Inhibition of antigen-induced T-cell proliferation by antibodies to endogenous AKR-MuLV

Lymph node cells from BALB/c mice immunized with ovalbumin or human γ-globulin were restimulated in vitro with these antigens and assayed for antigen-induced proliferation. The proliferative response was shown to be antigen specific and T cell dependent. A rabbit antiserum to envelope and core proteins of AKR murine leukemia virus was found to inhibit antigen-induced T-cell proliferation. The IgG fraction and F(ab′)₂ fragments of the antiserum were also inhibitory. The inhibition occurred after the initial step of antigen-T cell interaction and viral absorption studies showed the inhibition to be specific for anti-AKR virus antibodies. A hypothesis for the mechanism of inhibition is discussed in relation to a functional role for endogenous murine leukemia virus.     

Antibody-mediated targeting of liposomes to erythrocytes in the whole blood

F(ab′)₂ fragments derived from anti-rat erythrocyte antibody or normal rabbit serum IgG were covalently attached to the surface of liposomes consisting of equimolar amounts of egg phosphatidylcholine and cholesterol. These liposomes were interacted with rat, monkey or mouse blood, and their binding to both red and white blood cells was determined. Results of these studies show that coupling of liposomes to anti-rat erythrocyte F(ab′)₂ considerably enhances their binding to erythrocytes in rat blood. However, no such increase in the binding was observed with rat leukocytes or monkey and mouse erythrocytes. Besides, the interactions between the liposomes and target cells did not affect the permeability properties of the liposome bilayer. These observations indicate that liposomes coupled to cell-specific antibodies may serve as highly useful carriers for homing of drugs/enzymes to specific cells in biophase.     

The increased natural killer sensitivity of Epstein-Barr virus (EBV)-superinfected Raji cells is not due to the recognition of serologically determined EBV antigens by the effectors

The NK sensitivity of Raji cells was elevated after superinfection with the P3HR-1 strain of Epstein-Barr virus (EBV). The superinfected cells reacted with EBV antibody-positive sera and this was detectable by ADCC. F(ab′)₂ fragments from the antibody-positive sera blocked the ADCC reaction but did not affect attachment of lymphocytes and natural killing. This indicates that the EBV-determined membrane antigens that serve as targets for the reactivity with the sera are not responsible for the increased NK sensitivity.     

Evading pre-existing anti-hinge antibody binding by hinge engineering

Antigen-binding fragments (Fab) and F(ab′)₂ antibodies serve as alternative formats to full-length anti-bodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab′)₂ C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab′)₂ in vitro by proteolytic digestion when the binding of pre-existing AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T₂₂₅L variant and the natural C-terminal D₂₂₁ as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab′)₂ for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA.     

Neutralizing Activities of Early and Late IgG Fragments from Rabbits Immunized with Herpes Simplex Virus

Rabbits immunized with herpes virus were bled periodically and bivalent and univalent fragments of IgG from each serum sample were prepared by enzymatic digestion. The 2-week F(ab′)₂ showed a low neutralizing activity only after addition of anti-IgG. F(ab′)₂ of the 4-week serum retained almost all of the neutralizing activity of IgG, while its univalent fragments demonstrated none even when tested with anti-IgG. In contrast to these early IgG fragments, univalent fragments of the 9-week and 20-week IgG neutralized the virus to considerable extents in the absence of anti-IgG; after addition of anti-IgG the activity equaled that of intact IgG in the cases of Fab′ and Fab-II, though the activity of Fab-I was relatively low. The three univalent fragments were all sensitive to heating at 70 C and to ultraviolet irradiation, whereas intact IgG resisted these treatments. F(ab′)₂ was resistant to the heating and less sensitive to ultraviolet irradiation than univalent fragments. Neutralization kinetic curve experiments to test blocking effects of IgG fragments against the neutralization by intact IgG suggested that the early Fab′ did combine with the virus and that the late Fab′ exerted a higher blocking effect than the early Fab′.     

