Immunomodulation improves cancer cell clearance by T cells during multi-antigent T cell hybridizer therapy

Treatment of hematopoietic malignancies is challenging due to systemic side effects of current treatments and the possibility of relapse. Multi-antigen T cell hybridizer (MATCH) therapy is a novel treatment system composed of antibody fragments conjugated with morpholino oligonucleotides, which hybridize upon contact and direct autologous T cells against cancerous B cells. However, T cell exhaustion remains a challenging obstacle to the efficacy and duration of MATCH therapy. In this study, co-cultures of healthy human T cells and Raji B lymphoma cells were treated with MATCH therapy in vitro. Subsequent T cell activation, effector function, and exhaustion were assayed using immunohistochemistry and flow cytometry for corresponding cell surface markers. Interferon (IFN)-γ release was quantified using enzyme-linked immunosorbent assay. MATCH activation of T cells induced increases in PD-1 expression and IFN-γ release during the first 72 hours of co-culture and treatment. Multiple strategies aimed at reducing exhaustion and improving T cell ablation of lymphoma cells were then assessed. Ibrutinib pre-treatment of T cells moderately attenuated PD-1 expression but did not improve cancer cell clearance. Simultaneous administration of MATCH therapy with either PD-1 or IL-10 blockade reduced T cell PD-1 expression and markedly improved cancer cell clearance. These results suggest that immunomodulation, in the form of antibody blockades, has the potential to attenuate T cell exhaustion and enhance T cell effector functions during

All Rights Reserved ABSTRACT Treatment of hematopoietic malignancies is challenging due to systemic side effects of current treatments and the possibility of relapse. Multi-antigen T cell hybridizer (MATCH) therapy is a novel treatment system composed of antibody fragments conjugated with morpholino oligonucleotides, which hybridize upon contact and direct autologous T cells against cancerous B cells. However, T cell exhaustion remains a challenging obstacle to the efficacy and duration of MATCH therapy. In this study, co-cultures of healthy human T cells and Raji B lymphoma cells were treated with MATCH therapy in vitro. Subsequent T cell activation, effector function, and exhaustion were assayed using immunohistochemistry and flow cytometry for corresponding cell surface markers. Interferon (IFN)-γ release was quantified using enzyme-linked immunosorbent assay. MATCH activation of T cells induced increases in PD-1 expression and IFN-γ release during the first 72 hours of co-culture and treatment. Multiple strategies aimed at reducing exhaustion and improving T cell ablation of lymphoma cells were then assessed. Ibrutinib pre-treatment of T cells moderately attenuated PD-1 expression but did not improve cancer cell clearance. Simultaneous administration of MATCH therapy with either PD-1 or IL-10 blockade reduced T cell PD-1 expression and markedly improved cancer cell clearance. These results suggest that immunomodulation, in the form of antibody blockades, has the potential to attenuate T cell exhaustion and enhance T cell effector functions during ii MATCH therapy. Further, this study validates the ability of MATCH therapy to direct human T cells to ablate B lymphoma cells in vitro. iii TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 BACKGROUND 4 METHODS 15 RESULTS 24 DISCUSSION 33 REFERENCES 42 iv 1 INTRODUCTION Hematopoietic malignancies, including leukemia, lymphoma, and multiple myeloma, affect millions of individuals globally [1], [2]. Combating these complex cancers is difficult due to their systemic nature and the possibility of relapse [3]. Further, treatment for hematopoietic malignancies is often expensive and complicated, putting significant financial and emotional burdens on those suffering from them. Traditionally, small molecule drug treatment, also known as chemotherapy, has been considered the standard first-line option for treating such cancers [4]. However, this approach is complicated by systemic side effects and toxicity, which can be physically and emotionally devastating to patients. In response to these challenges, immunotherapy, or the directed activation of the immune system for therapeutic purposes, has gained widespread use in clinical settings [5]. Many current immunotherapy developments focus on recruiting a patient’s T cells. T cells target and eliminate other cells based on specific chemical markers and signals, especially in response to viral infections or cellular damage. T cell recruitment strategies, such as bispecific T cell engagers (BiTEs) [6] or chimeric antigen receptor (CAR) T cells [7], co-opt the preexisting immune defenses of T cells against damaged cells. However, T cell recruitment therapies are limited by the ability of cancer cells to undergo changes in expression of the target molecules, bypassing T cell target-specific functions, leading to relapse. Multi-antigen T cell hybridizer (MATCH) therapy is a novel treatment which readily overcomes the difficulty of changing target molecule expression and the possibility of relapse while maintaining a comparable ability to eliminate cancer cells. MATCH therapy is a two-component system: each component consists of an antibody fragment 2 conjugated with a short strand of modified deoxyribonucleic acid (a morpholino oligonucleotide). Antibody fragments with specificity for both T cell (effector cell) markers and B cell (target cell) markers have been successfully used to produce MATCH conjugates. Two MATCH conjugates are administered, and the interaction of their complementary nucleic acid sequences induces the effector cells to act upon and eliminate the target cells. Due to the multi-component structure of MATCH, treatment conjugates can be selected in patient-specific combinations to improve target cell identification and elimination. These conjugates can also be administered either simultaneously or consecutively. Additionally, MATCH therapy readily counters relapse because the conjugates can be easily alternated or exchanged during treatment, to match the changing cellular expression which is often observed in cancer cells after treatment has begun. While MATCH therapy is superior to other T cell-recruiting therapies with regard to this increased ability to counter relapse, it is nevertheless challenged (along with all other T cell-recruitment strategies) by the phenomenon of T cell exhaustion. T cell exhaustion is the process whereby the repeated stimulation of T cells during their recruitment against target cells induces: 1) a drastic reduction in T cell function and proliferation and 2) the appearance of external cellular exhaustion markers [8]. T cell exhaustion is poorly understood and due to the novelty of MATCH therapy, it has not yet been explored or quantified in response to MATCH treatment. The specific cellular pathways by which T cells are exhausted are not clearly established in general, nor which signals are necessary and sufficient to induce exhaustion [9]. It is also not known to what degree MATCH treatment induces exhaustion or how to potentially remediate and reverse these trends after MATCH treatment. Hypothetically, ‘co-treatments’ (directed at T cells) could be 3 introduced simultaneously with MATCH treatment to attenuate T cell exhaustion and improve MATCH therapy efficacy. This co-treatment strategy has been successfully employed for several other cancer therapies [10], [11], [12]. The aims of this study were twofold, including a general aim and a specific aim. First, the general aim of this study was to validate the efficacy of MATCH therapy as an alternative form of immunotherapy. Second, the specific aim of this study was to validate a paradigm for quantifying T cell exhaustion, to identify robust cellular exhaustion markers to measure T cell exhaustion after MATCH treatment, and to identify potential cotreatments for attenuating MATCH therapy-induced T cell exhaustion. To quantify T cell exhaustion in vitro, Raji cells (target cancer cells) and healthy human donor T cells (effector cells) were co-cultured and treated with MATCH multiple times over the course of several days. Cell markers for proliferation, effector function, and exhaustion were measured by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Cotreatments for the attenuation of T cell exhaustion were then administered to co-cultures of target and effector cells in conjunction with MATCH, and exhaustion markers were similarly quantified to determine if the co-treatment reduced exhaustion during MATCH treatment. These results will help the MATCH development team to dose treatments more effectively, and to explore co-treatments to improve MATCH efficacy. More generally, the development of MATCH therapy will provide clinicians an improved method for treating hematopoietic cancers with high specificity and a versatile potential for overcoming relapse. 