Increased tumor localization by monoclonal antibody A7 after F(ab')2 fragmentation in athymic nude mice bearing human pancreatic carcinomas

Much recent research has been directed toward the use of monoclonal antibodies (MoAbs) for the immunodetection of solid tumors. In pancreatic cancer, conventional immunoscintigraphy using intact MoAbs remains disappointing. In this study, 125I-labeled F(ab')2 fragments produced by pepsin digestion of MoAb A7 were injected intravenously into nude mice bearing human pancreatic cancer, HPC-YS, xenografts that have previously been shown to react specifically with MoAb A7. The tumor tissue/blood ratio of 125I-labeled F(ab')2 fragments of MoAb A7 increased with time and was much higher than those for normal tissues. Moreover, the tumor tissue/blood ratio of 125I-labeled F(ab')2 fragments was greater than that of intact MoAb A7, although the F(ab')2 accumulation was less than that of intact MoAb A7 in the tumor. These results suggest that F(ab')2 fragments of MoAb A7 may be suitable carriers of radionuclides for immunodetection of human pancreatic cancer.     

Accurate measurement of intrinsic positive end-expiratory pressure: how to detect and correct for expiratory muscle activity

The basic amino acid L-lysine was administered to mice in an attempt to circumvent unwanted renal accumulation of 67Cu-labelled F(ab')2 fragments derived from the anti-NCAM IgG1, SEN7 and anti-CEA IgG1 monoclonal antibody (MAb)35. In control experiments, significant renal uptake of both 67Cu-labelled F(ab')2 fragments was observed, radiolabel being primarily localised to proximal tubules in the renal cortex. Following optimised L-lysine dosing protocols, renal uptake of 67Cu-MAb35 F(ab')2 was inhibited by up to 42%. Surprisingly, little inhibition (< 10%) of 67Cu-SEN7 F(ab')2 uptake was observed. Experiments to investigate this differential inhibition indicated that inhibition of MAb35 F(ab')2 uptake was relatively short-lived (approx. 6 hr), whilst no apparent differences were found in blood clearance rates between either 67Cu-F(ab')2 fragment. L-lysine administration caused a significant diuresis with high levels of intact 67Cu-labelled SEN7 and MAb35 F(ab')2 appearing in the urine, possibly due to blockade of renal uptake and lysine-induced increases in glomerular membrane permeability. Iso-electric focusing studies failed to identify any charge differences between the 67Cu-labelled F(ab')2 fragments, although a cathodal migration of all 67Cu-labelled samples, presumably due to the net positive charge conferred by addition of 67Cu2+ ions, was observed. Our results demonstrate that in addition to net charge, other unidentified characteristics may influence renal accumulation of radiometal-labelled F(ab')2 fragments and their inhibition by L-lysine.     

Biodistribution of charged F(ab')2 photoimmunoconjugates in a xenograft model of ovarian cancer

The effect of charge modification of photoimmunoconjugates (PICs) on their biodistribution in a xenograft model of ovarian cancer was investigated. Chlorin(e6)c(e6) was attached site specifically to the F(ab')2 fragment of the murine monoclonal antibody OC125, directed against human ovarian cancer cells, via poly-1-lysine linkers carrying cationic or anionic charges. Preservation of immunoreactivity was checked by enzyme-linked immunosorbent assay (ELISA). PICs were radiolabelled with 125I and compared with non-specific rabbit IgG PICs after intraperitoneal (i.p.) injection into nude mice. Samples were taken from normal organs and tumour at 3 h and 24 h. Tumour to normal 125I ratios showed that the cationic OC125F(ab')2 PIC had the highest tumour selectivity. Ratios for c(e6) were uniformly higher than for 125I, indicating that c(e6) became separated from 125I. OC125F(ab')2 gave highest tissue values of 125I, followed by cationic OC125F(ab')2 PIC; other species were much lower. The amounts of c(e6) delivered per gram of tumour were much higher for cationic OC125F(ab')2 PIC than for other species. The results indicate that cationic charge stimulates the endocytosis and lysosomal degradation of the OC125F(ab')2-pl-c(e6) that has bound to the i.p. tumour. Positively charged PICs may have applications in the i.p. photoimmunotherapy of minimal residual ovarian cancer.     

