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* Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute,
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, and
Division of Bioengineering and Physical Sciences, Office of Research Services, Office of the Director, National Institutes of Health, Bethesda, MD 20892
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The carcinoembryonic Ag (CEA), a cell surface glycoprotein of
180-kDa molecular mass, is one of the most widely used human
tumor markers. It is expressed at high levels in the embryonic and
fetal digestive epithelial tissue and, to a lesser extent, in normal
adult colon and stomach epithelium (15). CEA is
overexpressed in 
95% of gastrointestinal and pancreatic cancers, as
well as in most non-small-cell lung carcinomas. Also, it is expressed
in breast carcinoma and squamous cell carcinoma of the head and neck
(reviewed in Refs. 11 and 16). A number of
murine anti-CEA mAbs have been generated (17) and used
for measuring CEA levels in blood (18) and for
immunohistopathology of tissues from cancer patients (11, 19). In addition, Abs directed to CEA were among the first to be
used in clinical trials to successfully localize tumors
(20, 21, 22, 23). Also, several clinical trials have been
conducted to evaluate the anti-tumor activity of anti-CEA mAbs
(24, 25, 26, 27). CEA has some degree of cross-reactivity with
several proteins, including nonspecific cross-reacting Ag-1 (NCA-1),
normal fecal Ag-1, and the NCA-related proteins present in human
granulocytes (17).
The mAb COL-1, an IgG2a, has a high affinity for CEA and has no detectable reactivity to granulocytes or to the CEA-related Ags NCA-1 and normal fecal Ag-1 (28). Murine COL-1 (mCOL-1) reacts with biopsies of colon and gastric carcinomas (80 and 88%, respectively) and with biopsies of human mammary and non-small-cell lung carcinomas (17, 29). Except for some reactivity to skin and gastric and colon mucosa, mCOL-1 does not react with normal tissues (17). In preclinical studies, 125I-labeled mCOL-1 achieved good tumor radiolocalization using LS-174T colon carcinoma xenograft in athymic mice (30). In patients, 131I-labeled mCOL-1 showed good tumor targeting of human gastrointestinal carcinomas in a phase I clinical trial (24). Not unexpectedly, mCOL-1 was found to induce HAMA response in patients (24, 31). Yu et al. (24) reported that 61 and 83% of patients treated with the Ab developed elevated HAMA levels by days 20 and 40, respectively. It was observed by Meredith et al. (31) that 93% of the patients developed Ig response, with most patients developing HAMA by day 14 and some as early as days 7–11 after mCOL-1 administration. The peak of Ab response generally occurred at 4–6 wk after exposure. An attempt has now been undertaken to reduce the immunogenicity of the mCOL-1 mAb by progressively reducing its murine content by genetic manipulation. This report describes the development and characterization of a mouse-human chimeric COL-1 (cCOL-1) mAb, a humanized COL-1 (HuCOL-1) mAb, and variants of HuCOL-1. A final variant of HuCOL-1 was developed by a previously suggested humanization protocol (14) based on transplantation of "abbreviated" xenogeneic CDRs onto the human Ab frameworks. This variant, which retains significant Ag-binding reactivity, is minimally reactive to sera from patients who were earlier administered mCOL-1 during clinical trials.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The long overlapping oligomers and oligonucleotide primers used for DNA amplifications were supplied by Lofstrand Labs (Gaithersburg, MD) and Midland Certified Reagent (Midland, TX). The sequences of the four primers that were used to generate DNA fragments encoding the VH and VL domains of the mCOL-1 mAb were as follows: 1) 5' VH: 5'-AGTAAGCTTCCACCATGGAGTGGTCCTGGGTCTTCCTCTTCTTCCTGTCCGTGACTACTGGAGTGCACTCCGAGGTTCAGCTGCAGCA-3'; 2) 3' VH: 5'-CGATGGGCCCGTAGTTTTGGCAGAGGAGACGGCGACCG-3'; 3) 5' VL: 5'-TAGCAAGCTTCCACCATGGATAGCCAGGCCCAGGTGCTCATGCTCCTGCTGCTGTGGGTGAGCGGCACATGCGGCGACATTGTGCTGACACA-3'; 4) and 3' VL: 5'-TGCAGCCGCGGCCCGTTTGATTTCCAGCTTGG-3'.