Rabbit antisera to human B cell alloantigens: effects on the mixed lymphocyte response.

Antisera were produced in rabbits to two glycoproteins (31,000 MW and 23,000 MW by SDS-gel electrophoresis) isolated from papain digests of membranes from a human B lymphoblastoid cell line (LCL). After minimal absorption with a T LCL the antisera reacted with two glycoproteins (35,000 MW and 27,000 MW) present in detergent-solubilized membranes from human B LCLs. The immunizing molecules are proposed to have arisen by proteolysis of the intact molecules in the detergent-solubilized membranes. The glycoproteins were detectable on human B LCLs and macrophages, but absent from T LCLs and fibroblasts. The molecules were identified as B cell alloantigens by their reactivity with alloantisera specific for human B cells. The rabbit antisera reacted with peripheral blood B lymphocytes, but not with T lymphocytes, platelets or erythrocytes.Pretreatment of human peripheral blood mononuclear cells with the rabbit antisera in a uni- or bidirectional mixed lymphocyte response (MLR) rendered them unable to stimulate, but they were able to respond. Addition of the antisera at various intervals during the MLR to block continuous stimulation indicated that cells were activated at different times. The presence of an F(ab′)₂ or Fab′ preparation of the antisera throughout the MLR did not inhibit the response at the concentrations tested. Further experiments suggest that, while a responding lymphocyte can replicate several times without restimulation, there is a delay between commitment to and commencement of division.     

Effect of antibody to detergent solubilized zonae pellucidae on in vivo and in vitro fertilization in the mouse

An antiserum was produced in one rabbit against mouse zonae pellucidae solubilized with 70 mM Na₂SO₃, 1% SDS, and 0.04 mM CuSO₄. An IgG of zona antibody completely inhibited fertilization both in vitro and in vivo in the mouse. F(ab′)₂ fragment obtained by pepsin digestion of IgG zona antibody inhibited fertilizability of eggs in vitro but did not inhibit fertilization in vivo after passive immunization.     

Neocarzinostatin: controlled release of chromophore and its interaction with DNA

A nonagglutinating derivative of wheat germ agglutinin has been prepared that binds to platelets and precipitates an antibody to the lectin. Platelets treated with this inactive derivative released serotonin when exposed to bivalent F(ab′)₂, but not monovalent Fab, fragments of the lectin antibody. Bridging of platelet-bound Fab by an antibody again induced secretion. The F(ab′)₂ or Fab fragments plus IgG, without the derivative, did not induce secretion. This secretion was not affected by indomethacin showing a direct activation of platelets. Platelets treated with con A followed by F(ab′)₂ to con A did not secrete. In addition, lentil lectin failed to release platelet serotonin. The receptors of the lectin derivative are mobile on the platelet surface and their redistribution may lead to secretion.     

Dual fluorescence confocal imaging of the accessibility and binding of F(ab′)2 to an EBA resin with various immobilised antigen densities

Dual fluorescence confocal laser scanning microscopy has been used to visualise the binding of a fluorescently labelled polyclonal ovine anti-fluorescein F(ab′)₂ antibody to immobilised fluorescein. The fluorescent ligand was immobilised on a Streamline quartz base agarose matrix; a resin used industrially for expanded bed chromatography, using two different fluorescein initial concentrations in order to obtain two batches of immunogen-affinity adsorbent with different immobilised ligand densities. The fluorescein specific F(ab′)₂ were purified from anti-fluorescein serum pepsin digest by adsorption on immobilised antigen chromatographic resin, followed by conjugation to the fluorescent probe Alexa Fluor 660. The dual fluorescence signals from the immobilised antigen and the immuno-specific F(ab′)₂ were used to map the progressive depth of the bound F(ab′)₂ layer within individual adsorbent beads. In addition, the labelled anti-fluorescein F(ab′)₂ was diluted to identical antigen binding activity concentrations in crude serum digest and in blank buffer and the resulting fluorescence intensity profiles were comparatively assessed for any detectable differences in binding patterns that might be caused by processing the more complex mixture of crude serum digests. It was observed that the relative immobilised ligand utilisation was higher when using the immuno-adsorbent with lower immobilised antigen density. Furthermore, the progression of the adsorbed F(ab′)₂ front inside the immuno-adsorbent beads displayed closer agreement with the postulates of the shrinking core mechanism (SCM) when the immuno-adsorbent with lower immobilised antigen was used. The confocal images did not reveal any differences between the depth of the adsorption fronts of crude serum digest and pre-purified F(ab′)₂ samples.     