4 BACKGROUND Leukemia, lymphoma, and multiple myeloma are three types of cancer originating in hematopoietic tissues. Due to the circulatory nature of blood, these malignancies are phenotypically different from ‘solid-tumor’ cancers. While some specific cases may be more localized to lymphoid and myeloid tissues, such as lymph nodes and bone marrow, the systemic impact is usually severe. Normal blood cells are crowded out and outcompeted by aggressive malignant cells, resulting in diminished immune function, fever and generalized pain [13]. To study the effect of novel therapies on cancerous cells, immortalized cells lines are used for experiments to control for cellular diversity. These cell lines arise from a cell acquired from a patient’s tumor and can be cultured indefinitely for experiments and observation outside of the body. Raji is an immortalized B cell lymphoma cell line and was the first human hematopoietic-derived cell line [14]. It is used as the model target cell for all experiments in this study. To combat these cancers, chemotherapies were developed with systemic toxicity and general specificity. Because cancerous cells are characterized by rapid proliferation and division, targeting these processes is likely to preferentially affect cancerous cells [15]. However, due to their general specificity, such treatments interfere with all cells undergoing such processes, including healthy cells. Thus, the side effects of systemic chemotherapy are pronounced in rapidly proliferating healthy cells, such as epithelial cells in the digestive system. A less systemically toxic and more specific therapy is desirable, and the solution arose in the form of monoclonal antibody therapy, from which MATCH is partially derived. 5 The development of monoclonal antibody therapy, which is one of the most common forms of immunotherapy, provided a powerful alternative to chemotherapy with less systemic toxicity and an increased ability to target cancer cells. By using antibodies with specificity for cancer cell markers, immunotherapy introduced a new approach to oncological treatments with greater specificity and less off-target systemic toxicity than traditional chemotherapy [16]. To date, monoclonal antibody therapy has been extensively studied and its mechanisms of cell targeting and elimination are well understood [17]. However, monoclonal antibody therapy is limited by the existence of a cancer cell-specific target, as well as the possibility for cancer cells to undergo changes in the expression of these markers, leading to relapse. Monoclonal antibodies are developed against a single specific molecular target by coopting the pre-existing mammalian cell machinery that produces antibodies in healthy organisms [18]. These antibodies also serve as one of the molecular starting points for the synthesis of MATCH conjugates, as explained below. Monoclonal antibodies are structurally homogeneous and can be administered intravenously to target human cells. For example, rituximab is a monoclonal antibody which targets the cell surface protein CD20, which is expressed on B cells during most of their maturation stages. Rituximab gained fame as the first antibody used in clinical settings in 1997 and has been used extensively as a therapy for a variety of medical conditions in the ensuing decades [19]. The MATCH conjugates targeting B cells used in this study are partially derived from rituximab antibodies. In general, the synthesis of any MATCH conjugate begins with acquiring a monoclonal antibody for the desired cellular target. 6 MATCH technology arose out of a strategy called drug-free macromolecular therapeutics (DFMT). Early DFMT experiments used synthetic water-soluble polymers to crosslink target receptors on cancerous cells, leading to cell death without the use of small molecule drugs [20], [21]. In the first iteration of DFMT, a monoclonal antibody fragment (Fab’) was conjugated to a specific peptide sequence and several identical copies of a second peptide sequence were grafted onto an N-(2-hydroxypropyl)methacrylamide (HPMA) polymer backbone (Figure 1-1). Upon contact, the two peptide sequences then interacted through hydrophobic and electrostatic interactions, creating a coiled-coil structure of antiparallel heterodimers. This system induced crosslinking of the Fab’targeted receptors, leading to target cell apoptosis (programmed cellular death) [22]. In other words, this system allowed cells to be programmed to die in a controlled way according to cell-specific markers on their surface, without directing other types of cells to die or using small molecule drugs. In the next generation of DFMT, the coiled-coil peptide components were replaced with complementary morpholino oligonucleotide sequences (MORF1 and MORF2) [23]. MORF1 and MORF2 spontaneously hybridize upon contact in a manner analogous to complementary strands of DNA (Figure 1-2). Targeting receptors on B cells using this modified construct successfully eliminated target cells [24]. The next major modification was replacing the HPMA copolymer backbone with human serum albumin (HSA) as an intermediary for crosslinking (Figure 1-3). Targeting and crosslinking receptors on B cells using this structure also induced apoptosis [25]. This final DFMT structure thus consisted of Fab’-MORF conjugates which were hybridized with complementary multivalent HSAMORF conjugates. However, researchers hypothesized that effector cells could be 7 Figure 1. The Four Generations of DFMT. DFMT induces cancer cell apoptosis without the use of small molecule drugs. (1) In the first generation, two short peptides were designed with complementary structures that formed a coiled-coil structure upon contact. One peptide was conjugated to a water-soluble polymer backbone (yellow), and the other was attached to an antibody fragment specific for a receptor on the target cell (red). Multiple binding events led to receptor ‘cross-linking’, killing the cancer cell. (2) In the second generation, the coiled-coil peptide motifs were replaced with two complementary morpholino oligonucleotides (MORF1 and MORF2). (3) In the third generation, the polymer-MORF backbone was replaced by HSA-MORF as the cross-linking mediator. (4) In the fourth and current generation of DFMT, the cross-linking function of HSA-MORF is replaced by T cell engagement. This is achieved by introducing a T cell-targeting Fab’-MORF conjugate, analogous to the original cancer cell-targeting conjugates. (5) A generic Fab’-MORF conjugate is composed of an antibody fragment (Fab’, right) which binds to a specific molecular target. This Fab’ is conjugated to a short morpholino oligonucleotide (MORF, left). Fab’-MORF conjugates with complementary MORF moieties (i.e. MORF1 and MORF2) spontaneously hybridize upon contact. recruited to interact with target B cell receptors, instead of simply cross-linking these receptors. Initial experiments demonstrated that switching the HSA-MORF component with a T cell-specific Fab’-MORF conjugate effectively and quickly eliminated target cells 8 through directed T cell activation. This system was subsequently named multi-antigen T cell hybridizers (MATCH) and is in current clinical development (Figure 1). Other strategies include T-cell engaging components as way of augmenting traditional antibody immunotherapy, similar to MATCH. For example, BiTEs build upon traditional monoclonal antibody therapy by targeting markers on both target cells and effector cells (cytotoxic “killer” T cells) with a single molecule to induce T cell-mediated death. These molecules are functionally bi-specific antibodies. Blinatumomab is a BiTE with specificity for CD3 (a global T cell marker) and CD19 (a global B cell marker). In clinical use for patients with acute lymphoblastic leukemia, the use of blinatumomab yielded an 80% remission rate for patients who had already experienced some degree of relapse [26]. Another analogous bi-specific molecule was developed with specificity for CS-1, a target on multiple myeloma cells, in place of CD19. This bispecific antibody induced type-1 cytokine production in activated T cells, as well as release of the cytotoxic molecule granzyme B [27]. Further, when a third novel BiTE with specificity for CD22 (another B cell marker) and CD3 was administered in conjunction with blinatumomab in vivo to treat leukemia in mice, the combination therapy outperformed either individual treatment [28]. MATCH therapy mirrors the structure and function of these BiTEs, with the notable difference that the targeting motifs are interchangeable in MATCH. Antibody and antibody-derived therapies are not the only successful forms of immunotherapy or T cell engagement. Another related approach is CAR T cell therapy. CAR T cells are biologically engineered T cells which are genetically programmed to express targeting receptors that recognize cancer cell markers [7], [29]. This leads to the genetically engineered direct killing of cancer cells by these modified CAR T cells. CAR 9 T cell therapy is an alternative and innovative approach to