Purpose: We had previously reported that certain of our murine (m) antihuman light chain monoclonal antibodies (mAbs) recognized an epitope common to AL and other types of amyloid fibrils. On the basis of this evidence, one such antibody, 11-1F4, was administered to mice bearing AL amyloidomas induced by s.c. injection of human AL extracts. The mAb bound to the amyloid and initiated an Fc-mediated cellular inflammatory response that led to rapid reduction in the tumor masses. To develop this reagent for clinical use, the 11-1F4 mAb was chimerized and its activity compared with that of the unmodified antibody.

Experimental Design: The chimeric (c) 11-1F4 mAb was produced in CHOdhfr-stable mammalian cell lines that had been transfected with a supervector DNA encoding the mouse 11-1F4 heavy and light chain variable regions (VH, VL) and human heavy and light chain constant regions (CH, CL). The antibody products were analyzed for their fibril binding activity and ability to effect amyloidolysis in two in vivo experimental models.

Results: The capability of the c11-1F4 mAb to interact with amyloid was demonstrated in vitro. Administration of this reagent into mice bearing human AL tumors or those with systemic AA deposits resulted in marked reduction in amyloid burden with no evidence of toxicity in the animals.

Conclusions: These results have led to the decision to produce GMP-grade c11-1F4 for a Phase I/II clinical trial in patients with primary (AL) amyloidosis where the effectiveness of the reagent could be determined. The use of amyloid-reactive antibodies would represent a novel approach in the treatment of individuals with this invariably fatal disorder.

Primary (AL) amyloidosis is a monoclonal plasma cell dyscrasia associated with the production of amyloidogenic immunoglobulin light chains that form fibrillar deposits in vital tissues; this relentless process leads to organ failure and death, usually within 9–36 months (1, 2, 3, 4, 5). Heretofore, treatment of patients with this disease had been limited to the use of antiplasma cell chemotherapy given in conventional amounts or in high doses combined with autologous stem cell transplantation (6, 7, 8, 9, 10, 11, 12). Such efforts have, in some cases, extended survival; however, the overall prognosis remains poor.

As part of ongoing studies on the pathogenesis of primary (AL) amyloidosis and the use of immunotherapy as a means to eliminate amyloid deposits, we discovered that certain of our m3 mAbs prepared against human light chain-related fibrils were capable of recognizing an amyloid-related, conformational epitope, as evidenced immunohistochemically and by ELISA. Furthermore, when one such reagent, the IgG1κ mAb 11-1F4, was administered to mice bearing human AL amyloidomas, the antibody bound to the amyloid and initiated an inflammatory response that led to elimination of the induced tumor (13).

To facilitate translation of these promising experimental findings into clinical practice, we requested assistance from the National Cancer Institute’s RAID Program. Subsequently, RAID contracted with AERES Biomedical Ltd. (Mill Hill, London, United Kingdom) to chimerize the m11-1F4 amyloid-reactive mAb. Three CHO dhfr-stable cell lines were transfected with a supervector DNA specifying the murine VH and VL and human CH and CL. The resulting c11-1F4 mAbs produced by these mammalian cells were harvested, purified, and tested for their capacity to interact with amyloid in several in vitro and in vivo experimental systems. We now report the results of our studies where we have shown comparable fibril binding and effective, although somewhat reduced, amyloidolytic activity of the modified antibody as compared with its murine (native) counterpart.

PCR Cloning and Sequencing of the m11-1F4 Antibody Heavy and Light Chain Variable Region Genes.