Natural antibodies against the immunoglobulin F(ab')2 fragment cause elimination of antigens recognized by the F(ab')2 from the circulation

A humanized monoclonal IgG1 antibody, designated hC4G1, recognizes the fibrinogen receptor glycoprotein (GP)IIb/IIIa on platelets and inhibits platelet aggregation. When the F(ab')2 fragment of hC4G1 (F(ab')2 hC4G1) was administered to cynomolgus monkeys, all the monkeys showed inhibition of platelet aggregation ex vivo. Unexpectedly, a significant decrease in platelet count was observed in 5 of 18 monkeys. Antibodies against F(ab')2 hC4G1 were detected in the plasma of these monkeys by ELISA. Antibody activity in the plasma of these monkeys was significantly correlated with the intensity of platelet decrease (r = 0.84). The natural monkey antibodies to F(ab')2 hC4G1 were directed against the C-terminal region of F(ab')2 fragment common to all human and humanized IgG antibodies. Natural homo-reactive antibodies were also detected in human plasma from 15 of 40 healthy volunteers. Specificity was closely similar to that of the monkey antibodies. Affinity-purified human homoreactive antibodies enhanced phagocytosis of platelets treated with the F(ab')2 hC4G1. Monkey plasma with high homo-reactive antibody activity was confirmed to decrease platelet count when administered together with F(ab')2 hC4G1 to a monkey with low antibody activity. These results suggest that F(ab')2 of humanized and human antibodies causes elimination of the corresponding antigens from the circulation by homo-reactive antibodies.     

Anti-idiotypic activity against anti-myeloperoxidase antibodies in pooled human immunoglobulin

We investigated the ability of six different pooled human immunoglobulin (PHIG) preparations to inhibit the binding of anti-myeloperoxidase (MPO) antibodies to MPO. All six PHIG preparations inhibited the binding of anti-MPO antibodies from six sera to MPO in a concentration-dependent manner in the concentration range 0.016-10 mg/ml. There was considerable variation in the ability of each PHIG preparation to inhibit the binding of anti-MPO antibody in a given serum. Further differences were seen in the ability of a given PHIG to inhibit anti-MPO binding in different sera. F(ab')2 fragments from two PHIG preparations also inhibited in a concentration-dependent manner anti-MPO binding to MPO in all six sera in the concentration range 0.002-2.65 mg/ml, with a maximum inhibition of 42%. Little inhibition was seen with F(ab')2 of normal human IgG from individual donors (1.8-12.2% at the maximum concentration of 2 mg/ml). F(ab')2 fragments from three anti-MPO containing sera and two affinity-purified anti-MPO antibodies were eluted by affinity chromatography from Sepharose-bound PHIG F(ab')2 and showed anti-MPO antibody activity. We have shown that PHIG and F(ab')2 fragments of PHIG inhibit anti-MPO binding to MPO, and further that F(ab')2 fragments of PHIG bind to F(ab')2 fragments of anti-MPO antibodies. These observations indicate binding between the variable regions of PHIG and the antigen binding site of anti-MPO antibodies, and are consistent with an anti-idiotypic reaction. The variability seen in the inhibitory effect of the different PHIG preparations in anti-MPO-positive sera implies differences in their anti-idiotype content, while the variability of the inhibitory effect of a particular PHIG preparation between different sera suggests heterogeneity in the idiotypic repertoire of anti-MPO antibodies. Such variations in the inhibitory effect of different PHIG preparations on antibody binding may be an important determinant of their therapeutic effect.     

