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.     

Immunological enhancement by mouse isoantibodies: the importance of complement fixation

Balb/c anti-E.L.4 serum was separated into seven fractions of increasing electrophoretic mobility by gradient elution from a column of DEAE-cellulose. The first four fractions, containing the bulk of the immunoglobulins, were tested for their effects in vivo on the growth of E.L.4 in Balb/c mice. The first fraction, which contained most of the cytotoxic activity in vitro, produced inhibition of growth, while the third fraction produced enhancement.

The activities of F(ab′)₂ and Fab′ fragments from A strain anti-E.L.4 serum were also studied. Both preparations were found to have lost cytotoxic activity on E.L.4 cells in vitro, but to be capable of blocking the effect of undigested IgG. F(ab′)₂ retained haemagglutinating activity on C57B1 red cells, whereas Fab′ did not. Fab′ was able to block the haemagglutinating effect of undigested IgG and F(ab′)₂. Both F(ab′)₂ and Fab′ could produce enhancement of the growth of E.L.4 in A strain mice.

It is suggested that non-complement-fixing antibodies are critical in the production of enhancement.

     

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.     

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.     

Natural Killer Cells Modulate Overt Autoimmunity to Homeostasis in Nonobese Diabetic Mice after Anti-CD3 F(ab′)2 Antibody Treatment through Secreting Transforming Growth Factor-β

Recently, the anti-CD3 antibody has been shown to be a promising candidate for the efficient treatment of overt autoimmunity. However, the mechanisms underlying this effect remain unclear. Our previous studies demonstrated that natural killer (NK)T cells and transforming growth factor (TGF)-β were key elements in anti-CD3 F(ab′)₂-mediated re-establishment of glucose homeostasis and restoration of self tolerance to islets in type 1 diabetes. In this report, we further investigate the regulatory pathways involved, especially the cellular source of TGF-β production. The treatment of new-onset nonobese diabetic mice with anti-CD3 F(ab′)₂ resulted in a significant increase in the numbers of NK cells in spleen and pancreatic lymph nodes that secrete TGF-β. Depletion of this cell population with a specific anti-AsGM1 antibody abrogated anti-CD3 F(ab′)₂ therapeutic effects and splenic TGF-β production. When fractionated from recovered mice after CD3 antibody therapy, these NK cells actively suppressed diabetogenic cell proliferation and prevented the cotransfer of diabetes into nonobese diabetic-severe combined immunodeficient mice in a TGF-β-dependent manner. In addition, the regulatory NKT cells from remitting mice were capable of causing NK cells to exhibit a TGF-β-producing phenotype by the secretion of the T helper 2 cytokines interleukins 4 and 10. Overall, these data indicate that NK cells are the main source of TGF-β production after anti-CD3 F(ab′)₂ treatment, which are controlled by a population of T helper 2-like NKT cells.Type 1 diabetes in human and nonobese diabetic (NOD) mice is an autoimmune disease in which pancreatic islet β cells are destroyed by the cellular immune system.¹ Based on our understanding of the pathogenesis of β cell destruction in type 1 diabetes, many strategies targeted to immune cells have been developed, including antibodies recognizing antigens expressed on the surface of T cells. CD3-specific antibodies have been believed to be promising candidates to treat overt diabetes.^2,3,4 Short-term administration of an anti-CD3 antibody resulted in acquisition of immune tolerance to islets and long-lasting normoglycemia. In surveying the underlying mechanisms, our previous study has identified that natural killer (NK)T cells are key players in the immunoregulation of autoimmunity after anti-CD3 F(ab′)₂ therapy.⁵ Furthermore, anti-CD3 F(ab′)₂ treatment heightened the level of production of transforming growth factor (TGF)-β, which is widely accepted as a critical immunoregulatory cytokine in controlling pathogenic cells and maintaining immune homeostasis.⁶ Interestingly, up-regulated TGF-β appears not to derive from NKT cells or CD4⁺CD25⁺ regulatory T cells, as depletion of this regulatory subset does not affect TGF-β secretion in tolerized NOD mice.⁶ Thus, it is necessary to clarify the identity of lymphocyte population responsible for producing TGF-β after CD3 antibody treatment.NK cells have been shown to be important components in bridging innate and adaptive immunity. Although this kind of cell plays an effector role in cleaning virally infected cells and rejection of allogenic grafts through cytotoxic capacity and producing pro-inflammatory cytokines,^7,8 in some settings, their role is regulatory, as they can also produce multiple immunomodulatory cytokines, eg, interferon-γ, TGF-β, and interleukin (IL)-10.⁹ Recently, the deficiency of NK cell function in NOD mice has been reported, which contributes to diabetes development.^10,11 Accordingly, it is conceivable to prevent the onset of diabetes by modulating NK cells. In fact, a recent study demonstrated that administration of the NK cell activator poly (I:C) in young NOD mice potentially reduced diabetes incidence and insulitis by secreting TGF-β.¹² Based on the regulatory function of NK cells in autoimmune disorders, this study examined the role of NK cells in anti-CD3 F(ab′)₂-mediated therapeutic effects. We found that anti-CD3 F(ab′)₂ antibody treatment increased the frequency and number of NK cells with a hallmark of producing TGF-β and depletion of NK cells abolished anti-CD3 F(ab′)₂ effects. Furthermore, NK cells from treated mice inhibited diabetogenic cell response to autoantigen stimulation in vitro and prevented the transfer of diabetes in vivo in a TGF-β-dependent manner.     

