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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

Supplementary figures and methods

Bisphosphonate-related osteonecrosis of the jaw is a serious complication of systemic bisphosphonate administration, the mechanism of which remains unclear. Many hypotheses regarding pathophysiology are discussed, including the most commonly cited: suppression of bone remodeling and suppression of angiogenesis, but none of these would explain all the unique features of bisphosphonaterelated jaw osteonecrosis. Bisphosphonates are potent inhibitors of osteoclasts, and recent studies have shown that osteoclasts are important for bone angiogenesis. Therefore, we hypothesize that bisphosphonates inhibit osteoclastic stimulation of angiogenesis, thereby contributing to the occurrence of osteonecrosis of the jaws. This theory would partially explain the pathophysiology of bisphosphonate-related osteonecrosis of the jaw to the unfathomable.

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

Introduction Benign jaw tumors or tumor-like swellings of the jaws can be classified as odontogenic or non-odontogenic. The type of surgery undertaken or the histological typing of the tumor could be the cause of the recurrence of these tumors. Our objective was to determine clinical and paraclinical features implicated in or that signal the recurrence of benign tumors. Material and methods We carried out a case control study during the period from January 2010 to April 2020 in the units of Stomatology and maxillofacial surgery, radiology and pathology units of Yaounde University Teaching Hospital, Yaounde Central Hospital and Yaounde Gynaecology, Obstetrics and Pediatrics Hospital. Participants who had been operated for a benign jaw tumor were included in this study. For the statistical analysis: the significant threshold was set at 0.05 that is all p values less than 0.05 were considered statistically significant. Results We retained 89 study participants with a recurrence rate of 7.51%. In our study, we had 61.8% females and 38.2% males with a sex ratio of 0.62. The localization at the level of the angle and ramus of the mandible, tooth preservation, type of anesthesia, radiological tumor aspect and histomorphology (ameloblastic fibroma and osteoma) were statistically significant for recurrence. Conclusion The recurrence of jaw tumors is associated with several clinical and paraclinical factors. These factors can help predict recurrences and hence reduce the complications associated with late treatment plan.

Objective The aim of this study was to evaluate the efficacy of 48-h antibiotic prophylaxis in the prevention of surgical site infections during simple dental extractions by conducting a clinical trial controlled by 7-day antibiotic therapy. Our hypothesis was that there was no difference between these two protocols in the prevention of surgical site infection. Method ology: This was a 6-month randomised multicentre clinical trial conducted in three health facilities in the city of Yaounde. The study included any patient aged 18 years or older who was scheduled for a simple dental extraction. The calculated sample size was 142 patients randomly divided into 2 groups. Group I consisted of patients who took 1 g of amoxicillin 1 h before surgery and then 1 g every 12 h for 48 h. Group 2 took amoxicillin only postoperatively at a rate of 1 g every 12 h for 7 days. The control was done at day and day 7. The primary endpoint was the occurrence of surgical site infection and the secondary endpoints were the occurrence of dry socket, oedema and pain. Results No statistically significant difference was found at the 5% level between the two protocols regarding the occurrence of pain, postoperative oedema and surgical site infection with respective p-values of 0.834; 0.414; 0.175. In addition, a statistically significant difference was found for dry socket disease (p-value = 0.0149) showing a reduced risk in the 48-h group. Conclusion It was found that there was no difference between these two protocols except for dry socket disease where the 48 h antibiotic prophylaxis protocol seems to be more effective. Furthermore, the question of the necessity of antibiotic prophylaxis for simple extractions remains, as only a randomised placebo-controlled clinical trial could provide answers.

