Immunological enhancement of a murine tumor allograft by passive alloantibody IgG and F(ab′)2

The F(ab′)₂ fraction of an anti-tumor IgG alloantiserum induced passive enhancement of tumor allografts in inbred mice about as well as did intact IgG; i.e. apparently the Fc fragment is not required for suppressing cellular immunity. Passive F(ab′)₂, however, was somewhat less efficient than intact IgG or whole alloantiserum in depressing the allohemagglutinin response. The latter finding is in accord with previous observations on the relative inefficiency of F(ab′)₂ in feedback inhibition of the humoral response of mice to a heteroantigen.     

Effective prophylaxis of influenza A virus pneumonia in mice by topical passive immunotherapy with polyvalent human immunoglobulins or F(ab′)2 fragments

The effectiveness of polyvalent plasma-derived human immunoglobulins (IVIG) in passive immunotherapy of influenza virus pneumonia was assessed, using the Strain Scotland (A/Scotland/74 (H3N2)) adapted to BALB/c mice by repeated lung passages. Haemagglutinin antibodies in two batches of IVIG at 10 mg/ml had a titre of 1/16. Intravenous injection of 1000–5000 μg of IVIG, 3 h after infection, gave 60–70% protection, whereas intranasal injection of 25–50 μg protected 90% of mice infected with a lethal dose of influenza virus. F (ab′)₂ fragments were at least as protective as intact IVIG, suggesting that complement or Fcγ receptor-bearing cells were not required. Topical passive immunotherapy with IVIG or F(ab′)₂ gave protection up to 8 h after infection, but not at 24 h, suggesting that anti-influenza A antibodies in IVIG, delivered locally, are only effective at early stages of the infectious process. The potential value of topical administration of IVIG or F(ab′)₂ fragments for influenza A pneumonia prophylaxis was further demonstrated by the protective effects of their intranasal administration 24 h before challenge.     

Increased Plasma Paraquat Levels in Intoxicated Mice Following Antiparaquat F(ab′)2 Treatment

Abstract

Death most often results from human acute poisonings due to paraquat, a widely used herbicide. It causes a quick and insidious accumulation in lungs. It was proposed to study the effects of the administration of antiparaquat F(ab′)₂ fragments in mice intoxicated with paraquat. Antisera against a paraquat acid derivative coupled to bovine serum albumin were prepared in rabbits, then purified using immunoaffinity chromatography columns and fragmented by pepsin. Antiparaquat F(ab′)₂ antibodies obtained were preventively injected to mice. After intravenous paraquat injection of 8 mg/kg, plasma paraquat levels were measured from 0.25 to 48 hours. Plasma from antiparaquat F(ab′)₂ pretreated mice as compared with non-specific immunoglobulin pretreated control mice showed a significant increase (p < 0.001) of the paraquat concentrations from the 4th (1.17 ± 0.06 versus 0.20 ± 0.01 μg/ml) to the 48th hour (0.47 ± 0.08 versus 0.02 ± 0.01 μg/ml). Although pulmonary paraquat concentrations presented no modification, it could be considered that these preliminary results would have to be studied thoroughly with a view to finding an efficient treatment in human acute poisonings with paraquat.     

Preparation of F(ab′)2 fragments from monoclonal mouse IgG1 suitable for use in radioimaging

Monoclonal antibodies of IgG1 subclass raised against purified human prostate-specific acid phosphatase were subjected to different procedures to produce F(ab′)₂ fragments suitable for radioimaging of prostatic cancer, following derivatization and labeling with radionuclides. The main aim was to obtain highly purified fragments with preserved immunological activity. Optimized pepsin digestion led to the formation of mainly F(ab′)₂ and Fab fragments, and, following Sephacryl S-200 gel filtration, the yield of pure F(ab′)₂ fragments was 24 ± 11% of the theoretical maximum. After papain digestion in the absence of thiols, no formation of Fab fragments was observed, and the F(ab′)₂ fragments formed could be efficiently separated from Fc fragments by chromatofocusing or ion exchange chromatography. The yield of F(ab′)₂ fragments from papain digestion was 50 ± 5% of the theoretical maximum. Both the above procedures gave F(ab′)₂ fragments with immunoreactivity and affinity identical to those of the precursor IgG1, despite the fact that isoelectric focusing profiles of the two F(ab′)₂ preparations differed, suggesting different digestion sites.     

