U.S. patent application number 10/787378 was filed with the patent office on 2004-12-23 for gylocosylated humanized b-cell specific antibodies.
This patent application is currently assigned to Immunomedics, Inc. Invention is credited to Hansen, Hans J., Leung, Shui-on, Qu, Zhengxing.
Application Number | 20040258682 10/787378 |
Document ID | / |
Family ID | 21761323 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040258682 |
Kind Code |
A1 |
Leung, Shui-on ; et
al. |
December 23, 2004 |
Gylocosylated humanized B-cell specific antibodies
Abstract
A humanized specific monoclonal antibody or antibody fragment,
especially a B-cell specific antibody or antibody fragment, is
engineered to contain a glycosylation site in the non-Fc constant
region. The glycosylated antibody is useful for diagnosis and/or
therapy whenever a targeting antibody or fragment is used,
especially for B-cell malignancies. The carbohydrate moiety allows
conjugation of labeling or therapeutic agents of increased size,
without affecting the binding affinity or specificity of the
antibody.
Inventors: |
Leung, Shui-on; (Morris
Township, NJ) ; Hansen, Hans J.; (Picayune, MS)
; Qu, Zhengxing; (Warren, NJ) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Immunomedics, Inc
|
Family ID: |
21761323 |
Appl. No.: |
10/787378 |
Filed: |
February 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10787378 |
Feb 27, 2004 |
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09894839 |
Jun 29, 2001 |
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09894839 |
Jun 29, 2001 |
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09155107 |
Nov 17, 1998 |
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6254868 |
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09155107 |
Nov 17, 1998 |
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PCT/US97/04196 |
Mar 19, 1997 |
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60013709 |
Mar 20, 1996 |
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Current U.S.
Class: |
424/130.1 ;
435/320.1; 435/328; 435/69.1; 530/388.26; 536/23.53 |
Current CPC
Class: |
C07K 2317/77 20130101;
C07K 2319/00 20130101; C07K 2317/41 20130101; A61P 35/00 20180101;
A61K 2039/505 20130101; C07K 2317/24 20130101; C07K 2317/54
20130101; C07K 16/2803 20130101; A61K 38/00 20130101 |
Class at
Publication: |
424/130.1 ;
435/069.1; 435/320.1; 435/328; 530/388.26; 536/023.53 |
International
Class: |
C07H 021/04; A61K
039/395; C07K 016/40; C12N 005/06 |
Claims
We claim:
1. A method of producing an antibody or antigen-binding fragment
thereof comprising: expressing said antibody or antigen-binding
fragment thereof which is engineered to contain a glycosylation
site in the non-Fc constant heavy chain region, wherein said
antibody or antigen-binding fragment is glycosylated in the CH1
region, or in the constant light chain region, wherein genes
encoding said heavy chain and light chain regions have been
engineered with a mutation such that a glycosylation site is
created in the CH1 region gene or the constant light chain gene,
and operably linked to expression control elements in an expression
vector, in a cell that allows glycosylation; and producing said
antibody or antibody fragment glycosylated in the CH1 region or the
light chain constant region in said cell.
2. The method of claim 1, wherein said expression vector comprises
an amplifiable dihydrofolate reductase (dhfr) gene.
3. The method of claim 2, wherein said expression vector is
pdHL2.
4. The method of claim 3, wherein said cell is a SP2/0 myeloma
cell.
5. The method of claim 1, wherein the antibody or fragment thereof
comprises a humanized antibody or antigen-binding fragment
thereof.
6. The method of claim 1, wherein the antibody or fragment thereof
comprises a humanized B-cell specific antibody or antigen-binding
fragment thereof.
7. The method of claim 6, wherein said glycosylation is located on
a site selected from the group consisting of the HCN1, HCN2, HCN3,
HCN4, and HCN5 sites (SEQ ID NOS: 10-14) of FIG. 12.
8. The method of claim 7, wherein said glycosylation site is the
HCN5 site (SEQ ID NO: 10) of FIG. 12.
9. The method of claim 7, wherein said glycosylation site is the
HCN1 site (SEQ ID NO: 10) of FIG. 12.
10. The method of claim 6, wherein the antibody or antigen-binding
fragment thereof is engineered to contain a glycosylation site is
an antibody or antigen-binding fragment thereof having the binding
specificity of the hLL2 antibody.
11. The method of claim 1, wherein said glycosylation is located at
a N-linked glycosylation site.
12. The method of claim 10, wherein said expression vector
comprises an amplifiable dihydrofolate reductase (dhfr) gene.
13. The method of claim 12, wherein said expression vector is
pdHL2.
14. The method of claim 13, wherein said cell is a SP2/0 myeloma
cell.
15. The method of claim 1, wherein said antibody or fragment
thereof is encoded by a DNA molecule comprising a DNA sequence
comprising an engineered glycosylation site in the DNA sequence
encoding the CH1 region or the constant light chain region.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/894,839, filed Jun. 29, 2001, incorporated herein by
reference in its entirety, which is a continuation of U.S.
application Ser. No. 09/155,107, filed Nov. 17, 1998, incorporated
herein by reference in its entirety, which is a National Stage
application under 35 U.S.C. .sctn.371 of International Application
No. PCT/US97/04196, filed Mar. 19, 1997, which is an application
claiming the benefit under 35 USC 119(e) of U.S. Application Ser.
No. 60/013,709, filed Mar. 20, 1996, all of which are incorporated
herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] The invention relates generally to immunoconjugates for
diagnostic and therapeutic uses in cancer. In particular, the
invention relates to recombinantly produced humanized monoclonal
antibodies directed against B-cell lymphoma and leukemia cells,
which antibodies can be covalently conjugated to a diagnostic or
therapeutic reagent without loss of antibody binding and
internalization function and with reduced production of human
anti-mouse antibodies.
[0003] Non-Hodgkins lymphoma (NHL) and chronic lymphocytic leukemia
are B-cell malignancies that remain important contributors to
cancer mortality. The response of these malignancies to various
forms of treatment is mixed. They respond reasonably well to
chemotherapy, and, in cases where adequate clinical staging of NHL
is possible, as for patients with localized disease, satisfactory
treatment may be provided using field radiation therapy (Hall et
al., Radiology for the Radiologist, Lippincott, Philadelphia, 1989,
pp 365-376). However, the toxic side effects associated with
chemotherapy and the toxicity to the hematopoietic system from
local, as well as whole body, radiotherapy, limits the use of these
therapeutic methods. About one-half of the patients die from the
disease (Posner et al., Blood, 61: 705 (1983)).
[0004] The use of targeting monoclonal antibodies conjugated to
radionuclides or other cytotoxic agents offers the possibility of
delivering such agents directly to the tumor site, thereby limiting
the exposure of normal tissues to toxic agents (Goldenberg, Semin.
Nucl. Med., 19: 332 (1989)). In recent years, the potential of
antibody-based therapy and its accuracy in the localization of
tumor-associated antigens have been demonstrated both in the
laboratory and clinical studies (see., e.g., Thorpe, TIBTECH, 11:
42 (1993); Goldenberg, Scientific American, Science & Medicine,
1: 64 (1994); Baldwin et al., U.S. Pat. Nos. 4,925,922 and
4,916,213; Young, U.S. Pat. No. 4,918,163; U.S. Pat. No. 5,204,095;
Irie et al., U.S. Pat. No. 5,196,337; Hellstrom et al., U.S. Pat.
Nos. 5,134,075 and 5,171,665). In general, the use of radio-labeled
antibodies or antibody fragments against tumor-associated markers
for localization of tumors has been more successful than for
therapy, in part because antibody uptake by the tumor is generally
low, ranging from only 0.01% to 0.001% of the total dose injected
(Vaughan et al., Brit. J. Radiol., 60: 567 (1987)). Increasing the
concentration of the radiolabel to increase the dosage to the tumor
is counterproductive generally as this also increases exposure of
healthy tissue to radioactivity.
[0005] LL2 (EPB2) is a highly specific anti-B-cell lymphoma and
anti-lymphocytic leukemia cell murine monoclonal antibody (mAb)
that is rapidly internalized by such cells and that can overcome
some of the aforementioned difficulties (Shih et al., Int. J.
Cancer, 56: 538 (1994)). LL2, which is of the IgG2a antibody type,
was developed using the Raji B-lymphoma cell line as the source of
antigen (Pawlak-Byczkowska et al., Cancer Res., 49: 4568 (1989)).
Murine LL2 (mLL2) is known to react with an epitope of CD22
(Belisle et al., Proc Amer. Assn. Clin. Res., 34: A2873 (1993)).
CD22 molecules are expressed in the cytoplasm of progenitor and
early pre-B cells, and appear in the cell surface of mature
B-cells.
[0006] By immunostaining of tissue sections, mLL2 was shown to
react with 50 of 51 B-cell lymphomas tested. mLL2 provides a highly
sensitive means of detecting B-cell lymphoma cell in vivo, as
determined by a radioimmunodetection method (Murthy et al., Eur. J.
Nucl. Med., 19: 394 (1992)). The Fab' fragment of mLL2 labeled with
.sup.99mTc localized to 63 of 65 known lesions in Phase II trial
patients with B-cell lymphoma (Mills et al., Proc. Amer. Assn.
Cancer Res., 14: A2857 (1993)). In addition, .sup.131I-labeled mLL2
was therapeutically effective in B-cell lymphoma patients
(Goldenberg et al., J. Clin. Oncol., 9: 548 (1991)). mLL2 Fab'
conjugated to the exotoxin PE38 KDEL induced complete remission of
measurable human lymphoma xenografts (CA-46) growing in nude mice
(Kreitman et al., Cancer Res., 53: 819 (1993)).
[0007] The clinical use of mLL2, just as with most other promising
murine antibodies, has been limited by the development in humans of
a human anti mouse antibody response (HAMA). While a HAMA response
is not invariably observed following injection of mLL2, in a
significant number of cases patients developed HAMA following a
single treatment with mLL2. This can limit the diagnostic and
therapeutic usefulness of such antibody conjugates, not only
because of the potential anaphylactic problem, but also as a major
portion of the circulating conjugate may be complexed to and
sequestered by the circulating anti-mouse antibodies. This is
exemplified by one study in which about 30% of the patients
developed low level HAMA response following a single injection of
about 6 mg of mLL2 .sup.131I-IgG and nearly all developed a strong
HAMA response with additional injections. On the other hand, with
mLL2 Fab' labeled with .sup.99mTc, no HAMA response was observed.
Such HAMA responses in general pose a potential obstacle to
realizing the full diagnostic and therapeutic potential of the mLL2
antibody.
[0008] As noted above, the use of fragments of mLL2, such as
F(ab').sub.2 and Fab', partially alleviates/circumvents these
problems of immunogenicity. However, there are circumstances in
which whole IgG is more desirable, such as when induction of
cellular immunity is intended for therapy, or where an antibody
with enhanced survival time is required.
[0009] For monoclonal antibodies to function as the delivery
vehicles for drugs and radionuclides, it is of prime importance to
develop methods for their site-specific conjugations, with minimal
perturbation of the resultant immunoreactivities. Most commonly,
the conjugation of drugs and radionuclides are accomplished through
their covalent attachments to side chains of amino acid residues.
Due to the non-site-restricted nature of these residues, it is
difficult to avoid undesirable couplings at residues that lie
within or are in close vicinity to the ABS, leading to reduced
affinity and heterogenous antigen-binding properties.
Alternatively, conjugation can be directed at sulfhydryl groups.
However, direct labeling relies on the reduction of S--S bonds,
with the possible risk of protein fragmentation.
[0010] U.S. patent application Ser. No. 08/289,576, now abandoned,
but refiled as continuation application, U.S. patent application
Ser. No. 08/690,102, now U.S. Pat. No. 5,789,554, issued on Aug. 4,
1998, the entire disclosure of which is incorporated herein by
reference, discloses a humanized mAb having a naturally occurring
N-linked glycosylation site found at amino acid positions 18-20 of
the LL2 VK domain for site-specific drug or chelate conjugation.
The attached carbohydrate moiety was positioned away from, and
demonstrated no physical contacts with, the antigen binding site
(ABS). The immunoreactivity of the antibody was not affected when
chelates such as DTPA were conjugated to the carbohydrate.
[0011] However, there are limitations to the usefulness of this
antibody. For one, it is not clear what size and type of chelates
can be attached before immunoreactivity is affected. We have
determined that attachment of larger chelates does affect the
binding affinity. Thus, attachment of an 18 kD Dox-dextran to the
carbohydrate at position 18-20 of the LL2 VK domain reduces
immunoreactivity to about 50%. Furthermore, it would be very
advantageous to engineer other antibodies to contain active
glycosylation sites. Engineering other antibodies so that
glycosylation sequences are present in the variable region is
difficult because the engineering steps would need to be repeated
for each antibody. Furthermore, the immunoreactivity of the
construct might be affected.
[0012] IgG glycosylation at Asn-297 in the CH2 Fc domain has been
well-characterized as important for the maintenance of antibody
stability and the appropriate structure for proper effector
functions. See Tao and Morrison, J. Immunol. 143: 2595 (1989). Due
to the restricted localization of immunoglobulin glycosylation
sites, which are distal to the ABS, oligosaccharide modification of
monoclonal antibodies was used to prepare conjugates. Conjugates
modified with .sup.131I coupled to a tyrosine-containing peptide,
which was then site-specifically attached to oxidized
oligosaccharides, exhibited greater targeting efficiency compared
to the conjugates that were modified nonselectively on tyrosine.
Because the use of Asn-297-associated carbohydrate requires the
presence of the Fc portion of the antibody, its use is limited.
There are certain applications employing antibody fragments in
which the Fc portion is not present.
