U.S. patent application number 17/643506 was filed with the patent office on 2022-05-26 for antibodies specific to fcrn.
This patent application is currently assigned to UCB BIOPHARMA SPRL. The applicant listed for this patent is UCB BIOPHARMA SPRL. Invention is credited to Paul Alan ATHERFOLD, Thomas Allen CESKA, Helene Margaret FINNEY, Lara KEVORKIAN, Kaushik SARKAR, Bryan John SMITH, Kerry Louise TYSON.
Application Number | 20220162311 17/643506 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-26 |
United States Patent
Application |
20220162311 |
Kind Code |
A1 |
ATHERFOLD; Paul Alan ; et
al. |
May 26, 2022 |
ANTIBODIES SPECIFIC TO FCRN
Abstract
The disclosure relates to antibodies specific to FcRn,
formulations comprising the same, use of each in therapy, processes
for expressing and optionally formulating said antibody, DNA
encoding the antibodies and hosts comprising said DNA.
Inventors: |
ATHERFOLD; Paul Alan;
(Slough, GB) ; CESKA; Thomas Allen; (Slough,
GB) ; FINNEY; Helene Margaret; (Slough, GB) ;
KEVORKIAN; Lara; (Slough, GB) ; SARKAR; Kaushik;
(Celltech, GB) ; SMITH; Bryan John; (Slough,
GB) ; TYSON; Kerry Louise; (Slough, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UCB BIOPHARMA SPRL |
Brussels |
|
BE |
|
|
Assignee: |
UCB BIOPHARMA SPRL
Brussels
BE
|
Appl. No.: |
17/643506 |
Filed: |
December 9, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16299407 |
Mar 12, 2019 |
11220547 |
|
|
17643506 |
|
|
|
|
15036209 |
May 12, 2016 |
10273302 |
|
|
PCT/EP2014/074409 |
Nov 12, 2014 |
|
|
|
16299407 |
|
|
|
|
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 47/68 20060101 A61K047/68; A61K 47/60 20060101
A61K047/60 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 12, 2013 |
GB |
1320066.2 |
Claims
1. A method of treating an autoimmune disease, the method
comprising administering to a patient a therapeutically effective
amount of an anti-FcRn antibody or FcRn-binding fragment thereof,
wherein the anti-FcRn antibody or FcRn-binding fragment thereof
comprises (i) a heavy chain or heavy chain fragment having a
variable region, and (ii) a complementary light chain or light
chain fragment having a variable region, wherein said heavy chain
variable region comprises three CDRs, wherein CDR H1 has the
sequence given in SEQ ID NO: 1, CDR H2 has the sequence given in
SEQ ID NO: 2, and CDR H3 has the sequence given in SEQ ID NO: 3,
and wherein said light chain variable region comprises three CDRs,
wherein CDR L1 has the sequence given in SEQ ID NO: 4, CDR L2 has
the sequence given in SEQ ID NO: 5 or SEQ ID NO: 7 and CDR L3 has
the sequence given in SEQ ID NO: 6.
2. The method of claim 1, wherein the heavy chain comprises the
sequence given in SEQ ID NO:12 and the light chain comprises the
sequence given in SEQ ID NO: 8.
3. The method of claim 1, wherein the anti-FcRn antibody or
FcRn-binding fragment thereof is humanized.
4. The method of claim 1, wherein the heavy chain comprises the
sequence given in SEQ ID NO: 25 and the light chain comprises the
sequence given in SEQ ID NO: 16.
5. The method of claim 1, wherein the heavy chain comprises the
sequence given in SEQ ID NO: 59 and the light chain comprises the
sequence given in SEQ ID NO: 51.
6. The method of claim 1, wherein the heavy chain comprises a
sequence having at least 80% identity to the sequence given in SEQ
ID NO: 25 and the light chain comprises a sequence having at least
80% identity to the sequence given in SEQ ID NO: 16.
7. The method of claim 1, wherein the anti-FcRn binding fragment
thereof is a scFv, Fv, Fab or Fab' fragment.
8. The method of claim 7, wherein the heavy chain comprises the
sequence given in SEQ ID NO: 33 and the light chain comprises the
sequence given in SEQ ID NO: 20.
9. The method of claim 7, wherein the FcRn-binding fragment is a
Fab fragment having a heavy chain comprising the sequence given in
SEQ ID NO: 29 and a light chain comprising the sequence given in
SEQ ID NO: 20.
10. The method of claim 7 wherein the FcRn-binding fragment is a
Fab' fragment having a heavy chain comprising the sequence given in
SEQ ID NO: 63 and a light chain comprising the sequence given in
SEQ ID NO: 55.
11. The method of claim 7 wherein the FcRn-binding fragment is a
Fab-dsFv fragment having a heavy chain comprising the sequence
given in SEQ ID NO: 42 and a light chain comprising the sequence
given in SEQ ID NO: 40.
12. The method of claim 1, wherein the antibody or binding fragment
is conjugated to a polymer comprising a starch, albumin or
polyethylene glycol.
13. The method of claim 12, wherein the polymer is PEG, comprising
a molecular weight in the range 5 to 50 kDa.
14. The method of claim 1 wherein the anti-FcRn antibody is a full
length antibody.
15. The method of claim 14 wherein the full length anti-FcRn
antibody is selected from the group consisting of an IgG1, IgG4,
and IgG4P.
16. The method according to claim 15, wherein the heavy chain
comprises the sequence given in SEQ ID NO: 37, SEQ ID NO:39 or SEQ
ID NO:73 and the light chain comprises the sequence given in SEQ ID
NO: 20.
17. A method comprising administering to a patient a
therapeutically effective amount of an anti-FcRn antibody or
FcRn-binding fragment thereof, wherein the anti-FcRn antibody or
FcRn-binding fragment thereof comprises (i) a heavy chain or heavy
chain fragment having a variable region, and (ii) a complementary
light chain or light chain fragment having a variable region,
wherein said heavy chain variable region comprises three CDRs,
wherein CDR H1 has the sequence given in SEQ ID NO: 1, CDR H2 has
the sequence given in SEQ ID NO: 2, and CDR H3 has the sequence
given in SEQ ID NO: 3, and said light chain variable region
comprises three CDRs, wherein CDR L1 has the sequence given in SEQ
ID NO: 4, CDR L2 has the sequence given in SEQ ID NO: 5 or SEQ ID
NO: 7 and CDR L3 has the sequence given in SEQ ID NO: 6; and the
patient has a disease selected from the group consisting of
myasthenia gravis, pemphigus vulgaris, neuromyelitis optica,
Guillain-Barre syndrome, lupus, idiopathic thrombocytopenic
purpura, thrombotic thrombocytopenic purpura, antiphospholipid
syndrome (APS), autoimmune urticarial, chronic inflammatory
demyelinating polyneuropathy (CIDP), Goodpasture's syndrome,
Graves' disease, hemolytic anemia, neutropenia, paraneoplastic
cerebellar degeneration, paraproteinemic polyneuropathies, primary
biliary cirrhosis, stiff person syndrome, vitiligo and warm
idiopathic haemolytic anaemia, or combinations thereof.
18. The method of claim 17, wherein the heavy chain comprises the
sequence given in SEQ ID NO: 25 and the light chain comprises the
sequence given in SEQ ID NO: 16.
19. The method of claim 17, wherein the FcRn-binding fragment is a
Fab fragment having a heavy chain comprising the sequence given in
SEQ ID NO: 29 and a light chain comprising the sequence given in
SEQ ID NO: 20.
20. A method of treating an autoimmune disease, the method
comprising administering to a patient a therapeutically effective
amount of a pharmaceutical composition comprising an anti-FcRn
antibody or FcRn-binding fragment thereof in combination with one
or more of a pharmaceutically acceptable excipient, diluent, or
carrier, wherein the anti-FcRn antibody or FcRn-binding fragment
thereof comprises (i) a heavy chain or heavy chain fragment having
a variable region, and (ii) a complementary light chain or light
chain fragment having a variable region, said heavy chain variable
region comprises three CDRs, wherein CDR H1 has the sequence given
in SEQ ID NO: 1, CDR H2 has the sequence given in SEQ ID NO: 2, and
CDR H3 has the sequence given in SEQ ID NO: 3, and said light chain
variable region comprises three CDRs, wherein CDR L1 has the
sequence given in SEQ ID NO: 4, CDR L2 has the sequence given in
SEQ ID NO: 5 or SEQ ID NO: 7 and CDR L3 has the sequence given in
SEQ ID NO: 6.
21. The method of claim 20, wherein the heavy chain comprises the
sequence given in SEQ ID NO: 25 and the light chain comprises the
sequence given in SEQ ID NO: 16.
22. The method of claim 20, wherein the FcRn-binding fragment is a
Fab fragment having a heavy chain comprising the sequence given in
SEQ ID NO: 29 and a light chain comprising the sequence given in
SEQ ID NO: 20.
23. The method of claim 20 wherein the pharmaceutical composition
comprises other active ingredients.
24. The method of claim 21 wherein the pharmaceutical composition
comprises other active ingredients.
25. The method of claim 22 wherein the pharmaceutical composition
comprises other active ingredients.
26. The method of claim 20 wherein the autoimmune disease is
selected from the group consisting of myasthenia gravis, pemphigus
vulgaris, neuromyelitis optica, Guillain-Barre syndrome, lupus,
idiopathic thrombocytopenic purpura or thrombotic thrombocytopenic
purpura, antiphospholipid syndrome (APS), autoimmune urticarial,
chronic inflammatory demyelinating polyneuropathy (CIDP),
Goodpasture's syndrome, Graves' disease, hemolytic anemia,
neutropenia, paraneoplastic cerebellar degeneration,
paraproteinemic polyneuropathies, primary biliary cirrhosis, stiff
person syndrome, vitiligo and warm idiopathic haemolytic anaemia,
or combinations thereof.
27. The method of claim 21 wherein the autoimmune disease is
selected from the group consisting of myasthenia gravis, pemphigus
vulgaris, neuromyelitis optica, Guillain-Barre syndrome, lupus,
idiopathic thrombocytopenic purpura or thrombotic thrombocytopenic
purpura, antiphospholipid syndrome (APS), autoimmune urticarial,
chronic inflammatory demyelinating polyneuropathy (CIDP),
Goodpasture's syndrome, Graves' disease, hemolytic anemia,
neutropenia, paraneoplastic cerebellar degeneration,
paraproteinemic polyneuropathies, primary biliary cirrhosis, stiff
person syndrome, vitiligo and warm idiopathic haemolytic anaemia,
or combinations thereof.
28. The method of claim 22 wherein the autoimmune disease is
selected from the group consisting of myasthenia gravis, pemphigus
vulgaris, neuromyelitis optica, Guillain-Barre syndrome, lupus,
idiopathic thrombocytopenic purpura or thrombotic thrombocytopenic
purpura, antiphospholipid syndrome (APS), autoimmune urticarial,
chronic inflammatory demyelinating polyneuropathy (CIDP),
Goodpasture's syndrome, Graves' disease, hemolytic anemia,
neutropenia, paraneoplastic cerebellar degeneration,
paraproteinemic polyneuropathies, primary biliary cirrhosis, stiff
person syndrome, vitiligo and warm idiopathic haemolytic anaemia,
or combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Divisional application under 35
U.S.C. .sctn. 120 of U.S. application Ser. No. 16/299,407, filed
Mar. 12, 2019, now U.S. Pat. No. 11,220,547, issued Jan. 11, 2022,
which is a Divisional application under 35 U.S.C. .sctn. 120 of
U.S. application Ser. No. 15/036,209, filed May 12, 2016, now U.S.
Pat. No. 10,273,302, issued Apr. 30, 2019, which is the U.S.
National Phase under 35 U.S.C. .sctn. 371 of PCT International
Application No. PCT/EP2014/074409, filed Nov. 12, 2014, which
claims priority to GB 1320066.2, filed Nov. 12, 2013, all of which
are incorporated herein by reference in their entirety.
[0002] The disclosure relates to antibodies specific to FcRn,
formulations comprising the same, use of each in therapy, processes
for expressing and optionally formulating said antibody, DNA
encoding the antibodies and hosts comprising said DNA.
[0003] FcRn is a non-covalent complex of membrane protein FcRn a
chain and (32 microglobulin (.beta.2M). In adult mammals FcRn plays
a key role in maintaining serum antibody levels by acting as a
receptor that binds and salvages antibodies of the IgG isotype. IgG
molecules are endocytosed by endothelial cells, and if they bind to
FcRn, are recycled transcytosed out into, for example circulation.
In contrast, IgG molecules that do not bind to FcRn enter the cells
and are targeted to the lysosomal pathway where they are degraded.
A variant IgG1 in which His435 is mutated to alanine results in the
selective loss of FcRn binding and a significantly reduced serum
half-life (Firan et al. 2001, International Immunology 13:993).
[0004] It is hypothesised that FcRn is a potential therapeutic
target for certain autoimmune disorders caused at least in part by
autoantibodies. The current treatment for certain such disorders
includes plasmapheresis. Sometimes the plasmapheresis is employed
along with immunosuppressive therapy for long-term management of
the disease. Plasma exchange offers the quickest short-term answer
to removing harmful autoantibodies. However, it may also be
desirable to suppress the production of autoantibodies by the
immune system, for example by the use of medications such as
prednisone, cyclophosphamide, cyclosporine, mycophenolate mofetil,
rituximab or a mixture of these.
[0005] Examples of diseases that can be treated with plasmapheresis
include: Guillain-Barre syndrome; Chronic inflammatory
demyelinating polyneuropathy; Goodpasture's syndrome;
hyperviscosity syndromes; cryoglobulinemia; paraproteinemia;
Waldenstrom macroglobulinemia; myasthenia gravis; thrombotic
thrombocytopenic purpura (TTP)/hemolytic uremic syndrome; Wegener's
granulomatosis; Lambert-Eaton Syndrome; antiphospholipid antibody
syndrome (APS or APLS); microscopic polyangiitis; recurrent focal
and segmental glomerulosclerosis in the transplanted kidney; HELLP
syndrome; PANDAS syndrome; Refsum disease; Behcet syndrome;
HIV-related neuropathy; Graves' disease in infants and neonates;
pemphigus vulgaris; multiple sclerosis, rhabdomyolysis and
alloimune diseases.
[0006] Plasmapheresis is sometimes used as a rescue therapy for
removal of Fc containing therapeutics, for example in emergencies
to reduced serious side effects.
[0007] Though plasmapheresis is helpful in certain medical
conditions there are potential risks and complications associated
with the therapy. Insertion of a rather large intravenous catheter
can lead to bleeding, lung puncture (depending on the site of
catheter insertion), and, if the catheter is left in too long, it
can lead to infection and/or damage to the veins giving limited
opportunity to repeat the procedure.
[0008] The procedure has further complications associated with it,
for example when a patient's blood is outside of the body passing
through the plasmapheresis instrument, the blood has a tendency to
clot. To reduce this tendency, in one common protocol, citrate is
infused while the blood is running through the circuit. Citrate
binds to calcium in the blood, calcium being essential for blood to
clot. Citrate is very effective in preventing blood from clotting;
however, its use can lead to life-threateningly low calcium levels.
This can be detected using the Chvostek's sign or Trousseau's sign.
To prevent this complication, calcium is infused intravenously
while the patient is undergoing the plasmapheresis; in addition,
calcium supplementation by mouth may also be given.
[0009] Other complications of the procedure include: hypotension;
potential exposure to blood products, with risk of transfusion
reactions or transfusion transmitted diseases, suppression of the
patient's immune system and bleeding or hematoma from needle
placement.
[0010] Additionally facilities that provide plasmapheresis are
limited and the procedure is very expensive.
[0011] An alternative to plasmapheresis is intravenous
immunoglobulin (IVIG), which is a blood product containing pooled
polyclonal IgG extracted from the plasma of over one thousand blood
donors. The therapy is administered intravenously and lasts in the
region of 2 weeks to 3 months.
[0012] Complications of the IVIG treatment include headaches,
dermatitis, viral infection from contamination of the therapeutic
product, for example HIV or hepatitis, pulmonary edema, allergic
reactions, acute renal failure, venous thrombosis and aseptic
meningitis.
[0013] Thus there is a significant unmet need for therapies for
autoimmune disorders which are less invasive and which expose the
patients to less medical complications.
[0014] Thus there is a significant unmet need for therapies for
immunological disorders and/or autoimmune disorders which are less
invasive and which expose the patients to less medical
complications.
[0015] Accordingly agents that block or reduce the binding of IgG
to FcRn may be useful in the treatment or prevention of such
autoimmune and inflammatory diseases. Anti-FcRn antibodies have
been described previously in WO2009/131702, WO2007/087289 and
WO2006/118772.
[0016] However, there remains a need for improved anti-FcRn
antibodies.
SUMMARY OF THE DISCLOSURE
[0017] Thus in one aspect there is provided an anti-FcRn antibody
or binding fragment thereof comprising a heavy chain or heavy chain
fragment having a variable region, wherein said variable region
comprises one, two or three CDRs independently selected from SEQ ID
NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, for example wherein CDR H1 is
SEQ ID NO: 1, CDR H2 is SEQ ID NO: 2 and/or CDR H3 is SEQ ID NO:
3.
[0018] Thus one embodiment CDR H1 is SEQ ID NO: 1 and CDR H2 is SEQ
ID NO: 2, or CDR H1 is SEQ ID NO: 1 and CDR H3 is SEQ ID NO: 3, or
CDR H2 is SEQ ID NO: 2 and CDR H3 is SEQ ID NO: 3.
[0019] In another aspect there is provided an antibody or fragment
comprising a sequence or combinations of sequences as defined
herein, for example a cognate pair variable region.
[0020] The antibodies of the disclosure block binding of IgG to
FcRn and are thought to be useful in reducing one or more
biological functions of FcRn, including reducing half-life of
circulating antibodies. This may be beneficial in that it allows
the patient to more rapidly clear antibodies, such as
autoantibodies. Accordingly antibodies of the disclosure reduce
binding of IgG to FcRn.
[0021] Importantly the antibodies of the present invention are able
to bind human FcRn, for example at both pH6 and pH7.4 with
comparable and high binding affinity. Advantageously therefore the
antibodies are able to continue to bind FcRn even within the
endosome, thereby maximising the blocking of FcRn binding to
IgG.
[0022] In one embodiment the antibodies or binding fragments
according to the present disclosure comprise a light chain or light
chain fragment having a variable region, for example comprising
one, two or three CDRs independently selected from SEQ ID NO: 4,
SEQ ID NO: 5 or SEQ ID NO: 7 and SEQ ID NO: 6, in particular
wherein CDR L1 is SEQ ID NO: 4, CDR L2 is SEQ ID NO: 5 or SEQ ID
NO: 7 and CDR L3 is SEQ ID NO: 6.
[0023] Thus one embodiment CDR L1 is SEQ ID NO: 4 and CDR L2 is SEQ
ID NO: 5 or SEQ ID NO: 7, or CDR L1 is SEQ ID NO: 1 and CDR L3 is
SEQ ID NO: 6, or CDR L2 is SEQ ID NO: 5 or SEQ ID NO: 7 and CDR L3
is SEQ ID NO:6.
[0024] In one embodiment the antibodies or binding fragments
according to the present disclosure comprise CDR sequences selected
from SEQ ID NOs: 1 to 7, for example wherein CDR H1 is SEQ ID NO:
1, CDR H2 is SEQ ID NO: 2, CDR H3 is SEQ ID NO: 3, CDR L1 is SEQ ID
NO: 4, CDR L2 is SEQ ID NO: 5 or SEQ ID NO: 7 and CDR L3 is SEQ ID
NO: 6.
[0025] Also provided is an antibody or binding fragment that binds
the same epitope as an antibody or binding fragment explicitly
disclosed herein. Accordingly there is provided an anti-FcRn
antibody or binding fragment thereof which binds an epitope of
human FcRn which comprises one, two, three, or four amino acids
selected from the group consisting of residues E115, W131, P132,
and E133 of human FcRn extracellular domain (SEQ ID NO: 48) and
wherein the anti-FcRn antibody or binding fragment thereof further
binds one or more (such as all) residues selected from the group
consisting of A81, G83, G84, K85, G86, P87, N113, L135, A136, and
Q139 and optionally further binds one or more residues selected
from the group consisting of L82, Y88, L112 and D130.
[0026] In one embodiment there is provided an antibody or binding
fragment that cross-blocks an antibody or binding fragment
explicitly disclosed herein to human FcRn, or is cross-blocked from
binding human FcRn by said antibody.
[0027] In one embodiment antibodies and binding fragments of the
present disclosure block binding of human IgG to human FcRn.
[0028] In one embodiment antibodies and binding fragments of the
present disclosure do not bind (32 microglobulin.
[0029] In one embodiment antibodies and binding fragments of the
present disclosure do not bind human (32 microglobulin
[0030] In one example antibodies and binding fragments of the
present disclosure do not reduce circulating albumin levels by more
than 50%, preferably by no more than 25%.
[0031] In one example antibodies and binding fragments of the
present disclosure do not reduce circulating albumin levels.
[0032] The disclosure also extends to a polynucleotide, such as
DNA, encoding an antibody or fragment as described herein, for
example where the DNA is incorporated into a vector.
[0033] Also provided is a host cell comprising said
polynucleotide.
[0034] Methods of expressing an antibody or fragment are provided
herein as are methods of conjugating an antibody or fragment to a
polymer, such as PEG.
[0035] The present disclosure also relates to pharmaceutical
compositions comprising said antibodies and fragments.
[0036] In one embodiment there is provided a method of treatment
comprising administering a therapeutically effective amount of an
antibody, fragment or composition as described herein.
[0037] The present disclosure also extends to an antibody, fragment
or composition according to the present disclosure for use in
treatment, particularly in the treatment of an immunological and/or
autoimmune disorder.
[0038] Thus the present disclosure provides antibodies, fragments
thereof and methods for removal of pathogenic IgG, which is
achieved by accelerating the body's natural mechanism for
catabolising IgG.
[0039] In essence the antibodies and fragments according to the
disclosure block the system that recycles IgG in the body.
[0040] The present therapy is likely to provide a replacement or
supplement for certain diseases where plasmapheresis is a therapy
or IVIg therapy, which is advantageous for patients.
BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1 Shows % hIgG in transgenic mice determined by
LC-MS/MS
[0042] FIG. 1A shows the effect of 1638 IgG4P format on the
concentration of human IVIg in serum of human FcRn-transgenic
mice.
[0043] FIG. 1B shows the effect of 1638 FabFv and Fab'PEG formats
on the concentration of human IVIg in human FcRn-transgenic
mice
[0044] FIG. 1C shows the pharmacokinetics of 1638 IgG4P format in
human FcRn-transgenic mice.
[0045] FIG. 1D shows the pharmacokinetics of 1638 FabFv and Fab'PEG
formats in human FcRn-transgenic mice
[0046] FIG. 1E The effect of 1638 FabFv and Fab'PEG formats on the
concentration of serum albumin in human FcRn-transgenic mice.
[0047] FIG. 1F The effect of 1638 IgG4P format on the concentration
of serum albumin in human FcRn-transgenic mice.
[0048] FIG. 2 shows representative binding curves for
CA170_1638.g49 IgG4. The mean K.sub.D values (n=3) were 0.20 nM in
neutral buffer, & 0.22 nM in acidic buffer, respectively
[0049] FIG. 3 shows CA170_1638.g49 IgG4 inhibits IgG recycling in
MDCK II clone 15 cells
[0050] FIG. 4 shows CA170_1638.g49 IgG4 inhibits IgG transcytosis
in MDCK II clone 15 cells.
[0051] FIG. 5 shows CA170_1638.g49 FabFv inhibits IgG transcytosis
in MDCK II clone 15 cells.
[0052] FIG. 6 shows representative binding curves for
CA170_1638.g49 IgG4. The mean K.sub.D values (n=3) were 0.3 in
neutral buffer, and 0.43 in acidic buffer, respectively (see Table
2).
[0053] FIG. 7 shows CA170_1638 CDR sequences
[0054] FIGS. 8A-L Antibody sequences (nucleic and amino acid)
according to the present disclosure: (A) SEQ ID NOs: 8-15; (B) SEQ
ID NOs: 16-21; (C) SEQ ID NOs: 22-27; (D) SEQ ID NOs: 28-32; (E)
SEQ ID NOs: 33-37; (F) SEQ ID NOs: 38-40; (G) SEQ ID NOs: 41-44;
(H) SEQ ID NOs: 45-52; (I) SEQ ID NOs: 53-57; (J) SEQ ID NOs:
58-63; (K) SEQ ID NOs: 64-66 and 72; (L) SEQ ID NOs: 73-74.
[0055] FIG. 9A Humanisation of antibody 1638.g49
[0056] FIG. 9B Humanisation of antibody 1638.g49
DETAILS OF THE DISCLOSURE
[0057] FcRn as employed herein refers to the non-covalent complex
between the human IgG receptor alpha chain, also known as the
neonatal Fc receptor, the amino acid sequence of which is in
UniProt under number P55899, the extracellular domain of which is
provided in FIG. 8 (SEQ ID NO:48), together with human (32
microglobulin (.beta.2M), the amino acid sequence of which is in
UniProt under number P61769 (provided herein with signal peptide
(SEQ ID NO:50), without signal peptide (SEQ ID NO:72)).
[0058] Antibody molecule as employed herein refers to an antibody
or binding fragment thereof.
[0059] The term `antibody` as used herein generally relates to
intact (whole) antibodies i.e. comprising the elements of two heavy
chains and two light chains. The antibody may comprise further
additional binding domains, for example as per the molecule DVD-Ig
as disclosed in WO 2007/024715, or the so-called (FabFv).sub.2Fc
described in WO2011/030107. Thus antibody as employed herein
includes bi, tri or tetra-valent full length antibodies.
[0060] Binding fragments of antibodies include single chain
antibodies (i.e. a full length heavy chain and light chain); Fab,
modified Fab, Fab', modified Fab', F(ab').sub.2, Fv, Fab-Fv,
Fab-dsFv, single domain antibodies (e.g. VH or VL or VHH), scFv,
dsscFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies,
tribodies, triabodies, tetrabodies and epitope-binding fragments of
any of the above (see for example Holliger and Hudson, 2005, Nature
Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design
Reviews--Online 2(3), 209-217). The methods for creating and
manufacturing these antibody fragments are well known in the art
(see for example Verma et al., 1998, Journal of Immunological
Methods, 216, 165-181). The Fab-Fv format was first disclosed in
WO2009/040562 and the disulphide stabilised versions thereof, the
Fab-dsFv was first disclosed in WO2010/035012. Other antibody
fragments for use in the present invention include the Fab and Fab'
fragments described in International patent applications
WO2005/003169, WO2005/003170 and WO2005/003171. Multi-valent
antibodies may comprise multiple specificities e.g. bispecific or
may be monospecific (see for example WO92/22583 and WO05/113605).
One such example of the latter is a Tri-Fab (or TFM) as described
in WO92/22583.
[0061] In one embodiment there is provided a Fab fragment.
[0062] In one embodiment there is provided a Fab' fragment.
[0063] A typical Fab' molecule comprises a heavy and a light chain
pair in which the heavy chain comprises a variable region V.sub.H,
a constant domain C.sub.H1 and a natural or modified hinge region
and the light chain comprises a variable region V.sub.L and a
constant domain C.sub.L.
[0064] In one embodiment there is provided a dimer of a Fab'
according to the present disclosure to create a F(ab').sub.2 for
example dimerisation may be through the hinge.
