U.S. patent application number 11/519585 was filed with the patent office on 2007-03-15 for biological products.
This patent application is currently assigned to Celltech R&D Limited. Invention is credited to Heather Margaret Ladyman, Andrew George Popplewell, Simon Peter Tickle.
Application Number | 20070059307 11/519585 |
Document ID | / |
Family ID | 9935990 |
Filed Date | 2007-03-15 |
United States Patent
Application |
20070059307 |
Kind Code |
A1 |
Popplewell; Andrew George ;
et al. |
March 15, 2007 |
Biological products
Abstract
There is disclosed antibody molecules containing at least one
CDR derived from a mouse monoclonal antibody having specificity for
human CD22. There is also disclosed a CDR grafted antibody wherein
at least one of the CDRs is a modified CDR. Further disclosed are
DNA sequences encoding the chains of the antibody molecules,
vectors, transformed host cells and uses of the antibody molecules
in the treatment of diseases mediated by cells expressing CD22.
Inventors: |
Popplewell; Andrew George;
(Slough, GB) ; Tickle; Simon Peter; (Slough,
GB) ; Ladyman; Heather Margaret; (Slough,
GB) |
Correspondence
Address: |
COZEN O'CONNOR, P.C.
1900 MARKET STREET
PHILADELPHIA
PA
19103-3508
US
|
Assignee: |
Celltech R&D Limited
Slough
GB
|
Family ID: |
9935990 |
Appl. No.: |
11/519585 |
Filed: |
September 11, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10428408 |
May 2, 2003 |
|
|
|
11519585 |
Sep 11, 2006 |
|
|
|
Current U.S.
Class: |
424/144.1 ;
435/320.1; 435/326; 435/69.1; 514/263.31; 514/44R; 530/388.22;
536/23.53 |
Current CPC
Class: |
C07K 2317/20 20130101;
A61K 2039/505 20130101; C07K 16/2851 20130101; C07K 16/2803
20130101; C07K 2317/76 20130101; C07K 2317/24 20130101; A61P 35/00
20180101; C07K 2317/567 20130101; C07K 2317/52 20130101; C07K
2317/92 20130101; C07K 2317/41 20130101; A61P 43/00 20180101; C07K
2317/56 20130101; C07K 2317/40 20130101; C07K 2317/565 20130101;
A61K 2039/507 20130101; C07K 16/3061 20130101; C07K 16/464
20130101 |
Class at
Publication: |
424/144.1 ;
530/388.22; 435/069.1; 435/326; 435/320.1; 514/044; 536/023.53;
514/263.31 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; A61K 39/395 20060101 A61K039/395; A61K 31/522 20060101
A61K031/522; C12N 5/06 20060101 C12N005/06 |
Foreign Application Data
Date |
Code |
Application Number |
May 2, 2002 |
GB |
0210121.0 |
Claims
1-4. (canceled)
5. The antibody molecule of claim 58, which is a CDR-grafted
antibody molecule.
6. The antibody molecule of claim 5, wherein the variable domain
comprises human acceptor framework regions and non-human donor
CDRs.
7. The antibody molecule of claims 6 or 47, wherein the human
acceptor framework regions of the variable domain of the heavy
chain are based on a human sub-group I consensus sequence and
comprise donor residues at positions 1, 28, 48, 71 and 93 that
correspond to the residues at those positions in SEQ ID NO:8.
8. The antibody molecule of claim 7, additionally comprising donor
residues at positions 67 and 69 that correspond to the residues at
those positions in SEQ ID NO:8.
9. The antibody molecule of claims 6 or 47, wherein the human
acceptor framework regions of the variable domain of the light
chain are based on a human sub-group I consensus sequence and
comprise donor residues at positions 2, 4, 37, 38, 45 and 60 that
correspond to the residues at those positions in SEQ ID NO:7.
10. The antibody molecule of claim 9, additionally comprising a
donor residue at position 3 that corresponds to the residue at that
position in SEQ ID NO:7.
11. An antibody molecule having specificity for human CD22,
comprising a heavy chain according to claim 7, and a light chain
according to claim 9.
12-18. (canceled)
19. A variant of the antibody molecule of any one of claims 58, 5
to 6, and 46 to 47, which has an improved affinity for CD22 as
compared to the murine monoclonal antibody from which it was
derived.
20. The variant of claim 19 which is obtained by an affinity
maturation protocol.
21-22. (canceled)
23. An antibody molecule having specificity for human CD22
comprising a hybrid CDR comprising a truncated donor CDR sequence
wherein the missing portion of the donor CDR is replaced by a
different sequence and forms a functional CDR.
24. The antibody molecule of claim 23, wherein the different
sequence is from the antibody from which the framework regions of
the antibody molecule are derived.
25. The antibody molecule of claim 24, wherein the different
sequence is derived from a germline antibody having consensus
framework regions.
26. The antibody molecule of any one of claims 23 to 25, wherein
CDR-H2 of the heavy chain is hybrid in the antibody molecule.
27. The antibody molecule of any one of claims 23 to 25, wherein
the truncation of the donor CDR is from 1 to 8 amino acids.
28. The antibody molecule of claim 27, wherein the truncation is
from 4 to 6 amino acids.
29. The antibody molecule of any one of claims 23 to 25, wherein
the truncation is made at the C-terminus of the CDR.
30-39. (canceled)
40. A composition comprising the antibody molecule of any one of
claims 58, 4 to 6, 23 to 25, and 46 to 47.
41. The composition according to claim 40, comprising a
pharmaceutically acceptable excipient, diluent or carrier.
42. The composition according to claim 40, additionally comprising
anti-T cell, anti-IFN.gamma. or anti-LPS antibodies, or a
non-antibody ingredients.
43-45. (canceled)
46. The antibody molecule of claim 58 which is a CDR-grafted
antibody molecule.
47. The antibody molecule of claim 46, wherein the variable domain
comprises human acceptor framework regions and non-human donor
CDRs.
48-57. (canceled)
58. An antibody molecule having specificity for human CD22
comprising a heavy chain wherein the variable domain comprises SEQ
ID NO:1 for CDR-H1; SEQ ID NO: 2 or SEQ ID NO:13 or SEQ ID NO:15 or
SEQ ID NO:16 or residues 50-65 of SEQ ID NO: 27 for CDR-H2; SEQ ID
NO:3 for CDR-H3; SEQ ID NO:4 for CDR-L1; SEQ ID NO:5 for CDR-L2;
and SEQ ID NO:6 for CDR-L3.
59. The antibody molecule according to claim 57 having residues
50-65 of SEQ ID NO:27 for CDR-H2.
Description
[0001] This application is a Continuation of application Ser. No.
10/428,408, filed May 2, 2003, and claims priority under 35 U.S.C.
.sctn.119(a)-(d) to United Kingdom Application No. GB 0210121.0,
filed May 2, 2002, both applications being incorporated by
reference herein in their entireties.
[0002] The present invention relates to an antibody molecule having
specificity for antigenic determinants of the B lymphocyte antigen,
CD22. The present invention also relates to the therapeutic uses of
the antibody molecule and methods for producing the antibody
molecule.
[0003] In a natural antibody molecule, there are two heavy chains
and two light chains. Each heavy chain and each light chain has at
its N-terminal end a variable domain. Each variable domain is
composed of four framework regions (FRs) alternating with three
complementarity determining regions (CDRs). The residues in the
variable domains are conventionally numbered according to a system
devised by Kabat et al. This system is set forth in Kabat et al.,
1987, in Sequences of Proteins of Immunological Interest, US
Department of Health and Human Services, NIH, USA (hereafter "Kabat
et al. (supra)"). This numbering system is used in the present
specification except where otherwise indicated.
[0004] The Kabat residue designations do not always correspond
directly with the linear numbering of the amino acid residues. The
actual linear amino acid sequence may contain fewer or additional
amino acids than in the strict Kabat numbering corresponding to a
shortening of, or insertion into, a structural component, whether
framework or CDR, of the basic variable domain structure. The
correct Kabat numbering of residues may be determined for a given
antibody by alignment of residues of homology in the sequence of
the antibody with a "standard" Kabat numbered sequence.
[0005] The CDRs of the heavy chain variable domain are located at
residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues
95-102 (CDR-H3) according to the Kabat numbering.
[0006] The CDRs of the light chain variable domain are located at
residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97
(CDR-L3) according to the Kabat numbering.
[0007] Construction of CDR-grafted antibodies is described in
European Patent Application EP-A-0239400, which discloses a process
in which the CDRs of a mouse monoclonal antibody are grafted onto
the framework regions of the variable domains of a human
immunoglobulin by site directed mutagenesis using long
oligonucleotides. The CDRs determine the antigen binding
specificity of antibodies and are relatively short peptide
sequences carried on the framework regions of the variable
domains.
[0008] The earliest work on humanising monoclonal antibodies by
CDR-grafting was carried out on monoclonal antibodies recognising
synthetic antigens, such as NP. However, examples in which a mouse
monoclonal antibody recognising lysozyme and a rat monoclonal
antibody recognising an antigen on human T-cells were humanised by
CDR-grafting have been described by Verhoeyen et al. (Science, 239,
1534-1536, 1988) and Riechmann et al. (Nature, 332, 323-324, 1988),
respectively.
[0009] Riechmann et al., found that the transfer of the CDRs alone
(as defined by Kabat (Kabat et al. (supra) and Wu et al., J. Exp.
Med., 132, 211-250, 1970)) was not sufficient to provide
satisfactory antigen binding activity in the CDR-grafted product.
It was found that a number of framework residues have to be altered
so that they correspond to those of the donor framework region.
Proposed criteria for selecting which framework residues need to be
altered are described in International Patent Application No. WO
90/07861.
[0010] A number of reviews discussing CDR-grafted antibodies have
been published, including Vaughan et al. (Nature Biotechnology, 16,
535-539, 1998).