immunotherapy, relying on genetic modification to produce target-specific functions. T cell recruitment directly activates cytotoxic T cells against target cells to improve the frequency and ease of cellmediated cytotoxicity. As a result, CAR T cells have been successfully implemented to combat lymphoma, and CAR T cells with anti-CD19 specificity have yielded long-term remission in such cases [30]. However, such therapy is complicated by the systemic expression of potential target markers (in other words, the danger of non-target cytotoxicity) and fratricide of other CAR T cells [31]. Additionally, producing CAR T cells requires the isolation and genetic reprogramming of T cells, which can be expensive and time-consuming. In comparison, MATCH therapy does not require genetic modification. The two MATCH conjugates used in this study were Fab’RTX-MORF1 and Fab’CD3MORF2. This nomenclature signifies that the former MATCH conjugate is rituximabderived, and therefore has specificity for CD20, as described above. CD20 is a surface membrane protein found exclusively on B cells [32]. As a result, CD20 is an ideal B cellspecific marker for targeted immunotherapies against such malignancies. Therefore, the Fab’RTX-MORF1 conjugate serves as the B cell targeting motif in this study. Because Raji cells express high levels of CD20, MATCH systems employing this rituximab-based MATCH conjugate are specific for and effective against Raji cells. The latter MATCH conjugate is derived from a generic αCD3 antibody and serves as a T cell-targeting motif. CD3 is a surface protein found on all T cells which complexes with the T-cell receptor (TCR) and serves as its co-receptor [33]. TCR binding with a major histocompatibility complex on another autologous cell initiates T cell activation [34]. As such, MATCH conjugates derived from αCD3 antibodies mimic the natural stimulation and activation of 10 T cells through binding the TCR co-receptor, CD3. In essence, MATCH conjugates artificially stimulate the TCR pathway as if the targeted molecule were presented through the major histocompatibility complex. However, as with other T cell-engaging therapies, T cell exhaustion remains a significant hurdle to MATCH therapy. T cell exhaustion is a complex cellular phenomenon, whereby both internal and external factors induce a reduction in T cell effector functions [8]. Importantly, exhausted T cells are not dead or completely incapable of demonstrating effector functions, and these cells often prevent further tumor growth in place of completely ablating the tumor [35]. Several responsible internal cellular factors have been hypothesized, such as T cell factor 1 (TCF1), which is a critical factor for T cell renewal and differentiation [36], [37]. TCF1+ T cells retain an ability to proliferate and renew themselves, whereas T cells with low or absent expression of TCF1 are terminally exhausted [38]. Another marker more specific to the exhaustion pathway is thymocyte selection-associated high mobility group box protein (TOX), which is upregulated as T cells become terminally exhausted [38]. Deletion of TOX in antigen-activated T cells is sufficient to reduce exhaustion [39]. The search for external cell surface markers associated with exhaustion has yielded several promising candidates. Three such candidates are the inhibitory receptors: 1) killercell lectin like receptor G1 (KLRG1), 2) programmed cell death protein 1 (PD-1), and 3) T-cell immunoglobulin and mucin-domain containing-3 (TIM-3). KLRG1 has already been connected to the global process of senescence and demonstrates inhibitory functions in T cells with the progression of time [40]. KLRG1 is also expressed on another type of effector cell, natural killer (NK) cells, and activation of NK cells induces expression of 11 KLRG1 on the cell surface [41]. Finally, KLRG1 expression is linked to T cell exhaustion within non-cancerous contexts. In patients with HIV, exhaustion is marked by increased levels of KLRG1, just as in therapeutically-engaged T cells, as well as by increased levels of PD-1 [42]. PD-1, a second exhaustion marker, is a surface protein present on the surface of T cells, whose activation induces apoptosis in effector T cells and prevents apoptosis in regulatory T cells [43]. PD-1 expression increases after TCR activation, and remains at high levels with repeated antigenic stimulation [44]. It is both a surface exhaustion marker, as well as a therapeutic target, and therefore will be discussed below. Similar to PD-1, TIM-3 is another inhibitory receptor that highly expressed in exhausted T cells [45]. Research has not fully elucidated how exhaustion differentially affects various subsets of T cells, nor whether exhaustion can be considered a proper deviation from normal T cell differentiation. Two of these subsets of T cells are called helper T cells and killer T cells, also known as CD4+ T cells and CD8+ T cells, respectively. Many studies focus on exhaustion in CD8+ exclusively. TCR activation leads to increased TOX expression and T cell exhaustion in CD8+ T cells [39]. Among CD8+ T cells, exhaustion is marked by the expression of PD-1 and lymphocyte-activation gene 3 (LAG3), reduced memory function, dysfunctional cellular metabolism, and a lack of cell proliferation [46]. Several attempts to reduce exhaustion induced by either cancer or T cell-recruiting strategies have been made with varying success. These therapies can be generally divided into the following categories, as outlined by Guan et al.: 1) blockade of external cell receptors, 2) therapies targeting transcription factors, 3) epigenetic therapy, 4) improving cellular metabolism, and 5) cytokine blockades [10]. Possible remediation therapies from group 1 and group 5 were included in this study. 12 Three targets for cell receptor blockade (group 1 therapy) include PD-1, TIM-3, and KLRG1. Perhaps the most common external cell receptor blockade is PD-1 blockade. PD-1 blockade uses monoclonal antibodies to block PD-1’s binding site without activating it, reducing the receptor’s inhibitory capacity. This strategy has improved patient outcomes in several cancer types [43], [47], [48]. Such a blockade has also been implemented in ex vivo studies with exhausted CD4+ T cells, leading to a modest restoration of IFN-γ production [49]. Interestingly, the natural ligand for PD-1, known as PD-L1, can also be targeted by molecular cross-linking, resulting in tumor inhibition [50]. TIM-3, the second of the previously mentioned exhaustion receptors, is another promising blockade target. TIM-3 blockade has been shown to reinvigorate T cell proliferation even more effectively than a PD-1 blockade in hepatitis C studies [51]. Blockade of KLRG-1, the third of the previously mentioned exhaustion receptors, in combination with PD-1 blockade effectively reduced tumor size in murine mammary carcinoma [12]. This study includes a PD-1 blockade; TIM-3 and KLRG-1 blockades were also performed, and the corresponding data will be presented in future publications. However, cellular signaling pathways can also be disrupted without using antibody blockades. For example, ibrutinib is a small molecule drug, which inhibits developmental, proliferation, and migration signaling pathways in B cells. Ibrutinib is called a Bruton’s tyrosine kinase inhibitor because it binds to and interferes with this kinase, which is a key component of immune cell signaling. Ibrutinib administration improves patient outcomes during the treatment of B cell cancers by directly acting on B cells to limit their growth and proliferation [52]. However, ibrutinib also influences T cells and has been shown to decrease PD-1 expression of T cells in patients with chronic lymphocytic leukemia (CLL) 13 [53]. Additionally, ibrutinib has been shown to increase interferon gamma (IFN-γ) output in CD8+ T cells after use in patients with CLL [11]. Blockade therapies can also target molecules which are not directly on the surface of cells. These non-effector cell blockades frequently target cytokines involved in lymphocytic signaling. One example of this strategy is IL-10 blockade. IL-10 is a wellknown inhibitory cytokine with anti-inflammatory properties. IL-10 signaling leads to reduced IFN-γ and IL-2 production. IL-10 blockade in patient-sample, colorectal-cancercell cultures significantly increased T cell-mediated target cell death [54]. On the contrary, IL-10 blockade therapy in a CLL mice model has demonstrated TCR signaling dysregulation, reduced T cell effector function, and increased T cell exhaustion phenotypes [55]. It is therefore unclear whether IL-10 blockade therapy has universally consistent results with regards to T cell exhaustion. Treatment design (in the form of dosage and treatment schedules) can also influence T cell exhaustion. A drug-free approach to remediating exhaustion and cellular inhibition is treatment-free intervals. Treatment-free intervals involve alternating periods of antigen activation and rest. In the activation phase, treatment is administered, and cellular interaction occurs. In rest phases, the treatment is either removed or the effector cells are isolated and separated from the target cells. This cycle can be repeated several times. Because antigen stimulation is widely agreed to initiate exhaustion pathways, a disturbance in repetitive activation reduces cellular activity and burden. Treatment-free intervals have successfully reduced exhaustion in T cells during treatment with bispecific antibodies in vitro [56]. 