Each of the 5' primers carries a HindIII site followed by a
sequence encoding a signal peptide. The 3' VH
primer carries an ApaI, whereas the 3'
VL primer has a SacII site. The four
119- to 133-bp-long oligonucleotides that were used to generate each of
the VH and VL genes of
HuCOL-1 are shown by long arrows in Fig. 1
. The sequence of the 20- to 21-bp-long
end primers used for DNA amplification were as follows: 5) 5'
VH: 5'-CGTAAGCTTCCACCATGGAG-3'; 6) 3'
VH: 5'-TGGGCCCTTGGTGGAGGCTGA-3';
7) 5' VL: 5'-GCAAGCTTCCACCATGGATA-3';
and 8) 3' VL:
5'-TGCAGCCGCGGTACGTTTGAT-3'.
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The sequences recognized by the restriction endonucleases are in italics, and the mutagenic changes are underlined.
DNA amplification
All PCRs were conducted in a final volume of 100 µl of PCR buffer containing 200 µM dNTPs, 3 U of Taq polymerase (Boehringer Mannheim, Indianapolis, IN), 0.2 µM each of the end primers, and 100 ng of DNA template. Initial denaturation at 94°C for 2 min was followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. It was followed by a final primer extension step at 72°C for 10 min. The VL and VH genes of the HuCOL-1 were synthesized by the overlap extension PCR that has previously been described (32). Primer-induced mutagenesis was conducted by a dual-step PCR as described by Landt et al. (33).
Expression vectors
Two different baculovirus transfer vectors, pAcUW51 (BD PharMingen, San Diego, CA) and pBAC4x-1 (Novagen, Madison, WI), have been used for generating the recombinant viruses and the subsequent coexpression of the Ig H and L chains. In pAcUW51 vector, one of the target genes can be cloned at the BamHI site located downstream of the polh promoter, whereas the other gene can be driven by the p10 promoter by inserting the gene at either the BglII or the EcoRI site located 3' to the promoter. The pBAC4x-1 baculovirus transfer plasmid contains two of each of the polh and p10 promoters, with a unique cloning site placed downstream of each promoter.
Assembly of the V and C region genes and generation of expression constructs
To generate constructs encoding the chimeric H and L chains of
mAb COL-1, the V region sequences of the H and L chain genes were PCR
amplified using the phagemid constructs of the cDNAs encoding the Fd
and the L chain of mCOL-1 as templates. Primers no. 3 and no. 4 were
used as forward and reverse primers, respectively, to amplify a 420-bp
sequence encoding the VL domain along with the
signal peptide located upstream. The 3' primer was designed to extend
the 3' end of the amplified sequence to a unique SacII site
located 10 bp downstream from the start of the human 
C region. A
DNA fragment encoding the human C region was excised from a
pre-existing construct pLNCXHuCC49HuK (32) by
SacII/ClaI treatment. The construct carried an
EcoRI site immediately upstream of ClaI site. The
V and the C regions of the L chain were joined to the
HindIII/ClaI linearized pBluescript II
S/K+ (pBSc) plasmid (Stratagene, La Jolla, CA) by
three-way ligation. Taking advantage of an EcoRI site
upstream of the HindIII site in pBSc, the entire L chain
sequence was then released from the construct by EcoRI
digestion and inserted into the baculovirus expression vector pAcUW51
at the EcoRI site located downstream from the p10
promoter.
For the assembly of the chimeric H chain, a 460-bp sequence encoding
the VH domain and its leader peptide was PCR
amplified using primers nos. 1 and 2 as 5' and 3' primers,
respectively. The design of the 3' primer facilitated amplification of
the VH sequence to extend to the ApaI
site located 17 bp downstream from the start of the
CH1 domain. To assemble the V and C regions, an
ApaI/ClaI DNA fragment carrying the human 
1 C
region was excised from a pre-existing construct pLgpCXHuCC49HuG1
(32). The ApaI/ClaI fragment along
with the 460-bp PCR product was inserted into the
HindIII/ClaI linearized pBSc. The DNA encoding
the entire H chain was released by HindIII/ClaI
treatment of the pBSc construct. The termini of the target DNA were
filled in using Klenow fragments of the DNA polymerase, and the DNA
fragment was subcloned in the L chain construct of pAcUW51 at the
blunt-ended BamHI site located downstream of the
polh promoter.
DNA manipulations similar to those described for cCOL-1 were conducted
to assemble the humanized and the variant V regions and their
respective C regions into pBSc for the subsequent subcloning in
baculovirus expression vector pBAC4x-1. After joining the V regions of
the H and L chains to their respective C regions in pBSc, the assembled
L chain of the HuCOL-1 or its variant was released from the pBSc
construct and cloned at the EcoRI site, downstream from the
p10 promoter. The entire H chain of the HuCOL-1 or its variant was
excised from its pBSc construct by HindIII/XhoI
treatment, and it was cloned unidirectionally in the L chain construct
of pBAC4x-1 at the HindIII/XhoI site, downstream
of polh promoter. Three expression constructs were
generated: one containing the variant L chain and the parental
humanized H chain, the second containing the variant H chain and the
parental humanized L chain, and the third carrying variants of both the
L and H chains (Table I).