Two clones (B2C4 and B2D6) from the SP2/0 hybridoma cell line producing the IgG1κ m11-1F4 amyloid-reactive mAb were furnished to AERES Biomedical Ltd. (Mill Hill). The cells were cultured in DMEM media (Life Technologies, Inc., Rockville, MD) supplemented with 20% (v/v) fetal bovine serum (Hyclone, Logan, UT), penicillin/streptomycin, and l-glutamine (Life Technologies, Inc.). After growth to 108 viable cells, total RNA was isolated from each clone (RNA Isolation kit; Stratagene, La Jolla, CA), and first-strand cDNA was synthesized using the furnished NotI-d(T)18 primer. The mVH and VL (Vκ) genes were then PCR amplified from each cDNA template, as described by Jones and Bendig (14) with AmpliTaq DNA polymerase. Separate PCRs were performed using different degenerate leader sequence-specific VH and Vκ and appropriate CH and CL primers (an equimolar mix of Cγ1, Cγ2a, Cγ2b, Cγ3, and Cκ). The products were identified by electrophoresis on 1% agarose/Tris borate (pH 8.8) gels containing 0.5 μg/ml ethidium bromide. Putatively positive products (∼450 bp) were cloned directly into the pCR2.1 vector provided in the Topo TA Cloning kit (Invitrogen, Carlsbad, CA) and transformed into TOP10-competent cells using the protocol described by the manufacturer. Colonies that contained the plasmid with the 450-bp insert were selected by PCR screening using 17- and 21-mer oligonucleotide primers according to the method of Güssow and Clackson (15) and the double-stranded DNA was sequenced with the ABI PRISM 310 Genetic Analyzer and the ABI Prism BigDye terminator. Specifically designed PCR primers were used to modify the 5′ and 3′ ends of the 11-1F4 VH and Vκ genes to obtain transient expression of their products in mammalian cells. The back primers introduced HindIII restriction and Kozak translation initiation sites, as well as an immunoglobulin leader sequence. The forward Vκ primer provided a splice donor and BamHI restriction site and that for the VH, the first 22 bp of the Cγ1gene, including an ApaI restriction site. The positive PCR products containing the correctly modified 11-1F4 VH and Vκ genes were identified as previously noted and subcloned into their respective expression vectors as HindIII-ApaI (human γ1, Gm1 allotype) and HindIII-BamHI (human Cκ, Km3 allotype) fragments. The ligated Vκ and VH constructs were then used to transform DH5α competent cells, and positive clones were selected by PCR screening.

Construction of a Single Supervector for Transient Expression of c11-1F4 mAb in COS Cells.

A single supervector expressing both chains of the c11-1F4 antibody was constructed by ligating the EcoRI restriction enzyme digestion products of the heavy and light chain expression cassettes.

Transient Expression of the c11-1F4 Antibody in COS Cells.

The c11-1F4 antibody was transiently expressed in ECACC/COS cells by cotransfection of each of the heavy and light chain vector constructs, as well as transfection of the single supervector construct. After incubation for 72-h, the medium was collected, spun to remove cell debris, and analyzed by ELISA for chimeric antibody production and antigen binding.

Quantification and Binding Analyses of the c11-1F4 Antibodies.

Whole IgG molecules present in COS cell supernatants were quantified using Nunc-Immuno MaxiSorp plates (Life Technologies, Inc., Gaithersburg, MD) in a capture ELISA. Antibody molecules were bound by an immobilized goat antihuman IgG Fcγ fragment-specific antibody and detected by an antihuman κ light chain peroxidase-conjugated antibody (Sigma Chemical Co., St. Louis, MO). A standard curve was generated using known concentrations of a control IgG protein on the same plate. To test the capability of the c11-1F4 antibodies to bind amyloid fibrils, a direct binding ELISA was used, as described previously (16).

Transfection of CHOdhfr Mammalian Cells.

CHOdhfr cells (DUKS B11) were grown first in a nonselective media consisting of α-MEM with ribonucleosides and deoxyribonucleosides (Life Technologies, Inc.), supplemented with 10% fetal clone II (Hyclone) and 50 μg/ml gentamicin (Life Technologies, Inc.) in a 37°C, 5% CO2 incubator. Aliquots of 107 cells/ml in PBS were transfected with 13 μg of the 11-1F4 supervector DNA at 1900V, 25 μFarad capacitance using a Bio-Rad Gene Pulser. After a 24-h incubation in nonselective media, the cells were grown in the presence or absence of 10−8 or 10−9m MTX in α-MEM without ribo- or deoxyribonucleosides (Life Technologies, Inc.) supplemented with 10% dialyzed fetal bovine serum and 50 mg/ml gentamicin. The selective media were changed every 3–4 days until foci appeared. After repeated subculture and additional rounds of MTX amplification, three cell lines were selected on the bases of optimum growth and antibody production rates.

