Download Molecular Modeling and Preclinical Evaluation of the Humanized

Vol. 5, 899 –908, April 1999
Clinical Cancer Research 899
Molecular Modeling and Preclinical Evaluation of the Humanized
NR-LU-13 Antibody1
Scott S. Graves,2 Stephen C. Goshorn,
Diane M. Stone, Don B. Axworthy, John M. Reno,
Becky Bottino, Stephen Searle, Andrew Henry,3
Jan Pedersen,4 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.]
ther 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-huNRLU-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 antimouse antibody response, allowing for repeated i.v. administration into humans.
INTRODUCTION
ABSTRACT
A mouse-human chimeric monoclonal antibody (chNRLU-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
CMVP1E. 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 ei-
Received 10/9/98; revised 1/8/99; accepted 1/13/99.
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.
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.
3
Present address: Oxford Molecular Ltd., The Medawar Centre, Oxford
Science Park, Sandford upon Thames, Oxon OX4 4GA, United Kingdom.
4
Present address: Receptor Technologies A/S, Fabriksparken 58, 2600
Glostrup, Denmark.
5
Present address: University of Washington, School of Medicine, Division of Medical Genetics, Department of Laboratory Medicine, Seattle, WA 98195.
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 (2–5).
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 –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 (12–16). 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
(17–19).
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.
Downloaded from clincancerres.aacrjournals.org on August 3, 2017. © 1999 American Association for Cancer
Research.
900 NR-LU-13 Humanization
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 NRLU-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
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 enhancerpromoter 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, R3.5H5G9CL (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 R3.5H5G9CL 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 R3.5H5G9CL 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 3 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
NxH23 NxH7 (Fig. 1A). The synthesized VH chain was digested with HindIII/BamHI, and the resulting 439-bp fragment
was inserted into the complementary site of vector p4gB, containing the genomic g1 constant heavy (gCH) chain region
obtained from the human B cell plasmacytoma cell line MC/
Downloaded from clincancerres.aacrjournals.org on August 3, 2017. © 1999 American Association for Cancer
Research.
Clinical Cancer Research 901
Fig. 1 Schematic of synthesis
of the humanized variable
chain. A, oligonucleotides
NxH13 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 p4gB containing the
genomic constant heavy chain
region. B, oligonucleotides
NxK13 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.
CAR (ATCC CRL 8083). The VL chain was synthesized using
primers NxK1 3 NxK8 in a manner similar to the procedure
used for the synthesis of the VH chain (Fig. 1B), 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 am-
plification of genomic DNA from peripheral blood lymphocytes. The intermediate vectors pVKE and p4gB 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 p4gB containing the VH-gCH1 3 H3 region to generate the
intermediate expression vector, pWE1A2 (Fig. 2A). Total RNA
extracted from pWE1A2-transfected CHO cells was subjected
Downloaded from clincancerres.aacrjournals.org on August 3, 2017. © 1999 American Association for Cancer
Research.
902 NR-LU-13 Humanization
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 p4gB 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 (CMVP1E) 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.
to reverse transcriptase-mediated PCR (RT-PCR) using primers
complementary to the 59 and 39 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).
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 mM hypoxanthine and 16 mM thymidine. Exponentially growing cells
were transfected with 5 mg 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 mg/mL G418. Heavy
and light chains were amplified by stepwise increases with
MTX to a maximum of 50 mM. 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
mm MTX until the culture reached a density of 1 3 106 cells/ml.
The 0.1-mm hollow fiber clarified supernatants containing the
secreted huNR-LU-13 antibody were purified by batch DEAESepharose (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 b-galactosidase as described
previously (37).
ELISAs. Goat F(ab9)2 anti-human IgG and peroxidaselabeled 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) 3 100.
