Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrate Improved Homogeneity and Other Pharmacological Properties over Conventional Heterogeneous ADCs.

Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures of chemically distinct molecules that vary in both drugs/antibody (DAR) and conjugation sites. Suboptimal properties of heterogeneous ADCs have led to new site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody engineering to identify optimal conjugation sites and introduce unique functional groups for conjugation with appropriately modified linkers. Alternative nonrecombinant methods have emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs containing four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect on the pharmacological properties of ADCs in vivo is unknown. In order to determine the relative impact of interchain cysteine cross-linking on the therapeutic window and other properties of ADCs in vivo, we synthesized a derivative of the known ADC payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC) linker. The DBM-MMAF derivative was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs containing predominantly four drugs/antibody. The pharmacological properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine cross-linking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs.

Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity.

Innate pattern recognition receptor agonists, including Toll-like receptors (TLRs), alter the tumor microenvironment and prime adaptive antitumor immunity. However, TLR agonists present toxicities associated with widespread immune activation after systemic administration. To design a TLR-based therapeutic suitable for systemic delivery and capable of safely eliciting tumor-targeted responses, we developed immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies. Systemically administered human epidermal growth factor receptor 2 (HER2)-targeted ISACs were well tolerated and triggered a localized immune response in the tumor microenvironment that resulted in tumor clearance and immunological memory. Mechanistically, ISACs required tumor antigen recognition, Fcγ-receptor-dependent phagocytosis and TLR-mediated activation to drive tumor killing by myeloid cells and subsequent T-cell-mediated antitumor immunity. ISAC-mediated immunological memory was not limited to the HER2 ISAC target antigen since ISAC-treated mice were protected from rechallenge with the HER2- parental tumor. These results provide a strong rationale for the clinical development of ISACs.

Alpha 4 integrins and vascular cell adhesion molecule-1 play a role in sympathetic innervation of the heart.

Sympathetic neurons innervate the heart early in postnatal development, an event that is crucial for proper modulation of blood pressure and cardiac function. However, the axon guidance cues that direct sympathetic neurons to the heart, and the neuronal receptors that recognize those cues, are poorly understood. Here we present evidence that interactions between the alpha4beta1 integrin on sympathetic neurons and vascular cell adhesion molecule-1 (VCAM-1) in the heart plays a role in cardiac innervation. The alpha4 subunit was detected on postnatal rat superior cervical ganglion (SCG) neurons in culture and in cryosections of SCG and heart. VCAM-1 immunoreactivity was detected on cardiac myocytes that associate with invading sympathetic neurons. Purified recombinant soluble VCAM-1 (rsVCAM-1) stimulated SCG neurite outgrowth at levels comparable with laminin 2/4 and fibronectin (Fn), and outgrowth on rs-VCAM-1 and Fn was blocked by antibodies specific for the alpha4 and beta1 integrin subunits. Intrathoracic injection of function-blocking antibodies to alpha4 and VCAM-1, as well as a small molecule inhibitor of alpha4 integrins, significantly reduced sympathetic innervation of the heart. These results indicate that the interaction between alpha4 integrin and VCAM-1 is important for sympathetic innervation of the heart.

Involvement of alpha4 integrins in maintenance of cardiac sympathetic axons.

Sympathetic neurons extend and maintain axons that innervate the myocardium, and proper innervation is important for cardiac function. However, the molecular basis for axon outgrowth and maintenance is not well understood. We have shown previously that the integrin alpha4beta1 is expressed on developing axons, and the alpha4 function is important for the development of innervation in vivo [Wingerd, K.L., Goodman, N.L., Tresser, J.W., Smail, M.M., Leu, S.T., Rohan, S.J., Pring, J.L., Jackson, D.Y., and Clegg, D.O., 2002. Alpha 4 integrins and vascular cell adhesion molecule-1 play a role in sympathetic innervation of the heart. J. Neurosci. 22,10772-10780]. Here we examine the function of alpha4beta1 integrins in the maintenance of cardiac sympathetic innervation in vitro and in vivo, and investigate integrin expression and function after myocardial infarction and in hypertensive rats. On substrates of vascular cell adhesion molecule-1 (VCAM-1), alpha4beta1 was required for both initial outgrowth and maintenance of neurites in vitro. On fibronectin substrates, initial outgrowth requires only alpha4 integrins, but maintenance requires both alpha4 integrins and RGD-dependent integrins. In vivo, in adult Long Evans rats, inhibition of alpha4 integrins resulted in decreased maintenance of sympathetic fibers innervating the apex of the heart. However, alpha4 integrins were not detected on most sympathetic axons that sprout after myocardial infarction, and alpha4 function was not required for sprouting. Spontaneously hypertensive rats (SHR) have increased numbers of cardiac sympathetic fibers compared to the parental Wistar strain, but many of these lack alpha4 expression, and alpha4 function is not required for maintenance of these fibers in the heart. These results suggest that developing sympathetic axons and sprouting sympathetic axons use different mechanisms of outgrowth, and that maintenance of cardiac sympathetic innervation involves alpha4 integrins in some rat strains.

