid or second expansion occurs at about day 3, day 4, day 5, day 6, or day 7 after the initiation of the rapid or second expansion.

[0400] In some embodiments, the splitting of the rapid or second expansion occurs at about day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15. or day 16 day 17, or day 18 after the initiation of the first expansion (i.e.. pre-REP expansion). In one exemplary embodiment, the splitting of the rapid or second expansion occurs at about day 1 after the initiation of the first expansion.

[0401] In some embodiments, the rapid or second expansion is further performed for a period of about 7 to 11 days after the splitting. In some embodiments, the rapid or second expansion is further performed for a period of about 5 days. 6 days. 7 days. 8 days. 9 days, 10 days, or 11 days after the splitting.

[0402] In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting comprises the same components as the cell culture medium used for the rapid or second expansion after the splitting. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting comprisesAttorney Docket No. 5134-WO different components from the cell culture medium used for the rapid or second expansion after the splitting.

[0403] In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting comprises IL-2, optionally OKT-3 and further optionally APCs, e.g., aAPCs. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting comprises IL-2, OKT-3, and further optionally APCs, e.g., aAPCs. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting comprises IL-2. OKT-3 and aAPCs as described herein. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting comprises IL-2, OKT-3 and LNPs as described herein.

[0404] In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting is generated by supplementing the cell culture medium in the first expansion with fresh culture medium comprising IL-2, optionally OKT-3 and further optionally APCs, e.g. aAPCs. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting is generated by supplementing the cell culture medium in the first expansion with fresh culture medium comprising IL-2, OKT-3 and aAPCs as described herein. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting is generated by supplementing the cell culture medium in the first expansion with fresh culture medium comprising IL-2, OKT-3 and LNPs as described herein. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting is generated by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, optionally OKT-3 and further optionally APCs, e.g. aAPCs. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting is generated by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, OKT-3 and aAPCs as described herein. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting is generated by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, OKT-3 and LNPs as described herein.

[0405] In some embodiments, the cell culture medium used for the rapid or second expansion after the splitting comprises IL-2, and optionally OKT-3. In some embodiments, the cell culture medium used for the rapid or second expansion after the splitting comprises IL-2, and OKT-3. In some embodiments, the cell culture medium used for the rapid or secondAttorney Docket No. 5134-WO expansion after the splitting is generated by replacing the cell culture medium used for the rapid or second expansion before the splitting with fresh culture medium comprising IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for the rapid or second expansion after the splitting is generated by replacing the cell culture medium used for the rapid or second expansion before the splitting with fresh culture medium comprising IL-2 and OKT-3.

[0406] In some embodiments, the splitting of the rapid expansion occurs in a closed system.

[0407] In some embodiments, the scaling up of the TIL culture during the rapid or second expansion comprises adding fresh cell culture medium to the TIL culture (also referred to as feeding the TILs). In some embodiments, the feeding comprises adding fresh cell culture medium to the TIL culture frequently. In some embodiments, the feeding comprises adding fresh cell culture medium to the TIL culture at a regular interval. In some embodiments, the fresh cell culture medium is supplied to the TILs via a constant flow. In some embodiments, an automated cell expansion system such as XURI W25 is used for the rapid expansion and feeding.1. Antigen Presenting Cells

[0408] In some embodiments, the second expansion procedures described herein (for example including expansion such as those described in Step D from Figure 1, as well as those referred to as REP) require an excess of antigen presenting cells during REP TIL expansion and / or during the second expansion. In some embodiments, the antigen presenting cells are the artificial antigen presenting cells (aAPCs) described herein. In some embodiments, the lipid nanoparticles (LNPs) are used as antigen presenting cells during REP TIL expansion and / or during the second expansion.