Regulation of the immune response: II. Further studies on differences in ability of F(ab′)2 and 7S antibodies to inhibit an antibody response

The ability of F(ab′)₂ antibody preparations to suppress an immune response is much less than that of intact 7S antibody. The activity possessed by F(ab′)₂ preparations withstood repurification procedures, hence contamination with intact 7S antibody is unlikely. Daily or thrice daily injections of antibody did not make equal the suppressive activities of F(ab′)₂ and intact antibody, indicating that rapid excretion of F(ab′)₂ antibody is not the sole factor involved in the difference in immunosuppressive potency between intact 7S and F(ab′)₂ antibody. Some possibilities for distinct differences in the mechanism of the immuno-suppressive action of F(ab′)₂ and 7S antibodies are raised and discussed.     

Complement fixation by the F(ab′)2-fragment of pepsin-treated rabbit antibody

The F(ab′)₂-fragments of rabbit anti-ovalbumin and anti-human serum albumin IgG fixed between 30 and 50 per cent of guinea-pig complement when they were aggregated with the appropriate antigen. The fixation took place at 37° but not at 4° and was completely inhibited by 0.005 M EDTA. The fixation was more efficient when preformed immune aggregates were used rather than allowing the aggregates to form in the fixation mixture. The loss in haemolytic activity was probably due to the fixation of one or more of the late components (C3 to C9) of guinea-pig complement since no significant fixation of the C1 and C2 components could be found. These results may help to explain previous conflicting reports on the ability of 5S antibody to fix complement. The results also suggest that rabbit antibody F(ab′)₂ may fix complement by a pathway which utilizes only the components C3 to C9 thus bypassing the more usual pathway initiated by C1 activation.     

Regulation of the immune response: VI. Inability of F(ab′)2 antibody to terminate established immune responses and its ability to interfere with IgG antibody-mediated immunosuppression

Intact IgG antibody can terminate established immune responses, whereas F(ab′)₂ antibody cannot do so. The difference between the two antibodies appears to be qualitative. F(ab′)₂ antibody, but not pepsin-digested normal serum, can interfere with the suppression and termination of immune responses induced by intact IgG antibody. These results are discussed in terms of the tripartite inactivation model.     

Efficacy of an anti-CD5-ricin a chain immunoconjugate in an improved human peripheral blood lymphocyte-reconstituted severe combined immunodeficient mouse model

Severe combined immunodeficient mice reconstituted with human peripheral blooldlymphocytes (hu-PBL-SCID mice) were used to evaluate in vivo efficacy of a mouse IgG1 monoclonal antibody (mAb)-ricin toxin A chain immunoconjugate (H65-RTA) directed against the CD5 cell surface antigen present on human T-cells. Initial studies demonstrated that plasma levels of human soluble interleukin-2 receptor (sIL-2R) are predictive of human T-cell engraftment in spleens and blood of SCID mice transplanted with human PBL. Therefore, chimeric mice with detectable plasma levels of human sIL-2R were used in subsequent studies. Systemic injection of such mice with H65-RTA resulted in a significant depletion of human T-cells from spleens, blood and bone marrow, and a decrease in plasma levels of human sIL-2R as compared to vehicle-treated control animals. The effect of H65-RTA was dose-dependent, treatment schedule-dependent, and mAb-specific, as an isotype-, linker- and toxin-matched immunoconjugate of irrelevant binding specificity was not efficacious. Moreover, human T-cells remained depleted from SCID tissues for at least 10 days after cessation of H65-RTA treatment, indicating that the cells were killed by the immunoconjugate. Unconjugated H65 mAb and an H65-derived F(ab′)₂-RTA conjugate, but not unconjugated F(ab′)₂, were also efficacious, suggesting that the Fc portion of the mAb and the toxin moiety may both play a role in the mechanism of human T-cell depletion by H65-RTA in this model. Results indicate that the hu-PBL-SCID mouse model can be used to compare in vivo efficacy of immunosuppressive agents specifically directed against human T-cells.     