SUMMARY OF THE INVENTION
[0013] The present invention extends those approaches by
engineering N-linked glycosylation sites into the Constant-kappa
(CK), a constant light chain domain and the constant-heavy (CH1)
domains. This has the following advantages:
[0014] 1. glycosylation will be on a different domain which is
physically more distant from the variable domains constituting the
ABS;
[0015] 2. high dosage conjugation of chelates or even bulky groups
which might affect the fine structure of the CK or CH1 domain would
be expected to have minimal effects, if any, on the VH and VK
domains forming the ABS;
[0016] 3. antibody fragments, a preferred format in some clinical
applications, contain both the CH1 and CK domains, and the
conjugation site should be suitable for use in antibody fragments
(e.g., Fab, F(ab').sub.2);
[0017] 4. unlike the VK-appended glycosylation site which would
have to be introduced (e.g. by site-directed mutagenesis) into
different antibodies on a case-by-case basis, the CK or CH1 domain
containing the carbohydrate addition sites, once identified as an
efficient conjugation handle, can easily be ligated to different
variable domains having different antigen specificities.
[0018] It is an object of this invention to provide humanized
antibodies, having glycosylation in the CK or CH1, domains, that
retain antigen binding specificity.
[0019] It is another object of this invention to provide conjugates
of the glycosylated mAbs containing therapeutic or diagnostic
modalities.
[0020] It is a further object of this invention to provide methods
of therapy and diagnosis that utilize the humanized mAbs of the
invention.
[0021] In order to achieve these objectives, in one aspect of the
invention, a monoclonal antibody or antibody fragment which is
engineered to contain a glycosylation site in the non-Fc constant
heavy-chain or light-chain region has been provided. In a preferred
embodiment, the monoclonal antibody or antibody fragment is a
humanized antibody or antibody fragment. In another preferred
embodiment, the humanized specific monoclonal antibody is a
humanized B-cell specific antibody or antibody fragment. In yet
another preferred embodiment, the glycosylation is located on a
site selected from the group consisting of the HCN1, HCN2, HCN3,
HCN4, and HCN5 sites of FIG. 12. In particularly preferred
embodiments, the glycosylation site is the HCN5 site or the HCN1
site of FIG. 12. In a further preferred embodiment, the antibody
which is engineered to contain a glycosylation site is an antibody
having the specificity of the hLL2 antibody.
[0022] In another aspect of the invention, an isolated DNA molecule
comprising an antibody heavy chain gene which comprises a sequence
within the CH1 region has been provided, which, when the gene is
coexpressed with a second gene for an antibody light chain in a
cell supporting glycosylation, will produce an antibody
glycosylated in the CH1 region.
[0023] In a further aspect, an isolated DNA molecule comprising an
antibody light chain gene which comprises a sequence within the
constant region has been provided, which, when said gene is
coexpressed with a second gene for an antibody heavy chain in a
cell supporting glycosylation, will produce an antibody
glycosylated in the constant K region.
[0024] In a yet further aspect of the invention, a method of
producing an antibody or antibody fragment glycosylated in the
constant K and/or CH1 region has been provided comprising
coexpressing light and heavy chain genes or portions thereof, which
have been engineered with a mutation such that a glycosylation site
is created in the constant K region or into the CH1 region of said
heavy chain gene or portions thereof, in a cell that allows
glycosylation, such that the antibody or antibody fragment
glycosylated in the constant K and/or CH1 region is produced, and
isolating the antibody or antibody fragment.
[0025] In a further still aspect of the invention, a method of
diagnosis or treatment of a patient has been provided, wherein a
monoclonal antibody or antibody fragment is used to target a
specific antigen, the antibody or fragment being used as such or
conjugated to a diagnostic or therapeutic agent,
[0026] the improvement wherein said antibody or fragment is a
humanized monoclonal antibody or antibody fragment engineered to
contain a glycosylation site in the non-Fc constant heavy-chain or
light-chain region. In a preferred embodiment, the antibody or
antibody fragment is a B-cell specific antibody or antibody
fragment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows a comparison of murine and humanized LL2 VK
(FIG. 1A, SEQ ID NOS: 2, 6 & 20) and VH (FIG. 1B, SEQ ID NOS:
4, 21 & 8) domains. Only hFR sequences (designated as REIHuVK
and EUHuVH) different than mFR sequences (designated as murine) are
shown, and designated by asterisks. CDRs are boxed. FR residues
shown by computer modeling to contact a CDR are underlined.
[0028] FIG. 2 shows the vicinal relationships of LL2 CDRs to their
framework regions (FRs). Separate energy-minimized models for the
VL and VH domains of mLL2 were constructed, and all FR residues
within a radius of 4.5 .ANG. or any CDR atom were identified as
potential CDR-FR contacts. CDRs of the light (L1, L2, and L3, FIG.
2A) and heavy (H1, H2, and H3, FIG. 2B) chains are shown as "ball
and stick" representations superimposed on their respective,
space-filling FRs.
[0029] FIG. 3A shows the light chain staging (VKpBR) and mammalian
expression (pKH) vectors, and FIG. 3B shows the heavy chain staging
(VHpBS) and mammalian expression (pG1g) vectors.
[0030] FIG. 4 shows the double-stranded DNA and amino acid
sequences of the LL2 VK domain (FIG. 4A, SEQ ID NOS: 1 & 2) and
the LL2 VH domain (FIG. 4B, SEQ ID NOS: 3 & 4). Amino acid
sequences encoded by the corresponding DNA sequences are given as
one letter codes. CDR amino acid sequences are boxed. The
Asn-glycosylation site located in FR1 of LL2VK (FIG. 4A) is shown
as the underlined NVT sequence.
[0031] FIG. 5A shows the double stranded DNA and corresponding
amino acid residues of the hLL2 VK domain (SEQ ID NOS: 5 & 6).
CDR amino acid sequences are boxed. The corresponding data for the
VH domain (SEQ ID NOS: 7 & 8) is shown in FIG. 5B.
[0032] FIG. 6 is a schematic diagram representation of the PCR/gene
synthesis of the humanized VH region and the subcloning into the
staging vector, VHpBS.
[0033] FIG. 7 shows the results of a comparative Raji cell
competitive antibody binding assay involving mLL2 and cLL2
antibodies competing for binding to cells against tracer
radiolabeled mLL2.
[0034] FIG. 8 shows the results of a comparative Raji cell
competitive antibody binding assay in which mixed
humanized/chimeric LL2s were compared to cLL2 (FIG. 8A), and two
versions of hLL2 compared to cLL2 (FIG. 8B).
[0035] FIG. 9 shows a comparison of antibody
internalization:surface binding ratios as a function of time for
cLL2, cLL2 (Q to V mutagenesis), hLL2 and mLL2 antibodies.
[0036] FIG. 10 shows the effect of deglycosylation of mLL2 on its
binding affinity to Raji cells.
[0037] FIG. 11 shows a competitive binding assay where peroxidase
conjugated mLL2 binding to WN was measured. hLL2 and glycosylated
derivatives in the heavy chain constant regions, at the indicated
concentrations, were used to compete with mLL2.
[0038] FIG. 12 shows the N-glycan acceptor sequences and positions
introduced into the CH.sub.1 and CK domains of hLL2 (SEQ ID NOS:
9-19). Site-directed mutagenesis were used to generate the
tri-peptide acceptor sequences (shown in bold letters). Partial
peptide sequences of the CH.sub.1 (H chain) and CK (K chain)
domains of hLL2 are shown and aligned according to sequence and
structure homology to indicate the locations of engineered
potential N-linked glycosylation sites (HCN1--HCN5 and KCN1-KCN4).
The .beta.-strand sequences (C--F) are boxed. The residues were
numbered according to Kabat's system; asterisk (*) indicate these
heavy chain aa residues which were numbered discontinuously from
the previous aa residue. The aa residues indicated by * are
numbered, from left to right, as 156, 162, 171, 182, 203, and 205,
respectively.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039] Glycosylation sites are engineered into CK and CH1
immunoglobulin domains to provide humanized immunoglobulin with
engineered glycosylation sites. By using site-directed mutagenesis,
glycosylation sites are engineered into the constant regions of the
heavy and light chains, specifically into the CK and CH1 domains.
The mutated CK and CH1 nucleotide sequences are then subcloned into
light and heavy chain expression vectors, respectively. The CH1
mutated heavy chain expression vector is coexpressed with a light
chain expression vector to produce mutated, humanized antibodies
with altered glycosylation sites in the CH1 domain. A similar
procedure is followed to produce mutated humanized antibodies with
altered glycosylation sites in the CK domain.
[0040] It should be noted that not all potential
carbohydrate-addition sequences can be used for oligosaccharide
attachment. A series of glycosylation mutants were generated by
introducing novel N-linked glycosylation sequences at the heavy
chain complementarity determining region 2 (CDR2) region of
anti-dextran and anti-dansyl antibodies, respectively. While
glycosylation as found at Asn 54 and Asn 60 of the anti-dextran
antibody, the carbohydrate addition site placed in a similar
position (Asn 55) in the anti-dansyl antibody, however, was not
utilized. This "position effect" is not well understood, but is
most likely to be related to the protein conformation and
accessibility of the carbohydrate acceptor sequence to
glycolyl-transferase.
[0041] In this specification, the expressions "hLL2" or "hLL2 mAb"
are intended to refer to the monoclonal antibody constructed by
joining or subcloning the complementarity determining regions
(CDRs) of murine VK and VH regions to human framework regions (FRs)
and joining or subcloning these to human constant light and heavy
chains, respectively.
[0042] Covalent conjugates between the mutated antibodies of the
invention and a diagnostic or chemotherapeutic reagent, formulated
in pharmaceutically acceptable vehicles (see, e.g., Remington's
Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton,
Pa., 1990) can be prepared. B cell lymphoma and leukemia specific
antibodies comprising glycosylated CK and CH1 domains conjugated to
a diagnostic or therapeutic reagent resulting in humanized mAbs
continue to have the ability to internalize into target cells, and
to rapidly liberate the diagnostic or chemotherapeutic reagent
intracellularly (thereby increasing effectiveness of the reagent),
and the added advantage of a reduction of the HAMA response in the
human patient.
[0043] Since the carbohydrate moiety of the engineered antibodies
of the invention is not involved in the binding of the antigen,
conjugates in which a reagent is bound to the antibody through
carbohydrate moieties can be used. For example, a reagent can be
conjugated to an oxidized carbohydrate derivative. Methods for the
production of such conjugates, and their use in diagnostics and
therapeutics are provided, for example, in Shih et al., U.S. Pat.
No. 5,057,313, Shih et al., Int. J. Cancer 41: 832 (1988), and
copending, commonly owned Hansen et al., U.S. Ser. No. 08/162,912,
now U.S. Pat. No. 5,443,953, issued on Aug. 22, 1995, the contents
of which are incorporated herein by reference. Direct linkage of a
reagent to oxidized carbohydrate without the use of a polymeric
carrier is described in McKearn et al., U.S. Pat. No. 5,156,840,
which is also incorporated by reference.
[0044] A wide variety of diagnostic and therapeutic reagents can be
advantageously conjugated to the antibodies of the invention. These
include: chemotherapeutic drugs such as doxorubicin, methotrexate,
taxol, and the like; chelator, such as DTPA, to which detectable
labels such as fluorescent molecules or cytotoxic agents such as
heavy metals or radionuclides can be complexed; and toxins such as
Pseudomonas exotoxin, and the like. Several embodiments of these
conjugates are described in the examples below.
[0045] Additional or alternative glycosylation sites (NXT/S) can be
designed and introduced into the Vk, Ck and CH domains of any
antibody according to the invention, for example hLL2 (here X
stands for any amino acid but proline or aspartate). The effects on
binding specificity, biodistribution in vivo, in test animals, and
efficiency of conjugation of drugs and chelates of the glycosylated
moieties can be assayed to determine useful glycosylation sites.
Likely sites for glycosylation may be identified by comparison with
glycosylation sites from known Ab of different species or isotypes,
by analysis of the known structures of human CK and CH1 domains by
computer modeling to identify exposed positions, or by random
shot-gun mutagenesis.
[0046] Cell lines and culture media used in the present invention
include LL2 (EPB-2) hybridoma cells (Pawlak-Byczkowska et al. 1989
above), Sp2/0-Ag12 myeloma cells (ATCC, Rockville, Md.) and Raji
cells. These cells are preferably cultured in Dulbecco's modified
Eagle's Medium (DMEM) supplemented with 10% FCS (Gibco/BRL,
Gaithersburg, Mass.), 2 mM L-glutamine and 75 .mu.g/ml gentamicin,
(complete DMEM). Transfectomas are grown in Hybridoma Serum Free
Medium, HSFM, (Gibco/BRL, Gaithersburg, Mass.) containing 10% of
FCS and 75 .mu.g/ml gentamicin (complete HSFM) or, where indicated,
in HSFM containing only antibiotics. Selection of the transfectomas
may be carried out in complete HSFM containing 500 .mu.g/ml of
hygromycin (Calbiochem, San Diego, Calif.). All cell lines are
preferably maintained at 37.degree. C. in 5% CO.sub.2.