Modulation of Fc receptors on human peripheral blood lymphocytes by antisera against β2 microglobulin, Ia or immunoglobulin

The association between Fc receptors and other surface molecules was examined by EA-rosette (EAR) inhibition experiments. Twenty to 30% EAR were detected in suspensions of peripheral blood lymphocytes from normal individuals. Anti-β₂ microglobulin (βMi) sera fully suppressed EAR whereas anti-Ig, anti-Ia sera and heat aggregated IgG inhibited only 50–60% EAR. Thus almost half of the detected EAR were apparently not surface Ig positive B cells. Rabbit and monkey anti-βMi sera suppressed EAR effectively whereas rat and chicken antisera, despite strong βMi binding capacity, inhibited EAR poorly. The latter result was ascribed on the basis of immunofluorescent analysis to inadequate capping of βMi. Incubation of PBL with whole antisera suppressed EAR to a similar degree at either 0° or 37°, whereas F(ab′)₂ fragments were inhibitory only at 37°. Taken together the results suggest that Fc receptors can be inhibited by antibodies with specificity against any cell surface antigen. The blocking mechanism may be due to steric hindrance by the Fc part of antibody molecules and/or F(ab′)₂ fragment mediated co-capping.     

Purification and properties of non-precipitating rabbit antibodies

Precipitating and non-precipitating anti-egg albumin and anti-dinitrophenyl rabbit antibodies were specifically purified from hyperimmunized sera. Both populations of antibody were similar with regard to electrophoretic mobility and molecular size. Non-precipitating antibodies brought about passive haemagglutination and PCA, although with less efficiency than precipitating antibodies. On the other hand, only precipitating antibodies fixed complement and produced a reverse Arthus reaction. The F(ab′)₂ fragment obtained from non-precipitating antibody did not precipitate with antigen. These results are compatible with the hypothesis that non-precipitability is due to a particular configuration of the molecule that makes it impossible for one molecule of antibody to combine with two different molecules of antigen simultaneously.     

Destruction of antibody idiotopes with ultra-low concentrations of reducing agents

The nature of the idiotopes present on F(ab')₂ fragments prepared from rabbit anti-micrococcal carbohydrate antibodies and the loss of idiotypic reactivity of these F(ab')₂ fragments upon iodination were examined. Rabbit anti-micrococcal idiotopes were shown to be exquisitely sensitive to treatment with very low concentrations of sodium metabisulfite or 2-mercaptoethanol. The treatment destroyed anti-micrococcal idiotopes, as shown by the loss of idiotopes on F(ab')₂ fragments after reduction; the allotype epitopes and the antigen binding capacity of the F(ab')₂ fragments were unaffected. The destruction of the idiotopes by very low concentrations of reducing agents indicated that an extremely labile disulfide bond is involved in the structure of the idiotope or in the maintenance of the conformation of the anti-microccal idiotopes. Identical reduction-sensitive anti-micrococcal idiotopes have been demonstrated in a number of related outbred rabbits, and in each case they induced a natural auto-anti-idiotype (AAI) antibody response. Recognition of the existence of these reduction-sensitive idiotopes and their properties could provide a basis for further study of these idiotopes and may lead to a better understanding of the idiotope network.     