[0409] In some embodiments, the aAPCs express one or more co-stimulatory proteins. In some embodiments, the aAPCs express one or more of CD64, CD86 (B7-2), 4-1 BBL (CD137L), and / or OX40L (CD134L). In embodiments, the aAPCs express CD64. In embodiments, the aAPCs express CD86. In embodiments, the aAPCs express 4-1BBL. In embodiments, the aAPCs express OX40L. In embodiments, the aAPCs express CD64 and CD86. In embodiments, the aAPCs express CD64 and 4-1 BBL. In embodiments, the aAPCs express CD64 and OX40L. In embodiments, the aAPCs express CD86 and 4-1BBL. In embodiments, the aAPCs express CD86 and OX40L. In embodiments, the aAPCs express 4- 1BBL and OX40L. In embodiments, the aAPCs express CD64, CD86, and 4-1BBL. InAttorney Docket No. 5134-WO embodiments, the aAPCs express CD64, CD86, and OX40L. In embodiments, the aAPCs express CD64, 4-1 BBL, and OX40L. In embodiments, the aAPCs express CD86, 4-1 BBL. and OX40L. In embodiments, the aAPCs express CD64, CD86, 4-1 BBL, and OX40L.

[0410] In some embodiments, the LNPs are associated with (e.g.. conjugated to) one or more co-stimulatory proteins. In some embodiments, the LNPs are associated with (e.g., conjugated to) one or more of CD64, CD86 (B7-2), 4-1BBL (CD137L), and / or OX40L (CD134L). In embodiments, the LNPs are associated with (e.g., conjugated to) CD64. In embodiments, the LNPs are associated with (e.g., conjugated to) CD86. In embodiments, the LNPs are associated with (e.g., conjugated to) 4-1BBL. In embodiments, the LNPs are associated with (e.g., conjugated to) OX40L. In embodiments, the LNPs are associated with (e.g., conjugated to) CD64 and CD86. In embodiments, the LNPs are associated with (e.g., conjugated to) CD64 and 4-1BBL. In embodiments, the LNPs are associated with (e.g., conjugated to) CD64 and OX40L. In embodiments, the LNPs are associated with (e.g.. conjugated to) CD86 and 4-1BBL. In embodiments, the LNPs are associated with (e.g., conjugated to) CD86 and OX40L. In embodiments, the LNPs are associated with (e.g., conjugated to) 4-1BBL and OX40L. In embodiments, the LNPs are associated with (e.g., conjugated to) CD64, CD86. and 4-1BBL. In embodiments, the LNPs are associated with (e.g., conjugated to) CD64, CD86, and OX40L. In embodiments, the LNPs are associated with (e.g., conjugated to) CD64, 4-1BBL, and OX40L. In embodiments, the LNPs are associated with (e.g., conjugated to) CD86, 4-1BBL, and OX40L. In embodiments, the LNPs are associated with (e.g.. conjugated to) CD64, CD86, 4-1BBL. and OX40L.

[0411] In some embodiments, the aAPCs or LNPs are cultured in the presence of 5-60 ng / mL OK.T3 antibody and 1000-6000 lU / rnL IL-2. In some embodiments, the aAPCs or LNPs are cultured in the presence of 10-50 ng / mL OKT3 antibody and 2000-5000 lU / mL IL- 2. In some embodiments, the aAPCs or LNPs are cultured in the presence of 20-40 ng / mL OKT3 antibody and 2000-4000 lU / mL IL-2. In some embodiments, the aAPCs or LNPs are cultured in the presence of 25-35 ng / mL OKT3 antibody and 2500-3500 lU / mL IL-2.

[0412] In some embodiments, the ratio of TILs to aAPCs or LNPs 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, 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 TILs to aAPCs or LNPs in the second expansion is between 1 to 50 and 1 to 300. InAttorney Docket No. 5134-WO some embodiments, the ratio of TILs to aAPCs or LNPs in the second expansion is between 1 to 100 and 1 to 200.