[0065] In one embodiment the antibody or binding fragment thereof
comprises a binding domain. A binding domain will generally
comprises 6 CDRs, three from a heavy chain and three from a light
chain. In one embodiment the CDRs are in a framework and together
form a variable region. Thus in one embodiment an antibody or
binding fragment comprises a binding domain specific for antigen
comprising a light chain variable region and a heavy chain variable
region.
[0066] It will be appreciated that one or more (for example 1, 2, 3
or 4) amino acid substitutions, additions and/or deletions may be
made to the CDRs or other sequences (e.g variable domains) provided
by the present invention without significantly altering the ability
of the antibody to bind to FcRn. The effect of any amino acid
substitutions, additions and/or deletions can be readily tested by
one skilled in the art, for example by using the methods described
herein, in particular in the Examples, to determine FcRn
binding/blocking.
[0067] In one or more (for example 1, 2, 3 or 4) amino acid
substitutions, additions and/or deletions may be made to the
framework region employed in the antibody or fragment provided by
the present invention and wherein binding affinity to FcRn is
retained or increased.
[0068] The residues in antibody 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.
[0069] 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 complementarity determining region (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.
[0070] 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 system. However,
according to Chothia (Chothia, C. and Lesk, A. M. J. Mol. Biol.,
196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from
residue 26 to residue 32. Thus unless indicated otherwise `CDR-H1`
as employed herein is intended to refer to residues 26 to 35, as
described by a combination of the Kabat numbering system and
Chothia's topological loop definition.
[0071] 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 system.
[0072] Antibodies and fragments of the present disclosure block
FcRn and may thereby prevent it functioning in the recycling of
IgG. Blocking as employed herein refers to physically blocking such
as occluding the receptor but will also include where the antibody
or fragments binds an epitope that causes, for example a
conformational change which means that the natural ligand to the
receptor no longer binds. Antibody molecules of the present
invention bind to FcRn and thereby decrease or prevent (e.g.
inhibit) FcRn binding to an IgG constant region.
[0073] In one embodiment the antibody or fragment thereof binds
FcRn competitively with respect to IgG.
[0074] In one example the antibody or binding fragment thereof
functions as a competitive inhibitor of human FcRn binding to human
IgG. In one example the antibody or binding fragment thereof binds
to the IgG binding site on FcRn. In one example the antibody blocks
the IgG binding site. In one example the antibody or binding
fragment thereof does not bind .beta.2M.
[0075] Antibodies for use in the present disclosure may be obtained
using any suitable method known in the art. The FcRn
polypeptide/protein including fusion proteins, cells (recombinantly
or naturally) expressing the polypeptide (such as activated T
cells) can be used to produce antibodies which specifically
recognise FcRn, alone or incombination with .beta.2M. The
polypeptide may be the `mature` polypeptide or a biologically
active fragment or derivative thereof. The human protein is
registered in Swiss-Prot under the number P55899. The extracellular
domain of human FcRn alpha chain is provided in SEQ ID NO: 48. The
sequence of mature human .beta.2M is provided in SEQ ID NO: 72.
[0076] In one embodiment the antigen is a mutant form of FcRn which
is engineered to present FcRn on the surface of a cell, such that
there is little or no dynamic processing where the FcRn is
internalised in the cell, for example this can be achieved by
making a mutation in the cytoplasmic tail of the FcRn alpha chain,
wherein di-leucine is mutated to di-alanine as described in Ober et
al 2001 Int. Immunol. 13, 1551-1559.
[0077] Polypeptides, for use to immunize a host, may be prepared by
processes well known in the art from genetically engineered host
cells comprising expression systems or they may be recovered from
natural biological sources. In the present application, the term
"polypeptides" includes peptides, polypeptides and proteins. These
are used interchangeably unless otherwise specified. The FcRn
polypeptide may in some instances be part of a larger protein such
as a fusion protein for example fused to an affinity tag or
similar.
[0078] Antibodies generated against the FcRn polypeptide may be
obtained, where immunisation of an animal is necessary, by
administering the polypeptides to an animal, preferably a non-human
animal, using well-known and routine protocols, see for example
Handbook of Experimental Immunology, D. M. Weir (ed.), Vol 4,
Blackwell Scientific Publishers, Oxford, England, 1986). Many
warm-blooded animals, such as rabbits, mice, rats, sheep, cows,
camels or pigs may be immunized. However, mice, rabbits, pigs and
rats are generally most suitable.
[0079] Monoclonal antibodies may be prepared by any method known in
the art such as the hybridoma technique (Kohler & Milstein,
1975, Nature, 256:495-497), the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72)
and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies
and Cancer Therapy, pp 77-96, Alan R Liss, Inc., 1985).
[0080] Antibodies for use in the invention may also be generated
using single lymphocyte antibody methods by cloning and expressing
immunoglobulin variable region cDNAs generated from single
lymphocytes selected for the production of specific antibodies by,
for example, the methods described by Babcook, J. et al., 1996,
Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO92/02551;
WO2004/051268 and International Patent Application number
WO2004/106377.
[0081] Screening for antibodies can be performed using assays to
measure binding to human FcRn and/or assays to measure the ability
to block IgG binding to the receptor. An example of a binding assay
is an ELISA, in particular, using a fusion protein of human FcRn
and human Fc, which is immobilized on plates, and employing a
secondary antibody to detect anti-FcRn antibody bound to the fusion
protein. Examples of suitable antagonistic and blocking assays are
described herein below.
[0082] Specific as employed herein is intended to refer to an
antibody that only recognises the antigen to which it is specific
or an antibody that has significantly higher binding affinity to
the antigen to which it is specific compared to binding to antigens
to which it is non-specific, for example at least 5, 6, 7, 8, 9, 10
times higher binding affinity. Binding affinity may be measured by
techniques such as BIAcore as described herein below. In one
example the antibody of the present invention does not bind (32
microglobulin (.beta.2M). In one example the antibody of the
present invention binds cynomolgus FcRn. In one example the
antibody of the present invention does not bind rat or mouse
FcRn.
[0083] The amino acid sequences and the polynucleotide sequences of
certain antibodies according to the present disclosure are provided
and form an aspect of the invention.
[0084] In one embodiment the antibodies or binding fragments
according to the present disclosure are fully human, for example
prepared from a phage library or similar.
[0085] In one example the antibodies are rodent, such as rat
derived and comprise the light chain variable domain sequence given
in SEQ ID NO:8 and the heavy chain variable domain sequence given
in SEQ ID NO:12.
[0086] In one embodiment the antibody or fragments according to the
disclosure are humanised.
[0087] Humanised antibodies (which include CDR-grafted antibodies)
are antibody molecules having one or more complementarity
determining regions (CDRs) from a non-human species and a framework
region from a human immunoglobulin molecule (see, e.g. U.S. Pat.
No. 5,585,089; WO91/09967). It will be appreciated that it may only
be necessary to transfer the specificity determining residues of
the CDRs rather than the entire CDR (see for example, Kashmiri et
al., 2005, Methods, 36, 25-34). Humanised antibodies may optionally
further comprise one or more framework residues derived from the
non-human species from which the CDRs were derived. The latter are
often referred to as donor residues.
[0088] Thus in one embodiment as used herein, the term `humanised
antibody molecule` refers to an antibody molecule wherein the heavy
and/or light chain contains one or more CDRs (including, if
desired, one or more modified CDRs) from a donor antibody (e.g. a
non-human antibody such as a murine monoclonal antibody) grafted
into a heavy and/or light chain variable region framework of an
acceptor antibody (e.g. a human antibody) optionally further
comprising one or more framework residues derived from the
non-human species from which the CDRs were derived (donor
residues). For a review, see Vaughan et al, Nature Biotechnology,
16, 535-539, 1998. In one embodiment rather than the entire CDR
being transferred, only one or more of the specificity determining
residues from any one of the CDRs described herein above are
transferred to the human antibody framework (see for example,
Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment only
the specificity determining residues from one or more of the CDRs
described herein above are transferred to the human antibody
framework. In another embodiment only the specificity determining
residues from each of the CDRs described herein above are
transferred to the human antibody framework.
[0089] When the CDRs or specificity determining residues 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.
[0090] Suitably, the humanised antibody according to the present
invention has a variable domain comprising human acceptor framework
regions as well as one or more of the CDRs provided specifically
herein. Thus, provided in one embodiment is blocking humanised
antibody which binds human FcRn wherein the variable domain
comprises human acceptor framework regions and non-human donor
CDRs.
[0091] 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; these are
available at http://www.imgt.org/
[0092] In a humanised antibody of the present invention, 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.
[0093] One such suitable framework region for the heavy chain of
the humanised antibody of the present invention is derived from the
human sub-group VH3 sequence IGHV3-7 together with JH3 (SEQ ID NO:
46 and 47).
[0094] Accordingly, in one example there is provided a humanised
antibody comprising the sequence given in SEQ ID NO: 1 for CDR-H1,
the sequence given in SEQ ID NO: 2 for CDR-H2 and the sequence
given in SEQ ID NO: 3 for CDRH3, wherein the heavy chain framework
region is derived from the human subgroup VH3 sequence IGHV3-7
together with JH3.
[0095] The sequence of human JH3 is as follows: (DAFDV)WGQGTMVTVS
(SEQ ID No: 69). The DAFDV (SEQ ID NO: 70) motif is part of CDR-H3
and is not part of framework 4 (Ravetch, J V. et al., 1981, Cell,
27, 583-591).
[0096] In one example the heavy chain variable domain of the
antibody comprises the sequence given in SEQ ID NO: 25 or 59, such
as 25.
[0097] A suitable framework region for the light chain of the
humanised antibody of the present invention is derived from the
human sub-group VK1 sequence IGKV1-27 sequence together with JK4
(SEQ ID NO: 44 and 45).
[0098] Accordingly, in one example there is provided a humanised
antibody comprising the sequence given in SEQ ID NO: 4 for CDR-L1,
the sequence given in SEQ ID NO: 5 or SEQ ID NO: 7 for CDR-L2 and
the sequence given in SEQ ID NO: 6 for CDRL3, wherein the light
chain framework region is derived from the human sub-group VK1
sequence IGKV1-27 together with JK4.
[0099] The JK4 sequence is as follows: (LT)FGGGTKVEIK (Seq ID No:
71). The LT motif is part of CDR-L3 and is not part of framework 4
(Hieter, P A., et al., 1982, J. Biol. Chem., 257, 1516-1522).
[0100] In one example the light chain variable domain of the
antibody comprises the sequence given in SEQ ID NO: 16 or 51, such
as 16.
[0101] In a humanised 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
(see Reichmann et al., 1998, Nature, 332, 323-324). 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 WO91/09967.
[0102] Thus in one embodiment 1, 2, 3, 4, or 5 residues in the
framework are replaced with an alternative amino acid residue.
[0103] Accordingly, in one example there is provided a humanised
antibody, wherein at least the residues at each of positions 48 and
78 of the variable domain of the heavy chain (Kabat numbering) are
donor residues, see for example the sequence given in SEQ ID NO:
25.
[0104] In one embodiment residue 48 of the heavy chain variable
domain is replaced with an alternative amino acid, for example
valine.
[0105] In one embodiment residue 78 of the heavy chain variable
domain is replaced with an alternative amino acid, for example
leucine.
[0106] In one embodiment residue 48 is valine and residue 78 is
leucine in the humanised heavy chain variable region according to
the present disclosure.
[0107] Accordingly, in one example there is provided a humanised
antibody, wherein at least the residues at each of positions 70 and
71 of the variable domain of the light chain (Kabat numbering) are
donor residues, see for example the sequence given in SEQ ID NO:
16.
[0108] In one embodiment residue 70 of the light chain variable
domain is replaced with an alternative amino acid, for example
aspartic acid.
[0109] In one embodiment residue 71 of the light chain variable
domain is replaced with an alternative amino acid, for example
phenylalanine.
[0110] In one embodiment residue 70 is aspartic acid and residue 71
is phenylalanine in the humanised light chain variable region
according to the present disclosure.
[0111] In one embodiment the disclosure provides an antibody
sequence which is 80% similar or identical to a sequence disclosed
herein, for example 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%
or 99% over part or whole of the relevant sequence, for example a
variable domain sequence, a CDR sequence or a variable domain
sequence, excluding the CDRs. In one embodiment the relevant
sequence is SEQ ID NO: 16 or 51. In one embodiment the relevant
sequence is SEQ ID NO: 25 or 59.
[0112] In one embodiment, the present invention provides an
antibody molecule which binds human FcRn comprising a heavy chain,
wherein the variable domain of the heavy chain comprises a sequence
having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%,
98% or 99% identity or similarity to a sequence herein, for example
the sequence given in SEQ ID NO: 25 or 59, such as 25.
[0113] In one embodiment, the present invention provides an
antibody molecule which binds human FcRn comprising a light chain,
wherein the variable domain of the light chain comprises a sequence
having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%,
98% or 99% identity or similarity to the sequence given in SEQ ID
NO: 16 or 51, such as 16.
[0114] In one embodiment the present invention provides an antibody
molecule which binds human FcRn wherein the antibody has a heavy
chain variable domain which is at least 90%, 91%, 92%, 93%, 94%,
95% 96%, 97%, 98% or 99% similar or identical to a sequence given
herein, for example the sequence given in SEQ ID NO: 25 but wherein
the antibody molecule has the sequence given in SEQ ID NO: 1 for
CDR-H1, the sequence given in SEQ ID NO: 2 for CDR-H2 and the
sequence given in SEQ ID NO: 3 for CDR-H3.
[0115] In one embodiment the present invention provides an antibody
molecule which binds human FcRn wherein the antibody has a light
chain variable domain which is at least 90%, 91%, 92%, 93%, 94%,
95% 96%, 97%, 98% or 99% similar or identical to a sequence given
herein, for example the sequence in SEQ ID NO:16 but wherein the
antibody molecule has the sequence given in SEQ ID NO: 4 for
CDR-L1, the sequence given in SEQ ID NO: 5 or SEQ ID NO: 7 for
CDR-L2 and the sequence given in SEQ ID NO: 6 for CDR-L3.
[0116] In one embodiment the present invention provides an antibody
molecule which binds human FcRn wherein the antibody has a heavy
chain variable domain which is at least 90%, 91%, 92%, 93%, 94%,
95% 96%, 97%, 98% or 99% similar or identical to a sequence given
herein, for example the sequence given in SEQ ID NO: 25 and a light
chain variable domain which is at least 90%, 91%, 92%, 93%, 94%,
95% 96%, 97%, 98% or 99% similar or identical to a sequence given
herein, for example the sequence given in SEQ ID NO:16 but wherein
the antibody molecule has the sequence given in SEQ ID NO: 1 for
CDR-H1, the sequence given in SEQ ID NO: 2 for CDR-H2, the sequence
given in SEQ ID NO: 3 for CDR-H3, the sequence given in SEQ ID NO:
4 for CDR-L1, the sequence given in SEQ ID NO: 5 or SEQ ID NO: 7
for CDR-L2 and the sequence given in SEQ ID NO: 6 for CDR-L3.
[0117] "Identity", as used herein, indicates that at any particular
position in the aligned sequences, the amino acid residue is
identical between the sequences. "Similarity", as used herein,
indicates that, at any particular position in the aligned
sequences, the amino acid residue is of a similar type between the
sequences. For example, leucine may be substituted for isoleucine
or valine. Other amino acids which can often be substituted for one
another include but are not limited to: [0118] phenylalanine,
tyrosine and tryptophan (amino acids having aromatic side chains);
[0119] lysine, arginine and histidine (amino acids having basic
side chains); [0120] aspartate and glutamate (amino acids having
acidic side chains); [0121] asparagine and glutamine (amino acids
having amide side chains); and [0122] cysteine and methionine
(amino acids having sulphur-containing side chains). Degrees of
identity and similarity can be readily calculated (Computational
Molecular Biology, Lesk, A. M., ed., Oxford University Press, New
York, 1988; Biocomputing. Informatics and Genome Projects, Smith,
D. W., ed., Academic Press, New York, 1993; Computer Analysis of
Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular
Biology, von Heinje, G., Academic Press, 1987, Sequence Analysis
Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New
York, 1991, the BLAST.TM. software available from NCBI (Altschul,
S. F. et al., 1990, J. Mol. Biol. 215:403-410; Gish, W. &
States, D. J. 1993, Nature Genet. 3:266-272. Madden, T. L. et al.,
1996, Meth. Enzymol. 266:131-141; Altschul, S. F. et al., 1997,
Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, T. L.
1997, Genome Res. 7:649-656).
[0123] The antibody molecules of the present invention may comprise
a complete antibody molecule having full length heavy and light
chains or a fragment thereof and may be, but are not limited to
Fab, modified Fab, Fab', modified Fab', F(ab').sub.2, Fv, single
domain antibodies (e.g. VH or VL or VHH), scFv, dsscFv, bi, tri or
tetra-valent antibodies, Bis-scFv, diabodies, triabodies,
tetrabodies and epitope-binding fragments of any of the above (see
for example Holliger and Hudson, 2005, Nature Biotech.
23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online
2(3), 209-217). The methods for creating and manufacturing these
antibody fragments are well known in the art (see for example Verma
et al., 1998, Journal of Immunological Methods, 216, 165-181).
Other antibody fragments for use in the present invention include
the Fab and Fab' fragments described in International patent
applications WO2005/003169, WO2005/003170 and WO2005/003171.
Multi-valent antibodies may comprise multiple specificities e.g
bispecific or may be monospecific (see for example WO 92/22853,
WO05/113605, WO2009/040562 and WO2010/035012).
[0124] In one embodiment the antibody molecule of the present
disclosure is an antibody Fab fragment comprising the variable
regions shown in SEQ ID NOs: 16 and 25, for example for the light
and heavy chain respectively. In one embodiment the antibody
molecule has a light chain comprising the sequence given in SEQ ID
NO: 20 and a heavy chain comprising the sequence given in SEQ ID
NO: 29.
[0125] In one embodiment the antibody molecule of the present
disclosure is an antibody Fab fragment comprising the variable
regions shown in SEQ ID NOs: 51 and 59, for example for the light
and heavy chain respectively.
[0126] In one embodiment the antibody molecule of the present
disclosure is an antibody Fab or Fab' fragment comprising the
variable regions shown in SEQ ID NOs: 16 and 25, for example for
the light and heavy chain respectively. In one embodiment the
antibody molecule has a light chain comprising the sequence given
in SEQ ID NO: 20 and a heavy chain comprising the sequence given in
SEQ ID NO: 29 (Fab) or SEQ ID NO: 33 (Fab').
[0127] In one embodiment the antibody molecule of the present
disclosure is an antibody Fab' fragment comprising the variable
regions shown in SEQ ID NOs: 51 and 59, for example for the light
and heavy chain respectively. In one embodiment the antibody
molecule has a light chain comprising the sequence given in SEQ ID
NO: 55 and a heavy chain comprising the sequence given in SEQ ID
NO: 63.
[0128] In one embodiment the antibody molecule of the present
disclosure is a full length IgG1 antibody comprising the variable
regions shown in SEQ ID NOs: 16 and 25, for example for the light
and heavy chain respectively. In one embodiment the antibody
molecule has a light chain comprising the sequence given in SEQ ID
NO: 20 and a heavy chain comprising the sequence given in SEQ ID
NO: 73.
[0129] In one embodiment the antibody molecule of the present
disclosure is a full length IgG1 comprising the variable regions
shown in SEQ ID NOs: 51 and 59.
[0130] In one embodiment the antibody molecule of the present
disclosure is a full length IgG4 format comprising the variable
regions shown in SEQ ID NOs: 16 and 25, for example for the light
and heavy chain respectively. In one embodiment the antibody
molecule has a light chain comprising the sequence given in SEQ ID
NO: 20 and a heavy chain comprising the variable region sequence
given in SEQ ID NO: 25.
[0131] In one embodiment the antibody molecule of the present
disclosure is a full length IgG4 format comprising the variable
regions shown in SEQ ID NOs: 51 and 59, for example for the light
and heavy chain respectively. In one embodiment the antibody
molecule has a light chain comprising the sequence given in SEQ ID
NO: 55 and a heavy chain comprising the variable region sequence
given in SEQ ID NO: 59.
[0132] In one embodiment the antibody molecule of the present
disclosure is a full length IgG4P format comprising the variable
regions shown in SEQ ID NOs: 16 and 25, for example for the light
and heavy chain respectively. In one embodiment the antibody
molecule has a light chain comprising the sequence given in SEQ ID
NO: 20 and a heavy chain comprising the sequence given in SEQ ID
NO: 37 or SEQ ID NO: 39.
[0133] In one embodiment the antibody molecule of the present
disclosure is a full length IgG4P format comprising the variable
regions shown in SEQ ID NOs: 51 and 59, for example for the light
and heavy chain respectively. In one embodiment the antibody
molecule has a light chain comprising the sequence given in SEQ ID
NO: 55 and a heavy chain comprising the variable region sequence
given in SEQ ID NO: 59.
[0134] IgG4P as employed herein is a mutation of the wild-type IgG4
isotype where amino acid 241 is replaced by proline, see for
example where serine at position 241 has been changed to proline as
described in Angal et al., Molecular Immunology, 1993, 30 (1),
105-108.
[0135] In one embodiment the antibody according to the present
disclosure is provided as an FcRn binding antibody fusion protein
which comprises an immunoglobulin moiety, for example a Fab or Fab'
fragment, and one or two single domain antibodies (dAb) linked
directly or indirectly thereto, for example as described in
WO2009/040562, WO2010035012, WO2011/030107, WO2011/061492 and
WO2011/086091 all incorporated herein by reference.
[0136] In one embodiment the fusion protein comprises two domain
antibodies, for example as a variable heavy (VH) and variable light
(VL) pairing, optionally linked by a disulphide bond.
[0137] In one embodiment the Fab or Fab' element of the fusion
protein has the same or similar specificity to the single domain
antibody or antibodies. In one embodiment the Fab or Fab' has a
different specificity to the single domain antibody or antibodies,
that is to say the fusion protein is multivalent. In one embodiment
a multivalent fusion protein according to the present invention has
an albumin binding site, for example a VH/VL pair therein provides
an albumin binding site. In one such embodiment the heavy chain
comprises the sequence given in SEQ ID NO: 42 and the light chain
comprises the sequence given in SEQ ID NO: 40.
[0138] In one embodiment the Fab or Fab' according to the present
disclosure is conjugated to a PEG molecule or human serum
albumin.
[0139] CA170_01638g49 and 1638.g49 are employed inchangeably herein
and are used to refer to a specific pair of antibody variable
regions which may be used in a number of different formats. These
variable regions are the heavy chain sequence given in SEQ ID NO:
25 and the light chain sequence given in SEQ ID NO: 16.
[0140] CA170_01638g28 and 1638.g28 are employed inchangeably herein
and are used to refer to a specific pair of antibody variable
regions which may be used in a number of different formats. These
variable regions are the heavy chain sequence given in SEQ ID NO:
59 and the light chain sequence given in SEQ ID NO: 51.
[0141] The constant region domains of the antibody molecule of the
present invention, if present, may be selected having regard to the
proposed function of the antibody molecule, and in particular the
effector functions which may be required. For example, the constant
region domains may be human IgA, IgD, IgE, IgG or IgM domains. In
particular, human IgG constant region domains may be used,
especially of the IgG1 and IgG3 isotypes when the antibody molecule
is intended for therapeutic uses and antibody effector functions
are required. Alternatively, IgG2 and IgG4 isotypes may be used
when the antibody molecule is intended for therapeutic purposes and
antibody effector functions are not required. It will be
appreciated that sequence variants of these constant region domains
may also be used. For example IgG4 molecules in which the serine at
position 241 has been changed to proline as described in Angal et
al., Molecular Immunology, 1993, 30 (1), 105-108 may be used. It
will also be understood by one skilled in the art that antibodies
may undergo a variety of posttranslational modifications. The type
and extent of these modifications often depends on the host cell
line used to express the antibody as well as the culture
conditions. Such modifications may include variations in
glycosylation, methionine oxidation, diketopiperazine formation,
aspartate isomerization and asparagine deamidation. A frequent
modification is the loss of a carboxy-terminal basic residue (such
as lysine or arginine) due to the action of carboxypeptidases (as
described in Harris, R J. Journal of Chromatography 705:129-134,
1995). Accordingly, the C-terminal lysine of the antibody heavy
chain may be absent.
[0142] In one embodiment the antibody heavy chain comprises a CH1
domain and the antibody light chain comprises a CL domain, either
kappa or lambda.
[0143] In one embodiment the light chain has the sequence given in
SEQ ID NO: 20 and the heavy chain has the sequence given in SEQ ID
NO: 29.
[0144] In one embodiment the light chain has the sequence given in
SEQ ID NO: 20 and the heavy chain has the sequence given in SEQ ID
NO: 33.
[0145] In one embodiment the light chain has the sequence given in
SEQ ID NO: 20 and the heavy chain has the sequence given in SEQ ID
NO: 37.
[0146] In one embodiment the light chain has the sequence given in
SEQ ID NO: 20 and the heavy chain has the sequence given in SEQ ID
NO: 74.
[0147] In one embodiment a C-terminal amino acid from the antibody
molecule is cleaved during post-translation modifications.
[0148] In one embodiment an N-terminal amino acid from the antibody
molecule is cleaved during post-translation modifications.
[0149] Also provided by the present invention is a specific region
or epitope of human FcRn which is bound by an antibody provided by
the present invention, in particular an antibody comprising the
heavy chain sequence gH33 (SEQ ID NO: 25) and/or the light chain
sequence gL7 (SEQ ID NO: 16 or an antibody comprising the heavy
chain sequence gH2 (SEQ ID NO: 59) and the light chain sequence gL2
(SEQ ID NO: 51).
[0150] This specific region or epitope of the human FcRn
polypeptide can be identified by any suitable epitope mapping
method known in the art in combination with any one of the
antibodies provided by the present invention. Examples of such
methods include screening peptides of varying lengths derived from
FcRn for binding to the antibody of the present invention with the
smallest fragment that can specifically bind to the antibody
containing the sequence of the epitope recognised by the antibody.
The FcRn peptides may be produced synthetically or by proteolytic
digestion of the FcRn polypeptide. Peptides that bind the antibody
can be identified by, for example, mass spectrometric analysis. In
another example, NMR spectroscopy or X-ray crystallography can be
used to identify the epitope bound by an antibody of the present
invention. In one example where X-ray crystallography is used, the
epitope is determined as those residues on the FcRn polypeptide
which are within 4 .ANG. of the antibody. In one example the
epitope is determined as those residues on the FcRn polypeptide
which are within 5 .ANG. of the antibody. Once identified, the
epitopic fragment which binds an antibody of the present invention
can be used, if required, as an immunogen to obtain additional
antibodies which bind the same epitope.
[0151] In one embodiment the antibody of the present disclosure
binds the human FcRn alpha chain extracellular sequence as shown
below:
TABLE-US-00001 (SEQ ID NO: 48) AESHLSLLYHLTAVSSPAPG TPAFWVSGWL
GPQQYLSYNS LRGEAEPCGA WVWENQVSWY WEKETTDLRI KEKLFLEAFK ALGGKGPYTL
QGLLGCELGPDNTSVPTAKFALNGEEFMNFDLKQGTWGGDWPEALAISQR WQQQDKAANK
ELTFLLFSCP HRLREHLERG RGNLEWKEPPSMRLKARPSSPGFSVLTCSAFSFYPPELQL
RFLRNGLAAG
TGQGDFGPNSDGSFHASSSLTVKSGDEHHYCCIVQHAGLAQPLRVELESPAKSS.