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Serum-free-adapted Sf9 insect cells (Life Technologies, Rockville, MD) were cultured at 27°C in Sf900-II medium (Life Technologies) with 50 µg/ml gentamicin. To develop transfectomas secreting cCOL-1, insect cells were cotransfected with the pAcUW51-derived expression construct and the linearized BaculoGold Baculovirus DNA (BD PharMingen). Transfectomas producing HuCOL-1 and its variants were generated by transfecting insect cells with one of the pBAC4x-1-derived expression constructs and the linearized BacVector2000 Baculovirus DNA (Novagen). A cationic liposome-mediated system (DOTAP; Boehringer Mannheim) was used for all transfections. Harvesting of the recombinant virus, screening for Ig expression, and Ag binding by ELISA have previously been described (13).
ELISA
ELISAs were conducted by coating 96-well polyvinyl microtiter
plates with CEA (100 ng/well; Research Diagnostic, Flanders, NJ) or
with Fc-fragment-specific goat anti-human IgG (100 ng/well)
(Jackson ImmunoResearch Laboratories, West Grove, PA). Anti-human
IgG or the CEA-coated plates were used to test for the production of Ig
by the insect cells or to assess its Ag reactivity, respectively. The
details of the assay procedure have been reported earlier
(34).
Purification of recombinant Abs
Three days after infection, the supernatants were collected and made free of cellular debris and any contaminating proteins, before using protein G agarose column (Life Technologies) to purify the desired protein as described earlier (13). The protein was concentrated using Centricon 30 (Amicon, Beverly, MA) and dialyzed in PBS buffer using a Slide-A-Lyzer cassette (Pierce, Rockford, IL). The protein concentration was determined by the method of Lowry et al. (35), and the purity of the eluted proteins was evaluated by SDS-PAGE under reducing and nonreducing conditions, using precast 4–20% Tris-glycine gel (Novex, San Diego, CA). The protein bands were visualized by Coomassie blue staining (Novex).
Competition RIA
The relative Ag binding of the mCOL-1 and the recombinant Abs derived from it were determined using competition RIA. Twenty-five microliters of serial dilutions of the Abs to be tested as well as the mCOL-1, prepared in 1% BSA in PBS, were added to microtiter plates containing 200 ng of CEA saturated with 5% BSA in PBS. 125I-labeled mCOL-1 or 125I-labeled HuCOL-1 (100,000 cpm in 25 µl) was then added to each well. After an overnight incubation at 4°C, the plates were washed and counted in a gamma-scintillation counter. The relative affinity constants were calculated by a modification of the Scatchard method (36).
Flow cytometric analysis
A previously described method (37) has been used for FACS analysis. To evaluate the ability of HuCOL-1 and its variants to bind to cell-surface CEA, 1 x 106 retrovirally transduced MC38 cells expressing CEA (38) were resuspended in cold Ca2+- and Mg2+-free Dulbecco’s PBS and incubated with the mCOL-1-derived Abs for 30 min on ice. A human IgG was used as an isotype control. After one washing cycle, the cell suspension was stained with FITC-conjugated mouse anti-human Ab (BD PharMingen) for 30 min on ice. A second washing cycle was performed, and then the samples were analyzed with a FACScan (BD Biosciences, Mountain View, CA) using CellQuest for Macintosh. Data from analysis of 10,000 cells were obtained.
Immunoadsorption of patient serum and detection of anti-V region Abs
Stored patients’ sera, from a phase I clinical trial
(24) that involved the administration of
131I-labeled mCOL-1 to gastrointestinal carcinoma
patients, were used to assess serum reactivity of the mCOL-1-derived
Abs. Several sera were tested for the presence of anti-V region Abs
to mAb COL-1. The sera, however, contain circulating Ag and
anti-murine Fc Abs, which could interfere with the binding of mAb
COL-1 and its derivative Abs to the anti-V region Abs. To
circumvent this problem, the circulating CEA and anti-murine Fc Abs
were removed by sequential preadsorption of the sera with purified
mCOL-6 and mCOL-4 mAbs, two Abs that react with epitopes of CEA
different from the one recognized by mCOL-1 (28). The mAb
mCOL-4 has the same isotype as that of mCOL-1. For preadsorption, serum
samples were added to mCOL-6 coupled to Reacti-gel according to the
method of Hearn et al. (39). The mixtures were incubated
overnight at 4°C with end-to-end rotation and were centrifuged at
1000 x g for 5 min. Preadsorption was repeated until
the supernatants displayed no detectable anti-murine Fc activity.