[0413] In some embodiments, the second expansion procedures described herein require a ratio of about 2.5xl09aAPCs or LNPs to about lOOxlO6TIL. In other embodiments, the second expansion procedures described herein require a ratio of about 2.5x109aAPCs or LNPs to about 50xl06TIL. In yet other embodiments, the second expansion procedures described herein require about 2.5xl09aAPCs or LNPs to about 25xl06TIL.

[0414] In some embodiments, aAPCs or LNPs are used in the second expansion as a replacement for, or in combination with, PBMCs.2. Cytokines and Other Additives

[0415] 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.

[0416] Alternatively, using combinations of cytokines for the rapid expansion and or second expansion of TILs is additionally 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 Al, the disclosure of which is incorporated by reference herein. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-2L 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.

[0417] In some embodiments, Step D may also include the addition of OKT-3 antibody or muromonab to the culture media, as described elsewhere herein. In some embodiments, Step D may also include the addition of a 4-1 BB agonist to the culture media, as described elsewhere herein. In some embodiments. Step D may also include the addition of an OX-40 agonist to the culture media, as described elsewhere herein. In addition, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists such as a thiazolidinedione compound, may be used in the culture media during Step D, as described in U.S. Patent Application Publication No. US 2019 / 0307796 Al, the disclosure of which is incorporated by reference herein.Attorney Docket No. 5134-WOE. STEP E: Harvest TILs

[0418] After the second expansion step, cells can be harvested. In some embodiments the TILs are harvested after one. two, three, four or more expansion steps, for example as provided in Figure 1. In some embodiments the TILs are harvested after two expansion steps, for example as provided in Figure 1.

[0419] TILs can be harvested in any appropriate and sterile manner, including for example by centrifugation. Methods for TIL harvesting are well known in the art and any such know methods can be employed with the present process. In some embodiments, TILs are harvested using an automated system.

[0420] Cell harvesters and / or cell processing systems are commercially available from a variety of sources, including, for example. Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell based harvester can be employed with the present methods. In some embodiments, the cell harvester and / or cell processing systems is a membrane-based cell harvester. In some embodiments, cell harvesting is via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term “LOVO cell processing system” also refers to any instrument or device manufactured by any vendor that can pump a solution comprising cells through a membrane or filter such as a spinning membrane or spinning filter in a sterile and / or closed system environment, allowing for continuous flow and cell processing to remove supernatant or cell culture media without pelletization. In some embodiments, the cell harvester and / or cell processing system can perform cell separation, washing, fluid-exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[0421] In some embodiments, the harvest, for example, Step E according to Figure 1, is performed from a closed system bioreactor. In some embodiments, a closed system is employed for the TIL 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.

[0422] In some embodiments, Step E according to Figure 1, is performed according to the processes described herein. In some embodiment...

Claims

Attorney Docket No. 5134-WOCLAIMSWe claim:

1. An artificial antigen presenting cell (aAPC) comprising a U937 cell expressing one or more co-stimulatory molecules.

2. The aAPC of Claim 1, wherein the one or more co-stimulatory molecules comprise one or more of a CD64 protein, a CD86 protein, a 4-1BBL protein, or an OX40L protein.

3. The aAPC of Claim 2, wherein the one or more co-stimulatory molecules comprise a CD64 protein.

4. The aAPC of Claim 2, wherein the one or more co-stimulatory molecules comprise a CD64 protein, and a CD86 protein.

5. The aAPC of Claim 2, wherein the one or more co-stimulatory molecules comprise a CD64 protein, and a 4-1 BBL protein.

6. The aAPC of Claim 2, wherein the one or more co-stimulatory molecules comprise a CD64 protein, and an OX40L protein.

7. The aAPC of Claim 2, wherein the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and a 4-1BBL protein.

8. The aAPC of Claim 2, wherein the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and an OX40L protein.

9. The aAPC of Claim 2, wherein the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, a 4-1BBL protein, and an OX40L protein.

