Application of In vitro transcytosis models to brain targeted biologics.

The blood brain barrier (BBB) efficiently limits the penetration of biologics drugs from blood to brain. Establishment of an in vitro BBB model can facilitate screening of central nervous system (CNS) drug candidates and accelerate CNS drug development. Despite many established in vitro models, their application to biologics drug selection has been limited. Here, we report the evaluation of in vitro transcytosis of anti-human transferrin receptor (TfR) antibodies across human, cynomolgus and mouse species. We first evaluated human models including human cerebral microvascular endothelial cell line hCMEC/D3 and human colon epithelial cell line Caco-2 models. hCMEC/D3 model displayed low trans-epithelial electrical resistance (TEER), strong paracellular transport, and similar transcytosis of anti-TfR and control antibodies. In contrast, the Caco-2 model displayed high TEER value and low paracellular transport. Anti-hTfR antibodies demonstrated up to 70-fold better transcytosis compared to control IgG. Transcytosis of anti-hTfR.B1 antibody in Caco-2 model was dose-dependent and saturated at 3 µg/mL. Enhanced transcytosis of anti-hTfR.B1 was also observed in a monkey brain endothelial cell based (MBT) model. Importantly, anti-hTfR.B1 showed relatively high brain radioactivity concentration in a non-human primate positron emission tomography study indicating that the in vitro transcytosis from both Caco-2 and MBT models aligns with in vivo brain exposure. Typically, brain exposure of CNS targeted biologics is evaluated in mice. However, antibodies, such as the anti-human TfR antibodies, do not cross-react with the mouse target. Therefore, validation of a mouse in vitro transcytosis model is needed to better understand the in vitro in vivo correlation. Here, we performed transcytosis of anti-mouse TfR antibodies in mouse brain endothelial cell-based models, bEnd3 and the murine intestinal epithelial cell line mIEC. There is a good correlation between in vitro transcytosis of anti-mTfR antibodies and bispecifics in mIEC model and their mouse brain uptake. These data strengthen our confidence in the predictive power of the in vitro transcytosis models. Both mouse and human in vitro models will serve as important screening assays for brain targeted biologics selection in CNS drug development.

Fibronectin extra domain A as a drug delivery targeting epitope for rheumatoid arthritis.

OBJECTIVES: To assess the ability of monoclonal antibodies (mAbs) specific for fibronectin extra-domain A (FnEDA) to target diseased tissues of mouse collagen induced arthritis (mCIA) models. To explore the parameters of the targeting exhibited by anti-FnEDA mAbs including timing and location. METHODS: Targeting capabilities of anti-FnEDA mAbs were demonstrated by biodistribution study where i.v. injected antibodies were detected by conjugated near-infrared (NIR) fluorophore, 125I label and immunohistochemistry (IHC) of the injected antibody. Location of FnEDA expression in both mCIA and human RA tissue were mapped by IHC. Quantification of anti-FnEDA mAbs targeted to disease tissue was measured by whole-body autoradiography (WBA). Timing of the targeting was interrogated with fluorescent and confocal microscopy using anti-FnEDA mAbs labeled with different fluorophores and injected at different times. RESULTS: Anti-FnEDA mAbs show specific targeting to diseased paws of mCIA animal. The targeting was focused on inflamed synovium which is consistent with FnEDA expression profile in both mCIA and human RA tissues. Anti-FnEDA mAbs accumulated in diseased tissue at pharmacologically relevant concentrations, the targeting was sustained for up to 14 days and FnEDA was able to support targeting of multiple doses of anti-FnEDA mAbs given 5 days apart. CONCLUSION: FnEDA is specifically upregulated in the inflamed tissues of mCIA. Antibodies specific for FnEDA can be useful as molecular delivery vehicles for disease specific targeting of payloads to inflamed joint tissue.

Fibronectin extra domain A as a drug delivery targeting epitope for rheumatoid arthritis

Abstract Objectives: To assess the ability of monoclonal antibodies (mAbs) specific for fibronectin extra-domain A (FnEDA) to target diseased tissues of mouse collagen induced arthritis (mCIA) models. To explore the parameters of the targeting exhibited by anti-FnEDA mAbs including timing and location. Methods: Targeting capabilities of anti-FnEDA mAbs were demonstrated by biodistribution study where i.v. injected antibodies were detected by conjugated near-infrared (NIR) fluorophore, 125I label and immunohistochemistry (IHC) of the injected antibody. Location of FnEDA expression in both mCIA and human RA tissue were mapped by IHC. Quantification of anti-FnEDA mAbs targeted to disease tissue was measured by whole-body autoradiography (WBA). Timing of the targeting was interrogated with fluorescent and confocal microscopy using anti-FnEDA mAbs labeled with different fluorophores and injected at different times. Results: Anti-FnEDA mAbs show specific targeting to diseased paws of mCIA animal. The targeting was focused on inflamed synovium which is consistent with FnEDA expression profile in both mCIA and human RA tissues. Anti-FnEDA mAbs accumulated in diseased tissue at pharmacologically relevant concentrations, the targeting was sustained for up to 14 days and FnEDA was able to support targeting of multiple doses of anti-FnEDA mAbs given 5 days apart. Conclusion: FnEDA is specifically upregulated in the inflamed tissues of mCIA. Antibodies specific for FnEDA can be useful as molecular delivery vehicles for disease specific targeting of payloads to inflamed joint tissue.