Anti-C1q receptor/calreticulin autoantibodies in patients with systemic lupus erythematosus (SLE)

SLE is a disease characterized by the presence of multiple autoantibodies and high levels of circulating immune complexes. We studied the presence and functional relevance of autoantibodies directed against a receptor for the collagen-like stalks of the first subcomponent of complement, also known as calreticulin (cC1qR/CaR), in patients with SLE. In a cross-sectional study it was found that higher titres of antibodies against cC1qR/CaR are present in sera of SLE patients compared with normal donors. No association between anti-cC1qR/CaR titres and SLE disease activity was found. Following gel filtration of SLE serum it was found that anti-cC1qR/CaR reactivity is associated with the peak of monomeric IgG. Purified IgG from patients was able to specifically immunoprecipitate cC1qR/CaR. Since we have shown previously that cC1qR/CaR is able to inhibit the haemolytic activity of C1q, we determined a possible pathogenic role for anti-cC1qR/CaR on complement regulation. IgG derived from SLE serum reversed the inhibitory capacity of cC1qR/CaR in a dose-dependent fashion up to 63%, whereas IgG from normal donors had no significant effect. With respect to the capacity of anti-cC1qR/CaR antibodies to activate neutrophils, it was found that incubation of normal neutrophils with F(ab′)₂ anti-cC1qR/CaR resulted in a very limited oxidative burst. However, cross-linking of F(ab′)₂ anti-cC1qR/CaR on the neutrophils clearly induced neutrophil activation. Pre-incubation of the SLE-derived F(ab′)₂ with cC1qR/CaR prevented activation of neutrophils up to 81 ± 5%. These results suggest that the presence of anti-cC1qR/CaR antibodies in patients with SLE may modulate complement and neutrophil activation.     

IgG2 and other immunoglobulin classes on the cell surface of rat lymphoid cells

The binding to rat lymphoid cells of the F(ab′)₂ fragments of purified rabbit antibodies specific for the Fc or Fd part of rat IgG₂ was studied. Both reagents heavily labeled about 15 % and 6 % of cells from spleen and cervical lymph nodes respectively of rats kept in a conventional animal house. In contrast to this there were very few IgG₂ positive cells in the thoracic duct lymph of any animals, or in the spleen of rats from an SPF animal house. Results on the quantitative binding of rabbit antibodies against rat Fab, IgM and IgA to rat lymphoid cells are also reported. IgM positive cells in the spleen bound much more anti-IgM relative to their binding of anti-Fab antibody, than IgM positive cells from thoracic duct lymph or cervical lymph node.     

The production of precipitating antiglobulin reagents specific for the subclasses of human IgG

Precipitating antisera against the four subclasses of human IgG were prepared by immunizing different species of animals (monkeys, rabbits, chickens, guinea-pigs and sheep) with whole IgG or Fc fragments. Freund''s complete and incomplete adjuvant and Al(OH)₃ were used to enhance antibody formation.In most of the experiments with rabbits, immunization against a particular subclass was accompanied by induction of unresponsiveness to the other subclasses.Different animal species showed distinct preferences for production of certain IgG subclass antisera. Anti-IgG₃ could quite easily be produced in each of the animal species tested (monkey, rabbit and guinea-pig). Anti-IgG₂ and anti-IgG₄ could be raised in monkeys and, with greater efforts, in rabbits. Anti-IgG₁ could easily be raised in guinea-pigs and with difficulty in rabbits. In our hands monkeys did not react to IgG₁, possibly because only Gm(f+) paraproteins were used; experiments with rabbits and guinea-pigs showed that antibody formation against IgG₁ was promoted by the presence of the Gm(a) marker. The Gm(n) marker was found to influence anti-IgG₂ formation to an even greater extent: no subclass-specific antibodies were obtained when Gm(n-) IgG₂ paraproteins were used as antigen. Apart from IgG subclass-specific antibodies the following additional antibodies were often found: antibodies directed to a combination of a certain heavy chain and a kappa light chain in monkeys; antibodies specific for one or more antigenic determinants common to IgG₂ and IgG₃ in monkeys and rabbits immunized with IgG₂; antibodies specific for one or more antigenic determinants common to IgG₁ and IgG₂ in guinea-pigs and rabbits immunized with Gm(f+) IgG₁.     