Mycoplasma PCR Screen and Sterility Tests.

Media from the chosen cell lines were screened for the presence of Mycoplasma using a PCR-based kit. Testing for bacterial or fungal contamination was done by the Alamar Blue sterility assay (Serotech, Ltd., Raleigh, NC). Additionally, sterile flasks containing tryptic soy, Sabaraud, or thioglycollate broth were inoculated with cell suspensions and cultured for 3 weeks. As a control, media from cells grown in the absence of gentamicin and MTX also were tested.

Determination of Production Rates of Selected CHOdhfr Stable Cell Lines during Exponential and Static Growth.

To measure antibody production under conditions of exponential growth, 75-cm2 tissue culture flasks were seeded in duplicate with each cell line at a concentration of 1 × 105 cells/ml (total medium volume = 20 ml) and incubated at 37°C under 5% CO2. After 24, 48, 72, and 96-h, the cell number was determined and the antibody concentration in the media measured by a quantitative ELISA (see above) and expressed as μg/106 cells. For stable growth, duplicate flasks were seeded with each cell line at a concentration of 1 × 106 cells/ml (total medium volume = 20 ml), and after 4 days, the cells were counted and the antibody concentration determined.

Production and Purification of c11-1F4 mAb.

To have sufficient quantities of the c11-1F4 antibodies available for in vitro and in vivo studies, the three CHOdhfr-stable cell lines (22C1, 22C5, and 22D2) were grown for 1 month in single Integra CL1000 production flasks (Integra Biosciences, Ijamsville, MD) containing α-MEM, 10−6m MTX, and 50 μg/ml gentamicin. The supernatants were concentrated to ∼5 ml, diluted 1:1 in Pierce Protein A binding buffer, and the antibody isolated using an Immunopure Plus Protein A Purification Kit (Pierce, Rockford, IL), as specified by the manufacturer. The eluted and neutralized material was then dialyzed in PBS overnight using Slide-A-Lyzer Dialysis cassettes (Pierce), sterile filtered, aliquoted into 1-ml volumes, and frozen at −20°C.

Immunohistochemistry.

Immunochemical analyses were performed by the avidin-biotin complex technique (Vector Laboratories, New Bedford, MA) on 4-μm thick deparaffinized sections of normal multitissue (BioGenex, San Ramon, CA) and AL-laden tissue mounted on poly-l-lysine-coated slides. The primary reagents included the m or c11-1F4 mAbs; affinity-purified goat antimouse or human IgG horseradish conjugates were used as secondary antibodies (Bio-Rad, Hercules, CA) with diaminobenzidine (Vector Laboratories) as substrate.

In Vivo Models.

Subcutaneous amyloidomas were induced in BALB/c mice by interscapular 100-mg injections of human amyloid fibrils extracted from the liver or spleen from patients with primary (AL) amyloidosis, as described previously (13). Systemic deposits of AA amyloid (secondary amyloidosis) were induced in BALB/c mice by s.c. 0.5-ml injections of AgNO3 (1%) on days 1 and 10; additionally, on day 1, the animals received i.v. 100-μg doses of AEF, which consisted of AA fibrils extracted from the livers of amyloidotic mice (17).

Generation of c11-1F4 Antibody.

