780 ALTA-002, a SIRPα-directed TLR9 agonist antibody conjugate activates myeloid cells and promotes anti-tumor immunity

BackgroundNovel therapies engaging both innate and adaptive immune responses may engender durable anti-tumor immunity. Activation of toll-like receptor 9 (TLR9) by unmethylated CpG oligonucleotides promotes innate inflammatory responses and induces adaptive immunity. Immune cells expressing TLR9 encompass B cells and myeloid cells (including dendritic cells and plasmacytoid dendritic cells). Recently, several TLR9 agonists have demonstrated clinical benefit in patients with melanoma when administered intra-tumorally.1 Specifically designed for systemic administration, we developed a novel Toll-like Receptor Agonist Antibody Conjugate (TRAAC) molecule comprised of a differentiated TLR-9 agonist (T-CpG) conjugated to an antibody against SIRPα (ALTA-002). Signal regulatory protein α (SIRPα) is a myeloid inhibitory receptor that suppresses immune activation following binding of its ligand CD47. Blockade of CD47-SIRPα myeloid checkpoint pathway has been shown to promote myeloid-mediated anti-tumor functions leading to the induction of adaptive immunity.2 Additionally, SIRPα is highly expressed in various tumor types including renal cell carcinoma and melanoma.3 Here we present preclinical data demonstrating that ALTA-002 delivers T-CpG to SIRPα expressing myeloid cells, triggering TLR9 signaling, cell activation and immune modulation resulting in robust anti-tumor efficacy.MethodsIn vitro activity of ALTA-002 was evaluated using human PBMCs co-cultured in the presence of SIRPα positive and negative tumor cells. Anti-tumor efficacy of mouse ALTA-002 surrogate was evaluated in multiple syngeneic tumor models with varying immunogenicity profiles.ResultsIn vitro co-culture of human PBMC and SIRPα positive or negative tumor cells with ALTA-002 stimulates myeloid cells, leading to increased IRF7 induction, expression of co-stimulatory molecules, and cytokine secretion. In vitro treatment with ALTA-002 led to enhanced phagocytic engulfment by human monocyte-derived macrophages across multiple SIRPα positive and negative tumor cell lines. Following systemic delivery of a mouse ALTA-002 surrogate, durable anti-tumor activity was observed in both SIRPα positive and negative expressing tumors. ALTA-002 treated mice with eradicated tumors suppressed tumor growth upon tumor re-challenge, indicating tumor-specific immune memory.ConclusionsThese results demonstrate the unique properties of systemically administered ALTA-002, which integrates TLR9 activation and blockade of CD47-SIRPα interaction on myeloid cells to engender both innate and adaptive anti-tumor immune responses. These data support the development of ALTA-002 as an anti-cancer therapeutic for a variety of tumor malignancies.ReferencesHamid O, Ismail R, Puzanov I. Intratumoral Immunotherapy-Update 2019. Oncologist 2020;25(3):e423-e4382.Kuo T, Chen A, Harrabi O. Targeting the myeloid checkpoint receptor SIRPα potentiates innate and adaptive immune responses to promote anti-tumor activity. J Hematol Oncol 2020;13:160–1783.Yanagita T, Murata Y, Tanaka D. Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy. JCI Insight 2017;2(1):e89140.Ethics ApprovalIn vivo studies were approved by the Institutional Animal Care and Use Committee of Tallac Therapeutics.

In Silico Designing of a Novel Antibody Conjugate as a Potential Immunotherapeutic for the Treatment of CD19-Positive Hematologic Malignancies