[0011] Malignant lymphomas are a diverse group of neoplasms. The
majority of cases occur in older people. Non-Hodgkins Lymphoma
(NHL) is a disease that currently affects 200,000 to 250,000
patients in the U.S. It is the second fastest rising cancer in the
U.S., rising at a rate of about 55,000 new cases per year. The
incidence is rising at a rate that is greater than can be accounted
for simply by the increasing age of the population and exposure to
known risk factors.
[0012] The classification of lymphoma is complex, and has evolved
in recent decades. In 1994 the Revised European-American Lymphoma
(REAL) classification was introduced. This classification organises
lymphomas of B cell (the most frequently identified), T cell and
unclassifiable origin into agreed subtypes. In everyday practice,
the grouping of NHLs into low, intermediate and high-grade
categories on the basis of their general histological appearance,
broadly reflects their clinical behaviour.
[0013] NHL predominantly affects the lymph nodes but, in individual
patients, the tumour may involve other anatomical sites such as the
liver, spleen, bone marrow, lung, gut and skin. The disease
commonly presents as a painless enlargement of lymph nodes.
Extranodal lymphoma most frequently affects the gut, although
primary lymphoma of virtually every organ has been documented.
Systemic symptoms include fever, sweats, tiredness and weight
loss.
[0014] Until recently, the Ann Arbor staging system, based entirely
upon the anatomical extent of disease, was the major determinant of
therapy in NHL. This information may be refined by incorporating
additional prognostic pointers, including age, serum lactate
dehydrogenase levels and performance status. Even so, knowledge of
the Ann Arbor staging system, together with the histological and
immunological subtype of the tumour, is still the major determinant
of treatment.
[0015] Low grade NHL has an indolent course, with a median patient
survival of 8 to 10 years. Survival is little impacted by currently
available therapy, although irradiation of local disease and
chemotherapy for systemic symptoms improves patients' quality of
life. Combination chemotherapy may be reserved for relapsed
disease. Intermediate disease and, especially, high grade disease
is extremely aggressive and tends to disseminate. Disease of this
grade requires urgent treatment. Radiotherapy may be a useful
component of treatment in patients with very bulky disease. Many
different chemotherapy regimens have been employed, and long-term
disease-free survival may be obtained in more than half of
patients. High dose therapy with stem cell support was introduced
initially for patients with relapsed or refractory disease, but is
now increasingly finding a place in first line therapy for patients
with poor-risk disease. The tendency in recent years for an
increasingly aggressive therapeutic approach must be balanced
against the generally elderly age and relative debility of many
patients with NHL, and by the need to match the toxicity of
treatment to the individual prognosis of each patient's
disease.
[0016] Improved treatments, that are more effective and better
tolerated, are needed. Agents recently introduced include new
cytotoxic drugs, progressively incorporated into combinations, and
the introduction of antibody-based therapies.
[0017] Non-Hodgkin's lymphoma encompasses a range of B cell
lymphomas. B cell antigens therefore represent suitable targets for
antibody therapy.
[0018] CD22 is a 135 kDa membrane glycoprotein belonging to a
family of sialic acid binding proteins called sialoadhesins. It is
detected in the cytoplasm early in B cell development, appears on
the cell surface simultaneously with IgD and is found on most
mature B cells. Expression is increased following B cell
activation. CD22 is lost with terminal differentiation and is
generally reported as being absent on plasma cells. Thus this
internalising antigen is present on the surface of pre-B cells and
mature B cells but not stem cells or plasma cells.
[0019] Two isoforms of CD22 exist in man. The predominant form
(CD22.beta.) contains 7 immunoglobulin-like (Ig-like) domains in
the extracellular region. The CD22.alpha. variant lacks Ig-like
domain 4 and may have a truncated cytoplasmic domain. Antibodies
which block CD22 adhesion to monocytes, neutrophils, lymphocytes
and erythrocytes have been shown to bind within the first or second
Ig-like domain.
[0020] The cytoplasmic domain of CD22 is tyrosine phosphorylated
upon ligation of the B cell antigen receptor and associates with
Lyk, Syk and phosphatidyl inositol 3-kinase. The function of CD22
is to down-modulate the B cell activation threshold. It can also
mediate cell adhesion through interaction with cells bearing the
appropriate sialoglycoconjugates.
[0021] CD22 is expressed in most B cell leukaemias and lymphomas,
including NHL, acute lymphoblastic leukaemia (B-ALL), chronic
lymphocytic leukaemia (B-CLL) and especially acute non-lymphocytic
leukaemia (ANLL).
[0022] Monoclonal antibodies against CD22 have been described in
the prior art. WO 98/41641 describes recombinant anti-CD22
antibodies with cysteine residues at V.sub.H44 and V.sub.L100. WO
96/04925 describes the V.sub.H and V.sub.L regions of the anti-CD22
antibody LL2. U.S. Pat. No. 5,686,072 describes combinations of
anti-CD22 and anti-CD19 immunotoxins. WO 98/42378 describes the use
of naked anti-CD22 antibodies for the treatment of B-cell
malignancies.
[0023] A number of antibody-based therapeutics have either been
recently licensed, eg. Rituxan (an unlabelled chimeric human
.gamma.1 (+m.gamma.1V-region) specific for CD20), or are in
clinical trials for this disease. These rely either on complement-
or ADCC-mediated killing of B cells or the use of radionuclides,
such as .sup.131I or .sup.90Y, which have associated preparation
and use problems for clinicians and patients. There is a need for
an antibody molecule to treat NHL which can be used repeatedly and
produced easily and efficiently. There is also a need for an
antibody molecule, which has high affinity for CD22 and low
immunogenicity in humans.
SUMMARY OF THE INVENTION
[0024] In a first aspect, the present invention provides an
antibody molecule having specificity for human CD22, comprising a
heavy chain wherein the variable domain comprises a CDR (as defined
by Kabat et al., (supra)) having the sequence given as H1 in FIG. 1
(SEQ ID NO:1) for CDR-H1, as H2 in FIG. 1 (SEQ ID NO:2) or an H2
from which a potential glycosylation site has been removed, or an
H2 in which the lysine residue at position 60 (according to the
Kabat numbering system) has been replaced by an alternative amino
acid, or an H2 in which both the glycosylation site and the
reactive lysine at position 60 have been removed for CDR-H2 or as
H3 in FIG. 1 (SEQ ID NO:3) for CDR-H3.
[0025] The antibody molecule of the first aspect of the present
invention comprises at least one CDR selected from H1, H2 and H3
(SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3) for the heavy chain
variable domain. Preferably, the antibody molecule comprises at
least two and more preferably all three CDRs in the heavy chain
variable domain.
[0026] In a second aspect of the present invention, there is
provided an antibody molecule having specificity for human CD22,
comprising a light chain wherein the variable domain comprises a
CDR (as defined by Kabat et al., (supra)) having the sequence given
as L1 in FIG. 1 (SEQ ID NO:4) for CDR-L1, L2 in FIG. 1 (SEQ ID
NO:5) for CDR-L2 or L3 in FIG. 1 (SEQ ID NO:6) for CDR-L3.
[0027] The antibody molecule of the second aspect of the present
invention comprises at least one CDR selected from L1, L2 and L3
(SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6) for the light chain
variable domain. Preferably, the antibody molecule comprises at
least two and more preferably all three CDRs in the light chain
variable domain.
[0028] The antibody molecules of the first and second aspects of
the present invention preferably have a complementary light chain
or a complementary heavy chain, respectively.
[0029] Preferably, the antibody molecule of the first or second
aspect of the present invention comprises a heavy chain wherein the
variable domain comprises a CDR (as defined by Kabat et al.,
(supra)) having the sequence given as H1 in FIG. 1 (SEQ ID NO:1)
for CDR-H1, as H2 in FIG. 1 (SEQ ID NO:2) or an H2 from which a
potential glycosylation site has been removed, or an H2 in which
the lysine residue at position 60 (according to the Kabat numbering
system) has been replaced by an alternative amino acid, or an H2 in
which both the glycosylation site and the reactive lysine at
position 60 have been removed for CDR-H2 or as H3 in FIG. 1 (SEQ ID
NO:3) for CDR-H3 and a light chain wherein the variable domain
comprises a CDR (as defined by Kabat et al., (supra)) having the
sequence given as L1 in FIG. 1 (SEQ ID NO:4) for CDR-L1, as L2 in
FIG. 1 (SEQ ID NO:5) for CDR-L2 or as L3 in FIG. 1 (SEQ ID NO:6)
for CDR-L3.
[0030] The CDRs given in SEQ IDS NOS:1 to 6 and in FIG. 1 referred
to above are derived from a mouse monoclonal antibody 5/44.
[0031] The complete sequences of the variable domains of the mouse
5/44 antibody are shown in FIG. 2 (light chain) (SEQ ID NO:7) and
FIG. 3 (heavy chain) (SEQ ID NO:8). This mouse antibody is also
referred to below as "the donor antibody" or the "murine monoclonal
antibody".
[0032] A first alternatively preferred embodiment of the first or
second aspect of the present invention is the mouse monoclonal
antibody 5/44 having the light and heavy chain variable domain
sequences shown in FIG. 2 (SEQ ID NO:7) and FIG. 3 (SEQ ID NO:8),
respectively. The light chain constant region of 5/44 is kappa and
the heavy chain constant region is IgG1.
[0033] In a second alternatively preferred embodiment, the antibody
according to either of the first and second aspects of the present
invention is a chimeric mouse/human antibody molecule, referred to
herein as the chimeric 5/44 antibody molecule. The chimeric
antibody molecule comprises the variable domains of the mouse
monoclonal antibody 5/44 (SEQ ID NOS:7 and 8) and human constant
domains. Preferably, the chimeric 5/44 antibody molecule comprises
the human C kappa domain (Hieter et al., Cell, 22, 197-207, 1980;
Genebank accession number J00241) in the light chain and the human
gamma 4 domains (Flanagan et al., Nature, 300, 709-713, 1982) in
the heavy chain, optionally with the serine residue at position 241
replaced by a proline residue.
