Glen M. Lang

Synthesis, isolation, and characterization of conjugates of ovalbumin with monomethoxypolyethylene glycol using cyanuric chloride as the coupling agent

The experimental conditions for the preparation of conjugates of ovalbumin (OA) and monomethoxypolyethylene glycol (mPEG) of a preselected average degree of conjugation, n, using cyanuric chloride as the coupling agent, have been investigated with emphasis on purification and characterization of the products. These conjugates served as prototypes of tolerogenic mPEG derivatives of antigenic proteins which were capable of suppressing in mammals the immunological response to the corresponding unmodified antigens. In other studies in this laboratory, the tolerogenicity of OA(mPEG)n conjugates was found to be a function of n. The reproducibility of the reaction leading to the production of OA(mPEG)n conjugates was shown to depend primarily on the reactivity of the mPEG-cyanuric chloride intermediate, which--for best results--had to be synthesized under completely anhydrous conditions. Isolation of the OA(mPEG)n conjugates was optimized by the use of ion-exchange chromatography whereby rapid removal of large amounts of uncoupled intermediate from the conjugate was achieved; the conditions of fractionation were affected by the degree of conjugation. This method of purification was superior to dialysis, ultrafiltration, and gel filtration. Furthermore, by the application of analytical hydrophobic interaction HPLC it was possible to differentiate among conjugates of different degrees of conjugation and to establish the absence of any detectable free OA in any of the preparations. The quantity of mPEG in the conjugates was determined directly by NMR.

Tolerogenic polyethylene glycol derivatives of xenogeneic monoclonal immunoglobulins

It is becoming apparent that the effectiveness of xenogeneic monoclonal antibodies (XIg), which are increasingly used for diverse therapeutic purposes in man, may be counteracted by their inherent immunogenicity. Since conjugates of proteins with monomethoxypolyethylene glycol (mPEG) have proved to be effective tolerogens in other systems, we have used an experimental model in mice to explore the tolerogenicity of mPEG conjugates of a human monoclonal IgG (HIgG), i.e. a myeloma protein. Administration of these conjugates prior to immunization with heat aggregated HIgG (ha-HIgG) resulted in specific tolerance, as manifested by a marked reduction in the level of antibodies to HIgG, which was related to the degree of conjugation and the dose of conjugate administered. Thus, administration of HIgG(mPEG)20 6 to 43 days prior to immunization with ha-HIgG resulted in an inhibition of anti-HIgG antibody formation of the order of 85-90%, in relation to the titres of mice receiving PBS in lieu of HIgG(mPEG)20; these results hold the promise that mPEG conjugates of XIg may prove therapeutically useful in man in relation to organ transplantation, localization of tumours by immuno-imaging and tumour destruction by immunotoxins.

The development of a radioallergosorbent test (RAST) for murine IgE antibodies

A purified monoclonal IgE preparation, isolated from the ascitic fluid of mice bearing a hybridoma secreting IgE with specificity to ovalbumin, was used for the production of goat anti-murine IgE (GAME) antiserum, which was then rendered monospecific for the epsilon chain. Another monoclonal hybridoma IgE preparation with specificity for the 2,4-dinitrophenyl group was isolated from ascitic fluid in a relatively pure state by affinity chromatography and used in the form of an immunosorbent to isolate antibodies from the monospecific goat serum. The GAME antibodies were 125I-labeled and used to develop a radioallergosorbent test (RAST) for the quantitation of murine IgE antibodies specifically adsorbed onto antigen-coupled paper discs. The RAST was specific for antibodies of the IgE class only and was as sensitive as and more accurate than PCA assay. RAST results on sera of mice treated with tolerogenic conjugates indicated a reduction in the affinity and concentration of the IgE antibody populations on suppression of the IgE response. The effect of interference by non-IgE antibody populations on the RAST curves has been discussed.

