CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to United States Provisional Patent Application serial number 62/408,547 filed on October 14, 2016, the entirety of which is hereby incorporated by reference.

BACKGROUND

The testing of therapeutic combinations between pharmaceuticals that act on checkpoint inhibitor pathways and intracellular targets currently requires complicated and time consuming mouse experiments. Mouse experiments are further limited as they cannot precisely replicate human physiology. Improved methods of testing therapeutic combination treatments in human- like systems are therefore desirable. SUMMARY OF THE INVENTION

In some aspects, the invention relates to an inducible T-cell system. An inducible T- cell system may comprise a T-cell engineered to inducibly express two or more checkpoint receptors. The T-cell may express the T-cell receptor (TCR) and a costimulatory receptor. The inducible T-cell system may comprise a ligand-presenting cell (e.g. , a B-cell) engineered to constitutively express one or more checkpoint receptor ligands. The ligand-presenting cell may express a costimulatory ligand. The inducible T-cell system may comprise an anti-CD3 antibody, e.g. , to stimulate TCR signaling.

The T-cell may be a Jurkat cell. The ligand-presenting cell may be a B-cell or an antigen-presenting cell. The ligand-presenting cell may be a Raji cell. The T-cell and/or B- cell may be derived from a human cell line.

The ligand-presenting cell may be derived from a human or non-human cell, and the one or more checkpoint receptor ligands may be human checkpoint receptor ligands. The ligand-presenting cell may be derived from a human or non-human cell, and the

costimulatory ligand may be a human costimulatory ligand.

The two or more checkpoint receptors may comprise PD-1 and CTLA-4. The one or more checkpoint receptor ligands may comprise PD-L1 and/or PD-L2. The two or more checkpoint receptors may comprise PD-1, CTLA-4, PD-1H, LAG- 3, BTLA, KIR2D, TIM-3, ADORA2A, CD 160, 2B4, TIGIT, and/or interleukin-10 receptor. The one or more checkpoint receptor ligands may comprise PD-L1, PD-L2, B7.1, B7.2, B7-H4, B7-H2, B7- H7, MHC class II, HVEM, CD155, galectin-9, and/or TIM-3 ligand.

The costimulatory receptor may be CD28. The costimulatory ligand expressed by the ligand-presenting cell may be B7.1 or B7.2.

The expression of the two or more checkpoint receptors may be controlled by a promoter inducible by doxycycline (dox).

The anti-CD3 antibody may be associated with a bead.

The inducible T-cell system may further comprise reagents to determine the expression of interleukin-2 or interferon γ by the T-cell. For example, the reagents may be selected from a primary antibody, an anti-interleukin-2 antibody, an anti-interferon γ antibody, a secondary antibody, an anti-IgG antibody, an antibody-fluorophore conjugate, an antibody-biotin conjugate, an antibody-enzyme conjugate, a colorimetric substrate for horseradish peroxidase, and a colorimetric substrate for alkaline phosphatase.

The T-cell may further comprise a gene knockout or knockdown. The gene knockout or knockdown may be a lymphoid lineage gene knockout or knockdown. A lymphoid lineage gene may be a T-cell gene. A knockout may be a CRISPR/Cas9-mediated knockout.

The inducible T-cell system may further comprise one or more pharmaceutical agents. The one or more pharmaceutical agents may comprise a small molecule compound and/or a large molecule (e.g. , an antibody).

In some aspects, the invention relates to a method of identifying small molecule T-cell activators. The method may comprise providing an inducible T-cell system comprising a T- cell engineered to inducibly express two or more checkpoint receptors. The T-cell may express the T-cell receptor (TCR) and a costimulatory receptor. The method may comprise providing a B-cell engineered to constitutively express one or more checkpoint receptor ligands. The B-cell may express a costimulatory ligand to provide a costimulatory signal. The method may comprise providing an anti-CD3 antibody to stimulate TCR signaling. The TCR signaling may be measurable by IL-2 expression of the T-cell (or interferon γ expression).

The method may comprise inducing the expression of the two or more checkpoint receptors in the T-cell. The method may comprise contacting the T-cell of the inducible T- cell system with one or more candidate compounds. The method may comprise determining the level of IL-2 expression (or interferon γ expression) of the T-cell as compared to a control level of IL-2 expression (or interferon γ expression) of a non-induced control T-cell, thereby identifying if the one or more candidate compounds are T-cell activators. In some aspects, the invention relates to a method of assessing interactions between T-cell activators and checkpoint inhibitory antibodies. The method may comprise providing an inducible T-cell system comprising a T-cell engineered to inducibly express two or more checkpoint receptors. The T-cell may express the T-cell receptor (TCR) and a costimulatory receptor. The method may comprise providing a B-cell engineered to constitutively express one or more checkpoint receptor ligands. The B-cell may express a costimulatory ligand to provide a costimulatory signal. The method may comprise providing an anti-CD3 antibody to stimulate TCR signaling. The TCR signaling may be measurable by IL-2 expression of the T-cell (or interferon γ expression).

The method may comprise inducing the expression of the two or more checkpoint receptors in the T-cell. The method may further comprise contacting the T-cell of the inducible T-cell system with one or more candidate compounds. The method may further comprise contacting the T-cell of the inducible T-cell system with one, two, or more checkpoint inhibitory antibodies. The method may further comprise determining the level of IL-2 expression (or interferon γ expression) of the T-cell as compared to the level of IL-2 expression (or interferon γ expression) of an otherwise identical inducible T-cell system but contacted with only one or more candidate compounds, thereby assessing the interaction between one or more candidate compounds and the one, two, or more checkpoint inhibitory antibodies.

The one, two, or more checkpoint inhibitory antibodies may comprise an FDA approved checkpoint inhibitory drug. The FDA approved checkpoint inhibitory drug may be a monoclonal antibody. The one or more candidate compounds may comprise a small molecule compound.

In some aspects, the invention relates to a method of validating a T-cell target gene. The method may comprise providing an inducible T-cell system comprising a T-cell engineered to inducibly express two or more checkpoint receptors. The T-cell may further comprise a candidate T-cell gene knockout. The T-cell may express the T-cell receptor (TCR) and a costimulatory receptor. The method may comprise providing a B-cell engineered to constitutively express one or more checkpoint receptor ligands. The B-cell may express a costimulatory ligand to provide a costimulatory signal. The method may comprise providing an anti-CD3 antibody to stimulate TCR signaling. The TCR signaling may be measurable by IL-2 expression of the T-cell (or interferon γ expression).

The method may comprise inducing the expression of the two or more checkpoint receptors in the T-cell. The method may comprise determining the level of IL-2 expression (or interferon γ expression) of the T-cell as compared to a control level of IL-2 expression (or interferon γ expression) of a non-induced control T-cell, thereby determining if the candidate T-cell gene is a T-cell checkpoint-independent target gene.

In some aspects, the invention relates to a method of interrogating ON or OFF target activity of a small molecule compound. The method may comprise providing an inducible T- cell system comprising a T-cell engineered to inducibly express two or more checkpoint receptors. The T-cell may further comprise a candidate T-cell gene knockout. The T-cell may express the T-cell receptor (TCR) and a costimulatory receptor. The method may comprise providing a B-cell engineered to constitutively express one or more checkpoint receptor ligands. The B-cell may express a costimulatory ligand to provide a costimulatory signal. The method may comprise providing an anti-CD3 antibody to stimulate TCR signaling. The TCR signaling may be measurable by IL-2 expression of the T-cell (or interferon γ expression).

The method may comprise inducing the expression of the two or more checkpoint receptors in the T-cell. The method may comprise contacting the T-cell of the inducible T- cell system with a small molecule compound. The method may comprise determining the level of IL-2 expression (or interferon γ expression) of the T-cell with the T-cell target gene knockout as compared to a control level of IL-2 expression (or interferon γ expression) of a control wild-type T-cell under otherwise identical conditions, thereby interrogating ON vs. OFF target activity of the small molecule compound.

In any of the foregoing methods, the T-cell may be a Jurkat cell line, the B-cell may be a Raji cell line, or the T-cell and/or the B-cell may be derived from a human cell line.

In any of the foregoing methods, the two or more checkpoint receptors may comprise PD-1 and CTLA-4. In any of the foregoing methods, the one or more checkpoint receptor ligands may comprise PD-L1 and/or PD-L2.

In any of the foregoing methods, the expression of the two or more checkpoint receptors may be controlled by a promoter inducible by doxycycline (dox). The inducing step may comprise introducing the dox to the T-cell.

In any of the foregoing methods, the costimulatory receptor may be CD28.

In any of the foregoing methods, the costimulatory ligand expressed by the B-cell may be B7.1 or B7.2.

In any of the foregoing methods, the anti-CD3 antibody may be associated with a bead. In some aspects, the invention relates to a plurality of inducible T-cell systems. Each system may comprise a T-cell (e.g. , a Jurkat cell) engineered to inducibly express two or more checkpoint receptors (e.g. , PD-1 and CTLA-4). Each system may comprise a ligand- presenting cell (e.g. , a B cell such as a Raji cell) that expresses at least one checkpoint receptor ligand (e.g. , PD-L1 , PD-L2, B7.1, and/or B7.2). Each system may comprise a costimulatory ligand (e.g. , an anti-CD3 antibody associated with a solid support).

At least one system of a plurality of systems may comprise a transcription inducer (e.g. , doxycycline) that induces the transcription of the two or more checkpoint inhibitors. At least one system of a plurality of systems may lack the transcription inducer.

At least one system of a plurality of systems may comprise a pharmaceutical agent

(e.g. , anti-PD- 1 antibody, anti-CTLA-4 antibody, kinase inhibitor). At least one system of a plurality of systems may lack the pharmaceutical agent.

The T-cell of at least one system of the plurality may comprise a gene knockout or gene knockdown (e.g. , a random knockout or a knockout/knockdown of a T-cell signaling protein). The T-cell of at least one system of the plurality may lack the gene knockout or gene knockdown.

The plurality of systems may comprise a system that comprises a transcription inducer, comprises a pharmaceutical agent, and comprises a T-cell comprising a gene knockout or gene knockdown. The plurality of systems may comprise a system that lacks a transcription inducer, lacks a pharmaceutical agent, and lacks a T-cell comprising a gene knockout or gene knockdown. The plurality of systems may comprise a system that comprises a transcription inducer, lacks a pharmaceutical agent, and lacks a T-cell comprising a gene knockout or gene knockdown. The plurality of systems may comprise a system that lacks a transcription inducer, comprises a pharmaceutical agent, and lacks a T- cell comprising a gene knockout or gene knockdown. The plurality of systems may comprise a system that lacks a transcription inducer, lacks a pharmaceutical agent, and comprises a T- cell comprising a gene knockout or gene knockdown. The plurality of systems may comprise a system that comprises a transcription inducer, comprises a pharmaceutical agent, and lacks a T-cell comprising a gene knockout or gene knockdown. The plurality of systems may comprise a system that comprises a transcription inducer, lacks a pharmaceutical agent, and comprises a T-cell comprising a gene knockout or gene knockdown. The plurality of systems may comprise a system that lacks a transcription inducer, comprises a pharmaceutical agent, and comprises a T-cell comprising a gene knockout or gene knockdown. The plurality of systems may comprise a system that comprises one small molecule compound and at least two large molecules. The plurality of systems may comprise a system that lacks a small molecule compound and lacks a large molecule. The plurality of systems may comprise a system that comprises one small molecule compound and lacks a large molecule. The plurality of systems may comprise a system that comprises both one small molecule compound and one large molecule. The plurality of systems may comprise a system that comprises at least two small molecule compounds and lacks a large molecule. The plurality of systems may comprise a system that comprises at least two small molecule compounds and one large molecule. The plurality of systems may comprise a system that lacks a small molecule compound and comprises at least two large molecules.

The at least one large molecule may comprise an antibody such as an anti-CTLA-4 antibody and/or an anti-PD- 1 antibody. The at least one large molecule may comprise avelumab, atezolizumab, durvalumab, ipilimumab, lambrolizumab, nivolumab,

pembrolizumab, pidilizumab, tremelimumab, and/or ticilimumab. The at least one large molecule may comprise

ipilimumab and/or nivolumab.

In some aspects, the invention relates to a multi-well plate comprising a plurality of inducible T-cell systems as described herein. The multi-well plate may be a 6 well, 12 well, 24 well, 96 well, 384 well, 1536 well, or 3,456 well plate.

In some aspects, the invention relates to a method of preparing the multi-well plate, comprising adding a T-cell to each well of a plurality of wells of the multi-well plate. The T- cell may be engineered to inducibly express the two or more checkpoint receptors. The method may comprise adding a transcription inducer to a first group of wells of the plurality of wells. The method may comprise adding a costimulatory ligand to the plurality of wells. The method may comprise adding a ligand-presenting cell to the plurality of wells, wherein the ligand-presenting cell expresses a checkpoint receptor ligand.

Adding a T-cell to each well of a plurality of wells may comprise adding a knockout or knockdown T-cell to a first group of wells of a plurality of wells and adding a wild type T- cell to a second group of wells of the plurality of wells. The knockout or knockdown T-cell may be engineered to inducibly express two or more checkpoint receptors, and the knockout or knockdown T-cell may comprise a gene knockout or knockdown. The wild type T-cell may be engineered to inducibly express the two or more checkpoint receptors, and the wild type T-cell may lack the gene knockout or knockdown. The plurality of wells may consist of the first group of wells and the second group of wells. In some embodiments, no well of the plurality of wells is included in both the first group of wells and the second group of wells. The group of wells to which the transcription inducer is added may include both wells of the first group of wells and wells of the second group of wells.

The costimulatory ligand may be added to the plurality of wells at least 24 hours after adding the transcription inducer to a group of wells. The method may further comprise washing the cells of each well of the plurality prior to adding the costimulatory ligand, e.g., to reduce the concentration of the transcription inducer in each well.

The ligand-presenting cell may be added to the plurality of wells at least 5 minutes after adding the costimulatory ligand to the plurality of wells.

The method may further comprise adding a first antibody to each well of a fourth group of wells of the plurality of wells. The method may further comprise adding a second antibody to each well of a fifth group of wells of the plurality of wells, wherein the fifth group of wells includes at least one well of the fourth group of wells. The first antibody and second antibody may be selected from antibodies that bind checkpoint receptors and antibodies that bind checkpoint receptor ligands such as anti-CTLA-4 antibodies and anti- PD-1 antibodies.

The method may further comprise adding a first small molecule compound to each well of a sixth group of wells of the plurality of wells. The first small molecule compound may be, for example, a kinase inhibitor. The method may further comprise adding a second small molecule compound to each well of a seventh group of wells of the plurality of wells, wherein the seventh group of wells includes at least one well of the sixth group of wells.

In some aspects, the invention relates to a method of discovering or validating a drug target. The method may comprise providing a plurality of inducible T-cell systems as described herein. The method may further comprise measuring the concentration of a biomarker associated with T-cell activation in at least two systems of the plurality, wherein the at least two systems include a system comprising a T-cell having a knockout or knockdown of a gene encoding the drug target.

