Bispecific Targeting of PD-1 and PD-L1 Enhances T-cell Activation and Antitumor Immunity.

The programmed cell death protein 1 receptor (PD-1) and programmed death ligand 1 (PD-L1) coinhibitory pathway suppresses T-cell-mediated immunity. We hypothesized that cotargeting of PD-1 and PD-L1 with a bispecific antibody molecule could provide an alternative therapeutic approach, with enhanced antitumor activity, compared with monospecific PD-1 and PD-L1 antibodies. Here, we describe LY3434172, a bispecific IgG1 mAb with ablated Fc immune effector function that targets both human PD-1 and PD-L1. LY3434172 fully inhibited the major inhibitory receptor-ligand interactions in the PD-1 pathway. LY3434172 enhanced functional activation of T cells in vitro compared with the parent anti-PD-1 and anti-PD-L1 antibody combination or respective monotherapies. In mouse tumor models reconstituted with human immune cells, LY3434172 therapy induced dramatic and potent antitumor activity compared with each parent antibody or their combination. Collectively, these results demonstrated the enhanced immunomodulatory (immune blockade) properties of LY3434172, which improved antitumor immune response in preclinical studies, thus supporting its evaluation as a novel bispecific cancer immunotherapy.

Characterization of 7A5: A Human CD137 (4-1BB) Receptor Binding Monoclonal Antibody with Differential Agonist Properties That Promotes Antitumor Immunity.

The CD137 receptor plays a key role in mediating immune response by promoting T cell proliferation, survival, and memory. Effective agonism of CD137 has the potential to reinvigorate potent antitumor immunity either alone or in combination with other immune-checkpoint therapies. In this study, we describe the discovery and characterization of a unique CD137 agonist, 7A5, a fully human IgG1 Fc effector-null monoclonal antibody. The biological properties of 7A5 were investigated through in vitro and in vivo studies. 7A5 binds CD137, and the binding epitope overlaps with the CD137L binding site based on structure. 7A5 engages CD137 receptor and activates NF-κB cell signaling independent of cross-linking or Fc effector function. In addition, T cell activation measured by cytokine IFNγ production is induced by 7A5 in peripheral blood mononuclear cell costimulation assay. Human tumor xenograft mouse models reconstituted with human immune cells were used to determine antitumor activity in vivo. Monotherapy with 7A5 inhibits tumor growth, and this activity is enhanced in combination with a PD-L1 antagonist antibody. Furthermore, the intratumoral immune gene expression signature in response to 7A5 is highly suggestive of enhanced T cell infiltration and activation. Taken together, these results demonstrate 7A5 is a differentiated CD137 agonist antibody with biological properties that warrant its further development as a cancer immunotherapy. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/4/988/F1.large.jpg.

Novel therapeutic approach for neurogenic erectile dysfunction: effect of neurotrophic tyrosine kinase receptor type 1 monoclonal antibody.

BACKGROUND: Erectile dysfunction (ED) is a major health issue in aged populations, and neurogenic ED is particularly difficult to treat. Novel therapeutic approaches are needed for treatment of neurogenic ED of peripheral origin. OBJECTIVE: To investigate the therapeutic effects of a neurotrophic tyrosine kinase receptor type 1 monoclonal antibody (TrkA-mAb) on erectile function and sexual behavior in a rat model of cavernous nerve injury (CNI). DESIGN, SETTING, AND PARTICIPANTS: In one experiment, 84 male rats were randomly assigned to seven groups. The groups underwent either CNI or sham surgery, subsequent injection into the major pelvic ganglion (IMPG) of phosphate-buffered saline (PBS), an immunoglobulin G (IgG) control, or TrkA-mAb, and then intracavernosal (IC) injection of either PBS or varying TrkA-mAb concentrations immediately after surgery and then 1 wk later. Erectile function was assessed and histologic/molecular analyses were performed at 6 wk after surgery. In a second experiment, 36 male rats were randomly divided into three groups. The groups underwent CNI or sham surgery and then IC injection of PBS, IgG, or TrkA-mAb immediately after surgery and for 5 wk thereafter. At 6 wk after surgery, the performance of the rats in sexual behavior tests was videotaped. INTERVENTION: CNI or sham surgery; IMPG of PBS, IgG, or TrkA-mAb; IC injection of PBS or TrkA-mAb. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The intracavernous pressure response to cavernous nerve electrostimulation was measured and midpenile cross-sections were histologically examined. Western blotting (WB) of cavernous tissue protein was performed. Rats were assessed for chasing, mounting, intromission, and ejaculation behaviors during sexual behavior tests. The data were analyzed using one-way analysis of variance followed by the Tukey-Kramer t test. RESULTS AND LIMITATIONS: Recovery of erectile function of varying degrees was observed in the TrkA-mAb groups. TrkA-mAb treatment significantly suppressed tyrosine hydroxylase-positive nerve fibers in the corpus cavernosum and enhanced neuronal nitric oxide synthase-positive fibers in the dorsal nerve. The ratio of smooth muscle to collagen in the corpus cavernosum was significantly improved in TrkA-mAb treatment groups compared to PBS vehicle and IgG control groups. WB confirmed these biological changes. There was a nonsignificant increase in the average number of intromissions and ejaculations in the TrkA-mAb group. The study limitations include small sample size, variability in sexual behavior, lack of data on the neuromuscular mechanism involved, and lack of information of the role of neurotrophins or cytokines in regeneration. CONCLUSIONS: TrkA-mAb successfully inhibits sympathetic nerve regeneration, leads to parasympathetic nerve regeneration, and has therapeutic effects on ED and sexual behavior disorder in a rat model of CNI. PATIENT SUMMARY: This report provides strong evidence that a neurotrophic tyrosine kinase receptor type 1 monoclonal antibody (TrkA-mAb) inhibits sympathetic nerve regeneration, leads to parasympathetic nerve regeneration, and has therapeutic effects on erectile dysfunction and sexual behavior disorder in a rat model of cavernous nerve injury. The results raise the possibility that human patients with neurogenic erectile dysfunction may respond to TrkA-mAb in a manner that parallels the response seen in our rodent study.