Brain uptake of multivalent and multi-specific DVD-Ig proteins after systemic administration.

Therapeutic monoclonal antibodies and endogenous IgG antibodies show limited uptake into the central nervous system (CNS) due to the blood-brain barrier (BBB), which regulates and controls the selective and specific transport of both exogenous and endogenous materials to the brain. The use of natural transport mechanisms, such as receptor-mediated transcytosis (RMT), to deliver antibody therapeutics into the brain have been studied in rodents and monkeys. Recent successful examples include monovalent bispecific antibodies and mono- or bivalent fusion proteins; however, these formats do not have the capability to bind to both the CNS target and the BBB transport receptor in a bivalent fashion as a canonical antibody would. Dual-variable-domain immunoglobulin (DVD-Ig) proteins offer a bispecific format where monoclonal antibody-like bivalency to both the BBB receptor and the therapeutic target is preserved, enabling independent engineering of binding affinity, potency, valency, epitope and conformation, essential for successful generation of clinical candidates for CNS applications with desired drug-like properties. Each of these parameters can affect the binding and transcytosis ability mediated by different receptors on the brain endothelium differentially, allowing exploration of diverse properties. Here, we describe generation and characterization of several different DVD-Ig proteins, specific for four different CNS targets, capable of crossing the BBB through transcytosis mediated by the transferrin receptor 1 (TfR1). After systemic administration of each DVD-Ig, we used two independent methods in parallel to observe specific uptake into the brain. An electrochemiluminescent-based sensitive quantitative assay and a semi-quantitative immunohistochemistry technique were used for brain concentration determination and biodistribution/localization in brain, respectively. Significantly enhanced brain uptake and retention was observed for all TfR1 DVD-Ig proteins regardless of the CNS target or the systemic administration route selected.

Targeting Anti-TGF-ß Therapy to Fibrotic Kidneys with a Dual Specificity Antibody Approach.

Targeted delivery of a therapeutic agent to a site of pathology to ameliorate disease while limiting exposure at undesired tissues is an aspirational treatment scenario. Targeting diseased kidneys for pharmacologic treatment has had limited success. We designed an approach to target an extracellular matrix protein, the fibronectin extra domain A isoform (FnEDA), which is relatively restricted in distribution to sites of tissue injury. In a mouse unilateral ureteral obstruction (UUO) model of renal fibrosis, injury induced significant upregulation of FnEDA in the obstructed kidney. Using dual variable domain Ig (DVD-Ig) technology, we constructed a molecule with a moiety to target FnEDA and a second moiety to neutralize TGF-ß After systemic injection of the bispecific TGF-ß + FnEDA DVD-Ig or an FnEDA mAb, chemiluminescent detection and imaging with whole-body single-photon emission computed tomography (SPECT) revealed significantly higher levels of each molecule in the obstructed kidney than in the nonobstructed kidney, the ipsilateral kidney of sham animals, and other tissues. In comparison, a systemically administered TGF-ß mAb accumulated at lower concentrations in the obstructed kidney and exhibited a more diffuse whole-body distribution. Systemic administration of the bispecific DVD-Ig or the TGF-ß mAb (1-10 mg/kg) but not the FnEDA mAb attenuated the injury-induced collagen deposition detected by immunohistochemistry and elevation in Col1a1, FnEDA, and TIMP1 mRNA expression in the obstructed kidney. Overall, systemic delivery of a bispecific molecule targeting an extracellular matrix protein and delivering a TGF-ß mAb resulted in a relatively focal uptake in the fibrotic kidney and reduced renal fibrosis.

The Effect of Antibody Size and Mechanical Loading on Solute Diffusion Through the Articular Surface of Cartilage.