In some aspects, the invention relates to a method of determining whether a small molecule compound affects T-cell activation. The method may comprise providing a plurality of inducible T-cell systems as described herein. The method may further comprise measuring the concentration of a biomarker associated with T-cell activation in at least two systems of the plurality, wherein the at least two systems include both a system comprising the small molecule compound and a system lacking the small molecule compound.

In some aspects, the invention relates to a method of assessing an interaction between a small molecule compound and an antibody that binds a checkpoint receptor. The method may comprise providing a plurality of inducible T-cell systems as described herein. The method may further comprise measuring the concentration of a biomarker associated with T- cell activation in at least two systems of the plurality, wherein the at least two systems include a system comprising both the small molecule compound and the antibody.

In some aspects, the invention relates to a method of assessing an interaction between a first small molecule compound and a second small molecule compound. The method may comprise providing a plurality of inducible T-cell systems as described herein. The method may further comprise measuring the concentration of a biomarker associated with T-cell activation in at least two systems of the plurality, wherein the at least two systems include a system comprising both the first small molecule compound and the second small molecule compound.

In some aspects, the invention relates to a method of assessing whether a protein (e.g. , a signaling protein such as a kinase or phosphatase) affects T-cell activation through a signaling pathway mediated by a checkpoint receptor. The method may comprise providing a plurality of inducible T-cell systems as described herein. The method may further comprise measuring the concentration of a biomarker associated with T-cell activation in at least two systems of the plurality, wherein the at least two systems include a system comprising both an antibody that specifically binds the checkpoint receptor and a T-cell comprising a knockout or knockdown of a gene encoding the protein.

In some aspects, the invention relates to a method of assessing whether a

pharmaceutical agent displays an ON target or OFF target affect. The method may comprise providing a plurality of inducible T-cell systems as described herein. The method may further comprise measuring the concentration of a biomarker associated with T-cell activation in at least two systems of the plurality, wherein the at least two systems include a system comprising both the pharmaceutical agent and a T-cell comprising a knockout or knockdown of a gene encoding the target. The pharmaceutical agent may be a small molecule compound or a large molecule (e.g. , antibody).

Various embodiments of the foregoing methods may further comprise comparing the relative concentration of the biomarker in at least one system of the plurality of systems to the relative concentration of the biomarker in at least one other system of the plurality of systems.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are for illustration purposes only and are not intended to limit the scope of the claims or invention.

Figure 1 depicts an exemplary embodiment of an inducible T-cell system. The drawing depicts a T-cell that inducibly expresses checkpoint receptors PD-1 and CTLA-4. The T-cell also expresses CD28 and the T-cell receptor (TCR). The drawing depicts a B-cell that expresses either PD-Ll, PD-L2, or both and either B7.1, B7.2, or both. The drawing also depicts beads coated with anti-CD3 antibodies.

Figures 2A-2B depict exemplary embodiments of an inducible T-cell system, similar to the embodiment depicted in Figure 1. Figure 2A shows two states of the inducible T-cell system. In a first state (2A, left), the transcription inducer doxycycline (dox) is not present, and thus, the T-cell does not express the PD-1 or CTLA-4 constructs. The T-cell may receive costimulatory signals from B7.1 or B7.2, which interact with CD28, and anti-CD3 antibodies, which interact with the T-cell receptor. The costimulatory signals result in interleukin-2 (IL2) production by the T-cell. In a second state (2 A, right), the transcription inducer doxycycline (dox) is added, and thus, the T-cell may express PD-1 and CTLA-4. The T-cell may receive costimulatory signals from B7.1 or B7.2 and the anti-CD3 antibodies. The T- cell may also receive inhibitory signals from PD-Ll or PD-L2, which interact with PD-1, and B7.1 or B7.2, which interact with CTLA-4. The inhibitory signals result in decreased interleukin-2 (IL2) production by the T-cell relative to the first state (2B).

Figures 3A-3B depict exemplary embodiments of an inducible T-cell system that demonstrate how the system may be utilized to analyze combinations of therapeutic antibodies targeting immune checkpoint receptors. Experiments "1" and "2" of Figures 3A and 3B are identical to Figures 2A and 2B. Experiment "3" depicts the addition of the anti- PD-1 antibody nivolumab (Nivo; N) to the system in addition to doxycycline. Experiment "4" depicts the addition of the anti-CTLA-4 antibody ipilimumab (Ipi; I) to the system in addition to doxycycline. Experiment "5" depicts the addition of nivolumab, ipilimumab, and doxycycline to the system. Nivolumab and ipilimumab both increased interleukin-2 (IL2) production relative to an IgG control, and the combination of nivolumab and ipilimumab displayed an additive effect (3B). Figures 4A-4B depict exemplary embodiments of an inducible T-cell system that demonstrate how the system may be utilized to probe molecular pathways and identify drug targets related to checkpoint receptors. The figures depict a T-cell system that is similar to Figures 1 and 2A-2B except that the T-cell of the system comprises a knockout of a gene encoding an intracellular protein. The gene knockout resulted in elevated interleukin-2 (IL2) production in the presence of doxycycline thereby suggesting that the gene product inhibits T-cell activation. The protein product of the knockout gene may therefore be a viable drug target, e.g. , small molecule inhibitors of the protein product may increase T-cell activation.

Figures 5A-5B depict exemplary embodiments of an inducible T-cell system that demonstrate how the system may be utilized to analyze combination therapies that affect immune checkpoint receptors and the molecular pathways that the therapies might act upon. Experiments "1," "2," and "3" are described above (e.g. , in Figures 3A-3B). Experiment "4" is described in Figures 4A-4B. Experiment "5" shows that a knockout may be used in combination with a drug or drug cocktail, for example, to assess whether the drug or drug cocktail acts on a molecular pathway associated with the knockout gene. In this case, nivolumab (Nivo; N) and ipilimumab (Ipi; I) increase the activation of knockout T-cells, which suggests that a drug that inhibits the product of the knockout gene will display an additive effect when used in combination with nivolumab and/or ipilimumab. Experiments "6" and "7" show that the inducible T-cell system may be used to probe the effect of small molecule therapeutics either alone (Experiment "6") or in combination with other therapies (Experiment "7"). Experiment "6" shows that the small molecule compounds "compound 1" and "compound 2" activate T-cells despite checkpoint receptor-mediated T-cell inhibition (5B). Experiment "7" shows that the combination of nivolumab (Nivo; N), ipilimumab (Ipi; I), and either small molecule compound 1 or compound 2 displays a synergistic effect (5B).

Figures 6A-6C depict exemplary embodiments of an inducible T-cell system that demonstrate how the system may be utilized to analyze whether a small molecule compound affects T-cell signaling through a target protein. Figure 6A is identical to experiment "6" of Figure 5A, and it depicts an inducible T-cell system that is contacted with a small molecule compound. Figure 6B is similar to Figure 6A except that the T-cell of figure 6B comprises a gene knockout. Figure 6C depicts experiments with T-cells comprising or lacking a gene knockout that were incubated with Raji cells, anti-CD3 beads, doxycycline, and either small molecule "compound 1" or small molecule "compound 2." Control experiments were performed with either compound 1, compound 2, or vehicle (DMSO) and T-cells that did not include the knockout. The dashed line indicates the level of T-cell activation in DMSO control samples either comprising (KO) or lacking (WT) the gene knockout (set at 100%). Both compound 1 and compound 2 increased T-cell activation relative to the vehicle control in T-cells that did not include the knockout. Neither compound 1 nor compound 2 increased T-cell activation relative to the vehicle control in T-cells that included the knockout. These results suggest that both compound 1 and compound 2, which are designed to target the protein encoded by the knockout gene, display an "ON" target effect.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a system comprising a T-cell engineered to inducibly express two or more checkpoint receptors, a B-cell engineered to constitutively express one or more checkpoint receptor ligands, and an anti-CD3 antibody (e.g. , anti-CD3 coated beads). This system allows for the analysis of pharmaceutical agents that target checkpoint receptors to determine whether the agents display an additive or synergistic effect. The system also allows for the analysis of molecular pathways related to checkpoint inhibition, e.g. , by selectively knocking out T-cell genes. Knockouts may provide insights into whether pharmaceutical agents that target checkpoint receptors target the same or different molecular pathways. Knockouts may also provide insights into whether a pharmaceutical agent displays an ON target or OFF target effect, e.g. , wherein the gene encoding the target is knocked out of a T-cell. Knockouts may also identify proteins that may be suitable drug targets that modulate T-cell activation. Importantly, the inducible T-cell system provides a useful control because baseline readouts (e.g. interleukin-2 expression) may be obtained for the system by simply performing an experiment in the absence of conditions that allow for expression of the checkpoint receptors. Additionally, the expression of two or more checkpoint receptors allows for the analysis of additive or synergistic effects among combinations of pharmaceutical agents that target different checkpoint receptor pathways.

I. T-CELLS THAT EXPRESS CHECKPOINT RECEPTORS

Various aspects of the invention relate to a T-cell. The T-cell may be a mammalian T-cell, such as a human T-cell. The T-cell may be immortalized. For example, the T-cell may be immortalized because the T-cell has been transformed with an Epstein-Barr virus. The T-cell may be derived from a T-cell leukemia or lymphoma. The T-cell may be derived from a cell line, such as a human cell line. The T-cell may be a Jurkat, HuT-78, CEM, Molt- 4, DND-41, T-ALL1, or HPB-ALL cell. The T-cell may be a ARR, CCRF-CEM, CML-T1, DEL, DND-41, DU.528, H-SB2, HUT 102, JB6, Karpas 299, Karpas 45, KE-37/SKW-3, K- Tl, Loucy, MOLT 13, MOLT 16/17, MOLT 3/4, MT-1, P12-Ichikawa, Peer/Bel3, PF-382, RPMI 8402, SU-DHL-1, SUP-T1, SUP-T3, or TALL-104 cell. The T-cell may be derived from a Jurkat cell line. The T-cell may be a Jurkat cell. The T-cell may express one or more of CD3, TCRa, TCR , TCRy, TCR5, CD4, CD8, CD28, CTLA-4, CD40L, CD2, and LFA-1. The T-cell may be a suspension cell or an adherent cell.

T-cells express the T-cell receptor (TCR). A TCR may transduce a costimulatory signal upon binding to an antigen presented by a B-cell or an antigen presenting cell (APC). Alternatively, a TCR may transduce a costimulatory signal upon binding an anti-CD3 antibody. An anti-CD3 antibody may be associated with a bead, multi-well plate, or tissue culture plate, or an anti-CD3 antibody may be crosslinked as an oligomer, such as a tetramer. An anti-CD3 antibody may be a bi-specific antibody, such as a bi-specific T-cell engager (BiTE®). An example of an anti-CD3 bi-specific antibody that may bind CD3 and thereby transduce a costimulatory signal is blinatumomab (BLINCYTO®), which is bi-specific for CD3 and CD19 (present on B-cells).

A T-cell may express a costimulatory receptor such as CD28. CD28 is a cell surface receptor protein capable of transducing a costimulatory signal that enables T-cell activation and survival. CD28 may transduce an intracellular signal upon binding B7.1 (CD80) or B7.2 (CD86), which may be present on a B-cell. Alternatively, CD28 may transduce a costimulatory signal upon binding an anti-CD28 antibody. An anti-CD28 antibody may be associated with a bead, multi-well plate, or tissue culture plate, or an anti-CD28 antibody may be crosslinked as an oligomer, such as a tetramer.

A T-cell may express 4-1BB (CD137), CD27, OX40 (CD134; TNFRSF4), B-cell activating factor receptor (BAFFR), transmembrane activator and CAML interactor (TACI), B-cell Maturation Antigen (BMCA), CD40 ligand (CD40L; CD154), CD28, inducible T-cell costimulator (ICOS; CD278), and/or glucocorticoid-induced TNFR-related protein (GITR; AITR; TNFRSF18). 4- IBB, CD27, OX40, BAFFR, CAML, BMCA, CD40L, or GITR may transduce a costimulatory signal that enables T-cell activation and survival independent of CD28. A. Checkpoint receptors

A T-cell may be engineered to express two or more checkpoint receptors. A T-cell may comprise a recombinant gene encoding a checkpoint receptor. A T-cell may comprise 1, 2, 3, 4, 5, or more recombinant genes, wherein each recombinant gene encodes a different checkpoint receptor. Each checkpoint receptor may be selected from cytotoxic T- lymphocyte antigen 4 (CTLA-4; CD152), programmed cell death 1 (PD-1; CD279), programmed death- 1 homolog (PD-1H), lymphocyte-activation gene 3 (LAG-3; CD223), B- and T-lymphocyte attenuator (BTLA; CD272), a killer-cell immunoglobulin-like receptor (KIR), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), adenosine A2a receptor (A2aR; ADORA2A), CD 160, 2B4 (CD244), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and interleukin-10 receptor. The two or more checkpoint receptors may be inhibitory checkpoint receptors. Nevertheless, a checkpoint receptor may be selected from stimulatory checkpoint receptors, such as 4-1BB (CD137), CD27, OX40 (CD134;

TNFRSF4), B-cell activating factor receptor (BAFFR), transmembrane activator and CAML interactor (TACI), B-cell Maturation Antigen (BMCA), CD40 ligand (CD40L; CD154), CD28, inducible T-cell costimulator (ICOS; CD278), and glucocorticoid-induced TNFR- related protein (GITR; AITR; TNFRSF18). A T-cell may be engineered to express two or more checkpoint receptors wherein the two or more checkpoint receptors comprise PD- 1 and CTLA-4.

A checkpoint receptor may be a human checkpoint receptor, e.g., the checkpoint receptor may have a human amino acid sequence. A checkpoint receptor may be encoded by a human nucleotide sequence. Nevertheless, the amino acid sequence of a checkpoint receptor or the nucleotide sequence encoding the checkpoint receptor may have slight variations from human sequences, which may be introduced, for example, to assist with the cloning or expression of a recombinant gene. For example, tyrosine 165 of CTLA-4 may be replaced with phenylalanine (Tyrl65Phe) to increase surface localization of CTLA-4. A nucleotide sequence encoding a checkpoint receptor may contain introns or lack introns. A nucleotide sequence encoding a checkpoint receptor may be a cDNA nucleotide sequence or a reverse complement thereof.

Each checkpoint receptor may be the target of an agent that specifically binds a checkpoint receptor, such as a therapeutic antibody. For example, lambrolizumab, nivolumab, pembrolizumab, and pidilizumab are therapeutic antibodies that specifically bind the checkpoint receptor PD-1. Ipilimumab, ticilimumab, and tremelimumab are therapeutic antibodies that specifically bind the checkpoint receptor CTLA-4. Lirilumab is a therapeutic antibody that specifically binds the checkpoint receptor KIR2D. In some embodiments, one or more checkpoint receptors are not targets of a therapeutic antibody or an agent that specifically binds a checkpoint receptor. For example, a stimulatory checkpoint receptor might not the target of a therapeutic antibody. A T-cell may comprise a first recombinant gene and a second recombinant gene, e.g. , wherein the two or more checkpoint receptors are encoded by the first recombinant gene and the second recombinant gene. The first recombinant gene may encode the checkpoint receptor PD-1 and the second recombinant gene may encode the checkpoint receptor CTLA- 4. A gene may be recombinant because the mRNA-encoding nucleotide sequence of the gene may be operably-linked to an inducible promoter, such as a promoter that is inducible by doxycycline. A gene may be recombinant because the gene was introduced into a T-cell by transfection and then integrated into the genome of the cell.