An anti-okadaic acid-anti-idiotypic antibody bearing an internal image of okadaic acid inhibits protein phosphatase PP1 and PP2A catalytic activity

Okadaic acid (OA), produced by marine phytoplankton, is the parent compound of a family of marine toxins responsible for diarrheic shellfish poisoning (DSP). A monoclonal antibody to OA (6/50) (Ab1) has been raised and in turn used for immunization of syngeneic animals. Mice inoculated with the 6/50 idiotype produced both anti-idiotypic antibodies (Ab2) and OA binding antibodies (Ab3). The selected anti-idiotypic antibody 1/59 bound to the immunizing 6/50 idiotype but not to F(ab')2 fragments of pooled normal mouse Ig. It inhibited the binding of OA to solid-phase attached F(ab')2 of 6/50 IgG as well as the binding of 6/50 IgG to a solid-phase bound OA. Like OA, 1/59 anti-idiotypic antibody inhibited protein phosphatase 1 and 2A catalytic subunits in a 32P-phosphorylase a phosphatase radioassay. Thus, 1/59 IgG is a novel internal image anti-idiotypic antibody (Ab2 beta) and can serve as a surrogate of OA in biological assays.     

Nonmitogenic anti-CD3F(ab')2 fragments inhibit lethal murine graft-versus-host disease induced across the major histocompatibility barrier

We have investigated the in vivo administration of nonmitogenic anti-CD3F(ab')2 fragments for the prevention of lethal graft-vs-host disease (GVHD) in irradiated recipients of fully allogeneic bone marrow cells plus splenocyte (BMS) inocula. Recipients of anti-CD3F(ab')2 fragments administered for 1 mo post-bone marrow transplantation (BMT) had 100% survival without clinical or histopathological evidence of GVHD. Controls given saline injections succumbed by 39 days post-BMT. Similar results were obtained in groups of recipient mice given BMS in which T cells were depleted by in vitro anti-Thy-1.2 plus C' treatment. Further studies were undertaken to define mechanistic differences in the two approaches. Using Ly-5 congenic sources of donor bone marrow and spleen, we determined that anti-CD3F(ab')2 fragments induced TCR modulation and T cell depletion. Mature splenic-derived CD4+ cells were depleted to a greater extent than CD8+ cells. Early post-BMT, recipients receiving injections with control saline had the highest number of CD4+ and CD8+ cells (which may cause GVHD) followed by recipients of anti-CD3F(ab')2 fragments, with the fewest CD8+ cells observed in the anti-Thy-1.2 + C' treated group. CD3+CD4-CD8- cells (which may suppress GVHD generation) were present in higher numbers early post-BMT in recipients given anti-CD3F(ab')2 fragments as compared to recipients given anti-Thy-1.2 + C'-treated BMS. In long term survivors, a mononuclear T cell containing infiltrate without evidence of destruction was observed in sites of GVHD (lung and liver), consistent with a "Quilty" effect, which was not observed in either of the other two groups. Although survivors were tolerant of donor skin grafts and rejected third party grafts, recipients given anti-CD3F(ab')2 fragments but not anti-Thy-1.2 + C'-treated BMS had vigorous anti-host proliferative responses. These results demonstrate that although in vitro anti-Thy-1.2 + C' treatment of BMS (which is highly depletionary) and in vivo administration of anti-CD3F(ab')2 fragments (which is modulatory and less depletionary) are both effective strategies for GVHD, the cellular events involved in achieving GVHD prevention are indeed different.     

Early development and progression of lymphocyte-stimulatory cross-reactive idiotypes expressed on antibodies to soluble egg antigens during Schistosoma mansoni infection of mice

