Humanization of Immu31, an alpha-fetoprotein-specific antibody.

Immu31 is a murine monoclonal antibody (Ab) specific for alpha-fetoprotein (AFP), a tumor-associated marker. The excellent tumor targeting ability of Immu31 has led to the development of a Immu31-based radioimmunodiagnostic agent, AFP-Scan, for hepatocellular carcinoma and other AFP-producing tumors. To enhance the capability of Immu31-based immunoconjugates being used in diagnostic and therapeutic procedures in humans, a humanized version of Immu31 (hImmu31) was constructed by grafting the complementarity determining regions (CDRs) of murine variable domains for the heavy (VH) and kappa (Vkappa) chain to the respective human VH and Vkappa framework regions (FRs). The cDNA encoding the VH and Vkappa of Immu31 was cloned by reverse transcription-PCR from hybridoma cells, and a chimeric Immu31 (cImmu31) composed of murine V and human C domains was constructed. Competitive ELISA assays showed identical AFP binding activity between the chimeric and murine Abs, confirming the authenticity of the cloned V genes. Based on sequence homology, the EU FR1, FR2, and FR3 and the NEWM FR4 were selected as the scaffold for grafting VH CDRs and REI FRs for Vkappa CDRs of Immu31. The amino acid residues in murine FRs that are considered to be in contact with the CDRs of the Ab were maintained in the humanized version. hImmu31, thus constructed and expressed, showed comparable immunoreactivity in a competitive binding ELISA assay to that of murine Immu31 and cImmu31. High-level production was achieved by expressing hImmu31 in a dhfr-based amplifiable system, and the productivity has exceeded 100 mg/liter in terminal cultures.

Generation and monitoring of cell lines producing humanized antibodies.

Antibody humanization has eliminated or reduced the human antimouse antibody response associated with the administration of murine antibodies. We have successfully humanized three different antibodies: (a) hMN-3 (granulocyte targeting); (b) hMu-9 (colorectal cancer targeting); and (c) hWI2 (anti-idiotype to the anti-carcinoembryonic antigen antibody MN-14). All humanized antibodies demonstrated immunoreactivities comparable to their parent counterparts. Previously, we reported the generation of high productivity cell lines for hMN-14 and hLL2 using the amplifiable vector pdHL2. Through amplification, selection, and cloning procedures, cell lines capable of large scale production were established, and further enhancement of production was achieved by a fed-perfusion bioreactor process. Using a similar and improved approach, we have enhanced the production of the above-mentioned humanized antibodies by gene amplification induced by a stepwise increase in the concentration of methotrexate in the culture media. A reliable IgG determination method is essential to monitor amplification, especially at the final cloning stage, for the selection of the subclones with the highest productivity. We found that measurement of humanized IgG concentration in culture media supplemented with more than 1 microM methotrexate by a standard ELISA assay could be unreliable and misleading. Whereas the determination of antibody by adsorption/elution on protein A from a 100-ml culture is accurate and reproducible, the method is time-consuming, tedious, and labor intensive. We have recently developed a Western blot assay that enables us to monitor the productivity of the cultures. The assay is simple and sensitive, and it makes simultaneous determinations of relative antibody production from individual clones at the 96-well stage feasible. With this method, amplification, cloning, and adaptation to serum-free conditions of multiple cell lines can be monitored in an efficient manner.

The effects of domain deletion, glycosylation, and long IgG3 hinge on the biodistribution and serum stability properties of a humanized IgG1 immunoglobulin, hLL2, and its fragments.

