U.S. patent application number 14/603219 was filed with the patent office on 2015-08-27 for antibody fc variants.
This patent application is currently assigned to Roche Glycart AG. The applicant listed for this patent is Roche Glycart AG. Invention is credited to Monika BAEHNER, Stefan JENEWEIN, Manfred KUBBIES, Ekkehard MOESSNER, Tilman SCHLOTHAUER.
Application Number | 20150239981 14/603219 |
Document ID | / |
Family ID | 45888223 |
Filed Date | 2015-08-27 |
United States Patent
Application |
20150239981 |
Kind Code |
A1 |
BAEHNER; Monika ; et
al. |
August 27, 2015 |
ANTIBODY FC VARIANTS
Abstract
The invention relates to engineered polypeptides comprising Fc
variants and their uses. More specifically, Fc variants are
described exhibiting reduced effector function. These variants
cause a benefit for a patient suffering from a disease which could
be treated with an antibody for which it is desirable to reduce the
effector function elicited by antibodies.
Inventors: |
BAEHNER; Monika; (Muenchen,
DE) ; JENEWEIN; Stefan; (Neustadt/Weinstrasse,
DE) ; KUBBIES; Manfred; (Penzberg, DE) ;
MOESSNER; Ekkehard; (Kreuzlingen, CH) ; SCHLOTHAUER;
Tilman; (Penzberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Glycart AG |
Schlieren |
|
CH |
|
|
Assignee: |
Roche Glycart AG
Schlieren
CH
|
Family ID: |
45888223 |
Appl. No.: |
14/603219 |
Filed: |
January 22, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13431489 |
Mar 27, 2012 |
8969526 |
|
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14603219 |
|
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Current U.S.
Class: |
424/134.1 ;
424/133.1; 530/387.3 |
Current CPC
Class: |
C07K 2317/71 20130101;
A61P 3/10 20180101; C07K 16/00 20130101; Y10S 435/81 20130101; A61P
29/00 20180101; C07K 2317/92 20130101; A61P 35/00 20180101; A61P
25/00 20180101; C07K 2317/734 20130101; C07K 2317/56 20130101; C07K
2317/73 20130101; C07K 2319/30 20130101; C07K 2317/52 20130101;
C07K 16/2896 20130101; A61P 7/02 20180101; C07K 2317/732
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 29, 2011 |
EP |
11160251.2 |
Claims
1. (canceled)
2. The method according to claim 12, wherein Pro329 of the
wild-type human Fc region is substituted with glycine or arginine
or an amino acid residue large enough to destroy the proline
sandwich within the Fc region.
3. The method according to claim 12, wherein said at least one
further amino acid substitution is selected from S228P, E233P,
L234A, L235A, L235E, N297A, N297D, and P331S.
4. The method according to claim 3, wherein said at least one
further amino acid substitution is L234A and L235A of the human
IgG1 Fc region or S228P and L235E of the human IgG4 Fc region.
5. The method according to claim 12, wherein the affinity to at
least one further human receptor is reduced compared to the
polypeptide comprising a wild-type human IgG Fc region, wherein the
further human receptor is selected from Fc.gamma.I, Fc.gamma.IIA,
and C1q.
6. The method according to claim 12, wherein the polypeptide
comprises a human IgG1 or IgG4 Fc region.
7. The method according to claim 12, wherein the polypeptide is an
antibody or an Fc fusion protein.
8. The method according to claim 12, wherein thrombocyte
aggregation induced by the polypeptide is reduced compared to the
thrombocyte aggregation induced by a polypeptide comprising a
wild-type human IgG Fc region.
9. The method according to claim 12, wherein CDC induced by the
polypeptide is strongly reduced compared to the CDC induced by a
polypeptide comprising a wild-type human IgG Fc region.
10. (canceled)
11. The method according to claim 12, wherein the polypeptide is an
anti-CD9 antibody comprising a heavy chain variable region
comprising SEQ ID NO:9 and a variable light chain region comprising
SEQ ID NO:8.
12. A method of treating a disease comprising administering a
polypeptide comprising an Fc variant of a wild-type human IgG Fc
region, said Fc variant comprising an amino acid substitution at
position Pro329 and at least one further amino acid substitution,
wherein the residues are numbered according to the EU index of
Kabat, and wherein said polypeptide exhibits a reduced affinity to
one or more of human Fc.gamma.RIIIA, Fc.gamma.RIIA, and Fc.gamma.RI
compared to the polypeptide comprising the wildtype IgG Fc region,
and wherein the antibody-dependent cell-mediated cytotoxicity
(ADCC) induced by said polypeptide is 0-20% of the ADCC induced by
the polypeptide comprising a wild-type human IgG Fc region, wherein
it is favorable for treating the disease that an effector function
of the polypeptide is strongly reduced compared to the effector
function induced by the polypeptide comprising a wild-type human
IgG Fc region.
13. (canceled)
14. A method of treating an individual having a disease, wherein it
is favorable that the effector function of the polypeptide
comprising an Fc variant of a wild-type human IgG Fc region is
strongly reduced compared to the effector function induced by the
polypeptide comprising a wildtype human Fc polypeptide, comprising
administering to an individual an effective amount of a polypeptide
comprising an Fc variant of a wild-type human IgG Fc region, said
Fc variant comprising an amino acid substitution at position Pro329
and at least one further amino acid substitution, wherein the
residues are numbered according to the EU index of Kabat, and
wherein said polypeptide exhibits a reduced affinity to one or more
of human Fc.gamma.RIIIA, Fc.gamma.RIIA, and Fc.gamma.RI compared to
the polypeptide comprising the wildtype IgG Fc region, and wherein
the antibody-dependent cell-mediated cytotoxicity (ADCC) induced by
said polypeptide is 0-20% of the ADCC induced by the polypeptide
comprising a wild-type human IgG Fc region.
15. A method for down-modulating ADCC comprising a polypeptide
comprising an Fc variant of a wild-type human IgG Fc region, said
polypeptide having Pro329 of the human IgG Fc region substituted
with glycine, wherein the residues are numbered according to the EU
index of Kabat, wherein said polypeptide exhibits a reduced
affinity to the human Fc.gamma.RIIIA and Fc.gamma.RIIA, wherein
ADCC is down-modulated to at least 20% of the ADCC induced by the
polypeptide comprising the wildtype human IgG Fc region.
16. The method according to any one of claim 15, 22, or 23 wherein
the Fc variant comprises at least two further amino acid
substitution at L234A and L235A of the human IgG1 Fc region or
S228P and L235E of the human IgG4 Fc region.
17. The method of claim 16, wherein thrombocyte aggregation induced
by the polypeptide is reduced compared to the thrombocyte
aggregation induced by the polypeptide comprising a wildtype human
Fc region, wherein the polypeptide is a platelet activating
antibody.
18. A method of treating an individual having a disease with a
polypeptide, said polypeptide having Pro329 of the human IgG Fc
region substituted with Gly, wherein the residues are numbered
according to the EU index of Kabat, wherein said polypeptide is
characterized by a strongly reduced binding to Fc.gamma.RIIIA and
Fc.gamma.RIIA compared to the polypeptide comprising a wildtype
human IgG Fc region, comprising administering to the individual an
effective amount of said polypeptide.
19. The method according to claim 18, wherein said polypeptide
comprises at least two further amino acid substitution at L234A and
L235A of the human IgG1 Fc region or S228P and L235E of the human
IgG4 Fc region.
20. The method according to claim 11, wherein the polypeptide is
formulated as a pharmaceutical formulation.
21. (canceled)
22. The method of claim 15 further comprising down-modulating
antibody-dependent cellular phagocytosis (ADCP).
23. A method for down-modulating ADCP comprising a polypeptide
comprising an Fc variant of a wild-type human IgG Fc region, said
polypeptide having Pro329 of the human IgG Fc region substituted
with glycine, wherein the residues are numbered according to the EU
index of Kabat, wherein said polypeptide exhibits a reduced
affinity to the human Fc.gamma.RIIIA and Fc.gamma.RIIA.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/431,489 which claims the benefit of priority under 35
USC .sctn.119 of European patent application number 11160251.2,
filed on Mar. 29, 2011, each of which is hereby incorporated by
reference in its entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
146392026510SEQLIST.txt, date recorded: May 6, 2015, size: 40
KB).
FIELD OF THE INVENTION
[0003] The present invention concerns polypeptides comprising
variants of an Fc region. More particularly, the present invention
concerns Fc region-containing polypeptides that have altered
effector function as a consequence of one or more amino acid
substitutions in the Fc region of the polypeptide.
SUMMARY
[0004] The present invention relates to the field of antibody
variants and provides polypeptides comprising Fc variants with a
decreased effector function, like decreased ADCC and/or C1q
binding.
[0005] In particular the invention provides a polypeptide
comprising an Fc variant of a wild-type human IgG Fc region, said
Fc variant comprising an amino acid substitution at position Pro329
and at least one further amino acid substitution, wherein the
residues are numbered according to the EU index of Kabat, and
wherein said polypeptide exhibits a reduced affinity to the human
Fc.gamma.RIIIA and/or Fc.gamma.RIIA and/or Fc.gamma.RI compared to
a polypeptide comprising the wildtype IgG Fc region, and wherein
the ADCC induced by said polypeptide is reduced to at least 20% of
the ADCC induced by the polypeptide comprising a wild-type human
IgG Fc region.
[0006] In a specific embodiment Pro329 of a wild-type human Fc
region in the polypeptide described above is substituted with
glycine or arginine or an amino acid residue large enough to
destroy the proline sandwich within the Fc/Fc.gamma. receptor
interface, that is formed between the proline329 of the Fc and
tryptophane residues Trp 87 and Trp 110 of FcgRIII (Sondermann et
al.: Nature 406, 267-273 (20 Jul. 2000)). In a further aspect of
the invention the at least one further amino acid substitution in
the Fc variant is S228P, E233P, L234A, L235A, L235E, N297A, N297D,
or P331S and still in another embodiment said at least one further
amino acid substitution is L234A and L235A of the human IgG1 Fc
region or S228P and L235E of the human IgG4 Fc region.
[0007] In another aspect of the invention the polypeptide provided
exhibits a reduced affinity to at least one further receptor of the
group comprising the human receptors Fc.gamma.I, Fc.gamma.IIA and
C1q compared to the polypeptide comprising a wild-type human IgG Fc
region. In still another aspect of the invention the polypeptide
comprises a human IgG1 or IgG4 Fc region. In still another aspect
of the invention the polypeptide is an antibody or an Fc fusion
protein.
[0008] In a further embodiment the thrombocyte aggregation induced
by the polypeptide comprising the Fc variant is reduced compared to
the thrombocyte aggregation induced by a polypeptide comprising a
wild-type human IgG Fc region. In still a further embodiment, the
polypeptide according to the invention exhibits a strongly reduced
CDC compared to the CDC induced by a polypeptide comprising a
wild-type human IgG Fc region.
[0009] In another embodiment of the invention polypeptides
comprising an Fc variant, as described above, are provided for use
as a medicament. In a specific embodiment the polypeptide is an
anti-CD9 antibody, which is characterized in that the polypeptide
comprising the wildtype Fc region comprises as heavy chain variable
region SEQ ID NO:9 and as variable light chain region SEQ ID
NO:8.
[0010] In another aspect of the invention the polypeptides as
described above are provided for use in treating a disease wherein
it is favorable that an effector function of the polypeptide
comprising the Fc variant is strongly reduced compared to the
effector function induced by a polypeptide comprising a wild-type
human IgG Fc region.
[0011] In another embodiment the use of the polypeptides as
described above is provided for the manufacture of a medicament for
the treatment of a disease, wherein it is favorable that the
effector function of the polypeptide comprising an Fc variant of a
wild-type human IgG Fc region is strongly reduced compared to the
effector function induced by a polypeptide comprising a wild-type
human IgG Fc region.
[0012] In still another aspect of the invention a method of
treating an individual having a disease is provided, wherein it is
favorable that the effector function of the polypeptide comprising
an Fc variant of a wild-type human IgG Fc region is strongly
reduced compared to the effector function induced by a polypeptide
comprising a wildtype human Fc polypeptide, comprising
administering to an individual an effective amount of the
polypeptide described above.
[0013] A further aspect of the invention is a use of a polypeptide
comprising an Fc variant of a wild-type human IgG Fc region, said
polypeptide having Pro329 of the human IgG Fc region substituted
with glycine, wherein the residues are numbered according to the EU
index of Kabat, wherein said polypeptide exhibits a reduced
affinity to the human Fc.gamma.RIIIA and Fc.gamma.RIIA for
down-modulation of ADCC to at least 20% of the ADCC induced by the
polypeptide comprising the wildtype human IgG Fc region, and/or for
down-modulation of ADCP.
[0014] Another aspect of the invention is use of a polypeptide
comprising an Fc variant of a wild-type human IgG Fc region, said
polypeptide having Pro329 of the human IgG Fc region substituted
with glycine and wherein the Fc variant comprises at least two
further amino acid substitutions at L234A and L235A of the human
IgG1 Fc region or S228P and L235E of the human IgG4 Fc region,
wherein the residues are numbered according to the EU index of
Kabat, wherein said polypeptide exhibits a reduced affinity to the
human Fc.gamma.RIIIA and Fc.gamma.RIIA, for down-modulation of ADCC
to at least 20% of the ADCC induced by the polypeptide comprising
the wildtype human IgG Fc region, and/or for down-modulation of
ADCP.
[0015] Another aspect of the invention is use of the polypeptide
described above, wherein the thrombocyte aggregation induced by the
polypeptide described above is reduced compared to the thrombocyte
aggregation induced by a polypeptide comprising a wildtype human Fc
region, wherein the polypeptide is a platelet activating
antibody.
[0016] In another aspect of the invention a method of treating an
individual having a disease is provided, wherein said individual is
treated with a polypeptide, said polypeptide having Pro329 of the
human IgG Fc region substituted with glycine, wherein the residues
are numbered according to the EU index of Kabat, wherein said
polypeptide is characterized by a strongly reduced binding
Fc.gamma.RIIIA and/or Fc.gamma.RIIA compared to a polypeptide
comprising a wildtype human IgG Fc region, comprising administering
to the individual an effective amount of said polypeptide.
[0017] In still another aspect of the invention the polypeptide
used in said method comprises at least two further amino acid
substitutions at L234A and L235A of the human IgG1 Fc region or
S228P and L235E of the human IgG4 Fc region.
BACKGROUND
[0018] Monoclonal antibodies have great therapeutic potential and
play an important role in today's medical portfolio. During the
last decade, a significant trend in the pharmaceutical industry has
been the development of monoclonal antibodies (mAbs) as therapeutic
agents for the treatment of a number of diseases, such as cancers,
asthma, arthritis, multiple sclerosis etc. Monoclonal antibodies
are predominantly manufactured as recombinant proteins in
genetically engineered mammalian cell culture.
[0019] The Fc region of an antibody, i.e., the terminal ends of the
heavy chains of antibody spanning domains CH2, CH3 and a portion of
the hinge region, is limited in variability and is involved in
effecting the physiological roles played by the antibody. The
effector functions attributable to the Fc region of an antibody
vary with the class and subclass of antibody and include binding of
the antibody via the Fc region to a specific Fc receptor ("FcR") on
a cell which triggers various biological responses.
[0020] These receptors typically have an extracellular domain that
mediates binding to Fc, a membrane spanning region, and an
intracellular domain that may mediate some signaling event within
the cell. These receptors are expressed in a variety of immune
cells including monocytes, macrophages, neutrophils, dendritic
cells, eosinophils, mast cells, platelets, B cells, large granular
lymphocytes, Langerhans' cells, natural killer (NK) cells, and T
cells. Formation of the Fc/Fc.gamma.R complex recruits these
effector cells to sites of bound antigen, typically resulting in
signaling events within the cells and important subsequent immune
responses such as release of inflammation mediators, B cell
activation, endocytosis, phagocytosis, and cytotoxic attack. The
ability to mediate cytotoxic and phagocytic effector functions is a
potential mechanism by which antibodies destroy targeted cells. The
cell-mediated reaction wherein nonspecific cytotoxic cells that
express Fc.gamma.Rs recognize bound antibody on a target cell and
subsequently cause lysis of the target cell is referred to as
antibody dependent cell-mediated cytotoxicity (ADCC) (Ravetch, et
al., Annu Rev Immunol 19 (2001) 275-290). The cell-mediated
reaction wherein nonspecific cytotoxic cells that express
Fc.gamma.Rs recognize bound antibody on a target cell and
subsequently cause phagocytosis of the target cell is referred to
as antibody dependent cell-mediated phagocytosis (ADCP). In
addition, an overlapping site on the Fc region of the molecule also
controls the activation of a cell independent cytotoxic function
mediated by complement, otherwise known as complement dependent
cytotoxicity (CDC).
[0021] For the IgG class of Abs, ADCC and ADCP are governed by
engagement of the Fc region with a family of receptors referred to
as Fc.gamma. receptors (Fc.gamma.Rs). In humans, this protein
family comprises Fc.gamma.RI (CD64); Fc.gamma.RII (CD32), including
isoforms Fc.gamma.RIIA, Fc.gamma.RIIB, and Fc.gamma.RIIC; and
Fc.gamma.RIII (CD16), including isoforms Fc.gamma.RIIIA and
Fc.gamma.RIIIB (Raghavan, and Bjorkman, Annu. Rev. Cell Dev. Biol.
12 (1996) 181-220; Abes, et al., Expert Reviews VOL 5(6), (2009)
735-747). Fc.gamma.Rs are expressed on a variety of immune cells,
and formation of the Fc/Fc.gamma.R complex recruits these cells to
sites of bound antigen, typically resulting in signaling and
subsequent immune responses such as release of inflammation
mediators, B cell activation, endocytosis, phagocytosis, and
cytotoxic attack. Furthermore, whereas Fc.gamma.RI,
Fc.gamma.RIIA/c, and Fc.gamma.RIIIA are activating receptors
characterized by an intracellular immunoreceptor tyrosine-based
activation motif (ITAM), Fc.gamma.RIIB has an inhibition motif
(ITIM) and is therefore inhibitory. Moreover, de Reys, et al.,
Blood, 81, (1993) 1792-1800 concluded that platelet activation and
aggregation induced by monoclonal antibodies, like for example CD9,
is initiated by antigen recognition followed by an Fc domain
dependent step, which involves the Fc.gamma.RII-receptor (sec also:
Taylor, et al., Blood 96 (2000) 4254-4260). While Fc.gamma.RI binds
monomeric IgG with high affinity, Fc.gamma.RIII and Fc.gamma.RII
are low-affinity receptors, interacting with complexed or
aggregated IgG.
[0022] The complement inflammatory cascade is a part of the innate
immune response and is crucial to the ability for an individual to
ward off infection. Another important Fc ligand is the complement
protein C1q. Fc binding to C1q mediates a process called complement
dependent cytotoxicity (CDC). C1q is capable of binding six
antibodies, although binding to two IgGs is sufficient to activate
the complement cascade. C1q forms a complex with the C1r and C1s
serine proteases to form the C1 complex of the complement
pathway.
[0023] In many circumstances, the binding and stimulation of
effector functions mediated by the Fc region of immunoglobulins is
highly beneficial, e.g. for a CD20 antibody, however, in certain
instances it may be more advantageous to decrease or even to
eliminate the effector function. This is particularly true for
those antibodies designed to deliver a drug (e.g., toxins and
isotopes) to the target cell where the Fc/Fc.gamma.R mediated
effector functions bring healthy immune cells into the proximity of
the deadly payload, resulting in depletion of normal lymphoid
tissue along with the target cells (Hutchins, et al., PNAS USA 92
(1995) 11980-11984; White, et al., Annu Rev Med 52 (2001) 125-145).
In these cases the use of antibodies that poorly recruit complement
or effector cells would be of a tremendous benefit (see also, Wu,
et al., Cell Immunol 200 (2000) 16-26; Shields, et al., J. Biol
Chem 276(9) (2001) 6591-6604; U.S. Pat. No. 6,194,551; U.S. Pat.
No. 5,885,573 and PCT publication WO 04/029207).
[0024] In other instances, for example, where blocking the
interaction of a widely expressed receptor with its cognate ligand
is the objective, it would be advantageous to decrease or eliminate
all antibody effector function to reduce unwanted toxicity. Also,
in the instance where a therapeutic antibody exhibited promiscuous
binding across a number of human tissues it would be prudent to
limit the targeting of effector function to a diverse set of
tissues to limit toxicity. Last but not least, reduced affinity of
antibodies to the Fc.gamma.RII receptor in particular would be
advantageous for antibodies inducing platelet activation and
aggregation via Fc.gamma.RII receptor binding, which would be a
serious side-effect of such antibodies.
[0025] Although there are certain subclasses of human
immunoglobulins that lack specific effector functions, there are no
known naturally occurring immunoglobulins that lack all effector
functions. An alternate approach would be to engineer or mutate the
critical residues in the Fc region that are responsible for
effector function. For examples see PCT publications WO 2009/100309
(Medimmune), WO 2006/076594 (Xencor), WO 1999/58572 (Univ.
Cambridge), US 2006/0134709 (Macrogenics), WO 2006/047350 (Xencor),
WO 2006/053301 (Xencor), U.S. Pat. No. 6,737,056 (Genentech), U.S.
Pat. No. 5,624,821 (Scotgen Pharmaceuticals), and US 2010/0166740
(Roche).
