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Phenotype of T-cells used in Xenograft Studies

Serum levels of human PSA from day 56 of the MDA-PCa-2b xenograft study (as shown in Figures 5C, 5D).

Supplementary Methods and Supplementary Figure Legends

4–1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4–1BB on tumor-specific cytotoxic T cells makes 4–1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4–1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4–1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4–1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4–1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4–1BB–mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.

Supplementary figures and methods

4–1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4–1BB on tumor-specific cytotoxic T cells makes 4–1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4–1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4–1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4–1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4–1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4–1BB–mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.

Background 4-1BB (CD137) and OX40 (CD134) are critical activation-induced co-stimulatory receptors that regulate immune responses of activated T and NK cells by enhancing proliferation, cytokine production, survival, and cytolytic activity. A superagonist 4-1BB antibody has shown clinical activity but severe toxicities. APVO603, is a 4-1BB x OX40 targeting bispecific antibody with conditional agonism, activating these receptors only when both are co-engaged. The Fc portion was mutated to eliminate FcγR-mediated interactions. Co-stimulation through 4-1BB and OX40 has the potential to amplify the cytotoxic function and the number of activated T and NK cells in multiple solid tumor indications.1–2 Methods scFv binding domains to 4-1BB and OX40 were optimized to increase affinity, function and stability, and then incorporated into the ADAPTIR™ bispecific antibody platform to produce the APVO603 lead candidate. NF-κB/luciferase reporter cell lines expressing OX40 or 4-1BB were initially used to assess the agonistic function of APVO603’s binding domains. Primary PBMC were sub-optimally stimulated with an anti-CD3 antibody and T and NK cell proliferation was assessed using Cell TraceTM-labelled PBMC. Cytokine secretion was measured at 48 hrs using Luminex-based assays. For in vitro tumor lysis studies, PBMC were co-cultured with tumor cells expressing a tumor-associated antigen (TAA) and activated with TAA x CD3 bispecific protein. 7-AAD expression was assessed on tumor cells at 72 hrs. The in vivo therapeutic efficacy of APVO603 was evaluated using a murine MB49 bladder cancer model in human 4-1BB and OX40 double knock-in mice. Results APVO603 stimulates 4-1BB and OX40 NF-κB/luciferase reporter activity in a dose-dependent manner, and is strictly dependent on engagement of the reciprocal receptor to elicit 4-1BB or OX40 activity. In primary PBMC assays, APVO603 induces synergistic proliferation of CD4+, CD8+ T and NK cells when compared to OX40 or 4-1BB monospecific molecules with a wt Fc, either individually or in combination. Additionally, APVO603 enhances proinflammatory cytokine production and granzyme B expression, and augments in vitro tumor cell lysis induced by a TAAx CD3 engager. In vivo, APVO603 reduces growth of established MB49 tumors in human 4-1BB and OX40 double knock-in mice. Conclusions APVO603 is a dual-agonistic bispecific antibody that augments the effector function of activated CD4+ and CD8+ T and NK cells in a dose-dependent manner, and reduces growth of established tumors in vivo. This preclinical data, demonstrates conditional dual stimulation of 4-1BB and OX40 and supports further development of APVO603, a promising immuno-oncology therapeutic with potential for benefit in solid tumors. Ethics Approval Treatment of study animals was in accordance with conditions specified in the Guide for the Care and Use of Laboratory Animals, and the study protocol (ACUP 20) was approved by the Institutional Animal Care and Use Committee (IACUC). References Bandyopadhyay S, Long M, Qui H, Hagymasi A, Slaiby A, Mihalyo M, Aguila H, Mittler R, Vella A, Adler A. Self-antigen prevents CD8 T cell effector differentiation by CD134 and CD137 dual costimulation. J Immunol 2008;181(11):7728–37. Ryan J, Mittal P, Menoret A, Svedova J, Wasser J, Adler A, Vella A. A novel biologic platform elicits profound T cell costimuloaroty activity and antitumor immunity in mice. Cancer Immunol Immunother 2018;67(4):605–613.