Multiple PCR reactions designed to clone the m11-1F4 VH and Vκ genes yielded products of the expected size (∼450 bp) from both the B2C4 and B2D6 hybridoma cell lines. Analyses of the PCR products from at least three clones from each yielded the expected heavy and light chain sequences. The modified murine VH and Vκ 11-1F4 gene constructs (see “Materials and Methods”), along with the human Cγ1 and Cκ constant region genes, were successfully cloned into their respective mammalian expression vectors and used for cotransfection of COS cells. A single supervector comprised of the immunoglobulin genes from both species also was prepared for this purpose. Because of the substantially higher expression levels achieved with the supervector, this construct was used to transfect CHOdhfr-mammalian cells that were subsequently cultured in media with or without MTX. Three of the amplified lines, 22C1, 22C5, and 22D2, had the highest rate of antibody production (50.6, 42.5, and 47 μg/106 cells/day, respectively) with approximate doubling times of 48–60 h. Although the three lines expanded at the same rate during exponential growth over 96 h, the production rate for 22C1 increased from 31 to 37 μg/106 cells/day over this period, whereas that of 22C5 and 22D2 decreased from 24 to 20 and 34 to 31 μg, respectively. During stable growth, the production rates, based on an average of the initial and 96-h viable cell count, were comparable (24.7, 21.5, and 20.5 μg/106 cells/day, respectively). Similarly, over the 1-month period of culture, the antibody concentrations in the media remained relatively stable. The three CHO cell lines (22C1, 22C5, and 22D2) and their products were uncontaminated by bacteria, fungi, or Mycoplasma and had acceptable endotoxin levels (0.7, 1.1, and 23.4 units/μg, respectively).

In Vitro Analyses.

The properties of the three c11-1F4 mAbs are summarized in Table 1. By SDS-PAGE (Fig. 1) and Western blot analyses, these molecules were shown to have the molecular masses expected for chimeric IgG1 heavy- and κ-light chains. These analyses also demonstrated the stability of the antibody products after storage for 1–4 h at 4°C, 37°C, and 70°C. In other experiments, the binding of the c and m11-1F4 mAbs to synthetic AL amyloid fibrils was measured by ELISA where it was found to be comparable (Fig. 2). Additionally, the results of size exclusion HPLC indicated that the chimeric antibody preparations contained no appreciable protein aggregates, i.e., they consisted almost entirely of IgG monomers (Fig. 3).

The c11-1F4 mAbs, as with the murine reagent, interacted only with fibrillar light chains but not with the soluble counterparts. As illustrated in Fig. 4, the 22C1 antibody reacted exclusively with the fibrils at concentrations as low as 1 μg/ml. A similar pattern was found with the m11-1F4 mAb. In studies of BJPs representative of the Vκ1, Vκ4, Vλ6, and Vλ8 subgroups, neither mAb recognized the κ1, λ6, and λ8 proteins; immunostaining of the κ4 component was expected because the original 11-1F4 reagent was produced by immunizing mice with Vκ4 fibrils (13).

The capability of the c11-1F4 mAb to interact with human AL-containing deposits was tested immunohistochemically on tissues obtained from two patients with primary (AL) amyloidosis. As illustrated in Fig. 5, both the c and m11-1F4 reagents immunostained the vascular green birefringent congophilic areas in uterine and pancreatic tissues that were shown to contain κ4- and λ8-related amyloid deposits, respectively (as demonstrated using anti-Vκ4 and Vλ8 subgroup-specific mAbs).

Additionally, the reactivities of the c and m11-1F4 mAbs were tested against 15 different human specimens, including liver, spleen, kidney, heart, lung, intestine, muscle, pancreas, prostate, thyroid, testis/ovary, bladder, uterus, and brain. Diffusely mild to focally intense staining of hepatocytes, proximal renal tubules, and myocytes was noted. In contrast, far greater immunostaining was found when these tissues were studied with the chimeric IgG1κ anti-CD20 mAb Rituximab (Ref. 18; data not illustrated).

In Vivo Analysis.

The efficacy of the IgG1κ c11-1F4 mAb to accelerate amyloidolysis in our in vivo amyloidoma model was compared with that of the murine parent. Because the 22C1-, 22C5-, and 22D2-derived c11-1F4 preparations exhibited equivalent reactivity with amyloid and there were limited quantities of each, the three were combined and used in our in vivo experiments. Given the relatively large amount of amyloid extract required to produce a readily palpable amyloidoma (dry weight, 100 mg) and the scarcity of autopsy-derived samples, the numbers of animals used in each study was necessarily restricted to one pair of mice. In the first experiment, four sets were injected s.c. with a 1-ml volume of solution containing 100 mg of a human ALκ extract (Ref. 19; patient HIG) that was comprised of fragments (∼16 and 18 kDa) representing the major portion of the amyloidogenic precursor κ1 light chain (BJP, HIG), as demonstrated by SDS-PAGE, Western blot, and chemical analyses. By dot blot, the c11-1F4 mAb, as with the murine reagent, reacted with amyloid HIG but not with the BJP. Forty-eight h after injection of the fibrillar extract, two of the four pairs were given an i.v. 100-μg injection of either the m or cmAb; this treatment was repeated on days 4, 6, 8, 10, and 12 (i.e., each mouse received a total of 600 μg of antibody). For control purposes, similar doses of a monoclonal isotype matched (IgG1κ) murine protein (MOPC 31C) were administered to the third set over the 12-day period, whereas the fourth was untreated. There were no obvious signs of toxicity in any of the mice.