[0047] Designing Glycosylation Sites in CH1 and CK
[0048] An important aspect of this invention is that antibody
conformations can be modeled by computer modeling (see, for
example, Dion, in Goldenberg et al. eds., Cancer Therapy With
Radiolabelled Antibodies, CRC Press, Boca Raton, Fla., 1994), which
is incorporated by reference. In general, the 3-D structures are
best modeled by homology, which is facilitated by the availability
of crystallographic data from the Protein Data Bank (PDR Code 1REI,
Bernstein et al., J. Mol. Biol. 112: 535 (1977)), which is
incorporated by reference. Similarly, the antibody EU (VH)
sequences (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL
INTEREST, 5th edition, US Dept. of Health and Human Services, US
Gov. Printing Office (1991)) can be selected as the modeling
counterparts for FR1 to FR3 of the mLL2 heavy chain; FR4 was based
on NEWM. Id. As X-ray coordinate data is currently lacking for the
EU sequence, NEWM structural data (PDR Code 3FAB) for FRs 1 to 4
can be used, and amino acid side groups can be replaced to
correspond to mLL2 or EU (hLL2) as needed. The CDR of the light
chain can be modeled from the corresponding sequence of 1MCP
Protein Data Bank (L1 and L2) and 1REI (L3). For heavy chain CDRs,
H1 and H2 can be based on 2HFL Protein Data Bankand 1MCP,
respectively, while H3 can be modeled de novo. Wherever possible,
side group replacements should be performed so as to maintain the
torsion angle between C.alpha. and C.beta.. Energy minimization may
be accomplished by the AMBER forcefield (Weiner et al, J. Amer.
Chem. Soc. 106: 765 (1984) using the convergent method. Potentially
critical FR-CDR interactions can be determined by initially
modeling the light and heavy variable chains of mLL2. All FR
residues within a 4.5 .ANG. radius of all atoms within CDRs can
thereby be identified and retained in the final design model of
hLL2.
[0049] The homologous molecular model of Fab fragment of hLL2 was
created with QUANTA protein modeling package using the x-ray
structure of humanized anti-p185her2 antibody fragments (1FVD) as
main template. See Carter et al., Proc. Natl. Acad. Sci. 89: 4285
(1992); Eizenbrot et al., J. Mol. Biol. 229: 969 (1993). The
sequence identity between the two antibodies is about 80%. The
insertion regions were modeled by searching available protein data
libraries. After all coordinates were generated and connection
regions were regularized, a series of energy minimizations were
applied to the model. This includes 100 step Steepest descent (SD)
and Conjugated Gradient (CG) EM for side chain atoms only, then 100
step SD and CG EM for all except C.alpha. atoms and finally 100
step SD and EM for all atoms. A distance related dielectric
constant, 4r (r is the atom-atom distance in A) was used for
electrostatic interactions. The RMS of atomic position for
equivalent main chain and side chain atoms between 1FVD and hLL2
were 1.46 .ANG. and 2.11 .ANG., respectively. Point mutations were
then applied to hLL2 to generate the models of mutant antibodies,
hLL2HCN1 and hLL2HCN5. Complex-type oligosaccharides were modeled
using the same program with the compositions and structures
elucidated from carbohydrate sequencing.
[0050] Each generated oligosaccharide chain was then anchored to
the corresponding N-linked glycosylation site with the 01 of the
terminal GlcNac superimposed to the N.sub.d of the Asn and 01C1
bond of the GlcNac co-lined with one of Nd-H bonds of the Asn. The
conformation of the attached oligosaccharide chain was sequentially
manipulated so that the longest branch was close to the variable
region of the heavy chain of hLL2. After each adjustment, 100 step
SD and CG EM were applied to sugar atoms with fixed anchor atoms
and hLL2 atoms.
[0051] The designs for the CK and CH1 glycosylation sites are based
on the following principles:
[0052] 1. A carbohydrate-addition-site with the sequence NXS/T was
chosen. X can be any amino acids except Proline and Aspartate.
Whenever possible, only single amino acid changes to install
potential glycosylation sites at a chosen position were attempted
so as to minimize perturbation of the domain structure.
[0053] 2. Potential CK or CH1-associated glycosylation sites can be
identified from known antibodies sequence of different species or
isotypes.
[0054] 3. Analyses of the known structures of human CK and CH1
domains by computer modeling to identify exposed positions where
potential Asn-glycosylation sites can be planted.
[0055] Based on computer modeling studies, the closest approach
distance between the VK-appended oligosaccharide and the CDRs was
estimated to be 20 .ANG.. A distance greater than 4.1 .ANG. is
considered to be free of interactions. Thus, glycosylation sites
which are 4.1 .ANG. or further away from the antigen binding site
are likely candidates for use as conjugation sites for antibody
fragments. Whenever possible, the mutations introduced into the CH1
and CK domains are conservative in nature, so as to maintain the
final tertiary structure of the protein domains. A conservative
mutation generally involves substitution of one for another by
similar size and clinical properties. Specifically, the desired
sequence is NXT/S. For example, replacement of a glutamine (Q) in
the original sequence with asparagine (N) would be considered a
conservative substitution. In this way, various CH1 and CK domain
mutations can be designed to produce inventive glycosylation
sites.
[0056] Only exposed sites will have the chance of being
glycosylated. Therefore, computer modeling to help locating
additional sites that are at potentially favorable positions was
employed. The glycosylation site HCN5 was predicted to be farther
away from the ABS and at the surface position; HCN5 site is located
at the bottom loop formed between the E and F-stands. Other sites,
which are "evenly" dispersed along the C.kappa. and CH1, domains
sequences, were randomly selected. In all cases, possible
perturbations in the final tertiary structure were minimized by
carefully choosing sequences that required only one single amino
acid substitution to become potential glycosylation site. A total
of five CH.sub.1, (HCN1-5) and four C.kappa. (KCN1-4)-appended
sites were introduced to the CH.sub.1, and C.kappa. domains,
respectively. None of these sites appeared to be "buried," or at
the interface between two juxtaposed domains, as confirmed by
computer modeling analyses.
[0057] N-glycosylation was described only as an example. The
principles involved are equally applicable to O-glycosylation. An
artisan skilled in the art would readily understand the application
of the modeling, the design of glycosylation sites, and alteration
of constant K, CH, and VK regions, to allow for O-glycosylation.
O-glycosylation is known to occur at either threoine or serine. The
acceptor sequence for O-linked glycosylation is relatively ill
defined (Wilson et al., Biochem. J. 275: 526 (1991). There could be
a bias for higher content of proline, serine and threonine in these
regions, but accessibility, rather than the exact primary sequence
determines whether a particular threonine or serine residue will be
O-glycosylated. Nevertheless, potential O-glycosylation sequences,
such as those identified in other antibodies known to have
O-glycosylation (Chandrashekarkan et al., J. Biol. Chem. 259: 1549
(1981); Smyth and Utsumi, Nature 216: 322 (1967); Kim et al., J.
Biol. Chem. 269: 12345 (1994), can be used as the standard
sequences for grafting into different positions in the antibodies
of interest. Those confirmed to contain extensive O-glycosylation
can then be tested as conjugation site.
[0058] Another important aspect of the invention is that once a
glycosylation site is identified, further identification of other
potential glycosylation sites is made easier. This is due to two
phenomena. For one, successful glycosylation confirms and helps
further refine the modeling of the relevant regions. Secondly, the
constant K and CH1 regions are understood to display considerable
symmetry. Therefore, identification of a site where glycosylation
occurs on, say CH1, leads to an expectation that the equivalent CK,
position would be a good glycosylation site.
[0059] Light chain mutations. Potential N-linked glycosylation
sequences have been identified in the kappa constant regions of
rabbit antibodies at aa position 161-163 and 174-176. Similar sites
can be introduced into the CK domain of hLL2. See FIG. 12 for
examples.
[0060] Heavy chain mutations. In CH1, a
carbohydrate-addition-sequence, Asn-Asn-Ser, has been identified at
a.a. positions 161-163 (Kabat's numbering; Kabat et al., 1991) in
some of the human IgM CH1 domains. Similarly, the sequence
Asn-Val-Thr, was positioned in a.a. positions 168-170 in the CH1
domain of human IgA. Examples of sequences which can be modified to
produce altered glycosylation sites are: mutating the human IgG1
sequence Asn-Ser-Gly to Asn-Ser-Val at a.a. positions 162-164,
Ala-Leu-Thr to Asn-Leu-Thr at a.a. positions 165-167, and
Leu-Thr-Ser to Asn-Thr-Ser at a.a. positions 166-168, respectively.
These three potential N-linked glycosylation sites, are analogous
to that of IgM and IgA and can be introduced into the CH1 domain of
human IgG1, with expectation of minimal interference on the
resultant structure. Such glycosylation sites may thus remain in a
"natural" position. The design of similar mutations is well within
one of skill in the art, based on the teachings in the
specification.
[0061] Site-Directed Mutagenesis
[0062] Detailed protocols for oligonucleotide-directed mutagenesis
and related techniques for mutagenesis of cloned DNA are
well-known. For example, see Sambrook et al., supra, and Ausubel et
al., supra.
[0063] Asn-linked glycosylation sites may be introduced into
antibodies using conventional site-directed oligonucleotide
mutagenesis reactions. For example, to introduce an Asn in position
18 of a kappa protein, one may alter codon 18 from AGG to AAC. To
accomplish this, a single stranded DNA template containing the
antibody light chain sequence is prepared from a suitable strain of
E. coli (e.g., dut.sup.- ung.sup.-) in order to obtain a DNA
molecule containing a small number of uracils in place of
thymidine. Such a DNA template can be obtained by M13 cloning or by
in vitro transcription using a SP6 promoter. See, for example,
Ausubel et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John
Wiley & Sons, NY, 1987. An oligonucleotide complementary to the
single stranded DNA, comprising the mutated sequence is synthesized
conventionally, annealed to the single-stranded template and the
product treated with T4 DNA polymerase and T4 DNA ligase to produce
a double-stranded DNA molecule. Transformation of wild type E. coli
(dut.sup.+ ung.sup.+) cells with the double-stranded DNA allows
recovery of mutated DNA.
[0064] Alternatively, an Asn-linked glycosylation site can be
introduced into an antibody light chain using an oligonucleotide
containing the desired mutation, any amplifying of the
oligonucleotide by PCR, and cloning it into the variable regions
for the VL chain, or by using RNA from cells that produce the
antibody of interest as a template. Also see, Huse, in ANTIBODY
ENGINEERING: A PRACTICAL GUIDE, Boerrebaeck, ed., W.H. Freeman
& Co., pp 103-120, 1992. Site-directed mutagenesis can be
performed, for example, using the TRANSFORMER.TM. kit (Clontech,
Palo Alto, Calif.) according to the manufacturer's
instructions.
[0065] Alternatively, a glycosylation site can be introduced by
synthesizing an antibody chain with mutually priming
oligonucleotides, one such containing the desired mutation. See,
for example, Uhlmann, Gene 71: 29 (1988); Wosnick et al., Gene 60:
115 (1988); Ausubel et al., above, which are incorporated by
reference.
[0066] Although the description above referred to the introduction
of an Asn glycosylation site in position 18 of the light chain of
an antibody, it will occur to the skilled artisan that it is
possible to introduce Asn-linked glycosylation sites elsewhere in
the light chain or in the heavy chain variable region, or in the
constant regions.
[0067] The presence of a glycosylation site, or the absence of such
site in a humanized Ab, where the site was glycosylated in the
murine counterpart, may or may not affect the binding affinity or
specificity of the antibody. Glycosylation sites therefore can be
introduced or removed, by methods described above, but their impact
on activity needs to be determined. For reasons discussed above,
engineering glycosylation sites in the CH1 or CK regions are
preferred.
[0068] General Techniques for RNA Isolation, cDNA Synthesis and
Amplification
[0069] RNA isolation, cDNA synthesis, and amplification can be
carried out as follows. Total cell RNA can be prepared from a LL2
hybridoma cell line, using a total of about 10.sup.7 cells,
according to Sambrook et al., (Molecular Cloning: A Laboratory
Manual, Second ed., Cold Spring Harbor Press, 1989), which is
incorporated by reference. First strand cDNA can be reverse
transcribed from total RNA conventionally, such as by using the
SuperScript preamplification system (Gibco/BRL., Gaithersburg,
Md.). Briefly, in a reaction volume of 20 .mu.l, 50 ng of random
primers can be annealed to 5 .mu.g of RNA in the presence of 2
.mu.l of 10.times. synthesis buffer [200 mM Tris-HCl (pH 8.4), 500
mM KCl, 25 mM MgCl.sub.2, 1 mg/ml BSA], 1 .mu.l of 10 mM dNTP mix,
2 .mu.l of 0.1 M DTT, and 200 units of SuperScript reverse
transcriptase. The elongation step is initially allowed to proceed
at room temperature for 10 min followed by incubation at 42.degree.
C. for 50 min. The reaction can be terminated by heating the
reaction mixture at 90.degree. C. for 5 min.
[0070] Constructing antibodies with engineered glycosylation sites
in the VL and VH regions
[0071] cDNAs encoding the VL and VH regions of the mLL2 mAb have
been isolated and recombinantly subcloned into mammalian expression
vectors containing the genes encoding kappa and IgG.sub.1 constant
regions, respectively, of human antibodies. Cotransfection of
mammalian cells with these two recombinant DNAs expressed a cLL2
mAb that, like the parent mLL2 mAb, bound avidly to, and was
rapidly internalized by B-lymphoma cells.
[0072] The CDRs of the VK and VH DNAs have been similarly
recombinantly linked to the framework (FR) sequences of the human
VK and VH regions, respectively, which are subsequently linked,
respectively, to the human kappa and IgG.sub.1 constant regions,
and expressed hLL2 in mammalian cells.