Idiotypes (Id) that stimulate immunoregulatory anti-Id T lymphocyte proliferation are expressed on murine and human antibodies (Ab) to soluble egg antigens (SEA) of Schistosoma mansoni. Kinetics of early expression of these stimulatory Id have now been studied using immunoaffinity-purified serum anti-SEA Ab from mice infected with S. mansoni for 6, 7, 8, 12, or 16 weeks. Rabbit anti-Id Ab specific for mouse anti-SEA Id expressed at 8 weeks post-infection (anti-8WkId) demonstrated the strongest interactions with Id present at 7 and 8 weeks post-infection by competitive enzyme-linked immunosorbent assay. Anti-8WkId Ab reacted progressively less well with 12 WkId, 6WkId, and 16WkId. Splenocytes from mice infected for 8 weeks demonstrated the highest blast transformation responses in vitro to anti-SEA Id from mice infected for 6 weeks, while 7, 8, 12, and 16 weeks post-infection Id preparations stimulated progressively less proliferation. These data indicate that although eventual Id-associated immunoregulatory events contribute to chronicity in this disease, production of anti-SEA Ab that express stimulatory cross-reactive immunoregulatory Id comprises a substantial portion of the initial, acute anti-SEA response in mice infected with Schistosoma mansoni. Furthermore, either this particular Id-expressing response is not maintained, or its proportional presence is greatly diminished by the cumulative production of other multiple anti-SEA Ab during the establishment of chronicity, perhaps in response to its immunoregulatory influence very early in infection.     

Repeated administration of a F(ab')2 fragment of an anti-tumor necrosis factor alpha monoclonal antibody in patients with severe sepsis: effects on the cardiovascular system and cytokine levels

In an uncontrolled clinical trial the effects of repeated administration of the F(ab')2 fragment of a murine monoclonal anti-tumor necrosis factor alpha (TNF alpha)-antibody (MAK 195F) on cytokine levels and the cardiovascular system were studied in 20 patients with severe sepsis. Patients were treated with a total of 11 single dosages of the anti-TNF alpha-antibody intravenously over 5 days using either 1 mg/kg (n = 10) or 3 mg/kg (n = 10). The anti-TNF alpha-antibody was well tolerated in all patients without signs of toxicity and without development of anti-murine antibodies. As assessed by cytokine levels (TNF alpha, Interleukin-6) and hemodynamics there was no evidence that the higher dosage of the anti-TNF alpha-antibody (3 mg/kg per dose) was more effective than the lower dosage (1 mg/kg per dose). Comparison of our data with recent data from phase I or II trials using a complete murine monoclonal anti-TNF alpha-antibody suggest that the F(ab')2 fragments of the murine monoclonal anti-TNF alpha-antibody may be of similar efficacy. Definitive conclusions, however, with respect to improvement of mortality and improvement of the cardiovascular system, await the results of larger ongoing placebo-controlled trials.     

Different effects of methylxanthines on central serotonergic postsynaptic neurons in a mouse behavioral model

We have used genetic engineering to obtain secretion of anti-human CD5 antibody fragments from Escherichia coli for conjugation to the 30-kDa form of ricin A chain (RTA30). This was accomplished by introducing stop codons at two positions in the hinge region of the human IgG1 gene so that coexpression of the truncated heavy-chain genes (Fd') with a light chain would result in Fab' and/or F(ab')2 proteins containing either one or two interheavy-chain cysteines. An Fd' gene encoding both interheavy-chain cysteines yielded a mixture of F(ab')2 and Fab', which could be separated by size-exclusion chromatography. An Fd' gene encoding only one interheavy-chain cysteine yielded primarily Fab'. Purified F(ab')2 protein was equivalent to unlabeled chimeric IgG in competing for binding of IgG with CD5 antigen, while the molar concentration of the monovalent Fab' required for 50% binding inhibition was 4- to 5-fold higher than IgG. An immunoconjugate was prepared with Fab' by direct coupling to the unique free cysteine on RTA30. The bivalent F(ab')2 was conjugated to RTA30 after derivatization with the crosslinking agent 5-methyl-2-iminothiolane. These immunoconjugates efficiently killed a CD5+ T-cell line and human peripheral blood T cells.     

Regulation of carotenoid biosynthesis during tomato development

N-succinimidyl 4-methyl-3-(tri-n-butylstannyl)benzoate (MATE) was synthesized in two steps from 4-methyl-3-iodobenzoic acid. Radioiododestannylation of MATE proceeded more slowly than N-succinimidyl 3-(tri-n-butylstannyl)benzoate (ATE), but for reaction periods of 10 min or more, identical yields were obtained. Paired-label biodistribution studies were performed in mice with an intact monoclonal antibody and an F(ab')2 fragment labeled using MATE, ATE and Iodogen. Thyroid uptake with MATE was low, comparable to that seen with ATE, and considerably lower than that observed when the Iodogen method was used. With the F(ab')2 fragment, kidney uptake using MATE was 8-fold higher than that observed when either the ATE or Iodogen methods were used.     

