The gibberellin class of plant hormones has been implicated in the control of flowering in several species. In Arabidopsis, severe reduction of endogenous gibberellins delays flowering in long days and prevents flowering in short days. We have investigated how the differential effects of gibberellins on flowering correlate with expression of LEAFY , a floral meristem identity gene. We have found that the failure of gibberellin-deficient ga1-3 mutants to flower in short days was paralleled by the absence of LEAFY promoter induction. A causal connection between these two events was confirmed by the ability of a constitutively expressed LEAFY transgene to restore flowering to ga1-3 mutants in short days. In contrast to short days, impairment of gibberellin biosynthesis caused merely a reduction of LEAFY expression when plants were grown in long days or with sucrose in the dark. As a first step toward identifying other small molecules that might regulate flowering, we have developed a rapid in vitro assay for LEAFY promoter activity. INTRODUCTIONThe most dramatic phase change that flowering plants undergo is the transition from vegetative to reproductive growth. For this transition to be successful, plants must integrate a variety of environmental signals with endogenous cues, such as plant age (Bernier, 1988).In the facultative long-day plant Arabidopsis, the transition to reproductive growth occurs rapidly in long days but much more slowly in short days. Several flowering-time mutants, in which the timing of this transition is changed, have been isolated. Analysis of the responses of different mutants to the environment together with studies of their genetic interactions have resulted in a two-pathway model showing how the transition to flowering is regulated (Martínez-Zapater et al., 1994;Weigel, 1995;Peeters and Koornneef, 1996). According to this model, long days induce flowering via a facultative and fast pathway, whereas under noninductive photoperiods, an autonomous and much slower pathway is rate limiting. The latter pathway is thought to be related to plant age.The gibberellin (GA) class of plant hormones plays a role in many processes during plant development, including seed germination, cell elongation, and flowering (Finkelstein and Zeevaart, 1994). In Arabidopsis, physiological and genetic experiments have implicated GAs specifically in the autonomous pathway of flowering. Exogenous application of GAs accelerates flowering in wild-type Arabidopsis, particularly in short days (Langridge, 1957). That there is a causal connection between endogenous GA levels and flowering in Arabidopsis has been confirmed with several GA biosynthesis and signaling mutants. Mutants in which GA levels are severely reduced, such as ga1-3 , are unable to flower in short days (Wilson et al., 1992). ga1-3 mutants carry a deletion of the gene encoding ent -copalyl diphosphate synthase (formerly ent -kaurene synthetase A), which controls a key step in early GA biosynthesis (Koornneef and Van der Veen, 1980; Zeevaart and Talón, 19...

BackgroundDisialoganglioside 2 (GD2) is expressed on neuroblastomas as well as melanomas, small cell lung cancers, and sarcomas. Anti-GD2 mAb (Dinutuximab) can be used to treat these cancers and is part of the standard care for neuroblastoma. While GD2 is expressed minimally on most normal tissues, it is expressed on some nerve cells, and anti-GD2 treatment can cause neuropathic pain. A separate tumor-antigen, B7H3, is overexpressed on multiple tumor types, including those listed above, with minimal expression on most normal cells and no expression on nerve cells. We developed a bispecific SNIPER antibody, INV721, to simultaneously target these 2 tumor antigens, with one arm specific to GD2 and the other arm to B7H3. The individual Fab arms targeting GD2 and B7H3 are each low to moderate affinity, such that INV721 will only bind with high affinity when both arms bind to their antigens on the same cell, resulting in high-specificity of the SNIPER to tumor cells.MethodsINV721 binding to GD2/B7H3-expressing tumors was confirmed by flow cytometry, as well as in tumor-bearing mice injected with 89Zr-labeled to monitor in vivo biodistribution via positron emission tomography imaging. Antibody-dependent cellular cytotoxicity (ADCC) testing of INV721 was performed on human neuroblastoma and melanoma cell lines with an Incucyte spheroid-killing-assay. In vivo efficacy studies were carried out in mice bearing GD2/B7H3-expressing melanoma tumors to test our in situ vaccine (ISV) regimen, which included testing combinations of external beam radiation therapy (RT, 12Gy) ± INV721 (40 ug/dose) ± IL2 (75K U/dose).ResultsINV721 showed binding by flow cytometry to tumors that express both GD2 and B7H3 but minimal binding to cells that don’t express both antigens. 89Zr-INV721 showed elevated and persistent accumulation in the tumor with minimal uptake in normal tissues. Incucyte spheroid-killing assays revealed that INV721 was capable of ADCC. The ISV combination of RT+INV721+IL2 was capable of curing mice bearing ~57 mm3 melanoma tumors (12/12 mice tumor free), with >70% of these mice exhibiting long-term immune memory.ConclusionsINV721 binds to cells that express both GD2 and B7H3, and these preliminary studies show that INV721 is effective in our ISV regimen at curing mice bearing tumors that express these antigens. We are continuing our efforts to determine if INV721 is associated with less pain than Dinutuximab. The goal of this SNIPER-antibody is to enhance the tumor-specific delivery of therapeutic mAbs, which may decrease toxicity and improve efficacy for cancers expressing both GD2 and B7H3.

