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.

CD40/anti-CD40 antibody complexes which illustrate agonist and antagonist structural switches.

BACKGROUND: CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications. RESULTS: To better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies. CONCLUSIONS: The structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323.

Characterization of ABBV-221, a Tumor-Selective EGFR-Targeting Antibody Drug Conjugate.

Depatuxizumab mafodotin (depatux-m, ABT-414) is a tumor-selective antibody drug conjugate (ADC) comprised of the anti-EGFR antibody ABT-806 and the monomethyl auristatin F (MMAF) warhead. Depatux-m has demonstrated promising clinical activity in glioblastoma multiforme (GBM) patients and is currently being evaluated in clinical trials in first-line and recurrent GBM disease settings. Depatux-m responses have been restricted to patients with amplified EGFR, highlighting the need for therapies with activity against tumors with nonamplified EGFR overexpression. In addition, depatux-m dosing has been limited by corneal side effects common to MMAF conjugates. We hypothesized that a monomethyl auristatin E (MMAE) ADC utilizing an EGFR-targeting antibody with increased affinity may have broader utility against tumors with more modest EGFR overexpression while mitigating the risk of corneal side effects. We describe here preclinical characterization of ABBV-221, an EGFR-targeting ADC comprised of an affinity-matured ABT-806 conjugated to MMAE. ABBV-221 binds to a similar EGFR epitope as depatux-m and retains tumor selectivity with increased binding to EGFR-positive tumor cells and greater in vitro potency. ABBV-221 displays increased tumor uptake and antitumor activity against wild-type EGFR-positive xenografts with a greatly reduced incidence of corneal side effects relative to depatux-m. ABBV-221 has similar activity as depatux-m against an EGFR-amplified GBM patient derived xenograft (PDX) model and is highly effective alone and in combination with standard-of-care temozolomide in an EGFRvIII-positive GBM xenograft model. Based on these results, ABBV-221 has advanced to a phase I clinical trial in patients with advanced solid tumors associated with elevated levels of EGFR. Mol Cancer Ther; 17(4); 795-805. ©2018 AACR.

Analysis of 5518 unique, productively rearranged human VH3-23*01 gene sequences reveals CDR-H3 length-dependent usage of the IGHD2 gene family.

Clear and accurate understanding of diversity in antibody complementarity-determining regions (CDRs) is critical for antibody discovery and engineering. Previous observations of antibody CDR-H3 diversity were based on analyzing available antibody sequences in the public databases. The results may not accurately reflect that of natural antibody repertoire due to erroneous species annotation and the presence of man-made CDR loop diversity in public antibody sequence databases. In this study, in a precisely controlled germline context, we explored the relationship between amino acid composition and CDR-H3 length using 5518 unique productively rearranged human VH3-23*01 gene sequences. CDR-H3 length-dependent usage of the Cys-Xn-Cys motif is reported here.

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.

Generation and characterization of ABBV642, a dual variable domain immunoglobulin molecule (DVD-Ig) that potently neutralizes VEGF and PDGF-BB and is designed for the treatment of exudative age-related macular degeneration.

Exudative age-related macular degeneration (AMD) is the most common cause of moderate and severe vision loss in developed countries. Intraocular injections of vascular endothelial growth factor (VEGF or VEGF-A)-neutralizing proteins provide substantial benefit, but frequent, long-term injections are needed. In addition, many patients experience initial visual gains that are ultimately lost due to subretinal fibrosis. Preclinical studies and early phase clinical trials suggest that combined suppression of VEGF and platelet-derived growth factor-BB (PDGF-BB) provides better outcomes than suppression of VEGF alone, due to more frequent regression of neovascularization (NV) and suppression of subretinal fibrosis. We generated a dual variable domain immunoglobulin molecule, ABBV642 that specifically and potently binds and neutralizes VEGF and PDGF-BB. ABBV642 has been optimized for treatment of exudative AMD based on the following design characteristics: 1) high affinity binding to all VEGF-A isoforms and both soluble and extracellular matrix (ECM)-associated PDGF-BB; 2) potential for extended residence time in the vitreous cavity to decrease the frequency of intraocular injections; 3) rapid clearance from systemic circulation compared with molecules with wild type Fc region for normal FcRn binding, which may reduce the risk of systemic complications; and 4) low risk of potential effector function. The bispecificity of ABBV642 allows for a single injection of a single therapeutic agent, and thus a more streamlined development and regulatory path compared with combination products. In a mouse model of exudative AMD, ABBV642 was observed to be more effective than aflibercept. ABBV642 has potential to improve efficacy with reduced injection frequency in patients with exudative AMD, thereby reducing the enormous disease burden for patients and society.

High efficiency and long-term foreign gene expression in cultured liver sinusoidal endothelial cells by retroviral transduction.