14 One of the difficulties of in vitro work with T cell engagement is modeling biological realities. To effectively replicate antigen re-stimulation in vitro, several research groups have implemented variations of the ‘rechallenge’ paradigm. Many in vitro experimental designs struggle to model lymphocytic realities, due to high effector-to-target ratios and short co-culture times, and this technique addresses such difficulties [57]. In the rechallenge paradigm, target and effector cells are co-cultured initially, and then additional target cells are introduced into the culture at specified intervals. This allows for partial or even complete clearance of target cells, followed by subsequent reactivation, which more clearly produce exhaustion states in vitro. The rechallenge paradigm has been used to model treatment-free intervals [56]. This approach has also demonstrated the phenotypic divergence of CD4+ and CD8+ T cell phenotypes over time during CAR T cell therapy [57]. The rechallenge paradigm serves as the basis for sizeable amounts of data collection in this research. 15 MATERIALS A. Materials and Chemicals For the synthesis of the Fab’-MORF conjugates, two complementary phosphorodiamidate morpholino oligonucleotide strands (MORF) were customized and purchased from Gene Tools (Philomath, OR, USA). The 25-base pair strands were modified with a primary amine at the 3’ termini. The sequences of these two strands are 5’-GAGTAAGCCAAGGAGAATCAATATA-NH2 3’ TATATTGATTCTCCTTGGCTTACTC-NH2-3’ (MORF2). (MORF1) and 5’- Tris(2-carboxyethyl) phosphine (TCEP), the heterobifunctional SM(PEG)2 linker, LysoTracker Green DND-26 and JC-1 (5,5’,6,6’-tetrachloro-1,1’3,3’-tetraethylbenzimidazoylcarbocyanine iodide) were purchased from ThermoFisher Scientific. Pepsin was purchased from Sigma Aldrich. RPMI 1640 medium was purchased from Gibco. A human IFN-γ ELISA detection kit was purchased from StemCell, which included all necessary reagents and materials for ELISA. An EasySep™ Human T Cell Isolation Kit was also purchased from StemCell, including an isolation magnet. T cell culture media and the T cell culture cocktail (αCD3/αCD28 recombinant monoclonal antibodies) and recombinant IL-2 for culture were also purchased from StemCell. Conjugated antibodies used for this study include: αPD-1-FITC (Clone: M1H4), αTIM3-APC (Clone: F38-2E2), αCD8-PE (Clone: RPA-T8), and αCD4-APC (Clone: RPA-T4), purchased from ThermoFisher; and αCD-10-PE (Clone: HI10a,) αCD19-APC (Clone: HIB) α TOX-PE (Clone: IM7) and αCD44-PE (Clone: MF-14), purchased from Biolegend. For the PD-1 blockade, purified anti-human PD-1 antibody (Clone: EH12.2H7) was purchased from Biolegend. 16 B. Synthesis of Fab’-MORF Conjugates The Fab’-MORF conjugates were previously synthesized and characterized by other members of the research group, according to published protocol [24], [25]. A short summary of the general synthesis is included here. The whole antibody to be used as the Fab’ (targeting) motif was enzymatically cleaved by 10% w/w pepsin in citric acid buffer (pH 4.0) at 37 °C for 1.5 hours. This cleavage removed the Fc region of the antibody, yielding a F(ab’)2 molecule after centrifugal ultrafiltration. The disulfide bonds connected the two Fab’ fragments of F(ab’) 2 were then reduced and cleaved using TCEP (10 mM) in citric acid buffer (pH 5.5) at 37 °C for 1 hour, and the products were purified by further centrifugal ultrafiltration. The morpholino oligonucleotide (MORF) components (known as MORF1 and MORF2) were ordered, as previously described, with NH2 groups at the 3’ termini. Each MORF strand was separately reacted with excess SM(PEG)2 in a 1:3 DMSO:PBS (pH 7.4) mixture at ambient temperature for 2 hours. This product (MORF-PEG2-maleimide) was then filtered and reacted with the appropriate purified Fab’ product at a 1:1.3 ratio (Fab’:MORF) in PBS (pH 6.5) for 3 hours at ambient temperature. The final product was then purified, again by centrifugal ultrafiltration. The whole antibodies used for the Fab’ synthesis for this study were rituximab (RTX, an αCD20 antibody) and an αCD3 antibody. The nomenclature used in this study for these final products is therefore Fab’RTX-MORF-1 and Fab’CD3-MORF2, with the former describing the B cell-targeting component and the latter describing the T cell-targeting component. 17 C. Cell lines and culturing The main human cell line used for this experiment was Raji (CCL-86), purchased from American Type Culture Collection. Additionally, Jurkat (Clone E6-1) cells were used for confocal microscopy only, which were also purchased from American Type Culture Collection. Both Raji and Jurkat cell lines were cultured in RPMI 1640 medium (supplemented with 10% FBS, penicillin (200 U/mL) and streptomycin (200 μg/mL)) at standard incubation conditions of 37 °C and 5% CO2. Healthy human T cells were acquired according to the protocol described below; peripheral blood mononuclear cells (PBMCs) used for T cell isolation were purchased from StemCell (cat# 70025.3, lot#211270301C). These cells were cultured in T cell media purchased from StemCell, which was supplemented by the accompanying T cell cocktail (recombinant CD3 and CD28 antibodies and recombinant IL-2) according to manufacturer protocol. Cell washing was performed as follows. Samples to be analyzed were placed into 1.7 mL vials and centrifugated for 5 minutes at 2,000 RPM. The supernatant was drawn off and discarded by micropipette, the remaining cells were resuspended in PBS (170 μL), and centrifugated for 5 minutes at 2,000 RPM. After drawing off the supernatant, the remaining cells were then resuspended in the amount of PBS dictated by the following procedure (50 μL for immunostaining or 300-400 μL for flow cytometry). D. T cell isolation All T cells used in this experiment are healthy human T cells (not acquired from patients). Healthy human donor T cells were isolated by acquiring PBMCs (see Section 18 C) and following the manufacturer protocol (StemCell) for T cell isolation, using negative selection. The isolation procedure is briefly described here. PBMCs in PBS suspension were added to polystyrene tubes. Isolation cocktail (50 μL) was added to the tube. The tubes were incubated for 5 minutes at room temperature. RapidSpheres™ (40 μL) were added to the tube. The tube was placed in the isolation magnet to incubate for 5 minutes. The magnet-tube apparatus was inverted above a collection tube and the negatively selected T cells (not bound by magnetic particles) flowed into the collection tube. E. Immunohistochemistry To aid in the quantification of cell states and protein expression, cells were stained with antibodies in a process known as immunohistochemistry. Staining refers to standard immunohistochemistry procedures as outlined here. Samples to be analyzed were washed according to standard washing procedures above. Following the wash, the remaining cells of each sample were resuspended in PBS (50 μL). Samples to be examined were then treated with the appropriate fluorophore-conjugated antibody (2 μL). These samples were then allowed to incubate in the absence of light for 30 minutes. Another round of washing was performed. At the end of this wash, samples were resuspended in PBS (400 μL) for flow cytometry analysis. F. Flow Cytometry Data Analysis To quantify the expression levels of cellular markers, flow cytometry was performed on cell samples using the following method. Using a plot of side-scatter (SSC) 19 against forward-scatter (FSC) for all detected cells and debris, the region of viable cells as determined by FSC and SSC data was identified and these cells were gated for further data analysis. Collected data metrics fell into two groups: 1) cell type proportions and 2) target molecule expression levels. For cell type proportion data, a histogram was created of the expression of a cell type-identifying molecule (CD3 for T cells, CD10 or CD19 for B cells). This most often produced a clear bimodal distribution, with the peak of higher expression corresponding to cells positive for that marker (thus, cells of that cell type), and the peak of lower expression corresponding to all other cells. Where two cell types were included in such a histogram, these cell events could then be gated and separated by their presence in either the higher or the lower peak. This method was used to separate cells by type for further cell analysis. The proportion of ‘cell events’ in each peak was then calculated and represents the overall proportion of that particular cell type at the end of culture. For target molecule