The procedure was then repeated using mCOL-4 coupled to Reacti-gel. To
detect anti-V region Abs by surface plasmon resonance (SPR), the
preadsorbed serum was used as a mobile reactant. Proteins were
immobilized on carboxymethylated dextran CM5 chips (BIAcore,
Piscataway, NJ) by amine coupling using standard procedure (40, 41). HuCOL-1 was immobilized on the surface of flow cell 1,
whereas the surface of flow cell 2 was coated with an unrelated
protein, rabbit globulin (Bio-Rad, Hercules, CA).
Sera reactivity
The reactivity of COL-1 variants to anti-V region Abs was
determined using a recently developed SPR-based competition assay
(58). Competition experiments were performed at 25°C
using a CM5 sensor chip containing either mCOL-1 or HuCOL-1 in flow
cell 1 and rabbit globulin (Bio-Rad), as a reference, in flow cell
2. Typically, mCOL-1, HuCOL-1, or its variants were used at different
concentrations, to compete with the Ab immobilized on the sensor chip
for binding to serum anti-V region Abs. Patient’s serum with or
without the competitor (mCOL-1, HuCOL-1, or its variants) was applied
across the sensor surface using a recently developed sample application
technique (42) at the unidirectional flow of 1 µl/min.
After the binding was measured for 1000 s, the samples were washed
from the surfaces with running buffer using a flow rate of 100
µl/min, and the surfaces were regenerated with 10 mM glycine (pH 2.0)
for the HuCOL-1 sensor chip or with HCl (pH 2.3) for the mCOL-1 sensor
chip. The percent binding at each Ab concentration was calculated as
follows: % binding = [slope of the signal obtained with
competitor (serum + mCOL-1, HuCOL-1, or HuCOL-1 variants)/slope of the
signal obtained without competitor (serum only)] x 100.
IC50 for each Ab, the concentration required for
50% inhibition of the binding of the serum to either mCOL-1 or
HuCOL-1, was calculated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The genes encoding the L chain and the Fd region of the H chain of
mAb COL-1 were generated by repertoire cloning methodology
(43), using synthetic oligonucleotides described in
Materials and Methods. The PCR products of the appropriate
size were cloned in 
phage vector, and phagemids carrying the target
genes were subsequently excised. The cloned genes were sequenced (data
not shown) before the phagemids were used as templates for the
subsequent PCR amplification.
Generation of genes encoding humanized COL-1 VL and VH domains
The mCOL-1 was humanized by grafting the CDRs of the L and H
chains onto the VL and VH
frameworks of the appropriate human Abs, but retaining those framework
residues that were deemed essential for preserving the structural
integrity of the combining site (44, 45, 46). The Ig CDRs have
been defined as comprising residues 31–35b, 50–65, and 95–102
in the H chain and residues 24–34, 50–56, and 89–97 in the L chain
(47). The framework residues that were deemed critical
were identified on the basis of the atomic coordinates of the Abs of
known structures available in the database (for example, see Ref.
48). The human Ab sequences that are most similar to
mCOL-1 are VJI'CL (49) (GenBank accession number
Z00022) for VL and MO30 (50)
(GenBank accession number A32483) for VH. The
alignment of the VL sequences of mCOL-1 and
VJI’CL, and the VH sequences of mCOL-1 and MO30
are shown in Fig. 2. Also indicated in
Fig. 2 are the locations of the framework residues that are critical
for Ag binding. The humanization protocols for the
VL and VH genes, shown in
Fig. 2, are based on putting the CDR sequences of mAb COL-1 together
with the frameworks of the human VL and
VH templates, while replacing some of the human
framework residues with those murine framework residues that may be
critical for Ag binding.
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that encompassed, on alternating strands, the entire sequence of either
the VL or VH region and its
leader and the respective end primers described in Materials and
Methods, DNA fragments encoding the humanized
VL or VH regions were
generated and amplified by overlap extension PCR technique
(32). The humanized VL or
VH regions thus generated were extended to the
ApaI and SacII sites located 10 and 17 bp
downstream from the 5' end of the C regions of the H and L chains,
respectively. The PCR products were cloned in pBSc to generate
pBScHuCOL-1VL and
pBScHuCOL-1VH constructs. The inserts were
sequenced to check the fidelity of the PCR products. Generation of genes encoding variants of HuCOL-1 VL and VH domains
Examination of the known structures of Ab-Ag complexes reveals
that only one-third of the CDR residues are involved in the interaction
with the Ag (48). This led to the proposal to redefine the
boundaries of the CDRs to 31–35b, 50–58, and 95–101 in the H chain
and 27d–34, 50–55, and 89–96 in the L chain (14).