[0073] Once the sequences for the hLL2 VK and VH domains are
designed, CDR engrafting can be accomplished by gene synthesis
using long synthetic DNA oligonucleotides as templates and
amplifying the long oligonucleotides by PCR, using short
oligonucleotides as primers. In most cases, the DNA encoding the VK
or VH domain will be approximately 350 base pairs (bp) long. By
taking advantage of codon degeneracy, a unique restriction site may
easily be introduced, without changing the encoded amino acids, at
regions close to the middle of the V gene DNA sequence. For
example, at DNA nucleotide positions 157-162 (amino acid positions
53 and 54) for the hLL2 VH domain, a unique AvrII site can be
introduced while maintaining the originally designed amino acid
sequence (FIG. 4B). Two long non-overlapping single-stranded DNA
oligonucleotides (.about.150 bp) upstream and downstream of the
AvrII site (see, for example, oligo A and oligo Bin in Example 3
below) can be generated by automated DNA oligonucleotide
synthesizer (Cyclone Plus DNA Synthesizer, Milligen-Biosearch). The
yields of full length DNA oligonucleotides such as oligos A and B
may be expected to be low. However, they can be amplified by two
pairs of flanking oligonucleotides in a PCR reaction. The primers
can be designed with the necessary restriction sites to facilitate
subsequent subcloning. Primers for oligo A and for oligo B should
contain overlapping sequence at the AvrII site so that the
resultant PCR product for oligo A and B, respectively, can be
joined in-frame at the AvrII site to form a full length DNA
sequence (ca 350 bp) encoding the hLL2 VH domain. The ligation of
the PCR products for oligo A (restriction-digested with PstI and
AvrII) and B (restriction-digested with AvrII and BstEII) at the
AvrII site and their subcloning into the PstII/BstEII sites of the
staging vector, VHpBS, can be completed in a single
three-fragment-ligation step. See for Example 3. The subcloning of
the correct sequence into VHpBS can be first analyzed by
restriction digestion analysis and subsequently confirmed by
sequencing reaction according to Sanger et al., Proc. Natl. Acad.
Sci. USA 74: 5463 (1977).
[0074] The HindIII/BamHI fragment containing the Ig promoter,
leader sequence and the hLL2 VH sequence can be excised from the
staging vector and subcloned to the corresponding sites in a
pSVgpt-based vector, pG1g, which contains the genomic sequence of
the human IgG constant region, an Ig enhancer and a gpt selection
marker, forming the final expression vector, hLL2pG1g. Similar
strategies can be employed for the construction of the hLL2 VK
sequence. The restriction site chosen for the ligation of the PCR
products for the long oligonucleotides (oligos C and D, see
examples below) can be NruI in this case.
[0075] The DNA sequence containing the Ig promoter, leader sequence
and the hLL2 VK sequence can be excised from the staging vector
VKpBR by treatment with BamH1/HindIII, and can be subcloned into
the corresponding sites of a pSVhyg-based vector, pKh, which
contains the genomic sequence of human kappa chain constant
regions, a hygromycin selection marker, an Ig and a kappa enhancer,
to form the final expression vector, hLL2pKh.
[0076] Humanization sometimes results in a reduction or even loss
of antibody affinity. Therefore, additional modification might be
required in order to restore the original affinity. See, for
example, Tempest et al., Bio/Technology 9: 266 (1991); Verhoeyen et
al., Science 239: 1534 (1988), which are incorporated by reference.
Knowing that cLL2 exhibits a binding affinity comparable to that of
its murine counterpart (see Example 5 below), defective designs, if
any, in the original version of hLL2 can be identified by mixing
and matching the light and heavy chains of cLL2 to those of the
humanized version. SDS-PAGE analysis of the different mix-and-match
humanized chimeric LL2 under non-reducing (the disulfide L-H chain
connections remain intact) and reducing conditions (the chains
separate) permits analyses of the relationships of the different
types of light and heavy chains on the properties of the molecule.
For example, migration as multiple bands or as a higher apparent
molecular size can be due to the presence of a glycan group at the
N-linked glycosylation site found in the FR1 region of the murine
VK domain of LL2. A discrete band migrating at about 25 kDa is the
expected molecular size for a non-glycosylated light chain.
[0077] In general, to prepare cLL2 mAb, VH and VK chains of mLL2
can be obtained by PCR cloning using DNA products and primers.
Orlandi et al., infra, and Leung et al., infra. The VK PCR primers
may be subcloned into a pBR327-based staging vector (VKpBR) as
described above. The VH PCR products may be subcloned into a
similar pBluescript-based staging vector (VHpBS) as described
above. The fragments containing the VK and VH sequences, along with
the promoter and signal peptide sequences, can be excised from the
staging vectors using HindIII and BamHI restriction endonucleases.
The VK fragments which are about 600 bp can be subcloned into a
mammalian expression vector, pKh for example, by conventional
methods. pKh is a pSVhyg-based expression vector containing the
genomic sequence of the human kappa constant region, an Ig
enhancer, a kappa enhancer and the hygromycin-resistant gene.
Similarly, the about 800 bp VH fragments can be subcloned into
pG1g, a pSVgpt-based expression vector carrying the genomic
sequence of the human IgG1 constant region, an Ig enhancer and the
xanthine-guanine phosphoribosyl transferase (gpt) gene. The two
plasmids may be transfected into mammalian expression cells, such
as Sp2/0-Ag14 cells, by electroporation and selected for hygromycin
resistance. Colonies surviving selection are expanded, and
supernatant fluids monitored for production of cLL2 mAb by an ELISA
method. A transfection efficiency of about 1-10.times.10.sup.6
cells is desirable. An antibody expression level of between 0.10
and 2.5 .mu.g/ml can be expected with this system.
[0078] General Techniques for RNA Isolation, cDNA Synthesis and
Amplification
[0079] RNA isolation, cDNA synthesis, and amplification can be
carried out as follows. Total cell RNA can be prepared from a LL2
hybridoma cell line, using a total of about 10.sup.7 cells,
according to Sambrook et al., (Molecular Cloning: A Laboratory
Manual, Second ed., Cold Spring Harbor Press, 1989), which is
incorporated by reference. First strand cDNA can be reverse
transcribed from total RNA conventionally, such as by using the
SuperScript preamplification system (Gibco/BRL., Gaithersburg,
Md.). Briefly, in a reaction volume of 20 .mu.l, 50 ng of random
primers can be annealed to 5 .mu.g of RNAs in the presence of 2
.mu.l of 10.times. synthesis buffer [200 mM Tris-HCl (pH 8.4), 500
mM KCl, 25 mM MgCl.sub.2, 1 mg/ml BSA], 1 .mu.l of 10 mM dNTP mix,
2 .mu.l of 0.1 M DTT, and 200 units of SuperScript reverse
transcriptase. The elongation step is initially allowed to proceed
at room temperature for 10 min followed by incubation at 42.degree.
C. for 50 min. The reaction can be terminated by heating the
reaction mixture at 90.degree. C. for 5 min.
[0080] Amplification of VH and VK sequences. The VK and VH
sequences for cLL2 or hLL2 can amplified by PCR as described by
Orlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989))
which is incorporated by reference. VK sequences may be amplified
using the primers CK3BH and VK5-3 (Leung et al., BioTechniques, 15:
286 (1993), which is incorporated by reference), while VH sequences
can be amplified using the primer CH1B which anneals to the CH1
region of murine 1gG, and VHIBACK (Orlandi et al., 1989 above). The
PCR reaction mixtures containing 10 .mu.l of the first strand cDNA
product, 9 .mu.l of 10.times. PCR buffer [500 mM KCl, 100 mM
Tris-HCl (pH 8.3), 15 mM MgCl2, and 0.01% (w/v) gelatin] (Perkin
Elmer Cetus, Norwalk, Conn.), can be subjected to 30 cycles of PCR.
Each PCR cycle preferably consists of denaturation at 94.degree. C.
for 1 min, annealing at 50.degree. C. for 1.5 min, and
polymerization at 72.degree. C. for 1.5 min. Amplified VK and VH
fragments can be purified on 2% agarose (BiORad, Richmond, Calif.).
See Example 3 for a method for the synthesis of an oligo A
(149-mer) and an oligo B (140-mer) on an automated Cyclone Plus DNA
synthesizer (Milligan-Biosearch).
[0081] PCR products for VK can be subcloned into a staging vector,
such as a pBR327-based staging vector VKpBR that contains an Ig
promoter, a signal peptide sequence and convenient restriction
sites to facilitate in-frame ligation of the VK PCR products. PCR
products for VH can be subcloned into a similar staging vector,
such as the pBluescript-based VHpBS. Individual clones containing
the respective PCR products may be sequenced by, for example, the
method of Sanger et al., Proc. Natl. Acad. Sci., USA, 74: 5463
(1977) which, is incorporated by reference.
[0082] Furthermore, it was found that the presence of glycosylation
sites, and therefore of appended carbohydrate (CHO) moieties causes
efficient and superior conjugation of drugs and chelates. This is
especially true when antibody fragments devoid of CH2-appended CHO
are being utilized.
[0083] The DNA sequences described herein include all alleles,
mutants and variants thereof, whether occurring naturally or
experimentally created.
[0084] Production of Antibodies with Mutated CH1 and CK Regions
[0085] CH1 and CK DNA sequences can be isolated, the protein
sequence modeled, and the DNA mutated by methodologies similar to
these described for the VK and VH sequences. Once the CH1 or CK
nucleotide sequence has been excised from a light or heavy chain
clone, and a glycosylation site inserted via mutagenesis, the
mutated CH1 or CK sequence can be re-inserted into the
corresponding heavy or light chain vector. In the case of a CH1
mutant, it can be coexpressed with a kappa chain expression vector,
such as hLL2pKh, into an appropriate cell, e.g., myeloma
Sp2/0-Ag14, and colonies can be selected for hygromycin resistance.
The supernatant fluids can be monitored for production of cLL2,
hLL2, or LL2 engineered with glycosylation sites in the non Fc
constant regions according to the invention by, for example, an
ELISA assay, as described below.
[0086] Transfection, and assay for antibody secreting clones by
ELISA, can be carried out as follows. About 10 .mu.g of hLL2pKh
(light chain expression vector) and 20 .mu.g of hLL2pG1g (heavy
chain expression vector) can be used for the transfection of
5.times.10.sup.6 SP2/0 myeloma cells by electroporation (BiORad,
Richmond, Calif.) according to Co et al., J. Immunol., 148: 1149
(1992) which is incorporated by reference. Following transfection,
cells may be grown in 96-well microtiter plates in complete HSFM
medium (GIBCO, Gaithersburg, Md.) at 37.degree. C., 5% CO.sub.2.
The selection process can be initiated after two days by the
addition of hygromycin selection medium (Calbiochem, San Diego,
Calif.) at a final concentration of 500 .mu.g/ml of hygromycin.
Colonies typically emerge 2-3 weeks post-electroporation. The
cultures can then be expanded for further analysis.
[0087] The level of expression of an Ig gene containing clone could
be enhanced by amplifying the copy number. This is typically done
by selection for a selectable marker linked to the gene of
interest, here the Ig gene. One skilled in the art would be
familiar with the use of such selection. Often the selective marker
is the dihydrofolate reductase gene (dhfr). Typically, a clone that
appears to contain an amplified copy number of the gene is
identified by its expression and amplification is confirmed by
nucleic acid hybridization experiments. Multiple rounds of
selection assay and confirmation by hybridization are typically
undertaken.
[0088] Transfectoma clones that are positive for the secretion of
cLL2, hLL2, or LL2 engineered with glycosylation sites in the non
Fc constant regions according to the invention can be identified by
ELISA assay. Briefly, supernatant samples (100 .mu.l) from
transfectoma cultures are added in triplicate to ELISA microtiter
plates precoated with goat anti-human (GAH)-IgG, F(ab').sub.2
fragment-specific antibody (Jackson ImmunoResearch, West Grove,
Pa.). Plates are incubated for 1 h at room temperature. Unbound
proteins are removed by washing three times with wash buffer (PBS
containing 0.05% polysorbate 20). Horseradish peroxidase (HRP)
conjugated GAH-IgG, Fc fragment-specific antibodies (Jackson
ImmunoResearch, West Grove, Pa.) are added to the wells, (100 .mu.l
of antibody stock diluted.times.10.sup.4, supplemented with the
unconjugated antibody to a final concentration of 1.0 .mu.g/ml).
Following an incubation of 1 h, the plates are washed, typically
three times. A reaction solution, [100 .mu.l, containing 167 .mu.g
of orthophenylene-diamine (OPD) (Sigma, St. Louis, Mo.), 0.025%
hydrogen peroxide in PBS], is added to the wells. Color is allowed
to develop in the dark for 30 minutes. The reaction is stopped by
the addition of 50 .mu.l of 4 N HCl solution into each well before
measuring absorbance at 490 nm in an automated ELISA reader
(Bio-Tek instruments, Winooski, Vt.). Bound antibodies are than
determined relative to an irrelevant chimeric antibody standard
(obtainable from Scotgen, Ltd., Edinburgh, Scotland).
[0089] Antibodies can be isolated from cell culture media as
follows. Transfectoma cultures are adapted to serum-free medium.
For production of chimeric antibody, cells are grown as a 500 ml
culture in roller bottles using HSFM. Cultures are centrifuged and
the supernatant filtered through a 0.2 micron membrane. The
filtered medium is passed through a protein A column (1.times.3 cm)
at a flow rate of 1 ml/min. The resin is then washed with about 10
column volumes of PBS and protein A-bound antibody is eluted from
the column with 0.1 M glycine buffer (pH 3.5) containing 10 mM
EDTA. Fractions of 1.0 ml are collected in tubes containing 10
.mu.l of 3 M Tris (pH 8.6), and protein concentrations determined
from the absorbencies at 280/260 nm. Peak fractions are pooled,
dialyzed against PBS, and the antibody concentrated, for example,
with the Centricon 30 (Amicon, Beverly, Mass.). The antibody
concentration is determined by ELISA, as before, and its
concentration adjusted to about 1 mg/ml using PBS. Sodium azide,
0.01% (w/v), is conveniently added to the sample as
preservative.