Background: Immunotherapy can now be considered as game changer of cancer treatment. So far, numerous monoclonal antibodies (mAbs) and their derivatives, such as antibody-drug conjugates (ADCs), have been approved by regulatory agencies for medical use. This implies that the recombinant or chemical conjugation of mAbs to cytotoxic agents can be regarded as a potential cancer treatment modality. Objectives: This study aimed to design an antibody conjugate through the recombinant conjugation of a humanized CD19-specific single-chain variable fragment (scFv), named HuFMC63, to granzyme B (GrB) using precise in silico approaches. Methods: Four different linker peptides were used for the conjugation of HuFMC63 to GrB, and the 3D structure of these antibody conjugates were predicted using GalaxyWEB. The antibody conjugate whose linker peptide had the least impact on the structural conformation of HuFMC63 and GrB was subsequently selected. Additionally, the solubility and melting temperature of the selected conjugate was compared with those of HuFMC6 and GrB, and its physicochemical properties and flexibility were also assessed. Ultimately, the binding capacity and the dissociation constant (Kd) of the selected conjugate to CD19 were compared with those of HuFMC63 (concisely referred to as Hu63), and then the residues that contributed to antigen binding were identified using LigPlot+ software. Results: The Hu63-(G4S)3-GrB conjugate, which is constructed using the (G4S)3 linker, was selected as the best conjugate. The solubility of Hu63-(G4S)3-GrB was predicted to be higher than HuFMC63 and GrB (from 60% in the unconjugated to 98% in the conjugated format). Moreover, it was elucidated that Hu63-(G4S)3-GrB binds CD19 in the same orientation as that of HuFMC63 and with the same Kd of 17 and 33 nM at 25.0°C and 37.0°C, respectively. Conclusions: In silico techniques, such as those employed in this study, could be utilized for the early development of immune-based therapeutics. Moreover, Hu63-(G4S)3-GrB could be introduced as a potent therapeutic for the elimination of CD19-positive malignant cells after careful preclinical and clinical evaluations.

Development of Polydiacetylene-Based Testosterone Detection as a Model Sensing Platform for Water-Insoluble Hormone Analytes

We have developed a polydiacetylene (PDA)-based sensing platform to detect testosterone (T) as a potential biomarker of preterm birth. The insolubility of the steroid hormone in water, where PDA assemblies are dispersed, poses a major issue, since they can hardly interact with each other. To overcome this challenge, acetonitrile was used as a suitable solvent. In addition, to minimize false signals of PDA assemblies caused by the solvent, a mixture of acetonitrile and distilled water was selected. To prove a concept of PDA-based sensing platform for targeting T hormone, we conjugated anti-T antibodies to surface of PDA assemblies to induce selective binding between T and anti-T antibodies. The fluorescence sensory signaling of the PDA-anti-T antibody conjugate was selectively generated for T, over 3.4 times higher sensitivity of the signaling compared to that from other sex steroid hormones studied (β-estradiol and progesterone).

Internalization of Foldamer-Based DNA Mimics through a Site-Specific Antibody Conjugate to Target HER2-Positive Cancer Cells

Inhibition of protein–DNA interactions represents an attractive strategy to modulate essential cellular functions. We reported the synthesis of unique oligoamide-based foldamers that adopt single helical conformations and mimic the negatively charged phosphate moieties of B-DNA. These mimics alter the activity of DNA interacting enzymes used as targets for cancer treatment, such as DNA topoisomerase I, and they are cytotoxic only in the presence of a transfection agent. The aim of our study was to improve internalization and selective delivery of these highly charged molecules to cancer cells. For this purpose, we synthesized an antibody-drug conjugate (ADC) using a DNA mimic as a payload to specifically target cancer cells overexpressing HER2. We report the bioconjugation of a 16-mer DNA mimic with trastuzumab and its functional validation in breast and ovarian cancer cells expressing various levels of HER2. Binding of the ADC to HER2 increased with the expression of the receptor. The ADC was internalized into cells and was more efficient than trastuzumab at inhibiting their growth in vitro. These results provide proof of concept that it is possible to site-specifically graft high molecular weight payloads such as DNA mimics onto monoclonal antibodies to improve their selective internalization and delivery in cancer cells.

Toward Homogenous Antibody Drug Conjugates Using Enzyme-Based Conjugation Approaches

In the last few decades, antibody-based diagnostic and therapeutic applications have been well established in medicine and have revolutionized cancer managements by improving tumor detection and treatment. Antibodies are unique medical elements due to their powerful properties of being able to recognize specific antigens and their therapeutic mechanisms such as blocking specific pathways, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity. Furthermore, modification techniques have paved the way for improving antibody properties and to develop new classes of antibody-conjugate-based diagnostic and therapeutic agents. These techniques allow arming antibodies with various effector molecules. However, these techniques are utilizing the most frequently used amino acid residues for bioconjugation, such as cysteine and lysine. These bioconjugation approaches generate heterogeneous products with different functional and safety profiles. This is mainly due to the abundance of lysine and cysteine side chains. To overcome these limitations, different site-direct conjugation methods have been applied to arm the antibodies with therapeutic or diagnostics molecules to generate unified antibody conjugates with tailored properties. This review summarizes some of the enzyme-based site-specific conjugation approaches.