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Clinical Cancer Research Vol. 5, 899-908, April 1999
© 1999 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Molecular Modeling and Preclinical Evaluation of the Humanized NR-LU-13 Antibody1

Scott S. Graves2, Stephen C. Goshorn, Diane M. Stone, Don B. Axworthy, John M. Reno, Becky Bottino, Stephen Searle, Andrew Henry3, Jan Pedersen4, Anthony R. Rees and Randell T. Libby5

Departments of Molecular Biology [S. S. G., S. C. G., D. M. S.], Pharmacology [D. B. A., J. M. R., B. B.], and Pathobiology [R. T. L], NeoRx Corporation, Seattle, Washington 98119, and Department of Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom [S. S., A. H., J. P., A. R. R.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A mouse-human chimeric monoclonal antibody (chNR-LU-13), specific for the EGP40 pancarcinoma antigen, was humanized through three-dimensional molecular modeling. Humanization of the chNR-LU-13 antibody is expected to enhance its use for patients undergoing immunotherapy. On the basis of the observed amino acid sequence identity, chNR-LU-13 complementary determining regions (CDRs) of the VL and VH regions were grafted onto the human anti-DNA-associated idiotype immunoglobulin clone, R3.5H5G'CL. Ten amino acids residues within the humanized framework were back-mutated to their corresponding chNR-LU-13 sequence, because they were predicted to disrupt the canonical classification of the CDRs or were within 5 Å of a CDR. Synthesis of the VL and VH regions was accomplished by recursive PCR, and the dual-chain expression vector p451.C4 was positioned under control of the CMVP+E. We observed by competitive ELISA that the recombinant humanized NR-LU-13 (huNR-LU-13) IgG1 antibody exhibited an indistinguishable immunoreactivity profile when compared with the murine monoclonal antibody (muNR-LU-10). The huNR-LU-13 antibody was effective in mediating both antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity when assayed against either the breast carcinoma cell line, MCF-7, or the colon adenocarcinoma cell line, SW1222. Biodistribution studies using i.v. coinjected 131I-muNR-LU-10 and 125I-huNR-LU-13 confirmed that the huNR-LU-13 specifically targets to the tumor in athymic BALB/c mice bearing the SW1222 human tumor xenograft. Humanization of the chNR-LU-13 antibody is expected to eliminate an undesired human anti-mouse antibody response, allowing for repeated i.v. administration into humans.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The use of antibodies for imaging and treatment of human tumors has been the subject of intense research for many years. Antibody research has focused, for the most part, on several areas: the development of appropriate tumor-specific antibodies, as well as methodologies for the attachment of diagnostic and toxic agents to the antibody. Stable attachment of compounds to the antibody is needed for efficient tumor localization and subsequent growth suppression in vivo (1) . However, a number of difficulties, including the strong HAMA6 response and rapid clearance of murine antibodies in patients undergoing immunotherapy, have greatly limited their therapeutic potential (2, 3, 4, 5) .

It has been suggested that these problems may be overcome through the use of hybrid chimeric antibodies in which the mouse variable and human constant regions are genetically fused. Recombinant human-mouse antibodies of this nature, while retaining the specificity of the original murine variable region, would be expected to assume the effector function of the human constant region. Chimeric antibodies constructed in this manner have thus far produced encouraging preliminary results (6, 7, 8, 9, 10) .

The murine IgG2b monoclonal antibody, NR-LU-10 (muNR-LU-10), has been shown previously to be reactive with epithelial tumors including carcinomas of the lung, colon, ovary, and breast (11) . It is one of a group of antibodies known as the small cell lung cancer cluster-2 antibodies, which reacts with a Mr 40,000 EGP (EGP40) antigen known as EGP-2, GA733–2, KSA, and 17-1A (12, 13, 14, 15, 16) . The antigen is nominally present on nonmalignant epithelia, yet is abundantly expressed on many carcinomas, suggesting an important role in epithelial structure and function. Numerous antibodies to this antigen, including KS1/4, have been used in clinical trials for the diagnosis and dose-dependent in vivo suppression of various adenocarcinomas (17, 18, 19) .