Accordingly, we have developed variants of HuCOL-1 H and L chains in
which these abbreviated CDRs have been grafted onto the VJI’CL and
MO30 frameworks (Table I).
Genes encoding the humanized VL and
VH domains of the variants
24,25,27L and 61H were
generated by primer-induced mutagenesis, using
pBScHuCOL-1VL and
pBScHuCOL-1VH constructs, respectively, as
templates. Variant 61H was generated by replacing
residue 61 of mCOL-1 H chain CDR2 (numbering convention of Kabat et al.
(47)) with the corresponding residues of mAb MO30 H chain
CDR2. For the generation of 24,25,27L variant,
residues 24, 25, and 27 of mCOL-1 L chain CDR1 were replaced with the
corresponding residues in L chain CDR1 of the human mAb VJI’CL (Table I). The V region sequences were synthesized by a dual-step PCR
procedure according to Landt et al. (33). For each L and H
chain V region, the mutagenic primer, containing the desired nucleotide
changes in the targeted CDR, was used as a 3' primer, whereas a 20-mer
end primer served as a 5' primer. The resulting PCR product was gel
purified and used as a 5' primer for the subsequent step of the PCR in
which a 21-mer oligonucleotide was used as a 3' primer. The PCR
products were cloned in pBSC vector and sequenced. The amino acid
sequences of the VL of the variant
24,25,27L and VH of the variant
61H are shown in Fig. 2, A and
B, respectively.
Immunoreactivity of the mAbs expressed in insect cells
The genes encoding the VL and
VH domains of mCOL-1, HuCOL-1, and the variants
61H and 24,25,27L were
assembled with the respective human C region genes (1 for the H and
for the L chain). The expression constructs were introduced into
Sf9 insect cells, and the supernatants harvested from the transfectants
were assayed for Ig production and Ag-binding reactivity by ELISA, as
described in Materials and Methods. All the transfectants
and the viral plaques, generated by infecting Sf9 cells with the
infectious supernatants, were found to be positive for Ig production as
assayed by ELISA. Results of an ELISA for Ag binding also showed that
all culture supernatants and the viral plaques were reactive with CEA,
albeit with varying degrees. To examine whether the different
constructs were expressing comparable levels of Ig molecules, viral
plaques were expanded and a large batch of Sf9 cells was freshly
infected, at a multiplicity of infection of 5, with infectious
supernatant derived from the highest producing clone of each construct,
and the infected cells were cultured under identical conditions. The
secreted Abs were purified from equal volumes of the culture
supernatants. The concentration of the secreted Abs was comparable
(2–3 µg/ml) in culture supernatants of all five transfectants.
SDS-PAGE of the secreted mAbs
The apparently lower Ag-binding reactivities of the variant mAbs
24,25,27L and
24,25,27L/61H than those of
cCOL-1, HuCOL-1, and the variant 61H could be
attributed either to any possible detrimental effect of genetic
manipulations of the combining site of the secreted Abs or to some
structural abnormality of the expressed Ig molecules. The latter may be
detected on SDS-PAGE by a change in size or mobility of the molecules.
To this end, the purified Abs from the culture supernatants and the
murine mAb COL-1 were analyzed by SDS-PAGE. The gel profile under
nonreducing conditions (data not shown) showed that the mobility of all
five recombinant Abs was identical with that of mCOL-1 mAb, which has a
molecular mass of 160 kDa. Under reducing conditions, all the
recombinant COL-1 Abs, like that of mCOL-1, yielded two protein bands
of 25–28 and 50–55 kDa (data not shown). These molecular masses
are in conformity with those of the Ig L and H chains. The results of
the SDS-PAGE analysis, together with the ELISA for CEA reactivity of
the serially diluted Abs, suggest that the reduced Ag reactivity of the
variants 24,25,27L and
24,25,27L/61H may be due to
some detrimental effect of the amino acid substitutions in the
combining site of HuCOL-1.