The autonomic nervous system and the immune system in juvenile rheumatoid arthritis

CD16, the low affinity receptor for monomeric IgG (Fc gamma RIIIA), is a well characterized activation molecule on NK cells. In this study we investigated the role of CD16 in NK cell-mediated regulation of immunoglobulin production. Cocultures of the CD16+ human NK clone CNK6 and highly purified SAC/IL-2-activated B lymphocytes with various CD16 antibodies showed significantly diminished NK-enhanced immunoglobulin production in a dose-dependent manner, indicating that CD16 is relevant in NK-B cell interaction. Similarly, recombinant soluble CD16 incubated with B cells before cultures, suppressed the NK cell-stimulated B cell antibody response. Enhanced immunoglobulin production was also inhibited by Fc-specific F(ab')2 anti-body fragments. Coculture of NK cells with B lymphocytes resulted in induction of mRNA for IFN-gamma and TNF-alpha. The accumulation of mRNA for these cytokines was prevented by addition of CD16 and Fc-specific antibodies. It is proposed that interaction of CD16 on NK cells with B cell bound immunoglobulin leads to induction of cytokines in NK cells which stimulate immunoglobulin production by B cells.     

Antibody response to pneumococcal polysaccharide vaccine in young, adult and old mice

The anti-pneumococcal antibody response was studied in young (5-week-old) and adult (10-week-old) BALB/c and CBA/J mice and in adult (9-10-week-old) and old (12-, 18- and 24-month-old) AB6F1 and B6D2F1 mice after s.c. immunization with a 23-valent pneumococcal polysaccharide vaccine. Both young and adult mice showed a significant IgM antibody response to the vaccine 6 days after immunization with 1-11 micrograms antigen. There were significant immune responses to serotypes 1, 2, 4 and 7F in contrast to small responses to serotypes 14, 19F and 23F after immunization with the vaccine. One month after immunization, there were only marginal differences in IgM anti-pneumococcal antibody levels to the vaccine (anti-PPS) between immunized and unimmunized BALB/c mice, whereas in CBA/J mice the anti-PPS remained higher in immunized than in unimmunized mice. Immunization of old mice induced a significant IgM antibody response 6 days after immunization, but the anti-PPS thereafter decreased rapidly towards preimmunization values in AB6F1 mice. A significant IgG anti-PPS was not detected in any of the mice studied. The IgA anti-PPS tended to vary over time with no consistent pattern. It is important to carefully consider age and strain of the mice used when studying the immune response to pneumococcal polysaccharide antigens.     

DNA vaccination with glutamic acid decarboxylase (GAD) generates a strong humoral immune response in BALB/c, C57BL/6, and in diabetes-prone NOD mice

The technique of DNA-based vaccination was used to generate a T-cell-dependent antibody response to glutamic acid decarboxylase (GAD) in BALB/c, C57BL/6, and non-obese diabetic (NOD) mice. Plasmids were constructed in which the expression of the rat GAD65 (rGAD65) or the rat GAD67 (rGAD67) gene was driven by the immediate early region promoter of the human cytomegalovirus (pCMV). This "naked" plasmid DNA was then injected into the regenerating muscles of the studied mice. In the vaccinated animals, antibody responses to GAD65 or to GAD67 were induced. Epitope recognition of GAD was studied by protein footprinting, a technique which makes use of a limited proteolysis of antibody-bound antigen. Different epitope recognition patterns were found, corresponding to strain-specific patterns. Mild trypsin treatment generated 50 kD, 46 kD, 40 kD, 30 kD, and 21 kD proteolytic fragments. In NOD mice, 50, 46 and 40 kD bands were the most prominent signals. In non-diabetes prone BALB/c mice, a faint 40 kD band appeared suggesting a rather weak protection of GAD from tryptic lysis. The pattern observed in C57BL/6 mice was more comparable to the NOD mice pattern with prominent 40 kD and 30 kD signals and a faint 21 kD fragment. Diabetes incidence was unchanged in NOD mice, and no diabetes was observed in C57BL/6 and BALB/c mice, respectively. The data demonstrate that genetic immunization is a suitable novel tool to stimulate and to manipulate an immune response against the diabetes-associated protein glutamic acid decarboxylase. Interestingly, our results indicate that, by genetic vaccination, distinct B-cell epitopes were generated in the various studied mouse strains.     