Approximately 11 days postamyloid induction, the tumors in the mAb-injected mice began to decrease in size. One from each pair was euthanized on day 15 where it was noted that the amyloidomas from the 11-1F4-treated animals were obviously smaller than those from the 2 controls (Fig. 6). On day 16, 3 of the 4 remaining mice were sacrificed (1 control was kept for additional observation to determine the time when the amyloidoma would resolve in an untreated animal). As shown in Table 2, the wet weights of the residual amyloidomas in the 2 mice that received the m11-1F4 mAb were somewhat less than those from the animals that received the chimeric counterpart. In both cases, however, there was a marked reduction in size as compared with the controls, i.e., an average of 83 and 70%, respectively. The amyloidoma in the untreated animal did not become palpably smaller until day 24. On day 30, the mouse was sacrificed, and the mass of the remaining amyloidoma was found to be similar to those removed from the 11-1F4 mAb-treated animals on day 16.

The excised amyloidomas from the treated and control mice were divided in half: one portion was frozen in ornithine carbamyl transferase and the other was fixed in formalin and embedded in paraffin. Tissue (liver, spleen, and kidneys) from each animal was processed in similar fashion. Microscopic examination of Congo red-stained sections of the amyloidomas revealed the presence of green birefringent material in all samples, albeit considerably less in those obtained from the m and c11-1F4 mAb-treated animals. The most notable difference was apparent in H&E- and chloresterase-treated specimens where the extent of macrophage, as well as activated neutrophil infiltration in the residual amyloidomas was far greater than that seen in controls. Furthermore, amyloid-bound c11-1F4 could be demonstrated immunohistochemically (data not illustrated). Other than the induced amyloid tumors, no gross pathological abnormalities were observed among the mice. Furthermore, the liver, spleen, and kidneys of the m and c11-1F4 mAb-treated and control animals appeared normal microscopically.

An identical protocol was used to compare the therapeutic efficacy of the c and m11-1F4 mAbs in mice injected with a human ALλ (patient BAL) extract that predominantly contained a 16-kDa light chain fragment of amyloidogenic λ3 BJP BAL. Because the amyloidoma in 1 of the m11-1F4 mAb-treated animals began to regress on day 12, 1 mouse from each of the four pairs was sacrificed on day 15. The masses of the amyloidomas removed from the m and c11-1F4 mAb-treated animals had decreased 75 and 15%, respectively, as compared with the control (Table 3). The remaining set was euthanized on day 21; in this case, the amyloidomas were reduced by 58 and 43%, respectively. The results of microscopic examination of the amyloidomas were similar to those described in the ALκ HIG study.

We had previously demonstrated that the amyloid-reactive m11-1F4 mAb accelerated amyloidolysis in mice bearing AA hepatic and splenic deposits (13, 20). Because of the unavailability of animals with systemic AL amyloidosis, we tested the therapeutic efficacy of the c11-1F4 mAb (which had been shown immunohistochemically and by ELISA to react with AA fibrils) in the in vivo AA model. This form of amyloid was induced over a 10-day period by a standard protocol that included injections into 10 mice of silver nitrate and AA-derived fibrils, i.e., AEF (Fig. 7). Five of these animals then received 100-μg i.v. doses of the antibody on days 14, 17, 20, and 23, whereas the remaining 5 served as untreated controls. The animals were sacrificed on day 24 and the liver, spleen, and kidneys examined histochemically. The most pronounced interstitial congophilic deposits in both control and treated animals occurred within the spleen. The amount of this material was quantitated using image analysis and spectral segmentation techniques from 4-μm thick Congo red-stained sections. As illustrated in Fig. 8, there was a statistically significant decrease (P = <.05) in the mean splenic amyloid burden of the c11-1F4 mAb-treated mice versus untreated controls (amyloid burden index, 0.450 and 1.195, respectively). This reduction in splenic amyloid was calculated to be 62%, as compared with 79% in the animals that received the m11-1F4 reagent.