A human monoclonal antibody targeting the stem cell factor receptor (c-Kit) blocks tumor cell signaling and inhibits tumor growth.

Stem cell factor receptor (c-Kit) exerts multiple biological effects on target cells upon binding its ligand stem cell factor (SCF). Aberrant activation of c-Kit results in dysregulated signaling and is implicated in the pathogenesis of numerous cancers. The development of more specific and effective c-Kit therapies is warranted given its essential role in tumorigenesis. In this study, we describe the biological properties of CK6, a fully human IgG1 monoclonal antibody against the extracellular region of human c-Kit. CK6 specifically binds c-Kit receptor with high affinity (EC 50 = 0.06 nM) and strongly blocks its interaction with SCF (IC 50 = 0.41 nM) in solid phase assays. Flow cytometry shows CK6 binding to c-Kit on the cell surface of human small cell lung carcinoma (SCLC), melanoma, and leukemia tumor cell lines. Furthermore, exposure to CK6 inhibits SCF stimulation of c-Kit tyrosine kinase activity and downstream signaling pathways such as mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), in addition to reducing tumor cell line growth in vitro. CK6 treatment significantly decreases human xenograft tumor growth in NCI-H526 SCLC (T/C% = 57) and Malme-3M melanoma (T/C% = 58) models in vivo. The combination of CK6 with standard of care chemotherapy agents, cisplatin and etoposide for SCLC or dacarbazine for melanoma, more potently reduces tumor growth (SCLC T/C% = 24, melanoma T/C% = 38) compared with CK6 or chemotherapy alone. In summary, our results demonstrate that CK6 is a c-Kit antagonist antibody with tumor growth neutralizing properties and are highly suggestive of potential therapeutic application in treating human malignancies harboring c-Kit receptor.

DEGA/AMIGO-2, a leucine-rich repeat family member, differentially expressed in human gastric adenocarcinoma: effects on ploidy, chromosomal stability, cell adhesion/migration and tumorigenicity.

We have discovered DEGA, a novel cDNA differentially expressed in human gastric adenocarcinomas. The DEGA gene product contains a signal peptide, five leucine-rich repeat motifs and a single IgG, and transmembrane domain, suggesting its residence on the plasma membrane. Transfection of 293 cells with a DEGA-GFP fusion construct confirmed its cell surface localization. Although the cytosolic portion of the DEGA gene product does not contain known protein domains, approximately one-fifth of these residues are either a serine or a threonine, suggesting that DEGA may play a role in signal transduction. BLAST searches revealed DEGA to be an exact match to AMIGO-2, a recently identified, but functionally uncharacterized protein related to AMIGO, a leucine-rich repeat containing cell adhesion molecule implicated in axon tract development. In this report, we show that DEGA/AMIGO-2 mRNA is differentially expressed in approximately 45% of tumor versus normal tissue from gastric adenocarcinoma patients. Stable expression of a DEGA/AMIGO-2 antisense construct in the gastric adenocarcinoma cell line, AGS, led to altered morphology, increased ploidy, chromosomal instability, decreased cell adhesion/migration, and a nearly complete abrogation of tumorigenicity in nude mice. These findings suggest a potential etiologic role for DEGA/AMIGO-2 in gastric adenocarcinoma.