Alpha 4 integrin antagonists.

The accumulation of leukocytes in various organs contributes to the pathogenesis of a number of human autoimmune diseases such as asthma, rheumatoid arthritis, Crohn s disease, ulcerative colitis, hepatitis C, and multiple sclerosis. The inflammatory processes leading to tissue damage and disease are mediated in part by the alpha4 integrins, alpha4beta1 and alpha4beta7, expressed on the leukocyte cell surface. These glycoprotein receptors modulate cell adhesion via interaction with their primary ligands, vascular cell adhesion molecule (VCAM) and mucosal addressin cell adhesion molecule (MAdCAM), expressed in the affected tissue. Upon binding, the combined integrin/CAM interactions at the cell surface result in firm adhesion of the leukocyte to the vessel wall followed by entry into the affected tissue. Elevated cell adhesion molecule (CAM) expression in various organs has been linked with several autoimmune diseases. Monoclonal antibodies specific for alpha4 integrins or their CAM ligands can moderate inflammation in animal models suggesting such inhibitors may be useful for treating human inflammatory diseases. The alpha4 integrins have become well validated drug targets for pharmaceutical companies and numerous publications describing alpha4 integrin antagonists have recently appeared. This article discusses the rationale for targeting alpha4 integrins for the treatment of autoimmune disorders and reviews some currently known antagonists. The methods used to identify lead molecules and the progress of selected antagonists toward becoming new drugs will is also discussed. (131 references).

Selective alpha4beta7 integrin antagonists and their potential as antiinflammatory agents.

The accumulation of leukocytes in various tissues contributes to the pathogenesis of numerous human autoimmune diseases. The integrin alpha4beta7, expressed on the surface of B and T lymphocytes, plays an essential role in lymphocyte trafficking throughout the gastrointestinal (GI) tract via interaction with its primary ligand, mucosal addressin cell adhesion molecule (MAdCAM). Elevated MAdCAM expression in the intestines and liver has been linked to GI-associated autoimmune disorders, including Crohn's disease, ulcerative colitis, and hepatitis C. Monoclonal antibodies that block the interaction of alpha4beta7 with MAdCAM inhibit lymphocyte homing to murine intestines without effecting migration to peripheral organs; this suggests that alpha4beta7-selective antagonists might be useful as GI specific antiinflammatory agents. Here, we report the discovery of highly potent and selective alpha4beta7 antagonists affinity selected from a random peptide-phage library. Subsequent optimization of initial peptide leads afforded alpha4beta7-selective heptapeptide inhibitors that competitively inhibit binding to MAdCAM in vitro and inhibit lymphocyte homing to murine intestines in vivo. Substitution of a single carboxylate moiety alters selectivity for alpha4beta7 by more than 500-fold to afford a potent and selective alpha4beta1 antagonist. The antagonists described here are the first peptides to demonstrate potency and selectivity for alpha4beta7 compared to other integrins.

Integrin alpha4beta1 function is required for cell survival in developing retina.

In the retina, integrins in the beta1 family have been shown to be important in many phases of neuronal development, particularly neuroblast migration and axon outgrowth. However, the functions of specific integrin heterodimers are not well defined. In this study, we investigated the functions of beta1 integrins in developing chicken retina by expression of a dominant-negative beta1A construct using a replication-competent retrovirus. Inhibition of integrins using this approach resulted in alteration of cell morphology and increased apoptosis, but did not preclude migration and axon elongation. In an attempt to identify which specific beta1 heterodimer was important, expression and function of the alpha4beta1 heterodimer were also investigated. At early developmental stages, alpha4 protein and mRNA were detected in undifferentiated neuroblasts throughout the retina. At later stages, expression was confined to retinal ganglion cells (RGCs) and amacrine cells. A small molecule antagonist of alpha4 integrins was shown to inhibit neurite outgrowth on recombinant soluble vascular cell adhesion molecule-1 (VCAM-1), a known ligand of alpha4beta1. Introduction of alpha4 antagonist in vivo gave rise to increased apoptosis and led to a thinning of the retina and reduced numbers of retinal ganglion cells (RGCs). We conclude that the integrin alpha4beta1 is important for survival of developing retinal neurons, including RGCs.

Solid-phase synthesis of dual alpha4beta1/alpha4beta7 integrin antagonists: two scaffolds with overlapping pharmacophores.

Two structural classes of dual alpha4beta1/alpha4beta7 integrin antagonists were investigated via solid-phase parallel synthesis. Using an acylated amino acid backbone, lead compounds containing biphenylalanine or tyrosine carbamate scaffolds were optimized for inhibition of alpha4beta1/VCAM and alpha4beta7/MAdCAM. A comparison of the structure-activity relationships in the inhibition of the alpha4beta7/MAdCAM interaction for substituted amines employed in both scaffolds suggests a similar binding mode for the compounds.