Background 4-1BB (CD137) is an activation-induced co-stimulatory receptor that regulates immune responses of activated CD8+ T cells and NK cells, by enhancing proliferation, survival, cytolytic activity and IFN-γ production. Its ability to induce potent anti-tumor CD8+ and NK cell activity makes 4-1BB an attractive target for designing novel therapeutics for immuno-oncology. However, clinical development of a monospecific 4-1BB agonistic antibody has been hampered by dose-limiting hepatic toxicities. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel 4-1BB x 5T4 bispecific antibody that stimulates 4-1BB function only when co-engaged with 5T4, a tumor-associated antigen. The combined preclinical dataset presented here provides an overview of the mechanism of action and the efficacy and safety profile of ALG.APV-527, supporting its advancement into the clinic. Methods ALG. APV-527 was built based the ADAPTIR™ platform with binding domains to 4-1BB and 5T4 generated using the ALLIGATOR-GOLD® human scFv library. ALG.APV-527 was tested using primary cells in the presence or absence of cells expressing 5T4. Cell Trace-labelled PBMC sub-optimally stimulated with anti-CD3, to induce 4-1BB expression, cells were gated using flow cytometry. T cell cytotoxicity was assessed by quantifying cell death in CD8+ T cell/tumor cell co-cultures, and images were obtained using a cell live imaging system (Cytation 5). For tumor inhibition studies, human 4-1BB knock-in mice were injected subcutaneously with MB49 cells transfected with human 5T4. Cured mice were subsequently used in a toxicity study and liver pathology was evaluated. Results In vitro, ALG.APV-527 enhances primary CD8+ T cell and NK cell function and proliferation in the presence of 5T4-expressing cells. Using imaging, ALG.APV-527 in combination with a bispecific T cell engager caused increased cell death in T cell/tumor cell co-cultures. ALG.APV-527 inhibited growth of established tumors at doses as low as 2 µg/mouse in a syngeneic bladder cancer model. Following recovery, mice exhibited a memory response when rechallenged with tumor. In a high dose safety study in human 4-1BB knock-in mice, ALG.APV-527 did not cause significant systemic immune activation, whereas urelumab analogue treated mice induced dermatitis, elevated serum cytokines, CD8+ T-cell liver infiltration and systemic T-cell proliferation. Conclusions ALG. APV-527 induces potent CD8+ T cell and NK cell co-stimulation and T-cell cytotoxicity and has potent in vivo anti-tumor activity, without inducing systemic toxicity. Based on preclinical data, ALG.APV-527 is a promising anti-cancer therapeutic for the treatment of a variety of 5T4-expressing solid tumors. Ethics Approval All studies were review and approved by the Internal Animal Care and Use Committee (IACUC) of Aptevo Therapeutics

CD137 (4-1BB; TNFSR9) is an activation-induced surface receptor that through costimulation effects provide antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being utilised for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cell is superior to that provided in-trans in terms of T cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mouse and human. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-κB signaling and provides a more robust induction of cell cycle and DNA damage repair gene expression programs. Here we report that the superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which may provide costimulation either in cis or in trans., Costimulation has been shown to be required for optimal activation of T cells and it could be delivered either in trans with respect to the source of CD3-TCR ligation or in cis on the same cell. Here the authors show that CD137 costimulation is more effective when delivered in cis to enhance T cell proliferation and activation.

Prostate-Specific Membrane Antigen (PSMA), is a tumor-associated antigen (TAA) that is expressed on prostate cancers, including metastatic castration-resistant prostate cancer (mCRPC). Current chemotherapeutic approaches for mCRPC are challenged by development of resistance resulting in limited clinical benefit. Immuno-oncology therapeutic candidates such as bispecifics re-directing T-cell responses to eliminate tumors, are a promising strategy to overcome the limitations of current approaches and provide benefit to patients with aggressive cancers. Here we present preclinical data demonstrating a potential new approach using low affinity targeting of CD3 and high affinity targeting of PSMA for treatment of a solid tumor cancer.APVO442 is Aptevo's bispecific candidate targeting PSMA and CD3. This candidate was designed to have unique pharmacokinetic and safety properties to potentially maximize potent anti-tumor responses against mCRPC. The APVO442 bispecific T-cell engager uses Aptevo's ADAPTIR-FLEX technology to generate a molecule with low-affinity monovalent CD3 engagement, paired with high-affinity bivalent PSMA binding designed to deliver a highly selective T-cell response at the tumor. The unique engineering of APVO442 reduces the potential of binding to CD3 expressed on peripheral T cells, thus minimizing the potential for on-target toxicity, such as cytokine release, and increasing the potential to deliver the effective concentration of the molecule localized to solid tumors.Preclinical testing demonstrated that APVO442 exhibits optimal manufacturability and functional characteristics for lead candidate selection. Anti-PSMA x anti-CD3 constructs with varying binding strengths to CD3 were evaluated for specificity of CD3 binding, ability to enhance T-cell activation, and ability to elicit T-cell-mediated cytotoxicity against PSMA-expressing tumor targets with varying levels of PSMA expression. APVO442 demonstrated 10-fold reduced binding to CD3 and EC50 compared to the highest affinity constructs tested while retaining equivalent binding to tumor cells expressing various levels of PSMA. The differences in CD3 affinity were associated in a slightly lower EC50 of potency however, the maximal responses for in vitro activation of CD4+ and CD8+ T cells including upregulation of CD25/CD69 expression, proliferation and in vitro tumor lysis were comparable between low and high affinity CD3 constructs. Finally, APVO442 induced reduced levels of multiple cytokines in vitro when compared to high affinity competitor molecules.In vivo, APVO442 elicited robust anti-tumor responses of human PSMA-expressing tumors in a murine xenograft tumor model. The in vivo activity of APVO442 was comparable to high affinity CD3 engaging comparators with similarly measured PK profiles. Additional in vivo characterization of APVO442 is ongoing and continued pre-clinical studies are planned for 2021. Citation Format: Rebecca Gottschalk, Robert E. Miller, Lynda Misher, Michelle H. Nelson, Allison Chunyk, Brian Woodruff, Elizabeth Haglin, Gabriela Hernandez-Hoyos, Peter Pavlik, Catherine McMahan, Hilario J. Ramos, David Bienvenue. APVO442: A bispecific T cell-engaging candidate utilizing the ADAPTIR-FLEXTMplatform technology with unique properties designed for optimal tumor distribution and cytotoxic response against PSMA-expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB172.

