ICOS-based chimeric antigen receptors program bipolar TH17/TH1 cells.

With the notable exception of B-cell malignancies, the efficacy of chimeric antigen receptor (CAR) T cells has been limited, and CAR T cells have not been shown to expand and persist in patients with nonlymphoid tumors. Here we demonstrate that redirection of primary human T cells with a CAR containing the inducible costimulator (ICOS) intracellular domain generates tumor-specific IL-17-producing effector cells that show enhanced persistence. Compared with CARs containing the CD3ζ chain alone, or in tandem with the CD28 or the 4-1BB intracellular domains, ICOS signaling increased IL-17A, IL-17F, and IL-22 following antigen recognition. In addition, T cells redirected with an ICOS-based CAR maintained a core molecular signature characteristic of TH17 cells and expressed higher levels of RORC, CD161, IL1R-1, and NCS1. Of note, ICOS signaling also induced the expression of IFN-γ and T-bet, consistent with a TH17/TH1 bipolarization. When transferred into mice with established tumors, TH17 cells that were redirected with ICOS-based CARs mediated efficient antitumor responses and showed enhanced persistence compared with CD28- or 4-1BB-based CAR T cells. Thus, redirection of TH17 cells with a CAR encoding the ICOS intracellular domain is a promising approach to augment the function and persistence of CAR T cells in hematologic malignancies.

Noninvasive bioluminescent imaging of primary patient acute lymphoblastic leukemia: a strategy for preclinical modeling.

The efficient engraftment in immune-deficient mice achieved with both acute lymphoblastic leukemia (ALL) cell lines and primary samples has facilitated identification of the antileukemia activity of a wide variety of agents. Despite widespread usage, however, little is known about the early ALL localization and engraftment kinetics in this model, limiting experimental read-outs primarily to survival and endpoint analysis at high disease burden. In this study, we report that bioluminescent imaging can be reproducibly achieved with primary human ALL samples. This approach provides a noninvasive, longitudinal measure of leukemia burden and localization that enhances the sensitivity of treatment response detection and provides greater insight into the mechanism of action of antileukemia agents. In addition, this study reveals significant cell line- and species-related differences in leukemia migration, especially early in expansion, which may confound observations between various leukemia models. Overall, this study demonstrates that the use of bioluminescent primary ALL allows the detection and quantitation of treatment effects at earlier, previously unquantifiable disease burdens and thus provides the means to standardize and expedite the evaluation of anti-ALL activity in preclinical xenograft studies.

Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains.

Mesothelin is a cell-surface molecule over-expressed on a large fraction of carcinomas, and thus is an attractive target of immunotherapy. A molecularly targeted therapy for these cancers was created by engineering T cells to express a chimeric receptor with high affinity for human mesothelin. Lentiviral vectors were used to express a single-chain variable fragment that binds mesothelin and that is fused to signaling domains derived from T-cell receptor zeta, CD28, and CD137 (4-1BB). When stimulated by mesothelin, lentivirally transduced T cells were induced to proliferate, express the antiapoptotic gene Bcl-X(L), and secrete multiple cytokines, all features characteristic of central memory T cells. When transferred intratumorally or intravenously into NOD/scid/IL2rgamma(-/-) mice engrafted with large pre-established tumors, the engineered T cells reduced the tumor burden, and in some cases resulted in complete eradication of the tumors at low effector-to-target ratios. Incorporation of the CD137 signaling domain specifically reprogrammed cells for multifunctional cytokine secretion and enhanced persistence of T cells. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of poorly immunogenic tumors. Genetically redirected T cells have promise of targeting T lymphocytes to tumor antigens, confer resistance to the tumor microenvironment, and providing immunosurveillance.

Time-dependent cytotoxic drugs selectively cooperate with IL-18 for cancer chemo-immunotherapy.