The nucleotide sequence encoding a checkpoint receptor may be operably-linked to a promoter, such as an inducible promoter. A recombinant gene may therefore comprise a nucleotide sequence encoding a checkpoint receptor and a promoter (e.g. , inducible promoter) operably-linked to the nucleotide sequence. Each nucleotide sequence encoding a checkpoint receptor may be operably-linked to a promoter (e.g. , an inducible promoter). A T-cell may comprise 1, 2, 3, 4, 5, or more recombinant genes, wherein each recombinant gene comprises both a nucleotide sequence encoding a checkpoint receptor and a promoter (e.g. , an inducible promoter) operably-linked to the nucleotide sequence. For example, a T- cell may comprise a first recombinant gene encoding the checkpoint receptor PD- 1 and a first inducible promoter, wherein the nucleotide sequence encoding PD- 1 is operably-linked to the first inducible promoter. The T-cell may comprise a second recombinant gene encoding the checkpoint receptor CTLA-4 and a second inducible promoter, wherein the nucleotide sequence encoding CTLA-4 is operably-linked to the second inducible promoter. The first inducible promoter and the second inducible promoter may comprise the same nucleotide sequence.

Each recombinant gene encoding a checkpoint receptor may comprise a promoter with the same nucleotide sequence, or each recombinant gene may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with one another. Each recombinant gene encoding a checkpoint receptor may comprise a promoter that is subject to control by the same stimulus.

The T-cell may be engineered to inducibly express two or more checkpoint receptors, wherein the two or more checkpoint receptors are controlled by an inducible promoter. The expression of a checkpoint receptor may be controlled by a promoter inducible by a transcription inducer such as doxycycline, tetracycline, a streptogramin, or a macrolide. The expression of a checkpoint receptor may be controlled by a promoter inducible by a dimerization (e.g. , ARGENT® or iDIMERIZE®). The expression of a checkpoint receptor may be controlled by a promoter inducible by a transcription inducer such as AP20187, AP21967, AP21998, or AP1510 (e.g., which induce dimerization of a transcription factor). The expression of a checkpoint receptor may be controlled by a hormone response element, such as an estrogen response element. The expression of a checkpoint receptor may be controlled by a promoter inducible by a transcription inducer such as estrogen. The two or more checkpoint receptors may be controlled by a promoter inducible by doxycycline (dox).

The expression of a checkpoint receptor may be controlled by a promoter inducible by doxycycline. Each nucleotide sequence encoding a checkpoint receptor may be operably- linked to a promoter that is inducible by doxycycline. A T-cell may comprise 1, 2, 3, 4, 5, or more recombinant genes, wherein each recombinant gene comprises a promoter that is inducible by doxycycline and operably-linked to a nucleotide sequence encoding a checkpoint receptor (e.g. , PD-1 and/or CTLA-4).

B. Genetic modifications

A T-cell may comprise one or more genetic modifications in addition to a

recombinant gene encoding a checkpoint receptor. A genetic modification may be a gene knockout, gene knockdown, expression cassette, or the modification of a regulatory element of a gene. For example, a T-cell may comprise a T-cell gene knockout or an antigen presenting cell gene knockout. Any gene may be knocked or knocked down, such as genes of the lymphoid lineage or T-cell specific genes.

Gene knockouts may be useful to determine whether the knockout gene affects a signaling pathway that is mediated by a checkpoint receptor. For example, PD- 1 may transduce signals through multiple different signaling pathways that implicate myriad signaling proteins, which may include SHP2, phosphoinositide 3-kinase (PI3K), protein kinase B (PKB; AKT), p27kip, and/or ZAP70. A T-cell may comprise a gene knockout of SHP2, PI3K, AKT, p27kip, and/or ZAP70.

A knockout of an individual signaling protein may enable the elucidation of whether a checkpoint receptor signals through the protein. For example, a knockout may be introduced into the T-cell of an inducible T-cell system as described herein, T-cell receptor signaling may be assessed in the absence of the checkpoint receptor (e.g. , IL2 production may be assessed in the absence of doxycycline-induced PD-l/CTLA-4 expression), and T-cell receptor signaling may be assessed in the presence of the checkpoint receptor (e.g. , IL2 production may be assessed in the presence of doxycycline-induced PD- l/CTLA-4 expression). If T-cell receptor signaling is similar in the absence and presence of the checkpoint receptor, then the knockout gene might be critical for downstream signaling through the checkpoint receptor. Such systems may be further probed, for example, by adding an antibody to the system that blocks T-cell receptor signaling (e.g. , nivolumab or pembrolizumab for PD-1; ipilimumab or tremelimumab for CTLA-4). If T-cell receptor signaling is similar in the presence of the checkpoint receptor, regardless of whether an antibody is present that blocks the checkpoint receptor, then the knockout gene is likely critical for signaling downstream of the checkpoint receptor. Polypeptides encoded by genes that are determined to be critical for T-cell receptor signaling may be viable drug targets, particularly if the polypeptide exists on the cell membrane or if the protein is secreted.

Intracellular polypeptides may also be viable drug targets (e.g. , for small molecule drugs). A genetic knockout identified using the above strategy might also be introduced into a lymphocyte used in an adoptive cell transfer therapy, for example, to suppress checkpoint inhibition of the lymphocyte.

Methods for knocking out a gene in a T-cell include homologous recombination and CRISPR/Cas9-mediated knockout. Other methods of generating specific knockouts include TALEN (transcription activator-like effector nucleases) and zinc finger nuclease strategies. A T-cell gene may thus be knocked out by CRISPR/Cas9, a zinc finger nuclease, or a TALEN. A T-cell may comprise a T-cell gene knockout, wherein the T-cell gene is knocked out by CRISPR/Cas9. A knockout may or may not replace the coding regions of the knockout gene with the coding regions of a reporter gene, e.g. , a T-cell may comprise a reporter gene. Additionally, a knockout may replace the promoter of a knockout gene with a different promoter, e.g. , a promoter that is operably-linked to a reporter gene.

A T-cell may comprise a gene for luciferase. A T-cell may comprise two gene fragments of β-galactosidase which may encode polypeptide products that may combine in the T-cell to form a protein with β-galactosidase activity. A T-cell may comprise a luciferase protein or a β-galactosidase protein.

A T-cell may be homozygous for a knockout (e.g. , wherein both copies of a gene are knocked out) or heterozygous for a knockout (e.g. , wherein a single copy of a gene is knocked out).

A T-cell may comprise a random gene knockout. Random knockouts may be generated by insertion mutagenesis such as by transfecting a T-cell with a retrovirus or transposable element (transposon). Random knockouts may be useful to identify genes or other genetic features that may be involved in checkpoint inhibition or T-cell activation. A T-cell may comprise a gene knockdown. A gene knockdown may be a transient knockdown (e.g. , wherein a T-cell is transfected with antisense RNA, short hairpin RNA, microRNA, or small interfering RNA) or a knockdown may be a stable knockdown (e.g. , wherein a T-cell is transfected with a vector encoding an antisense RNA, short hairpin RNA, microRNA, or small interfering RNA). A gene knockdown may be a conditional knockdown (e.g. , wherein the expression of an antisense RNA, microRNA, or small interfering RNA is controlled by an inducible promoter).

A T-cell may comprise a genetic modification that increases the expression of a gene, e.g. , in addition to the two or more checkpoint receptors. A T-cell may comprise a recombinant gene that does not encode a checkpoint receptor. A recombinant gene that does not encode a checkpoint receptor may instead encode a signaling protein, for example, such as a kinase or phosphatase. A recombinant gene that does not encode a checkpoint receptor may encode a human protein, such as a protein expressed by T-cells. The protein encoded by a recombinant gene may contain one or more mutations relative to the wild type protein. The one or more mutations may affect the expression, cellular localization, function, and/or activity of the protein. The one or more mutations may or may not be naturally-occurring mutations or mutations that are associated with disease, such as cancer or an autoimmune disease. The one or more mutations may or may not be associated with the efficacy of a drug that targets the protein, e.g. , a mutation may be associated with drug resistance or sensitivity.

A genetic modification that increases the expression of a gene may be introduced into a T-cell, for example, as an expression cassette comprising a promoter operably-linked to an open reading frame encoding a protein product. A T-cell may comprise a recombinant gene encoding a protein other than a checkpoint receptor, such as a signaling protein (e.g. , kinase or phosphatase). A nucleotide sequence encoding a protein other than a checkpoint receptor may be operably-linked to a promoter, such as a constitutive or inducible promoter.

A genetic modification may increase the expression of an endogenous gene of a T- cell. For example, the regulatory region of an endogenous gene of a T-cell may be modified to increase the expression of a gene. Modifications that increase expression include the replacement of an endogenous promoter with a strong promoter (or a promoter that is stronger than the endogenous promoter). An endogenous promoter may be replaced with an inducible promoter (e.g. , a doxycycline-inducible promoter) or a constitutive promoter (e.g. , cytomegalovirus promoter or SV40 promoter). Other modifications that increase gene expression include the mutation or deletion of a silencer nucleotide sequence. The modification of an endogenous gene may be accomplished using known techniques, such as homologous recombination, CRISPR/Cas9-mediated gene editing, TALEN strategies, and zinc-finger strategies.

II. CELLS EXPRESSING A CHECKPOINT RECEPTOR LIGAND

Various aspects of the invention relate to a checkpoint receptor ligand-presenting cell

(also referred to as a ligand-presenting cell). A checkpoint receptor ligand-presenting cell may be a B-cell. A B-cell may be a mammalian B-cell, such as a human B-cell. A B-cell may be immortalized. For example, a B-cell may be immortalized because the B-cell has been transformed with an Epstein-Barr virus. A B-cell may be derived from a B-cell leukemia or lymphoma. A B-cell may be derived from a cell line, such as a human cell line. A B-cell may be derived from a Raji cell line. A B-cell may be a Raji cell or a BLCL cell. A B-cell may be an Epstein-Barr virus-immortalized lymphoblast.

A B-cell may endogenously express B7.1 and/or B7.2. B7.1 and/or B7.2 may bind to CD28 present on a T-cell thereby transducing a costimulatory signal. B7.1 and/or B7.2 may bind to CTLA-4 present on a T-cell thereby suppressing the activation and proliferation of the T-cell. A B-cell may endogenously express PD-L1. PD-L1 may bind PD-1 present on a T- cell thereby suppressing the activation and proliferation of the T-cell. A B-cell may endogenously express major histocompatibility complex class II (MHC class II). MHC class II may bind LAG-3 present on a T-cell thereby suppressing the activation and proliferation of the T-cell.

A checkpoint receptor ligand-presenting cell may be a human or non-human cell. In some embodiments, the ligand-presenting cell is not a B-cell. For example, a ligand- presenting cell may be a HEK293 cell or a Chinese hamster ovary (CHO) cell. A ligand- presenting cell may be a 3T3-L1, 4T1, 9L, A172, A20, A253, A2780, A2780ADR, A2780cis, A431, A549, AHL-1, ALC, B16, B35, BCP-1, BEAS-2B, bEnd.3, BHK-21, BOSC23, BT- 20, BxPC3, C2C12, C3H-10T1/2, C6, Caco-2, Cal-27, CGR8, CHO, CML Tl, CMT12, COR-L23, COS-7, COV-434, CT26, D17, DH82, DU145, DuCaP, E14Tg2a, EL4, EM-2, EM-3, EMT6/AR1, FM3, GL261, H1299, HaCaT, HCA2, HEK293, HeLa, Hep G2, Hepalclc7, HL-60, HT-1080, HT-29, J558L, Jurkat, JY, K562, KBM-7, KCL-22, KG1, Ku812, KYO-1, L1210, L243, LNCaP, MA2.1, Ma-Mel, MC-38, MCF-IOA, MCF-7, MDA- MB-157, MDA-MB-231, MDA-MB-361, MDA-MB-468, MDCK II, MG63, Mono-Mac-6, MOR/0.2R, MRC-5, MTD-1A, MyEnd, NALM-1, NCI-H69, Neuro2a, NIH-3T3, NK-92, NTERA-2, NW-145, OK, OPCN, OPCT, P3X63Ag8, PC12, PC-3, Peer, PNT1A, PNT2, Pt K2, Raji, RBL-1, RenCa, RIN-5F, RMA-S, SaOS-2, SH-SY5Y, SiHa, SK-BR-3, SK-OV-3, T2, T-47D, T84, T98G, THP-1, U373, U87, U937, VCaP, Vera, VG-1, WM39, WT-49, YAC-1, or YAR cell. A checkpoint receptor ligand-presenting cell may be an adherent cell or a suspension cell.

A checkpoint receptor ligand-presenting cell may be derived from a human or non- human cell, and the ligand-presenting cell may be engineered to express a human checkpoint receptor ligand, such as B7.1, B7.2, PD-L1, or PD-L2. A ligand-presenting cell may be derived from a human or non-human cell, and the ligand-presenting cell may be engineered to express a human costimulatory ligand such as B7.1 or B7.2. A. Checkpoint receptor ligands

A checkpoint receptor ligand-presenting cell may be engineered to constitutively express one or more checkpoint receptor ligands. A ligand-presenting cell may comprise a recombinant gene encoding a checkpoint receptor ligand. A ligand-presenting cell may comprise 1, 2, 3, 4, 5, or more recombinant genes, wherein each recombinant gene encodes a different checkpoint receptor ligand. Each checkpoint receptor ligand may be selected from B7.1 (CD80), B7.2 (CD86), V-set domain-containing T-cell activation inhibitor 1 (VTCN1; B7-H4), programmed cell death-ligand 1 (PD-L1; B7-H1; CD274), B7-H2 (ICOS ligand; CD275), B7-H7, programmed cell death-ligand 2 (PD-L2; B7-DC; CD273), major histocompatibility complex class II (MHC class II), herpesvirus entry mediator (HVEM; TNFRSF14), CD155, galectin-9, and T-cell immunoglobulin and mucin-domain containing-3 ligand (TIM-3 ligand). The one or more checkpoint receptor ligands may comprise PD-L1 and/or PD-L2. A checkpoint receptor ligand may be a human checkpoint receptor ligand, e.g. , the checkpoint receptor ligand may have a human amino acid sequence. A checkpoint receptor ligand may be encoded by a human nucleotide sequence. Nevertheless, the amino acid sequence of a checkpoint receptor ligand or the nucleotide sequence encoding the checkpoint receptor ligand may have slight variations from human sequences, which may be introduced, for example, to assist with the cloning or expression of a recombinant gene. A nucleotide sequence encoding a checkpoint receptor ligand may contain introns or lack introns. A nucleotide sequence encoding a checkpoint receptor ligand may be a cDNA nucleotide sequence or a reverse complement thereof.