APVO603 is Aptevo's bispecific candidate targeting 4-1BB and OX40. It was designed to have unique properties with the potential to overcome some of the clinical challenges observed with monoclonal antibody targeting these receptors. APVO603 is engineered as an FcγR-signaling deficient bispecific antibody that utilizes Aptevo's ADAPTIR technology for a distinct approach for dual targeting of 4-1BB and OX40 in the absence of additional effector activity. The distinct characteristics of APVO603 may enable conditional activation of 4-1BB and OX40 via agonism of these receptors only when cross-linked via engagement of the other receptor via cis and/or trans cellular interactions. Thus, APVO603 is designed with the potential to overcome both the on-target toxicity and limited efficacy observed with 4-1BB and OX40 monoclonal antibody treatment in the clinic. Anti-4-1BB and OX40 binding domains were optimized to increase affinity, function and stability, then incorporated into the ADAPTIR bispecific antibody platform to produce the APVO603 lead candidate. APVO603 was found to augment 4-1BB and OX40 activity in a dose-dependent manner and is strictly dependent on engagement of the reciprocal receptor to elicit 4-1BB or OX40 signaling in vitro. In preclinical assays using PBMCs sub-optimally stimulated with anti-CD3, APVO603 induces synergistic proliferation of CD4+, CD8+ T and NK cells when compared to anti-OX40 or 4-1BB antibodies with a wt Fc, included either individually or in combination. Additionally, APVO603 enhances proinflammatory cytokine production, granzyme B expression, and reduces the T cell exhaustion phenotype. The mechanistic activity of APVO603 resulted in dose-dependent control of in vivo tumor growth in a preclinical humanized murine xenograft model using established murine MB49 bladder tumors in human 4-1BB and OX40 double knock-in mice. APVO603 is a dual-agonistic bispecific antibody that augments the effector function of activated CD4+ and CD8+ T cells and NK cells in a dose-dependent manner and reduces growth of established tumors in vivo. This preclinical data demonstrates conditional dual stimulation of 4-1BB and OX40 and supports further development of APVO603, a promising immuno-oncology therapeutic with potential for benefit in solid tumors. This program is progressing into IND-enabling studies later this year. Citation Format: Michelle H. Nelson, Robert E. Miller, Secil Franke-Welch, Ruth Chenault, Hang Fang, Allison Chunyk, Gabriela Hernandez-Hoyos, Hilario J. Ramos, David Bienvenue, Catherine McMahan. APVO603: A dual 4-1BB and OX40 bispecific approach utilizing ADAPTIRTMtechnology designed to deliver a conditional T cell/NK response against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB173.

Treatment of metastatic, castration-resistant prostate cancer (mCRPC) remains a highly unmet medical need and current therapies ultimately result in disease progression. Immunotherapy is a rapidly growing approach for treatment of cancer but has shown limited success to date in the treatment of mCRPC. We have developed a novel humanized bispecific antibody, MOR209/ES414, built on the ADAPTIR (modular protein technology) platform, to redirect T-cell cytotoxicity toward prostate cancer cells by specifically targeting T cells through CD3ϵ to prostate cancer cells expressing PSMA (prostate-specific membrane antigen). In vitro cross-linking of T cells with PSMA-expressing tumor cells by MOR209/ES414 triggered potent target-dependent tumor lysis and induction of target-dependent T-cell activation and proliferation. This activity occurred at low picomolar concentrations of MOR209/ES414 and was effective at low T-effector to tumor target cell ratios. In addition, cytotoxic activity was equivalent over a wide range of PSMA expression on target cells, suggesting that as few as 3,700 PSMA receptors per cell are sufficient for tumor lysis. In addition to high sensitivity and in vitro activity, MOR209/ES414 induced limited production of cytokines compared with other bispecific antibody formats. Pharmacokinetic analysis of MOR209/ES414 demonstrated a serum elimination half-life in NOD/SCID γ (NSG) mice of 4 days. Administration of MOR209/ES414 in murine xenograft models of human prostate cancer significantly inhibited tumor growth, prolonged survival, and decreased serum prostate-specific antigen levels only in the presence of adoptively transferred human T cells. On the basis of these preclinical findings, MOR209/ES414 warrants further investigation as a potential therapeutic for the treatment of CRPC. Mol Cancer Ther; 15(9); 2155–65. ©2016 AACR.