[0026] The binding of IgG to activating and inhibitory Fc.gamma.
receptors or the first component of complement (C1q) depends on
residues located in the hinge region and the CH2 domain. Two
regions of the CH2 domain are critical for Fc.gamma.Rs and
complement C1q binding, and have unique sequences. Substitution of
human IgG1 and IgG2 residues at positions 233-236 and IgG4 residues
at positions 327, 330 and 331 greatly reduced ADCC and CDC (Armour,
et al., Eur. J. Immunol. 29(8) (1999) 2613-2624; Shields, et al.,
J. Biol. Chem. 276(9) (2001) 6591-6604). Idusogie, et al., J.
Immunol 166 (2000) 2571-2575) mapped the C1q binding site for
rituxan and showed that Pro329Ala reduced the ability of Rituximab
to bind C1q and activate complement. Substitution of Pro329 with
Ala has been reported to lead to a reduced binding to the
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIIIA receptors (Shields, et
al., J. Biol. Chem. 276(9) (2001) 6591-6604) but this mutation has
also been described as exhibiting a wildtype-like binding to the
Fc.gamma.RI and Fc.gamma.RII and only a very small decrease in
binding to the Fc.gamma.RIIIA receptor (Table 1 and Table 2 in EP 1
068 241, Genentech).
[0027] Oganesyan, et al., Acta Cristallographica D64 (2008) 700-704
introduced the triple mutation L234F/L235E/P331S into the lower
hinge and C2H domain and showed a decrease in binding activity to
human IgG1 molecules to human C1q receptor, Fc.gamma.RI,
Fc.gamma.RII and Fc.gamma.RIIIA.
[0028] Still, there is an unmet need for antibodies with a strongly
decreased ADCC and/or ADCP and/or CDC. Therefore, the aim of the
current invention was to identify such antibodies. Surprisingly, it
has been found that mutating the proline residue at Pro329 to
glycine resulted in an unexpected strong inhibition of the
Fc.gamma.RIIIA and Fc.gamma.RIIA receptor and in a strong
inhibition of ADCC and CDC. Moreover, the combined mutation of
Pro329 and for example L234A and L235A (LALA) lead to an unexpected
strong inhibition of C1q, Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIIIA. Thus, a glycine residue appears to be unexpectedly
superior over other amino acid substitutions, like alanine, for
example, at position 329 in destroying the proline sandwich in the
Fc/Fc.gamma. receptor interlace.
DESCRIPTION OF THE FIGURES
[0029] FIGS. 1a-1e
[0030] Binding affinities of different Fc.gamma.Rs towards
immunoglobulins were measured by Surface Plasmon Resonance (SPR)
using a Biacore T100 instrument (GE Healthcare) at 25.degree.
C.
[0031] FIG. 1a) Fc.gamma.RI binding affinity was tested for GA101
(GA) antibody variants (IgG1-P329G, IgG4-SPLE and IgG1-LALA
mutation) and for P-selectin (PS) antibody variants (IgG1-P329G,
IgG1-LALA and IgG4-SPLE) as well as for the wildtype
antibodies.
[0032] FIG. 1b) Fc.gamma.RI binding affinity was tested for CD9
antibody variants (IgG1-wildtype, IgG1-P329G, IgG1-LALA, IgG4-SPLE,
IgG1-P329G/LALA, IgG4-SPLE/P329G) as well as for the wildtype
antibodies.
[0033] FIG. 1c) Fc.gamma.RIIA(R131) binding affinity was tested for
CD9 antibody variants (IgG1-wildtype, IgG1-P329G, IgG1-LALA,
IgG4-SPLE, IgG1-P329G/LALA, IgG4-SPLE/P329G) as well as for the
wildtype antibodies. A normalized response is shown as a function
of the concentration of the receptor.
[0034] FIG. 1d) Fc.gamma.RIIB binding affinity was tested for CD9
(named here: "TA") antibody variants (IgG1-wildtype,
IgG4-SPLE/P329G, IgG1-LALA, IgG1-LALA/P329G) and P-selectin (pSel)
antibody variants (IgG4-wildtype, IgG4-SPLE) as well as for the
wildtype antibodies.
[0035] FIG. 1e) Fc.gamma.RIIIA-V158 binding affinity was tested for
CD9 antibody variants (IgG1-wildtype, IgG4-SPLE, IgG1-LALA,
IgG4-SPLE/P329G, IgG1-P329G, IgG1-LALA/P329G) as well as for the
wildtype antibodies, a normalized response is shown as a function
of the concentration of the receptor.
[0036] FIG. 2
[0037] C1q binding was tested for P-selectin (PS) antibody variants
(IgG1 wildtype, P329G, IgG4-SPLE) and CD20 (GA) antibody variants
(IgG1-wildtype, P329G and IgG4-SPLE).
[0038] FIGS. 3a and 3b
[0039] Potency to recruit immune-effector cells depends on type of
Fc variant. Fc variants were coated on an ELISA plate and human
NK92 effector cells transfected with human Fc.gamma.RIIIA were
added. Induction of cytolytic activity of activated NK cells was
measured using an esterase assay.
[0040] FIG. 3a) CD20 (GA101) antibody variants (wildtype, LALA,
P329G, P329G/LALA) were analyzed. FIG. 3b) CD20 (GA101) antibody
variants (P329R or P329G mutations introduced) were analyzed. All
variants were produced in the glycoengineered version in order to
have a stronger signal for any effector cell recruitment
function.
[0041] FIGS. 4a and 4b
[0042] Potency to recruit immune-effector cells depends on type of
Fc variant, as measured by classical ADCC assay. Human NK92
cell-line transfected with human P.gamma.cRIIIA was used as
effector and CD20 positive Raji cells were used as target cells.
Different glycengineered CD20 antibody (GA101 G(2) and
non-glycoengineered CD20 antibody (GA101) variants (P329G, P329A or
LALA mutations introduced) were tested.
[0043] FIG. 4a) non-glycoengineered CD20 antibody: P329G, LALA and
P329G/LALA mutations, respectively, have been introduced into the
antibody, respectively.
[0044] FIG. 4b) glycoengineered CD20 antibody: P329G, P329A and
LALA mutations, respectively, have been introduced into the
antibody, respectively.
[0045] FIGS. 5a and 5b
[0046] Complement dependent cytotoxicity (CDC) assay. The different
Fc variants of a non-glycoengineered and glycoengineered CD20
(GA101) antibody were analyzed for their efficacy to mediate CDC on
SUDH-L4 target cells.
[0047] FIG. 5a) non-glycoengineered CD20: P329G, LALA and
P329G/LALA mutations, respectively, have been introduced into the
antibody, respectively.
[0048] FIG. 5b) glycoengineered CD20: P329G, P329A and LALA
mutations, respectively, have been introduced into the antibody,
respectively.
[0049] FIGS. 6a and 6b
[0050] FIG. 6a) Carbohydrate profile of Fc-associated glycans of
human IgG1 variants. The percentage of galactosylation on
Fc-associated oligosaccharides of hIgG1 containing the LALA, P329G,
P329A or P329G/LALA mutations only differs minimally from that of
wild type antibody.
[0051] FIG. 6b) Relative galactosylation: Four different IgGs with
introduced IgG1 P329G/LALA mutations. Four different V-domains were
compared for their amount of galactosylation when expressed in
Hek293 EBNA cells.
[0052] FIGS. 7a and 7b
[0053] Antibody-induced platelet aggregation in whole blood assay.
Murine IgG1 induced platelet aggregation as determined for two
donors differing in their response in dependence of the antibody
concentration.
[0054] FIG. 7a) Donor A, FIG. 7b) Donor B.
DETAILED DESCRIPTION OF THE INVENTION
[0055] Definitions
[0056] In the present specification and claims, the numbering of
the residues in an immunoglobulin heavy chain is that of the EU
index as in Kabat, et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991), expressly incorporated herein by
reference. The "EU index as in Kabat" refers to the residue
numbering of the human IgG1 EU antibody.
[0057] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen or an Fc receptor). Unless indicated otherwise, as used
herein, "binding affinity" refers to intrinsic binding affinity
which reflects a 1:1 interaction between members of a binding pair
(e.g., antibody/Fc receptor or antibody and antigen). The affinity
of a molecule X for its partner Y can generally be represented by
the dissociation constant (Kd). Affinity can be measured by common
methods known in the art, including those described herein.
Specific illustrative and exemplary embodiments for measuring
binding affinity are described in the following.
[0058] An "affinity matured" antibody refers to an antibody with
one or more alterations in one or more hypervariable regions
(HVRs), compared to a parent antibody which does not possess such
alterations, such alterations resulting in an improvement in the
affinity of the antibody for antigen.
[0059] An "amino acid modification" refers to a change in the amino
acid sequence of a predetermined amino acid sequence. Exemplary
modifications include an amino acid substitution, insertion and/or
deletion. The preferred amino acid modification herein is a
substitution. An "amino acid modification at" a specified position,
e.g. of the Fc region, refers to the substitution or deletion of
the specified residue, or the insertion of at least one amino acid
residue adjacent the specified residue. By insertion "adjacent" a
specified residue is meant insertion within one to two residues
thereof. The insertion may be N-terminal or C-terminal to the
specified residue.
[0060] An "amino acid substitution" refers to the replacement of at
least one existing amino acid residue in a predetermined amino acid
sequence with another different "replacement" amino acid residue.
The replacement residue or residues may be "naturally occurring
amino acid residues" (i.e. encoded by the genetic code) and
selected from the group consisting of: alanine (Ala); arginine
(Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys);
glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine
(His); isoleucine (Ile): leucine (Leu); lysine (Lys); methionine
(Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine
(Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val).
Preferably, the replacement residue is not cysteine. Substitution
with one or more non-naturally occurring amino acid residues is
also encompassed by the definition of an amino acid substitution
herein. A "non-naturally occurring amino acid residue" refers to a
residue, other than those naturally occurring amino acid residues
listed above, which is able to covalently bind adjacent amino acid
residues(s) in a polypeptide chain. Examples of non-naturally
occurring amino acid residues include norleucine, ornithine,
norvaline, homoserine and other amino acid residue analogues such
as those described in Ellman, et al., Meth. Enzym. 202 (1991)
301-336. To generate such non-naturally occurring amino acid
residues, the procedures of Noren, et al., Science 244 (1989) 182
and Ellman, et al., supra, can be used. Briefly, these procedures
involve chemically activating a suppressor tRNA with a
non-naturally occurring amino acid residue followed by in vitro
transcription and translation of the RNA.
[0061] An "amino acid insertion" refers to the incorporation of at
least one amino acid into a predetermined amino acid sequence.
While the insertion will usually consist of the insertion of one or
two amino acid residues, the present application contemplates
larger "peptide insertions", e.g. insertion of about three to about
five or even up to about ten amino acid residues. The inserted
residue(s) may be naturally occurring or non-naturally occurring as
disclosed above.
[0062] An "amino acid deletion" refers to the removal of at least
one amino acid residue from a predetermined amino acid
sequence.
[0063] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0064] The term "antibody variant" as used herein refers to a
variant of a wildtype antibody, characterized in that an alteration
in the amino acid sequence relative to the wildtype antibody occurs
in the antibody variant, e.g. introduced by mutations a specific
amino acid residues in the wildtype antibody.
[0065] The term "antibody effector function(s)," or "effector
function" as used herein refers to a function contributed by an Fc
effector domain(s) of an IgG (e.g., the Fc region of an
immunoglobulin). Such function can be effected by, for example,
binding of an Fc effector domain(s) to an Fc receptor on an immune
cell with phagocytic or lytic activity or by binding of an Fc
effector domain(s) to components of the complement system. Typical
effector functions are ADCC, ADCP and CDC.
[0066] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
hinds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain
antibody molecules (e.g. scFv); and multispecific antibodies formed
from antibody fragments.
[0067] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. An exemplary
competition assay is provided herein.
[0068] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express FcRs (e.g. Natural Killer (NK) cells,
neutrophils, and macrophages) recognize bound antibody on a target
cell and subsequently cause lysis of the target cell. The primary
cells for mediating ADCC, NK cells, express Fc.gamma.RIII only,
whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII FcR expression on hematopoietic cells is summarized
in Table 3 on page 464 of Ravetch, and Kinet, Annu. Rev. Immunol 9
(1991) 457-492.
[0069] The term "Antibody-dependent cellular phagocytosis" and
"ADCP" refer to a process by which antibody-coated cells are
internalized, either in whole or in part, by phagocytic immune
cells (e.g., macrophages, neutrophils and dendritic cells) that
bind to an immunoglobulin Fc region.
[0070] The term "binding domain" refers to the region of a
polypeptide that binds to another molecule. In the case of an FcR,
the binding domain can comprise a portion of a polypeptide chain
thereof (e.g. the a chain thereof) which is responsible for binding
an Fc region. One useful binding domain is the extracellular domain
of an FcR a chain.
[0071] The term "binding" to an Fc receptor used herein means the
binding of the antibody to a Fc receptor in a BIAcore.RTM. assay
for example (Pharmacia Biosensor AB, Uppsala, Sweden).
[0072] In the BIAcore.RTM. assay the Fc receptor is bound to a
surface and binding of the variant, e.g. the antibody variant to
which mutations have been introduced, is measured by Surface
Plasmon Resonance (SPR). The affinity of the binding is defined by
the terms ka (rate constant for the association of the antibody
from the antibody/Fc receptor complex), kd (dissociation constant),
and KD (kd/ka). Alternatively, the binding signal of a SPR
sensogram can be compared directly to the response signal of a
reference, with respect to the resonance signal height and the
dissociation behaviors.
[0073] "C1q" is a polypeptide that includes a binding site for the
Fc region of an immunoglobulin. C1q together with two serine
proteases, C1r and C1s, forms the complex C1, the first component
of the complement dependent cytotoxicity (CDC) pathway. Human C1q
can be purchased commercially from, e.g. Quidel, San Diego,
Calif.
[0074] The "CH2 domain" of a human IgG Fc region (also referred to
as "C.gamma.2" domain) usually extends from about amino acid 231 to
about amino acid 340. The CH2 domain is unique in that it is not
closely paired with another domain. Rather, two N-linked branched
carbohydrate chains are interposed between the two CH2 domains of
an intact native IgG molecule. It has been speculated that the
carbohydrate may provide a substitute for the domain-domain pairing
and help stabilize the CH2 domain (Burton, Molec. Immunol. 22
(1985) 161-206).
[0075] The "CH3 domain" comprises the stretch of residues
C-terminal to a CH2 domain in an Fc region (i.e. from about amino
acid residue 341 to about amino acid residue 447 of an IgG).
[0076] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include squamous cell
cancer, small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung, squamous carcinoma of the lung, cancer
of the peritoneum, hepatocellular cancer, gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney cancer, liver cancer, prostate
cancer, vulval cancer, thyroid cancer, hepatic carcinoma and
various types of head and neck cancer.
[0077] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived there
from without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
[0078] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0079] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0080] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu);
chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin,
vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents); growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; antibiotics; toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof; and the various antitumor or anticancer
agents disclosed below.
[0081] The term "complement-dependent cytotoxicity" or CDC refers
to a mechanism for inducing cell death in which an Fc effector
domain(s) of a target-bound antibody activates a series of
enzymatic reactions culminating in the formation of holes in the
target cell membrane. Typically, antigen-antibody complexes such as
those on antibody-coated target cells bind and activate complement
component C1q which in turn activates the complement cascade
leading to target cell death. Activation of complement may also
result in deposition of complement components on the target cell
surface that facilitate ADCC by binding complement receptors (e.g.,
CR3) on leukocytes.
[0082] A "disorder" is any condition that would benefit from
treatment with a polypeptide, like antibodies comprising an Fc
variant. This includes chronic and acute disorders or diseases
including those pathological conditions which predispose the mammal
to the disorder in question. In one embodiment, the disorder is
cancer.
[0083] "Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis (ADCP); down regulation of cell surface
receptors (e.g. B cell receptor); and B cell activation.
[0084] A "reduced effector function" as used herein refers to a
reduction of a specific effector function, like for example ADCC or
CDC, in comparison to a control (for example a polypeptide with a
wildtype Fc region), by at least 20% and a "strongly reduced
effector function" as used herein refers to a reduction of a
specific effector function, like for example ADCC or CDC, in
comparison to a control, by at least 50%.
[0085] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0086] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc regions and variant Fc regions. In one embodiment, a human IgG
heavy chain Fc region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat, et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991).
[0087] A "variant Fc region" comprises an amino acid sequence which
differs from that of a "native" or "wildtype" sequence Fc region by
virtue of at least one "amino acid modification" as herein defined.
Preferably, the variant Fc region has at least one amino acid
substitution compared to a native sequence Fc region or to the Fc
region of a parent polypeptide, e.g. from about one to about ten
amino acid substitutions, and preferably from about one to about
five amino acid substitutions in a native sequence Fc region or in
the Fc region of the parent polypeptide. The variant Fc region
herein will preferably possess at least about 80% homology with a
native sequence Fc region and/or with an Fc region of a parent
polypeptide, and most preferably at least about 90% homology
therewith, more preferably at least about 95% homology
therewith.
[0088] The term "Fc-variant" as used herein refers to a polypeptide
comprising a modification in an Fc domain. The Fc variants of the
present invention are defined according to the amino acid
modifications that compose them. Thus, for example, P329G is an Fc
variant with the substitution of proline with glycine at position
329 relative to the parent Fc polypeptide, wherein the numbering is
according to the EU index. The identity of the wildtype amino acid
may be unspecified, in which case the aforementioned variant is
referred to as P329G. For all positions discussed in the present
invention, numbering is according to the EU index. The EU index or
EU index as in Kabat or EU numbering scheme refers to the numbering
of the EU antibody (Edelman, et al., Proc Natl Acad Sci USA 63
(1969) 78-85, hereby entirely incorporated by reference.) The
modification can be an addition, deletion, or substitution.
Substitutions can include naturally occurring amino acids and
non-naturally occurring amino acids. Variants may comprise
non-natural amino acids. Examples include U.S. Pat. No. 6,586,207;
WO 98/48032; WO 03/073238; US 2004/0214988 A1; WO 05/35727 A2; WO
05/74524 A2; Chin, J. W., et al., Journal of the American Chemical
Society 124 (2002) 9026-9027; Chin, J. W., and Schultz, P. G.,
ChemBioChem 11 (2002) 1135-1137; Chin, J. W., et al., PICAS United
States of America 99 (2002) 11020-11024; and, Wang, L., and
Schultz, P. G., Chem. (2002) 1-10, all entirely incorporated by
reference.
[0089] The term "Fc region-containing polypeptide" refers to a
polypeptide, such as an antibody or immunoadhesin (see definitions
below), which comprises an Fc region.
[0090] The terms "Fc receptor" or "FcR" are used to describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor
tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
(see review in Daeron, M., Annu. Rev. Immunol. 15 (1997) 203-234).
FcRs are reviewed in Ravetch, and Kinet, Annu. Rev. Immunol 9
(1991) 457-492; Capel, et al., Immunomethods 4 (1994) 25-34; and de
Haas, et al., J. Lab. Clin. Med. 126 (1995) 330-41. Other FcRs,
including those to be identified in the future, are encompassed by
the term "FcR" herein. The term also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus (Guyer, et al., J. Immunol. 117 (1976) 587 and
Kim, et al., J. Immunol. 24 (1994) 249).
[0091] By "IgG Fc ligand" as used herein is meant a molecule,
preferably a polypeptide, from any organism that binds to the Fc
region of an IgG antibody to form an Fc/Fc ligand complex. Fc
ligands include but are not limited to Fc.gamma.Rs, Fc.gamma.Rs,
Fc.gamma.Rs, FcRn, C1q, C3, mannan binding lectin, mannose
receptor, staphylococcal protein A, streptococcal protein G, and
viral Fc.gamma.R. Fc ligands also include Fc receptor homologs
(FcRH), which are a family of Fc receptors that are homologous to
the Fc.gamma.Rs (Davis, et al., Immunological Reviews 190 (2002)
123-136, entirely incorporated by reference). Fc ligands may
include undiscovered molecules that bind Fc. Particular IgG Fc
ligands are FcRn and Fc gamma receptors. By "Fc ligand" as used
herein is meant a molecule, preferably a polypeptide, from any
organism that binds to the Fc region of an antibody to form an
Fc/Fc ligand complex.
[0092] By "Fc gamma receptor", "Fc.gamma.R" or "FcgammaR" as used
herein is meant any member of the family of proteins that bind the
IgG antibody Fc region and is encoded by an Fc.gamma.R gene. In
humans this family includes but is not limited to Fc.gamma.RI
(CD64), including isoforms Fc.gamma.RIA, Fc.gamma.RIB, and
Fc.gamma.RIC; Fc.gamma.RII (CD32), including isoforms Fc.gamma.RIIA
(including allotypes H131 and R131), Fc.gamma.RIIB (including
Fc.gamma.RIIB-1 and Fc.gamma.RIIB-2), and Fc.gamma.RIIc; and
Fc.gamma.RIII (CD16), including isoforms Fc.gamma.RIIIA (including
allotypes V158 and F158) and Fc.gamma.RIIIb (including allotypes
Fc.gamma.RIIB-NA1 and Fc.gamma.RIIB-NA2) (Jefferis, et al., Immunol
Lett 82 (2002) 57-65, entirely incorporated by reference), as well
as any undiscovered human Fc.gamma.Rs or Fc.gamma.R isoforms or
allotypes. An Fc.gamma.R may be from any organism, including but
not limited to humans, mice, rats, rabbits, and monkeys. Mouse
Fc.gamma.Rs include but are not limited to Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32), Fc.gamma.RIII (CD16), and Fc.gamma.RIII-2
(CD16-2), as well as any undiscovered mouse Fc.gamma.Rs or
Fc.gamma.R isoforms or allotypes.