On the basis of the results of the comparative immunological analyses (fibril binding, dot blot, and immunohistochemical assays), it was evident that the reactivity of the c11-1F4 mAb was identical to that of the original murine antibody. Moreover, as demonstrated in studies involving mice bearing human AL amyloidomas or murine AA splenic deposits, the modified reagent, in which the mouse heavy and light chain constant region domains were replaced by their human counterparts, also was capable of accelerating amyloidolysis. Although the precise mechanism(s) responsible for this response is subject to additional investigation, we have shown that the Fc region of the 11-1F4 antibody molecule is essential to initiation of an immune effector response that leads to amyloid degradation (13); thus, we attribute the lesser degree of resolution achieved with the chimeric reagent in the two mouse models to the presence of human, rather than murine protein, in the Fc portion of the immunoglobulin molecule.

Notably, both types of 11-1F4 mAbs were capable of accelerating removal of the ALκ and ALλ amyloidomas. However, as generally found in our previous in vivo studies involving the murine reagent and four κ-type and eight λ-type extracts, the latter took somewhat longer to resolve (13). The factor(s) responsible for this difference remains to be determined.

With regard to potential toxicity, the c11-1F4 mAb, as with its murine counterpart, specifically recognized AL fibrils and failed to bind native light chains, with the exception of κ4 molecules. Presumably, this reactivity would not have an adverse effect clinically, given the fact that immunoglobulins bearing κ4 light chains represent <5% of the total human Igκ population (21). Furthermore, there were no abnormalities noted microscopically in the liver, spleen, or kidneys of the c11-1F4 mAb-treated animals, and in contrast to the c mAb Rituximab, this reagent had little or no reactivity with human tissue.

The demonstration of the effective performance of the c11-1F4 mAb molecule in these in vitro and in vivo studies has formed the basis to proceed with full-scale GMP production of this antibody for an eventual Phase I/II clinical trial involving patients with primary (AL) amyloidosis.

1

Presented at the “Ninth Conference on Cancer Therapy with Antibodies and Immunoconjugates,” October 24–26, 2002, Princeton, NJ. Supported, in part, by United States Public Health Service Research Grant CA10056 from the National Cancer Institute, Contract 21X5034A from Science Applications International Corporation-Frederick, and the Aslan Foundation. A. S. is an American Cancer Society Clinical Research Professor.

3

The abbreviations used are: m, murine; c, chimeric; RAID, Rapid Access to Intervention Development; CH, heavy chain constant region; CL, light chain constant region; MTX, methotrexate; mAb, monoclonal antibody; VH, heavy chain variable region; VL, light chain variable region; AEF, amyloid-enhancing factor; BJP, Bence Jones protein.

Fig. 1.

Analyses by SDS-PAGE of c11-1F4 mAbs under reducing and nonreducing conditions (Coomassie blue-stained Novex bis tris 4–12% NuPage gels). Lane 1 contained protein standards of known molecular masses as indicated; Lane 2, a human IgG mAb; Lanes 46, 22C1, 22C5, and 22D2 mAbs, respectively. The locations of the immunoglobulin heavy and light chains are as indicated.

Fig. 1.

Analyses by SDS-PAGE of c11-1F4 mAbs under reducing and nonreducing conditions (Coomassie blue-stained Novex bis tris 4–12% NuPage gels). Lane 1 contained protein standards of known molecular masses as indicated; Lane 2, a human IgG mAb; Lanes 46, 22C1, 22C5, and 22D2 mAbs, respectively. The locations of the immunoglobulin heavy and light chains are as indicated.

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Fig. 2.

In vitro reactivity of c11-1F4 antibodies. The c11-1F4 mAbs 22C1, 22C5, and 22D2 and m11-1F4 mAb were tested in a binding ELISA against synthetic light chain fibrils (JTO5) coated onto an ELISA plate.