The ability to induce potent anti-tumor activity by stimulating 4-1BB (CD137), a key co-stimulatory receptor, makes 4-1BB an attractive immunotherapeutic target. However, a clinically tested, 4-1BB targeting monospecific antibody has been hampered by dose-limiting hepatic toxicities. To improve safety of 4-1BB targeting therapies we have developed a 4-1BB x 5T4 bispecific antibody designed to direct tumor-specific T cell responses to the tumor by stimulating 4-1BB only when co-engaged with 5T4, a tumor-associated antigen. The preclinical dataset presented here provides an overview of the mechanism of action and the efficacy and safety profile of ALG.APV-527, supporting its advancement into the clinic. ALG.APV-527 was built using the ADAPTIR™ platform with binding domains from the ALLIGATOR-GOLD® human scFv library. Its 5T4-dependent agonistic function was assessed using primary CD8+ T cells or NK cells in the presence of 5T4-expressing cells. Secretion of IFN-γ or granzyme B was measured at 72 hrs using ELISA. To measure proliferation, PBMCs were labelled with Cell Trace™ and gated CD8+ T cells were analyzed using flow cytometry. For tumor inhibition studies, the human 5T4-expressing colon carcinoma HCT116 xenograft model was used. 5T4 expression was evaluated in normal human tissues and different human tumors by immunohistochemistry. The preclinical safety profile of ALG.APV-527 was evaluated in a single and repeated dose, dose-range finding toxicology study in non-human primates (NHP). The study design included all the standard repeated dose toxicity parameters and in addition, pharmacokinetics, immunogenicity, and pharmacodynamic end-points. ALG.APV-527 induces a 5T4-dependent increase in IFN-γ and granzyme B production and enhances proliferation of T cells and NK cells. Furthermore, ALG.APV-527 inhibits tumor growth in a human 5T4-expressing colon carcinoma xenograft model. 5T4 is overexpressed in multiple solid tumors, potentially directing the activity of 4-1BB induced by ALG.APV-527 to 5T4-expressing tumors, improving the risk/benefit profile. Four doses (administered once weekly) did not cause any adverse events in the NHP toxicity study. In conclusion, ALG.APV-527 induces potent CD8+ T cell and NK co-stimulation but only in the presence of 5T4. Based on its efficacy and preclinical safety profile, ALG.APV-527 is a promising anti-cancer therapeutic for the treatment of multiple 5T4-expressing solid tumors. Citation Format: Anna Dahlman, Michelle Nelson, Jeannette Bannink, Starrla Johnson, Doreen Werchau, Anneli Nilsson, Lill Ljung, Gabriele Blahnik-Fagan, Robert Bader, Adnan Deronic, Peter Ellmark, Maria Askmyr, Gabriela Hernandez-Hoyos, Cathy McMahan, Sara Fritzell. Preclinical safety and efficacy of a tumor-directed T cell activating 4-1BB x 5T4 ADAPTIR™ bispecific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2380.

GATA-3 is necessary for the development of MHC class II-restricted CD4 T cells, and its expression is increased during positive selection of these cells. TCR signals drive this upregulation, but the signaling pathways that control this process are not well understood. Using genetic and pharmacological approaches, we show that GATA-3 upregulation during thymocyte-positive selection is the result of additive inputs from the Ras/MAPK and calcineurin pathways. This upregulation requires the presence of the transcription factor c-Myb. Furthermore, we show that TH-POK can also upregulate GATA-3 in double-positive thymocytes, suggesting the existence of a positive feedback loop that contributes to lock in the initial commitment to the CD4 lineage during differentiation.