expression, a histogram of the molecule in question was created for all cells of interest. The mean fluorescence intensity (MFI) value was then calculated for all cell events within the specified group. All reported fluorescence intensities (as MFIs) in this study have been normalized against the T cell negative control group, unless otherwise stated. G. Rechallenge Protocol To monitor the changes in expression of cellular markers and cytokines in response to repeated cellular activation, cells were cultured and repeatedly treated with MATCH using a ‘rechallenge protocol’. For all experiments using the rechallenge protocol, the following steps were observed. A six-well plate was seeded with 2M T cells and 400K Raji cells per well (yielding a 5:1 effector:target cell ratio) for all co-culture groups. The 20 experimental group received MATCH treatment. A negative-control, co-culture group received no treatment. Two T cell control groups were included (unless otherwise specified) which only consisted of 2M T cells per well, as explained below. At 0 hours, the experimental co-culture group was treated with Fab’RTX-MORF-1 (50 nM) and Fab’CD3MORF2 (50 nM), resulting in a 1:1 ratio for treatment conjugates. One of the T cell control groups received no treatment, and one received Fab’CD3-MORF2 (50 nM) to mimic onesided MATCH activation without any target cell. All groups were cultured in RPMI 1640 medium (4 mL total volume per well) for 24 hours at standard incubation conditions of 37°C and 5% CO2. Leftover T cells and Raji cells which had not been seeded or treated were prepared for immunohistochemistry to record baseline values of the appropriate metrics. At 24 hours, each sample was individually re-suspended, and an aliquot (1 mL) was removed and set aside for immunohistochemistry. The remainder of the sample was centrifugated for 5 minutes at 2000 RPM, and the supernatant was then removed. The samples were then resuspended in 4 mL of RPMI-1640 and seeded separately into a new six-well plate. Each group received its appropriate treatment, if applicable. The cells were then re-incubated for 24 hours for culture. At 48 hours, this procedure was repeated. At 72 hours this procedure was again repeated, with the modification that the cells remaining, after the aliquot was removed, were discarded and the experiment was ended. This protocol yielded an in vitro simulation of repeated antigenic stimulation by MATCH over the course of 72 hours, with four data collection time points (0, 24, 48, and 72 hours). 21 H. ELISA Protocol To quantify IFΝ-γ levels as a function of time, cell samples of a given ‘rechallenge experiment’ were spun down at each time point according to standard procedure, and the supernatant was then drawn off and stored under refrigeration, instead of being discarded. These supernatant samples were kept refrigerated until all samples had been collected, and then ELISA was performed on all samples simultaneously using the same ELISA plate. Manufacturer’s protocol (StemCell) was followed for ELISA, which is briefly summarized here: All reagents were brought to room temperature. Samples were diluted at a 1:1 ratio with ELISA diluent. A portion of each sample (100 μL) was placed into the ELISA well, and a diagram was drawn to indicate which sample was placed in which well. The plate was covered with the adhesive cover and was left at room temperature for 2 hours to incubate. Each well was then washed 5 times with wash buffer (300 μL) and blotted dry. Diluted detection antibody (12 μL detection antibody in 12 mL ELISA Diluent) was added to each well (100 μL). The plate was again covered with the adhesive cover and incubated for 1 hour at room temperature. The washing step was repeated (5 washes). Diluted SAHRP (100 μL) was added to each well. The plate was again covered with the adhesive cover and incubated for 1 hour at room temperature. The washing step was repeated (5 washes). The lights were turned off in the lab and the remainder of the steps were performed in the dark. TMB substrate (100 μL) was added to each well. The plate was incubated at room temperature for 15 minutes. Stop Solution (100 μL) was added to each well. Optical absorbance values were then detected at 450 nm with a microplate reader. 22 I. Ibrutinib Remediation Therapy To determine if small molecule signaling inhibitor drugs could remediate T cell exhaustion, T cells were pre-treated with ibrutinib and then co-cultured with target cells using the following protocol. As discussed above, ibrutinib has been suggested as a potential treatment for overactive BCR signaling. Because ibrutinib potentiates cellular inhibition for both T cells and B cells, pre-treatment of T cells with ibrutinib followed by cell washing before co-culturing was required to prevent unintentional B cell-ibrutinib interactions. A 24-well plate was seeded with 100K T cells per well in 800 μL of RPMI medium per well. Ibrutinib was then added to the appropriate samples at concentrations of 50 nM, 100 nM, 500 nM, 1μM and 5 μM, with control groups receiving no ibrutinib treatment. Cells were then incubated under standard conditions for 24 hours. All samples were individually centrifugated, washed, and counted to confirm that approximately 200K were present in each new well. Each well was then seeded with 600K Raji cells, to yield a 1:3 ratio, and all samples (except for negative control samples) received a 1:1 50 nM MATCH treatment. At 24 hours of co-culture, cells were stained with αCD19-APC and αPD-1-FITC antibodies and flow cytometry was performed to measure the percentage of B cells and T cells comprising the final culture population, as well as T cell PD-1 expression. J. Antibody Blockade Therapy To determine how a simultaneous PD-1 or IL-10 blockade modulated the effect of MATCH treatment, the following co-culture protocol was observed. A 24-well plate was seeded with 50K T cells and 150K B cells in 800 μL of RPMI-1640 medium per well, 23 yielding a 1:3 ratio. Lower cell numbers were used for this experiment to allow the cells to be cultured for 48 hours with sufficient nutrients, demonstrating a more prolonged exhaustion state. All samples except for the negative control group were treated with Fab’RTX-MORF-1 (50 nM) and Fab’CD3-MORF-2 (50 nM) at a 1:1 ratio. Experimental groups were treated with either 0.1 μg/mL, 1.0 μg/mL, or 10 μg/mL αPD-1 or αIL-10 monoclonal antibody (without fluorophore). After 48 hours of co-culture, cells were washed and stained with αCD19-APC (2 μL) and αPD-1-FITC (2 μL) antibodies and flow cytometry was performed to measure the percentage of B cells and T cells comprising the final culture population, as well as T cell PD-1 expression. K. Statistical Analysis Statistical analysis was performed using one-way ANOVA, unless otherwise stated. To test for normality, all flow cytometry data were plotted as histograms to check for visual symmetry and the presence of outliers. All data used in this study were visually symmetric and the cell counts exceeded n=100 for single flow cytometry samples. For this reason, according to standard flow cytometry practices, the data were parametric and oneway ANOVA tests were determined to be the most appropriate statistical analysis. Following one-way ANOVA analysis, Tukey post-hoc pair-wise mean comparisons were performed after checking for equal variances, and relevant mean comparisons are indicated graphically. The data in this study are presented as mean ± standard deviation. Relevant pvalues are represented graphically as follows: * (p < 0.05), ** (p < 0.01), and *** (p < 0.001). 24 RESULTS The first phase of data collection consisted of measuring proliferation, exhaustion, and effector functions of T cells in response to MATCH treatment. Initial experiments quantified the expression of PD-1 (a primary T cell exhaustion marker) over the course of a three-day rechallenge experiment. PD-1 expression was determined by flow cytometry and normalized against the T cell culture, which did not include MATCH therapy or target cancer cells. Separate T cell culture samples with the T cell-targeting MATCH component added into the culture (without any target cells) showed PD-1 levels similar to the T cell culture control. The untreated co-culture group, comprised of both T cells and target cells without any treatment added, showed PD-1 levels similar to the control group at 24 and 48 hours, with a nearly 3-fold rise in PD-1 expression at 72 hours. However, the treatment group receiving MATCH showed a nearly 3-fold rise in PD-1 expression at 24 hours, followed by a further increase to nearly 8 times the control levels at 48 hours, followed by a decrease to 6 times the control levels at 72 hours. These PD-1 expression levels were statistically significant in comparison to the untreated co-culture (Figure 2). A similar pattern was observed when rechallenge experiments were performed to test for a second exhaustion marker, TIM-3. For this second experiment, all TIM-3 levels (including control values) were normalized against