[0034] Preferably, the antibody of the present invention comprises
a heavy chain wherein the variable domain comprises as CDR-H2 (as
defined by Kabat et al., (supra)) an H2' in which a potential
glycosylation site sequence has been removed and which unexpectedly
increased the affinity of the chimeric 5/44 antibody for the CD22
antigen and which preferably has as CDR-H2 the sequence given as
H2' (SEQ ID NO:13).
[0035] Alternatively or additionally, the antibody of the present
invention may comprise a heavy chain wherein the variable domain
comprises as CDR-H2 (as defined by Kabat et al., (supra)) an H2''
in which a lysine residue at position 60, which is located at an
exposed position within CDR-H2 and which is considered to have the
potential to react with conjugation agents resulting in a reduction
of antigen binding affinity, is substituted for an alternative
amino acid to result in a conserved substitution. Preferably CDR-H2
has the sequence given as H2'' (SEQ ID NO:15).
[0036] Alternatively or additionally, the antibody of the present
invention may comprise a heavy chain wherein the variable domain
comprises as CDR-H2 (as defined by Kabat et al., (supra)) an H2'''
in which both the potential glycosylation site sequence and the
lysine residue at position 60, are substituted for alternative
amino acids. Preferably CDR-H2 has the sequence given as H2''' (SEQ
ID NO:16).
[0037] In a third alternatively preferred embodiment, the antibody
according to either of the first and second aspects of the present
invention is a CDR-grafted antibody molecule. The term "a
CDR-grafted antibody molecule" as used herein refers to an antibody
molecule wherein the heavy and/or light chain contains one or more
CDRs (including, if desired, a modified CDR) from a donor antibody
(e.g. a murine monoclonal antibody) grafted into a heavy and/or
light chain variable region framework of an acceptor antibody (e.g.
a human antibody).
[0038] Preferably, such a CDR-grafted antibody has a variable
domain comprising human acceptor framework regions as well as one
or more of the donor CDRs referred to above.
[0039] When the CDRs are grafted, any appropriate acceptor variable
region framework sequence may be used having regard to the
class/type of the donor antibody from which the CDRs are derived,
including mouse, primate and human framework regions. Examples of
human frameworks which can be used in the present invention are
KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al. (supra)).
For example, KOL and NEWM can be used for the heavy chain, REI can
be used for the light chain and EU, LAY and POM can be used for
both the heavy chain and the light chain. Alternatively, human
germline sequences may be used. The preferred framework region for
the light chain is the human germline sub-group sequence (DPK9+JK1)
shown in FIG. 5 (SEQ ID NO:17). The preferred framework region for
the heavy chain is the human sub-group sequence (DP7+JH4) shown in
FIG. 6 (SEQ ID NO:21).
[0040] In a CDR-grafted antibody of the present invention, it is
preferred to use as the acceptor antibody one having chains which
are homologous to the chains of the donor antibody. The acceptor
heavy and light chains do not necessarily need to be derived from
the same antibody and may, if desired, comprise composite chains
having framework regions derived from different chains.
[0041] Also, in a CDR-grafted antibody of the present invention,
the framework regions need not have exactly the same sequence as
those of the acceptor antibody. For instance, unusual residues may
be changed to more frequently-occurring residues for that acceptor
chain class or type. Alternatively, selected residues in the
acceptor framework regions may be changed so that they correspond
to the residue found at the same position in the donor antibody or
to a residue that is a conservative substitution for the residue
found at the same position in the donor antibody. Such changes
should be kept to the minimum necessary to recover the affinity of
the donor antibody. A protocol for selecting residues in the
acceptor framework regions which may need to be changed is set
forth in WO 91/09967.
[0042] Preferably, in a CDR-grafted antibody molecule according to
the present invention, if the acceptor light chain has the human
sub-group DPK9+JK1 sequence (shown in FIG. 2) (SEQ ID NO:17) then
the acceptor framework regions of the light chain comprise donor
residues at positions 2, 4, 37, 38, 45 and 60 and may additionally
comprise a donor residue at position 3 (according to Kabat et al.
(supra)).
[0043] Preferably, in a CDR-grafted antibody molecule of the
present invention, if the acceptor heavy chain has the human
DP7+JH4 sequence (shown in FIG. 3) (SEQ ID NO:21), then the
acceptor framework regions of the heavy chain comprise, in addition
to one or more donor CDRs, donor residues at positions 1, 28, 48,
71 and 93 and may additionally comprise donor residues at positions
67 and 69 (according to Kabat et al. (supra)).
[0044] Donor residues are residues from the donor antibody, i.e.
the antibody from which the CDRs were originally derived.
[0045] Preferably, the antibody of the present invention comprises
a heavy chain wherein the variable domain comprises as CDR-H2 (as
defined by Kabat et al., (supra)) an H2' in which a potential
glycosylation site sequence has been removed in order to increase
the affinity of the chimeric 5/44 antibody for the CD22 antigen and
which preferably has as CDR-H2 the sequence given as H2' (SEQ ID
NO:13).
[0046] Alternatively or additionally, the antibody of the present
invention may comprise a heavy chain wherein the variable domain
comprises as CDR-H2 (as defined by Kabat et al., (supra)) an H2''
in which a lysine residue at position 60, which is located at an
exposed position within CDR-H2 and which is considered to have the
potential to react with conjugation agents resulting in a reduction
of antigen binding affinity, is substituted for an alternative
amino acid. Preferably CDR-H2 has the sequence given as H2'' (SEQ
ID NO:15).
[0047] Alternatively or additionally, the antibody of the present
invention may comprise a heavy chain wherein the variable domain
comprises as CDR-H2 (as defined by Kabat et al., (supra)) an H2'''
in which both the potential glycosylation site sequence and the
lysine residue at position 60, are substituted for alternative
amino acids. Preferably CDR-H2 has the sequence given as H2''' (SEQ
ID NO:16).
[0048] The antibody molecule of the present invention may comprise:
a complete antibody molecule, having full length heavy and light
chains; a fragment thereof, such as a Fab, modified Fab, Fab',
F(ab').sub.2 or Fv fragment; a light chain or heavy chain monomer
or dimer; a single chain antibody, e.g. a single chain Fv in which
the heavy and light chain variable domains are joined by a peptide
linker. Similarly, the heavy and light chain variable regions may
be combined with other antibody domains as appropriate.
[0049] The antibody molecule of the present invention may have an
effector or a reporter molecule attached to it. For instance, it
may have a macrocycle, for chelating a heavy metal atom, or a
toxin, such as ricin, attached to it by a covalent bridging
structure. Alternatively, procedures of recombinant DNA technology
may be used to produce an antibody molecule in which the Fc
fragment (CH2, CH3 and hinge domains), the CH2 and CH3 domains or
the CH3 domain of a complete immunoglobulin molecule has (have)
been replaced by, or has (have) attached thereto by peptide
linkage, a functional non-immunoglobulin protein, such as an enzyme
or toxin molecule.
[0050] The antibody molecule of the present invention preferably
has a binding affinity of at least 0.85.times.10.sup.-10 M, more
preferably at least 0.75.times.10.sup.-10 M and most preferably at
least 0.5.times.10.sup.-10 M.
[0051] Preferably, the antibody molecule of the present invention
comprises the light chain variable domain 5/44-gL1 (SEQ ID NO:19)
and the heavy chain variable domain 5/44-gH7 (SEQ ID NO:27). The
sequences of the variable domains of these light and heavy chains
are shown in FIGS. 5 and 6, respectively.
[0052] The present invention also relates to variants of the
antibody molecule of the present invention, which have an improved
affinity for CD22. Such variants can be obtained by a number of
affinity maturation protocols including mutating the CDRs (Yang et
al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et
al., Bio/Technology, 10, 779-783, 1992), use of mutator strains of
E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA
shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733,
1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88,
1996) and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998).
Vaughan et al. (supra) discusses these methods of affinity
maturation.
[0053] The present invention also provides a DNA sequence encoding
the heavy and/or light chain(s) of the antibody molecule of the
present invention.
[0054] Preferably, the DNA sequence encodes the heavy or the light
chain of the antibody molecule of the present invention.
[0055] The DNA sequence of the present invention may comprise
synthetic DNA, for instance produced by chemical processing, cDNA,
genomic DNA or any combination thereof.
[0056] The present invention also relates to a cloning or
expression vector comprising one or more DNA sequences of the
present invention. Preferably, the cloning or expression vector
comprises two DNA sequences, encoding the light chain and the heavy
chain of the antibody molecule of the present invention,
respectively.
[0057] General methods by which the vectors may be constructed,
transfection methods and culture methods are well known to those
skilled in the art. In this respect, reference is made to "Current
Protocols in Molecular Biology", 1999, F. M. Ausubel (ed), Wiley
Interscience, New York and the Maniatis Manual produced by Cold
Spring Harbor Publishing.
[0058] DNA sequences which encode the antibody molecule of the
present invention can be obtained by methods well known to those
skilled in the art. For example, DNA sequences coding for part or
all of the antibody heavy and light chains may be synthesised as
desired from the determined DNA sequences or on the basis of the
corresponding amino acid sequences.
[0059] DNA coding for acceptor framework sequences is widely
available to those skilled in the art and can be readily
synthesised on the basis of their known amino acid sequences.
[0060] Standard techniques of molecular biology may be used to
prepare DNA sequences coding for the antibody molecule of the
present invention. Desired DNA sequences may be synthesised
completely or in part using oligonucleotide synthesis techniques.