CD137 (4-1BB) is a key costimulatory immune receptor (member of the TNFR-superfamily) that is expressed primarily on activated CD8+ T and NK cells. Stimulation of CD137 leads to enhanced proliferation, increased survival, intensified cytolytic activity of T cells and induced IFN-γ production. CD137 monoclonal antibody therapies have shown promising anti-tumor effects in the clinic, but systemic immune stimulation have induced dose-limiting hepatic toxicities. A novel bispecific antibody (ALG.APV-527) was developed based on ADAPTIR™ technology to direct the activation of T cells and NK cells to the tumor area, thereby minimizing systemic toxicity. ALG.APV-527 contains binding domains to the costimulatory molecule CD137 and the tumor-associated antigen 5T4. 5T4 is a tumor antigen expressed on a variety of solid tumor types. ALG.APV-527 was generated with binding domains from the Alligator-Gold® human scFv library, and has been optimized for binding and function. ALG.APV-527 increased CD8+ T-cell activation, as measured by IFN-γ production, but only in the presence of 5T4-expressing cells. Additionally, the CD137 ×5T4 bispecific antibody enhanced proliferation of primary T cells and increased potency in an NF-κB reporter assay. Of importance, the binding affinity and function of ALG.APV-527 retained a low Ec50 (nM range) regardless of the level of 5T4 expression on target cells. In conclusion, we have developed a novel ADAPTIR™ bispecific molecule that, based on preclinical data, has potential as a promising therapeutic for the treatment of a variety of 5T4-expressing solid tumors.

CD8+ T cells only require a brief stimulation with antigen in vitro to divide and differentiate into effector and memory cells upon transfer in vivo. The efficiency of clonal expansion and the functional characteristics of memory cells derived from briefly stimulated cells are poorly defined. We developed a system that allowed us to examine programming entirely in vivo. This was achieved by rapidly killing peptide-pulsed DCs carrying a diphtheria toxin receptor transgene with timed injections of diphtheria toxin without altering the course of an accompanying infection. The magnitude of clonal expansion, but not the functionality of the effector cells, correlated directly with the duration of antigen exposure. Furthermore, memory T cells were capable of mounting a secondary response, regardless of the length of antigen encounter during the primary response. These results indicate that the duration of initial antigen encounter influences the magnitude of the primary response, but does not program responsiveness during the secondary challenge.

T cell precursors undergo a series of developmental choices that progressively narrow their ability to give rise to different cell lineages. Evidence accumulated in the last few years suggests that Notch occupies a central place among the signal transduction pathways that regulate many of these choices, including the T/B, alphabeta/gammadelta and CD4/CD8 lineage decisions. Nevertheless the mechanisms by which Notch exerts its effects are not well understood, and in some cases the physiologic role is unclear. In this review we try to present succinctly the experiments and highlight the areas of controversy.

Immature double positive (DP) thymocytes bearing a T cell receptor (TCR) that interacts with self-major histocompatibility complex (MHC) molecules receive signals that induce either their differentiation (positive selection) or apoptosis (negative selection). Furthermore, those cells that are positively selected develop into two different lineages, CD4 or CD8, depending on whether their TCRs bind to MHC class II or I, respectively. Positive selection therefore involves rescue from the default fate (death), lineage commitment, and progression to the single positive (SP) stage. These are probably temporally distinct events that may require both unique and overlapping signals. Work in the past several years has started to unravel the signaling networks that control these processes. One of the first pathways identified as important for positive selection was Ras and its downstream effector, the Erk mitogen-activated protein kinase (MAPK) cascade. In this review we examine the factors that connect the TCR to the Ras/Erk cascade in DP thymocytes, as well as what we know about the downstream effectors of the Ras/Erk cascade important for positive selection. We also consider the possible role of this cascade in CD4/CD8 lineage development, and the possible interactions of the Ras/Erk cascade with Notch during these cell fate determination processes.

We have applied RNA interference (RNAi) technology to the analysis of genes involved in T-cell development, combining a reaggregate fetal thymic organ culture (rFTOC) system with retroviral delivery of short interfering RNA (siRNA) hairpins. The process involves the isolation of murine fetal liver or fetal thymocytes, infection with retroviral particles carrying the construct of interest, followed by reaggregation of the transduced precursors with fetal thymic stroma into lobes. Subsequently, individual lobes are harvested and analyzed for development at various time points. These reaggregate cultures recapitulate most features of T-cell development in vivo, including pre-TCR selection and expansion, positive selection of CD4 and CD8 T cells, and negative selection. In our hands, the combination of retroviral delivery of RNAi and rFTOCs is a quick alternative to conventional knockouts for the analysis of gene function during T-cell development. This chapter describes the methods we have developed to knock down gene expression in T-cell precursors, using retroviral delivery of siRNA hairpins.