[0152] The residues underlined are those known to be critical for
the interaction of human FcRn with the Fc region of human IgG.
Those in bold are residues of the human FcRn polypeptide involved
in binding the antibody comprising the heavy chain sequence given
in SEQ ID NO: 25 and the light chain sequence given in SEQ ID
NO:16, ie they are within 4 .ANG. of the antibody as determined by
X-ray crystallography. Residues in italic are those involved in
binding the same antibody at 5 .ANG..
[0153] In one aspect of the invention there is provided an
anti-FcRn antibody or binding fragment thereof which binds an
epitope of human FcRn which comprises one, two, three, or four
amino acids selected from the group consisting of residues E115,
W131, P132, and E133 of human FcRn extracellular domain (SEQ ID NO:
48), and wherein the anti-FcRn antibody or binding fragment thereof
further binds one or more residues, such as two, three, four, five,
six, seven, eight, nine or ten residues selected from the group
consisting of A81, G83, G84, K85, G86, P87, N113, L135, A136, and
Q139 and optionally further binds one or more residues selected
from the group consisting of L82, Y88, L112 and D130.
[0154] Accordingly in one example there is provided an anti-FcRn
antibody or binding fragment thereof which binds an epitope of
human FcRn which comprises one, two, three, or four amino acids
selected from the group consisting of residues E115, W131, P132,
and E133 and at least one residue, for example at least 2, 3, 4, 5,
6, 7, 8, 9, 10 residues selected from the group consisting of A81,
G83, G84, K85, G86, P87, N113, L135, A136, and Q139 and wherein
said anti-FcRn antibody or binding fragment thereof optionally
further binds one or more residues, for example at least 2, 3 or 4
residues selected from the group consisting of L82, Y88, L112 and
D130 of human FcRn extracellular domain (SEQ ID NO: 48).
[0155] In one example an antibody according to this aspect of the
invention does not bind V105, P106, T107, A108 and K109 of human
FcRn extracellular domain (SEQ ID NO: 48).
[0156] In one example an antibody according to this aspect of the
invention does not bind E116, F117, M118, N119, F120, D121, L122,
K123, Q124, G128 and G129 of human FcRn extracellular domain (SEQ
ID NO: 48).
[0157] In one example an antibody according to this aspect of the
invention does not bind V105, P106, T107, A108, K109, E116, F117,
M118, N119, F120, D121, L122, K123, Q124, G128, and G129 of human
FcRn extracellular domain (SEQ ID NO: 48).
[0158] In one example there is provided an anti-FcRn antibody or
binding fragment thereof which binds an epitope of human FcRn which
comprises one, two, three, or four amino acids selected from the
group consisting of residues E115, W131, P132, and E133 and at
least one residue, for example at least 2, 3, 4, 5, 6, 7 or 8
residues selected from the group consisting of A81, L82, G83, G84,
K85, G86, P87 and Y88 of human FcRn extracellular domain (SEQ ID
NO: 48)
[0159] In one example there is provided an anti-FcRn antibody or
binding fragment thereof which binds an epitope of human FcRn which
comprises one, two, three, or four amino acids selected from the
group consisting of residues E115, W131, P132, and E133 and at
least one residue, for example at least 2, 3, 4, 5 or 6 residues
selected from the group consisting of L112, N113, D130, L135, A136,
and Q139 of human FcRn extracellular domain (SEQ ID NO: 48)
[0160] In one example there is provided an anti-FcRn antibody or
binding fragment thereof which binds an epitope of human FcRn which
comprises one, two, three, or four amino acids selected from the
group consisting of residues E115, W131, P132, and E133 and at
least one residue selected from the group consisting of A81, L82,
G83, G84, K85, G86, P87, Y88, L112, N113, D130, L135, A136, and
Q139 of human FcRn extracellular domain (SEQ ID NO: 48).
[0161] In one example there is provided an anti-FcRn antibody or
binding fragment thereof which binds an epitope of human FcRn which
comprises residues E115, W131, P132, and E133 and at least one
residue selected from the group consisting of A81, L82, G83, G84,
K85, G86, P87, Y88, L112, N113, D130, L135, A136, and Q139 of human
FcRn extracellular domain (SEQ ID NO: 48).
[0162] In one example the present invention provides an anti-FcRn
antibody or binding fragment thereof which binds an epitope of
human FcRn which comprises or consists of residues A81, G83, G84,
K85, G86, P87, N113, E115, W131, P132, E133, L135, A136, and Q139
of human FcRn extracellular domain (SEQ ID NO: 48).
[0163] In one example the present invention provides an anti-FcRn
antibody or binding fragment thereof which binds an epitope of
human FcRn which comprises or consists of residues A81, L82, G83,
G84, K85, G86, P87, Y88, L112, N113, E115, D130, W131, P132, E133,
L135, A136, and Q139 of human FcRn extracellular domain (SEQ ID NO:
48).
[0164] In one embodiment the antibodies which bind the epitope
described herein above provided by the present invention are fully
human. In one embodiment they are humanised. In one example they
have an affinity for human FcRn of 150 pM or less, typically 130 pM
or less.
[0165] Antibodies which cross-block the binding of an antibody
molecule according to the present invention in particular, an
antibody molecule comprising the heavy chain sequence given in SEQ
ID NO: 25 and the light chain sequence given in SEQ ID NO:16 may be
similarly useful in blocking FcRn activity. Accordingly, the
present invention also provides an anti-FcRn antibody molecule,
which cross-blocks the binding of any one of the antibody molecules
described herein above to human FcRn and/or is cross-blocked from
binding human FcRn by any one of those antibodies. In one
embodiment, such an antibody binds to the same epitope as an
antibody described herein above. In another embodiment the
cross-blocking neutralising antibody binds to an epitope which
borders and/or overlaps with the epitope bound by an antibody
described herein above.
[0166] Cross-blocking antibodies can be identified using any
suitable method in the art, for example by using competition ELISA
or BIAcore assays where binding of the cross blocking antibody to
human FcRn prevents the binding of an antibody of the present
invention or vice versa. Such cross blocking assays may use
isolated natural or recombinant FcRn or a suitable fusion
protein/polypeptide. In one example binding and cross-blocking is
measured using recombinant human FcRn extracellular domain (SEQ ID
NO: 48). In one example the recombinant human FcRn alpha chain
extracellular domain is used in a complex with .beta.2
microglobulin (.beta.2M) (SEQ ID NO:72).
[0167] In one embodiment there is provided an anti-FcRn antibody
molecule which blocks FcRn binding to IgG and which cross-blocks
the binding of an antibody whose heavy chain comprises the sequence
given in SEQ ID NO: 25 and whose light chain comprises the sequence
given in SEQ ID NO: 16 to human FcRn. In one embodiment the
cross-blocking antibodies provided by the present invention inhibit
the binding of an antibody comprising the heavy chain sequence
given in SEQ ID NO: 25 and the light chain sequence given in SEQ ID
NO: 16 by greater than 80%, for example by greater than 85%, such
as by greater than 90%, in particular by greater than 95%
inhibition.
[0168] In one embodiment the cross-blocking antibodies provided by
the present invention are fully human. In one embodiment the
cross-blocking antibodies provided by the present invention are
humanised. In one embodiment the cross-blocking antibodies provided
by the present invention have an affinity for human FcRn of 150 pM
or less, 130 pM or less or 100 pM or less. In one embodiment the
cross-blocking antibodies provided by the present invention have an
affinity for human FcRn of 50 pM or less. Affinity can be measured
using the methods described herein below.
[0169] Biological molecules, such as antibodies or fragments,
contain acidic and/or basic functional groups, thereby giving the
molecule a net positive or negative charge. The amount of overall
"observed" charge will depend on the absolute amino acid sequence
of the entity, the local environment of the charged groups in the
3D structure and the environmental conditions of the molecule. The
isoelectric point (pI) is the pH at which a particular molecule or
solvent accessible surface thereof carries no net electrical
charge. In one example, the FcRn antibody and fragments of the
invention may be engineered to have an appropriate isoelectric
point. This may lead to antibodies and/or fragments with more
robust properties, in particular suitable solubility and/or
stability profiles and/or improved purification
characteristics.
[0170] Thus in one aspect the invention provides a humanised FcRn
antibody engineered to have an isoelectric point different to that
of the originally identified antibody. The antibody may, for
example be engineered by replacing an amino acid residue such as
replacing an acidic amino acid residue with one or more basic amino
acid residues. Alternatively, basic amino acid residues may be
introduced or acidic amino acid residues can be removed.
Alternatively, if the molecule has an unacceptably high pI value
acidic residues may be introduced to lower the pI, as required. It
is important that when manipulating the pI care must be taken to
retain the desirable activity of the antibody or fragment. Thus in
one embodiment the engineered antibody or fragment has the same or
substantially the same activity as the "unmodified" antibody or
fragment.
[0171] Programs such as **ExPASY
http://www.expasy.ch/tools/pi_tool.html, and
http://www/iut-arles.up.univ-mrs.fr/w3bb/d_abim/compo-p.html, may
be used to predict the isoelectric point of the antibody or
fragment. Alternatively or additionally, the pI can be measured
using any suitable standard laboratory technique.
[0172] The antibody molecules of the present invention suitably
have a high binding affinity, in particular in the nanomolar range.
Affinity may be measured using any suitable method known in the
art, including BIAcore, as described in the Examples herein, using
isolated natural or recombinant FcRn or a suitable fusion
protein/polypeptide. In one example affinity is measured using
recombinant human FcRn extracellular domain as described in the
Examples herein (SEQ ID NO: 48). In one example affinity is
measured using the recombinant human FcRn alpha chain extracellular
domain (SEQ ID NO: 48) in association with human (32 microglobulin
(.beta.2M) (SEQ ID NO: 72). Suitably the antibody molecules of the
present invention have a binding affinity for isolated human FcRn
of about 1 nM or lower. In one embodiment the antibody molecule of
the present invention has a binding affinity of about 500 pM or
lower (i.e. higher affinity). In one embodiment the antibody
molecule of the present invention has a binding affinity of about
250 pM or lower. In one embodiment the antibody molecule of the
present invention has a binding affinity of about 200 pM or lower.
In one embodiment the antibody molecule of the present invention
has a binding affinity of about 150 pM or lower. In one embodiment
the present invention provides an anti-FcRn antibody with a binding
affinity of about 100 pM or lower. In one embodiment the present
invention provides a humanised anti-FcRn antibody with a binding
affinity of about 100 pM or lower. In one embodiment the present
invention provides an anti-FcRn antibody with a binding affinity of
50 pM or lower.
[0173] In one embodiment the antibodies of the present invention
are able to bind human FcRn at both pH6 or lower pH (in particular
pH 6) and pH7.4 or higher pH (in particular pH7.4) with comparable
binding affinity. Advantageously therefore the antibodies are able
to continue to bind FcRn even within the endosome, thereby
maximising the blocking of FcRn binding to IgG.
[0174] In one embodiment the antibodies of the present invention
are able to bind human FcRn with a binding affinity of 150 pM or
lower when measured at pH6 and pH7.4. In one embodiment the
antibodies of the present invention are able to bind human FcRn
with a binding affinity of 130 pM or lower when measured at pH6 and
pH7.4. In one embodiment the antibodies of the present invention
are able to bind human FcRn with a binding affinity of 130 pM or
lower when measured at pH6 and a binding affinity of 50 pM or lower
when measured at pH7.4.
[0175] The affinity of an antibody or binding fragment of the
present invention, as well as the extent to which a binding agent
(such as an antibody) inhibits binding, can be determined by one of
ordinary skill in the art using conventional techniques, for
example those described by Scatchard et al. (Ann. KY. Acad. Sci.
51:660-672 (1949)) or by surface plasmon resonance (SPR) using
systems such as BIAcore. For surface plasmon resonance, target
molecules are immobilized on a solid phase and exposed to ligands
in a mobile phase running along a flow cell. If ligand binding to
the immobilized target occurs, the local refractive index changes,
leading to a change in SPR angle, which can be monitored in real
time by detecting changes in the intensity of the reflected light.
The rates of change of the SPR signal can be analyzed to yield
apparent rate constants for the association and dissociation phases
of the binding reaction. The ratio of these values gives the
apparent equilibrium constant (affinity) (see, e.g., Wolff et al,
Cancer Res. 53:2560-65 (1993)).
[0176] In the present invention affinity of the test antibody
molecule is typically determined using SPR as follows. The test
antibody molecule is captured on the solid phase and human FcRn
alpha chain extracellular domain in non-covalent complex with human
.beta.2M is run over the captured antibody in the mobile phase and
affinity of the test antibody molecule for human FcRn determined.
The test antibody molecule may be captured on the solid phase chip
surface using any appropriate method, for example using an anti-Fc
or anti Fab' specific capture agent. In one example the affinity is
determined at pH6. In one example the affinity is determined at
pH7.4.
[0177] It will be appreciated that the affinity of antibodies
provided by the present invention may be altered using any suitable
method known in the art. The present invention therefore also
relates to variants of the antibody molecules of the present
invention, which have an improved affinity for FcRn. 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.
[0178] In one embodiment the antibody molecules of the present
invention block human FcRn activity. Assays suitable for
determining the ability of an antibody to block FcRn are described
in the Examples herein. A suitable assay for determining the
ability of an antibody molecule to block IgG recycling in vitro is
described herein below.
[0179] If desired an antibody for use in the present invention may
be conjugated to one or more effector molecule(s). It will be
appreciated that the effector molecule may comprise a single
effector molecule or two or more such molecules so linked as to
form a single moiety that can be attached to the antibodies of the
present invention. Where it is desired to obtain an antibody
fragment linked to an effector molecule, this may be prepared by
standard chemical or recombinant DNA procedures in which the
antibody fragment is linked either directly or via a coupling agent
to the effector molecule. Techniques for conjugating such effector
molecules to antibodies are well known in the art (see, Hellstrom
et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds.,
1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and
Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123).
Particular chemical procedures include, for example, those
described in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and
WO 03/031581. Alternatively, where the effector molecule is a
protein or polypeptide the linkage may be achieved using
recombinant DNA procedures, for example as described in WO 86/01533
and EP0392745.
[0180] The term effector molecule as used herein includes, for
example, antineoplastic agents, drugs, toxins, biologically active
proteins, for example enzymes, other antibody or antibody
fragments, synthetic or naturally occurring polymers, nucleic acids
and fragments thereof e.g. DNA, RNA and fragments thereof,
radionuclides, particularly radioiodide, radioisotopes, chelated
metals, nanoparticles and reporter groups such as fluorescent
compounds or compounds which may be detected by NMR or ESR
spectroscopy.
[0181] Examples of effector molecules may include cytotoxins or
cytotoxic agents including any agent that is detrimental to (e.g.
kills) cells. Examples include combrestatins, dolastatins,
epothilones, staurosporin, maytansinoids, spongistatins, rhizoxin,
halichondrins, roridins, hemiasterlins, taxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof.
[0182] Effector molecules also include, but are not limited to,
antimetabolites (e.g. methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g. mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g. daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g. dactinomycin (formerly actinomycin), bleomycin,
mithramycin, anthramycin (AMC), calicheamicins or duocarmycins),
and anti-mitotic agents (e.g. vincristine and vinblastine).
[0183] Other effector molecules may include chelated radionuclides
such as .sup.111In and .sup.90Y, Lu.sup.177, Bismuth.sup.213,
Californium.sup.252, Iridium.sup.192 and
Tungsten.sup.188/Rhenium.sup.188; or drugs such as but not limited
to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and
suramin.
Other effector molecules include proteins, peptides and enzymes.
Enzymes of interest include, but are not limited to, proteolytic
enzymes, hydrolases, lyases, isomerases, transferases. Proteins,
polypeptides and peptides of interest include, but are not limited
to, immunoglobulins, toxins such as abrin, ricin A, pseudomonas
exotoxin, or diphtheria toxin, a protein such as insulin, tumour
necrosis factor, .alpha.-interferon, .beta.-interferon, nerve
growth factor, platelet derived growth factor or tissue plasminogen
activator, a thrombotic agent or an anti-angiogenic agent, e.g.
angiostatin or endostatin, or, a biological response modifier such
as a lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2),
granulocyte macrophage colony stimulating factor (GM-CSF),
granulocyte colony stimulating factor (G-CSF), nerve growth factor
(NGF) or other growth factor and immunoglobulins.
[0184] Other effector molecules may include detectable substances
useful for example in diagnosis. Examples of detectable substances
include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, radioactive
nuclides, positron emitting metals (for use in positron emission
tomography), and nonradioactive paramagnetic metal ions. See
generally U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics. Suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; suitable prosthetic
groups include streptavidin, avidin and biotin; suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride and phycoerythrin; suitable
luminescent materials include luminol; suitable bioluminescent
materials include luciferase, luciferin, and aequorin; and suitable
radioactive nuclides include .sup.125I, .sup.131I, .sup.111In and
.sup.99Tc.
[0185] In another example the effector molecule may increase the
half-life of the antibody in vivo, and/or reduce immunogenicity of
the antibody and/or enhance the delivery of an antibody across an
epithelial barrier to the immune system. Examples of suitable
effector molecules of this type include polymers, albumin, albumin
binding proteins or albumin binding compounds such as those
described in WO05/117984.
[0186] In one embodiment a half-life provided by an effector
molecule which is independent of FcRn is advantageous.
[0187] Where the effector molecule is a polymer it may, in general,
be a synthetic or a naturally occurring polymer, for example an
optionally substituted straight or branched chain polyalkylene,
polyalkenylene or polyoxyalkylene polymer or a branched or
unbranched polysaccharide, e.g. a homo- or
hetero-polysaccharide.
[0188] Specific optional substituents which may be present on the
above-mentioned synthetic polymers include one or more hydroxy,
methyl or methoxy groups.
[0189] Specific examples of synthetic polymers include optionally
substituted straight or branched chain poly(ethyleneglycol),
poly(propyleneglycol) poly(vinylalcohol) or derivatives thereof,
especially optionally substituted poly(ethyleneglycol) such as
methoxypoly(ethyleneglycol) or derivatives thereof.
[0190] Specific naturally occurring polymers include lactose,
amylose, dextran, glycogen or derivatives thereof.
[0191] In one embodiment the polymer is albumin or a fragment
thereof, such as human serum albumin or a fragment thereof.
[0192] "Derivatives" as used herein is intended to include reactive
derivatives, for example thiol-selective reactive groups such as
maleimides and the like. The reactive group may be linked directly
or through a linker segment to the polymer. It will be appreciated
that the residue of such a group will in some instances form part
of the product as the linking group between the antibody fragment
and the polymer.
[0193] The size of the polymer may be varied as desired, but will
generally be in an average molecular weight range from 500 Da to
50000 Da, for example from 5000 to 40000 Da such as from 20000 to
40000 Da. The polymer size may in particular be selected on the
basis of the intended use of the product for example ability to
localize to certain tissues such as tumors or extend circulating
half-life (for review see Chapman, 2002, Advanced Drug Delivery
Reviews, 54, 531-545). Thus, for example, where the product is
intended to leave the circulation and penetrate tissue, for example
for use in the treatment of a tumour, it may be advantageous to use
a small molecular weight polymer, for example with a molecular
weight of around 5000 Da. For applications where the product
remains in the circulation, it may be advantageous to use a higher
molecular weight polymer, for example having a molecular weight in
the range from 20000 Da to 40000 Da.
[0194] Suitable polymers include a polyalkylene polymer, such as a
poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol)
or a derivative thereof, and especially with a molecular weight in
the range from about 15000 Da to about 40000 Da.
[0195] In one example antibodies for use in the present invention
are attached to poly(ethyleneglycol) (PEG) moieties. In one
particular example the antibody is an antibody fragment and the PEG
molecules may be attached through any available amino acid
side-chain or terminal amino acid functional group located in the
antibody fragment, for example any free amino, imino, thiol,
hydroxyl or carboxyl group. Such amino acids may occur naturally in
the antibody fragment or may be engineered into the fragment using
recombinant DNA methods (see for example U.S. Pat. Nos. 5,219,996;
5,667,425; WO98/25971, WO2008/038024). In one example the antibody
molecule of the present invention is a modified Fab fragment
wherein the modification is the addition to the C-terminal end of
its heavy chain one or more amino acids to allow the attachment of
an effector molecule. Suitably, the additional amino acids form a
modified hinge region containing one or more cysteine residues to
which the effector molecule may be attached. Multiple sites can be
used to attach two or more PEG molecules.
[0196] Suitably PEG molecules are covalently linked through a thiol
group of at least one cysteine residue located in the antibody
fragment. Each polymer molecule attached to the modified antibody
fragment may be covalently linked to the sulphur atom of a cysteine
residue located in the fragment. The covalent linkage will
generally be a disulphide bond or, in particular, a sulphur-carbon
bond. Where a thiol group is used as the point of attachment
appropriately activated effector molecules, for example thiol
selective derivatives such as maleimides and cysteine derivatives
may be used. An activated polymer may be used as the starting
material in the preparation of polymer-modified antibody fragments
as described above. The activated polymer may be any polymer
containing a thiol reactive group such as an .alpha.-halocarboxylic
acid or ester, e.g. iodoacetamide, an imide, e.g. maleimide, a
vinyl sulphone or a disulphide. Such starting materials may be
obtained commercially (for example from Nektar, formerly Shearwater
Polymers Inc., Huntsville, Ala., USA) or may be prepared from
commercially available starting materials using conventional
chemical procedures. Particular PEG molecules include 20K
methoxy-PEG-amine (obtainable from Nektar, formerly Shearwater;
Rapp Polymere; and SunBio) and M-PEG-SPA (obtainable from Nektar,
formerly Shearwater).
[0197] In one embodiment, the antibody is a modified Fab fragment,
Fab' fragment or diFab which is PEGylated, i.e. has PEG
(poly(ethyleneglycol)) covalently attached thereto, e.g. according
to the method disclosed in EP 0948544 or EP1090037 [see also
"Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical
Applications", 1992, J. Milton Harris (ed), Plenum Press, New York,
"Poly(ethyleneglycol) Chemistry and Biological Applications", 1997,
J. Milton Harris and S. Zalipsky (eds), American Chemical Society,
Washington D.C. and "Bioconjugation Protein Coupling Techniques for
the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove
Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery
Reviews 2002, 54:531-545]. In one example PEG is attached to a
cysteine in the hinge region. In one example, a PEG modified Fab
fragment has a maleimide group covalently linked to a single thiol
group in a modified hinge region. A lysine residue may be
covalently linked to the maleimide group and to each of the amine
groups on the lysine residue may be attached a
methoxypoly(ethyleneglycol) polymer having a molecular weight of
approximately 20,000 Da. The total molecular weight of the PEG
attached to the Fab fragment may therefore be approximately 40,000
Da.
[0198] Particular PEG molecules include
2-[3-(N-maleimido)propionamido]ethyl amide of
N,N'-bis(methoxypoly(ethylene glycol) MW 20,000) modified lysine,
also known as PEG2MAL40K (obtainable from Nektar, formerly
Shearwater).
[0199] Alternative sources of PEG linkers include NOF who supply
GL2-400MA3 (wherein m in the structure below is 5) and GL2-400MA
(where m is 2) and n is approximately 450:
##STR00001##
[0200] That is to say each PEG is about 20,000 Da.
[0201] Thus in one embodiment the PEG is
2,3-Bis(methylpolyoxyethylene-oxy)-1-{[3-(6-maleimido-1-oxohexyl)amino]pr-
opyloxy} hexane (the 2 arm branched PEG,
--CH.sub.2).sub.3NHCO(CH.sub.2).sub.5-MAL, Mw 40,000 known as
SUNBRIGHT GL2-400MA3.
[0202] Further alternative PEG effector molecules of the following
type:
##STR00002##
are available from Dr Reddy, NOF and Jenkem.
[0203] In one embodiment there is provided an antibody which is
PEGylated (for example with a PEG described herein), attached
through a cysteine amino acid residue at or about amino acid 232 in
the chain, for example amino acid 232 of the heavy chain (by
sequential numbering), for example amino acid 232 of SEQ ID NO:
33.
[0204] In one embodiment the present disclosure provides a Fab'PEG
molecule comprising one or more PEG polymers, for example 1 or 2
polymers such as a 40 kDa polymer or polymers.
[0205] Fab'-PEG molecules according to the present disclosure may
be particularly advantageous in that they have a half-life
independent of the Fc fragment. In one example the present
invention provides a method treating a disease ameliorated by
blocking human FcRn comprising administering a therapeutically
effective amount of an anti-FcRn antibody or binding fragment
thereof wherein the antibody or binding fragment thereof has a half
life that is independent of Fc binding to FcRn.
[0206] In one embodiment there is provided a Fab' conjugated to a
polymer, such as a PEG molecule, a starch molecule or an albumin
molecule.
[0207] In one embodiment there is provided a scFv conjugated to a
polymer, such as a PEG molecule, a starch molecule or an albumin
molecule.
[0208] In one embodiment the antibody or fragment is conjugated to
a starch molecule, for example to increase the half life. Methods
of conjugating starch to a protein as described in U.S. Pat. No.
8,017,739 incorporated herein by reference.
[0209] In one embodiment there is provided an anti-FcRn binding
molecule (i.e an antibody or binding fragment thereof) which:
[0210] Causes 50-85% reduction, such as a 70% reduction of plasma
IgG concentration, [0211] With not more than 25% or 20% reduction
of plasma albumin concentration, and/or [0212] With the possibility
of repeat dosing to achieve long-term maintenance of low plasma IgG
concentration.
[0213] The present invention also provides an isolated DNA sequence
encoding the heavy and/or light chain(s) of an antibody molecule of
the present invention. Suitably, the DNA sequence encodes the heavy
or the light chain of an antibody molecule of the present
invention. The DNA sequence of the present invention may comprise
synthetic DNA, for instance produced by chemical processing, cDNA,
genomic DNA or any combination thereof.
[0214] DNA sequences which encode an 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.
[0215] 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.
[0216] 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.
[0217] Examples of suitable DNA sequences are provided in
herein.
[0218] Examples of suitable DNA sequences encoding the 1638.g49
light chain variable region are provided in SEQ ID NO: 17, SEQ ID
NO: 19 and SEQ ID NO: 21.
[0219] Examples of suitable DNA sequences encoding the 1638.g28
light chain variable region are provided in SEQ ID NO: 52 and SEQ
ID NO: 54.
[0220] Examples of suitable DNA sequences encoding the 1638.g49
heavy chain variable region are provided in SEQ ID NO: 26 and SEQ
ID NO: 28.
[0221] Examples of suitable DNA sequences encoding the 1638.g28
heavy chain variable region are provided in SEQ ID NO: 60 and
62.
[0222] Examples of suitable DNA sequences encoding the 1638.g49
light chain (variable and constant) are provided in SEQ ID NO: 21,
SEQ ID NO: 22 and SEQ ID NO: 24, and for 1638.g28 light chain the
sequence given in SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:58 and
1638.g28 heavy chain the sequence given in SEQ ID NO: 64 or SEQ ID
NO: 66.
[0223] Examples of suitable DNA sequences encoding the 1638.g49
heavy chain (variable and constant, depending on format) are
provided in SEQ ID NO: 30 (Fab), SEQ ID NO: 34 or 36 (Fab'), SEQ ID
NO: 38 (IgG4P), SEQ ID NO: 43 (FabFv) and SEQ ID NO:74 (IgG1).