Site-directed mutagenesis and polymerase chain reaction (PCR)
techniques may be used as appropriate.
[0061] Any suitable host cell/vector system may be used for
expression of the DNA sequences encoding the antibody molecule of
the present invention. Bacterial, for example E. Coli, and other
microbial systems may be used, in part, for expression of antibody
fragments such as Fab and F(ab').sub.2 fragments, and especially Fv
fragments and single chain antibody fragments, for example, single
chain Fvs. Eukaryotic, e.g. mammalian, host cell expression systems
may be used for production of larger antibody molecules, including
complete antibody molecules. Suitable mammalian host cells include
CHO, myeloma or hybridoma cells.
[0062] The present invention also provides a process for the
production of an antibody molecule according to the present
invention comprising culturing a host cell containing a vector of
the present invention under conditions suitable for leading to
expression of protein from DNA encoding the antibody molecule of
the present invention, and isolating the antibody molecule.
[0063] The antibody molecule may comprise only a heavy or light
chain polypeptide, in which case only a heavy chain or light chain
polypeptide coding sequence needs to be used to transfect the host
cells. For production of products comprising both heavy and light
chains, the cell line may be transfected with two vectors, a first
vector encoding a light chain polypeptide and a second vector
encoding a heavy chain polypeptide. Alternatively, a single vector
may be used, the vector including sequences encoding light chain
and heavy chain polypeptides.
[0064] The present invention also provides a therapeutic or
diagnostic composition comprising an antibody molecule of the
present invention in combination with a pharmaceutically acceptable
excipient, diluent or carrier.
[0065] The present invention also provides a process for
preparation of a therapeutic or diagnostic composition comprising
admixing the antibody molecule of the present invention together
with a pharmaceutically acceptable excipient, diluent or
carrier.
[0066] The antibody molecule may be the sole active ingredient in
the therapeutic or diagnostic composition or may be accompanied by
other active ingredients including other antibody ingredients, for
example anti-T cell, anti-IFN.gamma. or anti-LPS antibodies, or
non-antibody ingredients such as xanthines.
[0067] The pharmaceutical compositions preferably comprise a
therapeutically effective amount of the antibody of the invention.
The term "therapeutically effective amount" as used herein refers
to an amount of a therapeutic agent needed to treat, ameliorate or
prevent a targeted disease or condition, or to exhibit a detectable
therapeutic or preventative effect. For any antibody, the
therapeutically effective dose can be estimated initially either in
cell culture assays or in animal models, usually in rodents,
rabbits, dogs, pigs or primates. The animal model may also be used
to determine the appropriate concentration range and route of
administration. Such information can then be used to determine
useful doses and routes for administration in humans.
[0068] The precise effective amount for a human subject will depend
upon the severity of the disease state, the general health of the
subject, the age, weight and gender of the subject, diet, time and
frequency of administration, drug combination(s), reaction
sensitivities and tolerance/response to therapy. This amount can be
determined by routine experimentation and is within the judgement
of the clinician. Generally, an effective dose will be from 0.01
mg/kg to 50 mg/kg, preferably 0.1 mg/kg to 20 mg/kg, more
preferably about 15 mg/kg.
[0069] Compositions may be administered individually to a patient
or may be administered in combination with other agents, drugs or
hormones.
[0070] The dose at which the antibody molecule of the present
invention is administered depends on the nature of the condition to
be treated, the grade of the malignant lymphoma or leukaemia and on
whether the antibody molecule is being used prophylactically or to
treat an existing condition.
[0071] The frequency of dose will depend on the half-life of the
antibody molecule and the duration of its effect. If the antibody
molecule has a short half-life (e.g. 2 to 10 hours) it may be
necessary to give one or more doses per day. Alternatively, if the
antibody molecule has a long half life (e.g. 2 to 15 days) it may
only be necessary to give a dosage once per day, once per week or
even once every 1 or 2 months.
[0072] A pharmaceutical composition may also contain a
pharmaceutically acceptable carrier for administration of the
antibody. The carrier should not itself induce the production of
antibodies harmful to the individual receiving the composition and
should not be toxic. Suitable carriers may be large, slowly
metabolised macromolecules such as proteins, polypeptides,
liposomes, polysaccharides, polylactic acids, polyglycolic acids,
polymeric amino acids, amino acid copolymers and inactive virus
particles.
[0073] Pharmaceutically acceptable salts can be used, for example
mineral acid salts, such as hydrochlorides, hydrobromides,
phosphates and sulphates, or salts of organic acids, such as
acetates, propionates, malonates and benzoates.
[0074] Pharmaceutically acceptable carriers in therapeutic
compositions may additionally contain liquids such as water,
saline, glycerol and ethanol. Additionally, auxiliary substances,
such as wetting or emulsifying agents or pH buffering substances,
may be present in such compositions. Such carriers enable the
pharmaceutical compositions to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries and suspensions,
for ingestion by the patient.
[0075] Preferred forms for administration include forms suitable
for parenteral administration, e.g. by injection or infusion, for
example by bolus injection or continuous infusion. Where the
product is for injection or infusion, it may take the form of a
suspension, solution or emulsion in an oily or aqueous vehicle and
it may contain formulatory agents, such as suspending,
preservative, stabilising and/or dispersing agents. Alternatively,
the antibody molecule may be in dry form, for reconstitution before
use with an appropriate sterile liquid.
[0076] Once formulated, the compositions of the invention can be
administered directly to the subject. The subjects to be treated
can be animals. However, it is preferred that the compositions are
adapted for administration to human subjects.
[0077] The pharmaceutical compositions of this invention may be
administered by any number of routes including, but not limited to,
oral, intravenous, intramuscular, intra-arterial, intramedullary,
intrathecal, intraventricular, transdermal, transcutaneous (for
example, see WO98/20734), subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, intravaginal or rectal
routes. Hyposprays may also be used to administer the
pharmaceutical compositions of the invention. Typically, the
therapeutic compositions may be prepared as injectables, either as
liquid solutions or suspensions. Solid forms suitable for solution
in, or suspension in, liquid vehicles prior to injection may also
be prepared.
[0078] Direct delivery of the compositions will generally be
accomplished by injection, subcutaneously, intraperitoneally,
intravenously or intramuscularly, or delivered to the interstitial
space of a tissue. The compositions can also be administered into a
lesion. Dosage treatment may be a single dose schedule or a
multiple dose schedule.
[0079] It will be appreciated that the active ingredient in the
composition will be an antibody molecule. As such, it will be
susceptible to degradation in the gastrointestinal tract. Thus, if
the composition is to be administered by a route using the
gastrointestinal tract, the composition will need to contain agents
which protect the antibody from degradation but which release the
antibody once it has been absorbed from the gastrointestinal
tract.
[0080] A thorough discussion of pharmaceutically acceptable
carriers is available in Remington's Pharmaceutical Sciences (Mack
Publishing Company, N.J. 1991).
[0081] It is also envisaged that the antibody of the present
invention will be administered by use of gene therapy. In order to
achieve this, DNA sequences encoding the heavy and light chains of
the antibody molecule under the control of appropriate DNA
components are introduced into a patient such that the antibody
chains are expressed from the DNA sequences and assembled in
situ.
[0082] The present invention also provides the antibody molecule of
the present invention for use in treating a disease mediated by
cells expressing CD22.
[0083] The present invention further provides the use of the
antibody molecule according to the present invention in the
manufacture of a medicament for the treatment of a disease mediated
by cells expressing CD22.
[0084] The antibody molecule of the present invention may be
utilised in any therapy where it is desired to reduce the level of
cells expressing CD22 that are present in the human or animal body.
These CD22-expressing cells may be circulating in the body or be
present in an undesirably high level localised at a particular site
in the body. For example, elevated levels of cells expressing CD22
will be present in B cell lymphomas and leukaemias. The antibody
molecule of the present invention may be utilised in the therapy of
diseases mediated by cells expressing CD22.
[0085] The antibody molecule of the present invention is preferably
used for treatment of malignant lymphomas and leukaemias, most
preferably NHL.
[0086] The present invention also provides a method of treating
human or animal subjects suffering from or at risk of a disorder
mediated by cells expressing CD22, the method comprising
administering to the subject an effective amount of the antibody
molecule of the present invention.
[0087] The antibody molecule of the present invention may also be
used in diagnosis, for example in the in vivo diagnosis and imaging
of disease states involving cells that express CD22.
[0088] The present invention is further described by way of
illustration only in the following examples, which refer to the
accompanying Figures, in which:
[0089] FIG. 1 shows the amino acid sequence of the CDRs of mouse
monoclonal antibody 5/44 (SEQ ID NOS:1 to 6);
[0090] FIG. 2 shows the complete sequence of the light chain
variable domain of mouse monoclonal antibody 5/44;
[0091] FIG. 3 shows the complete sequence of the heavy chain
variable domain of mouse monoclonal antibody 5/44;
[0092] FIG. 4 shows the strategy for removal of the glycosylation
site and reactive lysine in CDR-H2;
[0093] FIG. 5 shows the graft design for the 5/44 light chain
sequence;
[0094] FIG. 6 shows the graft design for the 5/44 heavy chain
sequence;
[0095] FIG. 7 shows the vectors pMRR14 and pMRR10.1;
[0096] FIG. 8 shows the Biacore assay results of the chimeric 5/44
mutants;
[0097] FIG. 9 shows the oligonucleotides for 5/44 gH1 and gL1 gene
assemblies;
[0098] FIG. 10 shows the intermediate vectors pCR2.1(544gH1) and
pCR2.1(544gL1);
[0099] FIG. 11 shows the oligonucleotide cassettes used to make
further grafts;
[0100] FIG. 12 shows the competition assay between fluorescently
labelled mouse 5/44 antibody and grafted variants; and
[0101] FIG. 13 shows the full DNA and protein sequence of the
grafted heavy and light chains.