Immunoregulatory characteristics of the in vitro anti-ssDNA response

Continuous blockade of B-cell antigen receptors (BCRs) with Fab αsIg prevents the anti-ssDNA response of high, but not low, density B cells. Signaling via the BCRs, by prior exposure to crosslinking F(ab′)₂ αsIg, had no effect on the spontaneous anti-DNA response, but prevented a lipopolysaccharide-induced anti-DNA response. Pretreatment with intact αsIg, which provides exogenously derived Fc signals, reduced the response. An Fc-signal-blocking agent, F(ab′)₂ anti-IgG-Fc antibody, increased the number of anti-DNA antibody-forming cells produced in the absence of exogenous IgG anti-ssDNA antibody. Thus, activation is dependent on the availability of the BCRs, prior BCR crosslinking does not interfere with activation, and endogenous IgG anti-ssDNA antibody limits the activation of anti-ssDNA-specific B cells most of which are T-cell independent. These results indicate that the anti-ssDNA response is driven through the BCR.     

Intravenous Immunoglobulins for Therapeutic Use Contain Anti-idiotypes against Xenophile Antibodies and Prolong Discordant Graft Survival

Xenotransplantation between discordant species leads to a graft survival of a few minutes, due to binding of natural antibodies to the xenogeneic endothelial cells, complement activation, and endothelial cell activation. Polyclonal human immunoglobulins for intravenous use (IVIg) from normal donors have been proven effective in a variety of antibody-mediated disorders and contain anti-idiotypic antibodies directed against a number of disease-associated and natural antibodies. We have shown that administration of IVIg delays rejection of a guinea pig heart to a rat. We demonstrate herein that IVIg can inhibit the binding of xenoreactive rat IgG antibodies to guinea pig endothelial cells. This inhibition is likely due to the presence, among IVIg, of anti-idiotypic antibodies as F(ab′)₂fragments of IVIg were as effective as whole IVIg. In addition, natural anti-endothelium rat antibodies were retained on a column of F(ab′)₂fragments of IVIg coupled to Sepharose. The degree of inhibition of binding of IgG natural antibodies correlated with the survival of the xenograft when IVIg was administered prior to transplantation. Thus IVIg prolong xenograft survival through idiotypic–anti-idiotypic interactions with natural xenoreactive antibodies of the IgG isotype.     

Trypanosoma cruzi calreticulin: A novel virulence factor that binds complement C1 on the parasite surface and promotes infectivity

In Trypanosoma cruzi, calreticulin (TcCRT) translocates from the endoplasmic reticulum (ER) to the area of flagellum emergence. We propose herein that the parasite uses this molecule to capture complement C1, in an infective apoptotic mimicry strategy. Thus, TcCRT/C1 interactions, besides inhibiting the classical pathway of complement activation as previously shown in our laboratories, will also promote infectivity. This fact correlates with significant increases in TcCRT mRNA levels during early infection stages of a VERO cell line. In vitro, the collagenous and globular C1q domains simultaneously bind TcCRT and antigen aggregated Igs, respectively. Accordingly, mouse immunizations with TcCRT induced humoral responses that, after challenge, correlated with increased parasitemia. Thus, on the parasite surface, whole Igs anti-TcCRT promote C1 deposits on trypomastigotes while, as expected, F(ab′)₂ fragments decrease it. Likewise, pretreatment of the parasites with whole anti-TcCRT antibodies augmented parasitemia and mortality in mice. In contrast, pretreatment with F(ab′)₂ fragments anti-TcCRT, devoid of their capacity to provide additional C1q binding sites, was protective. Most important, while pretreatment of trypomastigotes with C1q increased infectivity in the RAW murine cell line, as well as mice mortality and parasitemia, the F(ab′)₂ fragments significantly interfered with the C1q-dependent infectivity. Differently from other surface molecules involved in infectivity, TcCRT uses C1 as an adaptor molecule to recognize host cells. As expected, since TcCRT is one of several cell surface parasite molecules participating in infectivity, attempts to interfere with the C1/TcCRT interactions with F(ab′)₂ fragments, were moderately but significantly effective, both in vitro and in vivo.     