the baseline TIM-3 expression levels to determine if the T cell culture itself experienced a change in TIM-3 expression over time. The T cell culture, which served as the negative control group, did experience a decrease in TIM-3 levels over 72 hours, to less than 25% of the original TIM-3 expression levels. Likewise, the T cell culture with the T cell-targeting component of MATCH also demonstrated an overall decrease in TIM-3 expression over 72 hours. The co-culture 25 Figure 2. PD-1 expression of CD8+ T cells during MATCH treatment. PD-1 expression increases in response to MATCH treatment but stays relatively constant for unactivated co-cultures and single-cell cultures. Over the course of 72 hours, the MATCHactivated T cells experience a sharp increase, followed by a moderate decline, in the expression of the exhaustion marker PD-1. Untreated co-cultures of T cells and B cells observe a moderate increase in T cell PD-1 expression at the end of culture. Data are represented as mean ± SD (n=1 for control groups; n=2 for groups with error bars). (without MATCH treatment) similarly showed decreases in TIM-3 expression, although the TIM-3 expression in this group was still slightly higher than the TIM-3 levels in the other two groups at 72 hours. However, the MATCH-treated co-culture T cells experienced an increase in TIM-3 expression at 24 hours (1.4-fold increase), followed by a further increase at 48 hours (2-fold increase compared with baseline levels), followed by a slight decrease at 72 hours (about 1.7-fold increase compared with baseline levels). This pattern of a 48-hour increase followed by a 24-hour decrease in TIM-3 expression mirrored the results for PD-1 expression (Figure 3). 26 Figure 3. TIM-3 expression during MATCH treatment. TIM3 expression increases in response to MATCH treatment. MATCH-activated T cells experience a 48-hour increase in TIM3 expression, followed by a slight decline over the next 24 hours. Untreated cocultured cells and control groups all experience a decrease in TIM3 expression in the absence of cell activation. Data are represented as mean ± SD (n=1 for control groups; n=2 for groups with error bars). A rechallenge experiment was then performed to test for CD44 expression levels over time. Since CD44 is a marker of T cell proliferation and migration in response to activation, it was hypothesized that these trends would mirror exhaustion trends over the same time period (as measured by PD-1 and TIM-3). Similar trends were observed, although the changes in expression were proportionally smaller than for the exhaustion markers. The data from this experiment were also normalized against the baseline levels (following the TIM-3 measurement protocol). The T cell culture, as well as the T cell culture with the T cell-targeting component of MATCH both experienced fairly stable levels of CD44 during the entire 72-hour period. However, the untreated co-culture group showed a modest decrease in CD44 levels by the end of the 72-hour period. In contrast, MATCH-treated cells experienced a 48-hour increase in CD44 expression (1.2 fold- 27 increase at 48 hours in comparison to baseline levels), followed by a drop to near baseline levels at 72 hours (Figure 4). Having demonstrated that the T cells showed a similar pattern of exhaustion and proliferation across the 72-hour rechallenge period, the direct effector output of T cells was next examined. This was performed by measuring the concentrations of IFN-γ, a primary effector molecule released by T cells, in the supernatant collected during the 72-hour rechallenge experiments. It was hypothesized that these concentration trends should mirror the observed levels of PD-1, TIM-3, and CD44. Since cells were washed at each 24-hour time point, the total concentration of IFN-γ at a given time point represents the total IFNγ produced during the previous 24 hours. The T cell culture, T cell culture with targeting component, and untreated co-culture groups all showed fairly constant levels of IFN-γ Figure 4. CD44 expression during MATCH treatment. CD44 expression moderately increases in response to MATCH treatment. The highest peak of CD44 expression was observed in MATCH-treated T cells at 48 hours, although this peak was only a 1.2-fold increase from baseline levels. In comparison, all other control groups experienced a decrease in CD44 levels in the absence of cell activation at the end of 72 hours. Data are represented as mean ± SD (n=1 for control groups; n=2 for groups with error bars). 28 levels across the 72 hours. However, T cells from the MATCH-treated samples showed an 11-fold increase in IFN-γ production at 24 hours, a nearly 35-fold increase at 48 hours, followed by a drop to a 15-fold increase at 72 hours (when compared with control levels). These increases are statistically significant in comparison to the baseline values. This trend again followed the previously observed pattern of a 48-hour increase and subsequent 24hour decrease in target molecule expression (Figure 5). The second phase of data collection included T cell PD-1 expression quantification in response to a variety of proposed exhaustion remediation treatments which were administered simultaneously with MATCH treatment. The new negative control group for these experiments consisted of untreated co-cultures of T cells and cancerous B cells. The positive control group consisted of MATCH-treated co-cultures without any exhaustion Figure 5. IFN-γ release during MATCH treatment. IFN-γ production is significantly increased by MATCH activation in T cells. IFN-γ production, measured by ELISA to determine the concentration of IFN-γ in cellular supernatant, experiences a 35-fold increase over the first 48 hours of co-culture with MATCH treatment. These levels drop to a 15-fold increase (compared to baseline concentrations) at the end of the 72-hour co-culture. In comparison, all other groups demonstrate comparably low levels throughout the culture. Data are represented as mean ± SD (n=4). 29 remediation therapy. All experimental groups were compared to these groups. Additionally, the fraction of residual B cells remaining at the end of the experiment was measured for all groups to determine the overall efficacy of exhaustion remediation therapies in augmenting T cell activation and cancer cell clearance. The first remediation therapy tested was pre-treatment of T cells with ibrutinib. At the end of the culturing phase, the MATCH-treated group demonstrated a 4-fold increase in PD-1 expression compared to the untreated group, similar to previously observed levels. Increasing titrations of ibrutinib gradually diminished the PD-1 expression to an overall 3.4-fold increase (compared to the untreated group) at the highest dose of ibrutinib. An outlier to this steadily decreasing PD-1 expression trend was observed at 100 nM ibrutinib, where the PD-1 expression was unexpectedly low (Figure 6). However, no ibrutinib dose was able to further decrease the residual cancer cell proportion at the end of culture in comparison to MATCH alone. In fact, several of the higher doses of ibrutinib resulted in higher amounts of residual B cells at the end of co-culture (Figure 7). 30 Figure 6. PD-1 expression in T cells during MATCH treatment following ibrutinib pre-treatment of T cells. PD-1 expression decreases with increased ibrutinib pretreatment concentrations. The highest PD-1 expression is observed in MATCH-treated cells without ibrutinib exhaustion remediation therapy. Increasing doses of ibrutinib show a consistent pattern in decreasing the PD-1 expression, with an outlier at the 100 nM dose of ibrutinib. Data are represented as mean ± SD (n=3). Figure 7. B cell residual populations following MATCH treatment with ibrutinibtreated T cells. Proportion of residual B cells after co-culture was minimally influenced by ibrutinib pre-treatment of T cells. A moderate decrease in the proportion of B cells remaining after treatment is observed in the positive control group (MATCH without ibrutinib). Increasing doses of ibrutinib generally increase the residual B cell population, with the highest dose showing a residual B cell population similar to untreated co-cultured cells. Data are represented as mean ± SD (n=3). 31 The second remediation therapy examined was a PD-1 blockade. This time the B cell ablation was more successful in the group receiving only MATCH treatment than in previous experiments, with a reduction from a final population consisting of 70% cancer cells in the untreated group to less than 15% in the treated group. The lowest dose of PD1 blockade did not noticeably alter the residual B cell population. The middle dose suppressed the residual B cell population to under 10%. The highest dose of PD-1 blockade further reduced the residual B cell population to 5% of the remaining total cell population, which was statistically significantly lower in comparison with the group receiving only MATCH treatment (Figure 8). The third remediation therapy examined was an IL-10 blockade. These results largely mirrored the results of the PD-1 blockade, although the initial cancer ablation by MATCH alone was markedly reduced. The lowest dose of IL-10 blockade did not Figure 8. B cell residual populations following simultaneous MATCH treatment and PD-1 blockade. PD-1 blockade attenuates exhaustion and improves rates of B cell ablation. MATCH treatment alone reduces the residual B cell population from nearly 75% to 13% in this trial. Increasing doses of PD-1 blockade further suppress that residual value to 5%. Data are represented as mean ± SD (n=3). 