Radioimmunodetection of human tumor xenografts by monoclonal antibody F(ab′)2 fragments

Experimental procedures are described for the radiolocalization of human tumors by murine monoclonal antibodies (MAb) in animal model systems. Visualization of tumor xenografts was clearer in nude mice as compared to experimentally immunosuppressed mice due to the higher viability of the tumors in nude mice. MAb localization in tumor tissue was greatly enhanced when F(ab′)₂ fragments rather than intact antibody molecules were used. Although tumors could be visualized with either ¹³¹I-, ¹²³I- or ¹¹¹In-labeled MAb fragments without using background subtraction, tumor-to-background ratios of radioactivity were highest for ¹³¹I-labeled fragments. ¹³¹I-labeled F(ab′)₂ fragments of eight MAb against human colorectal carcinoma, melanoma or lung carcinoma localized specifically only in those tumors that bound the MAb in vitro and not in unrelated tumors. Radiolabeled fragments of MAb with other specificities (anti-hepatitis virus MAb) did not localize in tumors. All MAb that inhibited tumor growth in nude mice effectively localized these tumors by γ-scintigraphy. On the other hand, some MAb were effective in localizing tumors but ineffective in inhibiting their growth. The ability of the specific radiolabeled F(ab′)₂ fragments to localize in tumor grafts correlated significantly with MAb binding affinity and density of antigenic sites on tumor cells together, but not with either in vitro binding parameter alone. Thus, Scatchard analysis of MAb binding to tumor cells may be an effective means to screen for MAb with tumor radiolocalization potential.     

A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo

We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095–2, displays specificity for IdeS-generated F(ab’)₂ fragments, but not for full-length IgG or for closely-related F(ab’)₂ fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095–2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab’)₂ fragment. Similarly, 2095–2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab’)₂ fragment. mAb 2095–2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab’)₂ fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095–2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095–2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab’)₂ fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095–2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab’)₂ fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095–2 to F(ab’)₂, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.     

Effects of antibodies to MHC gene products on different Fc-receptor-mediated cell functions

The effects of rabbit anti-HLA-DR and anti-β₂-microglobulin (anti-β₂m) antibodies on three different Fc-receptor-mediated cell functions of peripheral blood mononuclear leukocytes were studied. Both IgG and F(ab′)₂ fragments of anti-HLA-DR antibodies inhibited the cytotoxicity against a monolayer of antibody-sensitized sheep erythrocytes (plaque formation) whereas no effect was observed on the cytotoxicity against antibody-sensitized chicken erythrocytes in suspension (⁵¹Cr release), or on EA-rosette formation. On the other hand, all the three functions were inhibited by the IgG fraction of anti-β₂m antibodies but not by the corresponding F(ab′)₂ fragments. The results demonstrate that the plaque-forming cells (PFC) carry HLA-DR-like antigens. Furthermore, a closer association exists between the HLA-DR-like antigens and the Fc receptors than between the β₂m molecules and the Fc receptors on the PFC. The results further support our earlier investigations suggesting that the PFC are of monocytic origin.     