It has recently been demonstrated that Tc-99m-labeled muNR-LU-10 Fab fragment is an effective imaging and staging antibody for small cell lung carcinoma (20) . Therapeutic tumor suppression using 186Re whole murine antibody, however, requires multiple dosing regimens (21) . Reduced clinical efficacy in vivo has been attributed to rapid clearance from the blood, primarily due to the HAMA response (4 , 22 , 23) . A chimeric version of the murine NR-LU-10 antibody, designated NR-LU-13 (chNR-LU-13), targets tumors with reduced immunogenicity and delayed clearance, vis-à-vis the murine antibody (24) . Recent efforts to humanize antibodies have been undertaken in an effort to remove the HAMA response, thus eliminating the potential barrier for multiple dosing regimens.

In the present report, we describe a detailed construction and preclinical characterization of a humanized version of the chimeric NR-LU-13 antibody containing the murine CDRs grafted onto a human antibody framework. The construct we describe has been demonstrated by competitive ELISA to retain immunoreactivity, while exhibiting preferential localization to the tumor in BALB/C nude mice bearing s.c. SW1222 colon tumor xenografts. We further discuss the potential utility of the humanized NR-LU-13 (huNR-LU-13) antibody as an effective imaging and therapeutic agent for the diagnosis and treatment of antigen-positive solid tumors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Supplies.
DNA-modifying restriction enzymes and oligo(dT) columns were purchased from either Promega Corp. (Madison, WI) or Life Technologies, Inc. (Grand Island, NY). TaqI or Ultima DNA polymerase, as well as all PCR-related reagents, were purchased from Perkin-Elmer (Branchburg, NJ). The cDNA holding vector, pSPORT, as well as Escherichia coli host strain DH10B were purchased from Life Technologies, Inc. LipofectAce and IMDM, as well as hypoxanthine, thymidine, and G418, were obtained from Life Technologies, Inc. DMEM/F12 and FCS were obtained from BioWhittaker (Walkersville, MD). Dialyzed fetal bovine serum was obtained from Sigma Chemical Co. (St. Louis, MO). The mammalian vector, pCDNA3, containing the enhancer-promoter sequences from CMV, as well as bovine growth hormone polyadenylation signal were obtained from Invitrogen (Carlsbad, CA). Ampholytes were obtained from Pharmacia (Piscataway, NJ). All other standard reagents, including antibiotics and MTX, were of molecular biology grade and were obtained from Sigma. CHO cells deficient in the dhfr gene (CRL 9096), as well as SW1222 and MCF-7 cells, were obtained from the American Type Culture Collection. Probes specific for either the murine heavy or light chains were synthesized on an Applied Biosystems 381A DNA synthesizer. BALB/C and athymic nude mice used in the biodistribution studies were obtained from Batton and Kingen (Seattle, WA). Radioisotopes were obtained from the New England Nuclear Corp. (Wilmington, DE).

DNA Manipulations.
Cloning and general DNA manipulations were performed according to established procedures (25) . Oligonucleotides specific for either the heavy or light antibody chains were synthesized on an Applied Biosystems 381A DNA synthesizer (Foster City, CA), and desalted on NAP-25 columns (Pharmacia) according to the manufacturer’s recommendations. PCR amplifications were performed according to the manufacturer’s instructions on a Coy model 110A thermal cycler. Unless otherwise indicated, PCR reactions were preincubated for 5 min at 94°C, followed by 30 cycles of denaturation (0.5 min at 94°C), annealing (1 min at 55°C), and primer extension (1 min at 74°C), coupled with a final extension step for 5 min at 74°C. Reaction products were used in ligation reactions without further purification. DNA sequencing was performed by the dideoxy chain termination procedure (26) .

Rationale for Humanization of the Murine Chimeric Antibody.
The VH and VL regions from the murine chimeric antibody were subjected to dideoxy sequence analysis, and the inferred amino acid sequences were compared with a database of 485 human VL and 701 VH sequences of which several were from the same clone (27) . These sequences were scored for sequence identity using the screening program QUALIS (28) . The most identical human sequence, R3.5H5G'CL (29) , was selected to supply the framework for the grafted antibody: the sequence of the original chNR-LU-13 CDRs was then modeled onto the R3.5H5G'CL human framework, providing for the initial humanized Fv sequence. Molecular models were constructed based on the previously described combined algorithms of Martin et al. (29) and Pedersen et al. (30 , 31) , and wherever possible, CDRs were modeled from established canonical loops (32) . Remaining loops were modeled using a combination of database search and ab initio methods, using the conformational search program CONGEN (33) . In the case of the chNR-LU-13 CDRs, L1, L2, L3, H1, and H2 were built from canonical loops. CDR H3, on the other hand, was constructed using a database search at the base of the CDR and ab initio fragment generation for the central portion of the loop in an attempt to saturate conformational space. CDR H3 was built onto a combining site containing the backbone atoms of the canonical loop and all atoms for framework residues. All CDR side chains were reconstructed using CONGEN as described above. The models were energy minimized by maintaining fixed framework regions, while framework side chains and CDRs were allowed to move. At framework positions where the chimeric and human sequences differed, the chimeric residue was selected as a back mutation if it appeared to affect the conformation of the CDRs. Conformational effects were determined by changes in the classification of the CDR canonical structures and by modeling the effects of back mutating residues within 5 Å of the CDRs. Final selection of the variable region sequence was based on a visual comparison of models of the R3.5H5G'CL framework and the chNR-LU-13 sequence.