Antibody (Ab) fragments are preferred agents for imaging applications because of their rapid clearance from the blood, thereby providing high tumor:blood ratios within a few hours. Several preclinical studies have also suggested that Ab fragments might be preferred for therapeutic applications over an intact IgG. The purpose of this project was to develop engineered Ab fragments using a humanized anti-carcinoembryonic antigen and anti-CD22 Ab as the parent. Three types of variants were prepared: a deltaCH2 (deletion mutant missing the CH2), a gamma3 F(ab')2 containing the human IgG3 hinge, and three glycosylated variants. The gamma3 F(ab')2 and glycosylated variants were developed because of the potential for site-specific linkage to the Ab in its divalent or monovalent fragment. The gamma3 F(ab')2 variant contains 10 cysteine residues that could be used for direct coupling using thiol chemistry, whereas the glycosylated variants have N-linked glycosylation sites engineered in the CH1 domain (two variants) as well as the VK domain (one variant). All of these variants were successfully prepared and shown to react with the target antigen. All Abs could be purified to a single peak by size-exclusion HPLC, but the deltaCH2 variant showed two distinct peaks, which were believed to be both the divalent and monovalent forms of this fragment. The two CH1 glycosylated variants showed differences in the extent of glycosylation. Modeling studies suggest that one variant would be better suited for site-specific coupling than the other because the carbohydrate chain is extended further away from the antigen-binding site. The Abs were radioiodinated to determine their pharmacokinetic behavior in mice. All of the humanized Ab divalent fragments cleared nearly 20 times faster from the blood than the murine parent F(ab')2 over a 24-h period. The glycosylated fragments showed some added stability compared to the other fragments over 4 h, but by 24 h, they had cleared to the same extent. Size-exclusion high-performance liquid chromatography of blood samples indicated that the humanized Ab fragments were quickly degraded in the blood. Thus, there is an inherent instability of the divalent fragments from these humanized IgG1 constructs that may affect their utility in imaging or therapy applications.

90Yttrium-labeled complementarity-determining-region-grafted monoclonal antibodies for radioimmunotherapy: radiolabeling and animal biodistribution studies.

90Yttrium-labeled monoclonal antibodies (mAbs) are likely to be important to radioimmunotherapy (RAIT) of a variety of cancers. The goal of this study was to select and evaluate a form of [90Y]mAb suitable for RAIT and determine conditions for high-yield, reproducible radiolabelings. 90Y-Labelings, at 2-40 mCi levels, of cdr-grafted versions of anti-B-cell lymphoma (hLL2) and anti-CEA (hIMMU-14) mAbs were optimized to >90% incorporations using the macrocyclic chelator DOTA as the metal carrier. In in vitro challenge assays, the stability of mAbs labeled with [90Y]DOTA was better than that of the corresponding [90Y]benzyl-DTPA conjugates. The retention of [90Y]DOTA-hLL2 on Raji tumor cells in vitro was similar to that of the same mAb labeled with [90Y]benzyl-DTPA and was about twice as much as with [125I]hLL2, indicating residualization of metalated mAb. Both [90Y]hLL2 conjugates, prepared using DOTA and Bz-DTPA, had similar maximum tolerated doses of 125 muCi in BALB/c mice and showed no discernible chelator-induced immune responses. Animal biodistribution studies in nude mice bearing Ramos human B-cell lymphoma xenografts revealed similar tumor and tissue uptake over a 10 day period, with the exception of bone uptake which was up to 50% lower for [88Y]DOTA-hLL2 compared to [88Y]Bz-DTPA-hLL2 at time points beyond 24 h. With [90Y]DOTA-hLL2 fragments, in vivo animal tumor dosimetries were inferior to those for the IgG, and kidney uptake was relatively high even with D-lysine administration. The ability of [111In]DOTA-hLL2 to accurately predict [90Y]DOTA-hLL2 biodistribution was established. These preclinical findings demonstrate that [90Y]DOTA-(CDR-grafted) mAbs are suitable for examination in clinical RAIT.

Generation of a high-producing clone of a humanized anti-B-cell lymphoma monoclonal antibody (hLL2).