A checkpoint receptor ligand-presenting cell may comprise 1, 2, 3, 4, or more recombinant genes, wherein each recombinant gene encodes a checkpoint receptor ligand. A checkpoint receptor ligand may be selected from PD-L1, PD-L2, B7.1, and B7.2. A ligand- presenting cell may comprise a first recombinant gene wherein the first recombinant gene encodes the checkpoint receptor ligand PD-L1 or PD-L2. A ligand-presenting cell may comprise a second recombinant gene, wherein the second recombinant gene encodes the checkpoint receptor ligand B7.1 or B7.2. A gene may be recombinant because the mRNA- encoding nucleotide sequence of the gene is operably-linked to an inducible promoter, such as a promoter that is inducible by doxycycline. A gene may be recombinant because the gene was introduced into a ligand-presenting cell by transfection and then integrated into the genome of the cell.

The nucleotide sequence encoding a checkpoint receptor ligand may be operably- linked to a promoter such as a constitutive promoter. A recombinant gene may therefore comprise a nucleotide sequence encoding a checkpoint receptor ligand and a promoter (e.g. , constitutive promoter) operably-linked to the nucleotide sequence. Each nucleotide sequence encoding a checkpoint receptor ligand may be operably-linked to a promoter (e.g. , a constitutive promoter). A checkpoint receptor ligand-presenting cell may comprise 1, 2, 3, 4, 5, or more recombinant genes, wherein each recombinant gene comprises both a nucleotide sequence encoding a checkpoint receptor ligand and a promoter (e.g. , a constitutive promoter) operably-linked to the nucleotide sequence. For example, a ligand-presenting cell may comprise a recombinant gene encoding the checkpoint receptor ligand PD-L1 or PD-L2 and a constitutive promoter, e.g. , wherein the nucleotide sequence encoding PD-L1 or PD-L2 is operably-linked to the constitutive promoter. A ligand-presenting cell may comprise a recombinant gene encoding the checkpoint receptor ligand B7.1 or B7.2 and a constitutive promoter, e.g. , wherein the nucleotide sequence encoding B7.1 or B7.2 is operably-linked to the constitutive promoter.

A checkpoint receptor ligand-presenting cell may comprise a first recombinant gene encoding the checkpoint receptor ligand PD-L1 or PD-L2 and a first constitutive promoter, e.g. , wherein the nucleotide sequence encoding PD-L1 or PD-L2 is operably-linked to the first constitutive promoter. The ligand-presenting cell may comprise a second recombinant gene encoding the checkpoint receptor ligand B7.1 or B7.2 and a second constitutive promoter, e.g. , wherein the nucleotide sequence encoding B7.1 or B7.2 is operably-linked to the second constitutive promoter. The first constitutive promoter and the second constitutive promoter may comprise the same nucleotide sequence. Each recombinant gene encoding a checkpoint receptor ligand may comprise a promoter with the same nucleotide sequence or with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, a checkpoint receptor ligand-presenting cell is not engineered to constitutively express one or more checkpoint receptor ligands. For example, a ligand- presenting cell may endogenously express one or more of the checkpoint receptor ligands, e.g. , wherein the checkpoint receptor ligands are selected from B7.1, PD-L1, and MHC class II.

B. Costimulatory ligands

A checkpoint receptor ligand-presenting cell may express a costimulatory ligand. For example, a ligand-presenting cell may express B7.1 and/or B7.2, which are costimulatory ligands for CD28. A B-cell may endogenously express a costimulatory ligand, such as B7.1. A ligand-presenting cell may be engineered to constitutively or inducibly express a costimulatory ligand. For example, the ligand-presenting cell may be a HEK293 cell or a CHO cell, and the ligand-presenting cell may be engineered to express human B7.1 and/or human B7.2. Similarly, a ligand-presenting cell (e.g. , either a B-cell or non-B-cell) may be engineered to express an anti-CD28 antibody (e.g. , an anti-CD28 scFv antibody), which may optionally comprise a transmembrane domain that anchors the antibody to the ligand- presenting cell.

In some embodiments, the checkpoint receptor ligand-presenting cell does not comprise a costimulatory ligand or the inducible T-cell system does not comprise a ligand- presenting cell or B-cell. A costimulatory ligand may instead be provided as a reagent optionally associated with a solid support, such as a bead. For example, a costimulatory ligand may be an anti-human CD28 antibody, which may optionally be associated with a bead. III. SYSTEMS COMPRISING T-CELLS

Various aspects of the invention relate to a system comprising a T-cell as described in section I, supra. A system may be an inducible T cell system, e.g. , the system may comprise a T cell engineered to inducibly express two or more checkpoint receptors. A system may further comprise a checkpoint receptor ligand-presenting cell as described in section II, supra. A ligand-presenting cell may or may not be a B-cell. For example, the system may comprise an antigen presenting cell (APC) instead of a B-cell, e.g. , wherein the T-cell receptor costimulatory signal is provided by a MHC-peptide complex on the surface of the APC. A system may further comprise a second costimulatory ligand that is different from the costimulatory ligand expressed by a ligand-presenting cell, which may simulate T-cell receptor signaling. A second costimulatory ligand may be associated with a bead, multi-well plate, or tissue culture plate, or the second costimulatory ligand antibody may be crosslinked as an oligomer, such as a tetramer. A second costimulatory ligand may be, for example, an anti-CD3 antibody. An anti-CD3 antibody may be associated with a bead, e.g. , a system may further comprise an anti-CD3 antibody coated bead. An anti-CD3 antibody may be a bi- specific antibody, such as a bi-specific T-cell engager (BiTE®). An example of an anti-CD3 bi-specific antibody that may bind CD3 and thereby transduce a costimulatory signal is blinatumomab (BLINCYTO®), which is bi-specific for CD3 and CD19. C19 may be present on a B-cell, such as a B-cell of an inducible T-cell system.

A system may further comprise a transcription inducer. A transcription inducer may control the transcription of an inducible gene of a T-cell of a system. The transcription inducer may be doxycycline, tetracycline, a streptogramin, or a macrolide. The transcription inducer may be AP20187, AP21967, AP21998, or AP1510, which may mediate FKBP _F36 v- dimer mediated transcription (e.g. , in ARGENT® or iDIMERIZE®). The transcription inducer may be an estrogen, e.g. , which binds to a hormone response element.

A system may further comprise media, such as Roswell Park Memorial Institute (RPMI) media. A system may further comprise serum, such as fetal bovine serum (FBS). A system may further comprise an antibiotic such as a penicillin (e.g. , penicillin G), an aminoglycoside (e.g. , streptomycin, gentamicin), and/or an aminonucleoside (e.g. , puromycin).

A system may further comprise reagents to determine the expression level of a biomarker that is associated with T-cell activation. For example, a system may comprise an antibody that specifically binds the biomarker. The biomarker may be, for example, interleukin-2 or interferon γ. A system may further comprise reagents to determine the expression of interleukin-2 by the T-cell. For example, a system may comprise an anti- interleukin-2 antibody and/or a secondary antibody, such as an anti-IgG antibody. A system may further comprise reagents to determine the expression of interferon γ by the T-cell. For example, a system may comprise an anti-interferon γ antibody and/or a secondary antibody, such as an anti-IgG antibody. An anti-interleukin-2 antibody, anti-interferon γ antibody, or a secondary antibody may be conjugated to an enzyme (e.g. , alkaline phosphatase or horseradish peroxidase), a fluorophore (e.g. , fluorescein or Texas red), or a crosslinker (e.g. , biotin). A system may comprise a colorimetric alkaline phosphatase substrate or a colorimetric horseradish peroxidase substrate. A system may comprise a luciferase substrate such as BIO-GLO®.

A system may comprise one or more pharmaceutical agents such as two or more pharmaceutical agents. A pharmaceutical agent may or may not be an FDA approved pharmaceutical agent.

A pharmaceutical agent may be a small molecule compound or a large molecule. The one or more pharmaceutical agents may be selected from small molecule compounds and large molecules.

A pharmaceutical agent may be a candidate compound. Candidate compounds are typically small molecule compounds that are drug-like, including compounds of a drug screen (such as compounds used in high-throughput screening), rationally designed compounds, pre-lead compounds, lead compounds, clinical candidates, and repurposed drugs.

A small molecule compound may have a molecular weight of less than about 900 amu, such as less than about 800, 700, or 600 amu. A small molecule compound may have a molecular weight of about 3 amu to about 899.9 amu, such as about 100 to 800, 100 to 700, 100 to 600, 100 to 500, 200 to 800, 200 to 700, 200 to 600, 200 to 500, 300 to 800, 300 to 700, 300 to 600, 300 to 500, 400 to 800, 400 to 700, 400 to 600, 500 to 800, 500 to 700, or 600 to 800 amu. In certain embodiments, the small molecule compound has a molecular weight of about 400 amu to about 700 amu.

A small molecule compound may be an immunomodulatory imide drug (IMiD). A small molecule compound may be thalidomide, lenalidomide, pomalidomide, or apremilast.

A small molecule compound may be a kinase inhibitor such as a tyrosine kinase inhibitor, a serine/threonine kinase inhibitor, or a lipid kinase inhibitor.

A large molecule may have a molecular weight of at least about 900 amu, such as at least about 1 kDa, 10 kDa, 50 kDa, 100 kDa, or 150 kDa. A large molecule may have a molecular weight of about 900 amu to 5 kDa, 1 kDa to 50 kDa, 1 kDa to 30 kDa, 1 kDa to 25 kDa, 1 kDa to 20 kDa, 1 kDa to 10 kDa, 5 kDa to 30 kDa, 5 kDa to 25 kDa, 5 kDa to 20 kDa, 5 kDa to 15 kDa, 10 kDa to 30 kDa, 10 kDa to 25 kDa, 10 kDa to 20 kDa, 20 kDa to 80 kDa, 20 kDa to 40 kDa, 40 kDa to 60 kDa, 50 kDa to 100 kDa, 100 kDa to 200 kDa, 120 kDa to 180 kDa, 140 kDa to 160 kDa, 150 kDa to 300 kDa, or 150 kDa to 250 kDa. A cytokine, for example, may have a molecular weight of about 5 kDa to about 20 kDa. An antibody may have a molecular weight of about 120 kDa to about 180 kDa, such as about 140 kDa to about 160 kDa. A fragment antigen-binding (Fab) fragment of an antibody may have a molecular weight of about 20 kDa to about 80 kDa, such as about 40 kDa to about 60 kDa. A large molecule may be a hormone, cytokine, chemokine, lymphokine, interferon, or interleukin. A large molecule may be selected from interferon alfa, interferon alfacon-1, pegylated interferon alpfa-2a, pegylated interferon alfa- 2b, interferon βΐ, interferon jib, interleukin-2, interleukin-7, interleukin- 12, pifonakin, mobenakin, adargileukin alfa, aldesleukin, celmoleukin, denileukin diftitox, pegaldesleukin, teceleukin, tucotuzumab celmoleukin, daniplestim, muplestim, binetrakin, atexakin alfa, emoctakin, ilodecakin, oprelvekin, edodekin alfa, cintredekin besudotox, and iboctadekin.

A T-cell and/or ligand-presenting cell may produce a molecule with the same chemical structure as a large molecule or pharmaceutical agent, and the terms

"pharmaceutical agent" and "large molecule" are limited to molecules that are not produced by a cell of an inducible T-cell system. For example, a pharmaceutical agent (e.g. , large molecule) may be interleukin-2 or interferon γ, in which case the interleukin-2 or interferon γ pharmaceutical agent refers to interleukin-2 or interferon γ that is added to a T-cell system rather than interleukin-2 or interferon γ that is produced by a cell of the system. In some embodiments, the pharmaceutical agent does not have the same chemical structure as any molecule produced by a cell of a T-cell system, e.g. in some embodiments the pharmaceutical agent does not have the same chemical structure as interleukin-2, interferon γ, or any other molecule produced by a cell of an inducible T-cell system.

A large molecule may be interleukin-2 or interferon γ.

A large molecule may be selected from brain-derived neurotrophic factor (BDNF),

CCL11 (Eotaxin), CCL3 (MIP-1 alpha), CCL4 (MIP-1 beta), CCL5 (RANTES), CD31 (PECAM-1), CD62P (P-selectin), CXCL1 (GRO alpha), CXCL10 (IP-10), granulocyte- macrophage colony- stimulating factor (GM-CSF), hepatocyte growth factor (HGF), interferon a (IFNa), interferon γ (IFNy), interleukin- la, interleukin- 1β, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8 (cxcl8), interleukin-9, interleukin- 10, interleukin- 12, interleukin- 13, interleukin- 15, interleukin- 16, interleukin- 17a, interleukin-20, interleukin-21, interleukin- 1 receptor antagonist, leukemia inhibitory factor (LIF), monocyte chemoattractant protein 2 (MCP-2; CCL8), osteoprotegerin (OPG), platelet- derived growth factor subunit B dimer (PDGF-BB), sCD120b (sTNF Receptor II), stem cell factor (SCF), tumor necrosis factor a (TNFa), tissue plasminogen activator (t-PA), thymic stromal lymphopoietin (TSLP), vascular endothelial growth factor A (VEGF-A), and vascular endothelial growth factor A (VEGF-D).

A large molecule may be an antibody such as a therapeutic antibody. A therapeutic antibody is typically a monoclonal antibody, or an antigen-binding fragment thereof, which is either chimeric (e.g. , comprising human sequences), humanized, or fully human. An antibody may be a checkpoint inhibitory antibody. One or more pharmaceutical agents may comprise a small molecule compound, an antibody, and/or an FDA approved checkpoint inhibitory antibody. Two or more pharmaceutical agents may comprise a small molecule compound, an antibody, and/or an FDA approved checkpoint inhibitory antibody.

The term "antibody" encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. , bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen- binding activity. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments are well known in the art (see, e.g. , Nelson, MAbs (2010) 2(l):77-83) and include but are not limited to Fab, Fab', Fab'-SH, F(ab') ₂ , and Fv; diabodies; linear antibodies; single-chain antibody molecules including but not limited to single-chain variable fragments (scFv), fusions of light and/or heavy-chain antigen-binding domains with or without a linker (and optionally in tandem); and monospecific or multispecific antigen-binding molecules formed from antibody fragments (including, but not limited to multispecific antibodies constructed from multiple variable domains which lack Fc regions).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. , the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants, e.g. , containing naturally occurring mutations or that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method (see, e.g. , Kohler et al, Nature, 256:495 (1975)), recombinant DNA methods (see, e.g. , U.S. Patent No. 4,816,567), phage-display methods (e.g. , using the techniques described in Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol, 222:581-597 (1991)), and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. Specific examples of monoclonal antibodies herein include chimeric antibodies, humanized antibodies, and human antibodies, including antigen-binding fragments thereof. Two or more checkpoint inhibitory drugs may comprise an FDA approved checkpoint inhibitory drug wherein the FDA approved checkpoint inhibitory drug is a monoclonal antibody.

A "bi-specific antibody" is a multispecific antibody comprising an antigen-binding domain that is capable of specifically binding to two different epitopes on one biological molecule or is capable of specifically binding to epitopes on two different biological molecules.