Because of the heterogeneous nature of articular cartilage tissue, penetration of potential therapeutic molecules for osteoarthritis (OA) through the articular surface (AS) is complex, with many factors that affect transport of these solutes within the tissue. Therefore, the goal of this study is to investigate how the size of antibody (Ab) variants, as well as application of cyclic mechanical loading, affects solute transport within healthy cartilage tissue. Penetration of fluorescently tagged solutes was quantified using confocal microscopy. For all the solutes tested, fluorescence curves were obtained through the articular surface. On average, diffusivities for the solutes of sizes 200 kDa, 150 kDa, 50 kDa, and 25 kDa were 3.3, 3.4, 5.1, and 6.0 µm2/s from 0 to 100 µm from the articular surface. Diffusivities went up to a maximum of 16.5, 18.5, 20.5, and 23.4 µm2/s for the 200 kDa, 150 kDa, 50 kDa, and 25 kDa molecules, respectively, from 225 to 325 µm from the surface. Overall, the effect of loading was very significant, with maximal transport enhancement for each solute ranging from 2.2 to 3.4-fold near 275 µm. Ultimately, solutes of this size do not diffuse uniformly nor are convected uniformly, through the depth of the cartilage tissue. This research potentially holds great clinical significance to discover ways of further optimizing transport into cartilage and leads to effective antibody-based treatments for OA.

PK1/EG-VEGF induces monocyte differentiation and activation.

Macrophages exist as sentinels in innate immune response and react by expressing proinflammatory cytokines and up-regulating antigen-presenting and costimulatory molecules. We report a novel function for prokineticin-1 (PK1)/endocrine gland-derived vascular endothelial growth factor. Screening of murine tissue sections and cells for specific binding site leads to the identification of macrophages as an in vivo cellular target for PK1. We demonstrate PK1 induces differentiation of murine and human bone marrow cells into the monocyte/macrophage lineage. Human peripheral blood monocytes respond to PK1 by morphological changes and down-regulation of B7-1, CD14, CC chemokine receptor 5, and CXC chemokine receptor 4. Monocytes treated with PK1 have elevated interleukin (IL)-12 and tumor necrosis factor alpha and down-regulated IL-10 production in response to lipopolysaccharide. PK1 induces a distinct monocyte-derived cell population, which is primed for release of proinflammatory cytokines that favor a T helper cell type 1 response.

Anti GPVI human antibodies neutralizing collagen-induced platelet aggregation isolated from a combinatorial phage display library.

Glycoprotein VI is a type I membrane protein identified as a key platelet receptor for collagen. In vitro binding of the GPVI receptor with collagen leads to activation and ultimately to aggregation of platelets. In vivo, GPVI-collagen interactions could cause formation of occlusive thrombi within vessels with damaged endothelial barriers. GPVI antagonists are therefore important therapeutics in patients suffering from collagen-mediated ischemic disorders such as myocardial infarction or stroke. Polyclonal antibodies to GPVI prepared from one patient serum have previously been described. However, only their monovalent Fab fragments, incapable of receptor crosslinking, were found to protect platelets from collagen-mediated aggregation. Here we describe GPVI-neutralizing human antibodies derived from a combinatorial phage display library of single-chain antibodies. By selecting phage on GPVI-expressing U937 cells, we isolated five specific antibodies - A4, A9, A10, C3 and C9. Of the set A10 and C3 specifically blocked GPVI binding to collagen-rich adventitial layers in aorta sections. The higher affinity antibody A10 inhibited binding of snake-venom convulxin to GPVI. It also specifically protected human platelets from collagen-induced aggregation in vitro. A10-bound platelets could still be activated by ADP or thrombin suggesting that this human scFv may represent an original anti-platelet agent for the treatment of collagen-mediated thrombotic diseases.

Programmed death-1 targeting can promote allograft survival.

The recently identified CD28 homolog and costimulatory molecule programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2, which are homologs of B7, constitute an inhibitory regulatory pathway of potential therapeutic use in immune-mediated diseases. We examined the expression and functions of PD-1 and its ligands in experimental cardiac allograft rejection. In initial studies, we found that most normal tissues and cardiac isografts had minimal expression of PD-1, PD-L1, or PD-L2, but intragraft induction of all three molecules occurred during development of cardiac allograft rejection. Intragraft expression of all three genes was maintained despite therapy with cyclosporin A or rapamycin, but was prevented in the early posttransplant period by costimulation blockade using CD154 or anti-inducible costimulator mAb. We prepared PD-L1.Ig and PD-L2.Ig fusion proteins and showed that each bound to activated PD-1(+) T cells and inhibited T cell functions in vitro, thereby allowing us to test the effects of PD-1 targeting on allograft survival in vivo. Neither agent alone modulated allograft rejection in wild-type recipients. However, use of PD-L1.Ig administration in CD28(-/-) recipients, or in conjunction with immunosuppression in fully MHC-disparate combinations, markedly prolonged cardiac allograft survival, in some cases causing permanent engraftment, and was accompanied by reduced intragraft expression of IFN-gamma and IFN-gamma-induced chemokines. PD-L1.Ig use also prevented development of transplant arteriosclerosis post-CD154 mAb therapy. These data show that when combined with limited immunosuppression, or in the context of submaximal TCR or costimulatory signals, targeting of PD-1 can block allograft rejection and modulate T and B cell-dependent pathologic immune responses in vivo.