Engagement of the CD4 receptor inhibits the interleukin-2-dependent proliferation of human T cells transformed by Herpesvirus saimiri

Infection with Herpesvirus saimiri, a tumor virus of non-human primates, transformed human CD4⁺ T cell clones to permanent interleukin (IL)-2-dependent growth without need for restimulation with antigen and accessory cells. The IL-2-dependent proliferation of these cells was dramatically inhibited by soluble anti-CD4 whole antibodies, F(ab′)₂ and Fab fragments, and also by gp 120 of human immunodeficiency virus. The inhibition was not due to cell death and could be overcome by high concentrations of exogenous IL-2. Cell surface expression of CD4, and to a lesser degree the density of the IL-2 receptor α chain, were reduced upon anti-CD4 treatment. After long lasting (>12h) incubation with anti-CD4, abundance and activity of CD4-bound p56^lck were diminished while the free fraction of p56^lck remained unchanged. Since IL-2 binding to its receptor activated only the CD4-bound fraction of p56^lck, the IL-2-induced p56^lck activity was diminished after long-term CD4 ligation. Taken together, our results suggest a cross talk between CD4- and IL-2 receptor-mediated signaling via p56^lck.     

Human constant regions influence the antibody binding characteristics of mouse-human chimeric IgG subclasses

Although antibody affinity is primarily determined by immunoglobulin variable region structure human IgG antibodies of the four subclasses specific for the same antigen have been shown to differ in their affinity. To explore the influence of the immunoglobulin constant region on functional antibody affinity, a set of V region identical mouse–human chimeric IgG subclasses specific for TAG72 (tumour-associated glycoprotein) were studied. Biomolecular interaction analysis (BIA) was used to determine the binding kinetics of whole IgG subclasses and F(ab')₂ fragments. Despite identical V regions, binding kinetics differed for the four subclasses. The apparent dissociation rate constants of the intact immunoglobulins ranked IgG4 < IgG3 < IgG2 < IgG1. In contrast, analysis of the binding characteriztics of the F(ab')₂ fragments derived from IgG1, IgG2 and IgG4 revealed identical binding kinetics. The structure of the constant regions of the humanized IgG subclass antibodies clearly influenced functional antibody affinity, as has been described for the murine IgG subclasses. The exact mechanism for this phenomenon remains obscure but such differences should be taken into account when designing or choosing antibodies for therapeutic use.     

Immunoglobulins from rats that are resistant to adjuvant arthritis suppress the disease in arthritis-susceptible rats

The therapeutic effect of high doses of polyclonal immunoglobulins has been well established in various B cell-associated autoimmune diseases. In the present work we have examined the effect of low doses of immunoglobulins in adjuvant arthritis, a T cell-associated disease in the Lewis rat. Lewis rats were treated with purified rat immunoglobulins as well as their Fc and F(ab′)₂ fragments and their protective effect on adjuvant arthritis was evaluated. We found that early as well as late treatment with low doses of rat immunoglobulins induced refractoriness to disease induction. The effect was found to be carried out by the F(ab′)₂ part of the immunoglobulins and could be adsorbed by affinity purification on Mycobacterium tuberculosis. The protective antibodies were present in Fisher and BN rats that are resistant to adjuvant arthritis, but not in the arthritis susceptible Lewis and Wistar strains. We suggest that resistance to autoimmune arthritis is associated with the presence of protective immunoglobulins and that their effect is carried out through the antigen recognition part of the molecule.     