Cytotoxicity Assays. SW1222 (colon carcinoma) and
MCF-7 (breast carcinoma) cells were labeled with 200 mCi
51
Cr-chromate (DuPont NEN) at 2 3 106 cells/ml in DMEM/
F12 medium. After washing in medium containing 10% FCS,
1 3 104 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 C9MC 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 C9MC 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
Downloaded from clincancerres.aacrjournals.org on August 3, 2017. © 1999 American Association for Cancer
Research.
Clinical Cancer Research 903
percentage of cytotoxicity was determined according to the
following formula:
% cytotoxicity 5
Test release 2 spontaneous release
3 100
Total 2 spontaneous release
Spontaneous release for C9MC 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 3 106 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 mg/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 mg each of 131I-muNR-LU-10 and
125
I-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:
Tumor-specific antibody localization
5
% injected dose/gram of tumor
% injected dose/gram of blood
RESULTS
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 k 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 R3.5H5G9CL (29). The chimeric
sequence consists of a kV/hIIc chain pair, whereas the
R3.5H5G9CL clone is known to express an antibody that is a
kI/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-
Table 1
Region
Framework regions of homology
Identical residues
Homology
VH domain
FW
FW
FW
FW
I
II
III
IV
18/26
8/14
24/39
10/11
69%
57%
62%
91%
VL domain
FW
FW
FW
FW
I
II
III
IV
16/23
11/15
26/32
9/10
70%
73%
81%
90%
LU-13 and R3.5H5G9CL 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 39 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). The huNR-LU-13 model was generated by grafting the
chNR-LU-13 CDRs onto the human antibody framework of
R3.5H5G9CL, thus generating the humanized Fv sequence. The
initial humanized sequence was refined following three-dimensional modeling in which the original chNR-LU-13 and huNRLU-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).
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 chNRLU-13 antibody when analyzed on SDS-PAGE gels in the
presence or absence of 2 mM b-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 muNRLU-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), suggesting that the humanization process did not affect the binding
characteristics of the antibody.
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
Downloaded from clincancerres.aacrjournals.org on August 3, 2017. © 1999 American Association for Cancer
Research.
904 NR-LU-13 Humanization
Fig. 3 Comparison of the predicted amino acid sequence surrounding CDR1 3 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 R3.5H5G9CL 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).
Fig. 4 Competitive immunoreactivity profile in which the huNRLU-13 antibody (‚) and muNR-LU-10 antibody (f) 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.
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. 5, 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
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 1 NANaseIII glycosidic digestions; Lanes 3 and 6, PNGaseF
glycosidic digestion; Lanes 4 and 7, PNGaseF 1 b-galactosidase digestions.
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 b-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
Downloaded from clincancerres.aacrjournals.org on August 3, 2017. © 1999 American Association for Cancer
Research.
Clinical Cancer Research 905
(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. 5, Lanes 2 and 5). Further treatment of the oligos with
b-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. 5, 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.
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) or C9MC (Fig. 6B) 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) and C9MC (Fig. 7B).
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
125
I-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 tissuespecific 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). 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). It
is clear that full immunoreactivity is retained in the huNRLU-13 antibody, and that little effect in the overall non-target
tissue biodistribution has been imparted by the humanization
process.
DISCUSSION
Use of the chNR-LU-13 antibody for targeting of antigenpositive 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
Fig. 6 Evaluation of cytotoxic effector cell function of the muNRLU-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.
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,
R3.5H5G9CL, was selected for framework modeling. The original CDRs from the chNR-LU-13 antibody were grafted onto
the R3.5H5G9CL framework in an effort to retain the same
Downloaded from clincancerres.aacrjournals.org on August 3, 2017. © 1999 American Association for Cancer
Research.
906 NR-LU-13 Humanization
Fig. 7 Biodistribution of the
muNR-LU-10
and
huNRLU-13 antibodies in athymic
nude mice bearing the s.c.