PU.1 and GATA-3 are transcription factors that are required for development of T cell progenitors from the earliest stages. Neither one is a simple positive regulator for T lineage specification, however. When expressed at elevated levels at early stages of T cell development, each of these transcription factors blocks T cell development within a different, characteristic time window, with GATA-3 overexpression initially inhibiting at an earlier stage than PU.1. These perturbations are each associated with a distinct spectrum of changes in the regulation of genes needed for T cell development. Both transcription factors can interfere with expression of the Rag-1 and Rag-2 recombinases, while GATA-3 notably blocks PU.1 and IL-7Rα expression, and PU.1 reduces expression of HES-1 and c-Myb. A first-draft assembly of the regulatory targets of these two factors is presented as a provisional gene network. The target genes identified here provide insight into the basis of the effects of GATA-3 or PU.1 overexpression and into the regulatory changes that distinguish the developmental time windows for these effects.

The cytokine erythropoietin (Epo) promotes erythropoietic progenitor cell proliferation and is required for erythropoietic differentiation. We have found that the Epo gene is a direct transcriptional target gene of retinoic acid signaling during early erythropoiesis (prior to embryonic day E12.5) in the fetal liver. Mouse embryos lacking the retinoic acid receptor gene RXRα have a morphological and histological phenotype that is comparable with embryos in which the Epo gene itself has been mutated, and flow cytometric analysis indicates that RXRα-deficient embryos are deficient in erythroid differentiation. Epo mRNA levels are reduced substantially in the fetal livers of RXRα−/−embryos at E10.25 and E11.25, and genetic analysis shows that theRXRα and Epo genes are coupled in the same pathway. We furthermore show that the Epo gene is retinoic acid inducible in embryos, and that the Epo gene enhancer contains a DR2 sequence that represents a retinoic acid receptor-binding site and a retinoic acid receptor transcriptional response element. However, unlike Epo-deficient embryos that die from anemia, the erythropoietic deficiency in RXRα−/− embryos is transient; Epo mRNA is expressed at normal levels by E12.5, and erythropoiesis and liver morphology are normal by E14.5. We show that HNF4, like RXRα a member of the nuclear receptor family, is abundantly expressed in fetal liver hepatocytes, and is competitive with retinoic acid receptors for occupancy of the Epo gene enhancer DR2 element. We propose that Epo expression is regulated during the E9.5–E11.5 phase of fetal liver erythropoiesis by RXRα and retinoic acid, and that expression then becomes dominated by HNF4 activity from E11.5 onward. This transition may be responsible for switching regulation of Epo expression from retinoic acid control to hypoxic control, as is found throughout the remainder of life.

Although the promoter/enhancer of the IL-2 gene mediates inducible reporter gene expression in vitro, it cannot drive consistent expression in transgenic mice. The location and existence of any regulatory elements that could open the IL-2 locus in vivo have remained unknown, preventing analysis of IL-2 regulation in developmental contexts. In this study, we report the identification of such a regulatory region, marked by novel DNase-hypersensitive sites upstream of the murine IL-2 promoter in unstimulated and stimulated T cells. Inclusion of most of these sites in an 8.4-kb IL-2 promoter green fluorescent protein transgene gives locus control region-like activity. Expression is efficient, tissue specific, and position independent. This transgene is expressed not only in peripheral T cells, but also in immature thymocytes and thymocytes undergoing positive selection, in agreement with endogenous IL-2 expression. In contrast, a 2-kb promoter green fluorescent protein transgene, lacking the new hypersensitive sites, is expressed in only a few founder lines, and expression is dysregulated in CD8+ cells. Thus, the 6.4 kb of additional upstream IL-2 sequence contains regulatory elements that provide integration site independence and differential regulation of transgene expression in CD8 vs CD4 cells.

Thymocytes carrying MHC class I–restricted TCRs differentiate into CD8 T cells, while those recognizing MHC class II become CD4 T cells. The mechanisms underlying how MHC class recognition, coreceptor expression, and effector function are coordinated are not well understood. Since the tyrosine kinase Lck binds with more affinity to CD4 than CD8, it has been proposed as a candidate to mediate this process. By using transgenic mice with altered Lck activity, we show that thymocytes carrying a class II–restricted TCR develop into functional CD8 T cells when Lck activity is reduced. Conversely, thymocytes carrying a class I–restricted TCR develop into functional CD4 T cells when Lck activity is increased. These results directly show that quantitative differences in the Lck signal control the CD4/CD8 lineage decision.