Synthesis of the Humanized NR-LU-13 Construct.
On the basis of the amino acid sequence of the humanized model, a series of eight overlapping synthetic primers was used sequentially in recursive PCR sewing reactions (34) to synthesize the complete humanized VH chain. Primers NxH1 -> NxH8 were annealed and subjected to gap fill-in by PCR amplification in a reaction containing 30 pmol of the terminal oligonucleotides NxH1/NxH8 and 1 pmol each of the internal oligonucleotides NxH2-> NxH7 (Fig. 1A)Citation . The synthesized VH chain was digested with HindIII/BamHI, and the resulting 439-bp fragment was inserted into the complementary site of vector p4{gamma}B, containing the genomic {gamma}1 constant heavy (gCH) chain region obtained from the human B cell plasmacytoma cell line MC/CAR (ATCC CRL 8083). The VL chain was synthesized using primers NxK1 -> NxK8 in a manner similar to the procedure used for the synthesis of the VH chain (Fig. 1B)Citation , and the complete chain was digested with the restriction enzymes NotI/NheI. The resulting 426-bp VL fragment was inserted into the complementary site of vector pVKE containing the genomic light chain kappa constant (gCL) region obtained by PCR amplification of genomic DNA from peripheral blood lymphocytes. The intermediate vectors pVKE and p4{gamma}B were digested with BglII/EcoRI, and the 6-kbp fragment from pVKE containing the VL-gCL region was ligated with the 3.2-kbp fragment from p4{gamma}B containing the VH-gCH1 -> H3 region to generate the intermediate expression vector, pWE1A2 (Fig. 2A)Citation . Total RNA extracted from pWE1A2-transfected CHO cells was subjected to reverse transcriptase-mediated PCR (RT-PCR) using primers complementary to the 5' and 3' termini of both the gCL and gCH regions. The CL and CH chain cDNAs were restricted with NheI/ApaI and XbaI/BamHI, respectively, and inserted into the complementary sites of pWE1A2 generating the complete cDNA expression vector, p451.C4 (Fig. 2B)Citation .



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  		Fig. 1. Schematic of synthesis of the humanized variable chain. A, oligonucleotides NxH1-> NxH8 were synthesized and used sequentially in recursive PCR gap fill-in reactions to construct the VH immunoglobulin chain. The completed chain was digested with HindIII/BamHI, and the 439-bp fragment was inserted into the vector p4{gamma}B containing the genomic constant heavy chain region. B, oligonucleotides NxK1-> NxK8 were synthesized and similarly used in recursive PCR gap fill-in reactions to generate the VL immunoglobulin chain. The 426-bp amplification product was digested with NotI/NheI and inserted into vector pVKE containing the genomic constant light chain region.

 


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  		Fig. 2. Schematic diagrams of the humanized NR-LU-13 expression vectors. A, the intermediate expression vector, pWE1A2, contains the 3.2-kb fragment from p4{gamma}B consisting of the VH and genomic constant heavy (gCH) chain region, which is fused to the 6-kb fragment from pVKE containing the genomic constant light (gCL) ligated downstream of the VL region. Dual expression of both the heavy and light chain regions has been placed under control of the constitutive CMV promoter and enhancer (CMVP+E) elements, and polyadenylation signal as well as transcript termination was accomplished through use of the bovine growth hormone (BGH) signal. B, the final expression vector, p451.C4, replaces the gCH and gCL with its corresponding cDNA. Both plasmids contain the ColE1 ori and neomycin resistance gene for selection in CHO cells.