BACKGROUND: LL2 is a murine immunoglobulin (Ig)G2a-kappa anti-B-cell monoclonal antibody with proven targeting and therapeutic efficacy in the management of non-Hodgkin's lymphoma (NHL). The authors had previously generated a humanized LL2 (hLL2) that demonstrated binding properties identical to those of LL2. Nevertheless, the productivity of the cell line was insufficient for large-scale production of the antibody for clinical studies. Therefore, the authors chose an amplifiable system for the generation of hLL2. METHODS: The hLL2 sequences were ligated into the expression vector pdHL2, which has a dhfr amplifiable gene, and were incorporated into the SP2/0 cells by electroporation. A methotrexate (MTX) resistant clone producing hLL2 was identified. Stepwise increases in MTX concentrations, from 0.1 to 5 microM, and subcloning of the cells by limiting dilution were performed. RESULTS: By amplifying the dhfr and hLL2 genes with stepwise increases in the MTX concentration, the antibody production was enhanced from its original 1.4 to 70 +/- 5 mg per liter of culture media. Subsequent subcloning further improved the productivity. Immunoreactivity of the antibody was conserved, as proven by enzyme-linked immunosorbent assay and cell-binding assays. By isoelectrofocusing, the isoelectric point (pI) of the antibody was measured at approximately 9.6. The productivity of the clone was not affected by culture conditions or storage of the cells in liquid nitrogen. CONCLUSIONS: By means of gene amplification, the authors have generated a high-producing hLL2-IgG clone suitable for production of the quantity of antibody necessary for clinical diagnostic and therapeutic trials of NHL patients.

Chimerization of Mu-9: a colon-specific antigen-p antibody reactive with gastrointestinal carcinomas.

BACKGROUND: Mu-9 is a murine monoclonal antibody that is directed against affinity-purified colon-specific antigen-p (CSAp). CSAp is a tumor-associated antigen that is present in 60% of colorectal carcinomas. Preclinical and clinical studies have shown Mu-9 to have excellent targeting abilities. However, as administration of the murine immunoglobulin G (IgG) provoked a human anti-mouse antibody response, chimerization of Mu-9 is warranted for decreasing immunogenicity. METHODS: Polymerase chain reaction and cDNA library screening methods were used for the cloning of Mu-9 heavy and light chain variable regions for the construction of chimeric Mu-9. RESULTS: The functional chimeric Mu-9 antibody binds to the CSAp antigen in the GW-39 extracts. It has immunochemical properties similar to that of murine Mu-9. CONCLUSIONS: The V-region sequence information will be used for design of humanized Mu-9, which will be evaluated for targeting gastrointestinal carcinomas.

Site-specific modifications of light chain glycosylated antilymphoma (LL2) and anti-carcinoembryonic antigen (hImmu-14-N) antibody divalent f1agments.

Site-specific introduction of metal-chelating groups into F(ab')2 fragments of an antilymphoma antibody (LL2) possessing a natural Asn-linked light chain carbohydrate and an anti-carcinoembryonic antigen antibody (hImmu-14-N) grafted with a light chain carbohydrate site is described. For this purpose, four yttrium- (and indium)-chelating agents were used, containing a primary amino group for antibody binding and 1-(4-substituted benzyl)diethylenetriaminepentaacetic acid as the metal-chelator, separated by structurally different additional linkers. Conjugates were prepared by reacting excess chelator with oxidized carbohydrate of F(ab')2 fragments, with or without a subsequent reduction step. The conjugates, with up to an average of 5.5 chelating groups attached to a F(ab')2 fragment, were readily labeled with 90Y and 111In and were found to retain antigen-binding ability in in vitro assays. Tumor targeting was demonstrated using a 88Y-labeled hImmu-14-N F(ab')2 carbohydrate-modified conjugate. 2-Pyridyldithiopropionic hydrazide was conjugated to the carbohydrate region, and the disulfide was selectively deprotected to the thiol group, which is reactive with reduced 99mTc. These initial experiments establish that light chain carbohydrate modification of F(ab')2 is as facile as with the Fc-region carbohydrate of intact IgG, and thereby offer the possibility of designing site-specifically substituted F(ab')2 fragments with favorable pharmacokinetic properties.

Bacterial expression of a kemptide fusion protein facilitates 32P labeling of a humanized, anti-carcinoembryonic antigen (hMN-14) antibody fragment.