Context Facial osteomyelitis is an inflammatory process of the bones of the face. It is common in poor countries as a result of a weak health care system. The diagnosis and management of this pathology is complex in our context because of poor medical equipment. Aims The objective of this study was to establish a nosological framework for facial osteomyelitis. Settings and Design: This was a retrospective study of facial osteomyelitis collected in the Odontostomatology/Maxillo-Facial and Otolaryngology/Cervico-Facial Surgery Departments of the Yaounde University Hospital over a period of 15 years. Methods and material All patients diagnosed with facial osteomyelitis by one of the physicians of the above departments mentioned services were included. An epidemiological record (sex, age, ethnic origin, risk factors, clinical, origin of the disease, imaging and biology) was collected for each patient. Osteomyelitis was classified according to the Zurich classification. Statistical analysis used Descriptive statistics were performed and the Fisher test was used to indicate differences in radiographic characteristics between the types of facial osteomyelitis. A p-value of less than 0.05 was considered to indicate statistical significance. Results Of the ninety-nine facial osteomyelitis, eleven (12.3%) were acute and seventy-eight (87.6%) were chronic osteomyelitis, nine of which were primary and sixty-nine secondary. The presence of suppuration, fistula and or denudation were the prerogative of secondary chronic osteomyelitis. Heterogeneous spinal fibrosis and pseudodysplastic hyperplasia were pathognomonic of primary chronic osteomyelitis. Conclusion These results suggest that the main differences between the types of osteomyelitis reside: in the duration of disease progression; whether or not suppuration/stripping and fistula are present; the presence or absence of mixed sclerosis, sequestration and fistula heterogeneous spinal cord fibrosis and pseudodysplastic hyperplasia.

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.

Lipomas are benign tumors of adipose tissue with pathogenesis and a controversial etiology. An explicit link between soft tissue trauma and the occurrence of lipomas is difficult to establish. However, some cases have been described. These soft tissue tumors have been called "post-traumatic lipomas". Clinically oral lipomas appear as yellowish nodular masses less than 3 cm in diameter. Yet the oral lipoma can increase in size from 5 to 11 cm over several years. The reported case is the first referred in our service. It was a mass located on the inside of the left cheek painless, nodular, yellowish about 8 cm in diameter, secondary to trauma. The treatment was surgical under local anesthesia. In the presence of mature fat cells very similar in appearance to the surrounding normal fat associated with a detachment of the superficial layers of the oral epithelium, the suggested diagnosis was a posttraumatic intraoral lipoma.

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.

Introduction: Depletion of CD123 over-expressing tumor cells may improve patient outcomes in several hematological malignancies including AML, MDS, ALL, CML, HCL and BPDCN. CD123 is infrequently expressed by normal cells making it an attractive tumor target, currently being pursued using several different approaches including T-cell engaging immunotherapies. Cytokine release syndrome (CRS) is a concern with T-cell engaging immunotherapies that may require complex clinical development strategies to manage safely. We have developed APVO436, a bispecific anti-CD123 x anti-CD3 ADAPTIR molecule for redirecting T-cell cytotoxicity to CD123-expressing tumor cells, currently in Phase I clinical testing in AML and MDS. A potential advantage of the ADAPTIR platform is reduced cytokine release compared to other formats (Mol Cancer Ther; 15(9):2155-65). We previously compared APVO436 activity to another CD123 x CD3 bispecific antibody containing the amino acid sequence of MGD006. We also demonstrated that APVO436 induces activation of AML patient T cells to kill endogenous tumor cells. Here we extend these studies to test the capacity of APVO436 to induce memory T-cell generation and describe the results of repeat dose toxicology studies in cynomolgus macaques. Methods: Binding, T-cell activation and proliferation were assessed using flow cytometry. Cytotoxic activity was determined using chromium release and flow cytometry assays. Cytokine levels were assessed using multi-plex technology. Repeat dose toxicology studies were performed in cynomolgus macaques. Results: In vitro, APVO436 induced lower levels of several T-cell cytokines while demonstrating similar levels of tumor cell lysis potency compared to other CD123 x CD3 bispecific antibodies. APVO436 induced extensive T-cell proliferation and the development of fully functional memory T cells with cytolytic function. Repeat-dose administration of APVO436 in NHP for 4 weeks by IV bolus injection induced transient changes in T lymphocytes and CD123+ leukocytes. Terminal elimination half-life ranged up to 108 hours with dose proportional exposure after first and last dose; some impact on serum concentration was observed from anti-drug antibodies at late time points. Conclusions: APVO436 induced lower levels of T-cell cytokines associated with CRS compared to other CD123 x CD3 targeting bispecific antibodies and induced the generation of functional memory T cells. Repeat-dose toxicology studies in cynomolgus monkeys demonstrate that APVO436 has antibody-like clearance and volume of distribution parameters and a serum half-life of about 4.5 days. These data support clinical studies with APVO436 as a potential treatment for AML and other hematological malignancies with possible safety advantages over other CD123 targeting therapies. Citation Format: Michael R. Comeau, Rebecca Gottschalk, Mollie Daugherty, Toddy Sewell, Lynda Misher, Bannink Jeannette, Starrla Johnson, Lara Parr, John Kumer, David Jablonski, Melissa DeFrancesco, David Bienvenue, Gabriela H. Hoyos, Catherine J. McMahan, Jane A. Gross. APVO436, a bispecific anti-CD123 x anti-CD3 ADAPTIR™ molecule for redirected T-cell cytotoxicity with limited cytokine release, is well tolerated in repeat dose toxicology studies in cynomolgus macaques [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 LB-199.