A large molecule may be an antibody that specifically binds 2B4, 4- IBB, BAFF,

CCL2, CCR5, CD19, CD2, CD20, CD22, CD23, CD27, CD274, CD28, CD3, CD3, CD30, CD37, CD38, CD4, CD40, CD44, CD5, CD52, CD6, CD70, CD79B, CD80, CD134, CD152, CD154, CD160, CSF2, CTGF, CTLA-4, CXCR4, ICOSL, IFN-a, IFN-γ, interleukin-ΐβ, interleukin-4, interleukin-5, interleukin-6, interleukin-9, interleukin-12, interleukin-13, interleukin-17, interleukin-17A, interleukin-17F, interleukin-20, interleukin-22, interleukin- 23, interleukin-23A, KIR2D, LFA-1, L-selectin, MIF, OX40, PD-1, PD-IH, TIGIT, RANKL, T-cell receptor, TGF-β, TGF-βΙ, TGF- 2, or thymic stromal lymphopoietin.

A large molecule may be an antibody that specifically binds brain-derived

neurotrophic factor (BDNF), CCL11 (Eotaxin), CCL3 (MIP-1 alpha), CCL4 (MIP-1 beta), CCL5 (RANTES), CD31 (PECAM-1), CD62P (P-selectin), CXCL1 (GRO alpha), CXCL10 (IP- 10), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), interferon a (IFNa), interferon γ (IFNy), interleukin-la, interleukin-ΐβ, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8 (cxcl8), interleukin-9, interleukin-10, interleukin-12, interleukin-13, interleukin-15, interleukin-16, interleukin-17a, interleukin-20, interleukin-21, interleukin-1 receptor antagonist, leukemia inhibitory factor (LIF), monocyte chemoattractant protein 2 (MCP-2; CCL8), osteoprotegerin (OPG), platelet-derived growth factor subunit B dimer (PDGF-BB), sCD120b (sTNF Receptor II), stem cell factor (SCF), tumor necrosis factor a (TNFa), tissue plasminogen activator (t-PA), thymic stromal lymphopoietin (TSLP), vascular endothelial growth factor A (VEGF-A), or vascular endothelial growth factor A (VEGF-D).

A large molecule may be selected from Afasevikumab (which binds interleukin-17 A and interleukin-17F), Afutuzumab (which binds CD20), ALD518 (which binds interleukin- 6), Alemtuzumab (which binds CD52), Anrukinzumab (which binds interleukin-13), Aselizumab (which binds L-selectin), Atezolizumab (which binds CD274), Avelumab (which binds CD274), Basiliximab (which binds CD23), Bectumomab (which binds CD22), Belimumab (which binds BAFF), Bimekizumab (which binds interleukin-17A and interleukin-17F), Bivatuzumab (which binds CD44), Bleselumab (which binds CD40), Blinatumomab (which binds CD 19), Blontuvetmab (which binds CD20), Brazikumab (which binds interleukin-23), Brentuximab (which binds CD30), Briakinumab (which binds interleukin-12 and interleukin-23), Brodalumab (which binds interleukin-17), Canakinumab (which binds interleukin-ΐβ), Carlumab (which binds CCL2), Cedelizumab (which binds CD4), Clenoliximab (which binds CD4), Coltuximab (which binds CD 19), Dacetuzumab (which binds CD40), Daclizumab (which binds CD23), Dapirolizumab pegol (which binds CD154), Daratumumab (which binds CD38), Dectrekumab (which binds interleukin-13),

Denintuzumab (which binds CD19), Denosumab (which binds RANKL), Dupilumab (which binds interleukin-4), Durvalumab (which binds CD274), Efalizumab (which binds LFA-1), Elsilimomab (which binds interleukin-6), Enokizumab (which binds interleukin-9),

Epratuzumab (which binds CD22), FBTA05 (which binds CD20), Fezakinumab (which binds interleukin-22), Fletikumab (which binds interleukin-20), Fontolizumab (which binds IFN-γ), Foralumab (which binds CD3), Fresolimumab (which binds TGF-β), Galiximab (which binds CD80), Gevokizumab (which binds interleukin-ΐβ), Gomiliximab (which binds CD23), GSK3174998 (which binds OX40), Guselkumab (which binds interleukin-23), Ibalizumab (which binds CD4), Ibritumomab (which binds CD20), Imalumab (which binds MIF), Inebilizumab (which binds CD 19), Inolimomab (which binds CD23), Inotuzumab (which binds CD22), Ipilimumab (which binds CD152), Iratumumab (which binds CD30),

Isatuximab (which binds CD38), Itolizumab (which binds CD6), Ixekizumab (which binds interleukin-17 A), Keliximab (which binds CD4), Lambrolizumab (which binds PD-1), Lebrikizumab (which binds interleukin-13), Lenzilumab (which binds CSF2), Lerdelimumab (which binds TGF- 2), Lilotomab (which binds CD37), Lirilumab (which binds KIR2D), Lucatumumab (which binds CD40), Lulizumab pegol (which binds CD28), Lumiliximab (which binds CD23), Maslimomab (which binds T-cell receptor), MEDI6383 (which binds OX40), Mepolizumab (which binds interleukin-5), Metelimumab (which binds TGF-βΙ), Moxetumomab (which binds CD22), Muromonab-CD3 (which binds CD3), Namilumab (which binds CSF2), Naratuximab (which binds CD37), Nivolumab (which binds PD-1), Obinutuzumab (which binds CD20), Ocaratuzumab (which binds CD20), Ocrelizumab (which binds CD20), Odulimomab (which binds LFA-1), Ofatumumab (which binds CD20), Olokizumab (which binds interleukin-6), Otelixizumab (which binds CD3), Otlertuzumab (which binds CD37), Oxelumab (which binds OX-40), Pamrevlumab (which binds CTGF), Pascolizumab (which binds interleukin-4), Pembrolizumab (which binds PD-1), Perakizumab (which binds interleukin-17A), Pidilizumab (which binds PD- 1), Pinatuzumab (which binds CD22), Pogalizumab (which binds CD134), Polatuzumab (which binds CD79B),

Prezalizumab (which binds ICOSL), Priliximab (which binds CD4), PRO 140 (which binds CCR5), Reslizumab (which binds interleukin-5), Risankizumab (which binds interleukin- 23A), Rituximab (which binds CD20), Rontalizumab (which binds IFN-a), Ruplizumab (which binds CD 154), Sarilumab (which binds interleukin-6), Secukinumab (which binds interleukin-17A), SGN-CD19A (which binds CD19), Sifalimumab (which binds IFN-a), Siltuximab (which binds interleukin-6), Siplizumab (which binds CD2), Sirukumab (which binds interleukin-6), Tabalumab (which binds BAFF), Tamtuvetmab (which binds CD52), Taplitumomab (which binds CD 19), Teneliximab (which binds CD40), Teplizumab (which binds CD3), Tetulomab (which binds CD37), Tezepelumab (which binds thymic stromal lymphopoietin), TGN1412 (which binds CD28), Ticilimumab (which binds CTLA-4), Tildrakizumab (which binds interleukin-23), TNX-650 (which binds interleukin- 13), Toralizumab (which binds CD154), Tositumomab (which binds CD20), Tralokinumab

(which binds interleukin-13), Tregalizumab (which binds CD4), Tremelimumab (which binds CTLA-4), Ublituximab (which binds CD20), Ulocuplumab (which binds CXCR4), Urelumab (which binds 4- IBB), Ustekinumab (which binds interleukin- 12 and interleukin-23), Utomilumab (which binds 4-1BB), Varlilumab (which binds CD27), Veltuzumab (which binds CD20), Visilizumab (which binds CD3), Vorsetuzumab (which binds CD70),

Zanolimumab (which binds CD4), and Zolimomab (which binds CD5) or an antigen-binding fragment of any one of the foregoing. The large molecule may be selected from

YW243.55.S70, MED 14736, and MDX-1105 or an antigen-binding fragment of any one of the foregoing.

A system may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more pharmaceutical agents as described herein. A system may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10

pharmaceutical agents.

IV. PLURALITY OF SAMPLES

Various aspects of the invention relate to a plurality of systems. A system of the plurality may comprise an inducible T-cell, a checkpoint receptor ligand-presenting cell (e.g. , a B-cell), and second costimulatory ligand. A system may or may not further comprise a transcription inducer such as doxycycline. A system may further comprise 0, 1, 2, 3, or 4 of a first large molecule, a second large molecule, a first small molecule compound, and a second small molecule compound. A system may comprise an inducible T-cell, modified inducible T-cell, transcription inducer, checkpoint receptor ligand-presenting cell (e.g. , a B-cell), second costimulatory ligand, first large molecule, second large molecule, first small molecule compound, and second small molecule.

A plurality of systems may be arranged, for example, in a plate configured for high- throughput screening, e.g. , a multi-well plate, such as a 6 well, 12 well, 24 well, 96 well, 384 well, 1536 well, or 3,456 well plate. A plurality of systems may comprise 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 or more systems.

Each system of a plurality of systems may comprise a T-cell engineered to inducibly express two or more checkpoint receptors; a ligand-presenting cell that expresses at least one checkpoint receptor ligand; and a costimulatory ligand. The T-cell may be any T-cell described herein, such as a human T-cell (e.g. , a Jurkat cell). The ligand-presenting cell may be any ligand-presenting cell described herein, such as a human B-cell (e.g. , a Raji cell). The costimulatory ligand may be an anti-CD3 antibody or other ligand that specifically binds the T-cell receptor thereby activating T-cell receptor mediated signaling. An anti-CD3 antibody may be associated with a solid support, such as a bead.

At least one system of a plurality of systems may comprise a transcription inducer that induces the transcription of the two or more checkpoint inhibitors. At least one system of a plurality of systems may lack the transcription inducer. The transcription inducer may be a small molecule such as doxycycline, tetracycline, a streptogramin, a macrolide, a steroid (e.g. , an estrogen), AP20187, AP21967, AP21998, or AP1510.

At least one system of a plurality of systems may comprise a pharmaceutical agent. At least one system of a plurality of systems may lack the pharmaceutical agent. For example, at least one system may lack both a transcription inducer and a pharmaceutical agent; at least one system may comprise the transcription inducer and lack the pharmaceutical agent; at least one system may lack the transcription inducer and comprise the pharmaceutical agent; and/or at least one system may comprise both the transcription inducer and the pharmaceutical agent.

At least one system of a plurality of systems may comprise a T-cell comprising a gene knockout or gene knockdown (i.e. , wherein a T-cell comprising the gene knockout or gene knockdown is a T-cell engineered to inducibly express two or more checkpoint inhibitors). At least one system of a plurality of systems may comprise a T-cell lacking the gene knockout or gene knockdown (i.e. , wherein the T-cell lacking the gene knockout or gene knockdown is a T-cell engineered to inducibly express two or more checkpoint inhibitors). For example, at least one system may lack a transcription inducer and comprise a T-cell lacking the gene knockout or gene knockdown; at least one system may comprise the transcription inducer and comprise a T-cell lacking the gene knockout or gene knockdown; at least one system may lack the transcription inducer and comprise a T-cell comprising the gene knockout or gene knockdown; and/or at least one system may comprise the transcription inducer and comprise a T-cell comprising the gene knockout or gene knockdown. Similarly, at least one system may lack a pharmaceutical agent and comprise a T-cell lacking the gene knockout or gene knockdown; at least one system may comprise the pharmaceutical agent and comprise a T-cell lacking the gene knockout or gene knockdown; at least one system may lack the pharmaceutical agent and comprise a T-cell comprising the gene knockout or gene knockdown; and/or at least one system may comprise the pharmaceutical agent and comprise a T-cell comprising the gene knockout or gene knockdown

At least one system of a plurality of systems may comprise a T-cell engineered to express a recombinant gene in addition to the two or more checkpoint receptors. At least one system of a plurality of systems may comprise a T-cell lacking the recombinant gene. For example, at least one system may lack a transcription inducer and comprise a T-cell engineered to express the recombinant gene; at least one system may comprise the transcription inducer and comprise a T-cell engineered to express the recombinant gene; at least one system may lack the transcription inducer and comprise a T-cell lacking the recombinant gene; and/or at least one system may comprise the transcription inducer and comprise a T-cell lacking the recombinant gene. Similarly, at least one system may lack a pharmaceutical agent and comprise a T-cell engineered to express the recombinant gene; at least one system may comprise the pharmaceutical agent and comprise a T-cell engineered to express the recombinant gene; at least one system may lack the pharmaceutical agent and comprise a T-cell lacking the recombinant gene; and/or at least one system may comprise the pharmaceutical agent and comprise a T-cell lacking the recombinant gene.

A T-cell may be engineered to inducibly express two or more checkpoint receptors as described herein, such as two or more checkpoint receptors are selected from PD-1, CTLA-4, PD-1H, LAG-3, BTLA, KIR2D, TIM-3, ADORA2A, CD160, 2B4, TIGIT, and interleukin- 10 receptor. A T-cell may be engineered to inducibly express two or more checkpoint receptors selected from PD-1 and CTLA-4.

A ligand-presenting cell may express at least one checkpoint receptor ligand that is a ligand of the two or more checkpoint receptors of the T-cell. The ligand-presenting cell may endogenously express the at least one checkpoint receptor ligand or the ligand-presenting cell may be engineered to express the at least one checkpoint receptor ligand. The at least one checkpoint receptor ligand(s) may be selected from PD-L1, PD-L2, B7.1, B7.2, B7-H4, B7- H2, B7-H7, MHC class II, HVEM, CD 155, galectin-9, and TIM-3 ligand. For example, a ligand-presenting cell may express either PD-L1 or PD-L2 and either B7.1 or B7.2.

A ligand-presenting cell may express a second costimulatory ligand in addition to the costimulatory ligand of a system. The second costimulatory ligand may be a ligand of a costimulatory receptor, e.g. , wherein the costimulatory receptor is expressed by the T-cell of a system. The second costimulatory ligand may be a ligand of 4- IBB, CD27, OX40, BAFFR, TACI, BMCA, CD40L, CD28, ICOS, or GITR. For example, a ligand-presenting cell may express B7.1 and/or B7.2, which are ligands of CD28.

The costimulatory ligand may be, for example, an anti-CD3 antibody as described herein, which may be associated with a solid support, such as a bead, the surface of a tissue culture plate, or the surface of a multi-well plate.

A plurality of systems may be useful to determine whether a protein may be a suitable drug target or whether a pharmaceutical agent has an ON target or OFF target affect. For example, a plurality of systems may comprise an inducible T-cell system comprising a gene knockout or knockdown to determine whether a protein encoded by the gene is a suitable drug target. Similarly, a plurality of systems may comprise (1) a system comprising a pharmaceutical agent and a T-cell comprising a gene knockout or knockdown, (2) a system comprising the pharmaceutical agent and a T-cell lacking the gene knockout or knockdown, (3) a system lacking the pharmaceutical agent and a T-cell lacking the gene knockout or knockdown, and/or (4) a system lacking the pharmaceutical agent and a T-cell comprising the gene knockout or knockdown, which may be useful to determine whether the pharmaceutical agent affects T-cell activation through a pathway mediated by the protein product of the gene.