Despite the potential advantages of 32P over other isotopes for radioimmunotherapy, its development as a therapeutic has been hindered by the difficulty of the labeling chemistry. Recently, a heptapeptide [Kemptide (KPT)] has been chemically conjugated to antibodies, and the conjugates have successfully been labeled with 32P enzymatically by using bovine protein kinase. By using genetic engineering, we have produced a chimera (Fab.KPT) consisting of the Fab' moiety of the complementarity-determining region-grafted anti-carcinoembryonic antigen-monoclonal antibody, MN14, and a heptapeptide derivative of KPT (Trp-Arg-Arg-Ala-Ser-Leu-Gly). The recombinant protein was expressed in Escherichia coli as a soluble secretory product. The presence of the KPT derivative downstream of the COOH terminus of the hinge region did not impair the binding affinity of the antibody fragment. The Fab.KPT was enzymatically phosphorylated with 32P by bovine protein kinase, without significant effect on the resultant immunoreactivity; 100% of the 32P-labeled Fab.KPT was complexed with liquid carcinoembryonic antigen. The 32P-labeled humanized MN-14 Fab.KPT is expected to have longer blood circulation half-life, allowing for an improved therapeutic efficacy in radioimmunotherapy.

Development and evaluation of the specificity of a rat monoclonal anti-idiotype antibody, WN, to an anti-B-cell lymphoma monoclonal antibody, LL2.

Anti-idiotype monoclonal antibodies (Mabs) to mLL2, an anti-B-cell lymphoma and CD22-specific murine IgG2a-kappa Mab, were generated by hybridoma technology from splenocytes of Copenhagen rats immunized with mLL2 F(ab')2. Mab WN, an IgG2a-kappa, was selected based on its specific binding to mLL2 and not other IgG isotypes or anti-B-cell Mabs. In a radioimmunoassay, WN was found to inhibit the binding of 125I-labeled mLL2 to Raji cells and to have no effect on the binding of other B-cell-reactive antibodies. Using high performance liquid chromatography analysis, WN was shown to complex specifically with both mLL2 and mLL2 Fab'. Meanwhile, we have constructed chimeric (cLL2) and humanized (hLL2) versions of LL2. Both cLL2 and hLL2 were demonstrated to retain the original antigen specificity and affinity of mLL2 [S.O. Leung et al., Proc. Am. Assoc. Cancer Res., 2872 (abstract), 34: 481, 1993]. The specific binding of WN to either radioiodinated or peroxidase-conjugated mLL2 was inhibited in a dose-response manner, and to a similar extent by mLL2, cLL2, and hLL2. Since the mLL2 complementarity-determining regions are the only sequences common to mLL2, cLL2, and hLL2, the result confirms that WN is specific to the antigen-binding complementarity-determining regions. A WN binding assay is currently being evaluated as a substitute for the tedious, and sometimes inconsistent, Raji cell-binding assay for the determination of LL2 immunoreactivity. In conclusion, we have developed an anti-idiotype Mab, WN, to mLL2. Its potential use as a surrogate antigen for B-cell lymphoma is under investigation.

Engineering a unique glycosylation site for site-specific conjugation of haptens to antibody fragments.

A natural N-linked glycosylation site (Asn-Val-Thr) at amino acid positions 18-20 (Kabat's numbering) was identified in the framework-1 (FR-1) region of the light chain variable (V kappa) domain of a murine anti-B cell lymphoma Ab, LL-2. Our earlier studies demonstrated that no contact between the V kappa-appended oligosaccharide and the Ag binding site was evident, because glycosylation at this site did not affect the Ag binding property of the Ab. By using the murine LL-2 F(ab')2 fragment (which is devoid of constant region-appended oligosaccharide) as substrate, as much as five bifunctional chelator molecules per F(ab')2 fragment could be site specifically conjugated at the V kappa-appended carbohydrate moiety with no reduction in immunoreactivity. The resulting conjugates labeled efficiently with both 90Y and 111In, with no significant effect on Ab affinity. In contrast, conjugation of less than five chelates/Ab fragment randomly at lysine residues resulted in a three- to fivefold reduction in affinity. By a single Arg to Asn mutation, an N-linked glycosylation site similar to that of LL-2 was introduced in the FR-1 segment of a nonglycosylated, humanized anti-carcinoembryonic Ag (CEA) Ab, MN-14 (hMN-14). Glycosylation at the engineered carbohydrate-addition site was demonstrated by SDS-PAGE analysis. Neither glycosylation nor site-specific conjugation of chelate at the V kappa-appended carbohydrate moiety resulted in the loss of immunoreactivity. The glycosylated hMN-14 conjugate labeled efficiently with 90Y.