DETAILED DESCRIPTION OF THE INVENTION
Example 1
Generation of Candidate Antibodies
[0102] A panel of antibodies against CD22 were selected from
hybridomas using the following selection criteria: binding to Daudi
cells, internalisation on Daudi cells, binding to peripheral blood
mononuclear cells (PBMC), internalisation on PBMC, affinity
(greater than 10.sup.-9M), mouse .gamma.1 and production rate. 5/44
was selected as the preferred antibody.
Example 2
Gene Cloning and Expression of a Chimeric 5/44 Antibody
Molecule
[0103] Preparation of 5/44 Hybridoma Cells and RNA Preparation
Therefrom
[0104] Hybridoma 5/44 was generated by conventional hybridoma
technology following immunisation of mice with human CD22 protein.
RNA was prepared from 5/44 hybridoma cells using a RNEasy kit
(Qiagen, Crawley, UK; Catalogue No. 74106). The RNA obtained was
reverse transcribed to cDNA, as described below.
[0105] Distribution of CD22 on NHL Tumours
[0106] An immunohistochemistry study was undertaken to examine the
incidence and distribution of staining using the 5/44 anti-CD22
monoclonal antibodies. Control anti-CD20 and anti-CD79a antibodies
were included in the study to confirm B cell areas of tumours.
[0107] A total of 50 tumours were studied and these were
categorised as follows by using the Working Formulation and REAL
classification systems:
[0108] 7 B lymphoblastic leukaemia/lymphoma (High/i)
[0109] 4 B-CLL/small lymphocytic lymphoma (Low/A)
[0110] 3 lymphoplasmacytoid/Immunocytoma (Low/A)
[0111] 1 Mantle cell (Int/F)
[0112] 14 Follicle center lymphoma (Low to Int/D)
[0113] 13 Diffuse large cell lymphoma (Int to High/G,H)
[0114] 6 Unclassifiable (K)
[0115] 2 T cell lymphomas
[0116] 40 B cell lymphomas were positive for CD22 antigen with the
5/44 antibody at 0.1 .mu.g/ml and a further 6 became positive when
the concentration was increased to 0.5 .mu.g/ml. For the remaining
2 B cell tumours that were negative at 0.1 .mu.g/ml, there was
insufficient tissue remaining to test at the higher concentration.
However, parallel testing with another Celltech anti-CD22 antibody
6/13, which gave stronger staining than 5/44, resulted in all 48 B
cell lymphomas staining positive for CD22.
[0117] Thus, it is possible to conclude that the CD22 antigen is
widely expressed on B cell lymphomas and thus provides a suitable
target for immunotherapy in NHL.
[0118] PCR Cloning of 5/44 V.sub.H and V.sub.L
[0119] cDNA sequences coding for the variable domains of 5/44 heavy
and light chains were synthesised using reverse transcriptase to
produce single stranded cDNA copies of the mRNA present in the
total RNA. This was then used as the template for amplification of
the murine V-region sequences using specific oligonucleotide
primers by the Polymerase Chain Reaction (PCR).
[0120] a) cDNA Synthesis
[0121] cDNA was synthesised in a 20 .mu.l reaction volume
containing the following reagents: 50 mM Tris-HCl pH 8.3, 75 mM
KCl, 10 mM dithiothreitol, 3 mM MgCl.sub.2, 0.5 mM each
deoxyribonucleoside triphosphate, 20 units RNAsin, 75 ng random
hexanucleotide primer, 2 .mu.g 5/44 RNA and 200 units Moloney
Murine Leukemia Virus reverse transcriptase. After incubation at
42.degree. C. for 60 minutes, the reaction was terminated by
heating at 95.degree. C. for 5 minutes.
[0122] b) PCR
[0123] Aliquots of the cDNA were subjected to PCR using
combinations of primers specific for the heavy and light chains.
Degenerate primer pools designed to anneal with the conserved
sequences of the signal peptide were used as forward primers. These
sequences all contain, in order, a restriction site (V.sub.L SfuI;
V.sub.H HindIII) starting 7 nucleotides from their 5' ends, the
sequence GCCGCCACC (SEQ ID NO:50), to allow optimal translation of
the resulting mRNAs, an initiation codon and 20-30 nucleotides
based on the leader peptide sequences of known mouse antibodies
(Kabat et al., Sequences of proteins of immunological interest,
5.sup.th Edition, 1991, U.S. Department of Health and Human
Services, Public Health Service, National Institutes of
Health).
[0124] The 3' primers are designed to span the framework 4 J-C
junction of the antibody and contain a restriction site for the
enzyme BsiWI to facilitate cloning of the V.sub.L PCR fragment. The
heavy chain 3' primers are a mixture designed to span the J-C
junction of the antibody. The 3' primer includes an ApaI
restriction site to facilitate cloning. The 3' region of the
primers contains a mixed sequence based on those found in known
mouse antibodies (Kabat et al., 1991, supra).
[0125] The combinations of primers described above enable the PCR
products for V.sub.H and V1 to be cloned directly into an
appropriate expression vector (see below) to produce chimeric
(mouse-human) heavy and light chains and for these genes to be
expressed in mammalian cells to produce chimeric antibodies of the
desired isotype.
[0126] Incubations (100 .mu.l) for the PCR were set up as follows.
Each reaction contained 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM
KCl, 0.01% w/v gelatin, 0.25 mM each deoxyribonucleoside
triphosphate, 10 pmoles 5' primer mix, 10 pmoles 3' primer, 1 .mu.l
cDNA and 1 unit Taq polymerase. Reactions were incubated at
95.degree. C. for 5 minutes and then cycled through 94.degree. C.
for 1 minute, 55.degree. C. for 1 minute and 72.degree. C. for 1
minute. After 30 cycles, aliquots of each reaction were analysed by
electrophoresis on an agarose gel.
[0127] For the heavy chain V-region, an amplified DNA product was
only obtained when a primer pool annealing within the start of
framework I replaced the signal peptide primer pool. The fragments
were cloned into DNA sequencing vectors. The DNA sequence was
determined and translated to give a deduced amino acid sequence.
This deduced sequence was verified by reference to the N-terminal
protein sequence determined experimentally. FIGS. 2 and 3 shows the
DNA/protein sequence of the mature light and heavy chain V-regions
of mouse monoclonal 5/44 respectively.
[0128] c) Molecular Cloning of the PCR Fragments
[0129] The murine v-region sequences were then cloned into the
expression vectors pMRR10.1 and pMRR14 (FIG. 7). These are vectors
for the expression of light and heavy chain respectively containing
DNA encoding constant regions of human kappa light chain and human
gamma-4 heavy chain. The V.sub.L region was sub-cloned into the
expression vector by restriction digest and ligation from the
sequencing vector, using SfuI and BsiWI restriction sites, creating
plasmid pMRR10(544cL). The heavy chain DNA was amplified by PCR
using a 5' primer to introduce a signal peptide, since this was not
obtained in the cloning strategy--a mouse heavy chain antibody
leader from a different in-house hybridoma (termed 162) was
employed. The 5' primer had the following sequence: TABLE-US-00001
(SEQ ID NO:51)
.sup.5'GCGCGCAAGCTTGCCGCCACCATGGACTTCGGATTCTCTCTCGTGTTC
CTGGCACTCATTCTCAAGGGAGTGCAGTGTGAGGTGCAGCTCGTCGAGTC TGG.sup.3'.
[0130] The reverse primer was identical to that used in the
original V.sub.H gene cloning. The resultant PCR product was
digested with enzymes HindIII and ApaI, was sub-cloned, and its DNA
sequence was confirmed, creating plasmid pMRR14(544cH). Transient
co-transfection of both expression vectors into CHO cells generated
chimeric c5/44 antibody. This was achieved using the Lipofectamine
reagent according to the manufacturer's protocols (InVitrogen:Life
Technology, Groningen, The Netherlands. Catalogue no.
11668-027).
[0131] Removal of Glycosylation Site and Reactive Lysine
[0132] A potential N-linked glycosylation site sequence was
observed in CDR-H2, having the amino acid sequence N-Y-T (FIG. 3).
SDS-PAGE, Western blotting and carbohydrate staining of gels of
5/44 and its fragments (including Fab) indicated that this site was
indeed glycosylated (not shown). In addition, a lysine residue was
observed at an exposed position within CDR-H2, which had the
potential to reduce the binding affinity of the antibody by
providing an additional site for conjugation with an agent with
which the antibody may be conjugated.
[0133] A PCR strategy was used to introduce amino acid
substitutions into the CDR-H2 sequence in an attempt to remove the
glycosylation site and/or the reactive lysine, as shown in FIG. 4.
Forward primers encoding the mutations N55Q, T57A or T57V were used
to remove the glycosylation site (FIG. 4) and a fourth forward
primer containing the substitution K60R, was generated to remove
the reactive lysine residue (FIG. 4). A framework 4 reverse primer
was used in each of these PCR amplifications. The PCR products were
digested with the enzymes XbaI and ApaI and were inserted into
pMRR14(544cH) (also cleaved with XbaI and ApaI) to generate
expression plasmids encoding these mutants. The N55Q, T57A and T57V
mutations ablate the glycosylation site by changing the amino acid
sequence away from the consensus N-X-T/S whilst the K60R mutation
replaces the potentially reactive lysine with the similarly
positively charged residue arginine. The resultant cH variant
plasmids were co-transfected with the cL plasmid to generate
expressed chimeric antibody variants.
[0134] Evaluation of Activities of Chimeric Genes
[0135] The activities of the chimeric genes were evaluated
following transient transfection into CHO cells.
[0136] c) Determination of Affinity Constants by BiaCore
Analysis.