[0090] Comparative binding affinities of the antibodies thus
isolated may be determined by direct radioimmunoassay. An cLL2,
hLL2, or LL2 engineered with glycosylation sites in the non Fc
constant regions according to the invention can be used. Antibodies
can be labeled with .sup.131I, or .sup.125I, using the chloramine T
method (see, for example, Greenwood et al., Biochem. J, 89: 123
(1963) which is incorporated by reference). The specific activity
of the iodinated antibody is typically adjusted to about 10
.mu.Ci/.mu.g. Unlabeled and labeled antibodies are diluted to the
appropriate concentrations using reaction medium (HSFM supplemented
with 1% horse serum and 100 .mu.g/ml gentamicin). The appropriate
concentrations of both labeled and unlabeled antibodies are added
together to the reaction tubes in a total volume of 100 .mu.l. A
culture of Raji cells is sampled and the cell concentration
determined. The culture is centrifuged and the collected cells
washed once in reaction medium followed by resuspension in reaction
medium to a final concentration of about 10.sup.7 cells/ml. All
procedures are carried out in the cold at 4.degree. C. The cell
suspension, 100 .mu.l, is added to the reaction tubes. The reaction
is carried out at 4.degree. C. for 2 h with periodic gentle shaking
of the reaction tubes to resuspend the cells. Following the
reaction period, 5 ml of wash buffer (PBS containing 1% BSA) is
added to each tube. The suspension is centrifuged and the cell
pellet washed a second time with another 5 ml of wash buffer.
Following centrifugation, the amount of remaining radioactivity
remaining in the cell pellet is determined in a gamma counter
(Minaxi, Packard Instruments, Sterling, Va.).
[0091] The antigen-binding property of the antibodies of the
invention can be evaluated by competition binding with labeled mLL2
for an LL2 anti-idiotype antibody (WN).
[0092] The Raji cell surface antigen binding affinities of
mix-and-match and fully humanized antibodies can be compared to
that of cLL2 using various concentrations of mLL2 F(ab').sub.2
fragments devoid of the Fc portion as competitors, as evaluated by
flow cytometry assay. Residual surface-bound LL2 antibodies
carrying the human Fc portions (cLL2 and mix-and-match LL2) can be
detected by a FITC-labeled anti-human Fc specific antibody in a
flow cytometry assay. Where mix-and-match LL2 antibodies exhibit
antigen-binding affinities similar to that of cLL2, it can be
concluded that the original designs for the humanization of both
the light and heavy chains retain the mLL2 immunoreactivity.
[0093] The internalization of cLL2, hLL2, or LL2 engineered with
glycosylation sites in the non Fc constant regions according to the
invention into target cells can be followed by fluorescence
labeling, essentially according to the procedure of Pirker et al.,
J. Clin. Invest., 76: 1261 (1985), which is incorporated by
reference. Cultured Raji cells are centrifuged and the cells
resuspended in fresh medium to a concentration of about
5.times.10.sup.6 cells/ml. To each well of a 96-well microtiter
plate, 100 .mu.l of the cell suspension is added. The antibodies,
40 .mu.g/ml, in a volume of 100 .mu.l are added to the reaction
wells at timed intervals so as to terminate all reactions
simultaneously. The plate is incubated at 37.degree. C. in a
CO.sub.2 cell culture incubator. Unbound antibodies are removed by
washing the cells three times with cold 1% FCS/PBS at the end of
the incubation. The cells are then treated with 1 ml of
Formaid-Fresh [10% formalin solution (Fisher, Fair Lawn, N.J.)] for
15 min at 4.degree. C. After washing, antibodies present either on
the cell surface or inside the cells are detected by treatment with
FITC-labeled goat anti-mouse antibody (Tago, Burlingame, Calif.),
or FITC-labeled goat anti-human antibody (Jackson ImmunoResearch,
West Grove, Pa.), depending on whether the antibody being assayed
for is murine, chimeric, or humanized, respectively. Fluorescence
distributions are evaluated using a BH-2 fluorescence microscope
(Olympus, Lake Success, N.Y.).
[0094] The rate of antibody internalization can be determined
according to Opresko et al., (J. Biol. Chem., 262: 4116 (1987)),
using radio-iodinated antibody as tracer. Briefly, radiolabelled
antibodies (1.times.10.sup.4 cpm) are incubated with the Raji cells
(1.times.10.sup.6 cells/ml) at 4.degree. C. for 2 h in 0.5 ml of
DMEM medium containing 1% human serum. Following the reaction
interval, non-specifically bound antibodies are removed by washing
three times with 0.5 ml of DMEM medium. To each of the reaction
tubes 0.5 ml of DMEM medium is added and the suspension incubated
at 37.degree. C. for the determination of internalization. At timed
intervals, triplicates of cells are removed and chilled immediately
in an ice bath to stop further internalization. Cells are
centrifuged at 1000.times.g for 5 min at 4.degree. C. The
supernatant is removed and counted for radioactivity. The
surface-bound radioactivity is removed by treatment with 1 ml 0.1 M
acetate/0.1 M glycine buffer at pH 3.0 for 8 min. in the cold.
Radioactivity removed by the acid treatment, and that remaining
associated with the cells, are determined. The ratio of the
CPM.sub.internalization/CPM.sub.surface is plotted versus time to
determine the rate of internalization from the slope.
[0095] The representative embodiments described below are simply
used to illustrate the invention. Those skilled in these arts will
recognize that variations of the present materials fall within the
broad generic scope of the claimed invention. The contents of all
references mentioned herein are incorporated by reference.
EXAMPLE 1
[0096] Choice of Human Frameworks and Sequence Design for the
Humanization of LL2 Monoclonal Antibody
[0097] By comparing the murine variable (V) region framework (FR)
sequences of LL2 to that of human antibodies in the Kabat data base
(Kabat et al., Sequences of Proteins of Immunological Interest, 5th
ed., U.S. Department of Health and Human Services, U.S. Government
Printing Office, Washington, D.C.), which is incorporated by
reference, the human REI (FIG. 1A,) and EU (FIG. 1B) sequences were
found to exhibit the highest degree of sequence homology to the FRs
of VK and VH domains of LL2, respectively. Therefore, the REI and
EU FRs were selected as the human frameworks onto which the CDRs
for LL2 VK and VH were grafted, respectively. The FR4 sequence of
NEWM, however, rather than that of EU, was used to replace the EU
FR4 sequence for the humanization of LL2 heavy chain. Based on the
results of computer modeling studies (FIGS. 2A and 2B), murine FR
residues having potential CDR contacts, which might affect the
affinity and specificity of the resultant antibody, were retained
in the design of the humanized FR sequences (FIG. 1).
[0098] Two versions of humanized heavy chain were constructed. In
the first version (hLL2-1), the glutamine (Q) at amino acid
position 5 (Kabat numbering) was introduced to include a PstI
restriction site to facilitate its subcloning into the staging
vector (FIG. 3). This murine residue was converted, by
oligo-directed mutagenesis, to the human EU residue valine (V) in
hLL2-2. It should be noted that in the original murine kappa chain
variable sequence, a potential N-linked glycosylation site was
identified at positions 18-20 and was used for carbohydrate
addition. This glycosylation site was not included in the REI FR
sequence used for LL2 light chain humanization.
EXAMPLE 2
[0099] PCR Cloning and Sequence Elucidation for LL2 Heavy and Light
Chain Variable Regions
[0100] The variable regions for both heavy (VH) and light (VK)
chains of mLL2 (IgG2a) were obtained by PCR cloning using DNA
primers as described in general above and in greater detail in
Example 3, below. As PCR is prone to mutations, the variable region
sequence of multiple individual clones for either the heavy or
light chains was determined for six clones and confirmed to be
identical prior to use for the construction of the chimeric
antibody.
[0101] The PCR products for VK were subcloned into a pBR327-based
staging vector, VKpBR, which contained an Ig promoter, a signal
peptide sequence and convenient restriction sites to facilitate
in-frame ligation of the VK PCR products (FIG. 3A). The PCR
products for VH were subcloned into a similar pBluescript-based
staging vector, VHpBS (FIG. 3B).
[0102] As noted above, at least six individual clones containing
the respective PCR products were sequenced according to the method
of Sanger et al., 1977, above. All were shown to bear identical
sequences and their respective sequences were elucidated, as shown
in FIG. 4A for LL2 VK and in FIG. 4B for LL2 VH. No defective
mutations were identified within the sequences encoding the VK and
VH regions. Comparison of the PCR-amplified variable region
sequences of LL2 with the Kabat database (Kabat et al., above)
suggested that the VK and VH sequences of LL2 belong to subgroup 5
and 2B, respectively. Important residues such as Cys for
intra-domain disulfide linkage were retained at appropriate
positions.
[0103] In the FR1 framework region of VK, an N-linked carbohydrate
attachment site, Asn-Val-Thr, was identified at position 18-20
(FIG. 4A), suggesting that the VK of LL2 might be glycosylated. As
will be detailed below, SDS-PAGE analysis under reducing conditions
demonstrated that this Asn glycosylation site is indeed utilized
for carbohydrate addition. The presence of the glycosylation site
in the variable region does not, however, appear to affect the
immunoreactivity of the antibody. A comparison of the
immunoreactivity of mLL2 with that of cLL2 in a competitive RIA
showed that the two antibodies have nearly identical
activities.
EXAMPLE 3
[0104] PCR/Gene Synthesis of the Humanized V Genes
[0105] The designed sequence for the hLL2 VH domain, the
construction of the hLL2 VH domain by long oligonucleotides and
PCR, and the staging vector VHpBS containing the hLL2 VH domain are
summarized in the sketch shown in FIG. 6.
[0106] For the construction of the hLL2 VH domain, oligo A
(149-mer) and oligo B (140-mer) were synthesized on an automated
CYCLONE PLUS DNA synthesizer (Milligen Bioresearch).
[0107] Oligo A represents the minus strand of the hLL2 VH domain
complementary to nucleotides 24 to 172 (SEQ ID NO: 22): 5'-TAT AAT
CAT TCC TAG GAT TAA TGT ATC CAA TCC ATT CCA GAC CCT GTC CAG GTG CCT
GCC TGA CCC AGT GCA GCC AGT AGC TAG TAA AGG TGT AGC CAG AAG CCT TGC
AGG AGA CCT TCA CTG ATG ACC CAG GTT TCT TGA CTT CAG CC-3'.
[0108] Oligo B represents the minus strand of the hLL2 VH domain
complementary to nt 181 to 320 (SEQ ID NO: 23): 5'-CCC CAG TAG AAC
GTA GTA ATA TCC GCA CAA AAA TAA AAT GCC GTG TCC TCA GAC CTC AGG CTG
CTC AGC TCC ATG TAG GCT GTA TTG GTG GAT TCG TCT GCA GTT ATT GTG GCC
TTG TCC TTG AAG TTC TGA TT-3'
[0109] Oligos A and B were cleaved from the support and deprotected
by treatment with concentrated ammonium hydroxide. After the
samples were vacuum-dried (SpeedVac, Savant, Farmingdale, N.Y.) and
resuspended in 100 .mu.l of water, incomplete oligomers (less than
100-mer) were removed by centrifugation through a
CHROMOSPIN-100.TM. column (Clonetech, Palo Alto, Calif.) before the
DNA oligomers were amplified by PCR. All flanking primers for the
separate amplifications and PCR cloning of oligos A and B were
purified by SDS-PAGE essentially according to the methods of
Sambrook et al., 1989, above. From the CHROMASPIN-purified oligo A,
1 .mu.l of sample stock was PCR-amplified in a reaction volume of
100 .mu.l by adding 5 .mu.l of 5 .mu.M of oligo (SEQ ID NO: 24):
5'-CCA GCT GGT CCA ATC AGG GGC TGA AGT CAA GAA ACC TG-3' and of
oligo (SEQ ID NO: 25): 5'-AAG TGG ATC CTA TAA TCA TTC CTA GGA TTA
ATG-3' in the presence of 10 .mu.l of 10.times.PCR Buffer (500 mM
KCl, 100 mM Tris-HCL buffer, pH 8.3, 15 mM MgCl.sub.2) and 5 units
of AMPLITAQ.TM. DNA polymerase (Perkin Elmer Cetus, Norwalk,
Conn.). This reaction mixture was subjected to 30 cycles of PCR
reaction consisting of denaturation at 94.degree. C. for 1 minute,
annealing at 50.degree. C. for 1.5 minutes, and polymerization at
72.degree. C. for 1.5 minutes.
[0110] Oligo B was PCR-amplified by the primer pairs: 5'-TAA TCC
TAG GAA TGA TTA TAC TGA GTA CAA TCA GAA CTT CAA GGA CAA G-3' (SEQ
ID NO: 26) and: 5'-GGA GAC GGT GAC CGT GGT GCC TTG GCC CCA GTA GAA
CGT AGT AA-3' (SEQ ID NO: 27) under similar conditions.
[0111] Double-stranded PCR-amplified products for oligos A and B
were gel-purified, restriction-digested with PstI/AvrII (PCR
product of oligo A) and BstEII/AvrII (PCR product of oligo B), and
subcloned into the complementary PstI/BstEII sites of the heavy
chain staging vector, VHpBS. The humanized VH sequence was
subcloned into the pG1g vector, resulting in the final human IgG1
heavy chain expression vector, hLL2pG1g.