Background: Treatment of metastatic, castrate-resistant prostate cancer (CRPC) remains a highly unmet medical need. We have developed a humanized bispecific ADAPTIR™ (modular protein technology) molecule, ES414, which redirects T-cell cytotoxicity against cells expressing PSMA (Prostate Specific Membrane Antigen), a prostate cancer antigen. In previous preclinical studies, ES414 has been shown to induce cytotoxicity against prostate cancer cells in vitro when measured by target cell lysis and in vivo in multiple mouse xenograft models. ES414 was also previously shown to be well-tolerated in humanized mice and non-human primates. Here, we examine the effects of exposure to ES414 over several days on T-cell function in the presence of PSMA+ target cells.Materials and Methods: Long term cytotoxic activity induced by ES414 against PSMA(+) cell lines in the presence of purified human T cells was followed in vitro by use of high content microscopy. Cytotoxic activity was determined by incorporation of a label such as propidium iodide or 7-aminoactinomycin D. Binding to subsets of peripheral blood mononuclear cells (PBMC) and Fc(gamma) receptor-expressing cell lines was assessed by multi-color flow cytometry. T cells were assessed for activation and proliferation in a similar setting using multi-color flow cytometry. Cytokine release was measured at multiple timepoints using multiplex immunoassays. Results: ES414 selectively bound to T cells, and did not bind to other PBMC subsets nor to Fc (gamma) receptor expressing cell lines. T cells activated by ES414 in the presence of target cells rapidly induced target cell lysis within 4 hours and continued serial lysis for more than 48 hours. Target cell lysis occurred at low effector-to-target cell ratios. In the presence of target cells, ES414 also induced T-cell activation, as measured by upregulation of activation markers, and proliferation, but induced limited levels of cytokine secretion in comparison to control molecules. Conclusions: These preclinical in vitro studies show that ES414 selectively engages T cells, targets T cells towards tumor cells, and induces T-cell activation, with limited cytokine release. Limited cytokine release in the presence of redirected T-cell cytotoxicity is a desirable feature that warrants further investigation of ES414 as a potential therapeutic for CRPC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B249. Citation Format: Gabriela Hernandez-Hoyos, Toddy Sewell, Padma Ravikumar, Megan Aguilar, John Kumer, David Bienvenue, Catherine J. McMahan, Jane Gross, John W. Blankenship. Prolonged cytotoxic activity induced by anti-PSMA x anti-CD3 ADAPTIR™ molecule in the absence of significant cytokine release. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B249.

Ets family transcription factors control the expression of a large number of genes in hematopoietic cells. Here we show strikingly precise differential expression of a subset of these genes marking critical, early stages of mouse lymphocyte cell-type specification. Initially, the Ets family member factor Erg was identified during an arrayed cDNA library screen for genes encoding transcription factors expressed specifically during T cell lineage commitment. Multiparameter fluorescence-activated cell sorting for over a dozen cell surface markers was used to isolate 18 distinct primary-cell populations representing discrete T cell and B cell developmental stages, pluripotent lymphoid precursors, immature NK-like cells and myeloid hematopoietic cells. These populations were monitored for mRNA expression of the Erg, Ets-1, Ets-2, Fli-1, Tel, Elf-1, GABPα, PU.1 and Spi-B genes. The earliest stages in T cell differentiation show particularly dynamic Ets family gene regulation, with sharp transitions in expression correlating with specification and commitment events. Ets, Spi-B and PU.1 are expressed in these stages but not by later T-lineage cells. Erg is induced during T-lineage specification and then silenced permanently, after commitment, at the β-selection checkpoint. Spi-B is transiently upregulated during commitment and then silenced at the same stage as Erg. T-lineage commitment itself is marked by repression of PU.1, a factor that regulates B-cell and myeloid genes. These results show that the set of Ets factors mobilized during T-lineage specification and commitment is different from the set that maintains T cell gene expression during thymocyte repertoire selection and in all classes of mature T cells.

Known transcription factor-DNA interactions in the minimal enhancer of the murine interleukin-2 gene (IL-2) do not easily explain the T cell specificity of IL-2 regulation. To seek additional determinants of cell type specificity, in vivo methodologies were employed to examine chromatin structure 5' and 3' of the 300 bp IL-2 proximal promoter/enhancer region. Restriction enzyme accessibility revealed that until stimulation the IL-2 proximal promoter/enhancer exists in a closed conformation in resting T and non-T cells alike. Within this promoter region, DMS and DNase I genomic footprinting also showed no tissue-specific differences prior to stimulation. However, DNase I footprinting of the distal -600 to -300 bp region revealed multiple tissue-specific and stimulation-independent DNase I hypersensitive sites. Gel shift assays detected T cell-specific complexes binding within this region, which include TCF/LEF or HMG family and probable Oct family components. Upon stimulation, new DNase I hypersensitive sites appeared in both the proximal and distal enhancer regions, implying that there may be a functional interaction between these two domains. These studies indicate that a region outside the established IL-2 minimal enhancer may serve as a stable nucleation site for tissue-specific factors and as a potential initiation site for activation-dependent chromatin remodeling.