[0093] By "FcRn" or "neonatal Fc Receptor" as used herein is meant
a protein that binds the IgG antibody Fc region and is encoded at
least in part by an FcRn gene. The FcRn may be from any organism,
including but not limited to humans, mice, rats, rabbits, and
monkeys. As is known in the art, the functional FcRn protein
comprises two polypeptides, often referred to as the heavy chain
and light chain. The light chain is beta-2-microglobulin and the
heavy chain is encoded by the FcRn gene. Unless other wise noted
herein, FcRn or an FcRn protein refers to the complex of FcRn heavy
chain with beta-2-microglobulin.
[0094] By "wildtype or parent polypeptide" as used herein is meant
an unmodified polypeptide that is subsequently modified to generate
a variant. The wildtype polypeptide may be a naturally occurring
polypeptide, or a variant or engineered version of a naturally
occurring polypeptide. Wildtype polypeptide may refer to the
polypeptide itself, compositions that comprise the parent
polypeptide, or the amino acid sequence that encodes it.
Accordingly, by "wildtype immunoglobulin" as used herein is meant
an unmodified immunoglobulin polypeptide that is modified to
generate a variant, and by "wildtype antibody" as used herein is
meant an unmodified antibody that is modified to generate a variant
antibody. It should be noted that "wildtype antibody" includes
known commercial, recombinantly produced antibodies as outlined
below.
[0095] The term "fragment crystallizable (Fc) polypeptide" is the
portion of an antibody molecule that interacts with effector
molecules and cells. It comprises the C-terminal portions of the
immunoglobulin heavy chains.
[0096] The term "Framework" or "FR" refers to variable domain
residues other than hypervariable region (HVR) residues. The FR of
a variable domain generally consists of four FR domains: FR1, FR2,
FR3, and FR4. Accordingly, the HVR and FR sequences generally
appear in the following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0097] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0098] A "functional Fc region" possesses an "effector function" of
a native sequence Fc region. Exemplary "effector functions" include
C1q binding; complement dependent cytotoxicity; Fc receptor
binding; antibody-dependent cell-mediated cytotoxicity (ADCC);
phagocytosis; down regulation of cell surface receptors (e.g. B
cell receptor; BCR), etc. Such effector functions generally require
the Fc region to be combined with a binding domain (e.g. an
antibody variable domain) and can be assessed using various assays
as herein disclosed, for example.
[0099] "Hinge region" is generally defined as stretching from
Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22 (1985)
161-206). Hinge regions of other IgG isotypes may be aligned with
the IgG1 sequence by placing the first and last cysteine residues
forming inter-heavy chain S--S bonds in the same positions.
[0100] The "lower hinge region" of an Fc region is normally defined
as the stretch of residues immediately C-terminal to the hinge
region, i.e. residues 233 to 239 of the Fc region.
[0101] "Homology" is defined as the percentage of residues in the
amino acid sequence variant that are identical after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent homology. Methods and computer programs for the
alignment are well known in the art. One such computer program is
"Align 2", authored by Genentech, Inc., which was filed with user
documentation in the United States Copyright Office, Washington,
D.C. 20559, on Dec. 10, 1991.
[0102] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived there from without regard to the number of passages.
Progeny may not be completely identical in nucleic acid content to
a parent cell, but may contain mutations. Mutant progeny that have
the same function or biological activity as screened or selected
for in the originally transformed cell are included herein.
[0103] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0104] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source thereof, e.g. from blood or PBMCs as described
herein.
[0105] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0106] The term "hypervariable region" or "HVR," as used herein,
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops ("hypervariable loops"). Generally, native four-chain
antibodies comprise six HVRs; three in the VH (H1, H2, H3), and
three in the VL (L1, L2, L3). HVRs generally comprise amino acid
residues from the hypervariable loops and/or from the
"complementarity determining regions" (CDRs), the latter being of
highest sequence variability and/or involved in antigen
recognition. Exemplary hypervariable loops occur at amino acid
residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55
(H2), and 96-101 (H3) (Chothia, and Lesk, J. Mol. Biol. 196 (1987)
901-917). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2,
and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2,
89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat, et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). With the exception of CDR1 in VH, CDRs generally comprise
the amino acid residues that form the hypervariable loops. CDRs
also comprise "specificity determining residues," or "SDRs," which
are residues that contact antigen. SDRs are contained within
regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary
a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and
a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2,
89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3 (See
Almagro, and Fransson, Front. Biosci. 13 (2008) 1619-1633). Unless
otherwise indicated, HVR residues and other residues in the
variable domain (e.g., FR residues) are numbered herein according
to Kabat et al., supra.
[0107] "Immune complex" refers to the relatively stable structure
which forms when at least one target molecule and at least one
heterologous Fc region-containing polypeptide bind to one another
forming a larger molecular weight complex. Examples of immune
complexes are antigen-antibody aggregates and target
molecule-immunoadhesin aggregates. The term "immune complex" as
used herein, unless indicated otherwise, refers to an ex vivo
complex (i.e. other than the form or setting in which it may be
found in nature). However, the immune complex may be administered
to a mammal, e.g. to evaluate clearance of the immune complex in
the mammal.
[0108] An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s), including but not limited to a
cytotoxic agent.
[0109] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0110] An "isolated" antibody is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman, et al., J. Chromatogr. B 848 (2007) 79-87.
[0111] An "isolated" polypeptide is one that has been identified
and separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the polypeptide, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the polypeptide will be purified (1) to greater than
95% by weight of polypeptide as determined by the Lowry method, and
most preferably more than 99% by weight, (2) to a degree sufficient
to obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated polypeptide
includes the polypeptide in situ within recombinant cells since at
least one component of the polypeptide's natural environment will
not be present. Ordinarily, however, isolated polypeptide will be
prepared by at least one purification step.
[0112] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0113] "Isolated nucleic acid encoding an antibody refers to one or
more nucleic acid molecules encoding antibody heavy and light
chains (or fragments thereof), including such nucleic acid
molecule(s) in a single vector or separate vectors, and such
nucleic acid molecule(s) present at one or more locations in a host
cell.
[0114] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the polypeptide. The label may be itself be detectable (e.g.,
radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, may catalyze chemical alteration of a substrate
compound or composition which is detectable.
[0115] The term "ligand binding domain" as used herein refers to
any native cell-surface receptor or any region or derivative
thereof retaining at least a qualitative ligand binding ability of
a corresponding native receptor. In a specific embodiment, the
receptor is from a cell-surface polypeptide having an extracellular
domain that is homologous to a member of the immunoglobulin
supergenefamily. Other receptors, which are not members of the
immunoglobulin supergenefamily but are nonetheless specifically
covered by this definition, are receptors for cytokines, and in
particular receptors with tyrosine kinase activity (receptor
tyrosine kinases), members of the hematopoietin and nerve growth
factor receptor superfamilies, and cell adhesion molecules, e.g.
(E-, L- and P-) selectins.
[0116] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0117] A "naked antibody" refers to an antibody that is not
conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or
radiolabel. The naked antibody may be present in a pharmaceutical
formulation.
[0118] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant arid variable domain.
[0119] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. Native sequence human Fc regions include a native
sequence human IgG1 Fc region (non-A and A allotypes); native
sequence human IgG2 Fc region; native sequence human IgG3 Fc
region; and native sequence human IgG4 Fc region as well as
naturally occurring variants thereof.
[0120] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0121] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0122] By "position" as used herein is meant a location in the
sequence of a protein. Positions may be numbered sequentially, or
according to an established format, for example the EU index for
antibody numbering.
[0123] The terms "polypeptide" and "protein" are used
interchangeably to refer to a polymer of amino acid residues,
comprising natural or non-natural amino acid residues, and are not
limited to a minimum length.
[0124] Thus, peptides, oligopeptides, dimers, multimers, and the
like are included within the definition. Both full-length proteins
and fragments thereof are encompassed by the definition. The terms
also include post-translational modifications of the polypeptide,
including, for example, glycosylation, sialylation, acetylation,
and phosphorylation.
[0125] Furthermore, a "polypeptide" herein also refers to a
modified protein such as single or multiple amino acid residue
deletions, additions, and substitutions to the native sequence, as
long as the protein maintains a desired activity. For example, a
serine residue may be substituted to eliminate a single reactive
cysteine or to remove disulfide bonding or a conservative amino
acid substitution may be made to eliminate a cleavage site. These
modifications may be deliberate, as through site-directed
mutagenesis, or may be accidental, such as through mutations of
hosts which produce the proteins or errors due to polymerase chain
reaction (PCR) amplification.
[0126] The term "wildtype polypeptide" and "wildtype (human) Fc
region" as used herein refers to a polypeptide and Fc region,
respectively, comprising an amino acid sequence which lacks one or
more of the Fc region modifications disclosed herein, because they
have not been introduced, and serve for example as controls. The
wildtype polypeptide may comprise a native sequence Fc region or an
Fc region with pre-existing amino acid sequence modifications (such
as additions, deletions and/or substitutions).
[0127] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0128] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0129] A polypeptide with "altered" FcR binding affinity or ADCC
activity is one which has either enhanced or diminished FcR binding
activity and/or ADCC activity compared to a parent polypeptide or
to a polypeptide comprising a native sequence Fc region. The
polypeptide variant which "displays increased binding" to an FcR
binds at least one FcR with better affinity than the parent
polypeptide. The polypeptide variant which "displays decreased
binding" to an FcR, binds at least one FcR with worse affinity than
a parent polypeptide. Such variants which display decreased binding
to an FcR may possess little or no appreciable binding to an FcR,
e.g., 0-20% binding to the FcR compared to a native sequence IgG Fc
region, e.g. as determined in the Examples herein.
[0130] The polypeptide which hinds an FcR with "reduced affinity"
than a parent polypeptide, is one which binds any one or more of
the above identified FcRs with substantially reduced binding
affinity than the parent antibody, when the amounts of polypeptide
variant and parent polypeptide in the binding assay are essentially
the same. For example, the polypeptide variant with reduced FcR
binding affinity may display from about 1.15 fold to about 100
fold, e.g. from about 1.2 fold to about 50 fold reduction in FcR
binding affinity compared to the parent polypeptide, where FcR
binding affinity is determined, for example, as disclosed in the
Examples herein.
[0131] The polypeptide comprising an Fc variant which "mediates
antibody-dependent cell-mediated cytotoxicity (ADCC) in the
presence of human effector cells less effectively" than a parent or
wildtype polypeptide is one which in vitro or in vivo is
substantially less effective at mediating ADCC, when the amounts of
polypeptide variant and parent antibody used in the assay are
essentially the same. Generally, such variants will be identified
using the in vitro ADCC assay as herein disclosed, but other assays
or methods for determining ADCC activity, e.g. in an animal model
etc, are contemplated. The preferred variant is from about 1.5 fold
to about 100 fold, e.g. from about two fold to about fifty fold,
less effective at mediating ADCC than the parent, e.g. in the in
vitro assay disclosed herein.
[0132] A "receptor" is a polypeptide capable of binding at least
one ligand. The preferred receptor is a cell-surface receptor
having an extracellular ligand-binding domain and, optionally,
other domains (e.g. transmembrane domain, intracellular domain
and/or membrane anchor). The receptor to be evaluated in the assay
described herein may be an intact receptor or a fragment or
derivative thereof (e.g. a fusion protein comprising the binding
domain of the receptor fused to one or more heterologous
polypeptides). Moreover, the receptor to be evaluated for its
binding properties may be present in a cell or isolated and
optionally coated on an assay plate or some other solid phase.
[0133] The term "receptor binding domain" is used to designate any
native ligand for a receptor, including cell adhesion molecules, or
any region or derivative of such native ligand retaining at least a
qualitative receptor binding ability of a corresponding native
ligand. This definition, among others, specifically includes
binding sequences from ligands for the above-mentioned
receptors.
[0134] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of
the invention are used to delay development of a disease or to slow
the progression of a disease.
[0135] By "variant protein" or "protein variant", or "variant" as
used herein is meant a protein that differs from that of a parent
protein by virtue of at least one amino acid modification. Protein
variant may refer to the protein itself, a composition comprising
the protein, or the amino sequence that encodes it. Preferably, the
protein variant has at least one amino acid modification compared
to the parent protein, e.g. from about one to about seventy amino
acid modifications, and preferably from about one to about five
amino acid modifications compared to the parent. The protein
variant sequence herein will preferably possess at least about 80%
homology with a parent protein sequence, and most preferably at
least about 90% homology, more preferably at least about 95%
homology. Variant protein can refer to the variant protein itself,
compositions comprising the protein variant, or the DNA sequence
that encodes it. Accordingly, by "antibody variant" or "variant
antibody" as used herein is meant an antibody that differs from a
parent antibody by virtue of at least one amino acid modification,
"IgG variant" or "variant IgG" as used herein is meant an antibody
that differs from a parent IgG by virtue of at least one amino acid
modification, and "immunoglobulin variant" or "variant
immunoglobulin" as used herein is meant an immunoglobulin sequence
that differs from that of a parent immunoglobulin sequence by
virtue of at least one amino acid modification.
[0136] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt, et al., Kuby
Immunology, 6th ed., W.H. Freeman and Co. (2007) page 91). A single
VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano, et al., J. Immunol. 150 (1993)
880-887; Clackson, et al., Nature 352 (1991) 624-628.
[0137] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors."
[0138] The present application is directed to polypeptides that
include amino acid modifications that modulate binding to Fc
receptors, in particularly to Fc.gamma. receptors.
DETAILED DESCRIPTION
[0139] The invention herein relates to a method for making a
polypeptide comprising a Fc variant. The "parent", "starting",
"nonvariant" or wildtype polypeptide is prepared using techniques
available in the art for generating polypeptides or antibodies
comprising an Fc region. In the preferred embodiment of the
invention, the parent polypeptide is an antibody and exemplary
methods for generating antibodies are described in more detail in
the following sections. The parent polypeptide may, however, be any
other polypeptide comprising an Fc region, e.g. an immunoadhesin.
Methods for making immunoadhesins are elaborated in more detail
herein below.
[0140] In an alternative embodiment, a variant Fc region (Fc
variant) may be generated according to the methods herein disclosed
and this Fc variant can be fused to a heterologous polypeptide of
choice, such as an antibody variable domain or binding domain of a
receptor or ligand.
[0141] The wildtype polypeptide comprises an Fc region. Generally
the Fc region of the wildtype polypeptide will comprise a native or
wildtype sequence Fc region, and preferably a human native sequence
Fc region (human Fc region). However, the Fc region of the wildtype
polypeptide may have one or more pre-existing amino acid sequence
alterations or modifications from a native sequence Fc region. For
example, the C1q or Fc.gamma. binding activity of the Fc region may
have been previously altered (other types of Fc region
modifications are described in more detail below). In a further
embodiment the parent polypeptide Fc region is "conceptual" and,
while it does not physically exist, the antibody engineer may
decide upon a desired variant Fc region amino acid sequence and
generate a polypeptide comprising that sequence or a DNA encoding
the desired variant Fc region amino acid sequence.
[0142] In the preferred embodiment of the invention, however, a
nucleic acid encoding an Fc region of a wildtype polypeptide is
available and this nucleic acid sequence is altered to generate a
variant nucleic acid sequence encoding the Fc region variant.
[0143] DNA encoding an amino acid sequence variant of the starting
polypeptide is prepared by a variety of methods known in the art.
These methods include, but are not limited to, preparation by
site-directed (or oligonucleotide-mediated) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared DNA
encoding the polypeptide
[0144] Site-directed mutagenesis is a preferred method for
preparing substitution variants. This technique is well known in
the art (see, e.g., Carter, et al., Nucleic Acids Res. 13 (1985)
4431-4443 and Kunkel, et al., Proc. Natl. Acad. Sci. USA 82 (1985)
488). Briefly, in carrying out site-directed mutagenesis of DNA,
the starting DNA is altered by first hybridizing an oligonucleotide
encoding the desired mutation to a single strand of such starting
DNA. After hybridization, a DNA polymerase is used to synthesize an
entire second strand, using the hybridized oligonucleotide as a
primer, and using the single strand of the starting DNA as a
template. Thus, the oligonucleotide encoding the desired mutation
is incorporated in the resulting double-stranded DNA.
[0145] PCR mutagenesis is also suitable for making amino acid
sequence variants of the starting polypeptide. See Higuchi, in PCR
Protocols, Academic Press (1990) pp. 177-183; and Vallette, et al.,
Nuc. Acids Res. 17 (1989) 723-733. Briefly, when small amounts of
template DNA are used as starting material in a PCR, primers that
differ slightly in sequence from the corresponding region in a
template DNA can be used to generate relatively large quantities of
a specific DNA fragment that differs from the template sequence
only at the positions where the primers differ from the
template.
[0146] Another method for preparing variants, cassette mutagenesis,
is based on the technique described by Wells, et al., Gene 34
(1985) 315-323.
[0147] One embodiment of the invention encompasses polypeptides
comprising an Fc region of an antibody, comprising the addition,
substitution, or deletion of at least one amino acid residue to the
Fc region resulting in reduced or ablated affinity for at least one
Fc receptor. The Fc region interacts with a number of receptors or
ligands including but not limited to Fc Receptors (e.g.,
Fc.gamma.RI, Fc.gamma.RIIA, Fc.gamma.RIIIA), the complement protein
CIq, and other molecules such as proteins A and G. These
interactions are essential for a variety of effector functions and
downstream signaling events including, but not limited to, antibody
dependent cell-mediated cytotoxicity (ADCC), Antibody-dependent
cellular phagocytosis (ADCP) and complement dependent cytotoxicity
(CDC). Accordingly, in certain embodiments the variants of the
invention have reduced or ablated affinity for an Fc receptor
responsible for an effector function compared to a polypeptide
having the same amino acid sequence as the polypeptide comprising a
Fc variant of the invention but not comprising the addition,
substitution, or deletion of at least one amino acid residue to the
Fc region (also referred to herein as an "wildtype polypeptide").
In certain embodiments, polypeptide comprising a Fc variant of the
invention comprise at least one or more of the following
properties: reduced or ablated effector (ADCC and/or CDC and/or
ADCP) function, reduced or ablated binding to Fc receptors, reduced
or ablated binding to C1q or reduced or ablated toxicities. More
specifically, embodiments of the invention provide anti-CD20 (same
as GA101 or GA), anti-CD9 (same as TA) and anti-Selectin (pSel)
antibodies with reduced affinity for Fc receptors (e.g.
Fc.gamma.RI, Fc.gamma.RII, Fc.gamma.RIIIA) and/or the complement
protein C1q.
[0148] In one embodiment, antibodies of the invention comprise an
Fc region comprising at least one addition, substitution, or
deletion of an amino acid residue at position P329, wherein the
numbering system of the constant region is that of the EU index as
set forth in Kabat, et al., NIH Publication 91 (1991) 3242,
National Technical Information Service, Springfield, Va.
[0149] In a specific embodiment, polypeptides of the invention
comprise an Fc variant of a wild-type human Fc polypeptide said
variant comprising an amino acid substitution at position Pro329,
where the numbering of the residues in the IgG Fc region is that of
the EU index as in Kabat. In still another embodiment, said variant
comprises at least one further amino acid substitution.
[0150] In still another embodiment the polypeptide comprising a Fc
variant of a wild-type human Fc polypeptide has an amino acid
substitution, deletion or addition which destroys or diminishes the
function of the proline sandwich in the region and/or interface of
the Fc polypeptide with the Fc Gamma receptor.
[0151] In another embodiment Pro329 is substituted with an amino
acid which is either smaller or larger then proline. In still
another embodiment the substituted amino acid is Gly, Ala or Arg.
In a further aspect of the invention Pro329 of the Fc polypeptide
is substituted with glycine.
[0152] In still another embodiment said polypeptide comprising a Fc
variant has at least one further amino acid substitution, addition
of deletion. In still another embodiment, said variants exhibit a
reduced affinity to a human Fc receptor (Fc.gamma.R) and/or a human
complement receptor as compared to the polypeptide comprising the
wildtype Fc polypeptide.
[0153] In another embodiment said polypeptide comprising a Fc
variant exhibits a reduced affinity to a human Fc receptor
(Fc.gamma.R) and/or a human complement receptor as compared to the
polypeptide comprising the wildtype human Fc region. In a further
embodiment the affinity to at least one of the Fc.gamma.RI,
Fc.gamma.RII, Fc.gamma.RIIIA is reduced, in a still further
embodiment the affinity to the Fc.gamma.RI and Fc.gamma.RIIIA is
reduced, and in a still further embodiment the affinity to the
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIIIA is reduced, in still a
further aspect of the invention the affinity to the Fc.gamma.RI
receptor, Fc.gamma.RIIIA receptor and C1q is reduced, and in still
a further aspect of the invention the affinity to the Fc.gamma.RI,
Fc.gamma.RII, Fc.gamma.RIIIA and C1q receptor is reduced.
[0154] In still a further embodiment the ADCC induced by said
polypeptide comprising a Fc variant is reduced and in a preferred
embodiment the ADCC is reduced to at least 20% of the ADCC induced
by the polypeptide comprising the wildtype Fc polypeptide. In still
a further aspect of the invention, the ADCC and CDC induced by the
polypeptide comprising the wildtype Fc polypeptide is decreased or
ablated and in a still further aspect the polypeptide comprising a
Fc variant described above exhibit a decreased ADCC, CDC and ADCP
compared to the polypeptide comprising the wildtype Fc
polypeptide.
[0155] In one embodiment the at least one further amino acid
substitution in the polypeptide comprising the Fc variant is
selected from the group: S228P, E233P, L234A, L235A, L235E, N297A,
N297D, or P331S.
[0156] In a certain aspect of the invention the polypeptide
comprising a Fc variant comprises an antibody. In still another
aspect of the invention the polypeptide comprising a Fc variant
comprises a human IgG1 or IgG4 Fc region. In still a further aspect
of the invention the variants are IgG1 or IgG4 antibodies.