[0224] Accordingly in one example the present invention provides an
isolated DNA sequence encoding the heavy chain of an antibody Fab
or Fab' fragment of the present invention which comprises the
sequence given in SEQ ID NO: 30, 32, 34, 36, 64 or 66. Also
provided is an isolated DNA sequence encoding the light chain of an
antibody Fab or Fab' fragment of the present invention which
comprises the sequence given in SEQ ID NO: 21, 22 or 56.
[0225] In one example the present invention provides an isolated
DNA sequence encoding the heavy chain and the light chain of an
IgG4(P) antibody of the present invention in which the DNA encoding
the heavy chain comprises the sequence given in SEQ ID NO: 38 and
the DNA encoding the light chain comprises the sequence given in
SEQ ID NO: 22.
[0226] In one example the present invention provides an isolated
DNA sequence encoding the heavy chain and the light chain of an
IgG1 antibody of the present invention in which the DNA encoding
the heavy chain comprises the sequence given in SEQ ID NO: 74 and
the DNA encoding the light chain comprises the sequence given in
SEQ ID NO: 22.
[0227] In one example the present invention provides an isolated
DNA sequence encoding the heavy chain and the light chain of a
Fab-dsFv antibody of the present invention in which the DNA
encoding the heavy chain comprises the sequence given in SEQ ID NO:
43 and the DNA encoding the light chain comprises the sequence
given in SEQ ID NO: 41.
[0228] The present invention also relates to a cloning or
expression vector comprising one or more DNA sequences of the
present invention. Accordingly, provided is a cloning or expression
vector comprising one or more DNA sequences encoding an antibody of
the present invention. Suitably, 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 and suitable signal sequences. In one example the
vector comprises an intergenic sequence between the heavy and the
light chains (see WO03/048208).
[0229] 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.
[0230] Also provided is a host cell comprising one or more cloning
or expression vectors comprising one or more DNA sequences encoding
an antibody of the present invention. Accordingly the present
invention also provides a host cell for expression of an antibody
according to to the invention comprising: [0231] i) a DNA sequence
encoding the heavy chain of said antibody, and [0232] ii) a DNA
sequence encoding the light chain of said antibody
[0233] wherein the DNA sequences are provided in one or more
cloning or expression vectors.
[0234] 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 (especially for expressing antibody
fragments or eukaryotic, for example mammalian, host cell
expression systems may also be used (especially for expressing
full-length antibodies). Suitable mammalian host cells include CHO,
myeloma or hybridoma cells.
[0235] Suitable types of Chinese Hamster Ovary (CHO cells) for use
in the present invention may include CHO and CHO-K1 cells including
dhfr-CHO cells, such as CHO-DG44 cells and CHO-DXB11 cells, which
may be used with a DHFR selectable marker or CHOK1-SV cells which
may be used with a glutamine synthetase selectable marker. Other
cell types of use in expressing antibodies include lymphocytic cell
lines, e.g., NSO myeloma cells and SP2 cells, COS cells.
[0236] 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 or
vectors 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.
[0237] 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.
[0238] The antibodies and fragments according to the present
disclosure are expressed at good levels from host cells. Thus the
properties of the antibodies and/or fragments are conducive to
commercial processing.
[0239] Thus there is a provided a process for culturing a host cell
and expressing an antibody or fragment thereof, isolating the
latter and optionally purifying the same to provide an isolated
antibody or fragment. In one embodiment the process further
comprises the step of conjugating an effector molecule to the
isolated antibody or fragment, for example conjugating to a PEG
polymer in particular as described herein.
[0240] In one embodiment there is provided a process for purifiying
an antibody (in particular an antibody or fragment according to the
invention) comprising the steps: performing anion exchange
chromatography in non-binding mode such that the impurities are
retained on the column and the antibody is eluted.
[0241] In one embodiment the purification employs affinity capture
on an FcRn column.
[0242] In one embodiment the purification employs cibacron blue or
similar for purification of albumin fusion or conjugate
molecules.
Suitable ion exchange resins for use in the process include Q.FF
resin (supplied by GE-Healthcare). The step may, for example be
performed at a pH about 8.
[0243] The process may further comprise an intial capture step
employing cation exchange chromatography, performed for example at
a pH of about 4 to 5, such as 4.5. The cation exchange
chromatography may, for example employ a resin such as CaptoS resin
or SP sepharose FF (supplied by GE-Healthcare). The antibody or
fragment can then be eluted from the resin employing an ionic salt
solution such as sodium chloride, for example at a concentration of
200 mM.
[0244] Thus the chromatograph step or steps may include one or more
washing steps, as appropriate.
[0245] The purification process may also comprise one or more
filtration steps, such as a diafiltration step.
[0246] Thus in one embodiment there is provided a purified
anti-FcRn antibody or fragment, for example a humanised antibody or
fragment, in particular an antibody or fragment according to the
invention, in substantially purified from, in particular free or
substantially free of endotoxin and/or host cell protein or
DNA.
[0247] Purified form as used supra is intended to refer to at least
90% purity, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w/w or more
pure.
[0248] Substantially free of endotoxin is generally intended to
refer to an endotoxin content of 1 EU per mg antibody product or
less such as 0.5 or 0.1 EU per mg product.
[0249] Substantially free of host cell protein or DNA is generally
intended to refer to host cell protein and/or DNA content 400 .mu.g
per mg of antibody product or less such as 100 .mu.g per mg or
less, in particular 20 .mu.g per mg, as appropriate.
[0250] The antibody molecules of the present invention may also be
used in diagnosis, for example in the in vivo diagnosis and imaging
of disease states involving FcRn.
[0251] As the antibodies of the present invention are useful in the
treatment and/or prophylaxis of a pathological condition, the
present invention also provides a pharmaceutical or diagnostic
composition comprising an antibody molecule of the present
invention in combination with one or more of a pharmaceutically
acceptable excipient, diluent or carrier. Accordingly, provided is
the use of an antibody molecule of the invention for the
manufacture of a medicament. The composition will usually be
supplied as part of a sterile, pharmaceutical composition that will
normally include a pharmaceutically acceptable carrier. A
pharmaceutical composition of the present invention may
additionally comprise a pharmaceutically-acceptable excipient.
[0252] The present invention also provides a process for
preparation of a pharmaceutical or diagnostic composition
comprising adding and mixing the antibody molecule of the present
invention together with one or more of a pharmaceutically
acceptable excipient, diluent or carrier.
[0253] The antibody molecule may be the sole active ingredient in
the pharmaceutical or diagnostic composition or may be accompanied
by other active ingredients including other antibody ingredients or
non-antibody ingredients such as steroids or other drug molecules,
in particular drug molecules whose half-life is independent of FcRn
binding.
[0254] The pharmaceutical compositions suitably 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 amount 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.
[0255] The precise therapeutically 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, a
therapeutically effective amount will be from 0.01 mg/kg to 500
mg/kg, for example 0.1 mg/kg to 200 mg/kg, such as 100 mg/Kg.
[0256] Pharmaceutical compositions may be conveniently presented in
unit dose forms containing a predetermined amount of an active
agent of the invention per dose.
[0257] Therapeutic doses of the antibodies according to the present
disclosure show no apparent toxicology effects in vivo.
[0258] In one embodiment of an antibody or fragment according to
the invention a single dose may provide up to a 70% reduction in
circulating IgG levels. In one example of an antibody or fragment
according to the invention a single dose may provide up to a 80%
reduction in circulating IgG levels. In one example of an antibody
or fragment according to the invention a single dose may provide a
greater than 80% reduction in circulating IgG levels.
[0259] The maximal therapeutic reduction in circulating IgG may be
observed about 1 week after administration of the relevant
therapeutic dose. The levels of IgG may recover over the weeks
following dosing if further therapeutic doses are not delivered.
Recover as employed herein refers to levels returning to levels
similar to those observed before initial dosing commenced.
[0260] Advantageously, the levels of IgG in vivo may be maintained
at an appropriately low level by administration of sequential doses
of the antibody or fragments according to the disclosure.
[0261] Compositions may be administered individually to a patient
or may be administered in combination (e.g. simultaneously,
sequentially or separately) with other agents, drugs or
hormones.
[0262] Agents as employed herein refers to an entity which when
administered has a physiological affect.
[0263] Drug as employed herein refers to a chemical entity which at
a therapeutic dose has an appropriate physiological affect.
[0264] In one embodiment the antibodies or fragments according to
the present disclosure are employed with an immunosuppressant
therapy, such as a steroid, in particular prednisone.
[0265] In one embodiment the antibodies or fragments according to
the present disclosure are employed with Rituximab or other B cell
therapies.
[0266] In one embodiment the antibodies or fragments according to
the present disclosure are employed with any B cell or T cell
modulating agent or immunomodulator. Examples include methotrexate,
microphenyolate and azathioprine.
[0267] The dose at which the antibody molecule of the present
invention is administered depends on the nature of the condition to
be treated, the extent of the inflammation present and on whether
the antibody molecule is being used prophylactically or to treat an
existing condition.
[0268] The frequency of dosing will depend on the half life of the
antibody, its target-mediated disposition, the duration of its
effect, and the presence of anti-drug antibodies. If the antibody
has a short half life (a few hours) or a limited activity, and/or
if it is desirable to deliver small volumes of drug (e.g. for
subcutaneous injection), it may be necessary to dose frequently, as
frequently as once or more per day. Alternatively, if the antibody
has a long half life, has long duration of activity, or can be
dosed in large volumes (such as by infusion) dosing may be
infrequent, once per day, or every few days, weeks or months. In
one embodiment, sufficient time is allowed between doses to allow
anti-drug antibody levels to decline.
[0269] Half life as employed herein is intended to refer to the
duration of the molecule in circulation, for example in
serum/plasma.
[0270] Pharmacodynamics as employed herein refers to the profile
and in particular duration of the biological action of the molecule
according the present disclosure.
[0271] The pharmaceutically acceptable 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.
[0272] 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.
[0273] 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.
[0274] Suitable 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.
[0275] Once formulated, the compositions of the invention can be
administered directly to the subject. The subjects to be treated
can be animals. However, in one or more embodiments the
compositions are adapted for administration to human subjects.
Suitably in formulations according to the present disclosure, the
pH of the final formulation is not similar to the value of the
isoelectric point of the antibody or fragment, for example if the
pI of the protein is in the range 8-9 or above then a formulation
pH of 7 may be appropriate. Whilst not wishing to be bound by
theory it is thought that this may ultimately provide a final
formulation with improved stability, for example the antibody or
fragment remains in solution.
[0276] In one example the pharmaceutical formulation at a pH in the
range of 4.0 to 7.0 comprises: 1 to 200 mg/mL of an antibody
molecule according to the present disclosure, 1 to 100 mM of a
buffer, 0.001 to 1% of a surfactant, a) 10 to 500 mM of a
stabiliser, b) 10 to 500 mM of a stabiliser and 5 to 500 mM of a
tonicity agent, or c) 5 to 500 mM of a tonicity agent.
[0277] 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.
[0278] 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.
[0279] 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.
[0280] A thorough discussion of pharmaceutically acceptable
carriers is available in Remington's Pharmaceutical Sciences (Mack
Publishing Company, N.J. 1991).
[0281] In one embodiment the formulation is provided as a
formulation for topical administrations including inhalation.
[0282] Suitable inhalable preparations include inhalable powders,
metering aerosols containing propellant gases or inhalable
solutions free from propellant gases. Inhalable powders according
to the disclosure containing the active substance may consist
solely of the abovementioned active substances or of a mixture of
the abovementioned active substances with physiologically
acceptable excipient.
[0283] These inhalable powders may include monosaccharides (e.g.
glucose or arabinose), disaccharides (e.g. lactose, saccharose,
maltose), oligo- and polysaccharides (e.g. dextranes), polyalcohols
(e.g. sorbitol, mannitol, xylitol), salts (e.g. sodium chloride,
calcium carbonate) or mixtures of these with one another. Mono- or
disaccharides are suitably used, the use of lactose or glucose,
particularly but not exclusively in the form of their hydrates.
[0284] Particles for deposition in the lung require a particle size
less than 10 microns, such as 1-9 microns for example from 1 to 5
.mu.m. The particle size of the active ingredient (such as the
antibody or fragment) is of primary importance.
[0285] The propellent gases which can be used to prepare the
inhalable aerosols are known in the art. Suitable propellent gases
are selected from among hydrocarbons such as n-propane, n-butane or
isobutane and halohydrocarbons such as chlorinated and/or
fluorinated derivatives of methane, ethane, propane, butane,
cyclopropane or cyclobutane. The abovementioned propellent gases
may be used on their own or in mixtures thereof.
[0286] Particularly suitable propellent gases are halogenated
alkane derivatives selected from among TG 11, TG 12, TG 134a and
TG227. Of the abovementioned halogenated hydrocarbons, TG134a
(1,1,1,2-tetrafluoroethane) and TG227
(1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are
particularly suitable.
[0287] The propellent-gas-containing inhalable aerosols may also
contain other ingredients such as cosolvents, stabilisers,
surface-active agents (surfactants), antioxidants, lubricants and
means for adjusting the pH. All these ingredients are known in the
art.
[0288] The propellant-gas-containing inhalable aerosols according
to the invention may contain up to 5% by weight of active
substance. Aerosols according to the invention contain, for
example, 0.002 to 5% by weight, 0.01 to 3% by weight, 0.015 to 2%
by weight, 0.1 to 2% by weight, 0.5 to 2% by weight or 0.5 to 1% by
weight of active ingredient.
[0289] Alternatively topical administrations to the lung may also
be by administration of a liquid solution or suspension
formulation, for example employing a device such as a nebulizer,
for example, a nebulizer connected to a compressor (e.g., the Pan
LC-Jet Plus.RTM. nebulizer connected to a Pari Master.RTM.
compressor manufactured by Pari Respiratory Equipment, Inc.,
Richmond, Va.).
[0290] The antibody of the invention can be delivered dispersed in
a solvent, e.g., in the form of a solution or a suspension. It can
be suspended in an appropriate physiological solution, e.g., saline
or other pharmacologically acceptable solvent or a buffered
solution. Examples of buffered solutions known in the art may
contain 0.05 mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg NaCl,
0.15 mg to 0.25 mg polysorbate, 0.25 mg to 0.30 mg anhydrous citric
acid, and 0.45 mg to 0.55 mg sodium citrate per 1 ml of water so as
to achieve a pH of about 4.0 to 5.0. A suspension can employ, for
example, lyophilised antibody.
[0291] The therapeutic suspensions or solution formulations can
also contain one or more excipients. Excipients are well known in
the art and include buffers (e.g., citrate buffer, phosphate
buffer, acetate buffer and bicarbonate buffer), amino acids, urea,
alcohols, ascorbic acid, phospholipids, proteins (e.g., serum
albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and
glycerol. Solutions or suspensions can be encapsulated in liposomes
or biodegradable microspheres. The formulation will generally be
provided in a substantially sterile form employing sterile
manufacture processes.
[0292] This may include production and sterilization by filtration
of the buffered solvent/solution used for the formulation, aseptic
suspension of the antibody in the sterile buffered solvent
solution, and dispensing of the formulation into sterile
receptacles by methods familiar to those of ordinary skill in the
art.
[0293] Nebulizable formulation according to the present disclosure
may be provided, for example, as single dose units (e.g., sealed
plastic containers or vials) packed in foil envelopes. Each vial
contains a unit dose in a volume, e.g., 2 mL, of
solvent/solutionbuffer.
[0294] The antibodies disclosed herein may be suitable for delivery
via nebulisation.
[0295] It is also envisaged that the antibody of the present
invention may 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.
[0296] The present invention also provides an antibody molecule (or
compositions comprising same) for use in the control of autoimmune
diseases, for example Acute Disseminated Encephalomyelitis (ADEM),
Acute necrotizing hemorrhagic leukoencephalitis, Addison's disease,
Agammaglobulinemia, Alopecia areata, Amyloidosis, ANCA-associated
vasculitis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis,
Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune
aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis,
Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune
inner ear disease (AIED), Autoimmune myocarditis, Autoimmune
pancreatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic
purpura (ATP), Autoimmune thyroid disease, Autoimmune urticarial,
Axonal & nal neuropathies, Balo disease, Behcet's disease,
Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac
disease, Chagas disease, Chronic inflammatory demyelinating
polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis
(CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign
mucosal pemphigoid, Crohn's disease, Cogans syndrome, Cold
agglutinin disease, Congenital heart block, Coxsackie myocarditis,
CREST disease, Essential mixed cryoglobulinemia, Demyelinating
neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's
disease (neuromyelitis optica), Dilated cardiomyopathy, Discoid
lupus, Dressler's syndrome, Endometriosis, Eosinophilic
angiocentric fibrosis, Eosinophilic fasciitis, Erythema nodosum,
Experimental allergic encephalomyelitis, Evans syndrome, Fibrosing
alveolitis, Giant cell arteritis (temporal arteritis),
Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with
Polyangiitis (GPA) see Wegener's, Graves' disease, Guillain-Barre
syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis,
Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis,
Hypogammaglobulinemia, Idiopathic hypocomplementemic
tubulointestitial nephritis, Idiopathic thrombocytopenic purpura
(ITP), IgA nephropathy, IgG4-related disease, IgG4-related
sclerosing disease, Immunoregulatory lipoproteins, Inflammatory
aortic aneurysm, Inflammatory pseudotumour, Inclusion body
myositis, Insulin-dependent diabetes (type1), Interstitial
cystitis, Juvenile arthritis, Juvenile diabetes, Kawasaki syndrome,
Kuttner's tumour, Lambert-Eaton syndrome, Leukocytoclastic
vasculitis, Lichen planus, Lichen sclerosus, Ligneous
conjunctivitis, Linear IgA disease (LAD), Lupus (SLE), Lyme
disease, chronic, Mediastinal fibrosis, Meniere's disease,
Microscopic polyangiitis, Mikulicz's syndrome, Mixed connective
tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease,
Multifocal fibrosclerosis, Multiple sclerosis, Myasthenia gravis,
Myositis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia,
Ocular cicatricial pemphigoid, Optic neuritis, Ormond's disease
(retroperitoneal fibrosis), Palindromic rheumatism, PANDAS
(Pediatric Autoimmune Neuropsychiatric Disorders Associated with
Streptococcus), Paraneoplastic cerebellar degeneration,
Paraproteinemic polyneuropathies, Paroxysmal nocturnal
hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner
syndrome, Pars planitis (peripheral uveitis), Pemphigus vulgaris,
Periaortitis, Periarteritis, Peripheral neuropathy, Perivenous
encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis
nodosa, Type I, II, & III autoimmune polyglandular syndromes,
Polymyalgia rheumatic, Polymyositis, Postmyocardial infarction
syndrome, Postpericardiotomy syndrome, Progesterone dermatitis,
Primary biliary cirrhosis, Primary sclerosing cholangitis,
Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis,
Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon,
Reflex sympathetic dystrophy, Reiter's syndrome, Relapsing
polychondritis, Restless legs syndrome, Retroperitoneal fibrosis
(Ormond's disease), Rheumatic fever, Rheumatoid arthritis, Riedel's
thyroiditis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma,
Sjogren's syndrome, Sperm & testicular autoimmunity, Stiff
person syndrome, Subacute bacterial endocarditis (SBE), Susac's
syndrome, Sympathetic ophthalmia, Takayasu's arteritis, Temporal
arteritis/Giant cell arteritis, Thrombotic, thrombocytopenic
purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis,
Ulcerative colitis, Undifferentiated connective tissue disease
(UCTD), Uveitis, Vasculitis, Vesiculobullous dermatosis, Vitiligo,
Waldenstrom Macroglobulinaemia, Warm idiopathic haemolytic anaemia
and Wegener's granulomatosis (now termed Granulomatosis with
Polyangiitis (GPA).
[0297] Additional indications may also include hyperviscosity
syndromes; cryoglobulinemia; recurrent focal and segmental
glomerulosclerosis in the transplanted kidney; HELLP syndrome;
Refsum disease; HIV-related neuropathy; rhabdomyolysis and
alloimune diseases.
[0298] In one embodiment the antibodies or fragments according to
the disclosure are employed in the treatment or prophylaxis of
epilepsy or seizures.
[0299] In one embodiment the antibodies or fragments according to
the disclosure are employed in the treatment or prophylaxis of
multiple sclerosis.
[0300] In embodiment the antibodies and fragments of the disclosure
are employed in alloimmune disease/indications which includes:
[0301] Transplantation donor mismatch due to anti-HLA antibodies
[0302] Foetal and neonatal alloimmune thrombocytopenia, FNAIT (or
neonatal alloimmune thrombocytopenia, NAITP or NAIT or NAT, or
foeto-maternal alloimmune thrombocytopenia, FMAITP or FMAIT).
[0303] Additional indications include: rapid clearance of
Fc-containing biopharmaceutical drugs from human patients and
combination of anti-FcRn therapy with other therapies--IVIg,
Rituxan, plasmapheresis. For example anti-FcRn therapy may be
employed following Rituxan therapy. In addition anti-FcRn therapy
may be used to rapidly clear imaging agents such as radiolabelled
antibodies used in imaging tumors.
[0304] In embodiment the antibodies and fragments of the disclosure
are employed in a neurology disorder such as: [0305] Chronic
inflammatory demyelinating polyneuropathy (CIDP) [0306]
Guillain-Barre syndrome [0307] Paraproteinemic polyneuropathies
[0308] Neuromyelitis optica (NMO, NMO spectrum disorders or NMO
spectrum diseases), and [0309] Myasthenia gravis.
[0310] In embodiment the antibodies and fragments of the disclosure
are employed in a dermatology disorder such as: [0311] Bullous
pemphigoid [0312] Pemphigus vulgaris [0313] ANCA-associated
vasculitis [0314] Dilated cardiomyopathy
[0315] In embodiment the antibodies and fragments of the disclosure
are employed in an Immunology, haematology disorder such as: [0316]
Idiopathic thrombocytopenic purpura (ITP) [0317] Thrombotic
thrombocytopenic purpura (TTP) [0318] Warm idiopathic haemolytic
anaemia [0319] Goodpasture's syndrome [0320] Transplantation donor
mismatch due to anti-HLA antibodies
[0321] In one embodiment the disorder is selected from Myasthenia
Gravis, Neuro-myelitis Optica, CIDP, Guillaume-Barre Syndrome,
Para-proteinemic Poly neuropathy, Refractory Epilepsy, ITP/TTP,
Hemolytic Anemia, Goodpasture's Syndrome, ABO mismatch, Lupus
nephritis, Renal Vasculitis, Sclero-derma, Fibrosing alveolitis,
Dilated cardio-myopathy, Grave's Disease, Type 1 diabetes,
Auto-immune diabetes, Pemphigus, Sclero-derma, Lupus, ANCA
vasculitis, Dermatomyositis, Sjogren's Disease and Rheumatoid
Arthritis.
[0322] In one embodiment the disorder is selected from autoimmune
polyendocrine syndrome types 1 (APECED or Whitaker's Syndrome) and
2 (Schmidt's Syndrome); alopecia universalis; myasthenic crisis;
thyroid crisis; thyroid associated eye disease; thyroid
ophthalmopathy; autoimmune diabetes; autoantibody associated
encephalitis and/or encephalopathy; pemphigus foliaceus;
epidermolysis bullosa; dermatitis herpetiformis; Sydenham's chorea;
acute motor axonal neuropathy (AMAN); Miller-Fisher syndrome;
multifocal motor neuropathy (MMN); opsoclonus; inflammatory
myopathy; Isaac's syndrome (autoimmune neuromyotonia),
Paraneoplastic syndromes and Limbic encephalitis.
[0323] The antibodies and fragments according to the present
disclosure may be employed in treatment or prophylaxis.
[0324] The present invention also provides a method of reducing the
concentration of undesired antibodies in an individual comprising
the steps of administering to an individual a therapeutically
effective dose of an anti-FcRn antibody or binding fragment thereof
described herein.
[0325] The present invention further provides the use of an
antibody molecule according to the present invention in the
manufacture of a medicament for the treatment and/or prophylaxis of
a pathological disorder described herein such as an autoimmune
disease.
[0326] In one embodiment the present disclosure comprises use of
antibodies or fragments thereof as a reagent for diagnosis, for
example conjugated to a reporter molecule. Thus there is provided
antibody or fragment according to the disclosure which is labelled.
In one aspect there is provided a column comprising an antibody or
fragment according to the disclosure.
[0327] Thus there is provided an anti-FcRn antibody or binding
fragment for use as a reagent for such uses as: [0328] 1)
purification of FcRn protein (or fragments thereof)--being
conjugated to a matrix and used as an affinity column, or (as a
modified form of anti-FcRn) as a precipitating agent (e.g. as a
form modified with a domain recognised by another molecule, which
may be modified by addition of an Fc (or produced as full length
IgG), which is optionally precipitated by an anti-Fc reagent)
[0329] 2) detection and/or quantification of FcRn on cells or in
cells, live or fixed (cells in vitro or in vivo in tissue or cell
sections). Uses for this may include quantification of FcRn as a
biomarker, to follow the effect of anti-FcRn treatment. For these
purposes, the candidate might be used in a modified form (e.g. by
addition of an Fc domain, as in full length IgG, or some other
moiety, as a genetic fusion protein or chemical conjugate, such as
addition of a fluorescent tag used for the purposes of detection).
[0330] 3) purification or sorting of FcRn-bearing cells labeled by
binding to candidate modified by ways exemplified in (1) and
(2).
[0331] Also provided by the present invention is provided an assay
suitable for assessing the ability of a test molecule such as an
antibody molecule to block FcRn activity and in particular the
ability of the cells to recycle IgG. Such an assay may be useful
for identifying inhibitors of FcRn activity, such as antibody
molecules or small molecules and as such may also be useful as a
batch release assay in the production of such an inhibitor.
[0332] In one aspect there is provided an assay suitable for
assessing the ability of a test molecule such as an antibody
molecule to block human FcRn activity and in particular the ability
of human FcRn to recycle IgG, wherein the method comprises the
steps of: [0333] a) coating onto a surface non-human mammalian
cells recombinantly expressing human FcRn alpha chain and human (32
microglobulin (.beta.2M), [0334] b) contacting the cells under
mildly acidic conditions such as about pH5.9 with a test molecule
and an IgG to be recycled by the cell for a period of time
sufficient to allow binding of both the test molecule and the IgG
to FcRn, optionally adding the test molecule before the IgG to be
recycled and incubating for a period of time sufficient to allow
binding of the test molecule to FcRn. [0335] c) washing with a
slightly acidic buffer, and [0336] d) detecting the amount of IgG
internalised and/or recycled by the cells.
[0337] In one aspect there is provided an assay suitable for
assessing the ability of a test molecule such as an antibody
molecule to block human FcRn activity and in particular the ability
of human FcRn to recycle IgG, wherein the method comprises the
steps of: [0338] a) coating onto a surface non-human mammalian
cells recombinantly expressing human FcRn alpha chain and human (32
microglobulin (.beta.2M), [0339] b) contacting the cells under
mildly acidic conditions such as about pH5.9 with a test antibody
molecule and an IgG to be recycled by the cell for a period of time
sufficient to allow binding of both the test antibody molecule and
the IgG to FcRn, optionally adding the test antibody molecule
before the IgG to be recycled and incubating for a period of time
sufficient to allow binding of the test antibody molecule to FcRn.
[0340] c) washing with a slightly acidic buffer to remove unbound
IgG and test antibody molecule, and [0341] d) detecting the amount
of IgG recycled by the cells.