32 noticeably reduce the residual B cell population. However, the medium dose moderately reduced the residual B cell population after co-treatment. The highest dose of IL-10 blockade further suppressed the residual B cell population to half the proportion remaining after MATCH treatment alone (Figure 9). Figure 9. B cell residual populations following simultaneous MATCH treatment and IL-10 blockade. IL-10 blockade attenuates exhaustion and improves rates of B cell ablation. MATCH treatment alone reduces the residual B cell population from 70% to 40% in this trial. Increasing doses of IL-10 blockade further suppress that residual value to 20%. Data are represented as mean ± SD (n=3). 33 DISCUSSION T cell exhaustion poses a formidable challenge to the implementation of Multiantigen T cell hybridizer (MATCH) for the treatment of leukemia, lymphoma, and multiple myeloma. T cell exhaustion can severely limit the potency, duration, and success of any T cell recruitment strategy, such as MATCH, potentially rendering it ineffective. This study aimed to quantify trends of T cell activation and exhaustion in response to MATCH treatment and to identify a co-treatment to be simultaneously administered with MATCH to attenuate T cell exhaustion. These aims were accomplished by multi-day cell culturing, immunohistochemistry, and flow cytometry, with particular focus on measuring the exhaustion marker PD-1. PD-1 expression was observed to increase in response to MATCH treatment in a similar pattern to markers for cell proliferation (CD44) and T cell activity (IFN-γ). T cell exhaustion was attenuated, and MATCH treatment efficacy was improved upon simultaneous administration of either a PD-1 or IL-10 blockade, as measured by a decrease in residual cancer cells. These findings indicate that T cell exhaustion limits the efficacy of MATCH treatment for hematopoietic cancers. However, T cell exhaustion can be remediated during MATCH treatment by simultaneous ‘cotreatment’ with an immunomodulatory therapy directed at the T cells. Further, the success of MATCH in eliminating target cancer cells in vitro demonstrates the urgency to examine its success in vivo as a potential new oncological therapy. Expression levels of PD-1 in response to MATCH treatment indicate that MATCH induces T cell exhaustion over the course of repeated administration (Figure 2). Further, cells which receive only one MATCH conjugate (the T cell-targeting motif) but which do 34 not encounter target cells do not become exhausted. Additionally, co-culturing T cells and cancerous B cells without MATCH does not exhaust the T cells. MATCH is therefore both sufficient and necessary to induce T cell exhaustion. Interestingly, exhaustion levels decrease somewhat after 48 hours, remaining elevated in comparison to baseline values. It is not clear whether the cells showing high levels of exhaustion at 48 hours subsequently down-regulate PD-1 expression, as it is possible these cells are undergoing apoptosis or cellular anergy before the next time point. Importantly, TIM-3, CD44, and IFN-γ expression patterns are almost identical to the PD-1 expression pattern in response to MATCH treatment (Figures 3, 4, and 5). These markers indicate exhaustion, proliferation, and immunological activity, respectively. While the scale of expression is not identical, the harmonious expression trends support a logical narrative. To the same degree that T cells are activated (IFN-γ), they begin to proliferate (CD44), and to that same degree they also become exhausted (PD-1 and TIM-3). All four markers demonstrated a drop in expression after 72 hours. This is highly meaningful, considering that new target cells and new MATCH conjugates were administered daily, but T cells were not added at these subsequent time points. In other words, between 48 hours and 72 hours, T cells experience a reverse in their ability to proliferate and activate against target cells with repeated exposure. Again, it is unclear whether this is due to T cell death or anergy, but the consequence is clear: there is a limit to the extent of T cell recruitment through MATCH therapy without additional co-treatments. Further, IFN-γ release in response to MATCH treatment validates the ability of MATCH to directly induce T cell effector functions (Figure 5). At 48 hours, the supernatant of MATCH-treated cell co-cultures contained almost 35 times the amount of IFN-γ as the 35 untreated T cell culture. As one of the primary effector molecules of T cells during an immune response, IFN-γ is a direct measure of T cell recruitment and activation. MATCH conjugates are not just linking T cells and target cells in proximity. This binding is indeed inducing a cytotoxic interaction. These findings support the global aim of validating MATCH’s overall efficacy and mechanism of action. Pre-treatment of T cells with ibrutinib before co-culturing with target cells and treating with MATCH was ultimately unsuccessful as an exhaustion attenuation therapy. Ibrutinib pre-treatment produced a marked decrease in PD-1 expression, indicating successful attenuation of cellular exhaustion. PD-1 expression generally decreases with increased doses of ibrutinib (Figure 6). However, this decrease may not be clinically beneficial, as the actual ablation of cancer cells was not improved with ibrutinib cotreatment when compared to MATCH alone. The lowest doses of ibrutinib were able to maintain a similar level of cancer clearance in comparison to MATCH treatment alone, but increased doses of ibrutinib led to increased levels of residual cancer cells (Figure 7). Considering that higher doses of ibrutinib are toxic to immune cells, it is possible that these doses not only attenuated cell exhaustion, but also exerted toxic effects on the T cells. In contrast, PD-1 blockade both reduced cell exhaustion, as well as improved cancer cell clearance (Figure 8). The lowest dose of PD-1 blockade maintained the same level of cancer clearance and slightly decreased exhaustion (data not shown). A higher dose of PD-1 blockade maintained this reduced level of exhaustion and reduced the residual cancer cell population nearly by half. The highest dose of PD-1 blockade further reduced the residual cancer cell population. Similarly, IL-10 blockade also further suppressed the residual cancer cell proportion in comparison to MATCH alone (Figure 9). In both cases, 36 the residual proportion of cancerous B cells following MATCH treatment was reduced by half with the highest blockade dose, demonstrating the practical reduction of T cell exhaustion. With both PD-1 and IL-10 blockades, the immunomodulatory function of the blockade improved the efficacy of MATCH in clearing cancer cells. This study supports the well-documented examples of PD-1 blockade as an effective anti-exhaustion remedy [12], [47], [48]. However, one major divergence between this study and existing literature regards the efficacy of ibrutinib as an immunomodulatory agent. Previous research has indicated that ibrutinib has protective power against T cell senescence [53] and exhaustion [11] in patients with chronic lymphocytic leukemia. However, these results indicate that ibrutinib was ineffective at improving cancer cell clearance at best and diminished the immune response at worst during MATCH treatment. In a more global sense, this study also demonstrates the continued power of the immunotherapeutic agent rituximab and illustrates a novel expansion on its action through the MATCH conjugation strategy. For this study, the B cell-target motif is derived from rituximab, a CD20 antibody in widespread clinical use [19]. By isolating the CD20targeting moiety of rituximab through chemical cleavage and conjugating it with morpholino oligonucleotides, the epitope-binding specificity of rituximab is augmented by the ability to associate with a second cell-targeting conjugate. Additionally, MATCH is a development of previous strategies using cross-linking of CD20 and similar targets to induce cell death, known as drug-free macromolecular therapeutics [23], [24], [25]. This publication demonstrates the expansion of Fab’-MORF conjugates into a non-cross-linking paradigm. MATCH replaces the use of serum albumin as a cross-linking agent with a second, complementary Fab’-MORF conjugate which targets an effector cell. This 37 innovation dramatically expands the utility of Fab’-MORF conjugates from surface protein cross-linking agents to effector cell-inducing agents. This development