The liver sinusoidal endothelial cells (LSECs) constitute a very specialized endothelium. Due to their multiple functions and privileged location in the liver, these cells constitute an excellent target for gene therapy. In this work, the authors investigate the efficiency of retroviral gene transduction as a method for in vitro gene delivery into murine LSECs. Gene transduction into murine LSECs was performed using the PCMMP-eGFP/pIK-MLVgp retrovirus pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-g), containing eGFP as a reporter gene. Retroviral transduction resulted in a high efficiency of gene transfer (99%) and stable expression of eGFP in LSECs. The retroviral transduction protocol did not affect the morphology or expression of endothelial cell markers or the biological functions of LSECs. The authors have developed conditions for high-efficiency and stable retroviral gene transduction of LSECs. These results raise the possibility of liver gene therapy using LSECs as vehicle for the delivery of therapeutic proteins by means of retroviral vectors.

An improved yeast transformation method for the generation of very large human antibody libraries.

Antibody library selection by yeast display technology is an efficient and highly sensitive method to identify binders to target antigens. This powerful selection tool, however, is often hampered by the typically modest size of yeast libraries (approximately 10(7)) due to the limited yeast transformation efficiency, and the full potential of the yeast display technology for antibody discovery and engineering can only be realized if it can be coupled with a mean to generate very large yeast libraries. We describe here a yeast transformation method by electroporation that allows for the efficient generation of large antibody libraries up to 10(10) in size. Multiple components and conditions including CaCl(2), MgCl(2), sucrose, sorbitol, lithium acetate, dithiothreitol, electroporation voltage, DNA input and cell volume have been tested to identify the best combination. By applying this developed protocol, we have constructed a 1.4 x 10(10) human spleen antibody library essentially in 1 day with a transformation efficiency of 1-1.5 x 10(8) transformants/microg vector DNA. Taken together, we have developed a highly efficient yeast transformation method that enables the generation of very large and productive human antibody libraries for antibody discovery, and we are now routinely making 10(9) libraries in a day for antibody engineering purposes.

Ig knock-in mice producing anti-carbohydrate antibodies: breakthrough of B cells producing low affinity anti-self antibodies.

Natural Abs specific for the carbohydrate Ag Galalpha1-3Galbeta1-4GlcNAc-R (alphaGal) play an important role in providing protective host immunity to various pathogens; yet little is known about how production of these or other anti-carbohydrate natural Abs is regulated. In this study, we describe the generation of Ig knock-in mice carrying functionally rearranged H chain and L chain variable region genes isolated from a B cell hybridoma producing alphaGal-specific IgM Ab that make it possible to examine the development of B cells producing anti-carbohydrate natural Abs in the presence or absence of alphaGal as a self-Ag. Knock-in mice on a alphaGal-deficient background spontaneously developed alphaGal-specific IgM Abs of a sufficiently high titer to mediate rejection of alphaGal expressing cardiac transplants. In the spleen of these mice, B cells expressing alphaGal-specific IgM are located in the marginal zone. In knock-in mice that express alphaGal, B cells expressing the knocked in BCR undergo negative selection via receptor editing. Interestingly, production of low affinity alphaGal-specific Ab was observed in mice that express alphaGal that carry two copies of the knocked in H chain. We suggest that in these mice, receptor editing functioned to lower the affinity for self-Ag below a threshold that would result in overt pathology, while allowing development of low affinity anti-self Abs.

The influence of natural antibody specificity on antigen immunogenicity.

The natural antibody repertoire in humans, apes and Old World primates is distinct from the repertoire of all other placental mammals, and encodes antibodies specific for the carbohydrate epitope Galalpha1-3Galbeta1-4GlcNAc-R (alphaGal). Here, we examined whether conjugating antigens to the alphaGal epitope can augment their immunogenicity in alpha(1,3)galactosyltransferase knockout mice (GT0 mice) which, like humans, produce alphaGal-specific antibodies. Immunization of GT0 mice with BSA conjugated to alphaGal (alphaGal-BSA) led to significant production of anti-BSA IgG antibodies without the need for adjuvant. This response was dependent on the presence of alphaGal-reactive antibodies. Immunization of wild-type mice with alphaGal-BSA failed to induce an anti-BSA response. The presence of alphaGal-reactive antibodies also led to an increase in the T cell response to BSA following immunization with alphaGal-BSA when compared with mice that received BSA alone, resulting in an increased frequency of IFN-gamma- and IL-4-producing BSA-specific T cells. In addition, the ability to produce alphaGal-reactive antibodies enhanced the cytotoxic T lymphocyte anti-viral antigen response following vaccination with murine leukemia virus transformed cell lines that express alphaGal on their cell surface. Natural antibodies that bind alphaGal therefore play a key role in increasing the efficiency of priming to antigens decorated with alphaGal epitopes.

Ajoene inhibits the activation of human endothelial cells induced by porcine cells: implications for xenotransplantation.