[0137] The affinities of chimeric 5/44 or its variants, which have
had their glycosylation site or their reactive lysine removed, were
investigated using BIA technology for binding to CD22-mFc
constructs. The results are shown in FIG. 8. All binding
measurements were performed in the BIAcore.TM. 2000 instrument
(Pharmacia Biosensor AB, Uppsala, Sweden). The assay was performed
by capture of CD22mFc via the immobilised anti-mouse Fc. The
antibody was in the soluble phase. Samples, standard, and controls
(50 ul) were injected over immobilised anti-mouse Fc followed by
antibody in the soluble phase. After each cycle the surface was
regenerated with 50 ul of 40 mM HCl at 30 ul/min. The kinetic
analysis was performed using the BIAevaluation 3.1 software
(Pharmacia).
[0138] Removal of the glycosylation site in construct T57A resulted
in a slightly faster on-rate and a significantly slower off-rate
compared to the chimeric 5/44, giving an affinity improvement of
approximately 5-fold. The N55Q mutation had no effect on affinity.
This result was unexpected as it suggests that the removal of the
carbohydrate itself apparently has no effect on binding (as with
the N55Q change). The improved affinity was observed only with the
T57A change. One possible explanation is that, regardless of the
presence of carbohydrate, the threonine at position 57 exerts a
negative effect on binding that is removed on conversion of
threonine to alanine. The hypothesis that the small size of alanine
is important, and that the negative effect of threonine is related
to its size, is supported from the result obtained using the T57V
mutation: that replacement with valine at position 57 is not
beneficial (results not shown).
[0139] Removal of the lysine by the K60R mutation had a neutral
effect on affinity, i.e. the introduction of arginine removes a
potential reactive site without compromising affinity.
[0140] The mutations for removal of the glycosylation site and for
removal of the reactive lysine were therefore both included in the
humanisation design.
Example 2
CDR-Grafting of 5/44
[0141] The molecular cloning of genes for the variable regions of
the heavy and light chains of the 5/44 antibody and their use to
produce chimeric (mouse/human) 5/44 antibodies has been described
above. The nucleotide and amino acid sequences of the mouse 5/44
V.sub.L and V.sub.H domains are shown in FIGS. 2 and 3 (SEQ ID
NOS:7 and 8), respectively. This example describes the CDR-grafting
of the 5/44 antibody onto human frameworks to reduce potential
immunogenicity in humans, according to the method of Adair et al.,
(WO91/09967).
[0142] CDR-Grafting of 5/44 Light Chain
[0143] Protein sequence alignment with consensus sequences from
human sub-group I kappa light chain V region indicated 64% sequence
identity. Consequently, for constructing the CDR-grafted light
chain, the acceptor framework regions chosen corresponded to those
of the human VK sub-group I germline O12,DPK9 sequence. The
framework 4 acceptor sequence was derived from the human J-region
germline sequence JK1.
[0144] A comparison of the amino acid sequences of the framework
regions of murine 5/44 and the acceptor sequence is given in FIG. 5
and shows that there are 27 differences between the donor and
acceptor chains. At each position, an analysis was made of the
potential of the murine residue to contribute to antigen binding,
either directly or indirectly, through effects on packing or at the
V.sub.H/N.sub.L interface. If a murine residue was considered
important and sufficiently different from the human residue in
terms of size, polarity or charge, then that murine residue was
retained. Based on this analysis, two versions of the CDR-grafted
light chain, having the sequences given in SEQ ID NO:19 and SEQ ID
NO:20 (FIG. 5), were constructed.
[0145] CDR-Grafting of 5/44 Heavy Chain
[0146] CDR-grafting of 5/44 heavy chain was accomplished using the
same strategy as described for the light chain. The V-domain of
5/44 heavy chain was found to be homologous to human heavy chains
belonging to sub-group I (70% sequence identity) and therefore the
sequence of the human sub-group I germline framework VH1-3,DP7 was
used as an acceptor framework. The framework 4 acceptor sequences
were derived from human J-region germline sequence JH4.
[0147] A comparison of 5/44 heavy chain with the framework regions
is shown in FIG. 6 where it can be seen that the 5/44 heavy chain
differs from the acceptor sequence at 22 positions. Analysis of the
contribution that any of these might make to antigen binding led to
5 versions of the CDR-grafted heavy chains being constructed,
having the sequences given in SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, SEQ ID NO:26 and SEQ ID NO:27 (FIG. 6).
[0148] Construction of Genes for Grafted Sequences.
[0149] Genes were designed to encode the grafted sequences gH1 and
gL1, and a series of overlapping oligonucleotides were designed and
constructed (FIG. 9). A PCR assembly technique was employed to
construct the CDR-grafted V-region genes. Reaction volumes of 100
ul were set up containing 10 mM Tris-HCl pH8.3, 1.5 mM MgCl2, 50 mM
KCl, 0.001% gelatin, 0.25 mM each deoxyribonucleoside triphosphate,
1 pmole each of the `internal` primers (T1, T2, T3, B1, B2, B3), 10
pmole each of the `external` primers (F1, R1), and 1 unit of Taq
polymerase (AmpliTaq, Applied BioSystems, catalogue no. N808-0171).
PCR cycle parameters were 94.degree. C. for 1 minute, 55.degree. C.
for 1 minute and 72.degree. C. for 1 minute, for 30 cycles. The
reaction products were then run on a 1.5% agarose gel, excised and
recovered using QIAGEN spin columns (QIAquick gel extraction kit,
cat no. 28706). The DNA was eluted in a volume of 30 .mu.l.
Aliquots (1 .mu.l) of the gH1 and gL1 DNA were then cloned into the
InVitrogen TOPO TA cloning vector pCR2.1 TOPO (catalogue no.
K4500-01) according to the manufacturer's instructions. This
non-expression vector served as a cloning intermediate to
facilitate sequencing of a large number of clones. DNA sequencing
using vector-specific primers was used to identify correct clones
containing gH1 and gL1, creating plasmids pCR2.1 (544gH1) and
pCR2.1(544gL1) (FIG. 10).
[0150] An oligonucleotide cassette replacement method was used to
create the humanised grafts gH4,5,6 and 7, and gL2. FIG. 11 shows
the design of the oligonucleotide cassettes. To construct each
variant, the vector (pCR2.1(544gH1) or pCR2.1(544gL1)) was cut with
the restriction enzymes shown (XmaI/SacII for the heavy chain,
XmaI/BstEII for the light chain). The large vector fragment was gel
purified from agarose and was used in ligation with the
oligonucleotide cassette. These cassettes are composed of 2
complementary oligonucleotides (shown in FIG. 11), mixed at a
concentration of 0.5 pmoles/.mu.l in a volume of 200 .mu.l 12.5 mM
Tris-HCl pH 7.5, 2.5 mM MgCl.sub.2, 25 mM NaCl, 0.25 mM
dithioerythritol. Annealing was achieved by heating to 95.degree.
C. for 3 minutes in a waterbath (volume 500 ml) then allowing the
reaction to slow-cool to room temperature. The annealed
oligonucleotide cassette was then diluted ten-fold in water before
ligation into the appropriately cut vector. DNA sequencing was used
to confirm the correct sequence, creating plasmids pCR2.1
(5/44-gH4-7) and pCR2.1(5/44-gL2). The verified grafted sequences
were then sub-cloned into the expression vectors pMRR14 (heavy
chain) and pMR10.1 (light chain).
[0151] CD22 Binding Activity of CDR-Grafted Sequences
[0152] The vectors encoding grafted variants were co-transfected
into CHO cells in a variety of combinations, together with the
original chimeric antibody chains. Binding activity was compared in
a competition assay, competing the binding of the original mouse
5/44 antibody for binding to Ramos cells (obtained from ATCC, a
Burkitt's lymphoma lymphoblast human cell line expressing surface
CD22). This assay was considered the best way to compare grafts in
their ability to bind to cell surface CD22. The results are shown
in FIG. 8. As can be seen, there is very little difference between
any of the grafts, all performing more effectively than the
chimeric at competing against the murine parent. The introduction
of the 3 additional human residues at the end of CDR-H2 (gH6 and
gH7) does not appear to have affected binding.
[0153] The graft combination with the least number of murine
residues was selected, gL1gH7. The light chain graft gL1 has 6
donor residues. Residues V2, V4, L37 and Q45 are potentially
important packing residues. Residue H38 is at the V.sub.H/V.sub.L
interface. Residue D60 is a surface residue close to the CDR-L2 and
may directly contribute to antigen binding. Of these residues, V2,
L37, Q45 and D60 are found in germline sequences of human kappa
genes from other sub-groups. The heavy chain graft gH7 has 4 donor
framework residues (Residue R28 is considered to be part of CDR-H1
under the structural definition used in CDR-grafting (se Adair et
al (1991 WO91/09967)). Residues E1 and A71 are surface residues
close to the CDR's. Residue 148 is a potential packing residue.
Residue T93 is present at the V.sub.H/V.sub.L interface. Of these
residues, E1 and A71 are found in other germline genes of human
sub-group I. Residue 148 is found in human germline sub-group 4,
and T73 is found in human germline sub-group 3.
[0154] The full DNA and protein sequence of both the light chain
and heavy chain, including approximate position of introns within
the constant region genes provided by the vectors, are shown in
FIG. 13 and are given in SEQ ID NO:29 and SEQ ID NO:28 respectively
for the light chain and SEQ ID NO: 31 and SEQ ID NO:30 respectively
for the heavy chain.
[0155] DNA encoding these light and heavy chain genes was excised
from these vectors. Heavy chain DNA was digested at the 5' HindIII
site, then was treated with the Klenow fragment of E. coli DNA
polymerase I to create a 5' blunt end. Cleavage at the 3' EcoRI
site resulted in the heavy chain fragment which was purified from
agarose gels. In the same way, a light chain frament was produced,
blunted at the 5' SfuI site and with a 3' EcoRI site. Both
fragments were cloned into DHFR based expression vectors and used
to generate stable cell lines in CHO cells.