[0342] In one aspect there is provided an assay suitable for
assessing the ability of a test molecule such as an antibody
molecule to block human FcRn activity and in particular the ability
of human FcRn to recycle IgG, wherein the method comprises the
steps of: [0343] a) coating onto a surface non-human mammalian
cells recombinantly expressing human FcRn alpha chain and human (32
microglobulin (.beta.2M), [0344] b) contacting the cells under
mildly acidic conditions such as about pH5.9 with a test antibody
molecule and an IgG to be recycled by the cell for a period of time
sufficient to allow binding of both the test antibody molecule and
IgG to FcRn, optionally adding the test antibody molecule before
the IgG to be recycled and incubating for a period of time
sufficient to allow binding of the test antibody molecule to FcRn.
[0345] c) washing with a slightly acidic buffer to remove unbound
IgG and test antibody molecule, [0346] d) incubating the cells in a
neutral buffer such as about pH 7.2 [0347] e) detecting the amount
of IgG recycled by the cells by determining the amount of IgG
released into the supernatant.
[0348] Suitable cells include Madin-Darby Canine Kidney (MDCK) II
cells. Transfection of MDCKII cells with human FcRn alpha chain and
human (32 microglobulin (.beta.2M) has previously been described by
Claypool et al., 2002, Journal of Biological Chemistry, 277, 31,
28038-28050. This paper also describes recycling of IgG by these
transfected cells.
[0349] Media for supporting the cells during testing includes
complete media comprising MEM (Gibco #21090-022), 1.times.
non-essential amino acids (Gibco 11140-035), 1.times. sodium
pyruvate (Gibco #11360-039), and L-glutamine (Gibco #25030-024).
Acidic wash can be prepared by taking HBSS+(PAA #H15-008) and
adding 1M IVIES until a pH 5.9+/-0.5 is reached. BSA about 1% may
also be added (Sigma #A9647).
A neutral wash can be prepared by taking HBSS+(PAA #H15-008) and
adding 10M Hepes pH 7.2+/-0.5 is reached. BSA about 1% may also be
added (Sigma #A9647).
[0350] Washing the cells with acidic buffer removes the unbound
test antibody and unbound IgG and allows further analysis to be
performed. Acidic conditions used in step (b) encourage the binding
of the IgG to FcRn and internalisation and recycling of the
same.
[0351] The amount of test antibody or fragment and IgG on only the
surface of the cells may be determined by washing the cells with
neutral wash and analysing the supernatant/washings to detect the
quantity of test antibody or IgG. Importantly a lysis buffer is not
employed. To determine the amount of IgG internalised by the cells
the antibody may first be removed from the surface of the cell with
a neutral wash and the cells lysed by a lysis buffer and then the
internal contents analysed. To determine the amount of IgG recycled
by the cells the cells are incubated under neutral conditions for a
suitable period of time and the surrounding buffer analysed for IgG
content. If the surface and internal antibody content of the cell
is required then the cell can be washed with acid wash to maintain
the antibody presence on the cell surface, followed by cell lysis
and analysis of the combined material.
[0352] Where it is desired to measure both internalisation and
recycling of the IgG samples are run in duplicate and testing for
internalisation and recycling conducted separately.
[0353] A suitable lysis buffer includes 150 mM NaCl, 20 mM Tris, pH
7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton-X 100, for each 10 ml add
protease inhibitors/phosphate inhibitors as described in
manufacturer's guidelines.
[0354] Typically the IgG to be recycled is labelled, in one example
a biotinylated human IgG may be used. The IgG can then be detected
employing, for example a streptavidin sulfo-tag detection antibody
(such as MSD #r32ad-5) 25 mL at 0.2 ug/mL of MSD blocking buffer.
Blocking buffer may comprise 500 mM Tris, pH7.5. 1.5M NaCl and 0.2%
Tween-20 and 1.5% BSA.
[0355] Alternatively the IgG may be pre-labelled with a fluorophore
or similar label.
[0356] In one embodiment a suitable surface is a plastic plate or
well such as a 96 well plate or similar, a glass slide or a
membrane. In one example cells are coated onto the surface at a
density that results in the formation of a monolayer.
[0357] In one embodiment the assay described herein is not a
measurement of transcytosis of an antibody top to bottom across a
membrane with a pH gradient there-across, for example acid
conditions one side of the membrane and neutral conditions on the
underside of the membrane.
[0358] In one example the test antibody or fragment and IgG may be
incubated with the cells in step (b) for about 1 hour for example
at ambient temperature under acidic conditions to allow
binding.
[0359] In one example the test antibody or fragment may be
incubated with the cells in step (b) for about 1 hour for example
at ambient temperature under acidic conditions to allow binding
before addition of the IgG to be recycled. Subsequently the IgG to
be recycled by the cell may be incubated with the cells in step (b)
for about 1 hour for example at ambient temperature under acidic
conditions to allow binding.
[0360] Neutral conditions facilitate release of the IgG into the
supernatant.
[0361] Comprising in the context of the present specification is
intended to meaning including.
[0362] Where technically appropriate embodiments of the invention
may be combined.
[0363] Embodiments are described herein as comprising certain
features/elements. The disclosure also extends to separate
embodiments consisting or consisting essentially of said
features/elements.
[0364] Technical references such as patents and applications are
incorporated herein by reference.
[0365] The present invention is further described by way of
illustration only in the following examples, which refer to the
accompanying Figures, in which:
[0366] FIG. 1 Shows % hIgG in transgenic mice determined by
LC-MS/MS
[0367] FIG. 1a shows the effect of 1638 IgG4P format on the
concentration of human IVIg in serum of human FcRn-transgenic
mice.
[0368] FIG. 1b shows the effect of 1638 FabFv and Fab'PEG formats
on the concentration of human IVIg in human FcRn-transgenic
mice
[0369] FIG. 1c shows the pharmacokinetics of 1638 IgG4P format in
human FcRn-transgenic mice.
[0370] FIG. 1d shows the pharmacokinetics of 1638 FabFv and Fab'PEG
formats in human FcRn-transgenic mice
[0371] FIG. 1e The effect of 1638 FabFv and Fab'PEG formats on the
concentration of serum albumin in human FcRn-transgenic mice.
[0372] FIG. 1f The effect of 1638 IgG4P format on the concentration
of serum albumin in human FcRn-transgenic mice.
[0373] FIG. 2 shows representative binding curves for
CA170_1638.g49 IgG4. The mean K.sub.D values (n=3) were 0.20 nM in
neutral buffer, & 0.22 nM in acidic buffer, respectively
[0374] FIG. 3 shows CA170_1638.g49 IgG4 inhibits IgG recycling in
MDCK II clone 15 cells
[0375] FIG. 4 shows CA170_1638.g49 IgG4 inhibits IgG transcytosis
in MDCK II clone 15 cells.
[0376] FIG. 5 shows CA170_1638.g49 FabFv inhibits IgG transcytosis
in MDCK II clone 15 cells.
[0377] FIG. 6 shows representative binding curves for
CA170_1638.g49 IgG4. The mean K.sub.D values (n=3) were 0.3 in
neutral buffer, and 0.43 in acidic buffer, respectively (see Table
2).
[0378] FIG. 7 shows CA170_1638 CDR sequences
[0379] FIG. 8 Antibody sequences according to the present
disclosure
[0380] FIG. 9a Humanisation of antibody 1638.g49
[0381] FIG. 9b Humanisation of antibody 1638.g49
EXAMPLES
Abbreviations
[0382] .degree. C. temperature, degrees centigrade. [0383] ATR FTIR
Attenuated Total Reflectance Fourier Transform Infra-Red
Spectroscopy [0384] CH2 constant heavy chain region 2 [0385] cIEF
capillary isoelectric focusing [0386] DSC differential scanning
calorimetry [0387] G0F fucosylated aglactosyl biantennary glycan
[0388] H chain Heavy chain [0389] HPLC high performance liquid
chromatography [0390] IgG immunoglobulin G [0391] L chain Light
chain [0392] nLCMS nano-liquid chromatography mass spectrometry
[0393] PBS phosphate-buffered saline buffer [0394] pI isoelectric
point [0395] SD standard deviation [0396] SEC size exclusion
chromatography [0397] ToF time of flight [0398] T.sub.m melting
temperature [0399] TCEP tris(2-carboxyethyl)phosphine [0400] THP
Tris(hydroxypropyl)phosphine [0401] Tris
tris(hydroxymethyl)aminomethane
[0402] The following immunizations were performed in order to
generate material for B cell culture and antibody screening:
[0403] Sprague Dawley rats were immunized with three shots of
NIH3T3 mouse fibroblasts co-expressing mutant human FcRn (L320A;
L321A) (Ober et al., 2001 Int. Immunol. 13, 1551-1559) and mouse
.beta.2M with a fourth final boost of human FcRn extracellular
domain.
[0404] Sera were monitored for both binding to mutant FcRn on
HEK-293 cells and for its ability to prevent binding of Alexafluor
488-labelled human IgG. Both methods were performed by flow
cytometry. For binding, phycoerythrin (PE)-labelled anti mouse or
rat Fc specific secondary reagents were used to reveal binding of
IgG in sera.
[0405] B cell cultures were prepared using a method similar to that
described by Zubler et al. (1985). Briefly, B cells at a density of
approximately 5000 cells per well were cultured in bar-coded
96-well tissue culture plates with 200 .mu.l/well RPMI 1640 medium
(Gibco BRL) supplemented with 10% FCS (PAA laboratories ltd), 2%
HEPES (Sigma Aldrich), 1% L-Glutamine (Gibco BRL), 1%
penicillin/streptomycin solution (Gibco BRL), 0.1%
.beta.-mercaptoethanol (Gibco BRL), 2-5% activated rabbit
splenocyte culture supernatant and gamma-irradiated EL-4-B5 murine
thymoma cells (5.times.10.sup.4/well) for seven days at 37.degree.
C. in an atmosphere of 5% CO.sub.2.
[0406] The presence of FcRn-specific antibodies in B cell culture
supernatants was determined using a homogeneous fluorescence-based
binding assay using HEK-293 cells transiently transfected with
mutant FcRn (surface-stabilised) as a source of target antigen. 10
ul of supernatant was transferred from barcoded 96-well tissue
culture plates into barcoded 384-well black-walled assay plates
containing 5000 transfected HEK-293 cells per well using a Matrix
Platemate liquid handler. Binding was revealed with a goat anti-rat
or mouse IgG Fc.gamma.-specific Cy-5 conjugate (Jackson). Plates
were read on an Applied Biosystems 8200 cellular detection system.
From 3800.times.96-well culture plates, representing 38 different
immunized animals, 9800 anti-human FcRn binders were identified. It
was estimated that this represented the screening of approximately
2.5 billion B cells.
[0407] Following primary screening, positive supernatants were
consolidated on 96-well bar-coded master plates using an Aviso Onyx
hit-picking robot and B cells in cell culture plates frozen at -80
C. Master plates were then screened in a Biacore assay in order to
identify wells containing antibodies of high affinity and those
which inhibited the binding of human IgG to FcRn (see below).
Biomolecular interaction analysis using surface plasmon resonance
technology (SPR) was performed on a BIAcore T200 system (GE
Healthcare). Goat anti-rat IgG, Fc gamma (Chemicon International
Inc.) in 10 mM NaAc, pH 5 buffer was immobilized on a CM5 Sensor
Chip via amine coupling chemistry to a capture level of approx.
19500 response units (RU) using HBS-EP.sup.+ as the running buffer.
50 mM Phosphate, pH6+150 mM NaCl was used as the running buffer for
the affinity and blocking assay. B cell culture supernatants were
diluted 1 in 5 in 200 mM Phosphate, pH6+150 mM NaCl. A 600 s
injection of diluted B cell supernatant at 5 .mu.l/min was used for
capture by the immobilized anti-rat IgG, Fc. Human FcRn at 100 nM
was injected over the captured B cell culture supernatant for 180 s
at 30 .mu.l/min followed by 360 s dissociation. Human IgG (Jackson
ImmunoResearch) was injected over for 60 s with 180 s dissociation
at 30 .mu.l/min.
[0408] The data was analysed using T200 evaluation software
(version 1.0) to determine affinity constants (K.sub.D) of
antibodies and determine those which blocked IgG binding.
[0409] As an alternative assay, master plate supernatants were also
screened in a cell-based human IgG blocking assay. 25 ul of B cell
culture supernatant from master plates were added to 96 well
U-bottomed polypropylene plate. Mutant hFcRn-transfected HEK-293
cells (50,000 cells per well in 25 ul PBS pH6/1% FCS) were then
added to each well and incubated for 1 hour at 4.degree. C. Cells
were washed twice with 150 ul of PBS media. Cells were then
resuspended in 50 ul/well PBS/FCS media containing human IgG
labelled with Alexafluor 488 or 649 at 7.5 ug/ml and incubated 1
hour at 4.degree. C. Cells were then washed twice with 150 ul of
media and then resuspended in 35 ul/well of PBS/FCS media
containing 1% formaldehyde as fixative. Plates were then read on a
FACS Canto 2 flow cytometer.
[0410] To allow recovery of antibody variable region genes from a
selection of wells of interest, a deconvolution step had to be
performed to enable identification of the antigen-specific B cells
in a given well that contained a heterogeneous population of B
cells. This was achieved using the Fluorescent foci method.
Briefly, Immunoglobulin-secreting B cells from a positive well were
mixed with streptavidin beads (New England Biolabs) coated with
biotinylated human FcRn and a 1:1200 final dilution of a goat
anti-rat or mouse Fc.gamma. fragment-specific FITC conjugate
(Jackson). After static incubation at 37.degree. C. for 1 hour,
antigen-specific B cells could be identified due to the presence of
a fluorescent halo surrounding that B cell. These individual B
cells, identified using an Olympus microscope, were then picked
with an Eppendorf micromanipulator and deposited into a PCR tube.
Fluorescent foci were generated from 268 selected wells.
[0411] Antibody variable region genes were recovered from single
cells by reverse transcription polymerase chain reaction (RT)-PCR
using heavy and light chain variable region-specific primers. Two
rounds of PCR were performed on an Aviso Onyx liquid handling
robot, with the nested 2.degree. PCR incorporating restriction
sites at the 3' and 5' ends allowing cloning of the variable
regions into a mouse yl IgG (VH) or mouse kappa (VL) mammalian
expression vector. Paired heavy and light chain constructs were
co-transfected into HEK-293 cells using Fectin 293 (Invitrogen) and
cultured in 48-well plates in a volume of 1 ml. After 5-7 days
expression, supernatants were harvested and antibody subjected to
further screening.
[0412] PCR successfully recovered heavy and light chain cognate
pairs from single B cells from 156 of the selected wells. DNA
sequence analysis of the cloned variable region genes identified a
number of unique families of recombinant antibody. Following
expression, transient supernatants were interrogated in both human
IgG FACS blocking (described above) and IgG recycling assays. In
some cases, purified mouse .gamma.1 IgG was produced and tested
(data labeled accordingly).
[0413] The recycling assay used MDCK II cells (clone as described
in Examples 5, 6 and 7 below) over-expressing human FcRn and beta 2
microglobulin plated out at 25,000 cells per well of a 96 well
plate. These were incubated overnight at 37.degree. C., 5%
CO.sub.2. The cells were washed with HMS+Ca/Mg pH 7.2+1% BSA and
then incubated with 50 .mu.l of varying concentrations of HEK-293
transient supernatant or purified antibody for 1 hour at 37.degree.
C., 5% CO.sub.2. The supernatant was removed and 500 ng/ml of
biotinylated human IgG (Jackson) in 50 .mu.l of HBSS+Ca/Mg pH
5.9+1% BSA was added to the cells and incubated for 1 hour at
37.degree. C., 5% CO.sub.2. The cells were then washed three times
in HBSS+Ca/Mg pH 5.9 and 100 .mu.l of HBSS+Ca/Mg pH 7.2 added to
the cells and incubated at 37.degree. C., 5% CO.sub.2 for 2 hours.
The supernatant was removed from the cells and analysed for total
IgG using an MSD assay with an anti-human IgG capture antibody
(Jackson) and a streptavidin-sulpho tag reveal antibody (MSD). The
inhibition curve was analysed by non-linear regression to determine
IC50 values.
[0414] Based on performance in these assays a family of antibodies
was selected comprising the six CDRs given in SEQ ID NOs 1 to 6.
Antibody CA170_01638 had the best activity and was selected for
humanisation.
Example 1 Humanisation Method
[0415] Antibody CA170_01638 was humanised by grafting the CDRs from
the rat antibody V-regions onto human germline antibody V-region
frameworks. In order to recover the activity of the antibody, a
number of framework residues from the rat V-regions were also
retained in the humanised sequence. These residues were selected
using the protocol outlined by Adair et al. (1991) (Humanised
antibodies WO91/09967). Alignments of the rat antibody (donor)
V-region sequences with the human germline (acceptor) V-region
sequences are shown in FIGS. 9A and B, together with the designed
humanised sequences. The CDRs grafted from the donor to the
acceptor sequence are as defined by Kabat (Kabat et al., 1987),
with the exception of CDR-H1 where the combined Chothia/Kabat
definition is used (see Adair et al., 1991 Humanised antibodies.
WO91/09967). Human V-region IGKV1-27 plus JK4 J-region
(http://www.imgt.org/) was chosen as the acceptor for the light
chain CDRs. Human V-region IGHV3-7 plus JH3 J-region
(http://www.imgt.org/) was chosen as the acceptor for the heavy
chain CDRs.
[0416] Genes encoding a number of variant heavy and light chain
V-region sequences were designed and constructed by an automated
synthesis approach by Entelechon GmbH. Further variants of both
heavy and light chain V-regions were created by modifying the VH
and VK genes by oligonucleotide-directed mutagenesis. These genes
were cloned into a number of vectors to enable expression of
humanised 1638 Fab or IgG4 antibody in E. coli and mammalian cells,
respectively. The variant chains, and combinations thereof, were
assessed for their potency relative to the parent antibody, their
biophysical properties and suitability for downstream processing,
leading to the selection of the gL7 light chain graft and gH33
heavy chain graft. The final selected gL7 and gH33 graft sequences
are shown in FIGS. 9A and B, respectively. This V-region pairing
was named 1638.g49.
[0417] The light chain framework residues in graft gL7 are all from
the human germline gene, with the exception of residues 70 and 71
(Kabat numbering), where the donor residues Histidine (H70) and
Tyrosine (T71) were retained, respectively. Retention of these two
residues was important for full potency of the humanised antibody
or Fab. Residue 56 in CDRL2 of the gL7 graft was mutated from an
Aspartic acid (D56) to a Glutamic acid (E56) residue, thus removing
a potential Aspartic acid isomerization site from the gL7 sequence.
The heavy chain framework residues in graft gH33 are all from the
human germline gene, with the exception of residues 48 and 78
(Kabat numbering), where the donor residues Leucine (L48) and
Alanine (A78) were retained, respectively. Retention of these two
residues was essential for full potency of the humanised antibody
or Fab.
[0418] For expression of 1638.g49 Fab in E coli, the humanised
heavy and light chain V-region genes were cloned into the UCB
expression vector pTTOD, which contains DNA encoding the human
C-kappa constant region (K1m3 allotype) and the human gamma-1 CH1
region (with or without hinge region) (G1m17 allotype).
[0419] For expression of 1638.g49 IgG4 in mammalian cells, the
humanised light chain V-region gene was joined to a DNA sequence
encoding the human C-kappa constant region (K1m3 allotype), to
create a contiguous light chain gene. The humanised heavy chain
V-region gene was joined to a DNA sequence encoding the human
gamma-4 heavy chain constant region with the hinge stabilising
mutation S241P (Angal et al., Mol Immunol. 1993, 30(1):105-8), to
create a contiguous heavy chain gene. The heavy and light chain
genes were cloned into a mammalian expression vector.
[0420] Another earlier graft, 1638.g28 was used in Example 8A
described herein below and this contained more donor residues in
the heavy chain (gH2) than the 1638.g49 graft (F24, L48, K71, T73,
A78 and V93). Also the light chain of this antibody (gL2) contains
the unmodified CDRL2 given in SEQ ID NO: 5 rather than the modified
CDRL2 of SEQ ID NO: 7 which is used in 1638.g49. Sequences of both
sets of antibodies are given in FIG. 8. Antibody 1638.g28 was
expressed as a Fab' fragment as described above for 1638.g49.
Example 2 Preparation of 1638.g49 Fab'-PEG Conjugate
[0421] Fab' expressed in the periplasm of E. coli was extracted
from cells by heat extraction. Fab' purified by Protein G affinity
purification with an acid elution. Fab' reduced and PEGylated with
40 kDa PEG (SUNBRIGHT GL2-400MA3). PEG is covalently linked via a
maleimide group to one or more thiol groups in the antibody
fragment. PEGylation efficiency was confirmed by SE-HPLC. Fab'PEG
was separated from un-PEGylated Fab' and diFab' by cation exchange
chromatography. Fractions analyzed by SE-HPLC and SDS-PAGE. Pooling
carried out to minimize levels of impurities. Final sample
concentrated and diafiltered into desired buffer.
Example 3 Affinity for hFcRn Binding
[0422] Biomolecular interaction analysis using surface plasmon
resonance technology (SPR) was performed on a Biacore T200 system
(GE Healthcare) and binding to human FcRn extracellular domain
determined. Human FcRn extracellular domain was provided as a
non-covalent complex between the human FcRn alpha chain
extracellular domain (SEQ ID NO: 48) and (32 microglobulin
(.beta.2M) (SEQ ID NO: 72). Affinipure F(ab')2 fragment goat
anti-human IgG, Fc fragment specific (for IgG4 capture) (Jackson
ImmunoResearch Lab, Inc.) at 50 .mu.g/ml in 10 mM NaAc, pH 5 buffer
was immobilized on a CM5 Sensor Chip via amine coupling chemistry
to a capture level between 5000-6000 response units (RU) using
HBS-EP.sup.+ (GE Healthcare) as the running buffer.
[0423] 50 mM Phosphate, pH6+150 mM NaCl+0.05% P20 or HBS-1.sup.3+,
pH7.4 (GE Healthcare) was used as the running buffer for the
affinity assay. The antibody, 1638.g49 IgG4P was diluted to 1
.mu.g/ml in running buffer. A 60 s injection of IgG4 at 10
.mu.l/min was used for capture by the immobilized anti-human IgG,
Fc. Human FcRn extracellular domain was titrated from 20 nM to 1.25
nM over the captured anti-FcRn antibody (IgG4) for 300 s at 30
.mu.l/min followed by 1200 s dissociation. The surface was
regenerated by 2.times.60 s 50 mM HCl at 10 .mu.l/min for the
running buffer at pH6 or by 60 s 40 mM HCl and 30 s 10 mM NaOH for
the running buffer at pH7.4. The data was analysed using T200
evaluation software (version 1.0) using the 1:1 binding model with
local Rmax.
TABLE-US-00002 TABLE 1 Affinity data for anti-hFcRn 1638.g49 IgG4P
at pH 6.0 and pH 7.4 pH 6.0 Human FcRn 1638.g49 IgG4P ka
(M.sup.-1s.sup.-1) kd (s.sup.-1) KD (M) 1 1.10E+06 1.43E-04
1.29E-10 2 1.10E+06 1.39E-04 1.26E-10 3 1.11E+06 1.40E-04 1.27E-10
Mean 1.10E+06 1.41E-04 1.27E-10 pH 7.4 Human FcRn 1638.g49 IgG4P ka
(M.sup.-1s.sup.-1) kd (s.sup.-1) KD (M) 1 9.75E+05 2.51E-05
2.57E-11 2 9.62E+05 3.19E-05 3.32E-11 3 9.62E+05 2.82E-05 2.93E-11
Mean 9.67E+05 2.84E-05 2.94E-11
[0424] The affinity of 1638.49 g IgG4 was therefore determined to
be 127 pM at pH 6.0 and 29 pM at pH7.4.
Example 4
[0425] An IgG4P full length molecule and Fab-dsFv molecule where
the 1638.g49 variable region was incorporated into the Fab domain
of each format were analysed for biochemical integrity and
biophysical stability.
Methods and Results
1. Sequence Confirmation.
i) Protein Sequencing (Edman Chemical Method)
[0426] The N-terminal amino acid sequence of both IgG4 and Fab-dsFv
samples was obtained using an Applied Biosystems Procise 494
instrument. This was operated as recommended by the instrument
manufacturer. Approximately 100 pmoles of each sample was applied
to discs of polyvinylidene difluoride (Prosorb, used as per
manufacturer's recommendations) and subjected to 18 cycles which
included two blank runs and a standard hence resulting in the
analysis of the first 15 amino acid residues of the heavy and light
chains. Analysis was performed using SequencePro Data Analysis
Application V2.0.
[0427] For each sample, the observed sequence was a mixture of two,
approximately equally-abundant sequences, EVQLVESGGGLVQPG (SEQ ID
NO: 67) and DIQMTQSPSSLSASV (SEQ ID NO: 68) consistent with the
N-terminal sequences expected from the heavy and light chain gene
sequences respectively. The approximately equal abundance suggested
equal molar amounts of the 2 chains, with little to no significant
N-terminal blockage.
ii) Mass Spectrometry Analysis
a) Intact Mass Analysis
[0428] Intact mass spectrometry analysis was performed on two
batches of the IgG4 and the Fab-dsFv molecule after reduction with
20 mM TCEP for one hour. Masses were measured on an Agilent 6510
mass spectrometer equipped with a chip cube interface and a C8 chip
(43 mm Zorbax 300A C8 column+43 nL trap). All samples were diluted
to 0.1 mg/ml in 98% water/2% methanol/0.3% formic acid (solvent A)
prior to injection and 0.3 .mu.L was loaded onto the system.
Proteins were eluted from the chip into the mass spectrometer using
a gradient to 40% acetonitrile/0.1% formic at 350 nL/min. ToF-MS
data were collected in positive-ion mode between 500 and 5000 m/z
and processed using Agilent MassHunter software.
[0429] The observed masses for both the light chain and the heavy
chain for both formats is shown below (Table 2).
TABLE-US-00003 TABLE 2 Observed mass table of two IgG4 batches and
Fab-dsFv L-chain H-chain Expected.sup.1 Observed ppm Expected.sup.1
Observed ppm IgG4 23503.3 23505.8 106 50764.7 50768.9 83 Batch #1
IgG4 23505.7 102 50768.9 83 Batch #2 FabFv 36384.4 [L1] 27 38298.8
[H1] 60 36385.4 38301.1 [L2] 88 [H2] 99 36387.6 38302.6
.sup.1Expected mass calculated from the amino acid sequence with
the addition of IgG4: 2 L- and 4 H- intrachain disulphides, G0F
glycosylation and clipping of C-terminal Lys from the H-chain
FabFv: 3 L- and 3 H- intrachain disulphides.
[0430] The intact mass analysis of the TCEP reduced IgG4 was
consistent with the expected sequences with predominantly GOF
glycosylation and clipped C-terminal lysine (approximately 90%) on
the H-chain which is typical of recombinant IgG.
[0431] Similarly the intact mass spectra of the Fab-dsFv chains was
consistent with the sequence mass and expected number of
disulphides. There was heterogeneity in the observed mass of both
chains presumably due to partial reduction of the intra-chain
disulphides by TCEP.
b) Disulphide Mapping was Performed on IgG4 Only.