Interaction of antibodies with human glomerular epithelial cells

We tested the hypothesis that the pathogenesis of human idiopathic membranous glomerulonephritis is similar to that of Heymann glomerulonephritis, a model of membranous glomerulonephritis induced in rats by immunization with renal brush border preparations; the characteristic subepithelial deposits result from interaction of antibodies with a brush border antigen (gp330) expressed on the plasma membrane of glomerular visceral epithelial cells (GEC), followed by redistribution and shedding of gp330 immune complexes. The experiments were performed in cultured glomerular visceral epithelial cells, in living monkeys and rats, and in isolated perfused human, monkey, and rat kidneys. Antigens from plasma membranes of human renal brush border vesicles (HBBV) and GEC vesicles (HGECV) and their corresponding polyclonal and monoclonal antibodies reactive with human and monkey GEC were prepared. First, polyclonal antibodies to HGECV bound diffusely to cultured GEC; monoclonal antibody 8G5, recognizing a 60-kDa protein, mainly bound to the coated pits and apical invaginations; both polyclonal HGECV and 8G5 monoclonal antibodies induced antigen redistribution (capping) at 37 degrees C. Second, monkeys were actively or passively immunized, and isolated human and monkey kidneys were perfused with the antibodies. Active immunization with HBBV induced tubular immune deposits, whereas active immunization with HGECV did not provoke renal lesions. After passive immunization HBBV and HGECV antibodies bound diffusely to glomerular cells, and subepithelial deposits were observed during the autologous phase; in contrast, 8G5 induced early (day 3) granular deposits. Third, fine granular deposits developed in glomeruli of human and monkey kidneys perfused for 4 hours at 37 degrees C with 8G5; these deposits were more difficult to detect by electron microscopy than those occurring in kidneys of Lewis rats perfused with sheep antiHBBV. The results show that some antibodies redistribute antigens at the surface of human and monkey GEC in vitro, in vivo, and ex vivo and induce formation of granular deposits in human glomerular capillary walls. Failure to induce more severe lesions in human and monkey kidneys may be ascribed to lack of GEC antigens comparable to rat gp330, insufficient cross linking by monoclonal antibody, lack or insufficient concentration of epitope-specific antibodies, insufficient time of kidney perfusion, or a combination of these factors.     

Quantitative determination of humanized monoclonal antibody rhuMAb2H7 in cynomolgus monkey serum using a Generic Immunoglobulin Pharmacokinetic (GRIP) assay

Preclinical pharmacokinetic (PK) assays are important to help evaluate the safety and efficacy of a potential biotherapeutic before clinical studies. The assay typically requires a biotherapeutic-specific reagent to minimize matrix effects especially when the host species are non-human primates such as cynomolgus monkeys and the biotherapeutic is a humanized monoclonal antibody (MAb). Recombinant humanized mAb 2H7 (rhuMAb2H7) binds to the extracellular domain of CD20 that is expressed on B cells and results in B cell depletion. It is currently being evaluated for its therapeutic potential in rheumatoid arthritis (RA) in clinical studies. During the early development of rhuMAb2H7, a cynomolgus monkey PK assay was needed to help assess the pharmacokinetic parameters of rhuMAb2H7 in a pilot cynomolgus monkey study. However, development of a cynomolgus monkey PK assay was challenging due to lack of rhuMAb2H7-specific reagents. Here we describe an alternative method for detection of rhuMAb2H7 in cynomolgus monkey serum using polyclonal antibodies against human IgGs. This assay quantifies rhuMAb2H7 in 10% cynomolgus monkey serum with high sensitivity, accuracy, and precision. This assay successfully supported the rhuMAb2H7 development, and has the potential to be used to quantify other humanized MAb biotherapeutics in serum from a variety of non-human species.