places Fab’-MORF conjugates on par with other T cell recruitment strategies, such as CAR T cells [30] and bispecific T cell engagers [28]. The primary difference between MATCH and these other strategies, while not explicitly explored in this study, is that the target cell motif can be exchanged during treatment. Additionally, multiple target cell-specific MATCH conjugates can be simultaneously introduced, allowing MATCH to be bivalent, trivalent, or multivalent (which is generally not the case with other T cell recruitment strategies). Finally, the dose of each component can be altered, such that the target cell motif to effector cell motif ratio need not necessarily be one. Future work will examine if this ratio can be optimized for increased efficacy, decreased exhaustion, and decreased T cell toxicity. These studies are primarily limited by sample size. While each co-culture sample contained between 104 to 106 cells, each culture was performed using duplicate or triplicate samples per group. As such, the sample size was 2 or 3 for each group, considering that cells influence each other and are examined as populations. Each test was replicated with new cells to confirm the results due to this effectual small sample size, but that does not eliminate the possibility of random variation. Additionally, cell aliquots used to seed each plate were taken from larger cell cultures, so that any random variation existing in the culture itself would have been propagated throughout all experimental groups. MATCH conjugates are a novel technology and are still chemically synthesized in small quantities in a research laboratory setting. Therefore, conjugates used for this study were produced in several different syntheses. While the same conjugate synthesis protocol 38 was followed during each synthesis, variations in conjugate structure were possible across the duration of this research. These variations could include aberrant conjugation, variation in the Fab’-to-MORF ratio, or possible degradation of the chemical bond between the Fab’ and MORF moieties. Occasional differences in overall efficacy were observed between different batches of conjugates. While these differences were not serious enough to warrant concern on the part of the researchers, they do limit the power of the findings. However, studies were replicated with multiple batches of conjugates to address this limitation, and the general findings remained consistent across all replications. Finally, due to the novelty of the technology, the ratio of T cells to cancer cells in culture was modified over the course of the studies, as various results improved the understanding of MATCH’s mechanism and extent of action. As such, some initial experiments were performed with slightly different cell counts and ratios in comparison to later studies. These earlier studies will be repeated with appropriate cell counts and ratios to confirm that this variation did not meaningfully alter the results. These results imply that MATCH can be successfully augmented by co-treatment with an exhaustion remediation therapy. While the true impact of T cell exhaustion is most apparent over the course of weeks or months, these 72-hour co-cultures provide a microscopic insight into how exhaustion restricts T cell activity and proliferation upon MATCH activation. The success of PD-1 blockade as an exhaustion treatment in vitro implies that such a strategy can be employed in clinical settings when MATCH is translated into such use. These exhaustion studies also suggest that the full potential of MATCH has not yet been demonstrated. Considering that immunomodulation through PD-1 blockade or IL-10 blockade improves cancer cell ablation, it is possible that other strategies, such as 39 other small molecule drugs, dosing regimes, and time-dependent administration procedures, could also improve MATCH efficacy. It is highly probable that this study is only the first in a series of improvements on MATCH administration strategies, leading to continually improved in vitro and in vivo cancer cell ablation. Based on the apparent success of PD-1 blockade in attenuating exhaustion and improving cancer ablation, other cell surface exhaustion markers are promising blockade targets and blockades against these molecules will be attempted. These include the previously mentioned markers TIM3 and KLRG1. Similarly, based on the success of IL10 blockade in attenuating exhaustion, other cytokines should be examined as potential blockade targets. Finally other cell signaling inhibitory agents (analogous to ibrutinib) could be examined as ‘pre-treatments’ for T cells, to prevent overactivation in response to MATCH treatment. Beyond other exhaustion remediation therapies, the release of various cytokines upon MATCH activation as a function of time should be quantified, using ELISA or similar techniques. This information would provide additional insight into T cell mechanisms upon MATCH conjugate binding. Further, this data will play a central role in preparing for in vivo studies of MATCH, as well as evaluation of translation into clinical medicine. A similar examination of cytokine release in vivo, likely using a mouse model, will be essential to screen for the possibility of cytokine release syndrome. During initial MATCH efficacy assays, T cell culture lines (such as Jurkat) were used in the place of healthy human T cells to assess binding compatibility. While this data is not considered relevant enough to be presented in this text, these assays produced an interesting phenomenon. Cell lines arising from helper CD4+ T cells (such as Jurkat), 40 which have been traditionally assumed to lack direct cytotoxic activity, successfully eliminated target cells upon MATCH activation. It is unclear whether MATCH induced these cells to undergo a phenotype alteration, enabling them to directly release cytotoxic molecules. Were this to be true, it would alter the current understanding of the flexibility of T cell identities. To further explore this phenomenon, healthy human T cells will be cocultured with cancer cells, and they will be examined using confocal microscopy. Fluorescent stains will be introduced to distinguish separate T cell subpopulations (such as CD4+ and CD8+ cells) and to mark cytotoxic compounds (such as granzyme B). These images will confirm if MATCH is indeed sufficient to induce phenotype switching by observing if CD4+ ‘helper’ T cells are directly releasing cytotoxic molecules traditionally associated with CD8+ cells. Finally, once all exhaustion remediation therapies have been tested and the most effective dose of each possible therapy is determined, these optimal exhaustion remediation therapy doses will be examined side-by-side. In this study, each optimal dose will be examined against a co-culture group and a MATCH-treated group, and the amount of residual cancer cells and PD-1 expression will be quantified using flow cytometry. Supernatant will be collected at each time point and IFN-γ concentrations will be quantified at the end of the study using ELISA. This final in vitro exhaustion study will allow each possible therapy to be examined on the same cells, using the same conjugates, providing a clear indication of which therapy is most effective at reducing exhaustion and clearing cancer cells. A similar study will then be performed in vivo using a mouse model to verify that exhaustion remediation co-treatments prolong MATCH efficacy and improve cancer ablation in living organisms. 41 MATCH technology is a promising new development for the treatment of leukemia, lymphoma, and multiple myeloma. While the obstacle of T cell exhaustion initially hinders the progress of any T cell recruitment therapy, this study demonstrates that it is possible to attenuate this exhaustion and improve the rates of cancer clearance with immunomodulatory exhaustion ‘co-treatments’ during MATCH therapy. Further, this study also validates the cytotoxic capacity of MATCH to target and kill cancerous hematopoietic cells. With its two-component structure, enabling clinicians to alternate cancer targets during treatment, MATCH is superior to existing target-restricted therapies, such as bispecific antibodies. This new platform for cancer treatment will provide patients an additional treatment option for battling these complex diseases with less systemic toxicity and greater specificity than traditional chemotherapy. MATCH will likely serve as a versatile tool in overcoming treatment relapse and improving patient outcomes in the battle against leukemia, lymphoma, and multiple myeloma. 42 REFERENCES [1] J. Huang et al., “Disease Burden, Risk Factors, and Trends of Leukaemia: A Global Analysis,” Front. Oncol., vol. 12, p. 904292, Jul. 2022, doi: 10.3389/fonc.2022.904292. [2] V. Tietsche de Moraes Hungria et al., “Epidemiology of Hematologic Malignancies in Real-World Settings: Findings From the Hemato-Oncology Latin America Observational Registry Study,” J. Glob. 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