[0156] All references and patents cited herein are hereby
incorporated by reference in their entireties.
Sequence CWU 1
1
51 1 5 PRT mouse DOMAIN mouse monoclonal 5/44 CDR-H1 1 Asn Tyr Trp
Ile His 1 5 2 17 PRT mouse DOMAIN mouse monoclonal 5/44 CDR-H2 2
Gly Ile Asn Pro Gly Asn Asn Tyr Thr Thr Tyr Lys Arg Asn Leu Lys 1 5
10 15 Gly 3 12 PRT mouse DOMAIN mouse monoclonal 5/44 CDR-H3 3 Glu
Gly Tyr Gly Asn Tyr Gly Ala Trp Phe Ala Tyr 1 5 10 4 16 PRT mouse
DOMAIN mouse monoclonal 5/44 CDR-L1 4 Arg Ser Ser Gln Ser Leu Ala
Asn Ser Tyr Gly Asn Thr Phe Leu Ser 1 5 10 15 5 7 PRT mouse DOMAIN
mouse monoclonal 5/44 CDR-L2 5 Gly Ile Ser Asn Arg Phe Ser 1 5 6 9
PRT mouse DOMAIN mouse monoclonal 5/44 CDR-L3 6 Leu Gln Gly Thr His
Gln Pro Tyr Thr 1 5 7 113 PRT mouse DOMAIN mouse monoclonal 5/44 VL
domain 7 Asp Val Val Val Thr Gln Thr Pro Leu Ser Leu Pro Val Ser
Phe Gly 1 5 10 15 Asp Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Ala Asn Ser 20 25 30 Tyr Gly Asn Thr Phe Leu Ser Trp Tyr Leu
His Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gly Ile
Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Thr Ile Lys
Pro Glu Asp Leu Gly Met Tyr Tyr Cys Leu Gln Gly 85 90 95 Thr His
Gln Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110
Arg 8 121 PRT mouse DOMAIN mouse monoclonal 5/44 VH domain 8 Glu
Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala 1 5 10
15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asn Tyr
20 25 30 Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45 Gly Gly Ile Asn Pro Gly Asn Asn Tyr Thr Thr Tyr
Lys Arg Asn Leu 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Val Thr
Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Gly
Asn Tyr Gly Ala Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 9 13 PRT Artificial Sequence DOMAIN cH
9 Gly Asn Asn Tyr Thr Thr Tyr Lys Arg Asn Leu Lys Gly 1 5 10 10 13
PRT Artificial Sequence DOMAIN CDR-H2 MUTATION N55Q 10 Gly Asn Gln
Tyr Thr Thr Tyr Lys Arg Asn Leu Lys Gly 1 5 10 11 13 PRT Artificial
Sequence DOMAIN CDR-H2 MUTATION T57A 11 Gly Asn Asn Tyr Ala Thr Tyr
Lys Arg Asn Leu Lys Gly 1 5 10 12 13 PRT Artificial Sequence DOMAIN
CDR-H2 MUTATION T57V 12 Gly Asn Asn Tyr Val Thr Tyr Lys Arg Asn Leu
Lys Gly 1 5 10 13 17 PRT ARTIFICIAL SEQUENCE DOMAIN CDR-H2 MUTATION
(T57A) H' 13 Gly Ile Asn Pro Gly Asn Asn Tyr Ala Thr Tyr Lys Arg
Asn Leu Lys 1 5 10 15 Gly 14 13 PRT Artificial Sequence DOMAIN
CDR-H2 MUTATION K60R 14 Gly Asn Asn Tyr Thr Thr Tyr Arg Arg Asn Leu
Lys Gly 1 5 10 15 17 PRT ARTIFICIAL SEQUENCE DOMAIN CDR-H2 MUTATION
(K60R) H'' 15 Gly Ile Asn Pro Gly Asn Asn Tyr Thr Thr Tyr Arg Arg
Asn Leu Lys 1 5 10 15 Gly 16 17 PRT ARTIFICIAL SEQUENCE DOMAIN
CDR-H2 MUTATION (T57A K60R) H''' 16 Gly Ile Asn Pro Gly Asn Asn Tyr
Ala Thr Tyr Arg Arg Asn Leu Lys 1 5 10 15 Gly 17 70 PRT Homo
sapiens DOMAIN DPK9 HUMAN ACCEPTOR FRAMEWORK 17 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Trp Tyr Gln Gln Lys Pro Gly Lys Ala 20 25 30 Pro
Lys Leu Leu Ile Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 35 40
45 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp
50 55 60 Phe Ala Thr Tyr Tyr Cys 65 70 18 11 PRT Homo sapiens
DOMAIN JK1 HUMAN ACCEPTOR FRAMEWORK 18 Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 1 5 10 19 113 PRT Artificial Sequence CHAIN gL1 19
Asp Val Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Ala Asn
Ser 20 25 30 Tyr Gly Asn Thr Phe Leu Ser Trp Tyr Leu His Lys Pro
Gly Lys Ala 35 40 45 Pro Gln Leu Leu Ile Tyr Gly Ile Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Leu Gln Gly 85 90 95 Thr His Gln Pro Tyr
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg 20 113
PRT Artificial Sequence CHAIN gL2 20 Asp Val Val Val Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ser Ser Gln Ser Leu Ala Asn Ser 20 25 30 Tyr Gly Asn
Thr Phe Leu Ser Trp Tyr Leu His Lys Pro Gly Lys Ala 35 40 45 Pro
Gln Leu Leu Ile Tyr Gly Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu
Gln Gly 85 90 95 Thr His Gln Pro Tyr Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 110 Arg 21 80 PRT Homo sapiens CHAIN DP7
HUMAN ACCEPTOR FRAMEWORK 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Trp Val 20 25 30 Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met Gly Lys Phe Gln Gly 35 40 45 Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu 50 55 60 Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 65 70 75 80
22 11 PRT Homo sapiens DOMAIN JH4 HUMAN ACCEPTOR FRAMEWORK 22 Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 23 121 PRT
Artificial Sequence CHAIN gH1 23 Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Arg Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly
Ile Asn Pro Gly Asn Gln Tyr Thr Thr Tyr Lys Arg Asn Leu 50 55 60
Lys Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Gly Asn Tyr Gly Ala Trp Phe
Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 24 121 PRT Artificial Sequence CHAIN gH4 24 Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asn Tyr 20 25 30 Trp
Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Gly Ile Asn Pro Gly Asn Asn Tyr Ala Thr Tyr Arg Arg Asn Leu
50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr
Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Gly Asn Tyr Gly Ala
Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 25 121 PRT Artificial Sequence CHAIN gH5 25 Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asn Tyr 20 25
30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 Gly Gly Ile Asn Pro Gly Asn Asn Tyr Ala Thr Tyr Arg Arg
Asn Leu 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr
Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Gly Asn Tyr
Gly Ala Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 26 121 PRT Artificial Sequence CHAIN gH6 26 Glu
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asn Tyr
20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45 Gly Gly Ile Asn Pro Gly Asn Asn Tyr Ala Thr Tyr
Arg Arg Lys Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr
Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Tyr Gly
Asn Tyr Gly Ala Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 27 121 PRT Artificial Sequence CHAIN
gH7 27 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe
Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Gly Asn Asn Tyr
Ala Thr Tyr Arg Arg Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr
Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu
Gly Tyr Gly Asn Tyr Gly Ala Trp Phe Ala Tyr Trp Gly 100 105 110 Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 28 239 PRT Artificial
Sequence CHAIN Full sequence of grafted light chain 28 Met Lys Leu
Pro Val Arg Leu Leu Val Leu Leu Leu Phe Trp Ile Pro 1 5 10 15 Ala
Ser Arg Gly Asp Val Gln Val Thr Gln Ser Pro Ser Ser Leu Ser 20 25
30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser
35 40 45 Leu Ala Asn Ser Tyr Gly Asn Thr Phe Leu Ser Trp Tyr Leu
His Lys 50 55 60 Pro Gly Lys Ala Pro Gln Leu Leu Ile Tyr Gly Ile
Ser Asn Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr 100 105 110 Cys Leu Gln Gly Thr His
Gln Pro Tyr Thr Phe Gly Gln Gly Thr Lys 115 120 125 Val Glu Ile Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser
Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155
160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 225 230 235 29 781 DNA ARTIFICIAL CDS
Full DNA sequence of grafted light chain 29 ttcgaagccg ccaccatgaa
gttgcctgtt aggctgttgg tgcttctgtt gttctggatt 60 cctgcttccc
ggggtgacgt tcaagtgacc cagagcccat ccagcctgag cgcatctgta 120
ggagaccggg tcaccatcac ttgtagatcc agtcagagtc ttgcaaacag ttatgggaac
180 acctttttgt cttggtatct gcacaaacca ggtaaagccc cacaattgct
catctacgga 240 atctctaaca gatttagtgg tgtaccagac aggttcagcg
gttccggaag tggtactgat 300 ttcaccctca cgatctcgtc tctccagcca
gaagatttcg ccacttatta ctgtttacaa 360 ggtacacatc agccgtacac
attcggtcag ggtactaaag tagaaatcaa acgtacggta 420 gcggccccat