[0432] IgG4 (50 ug) were treated with 0.15% Rapigest in Tris-HCl
pH7.5 at 50.degree. C. for 15 minutes and any free cysteines
alkylated with iodoacetamide. Trypsin (1:25 w/w) was added and
proteins were hydrolysed overnight at room temperature and then the
reaction quenched by the addition of formic acid (5% v/v) and any
precipitate was removed by centrifugation. Samples were stored at
-20.degree. C. and diluted 1:1 with water before loading on the
LC-MS system. Aliquots (.about.3-5 ug) were loaded onto a
2.1.times.150 mm C18 column (Waters BEH1.7u) equilibrated with
water containing 0.2% formic acid and eluted with a gradient of
acetonitrile/1-propanol into a Waters Xevo mass spectrometer
operated in +ve-ion MS.sup.E mode. Data was analysed with MassLynx
and BioPharmaLynx software.
[0433] The results indicated that all the expected
disulphide-linked peptides were observed except the inter
H--H-chain peptide T19-SS-T19 species which was only observed with
a single disulphide bond and at low intensity. There was no
evidence for any scrambled disulphide species or
carbamidomethylated cysteine residues.
2. Biochemical Analysis
[0434] Size Exclusion Chromatography HPLC (SEC HPLC)
[0435] Size exclusion chromatography allowed analysis of monomeric
and oligomeric material. It was performed using a TSK G3000SW (7.7
mm I.D.times.30.0 cm L) column connected to an Agilent 1100 system.
The samples (25 .mu.l/25 .mu.g injection) were eluted isocratically
in 0.2 M sodium phosphate, pH 7 at 1.0 ml/min for 30 minutes,
30.degree. C. Elution was monitored by absorption at 280 nm.
[0436] The elution profiles showed that the IgG4 and Fab-dsFv were
homogeneous and eluted at expected retention times as judged by SEC
standards (BioRad 151-1901).
3. Molecular Charge.
[0437] Capillary isoelectric focusing (cIEF) was conducted to
estimate pI and acidic species content.
[0438] IgG4 and Fab-dsFv samples were diluted to 1 mg/ml in HPLC
grade water for analysis (non-reduced condition). The samples were
also subjected to reduction (2 mM THP/30 minutes) and alkylation
(20 mM iodoacetamide/80 minutes) to analyse for cysteine
adducts.
[0439] Samples were prepared by mixing the following: 30 .mu.l
protein sample, 0.35% methylcellulose, 4% pH3-10 ampholytes
(Pharmalyte), 1 .mu.l of each synthetic pI marker (4.65 and 9.77)
and HPLC grade water to make up the final volume to 100 .mu.l. The
mixture was then analysed using iCE280 IEF analyser (Convergent
Biosciences), pre-focusing at 1500 V for 1 minute followed by
focusing at 3000 V for 6 minutes. The calibrated electropherograms
were then integrated using Empower software (from Waters).
[0440] The pI was taken to be that of the main species (largest
peak).
[0441] For the IgG4 format, the main species had a pI of 7.3. This
was assumed to be the clipped parent molecule (removal of the C
terminal lysine, corroborated by mass spectrum analysis) which is
not atypical for IgG molecules. The clipped molecule would be more
acidic that the parent molecule (basic peak at 7.4). There was no
change to the pI profile pre- and post-reduction and alkylation,
indicating that there were no cysteine adducts.
[0442] For the Fab-dsFv format, the pI was taken to be that of the
main species (largest peak) which was 9.0. A more acidic species
(pI 8.8) was also evident which was less prominent post
reduction/alkylation indicating the presence of a reducible
adduct.
[0443] For both formats, minor peaks were present being either
acidic (to the left of the main peak) or basic (to the right of the
main peak). These species were presumed to be derivatives of the
main species, but were not characterised further.
4. Thermal Stability (T.sub.m)
[0444] When heated, a protein will tend to unfold, and the more
stably-folded a protein structure is, the more heat is required to
unfold it. Therefore, thermal stability (measured as melting
temperature, T.sub.m) is a measure of the stability of folding of a
protein, or resistance of a molecule to unfolding (denaturation),
which may be a prerequisite to aggregate formation. In a
temperature gradient, in defined conditions, the temperature at
which 50% of molecules are unfolded is T.sub.m. T.sub.m estimations
were made by two independent methods
[0445] i) Thermofluor Assay, measurement of 50% unfolding by
binding of a fluorescent dye (Sypro Orange) to exposed hydrophobic
surfaces that become exposed upon heat induced unfolding and
[0446] ii) Differential Scanning calorimetry (DSC).
[0447] Results from the two techniques generally correlate,
differing slightly in absolute value because methods employed are
different.
[0448] i) Thermofluor Assay
[0449] Samples were prepared as follows: 5 .mu.l of 30.times. sypro
orange was placed in a 96 well V-bottomed plate. Then, 45 .mu.l of
protein sample at 0.1 mg/ml was then added. This mix was pipetted,
in 10 .mu.l quadruplicates, into a 384 well plate. The format of
the 384 well plate was: sample 1: wells A1, B1, A2, B2; sample 2:
wells C1, D1, C2, D2. An inter-assay control was included, being an
irrelevant IgG4. This control, at 0.1 mg/ml (in PBS pH 7.4) was
added to 5 .mu.l of 30.times. concentrated dye, 10 .mu.l of this
master mix being placed into the 384 well in quadruplicate. The
plates were placed in a 7900HT fast real-time PCR system and heated
from 20.degree. C. to 99.degree. C. using a ramp rate of
1.1.degree. C./min; a CCD device simultaneously monitors the
fluorescence changes in the wells. A modified XE template (IDBS) is
used to process the intensity data and take into account multiple
transitions. Two unfolding transitions were evident for both the
IgG4 and the Fab-dsFv molecules. The T.sub.m 2 value for both
molecules represented the Fab unfolding domain and was shown to be
slightly lower for the IgG4 format. The T.sub.m1 value represented
the CH2 (constant heavy chain) domain and the dsFv domain of the
IgG4 and Fab-dsFv molecule respectively. The Fab-dsFv format was
shown to be more thermally stable than the IgG format in PBS, pH
7.4.
TABLE-US-00004 Sample T.sub.m 1 Mean (.degree. C.) T.sub.m 1 SD
T.sub.m 2 Mean (.degree. C.) T.sub.m 2 SD IgG4 65.4 0.1 81.1 0.4
Fab-dsFv 73.6 0.4 83.1 0.4
ii) DSC Method
[0450] DSC analysis was performed on the Fab-dsFv molecule only for
corroboration of the Thermofluor data and to determine the effect
of two different buffer types (PBS pH7.4 and 50 mM sodium
acetate/125 mM sodium chloride, pH 5.0) on the thermal
stability.
[0451] Samples at 1 mg/ml in PBS pH7.4 and 50 mM sodium acetate/125
mM sodium chloride, pH 5.0 with respective reference buffers were
loaded onto the MicroCal VP Capillary DSC instrument in triplicate.
The system settings included temperature scan from 20.degree. C. to
110.degree. C. and a scan rate of 60.degree. C./hr. The final
thermograms were processed using Origin software according to the
manufacturer's instructions. The T.sub.m was determined using
software's automated T.sub.m detection algorithm (for the main
transition) and manually peak picked for any other transitions that
was not automatically detected by the software.
[0452] Two distinct transitions could be observed in the two
buffers tested.
[0453] The lower infolding transition (T.sub.m 1) represented the
dsFv domain of the Fab-dsFv molecule and the higher transition
temperature (T.sub.m 2) represented the Fab domain.
[0454] The DSC data was in good agreement with the data obtained
from the Thermofluor assay. This technique was capable of being
able to discriminate between the two unfolding domains more easily
than the Thermofluor assay.
[0455] The Fab-dsFv molecule showed a slight increase in thermal
stability in the 50 mM sodium acetate/125 mM sodium chloride, pH
5.
TABLE-US-00005 Buffer T.sub.m1 mean (.degree. C.) SD T.sub.m 2 mean
(.degree. C.) SD Fab-dsFv (50 mM NaOAc/ 86.1 0 73.6 0.15 125 mM
NaCl, pH 5) Fab-dsFv (PBS, pH 7.4) 84.1 0.1 71.2 0.06
5. Molecular Structure: Attenuated Total Reflectance Fourier
Transform Infra-Red Spectroscopy (ATR FTIR) This technique was used
to compare the extent of interaction between .beta.-sheets within
the molecule (intra-.beta.-sheet) and between separate molecules
(inter-.beta.-sheet).
[0456] The analysis was performed using the Bruker Tensor 27 FTIR
spectrometer and the BIOATR II cell sampling accessory using a
resolution of 4 cm.sup.-1; 120 scans; aperture setting 6 mm and 20
.mu.L sample volume at 20.degree. C. where the following procedure
was performed for the analysis of the Fab-dsFv only. [0457] 1. Five
air background spectra were measured using the method BIOATR 10 06
10. xpm. [0458] 2. 20 .mu.L of sigma PBS pH7.4 was added to the
cell and then removed [0459] 3. 20 .mu.L of sigma PBS pH7.4 was
added to the cell and a spectrum was taken, the buffer was removed
and fresh buffer was added and a spectrum taken (in duplicate).
[0460] 4. 20 .mu.L of sample was added to the cell and a spectrum
was taken, the sample was then removed from the cell. [0461] 5. 20
.mu.L of sigma PBS pH7.4 was added to the cell and removed [0462]
6. 20 .mu.L of sample was added to the cell and a spectrum was
taken, the sample was then removed from the cell. (in duplicate)
[0463] 7. The cell was then cleaned following procedure below:
[0464] a. 20 .mu.L 1% SDS added to cell+cleaned with Q-tip [0465]
b. 20 .mu.L 1% SDS added to cell and removed [0466] c. 5 times 20
.mu.L H.sub.2O added to cell and removed [0467] d. 20 .mu.L buffer
added to cell and removed [0468] 8. The data was analysed to
produce the final data format in the following way. [0469] a.
Buffer spectrum 1 was subtracted from the Fab-dsFv spectrum 1 and
then repeated with buffer spectrum 2 and Fab-ds Fv spectrum 2.
[0470] b. The data was cut to 2200 cm-1 to 1000 cm-1 [0471] c. The
duplicate spectra were averaged. [0472] d. A second derivative was
then taken with a 25 point smoothing. This was the final data
format shown.
[0473] The results of the analysis showed that the Fab-dsFv had the
intra-beta sheet characteristics typical of antibody molecules.
Example 5 Cell-Based Potency
[0474] Cell-based assays were performed using Madin-Darby Canine
Kidney (MDCK) II cells which had been stably transfected with a
human FcRn and human B2M double gene vector with a Geneticin
selection marker. A stable cell clone was selected that was able to
recycle and transcytose human IgG and this was used for all
subsequent studies. It will be referred to as MDCK II clone 15.
[0475] Cell Based Affinity of CA170_1638.g49 IgG4 for Human
FcRn
[0476] Quantitative flow cytometry experiments were performed using
MDCK II clone 15 cells and AlexaFluor 488-labelled CA170_1638.g49
IgG4. Specific binding of antibody to FcRn across a range of
antibody concentrations was used to determine K.sub.D. The analyses
were performed in both neutral and acidic buffers to determine
whether environmental pH comparable to that found in blood plasma
(pH7.4) or endosomes (pH6) had any effect on the antibody
binding.
[0477] FIG. 2 shows representative binding curves for
CA170_1638.g49 IgG4 The mean K.sub.D values (n=3) were 0.20 in
neutral buffer, and 0.22 in acidic buffer, respectively (see Table
4).
TABLE-US-00006 TABLE 4 Mean K.sub.D values (nM) for CA170_1638.g49
IgG4 on MDCK II clone 15 cells. Antibody format Human FcRnpH 7.4
Human FcRnpH 6.0 1638.g49 IgG4 0.20 0.22
[0478] FIG. 2 shows CA170_1638.g49 IgG4 binding on MDCK II clone 15
cells in acidic and neutral pH.
[0479] MDCK II clone 15 cells were incubated in Facs buffer (PBS
with 0.2% w/v BSA, 0.09% w/v NaN3) for 30 mins prior to the
addition of Alexa-fluor 488-labelled CA170_1638.g49 IgG4 for 1 hour
in Facs buffer at either pH 7.4 or pH 6. The final antibody
concentrations ranged from 400 nM to 0.003 nM. The cells were
washed in ice cold Facs buffer then analysed by flow cytometry
using a Guava flow cytometer (Millipore, UK). Titration data sets
were also produced for isotype control antibodies for each antibody
format to determine non-specific binding. The number of moles of
bound antibody was calculated using interpolated values from a
standard curve generated from beads comprised of differing amounts
of fluorescent dye. Geometric mean fluorescence values were
determined in the flow cytometric analyses of cells and beads.
Non-specific binding was subtracted from the anti-FcRn antibody
values and the specific binding curve generated was analysed by
non-linear regression using a one-site binding equation (Graphpad
Prism.RTM.) to determine the K.sub.D. Data is representative of 3
experiments.
[0480] CA170_1638.g49 IgG4 can bind human FcRn expressed on cells
at both acidic and neutral pH
Example 6 Functional Cell Based Assays
[0481] FcRn expression is primarily intracellular (Borvak J et al.
1998, Int. Immunol., 10 (9) 1289-98 and Cauza K et al. 2005, J.
Invest. Dermatol., 124 (1), 132-139), and associated with endosomal
and lysosomal membranes. The Fc portion of IgG binds to FcRn at
acidic pH (<6.5), but not at a neutral physiological pH (7.4)
(Rhagavan M et al. 1995) and this pH-dependency facilitates the
recycling of IgG.
[0482] Once it is taken up by pinocytosis and enters the acidic
endosome, IgG bound to FcRn will be recycled along with the FcRn to
the cell surface, whereas at the physiologically neutral pH the IgG
will be released. (Ober R J et al. 2004, The Journal of Immunology,
172, 2021-2029). Any IgG not bound to FcRn will enter the lysosomal
degradative pathway.
[0483] An in vitro assay was established to examine the ability of
CA170_1638.g49 IgG4 to inhibit the IgG recycling capabilities of
FcRn. Briefly, MDCK II clone 15 cells were incubated with
biotinylated human IgG, in the presence and absence of 1638 IgG4 in
an acidic buffer (pH 5.9) to allow binding to FcRn. All excess
antibody was removed and the cells incubated in a neutral pH buffer
(pH 7.2) which allows release of surface-exposed, bound and
internalised IgG into the supernatant. The inhibition of FcRn was
followed using an MSD assay to detect the amount of IgG recycled
and thus released into the supernatant.
[0484] FIG. 3 shows CA170_1638.g49 IgG4 inhibits IgG recycling in
MDCK II clone 15 cells. MDCK II clone 15 cells were plated at
15,000 cells per well in a 96 well plate and incubated overnight at
37.degree. C., 5% CO.sub.2. The cells were incubated with 1 ug/ml
of biotinylated human IgG (Jackson) in the presence and absence of
CA170_1638.g49 IgG4 in HBSS.sup.+ (Ca/Mg) pH 5.9+1% BSA for 1 hour
at 37.degree. C., 5% CO.sub.2. The cells were washed with
HBSS.sup.+ pH 5.9 then incubated at 37.degree. C., 5% CO.sub.2 for
2 hours in HBSS.sup.+ pH 7.2. The supernatant was removed from the
cells and analysed for total IgG using an MSD assay (using an
anti-human IgG capture antibody (Jackson) and a streptavidin-sulpho
tag reveal antibody (MSD)). The inhibition curve was analysed by
non-linear regression (Graphpad Prism.RTM.) to determine the
EC.sub.50. The graph represents combined data from 3 experiments.
As shown in FIG. 3 CA170_1638.g49 IgG4 inhibits IgG recycling in a
concentration dependent manner with a mean EC.sub.50 value (n=3) of
0.31 nM.
[0485] CA170_1638.g49 IgG4 and FabFv Inhibits the Transcytosis of
Human IgG
[0486] FcRn can traffic IgG across polarised epithelial cell layers
in both the apical to basolateral and basolateral to apical
directions and thus plays an important role in permitting IgG to
move between the circulation and lumen at mucosal barriers
(Claypool et al. 2004 Mol Biol Cell 15(4):1746-59). FcRn can
traffic IgG across polarised epithelial cell layers in both the
apical to basolateral and basolateral to apical directions and thus
plays an important role in permitting IgG to move between the
circulation and lumen at mucosal barriers (Claypool et al. 2004 Mol
Biol Cell 15(4):1746-59). An in vitro assay was established to
examine the ability of CA170_1638.g49 IgG4 and FabFv to inhibit
FcRn dependent IgG transcytosis. Briefly, MDCK II clone 15 cells
were plated in a 24 well transwell plate and allowed to form
monolayers over 3 days. The cells were then incubated with
biotinylated human IgG in an acidic buffer which facilitates
binding to FcRn, on the apical side, in the presence and absence of
CA170_1638.g49 IgG4 or FabFv. The human IgG is transcytosed through
the cells from the apical to basolateral side and released into a
neutral buffer in the lower chamber. Levels of IgG on the
basolateral side were then measured using an MSD assay.
[0487] FIGS. 4 and 5 shows CA170_1638.g49 IgG4 and FabFv inhibits
apical to basolateral IgG transcytosis in MDCK II clone 15 cells.
MDCK II clone 15 cells were plated at 500,000 cells per well of a
24 well transwell plate and incubated for 3 days at 37.degree. C.,
5% CO.sub.2 until monolayers were formed. The pH of the apical
compartment was adjusted to 5.9 and the basolateral side to 7.2 in
a HBSS.sup.+ (Ca/Mg) buffer+1% BSA. Cells on the apical compartment
were incubated with 1 .mu.g/ml biotinylated human IgG (Jackson) in
the presence and absence of CA170_1638.g49 IgG4 or FabFv at the
indicated concentrations for 4 hours at 37.degree. C., 5% CO.sub.2.
The basolateral medium was then collected and total IgG measured by
MSD assay (using an anti-human IgG capture antibody (Jackson) and a
streptavidin-sulpho tag reveal antibody (MSD)). The inhibition
curve was analysed by non-linear regression (Graphpad Prism.RTM.)
to determine the EC.sub.50. The graph represents combined data from
3 experiments.
[0488] In summary FIGS. 4 and 5 shows that CA170_1638.g49 IgG4 and
FabFv can inhibit the apical to basolateral transcytosis of human
IgG in a concentration dependent manner with an EC.sub.50 value of
2.4 and 0.42 nM respectively (n=3).
[0489] Summary of In Vitro Effects of CA170_1638. g49 IgG4 and
FabFv
[0490] CA170_1638. g49 IgG4 and FabFv inhibit both IgG recycling
and transcytosis. The EC.sub.50 of 0.31 nM achieved in the IgG
recycling assay is comparable to the cell affinity binding data in
which K.sub.D values of 0.2 nM in neutral buffer and 0.22 nM in
acidic buffer were obtained. In the IgG transcytosis assay, an
EC.sub.50 of 2.4 nM and 0.42 nM was obtained for CA170_1638. g49
IgG4 and FabFv respectively, demonstrating a slight reduction in
potency between the IgG4 and the FabFv. However, the data in this
section have clearly shown that CA170_1638.g49 IgG4 and FabFv can
inhibit human FcRn function.
Example 7 Cross Reactivity of CA170_1638. g49 IgG4 with Non-Human
Primate FcRn
[0491] To validate the use of CA170_1638. g49 IgG4 in a non-human
primate PK/PD study and pre-clinical toxicology, its relative
affinity with cynomolgus macaque FcRn was examined. MDCK II cells
stably transfected with cynomolgus macaque FcRn and B2M (MDCKII
(Clone 40) was used in a cell based assay, alongside the previously
described MDCK II cells stably transfected with human FcRn and B2M
(MDCK II clone 15).
[0492] FIG. 6 shows CA170_1638. g49 IgG4 IgG4 binding on MDCK II
clone 40 cells in acidic and neutral pH. Specific binding of
antibody to FcRn across a range of antibody concentrations was used
to determine K.sub.D. The analyses were performed in both neutral
and acidic buffers to determine whether environmental pH comparable
to that found in blood plasma (pH7.4) or endosomes (pH6) had any
effect on the antibody binding.
[0493] FIG. 6 shows representative binding curves for CA170_1638.
g49 IgG4. The mean K.sub.D values (n=3) were 0.3 in neutral buffer,
and 0.43 in acidic buffer, respectively (see Table 5).
TABLE-US-00007 TABLE 5 Mean K.sub.D values (nM) for CA170_1638.g49
IgG4 on MDCK II clone 40 cells. Antibody format Cyno FcRnpH 7.4
Cyno FcRnpH 6.0 1638 IgG4 0.30 0.43
Example 8A Anti-FcRn Treatment Enhances the Clearance of hIgG In
Vivo in hFcRn Transgenic Mice
[0494] The effect of anti-FcRn molecules (CA170_01519.g57 Fab'PEG
(described in WO2014/019727) and CA170_01638.g28 Fab'PEG) on the
clearance of human IVIG was determined in human FcRn transgenic
mice (B6.Cg-Fcgrt.sup.tm1Dcr Tg(FCGRT)32Dcr/DcrJ, JAX Mice). Mice
were infused intravenously with 500 mg/kg human IgG (Human IgI 10%
Gamunex-c, Talecris Biotherapeutics). 24 hours later animals were
dosed with vehicle control (PBS) or anti-FcRn intravenously as a
single dose (100 mg/kg). Serial tail tip blood samples were taken
at -24, 8, 24, 48, 72, 96, 144 and 192 hours relative to anti-FcRn
treatment. Serum levels of human IgG in hFcRn mice were determined
by LC-MS/MS. Data presented in FIG. 1 are mean.+-.SEM with 5-6 mice
per treatment group. Blocking of hFcRn by each of the anti-FcRn
molecules tested resulted in accelerated clearance of hIVIG and
lower concentrations of total IgG were observed compared to control
mice.
Example 8B. Anti-FcRn Treatment Enhances the Clearance of hIgG In
Vivo in hFcRn Transgenic Mice
[0495] The anti-human FcRn antibody discovered bound to and
inhibited the binding of human IgG to human FcRn, but did not bind
or inhibit murine FcRn. Consequently, the effect of anti-FcRn
molecules in IgG4P format (1638.g49), Fab'PEG format (1638.g28),
and FabFv format on the clearance of human IVIg was determined in
human FcRn transgenic mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr/DcrJ,
JAX Mice). Mice were infused intravenously with 500 mg/kg human IgG
(Human IgI 10% Gamunex-c, Talecris Biotherapeutics). 24 hours later
animals were dosed with vehicle control (PBS) or anti-FcRn
intravenously as a single dose. Doses, sampling times and replicate
numbers were as indicated in the FIGS. 1a to 1e. Samples were
serial tail tip blood samples. Serum levels of human IgG,
endogenous mouse albumin and the anti-FcRn molecule itself were
determined by LC-MS/MS, with detection and quantification of
peptide sequences unique to each of those analytes. Data presented
in FIGS. 1a to 1e are each the Geometric mean and 95% confidence
interval.
[0496] Blockade of hFcRn by each of the three anti-FcRn molecules
tested resulted in clearance of hIVIg that was accelerated compared
to that in control mice that were treated with vehicle only, or
with a control Fab'PEG (A33, not anti-FcRn, conjugated to 40 kDa
PEG, as was 1638 Fab'PEG)--see FIGS. 1a and 1b. The effect was
dose-related--larger doses gave more prolonged periods during which
free anti-FcRn could be detected in serum (FIGS. 1c and 1d), this
leading to a more prolonged, and more profound clearance of human
IVIg from the mice. The 1638 Fab'PEG showed shorter
pharmacokinetics (disappeared more rapidly from free solution in
serum) than the control A33 Fab'PEG did, suggesting that the 1638
Fab'PEG had undergone target-mediated disposition--disappearing
from free solution by binding to FcRn target.
[0497] Although mouse IgG did not bind to the human FcRn present in
these transgenic mice, endogenous mouse albumin did bind and was
recycled by the human FcRn. Although binding of anti-human FcRn to
human FcRn did not block binding of albumin to the FcRn in in vitro
assay, if such inhibition occurred in vivo, it might have led to
accelerated clearance of endogenous mouse albumin. Data are shown
in FIG. 1e. Since albumin concentration in serum was somewhat
variable (from 16.6 to 59.9 mg/mL in a group of 30 mice, prior to
injection of anti-FcRn drug), to allow easier comparison of group
results, albumin data were normalised and given as a percentage of
the serum albumin concentration at time zero in FIG. 1e. A
recoverable effect on plasma albumin concentration might have
occurred after dosing with Fab'PEG or FabFv formats. Analysis of
variance (ANOVA) was carried out for repeated measurements, looking
at the treatment differences and the time differences
simultaneously. Each measurement of Fab'PEG or FabFv-treated animal
compared to the control in the same experiment at the same time
point, the controls being irrelevant (non-FcRn-binding) Fab'PEG or
vehicle only, respectively. These two formats showed a lowering of
albumin concentrations (at 5% level in the ANOVA analysis of data)
at around 48 to 72 hours post injection of drug, with levels
recovering to pre-dose levels thereafter. The maximum reduction of
plasma albumin concentration was about 10% after 100 mg/kg of the
Fab'PEG format (at 48 hours), or about 25% after 250 mg/kg FabFv at
144 hours. A similar ANOVA analysis was carried out on data showing
the effect of 1638 IgG4P on plasma albumin levels (shown in FIG.
1f). There was no significant difference between treated and
control animals, suggesting that treatment with the IgG4P format of
1638 did not affect plasma albumin concentration.
Example 9 Crystal Structure and Analysis of 1638.g49 Fab: FcRn
Complex
[0498] The 1638.g49 Fab was co-crystalised with hFcRn alpha chain
ECD region (SEQ ID NO: 48) and human beta 2 microglobulin (SEQ ID
NO: 72). The proteins were in 50 mM Sodium Acetate, 125 mM NaCl
pH6.0 and a crystallisation conditions were 0.1M Tris pH8.5, 40%
PEG400 and 0.2M LiSO.sub.4.H.sub.2O at a protein concentration of
10 mg/mL and a drop volume ratio of 0.4 .mu.L protein to 0.44,
reservoir in a sitting drop, vapour diffusion experiment. Crystalls
were allowed to grow for 8-21 days, followed by harvestingfrom the
drop, transfer to well buffer (since it already contained 40%
PEG400) and flash-frozen in liquid nitrogen (-180.degree. C.)
within 10 seconds.
[0499] X-ray data was collected at SOLEIL, using the oscillation
method. The cell dimensions of the crystals were a=101.49 .ANG.,
b=210.4 .ANG., c=101.49 .ANG.; alpha=90 degrees, beta=90 degrees
and gamma=90 degrees. The space group was determined to be P21212.
The molecular packing was determined using Phaser, and refinement
was carried out with Refmac, using data between 30 and 2.7 .ANG.,
to give a final R factor of 21.8% and Rfree of 27.2%. The results
are shown below:
[0500] The residues interacting with 1638.49 Fab' were all in the
FcRn a chain (not .beta.2M) and are indicated below in bold in the
FcRn extracellular domain sequence
TABLE-US-00008 (SEQ ID NO: 48) AESHLSLLYHLTAVSSPAPG TPAFWVSGWL
GPQQYLSYNS LRGEAEPCGA WVWENQVSWY WEKETTDLRI KEKLFLEAFK ALGGKGPYTL
QGLLGCELGPDNTSVPTAKFALNGEEFMNFDLKQGTWGGD WPEALAISQR WQQQDKAANK
ELTFLLFSCP HRLREHLERG RGNLEWKEPPSMRLKARPSSPGFSVLTCSA FSFYPPELQL
RFLRNGLAAG
TGQGDFGPNSDGSFHASSSLTVKSGDEHHYCCIVQHAGLAQPLRVELESPAKSS.