[0112] For constructing the full length DNA of the humanized VK
sequence, oligo E (150-mer) and oligo F (121-mer) were synthesized
as described above. Oligo E comprises (SEQ ID NO: 28): 5'-CCT AGT
GGA TGC CCA GTA GAT CAG CAG TTT AGG TGC TTT CCC TGG TTT CTG CTG GTA
CCA GGC CAA GTA GTT CTT GTG ATT TGC ACT GTA TAA AAC ACT TTG ACT GGA
CTT ACA GCT CAT AGT GAC CCT ATC TCC AAC AGA TGC GCT CAG-3'. It
represents the minus strand of the humanized VK domain
complementary to nt 31 to 180, and this sequence was PCR-amplified
by oligo (SEQ ID NO: 29): 5'-GAC AAG CTT CAG CTG ACC CAG TCT CCA
TCA TCT CTG AGC GCA TCT GTT GGA G-3' and oligo (SEQ ID NO: 30):
5'-AGA GAA TCG CGA AGG GAC ACC AGA TTC CCT AGT GGA TGC CCA
GTA-3'.
[0113] The Oligo F sequence (SEQ ID NO: 31) is 5'-GCA CCT TGG TCC
CTC CAC CGA ACG TCC ACG AGG AGA GGT ATT GGT GAC AAT AAT ATG TTG CAA
TGT CTT CTG GTT GAA GAG AGC TGA TGG TGA AAG TAA AAT CTG TCC CAG ATC
CGC TGC C-3'. It represents the minus strand of the humanized LL2
VK domain complementary to nt 208 to 328. It was PCR amplified by
oligo (SEQ ID NO:32): 5'-GAC AAG CTT TCG CGA TTC TCT GGC AGC GGA
TCT GGG ACA G-3' and oligo (SEQ ID NO: 33): 5'-GAC CGG CAG ATC TGC
ACC TTG GTC CCT CCA CCG-3'.
[0114] Gel-purified PCR products for oligos E and F were
restriction-digested with PvuII/NruI and NruI/BglIII, respectively.
The two PCR fragments E and F were then joined at the NruI site and
ligated to the complementary PvuI/BcII sites of the light chain
staging vector, VKpBR. The humanized VK sequence was subcloned into
vector pKh to form the final human kappa chain expression vector,
hLL2pKh.
[0115] To express the humanized antibodies, about 10 .mu.g of
linearized hLL2pKh and 20 .mu.g of linearized hLL2pG1g were used to
transfect 5.times.10.sup.6 SP2/0 cells by electroporation. The
transfectomas were selected with hygromycin at 500 .mu.g/ml and
secreted antibody was purified on a 1.times.3 cm column of protein
A. After concentrating the purified antibody by Centricon 30
centrifugation, antibody concentration was determined by ELISA. The
final concentration of the antibody was adjusted to 1 mg/ml in PBS
buffer containing 0.01% (w/v) sodium azide as a preservative.
[0116] FIG. 1 compares the amino acid sequence between murine and
humanized LL2 VK domains (FIG. 1A, SEQ ID NOS: 2, 6 & 20) and
between murine and humanized LL2 VH domains (FIG. 1B, SEQ ID NOS:
4, 21 & 8). In the VK chain, human REI framework sequences were
used for all FRs. In the VH chain, human EU framework sequences
were used for FR 1-3, and NEWM sequences were used for FR-4. Only
human FR sequences that are different from that of the mouse are
shown. Asterisks indicate murine FR sequences that are different
from that of the human FR at corresponding positions. Murine
residues at these positions were retained in the humanized
structure. CDRs are boxed.
[0117] In FIG. 4A (SEQ ID NOS: 1 & 2) there are shown the
double stranded DNA and corresponding amino acid sequences (shown
by single letter code) of the murine LL2 VK domain. CDR 1-3 amino
acid sequences are boxed. The corresponding display for VH is shown
in FIG. 4B (SEQ ID NOS: 3 & 4).
[0118] In FIG. 5A (SEQ ID NOS: 5 & 6) and FIG. 5B (SEQ ID NOS:
7 & 8) there are shown double-stranded DNA sequences and amino
acid sequences of humanized LL2 VK and LL2 VH, respectively. Amino
acid sequences are shown by the single-letter code, and CDR amino
acid sequences are boxed.
EXAMPLE 4
[0119] Construction, Expression and Purification of Chimeric LL2
Antibodies
[0120] The fragments containing the VK and VH sequences of LL2,
together with the promoter and signal peptide sequences, were
excised from LL2VKpBR and LL2VHpBS, respectively, by double
restriction digestion with HindIII and BamHI. The about 600 bp VK
fragments were then subcloned into the HindIII/BamHI site of a
mammalian expression vector, pKh (FIG. 3A). pKh is a pSVhyg-based
expression vector containing the genomic sequence of the human
kappa constant region, an Ig enhancer, a kappa enhancer and the
hygromycin-resistant gene. Similarly, the ca. 800 bp VH fragments
were subcloned into the corresponding HindIII/BamHI site of pG1g
(FIG. 3B), a pSVgpt-based expression vector carrying the genomic
sequence of the human IgG1 constant region, an Ig enhancer and the
xanthine-guanine phosphoribosyltransferase (gpt) gene. The final
expression vectors are designated as LL2pKh and LL2pG1g,
respectively.
[0121] The two plasmids were co-transfected into Sp2/0-Ag14 cells
by electroporation and selected for hygromycin resistance.
Supernatant from colonies surviving selection were monitored for
chimeric antibody secretion by ELISA assay (see above). The
transfection efficiency was approximately 1-10.times.10.sup.6
cells. The antibody expression level, in a terminal culture, was
found to vary in the range between <0.10 and 2.5/.mu.g/ml.
[0122] Protein A-purified mLL2 and cLL2 were analyzed by SDS-PAGE
under reducing and non-reducing conditions. The light chains of
both mLL2 and cLL2 showed a higher than expected apparent molecular
weight. As the human kappa constant region of cLL2 is known to
contain no potential glycosylation site, it can be inferred that
the potential glycosylation site identified in the FR1 region of
LL2 VK domain was utilized. Different versions of hLL2 and cLL2
antibodies were analyzed by SDS-PAGE under reducing and
non-reducing conditions. One hLL2 version was hLL2-1 (with seven
murine FR residues in the VH domain). Another hLL2 version was
hLL2-2 with 6 murine FR residues in the VH domain. The humanized
light chains migrated more rapidly and the bands were more discrete
bands when compared to the chimeric light chains.
[0123] Mix-and-match, cLL2 and hLL2 antibodies were analyzed by
SDS-PAGE, under reducing and non-reducing conditions. The
mix-and-match versions analyzed were the (hL/cH)LL2, the
(cL/hH)LL2-1, and the (cL/hH)LL-2. (cL/hH)LL2-1 and (cL/hH)LL-2
contain 7 and 6 murine residues in the FR regions of the heavy
chain, respectively. The migration observed for the (hL/cH)LL2
suggested that the humanized LL2 light chain did not undergo
glycosylation.
EXAMPLE 5
[0124] Binding of cLL2 Antibody to Raji Cell Surface Antigens
[0125] A competition cell binding assay was carried out to assess
the immunoreactivity of cLL2 relative to the parent mLL2. Using
.sup.131I-labeled mLL2 (0.025 .mu.g/ml) as a probe, Raji cells were
incubated with the antibodies and the relative binding to the cells
determined from the amount of cell-bound labeled mLL2 (see above).
As shown by the competition assays described in FIG. 7, both mLL2
and cLL2 antibodies exhibited similar binding activities.
[0126] The results were confirmed by a second competition assay
based on flow cytometry. Briefly, using Raji cells as before and
varying the concentration of one antibody relative to other, as
before, the amount of bound mLL2 or cLL2 was determined with
FITC-labeled anti-mouse Fc or anti-human Fc antibodies followed by
analysis using flow cytometry.
EXAMPLE 6
[0127] Binding of hLL2 Antibodies to Raji Cells
[0128] In experiments similar to those of Example 5, the antigen
binding affinities of the three different combinations of
mix-and-match or humanized LL2 were compared with that of cLL2 in
the flow cytometry assay.
[0129] Briefly, 1 .mu.g of cLL2, mix-and-match LL2, hLL2-1 or
hLL2-2 antibodies were incubated with 10.sup.8 Raji cells in the
presence of varying concentrations of mLL2 F(ab').sub.2 fragments
(as competitor) in a final volume of 100 .mu.l of PBS buffer
supplemented with 1% FCS and 0.01% sodium azide. The mixture was
incubated for 30 minutes at 4.degree. C., and washed three times
with PBS to remove unbound antibodies. By taking advantage of the
presence of human Fc portions in the antibodies, the binding levels
of the antibodies were assessed by adding a 20.times. diluted
FITC-labeled goat anti-human IgG1, Fc fragment-specific antibodies
(Jackson ImmunoResearch, West Grove, Pa.). The cells were washed
three times with PBS, and fluorescence intensities measured by a
FACSCAN fluorescence activated cell sorter (Becton-Dickinson,
Bedford, Mass.). The results are shown in FIG. 8A. Using the same
methods, cLL2 was compared to two versions of hLL2 (FIG. 8B).
[0130] The results shown in FIGS. 8A and B demonstrate that the
immunoreactivity of cLL2 is similar or identical to that of
humanized or mix-and-match antibodies. Taken together with the
comparison of cLL2 with mLL2 (FIG. 7), the authenticity of the
sequences for chimeric and humanized VK and VH obtained is
established, and the functionality of cLL2 and hLL2 confirmed.
EXAMPLE 7
[0131] Internalization of mLL2 and cLL2 by Raji Cells
[0132] One of the unique characteristics of the LL2 antibody is its
rapid internalization upon binding to Raji cells (Shih et al., 1994
above). Murine LL2 after internalization is likely to be rapidly
transferred to the Golgi apparatus and from there to the lysosome,
the organelle responsible for the degradation of a wide variety of
biochemicals (Keisari et al., Immunochem., 10: 565 (1973)).
[0133] Rates of antibody internalization were determined according
to Opresko et al., 1987 above. The ratio of
CPM.sub.intracellular/CPM.sub.su- rface was determined as a
function of time.
[0134] Rates of LL2 antibody internalization were determined by
incubating radiolabelled LL2 antibody (1.times.10.sup.6 cpm) with
0.5.times.10.sup.6 Raji cells in 0.5 ml of DMEM buffer containing
1% human serum for 2 hrs. at 4.degree. C. Excess human serum was
included to saturate Raji cell surface Fc receptors in order to
exclude or minimize non-antigen-specific internalization mediated
through the Fc receptors. Unbound radiolabelled LL2 antibodies were
removed from the cells by washing three times with 0.5 ml portions
of DMEM at 4.degree. C. Cells were then incubated at 37.degree. C.,
and, at timed intervals, aliquots of the cell suspension were
transferred to ice in order to stop internalization. The cells in
these aliquots were isolated by centrifugation at 1,000.times.g for
5 mins. at 4.degree. C., and surface bound radiolabelled LL2
stripped off cells with 1 ml of 0.1 M glycine acetate buffer, pH 3,
for 8 mins. at 4.degree. C. Radioactivity thus obtained (CPM
surface) and radioactivity remaining in the cells (CPM
intracellular) were determined. Rates of internalization were
calculated from the slope of the plot of intracellular: surface
radioactivity ratios as a function of time.
[0135] As shown in FIG. 9, mLL2, cLL2, cLL2Q and hLL2 antibodies
were internalized at a similar rate (Ke=0.107 (mLL2) to 0.1221
(cLL2Q, NVT to QVT mutation). Those numbers suggested that
approximately 50% of the surface-bound antibody could be
internalized in 10 min. The results show that neither chimerization
nor humanization nor deglycosylation by mutagenesis of mLL2
antibodies impair rates of internalization.
[0136] The pattern of internalization for mLL2, cLL2 and hLL2 was
also monitored by fluorescence microscopy on a time-course basis
using a FITC-labeled second antibody probe as described in the
specification. Internalization of both antibodies was observed in
at the earliest time point measurable. At 5 minutes, antibodies
were seen both on the cell surface and internalized in areas
immediately adjacent to the membrane as cytoplasmic micro-vesicles.
At 15 min. post-incubation, the fine dots dispersed around the
intramembrane began to merge into a group of granules, at locations
believed to be the Golgi apparatus. As more antibodies were being
internalized after 30 min. of incubation, redistribution of the
grouped antibodies to scattered locations, probably the lysosome in
which the antibodies were degraded, was observed. At 2 hrs
post-incubation, most of the antibodies were found inside the cell.
Only strong surface staining was observed when LL2 was incubated
for 20 min on ice. Both mLL2 and cLL2 were internalized with a
similar pattern. The internalization of LL2 was associated
specifically with antigen-antibody binding, as the irrelevant
control humanized antibody demonstrated only dull surface
staining.
[0137] The A103 antibody (an IgG2a antibody that binds to the
surface of all human epithelial cells but does not internalize
efficiently (Mattes et al., Hybridoma, 2: 253 (1983)) showed strong
membrane staining at up to 2 h, while the anti-transferrin receptor
antibody (SF9) internalized rapidly, just as did LL2.
EXAMPLE 8
[0138] Role of Glycosylation Site in FR1 Region of LL2 VK
Sequence
[0139] Of particular inventive interest is the identification of an
Asn-glycosylation site at position 18-20 within the FR1 region of
the LL2 NVT light chain sequence (FIG. 4A, SEQ ID NOS: 1 & 2).
As shown above, SDS-PAGE analysis under reducing condition suggests
that the Asn glycosylation site is utilized for carbohydrate
addition. In this example, the influence of the carbohydrate moiety
at position 18-20 on the functional activities of the light chains
was examined.
[0140] Murine and chimeric LL2 light chains, treated or untreated
with endoglycosidases F, were examined by SDS-PAGE under reducing
and non-reducing conditions. There was no distinction between the
antibody types as to electrophoretic behavior. In both cases,
deglycosylation reduced the rate of migration of the light
chain.
[0141] The effect of deglycosylation on the binding affinity to
Raji cells of the mLL2 antibody is shown in FIG. 10. Removing
carbohydrate by endoglycosidases F did not influence the binding
activity.