[0157] In another embodiment of the invention, polypeptides
comprising a Pro329 Fc variant variants further comprise at least
one addition, substitution, or deletion of an amino acid residue in
the Fc region that is correlated with increased stability of the
antibody. In still a further aspect of the invention the affinity
of the polypeptide comprising a Fc variant described above to the
Fcn receptor is only slightly, and for example not more than 10-20%
of the affinity of polypeptide comprising the wildtype Fc
polypeptide altered.
[0158] In one embodiment, the addition, substitution, or deletion
of an amino acid residue in a polypeptide comprising a Fc variant
is at position 228 and/or 235 of the Fc region, wherein the
numbering system of the constant region is that of the EU index as
set forth in Kabat, et al.
[0159] In a specific embodiment serine at position 228 and/or
leucine at position 235 in said polypeptide comprising a Fc variant
is substituted by another amino acid.
[0160] In a specific embodiment, polypeptides comprising a Fc
variant of the invention comprise an Fc region comprising an amino
acid substitution at position 228, wherein the serine residue is
substituted with proline.
[0161] In a specific embodiment, polypeptides comprising a Fc
variant of the invention comprise an Fc region comprising an amino
acid substitution at position 235, wherein the leucine residue is
substituted with glutamic acid.
[0162] In a specific embodiment the polypeptide comprising a Fc
variant comprises a triple mutation: an amino acid substitution at
position P329, a S228P and a L235E mutation (P329/SPLE).
[0163] In a further specific embodiment the polypeptide comprising
a Fc variant comprises a human IgG4 region.
[0164] In one embodiment, the addition, substitution, or deletion
of an amino acid residue is at position 234 and/or 235 of the Fc
region, wherein the numbering system of the constant region is that
of the EU index as set forth in Kabat et al.
[0165] In a specific embodiment leucine at position 234 and/or
leucine at position 235 in the polypeptide comprising a Fc variant
is substituted by another amino acid.
[0166] In a specific embodiment, polypeptides comprising a Fc
variant of the invention comprise an Fc region comprising an amino
acid substitution at position 234, wherein the leucine residue is
substituted with alanine.
[0167] In a specific embodiment, polypeptides comprising a Fc
variant of the invention comprise an Fc region comprising an amino
acid substitution at position 235, wherein the leucine residue is
substituted with serine.
[0168] In a specific embodiment the polypeptide comprising an Fc
variant of a wildtype human Fc polypeptide comprises a triple
mutation: an amino acid substitution at position Pro329, a L234A
and a L235A mutation (P329/LALA).
[0169] In a further specific embodiment the above mentioned
polypeptides comprise a human IgG1 region.
[0170] While it is preferred to alter binding to a Fc.gamma.R, Fc
region variants with altered binding affinity for the neonatal
receptor (FcRn) are also contemplated herein. Fc region variants
with improved affinity for FcRn are anticipated to have longer
serum half-lives, and such molecules will have useful applications
in methods of treating mammals where long half-life of the
administered polypeptide is desired, e.g., to treat a chronic
disease or disorder. Fc region variants with decreased FcRn binding
affinity, on the contrary, are expected to have shorter half-lives,
and such molecules may, for example, be administered to a mammal
where a shortened circulation time may be advantageous, e.g. for in
vivo diagnostic imaging or for polypeptides which have toxic side
effects when left circulating in the blood stream for extended
periods, etc. Fc region variants with decreased FcRn binding
affinity are anticipated to be less likely to cross the placenta,
and thus may be utilized in the treatment of diseases or disorders
in pregnant women.
[0171] Fc region variants with altered binding affinity for FcRn
include those comprising an Fc region amino acid modification at
any one or more of amino acid positions 238, 252, 253, 254, 255,
256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340,
356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424,
433, 434, 435, 436, 439 or 447. Those which display reduced binding
to FcRn will generally comprise an Fc region amino acid
modification at any one or more of amino acid positions 252, 253,
254, 255, 288, 309, 386, 388, 400, 415, 433, 435, 436, 439 or 447;
and those with increased binding to FcRn will usually comprise an
Fc region amino acid modification at any one or more of amino acid
positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317,
340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434.
[0172] In another embodiment, antibodies of the invention may be
any of any class (for example, but not limited to IgG, IgM, and
IgE). In certain embodiments, antibodies of the invention are
members of the IgG class of antibodies. In a specific embodiment,
antibodies of the invention are of the IgG1, IgG2 or IgG4 subclass.
In another specific embodiment, antibodies of the invention are of
the IgG1 subclass and comprise the following amino acid
substitutions: P329G and/or L234A and L235A of the Fc region. In
alternate embodiments, antibodies of the invention are of the IgG4
subclass. In a specific embodiment, antibodies of the invention are
of the IgG4 subclass and comprise the following amino acid
substitutions: P329G and/or S228P and L235E of the Fc region. In
certain embodiments, the modified antibodies of the present
invention may be produced by combining a variable domain, or
fragment thereof, with an Fc domain comprising one or more of the
amino acid substitutions disclosed herein. In other embodiments
modified antibodies of the invention may be produced by modifying
an Fc domain-containing antibody by introducing one or more of the
amino acid substitutions residues into the Fc domain.
[0173] Reduced Binding to Fc Ligands
[0174] One skilled in the art will understand that antibodies of
the invention may have altered (relative to an unmodified antibody)
Fc.gamma.R and/or C1q binding properties (examples of binding
properties include but are not limited to, binding specificity,
equilibrium dissociation constant (K.sub.D), dissociation and
association rates (k.sub.off and k.sub.on, respectively) binding
affinity and/or avidity) and that certain alterations are more or
less desirable. It is known in the art that the equilibrium
dissociation constant (K.sub.D) is defined as k.sub.off/k.sub.on.
One skilled in the art can determine which kinetic parameter is
most important for a given antibody application. For example, a
modification that reduces binding to one or more positive regulator
(e.g., Fc.gamma.RIIIA) and/or enhanced binding to an inhibitory Fc
receptor (e.g., Fc.gamma.RIIB) would be suitable for reducing ADCC
activity. Accordingly, the ratio of binding affinities (e.g.,
equilibrium dissociation constants (K.sub.D)) can indicate if the
ADCC activity of an antibody of the invention is enhanced or
decreased. Additionally, a modification that reduces binding to C1q
would be suitable for reducing or eliminating CDC activity. The
affinities and binding properties of an Fc region for its ligand,
may be determined by a variety of in vitro assay methods
(biochemical or immunological based assays) known in the art for
determining Fc-Fc.gamma.R interactions, i.e., specific binding of
an Fc region to an Fc.gamma.R including but not limited to,
equilibrium methods (e.g., enzyme-linked immuno absorbent assay
(ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE.RTM.
analysis), and other methods such as indirect binding assays,
competitive inhibition assays, fluorescence resonance energy
transfer (FRET), gel electrophoresis and chromatography (e.g., gel
filtration). These and other methods may utilize a label on one or
more of the components being examined and/or employ a variety of
detection methods including but not limited to chromogenic,
fluorescent, luminescent, or isotopic labels. A detailed
description of binding affinities and kinetics can be found in
Paul, W. E., ed., Fundamental Immunology, 4.sup.th Ed.,
Lippincott-Raven, Philadelphia (1999).
[0175] In one aspect of the invention a polypeptide comprising an
Fc variant of a wild-type human Fc region, said variant comprising
an amino acid substitution at position Pro329 and at least one
further amino acid substitution, exhibits a reduced affinity to a
human Fc receptor (Fc.gamma.R) and/or a human complement receptor
as compared to the polypeptide comprising the wildtype Fc
polypeptide. In one aspect polypeptides comprising an Fc variant of
the invention exhibit affinities for a Fc receptor that is at least
2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold,
or a least 10 fold, or at least 20 fold, or at least 30 fold, or at
least 40 fold, or at least 50 fold, or at least 60 fold, or at
least 70 fold, or at least 80 fold, or at least 90 fold, or at
least 100 fold, or at least 200 fold less than for a wildtype Fc
polypeptide.
[0176] In one aspect polypeptides comprising an Fc variant of the
invention exhibit reduced binding affinity for one or more Fc
receptors including, but not limited to Fc.gamma.RI (CD64)
including isoforms Fc.gamma.RIA, Fc.gamma.RII and Fc.gamma.RIII (CD
16, including isoforms Fc.gamma.RIIIA) as compared to an unmodified
antibody.
[0177] In one aspect polypeptides comprising an Fc variant of the
invention exhibit reduced binding affinity for Fc.gamma.RI (CD64)
Fc.gamma.RIIA and Fc.gamma.RIIIA as compared to an unmodified
antibody.
[0178] In one aspect polypeptides comprising an Fc variant of the
invention exhibit reduced binding affinity for Fc.gamma.RIIA and
Fc.gamma.RIIIA as compared to an unmodified antibody.
[0179] In one aspect polypeptides comprising an Fc variant of the
invention exhibit reduced binding affinity for Fc.gamma.RI (CD64)
and Fc.gamma.RIIIA as compared to an unmodified antibody.
[0180] In one aspect of the invention polypeptides comprising an Fc
variant of the invention exhibiting a reduced binding affinity for
the Fc receptors also exhibit a reduced affinity to the C1q
receptor.
[0181] In certain aspect polypeptides comprising an Fc variant of
the invention do not comprise a concomitant increase in binding to
the Fc.gamma.RIIIB receptor as compared to a wildtype polypeptide.
In certain aspects of the invention the polypeptides comprising an
Fc variant have a reduced affinity to the human receptor
Fc.gamma.IIIA, and to at least one further receptor of the group
comprising the human receptors Fc.gamma.IIA, Fc.gamma.IIIB, and C1q
compared to the polypeptide comprising the wildtype Fc polypeptide.
In further aspects of the invention polypeptides comprising an Fc
variant have a reduced affinity to the human receptor
Fc.gamma.IIIA, and to at two further receptors of the group
comprising the human receptors Fc.gamma.IIA, Fc.gamma.IIIB, and C1q
compared to the polypeptide comprising the wildtype Fc polypeptide.
In further aspect of the invention the polypeptides comprising an
Fc variant have a reduced affinity to the human Fc.gamma.RIA,
Fc.gamma.IIIA, Fc.gamma.IIA, Fc.gamma.IIIB, and C1q compared to the
polypeptide comprising the wildtype Fc polypeptide. In still
another aspect of the invention polypeptides comprising an Fc
variant have a reduced affinity to the human receptor Fc.gamma.RIA,
Fc.gamma.IIIA, Fc.gamma.IIA, Fc.gamma.IIIB, and C1q compared to the
polypeptide comprising the wildtype Fc polypeptide.
[0182] In one aspect of the invention polypeptides comprising an Fc
variant of the invention exhibit decreased affinities to
Fc.gamma.RI or Fc.gamma.RIIA relative to an unmodified antibody. In
one aspect of the invention polypeptides comprising an Fc variant
exhibit affinities for Fc.gamma.RI or Fc.gamma.RIIA that are at
least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7
fold, or a least 10 fold, or at least 20 fold, or at least 30 fold,
or at least 40 fold, or at least 50 fold, or at least 60 fold, or
at least 70 fold, or at least 80 fold, or at least 90 fold, or at
least 100 fold, or at least 200 fold less than that of a wildtype
polypeptide. In one aspect of the invention polypeptides comprising
an Fc variant exhibit affinity for the Fc.gamma.RI or Fc.gamma.RIIA
that are at least 90%, at least 80%, at least 70%, at least 60%, at
least 50%, at least 40%, at least 30%, at least 20%, at least 10%,
or at least 5% less than a than that of a wildtype polypeptide.
[0183] In one aspect of the invention polypeptides comprising an Fc
variant of the invention exhibit decreased affinity for the
Fc.gamma.RIIIA relative to an unmodified antibody. In one aspect
polypeptides comprising an Fc variant of the invention exhibit
affinities for Fc.gamma.RIIIA that are at least 2 fold, or at least
3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold,
or at least 20 fold, or at least 30 fold, or at least 40 fold, or
at least 50 fold, or at least 60 fold, or at least 70 fold, or at
least 80 fold, or at least 90 fold, or at least 100 fold, or at
least 200 fold less than that of a wildtype polypeptide.
[0184] In one aspect of the invention polypeptides comprising an Fc
variant of the invention exhibit affinities for Fc.gamma.RIIIA that
are at least 90%, at least 80%, at least 70%, at least 60%, at
least 50%, at least 40%, at least 30%, at least 20%, at least 10%,
or at least 5% less than that of a wildtype polypeptide.
[0185] It is understood in the art that the F1-58V allelic variant
of the Fc.gamma.RIIIA has altered binding characteristics to
antibodies. In one embodiment, polypeptides comprising an Fc
variant of the invention bind with decreased affinities to
Fc.gamma.RIIIA receptors relative to a wildtype polypeptide. In one
aspect polypeptides comprising an Fc variant of the invention
exhibit affinities for Fc.gamma.RIIIA (F1 58V) that are at least 2
fold, or at least 3 fold, or at least 5 fold, or at least 7 fold,
or a least 10 fold, or at least 20 fold, or at least 30 fold, or at
least 40 fold, or at least 50 fold, or at least 60 fold, or at
least 70 fold, or at least 80 fold, or at least 90 fold, or at
least 100 fold, or at least 200 fold less than that of a wildtype
polypeptide.
[0186] In one aspect of the invention polypeptides comprising an Fc
variant of the invention exhibit decreased affinity for the C1q
receptor relative to an unmodified antibody. In one aspect
polypeptides comprising an Fc variant of the invention exhibit
affinities for C1q receptor that are at least 2 fold, or at least 3
fold, or at least 5 fold, or at least 7 fold, or a least 10 fold,
or at least 20 fold, or at least 30 fold, or at least 40 fold, or
at least 50 fold, or at least 60 fold, or at least 70 fold, or at
least 80 fold, or at least 90 fold, or at least 100 fold, or at
least 200 fold less than that of a wildtype polypeptide.
[0187] In one aspect of the invention polypeptides comprising an Fc
variant of the invention exhibit affinities for C1q that are at
least 90%, at least 80%, at least 70%, at least 60%, at least 50%,
at least 40%, at least 30%, at least 20%, at least 10%, or at least
5% less than that of a wildtype polypeptide.
[0188] In one aspect of the invention polypeptides comprising an Fc
variant of the invention exhibit affinities for the human
Fc.gamma.RI, Fc.gamma.RIIA, Fc.gamma.RIIIA, Fc.gamma.RIIIA (F1 58V)
or C1q receptors that are at least 90%, at least 80%, at least 70%,
at least 60%, at least 50%, at least 40%, at least 30%, at least
20%, at least 10%, or at least 5% less than a wildtype
polypeptide.
[0189] In another aspect of the invention polypeptides comprising
an Fc variant of the invention exhibit affinities for the
Fc.gamma.RI, Fc.gamma.RIIA, Fc.gamma.RIIIA, Fc.gamma.RIIIA (F1 58V)
and/or C1q receptors, respectively, that are between about 10 nM to
100 nM, 10 nM to 1 .mu.M, 100 nM to about 100 .mu.M, or about 100
nM to about 10 .mu.M, or about 100 nM to about 1 .mu.M, or about 1
nM to about 100 .mu.M, or about 10 nM to about 100 .mu.M, or about
1 .mu.M to about 100 .mu.M, or about 10 .mu.M to about 100 .mu.M.
In certain embodiments, polypeptides comprising an Fc variant of
the invention exhibit affinities for the Fc.gamma.RI,
Fc.gamma.RIIA, Fc.gamma.RIIIA, Fc.gamma.RIIIA (F1-58V) or C1q
receptors that are greater than 100 nM, 500 nM, 1 .mu.M, greater
than 5 .mu.M, greater than 10 .mu.M, greater than 25 .mu.M, greater
than 50 .mu.M, or greater than 100 .mu.M.
[0190] In another aspect of the invention polypeptides comprising
an Fc variant of the invention exhibit increased affinities for the
Fc.gamma.RIIB as compared to a wildtype polypeptide. In another
aspect of polypeptides comprising an Fc variant of the invention
exhibit affinities for the Fc.gamma.RIIB that are unchanged or
increased by at least at least 2 fold, or at least 3 fold, or at
least 5 fold, or at least 7 fold, or a least 10 fold, or at least
20 fold, or at least 30 fold, or at least 40 fold, or at least 50
fold, or at least 60 fold, or at least 70 fold, or at least 80
fold, or at least 90 fold, or at least 100 fold, or at least 200
fold than that of an unmodified antibody. In another aspect
polypeptides comprising an Fc variant of the invention exhibit
affinities for the Fc.gamma.RIIB receptor that are increased by at
least 5%, at least 10%, at least 20%, at least 30%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, or at least 95% than a wildtype polypeptide.
[0191] In another aspect of the invention variants of the invention
exhibit affinities for the Fc.gamma.RI, Fc.gamma.RIIA
Fc.gamma.RIIIA, or Fc.gamma.RIIIA (F1 58V) or C1q receptors that
are less than 100 .mu.M, less than 50 .mu.M, less than 10 .mu.M,
less than 5 .mu.M, less than 2.5 .mu.M, less than 1 .mu.M, or less
than 100 nM, or less than 10 nM.
[0192] Reduced Effector Function
[0193] In a certain aspects of the invention polypeptides
comprising an Fc variant according to the invention modulate an
effector function as compared to the polypeptide comprising the
wildtype Fc polypeptide.
[0194] In still another aspect of the invention this modulation is
a modulation of ADCC and/or ADCP and/or CDC. In a further aspect of
the invention this modulation is down-modulation or reduction in
effect. In still another aspect of the invention this is a
modulation of ADCC and still in another aspect of the invention
this modulation is a down-modulation of ADCC. In still another
aspect this modulation is a down-modulation of ADCC and CDC, still
in another embodiment this is a down-modulation is ADCC only, in
still another embodiment this is a down-modulation of ADCC and CDC
and/or ADCP. In still another aspect of the invention the
polypeptides comprising an Fc variant according to the invention
down-modulate or reduce ADCC/CDC and ADCP.
[0195] In a further aspect of the invention the reduction or
down-modulation of ADCC or CDC or ADCP induced by the polypeptide
comprising the Fc variant, is a reduction to 0, 2.5, 5, 10, 20, 50
or 75% of the value observed for induction of ADCC, or CDC or ADCP,
respectively, by the polypeptide comprising the wildtype Fc
region.
[0196] In still further aspects of the invention the modulation of
ADCC induced by the polypeptides comprising an Fc variant according
to the invention is a decrease in potency such that the EC50 of
said Fc variant is approximately >10-fold reduced compared to
the polypeptide comprising the wildtype Fc polypeptide.
[0197] In still another aspect the variant according to the
invention is devoid of any substantial ADCC and/or CDC and/or ADCP
in the presence of human effector cells as compared to the
polypeptide comprising the wildtype Fc polypeptide.
[0198] In still another aspect of the invention the polypeptides
comprising an Fc variant of the invention exhibit a reduced, for
example reduction by at least 20%, or strongly reduced, for example
reduction by at least 50%, effector function, which could be a
reduction in ADCC (down-modulation), CDC and/or ADCP.
[0199] Reduced ADCC Activity
[0200] In vitro and/or in vivo cytotoxicity assays can be conducted
to confirm the reduction/depletion of CDC and/or ADCC activities.
For example, Fc receptor (FcR) binding assays can be conducted to
ensure that the antibody lacks Fc.gamma.R binding (hence likely
lacking ADCC activity), but retains FcRn binding ability. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, RII and RIII. FcR
expression on hematopoietic cells is summarized in Table 3 on page
464 of Ravetch, and Kinet, Annu. Rev. Immunol. 9 (1991) 457-492.
Non-limiting examples of in vitro assays to assess ADCC activity of
a molecule of interest is described in U.S. Pat. No. 5,500,362
(see, e.g. Hellstrom, I., et al., Proc. Nat'l Acad. Sci. USA 83
(1986) 7059-7063) and Hellstrom, I., et al., Proc. Nat'l Acad. Sci.
USA 82 (1985) 1499-1502; U.S. Pat. No. 5,821,337 (see Bruggemann,
M., et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively,
non-radioactive assays methods may he employed (see, for example,
ACTI.TM. non-radioactive cytotoxicity assay for flow cytometry
(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96.RTM.
non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful
effector cells for such assays include peripheral blood mononuclear
cells (PBMC) and Natural Killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be
assessed in vivo, e.g., in a animal model such as that disclosed in
Clynes, et al., Proc. Nat'l Acad. Sci. USA 95 (1998) 652-656. C1q
binding assays may also be carried out to confirm that the antibody
is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q
and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To
assess complement activation, a CDC assay may be performed (see,
for example, Gazzano-Santoro, et al., J. Immunol. Methods 202
(1996) 163; Cragg, M. S., et al., Blood 101 (2003) 1045-1052; and
Cragg, M. S., and Glennie, M. J., Blood 103 (2004) 2738-2743). FcRn
binding and in vivo clearance/half life determinations can also be
performed using methods known in the art (see, e.g., Petkova, S.
B., et al., Immunol. 18(12) (2006) 1759-1769).