Fig. 2.

In vitro reactivity of c11-1F4 antibodies. The c11-1F4 mAbs 22C1, 22C5, and 22D2 and m11-1F4 mAb were tested in a binding ELISA against synthetic light chain fibrils (JTO5) coated onto an ELISA plate.

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Fig. 3.

HPLC analyses of c11-1F4 mAbs. Elution profiles of 22D2, 22C5, and 22C1 mAbs (A, B, and C, respectively) and protein standards (D) that were passed through a Toso Haas G 3000 SWx1 size exclusion chromatography column (elution buffer, PBS).

Fig. 3.

HPLC analyses of c11-1F4 mAbs. Elution profiles of 22D2, 22C5, and 22C1 mAbs (A, B, and C, respectively) and protein standards (D) that were passed through a Toso Haas G 3000 SWx1 size exclusion chromatography column (elution buffer, PBS).

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Fig. 4.

Comparison by dot blot assay of the reactivities of m and c11-1F4 mAbs against fibrillar and soluble forms of light chains. Top: light chain fibrils were generated from a recombinant Vλ6 protein and applied as “dots” at the concentrations indicated, as were PBS and the soluble Vλ6 protein monomer. Bottom: human κ1, κ4, λ6, and λ8 Bence Jones proteins were likewise applied. Murine and c11-1F4 mAbs were used as primary reagents (5 μg/ml) and goat antimouse and goat antihuman immunoglobulin horseradish peroxidase-labeled antibodies, respectively, were the secondary reagents. The reaction was developed with diaminobenzidine.

Fig. 4.

Comparison by dot blot assay of the reactivities of m and c11-1F4 mAbs against fibrillar and soluble forms of light chains. Top: light chain fibrils were generated from a recombinant Vλ6 protein and applied as “dots” at the concentrations indicated, as were PBS and the soluble Vλ6 protein monomer. Bottom: human κ1, κ4, λ6, and λ8 Bence Jones proteins were likewise applied. Murine and c11-1F4 mAbs were used as primary reagents (5 μg/ml) and goat antimouse and goat antihuman immunoglobulin horseradish peroxidase-labeled antibodies, respectively, were the secondary reagents. The reaction was developed with diaminobenzidine.

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Fig. 5.

Reactivity of c11-1F4 mAb with human vascular AL-containing amyloid deposits. The left and right panels illustrate the results obtained when uterine and pancreatic tissues from patients with κ4- and λ8-associated AL amyloidosis, respectively, were studied. (top: Congo red (CR) stain; middle: anti-VL subgroup-specific mAb; bottom: c11-1F4 mAb; original magnification, ×200).

Fig. 5.

Reactivity of c11-1F4 mAb with human vascular AL-containing amyloid deposits. The left and right panels illustrate the results obtained when uterine and pancreatic tissues from patients with κ4- and λ8-associated AL amyloidosis, respectively, were studied. (top: Congo red (CR) stain; middle: anti-VL subgroup-specific mAb; bottom: c11-1F4 mAb; original magnification, ×200).

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Fig. 6.

Monoclonal amyloid antibody 11-1F4-mediated resolution of a human ALκ amyloidoma. Appearance of residual AL amyloid tumor (HIG) on day 15 in an untreated animal (control, left) and in mice given injections of m11-1F4 mAb, c11-1F4 mAb, or mIgG1κ protein MOPC 31C (control, right) 48 h after amyloidoma induction and then again on days 4, 6, 8, 10, and 12.

Fig. 6.

Monoclonal amyloid antibody 11-1F4-mediated resolution of a human ALκ amyloidoma. Appearance of residual AL amyloid tumor (HIG) on day 15 in an untreated animal (control, left) and in mice given injections of m11-1F4 mAb, c11-1F4 mAb, or mIgG1κ protein MOPC 31C (control, right) 48 h after amyloidoma induction and then again on days 4, 6, 8, 10, and 12.

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Fig. 7.