[0501] The residues underlined are those known to be critical for
the interaction of human FcRn with the Fc region of human IgG.
Those in bold are residues involved in binding the 1638.49 Fab'
antibody at 4 .ANG.. Residues in italic are those involved in
binding the same antibody at 5 .ANG..
[0502] The epitope defined by antibody residues closer than 4 .ANG.
was: A81, G83, G84, K85, G86, P87, N113, E115, W131, P132, E133,
L135, A136, Q139.
[0503] The epitope defined by antibody residues closer than 5 .ANG.
was: A81, G83, G84, K85, G86, P87, N113, E115, W131, P132, E133,
L135, A136, Q139, L82, Y88, L112, D130.
[0504] The ASCII text file named "CELL0018-371-DIV-2 Sequence
Listing," created on Dec. 7, 2021, comprising 91 kilobytes, is
hereby incorporated by reference in its entirety.
Sequence CWU 1
1
74112PRTArtificial SequenceCDRH1 1Gly Phe Ser Leu Ser Thr Tyr Gly
Val Gly Val Gly1 5 10216PRTArtificial SequenceCDRH2 2Asn Ile Trp
Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Glu Asn1 5 10
15313PRTArtificial SequenceCDRH3 3Thr Pro Ala Tyr Tyr Gly Ser His
Pro Pro Phe Asp Tyr1 5 10411PRTArtificial SequenceCDRL1 4Arg Thr
Ser Glu Asp Ile Tyr Thr Asn Leu Ala1 5 1057PRTArtificial
SequenceCDRL2 5Val Ala Lys Thr Leu Gln Asp1 569PRTArtificial
SequenceCDRL3 6Leu Gln Gly Phe Lys Phe Pro Trp Thr1
577PRTArtificial SequenceCDRL2 VARIANT 7Val Ala Lys Thr Leu Gln
Glu1 58107PRTArtificial SequenceRat Ab 1638 VL region 8Asp Ile Leu
Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly1 5 10 15Glu Thr
Ile Ser Ile Glu Cys Arg Thr Ser Glu Asp Ile Tyr Thr Asn 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ser Pro Gln Leu Leu Ile 35 40
45Tyr Val Ala Lys Thr Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr His Tyr Ser Leu Lys Ile Ser Gly Met Gln
Pro65 70 75 80Glu Asp Glu Gly Asp Tyr Phe Cys Leu Gln Gly Phe Lys
Phe Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100
1059321DNAArtificial SequenceRat Ab 1638 VL region 9gacatcctga
tgacacagtc tccagcttcc ctgtctgcat ctctgggaga aactatctcc 60atcgaatgtc
gaacaagtga agacatttac actaatttag cgtggtacca gcagaagtca
120gggaaatctc ctcaactcct gatctatgtt gcaaagacgt tgcaagatgg
ggtcccatca 180cggttcagtg gcagtggatc tggcacgcat tattctctca
agatcagcgg catgcaacct 240gaagatgaag gggattattt ctgtctgcag
ggtttcaagt ttccgtggac gttcggtgga 300ggcaccaagc tggaactgaa a
32110127PRTArtificial SequenceRat Ab 1638 VL region with signal
sequence underlined and italicised 10Met Asn Val Pro Thr Gln Phe
Leu Gly Leu Leu Leu Leu Trp Ile Thr1 5 10 15Asp Gly Ile Cys Asp Ile
Leu Met Thr Gln Ser Pro Ala Ser Leu Ser 20 25 30Ala Ser Leu Gly Glu
Thr Ile Ser Ile Glu Cys Arg Thr Ser Glu Asp 35 40 45Ile Tyr Thr Asn
Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ser Pro 50 55 60Gln Leu Leu
Ile Tyr Val Ala Lys Thr Leu Gln Asp Gly Val Pro Ser65 70 75 80Arg
Phe Ser Gly Ser Gly Ser Gly Thr His Tyr Ser Leu Lys Ile Ser 85 90
95Gly Met Gln Pro Glu Asp Glu Gly Asp Tyr Phe Cys Leu Gln Gly Phe
100 105 110Lys Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu
Lys 115 120 12511381DNAArtificial SequenceRat Ab 1638 VL region
with signal sequence underlined and italicised 11atgaatgtgc
ccactcaatt ccttgggttg ttgctgctgt ggataacaga tggcatatgc 60gacatcctga
tgacacagtc tccagcttcc ctgtctgcat ctctgggaga aactatctcc
120atcgaatgtc gaacaagtga agacatttac actaatttag cgtggtacca
gcagaagtca 180gggaaatctc ctcaactcct gatctatgtt gcaaagacgt
tgcaagatgg ggtcccatca 240cggttcagtg gcagtggatc tggcacgcat
tattctctca agatcagcgg catgcaacct 300gaagatgaag gggattattt
ctgtctgcag ggtttcaagt ttccgtggac gttcggtgga 360ggcaccaagc
tggaactgaa a 38112122PRTArtificial SequenceRat Ab 1638 VH region
12Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr
Tyr 20 25 30Gly Val Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly
Leu Glu 35 40 45Trp Leu Ala Asn Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ser 50 55 60Leu Glu Asn Arg Leu Thr Ile Ser Lys Asp Thr Ser
Asn Asn Gln Ala65 70 75 80Phe Leu Lys Ile Thr Asn Val Asp Thr Ala
Asp Ser Ala Thr Tyr Phe 85 90 95Cys Val Arg Thr Pro Ala Tyr Tyr Gly
Ser His Pro Pro Phe Asp Tyr 100 105 110Trp Gly Gln Gly Val Met Val
Thr Val Ser 115 12013366DNAArtificial SequenceRat Ab 1638 VH region
13caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg
60acttgcactt tctctgggtt ttcactgagt acttatggtg tgggtgtggg ctggattcgt
120cagccttcag ggaagggtct ggagtggctg gcaaacattt ggtgggatga
tgataagcgc 180tacaatccat ctctggaaaa ccgactcact atctccaagg
acacctccaa caaccaagca 240ttcctcaaga tcaccaatgt ggacactgca
gatagcgcca catacttctg tgttcggacc 300ccggcttact atggcagcca
tccccctttt gactactggg gccaaggagt catggtcaca 360gtctcg
36614141PRTArtificial SequenceRat Ab 1638 VH region with signal
sequence underlined and italicised 14Met Asp Arg Leu Thr Ser Ser
Phe Leu Leu Leu Ile Val Pro Ala Tyr1 5 10 15Val Leu Ser Gln Val Thr
Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln 20 25 30Pro Ser Gln Thr Leu
Ser Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu 35 40 45Ser Thr Tyr Gly
Val Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys 50 55 60Gly Leu Glu
Trp Leu Ala Asn Ile Trp Trp Asp Asp Asp Lys Arg Tyr65 70 75 80Asn
Pro Ser Leu Glu Asn Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn 85 90
95Asn Gln Ala Phe Leu Lys Ile Thr Asn Val Asp Thr Ala Asp Ser Ala
100 105 110Thr Tyr Phe Cys Val Arg Thr Pro Ala Tyr Tyr Gly Ser His
Pro Pro 115 120 125Phe Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val
Ser 130 135 14015423DNAArtificial SequenceRat Ab 1638 VH region
with signal sequence underlined and italicised 15atggacaggc
taacttcctc attcctactg ctgattgtcc ctgcatatgt cctgtctcag 60gttactctga
aagagtctgg ccctgggata ttgcagccct cccagaccct cagtctgact
120tgcactttct ctgggttttc actgagtact tatggtgtgg gtgtgggctg
gattcgtcag 180ccttcaggga agggtctgga gtggctggca aacatttggt
gggatgatga taagcgctac 240aatccatctc tggaaaaccg actcactatc
tccaaggaca cctccaacaa ccaagcattc 300ctcaagatca ccaatgtgga
cactgcagat agcgccacat acttctgtgt tcggaccccg 360gcttactatg
gcagccatcc cccttttgac tactggggcc aaggagtcat ggtcacagtc 420tcg
42316107PRTArtificial Sequence1638 gL7 V-region 16Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Thr Ser Glu Asp Ile Tyr Thr Asn 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr
Val Ala Lys Thr Leu Gln Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr His Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Gly Phe Lys Phe Pro
Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10517321DNAArtificial Sequence1638 gL7 V-region 17gatatccaga
tgacccagag tccaagcagt ctctccgcca gcgtaggcga tcgtgtgact 60attacctgtc
gcactagcga ggacatctac accaacctgg cgtggtatca gcagaaacca
120ggcaaagtgc cgaaactgct gatctacgtc gcgaaaaccc tccaggaagg
tgtaccgtct 180cgcttttccg gctctggtag cggtactcac tacaccctga
ccatctcttc cctccagccg 240gaagatgttg ctacctacta ttgcctccag
ggcttcaaat tcccgtggac tttcggtggc 300ggcacgaaag tggaaatcaa a
32118128PRTArtificial Sequence1638 gL7 V-region (E. coli
expression) 18Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala
Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu 20 25 30Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Thr Ser Glu 35 40 45Asp Ile Tyr Thr Asn Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Val 50 55 60Pro Lys Leu Leu Ile Tyr Val Ala Lys
Thr Leu Gln Glu Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr His Tyr Thr Leu Thr Ile 85 90 95Ser Ser Leu Gln Pro Glu
Asp Val Ala Thr Tyr Tyr Cys Leu Gln Gly 100 105 110Phe Lys Phe Pro
Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120
12519384DNAArtificial Sequence1638 gL7 V-region (E. coli
expression) 19atgaaaaaga cagctatcgc aattgcagtg gccttggctg
gtttcgctac cgtagcgcaa 60gctgatatcc agatgaccca gagtccaagc agtctctccg
ccagcgtagg cgatcgtgtg 120actattacct gtcgcactag cgaggacatc
tacaccaacc tggcgtggta tcagcagaaa 180ccaggcaaag tgccgaaact
gctgatctac gtcgcgaaaa ccctccagga aggtgtaccg 240tctcgctttt
ccggctctgg tagcggtact cactacaccc tgaccatctc ttccctccag
300ccggaagatg ttgctaccta ctattgcctc cagggcttca aattcccgtg
gactttcggt 360ggcggcacga aagtggaaat caaa 38420214PRTArtificial
Sequence1638 gL7 light chain (V + constant) 20Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Thr Ser Glu Asp Ile Tyr Thr Asn 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Val
Ala Lys Thr Leu Gln Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr His Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Gly Phe Lys Phe Pro Trp
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21021642DNAArtificial Sequence1638 gL7
light chain (V + constant, E. coli expression) 21gatatccaga
tgacccagag tccaagcagt ctctccgcca gcgtaggcga tcgtgtgact 60attacctgtc
gcactagcga ggacatctac accaacctgg cgtggtatca gcagaaacca
120ggcaaagtgc cgaaactgct gatctacgtc gcgaaaaccc tccaggaagg
tgtaccgtct 180cgcttttccg gctctggtag cggtactcac tacaccctga
ccatctcttc cctccagccg 240gaagatgttg ctacctacta ttgcctccag
ggcttcaaat tcccgtggac tttcggtggc 300ggcacgaaag tggaaatcaa
acgtacggta gcggccccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa
gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcac cagtaacaaa
aagttttaat agaggggagt gt 64222642DNAArtificial Sequence1638 gL7
light chain (V + constant, mammalian expression) 22gatatccaga
tgacccagag tccaagcagt ctctccgcca gcgtaggcga tcgtgtgact 60attacctgtc
gcactagcga ggacatctac accaacctgg cgtggtatca gcagaaacca
120ggcaaagtgc cgaaactgct gatctacgtc gcgaaaaccc tccaggaagg
tgtaccgtct 180cgcttttccg gctctggtag cggtactcac tacaccctga
ccatctcttc cctccagccg 240gaagatgttg ctacctacta ttgcctccag
ggcttcaaat tcccgtggac tttcggtggc 300ggcacgaaag tggaaatcaa
acggaccgtg gccgctccct ccgtgttcat cttcccaccc 360tccgacgagc
agctgaagtc cggcaccgcc tccgtcgtgt gcctgctgaa caacttctac
420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg
caactcccag 480gaatccgtca ccgagcagga ctccaaggac agcacctact
ccctgtcctc caccctgacc 540ctgtccaagg ccgactacga gaagcacaag
gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa
gtccttcaac cggggcgagt gc 64223235PRTArtificial Sequence1638 gL7
light chain (E. coli expression) 23Met Lys Lys Thr Ala Ile Ala Ile
Ala Val Ala Leu Ala Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Arg Thr Ser Glu 35 40 45Asp Ile Tyr Thr Asn
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val 50 55 60Pro Lys Leu Leu
Ile Tyr Val Ala Lys Thr Leu Gln Glu Gly Val Pro65 70 75 80Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr His Tyr Thr Leu Thr Ile 85 90 95Ser
Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Gly 100 105
110Phe Lys Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
115 120 125Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu 130 135 140Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe145 150 155 160Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln 165 170 175Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230
23524705DNAArtificial Sequence1638 gL7 light chain (E. coli
expression) 24atgaaaaaga cagctatcgc aattgcagtg gccttggctg
gtttcgctac cgtagcgcaa 60gctgatatcc agatgaccca gagtccaagc agtctctccg
ccagcgtagg cgatcgtgtg 120actattacct gtcgcactag cgaggacatc
tacaccaacc tggcgtggta tcagcagaaa 180ccaggcaaag tgccgaaact
gctgatctac gtcgcgaaaa ccctccagga aggtgtaccg 240tctcgctttt
ccggctctgg tagcggtact cactacaccc tgaccatctc ttccctccag
300ccggaagatg ttgctaccta ctattgcctc cagggcttca aattcccgtg
gactttcggt 360ggcggcacga aagtggaaat caaacgtacg gtagcggccc
catctgtctt catcttcccg 420ccatctgatg agcagttgaa atctggaact
gcctctgttg tgtgcctgct gaataacttc 480tatcccagag aggccaaagt
acagtggaag gtggataacg ccctccaatc gggtaactcc 540caggagagtg
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg
600acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt
cacccatcag 660ggcctgagct caccagtaac aaaaagtttt aatagagggg agtgt
70525122PRTArtificial Sequence1638 gH33 V-region 25Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30Gly Val
Gly Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp
Leu Ala Asn Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55
60Leu Glu Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Ala65
70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr 85 90 95Cys Ala Arg Thr Pro Ala Tyr Tyr Gly Ser His Pro Pro Phe
Asp Tyr 100 105 110Trp Gly Gln Gly Thr Met Val Thr Val Ser 115
12026366DNAArtificial Sequence1638 gH33 V-region 26gaggttcagc
tggtcgagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc 60tcttgtgcag
cgtccggctt ctctctgtct acctacggcg ttggtgttgg ttgggtacgt
120caggctccag gtaaaggtct ggaatggctc gcaaacatct ggtgggacga
cgataaacgc 180tacaacccgt ccctggagaa ccgcttcacc attagccgtg
ataacgcgaa aaactccgcg 240tatctccaga tgaactccct gcgtgccgaa
gacacggctg tgtactattg cgcgcgcact 300ccggcgtact atggctctca
cccaccgttt gattactggg gtcagggtac aatggttacc 360gtctcg
36627143PRTArtificial Sequence1638 gH33 V-region (E. coli
expression) 27Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala
Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu 20 25 30Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe 35 40 45Ser Leu Ser Thr Tyr Gly Val Gly Val Gly
Trp Val Arg Gln Ala Pro 50 55 60Gly Lys Gly Leu Glu Trp
Leu Ala Asn Ile Trp Trp Asp Asp Asp Lys65 70 75 80Arg Tyr Asn Pro
Ser Leu Glu Asn Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95Ala Lys Asn
Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110Thr
Ala Val Tyr Tyr Cys Ala Arg Thr Pro Ala Tyr Tyr Gly Ser His 115 120
125Pro Pro Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser 130
135 14028429DNAArtificial Sequence1638 gH33 V-region (E.coli
expression) 28atgaagaaga ctgctatagc aattgcagtg gcgctagctg
gtttcgccac cgtggcgcaa 60gctgaggttc agctggtcga gtctggaggc gggcttgtcc
agcctggagg gagcctgcgt 120ctctcttgtg cagcgtccgg cttctctctg
tctacctacg gcgttggtgt tggttgggta 180cgtcaggctc caggtaaagg
tctggaatgg ctcgcaaaca tctggtggga cgacgataaa 240cgctacaacc
cgtccctgga gaaccgcttc accattagcc gtgataacgc gaaaaactcc
300gcgtatctcc agatgaactc cctgcgtgcc gaagacacgg ctgtgtacta
ttgcgcgcgc 360actccggcgt actatggctc tcacccaccg tttgattact
ggggtcaggg taccatggtt 420accgtctcg 42929226PRTArtificial
Sequence1638 gH33 Fab heavy chain (V + human gamma-1 CH1) 29Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Tyr 20 25
30Gly Val Gly Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
35 40 45Trp Leu Ala Asn Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro
Ser 50 55 60Leu Glu Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Ala65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr 85 90 95Cys Ala Arg Thr Pro Ala Tyr Tyr Gly Ser His
Pro Pro Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr Met Val Thr Val
Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170
175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val 195 200 205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys 210 215 220Ser Cys22530678DNAArtificial
Sequence1638 gH33 Fab heavy chain (V + human gamma-1 CH1)
30gaggttcagc tggtcgagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc
60tcttgtgcag cgtccggctt ctctctgtct acctacggcg ttggtgttgg ttgggtacgt
120caggctccag gtaaaggtct ggaatggctc gcaaacatct ggtgggacga
cgataaacgc 180tacaacccgt ccctggagaa ccgcttcacc attagccgtg
ataacgcgaa aaactccgcg 240tatctccaga tgaactccct gcgtgccgaa
gacacggctg tgtactattg cgcgcgcact 300ccggcgtact atggctctca
cccaccgttt gattactggg gtcagggtac catggttacc 360gtctcgagcg
cttctacaaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc
420acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc
cgaaccggtg 480acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc
acaccttccc ggctgtccta 540cagtcctcag gactctactc cctcagcagc
gtggtgaccg tgccctccag cagcttgggc 600acccagacct acatctgcaa
cgtgaatcac aagcccagca acaccaaggt cgacaagaaa 660gttgagccca aatcttgt
67831247PRTArtificial Sequence1638 gH33 Fab heavy chain with (E.
coli expression) 31Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu
Ala Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu 20 25 30Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe 35 40 45Ser Leu Ser Thr Tyr Gly Val Gly Val
Gly Trp Val Arg Gln Ala Pro 50 55 60Gly Lys Gly Leu Glu Trp Leu Ala
Asn Ile Trp Trp Asp Asp Asp Lys65 70 75 80Arg Tyr Asn Pro Ser Leu
Glu Asn Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95Ala Lys Asn Ser Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110Thr Ala Val
Tyr Tyr Cys Ala Arg Thr Pro Ala Tyr Tyr Gly Ser His 115 120 125Pro
Pro Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135
140Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys145 150 155 160Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 165 170 175Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 180 185 190Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 195 200 205Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 210 215 220Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys225 230 235 240Lys
Val Glu Pro Lys Ser Cys 24532741DNAArtificial Sequence1638 gH33 Fab
heavy chain (E. coli expression) 32atgaagaaga ctgctatagc aattgcagtg
gcgctagctg gtttcgccac cgtggcgcaa 60gctgaggttc agctggtcga gtctggaggc
gggcttgtcc agcctggagg gagcctgcgt 120ctctcttgtg cagcgtccgg
cttctctctg tctacctacg gcgttggtgt tggttgggta 180cgtcaggctc
caggtaaagg tctggaatgg ctcgcaaaca tctggtggga cgacgataaa
240cgctacaacc cgtccctgga gaaccgcttc accattagcc gtgataacgc
gaaaaactcc 300gcgtatctcc agatgaactc cctgcgtgcc gaagacacgg
ctgtgtacta ttgcgcgcgc 360actccggcgt actatggctc tcacccaccg
tttgattact ggggtcaggg taccatggtt 420accgtctcga gcgcttctac
aaagggccca tcggtcttcc ccctggcacc ctcctccaag 480agcacctctg
ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg
540gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt
cccggctgtc 600ctacagtcct caggactcta ctccctcagc agcgtggtga
ccgtgccctc cagcagcttg 660ggcacccaga cctacatctg caacgtgaat
cacaagccca gcaacaccaa ggtcgacaag 720aaagttgagc ccaaatcttg t
74133234PRTArtificial Sequence1638 gH33 Fab' heavy chain (V + human
gamma-1 CH1 + hinge) 33Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Ser Leu Ser Thr Tyr 20 25 30Gly Val Gly Val Gly Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp Leu Ala Asn Ile Trp Trp Asp
Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60Leu Glu Asn Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Ala65 70 75 80Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg Thr
Pro Ala Tyr Tyr Gly Ser His Pro Pro Phe Asp Tyr 100 105 110Trp Gly
Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp
Lys Thr His Thr Cys Ala Ala225 23034702DNAArtificial Sequence1638
gH33 Fab' heavy chain (V + human gamma-1 CH1 + hinge) 34gaggttcagc
tggtcgagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc 60tcttgtgcag
cgtccggctt ctctctgtct acctacggcg ttggtgttgg ttgggtacgt
120caggctccag gtaaaggtct ggaatggctc gcaaacatct ggtgggacga
cgataaacgc 180tacaacccgt ccctggagaa ccgcttcacc attagccgtg
ataacgcgaa aaactccgcg 240tatctccaga tgaactccct gcgtgccgaa
gacacggctg tgtactattg cgcgcgcact 300ccggcgtact atggctctca
cccaccgttt gattactggg gtcagggtac catggttacc 360gtctcgagcg
cttctacaaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc
420acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc
cgaaccggtg 480acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc
acaccttccc ggctgtccta 540cagtcctcag gactctactc cctcagcagc
gtggtgaccg tgccctccag cagcttgggc 600acccagacct acatctgcaa
cgtgaatcac aagcccagca acaccaaggt cgacaagaaa 660gttgagccca
aatcttgtga caaaactcac acatgcgccg cg 70235255PRTArtificial
Sequence1638 gH33 Fab' heavy chain (E. coli expression) 35Met Lys
Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala1 5 10 15Thr
Val Ala Gln Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20 25
30Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45Ser Leu Ser Thr Tyr Gly Val Gly Val Gly Trp Val Arg Gln Ala
Pro 50 55 60Gly Lys Gly Leu Glu Trp Leu Ala Asn Ile Trp Trp Asp Asp
Asp Lys65 70 75 80Arg Tyr Asn Pro Ser Leu Glu Asn Arg Phe Thr Ile
Ser Arg Asp Asn 85 90 95Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp 100 105 110Thr Ala Val Tyr Tyr Cys Ala Arg Thr
Pro Ala Tyr Tyr Gly Ser His 115 120 125Pro Pro Phe Asp Tyr Trp Gly
Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys145 150 155 160Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170
175Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
180 185 190Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 195 200 205Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 210 215 220Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys225 230 235 240Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Ala Ala 245 250 25536765DNAArtificial
Sequence1638 gH33 Fab' heavy chain (E. coli expression)
36atgaagaaga ctgctatagc aattgcagtg gcgctagctg gtttcgccac cgtggcgcaa
60gctgaggttc agctggtcga gtctggaggc gggcttgtcc agcctggagg gagcctgcgt
120ctctcttgtg cagcgtccgg cttctctctg tctacctacg gcgttggtgt
tggttgggta 180cgtcaggctc caggtaaagg tctggaatgg ctcgcaaaca
tctggtggga cgacgataaa 240cgctacaacc cgtccctgga gaaccgcttc
accattagcc gtgataacgc gaaaaactcc 300gcgtatctcc agatgaactc
cctgcgtgcc gaagacacgg ctgtgtacta ttgcgcgcgc 360actccggcgt
actatggctc tcacccaccg tttgattact ggggtcaggg taccatggtt
420accgtctcga gcgcttctac aaagggccca tcggtcttcc ccctggcacc
ctcctccaag 480agcacctctg ggggcacagc ggccctgggc tgcctggtca
aggactactt ccccgaaccg 540gtgacggtgt cgtggaactc aggcgccctg
accagcggcg tgcacacctt cccggctgtc 600ctacagtcct caggactcta
ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 660ggcacccaga
cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtcgacaag
720aaagttgagc ccaaatcttg tgacaaaact cacacatgcg ccgcg
76537450PRTArtificial Sequence1638 gH33 IgG4 heavy chain (V + human
gamma-4P constant) 37Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Ser Leu Ser Thr Tyr 20 25 30Gly Val Gly Val Gly Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp Leu Ala Asn Ile Trp Trp Asp
Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60Leu Glu Asn Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Ala65 70 75 80Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg Thr
Pro Ala Tyr Tyr Gly Ser His Pro Pro Phe Asp Tyr 100 105 110Trp Gly
Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200 205Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
Gln Glu 260 265 270Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360
365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Arg Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445Gly Lys
450381350DNAArtificial Sequence1638 gH33 IgG4 heavy chain (V +
human gamma-4P constant) 38gaggttcagc tggtcgagtc tggaggcggg
cttgtccagc ctggagggag cctgcgtctc 60tcttgtgcag cgtccggctt ctctctgtct
acctacggcg ttggtgttgg ttgggtacgt 120caggctccag gtaaaggtct
ggaatggctc gcaaacatct ggtgggacga cgataaacgc 180tacaacccgt
ccctggagaa ccgcttcacc attagccgtg ataacgcgaa aaactccgcg
240tatctccaga tgaactccct gcgtgccgaa gacacggctg tgtactattg
cgcgcgcact 300ccggcgtact atggctctca cccaccgttt gattactggg
gtcagggtac aatggttacc 360gtctcgtctg cctccaccaa gggcccctcc
gtgttccctc tggccccttg ctcccggtcc 420acctccgagt ctaccgccgc
tctgggctgc ctggtcaagg actacttccc cgagcccgtg 480acagtgtcct
ggaactctgg cgccctgacc tccggcgtgc acaccttccc tgccgtgctg
540cagtcctccg gcctgtactc cctgtcctcc gtcgtgaccg tgccctcctc
cagcctgggc 600accaagacct acacctgtaa cgtggaccac aagccctcca
acaccaaggt ggacaagcgg 660gtggaatcta agtacggccc tccctgcccc
ccctgccctg cccctgaatt tctgggcgga 720ccttccgtgt tcctgttccc
cccaaagccc aaggacaccc tgatgatctc ccggaccccc 780gaagtgacct
gcgtggtggt ggacgtgtcc caggaagatc ccgaggtcca gttcaattgg
840tacgtggacg gcgtggaagt gcacaatgcc aagaccaagc ccagagagga
acagttcaac 900tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc
aggactggct gaacggcaaa 960gagtacaagt gcaaggtgtc caacaagggc
ctgccctcca gcatcgaaaa gaccatctcc 1020aaggccaagg gccagccccg
cgagccccag gtgtacaccc tgccccctag ccaggaagag 1080atgaccaaga
accaggtgtc cctgacctgt ctggtcaagg gcttctaccc ctccgacatt
1140gccgtggaat gggagtccaa cggccagccc gagaacaact acaagaccac
cccccctgtg 1200ctggacagcg acggctcctt cttcctgtac tctcggctga
ccgtggacaa gtcccggtgg 1260caggaaggca acgtcttctc ctgctccgtg
atgcacgagg ccctgcacaa ccactacacc 1320cagaagtccc tgtccctgag
cctgggcaag 135039449PRTArtificial Sequence1638 gH33 IgG4 heavy
chain (V + human gamma-4P constant mammalian, no c-terminal lys)
39Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr
Tyr 20 25 30Gly Val Gly Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu 35 40
45Trp Leu Ala Asn Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser
50 55 60Leu Glu Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Ala65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr 85 90 95Cys Ala Arg Thr Pro Ala Tyr Tyr Gly Ser His Pro
Pro Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr Met Val Thr Val Ser
Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185
190Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val