Introduction: Depletion of CD123 over-expressing malignant cells provides a potential new treatment option which may improve patient outcomes in several hematological malignancies. CD123 is over-expressed in AML, MDS, ALL, CML, HCL and BPDCN and infrequently expressed by normal cells making it an attractive target which is being pursued using a number of different approaches including T-cell engaging immunotherapy. Cytokine release syndrome is a significant concern with T-cell activating therapeutics which has led to severe complications in clinical trials. We have developed APVO436, a bispecific anti-CD123 x anti-CD3 ADAPTIR molecule for redirecting T-cell cytotoxicity to CD123-expressing tumor cells. A potential advantage of the ADAPTIR platform is reduced cytokine release upon T-cell engagement compared to other formats (Mol Cancer Ther. 2016 Sep;15(9):2155-65). Here we present in vitro and in vivo activity of APVO436 and compare the activity of APVO436 to another anti-CD123 x anti-CD3 bispecific containing the amino acid sequence of MGD006.Methods: Binding, T-cell activation and proliferation were assessed using multi-color flow cytometry. Cytotoxic activity was determined using chromium release assays and flow cytometry. PBMC samples were obtained from normal donors and AML patients. In vivo studies were performed using NSG mice transplanted with human PBMC's. The CD123 and CD3 binding domain sequences for flotuzumab (MGD006) were obtained from patent W02015026892 engineered in Macrogenic's dual-affinity re-targeting format as reported in Sci Transl Med. 2015 May 27;7(289):289ra82.Results: Dose-dependent cytotoxicity of CD123 expressing tumor cell lines and primary AML cells was induced by APVO436 at low effector to target ratios, accompanied by T-cell activation and proliferation. APVO436 induced significantly lower levels of several T-cell cytokines including IFNγ, IL-2, and TNFα compared to the molecule in the dual-affinity re-targeting format. In vivo, APVO436 significantly reduced established tumor burden in xenograft murine models.Conclusions: APVO436 potently induces T-cell activation, proliferation and CD123+ cell depletion with AML and normal donor samples and CD123 expressing tumor cell lines with limited levels of T-cell cytokine release compared to another CD123 x CD3 targeting bispecific format suggesting a potential safety advantage. APVO436 inhibits tumor-growth in sub-cutaneous tumor models with IV-implanted human T cells, indicating migration and engagement of T cells at the tumor site. These data are supportive of further investigation of APVO436 as a potential treatment option for AML and other hematological malignancies. GLP toxicology studies have been completed in non-human primates and APVO436 is advancing to clinical testing. Citation Format: Michael R. Comeau, Robert E. Miller, Robert Bader, Rebecca Gottschalk, Mollie Daugherty, Toddy Sewell, Lynda Misher, Lara Parr, Melissa DeFrancesco, David Bienvenue, Catherine J. McMahan, Gabriela H. Hoyos, Jane A. Gross. APVO436, a bispecific anti-CD123 x anti-CD3 ADAPTIR™ molecule for redirected T-cell cytotoxicity, induces potent T-cell activation, proliferation and cytotoxicity with limited cytokine release [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1786.

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.