10. The aAPC of any one of Claims 1-9. wherein the aAPC can stimulate and expand a T cell contacted with the aAPC.

11. The aAPC of any one of Claims 1-9. wherein the aAPC can stimulate and expand tumor infiltrating lymphocytes (TILs) contacted with the aAPC.

12. The aAPC of any one of Claims 1-9. wherein the aAPC expands a population of TILs by at least 50-fold over a period of 7 days in a cell culture medium comprising IL-2 at a concentration of about 3000 lU / mL and OKT-3 antibody at a concentration of about 30 ng / mL.

13. The aAPC of any one of Claims 1-12, wherein the CD64 protein comprises SEQ IDAttorney Docket No. 5134-WON0:4, or a sequence comprising one or more conservative amino acid substitutions thereof.

14. The aAPC of any one of Claims 1-13, wherein the CD86 protein comprises SEQ ID NO: 1, or a sequence comprising one or more conservative amino acid substitutions thereof.

15. The aAPC of any one of Claims 1-14, wherein the 4-1BBL protein comprises SEQ ID NO:2, or a sequence comprising one or more conservative amino acid substitutions thereof.

16. The aAPC of any one of Claims 1-15, wherein the OX40L protein comprises SEQ ID NO: 3, or a sequence comprising one or more conservative amino acid substitutions thereof.

17. The aAPC of any one of Claims 1-16, wherein the nucleic acid encoding CD64 comprises SEQ ID NO: 12.

18. The aAPC of any one of Claims 1-17, wherein the nucleic acid encoding CD86 comprises SEQ ID NO:9.

19. The aAPC of any one of Claims 1-18, wherein the nucleic acid encoding 4-1BBL comprises SEQ ID NOTO.

20. The aAPC of any one of Claims 1-19, wherein the nucleic acid encoding OX40L comprises SEQ ID NOT E21. A lipid nanoparticle (LNP) comprising one or more co-stimulatory molecules.

22. The LNP of Claim 21, wherein the one or more co-stimulatory molecules comprise one or more of a CD64 protein, a CD86 protein, a 4-1BBL protein, or an OX40L protein.

23. The LNP of Claim 22, wherein the one or more co-stimulatory molecules comprise a CD64 protein.

24. The LNP of Claim 22, wherein the one or more co-stimulatory molecules comprise a CD64 protein, and a CD86 protein.

25. The LNP of Claim 22, wherein the one or more co-stimulatory molecules comprise a CD64 protein, and a 4-1 BBL protein.

26. The LNP of Claim 22, wherein the one or more co-stimulatory molecules comprise a CD64 protein, and an OX40L protein.Attorney Docket No. 5134-WO27. The LNP of Claim 22, wherein the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and a 4-1 BBL protein.

28. The LNP of Claim 22, wherein the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and an OX40L protein.

29. The LNP of Claim 22, wherein the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, a 4-1 BBL protein, and an OX40L protein.

30. The LNP of any one of Claims 21-29, wherein the LNP can stimulate and expand a T cell contacted with the LNP.

31. The LNP of any one of Claims 21-29, wherein the LNP can stimulate and expand tumor infiltrating lymphocytes (TILs) contacted with the LNP.

32. The LNP of any one of Claims 21-29, wherein the LNP expands a population of TILs by at least 50-fold over a period of 7 days in a cell culture medium comprising IL-2 at a concentration of about 3000 lU / mL and OKT-3 antibody at a concentration of about 30 ng / mL.

33. The LNP of any one of Claims 21-32, wherein the CD64 protein comprises SEQ ID NO:4, or a sequence comprising one or more conservative amino acid substitutions thereof.

34. The LNP of any one of Claims 21-33, wherein the CD86 protein comprises SEQ ID NO: 1, or a sequence comprising one or more conservative amino acid substitutions thereof.

35. The LNP of any one of Claims 21-34, wherein the 4-1BBL protein comprises SEQ ID NO:2, or a sequence comprising one or more conservative amino acid substitutions thereof.