Abstract 3722 Background: Despite advances in treatments for B-cell leukemias and lymphomas, many patients ultimately relapse and succumb to disease following multiple courses of therapy. Bispecific antibody fragments that can simultaneously engage T cells and tumor cells have been shown, in the literature, to destroy tumor cells by effectively redirecting the cytotoxic function of T cells. T-cell engaging bispecific molecules linking anti-CD19 and anti-CD3 binding domains in the context of novel SCORPION™ (multi-specific protein therapeutic) proteins were evaluated both in vitro and in vivo for function and stability. Methods: Redirected T-cell cytotoxicity (RTCC) was measured by combining CD19 positive or negative cell lines with SCORPION proteins in the presence of human T cells. In a similar assay context, CFSE-labeled T cells were monitored for activation and proliferation. Functional RTCC assays were also used to analyze serum stability of SCORPION molecules in vitro and to complete an in vivo pharmacokinetic analysis. In vivo efficacy was assessed by monitoring the rate of tumor outgrowth of Ramos xenografts co-implanted with human peripheral blood mononuclear cells (PBMC) in NOD/SCID mice after treatment with SCORPION molecules. Results: SCORPION molecules potently mediate target-specific T-cell cytotoxicity toward tumor cell lines presenting cell surface CD19, with EC50 values for cytotoxicity at low pM concentrations. These molecules also demonstrate induction of T-cell activation and proliferation in the presence of target-bearing tumor cells but not in the absence of target expression. SCORPION molecules retain stable function following incubation at 37°C in mouse serum for up to a week in vitro, and pharmacokinetic analysis of SCORPION protein function in BALB/c mouse serum following intravenous administration resulted in half-life estimates of 69–84 hours. In efficacy studies conducted in NOD/SCID mice, SCORPION proteins significantly inhibited the outgrowth of Ramos tumor xenografts in the presence of human effector cells. Conclusion: SCORPION molecules targeting CD19 and CD3 effectively harness the cytotoxic activity of T cells to kill CD19 positive tumor cells both in vitro and in vivo and show potential for further investigation as possible therapeutic agents for B-cell malignancies. Disclosures: Chenault: Emergent BioSolutions: Employment. Gottschalk:Emergent BioSolutions: Employment. Hernandez-Hoyos:Emergent BioSolutions: Employment. Wiens:Emergent BioSolutions: Employment. Gordon:Emergent BioSolutions: Employment. Klee:Emergent BioSolutions: Employment, Equity Ownership. Bienvenue:Emergent BioSolutions: Employment. Dasovich:Emergent BioSolutions: Employment. Kumer:Emergent BioSolutions: Employment. Aguilar:Emergent BioSolutions: Employment. Bannink:Emergent BioSolutions: Employment, Equity Ownership. McMahan:Emergent BioSolutions: Employment, Equity Ownership. Natarajan:Emergent BioSolutions: Employment, Equity Ownership. Algate:Emergent BioSolutions: Employment, Equity Ownership. Blankenship:Emergent BioSolutions: Employment, Equity Ownership, Patents & Royalties.

GATA-3 is expressed at higher levels in CD4 than in CD8 SP thymocytes. Here we show that upregulation of GATA-3 expression in DP thymocytes is triggered by TCR stimulation, and the extent of upregulation correlates with the strength of the TCR signal. Overexpression of GATA-3 or a partial GATA-3 agonist during positive selection inhibits CD8 SP cell development but is not sufficient to divert class I-restricted T cell precursors to the CD4 lineage. Conversely, expression of the GATA-3 antagonist ROG or of a GATA-3 siRNA hairpin markedly enhances development of CD8 SP cells and reduces CD4 SP development. We propose that GATA-3 contributes to linking the TCR signal strength to the differentiation program of CD4 and CD8 thymocytes.

The essential hematopoietic transcription factor PU.1 is expressed in multipotent thymic precursors but downregulated during T lineage commitment. The significance of PU.1 downregulation was tested using retroviral vectors to force hematopoietic precursors to maintain PU.1 expression during differentiation in fetal thymic organ culture. PU.1 reduced thymocyte expansion and blocked development at the pro-T cell stage. PU.1-expressing cells could be rescued by switching to conditions permissive for macrophage development; thus, the inhibition depends on both lineage and developmental stage. An intact DNA binding domain was required for these effects. PU.1 expression can downregulate pre-Tα, Rag-1, and Rag-2 in a dose-dependent manner, and higher PU.1 levels induce Mac-1 and Id-2. Thus, downregulation of PU.1 is specifically required for progression in the T cell lineage.