Relative CEA-binding affinities of mAbs derived from COL-1
A competition RIA was performed to determine the relative
CEA-binding affinities of the COL-1-derived mAbs and the parental
mCOL-1 mAb. Serial dilutions of unlabeled Abs (murine, chimeric,
humanized COL-1 and its variants) were used to compete with the binding
of 125I-labeled HuCOL-1 (Fig. 3) or 125I-labeled
mCOL-1 (data not shown) to CEA. All of the COL-1-derived recombinant
Abs, like the parental mCOL-1, were able to completely inhibit the
binding of 125I-labeled mCOL-1 and
125I-labeled HuCOL-1 to CEA. The competition
profiles of cCOL-1, HuCOL-1, and the variant 61H
were comparable to that of the mCOL-1. In contrast, the competition
profiles of mAbs 24,25,27L and
24,25,27L/61H, although of
slopes similar to that of the parental mCOL-1 mAb, were shifted to the
right. The values of the relative Ka of cCOL-1,
HuCOL-1, and 61H mAbs, calculated from the linear
parts of the competition curves in Fig. 3, were 3.45 x
108 M-1, 2.82 x
108 M-1, and 2.64 x
108 M-1, respectively.
These relative affinities are 1.5- to 2-fold less than that of
mCOL-1 (Table I). The relative Ka values of
24,25,27L and
24,25,27L/61H were 1.2
x 108 M-1 and 1.03
x 108 M-1, respectively,
4.3- to 5-fold lower than that of parental mCOL-1 (Table I).
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Flow cytometric analysis was used to measure the binding of
HuCOL-1 and its variants (61H, 24, 25,
27L, and 24, 25,
27L/61H) to the CEA expressed on the
cell surface of a tumor cell line, MC38, that was retrovirally
transduced with CEA (38). No significant differences were
found in the mean fluorescence intensity or in the percentage of cells
that was reactive with HuCOL-1 and its variants (Fig. 4). The percentages of gated cells,
calculated after exclusion of irrelevant binding, were indeed between
54 and 56, whereas the mean fluorescence intensities were between 15
and 16 when 1 µg of each Ab was used.
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The immunogenicity of an Ab variant can be assessed only through
its clinical trial. A reasonable measure of the potential
immunogenicity of the variant Ab, however, is its in vitro reactivity
to sera from patients who were administered the parental Ab in a
clinical trial. To assess the potential immunogenicity of the mCOL-1,
HuCOL-1, and its variants in patients, the Abs were characterized for
their reactivity to sera from gastrointestinal carcinoma patients who
were administered 131I-labeled mCOL-1 in a phase
I clinical trial (24). As described in Materials and
Methods, any circulating CEA and anti-murine Fc Abs were
removed from the sera by immunoadsorption with mCOL-6 and mCOL-4, two
murine anti-CEA Abs of IgG1 and IgG2a isotypes, respectively. It
has been suggested on the basis of epitope mapping of CEA
(28) that mCOL-4 and mCOL-6 may react with CEA epitopes
that are different from each other and different from the one
recognized by mAb COL-1. Preadsorbed sera were tested for the presence
of anti-V region Abs to mAb COL-1. Specific binding profiles of
HuCOL-1 to the sera from patients EM, JS, and MB (data not shown) show
that all three sera have Abs against the variable regions of mCOL-1.
Serum reactivity of HuCOL-1 and HuCOL-1 variants was determined by
their ability to compete with mCOL-1 or HuCOL-1 immobilized on a sensor
chip for binding to the anti-variable region Abs to mCOL-1 present
in the serum. IC50, the concentration of the
competitor Ab required for 50% inhibition of the binding of mCOL-1 or
HuCOL-1 to the patient’s serum, was calculated by plotting the percent
inhibition as a function of competitor concentration. A higher
IC50 value indicates a decreased reactivity to
the serum, suggesting potentially reduced immunogenicity of the Abs in
patients. Fig. 5 shows the competition
profiles generated by HuCOL-1 and its variants when they were used to
compete with the HuCOL-1 immobilized on the sensor chip for binding to
the anti-V region Abs to COL-1 present in the sera of patients EM
(A), JS (B), and MB (C). The
competition profiles were used to calculate IC50
values that are presented in Table II.