Introduction: CD123 has emerged as a promising target for T-cell directed immunotherapy in acute myeloid leukemia due to its high level of expression on leukemic blasts and stem cells and low frequency of expression on normal cell types. CD123 is expressed by rare normal leukocyte populations, including basophils and plasmacytoid dendritic cells in circulation and hematopoietic progenitor cells in the bone marrow. We have developed APVO436, a bispecific anti-CD123 x anti-CD3 ADAPTIR molecule for redirecting T-cell cytotoxicity to CD123-expressing tumor cells. Results are presented that examine the in vitro and in vivo activity of APVO436 in preclinical models of AML, as well as pharmacokinetics and tolerability in nonhuman primates. Methods: CHO-produced APVO436 protein was used for in vitro functional studies with CD123+ AML tumor cell lines and primary human T-cell populations. Cytotoxic activity was determined using chromium release assays. On-cell binding, T-cell activation and proliferation were assessed using multicolor flow cytometry. Cytokine levels were measured using multiplex assay kits. In vivo studies to examine tumor growth inhibition activity were performed using NSG mice with established AML tumors followed by treatment with APVO436 coadministered with T cells. Tumor growth was assessed by bioluminescent imaging. In vivo studies to determine pharmacokinetics of APVO436 following single intravenous injections were performed in healthy BALB/c mice and cynomolgus monkeys. Results: APVO436 bound to CD123 and CD3 expressing cells with high affinity. APVO436 induced concentration-dependent lysis of CD123+ AML cell lines with primary human T cells at low effector-to-target ratios, accompanied by T-cell activation and proliferation. These activities were dependent on the expression of CD123 by the tumor target cells. Treatment of established disseminated tumors in mice with APVO436 resulted in a significant reduction in tumor burden. A single 10 mg/kg IV injection of APVO436 in normal BALB/c mice demonstrated an extended elimination serum half-life of approximately 300 hours. Single IV injections of APVO436 ranging from 0.25 mg/kg to 1 mg/kg were well tolerated in cynomolgus monkeys. The observed elimination half-life in cynomolgus serum was up to 84 hours with normal clearance and volume distribution. Conclusions: These data demonstrate APVO436 has potent in vitro activity against a number of CD123-expressing tumor cell lines and in vivo significantly reduces established tumor burden in xenograft models. APVO436 demonstrates an extended serum half-life in normal mice and cynomolgus monkeys. These data are supportive of further investigation of APVO436 as a potential treatment option for AML and other hematologic malignancies. Citation Format: Michael R. Comeau, Robert E. Miller, Jeannette Bannink, Starrla Johnson, Robert Bader, Rebecca Gottschalk, Lynda Misher, Danielle Mitchell, Lara Parr, Melissa DeFrancesco, David Bienvenue, Catherine J. McMahan, Gabriela H. Hoyos, Jane A. Gross. Characterization of APVO436, a bispecific anti-CD123 x anti-CD3 ADAPTIR™ molecule for redirected T-cell cytotoxicity, in preclinical models of AML and nonhuman primates [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B111.

Introduction: CD123 is a component of the IL-3 receptor expressed in several hematological malignancies including AML, ALL, HCL, and MDS. CD123 is a compelling target in AML due to its overexpression on AML blasts as well as leukemic stem cells, which are thought to be resistant to chemotherapy and may be responsible for relapse of disease following treatment. While CD123 is expressed by some normal leukocyte populations in circulation and hematopoietic progenitor cells in the bone marrow, the low frequency of expression on normal cell types provides a therapeutic window for targeting CD123 in tumor settings with the potential for durable response and reversible side effects. We have developed bispecific anti-CD123 x anti-CD3 ADAPTIR molecules APVO436 and APVO437 for redirecting T-cell cytotoxicity to CD123-expressing tumor cells. Results are presented that examine the in vitro and in vivo activity of these molecules in preclinical models of AML. Methods: APVO436 and APVO437 proteins were expressed in CHO cells. Affinity SPR studies were performed using recombinant CD123-ectodomain. In vitro functional studies were conducted with CD123+ AML tumor cell lines and primary human and cynomolgus macaque T-cell populations. Cytotoxic activity was determined using chromium release assays. On-cell binding, T-cell activation and proliferation were assessed using multi-color flow cytometry. Pharmacokinetic parameters were determined in BALB/c mice using a single IV dose of approximately 10 mg/kg. In vivo studies to examine tumor growth inhibition activity were performed with NOD/SCID mice co-implanted subcutaneously with AML tumor cells and human T-cells followed by treatment with APVO436 or APVO437. Tumor growth was assessed by measuring tumor volume and Bioluminescent Imaging. Results: APVO436 and APVO437 bound human CD123 protein with high affinity and binding to CD123 and CD3 expressing cell lines was confirmed by flow cytometry. Both APVO436 and APVO437 induced concentration-dependent lysis of CD123+ AML cell lines with primary human effector T-cells, accompanied by T-cell activation and proliferation. Comparable redirected T-cell cytotoxicity function was observed using primary cynomolgus macaque T cells. These activities were dependent on the expression of CD123 by the tumor target cells. APVO436 and APVO437 demonstrated an extended elimination half-life in mouse serum, typical of molecules capable of binding the neo-natal Fc receptor. In vivo, growth of AML tumor cells was inhibited by treatment with low doses of APVO436 and APVO437, significantly improving host survival. Conclusion: Taken together these data demonstrate potent in vitro and in vivo activity of APVO436 and APVO437 against CD123 expressing tumor cells and are supportive of further investigation of this approach as a potential treatment option for AML and other hematological malignancies. Citation Format: Michael R. Comeau, Danielle Mitchell, Rebecca Gottschalk, Lynda Misher, Mollie Daugherty, Lara Parr, Peter Pavlik, Brian Woodruff, Hang Fang, Megan Aguilar, Jeannette Bannink, Starrla Johnson, Gary Li, Robert E. Miller, Robert Bader, Nicole Zhang, Toddy Sewell, Maria Dasovich, Gabriela H. Hoyos, John W. Blankenship, Catherine McMahan, David Bienvenue, Jane A. Gross. Bispecific anti-CD123 x anti-CD3 ADAPTIR™ molecules for redirected T-cell cytotoxicity in hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 597. doi:10.1158/1538-7445.AM2017-597