[0201] It is contemplated that polypeptides comprising a Fc variant
of the invention are characterized by in vitro functional assays
for determining one or more Fc.gamma.R mediated effector cell
functions. In certain embodiments, antibodies of the invention have
similar binding properties and effector cell functions in in vivo
models (such as those described and disclosed herein) as those in
in vitro based assays. However, the present invention does not
exclude variants of the invention that do not exhibit the desired
phenotype in in vitro based assays but do exhibit the desired
phenotype in vivo. In one embodiment, polypeptides comprising a Fc
variant of the invention exhibit decreased ADCC activities as
compared to an unmodified wildtype Fc polypeptides. In another
aspect polypeptides comprising an Fc variant of the invention
exhibit ADCC activities that are at least 2 fold, or at least 3
fold, or at least 5 fold or at least 10 fold or at least 50 fold or
at least 100 fold less than that of an unmodified antibody. In
still another embodiment, antibodies of the invention exhibit ADCC
activities that are reduced by at least 10%, or at least 20%, or by
at least 30%, or by at least 40%, or by at least 50%, or by at
least 60%, or by at least 70%, or by at least 80%, or by at least
90%, or by at least 100%, relative to an unmodified antibody. In a
further aspect of the invention the reduction or down-modulation of
ADCC induced by the polypeptide comprising the Fc variant, is a
reduction to 0, 2.5, 5, 10, 20, 50 or 75% of the value observed for
induction of ADCC, or CDC or ADCP, respectively, by the polypeptide
comprising the wildtype Fc region. In certain embodiments,
polypeptides comprising an Fc variant of the invention have no
detectable ADCC activity. In specific embodiments, the reduction
and/or ablation of ADCC activity may be attributed to the reduced
affinity of the polypeptides comprising a Fc variant of the
invention for Fc ligands and/or receptors. In a specific embodiment
of the invention the down-modulation of ADCC is a decrease in
potency such that the EC50 of said polypeptide comprising an Fc
variant is approximately 10-fold or more reduced compared to the
wildtype Fc polypeptide.
[0202] In still another aspect the polypeptides comprising an Fc
variant according to the invention modulate ADCC and/or CDC and/or
ADCP. In a specific aspect the variants according to the inventions
show a reduced CDC and ADCC and/or ADCP activity.
[0203] Reduced CDC Activity
[0204] The complement activation pathway is initiated by the
binding of the first component of the complement system (C1q) to a
molecule, an antibody for example, complexed with a cognate
antigen. To assess complement activation, a CDC assay, e.g. as
described in Gazzano-Santoro, et al, J. Immunol. Methods 202 (1996)
163, may be performed.
[0205] The binding properties of the different variants to C1q can
be analyzed by an ELISA sandwich type immunoassay. The antibody
concentration at the half maximum response determines the EC.sub.50
value. This read-out is reported as relative difference to the
reference standard measured on the same plate together with the
coefficient of variation of sample and reference.
[0206] In one embodiment, polypeptides comprising an Fc variant
according to the invention exhibit decreased affinities to C1q
relative to a wildtype polypeptide. In another embodiment, of the
polypeptides comprising a Fc variant according to the invention
exhibit affinities for C1q receptor that are at least 2 fold, or at
least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10
fold, or at least 20 fold, or at least 30 fold, or at least 40
fold, or at least 50 fold, or at least 60 fold, or at least 70
fold, or at least 80 fold, or at least 90 fold, or at least 100
fold, or at least 200 fold less than the wildtype polypeptide.
[0207] In another embodiment, polypeptides comprising a Fc variant
according to the invention exhibit affinities for C1q that are at
least 90%, at least 80%, at least 70%, at least 60%, at least 50%,
at least 40%, at least 30%, at least 20%, at least 10%, or at least
5% less than that of the wildtype polypeptide. In another
embodiment, variants according to the invention exhibit affinities
for C1q that are between about 100 nM to about 100 .mu.M, or about
100 nM to about 10 .mu.M, or about 100 nM to about 1 .mu.M, or
about 1 nM to about 100 .mu.M, or about 10 nM to about 100 .mu.M,
or about 1 .mu.M to about 100 .mu.M, or about 10 .mu.M to about 100
.mu.M. In certain embodiments, polypeptides comprising an Fc
variant of the invention exhibit affinities for CIq that are
greater than 1 .mu.M, greater than 5 .mu.M, greater than 10 .mu.M,
greater than 25 .mu.M, greater than 50 .mu.M, or greater than 100
.mu.M.
[0208] In one embodiment polypeptide comprising an Fc variant of
the invention exhibit decreased CDC activities as compared to the
wildtype Fc polypeptide In another embodiment, polypeptide
comprising an Fc variant of the invention exhibit CDC activities
that are at least 2 fold, or at least 3 fold, or at least 5 fold or
at least 10 fold or at least 50 fold or at least 100 fold less than
that of a wildtype polypeptide. In still another embodiment
polypeptide comprising an Fc variant of the invention exhibit CDC
activities that are reduced by at least 10%, or at least 20%, or by
at least 30%, or by at least 40%, or by at least 50%, or by at
least 60%, or by at least 70%, or by at least 80%, or by at least
90%, or by at least 100%, or by at least 200%, or by at least 300%,
or by at least 400%, or by at least 500% relative to the wildtype
polypeptide. In certain aspects polypeptide comprising an Fc
variant of the invention exhibit no detectable CDC activities. In
specific embodiments, the reduction and/or ablation of CDC activity
may be attributed to the reduced affinity of the polypeptides
comprising an Fc variant for Fc ligands and/or receptors.
[0209] Reduced Antibody Related Toxicity
[0210] It is understood in the art that biological therapies may
have adverse toxicity issues associated with the complex nature of
directing the immune system to recognize and attack unwanted cells
and/or targets. When the recognition and/or the targeting for
attack do not take place where the treatment is required,
consequences such as adverse toxicity may occur. For example,
antibody staining of non-targeted tissues may be indicative of
potential toxicity issues.
[0211] In one aspect, polypeptide comprising an Fc variant of the
invention exhibit reduced staining of non-targeted tissues as
compared to the wildtype polypeptide. In another aspect, the
polypeptide comprising an Fc variant of the invention exhibit
reduced staining of non-targeted tissues that are at least 2 fold,
or at least 3 fold, or at least 5 fold, or at least 7 fold, or a
least 10 fold, or at least 20 fold, or at least 30 fold, or at
least 40 fold, or at least 50 fold, or at least 60 fold, or at
least 70 fold, or at least 80 fold, or at least 90 fold, or at
least 100 fold, or at least 200 fold less than that of to a
wildtype Fc polypeptide. In another embodiment, variants of the
invention exhibit reduced staining of non-targeted tissues that are
reduced by at least 10%, or at least 20%, or by at least 30%, or by
at least 40%, or by at least 50%, or by at least 60%, or by at
least 70%, or by at least 80%, or by at least 90%, or by at least
100%, or by at least 200%, or by at least 300%, or by at least
400%, or by at least 500% relative to the wildtype Fc
polypeptide.
[0212] In one embodiment, polypeptides comprising an Fc variant of
the invention exhibit a reduced antibody related toxicity as
compared to a wildtype polypeptide. In another embodiment,
polypeptide comprising an Fc variant of the invention exhibit
toxicities that are at least 2 fold, or at least 3 fold, or at
least 5 fold, or at least 7 fold, or a least 10 fold, or at least
20 fold, or at least 30 fold, or at least 40 fold, or at least 50
fold, or at least 60 fold, or at least 70 fold, or at least 80
fold, or at least 90 fold, or at least 100 fold, or at least 200
fold less than that of a wildtype polypeptide. In another aspect,
polypeptides comprising an Fc variant of the invention exhibit
toxicities that are reduced by at least 10%, or at least 20%, or by
at least 30%, or by at least 40%, or by at least 50%, or by at
least 60%, or by at least 70%, or by at least 80%, or by at least
90%, or by at least 100%, or by at least 200%, or by at least 300%,
or by at least 400%, or by at least 500% relative to the wildtype
polypeptide.
[0213] Thrombocyte Aggregation
[0214] In one aspect of the invention the wildtype polypeptide
induces platelet activation and/or platelet aggregation, and the
variants thereof, i.e. polypeptides, comprising Fc variants, show a
decreased or even ablated thrombocyte activation and/or
aggregation. In still another aspect of the invention these
wildtype polypeptides are antibodies targeting a platelet protein.
In yet another aspect the antibody is a CD9 antibody. In still
another embodiment this CD9 antibody has a mutation at position
P329G and/or at position L234A/L235A or S228P/L235E (P329G/LALA,
P329G/SPLE). In a further specific embodiment the antibody is
characterized by the SEQ ID NOs: 8-14.
[0215] It is understood in the art that biological therapies may
have as adverse effect thrombocyte aggregation. In vitro and in
vivo assays could be used for measuring thrombocyte aggregation. It
is assumed that the in vitro assay reflects the in vivo
situation.
[0216] In one aspect, polypeptides comprising an Fc variant of the
invention exhibit reduced thrombocyte aggregation in an in vitro
assay compared to the wildtype polypeptide. In another aspect,
polypeptides comprising an Fc variant of the invention exhibit
reduced thrombocyte aggregation in an in vitro assay that is at
least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7
fold, or a least 10 fold, or at least 20 fold, or at least 30 fold,
or at least 40 fold, or at least 50 fold, or at least 60 fold, or
at least 70 fold, or at least 80 fold, or at least 90 fold, or at
least 100 fold, or at least 200 fold less than that of the wildtype
polypeptide. In another embodiment, polypeptides comprising an Fc
variant of the invention exhibit reduced thrombocyte aggregation in
an in vitro assay that is reduced by at least 10%, or at least 20%,
or by at least 30%, or by at least 40%, or by at least 50%, or by
at least 60%, or by at least 70%, or by at least 80%, or by at
least 90%, or by at least 100%, or by at least 200%, or by at least
300%, or by at least 400%, or by at least 500% relative to the
wildtype polypeptide.
[0217] In still another aspect, polypeptides comprising an Fc
variant of the inventions exhibit a reduced in vivo thrombocyte
aggregation compared to the wildtype polypeptide. In another
aspect, variants of the invention exhibit reduced thrombocyte
aggregation in an in vivo assay that is at least 2 fold, or at
least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10
fold, or at least 20 fold, or at least 30 fold, or at least 40
fold, or at least 50 fold, or at least 60 fold, or at least 70
fold, or at least 80 fold, or at least 90 fold, or at least 100
fold, or at least 200 fold less than that of the wildtype Fc
polypeptide. In another embodiment, polypeptides comprising an Fc
variant of the invention exhibit reduced thrombocyte aggregation in
an in vivo assay that is reduced by at least 10%, or at least 20%,
or by at least 30%, or by at least 40%, or by at least 50%, or by
at least 60%, or by at least 70%, or by at least 80%, or by at
least 90%, or by at least 100%, or by at least 200%, or by at least
300%, or by at least 400%, or by at least 500% relative to the
wildtype polypeptide.
[0218] Internalizing Antibodies
[0219] Variants of the invention may bind to cell-surface antigens
that may internalize, further carrying the antibodies into the
cell. Once inside the cell, the variants may be released into the
cytoplasm, targeted to a specific compartment, or recycled to the
cell surface. In some embodiments, the variants of the invention
bind to a cell-surface antigen that internalizes. In other
embodiments, antibodies of the invention may be targeted to
specific organelles or compartments of the cell. In yet other
embodiments, the variants of the invention may be recycled to the
cell surface or periphery after internalization.
[0220] In a specific embodiment, the antibody of the invention is
specific for p-Selectin, CD9, CD19, CD81, CCR5 or CXCR5, IL17a or
Il-33.
[0221] Antibody Preparation
[0222] In the preferred embodiment of the invention, the Fc
region-containing polypeptide which is modified according to the
teachings herein is an antibody. Techniques for producing
antibodies follow:
[0223] Antigen Selection and Preparation
[0224] Where the polypeptide is an antibody, it is directed against
an antigen of interest. Preferably, the antigen is a biologically
important polypeptide and administration of the antibody to a
mammal suffering from a disease or disorder can result in a
therapeutic benefit in that mammal. However, antibodies directed
against nonpolypeptide antigens (such as tumor-associated
glycolipid antigens; see U.S. Pat. No. 5,091,178) are also
contemplated.
[0225] Where the antigen is a polypeptide, it may be a
transmembrane molecule (e.g. receptor) or ligand such as a growth
factor. Exemplary antigens include molecules such as renin; a
growth hormone, including human growth hormone and bovine growth
hormone; growth hormone releasing factor; parathyroid hormone;
thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;
insulin A-chain; insulin B-chain; proinsulin; follicle stimulating
hormone; calcitonin; luteinizing hormone; glucagon; clotting
factors such as factor VIIIC, factor IX, tissue factor (TF), and
von Willebrands factor; anti-clotting factors such as Protein C;
atrial natriuretic factor; lung surfactant; a plasminogen
activator, such as urokinase or human urine or tissue-type
plasminogen activator (t-PA); bombesin; thrombin; hemopoietic
growth factor; tumor necrosis factor-alpha and -beta;
enkephalinase; RANTES (regulated on activation normally T-cell
expressed and secreted); human macrophage inflammatory protein
(MIP-1-alpha); a serum albumin such as human serum albumin;
Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;
prorelaxin; mouse gonadotropin-associated peptide; a microbial
protein, such as beta-lactamase; DNase; IgE; a cytotoxic
T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin;
activin; vascular endothelial growth factor (VEGF); receptors for
hormones or growth factors; protein A or D; rheumatoid factors; a
neurotrophic factor such as bone-derived neurotrophic factor
(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6),
or a nerve growth factor such as NGF-.beta.; platelet-derived
growth factor (PDGF); fibroblast growth factor such as aFGF and
bFGF; epidermal growth factor (EGF); transforming growth factor
(TGF) such as TGF-alpha and TGF-beta, including TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, or TGF-.beta.5; insulin-like
growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain
IGF-I), insulin-like growth factor binding proteins; CD proteins
such as CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive
factors; immunotoxins; a bone morphogenetic protein (BMP); an
interferon such as interferon-alpha, -beta, and -gamma; colony
stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF;
interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase;
surface membrane proteins; decay accelerating factor; viral antigen
such as, for example, a portion of the AIDS envelope; transport
proteins; horning receptors; addressins; regulatory proteins;
integrins such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and
VCAM; a tumor associated antigen such as HER2, HER3 or HER4
receptor; and fragments of any of the above-listed
polypeptides.
[0226] Preferred molecular targets for antibodies encompassed by
the present invention include CD proteins such as CD4, CD8, CD19,
CD20 and CD34; members of the ErbB receptor family such as the EGF
receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such
as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, .alpha.4/.beta.7
integrin, and .alpha.v/.beta.3 integrin including either .alpha. or
.beta. subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11b
antibodies); growth factors such as VEGF; tissue factor (TF); alpha
interferon (.alpha.-IFN); an interleukin, such as IL-8; IgE; blood
group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl
receptor; CTLA-4; protein C etc.
[0227] Soluble antigens or fragments thereof, optionally conjugated
to other molecules, can be used as immunogens for generating
antibodies. For transmembrane molecules, such as receptors,
fragments of these (e.g. the extracellular domain of a receptor)
can be used as the immunogen. Alternatively, cells expressing the
transmembrane molecule can be used as the immunogen. Such cells can
be derived from a natural source (e.g. cancer cell lines) or may be
cells which have been transformed by recombinant techniques to
express the transmembrane molecule. Other antigens and forms
thereof useful for preparing antibodies will be apparent to those
in the art.
[0228] Polyclonal Antibodies
[0229] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hernocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or carbodiimide where R and R.sup.1
are different alkyl groups.
[0230] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with for example 1/10 of the original amount of
peptide or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later the animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Preferably, the animal is boosted
with the conjugate of the same antigen, but conjugated to a
different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
[0231] Monoclonal Antibodies
[0232] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler, et al., Nature, 256 (1975) 495, or may
be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
[0233] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster or macaque monkey, is immunized as
hereinabove described to elicit lymphocytes that produce or are
capable of producing antibodies that will specifically bind to the
protein used for immunization. Alternatively, lymphocytes may be
immunized in vitro. Lymphocytes then are fused with myeloma cells
using a suitable fusing agent, such as polyethylene glycol, to form
a hybridoma cell (Goding, Monoclonal Antibodies: Principles and
Practice, Academic Press (1986) pp. 59-103).
[0234] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0235] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J., Immunol. 133 (1984) 3001; Brodeur, et al., Monoclonal
Antibody Production Techniques and Applications, Marcel Dekker,
Inc., New York (1987) pp. 51-63).
[0236] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0237] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, Academic Press (1986) pp. 59-103). Suitable culture media
for this purpose include, for example, D-MEM or RPMI-1640 medium.
In addition, the hybridoma cells may be grown in vivo as ascites
tumors in an animal.
[0238] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0239] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. Recombinant production of antibodies will be described
in more detail below.
[0240] In a further embodiment, antibodies or antibody fragments
can be isolated from antibody phage libraries generated using the
techniques described in McCafferty, J., et al., Nature 348 (1990)
552-554. Clackson, et al., Nature 352 (1991) 624-628 and Marks, et
al., J. Mol. Biol. 222 (1991) 581-597 describe the isolation of
murine and human antibodies, respectively, using phage libraries.
Subsequent publications describe the production of high affinity
(nM range) human antibodies by chain shuffling (Marks, et al.,
Bio/Technology 10 (1992) 779-783), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse, et al., Nuc. Acids. Res. 21
(1993) 2265-2266). Thus, these techniques are viable alternatives
to traditional monoclonal antibody hybridoma techniques for
isolation of monoclonal antibodies.
[0241] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81 (1984)
6851-6855), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0242] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0243] Antibody Affinity
[0244] In certain embodiments, an antibody provided herein has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-8 M or less, e.g. from 10.sup.-8 M
to 10.sup.-13 M, e.g., from 10.sup.-9 M to 10.sup.-13 M).
[0245] In one embodiment, Kd is measured by a radiolabeled antigen
or Fc receptor binding assay (RIA) performed with the Fab version
of an antibody of interest and its antigen as described by the
following assay. Solution binding affinity of Fabs for antigen is
measured by equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled antigen in the presence of a titration series
of unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (see, e.g., Chen, et al., J. Mol. Biol. 293
(1999) 865-881). To establish conditions for the assay,
MICROTITER.RTM. multi-well plates (Thermo Scientific) are coated
overnight with 5 .mu.g/ml of a capturing anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked
with 2% (w/v) bovine serum albumin in PBS for two to five hours at
room temperature (approximately 23.degree. C.). In a non-adsorbent
plate (Nunc #269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed
with serial dilutions of a Fab of interest (e.g., consistent with
assessment of the anti-VEGF antibody, Fab-12, in Presta, et al.,
Cancer Res. 57 (1997) 4593-4599). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., about 65 hours) to ensure that equilibrium is
reached.
[0246] Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
polysorbate 20 (TWEEN-20.RTM.) in PBS. When the plates have dried,
150 .mu.l/well of scintillant (MICROSCINT-20.TM.; Packard) is
added, and the plates are counted on a TOPCOUNT.TM. gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.
[0247] According to another embodiment, Kd is measured using
surface plasmon resonance assays using a BIACORE.RTM.-2000 or a
BIACORE.RTM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree.
C. with immobilized antigen or Fc receptor CM5 chips at .about.10
response units (RU). Briefly, carboxymethylated dextran biosensor
chips (CM5, BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% polysorbatc 20 (TWEEN-20.TM.)
surfactant (PBST) at 25.degree. C. at a flow rate of approximately
25 .mu.l/min. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIACORE.RTM. Evaluation Software version 3.2) by
simultaneously fitting the association and dissociation sensograms.
The equilibrium dissociation constant (Kd) is calculated as the
ratio k.sub.off/k.sub.on. See, e.g., Chen, et al., J. Mol. Biol.
293 (1999) 865-881. If the on-rate exceeds 10.sup.6 M.sup.-1
s.sup.-1 by the surface plasmon resonance assay above, then the
on-rate can be determined by using a fluorescent quenching
technique that measures the increase or decrease in fluorescence
emission intensity (excitation=295 nm; emission=340 nm, 16 nm
band-pass) at 25.degree. C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations
of antigen as measured in a spectrometer, such as a stop-flow
equipped spectrophometer (Aviv Instruments) or a 8000-series
SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic) with a stirred
cuvette.
[0248] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab').sub.2, Fv, and scFv fragments, and
other fragments described below. For a review of certain antibody
fragments, see Hudson, et al., Nat. Med. 9 (2003) 129-134. For a
review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology
of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York), pp. 269-315 (1994); see also WO
93/16185; and U.S. Pat. No. 5,571,894 and U.S. Pat. No. 5,587,458.
For discussion of Fab and F(ab').sub.2 fragments comprising salvage
receptor binding epitope residues and having increased in vivo
half-life, see U.S. Pat. No. 5,869,046.
[0249] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP 0
404 097; WO 1993/01161; Hudson, et al., Nat. Med. 9 (2003) 129-134;
and Hollinger, et al., Proc. Natl. Acad. Sci. USA 90 (1993)
6444-6448. Triabodies and tetrabodies are also described in Hudson,
et al., Nat. Med. 9 (2003) 129-134.
[0250] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516 B1).
[0251] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0252] Chimeric and Humanized Antibodies
[0253] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl. Acad.
Sci. USA, 81 (1984) 6851-6855). In one example, a chimeric antibody
comprises a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) and a human constant region. In a further
example, a chimeric antibody is a "class switched" antibody in
which the class or subclass has been changed from that of the
parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0254] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0255] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro, and Fransson, Front. Biosci. 13 (2008)
1619-1633, and are further described, e.g., in Riechmann, et al.,
Nature 332 (1988) 323-329; Queen, et al., Proc. Nat'l Acad. Sci.
USA 86 (1989) 10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791,
6,982,321, and 7,087,409; Kashmiri, et al., Methods 36 (2005) 25-34
(describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28 (1991)
489-498 (describing "resurfacing"); Dall'Acqua, et al., Methods 36
(2005) 43-60 (describing "FR shuffling"); and Osbourn, et al.,
Methods 36 (2005)61-68 and Klimka, et al., Br. J. Cancer, 83 (2000)
252-260 (describing the "guided selection" approach to FR
shuffling).