Red cell Fc receptors and their participation in the passive haemagglutination mediated by non-precipitating antibodies

A study has been made of the fixation to sheep, rabbit, guniea-pig and human red cells (both untreated and trypsin-treated cells) brought about by different non-precipitating antibodies and their corresponding F(ab′)₂ fragments, as well as by human IgG and its corresponding fragments F(ab′)₂, Fab and Fc.

The results obtained with the antiglobulin test seem to indicate that there exists on the surface of the red cells Fc receptors which exhibit a greater affinity for the Fc fragment which has undergone structural changes consequent upon either the immunoglobulin-binding interaction or as a result of enzymic degradation.

The Fc receptors would appear to be superficial in the majority of animal species studied. In the case of human red cells their liberation depends upon prior treatment with trypsin

On the basis of these investigations consideration should be given to the theory that the passive haemagglutination mediated by non-precipitating antibodies could be of the mixed type, with the participation of both Fab and Fc fragments.

     

Affinity labelling of isoelectrofocused fractions from a DNP antibody preparation with the photoactive labels 2,4-dinitrophenyl-1-azide and 2,4-dinitrophenyl-ε-aminocaproyldiazoketone

Isoelectric (I.E.F.) fractions from two DNP antibody preparations have been affinity labelled with the photo-active labels 2,4-dinitrophenyl-1-azide (DNP—N₃) and 2,4-dinitrophenyl-ε-aminocaproyl diazoketone (DNP—EACDK). The ratios of heavy to light chains labelled for I.E.F. fractions A, B and C of antibody preparation A-2, labelled with DNP-N₃, were 4.5, 3.1 and 2.4 respectively. In excellent agreement with these results were the ratios found for the labelling of I.E.F. fractions A, B and C of a second antibody preparation A-1 pool I, which were 4.4, 3.8 and 2.0 respectively. A second fraction of DNP antibody preparation A-1 (pool II) was isoelectrically focused, and the I.E.F. fractions A′, B′ and C′ were affinity labelled with the label DNP—EACDK. The ratios of heavy to light chains labelled for the I.E.F. fractions A′, B′ and C′ were 3.4, 0.12 and 5.6 respectively. Mass spectral analyses of the predominant radioactive fraction isolated from the nagarse digestion of the DNP-N₃ labelled heavy chains from I.E.F. fractions A-2 A and A-2 C indicated that fraction A was labelled primarily on a phenyl-alanine residue and that fraction C contained most of the label on an alanine residue. The labelling results support the view that the combining sites of anti-DNP antibodies are distinct and differ substantially and the `characteristic' heavy/light chain ratio of 2 to 4 represents an average of individually homogeneous sites which are labelled by a given affinity reagent exclusively on either light or heavy chains but not both.     

C1q,a subunit of the first component of complement,enhances antibody-mediated apoptosis of cultured rat glomerular mesangial cells