The development of agonistic antibodies that activate T-cell co-stimulatory pathways represents a therapeutic strategy with significant clinical potential. However, challenges remain for the translation from in vitro efficacy to clinical success. OX40 and other tumor necrosis factor receptor (TNFR) superfamily members are notorious for requiring high-order receptor clustering in order to achieve full activity. For monoclonal antibodies, this high-order clustering is generally achieved through secondary cross-linking strategies. In vivo, this secondary cross-linking is often supplied through internal immune effector cells via Fc engagement. Bispecific and biparatopic antibodies represent an emerging class of drug molecules that enable unique mechanisms of action relative to their monoclonal counterparts. Here, we describe the use of our bispecific platform for the generation of large panels of biparatopic antibodies which enabled high-throughput screening for the discovery of an array of OX40 agonistic molecules. We have optimized these multivalent antibodies that exceed the potency of the OX40 ligand in NFkB activation without the need for secondary cross-linking. These agonist antibodies have additionally been characterized using primary T cell assays to monitor the kinetics of growth proliferation and cytokine secretion, outperforming cross-linked antibodies currently being tested in clinical trials. In co-culture systems, these agonist antibodies were effective in inhibiting immuno-suppressive properties of Tregs. Our lead OX40 agonist antibody has been optimized for activity and developability and has entered stable cell line development to further support pre-clinical activities. Citation Format: Bonnie Hammer, Mandar Bawadekar, Matthew Bissen, John Painter, Lauren Lehmann, Francis Qufei Li, Lucas Bailey, Bryan Glaser, Roland Green. A biparatopic agonist antibody for OX40 that exhibits superior activity without secondary crosslinking [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 563.

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Introduction: GD2 is expressed on neuroblastoma, melanomas, small cell lung cancers and sarcomas. While it is expressed minimally on normal tissues, it is expressed on nerve cells and, anti-GD2 (dinutuximab) treatment can cause neuropathic pain. To increase tumor specificity, we developed a bispecific SNIPER antibody to simultaneously target two tumor antigens (GD2 and B7H3). B7H3 is overexpressed on multiple tumor types, with minimal expression on most normal cells and is absent on nerve cells. The goal of this SNIPER is to enhance tumor-specificity, while reducing toxicity, ultimately improving efficacy for cancers expressing both targets. Experimental Procedures: SNIPER specificity was tested by flow cytometry for binding to GD2 +/- B7H3 expressing cells. Using an Incucyte S3, we tested antibody internalization properties [compared to anti-B7H3 and dinutuximab monoclonal antibodies (mAbs)] and antibody dependent cellular-cytotoxicity (ADCC) capabilities. Mice bearing variants of GD2/B7H3-expressing tumors were intravenously injected with 89Zr- radiolabeled SNIPER and its longitudinal in vivo biodistribution was monitored via positron emission tomography imaging. In vivo efficacy studies of SNIPER were tested against mice bearing either melanoma or neuroblastoma tumors that express both GD2 and B7H3. Summary of Data: In vitro and in vivo tumor specificity testing confirmed that SNIPER specifically targets B7H3+/GD2+ cells, but it does not bind to GD2+/B7H3- cells (which simulate nerve cells). We observed high internalization of anti-GD2 mAbs; we did not observe antibody internalization of either the SNIPER or anti-B7H3 mAbs. SNIPER was as effective at ADCC as the dinutuximab, but we saw minimal ADCC with the anti-B7H3 mAbs. An afucosylated version of SNIPER showed significantly enhanced ADCC compared to dinutuximab. Our in vivo efficacy studies found that SNIPER was as as dinutuximab when given at the same dose. Conclusions: The Fab arms of SNIPER targeting GD2 and B7H3 each have low-to-moderate affinity. SNIPER binds with strong avidity when both arms bind to their antigens on the same cell. Stronger avidity through both arms binding results in high-tumor specificity. Because SNIPER should not bind to nerves, it may be possible to administer increased doses of SNIPER beyond the tolerable dose of dinutuximab, which could further improve efficacy. Ongoing studies include antitumor efficacy testing, nerve binding assays and assessing reduction of pain in vivo with SNIPER. Citation Format: Amy K. Erbe, Arika Feils, Zack Rosenkrans, Jessica Wiwczar, Daniel Gerhardt, Bonnie Hammer, Mildred Felder, Mark Bercher, Alina Hampton, Lizzie Frankel, Dan Spiegelman, Noah Tsarovsky, Alexander Rakhmilevich, Jacquelyn Hank, Bryan Glaser, Reinier Hernandez, Roland Green, Paul M. Sondel. Tumor-targeting and efficacy of B7H3/GD2 bispecific SNIPER antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2019.