[0256] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims, et al., J. Immunol. 151
(1993) 2296); framework regions derived from the consensus sequence
of human antibodies of a particular subgroup of light or heavy
chain variable regions (see, e.g., Carter, et al., Proc. Natl.
Acad. Sci. USA, 89 (1992) 4285; and Presta, et al., J. Immunol.,
151 (1993) 2623); human mature (somatically mutated) framework
regions or human germline framework regions (see, e.g., Almagro,
and Fransson, Front. Biosci. 13 (2008) 1619-1633); and framework
regions derived from screening FR libraries (see, e.g., Baca, et
al., J. Biol. Chem. 272 (1997) 10678-10684 and Rosok, et al., J.
Biol. Chem. 271 (1996) 22611-22618).
[0257] Human Antibodies
[0258] In certain embodiments, an antibody provided herein is a
human antibody. Human antibodies can he produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk, and van de Winkel, Curr. Opin. Pharmacol. 5
(2001) 368-74 and Lonberg, Curr. Opin. Immunol. 20 (2008)
450-459.
[0259] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23 (2005) 1117-1125. See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HUMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELOCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0260] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor, J. Immunol., 133 (1984) 3001; Brodeur, et al.,
Monoclonal Antibody Production Techniques and Applications, Marcel
Dekker, Inc., New York, (1987) pp. 51-63; and Boerner, et al., J.
Immunol., 147 (1991) 86.) Human antibodies generated via human
B-cell hybridoma technology are also described in Li, et al., Proc.
Natl. Acad. Sci. USA, 103 (2006) 3557-3562. Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826
(describing production of monoclonal human IgM antibodies from
hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4) (2006)
265-268 (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers, and
Brandlein, Histology and Histopathology 20(3) (2005) 927-937 and
Vollmers, and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology 27(3) (2005) 185-91.
[0261] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0262] Library-Derived Antibodies
[0263] Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom, H. R., et al., in
Methods in Molecular Biology 178 (2002) 1-37 (O'Brien et al., ed.,
Human Press, Totowa, N.J., 2001) and further described, e.g., in
the McCafferty, J., et al., Nature 348 (1990) 552-554; Clackson, et
al., Nature 352 (1991) 624-628; Marks, et al., J. Mol. Biol. 222
(1992) 581-597; Marks, and Bradbury, in Methods in Molecular
Biology 248 161-175 (Lo, ed., Human Press, Totowa, N.J., 2003);
Sidhu, et al., J. Mol. Biol. 338(2) (2004) 299-310; Lee, et al., J.
Mol. Biol. 340(5) (2004) 1073-1093; Fellouse, Proc. Natl. Acad.
Sci. USA 101(34) (2004) 12467-12472; and Lee, et al., J. Immunol.
Methods 284(1-2) (2004) 119-132.
[0264] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter, et al., Ann. Rev.
Immunol., 12 (1994) 433-455. Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths, et al., EMBO J, 12 (1993)
725-734. Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom, and Winter, J. Mol. Biol., 227 (1992) 381-388.
Patent publications describing human antibody phage libraries
include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0265] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0266] Multispecific Antibodies
[0267] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, one of the binding specificities is for a specific
antigen and the other is for any other antigen. In certain
embodiments, bispecific antibodies may bind to two different
epitopes of the antigen. Bispecific antibodies may also be used to
localize cytotoxic agents to cells which express the antigen to
which the antibody binds. Bispecific antibodies can be prepared as
full length antibodies or antibody fragments.
[0268] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein, and Cuello, Nature 305 (1983) 537, WO 93/08829, and
Traunecker, et al., EMBO J. 10 (1991) 3655), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific
antibodies may also be made by engineering electrostatic steering
effects for making antibody Fc-heterodimeric molecules (WO
2009/089004 A1); cross-linking two or more antibodies or fragments
(see, e.g., U.S. Pat. No. 4,676,980, and Brennan, et al., Science,
229 (1985) 81); using leucine zippers to produce bi-specific
antibodies (see, e.g., Kostelny, et al., J. Immunol., 148(5) (1992)
1547-1553); using "diabody" technology for making bispecific
antibody fragments (see, e.g., Hollinger, et al., Proc. Natl. Acad.
Sci. USA, 90 (1993) 6444-6448); and using single-chain Fv (sFv)
dimers (see, e.g. Gruber, et al., J. Immunol., 152 (1994) 5368);
and preparing trispecific antibodies as described, e.g., in Tutt,
et al., J. Immunol. 147 (1991) 60.
[0269] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g. US 2006/0025576 A1).
[0270] The antibody or fragment herein also includes a "Dual Acting
FAb" or "DAF" comprising an antigen binding site that binds to a
specific antigen as well as another, different antigen (see, US
2008/0069820, for example).
[0271] Antibody Variants with Altered Binding Affinity to the
Antigen
[0272] In certain embodiments, it may be desirable to improve the
binding affinity to the antigen and/or other biological properties
of the antibody. Amino acid sequence variants of an antibody may be
prepared by introducing appropriate modifications into the
nucleotide sequence encoding the antibody, or by peptide synthesis.
Such modifications include, for example, deletions from, and/or
insertions into and/or substitutions of residues within the amino
acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0273] Substitution, Insertion, and Deletion Variants
[0274] In certain embodiments, polypeptides comprising Fc variants
additionally have one or more amino acid substitutions at other
parts than the Fc part, are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table 1 under the heading of
"conservative substitutions." More substantial changes are provided
in Table 1 under the heading of "exemplary substitutions," and as
further described below in reference to amino acid side chain
classes. Amino acid substitutions may he introduced into an
antibody of interest and the products screened for a desired
activity, e.g., retained/improved antigen binding, or decreased
immunogenicity.
TABLE-US-00001 TABLE 1 Original Exemplary Conservative Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0275] grouped according to common side-chain properties: [0276]
(1) hydrophobic: Ile, Met, Ala, Val, Leu, Ile; [0277] (2) neutral
hydrophilic: Cys, Ser, Thr, Asn, Gln; [0278] (3) acidic: Asp, Glu;
[0279] (4) basic: His, Lys, Arg; [0280] (5) residues that influence
chain orientation: Gly, Pro; [0281] (6) aromatic: Trp, Tyr,
Phe.
[0282] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0283] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
[0284] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mol. Biol. 207 (2008) 179-196), and/or SDRs
(a-CDRs), with the resulting variant VH or VL being tested for
binding affinity. Affinity maturation by constructing and
reselecting from secondary libraries has been described, e.g., in
Hoogenboom, et al., in Methods in Molecular Biology 178 (2002) 1-37
(O'Brien et al., ed., Human Press, Totowa, N.J., (2001)). In some
embodiments of affinity maturation, diversity is introduced into
the variable genes chosen for maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0285] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may be outside of HVR "hotspots" or SDRs. In certain
embodiments of the variant VH and VL sequences provided above, each
HVR either is unaltered, or contains no more than one, two or three
amino acid substitutions.
[0286] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham, and Wells,
Science 244 (1989) 1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0287] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0288] Glycosylation Variants
[0289] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0290] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright, et
al., TIBTECH 15 (1997) 26-32. The oligosaccharide may include
various carbohydrates, e.g., mannose, N-acetyl glucosamine
(GlcNAc), galactose, and sialic acid, as well as a fucose attached
to a GlcNAc in the "stem" of the biantennary oligosaccharide
structure. In some embodiments, modifications of the
oligosaccharide in an antibody of the invention may be made in
order to create antibody variants with certain improved
properties.
[0291] Polypeptides comprising Fc variants are further provided
with sialylated oligosaccharides, e.g., in which a differential
sialylation of the Fc core oligosaccharide attached to the Fc
region of the antibody is provided. Such polypeptides may have
increased sialylation and/or decreased ADCC function. Examples of
such antibody variants are described e.g. by Kaneko, et al.,
Science 313 (2006) 670-673.
[0292] Cysteine Engineered Antibody Variants
[0293] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
S400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0294] Antibody Derivatives
[0295] In certain embodiments, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0296] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam, et al., Proc. Natl. Acad. Sci.
USA 102 (2005) 11600-11605). The radiation may be of any
wavelength, and includes, but is not limited to, wavelengths that
do not harm ordinary cells, but which heat the nonproteinaceous
moiety to a temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
[0297] Recombinant Methods and Compositions
[0298] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an antibody variant
described herein is provided. Such nucleic acid may encode an amino
acid sequence comprising the VL and/or an amino acid sequence
comprising the VH of the antibody (e.g., the light and/or heavy
chains of the antibody). In a further embodiment, one or more
vectors (e.g., expression vectors) comprising such nucleic acid are
provided. In a further embodiment, a host cell comprising such
nucleic acid is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with): (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and an amino acid sequence
comprising the VH of the antibody, or (2) a first vector comprising
a nucleic acid that encodes an amino acid sequence comprising the
VL of the antibody and a second vector comprising a nucleic acid
that encodes an amino acid sequence comprising the VH of the
antibody. In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making an antibody
variant is provided, wherein the method comprises culturing a host
cell comprising a nucleic acid encoding the antibody, as provided
above, under conditions suitable for expression of the antibody,
and optionally recovering the antibody from the host cell (or host
cell culture medium).
[0299] For recombinant production of an antibody variant, nucleic
acid encoding an antibody, e.g., as described above, is isolated
and inserted into one or more vectors for further cloning and/or
expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0300] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular
Biology 248 (2003) 245-254 (B. K. C. Lo, ed., Humana Press, Totowa,
N.J.), describing expression of antibody fragments in E. coli.)
After expression, the antibody may be isolated from the bacterial
cell paste in a soluble fraction and can be further purified.
[0301] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, Nat. Biotech. 22 (2004)
1409-1414, and Li, et al., Nat. Biotech. 24 (2006) 210-215.
[0302] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0303] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0304] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or kidney cells (BHK); mouse sertoli cells (TM4
cells as described, e.g., in Mather, Biol. Reprod. 23 (1980)
243-251); monkey kidney cells (CV1); African green monkey kidney
cells (VERO-76); human cervical carcinoma cells (HELA); canine
kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung
cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT
060562); TRI cells, as described, e.g., in Mather, et al., Annals
N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other
useful mammalian host cell lines include Chinese hamster ovary
(CHO) cells, including DHFR.sup.- CHO cells (Urlaub, et al., Proc.
Natl. Acad. Sci. USA 77 (1980) 4216); and myeloma cell lines such
as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell
lines suitable for antibody production, see, e.g., Yazaki, and Wu,
Methods in Molecular Biology 248 (2003) 255-268 (B. K. C. Lo, ed.,
Humana Press, Totowa, N.J.).
[0305] Assays
[0306] Antibodies provided herein may be identified, screened for,
or characterized for their physical/chemical properties and/or
biological activities by various assays known in the art.
[0307] Binding Assays and Other Assays
[0308] In one aspect, an antibody of the invention is tested for
its antigen binding activity, e.g., by known methods such as ELISA,
Western blot, etc.
[0309] In an exemplary competition assay, immobilized antigen is
incubated in a solution comprising a first labeled antibody that
binds to the antigen (e.g.,) and a second unlabeled antibody that
is being tested for its ability to compete with the first antibody
for binding to the antigen. The second antibody may be present in a
hybridoma supernatant. As a control, immobilized antigen is
incubated in a solution comprising the first labeled antibody but
not the second unlabeled antibody. After incubation under
conditions permissive for binding of the first antibody to the
antigen, excess unbound antibody is removed, and the amount of
label associated with immobilized antigen is measured. If the
amount of label associated with immobilized antigen is
substantially reduced in the test sample relative to the control
sample, then that indicates that the second antibody is competing
with the first antibody for binding to the antigen (See Harlow, and
Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.).
[0310] Immunoconjugates
[0311] The invention also provides immunoconjugates comprising an
antibody herein conjugated to one or more cytotoxic agents, such as
chemotherapeutic agents or drugs, growth inhibitory agents, toxins
(e.g., protein toxins, enzymatically active toxins of bacterial,
fungal, plant, or animal origin, or fragments thereof), or
radioactive isotopes.
[0312] In one embodiment, an immunoconjugate is an antibody-drug
conjugate (ADC) in which an antibody is conjugated to one or more
drugs, including but not limited to a maytansinoid (see U.S. Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and
7,498,298); a dolastatin; a calicheamicin or derivative thereof
(see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman, et al.,
Cancer Res. 53 (1993) 3336-3342; and Lode, et al., Cancer Res. 58
(1998) 2925-2928); an anthracycline such as daunomycin or
doxorubicin (see Kratz, et al., Current Med. Chem. 13 (2006)
477-523; Jeffrey, et al., Bioorganic & Med. Chem. Letters 16
(2006) 358-362; Torgov, et al., Bioconj. Chem. 16 (2005) 717-721;
Nagy, et al., Proc. Natl. Acad. Sci. USA 97 (2000) 829-834;
Dubowchik, et al., Bioorg. & Med. Chem. Letters 12 (2002)
1529-1532; King, et al., J. Med. Chem. 45 (2002) 4336-4343; and
U.S. Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as
docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC1065.
[0313] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to an enzymatically active
toxin or fragment thereof, including but not limited to diphtheria
A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,
dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes.
[0314] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to a radioactive atom to
form a radioconjugate. A variety of radioactive isotopes are
available for the production of radioconjugates. Examples include
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive
isotopes of Lu. When the radioconjugate is used for detection, it
may comprise a radioactive atom for scintigraphic studies, for
example tc99m or I123, or a spin label for nuclear magnetic
resonance (NMR) imaging (also known as magnetic resonance imaging,
mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or iron.
[0315] Conjugates of an antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta, et al., Science 238 (1987) 1098. Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO 94/11026. The linker may be
a "cleavable linker" facilitating release of a cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari, et al., Cancer Res. 52 (1992) 127-131; U.S. Pat. No.
5,208,020) may be used.
[0316] The immunuoconjugates or ADCs herein expressly contemplate,
but are not limited to such conjugates prepared with cross-linker
reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS,
LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
[0317] Methods and Compositions for Diagnostics and Detection
[0318] In certain embodiments, any of the antibody variants
provided herein is useful for detecting the presence of the antigen
binding to that antibody in a biological sample. The term
"detecting" as used herein encompasses quantitative or qualitative
detection. In certain embodiments, a biological sample comprises a
cell or tissue.
[0319] In one embodiment, an antibody variant for use in a method
of diagnosis or detection is provided. In a further aspect, a
method of detecting the presence of the antigen to which said
antibody variant binds in a biological sample is provided. In
certain embodiments, the method comprises contacting the biological
sample with an antibody as described herein under conditions
permissive for binding of the antibody to the antigen, and
detecting whether a complex is formed between the antibody and the
antigen. Such method may be an in vitro or in vivo method. In one
embodiment, an antibody variant is used to select subjects eligible
for therapy with an antibody, e.g. where the antigen to which said
antibody binds is a biomarker for selection of patients.
[0320] Exemplary disorders that may he diagnosed using an antibody
of the invention include cancer, cardiovascular diseases, neuronal
disorders and diabetes.
[0321] In certain embodiments, labeled antibody variants are
provided. Labels include, but are not limited to, labels or
moieties that are detected directly (such as fluorescent,
chromophoric, electron-dense, chemiluminescent, and radioactive
labels), as well as moieties, such as enzymes or ligands, that are
detected indirectly, e.g., through an enzymatic reaction or
molecular interaction. Exemplary labels include, but are not
limited to, the radioisotopes .sup.32P, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I, fluorophores such as rare earth chelates or
fluorescein and its derivatives, rhodamine and its derivatives,
dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and
bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),
alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as
uricase and xanthine oxidase, coupled with an enzyme that employs
hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.
[0322] Pharmaceutical Formulations
[0323] Pharmaceutical formulations of an antibody variant as
described herein are prepared by mixing such antibody having the
desired degree of purity with one or more optional pharmaceutically
acceptable carriers (Remington's Pharmaceutical Sciences 16th
edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous solutions. Pharmaceutically acceptable
carriers are generally nontoxic to recipients at the dosages and
concentrations employed, and include, but are not limited to:
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride; benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene glycol (PEG). Exemplary pharmaceutically acceptable
carriers herein further include insterstitial drug dispersion
agents such as soluble neutral-active hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase
glycoproteins, such as rHuPH20 (HYLENEX.RTM., Baxter International,
Inc.). Certain exemplary sHASEGPs and methods of use, including
rHuPH20, are described in US Patent Publication Nos. 2005/0260186
and 2006/0104968. In one aspect, a sHASEGP is combined with one or
more additional glycosaminoglycanases such as chondroitinases.
[0324] Exemplary lyophilized antibody formulations are described in
U.S. Pat. No. 6,267,958. Aqueous antibody formulations include
those described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the
latter formulations including a histidine-acetate buffer.
[0325] The formulation herein may also contain more than one active
ingredients as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. Such active ingredients are suitably
present in combination in amounts that are effective for the
purpose intended.
[0326] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0327] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules.
[0328] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0329] Therapeutic Methods and Compositions
[0330] Any of the polypeptides provided herein may be used in
therapeutic methods.
[0331] In a specific aspect of the invention the polypeptide
according to the invention are used for treating a disease. In a
more specific aspect, the disease is such, that it is favorable
that the effector function of the variant is strongly, at least by
50%, reduced compared to the polypeptide comprising the wildtype Fc
polypeptide.
[0332] In a specific aspect the polypeptide according to the
invention is used in the manufacture of a medicament for the
treatment of a disease, wherein it is favorable that the effector
function of the polypeptide is strongly reduced compared to a
wildtype Fc polypeptide. In a further specific aspect the
polypeptide according to the invention is used in the manufacture
of a medicament for the treatment of a disease, wherein it is
favorable that the effector function of the polypeptide is reduced
compared to a wildtype Fc polypeptide, by at least 20%.
[0333] A further aspect is a method of treating an individual
having a disease, wherein it is favorable that the effector
function of the variant is strongly reduced compared to a wildtype
Fc polypeptide, comprising administering to the individual an
effective amount of the polypeptide according to the invention.
[0334] A strong reduction of effector function is a reduction of
effector function by at least 50% of the effector function induced
by the wildtype polypeptide.
[0335] Such diseases are for example all diseases where the
targeted cell should not be destroyed by for example ADCC, ADCP or
CDC. Moreover, this is true for those antibodies that are designed
to deliver a drug (e.g., toxins and isotopes) to the target cell
where the Fc/Fc.gamma.R mediated effector functions bring healthy
immune cells into the proximity of the deadly payload, resulting in
depletion of normal lymphoid tissue along with the target cells
(Hutchins, et al, PNAS USA 92 (1995) 11980-11984; White, et al,
Annu Rev Med 52 (2001) 125-145). In these cases the use of
antibodies that poorly recruit complement or effector cells would
be of tremendous benefit (see for example, Wu, et al., Cell Immunol
200 (2000) 16-26; Shields, et al., J. Biol Chem 276(9) (2001)
6591-6604; U.S. Pat. No. 6,194,551; U.S. Pat. No. 5,885,573 and PCT
publication WO 04/029207).
[0336] In other instances, for example, where blocking the
interaction of a widely expressed receptor with its cognate ligand
is the objective, it would be advantageous to decrease or eliminate
all antibody effector function to reduce unwanted toxicity. Also,
in the instance where a therapeutic antibody exhibited promiscuous
binding across a number of human tissues it would be prudent to
limit the targeting of effector function to a diverse set of
tissues to limit toxicity.
[0337] Also for agonist antibodies it would be very helpful if
these antibodies exhibit reduced effector function.
[0338] The conditions which can be treated with the polypeptide
variant are many and include cancer (e.g. where the antibody
variant binds the HER2 receptor, angiopoietin receptor or vascular
endothelial growth factor (VEGF)); allergic conditions such as
asthma (with an anti-IgE antibody); and LFA-1-mediated disorders
(e.g. where the polypeptide variant is an anti-LFA-1 or anti-ICAM-1
antibody), neurological and metabolic disorders.
[0339] Where the antibody binds the HER2 receptor, the disorder
preferably is HER2-expressing cancer, e.g. a benign or malignant
tumor characterized by overexpression of the HER2 receptor. Such
cancers include, but are not limited to, breast cancer, squamous
cell cancer, small-cell lung cancer, non-small cell lung cancer,
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, bladder cancer, hepatoma, colon cancer,
colorectal cancer, endometrial carcinoma, salivary gland carcinoma,
kidney cancer, liver cancer, prostate cancer, vulval cancer,
thyroid cancer, hepatic carcinoma and various types of head and
neck cancer.
[0340] The polypeptide or antibody variant is administered by any
suitable means, including parenteral, subcutaneous,
intraperitoneal, intrapulmonary, and intranasal, and, if desired
for local immunosuppressive treatment, intralesional
administration. Parenteral infusions include intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous
administration. In addition, the antibody variant is suitably
administered by pulse infusion, particularly with declining doses
of the polypeptide variant. Preferably the dosing is given by
injections, most preferably intravenous or subcutaneous injections,
depending in part on whether the administration is brief or
chronic.
[0341] For the prevention or treatment of disease, the appropriate
dosage of polypeptide or antibody variant will depend on the type
of disease to be treated, the severity and course of the disease,
whether the polypeptide variant is administered for preventive or
therapeutic purposes, previous therapy, the patient's clinical
history and response to the polypeptide variant, and the discretion
of the attending physician. The polypeptide variant is suitably
administered to the patient at one time or over a series of
treatments.
