Pharmacologic Characterization of ALD403, a Potent Neutralizing Humanized Monoclonal Antibody Against the Calcitonin Gene-Related Peptide.

ALD403 is a genetically engineered, humanized immunoglobulin G1 monoclonal antibody that inhibits the action of human calcitonin gene-related peptide (CGRP). Clinical trial data indicate that ALD403 is effective as a preventive therapy for migraine and has an acceptable safety profile. For preclinical characterization of ALD403, rabbit antibodies targeting α-CGRP were humanized and modified to eliminate fragment crystallizable (Fc) γ receptor (FcγR) and complement interactions. The ability of ALD403 to inhibit CGRP-induced cAMP production was assessed using a cAMP bioassay (Meso Scale Discovery). The IC50 for inhibition of cAMP release was 434 and 288 pM with the rabbit-human chimera antibody and the humanized ALD403, respectively. ALD403 inhibited α-CGRP binding with an IC50 of 4.7 × 10-11 and 1.2 × 10-10 M for the α-CGRP and AMY1 receptors, respectively. ALD403 did not induce antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity and did not stably interact with any of the FcγR mediating these functions, exhibiting only weak binding to FcγRI. ALD403 significantly lowered capsaicin-induced blood flow responses in rodents at all time points starting at 5 minutes postapplication in a dose-dependent manner. In conclusion, ALD403 is a potent functional ligand inhibitor of α-CGRP‒driven pharmacology. SIGNIFICANCE STATEMENT: α-Calcitonin gene-related peptide blockade by ALD403 was assessed via radiolabeled ligand displacement, in vitro inhibition of cell signaling, and in vivo inhibition of capsaicin-induced vasodilation. Lack of engagement of fragment crystallizable-mediated immune-effector functions by ALD403 was shown.

Pharmacologic Characterization of ALD1910, a Potent Humanized Monoclonal Antibody against the Pituitary Adenylate Cyclase-Activating Peptide.

Migraine is a debilitating disease that affects almost 15% of the population worldwide and is the first cause of disability in people under 50 years of age, yet its etiology and pathophysiology remain incompletely understood. Recently, small molecules and therapeutic antibodies that block the calcitonin gene-related peptide (CGRP) signaling pathway have reduced migraine occurrence and aborted acute attacks of migraine in clinical trials and provided prevention in patients with episodic and chronic migraine. Heterogeneity is present within each diagnosis and patient's response to treatment, suggesting migraine as a final common pathway potentially activated by multiple mechanisms, e.g., not all migraine attacks respond to or are prevented by anti-CGRP pharmacological interventions. Consequently, other unique mechanisms central to migraine pathogenesis may present new targets for drug development. Pituitary adenylate cyclase-activating peptide (PACAP) is an attractive novel target for treatment of migraines. We generated a specific, high-affinity, neutralizing monoclonal antibody (ALD1910) with reactivity to both PACAP38 and PACAP27. In vitro, ALD1910 effectively antagonizes PACAP38 signaling through the pituitary adenylate cyclase-activating peptide type I receptor, vasoactive intestinal peptide receptor 1, and vasoactive intestinal peptide receptor 2. ALD1910 recognizes a nonlinear epitope within PACAP and blocks its binding to the cell surface. To test ALD1910 antagonistic properties directed against endogenous PACAP, we developed an umbellulone-induced rat model of neurogenic vasodilation and parasympathetic lacrimation. In vivo, this model demonstrates that the antagonistic activity of ALD1910 is dose-dependent, retaining efficacy at doses as low as 0.3 mg/kg. These results indicate that ALD1910 represents a potential therapeutic antibody to address PACAP-mediated migraine.

Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation.