Blockade of epidermal growth factor receptor function by bivalent and monovalent fragments of 225 anti-epidermal growth factor receptor monoclonal antibodies

We have previously described anti-epidermal growth factor (EGF) receptor monoclonal antibodies (MAbs) which can block binding of transforming growth factor alpha (TGF-alpha) and EGF to receptors and inhibit activation of receptor tyrosine kinase. Studies with these MAbs involving cell cultures and nude mouse xenografts demonstrated their capacity to inhibit the growth of a variety of tumor cell lines, which express EGF receptors and TGF-alpha and appear to depend upon receptor activation for cell proliferation. To explore the mechanism(s) by which anti-EGF receptor 225 MAb inhibits cell proliferation, we have compared the activity of native 225 MAb with the response to bivalent 225 F(ab')2 and monovalent 225 Fab' fragments. Both native 225 MAb and its fragments could inhibit the binding of 125I-EGF to EGF receptors. Scatchard analysis revealed that the Kd of 225 F(ab')2 is comparable to that of 225 MAb (1 nM), whereas the Kd of 225 Fab' is 5 nM. Both bivalent 225 MAb and 225 F(ab')2 and monovalent 225 Fab' were able to completely inhibit TGF-alpha-induced EGF receptor tyrosine kinase activation, as assayed by autophosphorylation of tyrosine residues of EGF receptors on MCF10A nonmalignant human mammary cells, MDA468 human breast adenocarcinoma cells, and A431 human vulvar squamous carcinoma cells. The bivalent forms of MAb could inhibit proliferation stimulated by endogenous (autocrine) TGF-alpha in cultures of these three cell lines. They also blocked growth stimulation by added exogenous TGF-alpha in cultures of MCF10A cells and the growth-inhibitory effect of exogenous TGF-alpha upon MDA468 and A431 cell cultures. Monovalent 225 Fab' had weaker inhibitory effects upon the proliferation of these cell lines. To determine whether the in vivo antiproliferative activity of anti-EGF receptor MAb can occur without the participation of the Fc portion of MAb, the capacities of 225 F(ab')2 and native 225 MAb to inhibit growth of s.c. A431 cell xenografts were compared. Equimolar amounts of either 225 MAb or 225 F(ab')2 were administered at intervals equivalent to the half-lives of the molecules, to attempt to maintain comparable plasma levels. Both 225 MAb and 225 F(ab')2 inhibited A431 cell xenograft growth in a dose-dependent manner, with a more sustained response in the case of the intact antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