Radioactive gold cluster immunoconjugates: Potential agents for cancer therapy

An 11 gold atom (undecagold) cluster was covalently attached to specific sites on Fab′, F(ab′)₂ and whole IgG molecules such that each carried 11–33 gold atoms without significant loss of native immunospecificity. Gold cluster labeled 17-1A monoclonal F(ab′)₂ antibody fragments showed 80% immunoreactivity compared to native antibody fragments in binding to human colon carcinoma cells in vitro. Radioactive gold in vivo biodistributions in nude mice with human tumors are also reported. By using clusters, potentially a larger destructive payload can be carried per antibody.     

Central role of T lymphocytes in specific recognition of tumor antigens in 51Cr-leukocyte adherence inhibition

The plaque-forming cell (PFC) response of C57BL/6 mice to NP-coupled sheep red blood cells can be inhibited by administration of microgram doses of a variety of monoclonal anti-NP antibodies of various isotypes prior to immunization. NP-specific PFC are most strongly affected but a drastic reduction of the SRC-specific response also occurs. The latter depends on physical association of the immunizing red cells with NP groups. Inhibition was obtained with antibodies of all IgG subclasses, but was marginal or absent when up to 100 μg IgM or IgD antibodies or F(ab′)₂ fragments were administered. Our experiments lead us to conclude that antibody feedback inhibition is not the function of a particular IgG subclass and that the role of the variable region in feedback inhibition is sufficiently explained by its affinity for the antigen. It was noted that small doses of a monoclonal NP-specific IgM antibody failed to enhance the response to NP-SRC.     

Suppression and augmentation of the primary in vitro immune response by different classes of antibodies

The capacity of purified γG₁ and γG₂ anti-sheep red blood cell (SRBC) antibodies to exert antigen-specific feedback regulations on the primary in vitro immune response to SRBC was studied. Antibodies were administered to the culture in the native form, as sheep erythrocyte-antibody complexes or as pepsin-derived F(ab′)₂ antibody fragments. Marked differences in the feedback regulatory effects of γG₁ and γG₂ antibodies were found. Antibodies of the γG₁ class suppressed the immune response to SRBC, whereas γG₂ antibodies isolated from the same serum exerted an augmenting effect on antibody synthesis. These opposing feedback effects on in vitro antibody synthesis were immunologically specific, relatively insensitive to changes in antigen concentrations, and could be elicited by either adding antibodies and antigen separately to the culture or as preformed antigen-antibody complexes. Experiments comparing the activities of the F(ab′)₂ antibody fragments with the parent γG₁ and γG₂ antibodies suggested that the Fc fragments may be involved in these regulatory effects on the immune response. It is concluded that the antigen-specific suppressive and augmenting effects on antibody synthesis shown here are determined by the antibody class. In addition, we suggest that these opposing antibody-mediated feedback effects may represent one of the important elements of the immune response.     

Targeting of the antiviral protein from Phytolaccaamericana with an antibody

The antiviral protein (PAP) of $\text{Phytolacca}\text{americana}$ was conjugated with the Fab' fragment of IgG from a rabbit antiserum against murine leukemia L1210 cells via a disulfide bond employing $\text{N}$-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as the coupling agent. The conjugate showed a potent $\text{in}\text{vitro}$ cytotoxicity against L1210 cells which was competitively blocked by F(ab′)₂ directed against L1210 cells. PAP itself did not exhibit the cytotoxicity at the concentration corresponding to the PAP content in the conjugate concurrently tested.     

Infarcted heart uptake and biodistribution of radiolabelled anti-myosin monoclonal antibody in rat and dog myocardial infarct models

A new mouse monoclonal antibody that recognizes α- and β-heavy chains of human atrial and ventricular myosin and β-heavy chain of human slow skeletal muscle myosin was obtained. The ¹²⁵I- and ¹¹¹In-labelled antibody, and its F(ab′)₂ and Fab fragments localize in isoproterenol induced infarcted rat heart, with the F(ab′)₂ fragment showing the highest uptake. Comparison with ^99mTc-pyrophosphate uptake in infarcted dog heart, induced by selective obstruction of a coronary artery, suggest that the ¹¹¹In-labelled F(ab′)₂ localizes specifically in infarcted myocardium only.     