The Spike of SARS-CoV-2 recognizes a transmembrane protease, angiotensin-converting enzyme 2 (ACE2), on host cells to initiate infection. Soluble derivatives of ACE2, in which Spike affinity is enhanced and the protein is fused to Fc of an immunoglobulin, are potent decoy receptors that reduce disease in animal models of COVID-19. Mutations were introduced into an ACE2 decoy receptor, including adding custom N-glycosylation sites and a cavity-filling substitution together with Fc modifications, which increased the decoy's catalytic activity and provided small to moderate enhancements of pharmacokinetics following intravenous and subcutaneous administration in humanized FcRn mice. Most prominently, sialylation of native glycans increases exposures by orders of magnitude, and the optimized decoy is therapeutically efficacious in a mouse COVID-19 model. Ultimately, an engineered and highly sialylated decoy receptor produced using methods suitable for manufacture of representative drug substance has high exposure with a 5- to 9-day half-life. Finally, peptide epitopes at mutated sites in the decoys generally have low binding to common HLA class II alleles and the predicted immunogenicity risk is low. Overall, glycosylation is a critical molecular attribute of ACE2 decoy receptors and modifications that combine tighter blocking of Spike with enhanced pharmacokinetics elevate this class of molecules as viable drug candidates.

NL-201 Upregulates MHC-I Expression and Intratumoral T-cell Receptor Diversity, and Demonstrates Robust Antitumor Activity as Monotherapy and in Combination with PD-1 Blockade.

Cytokine engineering has shown promise as a means to create novel immunomodulatory agents or to improve upon the therapeutic potential of natural cytokines. NL-201, a de novo, hyperstable, IL2 receptor alpha (IL2Rα)-independent agonist of the receptors for IL2 and IL15, elicits robust preclinical activity in syngeneic murine cancer models, including those resistant to immune checkpoint inhibitors (ICI). Here, we report that NL-201 monotherapy converts 'cold' tumor microenvironments (TME) to immunologically 'hot' states by driving pro-inflammatory gene expression, enhancing IFNγ-dependent MHC-I expression, and expanding both T-cell number and clonal diversity. In addition, the combination of NL-201 and anti-PD-1 resulted in complementary antitumor activity in the immunologically 'cold' and ICI resistant B16F10, EMT6, and Renca syngeneic models. In the B16F10 model, treatment with NL-201 plus anti-PD-1 increased the abundance of CD4+ and CD8+ effector T cells in the TME. These findings reveal an important mechanistic basis for the antitumor activity of NL-201 both as a monotherapy and in combination with PD-1 antagonists, and provide further context for the role of IL2Rα-based signaling in ICI-resistant tumors.

ALD1613, a Novel Long-Acting Monoclonal Antibody to Control ACTH-Driven Pharmacology.

Adrenocorticotropic hormone (ACTH) is the primary regulator of adrenal glucocorticoid production. Elevated levels of ACTH play a critical role in disease progression in several indications, including congenital adrenal hyperplasia and Cushing disease. We have generated a specific, high-affinity, neutralizing monoclonal antibody (ALD1613) to ACTH. In vitro, ALD1613 neutralizes ACTH-induced signaling via all 5 melanocortin receptors and inhibited ACTH-induced cyclic adenosine monophosphate accumulation in a mouse adrenal cell line (Y1). ALD1613 administration to wild-type rats significantly reduced plasma corticosterone levels in a dose-dependent manner. In rodent models with either chronic infusion of ACTH or acute restraint stress-induced ACTH, corticosterone levels were significantly reduced by ALD1613. Administration of ALD1613 to nonhuman primates on days 1 and 7 stably reduced plasma cortisol levels >50% for 57 days. ALD1613 demonstrates the potential of a monoclonal antibody to be an effective therapeutic for conditions with elevated ACTH levels.

In vivo efficacy of folate-targeted lipid-protamine-DNA (LPD-PEG-Folate) complexes in an immunocompetent syngeneic model for breast adenocarcinoma.