The antigen-binding domain of a human IgG-anti-F(ab')2 autoantibody

Recent studies revealed an immunoregulatory role of natural IgG-anti-F(ab')2 antibodies in both healthy individuals and patients with certain diseases. The implication of anti-F(ab')2 antibodies in the pathogenesis of diseases prompted us to study the gene segment structure of their antigen-binding domains and their binding characteristics. cDNA was prepared from the lymphocytes of a patient with a high IgG-anti-F(ab')2 serum titer. Variable heavy and light gene segments were amplified by PCR and inserted into a phagemid surface expression vector. Single-chain antibodies displayed on the phage surface were screened for binding to F(ab')2 fragments. The subsequent analysis of 95 single clones demonstrated that they all bound specifically to F(ab')2. Sequence analyses of 12 clones showed that 11 were identical and 1 contained a silent point mutation in the heavy chain and three amino acid exchanges in the light chain. The heavy chains belonged to the V(H)3 and the light chains to the V(kappa)2 gene family. The 11 identical light-chain genes were completely homologous to a germ-line sequence (DPK-15). Binding assays showed that the single-chain antibodies bind to F(ab')2, but not to Fab, Fc, or intact IgG. This binding pattern was confirmed by surface plasmon resonance studies, which revealed a relatively high affinity (Ka = 2.8 x 10(7) M(-1)). The strong binding capacity was further demonstrated by competitive inhibition of the serum anti-IgG antibody's interaction with antigen. The present study defines for the first time to our knowledge the gene segment structure of the antigen-binding domain of two human IgG-anti-F(ab')2 autoantibody clones and describes the binding kinetics of the purified monomeric fragments.     

Host-mediated antibody-dependent cellular cytotoxicity contributes to the in vivo therapeutic efficacy of an anti-CD7-saporin immunotoxin in a severe combined immunodeficient mouse model of human T-cell acute lymphoblastic leukemia

We have investigated the anti-leukemia effect that is exerted by the murine anti-CD7 antibody HB2 in a severe combined immunodeficient (SCID) mouse model of human T-cell acute lymphoblastic leukemia (T-ALL) and determined the contribution that this antibody effect makes to the therapeutic potency of a saporin immunotoxin (IT) constructed with the same antibody. The anti-leukemia effect is not exerted through complement-mediated lysis or through direct growth-inhibitory signaling after binding of antibody to the CD7 molecule on the T-ALL cell surface but rather through antibody-dependent cellular cytotoxicity (ADCC). Thus, the in vivo depletion of SCID mice of their natural killer cells almost completely abolishes the therapeutic effect of native HB2 anti-CD7 antibody and moreover significantly reduces the in vivo therapeutic performance of the anti-CD7 HB2-SAPORIN IT. Furthermore, an IT constructed with the F(ab')2 fragment of the same anti-CD7 antibody (HB2-F(ab')2-SAPORIN), which is incapable of recruiting natural killer cells, performed significantly less well therapeutically than HB2-SAPORIN IT. There was also a significant improvement in the therapeutic performance of the HB2-F(ab')2-SAPORIN IT in SCID-HSB-2 mice when used in combination with intact HB2 antibody, presumably through restoration of an ADCC attack on the target HSB-2 cell. These combined data indicate that ADCC in the SCID mouse does contribute additively together with toxin to the in vivo therapeutic potency of the HB2-SAPORIN IT directed against this human T-ALL cell line and that this has potentially important implications for the utility of this and other related classes of immunotherapeutic in human therapy.     

Dextran modification of a Fab'--beta-lactamase conjugate modulated by variable pretreatment of Fab' with amine-blocking reagents

The physical and pharmacological properties of proteins can be altered by chemical modification with polymers. Preliminary studies showed that attachment of oxidized dextran to the bacterial protein, beta-lactamase (beta L) effectively reduced in vivo immunogenicity in mice with no loss of enzymatic activity. This report describes a general method for differentially dextran modifying the Fab' component of a Fab'--beta-lactamase conjugate by the use of amine-blocking reagents. Methyl acetimidate (MeAcm) and the N-succinimidyl derivative of (methylsulfonyl)ethyl carbonate (NHS-Msc), reagents which can reversibly block primary amines, were used in model studies to modulate the level of available reactive amines on the F(ab')2 fragments of both the anti-carcinoembryonic antigen antibody, ZCE025, and the antitumor-associated glycoprotein-72 antibody, CC49. MeAcm had little or no effect on immunoreactivity and was maximally effective in modulating dextran attachment, while NHS-Msc was much less effective. A comparison of NHS-Msc and MeAcm is described. Treatment of F(ab')2 with 5-300 mM MeAcm prior to dextran treatment showed a proportional decline in the level of dextran attachment as well as intramolecular cross-linking of the protein by the dextran polymers (6 kDa or 33-mer). A conjugate of beta L coupled to MeAcm-treated ZCE025 Fab' [reduced F(ab')2] was constructed under standard conditions using sulfosuccinimidyl N-[(4-carboxycyclohexyl)methyl]maleimide. After dextran modification, this conjugate maintained good immunoreactivity and enzymatic activity. Biodistribution studies in tumor-bearing nude mice of dextranated and nondextranated conjugate showed comparable overall distribution profiles except that the clearance of the dextranated conjugate from both blood and tumor was delayed about 48-72 h.     

