Elina Aizenshtein

The impact of PEGylation on protein immunogenicity

Covalent attachment of PEG (PEGylation) is widely used to improve the pharmaceutical properties of therapeutic proteins. The applicability and safety of this method have been proven by the use of various PEGylated pharmaceutical proteins approved by the Food and Drug Administration (FDA). One of the properties attributed to PEGylation is immunogenicity reduction of the PEGylated protein. In this study, the impact of PEGylation on immunogenicity was tested and compared for two proteins (chicken IgY and horse IgG) in two strains of mice (Balb/c and C57BL/6) for two routes of administration (i.v. and i.m.) and two sizes of PEG (5 kD and 20 kD). The influence of PEG was shown to be inconsistent between the mouse strains and routes of administration, even with the same tested protein. Consequently, immunogenicity reduction by PEGylation cannot be predicted or assumed; it must be tested on an individual case basis.

Overcoming the susceptibility gap between maternal antibody disappearance and auto-antibody production.

In the first 10-14 days of a chicks life, protection is conferred by maternal antibodies. Further broiler protection is achieved by active vaccination. However, the high level of maternal antibodies interferes with the induction of an effective immune response by vaccination at a young age. As a result, there is a gap between the reduction in protective maternal antibodies and elevation of self-produced antibodies following active vaccination. The major aim of this study was to test an approach consisting of passive and active vaccination to overcome this gap and to provide continuous resistance to infectious viral diseases during the broilers growth period. Newcastle disease virus (NDV), which is one of the worlds most prevalent infectious diseases of poultry, was tested as a model. Following subcutaneous injection of 18 hemagglutination-inhibiting (HI) units of anti-NDV immunoglobulin Y per 1-day-old chick, protective log2 antibody titers above 4 could be detected to at least 17 days of age. The combination of passive immunization on day 1 of age with attenuated live vaccination on day 10 led to high protective titers throughout the entire growth period, up to 41 days of age. Moreover, the HI titers in the group of birds immunized with the combined vaccination were significantly more homogeneous than those in the group vaccinated only with live virus. Thus, full protection against NDV of all broilers in flock during their entire growth period was achieved by a vaccination regime that combines passive immunization and live vaccination.

The effect of haptens on protein‐carrier immunogenicity

The immune response against hapten is T‐cell‐dependent, and so requires the uptake, processing and presentation of peptides on MHC class II molecules by antigen‐presenting cells to the specific T cell. Some haptens, following conjugation to the available free amines on the surface of the carrier protein, can reduce its immunogenicity. The purpose of this study was to explore the mechanism by which this occurs. Four proteins were tested as carriers and six molecules were used as haptens. The immune response to the carrier proteins was reduced > 100‐fold by some of the haptens (termed carrier immunogenicity reducing haptens – CIRH), whereas other haptens did not influence the protein immunogenicity (carrier immunogenicity non‐reducing haptens – nCIRH). Conjugation of the protein to a CIRH affected protein degradation by lysosomal cathepsins, leading to the generation of peptides that differ in length and sequence from those derived from the same native protein or that protein modified with nCIRH. Injection of CIRH‐conjugated protein into mice induced an increase in the population of regulatory T cells. The results of this study provide a putative mechanism of action for the reduction of immune response to haptenated proteins.

Melanoma antigens and related immunological markers

Melanoma is a highly lethal cancer deriving from transformed dermal melanocytes. Early diagnosed primary melanoma may be curable, but the cure-rate of more advanced stages is limited, with high mortality rate. With the progression of the tumor, the melanocytes overexpress intracellular or cell-surface molecules, including ectopic normal and tumor-specific proteins. Some of these induce a specific immune response by T and B lymphocytes. Antibodies raised against melanoma antigens were proposed for differential disease diagnosis, staging, prognosis and evaluation of treatment efficiency. Nevertheless, treatments based on stimulation of specific anti-melanoma immune responses have had only limited success. It seems that efficient immunotherapy should become more feasible pending on finding new adequate antigens to target. New insights into immune regulation of the tumor microenvironment and its progression may help the development of more successful treatments. We present here up-to-date information on known major melanoma-associated antigens, which could serve as tools for diagnosis as well as for clinical immunotherapy. This approach with promising results for treating some other selected malignancies is still experimental with a very limited success in melanoma. The development of new immune modulators of the tumor microenvironment and neo-antigens may be additional promising directions and may open new opportunities for the immunotherapy of melanoma.

Practical aspects in the use of passive immunization as an alternative to attenuated viral vaccines.

Passive immunization as a method to protect birds has been tested for many years and shown to be effective. Its advantages over active vaccination include no use of partially virulent viruses, overcoming the gap in the level of protection at young age due to interference of maternal antibodies to raise self-immune response following active vaccination and the possible immunosuppressive effect of attenuated vaccine viruses. However, a major obstacle to its implementation is its relatively high cost which is dependent, among other things, mainly on two factors: the efficacy of antibody production, and the use of specific pathogen-free (SPF) birds for antibody production to avoid the possible transfer of pathogens from commercial layers. In this study we show efficient production of immunoglobulin Y (IgY) against four different pathogens simultaneously in the same egg, and treatment of the extracted IgY with formalin to negate the need for SPF birds. Formalin, a common registered sterilization compound in vaccine production, was shown not to interfere with the Fab specific antigen binding or Fc-complement activation of the antibody. Following injection of 1-day-old broilers with antibodies against infectious bursal disease virus, protective antibody levels were acquired for the entire period of sensitivity to this pathogen (35 days). Passive vaccination with formalin-sterilized IgY against multiple antigens extracted from one commercial egg may be a cost-effective and advantageous complementary or alternative to attenuated vaccines in poultry.