Gene therapy utilizing lipid-based delivery systems holds tremendous promise for the treatment of cancer. However, due to the potential adverse inflammatory and/or immune effects upon systemic administration, treatments thus far have been predominantly limited to intratumoral or regional treatment. Previous studies from our group have demonstrated the antitumor efficacy of systemically administered, folate-targeted, lipid-protamine-DNA complexes (LPD-PEG-Folate) against breast cancer using an immunodeficient xenogenic murine model. In the current study, the antitumor efficacy of LPD-PEG-Folate in a syngeneic, immune competent, murine model of breast cancer was examined. In this model, the potential inflammatory or immune responses and their effects on systemic delivery can be addressed. The 410.4 murine breast adenocarcinoma cell line was initially evaluated in vitro for its interactions with LPD-PEG-Folate and control LPD-PEG formulations. Utilizing fluorescently labeled formulations and fluorescence-activated cell sorting (FACS) analysis, a 1.6-fold enhancement of binding and internalization of LPD-PEG-Folate over LPD-PEG formulations was observed, suggestive of specific receptor interaction. Increased binding was manifested as 5-26-fold increases in luciferase gene expression in 410.4 cell transfection when comparing LPD-PEG-Folate to LPD-PEG. Moreover, in vivo treatment of 410.4 breast tumors in BALB/c mice with i.v. injected LPD-PEG-Folate delivering the HSV-1 thymidine kinase (TK) gene, in combination with gancyclovir treatment, resulted in a significant reduction in mean tumor volume (260.1 mm3) compared to the LPD-PEG-TK (914.1 mm3), as well as the vehicle (749.7 mm3) and untreated (825.3 mm3) control groups (day 25, P<.019). In addition to a reduced tumor volume, LPD-PEG-Folate-TK treatment also increased median survival from 25 days in the nontargeted LPD-PEG-TK groups to 31 days (P=.0011), which correlated with the termination of treatment. Together, these results demonstrate that in the context of a fully functional immune system, LPD-PEG-Folate-TK treatment possesses significant specific antitumor efficacy and the potential for further preclinical development.

Calculating expected lung deposition of aerosolized administration of AAV vector in human clinical studies.

BACKGROUND: Cystic fibrosis is an autosomal recessive disease affecting approximately 1 in 2500 live births. Introducing the cDNA that codes for normal cystic fibrosis transmembrane conductance regulator (CFTR) to the small airways of the lung could result in restoring the CFTR function. A number of vectors for lung gene therapy have been tried and adeno-associated virus (AAV) vectors offer promise. The vector is delivered to the lung using a breath-actuated jet nebulizer. The purpose of this project was to determine the aerosolized AAV (tgAAVCF) particle size distribution (PSD) in order to calculate target doses for lung delivery. METHODS: A tgAAVCF solution was nebulized using the Pari LC Plus (n = 3), and the PSD was determined by coupling laser diffraction and inertial impaction (NGI) techniques. The NGI allowed for quantification of the tgAAVCF at each stage of impaction, ensuring that rAAV-CFTR vector is present and not empty particles. Applying the results to mathematical algorithms allowed for the calculation of expected pulmonary deposition. RESULTS: The mass median diameter (MMD) for the tgAAVCF was 2.78 +/- 0.43 microm. If the system works ideally and the patient only receives aerosol on inspiration, the patient would receive 47 +/- 0% of the initial dose placed in the nebulizer, with 72 +/- 0.73% of this being deposited beyond the vocal cords. CONCLUSIONS: This technology for categorizing the pulmonary delivery system for lung gene therapy vectors can be adapted for advanced aerosol delivery systems or other vectors.

Targeting of lipid-protamine-DNA (LPD) lipopolyplexes using RGD motifs.

The incorporation of pegylated lipid into Lipid-Protamine-DNA (LPD-PEG) lipopolyplexes causes a decrease of their in vitro transfection activity. This can be partially attributed to a reduction in particle binding to cells. To restore particle binding and specifically target LPD formulations to tumor cells, the lipid-peptide conjugate DSPE-PEG5K-succinyl-ACDCRGDCFCG-COOH (DSPE-PEG5K-RGD-4C) was generated and incorporated into LPD formulations (LPD-PEG-RGD). LPD-PEG-RGD was characterized with respect to its biophysical and biological properties. The Incorporation of DSPE-PEG5K-RGD-4C ligands into LPD formulations results in a 5 and a 15 fold increase in the LPD-PEG-RGD binding and uptake, respectively, over an LPD-PEG formulation. Enhancement of binding and uptake resulted in a 100 fold enhancement of transfection activity. Moreover, this transfection enhancement was specific to cells expressing appropriate integrin receptors (MDA-MB-231). Huh7 cells, known for their low level of alphavbeta3 and alphavbeta5 integrin expression, failed to show RGD mediated transfection enhancement. This transfection enhancement can be abolished in a competitive manner using free RGD peptide, but not an RGE control peptide. Results demonstrated RGD mediated enhanced LPD-PEG cell binding and transfection in cells expressing the integrin receptor. These formulations provide the basis for effective, targeted, systemic gene delivery.