Suppression of antibody responses in rats to murine anti-CD4 monoclonal antibodies by conjugates with monomethoxypolyethylene glycol

The effectiveness of therapeutically relevant xenogeneic monoclonal antibodies (MoAb) may be counteracted by their inherent immunogenicity. Since conjugates of diverse proteins with mono-methoxypolyethylene glycol (mPEG) were shown to induce Ag-specific tolerance in mice and rats, we used outbred rats in this study as an experimental model for establishing the tolerogenicity of mPEG conjugates of murine MoAb. The results demonstrate that: (i) murine anti-rat CD4 MoAb (W3/25) were more immunogenic than murine anti-human CD4 MoAb (MAX.16H5) in rats; (ii) W3/25 preferentially induced an anti-idiotypic (anti-id) antibody response; and (iii) antibodies to both common and idiotypic determinants could be suppressed in rats by treatment with W3/25(mPEG)28.

Inhibition of the Effector Phase of IgE-Mediated Allergies by Tolerogenic Conjugates of Allergens and Monomethoxypolyethylene Glycol

In this study we established that the conjugate of ovalbumin (OVA), which was used as a model allergen, with monomethoxypolyethylene glycol (mPEG), i.e., OVA(mPEG)11, was not only tolerogenic and essentially non-allergenic, but also capable of inactivating mast cells sensitized with anti-OVA IgE antibodies (Abs). Moreover, mast cells sensitized with a mixture of anti-OVA and anti-DNP IgE Abs were also desensitized by OVA(mPEG)11 with respect to both sensitivities. Most importantly, treatment of OVA-sensitive mice by OVA(mPEG)11 protected them from systemic anaphylaxis on challenge with OVA. The possibility of inactivating IgE-sensitized mast cells with mPEG conjugates of single epitopes of a given allergen is being investigated.

Specific immunosuppression of human anti-murine antibody responses in hu-PBL-SCID mice

Severe combined immunodeficient (SCID) mice were reconstituted with normal human peripheral blood leukocytes (PBLs) and were shown to produce a human anti-mouse immunoglobulin antibody response on immunization with heat-treated murine monoclonal IgG1 antibody to ovalbumin, referred to as ha-Mab-2. The human anti-mouse antibody response was proportional tot the number of B cells and mononuclear cells transferred from a given batch of PBL. However, pretreatment of hu-PBL-SCID mice with a tolerogenic covalent conjugate of monomethoxypolyethylene glycol (mPEG) and Mab-2 suppressed this response on subsequent injections of ha-Mab-2, and this suppression was shown to be antigen-specific, i.e., it did not suppress the antibody response to ovalbumin and did not affect the level of production of human immunoglobulin of hu-PBL-SCID mice. The suppression was due to the generation of human suppressor CD8+ T (Ts) cells, which down regulated CD4+ helper T cells in an antigen and HLA class I specific manner, i.e., these findings were in accord with the previously shown immunosuppressive effect of tolerogenic mPEG conjugates in normal mice.

Potential Therapeutic Efficacy of Allergen-Monomethoxypolyethylene Glycol Conjugates for in vivo Inactivation of Sensitized Mast Cells Responsible for Common Allergies and Asthma

Skin sites of rats, which had been systemically sensitized to ovalbumin (OVA) were injected intradermally with murine anti-DNP IgE mAbs or with murine polyspecific IgE to recombinant Bet v 1. Injection of OVA(mPEG)10-11 conjugates into these skin sites inhibited passive cutaneous anaphylaxis (PCA) on subsequent intravenous challenge with DNP44-BSA and rBet v 1; by contrast, neither OVA nor an unrelated mPEG conjugate affected the PCA reactions. In dogs sensitized to both OVA and ragweed pollen extract (RAG), inhalation of either allergen (AL) caused a dramatic increase in airway resistance (Rrs). By contrast, administration of an aerosol of OVA(mPEG) caused no change in Rrs. Moreover, thereafter, (1) in spite of repeated challenges with aerosolized OVA over many months, the increase in Rrs on inhalation of OVA was blocked and (2) insufflation of RAG resulted in increase in Rrs of only about 50% in relation to that prior to inhalation of the conjugate; this dogs anti-RAG hyperreactivity remained blunted over many months. It is concluded that AL-mPEG conjugates of optimal composition inactivate sensitized mast cells and basophils, as manifested by a significant decrease of cutaneous or airway responses on subsequent challenge with the respective AL(s).

