Search

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

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.

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.

Background : TRU-015 is a small modular immunopharmaceutical protein drug that binds to CD20 and effectively depleted B cells in nonhuman primates. Objective : The aim of this clinical study was to determine the pharmacokinetic (PK) and pharmacodynamic (PD) properties, immunogenicity, and tolerability of TRU-015 in patients with rheumatoid arthritis (RA). Methods : This Phase I, open-label, dose-escalation clinical study was conducted at 4 medical centers in the United States. Patients with RA who were receiving stable-dose methotrexate were enrolled in 1 of 8 dose groups and received TRU-015 as a single IV dose of 0.015, 0.05, 0.15, 0.5, 1.5, 5, or 15, or 2 IV doses of 15 mg/kg, administered 7 days apart (30 mg/kg). Patients were enrolled in the next higher dose cohort based on the tolerability observed in the prior cohort. Prior to TRU-015 infusion, patients were premedicated with an antihistamine and acetaminophen and may have received a corticosteroid at the investigator's discretion. Serum samples were collected for analysis of PK properties (serum t ½ ) and neutralizing antibodies to TRU-015; enzyme-linked immunosorbent assays and a cell-based neutralizing assay were used to evaluate samples from patients. PD response was measured using B-cell (CD19 + -cell) count using flow cytometry at prespecified time points. Tolerability was assessed during drug infusion and at prespecified time points after infusion using physical examination and laboratory analysis. Patients were followed for ≥4 weeks and until B-cell recovery. Results : Thirty-seven patients were enrolled. Most were female (81%) and white (95%); the mean age was 53 years. Serum t ½ ranged from 12 to 19 days. B-cell depletion generally increased in degree and duration with increasing doses. No neutralizing antibodies to TRU-015 were detected. Mild adverse events (AEs) included back pain, headache, peripheral edema, and upper respiratory infection (5 patients each). Mild urticaria occurred in 1 patient. Grade 3 AEs included hypertension, arthralgia, and urticaria and bronchospasm (1 patient each). No dose-limiting toxicity was found. Conclusions : In this small population of patients with RA, the C max and the AUC appeared to increase in a dose-proportional manner. The mean t ½ ranged from 12 to 19 days. TRU-015 was associated with dose-dependent B-cell depletion and an acceptable tolerability profile.

Monoclonal antibodies have begun to show great clinical promise for the treatment of cancer. Antibodies that can directly affect a tumor cell's growth and/or survival are of particular interest for immunotherapy. Previously, we described monoclonal antibody DMF10.62.3 that had antiproliferative and proapoptotic effects when it bound an antigen of unknown identity on tumor cells in vitro. In this report, we determined that DMF10.62.3 and a clonally related antibody DMF10.167.4 recognize the ganglioside GM2. These antibodies react with a GM2 epitope that is expressed on a large number of tumor cell lines, including human melanoma and small cell lung carcinoma, but not on normal primary lines or most normal tissues. Interestingly, this pattern of cellular reactivity is distinct from that reported for other previously described GM2 antibodies, a difference that is presumably due to DMF10.167.4's binding to a unique GM2-associated epitope. Additional characterization of DMF10.167.4 revealed that this antibody was able to induce apoptosis and/or block cellular proliferation when cultured in vitro with the human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines. In vivo, DMF10.167.4 antibody was well tolerated in mice and did not detectably bind to or damage normal tissues. However, this antibody was able to prevent murine E710.2.3 lymphoma, human CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines from establishing tumors in vivo and blocked progression of established CHL-1 and SBC-3 tumors in vivo. Therefore, monoclonal antibody DMF10.167.4 has immunotherapeutic potential.

With few exceptions, humans are the only species known to develop acquired immunodeficiency syndrome (AIDS) after human immunodeficiency virus (HIV) infection. We report here that an isolate of HIV type 2, EHO, readily established persistent infection in 100% of Macaca nemestrina in three consecutive transmission studies. Of the eight infected animals, five showed persistently high virus load and six developed AIDS-like diseases or CD4+ cell depletion within 4 years of infection. The pathology and clinical signs closely parallel those of HIV-1 infection of humans, including lymphadenopathy, anemia, CD4+ cell depletion, and opportunistic infections. A cell-free virus stock was established from the lymph nodes of an animal that developed AIDS-like diseases. This virus, HIV-2/287, was highly pathogenic in M. nemestrina, causing CD4+ cell depletion within 2–8 weeks post-infection. While both HIV-2 EHO and HIV-2/287 use predominantly CXCR4, the latter shows greatly enhanced replicative capacity in macaque peripheral blood mononuclear cells (PBMCs). The establishment of a human immunodeficiency virus that causes rapid and reproducible CD4+ cell depletion in macaques could facilitate the study of HIV pathogenesis and the development of effective vaccines and therapy against AIDS.

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

The present study utilizes an in vivo murine tumor expressing human Her-2/neu to evaluate potential Her-2/neu vaccines consisting of either full length or various subunits of Her-2/neu delivered in either protein or plasmid DNA form. Our results demonstrate that protective immunity against Her-2/neu-expressing tumor challenge can be achieved by vaccination with plasmid DNA encoding either full length or subunits of Her-2/neu. Partial protective immunity was also observed following vaccination with the intracellular domain (ICD), but not extracellular domain (ECD), protein subunit of Her-2/neu. The mechanism of protection elicited by plasmid DNA vaccination appeared to be exclusively CD4 dependent, whereas the protection observed with ICD protein vaccination required both CD4 and CD8 T cells.

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.