Immunological complex for enhancement of innate immune response in passive vaccination

Passive vaccination is used to treat a wide range of infections and cancer. However, this approach has some limitations. An immune complex termed Y-complex was developed to intensify the effect of the passive vaccine. The complex is composed of a microbead that carries specific antibodies and an inducer. It enables targeting of pathogen or abnormal cells, and stimulation of a desired response by innate immune cells, depending on the inducer. The production and efficacy of Y-complex as a passive immune prophylaxis is demonstrated in this study by its use in treating cow mastitis. In an in vitro assay, Y-complex inhibited propagation and induced phagocytosis of bacteria. In challenge experiments, cows were inoculated through the udder with Escherichia coli or Streptococcus dysgalactiae. Following treatment with Y-complex, no bacteria were isolated in the milk and N-acetyl-ß-D-glucosaminidase activity had returned to normal levels. Thus the Y-complex approach can be used as an effective treatment for mastitis. Due to its modularity, this approach may serve as a treatment for a variety of disease agents.

Coated cross-species antibodies by mannosamine-biotin adduct confer protection against snake venom without eliciting humoral immune response.

Passive immunization with cross-species antibodies triggers the patients immune response, thereby preventing repeated treatment. Mannosamine-biotin adduct (MBA) has been described as a masking agent for immunogenic reduction and here, the immunogenicity and biological activity of MBA-coated horse anti-viper venom (hsIgG) were compared to those of uncoated or PEGylated hsIgG. In in vitro tests, hsIgG binding was not affected by MBA conjugation. The immune response to hsIgG-MBA was about 8-fold and 32-fold lower than to PEG-coated and uncoated hsIgG, respectively. In vivo, hsIgG-MBA showed efficient venom-neutralization activity. We thus demonstrate the feasibility of using MBA as a masking agent for passive immunization with cross-species antibodies.

Formation of multimeric antibodies for self-delivery of active monomers

Abstract Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.

Modulating the innate immune activity in murine tumor microenvironment by a combination of inducer molecules attached to microparticles

Targeted cancer immunotherapy is challenging due to the cellular diversity and imposed immune tolerance in the tumor microenvironment (TME). A promising route to overcome those drawbacks may be by activating innate immune cells (IIC) in the TME, toward tumor destruction. Studies have shown the ability to “re-educate” pro-tumor-activated IIC toward antitumor responses. The current research aims to stimulate such activation using a combination of innate activators loaded onto microparticles (MP). Four inducers of Toll-like receptors 4 and 7, complement C5a receptor (C5aR) and gamma Fc receptor and their combinations were loaded on MP, and their influence on immune cell activation evaluated. MP stimulation of immune cell activation was tested in vitro and in vivo using a subcutaneous B16-F10 melanoma model induced in C57BL6 mice. Exposure to the TLR4 ligand lipopolysaccharide (LPS) bound to MP-induced acute inflammatory cytokine and chemokine activity in vitro and in vivo, with the elevation of CD45+ leukocytes in particular GR-1+ neutrophils and F4/80 macrophages in the TME. Nevertheless, LPS alone on MP was insufficient to significantly delay tumor progression. LPS combined with the C5aR ligand C5a-pep on the same MP resulted in a similar inflammation activation pattern. However, interleukin-10 levels were lower, and tumor growth was significantly delayed. Mixtures of these two ligands on separate MP did not yield the same cytokine activation pattern, demonstrating the importance of the cells’ dual activation. The results suggest that combining inducers of distinct innate immune activation pathways holds promise for successful redirection of TME-residing IIC toward anti-tumoral activation.

Abstract 3633: Combination of innate immune activators stimulates leukocyte populations in the tumor microenvironment

Tumor microenvironment (TME) harbors a multitude of immune cells which can comprise up to 50% of the tumor. The most abundant cells in the TME often are tumor associated macrophages (TAM) and tumor associated neutrophils (TAN). These cells potentially possess anti-tumoral cytotoxic effects, and have the potential to induce a broad range of cellular immune responses. However, in the developing tumor, due to elements secreted by the tumor cells, TAM and TAN are altered to act in a pro-tumor manner. The purpose of our study was the optimization and characterization of a treatment aimed at inducing an anti-tumoral response of innate immune cells in the TME. In a melanoma B16 mice model, micro-particles carrying targeting and induction components were injected intra-tumorally and the responses of the tumors, including immune cells populations were monitored. Without treatment the CD45+ cells ratio of total TME decreased as tumor progressed. Particles carrying Toll-like receptor ligands inhibited tumor growth, inducing significant elevation of CD45+ cells in the TME. Using different inducing molecules (inducers) or simultaneous use of several inducers enabled the stimulation of specific immune cell populations within the tumor. The current study suggests that by using tumor targeted particles with adequate inducers, one could change the tumor microenvironment innate immune cells toward an anti-tumor response. Citation Format: Ehud Shahar, Raphael Gorodetsky, Elina Aizenshtein, Jacob Pitcovski. Combination of innate immune activators stimulates leukocyte populations in the tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3633. doi:10.1158/1538-7445.AM2014-3633