We have shown previously that IgG2a anti-Thy-1 MoAb (ER4G) induces apoptosis of rat mesangial cells (GMC) in vitro. Since the classical complement pathway plays an essential role in Thy-1 nephritis, we analysed whether C1q, a subunit of the first component of complement, enhances the ER4G-mediated apoptosis of rat GMC. Two different subclasses of anti-Thy-1 MoAb, ER4G (IgG2a) and ER14 (IgG1), were used. It was established that ER4G binds C1q efficiently, while ER14 reacts poorly with C1q. For the experiments of apoptosis, quiescent rat GMC were exposed for 1 h at 37°C to a fixed concentration of anti-Thy-1 MoAb and incubated further for 16 h at 37°C in the presence or absence of C1q. GMC exposed to medium (M-GMC) followed by incubation of the cells with medium alone was used as controls. Apoptosis was assessed by morphological studies and quantitative analysis on FACS using FITC-annexin V (the annexin V methods) or bicolour FACS analysis using FITC-annexin V and propidium iodide (the annexin V/PI method). With the annexin V method, M-GMC revealed 9.4 ± 1.4% apoptosis. C1q had only marginal effects on apoptosis of M-GMC. GMC exposed to ER4G (ER4G-GMC) and further incubated with medium in the absence of C1q resulted in 25.7 ± 5.7% apoptosis (P < 0.01 relative to control). Incubation of ER4G-GMC together with 100 μg/ml of C1q significantly increased GMC-apoptosis up to 39.4 ± 4.9% (P < 0.01 relative to ER4G-GMC incubated in the absence of C1q). This enhancing effect of C1q on apoptosis of ER4G-GMC was time- and dose-dependent. In contrast, C1q did not significantly alter the apoptosis of either GMC exposed to ER14 (ER14-GMC) or to F(ab′)₂-ER4G (F(ab′)₂-ER4G-GMC), while ER14-GMC or F(ab′)₂-ER4G-GMC incubated with medium resulted in significant apoptosis compared with control. These results were supported by morphological studies and bicolour FACS analyses in time course experiments using the annexin V/PI method. The effect of C1q is dependent on the presence of intact C1q-containing globular heads and does not occur with collagen-like fragments of C1q. Furthermore, incubation of ER4G-GMC with anti-mouse κ-chain antibodies also increased ER4G-mediated GMC-apoptosis. These results indicate for the first time that C1q enhances antibody-mediated apoptosis of rat GMC in vitro, presumably by its binding to ER4G and probably by additional cross-linking of Thy-1 on the surface of GMC.     

Concomitant but Not Causal Association Between Surface Charge and Inhibition of Phagocytosis by Cryptococcal Polysaccharide

The mechanism by which capsular polysaccharides inhibit phagocytosis is not clearly understood. We investigated the association between a negative surface charge and inhibition of phagocytosis by the capsular polysaccharide of Cryptococcus neoformans. A two-polymer aqueous-phase system containing phosphate ions was used to assess surface charge. Opsonins such as normal bovine serum and normal human immunoglobulin G reduced the surface charge on non-encapsulated cryptococci and simultaneously enhanced phagocytosis. These same opsonins had no effect on phagocytosis or surface charge of encapsulated cryptococci. F (ab′)₂ fragments of normal human immunoglobulin G neither enhanced phagocytosis nor altered the surface charge of non-encapsulated cryptococci. Addition of purified cryptococcal polysaccharide to non-encapsulated cells inhibited phagocytosis of the yeast and induced a strong negative charge at the yeast surface. Chemical modification to reduce the surface charge of either purified cryptococcal polysaccharide or intact encapsulated cryptococci produced a small loss of phagocytosis-inhibiting activity; however, all treated polysaccharide preparations retained a significant ability to inhibit phagocytosis of the yeast. These results indicated that the association between surface charge and inhibition of phagocytosis was largely circumstantial, and presence of a negative surface charge could not account for the powerful antiphagocytic action of cryptococcal polysaccharide.     

The alternate pathway of complement activation: The role of C3 and its inactivator (KAF)

The immunochemical depletion from serum of C3b inactivator (KAF) has been performed using purified F(ab′)₂ antibody. In vitro KAF depletion leads to spontaneous activation of the `alternate pathway of complement fixation' as evidenced by conversion of glycine-rich β-glycoprotein, depletion of cobra venom factor—C3 proactivator and conversion of C3.

The status of the complement system following in vitro KAF depletion accurately mimics that found in vivo in the unique KAF-deficient patient.

The activation of the alternative pathway whether by KAF depletion or by conventional alternative pathway activators is prevented by the immunochemical depletion of C3. It therefore appears that the alternative pathway is essentially a C3b-feedback pathway which is normally controlled by the activity of KAF.