36. The LNP of any one of Claims 21-35, wherein the LNP comprises:(a) at least one ionizable lipid;(b) at least one neutral lipid;(c) cholesterol and / or a modified derivative thereof; and(d) at least one polymer conjugated lipid and / or a modified derivative thereof.

37. The LNP of any one of Claims 21-36, wherein the one or more co-stimulatory moleculesAttorney Docket No. 5134-WO are covalently conjugated to at least one component of the LNP.

38. The LNP of Claim 37, wherein the component to which the one or more co-stimulatory molecules are covalently conjugated is the modified derivative of the polymer conjugated lipid.

39. The LNP of Claim 38, wherein the covalent conjugation comprises a covalent bond forming reaction selected from the group consisting of a [1,4] -conjugate addition (i.e., Michael addition), [4+2] cycloaddition, [3+2] dipolar cycloaddition, nucleophilic addition, transition metal-catalyzed cross-coupling reaction, carbonyl condensation reaction, and reductive amination.

40. The LNP of Claim 39, wherein the covalent conjugation reaction comprises a [1,4]- conjugate addition reaction (i.e., Michael addition).

41. The LNP of Claim 39 or 40, wherein the [1,4] -conjugate addition occurs between the modified derivative of the polymer conjugated lipid which is further conjugated to a maleimide moiety and a cysteine thiol of a polypeptide.

42. The LNP of Claim 41, wherein the cystine thiol of the polypeptide is derived from a reduced disulfide bridge of the one or more co-stimulatory molecules.

43. A method of expanding tumor infiltrating lymphocytes (TILs), the method comprising a step of contacting a population of TILs with a population of aAPCs according to any one of Claims 1-20 or a population of LNPs according to any one of Claims 21-42 in a cell culture medium, wherein the population of TILs is expanded.

44. The method of Claim 43, wherein the cell culture medium further comprises IL-2 at an initial concentration of about 3000 lU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL.

45. The method of Claims 43 or 44, wherein the population of TILs is expanded by at least 50-fold over a period of 11 days.

46. The method of Claims 43 or 44, wherein the population of TILs is expanded by at least 200-fold over a period of 11 days.

47. The method of Claims 43 or 44, wherein the population of TILs is expanded by at least 500-fold over a period of 11 days.

48. The method of any one of Claims 43-45, wherein the expansion is performed using a gasAttorney Docket No. 5134-WO permeable container.

49. The method of any one of Claims 43-46, wherein the ratio of the population of TILs to the population of aAPCs or the population of LNPs is between 1 to 100 and 1 to 500.

50. The method of Claim 47, wherein the ratio of the population of TILs to the population of aAPCs or the population of LNPs is about 1 to 250.

51. A method of making a therapeutic population of tumor infdtrating lymphocytes (TILs), wherein the method comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of artificial antigen presenting cells (aAPCs) modified to express one or more co-stimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs.

52. A method of making a therapeutic population of tumor infiltrating lymphocytes (TILs), wherein the method comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2. OKT-3, and a population of lipid nanoparticles (LNPs) conjugated to one or more co-stimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs.Attorney Docket No. 5134-WO53. The method of claim 51 or 52, further comprising:(d) harvesting therapeutic population of TILs.

54. The method of claim 53, further comprising:(e) transferring the harvested therapeutic population of TILs into an infusion bag.

55. The method of claim 54, further comprising:(!) cry opreserving the infusion bag comprising the harvested therapeutic population of TILs using a cryopreservation process.

56. The method of any one of claims 51-55, wherein the first expansion is performed over a period of about 3-11 days or about 3-12 days.

57. The method of any one of claims 51 -55, wherein the first expansion is performed over a period of about 11 days or about 12 days.

58. The method of any one of claims 51-56, wherein the second expansion is performed over a period of about 7-11 days or about 7-12 days.

59. The method of any one of claims 51-56, wherein the second expansion is performed over a period of about 11 days or about 12 days.