195 200 205Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys 210 215 220Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
Phe Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu 260 265 270Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310
315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425
430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
435 440 445Gly40341PRTArtificial Sequence1638 gL7 FabFv light chain
40Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asp Ile Tyr Thr
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu
Leu Ile 35 40 45Tyr Val Ala Lys Thr Leu Gln Glu Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr His Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln
Gly Phe Lys Phe Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 210 215 220Gly Gly Gly Gly Ser Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Val225 230 235 240Ser Ala Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro 245 250 255Ser Val Trp
Ser Asn Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys 260 265 270Ala
Pro Lys Leu Leu Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val 275 280
285Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
290 295 300Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gly Gly305 310 315 320Gly Tyr Ser Ser Ile Ser Asp Thr Thr Phe Gly
Cys Gly Thr Lys Val 325 330 335Glu Ile Lys Arg Thr
340411023DNAArtificial Sequence1638 gL7 FabFv light chain
41gatatccaga tgacccagag tccaagcagt ctctccgcca gcgtaggcga tcgtgtgact
60attacctgtc gcactagcga ggacatctac accaacctgg cgtggtatca gcagaaacca
120ggcaaagtgc cgaaactgct gatctacgtc gcgaaaaccc tccaggaagg
tgtaccgtct 180cgcttttccg gctctggtag cggtactcac tacaccctga
ccatctcttc cctccagccg 240gaagatgttg ctacctacta ttgcctccag
ggcttcaaat tcccgtggac tttcggtggc 300ggcacgaaag tggaaatcaa
acgtacggta gcggccccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa
gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gtggtggagg tggctctggc 660ggtggtggct
ccggaggcgg aggaagcgac atccagatga cccagagccc ttcctctgta
720agcgccagtg tcggagacag agtgactatt acctgccaaa gctccccttc
agtctggtcc 780aattttctat cctggtatca gcaaaagccc ggaaaggctc
ctaaattgct gatctacgaa 840gcaagcaaac tcaccagcgg cgtgcccagc
aggttcagcg gcagtgggtc tggaactgac 900tttaccctga caatctcctc
actccagccc gaggacttcg ccacctatta ctgcggtgga 960ggttacagta
gcataagtga tacgacattt ggatgcggca ctaaagtgga aatcaagcgt 1020acc
102342363PRTArtificial Sequence1638 gH33 FabFv heavy chain 42Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Tyr
20 25 30Gly Val Gly Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu 35 40 45Trp Leu Ala Asn Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn
Pro Ser 50 55 60Leu Glu Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Ala65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg Thr Pro Ala Tyr Tyr Gly Ser
His Pro Pro Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr Met Val Thr
Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170
175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val 195 200 205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys 210 215 220Ser Cys Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Thr Gly Gly Gly225 230 235 240Gly Ser Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro 245 250 255Gly Gly Ser Leu Arg
Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser 260 265 270Asn Tyr Ala
Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu 275 280 285Trp
Ile Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp 290 295
300Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Val305 310 315 320Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr 325 330 335Cys Ala Arg Thr Val Pro Gly Tyr Ser Thr
Ala Pro Tyr Phe Asp Leu 340 345 350Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 355 360431089DNAArtificial Sequence1638 gH33 FabFv
heavy chain 43gaggttcagc tggtcgagtc tggaggcggg cttgtccagc
ctggagggag cctgcgtctc 60tcttgtgcag cgtccggctt ctctctgtct acctacggcg
ttggtgttgg ttgggtacgt 120caggctccag gtaaaggtct ggaatggctc
gcaaacatct ggtgggacga cgataaacgc 180tacaacccgt ccctggagaa
ccgcttcacc attagccgtg ataacgcgaa aaactccgcg 240tatctccaga
tgaactccct gcgtgccgaa gacacggctg tgtactattg cgcgcgcact
300ccggcgtact atggctctca cccaccgttt gattactggg gtcagggtac
aatggttacc 360gtctcgtccg cttctacaaa gggcccatcg gtcttccccc
tggcaccctc ctccaagagc 420acctctgggg gcacagcggc cctgggctgc
ctggtcaagg actacttccc cgaaccggtg 480acggtgtcgt ggaactcagg
cgccctgacc agcggcgtgc acaccttccc ggctgtccta 540cagtcctctg
gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc
600acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt
ggacaagaaa 660gttgagccca aatcttgttc cggaggtggc ggttccggag
gtggcggtac aggtggcggt 720gggtccgaag tccagctgct tgaatccgga
ggcggactcg tgcagcccgg aggcagtctt 780cgcttgtcct gcgctgtatc
tggaatcgac ctgagcaatt acgccatcaa ctgggtgaga 840caggcacctg
ggaaatgcct cgaatggatc ggcattatat gggctagtgg gacgaccttt
900tatgctacat gggcgaaggg tagattcaca atctcacggg ataatagtaa
gaacacagtg 960tacctgcaga tgaactccct gcgagcagag gataccgccg
tttactattg tgctcgcact 1020gtcccaggtt atagcactgc accctacttt
gatctgtggg ggcagggcac tctggtcacc 1080gtctcgtcc
108944107PRTArtificial SequenceHuman IGKV1-27 JK4 acceptor
framework 44Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro
Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys
Gln Lys Tyr Asn Ser Ala Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 10545321DNAArtificial SequenceHuman IGKV1-27
JK4 acceptor framework 45gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcgagtca gggcattagc
aattatttag cctggtatca gcagaaacca 120gggaaagttc ctaagctcct
gatctatgct gcatccactt tgcaatcagg ggtcccatct 180cggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct
240gaagatgttg caacttatta ctgtcaaaag tataacagtg cccctctcac
tttcggcgga 300gggaccaagg tggagatcaa a 32146114PRTArtificial
SequenceHuman IGHV3-7 JH3 acceptor framework 46Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asn
Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Ala Phe Asp Val Trp Gly Gln Gly Thr Met Val Thr
Val 100 105 110Ser Ser47342DNAArtificial SequenceHuman IGHV3-7 JH3
acceptor framework 47gaggtgcagc tggtggagtc tgggggaggc ttggtccagc
ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagt agctattgga
tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtggccaac
ataaagcaag atggaagtga gaaatactat 180gtggactctg tgaagggccg
attcaccatc tccagagaca acgccaagaa ctcactgtat 240ctgcaaatga
acagcctgag agccgaggac acggctgtgt attactgtgc gagagatgct
300tttgatgtct ggggccaagg gacaatggtc accgtctctt ca
34248274PRTArtificial SequenceHuman FcRn alpha chain extracellular
sequence 48Ala Glu Ser His Leu Ser Leu Leu Tyr His Leu Thr Ala Val
Ser Ser1 5 10 15Pro Ala Pro Gly Thr Pro Ala Phe Trp Val Ser Gly Trp
Leu Gly Pro 20 25 30Gln Gln Tyr Leu Ser Tyr Asn Ser Leu Arg Gly Glu
Ala Glu Pro Cys 35 40 45Gly Ala Trp Val Trp Glu Asn Gln Val Ser Trp
Tyr Trp Glu Lys Glu 50 55 60Thr Thr Asp Leu Arg Ile Lys Glu Lys Leu
Phe Leu Glu Ala Phe Lys65 70 75 80Ala Leu Gly Gly Lys Gly Pro Tyr
Thr Leu Gln Gly Leu Leu Gly Cys 85 90 95Glu Leu Gly Pro Asp Asn Thr
Ser Val Pro Thr Ala Lys Phe Ala Leu 100 105 110Asn Gly Glu Glu Phe
Met Asn Phe Asp Leu Lys Gln Gly Thr Trp Gly 115 120 125Gly Asp Trp
Pro Glu Ala Leu Ala Ile Ser Gln Arg Trp Gln Gln Gln 130 135 140Asp
Lys Ala Ala Asn Lys Glu Leu Thr Phe Leu Leu Phe Ser Cys Pro145 150
155 160His Arg Leu Arg Glu His Leu Glu Arg Gly Arg Gly Asn Leu Glu
Trp 165 170 175Lys Glu Pro Pro Ser Met Arg Leu Lys Ala Arg Pro Ser
Ser Pro Gly 180 185 190Phe Ser Val Leu Thr Cys Ser Ala Phe Ser Phe
Tyr Pro Pro Glu Leu 195 200 205Gln Leu Arg Phe Leu Arg Asn Gly Leu
Ala Ala Gly Thr Gly Gln Gly 210 215 220Asp Phe Gly Pro Asn Ser Asp
Gly Ser Phe His Ala Ser Ser Ser Leu225 230 235 240Thr Val Lys Ser
Gly Asp Glu His His Tyr Cys Cys Ile Val Gln His 245 250 255Ala Gly
Leu Ala Gln Pro Leu Arg Val Glu Leu Glu Ser Pro Ala Lys 260 265
270Ser Ser4999PRTArtificial SequenceRat ?2M 49Ile Gln Lys Thr Pro
Gln Ile Gln Val Tyr Ser Arg His Pro Pro Glu1 5 10 15Asn Gly Lys Pro
Asn Phe Leu Asn Cys Tyr Val Ser Gln Phe His Pro 20 25 30Pro Gln Ile
Glu Ile Glu Leu Leu Lys Asn Gly Lys Lys Ile Pro Asn 35 40 45Ile Glu
Met Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Ile 50 55 60Leu
Ala His Thr Glu Phe Thr Pro Thr Glu Thr Asp Val Tyr Ala Cys65 70 75
80Arg Val Lys His Val Thr Leu Lys Glu Pro Lys Thr Val Thr Trp Asp
85 90 95Arg Asp Met50119PRTArtificial SequenceHuman ?2M including
signal sequence 50Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu
Leu Ser Leu Ser1 5 10 15Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile
Gln Val Tyr Ser Arg 20 25 30His Pro Ala Glu Asn Gly Lys Ser Asn Phe
Leu Asn Cys Tyr Val Ser 35 40 45Gly Phe His Pro Ser Asp Ile Glu Val
Asp Leu Leu Lys Asn Gly Glu 50 55 60Arg Ile Glu Lys Val Glu His Ser
Asp Leu Ser Phe Ser Lys Asp Trp65 70 75 80Ser Phe Tyr Leu Leu Tyr
Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp 85 90 95Glu Tyr Ala Cys Arg
Val Asn His Val Thr Leu Ser Gln Pro Lys Ile 100 105 110Val Lys Trp
Asp Arg Asp Met 11551107PRTArtificial Sequence1638gL2 V-region
51Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asp Ile Tyr Thr
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu
Leu Ile 35 40 45Tyr Val Ala Lys Thr Leu Gln Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr His Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu
Asp Val Ala Thr Tyr Tyr Cys Leu Gln Gly Phe Lys Phe Pro Trp 85 90
95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10552321DNAArtificial Sequence1638gL2 V-region 52gatatccaga
tgacccagag tccaagcagt ctctccgcca gcgtaggcga tcgtgtgact 60attacctgtc
gcactagcga ggacatctac accaacctgg cgtggtatca gcagaaacca
120ggcaaagtgc cgaaactgct gatctacgtc gcgaaaaccc tccaggacgg
tgtaccgtct 180cgcttttccg gctctggtag cggtactcac tacaccctga
ccatctcttc cctccagccg 240gaagatgttg ctacctacta ttgcctccag
ggcttcaaat tcccgtggac tttcggtggc 300ggcacgaaag tggaaatcaa a
32153128PRTArtificial Sequence1638 gL2 V-region (E. coli
expression) 53Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala
Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu 20 25 30Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Thr Ser Glu 35 40 45Asp Ile Tyr Thr Asn Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Val 50 55 60Pro Lys Leu Leu Ile Tyr Val Ala Lys
Thr Leu Gln Asp Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr His Tyr Thr Leu Thr Ile 85 90 95Ser Ser Leu Gln Pro Glu
Asp Val Ala Thr Tyr Tyr Cys Leu Gln Gly 100 105 110Phe Lys Phe Pro
Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120
12554384DNAArtificial Sequence1638 gL2 V-region (E. coli
expression) 54atgaaaaaga cagctatcgc aattgcagtg gccttggctg
gtttcgctac cgtagcgcaa 60gctgatatcc agatgaccca gagtccaagc agtctctccg
ccagcgtagg cgatcgtgtg 120actattacct gtcgcactag cgaggacatc
tacaccaacc tggcgtggta tcagcagaaa 180ccaggcaaag tgccgaaact
gctgatctac gtcgcgaaaa ccctccagga cggtgtaccg 240tctcgctttt
ccggctctgg tagcggtact cactacaccc tgaccatctc ttccctccag
300ccggaagatg ttgctaccta ctattgcctc cagggcttca aattcccgtg
gactttcggt 360ggcggcacga aagtggaaat caaa 38455214PRTArtificial
Sequence1638 gL2 light chain (V + constant) 55Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Thr Ser Glu Asp Ile Tyr Thr Asn 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Val
Ala Lys Thr Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr His Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Gly Phe Lys Phe Pro Trp
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21056642DNAArtificial Sequence1638 gL2
light chain (V + constant, codon optimized for E. coli expression)
56gatatccaga tgacccagag tccaagcagt ctctccgcca gcgtaggcga tcgtgtgact
60attacctgtc gcactagcga ggacatctac accaacctgg cgtggtatca gcagaaacca
120ggcaaagtgc cgaaactgct gatctacgtc gcgaaaaccc tccaggacgg
tgtaccgtct 180cgcttttccg gctctggtag cggtactcac tacaccctga
ccatctcttc cctccagccg 240gaagatgttg ctacctacta ttgcctccag
ggcttcaaat tcccgtggac tttcggtggc 300ggcacgaaag tggaaatcaa
acgtacggta gcggccccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa
gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcac cagtaacaaa
aagttttaat agaggggagt gt 64257235PRTArtificial Sequence1638 gL2
light chain (E. coli expression) 57Met Lys Lys Thr Ala Ile Ala Ile
Ala Val Ala Leu Ala Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Arg Thr Ser Glu 35 40 45Asp Ile Tyr Thr Asn
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val 50 55 60Pro Lys Leu Leu
Ile Tyr Val Ala Lys Thr Leu Gln Asp Gly Val Pro65 70 75 80Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr His Tyr Thr Leu Thr Ile 85 90 95Ser
Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Gly 100 105
110Phe Lys Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
115 120 125Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu 130 135 140Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe145 150 155 160Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln 165 170 175Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230
23558705DNAArtificial Sequence1638 gL2 light chain (E. coli
expression) 58atgaaaaaga cagctatcgc aattgcagtg gccttggctg
gtttcgctac cgtagcgcaa 60gctgatatcc agatgaccca gagtccaagc agtctctccg
ccagcgtagg cgatcgtgtg 120actattacct gtcgcactag cgaggacatc
tacaccaacc tggcgtggta tcagcagaaa 180ccaggcaaag tgccgaaact
gctgatctac gtcgcgaaaa ccctccagga cggtgtaccg 240tctcgctttt
ccggctctgg tagcggtact cactacaccc tgaccatctc ttccctccag
300ccggaagatg ttgctaccta ctattgcctc cagggcttca aattcccgtg
gactttcggt 360ggcggcacga aagtggaaat caaacgtacg gtagcggccc
catctgtctt catcttcccg 420ccatctgatg agcagttgaa atctggaact
gcctctgttg tgtgcctgct gaataacttc 480tatcccagag aggccaaagt
acagtggaag gtggataacg ccctccaatc gggtaactcc 540caggagagtg
tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg
600acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt
cacccatcag 660ggcctgagct caccagtaac aaaaagtttt aatagagggg agtgt
70559122PRTArtificial Sequence1638gH2 V-region 59Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30Gly Val
Gly Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp
Leu Ala Asn Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55
60Leu Glu Asn Arg Phe Thr Ile Ser Lys Asp Thr Ala Lys Asn Ser Ala65
70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr 85 90 95Cys Val Arg Thr Pro Ala Tyr Tyr Gly Ser His Pro Pro Phe
Asp Tyr 100 105 110Trp Gly Gln Gly Thr Met Val Thr Val Ser 115
12060366DNAArtificial Sequence1638gH2 V-region 60gaggttcagc
tggtcgagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc 60tcttgtgcat
tctccggctt ctctctgtct acctacggcg ttggtgttgg ttgggtacgt
120caggctccag gtaaaggtct ggaatggctc gcaaacatct ggtgggacga
cgataaacgc 180tacaacccgt ccctggagaa ccgcttcacc attagcaaag
ataccgcgaa aaactccgcg 240tatctccaga tgaactccct gcgtgccgaa
gacacggctg tgtactattg cgttcgcact 300ccggcgtact atggctctca
cccaccgttt gattactggg gtcagggtac catggttacc 360gtctcg
36661143PRTArtificial Sequence1638 gH2 V-region with signal
sequence underlined and italicized (E. coli expression) 61Met Lys
Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala1 5 10 15Thr
Val Ala Gln Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20 25
30Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe
35 40 45Ser Leu Ser Thr Tyr Gly Val Gly Val Gly Trp Val Arg Gln Ala
Pro 50 55 60Gly Lys Gly Leu Glu Trp Leu Ala Asn Ile Trp Trp Asp Asp
Asp Lys65 70 75 80Arg Tyr Asn Pro Ser Leu Glu Asn Arg Phe Thr Ile
Ser Lys Asp Thr 85 90 95Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp 100 105 110Thr Ala Val Tyr Tyr Cys Val Arg Thr
Pro Ala Tyr Tyr Gly Ser His 115 120 125Pro Pro Phe Asp Tyr Trp Gly
Gln Gly Thr Met Val Thr Val Ser 130 135 14062429DNAArtificial
Sequence1638 gH2 V-region with signal sequence underlined and
italicized (E. coli expression) 62atgaagaaga ctgctatagc aattgcagtg
gcgctagctg gtttcgccac cgtggcgcaa 60gctgaggttc agctggtcga gtctggaggc
gggcttgtcc agcctggagg gagcctgcgt 120ctctcttgtg cattctccgg
cttctctctg tctacctacg gcgttggtgt tggttgggta 180cgtcaggctc
caggtaaagg tctggaatgg ctcgcaaaca tctggtggga cgacgataaa
240cgctacaacc cgtccctgga gaaccgcttc accattagca aagataccgc
gaaaaactcc 300gcgtatctcc agatgaactc cctgcgtgcc gaagacacgg
ctgtgtacta ttgcgttcgc 360actccggcgt actatggctc tcacccaccg
tttgattact ggggtcaggg taccatggtt 420accgtctcg 42963234PRTArtificial
Sequence1638 gH2 Fab' heavy chain (V + human gamma-1 CH1 + hinge)
63Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr
Tyr 20 25 30Gly Val Gly Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu 35 40 45Trp Leu Ala Asn Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ser 50 55 60Leu Glu Asn Arg Phe Thr Ile Ser Lys Asp Thr Ala
Lys Asn Ser Ala65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr 85 90 95Cys Val Arg Thr Pro Ala Tyr Tyr Gly
Ser His Pro Pro Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155
160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp Lys Thr His Thr Cys
Ala Ala225 23064702DNAArtificial Sequence1638 gH2 Fab' heavy chain
(V + human gamma-1 CH1 + hinge) 64gaggttcagc tggtcgagtc tggaggcggg
cttgtccagc ctggagggag cctgcgtctc 60tcttgtgcat tctccggctt ctctctgtct
acctacggcg ttggtgttgg ttgggtacgt 120caggctccag gtaaaggtct
ggaatggctc gcaaacatct ggtgggacga cgataaacgc 180tacaacccgt
ccctggagaa ccgcttcacc attagcaaag ataccgcgaa aaactccgcg
240tatctccaga tgaactccct gcgtgccgaa gacacggctg tgtactattg
cgttcgcact 300ccggcgtact atggctctca cccaccgttt gattactggg
gtcagggtac catggttacc 360gtctcgagcg cttctacaaa gggcccatcg
gtcttccccc tggcaccctc ctccaagagc 420acctctgggg gcacagcggc
cctgggctgc ctggtcaagg actacttccc cgaaccggtg 480acggtgtcgt
ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta
540cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag
cagcttgggc 600acccagacct acatctgcaa cgtgaatcac aagcccagca
acaccaaggt cgacaagaaa 660gttgagccca aatcttgtga caaaactcac
acatgcgccg cg 70265255PRTArtificial Sequence1638 gH2 Fab' heavy
chain (E. coli expression) 65Met Lys Lys Thr Ala Ile Ala Ile Ala
Val Ala Leu Ala Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu 20 25 30Val Gln Pro Gly Gly Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe 35 40 45Ser Leu Ser Thr Tyr Gly
Val Gly Val Gly Trp Val Arg Gln Ala Pro 50 55 60Gly Lys Gly Leu Glu
Trp Leu Ala Asn Ile Trp Trp Asp Asp Asp Lys65 70 75 80Arg Tyr Asn
Pro Ser Leu Glu Asn Arg Phe Thr Ile Ser Lys Asp Thr 85 90 95Ala Lys
Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105
110Thr Ala Val Tyr Tyr Cys Val Arg Thr Pro Ala Tyr Tyr Gly Ser His
115 120 125Pro Pro Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val
Ser Ser 130 135 140Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys145 150 155 160Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 165 170 175Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 180 185 190Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 195 200 205Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 210 215 220Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys225 230
235 240Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Ala Ala
245 250 25566765DNAArtificial Sequence1638 gH2 Fab' heavy chain d
(E. coli expression) 66atgaagaaga ctgctatagc aattgcagtg gcgctagctg
gtttcgccac cgtggcgcaa 60gctgaggttc agctggtcga gtctggaggc gggcttgtcc
agcctggagg gagcctgcgt 120ctctcttgtg cattctccgg cttctctctg
tctacctacg gcgttggtgt tggttgggta 180cgtcaggctc caggtaaagg
tctggaatgg ctcgcaaaca tctggtggga cgacgataaa 240cgctacaacc
cgtccctgga gaaccgcttc accattagca aagataccgc gaaaaactcc
300gcgtatctcc agatgaactc cctgcgtgcc gaagacacgg ctgtgtacta
ttgcgttcgc 360actccggcgt actatggctc tcacccaccg tttgattact
ggggtcaggg taccatggtt 420accgtctcga gcgcttctac aaagggccca
tcggtcttcc ccctggcacc ctcctccaag 480agcacctctg ggggcacagc
ggccctgggc tgcctggtca aggactactt ccccgaaccg 540gtgacggtgt
cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc
600ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc
cagcagcttg 660ggcacccaga cctacatctg caacgtgaat cacaagccca
gcaacaccaa ggtcgacaag 720aaagttgagc ccaaatcttg tgacaaaact
cacacatgcg ccgcg 7656715PRTArtificial SequenceN-termial sequence
Heavy chain 67Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly1 5 10 156815PRTArtificial SequenceN-terminal sequence Light
chain 68Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val1 5 10 156915PRTArtificial Sequencehuman JH3 69Asp Ala Phe Asp
Val Trp Gly Gln Gly Thr Met Val Thr Val Ser1 5 10
15705PRTArtificial Sequencepart of CDR-H3 70Asp Ala Phe Asp Val1
57112PRTArtificial SequenceJK4 sequence 71Leu Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys1 5 107299PRTArtificial SequenceHuman
?2-microglobulin 72Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg
His Pro Ala Glu1 5 10 15Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val
Ser Gly Phe His Pro 20 25 30Ser Asp Ile Glu Val Asp Leu Leu Lys Asn
Gly Glu Arg Ile Glu Lys 35 40 45Val Glu His Ser Asp Leu Ser Phe Ser
Lys Asp Trp Ser Phe Tyr Leu 50 55 60Leu Tyr Tyr Thr Glu Phe Thr Pro
Thr Glu Lys Asp Glu Tyr Ala Cys65 70 75 80Arg Val Asn His Val Thr
Leu Ser Gln Pro Lys Ile Val Lys Trp Asp 85 90 95Arg Asp
Met73453PRTArtificial Sequence1638gH33
IgG1 heavy chain (V + human gamma-1 constant) 73Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30Gly Val Gly
Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp Leu
Ala Asn Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60Leu
Glu Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Ala65 70 75
80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
85 90 95Cys Ala Arg Thr Pro Ala Tyr Tyr Gly Ser His Pro Pro Phe Asp
Tyr 100 105 110Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser
Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200
205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu225 230 235 240Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val 260 265 270Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu305 310 315
320Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro 340 345 350Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln 355 360 365Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala 370 375 380Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr385 390 395 400Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440
445Leu Ser Pro Gly Lys 450741965DNAArtificial Sequence1638gH33 IgG1
heavy chain (V + human gamma-1 constant, exons underlined)
74gaggttcagc tggtcgagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc
60tcttgtgcag cgtccggctt ctctctgtct acctacggcg ttggtgttgg ttgggtacgt
120caggctccag gtaaaggtct ggaatggctc gcaaacatct ggtgggacga
cgataaacgc 180tacaacccgt ccctggagaa ccgcttcacc attagccgtg
ataacgcgaa aaactccgcg 240tatctccaga tgaactccct gcgtgccgaa
gacacggctg tgtactattg cgcgcgcact 300ccggcgtact atggctctca
cccaccgttt gattactggg gtcagggtac aatggttacc 360gtctcgagcg
cttctacaaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc
420acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc
cgaaccggtg 480acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc
acaccttccc ggctgtccta 540cagtcctcag gactctactc cctcagcagc
gtggtgaccg tgccctccag cagcttgggc 600acccagacct acatctgcaa
cgtgaatcac aagcccagca acaccaaggt cgacaagaaa 660gttggtgaga
ggccagcaca gggagggagg gtgtctgctg gaagccaggc tcagcgctcc
720tgcctggacg catcccggct atgcagcccc agtccagggc agcaaggcag
gccccgtctg 780cctcttcacc cggaggcctc tgcccgcccc actcatgctc
agggagaggg tcttctggct 840ttttccccag gctctgggca ggcacaggct
aggtgcccct aacccaggcc ctgcacacaa 900aggggcaggt gctgggctca
gacctgccaa gagccatatc cgggaggacc ctgcccctga 960cctaagccca
ccccaaaggc caaactctcc actccctcag ctcggacacc ttctctcctc
1020ccagatctga gtaactccca atcttctctc tgcagagccc aaatcttgtg
acaaaactca 1080cacatgccca ccgtgcccag gtaagccagc ccaggcctcg
ccctccagct caaggcggga 1140caggtgccct agagtagcct gcatccaggg
acaggcccca gccgggtgct gacacgtcca 1200cctccatctc ttcctcagca
cctgaactcc tggggggacc gtcagtcttc ctcttccccc 1260caaaacccaa
ggacaccctc atgatctccc ggacccctga ggtcacatgc gtggtggtgg
1320acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc
gtggaggtgc 1380ataatgccaa gacaaagccg cgggaggagc agtacaacag
cacgtaccgt gtggtcagcg 1440tcctcaccgt cctgcaccag gactggctga
atggcaagga gtacaagtgc aaggtctcca 1500acaaagccct cccagccccc
atcgagaaaa ccatctccaa agccaaaggt gggacccgtg 1560gggtgcgagg
gccacatgga cagaggccgg ctcggcccac cctctgccct gagagtgacc
1620gctgtaccaa cctctgtccc tacagggcag ccccgagaac cacaggtgta
caccctgccc 1680ccatcccggg atgagctgac caagaaccag gtcagcctga
cctgcctggt caaaggcttc 1740tatcccagcg acatcgccgt ggagtgggag
agcaatgggc agccggagaa caactacaag 1800accacgcctc ccgtgctgga
ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1860gacaagagca
ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg
1920cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaa 1965
* * * * *
References