[0342] Depending on the type and severity of the disease, about 1
.mu.g/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of polypeptide or
antibody variant is an initial candidate dosage for administration
to the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. A typical daily dosage
might range from about 1 .mu.g/kg to 100 mg/kg or more, depending
on the factors mentioned above. For repeated administrations over
several days or longer, depending on the condition, the treatment
is sustained until a desired suppression of disease symptoms
occurs. However, other dosage regimens may be useful. The progress
of this therapy is easily monitored by conventional techniques and
assays.
[0343] In certain embodiments, the invention provides an antibody
variant or polypeptide for use in a method of treating an
individual having cancer comprising administering to the individual
an effective amount of the antibody variant. In one such
embodiment, the method further comprises administering to the
individual an effective amount of at least one additional
therapeutic agent, e.g., as described below. In further
embodiments, the invention provides an antibody variant for use in
inhibiting angiogenesis, inhibiting cell proliferation or depleting
B-cells in an individual comprising administering to the individual
an effective of the antibody variant to inhibit angiogenesis,
inhibit cell proliferation or deplete B-cells in an "individual"
according to any of the above embodiments is preferably a
human.
[0344] In a further aspect, the invention provides for the use of
an antibody variant or polypeptide in the manufacture or
preparation of a medicament. In one embodiment, the medicament is
for treatment of cancer or inflammatory diseases. In a further
embodiment, the medicament is for use in a method of treating
cancer, diabetes, neuronal disorders or inflammatory comprising
administering to an individual having cancer, diabetes, neuronal
disorders or inflammatory an effective amount of the medicament. In
one such embodiment, the method further comprises administering to
the individual an effective amount of at least one additional
therapeutic agent, e.g., as described below. In a further
embodiment, the medicament is for inhibiting angiogenesis,
inhibiting cell proliferation or depleting B-cells.
[0345] In a further embodiment, the medicament is for use in a
method of inhibiting angiogenesis, inhibiting cell proliferation or
depleting B-cells
[0346] In an individual comprising administering to the individual
an amount effective of the medicament to inhibit angiogenesis,
inhibit cell proliferation or deplete B-cells. An "individual"
according to any of the above embodiments may be a human.
[0347] In a further aspect, the invention provides pharmaceutical
formulations comprising any of the antibody variants provided
herein, e.g., for use in any of the above therapeutic methods. In
one embodiment, a pharmaceutical formulation comprises any of the
antibody variants provided herein and a pharmaceutically acceptable
carrier. In another embodiment, a pharmaceutical formulation
comprises any of the antibody variants provided herein and at least
one additional therapeutic agent, e.g., as described below.
[0348] Antibodies of the invention can be used either alone or in
combination with other agents in a therapy. For instance, an
antibody of the invention may be co-administered with at least one
additional therapeutic agent.
[0349] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the antibody of the invention can
occur prior to, simultaneously, and/or following, administration of
the additional therapeutic agent and/or adjuvant. Antibodies of the
invention can also be used in combination with radiation
therapy.
[0350] An antibody of the invention (and any additional therapeutic
agent) can be administered by any suitable means, including
parenteral, intrapulmonary, and intranasal, and, if desired for
local treatment, intralesional administration. Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal,
or subcutaneous administration. Dosing can be by any suitable
route, e.g. by injections, such as intravenous or subcutaneous
injections, depending in part on whether the administration is
brief or chronic. Various dosing schedules including but not
limited to single or multiple administrations over various
time-points, bolus administration, and pulse infusion are
contemplated herein.
[0351] Antibodies of the invention would be formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The antibody need not be, but is
optionally formulated with one or more agents currently used to
prevent or treat the disorder in question. The effective amount of
such other agents depends on the amount of antibody present in the
formulation, the type of disorder or treatment, and other factors
discussed above. These are generally used in the same dosages and
with administration routes as described herein, or about from 1 to
99% of the dosages described herein, or in any dosage and by any
route that is empirically/clinically determined to be
appropriate.
[0352] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with one or more other additional therapeutic agents)
will depend on the type of disease to be treated, the type of
antibody, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg)
of antibody can be an initial candidate dosage for administration
to the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the antibody would be in the range from about 0.05 mg/kg
to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be
administered to the patient. Such doses may be administered
intermittently, e.g. every week or every three weeks (e.g. such
that the patient receives from about two to about twenty, or e.g.
about six doses of the antibody). An initial higher loading dose,
followed by one or more lower doses may be administered. However,
other dosage regimens may be useful. The progress of this therapy
is easily monitored by conventional techniques and assays.
[0353] It is understood that any of the above formulations or
therapeutic methods may be carried out using an immunoconjugate of
the invention in place of or in addition to an antibody according
to the invention.
[0354] Articles of Manufacture
[0355] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antibody of the invention. The label
or package insert indicates that the composition is used for
treating the condition of choice. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises an antibody of
the invention; and (b) a second container with a composition
contained therein, wherein the composition comprises a further
cytotoxic or otherwise therapeutic agent. The article of
manufacture in this embodiment of the invention may further
comprise a package insert indicating that the compositions can be
used to treat a particular condition. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0356] It is understood that any of the above articles of
manufacture may include an immunoconjugate of the invention in
place of or in addition to an antibody variant.
[0357] Non-Therapeutic uses for the Polypeptide
[0358] The antibody variant of the invention may be used as an
affinity purification agent. In this process, the antibody variant
is immobilized on a solid phase such a Sephadex resin or filter
paper, using methods well known in the art. The immobilized
polypeptide variant is contacted with a sample containing the
antigen to be purified, and thereafter the support is washed with a
suitable solvent that will remove substantially all the material in
the sample except the antigen to be purified, which is bound to the
immobilized antibody variant. Finally, the support is washed with
another suitable solvent, such as glycine buffer, pH 5.0, that will
release the antigen from the polypeptide variant.
[0359] The antibody variant may also be useful in diagnostic
assays, e.g., for detecting expression of an antigen of interest in
specific cells, tissues, or serum.
[0360] For diagnostic applications, the antibody variant typically
will be labeled with a detectable moiety. Numerous labels are
available which can be generally grouped into the following
categories:
[0361] (a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I. The polypeptide variant can be labeled with
the radioisotope using the techniques described in Coligen, et al.,
Current Protocols in Immunology, Volumes 1 and 2, Ed.
Wiley-Interscience, New York, N.Y., Pubs. (1991) for example and
radioactivity can be measured using scintillation counting.
[0362] (b) Fluorescent labels such as rare earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. The fluorescent labels can be conjugated to the
polypeptide variant using the techniques disclosed in Current
Protocols in Immunology, supra, for example. Fluorescence can be
quantified using a fluorimeter.
[0363] (c) Various enzyme-substrate labels are available and U.S.
Pat. No. 4,275,149 provides a review of some of these. The enzyme
generally catalyzes a chemical alteration of the chromogenic
substrate that can be measured using various techniques. For
example, the enzyme may catalyze a color change in a substrate,
which can be measured spectrophotometrically. Alternatively, the
enzyme may alter the fluorescence or chemiluminescence of the
substrate. Techniques for quantifying a change in fluorescence are
described above. The chemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit
light which can be measured (using a chemiluminometer, for example)
or donates energy to a fluorescent acceptor. Examples of enzymatic
labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as
unease and xanthine oxidase), lactoperoxidase, microperoxidase, and
the like. Techniques for conjugating enzymes to antibodies are
described in O'Sullivan, et al., Methods for the Preparation of
Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in
Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic
press, New York, 73:147-166 (1981).
[0364] Examples of enzyme-substrate combinations include, for
example:
[0365] (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase
as a substrate, wherein the hydrogen peroxidase oxidizes a dye
precursor (e.g., orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));
[0366] (ii) alkaline phosphatase (AP) with para-Nitrophenyl
phosphate as chromogenic substrate; and
[0367] (iii) .beta.-D-galactosidase (.beta.-D-Gal) with a
chromogenic substrate (e.g., p-nitrophenyl-.beta.-D-galactosidase)
or fluorogenic substrate
4-methylumbelliferyl-.beta.-D-galactosidase.
[0368] Numerous other enzyme-substrate combinations are available
to those skilled in the art. For a general review of these, see
U.S. Pat. Nos. 4,275,149 and 4,318,980.
[0369] Sometimes, the label is indirectly conjugated with the
polypeptide variant. The skilled artisan will be aware of various
techniques for achieving this. For example, the polypeptide variant
can be conjugated with biotin and any of the three broad categories
of labels mentioned above can be conjugated with avidin, or vice
versa.
[0370] Biotin binds selectively to avidin and thus, the label can
be conjugated with the polypeptide variant in this indirect manner.
Alternatively, to achieve indirect conjugation of the label with
the polypeptide variant, the polypeptide variant is conjugated with
a small hapten (e.g., digoxin) and one of the different types of
labels mentioned above is conjugated with an anti-hapten
polypeptide variant (e.g., anti-digoxin antibody). Thus, indirect
conjugation of the label with the polypeptide variant can be
achieved.
[0371] In another embodiment of the invention, the antibody variant
need not be labeled, and the presence thereof can be detected using
a labeled antibody which binds to the polypeptide variant.
[0372] The antibody variant of the present invention may be
employed in any known assay method, such as competitive binding
assays, direct and indirect sandwich assays, and
immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual
of Techniques, (1987) pp. 147-158, CRC Press, Inc.
[0373] The antibody variant may also be used for in vivo diagnostic
assays. Generally, the polypeptide variant is labeled with a
radionuclide (such as .sup.111In, .sup.99Tc, .sup.14C, .sup.131I,
.sup.125I, .sup.3H, .sup.32P or .sup.35S) so that the antigen or
cells expressing it can be localized using immunoscintiography.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, the descriptions and examples should not be
construed as limiting the scope of the invention.
DESCRIPTION OF THE SEQUENCE LISTING
[0374] SEQ ID NO:1 Human kappa light chain [0375] SEQ ID NO:2 Human
lambda light chain [0376] SEQ ID NO:3 Human IgG1 (Caucasian
Allotype) [0377] SEQ ID NO:4 Human IgG1 (Afroamerican Allotype
[0378] SEQ ID NO:5 Human IgG1 LALA-Mutant (Caucasian Allotype)
[0379] SEQ ID NO:6 Human IgG4 [0380] SEQ ID NO:7 Human IgG4
SPLE-Mutant which represent exemplary human sequences for the kappa
light chain, lambda light chain, IgG1 and IgG4 which could serve as
basis for generating the variants according to the invention.
[0381] In sequence Id Nos 3-5, the sequence of human IgG1
allotypes, the P329 region according to Kabat EU index is located
at position 212, whereas said P329 region in sequence Id Nos 6 and
7 can be find at position 209. [0382] SEQ ID NO:8 Kappa light chain
of mAb 40A746.2.3 [0383] SEQ ID NO:9 Heavy chain of wildtype IgG1
of mAb 40A746.2.3 [0384] SEQ ID NO:10 Heavy chain of IgG1 P329G of
mAb 40A746.2.3 [0385] SEQ ID NO:11 Heavy chain of IgG1 LALA/P329G
of mAb 40A746.2.3 [0386] SEQ ID NO:12 Heavy chain of IgG4 SPLE of
mAb 40A746.2.3 [0387] SEQ ID NO:13 Heavy chain of IgG4 SPLE/P329G
of mAb 40A746.2.3 [0388] SEQ ID NO:14 Heavy chain of IgG1 LALA of
mAb 40A746.2.3
EXAMPLES
[0389] The following seven examples are examples of methods and
compositions of the invention. It is understood that various other
embodiments may be practiced, given the general description
provided above.
[0390] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
Example 1
[0391] Antibodies
[0392] For the experiments described below antibodies against CD9
(see SEQ IDs 8-14), P-selectin (sequences described in WO
2005/100402) and CD20 (synonym: GA101, sequences described in EP 1
692 182) were used.
[0393] All variants described herein, e.g. P329G, P329A, P329R
SPLE, LALA, P329G/LALA, P329G/SPLE variants of the selectin, CD9,
CD20 (GA101) and CD20 (GA101)-glycoengineered binding antibody
(numbering according to EU nomenclature) were generated using PCR
based mutagenesis. IgG molecules were expressed in the HEK-EBNA or
HEK293 (CD9 Fc variants) system, and purified using protein A and
size exclusion chromatography.
Example 2
[0394] Determination of the Binding Affinities of Different
Fc.gamma. Receptors to Immunoglobulins
[0395] Binding affinities of different Fc.gamma.Rs towards
immunoglobulins were measured by Surface Plasmon Resonance (SPR)
using a Biacore T100 instrument (GE Healthcare) at 25.degree.
C.
[0396] The BIAcore.RTM. system is well established for the study of
molecule interactions. It allows a continuous real-time monitoring
of ligand/analyte bindings and thus the determination of
association rate constants (k.sub.a), dissociation rate constants
(k.sub.d), and equilibrium constants (K.sub.D). Changes in the
refractive index indicate mass changes on the surface caused by the
interaction of immobilized ligand with analyte injected in
solution. If molecules bind immobilized ligands on the surface the
mass increases, in case of dissociation the mass decreases.
[0397] For a 1:1 interaction no difference in the results should be
seen if a binding molecule is either injected over the surface or
immobilized onto a surface. Therefore different settings were used
(with Fc.gamma. receptor as ligand or analyte respectively),
depending on solubility and availability of ligand or corresponding
analyte.
[0398] For Fc.gamma.RI 10000 resonance units (RU) of a capturing
system recognizing a polyhistidine sequence (pentaHis monoclonal
antibody, Qiagen Hilden, cat. no. 34660) was immobilized by the use
of an amine coupling kit supplied by the GE Healthcare and a CM5
chip at pH 4.5. Fc.gamma.RI was captured at a concentration of 5
.mu.g/ml by with a pulse of 60 sec at a flow of 5 .mu.l/min.
Different concentrations of antibodies ranging from 0 to 100 nM
were passed with a flow rate of 30 .mu.l/min through the flow cells
at 298 K for 120 sec to record the association phase. The
dissociation phase was monitored for up to 240 sec and triggered by
switching from the sample solution to running buffer. The surface
was regenerated by 2 min washing with a glycine pH 2 solution at a
flow rate of 30 ml/min. For all experiments HBS-P+ buffer supplied
by GE Healthcare was chosen (10 mM HEPES, pH 7.4, 150 mM NaCl,
0.05% (v/v) Surfactant P20). Bulk refractive index differences were
corrected for by subtracting the response obtained from a surface
without captured Fc.gamma.RI. Blank injections are also subtracted
(=double referencing).
[0399] The equilibrium dissociation constant (K.sub.D), defined as
k.sub.a/k.sub.d, was determined by analyzing the sensogram curves
obtained with several different concentrations, using BIAevaluation
software package. The fitting of the data followed a suitable
binding model.
[0400] For Fc.gamma.RIIA and Fc.gamma.RIIIAV158 10000 resonance
units (RU) of a monoclonal antibody to be tested was immobilized
onto a CM5 chip by the use of an amine coupling kit supplied by the
GE (pH 4.5 at a concentration of 10 .mu.g/ml).
[0401] Different concentrations of Fc.gamma.RIIA and IIIA ranging
from 0 to 12800 nM were passed with a flow rate of 5 .mu.l/min
through the flow cells at 298 K for 120 sec to record the
association phase. The dissociation phase was monitored for up to
240 sec and triggered by switching from the sample solution to
running buffer. The surface was regenerated by 0.5 mM washing with
a 3 mM NaOH/1M NaCl solution at a flow rate of 30 ml/min. For all
experiments HBS-P+ buffer supplied by GE Healthcare was chosen (10
mM HEPES, pH 7.4, 150 mM NaCl, 0.05% (v/v) Surfactant P20).
[0402] Bulk refractive index differences were corrected for by
subtracting the response obtained from a surface without captured
antibody. Blank injections are also subtracted (=double
referencing).
[0403] The equilibrium dissociation constant (K.sub.D), was
determined by analyzing the sensogram curves obtained with several
different concentrations, using BIA evaluation software package.
The fitting of the data followed a suitable binding model using
steady state fitting
[0404] For Fc.gamma.RIIB 10000 resonance units (RU) of a capturing
system recognizing a polyhistidine sequence (pentaHis monoclonal
antibody, Qiagen Hilden, cat. no. 34660) was immobilized by the use
of an amine coupling kit supplied by the GE Healthcare and a CM5
chip at pH 4.5. Fc.gamma.RIIB was captured at a concentration of 5
.mu.g/ml by with a pulse of 120 sec at a flow of 5 .mu.l/min.
Different antibodies were passed at a concentration of 1340 nM with
a flow rate of 5 .mu.l/min through the flow cells at 298 K for 60
sec to record the association phase. The dissociation phase was
monitored for up to 120 sec and triggered by switching from the
sample solution to running buffer. The surface was regenerated by
0.5 min washing with a glycine pH2.5 solution at a flow rate of 30
ml/min. For all experiments HBS-P+ buffer supplied by GE Healthcare
was chosen (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% (v/v)
Surfactant P20).
[0405] Bulk refractive index differences were corrected for by
subtracting the response obtained from a surface without captured
Fc.gamma.RIIB. Blank injections are also subtracted (=double
referencing).
[0406] Due to the very low intrinsic affinity of Fc.gamma.RIIB to
wildtype IgG1 no affinity was calculated rather a qualitative
binding was assessed.
[0407] The following tables summarize the effects of introducing a
mutation into the Fc part on binding to Fc.gamma.RI, Fc.gamma.RIIA,
Fc.gamma.RIIB, and Fc.gamma.RIIIAV1-58 (A) as well as the effect on
ADCC (measured without (BLT) and with target cells (ADCC)) and on
C1q binding (B)
TABLE-US-00002 TABLE 2A Fc.gamma.RI Fc.gamma.RIIaR131
Fc.gamma.RIIIAV158 Fc.gamma.RIIB WT IgG1 ++ (5 nM) ++ (2 .mu.M)
.sup. + (0.7 .mu.M) ++ IgG4 SPLE - +/- (10 .mu.M) .sup. - (>20
.mu.M) + IgG1 P329G ++ (6 nM) - (>20 .mu.M) - (>20 .mu.M) -
IgG1 P329A ge ++ (8 nM) + (4.4 .mu.M) + (1.8 .mu.M) + IgG1 P329G
LALA - - (>20 .mu.M) - (>20 .mu.M) - IgG1 P329G ge ++ (10 nM)
- (>20 .mu.M) - (>10 .mu.M) - *++ for ge IgG1 30 nM
TABLE-US-00003 TABLE 2B ADCC without ADCC with Mutant Fc.gamma.RI
Fc.gamma.RII Fc.gamma.RIII C1q target cells target cells Assay
Biacore Biacore Biacore CDC C1q BLT ADCC P329G + -- -- -- -- -- --
P329R n.d. n.d. n.d. n.d. n.d. -- -- LALA - n.d. - - n.d. n.d. --
IgG1_P329G/LALA -- -- -- n.d. n.d. n.d. n.d. IgG4_SPLE -- - -- --
-- n.d. n.d. -- strongly reduced/inactive in contrast to wt, -
reduced in contrast to wt, + comparable to wt interaction, n.d. not
determined, / no result
[0408] In more detail the following results have been obtained:
[0409] Affinity to the Fc.gamma.RI Receptor
[0410] P329G, P329A, SPLE and LALA mutations have been introduced
into the Fc polypeptide of a P-selectin, CD20 and CD9 antibody, and
the binding affinity to Fc.gamma.RI was measured with the Biacore
system. Whereas the antibody with the P329G mutation still binds to
Fc.gamma.R1 (FIGS. 1a and 1b), introduction of triple mutations
P329G/LALA and P329G/SPLE, respectively, resulted in antibodies for
which nearly no binding could be detected (FIG. 1b). The LALA or
SPLE mutations decreased binding to the receptor more than P329G
alone but less than in combination with P329G (FIGS. 1a and 1b).
Thus, the combination of P329G with either LALA or SPLE mutations
is much more effective than the P329G mutation or the double
mutations LALA or SPLE alone. The kd value for the CD20 IgG1
wildtype antibody was 4.6 nM and for the P329G mutant of the same
antibody 5.7 nM, but for the triple mutant P329G/LALA no kd value
could be determined due to the nearly undetectable binding of the
antibody to the Fc.gamma.RI receptor. The antibody itself, i.e.
whether a CD9 or CD20 or P-selectin was tested, has a minor effect
on the binding affinities.
[0411] Affinity to the Fc.gamma.RIIA Receptor
[0412] P329G, SPLE and LALA mutations, respectively, have been
introduced into the Fc polypeptide of the CD9 antibody and the
binding affinity to the Fc.gamma.RIIA-R131 receptor was measured
with the Biacore system. Binding level is normalized such as
captured mAb represents 100 RU. So not more than approximately 20
RU is expected for a 1:1 stoichiometry. FIG. 1c shows that the
binding to the Fc.gamma.RIIA receptor is strongly reduced by
introducing the LALA, SPLE/P329G, P329G and LALA/P329G mutation
into the Fc variant. In contrast to binding to the Fc.gamma.R1
receptor, the introduction of the P329G mutation alone is able to
very strongly block the binding to said receptor, more or less to a
similar extent as the triple mutation P329G/LALA (FIG. 1c).
[0413] Affinity to the Fc.gamma.RIIB Receptor
[0414] SPLE, LALA, SPLE/P329G and LALA/P329G mutations,
respectively, have been introduced into the Fc polypeptide of the
CD9 and P-selectin antibody and the binding affinity to
Fc.gamma.RIIB receptor was measured with the Biacore system. FIG.