SW1222 human tumor xenograft. Equimolar quantities of
131
I-muNR-LU-10 and 125I-NRLU-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 125IhuNR-LU-13 tumor:blood ratio
analyzed through 192 h after injection. Bars, SD.
antigen recognition site as the panreactive murine monoclonal
antibody, NR-LU-10 (11, 23). Three-dimensional modeling of
the initial humanized sequence revealed several R3.5H5G9CL
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 R3.5H5G9CL 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
R3.5H5G9CL 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
Downloaded from clincancerres.aacrjournals.org on August 3, 2017. © 1999 American Association for Cancer
Research.
Clinical Cancer Research 907
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– 45). Because the degree of antibody
glycosylation has been demonstrated to alter both the effector
function and tissue localization (46), we examined their Nlinked 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). 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. b-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 C9MC effector function consistent with the
human IgG1 isotype. In biodistribution studies, 131I-muNRLU-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-huNRLU-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 R3.5H5G9CL 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
7
S. Graves, personal communication.
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.
REFERENCES
1. Britton, K., and Granowska, M. Radioimmunoscintigraphy in tumor
identification. Cancer Surv., 6: 247–267, 1987.
2. Morell, A., Terry, W. D., and Waldman, T. A. Metabolic properties
of IgG subclasses in man. J. Clin. Investig., 49: 673– 680, 1970.
3. Chatenoud, L., Baudrihaye, M. F., Chkoff, N., Kreis, H., Goldstein,
G., and Bach, J-F. Restriction of the human in vivo immune response
against the mouse monoclonal antibody OKT3. J. Immunol., 137: 830 –
838, 1986.
4. Khazaeli, M. B., Saleh, M. N., Wheeler, R. H., Huster, W. J., Holden,
H., Carranto, R., and LoBuglio, A. F. Phase I trial of multiple large
doses of murine monoclonal antibody CO17–1A. II. Pharmacokinetics
and immune response. J. Natl. Cancer Inst., 80: 937–942, 1988.
5. Seccamani, E., Tattanelli, M., Mariani, M., Spranzi, E., Scassellati,
G. A., and Siccardi, A. G. A simple qualitative determination of human
antibodies to murine immunoglobulins (HAMA) in serum samples. Int.
J. Radiat. Appl. Instrum., 16: 167–170, 1989.
6. Sahagan, B. G., Dorai, H., Saltzgaber-Muller, J., Toneguzzo, F.,
Guindon, C. A., Lilly, S. P., McDonald, K. W., Morrissey, D., Stone,
B. A., Davis, G. L., McIntosh, P. K., and Moore, G. P. A genetically
engineered murine/human chimeric antibody retains specificity for human tumor-associated antigen. J. Immunol., 137: 1066 –1074, 1986.
7. Sun, L. K., Curtis, P., Radowicz-Szulczynska, E., Ghrayeb, J.,
Chang, N., Morrison, S. L., and Kaprowski, H. Chimeric antibody with
human constant regions and mouse variable regions directed against
carcinoma-associated antigen 17-1A. Proc. Natl. Acad. Sci. USA, 84:
214 –218, 1987.
8. Nishimura, Y., Yokoyama, M., Araki, K., Ueda, R., Kudo, A., and
Watanabe, T. Recombinant human-mouse chimeric monoclonal-antibody specific for common acute lymphocytic leukemia antigen. Cancer
Res., 47: 999 –1005, 1987.
9. Liu, A. Y., Robinson, R. R., Hellstrom, K. E., Murray, E. D., Chang,
C. P., and Hellstrom, I. Chimeric mouse-human IgG1 antibody that can
mediate lysis of cancer cells. Proc. Natl. Acad. Sci. USA, 84: 3439 –
3443, 1987.
10. Beidler, C. B., Ludwig, J. R., Cardenas, J., Phelps, J., Papworth,
C. G., Melcher, E., Sierzega, M., Myers, L. J., Unger, B. W., Fisher, M.,
David, G. S., and Johnson, M. J. Cloning and high level expression of
a chimeric antibody with specificity for human carcinoembryonic antigen. J. Immunol., 141: 4053– 4060, 1988.