Effect of VK framework-1 glycosylation on the binding affinity of lymphoma-specific murine and chimeric LL2 antibodies and its potential use as a novel conjugation site.

A potential asparagine (Asn)-linked glycosylation site was identified in the VK FRI sequence of an anti-B lymphoma monoclonal antibody (MAb), LL2.SDS-PAGE analysis and endo-F treatment of both murine and chimeric LL2 antibodies indicated that this site was glycosylated; however, no differences in the binding affinity to Raji cells were observed between the native murine LL2 and the endo-F-deglycosylated murine LL2 antibodies. Elimination of the glycosylation site from the chimeric LL2 antibody was accomplished by an Asn to Gln mutation in the tri-acceptor site found in the light chain. The resultant aglycosylated chimeric LL2 exhibited a similar Raji cell binding affinity to that of the glycosylated form. The results are in agreement with computer modeling studies which suggested the lack of interactions between the oligosaccharide moiety and the CDRs. The finding is interesting because it enables a wider choice of human framework sequences, which in most cases do not have a corresponding glycosylation site, for the humanization of the LL2 VK domain, as well as a greater latitude of host expression systems. Most importantly, the LL2 VK carbohydrate moiety might be used as a novel conjugation site for drugs and radionuclides without compromising the immunoreactivity of the antibody.

D-penicillamine- and quinidine-induced antinuclear antibodies in A.SW (H-2s) mice: similarities with autoantibodies in spontaneous and heavy metal-induced autoimmunity.

Ten percent of human lupus syndromes occur in patients as a result of treatment with certain medications. H-2s mice can produce autoantibodies following treatment with various drugs or heavy metals and they are a potential animal model of drug-induced lupus. We have examined nine anti-chromatin monoclonal antibodies (mAb) from A.SW mice that had been treated with either D-penicillamine or quinidine, two lupus-inducing drugs in humans. These mAb are specific either for DNA or histone-DNA complexes corresponding to nucleo-specific either for DNA or histone-DNA complexes corresponding to nucleosomes or subnucleosome particles. Only one mAb reacts with an unknown chromatin antigen. The V region sequences of six of these mAb were studied and are notable by several features. As previously observed in spontaneous autoantibodies to DNA or histone-DNA complexes, arginine or asparagine residues are found at critical locations throughout the V regions. Many of these residues, potentially important for binding to DNA or DNA-histone complexes, result either from somatic mutations or atypical VH-D-JH rearrangements. Another significant characteristic is that the VH genes of several D-penicillamine- or quinidine-induced mAb are most similar to those of anti-nucleolar mAb obtained from mercury-injected A.SW mice. The implications of these findings for the pathogenesis of spontaneous or induced autoimmunity are discussed.

Mimicry of a carcinoembryonic antigen epitope by a rat monoclonal anti-idiotype antibody.

Anti-idiotype antibodies (Ab2) that immunologically mimic tumor antigens are auspicious agents for the active immunization of cancer patients. We have developed W12, a rat monoclonal IgG1 Ab2 to MN-14, a murine anti-carcinoembryonic antigen (CEA) monoclonal antibody. W12 is specific for MN-14 and does not react with other isotype-matched anti-CEA monoclonal antibodies. Moreover, W12 inhibits the binding between MN-14 and CEA. Anti-CEA antibodies can be induced by immunization with W12 (but not with control rat IgG) in xenogenic animals (mice or rabbits). Immunoblotting studies indicate that the internal image determinant borne by W12 is conformational and requires the association of the heavy and light chains of the Ab2 molecule. This study indicates that W12 is a potential idiotype vaccine in patients with CEA-producing cancers.