 
Generation of the Humanized Antibody.
CHO cells deficient in the dhfr gene were grown in IMDM containing 10% dialyzed fetal bovine serum supplemented with 100 µM hypoxanthine and 16 µM thymidine. Exponentially growing cells were transfected with 5 µg of BglII linearized vector p451.C4 using LipofectAce according to the manufacturer’s recommendations and incubated for 48 h at 37°C. Transfected cells were grown in selective media containing 800 µg/mL G418. Heavy and light chains were amplified by stepwise increases with MTX to a maximum of 50 µM. The highest producing clone, designated NRX451, was subjected to two additional rounds of limiting dilution cloning.

Purification and Characterization.
Clone NRX451 was expanded over six passages to multiple 1-liter spinner flasks in IMDM containing 10% dialyzed fetal bovine serum and 50 µm MTX until the culture reached a density of 1 x 106 cells/ml. The 0.1-µm hollow fiber clarified supernatants containing the secreted huNR-LU-13 antibody were purified by batch DEAE-Sepharose (Pharmacia), followed by affinity chromatography on rProtein A-Sepharose (Pharmacia) according to the manufacturer’s recommendations. Final purity of the humanized antibody was determined by size exclusion high-performance liquid chromatography at an absorbance wavelength of 280 nm. Carbohydrate analysis and linkage specificity was determined by FACE as described previously (35 , 36) using the deglycosylating enzymes PNGaseF, NANaseIII, and ß-galactosidase as described previously (37) .

ELISAs.
Goat F(ab')2 anti-human IgG and peroxidase-labeled goat anti-human kappa (Tago) were used in a sandwich ELISA as capture/detection antibodies for the quantitation of the whole antibody according to established procedures. Regression analysis was performed using the chNR-LU-13 antibody as a standard.

Immunoreactivity was assessed in a competitive binding ELISA where serial dilutions of both the humanized and native murine antibodies were allowed to compete with peroxidase-labeled murine NR-LU-10 for binding to an 0.1% NP40 extract from the LS-174T human colon adenocarcinoma cell line (38) . After a log-logit transformation of the data, where curves were fit to the same slope, the concentration of competitor antibody required to achieve 50% inhibition (k) was calculated. The percentage of immunoreactivity was determined according to the following formula: k (murine standard)/k (test) x 100.

Cytotoxicity Assays.
SW1222 (colon carcinoma) and MCF-7 (breast carcinoma) cells were labeled with 200 µCi 51Cr-chromate (DuPont NEN) at 2 x 106 cells/ml in DMEM/F12 medium. After washing in medium containing 10% FCS, 1 x 104 cells were incubated in triplicate in 96-well microtiter plates with either the muNR-LU-10 or huNR-LU-13 antibody for 15 min. Human serum was added at a final concentration of 10% as a complement source for evaluating C'MC activity. Human peripheral blood mononuclear cells from healthy donors were added for ADCC at an E:T cell ratio of 20:1, whereas antibody was titrated for both C'MC and ADCC at 10-fold dilutions. After centrifugation, labeled target cells with antibody and complement, or antibody and effector cells, were incubated at 37°C in a humidified chamber containing 5% CO2 for 3.5 h. Supernatants were removed from microtiter wells, and cpm was determined in a Packard Model Cobra II Gamma Counter. The percentage of cytotoxicity was determined according to the following formula:

Spontaneous release for C'MC was calculated from the supernatants of the target cells containing 10% human serum.

Biodistribution.
Female BALB/C nu/nu mice or female BALB/C mice, 8 weeks of age, housed four per cage in temperature and humidity-controlled microisolators, were maintained on sterilized Lab Blox chow and water ad libitum. All treated animals were isolated in a shielded area, and studies were conducted in an accredited facility in accordance with the recommendations set forth by the Association for the Assessment and Accreditation of Laboratory Animal Care International. Treated animals were injected s.c. in the left flank with ~5 x 106 SW1222 colon carcinoma cells. Tumors were allowed to grow for 10–14 days until tumor volumes reached 100–200 mm3. The muNR-LU-10 and huNR-LU-13 antibodies were radiochemically labeled with 131I or 125I, respectively, according to established procedures (39) , and the injected dose was standardized to 25 µg/mouse of each antibody per mouse. Biodistribution studies were conducted to compare the blood clearance, as well as tumor xenograft and non-target organ localization of the muNR-LU-10 and huNR-LU-13 antibodies. After i.v. coinjection of 25 µg each of 131I-muNR-LU-10 and 125I-huNR-LU-13 antibodies into nude mice bearing the SW1222 colon carcinoma, the percentage of injected dose was calculated from a dilution standard, and the weighted volume of the material was injected. Groups of four mice per time point were weighed, bled via retroorbital plexus, and sacrificed by cervical dislocation at 4, 24, 48, and 192 h after injection. Tissues were blotted and weighed, and the count rate was determined in a Packard Cobra II dual-channel gamma counter (Laguna Hills, CA). Tumor-specific antibody localization was determined based on the following formula:


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Modeling of the Humanized CDR Regions.
The deduced amino acid sequence of both the VL and VH domains from the chNR-LU-13 sequence was compared with the immunoglobulin sequence database (27) , and the most identical human antibody was selected to provide the framework sequence for the grafted antibody. The chNR-LU-13 amino acid sequence was compared with 58 {kappa} heavy chain pairs, which are known to be expressed together as functional antibodies, and the most identical sequence pair was found to be associated with the human immunoglobulin clone R3.5H5G'CL (29) . The chimeric sequence consists of a {kappa}V/hIIc chain pair, whereas the R3.5H5G'CL clone is known to express an antibody that is a {kappa}I/hI chain pair, suggesting that both light and heavy chains were selected from the most homologous human classes. Comparison of the sequence surrounding the CDRs in the chNR-LU-13 and R3.5H5G'CL clone revealed ~55–70% homology within the FW I and FW III region in the VH domain, whereas somewhat higher sequence identities were observed for the same comparable regions of the VL domain. The region immediately 3' to CDR3, represented as FW IV in both the VH and VL domain, exhibited >90% sequence identity between the donor framework region and the chNR-LU-13 sequence (Table 1)Citation . The huNR-LU-13 model was generated by grafting the chNR-LU-13 CDRs onto the human antibody framework of R3.5H5G'CL, thus generating the humanized Fv sequence. The initial humanized sequence was refined following three-dimensional modeling in which the original chNR-LU-13 and huNR-LU-13 were visually compared. Several residues within the humanized model, which were observed to perturb the structure of the CDRs, were back mutated to the chimeric sequence when any residue within 5 Å of a CDR contained an altered residue type. Thus, immediately surrounding the CDRs, seven residues in the heavy chain and three residues in the light chain were back mutated to the chimeric sequence to preserve the CDR region and surrounding structure in the humanized version (Fig. 3)Citation .


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  		Table 1 Framework regions of homology

 


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  		Fig. 3. Comparison of the predicted amino acid sequence surrounding CDR1 -> CDR3. The standard single-letter symbols for amino acid residues are used. Amino acid residues in the chNR-LU-13 antibody are shown aligned with their corresponding region in the huNR-LU-13 model as well as the R3.5H5G'CL sequence. Underlined residues indicate those positions in the huNR-LU-13 model that were back mutated to their corresponding residues in the chNR-LU-13 sequence. Dots between sequences indicate regions of homology. Numbering is according to Kabat et al. (27) .

 
Expression and Characterization of the Humanized NRX451 Antibody.
dhfr-deficient CHO cells transfected with the linearized vector pNX451.C4 expressed ~10 pmol/cell/day of the huNR-LU-13. The humanized antibody exhibited profiles essentially comparable with both the muNR-LU-10 and chNR-LU-13 antibody when analyzed on SDS-PAGE gels in the presence or absence of 2 µM ß-mercaptoethanol (data not shown). On the basis of the changes introduced into the primary structure of the humanized antibody, a pI value in the range of ~9.0 was predicted from the sequence. Although the muNR-LU-10 and chNR-LU-13 exhibited pIs in the range of 5.85 to 6.85 and 7.35 to 8.00, respectively, the huNR-LU-13 exhibited a pI in the range of 8.65 to 9.2, consistent with its predicted theoretical pI (data not shown). However, the huNR-LU-13 antibody exhibited an immunoreactivity profile that was indistinguishable from the muNR-LU-10 antibody (Fig. 4)Citation , suggesting that the humanization process did not affect the binding characteristics of the antibody.



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  		Fig. 4. Competitive immunoreactivity profile in which the huNR-LU-13 antibody ({triangleup}) and muNR-LU-10 antibody ({blacksquare}) was allowed to compete with a peroxidase-labeled muNR-LU-10 antibody for binding to an 0.1% NP40 antigen extract from the LS-174T human colon adenocarcinoma cell line.