BACKGROUND: Time-dependent chemotherapeutic agents can selectively target tumor cells in susceptible phases of the cell cycle however a fraction of tumor cells in non-vulnerable cell cycle phases remain drug-resistant. Immunotherapy represents a promising approach to overcome the limitation of phase-specific drugs and improve their clinical efficacy. Here, we investigated the potential use of anticancer chemotherapeutic drugs in combination with IL-18, a cytokine with strong immunostimulatory properties. METHODS: Four chemotherapeutic drugs commonly used in ovarian cancer were first tested for the ability to increase the immunogenicity and killing of the murine ovarian cancer cell line ID8 in vitro. Chemotherapeutric agents with measured time-dependent immune-enhancing effects were then tested for antitumor effectiveness in vivo in combination with IL-18 immunotherapy using the ID8-Vegf ovarian cancer model. RESULTS: Paclitaxel or topotecan exposure alone mediated incomplete, time-dependent killing against the murine ovarian cancer cell line ID8 in vitro, whereas carboplatin or gemcitabine mediated comprehensive, dose-dependent killing. In the plateau phase of the time-dependent killing by topotecan or paclitaxel, drug-resistant ID8 cells were more immunogenic with elevated expression of MHC-I and Fas, and increased sensitivity to CTL and Fas agonistic antibody in vitro. Moreover, the antitumor effectiveness of time-dependent agents in vivo was significantly improved with the addition of IL-18 through a T cell-dependent mechanism, while the effectiveness of drugs without significant phase specificity were not. CONCLUSIONS: Tumor immunotherapy with IL-18 can significantly augment the killing fraction of phase-specific chemotherapeutic drugs and provide survival benefit. The safety profile of IL-18 and its positive interactions with select anticancer chemotherapeutic agents strongly supports the clinical investigation of this combinatorial approach.

Dichotomous impact of affinity on the function of T cell engaging bispecific antibodies.

BACKGROUND: Bispecific T cell engagers represent the majority of bispecific antibodies (BsAbs) entering the clinic to treat metastatic cancer. The ability to apply these agents safely and efficaciously in the clinic, particularly for solid tumors, has been challenging. Many preclinical studies have evaluated parameters related to the activity of T cell engaging BsAbs, but many questions remain. MAIN BODY: This study investigates the impact of affinity of T cell engaging BsAbs with regards to potency, efficacy, and induction of immunomodulatory receptors/ligands using HER-2/CD3 BsAbs as a model system. We show that an IgG BsAb can be as efficacious as a smaller BsAb format both in vitro and in vivo. We uncover a dichotomous relationship between tumor-associated antigen (TAA) affinity and CD3 affinity requirements for cells that express high versus low levels of TAA. HER-2 affinity directly correlated with the CD3 engager lysis potency of HER-2/CD3 BsAbs when HER-2 receptor numbers are high (~200 K/cell), while the CD3 affinity did not impact potency until its binding affinity was extremely low (<600 nM). When HER-2 receptor numbers were lower (~20 K/cell), both HER-2 and CD3 affinity impacted potency. The high affinity anti-HER-2/low CD3 affinity BsAb also demonstrated lower cytokine induction levels in vivo and a dosing paradigm atypical of extremely high potency T cell engaging BsAbs reaching peak efficacy at doses >3 mg/kg. This data confirms that low CD3 affinity provides an opportunity for improved safety and dosing for T cell engaging BsAbs. T cell redirection also led to upregulation of Programmed cell death 1 (PD-1) and 4-1BB, but not CTLA-4 on T cells, and to Programmed death-ligand 1 (PD-L1) upregulation on HER-2HI SKOV3 tumor cells, but not on HER-2LO OVCAR3 tumor cells. Using this information, we combined anti-PD-1 or anti-4-1BB monoclonal antibodies with the HER-2/CD3 BsAb in vivo and demonstrated significantly increased efficacy against HER-2HI SKOV3 tumors via both combinations. CONCLUSIONS: Overall, these studies provide an informational dive into the optimization process of CD3 engaging BsAbs for solid tumors indicating that a reduced affinity for CD3 may enable a better therapeutic index with a greater selectivity for the target tumor and a reduced cytokine release syndrome. These studies also provide an additional argument for combining T cell checkpoint inhibition and co-stimulation to achieve optimal efficacy.

Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo.