[0142] A mutation was introduced at position 18 of the light chain
so that the Asn was replaced with Gln to produce LL2Q VK FR1.
SDS-PAGE analyses demonstrated that the NVT to QVT mutation
abolished glycosylation of the antibody. Comparison of the Raji
cell binding affinity for cLL2 with and without light chain VK
glycosylation demonstrated that the carbohydrate moiety did not
influence binding of the antibody to these cells.
[0143] It can be concluded that the presence of the carbohydrate
site in the variable region does not affect the immunoreactivity of
the antibody. Computer modeling studies suggested that the VK
carbohydrate moiety in LL2 is remotely positioned from the CDRs and
forms a "cap" over the bottom loops of the FR-associated 13-barrels
supporting the CDRs. Humanization without inclusion of the original
glycosylation site resulted in a CDR-grafted LL2 antibody with
immunoreactivity comparable to that of its murine counterpart.
These characteristics indicate that the glycosylation site can be
used for conjugating therapeutic or diagnostic agents to LL2
without compromising the ability of the antibody to bind and
internalize in B-lymphoma or leukemia cells.
EXAMPLE 9
[0144] Conjugation of LL2 at its VK Region Carbohydrate-Bearing
Site
[0145] The apparent lack of involvement of the variable region
carbohydrate moiety in the functional activities of mLL2, cLL2 and
hLL2 mAbs indicates that this moiety could profitably be used as
the site of attachment of cytotoxic or detection agents such as
radionuclides or toxins, and thereby avoid potential interference
with the binding of the conjugate to a cell surface.
[0146] Using procedures described in Shih et al., U.S. Pat. No.
5,057,313 (which is incorporated by reference) for preparing
antibody conjugates through an oxidized carbohydrate moiety of the
antibody and a primary alkylamine group of a polymeric carrier to
which are covalently one or more of a variety of drugs, toxins,
chelator and detectable labels, a doxorubicin-dextran-LL2 antibody
fragment devoid of appended glycan was produced containing multiple
copies of the drug. The carbohydrate moieties of the cLL2 VK FR1
region involved were those covalently bound to the Asn
glycosylation site.
[0147] In one synthesis, dextran (18-40 kDa) was converted to an
amino dextran by oxidation of the dextran by NaIO.sub.4, Schiff
base formation with NH.sub.2--CH.sub.2--CHOH--CH.sub.2--NH.sub.2,
and reduction with NaBH.sub.4. The amino dextran was then condensed
with doxorubicin (DOX) in the presence of succinic anhydride and
1-ethyl-3-(3-dimethylaminopropy- l)carbodiimide to produce
DOX-aminodextran. The latter was then condensed with an aldehydic
group on LL2 VK FR-1 produced by oxidizing the carbohydrate moiety
of the antibody fragment with NaIO.sub.4.
[0148] In one preparation of DOX-LL2, the number of moles of DOX
attached to dextran was 14 moles per mole dextran, and the number
of moles of doxorubicin per mole F(ab').sub.2 was 8.9. The
immunoreactivity in the Raji cell binding assay above was about 80%
of control values. This conjugation system is not limited to the
mLL2 antibody. In a comparative study, 15-19 moles of DOX were
bound per mole of cLL2.
[0149] The conjugation possibilities are not limited to the use of
a carrier dextran as in the example above. For example, the
carbohydrate moiety of the LL2 VK FR1 region can be oxidized to
produce aldehydic groups. These in turn can be reacted with an
amino group on any drug to produce a Schiff base which, upon
reduction, produces multiple copies of the drug stably linked to
the antibody via alkylamine groups.
[0150] For example, where the drug is aminohexyl DTPA (a chelating
agent), there is produced a LL2 covalently bound to a chelator. The
chelator can be used to deliver to target tissues, for example, a
radionuclide or paramagnetic metal ion, with a potential for
diagnostic and therapeutic uses. DTPA-LL2 conjugates were produced
containing 5.5 moles of the chelator/mole of antibody which, in
turn, chelated 47.3% of Y-90 and 97.4% In-111
EXAMPLE 10
[0151] Enhanced Production of a Humanized Anti-B-Cell Lymphoma
Antibody.
[0152] Despite a demonstrated efficacy for murine LL2 in the
treatment and diagnosis of non-Hodgkin's B-cell lymphoma, a
thorough study of the clinical significance of its humanized
version (hLL2), however, was rendered difficult due to the low hLL2
productivity of the original transfectoma (ca. 1 mg/liter in a
terminal culture). By re-ligating the hLL2 heavy and light chain
sequences into an expression vector containing an amplifiable
dihydrofolate reductase gene (dhfr)(hLL2pdHL2), we were able to
transfect the vector into SP2/0 cells by electroporation and
generate a methotrexate (MTX) resistant and hLL2 producing clone.
At a MTX concentration of 0.1 .mu.M, 1.4 mg of hLL2 were purified
from a one-liter terminal culture. The level of hLL2 production
rose with stepwise increases in the concentration of MTX in the
culture media, and reached a production plateau of 70+/-5 mg/liter
at 3 .mu.M of MTX. The hLL2 thus purified exhibited a PI of 10.3
with conserved immunoreactivity. Furthermore, complete removal of
MTX selection, and freezing and thawing did not appear to affect
the high level productivity of the established clone, suggesting
that the amplified genes were stably integrated into the
chromosome.
EXAMPLE 11
[0153] Construction of N-Linked Glycosylation Sites into the
Constant Region of hLL2 Antibody
[0154] 1. Designing N-linked glycosylation site mutations.
[0155] (1) Light Chain Mutations.
[0156] Potential N-linked glycosylation sequences have been
identified in the kappa constant regions of rabbit antibodies at
a.a. position 161-163 and 174-176. Similar sites can be introduced
into the CK domain of hLL2, designated as sites KCNI and KCN2,
respectively. Additionally, three other CK mutants, namely KCN3 and
KCN4 were designed, as listed in FIG. 12.
[0157] (2) Heavy Chain Mutations.
[0158] Human IgM contains potential carbohydrate-addition-sequence,
NNS, in the CH1 domain at amino acid position 161-163. Similarly,
the sequence, NVT, was positioned at the residues 168-170 in the
CH, domain of human IgA. By the same rationale used in the designs
of light chain mutations, certain heavy chain mutations also were
introduced (FIG. 12).
[0159] Carbohydrate-addition-sequence, Asn-Asn-Ser, was identified
at a.a. positions 161-163 (Kabat's numbering; Kabat et al., 1991)
in some of the human IgM CH1 domains. Similarly, the sequence,
Asn-Val-Thr, was positioned in a.a. positions 168-170 in the CH1
domain of human IgA. By mutating the human IgG1 sequence
Asn-Ser-Gly to Asn-Ser-Val at a.a. positions 162-164, Ala-Leu-Thr
to Asn-Leu-Thr at a.a. positions 165-167, and Leu-Thr-Ser to
Asn-Thr-Ser at a.a. positions 166-168, respectively, three
potential N-linked glycosylation sites, most analogous to that of
IgM and IgA, were introduced into the CH1 domain of human IgG1,
with minimal interference on the resultant structure. Such
glycosylation sites may thus remain in a "natural" position. Other
glycosylation acceptor sequences were introduced based on their
surface accesibility as predicated by computer modeling (HCM5, for
example). Yet other sites were chosen randomly, by facility to
mutate the sequence, without modeling.
[0160] 2. Engineering Mutation Constructs for Expression.
[0161] (1) Design and Synthesis of Primers for Mutagenesis.
[0162] Oligonucleotide-directed site specific mutagenesis was used
to introduce the designed potential N-linked glycosylation sites in
hLL2 antibody. The oligonucleotide primers corresponding to each CK
and CHI mutation were synthesized and used for in vitro
mutagenesis. Each of these primers also introduced into the target
DNA fragment a restriction cleavage site (Table 1, underlined
sequences) to facilitate subsequent screening process. In Table 1,
the bold letters indicate the mutated bases.
[0163] Table 1
1 CK mutation primers: CKN1 (SEQ ID NO: 34)
5'-CCAATCGGGTAATTCGAATGAGAGTGTCACAGAG-3' CKN2 (SEQ ID NO: 35)
5'-GGACAGCACCTACAACTTAAGCAGCACCCTGAC-3' CKN3 (SEQ ID NO: 36)
5'-GGAAGGTGGATAACGCGTCCCAATCGGGTAA-3' CKN4 (SEQ ID NO: 37)
5'-AGCAGCACCCTAAATTTGAGCAAAGCAGACT-3' CKN5 (SEQ ID NO: 38)
5'-GAGTGTCACAGAGAACGTTAGCAAGGACAGCACC-3' CH.sub.1 mutation primers:
HCN1 (SEQ ID NO: 39) 5'-GTGTCGTGGAACTCAAGCGCTCTGACCAGCGGC-3' HCN2
(SEQ ID NO: 40) 5'-TTCCCGGCTGTCCTGAATTCCTCAGGACTCTACT-3' HCN3 (SEQ
ID NO: 41) 5'-CCTCAGGACTCTACTCGAATTCCAGCGTGGTGACCGT-3' HCN4 (SEQ ID
NO: 42) 5'-GTGGTGACCGTCCCGAATTCCAGCTTGGGCACC-3' HCN5 (SEQ ID NO:
43) 5'-GCCCTCCAGCAGCAACGGTACCCAGACCTACATCTGC-3'
[0164] (2) Construction of Expression Vectors.
[0165] By in vitro site-specific mutagenesis, potential N-linked
glycosylation sequences were introduced into the genes encoding the
light and heavy chain of hLL2. The sequences were confirmed by DNA
sequencing. Each mutated gene was then subcloned into the
corresponding expression vector (hLL2pKh for the kappa chain and
hLL2pG1g for the heavy chain).
[0166] The CH1 domain of human IgG1 was first excised from the
expression vector LL2pG1g containing the human genomic IgG1
constant region sequence (Leung et al., 1994b) by digestion with
the restriction enzymes BamHI and BstXI, and subcloned into the
corresponding sites of the pBluescript SK vector (Stratagene, La
Jolla, Calif.) for further manipulations. The resultant vector is
designated as CH1pBS.
[0167] Mutations were accomplished using the Transformer.TM.
Site-Directed Mutagenesis Kit (CLONTECH, Palo Alto, Calif.)
according to the manufacturer.quadrature.s specifications. The
selection primer, MutKS (5'-ACG GTA TCG ATA TGC ATG ATA TCG AAT
T-3'), is designed for use in conjunction with the respective
mutation primers in all cases. It was chosen to convert the HindIII
restriction site in the cloning sequence of pBluescript to a NsiI
restriction site (underlined).
[0168] To mutate Asn-Ser-Gly to Asn-Ser-Thr at a.a. positions
162-164, the selection primer MutKS and the primer CHO162 (5'-GTG
TCG TGG AAT TCA ACC GCC CTG ACC AGC GGC-3') were used to change the
Gly at position 164 will be mutated to Thr. An EcORI site
(underlined) is also included in the mutagenic primer as a
diagnostic site.
[0169] To mutate Ala-Leu-Thr to Asn-Leu-Thr at a.a. positions
165-167, the selection primer MutKS and the mutation primer CHO165
(5'-GTG TCG TGG AAT TCA GGC AAC CTG ACC AGC GGC-3') are used to
change the Ala-165 to Asn-165. An EcORI site (underlined) is
included in the mutagenic primer as a diagnostic site.
[0170] To mutate Leu-Thr-Ser to Asn-Thr-Ser at a.a. position
166-168, the selection primer MutKS and the mutation primer CHO166
(5'-TGG AAC TCA GGC GCG AAT ACC AGC GGC GTG CAC-3') were used to
change the Leu-166 to Asn-166. The KasI site (GGC GCC) in the
original CH1 sequence of human IgG1 is deliberately eliminated by
changing the 3.degree. C. into a G for diagnostic purposes.
[0171] The phosphorylated primer pairs (selection and the
respective mutation primers) at 100 ng each are annealed to 100 ng
of the staging vector, CH1pBS, in 20 mM Tris-CH1 (pH 7.5), 10 mM
MgCl.sub.2, 50 mM NaCl in a final volume of 20 .mu.l by incubation
at 95.degree. C. for 3 min, and then chilling on ice for 5 min. To
the mixture, 2 to 4 units of T4 DNA polymerase, 4 to 6 units of T4
DNA ligase together with 3 l of 10.times.synthesis buffer
(CLONTECH, Palo Alto, Calif.) are added. After an incubation period
of 2 hr at 37.degree. C., the polymerization and ligation reactions
are terminated by heating at 65.degree. C. for 5 min in the
presence of 3 l of prewarmed stop solution (0.25% SDS, 5 mM EDTA).
DNA from the mixture is used to transform electrocompetent E. coli
cells, BMH71-18 mutS (repair deficient), by the method of
electroporation. Transformants are then pooled and grown overnight
in SOC (20 mg/ml bacto-tryptone, 5 mg/ml bacto-yeast extract, 8.6
mM NaCl, 2.5 mM KCl, 20 mM glucose) with 50 g/ml ampicillin at
37.degree. C. Mini-plasmid DNA preparations from the pooled
transformants are digested with HindIII to linearize DNA not
mutated with the selection primer. After the enzymes are removed by
phenol extraction, the DNA is used for a second transformation with
competent DH5 cells. Plasmid DNA that fails to be digested with
HindIII is examined for the presence of the EcORI diagnostic site
(in the case of Gly to Thr, and Ala to Asn mutations), or the
absence of the KasI diagnostic site (in the case of the Leu to Asn
mutation). Final confirmation of the mutation is accomplished by
Sanger's dideoxy sequencing (Sanger et al., 1977). The CH1 region
confirmed to have the desired mutations is then excised with
BamHI/BstXI enzymes and cloned into the corresponding site of the
final heavy chain expression vectors for hLL2, hLL2pG1g.