Histone-reactive IgA antibodies in adult IgA nephropathy and other primary glomerulonephritis.

The levels of histone-reactive IgA antibodies in the sera of adult patients with IgA mesangial glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis and idiopathic nephrotic syndrome (minimal change disease+segmental glomerulosclerosis+IgM nephropathy) were evaluated by an enzyme-linked immunosorbent assay. Increased levels of IgA antibodies to all five major histones (H1, H2A, H2B, H3, H4) were found in all four disease groups when compared to normal controls. These histone-reactive IgA antibodies were restricted to the IgA1 subclass and their levels did not correlate with the levels of total serum IgA, nor with serum creatinine, creatinine clearance, and 24-hour proteinuria. Increasing ionic strength resulted in only partial inhibition of the binding to histones and, in individual patients, levels of reactivity with individual histones were usually correlated. This study shows that elevated levels of IgA antibodies reactive with self antigens are present in primary glomerulonephritis and extends previous observations indicating that anomalies of the IgA system occur in various forms of primary glomerulonephritis and are not limited to IgA nephropathy.

Molecular analysis of mercury-induced antinucleolar antibodies in H-2S mice.

In H-2S mice, the administration of mercuric chloride results in the development of antinucleolar autoantibodies. These mice represent a valuable model to study the role of environmental factors in the development of systemic autoimmunity. We have obtained seven antinucleolar mAb from mercury-injected A.SW mice and characterized their specificities and V genes. All mAb immunoprecipitate the U3 and U8 ribonucleoprotein particles (RNP) and some (but not all) react with fibrillarin, the only currently characterized protein component of mammalian nucleolar RNP. Several VH and V kappa genes are recurrently used by these antinucleolar RNP mAb and their H chain CDR3 segments contain several acidic residues that may be important for binding to the cationic proteins composing the nucleolar RNP. Our results support the concept that in H-2S mice administration of mercury induces a specific loss of tolerance to nucleolar RNP.

Structure and binding properties of monoclonal antibodies to core histones from autoimmune mice.

Histones are frequent targets of self-reactive antibodies during autoimmune syndromes. We report the specificities and V region genes of three IgG anti-histone MAbs obtained from autoimmune mice. Each of the MAbs, named LG2-1, LG2-2 and BWA3, is directed against a different determinant located in the basic amino-terminal domain of core histones. LG2-1 reacts with a peptide from histone H3 (residues 30-45), LG2-2 recognizes the amino-terminus of H2B (residues 1-13) and BWA3 binds an epitope corresponding to a region of high sequence similarity between H2A and H4 (residues 1-20 and 1-29, respectively). The analysis of their V region sequences indicates that the H chain CDRs of these MAbs are remarkable for the presence of negatively charged amino acid residues that may play a role in the binding to cationic histones. The H chain importance in conferring reactivity to histones is corroborated by the observation that each of the VH gene segments of these MAbs is very similar to VH genes of previously described murine anti-histone antibodies.

Relationships among antinuclear antibodies from autoimmune MRL mice reacting with histone H2A-H2B dimers and DNA.

The histone H2A-H2B dimer is a component of nucleosomes in chromatin and a frequent target of autoantibodies in spontaneous and drug-induced lupus. We obtained a panel of several lgG mAbs reacting with H2A-H2B or DNA from MRL mice which develop a spontaneous lupus-like syndrome. Several of these antibodies do not react with individual histones, but bind strongly to the H2A-H2B dimer and some bind even more strongly to the H2A-H2B-DNA complex. Moreover, these antibodies not only bind to H2A-H2B dimers in the absence of DNA, but also exhibit significant binding to DNA in the absence of histones, indicating an overlap between the anti-histone and anti-DNA specificities. The analysis of the variable region gene sequences of these antibodies shows a recurrent usage of similar VH genes, suggesting a dominant role for the heavy chain in determining binding specificity. The heavy chain third complementarity determining regions of these antibodies are also remarkable for their frequency of D-D fusions and of D segments read in unusual reading frames and for many arginine residues that may contribute to DNA binding. In addition, several antibodies obtained from an individual mouse are clonally related and some differ through somatic mutations, indicating that autoreactive clones are positively selected by nuclear antigens.