Each system of a plurality of systems may be selected from: a system that lacks a transcription inducer, lacks a pharmaceutical agent, and lacks a T-cell comprising a gene knockout or gene knockdown; a system that comprises a transcription inducer, lacks a pharmaceutical agent, and lacks a T-cell comprising a gene knockout or gene knockdown; a system that lacks a transcription inducer, comprises a pharmaceutical agent, and lacks a T- cell comprising a gene knockout or gene knockdown; a system that comprises a transcription inducer, comprises a pharmaceutical agent, and lacks a T-cell comprising a gene knockout or gene knockdown; a system that lacks a transcription inducer, lacks a pharmaceutical agent, and comprises a T-cell comprising a gene knockout or gene knockdown; a system that comprises a transcription inducer, lacks a pharmaceutical agent, and comprises a T-cell comprising a gene knockout or gene knockdown; a system that lacks a transcription inducer, comprises a pharmaceutical agent, and comprises a T-cell comprising a gene knockout or gene knockdown; and/or a system that comprises a transcription inducer, comprises a pharmaceutical agent, and comprises a T-cell comprising a gene knockout or gene knockdown.

A plurality of systems may comprise a system comprising two pharmaceutical agents. The two pharmaceutical agents may be selected from at least one small molecule compound and at least one large molecule. For example, the two pharmaceutical agents may be two small molecule compounds, two large molecules (e.g. , two antibodies), or a small molecule compound (e.g. , a kinase inhibitor) and a large molecule (e.g. , an antibody). Each system of a plurality of systems may be selected from: a system that lacks a small molecule compound and lacks a large molecule; a system that comprises one small molecule compound and lacks a large molecule; a system that comprises one small molecule compound and one large molecule; a system that comprises at least two small molecule compounds and lacks a large molecule; a system that comprises at least two small molecule compounds and one large molecule; a system that lacks a small molecule compound and comprises at least two large molecules; and/or a system that comprises one small molecule compound and at least two large molecules.

The at least one large molecule may comprise an antibody such as an anti-CTLA-4 antibody and an anti-PD- 1 antibody. The plurality of systems may comprise a system comprising two pharmaceutical agents wherein the two pharmaceutical agents are an anti- CTLA-4 antibody and an anti-PD- 1 antibody. The at least one large molecule may comprise 1 or 2 of avelumab, atezolizumab, durvalumab, ipilimumab, lambrolizumab, nivolumab, pembrolizumab, pidilizumab, tremelimumab, and ticilimumab. The plurality of systems may comprise a system comprising two pharmaceutical agents wherein the two pharmaceutical agents are selected from avelumab, atezolizumab, durvalumab, ipilimumab, lambrolizumab, nivolumab, pembrolizumab, pidilizumab, tremelimumab, and ticilimumab. For example, the at least one large molecule may comprise ipilimumab and/or nivolumab. The plurality of systems may comprise a system comprising two pharmaceutical agents wherein the two pharmaceutical agents are ipilimumab and nivolumab.

Each system of a plurality of inducible T-cell systems may comprise tissue culture media, such as RPMI media. A system may further comprise blood serum (e.g. , FBS), which may be present at a concentration of about 1% to about 20% serum, such as about 5% to about 15%, such as about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. Each system may further comprise an antibiotic such as a penicillin (e.g. , penicillin G), an aminoglycoside (e.g. , streptomycin, gentamicin), and/or an aminonucleoside (e.g. , puromycin).

Various aspects of the invention relate to a plurality of samples corresponding to a plurality of systems as described herein. A plurality of samples may comprise the supernatants of each system of a plurality of systems. A plurality of samples may or may not comprise the cells of an inducible T-cell system. A plurality of samples may be substantially free of the cells of a plurality of inducible T-cell systems (i.e. , T-cells and ligand-presenting cells).

A plurality of samples may comprise the supernatant of a plurality of inducible T-cell systems as described herein and reagents for an enzyme-linked immunosorbent assay (ELISA) or other colorimetric immunoassay. Each sample of a plurality of samples may consist essentially of the supernatant of an inducible T-cell system (e.g. , of a plurality of systems) as described herein and reagents for an (ELISA) or other colorimetric immunoassay.

Reagents for an ELISA or other colorimetric immunoassay may include one or more a primary antibody (e.g. , an anti-interleukin-2 antibody or an anti-interferon γ antibody), a secondary antibody (e.g. , an anti-IgG antibody, anti-IgA antibody, anti-IgY antibody, anti- IgM antibody, anti-IgE antibody, anti-IgD antibody, an anti-Rabbit IgG antibody, an anti-Rat IgG antibody, an anti-Mouse IgG antibody, an anti-Human IgG antibody), an antibody- fluorophore conjugate (e.g. , an antibody- Alexa Fluor 488 conjugate, an antibody-Texas Red conjugate, an antibody-fluorescein conjugate), an antibody-biotin conjugate, an antibody- enzyme conjugate (e.g. , an antibody- horseradish peroxidase conjugate or an antibody- alkaline phosphatase conjugate), a colorimetric substrate for horseradish peroxidase (e.g. , ABTS, OPD, AmplexRed, DAB, AEC, TMB, homovanillic acid, luminol), and a colorimetric substrate for alkaline phosphatase (e.g. , nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate).

A plurality of systems or plurality of samples may be arranged in a multi-well plate such as a 6 well, 12 well, 24 well, 96 well, 384 well, 1536 well, or 3,456 well plate. A multi- well plate may comprise, for example, U-bottom wells.

V. APPLICATIONS

Various aspects of the invention relate to methods of preparing an inducible T-cell system as described herein or a plurality of inducible T-cell systems. A method may utilize a multi-well plate, although a method may be adapted to other formats including individual tissue culture plates, tissue culture flasks, test tubes, microfluidics platforms, or lab-on-chip platforms. A method may be a method of preparing a multi-well plate.

The following methods generally describe the addition of cells, beads, transcription inducers, pharmaceutical agents, etc. to "systems." The addition of a composition (e.g. , cell, bead, transcription inducer, pharmaceutical agent) to a "system" may comprise the addition of the composition to a well of a multi-well plate. A plurality of systems may be arranged, for example, in a plurality of wells of a multi-well plate (or a plurality of wells on multiple multi-well plates). A group of systems may correspond to a group of wells of a plurality of wells.

A method may comprise adding a T-cell as described herein to each well of a plurality of wells of a multi-well plate. A method may comprise adding a T-cell as described herein to each system of a plurality of systems. The cells may be added in tissue culture media such as RPMI media, which may comprise serum (e.g. FBS). The tissue culture media may optionally comprise L-glutamine. The tissue culture media may optionally comprise an antibiotic, such as a penicillin (e.g. , penicillin G), an aminoglycoside (e.g. , streptomycin, gentamicin), and/or an aminonucleoside (e.g. , puromycin).

A T-cell may or may not comprise a genetic modification such as a gene knockout, gene knockdown, or a recombinant gene (i.e. , in addition to the two or more recombinant genes encoding checkpoint receptors). Each T-cell added to a system may be from the same species or cell line. For example, each T-cell may be a Jurkat cell. Each T-cell added to a system of a plurality of systems may comprise recombinant gene(s) encoding the same checkpoint receptors, e.g. , each T-cell may be descendant from the same parent cell, wherein the cell lineage defined by the parent cell is engineered to inducibly express two or more checkpoint receptors. The T-cells of individual systems may nevertheless vary. For example, a T-cell may further comprise a gene knockout, gene knockdown, or additional recombinant gene that has been engineered into the T-cell on top of the genes encoding the two or more checkpoint receptors.

A method may comprise adding a knockout T-cell to a first group of systems of a plurality of systems. The knockout T-cell may be engineered to inducibly express two or more checkpoint receptors and the knockout T-cell may comprise a gene knockout. The method may further comprise adding a wild type T-cell to a second group of systems of the plurality of systems. The wild type T-cell may be engineered to inducibly express the two or more checkpoint receptors and the wild type T-cell may lack the gene knockout. A wild type T-cell and knockout T-cell may be descendent from the same parent cell, e.g. , wherein the parent cell comprised genes encoding the two or more checkpoint receptors and wherein the parent cell lacked the gene knockout. A plurality of systems may consist of the first group of systems and the second group of systems. In some embodiments, the first group and second group do not overlap.

A method may further comprise adding a transcription inducer to a group of systems (e.g. , a third group of systems). The transcription inducer may be doxycycline, which may be added at a concentration of about 50 ng/mL to about 1000 ng/mL, such as about 100 ng/mL to about 500 ng/mL or about 200 ng/mL to about 300 ng/mL. The group of systems may be a subset of a plurality of systems. Systems that do not comprise the transcription inducer may serve as relevant controls.

A method may further comprise incubating the T-cells of a plurality of systems (e.g. , after adding the T-cells to a plurality of systems and after adding the transcription inducer to a group of systems). The T-cells may be incubated at about 37°C. The T-cells may be incubated for about 6 hours to about 7 days, such as about 12 hours to about 96 hours, about 24 hours to about 72 hours, or about 36 hours to about 50 hours. The T-cells may be incubated for at least about 6 hours, 12 hours, 24 hours, 30 hours, 36 hours, 42 hours, or 48 hours.

A method may further comprise washing the T-cells of a plurality of systems (e.g. , after adding a transcription inducer to a group of systems and/or prior to adding a costimulatory ligand). The T-cells may be washed in tissue culture media. Washing the T- cells may reduce the concentration of a transcription inducer in the tissue culture media.

A method may further comprise adding a costimulatory ligand as described herein (e.g. , anti-CD3 antibody-coated beads) to each system of a plurality of systems. The costimulatory ligand may be added to each system of a plurality of systems about 6 hours to about 7 days after adding the transcription inducer, such as about 12 hours to about 96 hours, about 24 hours to about 72 hours, or about 36 hours to about 50 hours. The costimulatory ligand may be added to each system of a plurality of systems at least about 6 hours, 12 hours, 24 hours, 30 hours, 36 hours, 42 hours, or 48 hours after adding the transcription inducer to a group of systems. The costimulatory ligand may be added to each system of a plurality of systems after washing the T-cells of a plurality of systems to reduce the concentration of a transcription inducer.

A method may comprise adding one or more pharmaceutical agents to systems of the plurality. For example, a method may comprise adding a first pharmaceutical agent to each system of a group of systems (e.g. , a fourth group of systems). A first pharmaceutical agent may be a large molecule (e.g. , antibody, such as an anti-PD-1 or anti-CTLA-4 antibody) or a small molecule compound (e.g. , kinase inhibitor). A first pharmaceutical agent may be added to a system that was contacted with a transcription inducer, e.g. , to determine whether the first pharmaceutical agent can increase the activation state of a T-cell expressing two or more checkpoint receptors in the presence of checkpoint receptor ligands. A first pharmaceutical agent may be added to a system that was not contacted with a transcription inducer, e.g. , as a control. A first pharmaceutical agent may be added to a system comprising a knockout T- cell, e.g. , to determine whether the first pharmaceutical agent increases T-cell activation through a signaling pathway mediated by the knockout gene. A first pharmaceutical agent may be added to a system comprising a wild type T-cell, e.g. , as a control.

A method may comprise adding a second pharmaceutical agent to a group of systems (e.g. , a fifth group of systems). A second pharmaceutical agent may be a large molecule or a small molecule compound. For example, the first pharmaceutical agent and the second pharmaceutical agent may be antibodies, e.g. , the first pharmaceutical agent may be an anti- PD- 1 antibody and the second pharmaceutical agent may be an anti-CTLA-4 antibody. The first pharmaceutical agent and the second pharmaceutical agent may both be small molecule compounds, or the first pharmaceutical agent may be a large molecule and the second pharmaceutical agent may be a small molecule compound. The first pharmaceutical agent and the second pharmaceutical agent may both be added to at least one discrete system, e.g. , to assess whether the two pharmaceutical agents display an additive or synergistic effect. The at least one system may comprise a system that was contacted with the transcription inducer. The at least one system may comprise a system that was not contacted with the transcription inducer, e.g. , to serve as a control. The first pharmaceutical agent may be added to at least one system to which the second pharmaceutical agent is not added, e.g. to serve as a control. The second pharmaceutical agent may be added to at least one system to which the first pharmaceutical agent is not added, e.g. to serve as a control. A plurality of systems may comprise a system to which neither the first pharmaceutical agent nor the second

pharmaceutical agent is added, e.g. , to serve as a control.

A method may comprise adding a third pharmaceutical agent to a group of systems

(e.g. , a sixth group of systems). A third pharmaceutical agent may be a large molecule or a small molecule compound. For example, the first pharmaceutical agent and the second pharmaceutical agent may be antibodies (e.g. , anti-PD-1 and anti-CTLA-4 antibodies, respectively), and the third pharmaceutical agent may be a small molecule compound (e.g. , a kinase inhibitor). The first pharmaceutical agent, second pharmaceutical agent, and third pharmaceutical agent may each be added to at least one discrete system, e.g. , to assess whether the three pharmaceutical agents display an additive or synergistic effect. The at least one system may comprise a system that was contacted with the transcription inducer. The at least one system may comprise a system that was not contacted with the transcription inducer, e.g. , to serve as a control. The first pharmaceutical agent may be added to at least one system to which the second pharmaceutical agent and third pharmaceutical agent are not added, e.g. to serve as a control. The second pharmaceutical agent may be added to at least one system to which the first pharmaceutical agent and third pharmaceutical agent are not added, e.g. to serve as a control. The third pharmaceutical agent may be added to at least one system to which the second pharmaceutical agent and first pharmaceutical agent are not added, e.g. to serve as a control. A plurality of systems may comprise a system to which neither the first pharmaceutical agent, second pharmaceutical agent, nor third pharmaceutical agent is added, e.g. , to serve as a control. Each permutation of the three pharmaceutical agents may be added to systems that were contacted with the transcription inducer. Various permutations of the three pharmaceutical agents may be added to systems that were not contacted with the transcription inducer, e.g. , to serve as controls.

A method may comprise adding a fourth pharmaceutical agent to a group of systems (e.g. , a seventh group of systems). A fourth pharmaceutical agent may be a large molecule or a small molecule compound. For example, the third pharmaceutical agent may be a small molecule compound and the fourth pharmaceutical agent may be a small molecule compound.

A pharmaceutical agent may be added at a concentration of about 0.1 μg/mL to about 100 μg/mL, such as about 1 μg/mL to about 50 μg/mL, about 0.1 μg/mL to about 10 μg/mL, about 0.1 μg/mL to about 1 μg/mL, about 1 μg/mL to about 10 μg/mL, about 5 μg/mL to about 20 μg/mL, about 10 μg/mL to about 30 μg/mL, or about 20 μg/mL to about 50 μg/mL (e.g. , wherein the pharmaceutical agent is a large molecule, such as an antibody).