Persistence of T cells engineered with chimeric antigen receptors (CARs) has been a major barrier to use of these cells for molecularly targeted adoptive immunotherapy. To address this issue, we created a series of CARs that contain the T cell receptor-zeta (TCR-zeta) signal transduction domain with the CD28 and/or CD137 (4-1BB) intracellular domains in tandem. After short-term expansion, primary human T cells were subjected to lentiviral gene transfer, resulting in large numbers of cells with >85% CAR expression. In an immunodeficient mouse xenograft model of primary human pre-B-cell acute lymphoblastic leukemia, human T cells expressing anti-CD19 CARs containing CD137 exhibited the greatest antileukemic efficacy and prolonged (>6 months) survival in vivo, and were significantly more effective than cells expressing CARs containing TCR-zeta alone or CD28-zeta signaling receptors. We uncovered a previously unrecognized, antigen-independent effect of CARs expressing the CD137 cytoplasmic domain that likely contributes to the enhanced antileukemic efficacy and survival in tumor bearing mice. Furthermore, our studies revealed significant discrepancies between in vitro and in vivo surrogate measures of CAR efficacy. Together these results suggest that incorporation of the CD137 signaling domain in CARs should improve the persistence of CARs in the hematologic malignancies and hence maximize their antitumor activity.

Characterization of 7A5: A Human CD137 (4-1BB) Receptor Binding Monoclonal Antibody with Differential Agonist Properties That Promotes Antitumor Immunity.

The CD137 receptor plays a key role in mediating immune response by promoting T cell proliferation, survival, and memory. Effective agonism of CD137 has the potential to reinvigorate potent antitumor immunity either alone or in combination with other immune-checkpoint therapies. In this study, we describe the discovery and characterization of a unique CD137 agonist, 7A5, a fully human IgG1 Fc effector-null monoclonal antibody. The biological properties of 7A5 were investigated through in vitro and in vivo studies. 7A5 binds CD137, and the binding epitope overlaps with the CD137L binding site based on structure. 7A5 engages CD137 receptor and activates NF-κB cell signaling independent of cross-linking or Fc effector function. In addition, T cell activation measured by cytokine IFNγ production is induced by 7A5 in peripheral blood mononuclear cell costimulation assay. Human tumor xenograft mouse models reconstituted with human immune cells were used to determine antitumor activity in vivo. Monotherapy with 7A5 inhibits tumor growth, and this activity is enhanced in combination with a PD-L1 antagonist antibody. Furthermore, the intratumoral immune gene expression signature in response to 7A5 is highly suggestive of enhanced T cell infiltration and activation. Taken together, these results demonstrate 7A5 is a differentiated CD137 agonist antibody with biological properties that warrant its further development as a cancer immunotherapy. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/4/988/F1.large.jpg.

Bispecific Targeting of PD-1 and PD-L1 Enhances T-cell Activation and Antitumor Immunity.

The programmed cell death protein 1 receptor (PD-1) and programmed death ligand 1 (PD-L1) coinhibitory pathway suppresses T-cell-mediated immunity. We hypothesized that cotargeting of PD-1 and PD-L1 with a bispecific antibody molecule could provide an alternative therapeutic approach, with enhanced antitumor activity, compared with monospecific PD-1 and PD-L1 antibodies. Here, we describe LY3434172, a bispecific IgG1 mAb with ablated Fc immune effector function that targets both human PD-1 and PD-L1. LY3434172 fully inhibited the major inhibitory receptor-ligand interactions in the PD-1 pathway. LY3434172 enhanced functional activation of T cells in vitro compared with the parent anti-PD-1 and anti-PD-L1 antibody combination or respective monotherapies. In mouse tumor models reconstituted with human immune cells, LY3434172 therapy induced dramatic and potent antitumor activity compared with each parent antibody or their combination. Collectively, these results demonstrated the enhanced immunomodulatory (immune blockade) properties of LY3434172, which improved antitumor immune response in preclinical studies, thus supporting its evaluation as a novel bispecific cancer immunotherapy.

Increased immunogenicity of surviving tumor cells enables cooperation between liposomal doxorubicin and IL-18.

BACKGROUND: Liposomal doxorubicin (Doxil) is a cytotoxic chemotherapy drug with a favorable hematologic toxicity profile. Its active drug, doxorubicin, has interesting immunomodulatory properties. Here, the effects of Doxil on surviving tumor cell immunophenotype were investigated. METHODS: Using ID8 murine ovarian cancer cells, the immunomodulatory effects of Doxil were studied by measuring its impact on ovarian cancer cell expression of MHC class-I and Fas, and susceptibility to immune attack in vitro. To evaluate the ability of Doxil to cooperate with cancer immunotherapy, the interaction between Doxil and Interleukin 18 (IL-18), a pleiotropic immunostimulatory cytokine, was investigated in vivo in mice bearing ID8-Vegf tumors. RESULTS: While Doxil killed ID8 tumor cells in a dose-dependent manner, tumor cells escaping Doxil-induced apoptosis upregulated surface expression of MHC-I and Fas, and were sensitized to CTL killing and Fas-mediated death in vitro. We therefore tested the hypothesis that the combination of immunotherapy with Doxil provides positive interactions. Combination IL-18 and Doxil significantly suppressed tumor growth compared with either monotherapy in vivo and uniquely resulted in complete tumor regression and long term antitumor protection in a significant proportion of mice. CONCLUSION: These data demonstrate that Doxil favorably changes the immunophenotype of a large fraction of the tumor that escapes direct killing thus creating an opportunity to expand tumor killing by immunotherapy, which can be capitalized through addition of IL-18 in vivo.