Polyreactive IgM antibodies generated from autoimmune mice and selected for histone-binding activity.

A panel of histone-reactive IgM mAbs was obtained from mice belonging to various spontaneously autoimmune strains. Most of these antibodies were polyreactive, i.e. they showed binding to other cationic antigens (poly-L-lysine, lysozyme, cytochrome c) or to cytoskeletal proteins (actin, myosin, vimentin). The variable regions of these antibodies were encoded by V genes and gene segments belonging to various families. Their H chain third hypervariable regions were unusual in that the D segments were read in all three possible reading frames in contrast to most conventional antibodies and other polyreactive antibodies obtained from normal mice.

Enhanced clearance of radiolabeled murine monoclonal antibody by a syngeneic anti-idiotype antibody in tumor-bearing nude mice.

A syngeneic anti-idiotype monoclonal antibody (MAb) (CM-11) directed against an anti-carcinoembryonic antigen (CEA) murine MAb (NP-4) was evaluated as a second antibody (SA) to promote the rapid clearance of radiolabeled NP-4 from the blood. Initial studies confirmed that CM-11 IgG removed 131I-NP-4 IgG from the blood as effectively as a polyclonal donkey anti-goat IgG removed 131I-goat IgG. However, use of an F(ab')2 in place of either the NP-4 or CM-11 IgG was not as effective in removing primary radiolabeled antibody, despite the formation of high-molecular-weight complexes. In accordance with previous results, the timing and dose of the SA injection was critical for optimizing tumor uptake and improving tumor/non-tumor ratios. In nude mice bearing GW-39 human colonic tumor xenografts, a delay in the injection of CM-11 by 48 hr after injection of radiolabeled NP-4 was optimal, since this allowed maximum tumor accretion. At a 200:1 CM-11:NP-4 ratio, tumor uptake was reduced, suggesting inhibition of NP-4 binding to CEA within the tumor. Despite optimizing tumor uptake by delaying SA injection and adjusting its dose, the percentage of 131I-NP-4 in the tumor decreased 2- to 3-fold within 2 days after CM-11 injection. A similar effect was seen for 111In-labeled NP-4 IgG with CM-11. Injection of excess unlabeled NP-4 given to block CM-11 shortly after its injection failed to curtail the loss of NP-4 from the tumor. Our results suggest that high blood levels of MAb are important for sustaining NP-4 in the tumor. Radiation-dose predictions derived from biodistribution studies indicate that a higher tumor dose may be delivered using the SA method than with either 131I-NP-4 IgG or F(ab')2 alone. Use of the SA method with 90Y-labeled NP-4 IgG, as modeled from biodistribution studies with 111In-NP-4 IgG, would likely be limited by liver toxicity.

Monoclonal autoantibodies to subnucleosomes from a MRL/Mp(-)+/+ mouse. Oligoclonality of the antibody response and recognition of a determinant composed of histones H2A, H2B, and DNA.

MRL/Mp(-)+/+ mice produce antinuclear antibodies and develop a spontaneous autoimmune syndrome with lupus-like nephritis. We obtained a panel of seven histone-reactive IgG mAb from a single MRL/Mp(-)+/+ mouse. These antibodies do not react significantly with DNA or individual histones, but bind strongly to the histone H2A-H2B dimer and even more strongly to the H2A-H2B-DNA complex. These antibodies also bind to whole nuclei when tested by immunofluorescence, indicating that they recognize an epitope accessible in chromatin. The V region sequences of these antibodies have been determined. The H chain third complementarity-determining regions of these antibodies are similar to those found in anti-DNA antibodies even though the antibodies in our panel do not react with DNA in the absence of histones, suggesting that DNA is part of the subnucleosome epitope. Several of these antibodies are clonally related, supporting the hypothesis that the activation of these clones is Ag-driven. Analysis of the sequences of these antibodies indicates that they derive from autoreactive B cells that were clonally expanded and whose V region genes have undergone numerous somatic mutations.