     

Development and evaluation of candidate vaccine and antitoxin against botulinum neurotoxin serotype F

To produce a vaccine suitable for human use, a recombinant non His-tagged isoform of the Hc domain of botulinum neurotoxin serotype F (rFHc) was expressed in Escherichia coli and purified by sequential chromatography. The rFHc was evaluated as a subunit vaccine candidate in mouse model of botulism. A dose-response was observed in both antibody titer and protective efficacy with increasing dosage of rFHc and number of vaccinations. These findings suggest that the rFHc is an effective botulism vaccine candidate. Further, we developed a new antitoxin against botulinum neurotoxin serotype F (BoNT/F) by purifying F(ab′)₂ fragments from pepsin digested serum IgGs of horses inoculated with rFHc. The protective effect of the F(ab′)₂ antitoxin against BoNT/F was determined both in vitro and in vivo. The results showed that the F(ab′)₂ antitoxin could prevent botulism in mice challenged with BoNT/F and effectively delayed progression of paralysis from botulism in the therapeutic setting. Thus, our results provide valuable experimental data for this new antitoxin as a potential candidate for treatment of botulism caused by BoNT/F.     

Anti-CD45 antibody enhances lipoxygenase pathway of human naïve mononuclear cells and cyclooxygenase pathway of neutrophils

Objective and Design: Anti-CD45 antibody exhibits multiple biological effects on human mononuclear cells (MNC) and polymorphonuclear neutrophils (PMN). We intended to determine whether anti-CD45 antibody could affect arachidonic acid metabolism and thereby, the interactions between human na?ve MNC and PMN. Materials and Methods: Human na?ve MNC and PMN were incubated with monoclonal anti-human CD45 IgG F(ab’)₂ antibody or non-specific IgG F(ab’)₂ for 30 min. The mRNA expression of cyclooxygenase type 1 (COX-1), type 2 (COX-2), 5-lipoxygenase (5-LOX) and leukotriene A₄ hydrolase (LTA₄ hydrolase) in both cells was detected by RT-PCR and quantified by densitometric determination. The presence of COX-1 and COX-2 molecules in the cells was detected by Western blot. The concentration of PGE₂ and LTB₄ in cultured supernatants was measured by EIA kits. Results: Anti-CD45 IgG F(ab’)₂ up-regulated LTA₄ hydrolase mRNA expression and LTB₄ production, but down-regulated COX-1 and COX-2 mRNA expression and PGE2 production, of na?ve MNC compared to non-specific IgG F(ab’)₂. In contrast, a reverse modulation by the specific antibody on PMN was observed including up-regulation of cyclooxygenase pathway and down-regulation of lipoxygenase pathway. Conclusions: A novel activity of anti-CD45 with reverse modulation on cyclooxygenase/lipoxygenase pathways was found such that the expression of COX-1 and COX-2 in PMN, and 5-LOX and LTA₄ hydrolase in MNC were enhanced. Received 4 May 2005; returned for revision 29 June 2005; returned for final revision 12 October 2005; accepted by M. Katori 16 November 2005     

Studies on the products of peptic digestion of IgA

Proteolytic digestion of IgA was studied using pepsin. Only minor differences were found between the peptic digestion products of secretory and serum IgA. The major products of proteolysis were a 4.8S—5.8S protein and small polypeptides. The 4.8S—5.8S fragment strongly resembled the F(ab′)₂ portion of other immunoglobulins in size, electrophoretic mobility, content of heavy and light chain antigens, formation of lighter proteins after reduction and alkylation and susceptibility to progressive digestion. In addition, a smaller 3.8S fragment which also contained heavy and light chain antigens was formed when IgA was digested by pepsin in the presence of a reducing agent. The antibody combining site of an IgA M-component with cold agglutinin activity appeared to reside on this 4.8S to 5.8S fragment of the molecule. These findings justify the conclusion that peptic digestion of IgA yields an F(ab′)_2α fragment similar to that of the other immunoglobulin classes.When IgA polymers were digested, another product was found which was larger than 7S but smaller than the undigested IgA. This protein, which is formed as an intermediate between IgA polymers and F(ab′)_2α, appears to be a digestion product unique to IgA.     