ctgtcttcat cttcccgcca tctgatgagc agttgaaatc tggaactgcc 480
tctgttgtgt gcctgctgaa taacttctat cccagagagg ccaaagtaca gtggaaggtg
540 gataacgccc tccaatcggg taactcccag gagagtgtca cagagcagga
cagcaaggac 600 agcacctaca gcctcagcag caccctgacg ctgagcaaag
cagactacga gaaacacaaa 660 gtctacgcct gcgaagtcac ccatcagggc
ctgagctcgc ccgtcacaaa gagcttcaac 720 aggggagagt gttagaggga
gaagtgcccc cacctgctcc tcagttccag cctgggaatt 780 c 781 30 467 PRT
Artificial Sequence CHAIN Full sequence of grafted heavy chain 30
Met Asp Phe Gly Phe Ser Leu Val Phe Leu Ala Leu Ile Leu Lys Gly 1 5
10 15 Val Gln Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Arg Phe 35 40 45 Thr Asn Tyr Trp Ile His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Gly Ile Asn Pro Gly
Asn Asn Tyr Ala Thr Tyr Arg 65 70 75 80 Arg Lys Phe Gln Gly Arg Val
Thr Met Thr Ala Asp Thr Ser Thr Ser 85 90 95 Thr Val Tyr Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys
Thr Arg Glu Gly Tyr Gly Asn Tyr Gly Ala Trp Phe Ala 115 120 125 Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135
140 Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
145 150 155 160 Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys Asn 210 215 220 Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser 225 230 235 240 Lys Tyr
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly 245 250 255
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260
265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
Gln 275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 340 345 350 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385
390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser
Arg Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Leu Gly Lys 465 31 2160
DNA Artificial Sequence CDS Full DNA sequence of grafted heavy
chain 31 aagcttgccg ccaccatgga cttcggattc tctctcgtgt tcctggcact
cattctcaag 60 ggagtgcagt gtgaggtgca attggtccag tcaggagcag
aggttaagaa gcctggtgct 120 tccgtcaaag tttcgtgtaa ggctagcggc
tacaggttca caaattattg gattcattgg 180 gtcaggcagg ctccgggaca
aggcctggaa tggatcggtg gcattaatcc cgggaataac 240 tacgctacat
ataggagaaa attccagggc agagttacga tgaccgcgga cacctccaca 300
agcactgtct acatggagct gtcatctctg agatccgagg acaccgcagt gtactattgt
360 actagagaag gctacggtaa ttacggagcc tggttcgcct actggggcca
gggtacccta 420 gtcacagtct cctcagcttc tacaaagggc ccatccgtct
tccccctggc gccctgctcc 480 aggagcacct ccgagagcac agccgccctg
ggctgcctgg tcaaggacta cttccccgaa 540 ccggtgacgg tgtcgtggaa
ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 600 gtcctacagt
cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 660
ttgggcacga agacctacac ctgcaacgta gatcacaagc ccagcaacac caaggtggac
720 aagagagttg gtgagaggcc agcacaggga gggagggtgt ctgctggaag
ccaggctcag 780 ccctcctgcc tggacgcacc ccggctgtgc agccccagcc
cagggcagca aggcatgccc 840 catctgtctc ctcacccgga ggcctctgac
caccccactc atgcccaggg agagggtctt 900 ctggattttt ccaccaggct
ccgggcagcc acaggctgga tgcccctacc ccaggccctg 960 cgcatacagg
ggcaggtgct gcgctcagac ctgccaagag ccatatccgg gaggaccctg 1020
cccctgacct aagcccaccc caaaggccaa actctccact ccctcagctc agacaccttc
1080 tctcctccca gatctgagta actcccaatc ttctctctgc agagtccaaa
tatggtcccc 1140 catgcccacc atgcccaggt aagccaaccc aggcctcgcc
ctccagctca aggcgggaca 1200 ggtgccctag agtagcctgc atccagggac
aggccccagc cgggtgctga cgcatccacc 1260 tccatctctt cctcagcacc
tgagttcctg gggggaccat cagtcttcct gttcccccca 1320 aaacccaagg
acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 1380
gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat
1440 aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt
ggtcagcgtc 1500 ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt
acaagtgcaa ggtctccaac 1560 aaaggcctcc cgtcctccat cgagaaaacc
atctccaaag ccaaaggtgg gacccacggg 1620 gtgcgagggc cacatggaca
gaggtcagct cggcccaccc tctgccctgg gagtgaccgc 1680 tgtgccaacc
tctgtcccta cagggcagcc ccgagagcca caggtgtaca ccctgccccc 1740
atcccaggag gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta
1800 ccccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca
actacaagac 1860 cacgcctccc gtgctggact ccgacggctc cttcttcctc
tacagcaggc taaccgtgga 1920 caagagcagg tggcaggagg ggaatgtctt
ctcatgctcc gtgatgcatg aggctctgca 1980 caaccactac acacagaaga
gcctctccct gtctctgggt aaatgagtgc cagggccggc 2040 aagcccccgc
tccccgggct ctcggggtcg cgcgaggatg cttggcacgt accccgtcta 2100
catacttccc aggcacccag catggaaata aagcacccac cactgccctg gctcgaattc
2160 32 94 DNA Artificial Sequence CDS 544gH1 T1 32 agtgtgaggt
gcaattggtc cagtcaggag cagaggttaa gaagcctggt gcttccgtca 60
aagtttcgtg taaggctagc ggctacaggt tcac 94 33 96 DNA Artificial
Sequence CDS 544gH1 T2 33 gtggcattaa tcccgggaat cagtacacta
catataaaag aaatctaaag ggcagagcaa 60 cgctgaccgc ggacacctcc
acaagcactg tctaca 96 34 95 DNA Artificial Sequence CDS 544gH1 T3 34
agagaaggct acggtaatta cggagcctgg ttcgcctact ggggccaggg taccctagtc
60 acagtctcct cagcttctac aaagggccca agaaa 95 35 94 DNA Artificial
Sequence CDS 544 gH1 B1 35 ggaccaattg cacctcacac tgcactccct
tgagaatgag tgccaggaac acgagagaga 60 atccgaagtc catggtggcg
gcaagctttt attc 94 36 97 DNA Artificial Sequence CDS 544gH1 B2 36
gattcccggg attaatgcca ccgatccatt ccaggccttg tcccggagcc tgcctgaccc
60 aatgaatcca ataatttgtg aacctgtagc cgctagc 97 37 93 DNA Artificial
Sequence CDS 544gH1 B3 37 cgtaattacc gtagccttct ctagtacaat
agtacactgc ggtgtcctcg gatctcagag 60 atgacagctc catgtagaca
gtgcttgtgg agg 93 38 21 DNA Artificial Sequence CDS 544gH1 F1 38
gaataaaagc ttgccgccac c 21 39 22 DNA Artificial Sequence CDS 544gH1
R1 39 tttcttgggc cctttgtaga ag 22 40 87 DNA Artificial Sequence CDS
544 gL1 T1 40 gcttcccggg gtgacgttca agtgacccag agcccatcca
gcctgagcgc atctgtagga 60 gaccgggtca ccatcacttg tagatcc 87 41 90 DNA
Artificial Sequence CDS 544 gL1 T2 41 tatctgcaca aaccaggtaa
agccccacaa ttgctcatct acggaatctc taacagattt 60 agtggtgtac
cagacaggtt cagcggttcc 90 42 91 DNA Artificial Sequence CDS 544gL1
T3 42 agatttcgcc acttattact gtttacaagg tacacatcag ccgtacacat
tcggtcaggg 60 tactaaagta gaaatcaaac gtacggcgtg c 91 43 88 DNA
ARTIFICIAL SEQUENCE CDS 544gL1 B1 43 gaacgtcacc ccgggaagca
ggaatccaga acaacagaag caccaacagc ctaacaggca 60 acttcatggt
ggcggcttcg aatcatcc 88 44 88 DNA Artificial Sequence CDS 544gL1 B2
44 ctttacctgg tttgtgcaga taccaagaca aaaaggtgtt cccataactg
tttgcaagac 60 tctgactgga tctacaagtg atggtgac 88 45 90 DNA
Artificial Sequence CDS 544gL1 B3 45 aacagtaata agtggcgaaa
tcttctggct ggagagacga gatcgtgagg gtgaaatcag 60 taccacttcc
ggaaccgctg aacctgtctg 90 46 20 DNA Artificial Sequence CDS 544gL1
F1 46 ggatgattcg aagccgccac 20 47 21 DNA ARTIFICIAL SEQUENCE CDS
544gL1 R1 47 gcacgccgta cgtttgattt c 21 48 339 DNA mouse CDS DNA
sequence of mouse monoclonal 5/44 VL 48 gatgttgtgg tgactcaaac
tccactctcc ctgcctgtca gctttggaga tcaagtttct 60 atctcttgca
ggtctagtca gagtcttgca aacagttatg ggaacacctt tttgtcttgg 120
tacctgcaca agcctggcca gtctccacag ctcctcatct atgggatttc caacagattt
180 tctggggtgc cagacaggtt cactggcagt ggttcaggga cagatttcac
actcaagatc 240 agcacaataa agcctgagga cttgggaatg tattactgct
tacaaggtac acatcagccg 300 tacacgttcg gaggggggac caagctggaa
ataaaacgt 339 49 363 DNA mouse CDS DNA sequence of mouse monoclonal
5/44 VH 49 gaggtccaac tgcagcagtc tgggactgta ctggcaaggc ctggggcttc
cgtgaagatg 60 tcctgcaagg cttctggcta caggtttacc aactactgga
ttcactgggt aaaacagagg 120 cctgggcagg gtctagaatg gattggtggt
attaatcctg gaaataatta tactacgtat 180 aagaggaact tgaagggcaa
ggccacactg actgcagtca catccgccag cactgcctac 240 atggacctca
gcagcctgac aagtgaggac tctgcggtct attactgtac aagagagggc 300
tatggtaact acggggcctg gtttgcttac tggggccagg ggactctggt caccgtctcc
360 tca 363 50 9 DNA Artificial Sequence PRIMER sequence within
oligonucleotide primer 50 gccgccacc 9 51 101 DNA Artificial
Sequence PRIMER 5' oligonucleotide primer 51 gcgcgcaagc ttgccgccac
catggacttc ggattctctc tcgtgttcct ggcactcatt 60 ctcaagggag
tgcagtgtga ggtgcagctc gtcgagtctg g 101
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