[0172] (3) Expression Vector for Gene Amplification.
[0173] In order to facilitate down stream process of antibody
production, it is desirable to utilize a gene amplification system
for antibody expression. After an antibody variant is proved to
have industrial potential, high level production could be achieved
by gene amplification. From this consideration, we planned to
construct these N-linked glycosylation site mutants in the
hLL2pdHL2 high level expression vector, a dhfr mini gene based
amplification system. Heavy chain mutations, HCN3, HCN4, and HCN5,
were subcloned into this vector for expression.
[0174] The final expression constructs for these mutations were
designated as hLL2HCN3pdHL2, hLL2HCN4 and hLL2HCN5pdHL2,
respectively.
[0175] 3. Expression of mutant hLL2 and glycosylation at engineered
sites. The constant domains containing the engineered glycosylation
sites were ligated to the respective variable (V) regions of hLL2.
The different glycosylation mutants were expressed in murine SP2/0
myeloma cells which were transfected with the heavy and light chain
expression vectors by electroporation. The engineered antibodies
were purified from the culture supernatant of the stable
antibody-producing cells by protein A columns, and the purified
proteins analyzed on SDS-PAGE under reducing conditions. The heavy
chains of the glycosylation mutants migrated at different rates
compared to that of the control antibody, hLL2, whose CH1 domain
did not contain any potential glycosylation sites. Since the
SDS-PAGE migration rate is inversely proportional to the molecular
sizes of the engineered oligosaccharides, the extent of
glycosylation at the different sites should be in the order of
HCN5>HCN1>HCN3>HCN2>HCN4 with hLL2HCN5 and hLL2HCN1
being the two most highly glycosylated Ab. By contrast, judging
from the lack of migration retardation in the light chains for the
mutants KCN1-4 we concluded that these CK-associated sites were
either not glycosylated at all, or glycosylated at an insignificant
level.
[0176] 4. hLL2HCN1 and hLL2HCN5 are N-glycosylated in the CH.sub.1
domain. The antibodies hLLHCN1, hLL2HCN5 and hLL2 were treated with
N-glycosidase F (PNGase F), which specifically cleaves all types of
Asn-bound glycan from peptides, and were analyzed on reducing
SDS-PAGE. The higher apparent molecular masses for the heavy chains
of hLL2HCN1 and hLL2HCN5 were reduced to that of hLL2 after PNGase
F digestion indicating that the size difference between these Abs
were attributed to the heavy chain associated N-linked CHOs. It
should be noted that, all human IgG.sub.1, Abs are naturally
glycosylated in the CH.sub.2 domain at Asn297. The size differences
observed might be due to differential glycosylation at the CH2
site, rather than at the engineered sites, as a result of
variations in the culture condition. We therefore prepared
F(ab').sub.2 fragments of hLL2HCN1, hLL2HCN5 and hLL2, and analyzed
these fragments on reducing SDS-PAGE. The size differences between
the Abs were shown to be associated with the Fd fragments
(VH-CH.sub.1), which are devoid of the Fc portion and the appended
oligosaccharides, the molecular size for Fd fragments of hLL2HCN5
being larger than that of hLL2HCN1. When fragments were
deglycosylated by PNGcase F treatment, these size differences were
eliminated and all Fd fragments migrated at the same position as
the unglycosylated hLL2, suggesting that the engineered sites were
actually used for glycosylation and the extent of glycosylation for
HCN5 site was larger than that of HCN1.
[0177] The N-linked oligosaccharide moieties in the CH.sub.1,
domain of hLL2HCN1 were directly visualized by CHO-specific
labeling. The oligosaccharide moieties attached to the were first
periodate oxidized. The aldehydes groups generated were then
covalently conjugated with biotin, which was probed and visualized
by streptavidin-peroxidase in a western blotting analysis. As we
anticipated, only the heavy chain but not light chains of both hLL2
and hLL2HCN1 were visible with CHO labeling. When quantified with
densitometry, the intensity of labeled CHOs in hLL2HCN1 was
approximately 2.5-fold of that in hLL2. The protein contents of the
different Abs analyzed were comparable, as shown by coomassie
blue-stained SDS-PAGE. We attributed this difference in intensity
to be the result of additional glycosylation in the engineered HCN1
site. This was confirmed when the F(ab').sub.2 fragments were
subjected to the same analysis: only the Fd fragment of hLL2HCN1
but not that of hLL2 demonstrated CHO specific labeling. By
contrast, potential CK glycosylation sites were not found to be
glycosylated.
[0178] It should be noted that, unlike the VK-appended
glycosylation site which exhibited heterogeneity in the degree of
glycosylation, only one discrete band was observed in the SDS-PAGE
analysis for hLL2(HCN1) Fd fragment. It is speculated that almost
all of the Fd fragments of hLL2(HCN1) were glycosylated, and the
degree of glycosylation was relatively homogenous, a desirable
property that would facilitate their subsequent characterizations
and applications.
[0179] 5. WN competitive binding assay. The antigen-binding
property of these two antibodies was evaluated by competition
binding with mLL2 to an LL2 anti-idiotype antibody (WN). This assay
showed that the binding activity of hLL2HCN1 and hLL2HCN2 to WN is
indistinguishable from that of hLL2. (FIG. 11).
EXAMPLE 12
[0180] Site-Specific Conjugation of Aminobenzyl DTPA and
Dextran-Doxorubicin to hLL2HCN1 and hLL2HCN5.
[0181] The site-specific modification of the F(ab').sub.2 fragments
of antibodies with DTPA was as described. See Leung et al., J.
Immunol. 154:5919 (1995). F(ab').sub.2 fragment (.about.1 mg/ml)
was oxidized with 15 mM of sodium metaperiodate at 4.degree. C. for
1 h. The oxidized material was purified, mixed with 545-fold molar
excess of aminobenzyl DTPA and the pH was adjusted to 5.97. The
mixture was incubated in the dark at ambient temperature for 5 h,
and then kept at 4.degree. C. for 18 h. The conjugates were
stabilized with 1O mM of sodium cyanoborohydride, purified and
concentrated. The chelator:F(ab').sub.2 ratio was determined by
metal binding assays and use of indium acetate spiked with
.sup.111In. See Meares et al., Anal. Biochem. 142:68 (1984).
Radiolabeling was performed as described. See Leung et al., (1995),
supra. The number of DTPA molecules conjugated to F(ab').sub.2
fragment was determined by metal-binding assay using In/In-111
system. Briefly, 40 .mu.g of the conjugates was incubated for 30
min with a known excess of indium acetate, spiked with In-111
acetate. The solution was made 10 mM in EDTA, and incubated for
further 10 min. The labeling was analyzed by ITLC using 10 mM EDTA
for development. DOX-dextran conjugate was prepared as described by
Shih et al., Cancer Res. 51: 4192 (1991), using amino-dextran of 18
kDa as the intermediate carrier. The intermediate conjugate
possessed a substitution level of 10.5 DOX molecules per dextran
polymer. DOX-dextran was then conjugated with the F(ab').sub.2
fragment of hLL2HCN1 or hLL2HCN5. Briefly, the antibody fragment
was concentrated to 1O mg/ml in 0.1 M sodium acetate buffer, pH
5.5, and treated with 20 mM of sodium metaperiodate in the dark at
4.degree. C. for 60 min. The oxidized antibody was purified on a
Bio-Spin column (Bio-Rad) that was pre-equilibrated in 0.05 M HEPES
buffer, pH 8.0, containing 0.1 M NaCl, and then treated with
DOX-dextran (4 equivalents) at room temperature for 24 h. After
sodium borohydride reduction, the conjugated product was purified
on a Bio-gel A-0.5 m gel column (Bio-Rad). The protein fractions
were pooled and concentrated in Centricon 50 concentrator (Amicon,
Beverly, Mass.). The trace amount of intermediates in the protein
conjugates was removed by repetitive washing with the conjugation
buffer as evaluated by HPLC on Bio-Sil Sec size exclusion column
(Bio-Rad).
EXAMPLE 13
[0182] CH.sub.1-Appended Oligosaccharides can be Used as Efficient
Conjugation Sites for Chelates and/or Drugs.
[0183] Under mild chemical conditions, an average of 1.6 and 2.97
molecules of DTPA were conjugated onto each F(ab').sub.2 fragment
of hLL2HCN1 and hLL2HCN5, respectively (Table 2). Both conjugates
demonstrated high efficiencies in .sup.111In incorporation (92% for
hLL2HCN1, 91% for hLL2HCN5). No significant changes in
immunoreactivities were observed before and after DTPA conjugation
of the glycosylation mutant fragments, as evaluated in a WN
competitive blocking assay. HCN5-appended CHO appeared to be more
reactive for chelate conjugation when compared to the HCN1-appended
CHO; almost twice as many DTPA molecules could be incorporated into
the HCN5 site.
[0184] Leung et al. (1995), supra, has shown that the VK-appended
CHO found in murine LL2 can be used as a site-specific conjugation
site for small chelates without reducing the Ag binding property of
the Ab. The effect of conjugating this VK-appended CHO with
dextran-DOX complex on immunoreactivity was examined. The
dextran-DOX complex was generated by chemically incorporating an
average of 10 DOX molecules onto an 18 kDa amino-dextran polymer.
Using the amino-dextran as the carrier for DOX, approximately 5.1
DOX molecules on average were incorporated onto the VK-appended CHO
of murine LL2, and a reduction of close to 60% of immunoreactivity
as evaluated by cell binding and ELISA assays, was observed. See
Table 3. Conjugation of slightly higher number of DOX molecules
(6.8) onto the HCN1 CHO, however, was comparatively less
detrimental in term of its effect on immunoreactivity; only 30%
reduction in the resultant binding affinity was noted. In contrast,
no significant changes in Ag binding property (less than 5%
reduction) were apparent when similar number of DOX molecules (7.2)
was conjugated at the HCN5 CHO. See Table 3.
[0185] The molecular masses of the F(ab').sub.2 fragments of hLL2,
hLL2HCN1 and hLL2HCN5 determined by mass spectrometry analysis
(Mass Consortium, San Diego, Calif.) were 99,000, 102,400 and
103,800, respectively since these fragments are identical in
sequences, except at the engineered site (one amino acid
difference), and the fragments did not carry the glycosylated Fc
portion, the molecular mass difference between the F(ab').sub.2 of
hLL2 and the glycosylation mutant should represent the molecular
weights of the different CH1-appended CHOs, i.e., 3.4 and 4.8 kD
for the CHOs at the HCN1 and the HCN5 sites, respectively.
[0186] By PNGase F digestion, the CH1-appended CHOs of hLL2HCN1 and
hLL2HCN5 were released for profiling and sequencing analyses using
fluoropore-assisted carbohydrate electrophoresis (FACE).
Heterogenous populations of CH1-appended CHO species were
identified. About 60% of the oligosaccharides from HCN5 site were
of the larger tri-antennary structure, while that from HCN1 were
mainly bi-antennary (>90%). These results are consistent with
the mass spectrometry studies indicating a larger average molecular
size of the CHO at the HCN5 sites compared to that of HCN1.
[0187] It should be emphasized that the above-described examples
merely describe several specific embodiments of the invention, and
applicants do not intend to be limited as to scope of claims by
these specific examples. Applicants also incorporate by reference
all publications and patents cited in the specification.
2TABLE 2 Site-specific conjugation of DTPA and radiolabeling.
Antibody (%) Efficiency.sup.a .sup.111In labeling Immunoreactivity
F(ab').sub.2 DTPA DTPA//F(ab').sub.2 % Incorp..sup.b
.mu.Ci/.mu.g.sup.c ID.sub.50 % of hLL2.sup.d hLL2 Non-conj. NA NA
NA 0.384 100 (.+-.0.021) hLL2HCN1 Non-conj. NA NA NA 0.355 100
(.+-.0.038) Conjugated 1.6 92 6 0.387 100.8 (.+-.0.042) hLL2HCN5
Non-conj. NA NA NA 0.443 115.4 (.+-.0.039) Conjugated 2.97 91 5.6
0.356 92.7 (.+-.0.077) .sup.aA control experiment using hMN14
F(ab').sub.2 (non-glycosylated) yield a negligible
chelate/F(ab').sub.2 ratio of 0.075, confirming that conjugation
were indeed directed to the carbohydrate moieties. .sup.bDetermined
by cobalt/cobalt-57 or indium/indium-111 assays (Meares et al.,
Anal. Biochem. 142:68, 1984). .sup.cHPLC yields; percentage of
labeling in each case was higher by using ITLC analysis; colloidal
metal was less than 1% in all labeling. .sup.dOn the basis of
comparisons to the ID.sub.50 of unmodified control F(ab').sub.2 in
competitive binding assays. ND: not determined
[0188]
3TABLE 3 Site-specific conjugation of doxorubicin. Immunoreactivity
Antibody (%) F(ab').sub.2 Yield.sup.a (DOX/ Cell ELISA.sup.d
Dextran-DOX (%) Efficiency.sup.b F(ab').sub.2) binding.sup.c mLL2
Non-conj. NA NA 100 100 Conjugated 55 5.1 41.9 42.2 hLL2HCN1
Non-conj. NA NA 100 100 Conjugated 30 6.8 70 70.6 hLL2HCN5
Non-conj. NA NA ND 100 Conjugated 80 7.2 ND 94.8 .sup.aDetermined
by spectrophotometry. .sup.bDetermined and calculated by
spectrophotometry. .sup.cActivity determined by a cell surface
binding assay as described in and calculated from the ID50 values.
.sup.dImmunoreactivity Calculated from the ID50 values.
* * * * *