 
It has been known that IgG proteins are glycosylated at the conserved Asn-297 site within the CH2 region and is believed to stabilize the molecule and contribute to the tertiary structure of the Fc region (40) . To compare glycosylation patterns of the huNR-LU-13 and muNR-LU-10 antibodies, N-linked oligosaccharide profiling was performed. Release of N-linked oligosaccharides from the proteins was carried out with the deglycosylating enzyme PNGase F, known to cleave Asn-linked hybrid, complex, and high mannose oligosaccharides from glycoproteins. Cleavage of the humanized and murine antibodies with PNGase F, followed by FACE electrophoresis, revealed the presence of bands migrating at the location of the G6 and G9 polymers of a partial wheat starch digest, typical of N-linked oligosaccharides (Fig. 5Citation , Lanes 3 and 6). The two antibodies contain virtually identical profiles in terms of band migration and general appearance. An exception is the slowest migrating N-linked oligosaccharide, greatly reduced in the humanized version, at approximately the G8 position. Structural analysis of released carbohydrates was then analyzed with the exoglycosidases NANase III and ß-galactosidase. Digestion with these enzymes results in the removal of either terminal sialic acid or galactose residues. Neuraminidase digestions were then performed on the released carbohydrates to determine the presence of terminal sialic. The charged sialic acid residues result in an increased mobility during FACE electrophoresis as compared with desialyated oligosaccharides. Thus, loss of sialic acid (charge) retards mobility within the gel, having a greater effect on band position than loss of molecular weight. The profiles before and after NANase III treatments remained the same in both cases; none of the bands shifted, confirming that neither the human or murine antibodies contain sialylated oligosaccharides (Fig. 5Citation , Lanes 2 and 5). Further treatment of the oligos with ß-galactosidase resulted in a lost of uncharged galactose residues, resulting in the production of a single band that comigrated with the fast migrating band in the original N-linked profile (Fig. 5Citation , Lanes 4 and 7). The apparent comigration with an asialo, agalacto oligosaccharide suggests that one of the oligosaccharides in both the muNR-LU-10 and huNR-LU-13 glycoproteins contain terminal N-acetylglucosamine residues.



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  		Fig. 5. N-Linked muNR-LU-10 and huNR-LU-13 carbohydrate analysis after glycosidic digestion and FACE electrophoresis. Lanes 1 and 8, glucose polymers from partial wheat starch digest; Lanes 2 and 5, PNGaseF + NANaseIII glycosidic digestions; Lanes 3 and 6, PNGaseF glycosidic digestion; Lanes 4 and 7, PNGaseF + ß-galactosidase digestions.

 
Effector Function.
Both murine and humanized antibodies were evaluated for their cytotoxic effector function against the breast carcinoma cell line MCF-7 and the colon adenocarcinoma cell line SW1222, using a 51Cr release assay (41) . The murine NR-LU-10 antibody failed to mediate either ADCC (Fig. 6A)Citation or C'MC (Fig. 6B)Citation with either the MCF or SW1222 cell lines at all antibody concentrations that were examined. In contrast, huNR-LU-13 antibody was effective in mediating high levels of cytotoxicity when assayed against either cell line for both ADCC (Fig. 7A)Citation and C'MC (Fig. 7B)Citation .



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  		Fig. 6. Evaluation of cytotoxic effector cell function of the muNR-LU-10 and huNR-LU-10 antibodies via 51Cr-release assay. A, ADCC with either muNR-LU-10 or the huNR-LU-13 antibody and human peripheral blood mononuclear cells for 3.5 h. B, compliment-mediated cytotoxicity analysis of either the muNR-LU-10 or huNR-LU-13 antibody and human serum for 3.5 h. Open symbols, MCF-7 cells; closed symbols, SW1222 cells; circles, muNR-LU-10 antibody; squares, huNR-LU-13 antibody.