11. Varki, N. M., Reisfield, R. A., and Walker, L. E. Antigens associated with a human lung adenocarcinoma defined by monoclonal antibodies. Cancer Res., 11: 638 – 687, 1984.
12. Helfrich, W., Koning, P. W., The, T. H., and DeLeij, L. Epitope
mapping of SCLC-Cluster-2 Mabs and generation of antibodies directed
against new EGP-2 epitopes. Int. J. Cancer Suppl., 8: 64 – 69, 1994.
13. deLeij, L., Helrich, W., Stein, R., and Mattes, M. J. SCLC-Cluster-2
antibodies detect the pancarcinoma/epithelial glycoprotein EGP-2. Int.
J. Cancer Suppl., 8: 60 – 63, 1994.
14. Szala, S., Froehlich, M., Scollon, M., Kasai, Y., Steplewski, Z.,
Koprowski, H., and Linnenbach, A. Molecular cloning of cDNA for the
Downloaded from clincancerres.aacrjournals.org on August 3, 2017. © 1999 American Association for Cancer
Research.
908 NR-LU-13 Humanization
carcinoma-associated antigen GA733-2. Proc. Natl. Acad. Sci. USA, 87:
3542–3546, 1990.
15. Strand, J., Hamilton, A. E., Beavers, L. S., Gamboa, G. C., Apelgren, L. D., Taber, L. D., Sportsman, J. R., Bumol, T. F., Sharp, J. D.,
and Gadski, R. A. Molecular cloning and characterization of a human
adenocarcinoma/epithelial cell surface antigen complementary DNA.
Cancer Res., 49: 314 –317, 1989.
16. Herlyn, M., Steplewski, Z., Herlyn, D., and Koprowski, H. CO
17-1A and related monoclonal antibodies: their production and characterization. Hybridoma, 5: S3–S10, 1986.
17. Spearman, M. E., Goodwin, R. M., Apelgren, L. D., and Bumol,
T. F. Disposition of the monoclonal antibody-Vinca alkaloid conjugate
KS1/4-DAVLB (LY256787) and free 4-desacetylvinblastine in tumorbearing nude mice. J. Pharmacol. Exp. Ther., 241: 695–703, 1987.
18. Bumol, T. F. Workshop summary: monoclonal antibodies conjugates with drugs or toxins. In: R. A. Reisfield and S. Sell (eds.),
Monoclonal Antibodies and Cancer Therapy, pp. 257–259. New York:
Alan R. Liss, Inc., 1985.
19. Bumol, T. F., Parrish, J., De Herdt, S. V., Spearman, M. E.,
Pohland, R., Borowitz, M. J., Briggs, S. L., Baker, A. L., Marder, P., and
Apelgren, L. D. Studies on a epithelial/epithelial malignancy associated
antigen associated with human adenocarcinomas. In: R. L. Ceriani (ed.),
Immunological Approaches to the Diagnosis and Therapy of Breast
Cancer, pp. 205–215. New York: Plenum Publishing Corp., 1987.
20. Balaban, E. P., Walker, B. S., Cox, J. V., Bordlee, R. P., Salk, D.,
Abrams, P. G., Sheehan, R. G., and Frenkel, E. P. Detection and staging
of small lung cell carcinoma with a technetium-labeled monoclonal
antibody: a comparison with standard staging methods. Clin. Nucl.
Med., 17: 439 – 445, 1992.
21. Breitz, H. B., Weiden, P. L., Vanderheyden, J-L., Appelbaum,
J. W., Bjorn, M. J., Fer, M. F., Wolf, S. B., Ratliff, B. A., Seiler, C. A.,
Foisie, D. C., Fisher, D. R., Schroff, R. W., Fritzberg, A. R., and
Abrams, P. G. Clinical experience with rhenium-186-labeled monoclonal antibodies for radioimmunotherapy: results of Phase I trials.