For serum MB, the IC50 values of all three
variants are 2- to 3-fold higher than that of HuCOL-1. Studies with the
serum from patient EM show that the variants
24,25,27L and
24,25,27L/61H have 50%
higher IC50 values, whereas the variant
61H has a significantly lower
IC50 value than that of HuCOL-1. For the serum
from patient JS, the IC50 of the variant
24,25,27L/61H is twice as
much as that of HuCOL-1, whereas the IC50 values
of the variant 61H and
24,25,27L are comparable to that of parental
HuCOL-1. When mCOL-1 was immobilized on the sensor chip and mCOL-1,
HuCOL-1, and the variant
24,25,27L/61H were used to
compete with it for binding to the anti-V region Abs in the serum
of patient MB, the competition profiles shown in Fig. 6 were generated. The data show that the
concentration of HuCOL-1 required for 50% inhibition of the binding of
the patient’s serum to mCOL-1 is 3-fold higher than that of mCOL-1,
whereas the concentration of the variant
24,25,27L/61H required to
attain 50% inhibition of the binding of mCOL-1 to the patient’s serum
is 5.5- and 17-fold higher than those of HuCOL-1 and mCOL-1,
respectively. Moreover, it should be pointed out that the slope of the
competition profile of the variant
24,25,27L/61H is quite
different from that of mCOL-1. Indeed, there was >2 log differential
in the concentrations of the
24,25,27L/61H and mCOL-1
mAbs required for the 60% inhibition of the binding of the sera
anti-V region Abs to mCOL-1 immobilized on the sensor chip (Fig. 6). Sera from two other patients, JS and EM, were used to compare serum
reactivity of mCOL-1 and HuCOL-1. Although the
IC50 value of HuCOL-1 was 6-fold higher than
that of mCOL-1 for JS serum, it was not possible to evaluate the
difference in the reactivity of the two Abs to EM serum; even 1000 nM
HuCOL-1 was unable to attain 50% inhibition of the binding of the
serum to mCOL-1 (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Development of a mouse-human chimeric mAb is an approach that has been
widely used to minimize HAMA response (52). Accordingly, a
cCOL-1 mAb was developed by replacing the C regions of the L and H
chains of the mCOL-1 with the human and 1 constant regions. The
Ag-binding affinity of the cCOL-1 (3.45 x
108 M-1) was found to be
comparable to that of murine Ab (5.17 x 108
M-1). A chimeric Ab, though likely to be less
immunogenic than the murine Ab, may evoke anti-V region response in
patients, because the V region of a murine Ab is potentially
immunogenic. Anti-V region responses have been reported after the
administration of chimeric Abs in patients (53, 54 ;
reviewed in Ref. 55). To attempt to reduce this problem,
mAb COL-1 has been humanized following a procedure that involves
grafting of the CDRs of a xenogeneic Ab onto the human Ig frameworks
(reviewed in Ref. 8).
The most important consideration in humanizing an Ab is the preservation of its Ag-binding property, which depends on the structural integrity of the combining site. This requires selecting the most appropriate human templates for humanization and grafting the CDRs of the target Ab onto the human scaffold, while retaining those murine framework residues that may be involved in ligand contact directly or through their interaction with the CDRs. In the absence of a three-dimensional structure of mCOL-1, the identification of the crucial frameworks residue was facilitated by a search of the database that yielded the V regions of two human Abs, VJI’CL and MO30, respectively, whose sequences were most similar to the mCOL-1 VL and VH sequences. There are 76 identities among the 112 overall residues of the VL of the VJI’CL and mCOL-1, and when comparing the 80 framework residues only, there are 65 identities. Among the 127 overall VH residues of the MO30 and COL-1 mAbs, there are 75 identities, while there are 57 identities in their 87 frameworks residues. Because the VL frameworks of VJI’CL and mCOL-1 are so extensively homologous, they differ only by five residues among those deemed crucial for Ag binding. The VH frameworks of COL-1, however, differ from MO30 at 17 positions that are most probably essential for the ligand binding. This approach of selecting the human frameworks to be used as templates had been successful for the humanization of several Abs, including mAb AUK12–2 (56), mAb 1B4 (57), and mAb CC49 (32). Based on these considerations, humanization of mCOL-1 was conducted using VJI’CL and MO30 as human templates, and the resulting HuCOL-1 showed only 2.5-fold lower affinity than that of the murine COL-1.
Because the murine CDRs of a humanized Ab could still evoke an
anti-V region response in patients, CDR grafting is not an ultimate
solution of the potential immunogenicity of a xenogeneic Ab. Padlan et
al. (14) suggested that not all of the CDR residues are
involved in the ligand contact. Tamura et al. (13)
humanized the anti-tumor-associated glycoprotein-72 mAb CC49 by
grafting only its SDRs onto the human Ab frameworks. The HuCC49
variant, designated HuCC49V10, that was developed by this approach
retained the Ag-binding properties of the parental mAb, while reacting
with the patients’ sera only minimally. The immunogenicity of an Ab,
therefore, could be reduced by transplanting only those parts of the
CDRs that contain the SDRs. The "abbreviated" CDRs have been
defined (14). Based on this rationale, a variant of each
of the L and H chains of the HuCOL-1 was developed. In L chain variant,
residues 24, 25, and 27 of the L chain CDR1 were replaced with the
corresponding residues of the human Ab VJI’CL, whereas in H chain
variant, residue 61 of the H-chain CDR2 was replaced with the residue
of the human Ab MO30, located at the same position. The Ag-binding
affinity of the H chain variant was comparable to that of the parental
HuCOL-1, whereas that of the L chain variant was 2.4-fold lower. The
slight loss in the affinity of the L chain variant was reflected in the
affinity of the HuCOL-1 variant (2.7-fold lower than that of the
parental HuCOL-1) that was generated by combining the H and the L chain
variants. Flow cytometric analysis of the CEA-transduced MC38 cells
that were treated with the variant Abs, however, shows that the slight
losses in the relative affinities of the variants had no effect on
their binding ability to the CEA expressed on the cell surface.