A pharmaceutical agent may be added at a concentration of about 100 pM to about 10 μΜ, such as about 100 pM to about 10 nM, about 500 pM to about 50 nM, about 1 nM to about 100 nM, about 5 nM to about 500 nM, about 10 nM to about 1 μΜ, about 50 nM to about 5 μΜ, about 100 pM to about 10 nM, about 500 pM to about 100 nM, about 1 nM to about 500 nM, about 1 nM to about 1 μΜ, or about 1 nM to about 5 μΜ (e.g. , for small molecule compounds). A method may further comprise adding a ligand-presenting cell to each system of a plurality of systems. The same ligand-presenting cell (e.g., different cells from the same cell line) may be added to each system. The ligand-presenting cell may be a B-cell, such as a Raji cell. The ligand-presenting cell may be a cell that is engineered to express a ligand of a checkpoint receptor (e.g. , PD-L1, PD-L2, B7.1, B7.2). The ligand-presenting cell may be added at a ratio of about 1: 10 to 10: 1 relative to the T-cell, such as about 1 :5 to about 5: 1, about 1 :4 to about 4: 1, about 1 :3 to about 3: 1, or about 1 :2 to about 2: 1. The ligand- presenting cell may be added after adding the costimulatory ligand. The ligand-presenting cell may be added after adding the one or more pharmaceutical agents.

A method may comprise measuring a concentration of a biomarker. Measuring the concentration of the biomarker may comprise measuring the concentration of the biomarker in the supernatant of a system. Measuring may comprise performing an enzyme-linked immunosorbent assay (ELISA) or other colorimetric immunoassay. The biomarker may be interleukin-2 or interferon γ. A method may comprise contacting a system or supernatant thereof with one or more of a primary antibody, an anti-interleukin-2 antibody, an anti- interferon γ antibody, a secondary antibody, an anti-IgG antibody, an antibody-fluorophore conjugate, an antibody-biotin conjugate, an antibody-enzyme conjugate, a colorimetric substrate for horseradish peroxidase, and a colorimetric substrate for alkaline phosphatase. A method may comprise contacting each system of a plurality of systems (or the supernatants thereof) with one or more of a primary antibody, an anti-interleukin-2 antibody, an anti- interferon γ antibody, a secondary antibody, an anti-IgG antibody, an antibody-fluorophore conjugate, an antibody-biotin conjugate, an antibody-enzyme conjugate, a colorimetric substrate for horseradish peroxidase, and a colorimetric substrate for alkaline phosphatase. A method may further comprise comparing the relative concentration of the biomarker in at least one system of the plurality of systems to the relative concentration of the biomarker in at least one other system of the plurality of systems.

A. Discovery and validation of novel targets

Various aspects of the invention relate to methods of validating a T-cell target gene. A method may comprise providing an inducible T-cell system as described herein. The inducible T-cell system may comprise a T-cell engineered to inducibly express two or more checkpoint receptors; a checkpoint receptor ligand-presenting cell engineered to

constitutively express one or more checkpoint receptor ligands; and/or an anti-CD3 antibody to stimulate TCR signaling. The T-cell may express the T-cell receptor (TCR) and a costimulatory receptor such as CD28. The T-cell may be a Jurkat cell. The ligand-presenting cell may express a costimulatory ligand (e.g. , B7.1 and/or B7.2). The costimulatory ligand may be useful to provide a costimulatory signal. The ligand-presenting cell may be a B-cell, such as a Raji cell.

The T-cell may comprise a gene knockout or knockdown, as described herein, supra.

A gene knockout may be made, for example, using a CRISPR/Cas9 strategy. The T-cell engineered to inducibly express two or more checkpoint receptors may therefore be a T-cell comprising a gene knockout or knockdown, such as a gene knockout generated using a CRISPR/Cas9 strategy. A method may comprise providing a CRISPR/Cas9 system to knockout a candidate T-cell gene.

A method may further comprise inducing the expression of the two or more checkpoint receptors in the T-cell. For example, the two or more checkpoint receptors may be operably-linked to a promoter that is inducible by doxycycline, and inducing the expression of two or more checkpoint receptors may comprise contacting the T-cell (or the inducible T-cell system) with doxycycline.

TCR signaling (e.g. , T-cell activation) is measurable by monitoring the interleukin-2 (IL2) expression of a T-cell. A method may further comprise determining the level of IL2 expression of the T-cell as compared to a control level of IL2 expression in a non-induced control T-cell. A method may further comprise determining the level of IL2 expression of the T-cell as compared to a control level of IL2 expression in an induced control T-cell that does not comprise the gene knockout or gene knockdown.

TCR signaling is measurable by monitoring other molecules, such as interferon γ (INFy), and thus, a method may further comprise determining the level of INFy expression of the T-cell as compared to a control level of INFy expression in a non-induced control T-cell. A method may further comprise determining the level of INFy expression of the T-cell as compared to a control level of INFy expression in an induced control T-cell that does not comprise the gene knockout or gene knockdown.

A method may thereby identify if the candidate T-cell gene is a T-cell checkpoint target gene.

Various aspects of the invention relate to a method of discovering or validating a drug target, comprising providing a plurality of inducible T-cell systems as described herein, supra, and measuring the concentration of a biomarker associated with T-cell activation (e.g. , interleukin-2 or interferon γ) in at least two systems of the plurality. For example, a plurality of inducible T-cell systems may include a system comprising a T-cell having a knockout or knockdown of a gene encoding the drug target. The plurality may further include a system comprising a T-cell lacking the knockout or knockdown of the gene. The method may further comprise comparing the relative concentration of the biomarker in the T-cell system comprising the knockout or knockdown and the T-cell system lacking the knockout or knockdown. The drug target is validated if the system comprising the T-cell having the knockout or knockdown displays increased T-cell activation relative to the system

comprising the T-cell lacking the knockout or knockdown.

B. Small molecule screening and validation

Various aspects of the invention relate to methods of identifying checkpoint inhibitors. A method may comprise providing an inducible T-cell system as described herein. The inducible T-cell system may comprise a T-cell engineered to inducibly express two or more checkpoint receptors; a checkpoint receptor ligand-presenting cell engineered to constitutively express one or more checkpoint receptor ligands; and/or an anti-CD3 antibody to stimulate TCR signaling. The T-cell may express the T-cell receptor (TCR) and a costimulatory receptor such as CD28. The T-cell may be a Jurkat cell. The ligand-presenting cell may express a costimulatory ligand (e.g. , B7.1 and/or B7.2). The costimulatory ligand may be useful to provide a costimulatory signal. The ligand-presenting cell may be a B-cell, such as a Raji cell.

A method may further comprise inducing the expression of the two or more checkpoint receptors in the T-cell. For example, the two or more checkpoint receptors may be operably-linked to a promoter that is inducible by doxycycline, and inducing the expression of two or more checkpoint receptors may comprise contacting the T-cell (or the inducible T-cell system) with doxycycline.

A method may further comprise contacting the T-cell (or inducible T-cell system) with one or more candidate compounds. A candidate compound may be, for example, a pharmaceutical agent, such as a small molecule compound or a large molecule as described herein, supra.

TCR signaling (e.g. , T-cell activation) is measurable by monitoring the interleukin-2 (IL2) expression of a T-cell. A method may further comprise determining the level of IL2 expression of the T-cell as compared to a control level of IL2 expression in a non-induced control T-cell. A method may further comprise determining the level of IL2 expression of the T-cell as compared to a control level of IL2 expression in an induced control T-cell that is not contacted with the one or more candidate compounds. TCR signaling is measurable by monitoring other molecules, such as interferon γ (INFy), and thus, a method may further comprise determining the level of INFy expression of the T-cell as compared to a control level of INFy expression in a non-induced control T-cell. A method may further comprise determining the level of INFy expression of the T-cell as compared to a control level of INFy expression in an induced control T-cell that is not contacted with the one or more candidate compounds.

A method may thereby identify if the one or more candidate compounds are checkpoint inhibitors.

Various aspects of the invention relate to a method of determining whether a small molecule compound affects T-cell activation, comprising providing a plurality of inducible T- cell systems as described herein, supra, and measuring the concentration of a biomarker associated with T-cell activation (e.g. , interleukin-2 or interferon γ) in at least two systems of the plurality. For example, a plurality of inducible T-cell systems may include a system comprising the small molecule compound. The plurality may further include a system lacking the small molecule compound. The method may further comprise comparing the relative concentration of the biomarker in the T-cell system comprising the small molecule compound and the T-cell system lacking the small molecule compound. The small molecule compound affects T-cell activation if the system comprising the small molecule compound displays different T-cell activation relative to the system lacking small molecule compound. A small molecule compound may be a viable drug candidate for use in treating cancer or infection, for example, if the system comprising the small molecule compound displays increased T-cell activation relative to the system lacking small molecule compound. A small molecule compound may be a viable drug candidate for use in autoimmune disease, for example, if the system comprising the small molecule compound displays attenuated T-cell activation relative to the system lacking small molecule compound.

C. Drug interaction screening

Various aspects of the invention relate to methods of assessing interactions between checkpoint inhibitors. A method may comprise providing an inducible T-cell system as described herein. The inducible T-cell system may comprise a T-cell engineered to inducibly express two or more checkpoint receptors; a checkpoint receptor ligand-presenting cell engineered to constitutively express one or more checkpoint receptor ligands; and/or an anti- CD3 antibody to stimulate TCR signaling. The T-cell may express the T-cell receptor (TCR) and a costimulatory receptor such as CD28. The T-cell may be a Jurkat cell. The ligand- presenting cell may express a costimulatory ligand (e.g. , B7.1 and/or B7.2). The

costimulatory ligand may be useful to provide a costimulatory signal. The ligand-presenting cell may be a B-cell, such as a Raji cell.

A method may further comprise inducing the expression of the two or more checkpoint receptors in the T-cell. For example, the two or more checkpoint receptors may be operably-linked to a promoter that is inducible by doxycycline, and inducing the expression of two or more checkpoint receptors may comprise contacting the T-cell (or the inducible T-cell system) with doxycycline.

A method may further comprise contacting the T-cell (or inducible T-cell system) with two or more checkpoint inhibitors. A checkpoint inhibitory drug may be, for example, an FDA-approved checkpoint inhibitory drug. A checkpoint inhibitory drug may be a monoclonal antibody, such as an anti-CTLA or anti-PD-1 antibody. The two or more checkpoint inhibitors may comprise a small molecule compound as described herein, supra.

TCR signaling (e.g. , T-cell activation) is measurable by monitoring the interleukin-2 (IL2) expression of a T-cell. A method may further comprise determining the level of IL2 expression of the T-cell as compared to a control level of IL2 expression in a non-induced control T-cell. A method may further comprise determining the level of IL2 expression of the T-cell as compared to a control level of IL2 expression in an induced control T-cell that is contacted with less than two or more checkpoint inhibitors, such as a single individual checkpoint inhibitor.

TCR signaling is also measurable by monitoring other molecules, such as interferon γ (INFy), and thus, a method may further comprise determining the level of INFy expression of the T-cell as compared to a control level of INFy expression in a non-induced control T-cell. A method may further comprise determining the level of INFy expression of the T-cell as compared to a control level of INFy expression in an induced control T-cell that is contacted with less than two or more checkpoint inhibitors, such as a single individual checkpoint inhibitor.

A method may thereby assess the interaction between the one or more checkpoint inhibitors.

Various aspects of the invention relate to a method of assessing an interaction between a small molecule compound and an antibody that binds a checkpoint receptor. The method may comprise providing a plurality of inducible T-cell systems as described herein, supra, and measuring the concentration of a biomarker associated with T-cell activation (e.g. , interleukin-2 or interferon γ) in at least two systems of the plurality. For example, a plurality of inducible T-cell systems may include a system comprising both the small molecule compound and the antibody. The plurality may further include a system comprising the small molecule compound and lacking the antibody, a system lacking the small molecule compound and comprising the antibody, and/or a system lacking the small molecule compound and lacking the antibody. The method may further comprise comparing the relative concentration of the biomarker in the T-cell system comprising both the small molecule compound and the antibody with a T-cell system lacking either the small molecule compound, the antibody, or both. Assessing an interaction between the small molecule compound and the antibody may comprise determining whether the two pharmaceutical agents display an additive or synergistic effect, e.g., based on one or more comparisons.

Various aspects of the invention relate to a method of assessing an interaction between a first small molecule compound and a second small molecule compound. The method may comprise providing a plurality of inducible T-cell systems as described herein, supra, and measuring the concentration of a biomarker associated with T-cell activation (e.g. , interleukin-2 or interferon γ) in at least two systems of the plurality. For example, a plurality of inducible T-cell systems may include a system comprising the first small molecule compound and the second small molecule compound. The plurality may further include a system comprising the first small molecule compound and lacking the second small molecule compound, a system lacking the first small molecule compound and comprising the second small molecule compound, and/or a system lacking both the first small molecule compound and the second small molecule compound. The method may further comprise comparing the relative concentration of the biomarker in the T-cell system comprising both the first small molecule compound and the second small molecule compound with a T-cell system lacking either the first small molecule compound, the second small molecule compound, or both. Assessing an interaction between the first small molecule compound and the second small molecule compound may comprise determining whether the two small molecule compounds display an additive or synergistic effect, e.g. , based on one or more comparisons.

Various aspects of the invention relate to a method of assessing an interaction between a first large molecule and a second large molecule. The method may comprise providing a plurality of inducible T-cell systems as described herein, supra, and measuring the concentration of a biomarker associated with T-cell activation (e.g. , interleukin-2 or interferon γ) in at least two systems of the plurality. For example, a plurality of inducible T- cell systems may include a system comprising both the first large molecule and the second large molecule. The plurality may further include a system comprising the first large molecule and lacking the second large molecule, a system lacking the first large molecule and comprising the second large molecule, and/or a system lacking both the first large molecule and the second large molecule. The method may further comprise comparing the relative concentration of the biomarker in the T-cell system comprising both the first large molecule and the second large molecule with a T-cell system lacking either the first large molecule, the second large molecule, or both. Assessing an interaction between the first large molecule and the second large molecule may comprise determining whether the two large molecules display an additive or synergistic effect, e.g. , based on one or more comparisons. D. Analysis of mechanism of action

Various aspects of the invention relate to methods of interrogating ON or OFF target activity of a small molecule compound. A method may comprise providing an inducible T- cell system as described herein. The inducible T-cell system may comprise a T-cell engineered to inducibly express two or more checkpoint receptors; a checkpoint receptor ligand-presenting cell engineered to constitutively express one or more checkpoint receptor ligands; and/or an anti-CD3 antibody to stimulate TCR signaling. The T-cell may express the T-cell receptor (TCR) and a costimulatory receptor such as CD28. The T-cell may be a Jurkat cell. The ligand-presenting cell may express a costimulatory ligand (e.g. , B7.1 and/or B7.2). The costimulatory ligand may be useful to provide a costimulatory signal. The ligand-presenting cell may be a B-cell, such as a Raji cell.