Background: Effective treatment of metastatic, triple-negative breast cancer (TNBC) remains a highly unmet medical need. We have developed ES425, a bispecific ADAPTIR™ (modular protein technology) molecule that redirects T-cell cytotoxicity to tumor cells expressing ROR1 (receptor tyrosine kinase-like orphan receptor 1), an oncofetal antigen expressed on TNBC and other malignancies. Results are presented for studies run to examine in vitro and in vivo activity of ES425 in preclinical models of TNBC. Materials and Methods: Target-dependent cytotoxic activity was examined in vitro by treating ROR1(+) cell lines and ROR1(−) cell lines with ES425 in the presence of purified human T cells or human peripheral blood mononuclear cells (PBMCs). Cytotoxic activity was determined using chromium release assays. T cells were assessed for activation and proliferation using multi-color flow cytometry. Pharmacokinetics of ES425 in NOD/SCID gamma (NSG) mice was determined using single intravenous dose of approximately 10 mg/kg. Serum concentrations at time points ranging from 15 minutes to 504 hours were used to calculate the terminal elimination half-life of ES425. To assess activity in vivo, NOD/SCID mice were implanted subcutaneously with the ROR1(+) TNBC tumor cell line MDA-MB-231 and purified human T cells and treated with ES425. This model was run twice with T cells from different human donors. Tumor growth was assessed by measuring tumor volume. Results: ES425 efficiently redirected T cell cytotoxicity against ROR1(+) cell lines at low picomolar concentrations in vitro. Cytotoxic activity was dependent on expression of ROR1 by the target cells. T cells were activated and proliferated in response to ES425 in the presence of ROR1(+) target cells; proliferation was not observed in response to ROR1(−) cells. In vivo, pharmacokinetic analysis showed a serum half-life of approximately 7 days in NSG mice, and ES425 inhibited growth of MDA-MB-231 tumors in mouse xenografts. Repeat experiments showed similar inhibition of tumor growth and an improvement in overall survival. Conclusions: These studies show that ES425 may be an efficient agent for redirecting T cell cytotoxicity in preclinical TNBC models and merits investigation as a potential therapeutic in TNBC and other malignancies. Citation Format: John W. Blankenship, Lynda Misher, Danielle Mitchell, Nicole Zhang, Philip Tan, Gabriela H. Hoyos, Padma Ravikumar, Robert Bader, Catherine J. McMahan, Robert E. Miller, Jeannette Bannink, Hang Fang, Lara Parr, Maria Dasovich, David Bienvenue, Megan Aguilar, Carina Xu, Mollie Daugherty, Brian Woodruff, Jane A. Gross. anti-ROR1 x anti-CD3 ADAPTIR™ molecule, ES425, redirects T-cell cytotoxicity and inhibits tumor growth in preclinical models of triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4995.