Humanization by transplanting the "abbreviated" CDRs was undertaken to eliminate any possible idiotopes, present in the CDR-grafted HuCOL-1, that could be the potential targets of patients’ immune response. However, it should be pointed out that engineering of this variant may have generated new idiotopes on the combining site of HuCOL-1 that may be targeted by the patient’s immune response. The immunogenicity of the COL-1-derived Abs can be evaluated only through their clinical trials in patients. Whether the HuCOL-1 variants, generated by grafting of the "abbreviated" CDRs, have reduced the potential to evoke anti-V region response in patients compared with the CDR-grafted HuCOL-1 was assessed by comparing their reactivity to sera from gastrointestinal cancer patients who were administered 131I-labeled mCOL-1 in a phase I clinical trial (24). The sera were shown to carry anti-V region Abs to COL-1. In lieu of clinical trials, serum reactivity of the variant Ab is as good a measure of its potential immunogenicity as one can get, without administering the variant in a patient. The results confirm an earlier observation using another Ab (13), that the pattern of the anti-V region responses differs from patient to patient. Some patients may elicit more vigorous response to certain idiotopes than other patients; nevertheless, the variant 24,25,27L/61H, compared with HuCOL-1, shows 1.5-, 2-, and 3-fold lower reactivity to EM, JS, and MB sera, respectively. The reduction in serum reactivity of the variant is much more significant than these numbers suggest, because the reactivity of HuCOL-1 to serum MB is 3-fold lower than that of mCOL-1. The lower serum reactivity of the variant 24,25,27L/61H may be due to the elimination of the immunogenic idiotopes from the L chain variant 24,25,27L, which shows 2- to 3-fold lower reactivity to EM and MB sera than does HuCOL-1. These results suggest that the variant 24,25,27L/61H, compared with HuCOL-1, is significantly less reactive to sera from patients who were administered 131I-labeled mCOL-1 in a clinical trial and, hopefully, substantially less immunogenic in patients.
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Acknowledgments |
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Footnotes |
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2 Current address: Tohoku University School of Medicine, 1-1 Seiro-Machi, Aoba-Ku, Sendai 980-77, Japan.
3 Current address: Marine Science Institute, College of Science, University of the Philippines, Diliman, Quezon City, Philippines.
4 Current address: Institute of Molecular Biology and Biotechnology, College of Science, University of the Philippines, Diliman, 1101 Quezon City, Philippines.
5 Current address: Dipartimento Medicina Sperimentale, Sezione di Biotecnologie e Biologia Molecolare, Facolta’ di Medicina e Chirurgia, Seconda Universita’ degli Studi di Napoli, Via Costantinopoli 16, 80128 Naples, Italy.
6 Address correspondence and reprint requests to Dr. Jeffrey Schlom, Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. E-mail address: js141c{at}nih.gov
7 Abbreviations used in this paper: HAMA, human anti-murine Ab; CDR, complementarity-determining region; SDR, specificity-determining residue; CEA, carcinoembryonic Ag; NCA, nonspecific cross-reacting Ag; m, murine; c, chimeric; Hu, humanized; pBSc, pBluescript II S/K+; SPR, surface plasmon resonance.
Received for publication April 12, 2002. Accepted for publication July 3, 2002.
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References |
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. Cancer Res. 50:6248.. Methods: A Companion to Methods in Enzymology 2:111.
: clinical and immunological data. Cancer Immunol. Immunother. 32:185.[Medline]
4) monoclonal antibody in humans. Cancer Res. 51:5461.
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), cCOL-1 (
), HuCOL-1 (
),
24,25,27L (
), 61H (
),
24,25,27L/61H (•), and HuIgG (
) were used
to compete for the binding of 125I-labeled HuCOL-1 to 200
ng of CEA coated in each well. Dashed line indicates competitor
HuCOL-1. The assay was done in triplicate and the error bars denote the
SD from the mean value of the data in triplicate.