Downregulation of helper T cells by an antigen-specific monoclonal Ts factor

The findings of previous studies in this laboratory demonstrating that conjugates of human monoclonal (myeloma) IgG (HIgG) and monomethoxypolyethylene glycol (mPEG) were able to induce in mice antigen-specific tolerance and CD8+ suppressor T (Ts) cells were confirmed in the present study. An extract (TsF) of a nonhybridized clone of Ts cells (viz., clone 23.32), which had been derived from spleen cells of mice tolerized with HIgG(mPEG)26, was shown to possess antigen-specific suppressive activity. This monoclonal TsF was able to specifically suppress in vitro antibody formation only if it was present from the beginning of the culture. From the results of the cellular dissection of the system used it was concluded that (i) the TsF had no effect on fully differentiated primed B cells or plasma cells, and (ii) the TsF inactivated carrier-primed Th cells when the culture contained concomitantly naive CD8+ T cells, accessory cells, and antigen. These data support the view that the monoclonal TsF exerted its downregulating effect on Th cells only if it could first interact with a CD8+ T cell, in the presence of accessory cells and antigen.

Induction of in vivo helper activity for murine antibody responses by macrophages pulsed with ovalbumin-monomethoxypolyethylene glycol (OA-mPEG) conjugates

Conjugates of protein antigens with an optimal number of monomethoxypolyethylene glycol (mPEG) chains of an appropriate molecular weight had been shown to suppress murine IgE responses to the unmodified antigen. To investigate the possibility that the tolerogenic capacity of these mPEG conjugates is attributable to a defect in macrophage (M phi) presentation of their antigenic determinants, the activity of ovalbumin (OA)-mPEG conjugates when pulsed onto mouse peritoneal adherent cells (M phi) was compared in this study with their activity in solution. Surprisingly, in contrast to the suppressogenic capacity of mPEG conjugates in solution, the OA-mPEG pulsed M phi appeared to exert a helper effect when injected intraperitoneally (ip), i.e., after subsequent immunization with dinitrophenylated OA (DNP3-OA) in Al(OH)3, the mice showed accelerated IgE and IgG1 antibody responses to OA and DNP. However, when M phi were exposed to limiting concentrations of OA or OA-mPEG, markedly higher concentrations of OA-mPEG were required to yield pulsed M phi, exerting a significant helper effect. It was concluded that although M phi were capable of presenting the OA determinants of OA-mPEG conjugates to helper T (Th) cells, the preparations of modified antigen were presented less effectively than native OA.

Suppression of human anti-mouse idiotypic antibody responses in hu-PBL-SCID mice

Severe combined immunodeficient (SCID) mice were engrafted with appropriate numbers of T cells and B plus mononuclear cells, which had been fractionated from normal human peripheral blood leukocytes (hu-PBLs). Treatment of these hu-PBL-SCID mice with a tolerogenic covalent conjugate of monomethoxypolyethylene glycol (mPEG) and an anti-ovalbumin, IgG1 murine monoclonal antibody, Mab-2, suppressed the human anti-mouse antibody responses to both the common (gamma 1,kappa) and the idiotypic determinants of Mab-2. Moreover, the Mab-2(mPEG)36 conjugate suppressed the immune responses of hu-PBL-SCID mice to the common and idiotypic determinants of murine monoclonal antibodies to the 2,4-dinitrophenyl residue and to human CD4, consisting also of gamma 1 and kappa chains. It is concluded that a tolerogenic mPEG conjugate of a murine monoclonal antibody induces pan-suppression of the human lymphoid system with respect to other murine monoclonal antibodies that share the isotypic determinants of the original one (here, Mab-2) incorporated in the conjugate. Hence, it may be anticipated that human anti-mouse antibody responses to any murine IgG monoclonal antibody would be suppressed by one of eight mPEG conjugates, each incorporating one of the four subclasses of IgG and one of the two light chains.