J. Nucl. Med., 33: 1099 –1109, 1992.
22. Schroff, R. W., and Stevenson, H. C. Human immune responses to
murine monoclonal antibodies. In: K. A. Foon and A. C. Morgan (eds.),
Monoclonal Antibody Therapy of Human Cancer, pp. 121–138. The
Hague: Nijhoff, 1985.
23. Saleh, M. N., LoBuglio, A. F., Wheeler, R. H., Rogers, K. J.,
Haynes, A., Lee, J. Y., and Khazael, M. B. A Phase II trial of murine
monoclonal antibody 17-1A and interferon-g: clinical and immunological data. Cancer Immunol. Immunother., 32: 185–190, 1990.
24. Weiden, P. L., Breitz, H. B., Seiler, C. A., Bjorn, M. J., Ratliff,
B. A., Mallett, R., Beaumier, P. L., Appelbaum, J. W., Fritzberg, A. R.,
and Salk, D. Rhenium-186-labeled chimeric antibody NR-LU-13: pharmacokinetics, biodistribution and immunogenicity relative to murine
analog NR-LU-10. J. Nucl. Med., 34: 2111–2119, 1993.
25. Maniatis, T. Fritsch, E. F., and Sambrook, J. Molecular Cloning: A
Laboratory Manual, pp. 1.25, 1.74 –1.76, 6.30 – 6.34, 7.12–7.15, 7.26 –
7.29. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982.
26. Sanger, F., Nicklen, S., and Coulson, A. R. DNA sequencing with
chain terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74: 5463–
5467, 1977.
27. Kabat, E. A., Wu, T. T., Reid-Miller, M., Perry, H. M., and
Gottesman, K. S. Sequence of Proteins of Immunological Interest, Ed. 5.
Washington, DC: U. S. Department of Health and Human Services,
1991.
28. Pedersen, J. T., Henry, A. H., Searle, S. J., Guild, B. C., Roguska,
M., and Rees, A. R. Comparison of surface accessible residues in human
and murine immunoglobulin Fv domains: implication for humanization
of murine antibodies. J. Mol. Biol., 235: 959 –973, 1994.
29. Manheimer-Lory, A., Katz, J. B., Pillinger, M., Ghossein, C.,
Smith, A., and Diamond, B. Molecular characteristics of antibody
bearing an anti-DNA-associated idiotype. J. Exp. Med., 174: 1639 –
1652, 1991.
30. Martin, R. C., Cheetham, J. C., and Rees, A. R. Modeling antibody
hypervariable loops: a combined algorithm. Proc. Natl. Acad. Sci. USA,
86: 9268 –9272, 1989.
31. Pedersen, J., Searle, S., Henry, A., and Rees, A. R. Antibody
modeling: beyond homology. Immunomethods, 1: 126 –136, 1992.
32. Chothia, C., Lesk, A. M., Tramontano, A., Levitt, M., Smith-Gill,
S. J., Air, G., Sheriff, S., Padlan, E. A., Davies, D., Tulip, W. R.,
Colman, P. M., Spinelli, S., Alizari, P. M., and Poljak, R. J. Conformations of immunoglobulin hypervariable regions. Nature (Lond.), 342:
877– 883, 1989.
33. Bruccoleri, R. E., and Karplus, M. Prediction of the folding of short
polypeptide segments by uniform conformational sampling. Biopolymers, 26: 137–168, 1987.
34. Prodromou, C., and Pearl, L. H. Recursive PCR: a novel technique
for total gene synthesis. Protein Eng., 5: 827– 829, 1992.
35. Jackson, P. Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 2-aminoacridone: subpicomolar detection using an imaging system based on a cooled charged-coupled
device. Anal. Biochem., 196: 238 –244, 1991.
36. Jackson, P., and Williams, G. R. Polyacrylamide gel electrophoresis
of reducing saccharides labeled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid: applications to the enzymological structural
analysis of oligosaccharides. Electrophoresis, 12: 94 –96, 1991.