Recombinant F(ab') C242-Pseudomonas exotoxin, but not the whole antibody-based immunotoxin, causes regression of a human colorectal tumor xenograft

We have previously made a Mr 195,000 immunotoxin (IT) composed of mAb C242 coupled to Pseudomonas exotoxin A. This IT inhibited growth but did not cause regression of a human colorectal cancer xenograft growing in nude mice (W. Debinski et al., J. Clin. Invest., 90: 405-411, 1992). Since smaller proteins penetrate into tissues and tumors better than larger proteins, we have made a smaller recombinant (r) IT to overcome the hypothesized poor tumor penetration of the Mr 195,000 conjugate. This was accomplished by making a C242rF(ab')-based Mr 86,000 IT. To make rF(ab')-IT, the Fd and kappa chains of mAb C242 were cloned, and kappa was fused at the gene level to PE38QQR, a mutant form of Pseudomonas exotoxin. Both C242Fd and C242kappa-PE38QQR were expressed in a bacterial expression system, and large amounts of the C242Fd attached via a disulfide bond to the C242kappa-PE38QQR were obtained. The C242rF(ab')-IT covalently linked heterodimer has a 50% inhibitory concentration of 0.2-2.0 ng/ml (2-20 pm, respectively) on human colon adenocarcinoma cell lines that express the C242 antigen. When injected into mice bearing Colo205 tumors, the C242rF(ab')-PE38QQR caused an immediate regression of the tumors, while the C242-PE38QQR conjugate had only a growth inhibitory effect. In addition, several cures were obtained. Our results indicate that rIT C242F(ab')-PE38QQR is a much more potent antitumor agent than an IgG conjugate.     

Use of anti-CD3 epsilon F(ab')2 fragments in vivo to modulate graft-versus-host disease without loss of graft-versus-leukemia reactivity after MHC-matched bone marrow transplantation

The use of T cell-specific mAb in vivo for prevention and treatment of graft-vs-host disease (GVHD) and its impact on graft-vs-leukemia (GVL) reactivity was examined in a murine model of MHC-matched bone marrow transplantation (BMT). F(ab')2 fragments of a CD3 epsilon-specific mAb were administered to irradiated AKR (H-2k) hosts after transplantation of BM plus spleen cells from B10.BR donors (BMS chimeras). The effects on GVH and GVL reactivity were Ab dose- and schedule-dependent. A short course of mAb (qe2d, days 0 to 8) prevented clinical evidence of GVHD and mortality. Anti-CD3 F(ab')2 mAb reversed clinical symptoms of acute GVHD when delayed up to 18 days post-transplant. Anti-host (Mls-1a)-specific V beta 6+ cells were absent from the spleens of GVH-negative control mice, but persisted in Ab-treated BMS chimeras despite the absence of GVHD. Leukemic mice given 16.7 micrograms of Ab on days 0, 2, and 4 survived leukemia-free without developing severe GVHD. A longer course of Ab completely prevented GVHD, but led to leukemia relapse in tumor-bearing hosts, despite engraftment of donor T cells. The GVL effect was quantitatively stronger when Ab was used for GVH therapy as compared with GVH prevention. Some Ab-treated, GVH-free chimeras relapsed with lymphomas in unusual sites, suggesting that occult tumor cells may persist in nonlymphoid tissues. These experiments demonstrate that T cell-specific mAb can be used successfully in vivo to avoid severe GVHD, but that excessive or ill-timed administration of Ab may eliminate GVL reactivity.