 


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  		Fig. 7. Biodistribution of the muNR-LU-10 and huNR-LU-13 antibodies in athymic nude mice bearing the s.c. SW1222 human tumor xenograft. Equimolar quantities of 131I-muNR-LU-10 and 125I-NR-LU-13 antibody were i.v. coinjected, and animals were sacrificed at 4, 24, 48, and 192 h after injection. A, biodistribution represented as the percentage of injected dose/gram of tissue. BL, blood; TA, tail; SK, skin; MU, muscle; BO, bone; LU, lung; LV, liver; SP, spleen; ST, stomach; KD, kidney; INT, intestine; TU, tumor. Bars, SD. B,131I-muNR-LU-10 and 125I-huNR-LU-13 tumor:blood ratio analyzed through 192 h after injection. Bars, SD.

 
Biodistribution Studies.
Athymic nude mice bearing the s.c. SW1222 human tumor xenograft were used to evaluate potential differences in the pharmacokinetic, as well as in vivo immunoreactivity properties of the murine and humanized antibodies. Equimolar quantities of both 131I-muNR-LU-10 and 125I-huNR-LU-13 antibody preparations were coinjected to control for variability in the biodistribution data between animals, as well as to allow for the direct in vivo comparison of the targeting specificity. A consistent decline in the level of tissue-specific antibody was observed in the blood as well as soft tissues, as measured by the percentage of injected dose/gram throughout the time course examined (Fig. 7A)Citation . All tissues examined displayed similar clearance profiles 4 h after injection. Only the tumor site continued to target elevated levels of both the muNR-LU-10 and huNR-LU-13 antibody through 48 h after injection. Although the percentage of injected dose of both antibodies exhibited similar profiles, the murine antibody preparation appeared to target to the tumor at slightly higher levels. After correction for variances in blood pool concentrations, both antibodies were found to exhibit indistinguishable biodistribution profiles at all time points examined (data not shown). Moreover, when one examines the radiolocalization of the antibody, a nearly identical increase in antibody localization to the tumor was observed for the muNR-LU-10 and huNR-LU-13 antibody preparations through 192 h after injection (Fig. 7B)Citation . It is clear that full immunoreactivity is retained in the huNR-LU-13 antibody, and that little effect in the overall non-target tissue biodistribution has been imparted by the humanization process.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Use of the chNR-LU-13 antibody for targeting of antigen-positive solid tumors has been hampered by the potential HAMA response upon repeated administration. Such responses have been reported to contribute to the rapid clearance of murine and recombinant antibodies, resulting in a reduction in the dose required for effective targeting (37) .

In an effort to reduce potential HAMA responses and thus increase its therapeutic efficacy, we have synthesized a humanized version of chNR-LU-13 based on molecular modeling procedures. Initially, 24 structural models representing intermediates of the grafting process were generated based on data obtained from the Immunoglobulin Sequence Database (27) , as well as Brookhaven Protein Database (42) , prior to establishment of an optimal humanized sequence. The most identical human immunoglobulin framework sequence obtained from the anti-DNA-associated idiotype immunoglobulin clone, R3.5H5G'CL, was selected for framework modeling. The original CDRs from the chNR-LU-13 antibody were grafted onto the R3.5H5G'CL framework in an effort to retain the same antigen recognition site as the panreactive murine monoclonal antibody, NR-LU-10 (11 , 23) . Three-dimensional modeling of the initial humanized sequence revealed several R3.5H5G'CL framework residues that either disrupted the structure of the CDRs within the VH domain or resulted in significant alterations in either the side chain or backbone configuration. Only one position representing the determinant for CDR H2 was observed where the R3.5H5G'CL residue resulted in a change in the canonical classification of the CDR. The original specificity of CDR H2 was retained by back mutating Arg-71 in the R3.5H5G'CL structure to Ala-71 in the chimeric antibody. Additionally, Thr-24, Tyr-59, Ala-60, Met-69, Ser-76, and Ala-93 in the VH domain, as well as Ser-49 in the VL domain, were back mutated to the chNR-LU-13 residue to correct for structural distortions in the molecule. Lastly, Thr-69 and Phe-71 in the VL domain were back mutated to their corresponding chimeric residue to avoid potential structural and functional distortion of the nearby CDRs. Most of the differing residues were positioned at the base of the FV domain, toward the COOH-terminal portion of the Fab fragment, consistent with the prediction that certain CDR conformations are determined by relatively few residues near the CDR or within the framework region itself (31) .