60. The method of any one of claims 51-55, wherein the first expansion is performed over a period of about 11 days, and the second expansion is performed over a period of about 11 days or about 12 days.

61. The method of any one of claims 51-60, wherein step (b) and step (c) are performed in a closed system, wherein the transition from step (b) to step (c) occurs without opening the closed system.

62. The method of claim 61, wherein the transition from step (c) to step (d) occurs without opening the closed system.

63. The method of claim 61 or 62, wherein the transition from step (d) to step (e) occurs without opening the closed system.

64. The method of any one of claims 51-63, wherein the one or more co-stimulatory molecules comprise one or more of: a CD64 protein, a CD86 protein, a 4-1BBL protein, or an OX40L protein.

65. The method of claim 64, wherein the one or more co-stimulatory molecules comprise aAttorney Docket No. 5134-WOCD64 protein.

66. The method of claim 64, wherein the one or more co-stimulatory molecules comprise a CD64 protein, and a CD86 protein.

67. The method of claim 64, wherein the one or more co-stimulatory molecules comprise a CD64 protein, and a 4-1 BBL protein.

68. The method of claim 64, wherein the one or more co-stimulatory molecules comprise a CD64 protein, and an OX40L protein.

69. The method of claim 64, wherein the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and a 4-1BBL protein.

70. The method of claim 64. wherein the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and an OX40L protein.

71. The method of claim 64. wherein the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, a 4-1 BBL protein, and an OX40L protein.

72. A therapeutic population of TILs made by the method of any one of claims 43-71.

73. A pharmaceutical composition comprising the therapeutic population of TILs of claim 72.

74. A method of treating a cancer patient with a therapeutic population of tumor infiltrating lymphocytes (TILs) comprising the steps of:(a) obtaining and / or receiving a first population of TILs from a tumor resected from the patient;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of artificial antigen presenting cells (aAPCs) modified to express one or more co-stimulatory molecules, to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs; and(d) administering a therapeutically effective dosage of the therapeutic population of TILs to the patient with the cancer.Attorney Docket No. 5134-WO75. A method of treating a cancer patient with a therapeutic population of tumor infiltrating lymphocytes (TILs) comprising the steps of:(a) obtaining and / or receiving a first population of TILs from a tumor resected from the patient;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and a population of lipid nanoparticles (LNPs) conjugated to one or more co-stimulatoiy molecules, to produce a third population of TILs. wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs; and(d) administering a therapeutically effective dosage of the therapeutic population of TILs to the patient with the cancer.

76. The method of any one of claims 74 or 75, wherein the cancer is selected from the group consisting of anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papilloma virus (HPV), central nervous system associated cancer (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma, and primitive neuroectodermal tumor), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC), hypopharynx cancer, larynx cancer, nasopharynx cancer, oropharynx cancer, and phary nx cancer), kidney cancer, liver cancer, lung cancer (including non-small-cell lung cancer (NSCLC) and small-cell lung cancer), melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), penile cancer, rectal cancer, renal cancer, renal cell carcinoma, sarcoma (including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other boneAttorney Docket No. 5134-WO and soft tissue sarcomas), thyroid cancer (including anaplastic thyroid cancer), uterine cancer, and vaginal cancer.

77. The method of any one of claims 74-76, further comprising the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the second population of TILs to the patient.

78. The method of claim 77, 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 five days.

79. The method of claim 77, 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.

80. The method of any one of claims 77 or 78, wherein the cyclophosphamide is administered with mesna.

81. The method of any one of claims 74-80, further comprising the step of treating the patient with an IL-2 regimen starting on the day after the administration of the TILs to the patient.

82. The method of any one of claims 74-80, further comprising the step of treating the patient with an IL-2 regimen starting on the same day as administration of the TILs to the patient.

83. The method of claim 81 or 82, wherein the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 lU / kg of aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerance.

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