1d shows that the binding to the Fc.gamma.RIIB receptor is strongly
reduced in the LALA and triple mutants P329G/LALA, P329G/SPLE
[0415] Affinity to the Fc.gamma.RIIIA Receptor
[0416] P329G, LALA, SPLE, P329G/LALA, and SPLE/P329G mutations have
been introduced into the Fc polypeptide of the CD9 and the binding
affinity to Fc.gamma.RIIIA-V158 receptor was measured with the
Biacore system. The P329G mutation and the triple mutation
P329G/LALA reduced binding to the Fc.gamma.RIIIA receptor most
strongly, to nearly undetectable levels. The P329G/SPLE also lead
to a strongly reduced binding affinity, the mutations SPLE and
LALA, respectively, only slightly decreased the binding affinity to
the Fc.gamma.RIIIA receptor (FIG. 1e).
Example 3
[0417] C1Q ELISA
[0418] The binding properties of the different polypeptides
comprising Fc variants to C1q were analyzed by an ELISA sandwich
type immunoassay. Each variant is coupled to a hydrophobic Maxisorp
96 well plate at 8 concentrations between 10 .mu.g/ml and 0
.mu.g/ml. This coupling simulates complexes of antibodies, which is
a prerequisite for high affinity binding of the C1q molecule. After
washing, the samples are incubated to allow C1q binding. After
further washing the bound C1q molecule is detected by a polyclonal
rabbit anti-hC1q antibody. Following the next washing step, an
enzyme labelled anti-rabbit-Fc.gamma. specific antibody is added.
Immunological reaction is made visible by addition of a substrate
that is converted to a coloured product by the enzyme. The
resulting absorbance, measured photometrically, is proportional to
the amount of C1q bound to the antibody to be investigated.
EC.sub.50 values of the variant-C1q interaction were calculated.
The absorption units resulting from the coloring reaction are
plotted against the concentration of the antibody. The antibody
concentration at the half maximum response determines the EC.sub.50
value. This read-out is reported as relative difference to the
reference standard measured on the same plate together with the
coefficient of variation of sample and reference.
[0419] The P329G mutation introduced into the P-selectin or CD20
antibody strongly reduced binding to C1q, similar to the SPLE
mutation (FIG. 2). Table 3 summarizes the calculated EC 50 values
for binding of the variants to the C1q receptor. C1q belongs to the
complement activation proteins and plays a major role in the
activation of the classical pathway of the complement, which leads
to the formation of the membrane attack complex. C1q is also
involved in other immunological processes such as enhancement of
phagocytosis, clearance of apoptotic cells or neutralization of
virus. Thus, it can be expected that the mutants shown here to
reduce binding to C1q, e.g. P329G and SPLE, as well as very likely
also the triple mutations comprising the aforementioned single
mutations, strongly reduces the above mentioned functions of
C1q.
TABLE-US-00004 TABLE 3 Antibody EC50 value P-Selectin IgG1 wt 1.8
GA101 IgG1 wt 2.4 P-Selectin IgG1_P329G 2.7 P-Selectin IgG4 SPLE
3.0 GA101 IgG1 P329G 5.5 GA101 IgG4 SPLE >10
Example 4
[0420] ADCC without Target Cells, BLT Assay
[0421] The antibodies to be tested (CD20 (GA101) and CD9) were
coated in PBS over night at 4.degree. C. in suitable 96-flat bottom
well plates. After washing the plate with PBS, the remaining
binding sites were blocked with PBS/1% BSA solution for 1 h at RT.
In the meantime, the effector cells (NK-92 cell line transfected to
express low or high affine human Fc.gamma.RIII) were harvested and
200 000 living cells/well were seeded in 100 .mu.l/well AIM V
medium into the wells after discarding the blocking buffer. 100
.mu.l/well saponin buffer (0.5% saponin+1% BSA in PBS) was used to
determine the maximal esterase release by the effector cells. The
cells were incubated for 3 h at 37.degree. C., 5% CO2 in a
incubator. After 3 h, 20 .mu.l/well of the supernatants were mixed
with 180 .mu.l/well BLT substrate (0.2 mM BLT+0.11 mM DTNB in 0.1 M
Tris-HCL, pH 8.0) and incubated for 30 min at 37.degree. C. before
reading the plate at 405 nm in a microplate reader. The percentage
of esterase release was determined setting the maximal release
(saponin-treated cells) to 100% and the unstimulated cells (no ab
coated) to 0% release.
[0422] The wildtype CD20 antibody (GA101 wt (1)) shows strong
induction of cytolytic activity. The LALA variant shows a marked
reduction in esterase release, whereas the P329G and the P329G/LALA
variant do not show any ADCC activity (FIG. 3a). FIG. 3b shows that
not only an exchange of G at position P329 leads to markedly
reduced cytosolic activity but also an exchange of P329 to R329
(CD20 antibody). Thus arginine appears to destroy the function of
the proline sandwich in the antibody, similar to glycine. The
strongly reduced ADCC observed here for the P329G mutant most
likely resulted from the strongly reduced binding to the
Fc.gamma.RIIA and Fc.gamma.RIIIA receptor (see FIG. 1c and FIG.
1e).
Example 5
[0423] ADCC with Target Cells
[0424] Human peripheral blood mononuclear cells (PBMC) were used as
effector cells and were prepared using Histopaque-1077 (Sigma
Diagnostics Inc., St. Louis, Mo. 63178 USA) and following
essentially the manufacturer's instructions. In brief, venous blood
was taken with heparinized syringes from volunteers. The blood was
diluted 1:0.75-1.3 with PBS (not containing Ca++ or Mg++) and
layered on Histopaque-1077. The gradient was centrifuged at
400.times.g for 30 min at room temperature (RT) without breaks. The
interphase containing the PBMC was collected and washed with PBS
(50 ml per cells from two gradients) and harvested by
centrifugation at 300.times.g for 10 minutes at RT. After
resuspension of the pellet with PBS, the PBMC were counted and
washed a second time by centrifugation at 200.times.g for 10
minutes at RT. The cells were then resuspended in the appropriate
medium for the subsequent procedures. The effector to target ratio
used for the ADCC assays was 25:1 and 10:1 for PBMC and NK cells,
respectively. The effector cells were prepared in AIM-V medium at
the appropriate concentration in order to add 50 ml per well of
round bottom 96 well plates. Target cells were human B lymphoma
cells (e.g., Raji cells) grown in DMEM containing 10% FCS. Target
cells were washed in PBS, counted and resuspended in AIM-V at 0.3
million per ml in order to add 30'000 cells in 100 ml per
microwell. Antibodies were diluted in AIM-V, added in 50 ml to the
pre-plated target cells and allowed to bind to the targets for 10
minutes at RT. Then the effector cells were added and the plate was
incubated for 4 hours at 37.degree. C. in a humified atmosphere
containing 5% CO.sub.2. Killing of target cells was assessed by
measurement of lactate dehydrogenase (LDH) release from damaged
cells using the Cytotoxicity Detection kit (Roche Diagnostics,
Rotkreuz, Switzerland). After the 4-hour incubation the plates were
centrifuged at 800.times.g. 100 ml supernatant from each well was
transferred to a new transparent flat bottom 96 well plate. 100 ml
color substrate buffer from the kit were added per well. The
V.sub.max values of the color reaction were determined in an ELISA
reader at 490 nm for at least 10 min using SOFTmax PRO software
(Molecular Devices, Sunnyvale, Calif. 94089, USA). Spontaneous LDH
release was measured from wells containing only target and effector
cells but no antibodies. Maximal release was determined from wells
containing only target cells and 1% Triton X-100. Percentage of
specific antibody-mediated killing was calculated as follows:
((x-SR)/(MR-SR)*100, where x is the mean of Vmax at a specific
antibody concentration, SR is the mean of Vmax of the spontaneous
release and MR is the mean of V.sub.max of the maximal release.
[0425] The potency to recruit immune-effector cells depends on type
of Fc variant as measured by classical ADCC assay. Here, human NK92
cell-line transfected with human FcgRIIIA was used as effector and
CD20 positive Raji cells were used as target cells. As can be seen
in FIG. 4a the ADCC is strongly reduced in GA101 (CD20) Fc variants
wherein glycine replaces proline (P329G) and also, to a similar
extent, in the double mutant P329G/LALA. In contrast the ADCC
decrease was less strong with the LALA mutation. In order to better
distinguish between the different variants, the variants were also
produced in the glycoengineered version to enhance the ADCC
potential. It can be observed that the parental molecule (GA101
(CD20)) shows strong ADCC as expected. The LALA version is strongly
impaired in its ADCC potential. The P329G mutant very strongly
decreased the ADCC; much more than a P329A variant of the GA101
(CD20) antibody (FIG. 4b).
Example 6
[0426] Complement Activity
[0427] Target cells were counted, washed with PBS, resuspended in
AIM-V (Invitrogen) at 1 million cells per ml. 50 ml cells were
plated per well in a flat bottom 96 well plate. Antibody dilutions
were prepared in AIM-V and added in 50 ml to the cells. Antibodies
were allowed to bind to the cells for 10 minutes at room
temperature. Human serum complement (Quidel) was freshly thawed,
diluted 3-fold with AIM-V and added in 50 ml to the wells. Rabbit
complement (Cedarlane Laboratories) was prepared as described by
the manufacturer, diluted 3-fold with AIM-V and added in 50 ml to
the wells. As a control, complement sources were heated for 30 min
at 56.degree. C. before addition to the assay. The assay plates
were incubated for 2 h at 37.degree. C. Killing of cells was
determined by measuring LDH release. Briefly, the plates were
centrifuged at 300.times.g for 3 min. 50 ml supernatant per well
were transferred to a new 96 well plate and 50 ml of the assay
reagent from the Cytotoxicity Kit (Roche) were added. A kinetic
measurement with the ELISA reader determined the Vmax corresponding
with LDH concentration in the supernatant. Maximal release was
determined by incubating the cells in presence of 1% Triton
X-100.
[0428] The different Fc variants were analyzed to mediate CDC on
SUDH-L4 target cells. The non-glycoengineered GA101 molecule shows
clear induction of CDC. The LALA variant shows activity only at the
highest concentration, whereas and the P329G and P329G/LALA
variants do not show any CDC activity (FIG. 5a). Moreover, the LALA
variant as well as the P329G and P329A variants of a
glycoengineered GA101 molecule do not show any CDC activity (FIG.
5b).
Example 7
[0429] Carbohydrate Profile of Human IgG1
[0430] The carbohydrate profiles of human IgG1 antibodies
containing mutations within the Fc, aimed at abrogating the binding
to Fc.gamma. receptors, were analyzed by MALDI/TOF-MS in positive
ion mode (neutral oligosaccharides).
[0431] Human (h) IgG1 variants were treated with sialidase (QA-Bio)
following the manufacturer's instructions to remove terminal sialic
acid. The neutral oligosaccharides of hIgG1 were subsequently
released by PNGase F (QA-Bio) digestion as previously described
(Ferrara, C. et al., Biotech. Bioeng. 93 (2006) 851-861). The
carbohydrate profiles were analyzed by mass spectrometry (Autoflex,
Bruker Daltonics GmbH) in positive ion mode as previously described
(Ferrara, C. et al., Biotech. Bioeng. 93 (2006) 851-861).
[0432] The carbohydrate profile of the neutral Fc-associated
glycans of human IgG1 is characterized by three major m/z peaks,
which can be assigned to fucosylated complex oligosaccharide with
none (G0), one (G1) or two (G2) terminal galactose residues.
[0433] The carbohydrate profiles of hIgG1 containing mutations
within the Fc, aimed at abrogating binding to Fc receptors, were
analyzed and compared to that obtained for the wild type antibody.
The IgG variants containing one of the mutations within the Fc
(P329G, LALA, P329A, P329G/LALA) show similar carbohydrate profiles
to the wild type antibody, with the Fc-associated glycans being
fucosylated complex oligosaccharides (data not shown). Mutation
within the Fc can affect the level of terminal galactosylation and
terminal sialylation, as observed by replacing amino acid at
positions 241, 243, 263, 265, or 301 by alanine (Lund, J. et al.,
J. Immunol. 157 (1996) 4963-4969).
[0434] FIG. 6a shows the relative percentage of galactosylation for
the different hIgG1 Fc-variants described here. Slight variations
can be observed when the antibodies are expressed in a different
host, but no significant difference in terminal galactosylation
could be observed.
[0435] FIG. 6b indicates the variability in galactosylation content
for wild type and IgG1-P329G/LALA for 4 different antibodies, where
four different V-domains were compared for their amount of
galactosylation when expressed in Hek293 EBNA cells.
Example 8
[0436] Antibody-Induced Platelet Aggregation in Whole Blood
Assay.
[0437] Whole blood platelet aggregation analysis using the
Multiplate instrument from Dynabyte. First, 20 ml blood from normal
human donors are withdrawn and transferred into hiruidin tubes
(Dynabyte Medical, #MP0601). Plug minicell impedance device
(Dynabead #MP0021) into the Multiplate instrument was used for the
assay. Then, 175 .mu.l 0.9% NaCl were added to the minicell.
Antibody was added to minicell to obtain the final test
concentration. Then, 175 .mu.l human blood were added and incubated
for 3 min at 37.degree. C. Automated start of impedance analysis
for additional 6 min at 37.degree. C. The data were analyzed by
quantification of area-under-the-curve as a measure of platelet
aggregation.
[0438] The CD9 antibody has been shown to induce platelet
activation and platelet aggregation (Worthington, et al., Br. J.
Hematol. 74(2) (1990) 216-222). Platelet aggregation induced by
antibodies binding to platelets previously has been shown to
involve binding to Fc.gamma.RIIA (de Reys, et al., Blood 81 (1993)
1792-1800). As shown above the mutations LALA, P329G, P329G/LALA
and P329G/SPLE introduced into the CD9 antibody strongly reduced
binding of the CD9 antibody to the Fc.gamma.RIIA receptor (FIG.
1c).
[0439] The activation (measured by Ca efflux, data not shown) as
well as platelet aggregation induced by a CD9 antibody was
eliminated by introducing the P329G and LALA triple mutation into
the antibody such that the Fc.gamma.RIIA binding is strongly
reduced compared to the wildtype antibody (see FIGS. 7a and 7b).
Murine IgG1 induced platelet aggregation at low antibody
concentrations (0.1-1 .mu./ml). At higher concentrations
overstimulation of platelets leads to silencing of the aggregation
response (3-30 .mu.g/ml). Donor variability was observed with
chim-hu-IgG4-SPLE. In FIG. 6a data for a chim-hu-IgG4-SPLE
responder at higher antibody concentrations and in FIG. 6b data for
a chim-hu-IgG4-SPLE non-responder is shown. None of the blood
samples showed any aggregation response with the antibody variants
chim-hu-IgG1-LALA, chim-hu-IgG-WT-P329G, chim-hu-IgG1-LALA-P329G,
chim-hu-IgG4-SPLE-P329G, chim-hu-IgG4-SPLE-N297Q. Controls:
spontaneous aggregation in untreated blood sample (background);
ADP-induced (ADP) and Thrombin analogon-induced (TRAP6) platelet
aggregation. Isotype controls: Murine IgG1 (murine Isotype) and
human IgG4-SPLE (hu-IgG4-SPLE Isotype).
[0440] One possible interpretation of these data is that the
decreased binding of the CD9 antibody with the triple mutations to
the Fc.gamma.RIIA receptor is the reason for the diminished
platelet aggregation observed with these kind of mutant antibodies.
In principle, prevention of thrombocyte aggregation, as a toxic
side-effect of an antibody treatment, might thus be possible by
introducing the above mentioned mutations, capable of reducing
binding to the Fc.gamma.RIIA receptor, into the Fc part of an
antibody.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 14 <210> SEQ ID NO 1 <211> LENGTH: 107 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
1 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1
5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 100 105 <210> SEQ ID NO 2 <211>
LENGTH: 105 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 2 Gln Pro Lys Ala Ala Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln Ala Asn Lys Ala
Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala Val
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45 Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65 70
75 80 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
Glu 85 90 95 Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105
<210> SEQ ID NO 3 <211> LENGTH: 330 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3 Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145
150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 330 <210> SEQ ID NO 4 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 5
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 5 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 6 <211> LENGTH: 327 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 6 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150
155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275
280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325
<210> SEQ ID NO 7 <211> LENGTH: 327 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 7 Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10
15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Glu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145
150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265
270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325
<210> SEQ ID NO 8 <211> LENGTH: 214 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: LC_1_1-PEP <400> SEQUENCE: 8
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5
10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Asn Val Lys
Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu
Thr Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln Tyr Ser Lys Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
<210> SEQ ID NO 9 <211> LENGTH: 449 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: 8201_HC_IGG1-PEP <400>
SEQUENCE: 9 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser
Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro
Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly
Phe Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile
Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Ser
Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Glu Gly
Gly Asn Tyr Arg Tyr Ser Trp Phe Pro Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235
240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360
365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> SEQ
ID NO 10 <211> LENGTH: 449 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: 8206_HC_IGG1-P329G-PEP <400> SEQUENCE: 10
Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5
10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser
Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys
Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp
Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Ser Ser Val Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Glu Gly Gly Asn Tyr
Arg Tyr Ser Trp Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Gly Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> SEQ ID NO 11
<211> LENGTH: 449 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 8207_HC_IGG1-LALA-P329G-PEP <400> SEQUENCE: 11
Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5
10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser
Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys
Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp
Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Ser Ser Val Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Glu Gly Gly Asn Tyr
Arg Tyr Ser Trp Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Gly Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> SEQ ID NO 12
<211> LENGTH: 446 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 8202_HC_IGG4-SPLE-PEP <400> SEQUENCE: 12 Asp Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20
25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu
Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn Tyr Asn
Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser
Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Ser Ser Val Thr Thr Glu
Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Glu Gly Gly Asn Tyr Arg Tyr
Ser Trp Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val
Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150
155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro
Glu Phe Glu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275
280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395
400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
Gly Lys 435 440 445 <21 0> SEQ ID NO 13 <211> LENGTH:
446 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION:
8208_HC_IGG4-SPLE-P329G-PEP <400> SEQUENCE: 13 Asp Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser
Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25
30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn Tyr Asn Pro
Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys
Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Ser Ser Val Thr Thr Glu Asp
Thr Ala Thr Tyr Phe Cys 85 90 95 Glu Gly Gly Asn Tyr Arg Tyr Ser
Trp Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val
Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu
Phe Glu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280
285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Gly Ser Ser Ile
Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405
410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys 435 440 445 <2 10> SEQ ID NO 14 <211> LENGTH: 449
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION:
8205_HC_IGG1_LALA-PEP <400> SEQUENCE: 14 Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser
Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr
Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40
45 Met Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu
50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln
Phe Phe 65 70 75 80 Leu Gln Leu Ser Ser Val Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95 Glu Gly Gly Asn Tyr Arg Tyr Ser Trp Phe
Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295
300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 440 445 Lys
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 14 <210>
SEQ ID NO 1 <211> LENGTH: 107 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 1 Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20
25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 105 <210> SEQ ID NO 2 <211> LENGTH:
105 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45 Lys Ala Gly Val Glu
Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr Ala Ala
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65 70 75 80 His
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90
95 Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> SEQ ID
NO 3 <211> LENGTH: 330 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 3 Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 4 <211> LENGTH: 330 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145
150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 330 <210> SEQ ID NO 5 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 5 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 6 <211> LENGTH: 327 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 6 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150
155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275
280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325
<210> SEQ ID NO 7 <211> LENGTH: 327 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 7 Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10
15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Glu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145
150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265
270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325
<210> SEQ ID NO 8 <211> LENGTH: 214 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: LC_1_1-PEP <400> SEQUENCE: 8
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5
10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Asn Val Lys
Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu
Thr Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln Tyr Ser Lys Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
<210> SEQ ID NO 9 <211> LENGTH: 449 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: 8201_HC_IGG1-PEP <400>
SEQUENCE: 9 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser
Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro
Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly
Phe Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile
Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Ser
Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Glu Gly
Gly Asn Tyr Arg Tyr Ser Trp Phe Pro Tyr Trp Gly Gln Gly 100 105
110
Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235
240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360
365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> SEQ
ID NO 10 <211> LENGTH: 449 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: 8206_HC_IGG1-P329G-PEP <400> SEQUENCE: 10
Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5
10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser
Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys
Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp
Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Ser Ser Val Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Glu Gly Gly Asn Tyr
Arg Tyr Ser Trp Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Gly Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> SEQ ID NO 11
<211> LENGTH: 449 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 8207_HC_IGG1-LALA-P329G-PEP <400> SEQUENCE: 11
Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5
10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser
Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys
Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp
Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Ser Ser Val Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Glu Gly Gly Asn Tyr
Arg Tyr Ser Trp Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Gly Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly
435 440 445 Lys <210> SEQ ID NO 12 <211> LENGTH: 446
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION:
8202_HC_IGG4-SPLE-PEP <400> SEQUENCE: 12 Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser
Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr
Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40
45 Met Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu
50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln
Phe Phe 65 70 75 80 Leu Gln Leu Ser Ser Val Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95 Glu Gly Gly Asn Tyr Arg Tyr Ser Trp Phe
Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu
Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295
300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420
425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435
440 445 <21 0> SEQ ID NO 13 <211> LENGTH: 446
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION:
8208_HC_IGG4-SPLE-P329G-PEP <400> SEQUENCE: 13 Asp Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser
Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25
30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn Tyr Asn Pro
Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys
Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Ser Ser Val Thr Thr Glu Asp
Thr Ala Thr Tyr Phe Cys 85 90 95 Glu Gly Gly Asn Tyr Arg Tyr Ser
Trp Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val
Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu
Phe Glu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280
285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Gly Ser Ser Ile
Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405
410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys 435 440 445 <2 10> SEQ ID NO 14 <211> LENGTH: 449
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION:
8205_HC_IGG1_LALA-PEP <400> SEQUENCE: 14 Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser
Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr
Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40
45 Met Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu
50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln
Phe Phe 65 70 75 80 Leu Gln Leu Ser Ser Val Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95 Glu Gly Gly Asn Tyr Arg Tyr Ser Trp Phe
Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275
280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395
400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440 445 Lys
* * * * *