huNR-LU-13, which retains the murine CDRs specific for the EGP40 antigen, was expressed in CHO cells from the dual chain vector, p451.C4. These cells have been exploited previously to produce several therapeutic proteins for research and clinical applications (43, 44, 45) . Because the degree of antibody glycosylation has been demonstrated to alter both the effector function and tissue localization (46) , we examined their N-linked oligosaccharide profiles. Digestion with PNGaseF confirmed the presence of N-linked oligosaccharides as determined by the presence of bands generally between the G6 and G9 polymers of a partial wheat starch digest, typical of N-linked oligosaccharides (Fig. 5)Citation . A single minor N-linked oligosaccharide observed in the murine NR-LU-10, however, was not detected in the humanized antibody. Because the loss of sialic acid residues have been attributed to rapid blood clearance and enhanced localization of recombinant chimeric anti-carcinoma antibodies to the liver (46) , we performed exoenzyme digests on PNGaseF profiles to determine whether the murine and humanized antibodies contain sialic acid residues. It is apparent that although minor variations in band position were observed after NANaseIII digestion of PNGase profiles, the majority of bands in both the murine and humanized antibody preparations did not shift and thus are not sialylated. The two slowest migrating bands in both the ch- and huNR-LU-10 antibodies are digalacto and monogalacto biantennary oligosaccharides. ß-Galactosidase digestions of the PNGase profiles confirmed that both proteins contained terminal N-acetylglucosamine residues consistent with other humanized antibodies (47) .

Thus, the humanization process, including back mutations surrounding the grafted CDRs, did not disrupt the immunoreactivity of the antibodies with the LS174T colon adenocarcinoma cell line. In addition, the humanized antibody demonstrated ADCC and C'MC effector function consistent with the human IgG1 isotype. In biodistribution studies, 131I-muNR-LU-10 and 125I-huNR-LU-13 were capable of efficiently targeting to the tumor. Radiolocalization of both antibodies displayed nearly identical biodistribution profiles after correction for blood pool concentrations. All non-tumor-bearing tissues failed to accumulate either the 131I-muNR-LU-10 or 125I-huNR-LU-13 antibodies. The near identical biodistribution profiles observed between the murine and humanized antibodies further support our position that the humanization process resulted in retention of both the binding and tumor-targeting specificity conferred to the R3.5H5G'CL antibody by the grafted CDRs. Preliminary clinical studies demonstrate that although an expected strong HAMA response was observed in those patients receiving the muNR-LU-10 antibody, patients receiving the huNR-LU-13 antibody failed to demonstrate an immune response.7

These studies demonstrate that molecular modeling practices can be used to select the most appropriate humanized framework to graft CDRs specific for developing therapeutic antibodies to tumors bearing the well-characterized EGP40 antigen. In the future, the huNR-LU-13 will serve as a platform for the development of pretargeting antibodies useful in the clinical diagnosis and therapy of cancer.


   ACKNOWLEDGMENTS
 
We thank Doug Woodle for expert technical assistance in the construction of the humanized antibody. We acknowledge Glyko (Novato, CA) for their assistance in FACE technology and are especially grateful to Chuck Hague for critical review of the N-linked oligosaccharide profiling experiments. Special thanks are extended to Lawrence Loeb for review of the manuscript. We are grateful to Andrea Fahlenkamp Kiesel for skillful assistance in the preparation of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by NeoRx Corporation. Back

2 To whom requests for reprints should be addressed, at NeoRx Corporation, 410 W. Harrison St., Seattle, WA 98119. Phone: (206) 281-7001; Fax: (206) 298-9442. Back

3 Present address: Oxford Molecular Ltd., The Medawar Centre, Oxford Science Park, Sandford upon Thames, Oxon OX4 4GA, United Kingdom. Back

4 Present address: Receptor Technologies A/S, Fabriksparken 58, 2600 Glostrup, Denmark. Back

5 Present address: University of Washington, School of Medicine, Division of Medical Genetics, Department of Laboratory Medicine, Seattle, WA 98195. Back

6 The abbreviations used are: HAMA, human anti-mouse antibody; EGP, epithelial glycoprotein; CMV, cytomegalovirus; MTX, methotrexate; CHO, Chinese hamster ovary; VL, variable light; VH, variable heavy; IMDM, Iscove’s modified Dulbecco’s medium; FACE, Fluorophore-assisted carbohydrate electrophoresis; ADCC, antibody-dependent cytotoxicity; ch, chimeric; hu, humanized; CDR, complementary determining region; FW, framework region. Back

7 S. Graves, personal communication. Back

Received 10/ 9/98; revised 1/ 8/99; accepted 1/13/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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