37. Reynolds, J. C., Del Vecchio, S., Sakahara, H., Lora, M. E., Carrasquillo, J. A., Neumann, R. D., and Larson, S. M. Anti-murine
antibody response to mouse monoclonal antibodies: clinical findings
and implications. Nucl. Med. Biol., 16: 121–128, 1989.
38. Tom, B. H., Rutzky, L. P., Jakstys, M. M., Oyasu, R., Kaye, C. I.,
and Kahan, B. Human colonic adenocarcinoma cells. I. Establishment
and description of a new line. In Vitro (Rockville), 12: 180 –191, 1976.
39. Wilbur, D. S., Hadley, S. W., Hylarides, M. D., Abrams, P. G.,
Beaumier, P. A., Morgan, A. C., Reno, J. M., and Fritzberg, A. R.
Development of a stable radioiodinating reagent to label monoclonal
antibodies for radiotherapy of cancer. J. Nucl. Med., 30: 216 –226, 1989.
40. Deisenhofer, J. Crystallographic refinement and atomic models of a
human Fc fragment and its complex with fragment B of protein A from
Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry, 20:
2361–2370, 1981.
41. Morgan, A. C., Sullivan, W., Graves, S., and Woodhouse, C. S.
Murine monoclonal antibody IgG3 to human colorectal tumor associated antigens: augmentation of antibody dependent cell mediated cytotoxicity (ADCC) by IL-2. Cancer Res., 49: 2773–2776, 1989.
42. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F.,
Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T., and Tasumi,
M. The Protein Databank: a computer based archival file for macromolecular structures. J. Mol. Biol., 112: 535–542, 1977.
43. Kaufman, R. J., Wasley, L. C., Spilotes, S. D., Gossels, S. D., Latt,
S. A., Larsen, G. R., and Kay, R. M. Coamplification and coexpression
of human tissue type plasminogen activator and murine dihydrofolate
reductase sequences in Chinese hamster ovary cells. Mol. Cell. Biol., 5:
1750 –1759, 1985.
44. Zettlmeissl, G., Ragg, H., and Karges, H. E. Expression of biologically active antithrombin III in Chinese hamster ovary cells. BioTechnology, 5: 720 –725, 1987.
45. Page, M. J., and Sydenham, M. A. High level expression of the
humanized monoclonal antibody campath-1H in Chinese hamster ovary
cells. BioTechnology, 9: 64 – 68, 1991.
46. Hand, P. H., Calvo, B., Milenic, D., Yokota, T., Finch, M., Snoy, P.,
Garmestani, K., Gansow, O., Schlom, J., and Kashmiri, S. V. S. Comparative biological properties of a recombinant chimeric anti-carcinoma
MAb and a recombinant aglycosylated variant. Cancer Immunol. Immunother., 35: 165–174, 1992.
47. Qu, Z., Sharkey, R. M., Hansen, H. J., Goldenberg, D. M., and
Leung, S. Structure determination of N-linked oligosaccharides engineered at the CH1 domain of humanized LL2. Glycobiology, 7: 803–
809, 1997.
Downloaded from clincancerres.aacrjournals.org on August 3, 2017. © 1999 American Association for Cancer
Research.
Molecular Modeling and Preclinical Evaluation of the
Humanized NR-LU-13 Antibody
Scott S. Graves, Stephen C. Goshorn, Diane M. Stone, et al.
Clin Cancer Res 1999;5:899-908.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://clincancerres.aacrjournals.org/content/5/4/899
This article cites 38 articles, 17 of which you can access for free at:
http://clincancerres.aacrjournals.org/content/5/4/899.full#ref-list-1
This article has been cited by 1 HighWire-hosted articles. Access the articles at:
http://clincancerres.aacrjournals.org/content/5/4/899.full#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from clincancerres.aacrjournals.org on August 3, 2017. © 1999 American Association for Cancer
Research.