Structural requirements of rabbit IgG F(ab')2 fragment for activation of the complement system through the alternative pathway—II. Ionic and indole groups

The structural features of rabbit IgG F(ab')₂ fragments required for complement activation through the alternative pathway have been investigated by means of chemical modification of specific groups. These included ionic (amino and carboxyl) and indole groups.Potassium cyanate, glycine-ethyl ester in the presence of EDC,2 and NBB were used as modifying agents of the amino, carboxyl and indole groups respectively. Three different aspects of the modified protein were analysed: the conformational integrity of the protein (by CD and antigenic reactivity), the antibody activity (by the haemaglutination test and radiobinding to the erythrocyte surface) and the capacity to activate complement through-the alternative pathway (by direct haemolysis in conditions in which the classical pathway was blocked).Changes in protein conformation caused by the chemical treatment applied were not detected by the techniques used. An enhancement of the antibody activity of the F(ab')₂ fragment against the sheep erythrocyte membrane was observed when it was amidated by glycine-ethyl ester and a decrease when it was carbamylated or two tryptophanyl residues were altered per molecule. The complement-activating capability was preserved after tryptophan modification. On the contrary, modification of amino groups produced a partial decrease in the complement-activating ability and almost total loss of the ability when 34 carboxyl groups were amidated.     

Tumour cell killing using chemically engineered antibody constructs specific for tumour cells and the complement inhibitor CD59

Immunotherapy using MoAbs is inefficient due to limited activation of human effectors by mouse antibodies and multiple protective mechanisms available to host cells against autologous complement. We have used chemically engineered antibody constructs and human complement in vitro to specifically target and kill neoplastic B lymphoid cells (Raji). Fab′γFcγ² chimaeric antibody (specific for human CD37) was used to activate the classical pathway of human complement on Raji cells, whilst CD59 was neutralized using one of two different bispecific F(ab′γ)² antibody constructs which contained both cell-targeting (anti-CD19 or anti-CD38) and CD59-neutralizing moieties. When either bispecific construct was used to neutralize CD59, 15–25% of cells were lysed. If CD55 was also neutralized using specific antibody, Raji cells were efficiently killed (70% lysis). When added to a mixture of target (Raji) and bystander (K562) cells, one bispecific antibody (anti-CD38 × anti-CD59) could be specifically delivered to Raji, avoiding significant uptake on CD59-expressing bystander cells (K562). The second bispecific antibody (anti-CD19 × anti-CD59) bound equally well to either cell type. Cell-specific targeting was dependent upon combination of a low-affinity anti-CD59 Fab′γ with a high-affinity anti-tumour cell Fab′γ. When Raji and K562 cells were mixed and incubated with a combination of the engineered constructs and anti-CD55 antibodies, Raji cell lysis (30–40%) was observed in the absence of K562 killing. We propose that combinations of these constructs may be of use for treatments such as ex vivo purging of autologous bone marrow or in vivo targeting of tumour cells.     

Cytotoxicity of human lymphocytes induced by rabbit antibodies to chicken erythrocytes. Inhibition by normal IgG and by human myeloma proteins of different IgG subclasses

Normal IgG preparations of human, rabbit or guinea-pig origin (IgG2) were tested for their capacity to inhibit the cytotoxicity of purified human lymphocytes, as induced by rabbit IgG antibodies to chicken erythrocytes. All IgGs were found to be about equally efficient inhibitors. Human F(ab′)₂ used for control, gave no inhibition. Human myeloma proteins of subclasses IgG1, IgG2 and IgG3, were about equally efficient inhibitors. In contrast, the inhibitory action of myeloma proteins belonging to subclass IgG4 was weak and more irregular. In this assay system, a large excess (∼ 10⁶ ×) of normal IgG over antibodies had to be added in order to achieve ≥50 per cent inhibition. Heating of the inhibitors to 63° for 30 minutes did not significantly enhance their inhibitory capacity.For comparison, the same human IgG preparations and myeloma proteins were also tested for their capacity to inhibit phagocytosis by human blood monocytes of chicken erythrocytes sensitized with rabbit IgG antibody. As was to be expected in this system, only HGG, IgG1 and IgG3 caused inhibition whereas F(ab′)₂, IgG2 and IgG4 were completely negative.