Protocol for systemic AA amyloid induction and immunotherapy. AA amyloid was induced in mice by injections of silver nitrate (AgNO3) on days 1 and 10 and AA fibrils (AEF) on day 1. Groups of animals received either m or c11-1F4 mAbs at the times indicated. Another (control) group remained untreated.

Fig. 7.

Protocol for systemic AA amyloid induction and immunotherapy. AA amyloid was induced in mice by injections of silver nitrate (AgNO3) on days 1 and 10 and AA fibrils (AEF) on day 1. Groups of animals received either m or c11-1F4 mAbs at the times indicated. Another (control) group remained untreated.

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Fig. 8.

Chimeric 11-1F4 mAb-induced resolution of systemic murine AA amyloid. Graphic representation of percent of AA amyloid remaining in splenic tissue obtained from mice that were injected with the c11-1F4 (treated) and those that received no antibody (control). The amyloid burden index (ABI) was calculated according to the following formula: ABI = CR + area/total area × 100.

Fig. 8.

Chimeric 11-1F4 mAb-induced resolution of systemic murine AA amyloid. Graphic representation of percent of AA amyloid remaining in splenic tissue obtained from mice that were injected with the c11-1F4 (treated) and those that received no antibody (control). The amyloid burden index (ABI) was calculated according to the following formula: ABI = CR + area/total area × 100.

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Table 1

Properties of CHOdhfr cell line mAb products

Cell lineStability (4°C, 37°C, −70°C)aSDS PAGEa,bFibril binding assaya,bAggregationbDot blotcImmunohistochemistryc
22C1 +,+,+ 
22C5 +,+,+ NDd 
22D2 +,+,+ ND 
Cell lineStability (4°C, 37°C, −70°C)aSDS PAGEa,bFibril binding assaya,bAggregationbDot blotcImmunohistochemistryc
22C1 +,+,+ 
22C5 +,+,+ NDd 
22D2 +,+,+ ND 
a

AERES data.

b

RAID data.

c

UT data.

d

ND, not determined.

Table 2

ALκ (HIG) amyloidoma study

Antibody treatmentWeight of residual amyloidoma (g)aPercentage of reductionb
Murine 11-1F4 mAb 0.02c 0.17d 97c 68d 
Chimeric 11-1F4 mAb 0.16 0.18 73 66 
Murine IgG1κ MOPC 31C 0.59 0.53  
None 0.56  
Antibody treatmentWeight of residual amyloidoma (g)aPercentage of reductionb
Murine 11-1F4 mAb 0.02c 0.17d 97c 68d 
Chimeric 11-1F4 mAb 0.16 0.18 73 66 
Murine IgG1κ MOPC 31C 0.59 0.53  
None 0.56  
a

Wet weight.

b

Compared with MOPC controls.

c,d

Days 15 and 16, respectively.

Table 3

ALλ (BAL) amyloidoma study

Antibody treatmentWeight of residual amyloidoma (g)aPercentage of reductionb
Murine 11-1F4 mAb 0.13c 0.22d 75c 58d 
Chimeric 11-1F4 mAb 0.44 0.30 15 43 
Murine IgG1κ MOPC 31C 0.52 0.53  
None 0.45  
Antibody treatmentWeight of residual amyloidoma (g)aPercentage of reductionb
Murine 11-1F4 mAb 0.13c 0.22d 75c 58d 
Chimeric 11-1F4 mAb 0.44 0.30 15 43 
Murine IgG1κ MOPC 31C 0.52 0.53  
None 0.45  
a

Wet weight.

b

Compared with MOPC controls.

c,d

Days 15 and 21, respectively.

We thank Dr. Siobhán O’Brien and Dr. Tarran Jones of AERES Biomedical Ltd. for preparing the c11-1F4 mAbs used in this study. We also thank Drs. Morris Kelsey, Barry Kobrin, Aparna Kolehkar, and other staff of the Biological Resource Branch and Biopharmaceutical Development Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute-Frederick Cancer Research and Development Center for their scientific input and project oversight. We also thank Ms. Ronda Reed for secretarial assistance and Sallie Macy, Charles, L. Murphy, Dr. Lian Tang, Teresa Williams, Dennis Wolfenbarger, and Craig Wooliver for their technical contributions.

1
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