Ajoene, is an organosulfur compound derived from garlic that strongly inhibit platelet aggregation, proliferation of human lymphocytes induced by phytohemagglutinin, and in general, blocks membrane-mediated signaling of cell activation. As a thrombotic microangiopathy frequently complicates procedures designed to induce pig-to-baboon chimerism by infusion of large amounts of pig progenitor cells in baboons, it was thought that ajoene might be useful to prevent such complication. For such purpose, we studied the effects of ajoene on the activation of human umbilical vein endothelial cells (HUVEC) induced by pig peripheral blood mononuclear cells (p-PBMC). Co-cultures of p-PBMC with HUVEC results in activation of the HUVEC as shown by over-expression of E-selectin and vascular cells adhesion molecule-1 (VCAM-1). Ajoene (25 microm) strongly inhibits HUVEC activation induced by tumor necrosis factor-alpha (TNF-alpha) or p-PBMC as shown by a down regulation of VCAM-1 and of E-selectin expression. After 5 or 8 h of pre-treatment with Ajoene, HUVEC incubated with TNF and p-PBMC showed an E-selectin or VCAM-1 expression, respectively, at levels similar to the positive control indicating that the inhibitory effect is transient. Ajoene at concentration of 25 microm or lower did not affect HUVEC viability. Based on the finding that Ajoene has a strong, although transient, inhibitory effect on the activation of the endothelium induced by pig cells and its known anti-platelet activity, it is suggested that this garlic compound could be useful to prevent the development of microangiopathy and thrombotic disorders seen in primates infused with pig cells.

Aggregation of human platelets in plasma by porcine blood cells in vitro is probably mediated by thrombin generation.

The infusion of pig progenitor cells into baboons is associated with a thrombotic microangiopathy probably related to the interaction of these cells with the baboon endothelial cells and platelets. We have shown previously that pig peripheral blood mononuclear cells (p-PBMC), are able to activate the human coagulation cascade as they are able to generate thrombin when added to defibrinated plasma. In this work, we have tested the interaction of p-PBMC with human platelets to assess the capacity of p-PBMC to cause platelet aggregation and the possible role of complement activation in this aggregation. Human platelet aggregation assays, using collagen (1 or 2 microg/ml), were performed with platelets in platelet-rich plasma (PRP) or platelets washed by filtration. PRP or washed platelets were also incubated with p-PBMC or human PBMC (h-PBMC) at several concentrations and aggregation was measured. The effect of Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA), an inhibitor of thrombin, was studied on platelet aggregation caused by the pig cells. Complement activation was measured by deposition of fragment c derived from C3 splitting (C3c) on pig cells incubated with citrated platelet poor plasma (PPP). When human PRP was incubated with p-PBMC, aggregation was a consistent event quantitatively similar to that induced by collagen. No aggregation of washed platelets was observed when these were incubated with p-PBMC or h-PBMC. Aggregation of human platelets in PRP, induced by p-PBMC, was inhibited when DAPA (100 microm) was added to the incubation mixture (23%), indicating that the thrombin inhibitor blocked the capacity of p-PBMC to aggregate human platelets. No deposition of C3c fragments on p-PBMC was detected when the porcine cells were incubated for up to 20 min with citrated PPP. The fact is that p-PBMC induces human platelet aggregation in plasma being thrombin generation a likely explanation for this observation. Our data suggest that, in the system assayed, complement activation is not a cause of platelet aggregation. These findings are relevant for the clarification of the reported thrombotic microangiopathy complicating the intravenous infusion of pig cells in primates in attempts to induce pig tolerance in baboons.

Pig peripheral blood mononuclear cells are directly associated with the thrombotic microangiopathy that complicates the induction of chimerism in pig-to-baboon xenotransplantation.

The infusion of large numbers of porcine cells into primates in order to induce specific immunologic tolerance by mixed hematopoietic chimerism, results in thrombotic microangiopathy that can be fatal. For this reason, it is important to study in vitro the interaction of primate endothelial cells with pig cells. We show that pig peripheral blood mononuclear cells (p-PBMC) activate human endothelial cells (hECs) through direct contact. Thus, when endothelial cells are cultured in the presence of p-PBMC, overexpression of VCAM-1 and E-selectin adhesion molecules occurs within 3 h of culture and continues for at least 9 h. The co-culture of p-PBMC and hECs also results in an important adhesion of human platelets to both types of cell. Thus, viewed with the microscope, platelets aggregate above the endothelial cells and also around the pig cells. We present data that suggest that the presence of p-PBMC may be more important with regard to the increase of platelet adhesion to the endothelial cells than the activation alone of the cells. Our results also show that p-PBMC, and not the activated endothelia or the culture supernatant of activated hECs, are able to activate the coagulation cascade because they are able to generate thrombin when added to defibrinated human plasma. Overall, these findings suggest that p-PBMC are of primary importance for the development of the thrombotic disorders that occur in primates transplanted with pig progenitor cells.