The Folate Pathway Inhibitor Pemetrexed Pleiotropically Enhances Effects of Cancer Immunotherapy.

PURPOSE: Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non-small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown. EXPERIMENTAL DESIGN: Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis. RESULTS: Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell-intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1. CONCLUSIONS: Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell-intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade.See related commentary by Buque et al., p. 6890.

Genetic engineering of T cells for adoptive immunotherapy.

To be effective for the treatment of cancer and infectious diseases, T cell adoptive immunotherapy requires large numbers of cells with abundant proliferative reserves and intact effector functions. We are achieving these goals using a gene therapy strategy wherein the desired characteristics are introduced into a starting cell population, primarily by high efficiency lentiviral vector-mediated transduction. Modified cells are then expanded using ex vivo expansion protocols designed to minimally alter the desired cellular phenotype. In this article, we focus on strategies to (1) dissect the signals controlling T cell proliferation; (2) render CD4 T cells resistant to HIV-1 infection; and (3) redirect CD8 T cell antigen specificity.

Discovery and preclinical characterization of the antagonist anti-PD-L1 monoclonal antibody LY3300054.

BACKGROUND: Modulation of the PD-1/PD-L1 axis through antagonist antibodies that block either receptor or ligand has been shown to reinvigorate the function of tumor-specific T cells and unleash potent anti-tumor immunity, leading to durable objective responses in a subset of patients across multiple tumor types. RESULTS: Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ monoclonal antibody that binds to human PD-L1 with high affinity and inhibits interactions of PD-L1 with its two cognate receptors PD-1 and CD80. The functional activity of LY3300054 on primary human T cells is evaluated using a series of in vitro T cell functional assays and in vivo models using human-immune reconstituted mice. LY3300054 is shown to induce primary T cell activation in vitro, increase T cell activation in combination with anti-CTLA4 antibody, and to potently enhance anti-tumor alloreactivity in several xenograft mouse tumor models with reconstituted human immune cells. High-content molecular analysis of tumor and peripheral tissues from animals treated with LY3300054 reveals distinct adaptive immune activation signatures, and also previously not described modulation of innate immune pathways. CONCLUSIONS: LY3300054 is currently being evaluated in phase I clinical trials for oncology indications.

Correction to: Discovery and preclinical characterization of the antagonist anti-PD-L1 monoclonal antibody LY3300054.

Unfortunately, after publication of this article [1], it was noticed that corrections to the legends of Figs. 1 and 2 were not correctly incorporated. The correct legends can be seen below.

The CDK4/6 Inhibitor Abemaciclib Induces a T Cell Inflamed Tumor Microenvironment and Enhances the Efficacy of PD-L1 Checkpoint Blockade.

Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic tumor models and in vitro assays to investigate the impact of abemaciclib on T cells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased T cell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and enhanced cell cycle control. In vitro, treatment with abemaciclib resulted in increased activation of human T cells and upregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate T cell anti-tumor immunity.

Human CD26high T cells elicit tumor immunity against multiple malignancies via enhanced migration and persistence.

CD8+ T lymphocytes mediate potent immune responses against tumor, but the role of human CD4+ T cell subsets in cancer immunotherapy remains ill-defined. Herein, we exhibit that CD26 identifies three T helper subsets with distinct immunological properties in both healthy individuals and cancer patients. Although CD26neg T cells possess a regulatory phenotype, CD26int T cells are mainly naive and CD26high T cells appear terminally differentiated and exhausted. Paradoxically, CD26high T cells persist in and regress multiple solid tumors following adoptive cell transfer. Further analysis revealed that CD26high cells have a rich chemokine receptor profile (including CCR2 and CCR5), profound cytotoxicity (Granzyme B and CD107A), resistance to apoptosis (c-KIT and Bcl2), and enhanced stemness (ß-catenin and Lef1). These properties license CD26high T cells with a natural capacity to traffic to, regress and survive in solid tumors. Collectively, these findings identify CD4+ T cell subsets with properties critical for improving cancer immunotherapy.

Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials.

Alternative splicing as a novel of means of regulating the expression of therapeutic genes.

In order to determine the potential of alternative splicing as a means of targeting the expression of therapeutic genes to tumor cells in vivo, a series of episomal plasmid-based "splice-activated gene expression" (pSAGE) vectors was generated, which contain minigene cassettes composed of various combinations of the three alternatively spliced exons present in the differentially expressed adhesion protein CD44R1 (v8, v9, and v10) with or without their corresponding intronic sequences, positioned in-frame between the CD44 leader sequence and a "leaderless" human liver/bone/kidney alkaline phosphatase (ALP) cDNA. Because both the v8-v9 and v9-v10 introns contain multiple in-frame stop codons, the expression and enzymatic activity of ALP are dependent upon the accurate removal of intronic sequences from the pre-mRNA transcripts encoded by these constructs. The various pSAGE constructs were introduced into CD44H-positive (T24) and CD44R1-positive (PC3) target cells by electroporation and transfectants selected in hygromycin B. ALP expression was determined by staining with the ALP substrate, BCIP/INT, and the transfected cells tested for their sensitivity to the inactive prodrug, etoposide phosphate. ALP-mediated dephosphorylation of etoposide phosphate generates the potent topoisomerase II inhibitor etoposide. The data obtained indicate that whereas the v8-v9 intron is spliced in both CD44H- and CD44R1-positive cells, the v9-v10 intron is efficiently and accurately removed only in CD44R1-positive cells. Furthermore, only CD44R1-positive cells were sensitized to etoposide phosphate when transfected with the v9-v10.ALP construct. These data emphasize the potential usefulness of alternative splicing as a novel means of targeting gene expression to tumor cells in vivo.

Exploiting the differential production of angiogenic factors within the tumor microenvironment in the design of a novel vascular-targeted gene therapy-based approach to the treatment of cancer.

PURPOSE: The aim of this study is to explore a novel strategy through which the differential production of pro-angiogenic cytokines within the tumor microenvironment can be exploited as a means of selectively killing the vascular endothelial cells upon which the survival and growth of a tumor depend. METHODS AND MATERIALS: Adenoviral vectors encoding a chimeric cell surface receptor composed of the extracellular domain of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR fused in frame to the membrane spanning and cytoplasmic domain of Fas were constructed and used to transduce primary human endothelial cells in vitro. The apoptotic response of these cells induced upon ligation of the chimeric receptor with VEGF was determined by measuring caspase-3 activation, AnnexinV-FITC binding, and the release of glucose-6-phosphate dehydrogenase. RESULTS: The chimeric Flk-1/Fas protein is stable and expressed at high levels on the surface of adenovirally transduced cells. Upon the addition of exogenous VEGF, these cells undergo rapid apoptosis. CONCLUSIONS: Receptor/Fas chimeras that recognize and bind pro-angiogenic cytokines represent a novel means by which the signal transduction events normally triggered in vascular endothelial cells upon the binding of angiogenic cytokines may be redirected toward the induction of apoptotic cell death. It is proposed that these constructs will prove of value in the further development of safe and effective vascular-targeted gene therapy-based approaches to the treatment of cancer.

Concise review: guidance in developing commercializable autologous/patient-specific cell therapy manufacturing.

Cell therapy is poised to play an enormous role in regenerative medicine. However, little guidance is being made available to academic and industrial entities in the start-up phase. In this technical review, members of the International Society for Cell Therapy provide guidance in developing commercializable autologous and patient-specific manufacturing strategies from the perspective of process development. Special emphasis is placed on providing guidance to small academic or biotech researchers as to what simple questions can be addressed or answered at the bench in order to make their cell therapy products more feasible for commercial-scale production. We discuss the processes that are required for scale-out at the manufacturing level, and how many questions can be addressed at the bench level. The goal of this review is to provide guidance in the form of topics that can be addressed early in the process of development to better the chances of the product being successful for future commercialization.