Localization by whole-body autoradiography of intact and fragmented radiolabeled antibodies in a metastatic human colonic cancer model

In this report, we have employed macroautoradiography to compare the tumor targeting of ¹²⁵I-labeled anti-carcinoembryonic antigen (CEA) MAb (NP-4) to ¹²⁵I-labeled anti-colon-specific antigen-p (CSAp) MAb (Mu-9) and their labeled F(ab′)₂ and Fab′ fragments, in nude mice each bearing large dorsal human colonic tumor xenografts, and small nodular tumors in the liver and lungs. Using intact MAbs (NP-4 and Mu-9), clearance of background radioactivity was delayed to 3–7 days post-treatment. Treatment with F(ab′)₂ and Fab′ fragments of both NP-4 and Mu-9 MAbs, however, promoted clearance of background ¹²⁵I-radioactivity which was well advanced by 6–24 h and complete by 24–48 h after injection. Localization of ¹²⁵I-radioactivity in large and micrometastatic tumor perimeters was the most characteristic uptake pattern observed for both intact and fragmented MAbs. Qualitative analysis of macroautoradiographic images and quantitative densitometry indicated that the higher tumor-to-blood ratios achieved with labeled F(ab′)₂ and Fab′ fragments at early time points, compared to labeled whole immunoglobulin, appeared to be more a function of rapid plasma clearance, tumor mass, location of xenografts and specific tumor growth patterns than increased tumor penetrance by lower molecular weight univalent and bivalent immune fragments.     

Effects of anti-beta-2 microglobulin antibodies on human lymphocytes.

Anti-β2 microglobulin antisera prepared in rabbits immunized with β2m purified from the urine of a single patient were cytotoxic for human T and B lymphocytes of all donors tested; lymphocytotoxicity could be fully inhibited by all human sera tested, not by serum from other animal species. Anti-β2 microglobulin antibodies and their F(ab′)2 fragments had little effect on E and EAC rosette formation, suggesting that β2m is not closely associated with receptors for sheep erythrocytes on T lymphocytes or receptors for C3 on B cells. Anti-β2m IgG and F(ab′)2 fragments inhibited EA rosette formation though the latter did not impair lysis of antibody-coated xenogeneic erythrocytes by lymphocytes bearing receptors for the Fc portion of IgG. Some of the antisera had a mild mitogenic effect, all of them inhibited mitogen and antigen-induced lymphocyte proliferation at high concentrations whereas they potentiated these responses at low concentrations. In mixed lymphocyte cultures pretreatment of responding cells markedly depressed the response whereas coating of stimulating cells with β2m antibodies had little or no effect.     

Transchelation of 99mTc from low affinity sites to high affinity sites of antibody

An exclusive labeling of high affinity sites of IgG and its F(ab′)₂ fragments with ^99mTc was accomplished. Antibody was first labeled in 0.1 M acetate buffer at pH 4.5, using stannous chloride as a reducing agent. Thus, high capacity, low affinity sites and low capacity, high affinity sites were both labeled. These ^99mTc complexes were stable at pH 4.5 and 7.0; however, they became destabilized at pH 8.2 and 9.0. Transchelation of ^99mTc to DTPA took place at the higher pH values and leveled off at 54% ^99mTc-F(ab′)₂ and 73% ^99mTc-IgG. These results indicate that the majority of ^99mTc bound to the low affinity sites was transchelated to the high affinity sites rather than to DTPA since low affinity sites account for 84% of total F(ab′)₂ sites and 76% of IgG sites. Biodistribution data in mice at 2.5 h postinjection were consistent with this hypothesis in that tissue concentrations of ¹¹¹In-DTPA-F(ab′)₂ were similar to the reequilibrated ^99mTc-F(ab′)₂ but were much higher than that of the unequilibrated ^99mTc-F(ab′)₂.