The T-cell may comprise a gene knockout or knockdown, as described herein, supra. A gene knockout may be made, for example, using a CRISPR/Cas9 strategy. The T-cell engineered to inducibly express two or more checkpoint receptors may therefore be a T-cell comprising a gene knockout or knockdown, such as a gene knockout generated using a CRISPR/Cas9 strategy. A method may comprise providing a CRISPR/Cas9 system to knockout a candidate T-cell gene.

A method may further comprise inducing the expression of the two or more checkpoint receptors in the T-cell. For example, the two or more checkpoint receptors may be operably-linked to a promoter that is inducible by doxycycline, and inducing the expression of two or more checkpoint receptors may comprise contacting the T-cell (or the inducible T-cell system) with doxycycline.

A method may further comprise contacting the T-cell (or the inducible T-cell system) with a pharmaceutical agent, such as a small molecule compound. The pharmaceutical agent may specifically bind and/or inhibit the product encoded by the knockout gene or knockdown gene, or the pharmaceutical agent may specifically bind and/or inhibit a molecule upstream or downstream of the product in a signaling pathway that includes the product. For example, the gene knockout or gene knockdown may be a kinase and the pharmaceutical agent may be a kinase inhibitor. Similarly, the gene knockout or gene knockdown may be a substrate of a kinase and the pharmaceutical agent may be a kinase inhibitor.

TCR signaling (e.g. , T-cell activation) is measurable by monitoring the interleukin-2 (IL2) expression of a T-cell. A method may further comprise determining the level of IL2 expression of the T-cell as compared to a control level of IL2 expression in a non-induced control T-cell. A method may further comprise determining the level of IL2 expression of the T-cell as compared to a control level of IL2 expression in an induced control T-cell that does not comprise the gene knockout or gene knockdown.

TCR signaling is measurable by monitoring other molecules, such as interferon γ (INFy), and thus, a method may further comprise determining the level of INFy expression of the T-cell as compared to a control level of INFy expression in a non-induced control T-cell. A method may further comprise determining the level of INFy expression of the T-cell as compared to a control level of INFy expression in an induced control T-cell that does not comprise the gene knockout or gene knockdown.

A method may thereby identify if the candidate T-cell gene is a T-cell checkpoint target gene.

Various aspects of the invention relate to a method of assessing whether a protein affects T-cell activation through a signaling pathway mediated by a checkpoint receptor. The protein may be, for example, a kinase or phosphatase. The method may comprise providing a plurality of inducible T-cell systems as described herein, supra, and measuring the concentration of a biomarker associated with T-cell activation (e.g., interleukin-2 or interferon γ) in at least two systems of the plurality. For example, a plurality of inducible T- cell systems may include a system comprising a checkpoint inhibitory antibody and a T-cell having a knockout or knockdown of a gene encoding the protein. The plurality may further include a system comprising the checkpoint inhibitory antibody and comprising a T-cell lacking the knockout or knockdown of the gene, a system lacking the checkpoint inhibitory antibody and comprising a T-cell having the knockout or knockdown of the gene, and/or a system lacking the checkpoint inhibitory antibody and comprising a T-cell lacking the knockout or knockdown of the gene. The method may further comprise comparing the relative concentration of the biomarker in the T-cell system comprising the checkpoint inhibitor antibody and T-cell comprising the knockout or knockdown with a T-cell system lacking either the checkpoint inhibitor antibody, the knockout/knockdown, or both. A protein affects T-cell activation through a signaling pathway mediated by the checkpoint receptor targeted by the checkpoint inhibitory antibody if T-cell activation in the system comprising the checkpoint inhibitory antibody and knockout/knockdown is different from the system comprising the checkpoint inhibitory antibody and lacking the knockout/knockdown.

Various aspects of the invention relate to a method of assessing whether a pharmaceutical agent displays an ON target or OFF target effect. The method may comprise providing a plurality of inducible T-cell systems as described herein, supra, and measuring the concentration of a biomarker associated with T-cell activation (e.g. , interleukin-2 or interferon γ) in at least two systems of the plurality. For example, a plurality of inducible T- cell systems may include a system comprising both a pharmaceutical agent and a T-cell having a knockout or knockdown of a gene encoding the target. The plurality may further include a system comprising the pharmaceutical agent and a T-cell lacking the knockout or knockdown of the gene, a system lacking the pharmaceutical agent and comprising a T-cell having the knockout or knockdown of the gene, and/or a system lacking the pharmaceutical agent and comprising a T-cell lacking the knockout or knockdown of the gene. The method may further comprise comparing the relative concentration of the biomarker in the T-cell system comprising both the pharmaceutical agent and the T-cell comprising the knockout or knockdown with a T-cell system lacking either the pharmaceutical agent, the

knockout/knockdown, or both. The pharmaceutical agent may display an ON target effect, for example, if the system comprising the pharmaceutical agent and knockout/knockdown displays similar T-cell activation as the system comprising the pharmaceutical agent and lacking the knockout/knockdown and/or similar T-cell activation as the system lacking the pharmaceutical agent and comprising the knockout/knockdown.

EXEMPLIFICATION

While certain cells, compositions, and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same.

Example 1. T-cells expressing doxy cycline -inducible PD-1 and CTLA-4

Jurkat cells, which are immortalized human T-cells, were transfected with a gene for checkpoint receptor PD- 1 operably-linked to a doxycycline-inducible promoter and a gene for checkpoint receptor CTLA-4 operably-linked to a doxycycline-inducible promoter. Briefly, human checkpoint receptor cDNAs for PD- 1 and CTLA-4 were cloned into a pLVX- TetOne vector (Clontech) under the control of the doxycycline inducible TRE3G promoter. Jurkat cells were infected with lenti viral vectors pLVX-TetOne-PD-1 and pLVX-TetOne- CTLA-4 thereby generating a T-cell line configured to inducibly express PD-1 and CTLA-4.

Jurkat cells endogenously express CD28 and the T-cell receptor (TCR). Figure 1 includes a drawing of a T-cell expressing PD-1 and CTLA-4 transgenes as well as CD28 and the T-cell receptor (TCR). CD28 is a costimulatory receptor required for T-cell activation and survival. T-cell stimulation through CD28 in addition to the T-cell receptor can provide a potent signal for the production of various cytokines including interleukin-2 (IL2). CD28 may be activated by ligands B7.1 (CD80) and B7.2 (CD86) as well as certain anti-CD28 antibodies. The T-cell receptor may be activated by engaging an antigen bound to a major histocompatibility complex (MHC) molecule or by engaging an anti-TCR antibody such as an anti-CD3 antibody.

Example 2. B-cells constitutively expressing PD-L1 or PD-L2

Raji cells, which are immortalized human B-cells, were transfected with a gene for either PD-L1 or PD-L2. Briefly, human checkpoint ligand cDNAs for PD-L1 and PD-L2 were each cloned into a pLVX-IRES-Puro vector (Clontech) under expression of a constitutive CMV promoter. Raji cells were infected with a lentiviral vector pLVX-IRES- Puro-PD-Ll or pLVX-IRES-Puro-PD-L2, thereby generating B-cells that constitutively express either PD-L1 or PD-L2.

PD-L1 and PD-L2 are checkpoint receptor ligands, which bind to checkpoint receptor PD-1, thereby inhibiting T-cell activation. Raji cells also endogenously express checkpoint receptor ligands B7.1 (CD80) and B7.2 (CD86), which bind to checkpoint receptor CTLA-4. B7.1 and B7.2 may also activate T-cells by binding CD28. Figure 1 includes a drawing of a B-cell expressing either a PD-L1 transgene or a PD-L2 transgene (PD-L1/PD-L2) as well as B7.1 and B7.2 (B-7.1/B-7.2).

Example 3. Expression of PD-1 and CTLA-4 checkpoint receptors in the presence of a PD-1 checkpoint ligand (PD-L1 or PD-L2) and a CTLA-4 checkpoint ligand (B7.1 or B7.2) decreases Ί -cell-mediated interleukin-2 production

The Jurkat cells expressing inducible PD-1 and CTLA-4 transgenes described in Example 1 were combined with (a) the Raji cells constitutively expressing either a PD-L1 or PD-L2 transgene as well as B7.1 and B7.2 as described in Example 2 and (b) anti-CD3 beads (CD3 Ab). The anti-CD3 beads were prepared by coating anti-human CD3 antibodies from eBioscience to Dynabeads M-450 Epoxy beads from Invitrogen using Invitrogen' s coating protocol. Briefly, 700 of Dynabeads M-450 Epoxy beads were separated from

supernatant using a magnet. 616 of phosphate-buffered saline (PBS, pH 7.4) was added to the beads followed by 140 of anti-human CD3 antibodies from eBioscience. The mixture was incubated at room temperature for 15 minutes on a rotary mixer. 84 μΐ _^ οί 1% bovine serum albumin (BSA) in PBS (pH 7.4) was added to a final concentration of 0.1% BSA. The mixture was incubated at room temperature for 16-24 hours on a rotary mixer. The beads were then washed 3 times with 1 mL of 0.1% BSA in PBS (pH 7.4). The beads were resuspended in 1120 μΐ, of 0.1% BSA in PBS (pH 7.4) resulting in a lOx stock solution of 200,000 beads per 10 μΐ,. Prior to adding the beads to cells, the PBS is removed and the beads were diluted with RPMI media containing 10% fetal bovine serum (FBS).

The inducible Jurkat cells described in Example 1 were induced with 250 ng/niL doxycycline for 2 days. Control samples without doxycycline were cultured in parallel. After the 2 days, the cell media was changed to wash out the doxycycline, and the cells were diluted to 1,000,000 cells in 900 μΐ, of RPMI media containing 10% fetal bovine serum (FBS). Raji cells constitutively expressing either PD-Ll or PD-L2 as described in Example 2 were diluted to 1,000,000 cells in 1000 μΐ, of RPMI media containing 10% FBS. 2,000,000 anti-CD3 beads in 50 μΐ, were added to 450 μΐ, of the Jurkat cells (500,000 cells), i.e. , at a ratio of four anti-CD3 beads to one Jurkat cell (4: 1). The Jurkat cells and beads were incubated at room temperature for 30 minutes. 500 μΐ, of the Raji cells (500,000 cells) were then added to the Jurkat cells (500,000 cells), and beads (2,000,000 beads), i.e. , at a ratio of one Raji cell to one Jurkat cell to 4 beads (1: 1 :4), thereby resulting in 1000 μΐ, total volume. 200 μΐ, of each sample was then transferred into a 96-well U-bottom plate, and the plate was incubated at 37°C for 22 hours.

After 22 hours, the 96-well plates were centrifuged at 1000 rpm for 5 minutes. 150 μΐ _^ of supernatant was transferred into a new 96-well U-bottomed plate. The new 96-well plate was optionally frozen. 100 μΐ, of supernatant was then used in an interleukin-2 (IL2) ELISA to determine the concentration of IL2 secreted by the Jurkat cells.

The Jurkat cells secreted IL2 in the absence of doxycycline (- dox), and interleukin-2 secretion decreased upon the addition of doxycycline (+ dox). This finding suggests that the doxycycline-inducible expression of checkpoint receptors PD-1 and CTLA-4 decreases T-cell activation in the presence of PD- 1 and CTLA-4 checkpoint ligands, which is consistent with canonical checkpoint inhibition (Figure 2). Example 4. T-cells expressing inducible PD-1 and CTLA-4 display increased interleukin-2 secretion in the presence of anti-PD-1 antibody nivolumab and anti-CTLA-4 antibody ipilimumab

The anti-PD-1 antibody nivolumab (Nivo; N) and/or the anti-CTLA-4 antibody ipilimumab (Ipi; I) were added to the Jurkat/Raji/anti-CD3 bead system described in Example 3. Briefly, 5-25 μg of nivolumab and/or ipilimumab was added to Jurkat cells in a protocol similar to that described in Example 3. The antibodies were added to the system after adding the anti-CD3 beads to the Jurkat cells and before incubating the Jurkat cells for 30 minutes at room temperature. A control IgG antibody (IgG) was added to a different sample as a negative control. Individually, the anti-PD-1 antibody and the anti-CTLA-4 antibody partially restored interleukin-2 (IL2) secretion in the presence of doxycycline-induced PD-1 and CTLA-4 expression (Figure 3B, experiments "3" and "4"). The combination of the anti- PD-1 antibody and the anti-CTLA-4 antibody displayed an additive effect (Figure 3B, experiment "5").

Example 5. T-cells expressing inducible PD-1 and CTLA-4 and comprising a gene knockout display increased interleukin-2 expression

A gene involved in the negative regulation of T-cells was knocked out of the Jurkat cells described in Example 1 using a CRISPR/Cas9 knockout strategy. Briefly, single guide RNAs (sgRNAs) and Cas9 were co-delivered to the Jurkat cells described in Example 1 using a single lentiviral vector. The knockout cells were incubated with Raji cells, anti-CD3 beads, and doxycycline as described in Example 3. The knockout cells displayed high interleukin-2 (IL2) secretion, suggesting that the knockout gene could be targeted to activate T-cells in the context of immune checkpoint inhibition (Figure 4).

The knockout cells were incubated with Raji cells, anti-CD3 beads, doxycycline, anti- PD-1 antibody nivolumab (Nivo; N), and anti-CTLA-4 antibody ipilimumab (Ipi; I) as described in Example 4. The gene knockout and the anti-PD- 1 and anti-CTLA-4 antibodies displayed an additive effect on interleukin-2 (IL2) secretion (Figure 5, experiments "3," "4," and "5"). This finding suggests that a drug that inhibits the product of the knockout gene will display an additive effect when used in combination with nivolumab and/or ipilimumab. Example 6. Small molecules "compound 1 " and "compound 2 " display an additive effect in combination with anti-PD-1 antibody nivolumab and anti-CTLA-4 antibody ipilimumab on T- cell mediated interleukin-2 secretion in the presence of checkpoint inhibitors

Jurkat cells, Raji cells, and anti-CD3 beads were incubated in the presence of doxycycline as described in Example 3 and either (a) anti-PD-1 antibody nivolumab and anti- CTLA-4 antibody ipilimumab (b) small molecule compound 1 or small molecule compound 2, or (c) nivolumab, ipilimumab, and either compound 1 or compound 2. The small molecules compound 1 and compound 2 displayed a synergistic effect on interleukin-2 (IL2) secretion in combination with both nivolumab and ipilimumab (Figure 5, experiments "3," "6," and "7").

Example 7. The effect of small molecules on T-cell mediated interleukin-2 secretion disappears in T-cells comprising a gene knockout

Inducible T-cells comprising or lacking a gene knockout were incubated with Raji cells, anti-CD3 beads, doxycycline, and either compound 1, compound 2, or vehicle

(DMSO). Control experiments were performed with either compound 1, compound 2, or vehicle and T-cells that did not include the knockout. Both compound 1 and compound 2 increased T-cell activation relative to the vehicle control in T-cells that did not include the knockout (Figure 6C). Neither compound 1 nor compound 2 increased T-cell activation relative to the vehicle control in T-cells that included the knockout (Figure 6C). These results suggest that both compound 1 and compound 2, which are designed to target the protein encoded by the knockout gene, display an "ON" target effect, i. e. , that compound 1 and compound 2 specifically target the protein product of the knockout gene. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims: