U.S. patent application number 15/046379 was filed with the patent office on 2016-06-09 for antibodies with enhanced or suppressed effector function.
The applicant listed for this patent is Zymeworks Inc.. Invention is credited to Dustin Bleile, Igor D'Angelo, Surjit B. Dixit, Eric Escobar-Cabrera, Paula I. Lario, Anders Ohrn, David K.Y. Poon, Stacey A.L. Tom-Yew.
Application Number | 20160159921 15/046379 |
Document ID | / |
Family ID | 44711257 |
Filed Date | 2016-06-09 |
United States Patent
Application |
20160159921 |
Kind Code |
A1 |
D'Angelo; Igor ; et
al. |
June 9, 2016 |
ANTIBODIES WITH ENHANCED OR SUPPRESSED EFFECTOR FUNCTION
Abstract
Rationally designed antibodies and polypeptides that comprise
multiple Fc region amino acid substitutions that synergistically
provide enhanced selectivity and binding affinity to a target Fc
receptor are provided. The polypeptides are mutated at multiple
positions to make them more effective when incorporated in antibody
therapeutics than those having wild-type Fc components.
Inventors: |
D'Angelo; Igor; (Port Moody,
CA) ; Bleile; Dustin; (Vancouver, CA) ;
Tom-Yew; Stacey A.L.; (New Westminster, CA) ;
Escobar-Cabrera; Eric; (Burnaby, CA) ; Lario; Paula
I.; (Vancouver, CA) ; Ohrn; Anders;
(Vancouver, CA) ; Poon; David K.Y.; (Richmond,
CA) ; Dixit; Surjit B.; (Richmond, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zymeworks Inc. |
Vancouver |
|
CA |
|
|
Family ID: |
44711257 |
Appl. No.: |
15/046379 |
Filed: |
February 17, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13638362 |
Dec 19, 2012 |
9296815 |
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PCT/CA2011/000321 |
Mar 28, 2011 |
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15046379 |
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61436584 |
Jan 26, 2011 |
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61318583 |
Mar 29, 2010 |
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Current U.S.
Class: |
424/133.1 ;
435/320.1; 435/69.6; 506/8; 530/387.3; 536/23.53; 702/19 |
Current CPC
Class: |
C07K 2317/524 20130101;
C07K 2317/52 20130101; A61P 37/00 20180101; C07K 16/32 20130101;
A61P 29/00 20180101; C07K 2317/732 20130101; C07K 16/2803 20130101;
C07K 16/2887 20130101; C07K 16/28 20130101; C07K 14/70535 20130101;
C07K 2317/24 20130101; C07K 16/2851 20130101; A61P 35/00 20180101;
G16B 15/00 20190201; C07K 16/283 20130101; C07K 2317/72 20130101;
C07K 2317/76 20130101; C07K 2317/92 20130101; C07K 16/082 20130101;
C07K 16/464 20130101; C07K 16/00 20130101 |
International
Class: |
C07K 16/32 20060101
C07K016/32; C07K 16/08 20060101 C07K016/08; C07K 16/28 20060101
C07K016/28 |
Claims
1. A polypeptide comprising a variant Fc region, wherein said
variant Fc region comprises three or more amino acid modifications
relative to a wild-type Fc region, and has an altered effect
relative to a polypeptide comprising a wild-type Fc region or
variant Fc region having only one of the three or more amino acid
modifications; wherein at least two of the three or more
modifications provide a synergistic effect compared to single
position modifications at the at least two positions thereby
exhibiting a selected binding profile to Fc.gamma. receptors.
2. The polypeptide of claim 1 wherein the amino acid modifications
produce amino acid interactions and dynamics that result in
enhanced binding affinity and/or specificity to a first Fc.gamma.
receptor while diminishing binding affinity and/or specificity to a
second Fc.gamma. receptor compared to a polypeptide that lacks the
at least three or more amino acid modifications.
3. The polypeptide of claim 2 wherein the first Fc.gamma. receptor
is Fc.gamma.RIIIa receptor and the second Fc.gamma. receptor is
Fc.gamma.RIIa or Fc.gamma.RIIb.
4. The polypeptide of claim 1 wherein the amino acid modifications
produce favorable Fc.gamma.RIIIa-specific interactions and/or
unfavorable interactions with Fc.gamma.RIIa and/or Fc.gamma.RIIb
receptors.
5. The polypeptide of claim 1 wherein the amino acid modifications
have minimal impact on the Fc.gamma.RIIIa receptor while producing
detrimental effects on binding of the polypeptide to Fc.gamma.RIIa
and/or Fc.gamma.RIIb.
6. The polypeptide of claim 1 wherein the polypeptide comprises
modifications S239E/D265S/I332E.
7. The polypeptide of claim 1 wherein the polypeptide comprises
modifications H268D/E269L/S298A/K326A/A327H.
8. The polypeptide of claim 2 wherein the first Fc.gamma. receptor
is Fc.gamma.RIIa receptor and the second Fc.gamma. receptor is
Fc.gamma.RIIIa or Fc.gamma.RIIb.
9. The polypeptide of claim 1 wherein the amino acid modifications
produce favorable Fc.gamma.RIIa-specific interactions and/or
unfavorable interactions with Fc.gamma.RIIIa and/or Fc.gamma.RIIb
receptors.
10. The polypeptide of claim 1 wherein the amino acid modifications
have minimal impact on the Fc.gamma.RIIa receptor while producing
detrimental effects on binding of the polypeptide to Fc.gamma.RIIIa
and/or Fc.gamma.RIIb.
11. The polypeptide of claim 2 wherein the first Fc.gamma. receptor
is Fc.gamma.RIIb receptor and the second Fc.gamma. receptor is
Fc.gamma.RIIIa receptor or Fc.gamma.RIIa receptor.
12. The polypeptide of claim 1 wherein the amino acid modifications
produce favorable Fc.gamma.RIIb-specific interactions and/or
unfavorable interactions with Fc.gamma.RIIIa and/or Fc.gamma.RIIa
receptors.
13. The polypeptide of claim 1 wherein the amino acid modifications
have minimal impact on the Fc.gamma.RIIb receptor while producing
detrimental effects on binding of the polypeptide to Fc.gamma.RIIIa
and/or Fc.gamma.RIIa.
14. The polypeptide of claim 1 wherein the polypeptide comprises
modifications D265E/S267D/A330S.
15. The polypeptide of claim 1 wherein the polypeptide comprises
modifications G237F/A327L/A330I.
16. The polypeptide of claim 1 wherein the polypeptide comprises
modifications G237F/S239E/H268D.
17. The polypeptide of claim 1 wherein the polypeptide comprises
modifications S239E/S267E/H268D.
18. The polypeptide of claim 1 wherein the polypeptide comprises
modifications S239E/S298A/K326A/A327H.
19. The polypeptide of claim 1 wherein the polypeptide comprises
modifications G237F/S298A/A330L/I332E.
20. The polypeptide of claim 1 wherein the polypeptide comprises
modifications G237F/S239E/A327H.
21. The polypeptide of claim 1 wherein the polypeptide comprises
modifications L235A/S239E/D265E/A327H.
22. The polypeptide of claim 1 wherein the polypeptide comprises
modifications G237F/S239E/D270N.
23. The polypeptide of claim 1 wherein binding of the polypeptide
comprising a wild-type Fc region to Fc.gamma. receptors is
detectable by an in vitro assay.
24. The polypeptide of claim 1 wherein the three or more
modifications comprise the mutation S239E wherein the polypeptide
has higher selectivity in binding to the Fc.gamma.RIIIa receptor
compared to a polypeptide that lacks the S239E mutation.
25. The polypeptide of claim 1 wherein the three or more
modifications comprise the mutation S298A wherein the polypeptide
has reduced binding affinity to Fc.gamma.RIIa and Fc.gamma.RIIb
receptors compared to a polypeptide that lacks the S298A
mutation.
26. The polypeptide of claim 1 wherein the polypeptide comprises
the amino acid modifications S298A/K326A/A327H wherein the
polypeptide has improved binding selectivity to Fc.gamma.RIIIa
receptor as compared to a polypeptide lacking the S298A/K326A/A327H
modifications.
27. The polypeptide of claim 1 wherein the polypeptide comprises
the amino acid modifications S239E/S298A/K326A/A327H wherein the
polypeptide has improved binding selectivity to Fc.gamma.RIIIa
receptor as compared to a polypeptide lacking the
S239E/S298A/K326A/A327H modifications.
28. The polypeptide of claim 1 wherein the polypeptide comprises
the amino acid modifications G236A/S239D/D270L/I332E, wherein the
polypeptide has improved binding selectivity to Fc.gamma.RIIIa
receptor as compared to a polypeptide lacking the
G236A/S239D/D270L/I332E modifications.
29. The polypeptide of claim 1 wherein the polypeptide comprises
three amino acid modifications, said mutations selected from
D270L/Y300L/A330K, G237F/S267G/N325F, G237F/V266L/S267D,
L234F/S267G/N325L, and L234F/S267E/N325L.
30. The polypeptide of claim 1 wherein the polypeptide comprises
modifications of at least three amino acids, said modifications
selected from G237F/S239E/A327H, G237F/A327L/A330I,
S239E/A327L/A330I, S239E/S267E/H268D, G237F/S239E/D270N,
G236E/G237F/S239E, S239E/D265S/I332E, G237F/S239E/D265E,
G237F/S239E/H268D, H268E/D270E/S267G, H268D/K326A/A327H,
D265E/S267D/A330S, L235A/S239E/D265E, A327H/E269L/K236A,
G237F/D270Q/S239E, A330V/I332L/K326, and G236S/A327H/A330I.
31. The polypeptide of claim 1 wherein the polypeptide comprises
simultaneous modification of at least four amino acids, said
modifications selected from L235A/S239E/D265E/A327H,
S239E/D265S/H268D/I332E, S239D/D265S/S298A/I332E,
S239E/S298A/K326A/A327H, G237F/S298A/A330L/I332E, and
G236E/D270N/A327V/I332E, and G236A/S239D/D270L/I332E.
32. The polypeptide of claim 1 wherein the polypeptide comprises
the amino acid modifications H268D/E269L/S298A/K326A/A327H.
33. The polypeptide of any of claims 1-32 wherein the polypeptide
comprises the antibody trastuzumab or an antigen binding portion
thereof.
34. The polypeptide of claim 1, wherein one or more amino acid
modifications are located between positions 234-330 according to
the EU index.
35. The polypeptide of claim 1 wherein at least three amino acid
modifications are selected from the group consisting of: L234Q,
L234N, L235A, G236E, E236L, E236D, G237F, G237N, S239E, S239D,
D265E, D265S, S267E, S267D, S267G, H268D, H268E, E269L, E269L,
D270N, D270I, D270E, S298A, K326A, K326D, A327H, A327V, A327L,
A327T, A330V, A330L, A330W, A330I, A330S, I332L, I332D, and
1332E.
36. The polypeptide of claim 1 wherein said variant Fc region
comprises a combination of amino acid modifications wherein said
combination is selected from the group consisting of:
L235A/S239E/D265E; L235A/G237F/D265E; S239E/E269D/A327H;
S239E/G237N/A327H; S239E/G237F/A327V; G237F/D270I/S239E;
G237F/A327L/S239E; A327H/E269L/K326A; A330V/I332L/S239E;
A327T/E269L/K326A; D270N/A327T/K326A; A330V/I332L/S239E;
A330W/I332D/S239E; G236E/D265E/A327H/A330I; D270N/S298A/A327V;
G236E/D265E/D270N/A327H/A330I; G236E/D270N/A327H/A330I;
G236E/D270N/A327V/I332E; G236E/D270N/A327V/G237F;
L234N/S239E/A330I/I332E; L234Q/S239E/A330I/I332E;
L234Q/S239E/A330I/I332E/S298A; G237F/S239D/D265E/D270N/S298A;
G237F/S239E/D270N/A330L/I332E; G237F/S239E/D270N/A330L/I332E/S298A;
S239E/G237F/A327H; G237F/A327L/A330I; S239E/A330I/A327L;
D265E/S239E/L235A/A327H; S267E/S239E/H268D; G237F/D270N/S239E;
S239E/G237F/G236E; 1332E/D265S/S239E/H268D; I332E/D265S/S239E;
D265E/S239E/G237F; S239E/H268D/G237F; S298A/D265S/S239D/I332E;
S298A/K326A/A327H/S239E; S298A/G237F/A3330L/I332E;
H268E/D270E/S267G; H268D/K326A/A327H;
H268D/K326A/A327H/E269L/S298A; and A330S/D265E/S267D.
37. The polypeptide of claim 1 wherein said variant Fc region
comprises a combination of amino acid modifications wherein said
combination is selected from the group consisting of
S239E/S267E/H268D; S237F/S239E/D265E and H268E/D270/E/S267G.
38. The polypeptide of claim 1, wherein said variant Fc region
comprises amino acid modification H268D and not modification
S267E.
39. The polypeptide of claim 1, wherein the Fc region of the parent
polypeptide is a human IgG Fc region.
40. The polypeptide of claim 39, wherein the human IgG Fc region is
a human IgG1, IgG2, IgG3, or IgG4 Fc region.
41. The polypeptide of claim 1 wherein said polypeptide is an
antibody.
42. The antibody of claim 41, wherein said antibody is a monoclonal
antibody, a humanized antibody, or a human antibody.
43. A nucleic acid comprising a nucleotide sequence encoding the
polypeptide of claim 1.
44. A vector comprising the nucleic acid of claim 43.
45. A method for producing the polypeptide of claim 1, said method
comprising: (i) culturing in a medium a host cell comprising a
nucleic acid encoding said polypeptide, under conditions suitable
for the expression of said polypeptide; and (ii) recovering the
polypeptide from said medium.
46. A therapeutic antibody that specifically binds a cancer target
antigen, said therapeutic antibody comprising a variant Fc region
of claim 1.
47. The therapeutic antibody of claim 46, wherein said therapeutic
antibody is selected from the group consisting of abagovomab,
adalimumab, alemtuzumab, aurograb, bapineuzumab, basiliximab,
belimumab, bevacizumab, briakinumab, canakinumab, catumaxomab,
certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab,
galiximab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan,
infliximab, ipilimumab, lumiliximab, mepolizumab, motavizumab,
muromonab, mycograb, natalizumab, nimotuzumab, ocrelizumab,
ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab,
ranibizumab, reslizumab, rituximab, teplizumab,
tocilizumab/atlizumab, tositumomab, trastuzumab, Proxinium.TM.,
Rencarex.TM., ustekinumab, zalutumumab, and any other
antibodies.
48. The therapeutic antibody of claim 46, wherein said target
antigen is selected from the group consisting of a-chain (CD25) of
IL-2R, Amyloid beta, anti-EpCAM.times.anti-CD3, BLyS (or BAFF),
CD11a, CD20, CD22, CD23, CD3, CD4, CD52, CD80, CTLA-4, EGFR, EpCAM,
F protein of RSV, G250, glycoprotein IIb/IIIa R, HER2, HER2/neu R,
Hsp90, IgE antibody, IL-12/IL-23, IL-1b, IL-5, IL-6 receptor,
Integrin alpha-4/beta-1, Mucin 16/CA-125, RANKL, TNF alpha, VEGF-A,
and other therapeutically advantageous targets.
49. A method of treating cancer in a patient having a cancer
characterized by a cancer antigen, said method comprising
administering to said patient a therapeutically effective amount of
a therapeutic antibody of claim 46.
50. The method of claim 49, wherein said patient is human.
51. A method of treating immune disorders in a patient having an
immune disorder characterized by an immune antigen, said method
comprising administering to said patient a therapeutically
effective amount of a therapeutic antibody of claim 46.
52. A pharmaceutical composition comprising a therapeutically
effective amount of the polypeptides of claim 1, and a
pharmaceutically acceptable carrier.
53. The polypeptide of claim 1 wherein the polypeptide comprising a
variant Fc region is more effective at mediating ADCC relative to
wild type.
54. The polypeptide of claim 53 wherein the polypeptide comprising
a variant Fc region is about 1.5 to about 100 fold more effective
in mediating ADCC relative to wild type.
55. The polypeptide of claim 53 wherein the polypeptide comprising
a variant Fc region is about 2 to about 50 fold more effective in
mediating ADCC relative to wild type.
56. The polypeptide of claim 1 wherein the polypeptide comprising a
variant Fc region is more effective at mediating inhibition of
inflammatory immune responses relative to wild type.
57. The polypeptide of claim 56 wherein the polypeptide comprising
a variant Fc region is about 2 fold more effective in mediating
inhibition of inflammatory immune responses relative to wild
type.
58. The polypeptide of claim 56 wherein the polypeptide comprising
a variant Fc region is about 10 fold more effective in mediating
inhibition of inflammatory immune responses relative to wild
type.
59. The polypeptide of claim 56 wherein the polypeptide comprising
a variant Fc region is about 50 fold more effective in mediating
inhibition of inflammatory immune responses relative to wild
type.
60. The polypeptide of claim 56 wherein the polypeptide comprising
a variant Fc region is about 100 fold more effective in mediating
inhibition of inflammatory immune responses relative to wild
type.
61. A method for identifying Fc variant polypeptides in silico
based on calculated binding affinities to Fc.gamma.RIIa,
Fc.gamma.RIIb and/or Fc.gamma.RIIIa.
62. The method of claim 61 comprising further calculating in silico
electrostatics, solvation, packing, packing density, hydrogen
binding, and entropic effects of said Fc variant polypeptides.
63. The method of claim 61 further comprising constructing said Fc
variant polypeptides and expressing said polypeptides in the
context of an antibody in mammalian cells.
64. The polypeptide of claim 1, wherein amino acid modifications do
not comprise simultaneous modifications at positions 317 and 353
according to the EU index.
65. The polypeptide of claim 1, wherein amino acid modifications do
not encompass a substitution at position 332 according to the EU
index.
Description
CROSS-REFERENCE
[0001] This application claims priority to U.S. Provisional
Application No. 61/436,584, filed Jan. 26, 2011, and U.S.
Provisional Application No. 61/318,583, filed Mar. 29, 2010. The
contents of both provisional applications are incorporated in their
entirety by reference herein.
FIELD OF INVENTION
[0002] The present invention relates to antibodies, fusion proteins
and polypeptides. Specifically, the instant invention relates to
designed antibodies and polypeptides that comprise Fc region amino
acid substitutions that synergistically provide enhanced
selectivity and binding affinity to a target Fc receptor.
BACKGROUND OF THE INVENTION
[0003] Antibodies are proteins which exhibit binding specificity to
a specific antigen. Native antibodies are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies between the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end;
the constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light and heavy chain variable domains.
[0004] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are responsible for the binding specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed through the variable domains
of antibodies. It is concentrated in three segments called
complementarity determining regions (CDRs) both in the light chain
and the heavy chain variable domains. The more highly conserved
portions of the variable domains are called the framework regions
(FRs). The variable domains of native heavy and light chains each
comprise four FRs, largely adopting a .beta.-sheet configuration,
connected by three CDRs, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The CDRs in each
chain are held together in close proximity by the FRs and, with the
CDRs from the other chain, contribute to the formation of the
antigen binding site of antibodies (see Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)).
[0005] The constant domains are not involved directly in binding an
antibody to an antigen, but exhibit various effector functions.
Depending on the amino acid sequence of the constant region of
their heavy chains, antibodies or immunoglobulins can be assigned
to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these
may be further divided into subclasses (isotypes), e.g. IgG1, IgG2,
IgG3, and IgG4; IgA1 and IgA2. The heavy chain constant regions
that correspond to the different classes of immunoglobulins are
called .alpha., .delta., .epsilon., .gamma. and .mu., respectively.
Of the various human immunoglobulin classes, only human IgG1, IgG2,
IgG3 and IgM are known to activate complement; and human IgG1 and
IgG3 mediate ADCC more effectively than IgG2 and IgG4
[0006] Papain digestion of antibodies produces two identical
antigen binding fragments, called Fab fragments, each with a single
antigen binding site, and a residual "Fc" fragment, whose name
reflects its ability to crystallize readily. The crystal structure
of the human IgG Fc region has been determined (Deisenhofer,
Biochemistry 20:2361-2370 (1981)). In human IgG molecules, the Fc
region is generated by papain cleavage N-terminal to Cys 226. The
Fc region is central to the effector functions of antibodies.
[0007] The effector functions mediated by the antibody Fc region
can be divided into two categories: (1) effector functions that
operate after the binding of antibody to an antigen (these
functions involve the participation of the complement cascade or Fc
receptor (FcR)-bearing cells); and (2) effector functions that
operate independently of antigen binding (these functions confer
persistence in the circulation and the ability to be transferred
across cellular barriers by transcytosis). Ward and Ghetie,
Therapeutic Immunology 2:77-94 (1995).
[0008] While binding of an antibody to the requisite antigen has a
neutralizing effect that might prevent the binding of a foreign
antigen to its endogenous target (e.g. receptor or ligand), binding
alone may not remove the foreign antigen. To be efficient in
removing and/or destructing foreign antigens, an antibody should be
endowed with both high affinity binding to its antigen, and
efficient effector functions.
Fc Receptor (FcR)
[0009] The interaction of antibodies and antibody-antigen complexes
with cells of the immune system effects a variety of responses,
including antibody-dependent cell-mediated cytotoxicity (ADCC) and
complement dependent cytotoxicity (CDC) (reviewed in Daeron, Annu.
Rev. Immunol. 15:203-234 (1997); Ward and Ghetie, Therapeutic
Immunol. 2:77-94 (1995); as well as Ravetch and Kinet, Annu. Rev.
Immunol. 9:457-492 (1991)).
[0010] Several antibody effector functions are mediated by Fc
receptors (FcRs), which bind the Fc region of an antibody. FcRs are
defined by their specificity for immunoglobulin isotypes; Fc
receptors for IgG antibodies are referred to as Fc.gamma.R, for IgE
as Fc.epsilon.R, for IgA as Fc.alpha.R and so on. Three subclasses
of Fc.gamma.R have been identified in humans: Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16). Because each
Fc.gamma.R subclass is encoded by two or three genes, and
alternative RNA spicing leads to multiple transcripts, a broad
diversity in Fc.gamma.R isoforms exists. The three genes encoding
the Fc.gamma.RI subclass (Fc.gamma.RIA, Fc.gamma.RIB and
Fc.gamma.RIC) are clustered in region 1q21.1 of the long arm of
chromosome 1; the genes encoding Fc.gamma.RII isoforms
(Fc.gamma.RIIA, Fc.gamma.RIIB and Fc.gamma.RIIC) and the two genes
encoding Fc.gamma.RIII (Fc.gamma.RIIIA and Fc.gamma.RIIIB) are all
clustered in region 1q22. These different FcR subtypes are
expressed on different cell types (reviewed in Ravetch and Bollard,
Annu. Rev. Immunol. 19:275-290 (2001). For example, in humans,
Fc.gamma.RIIIB is found only on neutrophils, whereas Fc.gamma.RIIIA
is found on macrophages, monocytes, natural killer (NK) cells, and
a subpopulation of T-cells. Notably, Fc.gamma.RIIIA is the only FcR
present on NK cells, one of the cell types implicated in ADCC.
[0011] Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII are
immunoglobulin superfamily (IgSF) receptors; Fc.gamma.RI has three
IgSF domains in its extracellular domain, while Fc.gamma.RII and
Fc.gamma.RIII have only two IgSF domains in their extracellular
domains.
[0012] Another type of Fc receptor is the neonatal Fc receptor
(FcRn). FcRn is structurally similar to major histocompatibility
complex (MHC) and consists of an .alpha.-chain noncovalently bound
to .beta.2-microglobulin.
[0013] Fc.gamma.RII (CD32), has several isoforms, IIa, IIb1, Iib2,
IIb3 and IIc, and is the most widely distributed human Fc.gamma.R
type, being expressed on most types of blood leukocytes, dendritic
cells and platelets. Fc.gamma.RII is a low affinity receptor that
only binds to aggregated IgG. It is the only Fc.gamma.R class to be
able to bind to IgG2. The Fc.gamma.RIIa is expressed on a range of
cell types, including monocytes, macrophages, neutrophils,
eosinophils and basophils, which it can co-activate in combination
with other immunoglobulin receptors through its ITAM motifs.
Fc.gamma.RIIa binds IgG antibodies attached to cells and causes
lysis of those cells. This process is called antibody-dependant
cell mediated cytotoxicity. The Fc.gamma.RIIb is also widely
expressed but bears an immunoreceptor tyrosine-based inhibitory
motif (ITIM) which is necessary for its inhibitory effects.
Fc.gamma.RIIb can suppress activation of B cells by invoking
negative signaling when it is cross-linked to surface
immunoglobulin via Ab-Ag complexes. The activation of macrophages
and monocytes via Fc.gamma.R is suppressed by co-ligation of
Fc.gamma.RIIb. (Armour et al. (2003) "Differential binding to human
Fc.gamma.RIIa and Fc.gamma.RIIb receptors by human IgG wildtype and
mutant antibodies," Mol. Immunol., 40:585-593). Cells transfected
to express both Fc.gamma.RIIa and Fc.gamma.RIIb show reduced
phagocytosis relative to cells bearing Fc.gamma.RIIa alone and the
cytotoxicity of anti-tumour Ab was enhanced in
Fc.gamma.RIIb-deficient mice. (Hunter et al. (1998) "Inhibition of
Fc.gamma. receptor mediated phagocytosis by nonphagocytic Fc.gamma.
receptor." Blood, 91:1762-1768; and Clynes et al. (2000)
"Inhibitory Fc receptors modulate in vivo cytotoxicity against
tumour targets." Nat. Med. 6:443-446). Thus, shifting relative
binding affinity of Fc.gamma.R from Fc.gamma.RIIa to Fc.gamma.RIIb,
for example would suppress activation of B cells and or macrophages
and monocytes and would be useful to dampen immune responses in
inflammatory disorders such in various autoimmune diseases.
Conversely, shifting the relative binding affinity of Fc.gamma.
from Fc.gamma.RIIb to Fc.gamma.RIIa would lead to an enhancement of
ADCC, and would be useful to enhance tumor cell killing, for
example in the treatment of cancer.
[0014] Fc.gamma.RIII (CD16) has two isoforms which are able to bind
to IgG1 and IgG3. The Fc.gamma.RIIIa has an intermediate affinity
for IgG and is expressed on macrophages, monocytes, NK cells and
subsets of T cells. Fc.gamma.RIIIb is a low-affinity receptor which
is selectively expressed on neutrophils. Fc.gamma.RIIIa, like
FC.gamma.RIIa, binds to IgG antibodies attached to cells and causes
the lysis of those cells by ADCC. Fc.gamma.RIIIa binds clustered
IgG molecules bound to cell surfaces and does not bind to monomeric
IgG. Therefore, ADCC occurs only when the target cell is coated
with antibody. Engagement of Fc.gamma.RIIIa by antibody-coated
target cells activates NK cells to synthesize and secrete cytokines
such as IFN-.gamma., as well as discharge the contents of their
granules, which mediate the cytolytic functions of this cell
type.
[0015] Altering the effector activity of antibodies by shifting
effector function from otherwise inhibitory immune response to
inducing ADCC and vice versa is desirable for bettering treatment
outcomes in a variety of diseases and conditions. Lazar et al.
(2006) Proc. Natl. Acad. Sci. U.S.A., 103:4005, Stavenhagen et al.
(2007) Cancer Res. 67:8882, Oganesyan et al. (2008) Mol. Immunol.
45:1872, Veri et al. (2007) Immunology, 121:392, and Shields, et
al. (2001) J. Biol. Chem. 276:6591 disclose efforts in this
research area.
[0016] Presta et al. (U.S. Pat. No. 6,737,056) discloses
polypeptides comprising a variant Fc region. The disclosed
variations are based on single position mutations. By performing
alanine scans, Presta et al. discloses single position Fc region
amino acid modification at positions 238, 265, 269, 270, 292, 294,
295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416,
419, 435, 438 or 439; these single position mutations are purported
to result in reduced binding to Fc.gamma.RII. Presta et al. also
discloses that systematic alanine scans of the entire Fc region
purportedly show that an Fc region amino acid modification at any
one of amino acid positions 238, 239, 248, 249, 252, 254, 265, 268,
269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327,
329, 338, 340, 373, 376, 382, 388, 389, 416, 434, 435 or 437
results in reduced binding to Fc.gamma.RIII. Presta et al. also
discloses polypeptides with increased binding to an Fc.gamma.RII
comprising single position amino acid modifications at any one of
amino acid positions 255, 256, 258, 267, 268, 272, 276, 280, 283,
285, 286, 290, 301, 305, 307, 309, 312, 315, 320, 322, 326, 330,
331, 337, 340, 378, 398 or 430 of the Fc region identified by
alanine scans as well. Presta et al. discloses one single instance
where two Fc amino acid positions are mutated at the same time
(i.e., modifications S317A and K353A), however Presta et al. does
not suggest that these double mutations provide any synergistic
effect in obtaining a desired binding profile of the modified
polypeptide to Fc.gamma.RII. Presta et al. is completely silent on
potential synergism of simultaneous modifications at more than a
single amino acid position.
[0017] Lazar et al. (U.S. Pat. No. 7,317,091) discloses antibodies
comprising an amino acid modification at position 332 in the Fc
region purportedly resulting in altered binding to an Fc.gamma.R.
Lazar et al. discloses that individual substitutions in positions
234, 235, 239, 240, 243, 264, 266, 272, 274, 278, 325, 328, 330,
and 332 purportedly effect the binding to an Fc.gamma.R. While
Lazar al. purports to disclose synergy of Fc variants when combined
with engineered glycoforms, Lazar et al. is silent on potential
synergism that may be provided by a selection of simultaneously
modified Fc amino acid positions, regardless of additional
synergism that may be provided by engineered glycoforms.
[0018] Stavenhagen (U.S. Pat. No. 7,632,497) discloses molecules
having a variant Fc region, wherein the variant Fc region comprises
at least one amino acid modification relative to a wild-type Fc
region. These modified molecules purportedly confer an effector
function to a molecule, where the parent molecule does not
detectably exhibit this effector function.
[0019] Current approaches to optimize the Fc region in therapeutic
monoclonal antibodies and soluble polypeptides fused to Fc regions
have focused on a limited number of single amino acid changes based
on alanine screens, site-directed mutagenesis etc. Other approaches
in engineering Fc regions have focused on the glycosylation of the
Fc region to optimize Fc region function. Still other approaches
have focused on Fe modifications that purportedly confer effector
function by modifying wild type molecules that lack effector
function, but do not purport to increase or otherwise modify
existing effector function of a wild type molecule.
[0020] There is currently no known Fc protein or polypeptide that
is optimized to bind a particular Fc.gamma.R of interest with very
high specificity as compared to other Fc.gamma. receptors. Although
the effect of individual Fe region amino acid mutations on the
binding with certain Fc.gamma. receptors is well understood,
previous studies fail to describe the effect of simultaneous
changes to multiple amino acids. Additionally, the prior art does
not suggest suitable replacements for substituted Fc region amino
acids to obtain optimal binding to the Fc.gamma.R of interest,
including modification of effector function of a wild type molecule
that already has detectable effector function.
[0021] Hence, there is a need in the art for polypeptides and
antibodies that comprise multiple, synergistic amino acid
substitutions in the Fc region such that the antibody or fusion
protein is optimized for binding to the Fc.gamma.R isoform of
choice.
SUMMARY OF THE INVENTION
[0022] The instant compositions provide polypeptides, fusion
protein and antibodies comprising Fc region that bind a target
Fc.gamma.R with very high specificity. These polypeptides and
antibodies are designed by rational analysis of the binding of each
Fc.gamma.R with the Fc region and by subsequent design of multiple
(three or more) amino acid substitutions that synergistically
provide desired selectivity and binding affinity for the target Fc
receptor. Accordingly, the polypeptides and proteins provided
herein comprise multiple variations in the Fc region as compared to
the wild type Fc region, said variations tailored to improving the
specificity for the Fc.gamma.R under consideration, and for
obtaining a selected binding profile based on enthalpic and
entropic factors optimized by the choice of the most favorable
amino acid for each position.
[0023] One embodiment provides a polypeptide comprising a variant
Fc region, wherein said variant Fc region comprises three or more
amino acid modifications relative to a wild-type Fc region, and has
an altered effect relative to a polypeptide comprising a wild-type
Fc region; wherein at least two of the three or more modifications
provide a synergistic effect compared to single position
modifications at the at least two positions thereby exhibiting a
selected binding profile to Fc.gamma. receptors.
[0024] Another embodiment provides a polypeptide wherein the amino
acid modifications produce amino acid interactions and dynamics
that result in enhanced binding free energy to a first Fc.gamma.
receptor while diminishing binding affinity to a second Fc.gamma.
receptor compared to a polypeptide that lacks the at least three or
more amino acid modifications. The first Fc.gamma. receptor may be
Fc.gamma.RIIIa receptor and the second Fc.gamma. receptor may be
Fc.gamma.RIIa or Fc.gamma.RIIb.
[0025] Another embodiment provides a polypeptide wherein the amino
acid modifications produce favorable Fc.gamma.RIIIa-specific
interactions and/or unfavorable interactions with Fc.gamma.RIIa
and/or Fc.gamma.RIIb receptors.
[0026] Another embodiment provides a polypeptide wherein the amino
acid modifications have minimal impact on the Fc.gamma.RIIIa
receptor while producing detrimental effects on binding of the
polypeptide to Fc.gamma.RIIa and/or Fc.gamma.RIIb.
[0027] One embodiment provides a polypeptide wherein the first
Fc.gamma. receptor is Fc.gamma.RIIa receptor and the second
Fc.gamma. receptor is Fc.gamma.RIIIa or Fc.gamma.RIIb.
[0028] One embodiment provides a polypeptide wherein the amino acid
modifications produce favorable Fc.gamma.RIIa-specific interactions
and/or unfavorable interactions with Fc.gamma.RIIIa and/or
Fc.gamma.RIIb receptors.
[0029] One embodiment provides a polypeptide wherein the amino acid
modifications have minimal impact on the Fc.gamma.RIIa receptor
while producing detrimental effects on binding of the polypeptide
to Fc.gamma.RIIIa and/or Fc.gamma.RIIb.
[0030] One embodiment provides a polypeptide wherein the Fc.gamma.
receptor with enhanced binding free energy is Fc.gamma.RIIb
receptor and the Fc.gamma. receptor with diminished binding
affinity is Fc.gamma.RIIIa receptor or Fc.gamma.RIIa receptor.
[0031] One embodiment provides a polypeptide wherein the amino acid
modifications produce favorable Fc.gamma.RIIb-specific interactions
and/or unfavorable interactions with Fc.gamma.RIIIa and/or
Fc.gamma.RIIa receptors.
[0032] One embodiment provides a polypeptide wherein the amino acid
modifications have minimal impact on the Fc.gamma.RIIb receptor
while producing detrimental effects on binding of the polypeptide
to Fc.gamma.RIIIa and/or Fc.gamma.RIIa.
[0033] One embodiment provides a polypeptide wherein the amino acid
substitutions reduce or disrupt the binding affinity to the
Fc.gamma.RIIa receptor, the Fc.gamma.RIIIa receptor and the
Fc.gamma.RIIb receptor when compared to the wild-type
polypeptide.
[0034] One embodiment provides a polypeptide wherein binding of the
polypeptide comprising a wild-type Fc region to Fc.gamma. receptors
is detectable by an in vitro assay.
[0035] In certain embodiments of the instant invention, are methods
for engineering optimized Fc polypeptides and proteins. It is an
object of the present disclosure to provide design strategies that
may be used to guide Fe optimization. It is a further object of the
present disclosure to provide computational screening methods that
may be used to design Fc proteins. It is a further object of the
present disclosure to provide methods for generating libraries for
experimental testing. It is a further object of the present
disclosure to provide experimental production and screening methods
for obtaining optimized Fe proteins.
[0036] In certain embodiments are provided isolated nucleic acids
encoding the Fc proteins described herein. In certain embodiments
are provided vectors comprising said nucleic acids, optionally,
operably linked to control sequences. In certain embodiments are
provided host cells containing the vectors, and methods for
producing and optionally recovering the Fc proteins.
[0037] In certain embodiments are provided antibodies and
polypeptides that comprise the Fc proteins disclosed herein. Said
antibodies and polypeptides may find use in a therapeutic
product.
[0038] The present disclosure provides compositions comprising
antibodies and polypeptides that comprise the Fc proteins described
herein, and a physiologically or pharmaceutically acceptable
carrier or diluent.
[0039] In certain embodiments are provided therapeutic and
diagnostic uses for antibodies and polypeptides that comprise the
Fc proteins disclosed herein.
[0040] Provided herein are polypeptides comprising a variant Fc
region, wherein said variant Fc region comprises at least three
amino acid modifications relative to a wild-type Fc region, and has
an altered effect relative to a polypeptide comprising a wild-type
Fc region or variant Fc region comprising only one or two amino
acid modifications; and wherein at least two of the modifications
provide a synergistic effect compared to single position
modifications thereby exhibiting a selected binding profile to
Fc.gamma. receptors. In certain embodiments, one or both amino acid
modifications are located between positions 234-330 according to
the EU index. In certain embodiments, the modifications do not
comprise simultaneous substitution at positions 317 and 353
according to the EU index. In some embodiments, the amino acid
modifications do not comprise a substitution at position 332
according to the EU index. In certain embodiments, the polypeptides
comprise the modifications L235A/S239E/D265E. In some embodiments,
the polypeptides comprise the modifications A327H/E269L/K236A. In a
certain embodiments, the polypeptides comprise the modifications
G237F/D270Q/S239E. In some other embodiments, the polypeptides
comprise the modifications A330V/I332L/K326. In one embodiment, the
polypeptides comprise the modifications G236S/A327H/A330I.
[0041] In certain embodiments of the polypeptides described herein,
the amino acid modifications produce amino acid interactions and
dynamics that result in enhanced binding affinity and/or
specificity to a first Fc.gamma. receptor while diminishing binding
affinity and/or specificity to a second Fc.gamma. receptor compared
to a polypeptide that lacks the three or more amino acid
modifications. In some of these embodiments, the first Fc.gamma.
receptor is Fc.gamma.RIIIa receptor and the second Fc.gamma.
receptor is Fc.gamma.RIIa or Fc.gamma.RIIb. In certain embodiments,
the amino acid modifications produce favorable
Fc.gamma.RIIIa-specific interactions and/or unfavorable
interactions with Fc.gamma.RIIa and/or Fc.gamma.RIIb receptors. In
some embodiments, the amino acid modifications have minimal impact
on the Fc.gamma.RIIIa receptor while producing detrimental effects
on binding of the polypeptide to Fc.gamma.RIIa and/or
Fc.gamma.RIIb.
[0042] Provided herein are polypeptides comprising a variant Fc
region, wherein said variant Fc region comprises at least three
amino acid modifications relative to a wild-type Fc region, wherein
the amino acid modifications produce amino acid interactions and
dynamics that result in enhanced binding affinity and/or
specificity to a Fc.gamma.RIIIa receptor while diminishing binding
affinity and/or specificity to Fc.gamma.RIIa or Fc.gamma.RIIb
receptor compared to a polypeptide that lacks the two amino acid
modifications. In some embodiments, the polypeptide comprises
modifications S239E/D265S/I332E. In certain embodiments, the
polypeptide comprises the modifications G237F/S239E/A327H. In
certain other embodiments, the polypeptide comprises modifications
H268D/E269L/S298A/K326A/A327H. In some embodiments, the polypeptide
comprises the modifications L235A/S239E/D265E/A327H. In an
embodiment, the polypeptide comprises the modifications
G237F/S239E/D270N. In some embodiments, the polypeptide comprises
the modifications G236E/G237F/S239E. In an embodiment, the
polypeptide comprises the modifications S239E/D265SI332E and
alternatively H268D. In certain embodiments, the polypeptide
comprises modifications selected from the group of
G237F/S239E/D265E, S239E/S298A/K326A/A327H, and
G236E/D270N/A327V/I332E. In certain embodiments, the polypeptide
comprises the modifications S298A/K326A/A327H wherein the
polypeptide has improved binding selectivity to Fc.gamma.RIIIa
receptor as compared to a polypeptide lacking the S298A/K326A/A327H
modifications.
[0043] In an aspect, provided herein are polypeptides comprising a
variant Fc region, wherein said variant Fc region comprises at
least three amino acid modifications relative to a wild-type Fc
region, wherein the amino acid modifications produce amino acid
interactions and dynamics that result in enhanced binding affinity
and/or specificity to a Fc.gamma.RIIa receptor while diminishing
binding affinity and/or specificity to Fc.gamma.RIIIa or
Fc.gamma.RIIb receptor compared to a polypeptide that lacks at
least one of the amino acid modifications. In some embodiments, the
polypeptide comprises modifications G237F, A327L and A330I. In
certain embodiments, the amino acid modifications produce favorable
Fc.gamma.RIIa-specific interactions and/or unfavorable interactions
with Fc.gamma.RIIIa and/or Fc.gamma.RIIb receptors. In certain
embodiments, the amino acid modifications have minimal impact on
the Fc.gamma.RIIa receptor while producing detrimental effects on
binding of the polypeptide to Fc.gamma.RIIIa and/or Fc.gamma.RIIb.
In certain embodiments, the polypeptide comprises modifications
G237F, S239E and H268D. In some embodiments, the polypeptide
comprises modifications D265E/S267D/A330S.
[0044] In a further aspect, provided herein are polypeptides
comprising a variant Fc region, wherein said variant Fc region
comprises at least three amino acid modifications relative to a
wild-type Fc region, wherein the amino acid modifications produce
amino acid interactions and dynamics that result in enhanced
binding affinity and/or specificity to a Fc.gamma.RIIb receptor
while diminishing binding affinity and/or specificity to
Fc.gamma.RIIIa or Fc.gamma.RIIa receptor compared to a polypeptide
that lacks at least one of the amino acid modifications. In certain
embodiments, the amino acid modifications produce favorable
Fc.gamma.RIIb-specific interactions and/or unfavorable interactions
with Fc.gamma.RIIIa and/or Fc.gamma.RIIa receptors. In certain
embodiments, the amino acid modifications have minimal impact on
the Fc.gamma.RIIb receptor while producing detrimental effects on
binding of the polypeptide to Fc.gamma.RIIIa and/or Fc.gamma.RIIa.
In certain embodiments, the polypeptide comprises modifications
S239D/D265S/S298A/I332E. In some other embodiments, the polypeptide
comprises the modifications G237F/S298A/A330L/I332E. In some
embodiments the polypeptide comprises the modifications H268D,
K326A, A327H and alternatively one or both of E269L and S298A. In
an embodiment, the polypeptide comprises the modifications
G237F/V266L/S267D. In some embodiments, the polypeptides comprise
the modifications L234F/S267G/N325L or L234F/S267E/N325L. In an
embodiment, the polypeptide comprises modifications
G236A/S239D/D270L/I332E.
[0045] In certain aspects described herein, the binding of the
polypeptide comprising a wild-type Fc region to Fc.gamma. receptors
is detectable by an in vitro assay.
[0046] In an aspect, provided herein are polypeptides comprising a
variant Fc region, wherein said variant Fc region comprises at
least three amino acid modifications relative to a wild-type Fc
region, wherein one of the modifications comprises the mutation
S239E wherein the polypeptide has higher selectivity in binding to
the Fc.gamma.RIIIa receptor compared to a polypeptide that lacks
the S239E mutation.
[0047] In an aspect, provided herein are polypeptides comprising a
variant Fc region, wherein said variant Fc region comprises at
least three amino acid modifications relative to a wild-type Fc
region, wherein one of the modifications comprises the mutation
S239E one of the modifications comprises the mutation S298A wherein
the polypeptide has reduced binding affinity to Fc.gamma.RIIa and
Fc.gamma.RIIb receptors compared to a polypeptide that lacks the
S298A mutation.
[0048] In an aspect provided herein are polypeptides comprising a
variant Fc region, wherein said variant Fc region comprises at
least three amino acid modifications relative to a wild-type Fc
region, said modifications selected from D270L/Y300L/A330K,
G237F/S267G/N325F, G237F/V266L/S267D, L234F/S267G/N325L,
L234F/S267E/N325L, G237F/S239E/A327H, G237F/A327L/A330I,
S239E/A327L/A330I, S239E/S267E/H268D, G237F/S239E/D270N,
G236E/G237F/S239E, S239E/D265S/I332E, G237F/S239E/D265E,
G237F/S239E/H268D, H268E/D270E/S267G, H268D/K326A/A327H,
D265E/S267D/A330S, L235A/S239E/D265E, A327H/E269L/K236A,
G237F/D270Q/S239E, A330V/I332L/K326, and G236S/A327H/A330I.
[0049] In an aspect provided herein are polypeptides comprising a
variant Fc region, wherein said variant Fc region comprises at
least four amino acid modifications relative to a wild-type Fc
region, said modifications selected from L235A/S239E/D265E/A327H,
S239E/D265S/H268D/I332E, S239D/D265S/S298A/1332E,
S239E/S298A/K326A/A327H, G237F/S298A/A330L/I332E, and
G236E/D270N/A327V/I332E, G236A/S239D/D270L/I332E and
H268D/E269L/S298A/K326A/A327H.
[0050] In an aspect provided herein are polypeptides comprising a
variant Fc region, wherein said variant Fc region comprises at
least three amino acid modifications relative to a wild-type Fc
region, wherein the at least three amino acid modifications are
selected from the group consisting of: L234Q, L234N, L235A, G236E,
E236L, E236D, G237F, G237N, S239E, S239D, D265E, D265S, S267E,
S267D, S267G, H268D, H268E, E269L, E269L, D270N, D270I, D270E,
S298A, K326A, K326D, A327H, A327V, A327L, A327T, A330V, A330L,
A330W, A330I, A330S, I332L, I332D, and 1332E.
[0051] In an aspect provided herein are polypeptides comprising a
variant Fc region, said variant Fc region comprises a combination
of amino acid modifications wherein said combination is selected
from the group consisting of: L235A/S239E/D265E; L235A/G237F/D265E;
S239E/E269D/A327H; S239E/G237N/A327H; S239E/G237F/A327V;
G237F/D270I/S239E; G237F/A327L/S239E; A327H/E269L/K326A;
A330V/I332L/S239E; A327T/E269L/K326A; D270N/A327T/K326A;
A330V/I332L/S239E; A330W/I332D/S239E; G236E/D265E/A327H/A330I;
D270N/S298A/A327V; G236E/D265E/D270N/A327H/A330I;
G236E/D270N/A327H/A330I; G236E/D270N/A327V/I332E;
G236E/D270N/A327V/G237F; L234N/S239E/A330I/I332E;
L234Q/S239E/A330I/I332E; L234Q/S239E/A330I/I332E/S298A;
G237F/S239D/D265E/D270N/S298A; G237F/S239E/D270N/A330L/I332E;
G237F/S239E/D270N/A330L/I332E/S298A; S239E/G237F/A327H;
G237F/A327L/A330I; S239E/A330I/A327L; D265E/S239E/L235A/A327H;
S267E/S239E/H268D; G237F/D270N/S239E; S239E/G237F/G236E;
1332E/D265S/S239E/H268D; I332E/D265S/S239E; D265E/S239E/G237F;
S239E/H268D/G237F; S298A/D265S/S239D/1332E;
S298A/K326A/A327H/S239E; S298A/G237F/A3330L/I332E;
H268E/D270E/S267G; H268D/K326A/A327H;
H268D/K326A/A327H/E269L/S298A; A330S/D265E/S267D;
S239E/S267E/H268D; S237F/S239E/D265E and H268E/D270/E/S267G
[0052] In an aspect, provided herein are polypeptides that comprise
a variant Fc region, wherein said variant Fc region comprises at
least three amino acid modifications relative to a wild-type Fc
region wherein when said variant Fc region comprises amino acid
modification H268D, said variant does not comprise the modification
S267E.
[0053] In certain embodiments of the polypeptides described herein,
the Fc region of the parent polypeptide is a human IgG Fc region.
In some of these embodiments, the human IgG Fc region is a human
IgG1, IgG2, IgG3, or IgG4 Fc region.
[0054] In certain embodiments of the polypeptides described herein,
the polypeptide is an antibody. In some embodiments, the antibody
is a monoclonal antibody, a humanized antibody, or a human
antibody.
[0055] In an aspect is a nucleic acid comprising: a nucleotide
sequence encoding a polypeptide described herein. In certain
embodiments is a vector, comprising the nucleic acid.
[0056] In an aspect is a method for producing a polypeptide or
protein described herein, said method comprising: (i) culturing in
a medium a host cell comprising a nucleic acid encoding said
polypeptide, under conditions suitable for the expression of said
polypeptide; and (ii) recovering the polypeptide from said
medium.
[0057] In an aspect described herein is a therapeutic antibody that
specifically binds a cancer target antigen, said therapeutic
antibody comprising a variant Fc region polypeptide described
herein. In certain embodiments, the therapeutic antibody is
selected from the group consisting of abagovomab, adalimumab,
alemtuzumab, aurograb, bapineuzumab, basiliximab, belimumab,
bevacizumab, briakinumab, canakinumab, catumaxomab, certolizumab
pegol, cetuximab, daclizumab, denosumab, efalizumab, galiximab,
gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, infliximab,
ipilimumab, lumiliximab, mepolizumab, motavizumab, muromonab,
mycograb, natalizumab, nimotuzumab, ocrelizumab, ofatumumab,
omalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab,
reslizumab, rituximab, teplizumab, tocilizumab/atlizumab,
tositumomab, trastuzumab, Proxinium.TM., Rencarex.TM., ustekinumab,
zalutumumab, and any other antibodies. In certain embodiments, the
target antigen is selected from the group consisting of a-chain
(CD25) of IL-2R, Amyloid beta, anti-EpCAM.times.anti-CD3, BLyS (or
BAFF), CD11a, CD20, CD22, CD23, CD3, CD4, CD52, CD80, CTLA-4, EGFR,
EpCAM, F protein of RSV, G250, glycoprotein IIb/IIIa R, HER2,
HER2/neu R, Hsp90, IgE antibody, IL-12/IL-23, IL-1b, IL-5, IL-6
receptor, Integrin alpha-4/beta-1, Mucin 16/CA-125, RANKL, TNF
alpha, VEGF-A, and other therapeutically advantageous targets.
[0058] In an aspect described herein is a method of treating cancer
in a patient having a cancer characterized by a cancer antigen,
said method comprising administering to said patient a
therapeutically effective amount of a therapeutic antibody
described herein. In certain embodiments, the patient is human.
[0059] In an aspect described herein is a method of treating immune
disorders in a patient having an immune disorder characterized by
an immune antigen, said method comprising administering to said
patient a therapeutically effective polypeptide, antibody or
protein described herein.
[0060] In an aspect is a pharmaceutical composition, said
composition comprising a therapeutically effective amount of a
polypeptide described herein, and a pharmaceutically acceptable
carrier.
[0061] In an aspect described herein are polypeptides, said
polypeptides comprising a variant Fc region with at least three
amino acid substitutions, and wherein said polypeptides are more
effective at mediating antibody-dependent cellular cytotoxicity
(ADCC) relative to wild type. In certain embodiments, the
polypeptide comprising a variant Fc region is about 1.5 to about
100 fold more effective in mediating ADCC relative to wild type. In
certain embodiments, the polypeptide comprising a variant Fc region
is about 2 to about 50 fold more effective in mediating ADCC
relative to wild type.
[0062] In an aspect described herein are polypeptides, said
polypeptides comprising a variant Fc region with at least three
amino acid substitutions, wherein the polypeptide is more effective
at mediating inhibition of inflammatory immune responses relative
to wild type. In certain embodiments, the polypeptide comprising a
variant Fc region is about 2 fold more effective in mediating
inhibition of inflammatory immune responses relative to wild type.
In some embodiments, the polypeptide comprising a variant Fc region
is about 10 fold more effective in mediating inhibition of
inflammatory immune responses relative to wild type. In certain
embodiments, the polypeptide comprising a variant Fc region is
about 50 fold more effective in mediating inhibition of
inflammatory immune responses relative to wild type. In certain
embodiments, the polypeptide comprising a variant Fc region is
about 100 fold more effective in mediating inhibition of
inflammatory immune responses relative to wild type.
[0063] Also provided herein is a method for identifying Fc variant
polypeptides in silico based on calculated binding affinities to
Fc.gamma.RIIa, Fc.gamma.RIIb and/or Fc.gamma.RIIIa. In certain
embodiments, the method of identifying Fc variant polypeptides in
silico further calculates in silico electrostatics, solvation,
packing, packing density, hydrogen binding, and entropic effects of
said Fc variant polypeptides. In certain embodiments, the method of
identifying Fc variant polypeptides in silico further comprises
constructing the identified Fc variant polypeptides and expressing
said polypeptides in the context of an antibody in mammalian
cells.
[0064] Other aspects and features of the present invention will
become apparent to those ordinarily skilled in the art upon review
of the following description of specific embodiments of the
invention.
INCORPORATION BY REFERENCE
[0065] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0066] The features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0067] FIG. 1: Effects of the A327H and S239E mutations: A portion
of the binding interface is shown between the chain-B of the
antibody Fc (green) and the various Fc.gamma. receptors (wheat).
The wild-type Fc to Fc.gamma.RIIIa structure is shown on top with
Fc variant-induced changes in structure and dynamics for each
receptor shown on the bottom. Side-chain positions to be mutated
are colored in dark green and the residue labels are in red. Dashed
lines denote electrostatic interactions with the distances shown in
angstroms (.ANG.). For the Fc.gamma.RIIa, the two displayed
conformations of His134 are the maximum allowable conformational
movements with the intermediate conformers omitted for clarity.
[0068] FIG. 2: Effects of the S239E mutation: A portion of the
binding interface is shown between the chain-A of the antibody Fc
(green) and the receptors (wheat) Fc.gamma.RIIIa (left) and FcgRIIa
& IIb (right). Mutated side-chain positions are colored in dark
green with the residue labels in red. Dashed lines denote
electrostatic interactions with the distances shown in angstroms
(.ANG.). Fc.gamma.RIIa and Fc.gamma.RIIb both contain conserved
Tyr160 and Thr161 residues and are shown together for clarity.
[0069] FIG. 3: Effects of the S298A mutation: A portion of the
binding interface is shown between the chain-B of the antibody Fc
(green) and the various Fc.gamma. receptors (wheat). The wild-type
Fc is shown on the left with the variant Fc shown on the right.
Interactions with the Fc.gamma.RIIa and IIb receptors are shown on
the top while the Fc.gamma.RIIIa is shown on the bottom. The S298A
mutation is labeled in red with the dashed lines denoting
electrostatic interactions with the intermolecular distances shown
in angstroms (.ANG.). For the Fc.gamma.RIIIa system, the displayed
conformations for Lys128 and Asn126 are the maximum allowable
conformational movements with the intermediate conformers omitted
for clarity.
[0070] FIG. 4: In vitro binding profiles to the three Fc.gamma.
receptors: The in vitro binding (green) of the
S239E/S298A/K326A/A327H variant (left) and the S239E control
(right) were measured using surface plasmon resonance with binding
reported as the association constant (K.sub.A) in molar [M] in
comparison to the wild-type Herceptin antibody (blue).
DETAILED DESCRIPTION OF THE INVENTION
[0071] Embodiments disclosed herein are drawn to polypeptides,
fusion protein and antibodies comprising at least three
substitutions in the Fc region wherein the polypeptide binds a
target Fc.gamma.R receptor with higher specificity compared to a
polypeptide that lacks the at least three substitutions. These
polypeptides and antibodies are designed by rational analysis of
the binding of each Fc.gamma.R with the Fc region and by subsequent
design of multiple amino acid substitutions that synergistically
provide enhanced selectivity and binding affinity for the target Fc
receptor. Accordingly, the polypeptides and proteins provided
herein comprise multiple variations in the Fc region as compared to
the wild type Fc region, said variations tailored to improving the
specificity for the Fc.gamma.R under consideration, and for
obtaining maximum energetically favorable binding based on
enthalpic and entropic factors optimized by the choice of the most
favorable amino acid for each substituted position.
[0072] One embodiment provides a polypeptide comprising a variant
Fc region, wherein said variant Fc region comprises three or more
amino acid modifications relative to a wild-type Fc region, and has
an altered effect relative to a polypeptide comprising a wild-type
Fc region; wherein at least two of the three or more modifications
provide a synergistic effect compared to single position
modifications at the at least two positions thereby exhibiting a
selected binding profile to Fc.gamma. receptors.
[0073] Another embodiment provides a polypeptide wherein the amino
acid modifications produce amino acid interactions and dynamics
that result in enhanced binding free energy to a first Fey receptor
while diminishing binding affinity to a second Fc.gamma. receptor
compared to a polypeptide that lacks the at least three or more
amino acid modifications. In certain embodiments, the first
Fc.gamma. receptor is Fc.gamma.RIIIa receptor and the second
Fc.gamma. receptor is Fc.gamma.RIIa or Fc.gamma.RIIb.
[0074] Another embodiment provides a polypeptide wherein the amino
acid modifications produce favorable Fc.gamma.RIIIa-specific
electrostatic interactions and steric repulsion to Fc.gamma.RIIa
and/or Fc.gamma.RIIb receptors.
[0075] Another embodiment provides a polypeptide wherein the amino
acid modifications have minimal impact on the Fc.gamma.RIIIa
receptor while producing detrimental effects onbinding of the
polypeptide to Fc.gamma.RIIa and/or Fc.gamma.RIIb.
[0076] Another embodiment provides a polypeptide of wherein the
amino acid substitutions preserve the binding interface and the
protein-protein interactions with the Fc.gamma.RIIIa when compared
to the wild-type Fc, and result in disruption of binding of the
polypeptide to the Fc.gamma.RIIa receptor is disrupted.
[0077] Any patent, application or other reference cited or repeated
below, or above, is incorporated by references in its entirety for
all purposes as well as for the proposition that is recited.
[0078] The following definitions may be used to understand the
compositions and methods provided herein, but are meant to
encompass scientific equivalents.
[0079] Throughout 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.
[0080] A "parent polypeptide" is a polypeptide comprising an amino
add sequence which lacks one or more of the Fc region modifications
disclosed herein and which differs in effector function compared to
a polypeptide variant as herein disclosed. The parent 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).
[0081] As used herein, "synergistic" means that the Fc.gamma.R
binding of the Fc protein designed with multiple amino acid
substitutions is greater than their additive binding observed for
individual substitutions.
[0082] The term "Fc region" is used to define a C-terminal region
of an immunoglobulin heavy chain. The "Fc region" may be a native
sequence Fc region or a variant Fc region. Although the boundaries
of the Fc region of an immunoglobulin heavy chain might vary, the
human IgG heavy chain Fc region is usually defined to stretch from
an amino acid residue at position Cys226, or from Pro230, to the
carboxyl-terminus thereof. The Fc region of an immunoglobulin
generally comprises two constant domains, CH2 and CH3.
[0083] 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.
22:161-206 (1985).
[0084] 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)
[0085] A "functional Fc region" possesses an "effector function" of
a native sequence Fc region. Exemplary "effector functions" include
Clq 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.
[0086] A "native sequence Fc region" comprises an amino add
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.
[0087] A "variant Fc region" comprises an amino acid sequence which
differs from that of a native sequence Fc region by virtue of at
least one "amino acid modification" as herein defined. 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. In certain embodiments, the
variant Fc region herein possesses 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] "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.
[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, in certain
embodiments, the 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 M. in Daeron, Annu. Rev. Immunol.
15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu.
Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34
(1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).
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
materal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976)
and Kim et al., J. Immunol. 24:249 (1994)).
[0091] "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
9:457-92 (1991).
[0092] "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.
[0093] 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 which "displays increased binding" to an FcR binds at
least one FcR with better affinity than the parent polypeptide. The
polypeptide 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.
[0094] The polypeptide which binds an FcR with "better affinity"
than a parent polypeptide, is one which binds any one or more of
the above identified FcRs with substantially better binding
affinity than the parent antibody, when the amounts of polypeptide
and parent polypeptide in the binding assay are essentially the
same. For example, the polypeptide with improved 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 improvement in FcR binding
affinity compared to the parent polypeptide, where FcR binding
affinity is determined, for example, as disclosed in the Examples
herein.
[0095] The polypeptide which "mediates antibody-dependent
cell-mediated cytotoxicity (ADCC) in the presence of human effector
cells more effectively" than a parent antibody is one which in
vitro or in vivo is substantially more effective at mediating ADCC,
when the amounts of polypeptide variant and parent antibody used in
the assay are essentially the same. Generally, such polypeptides
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 polypeptide is from about 1.5 fold to about 100 fold,
e.g. from about two fold to about fifty fold, more effective at
mediating ADCC than the parent, e.g. in the in vitro assay
disclosed herein.
[0096] 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. In certain embodiments the amino acid modification herein
is a substitution.
[0097] 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. In certain embodiments the insertion is N-terminal or
C-terminal to the specified residue.
[0098] 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, omithine,
norvaline, homoserine and other amino acid residue analogues such
as those described in Ellman et al. Meth. Enzym. 202:301-336
(1991). To generate such non-naturally occurring amino acid
residues, the procedures of Noren et al. Science 244:182 (1989) 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.
[0099] An "amino acid insertion" refers to the incorporation of at
least one amino acid into a predetermined amino acid sequence. In
certain embodiments, the insertion consists of the insertion of one
or two amino acid residues. In certain other embodiments, are
larger "peptide insertions", e.g. insertion of about three to about
five or even up to about ten amino acid residues. In these
embodiments the inserted residue(s) are naturally occurring or
non-naturally occurring as disclosed above.
[0100] An "amino acid deletion" refers to the removal of at least
one amino acid residue from a predetermined amino acid
sequence.
[0101] "Hinge region" is generally defined as stretching from
Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206
(1985)). 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.
[0102] 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. Prior to the
present disclosure, Fc.gamma.R binding was generally attributed to
amino acid residues in the lower hinge region of an IgG Fc
region.
[0103] "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.
[0104] 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 .alpha. chain thereof) which is responsible for
binding an Fc region. One useful binding domain is the
extracellular domain of an FcR.alpha.chain.
[0105] The term "antibody" is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired biological activity.
[0106] "Antibody fragments", as defined herein, comprise a portion
of an intact antibody, generally including the antigen binding or
variable region of the intact antibody or the Fc region of an
antibody which retains FcR binding capability. Examples of antibody
fragments include linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments. In certain embodiments, the antibody fragments retain at
least part of the hinge and optionally the CH1 region of an IgG
heavy chain. In some embodiments the antibody fragments retain the
entire constant region of an IgG heavy chain, and include an IgG
light chain.
[0107] 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 except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations that typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. 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. In certain
embodiments the monoclonal antibodies to be used in accordance with
the present disclosure are made by the hybridoma method first
described by Kohler et al., Nature 256:495 (1975), or may be made
by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). In
some embodiments "monoclonal antibodies" are isolated from phage
antibody libraries using the techniques described in Clackson et
al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.
222:581-597 (1991), for example.
[0108] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; and Morrison
et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
[0109] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
Immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0110] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (ie.
residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable domain; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (i.e. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the light chain variable domain and 26-32 (H1),
53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;
Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework" or
"FR" residues are those variable domain residues other than the
hypervariable region residues as herein defined.
[0111] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the "binding domain" of a
heterologous "adhesin" protein (e.g. a receptor, ligand or enzyme)
with an immunoglobulin constant domain. Structurally, the
immunoadhesins comprise a fusion of the adhesin amino acid sequence
with the desired binding specificity which is other than the
antigen recognition and binding site (antigen combining site) of an
antibody (i.e. is "heterologous") and an immunoglobulin constant
domain sequence.
[0112] 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.
[0113] 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.
[0114] An "antibody-immunoadhesin chimera" comprises a molecule
that combines at least one binding domain of an antibody (as herein
defined) with at least one immunoadhesin (as defined in this
application). Exemplary antibody-immunoadhesin chimeras are the
bispecific CD4-IgG chimeras described in Berg et al., PNAS (USA)
88:4723-4727 (1991) and Chamow et al., J. Immunol. 153:4268
(1994).
[0115] 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 nonproteinadeous solutes. In certain 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.
[0116] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented.
[0117] A "disorder" is any condition that would benefit from
treatment with the polypeptide 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.
[0118] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the polypeptide. In certain embodiments the label is itself
detectable (e.g., radioisotope labels or fluorescent labels). In
some other embodiments, the label catalyzes chemical alteration of
a substrate compound or composition which is detectable. An
exemplary embodiment comprises an enzymatic label that catalyzes a
chemical alteration of a substrate compound or composition which is
detectable.
[0119] An "isolated" nucleic acid molecule is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the polypeptide nucleic acid.
An isolated nucleic acid molecule is other than in the form or
setting in which it is found in nature. Isolated nucleic acid
molecules therefore are distinguished from the nucleic acid
molecule as it exists in natural cells. However, an isolated
nucleic acid molecule includes a nucleic acid molecule contained in
cells that ordinarily express the polypeptide where, for example,
the nucleic acid molecule is in a chromosomal location different
from that of natural cells.
[0120] The expression "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0121] 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.
[0122] 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
therefrom 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.
[0123] The term "molecular complex" when used herein refers to the
relatively stable structure which forms when two or more
heterologous molecules (e.g. polypeptides) bind (preferably
noncovalently) to one another. The preferred molecular complex
herein is an immune complex.
[0124] "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.
[0125] The term "target molecule" refers to a molecule, usually a
polypeptide, which is capable of being bound by a heterologous
molecule and has one or more binding sites for the heterologous
molecule. The term "binding site" refers to a region of a molecule
to which another molecule can bind. The "first target molecule"
herein comprises at least two distinct binding sites (for example,
two to five separate binding sites) for an analyte (e.g. an Fc
region-containing polypeptide) such that at least two analyte
molecules can bind to the first target molecule. In a preferred
embodiment, the two or more binding sites are identical (e.g.
having the same amino acid sequence, where the target molecule is a
polypeptide). An "analyte" is a substance that is to be analyzed.
The preferred analyte is an Fc region-containing polypeptide that
is to be analyzed for its ability to bind to an Fc receptor.
[0126] 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.
[0127] The phrase "low affinity receptor" denotes a receptor that
has a weak binding affinity for a ligand of interest, e.g. having a
binding constant of about 50 nM or worse affinity. Exemplary low
affinity receptors include Fc.gamma.RII and Fc.gamma.RIII.
[0128] By "effector function" as used herein is meant a biochemical
event that results from the interaction of an antibody Fc region
with an Fc receptor or ligand. Effector functions include but are
not limited to ADCC, ADCP, and CDC. By "effector cell" as used
herein is meant a cell of the immune system that expresses one or
more Fc receptors and mediates one or more effector functions.
Effector cells include but are not limited to monocytes,
macrophages, neutrophils, dendritic cells, eosinophils, mast cells,
platelets, B cells, large granular lymphocytes, Langerhans' cells,
natural killer (NK) cells, and .gamma..gamma. T cells, and may be
from any organism including but not limited to humans, mice, rats,
rabbits, and monkeys. By "library" herein is meant a set of Fc
proteins in any form, including but not limited to a list of
nucleic acid or amino acid sequences, a list of nucleic acid or
amino acid substitutions at variable positions, a physical library
comprising nucleic acids that encode the library sequences, or a
physical library comprising the Fc protein proteins, either in
purified or unpurified form.
[0129] By "Fc-fusion" as used herein is meant a protein wherein one
or more polypeptides is operably linked to an Fc region or a
derivative thereof. Fc fusion is herein meant to be synonymous with
the terms "immunoadhesin", "Ig fusion", "Ig chimera", and "receptor
globulin" (sometimes with dashes) as used in the prior art (Chamow
et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997,
Curr Opin Immunol 9:195-200). An Fc fusion combines the Fc region
of an immunoglobulin with a fusion partner, which in general can be
any protein or small molecule. The role of the non-Fc part of an Fc
fusion, i.e. the fusion partner, is to mediate target binding, and
thus it is functionally analogous to the variable regions of an
antibody. Virtually any protein or small molecule may be linked to
Fc to generate an Fc fusion. Protein fusion partners may include,
but are not limited to, the target-binding region of a receptor, an
adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or
some other protein or protein domain. Small molecule fusion
partners may include any therapeutic agent that directs the Fc
fusion to a therapeutic target. Such targets may be any molecule,
preferrably an extracellular receptor, that is implicated in
disease. Two families of surface receptors that are targets of a
number of approved small molecule drugs are G-Protein Coupled
Receptors (GPCRs), and ion channels, including K+, Na+, Ca+
channels. Nearly 70% of all drugs currently marketed worldwide
target GPCRs. Thus the Fc proteins described herein may be fused to
a small molecule that targets, for example, one or more GABA
receptors, purinergic receptors, adrenergic receptors,
histaminergic receptors, opiod receptors, chemokine receptors,
glutamate receptors, nicotinic receptors, the 5HT (serotonin)
receptor, and estrogen receptors. A fusion partner may be a
small-molecule mimetic of a protein that targets a therapeutically
useful target. Specific examples of particular drugs that may serve
as Fc fusion partners can be found in L. S. Goodman et at, Eds.,
Goodman and Gilman's The Pharmacological Basis of Therapeutics
(McGraw-Hill, New York, ed. 9, 1996). Fusion partners include not
only small molecules and proteins that bind known targets for
existing drugs, but orphan receptors that do not yet exist as drug
targets. The completion of the genome and proteome projects are
proving to be a driving force in drug discovery, and these projects
have yielded a trove of orphan receptors. There is enormous
potential to validate these new molecules as drug targets, and
develop protein and small molecule therapeutics that target them.
Such protein and small molecule therapeutics are contemplated as Fc
fusion partners that employ the Fc proteins described herein. A
variety of linkers, defined and described below, may be used to
covalently link Fc to a fusion partner to generate an Fc
fusion.
[0130] 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-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., 2002, Immunological Reviews
190:123-136). Fc ligands may include undiscovered molecules that
bind Fc.
[0131] By "IgG" as used herein is meant a polypeptide belonging to
the class of antibodies that are substantially encoded by a
recognized immunoglobulin gamma gene. In humans this class
comprises IgG1, IgG2, IgG3, and IgG4. In mice this class comprises
IgG1, IgG2a, IgG2b, IgG3. By "immunoglobulin (Ig)" herein is meant
a protein consisting of one or more polypeptides substantially
encoded by immunoglobulin genes. Immunoglobulins include but are
not limited to antibodies. Immunoglobulins may have a number of
structural forms, including but not limited to full length
antibodies, antibody fragments, and individual immunoglobulin
domains. By "immunoglobulin (Ig) domain" herein is meant a region
of an immunoglobulin that exists as a distinct structural entity as
ascertained by one skilled in the art of protein structure. Ig
domains typically have a characteristic .quadrature.-sandwich
folding topology. The known Ig domains in the IgG class of
antibodies are V.sub.H, C.gamma.1, C.gamma.2, C.gamma.3, V.sub.L,
and C.sub.L.
[0132] By "parent polypeptide" or "precursor polypeptide"
(including Fc parent or precursors) as used herein is meant a
polypeptide that is subsequently modified to generate a variant.
Said parent polypeptide may be a naturally occurring polypeptide,
or a variant or engineered version of a naturally occurring
polypeptide. Parent polypeptide may refer to the polypeptide
itself, compositions that comprise the parent polypeptide, or the
amino acid sequence that encodes it. Accordingly, by "parent Fc
polypeptide" as used herein is meant an unmodified Fc polypeptide
that is modified to generate a variant, and by "parent antibody" as
used herein is meant an unmodified antibody that is modified to
generate a variant antibody.
[0133] By "residue" as used herein is meant a position in a protein
and its associated amino acid identity. For example, Asparagine 297
(also referred to as Asn297, also referred to as N297) is a residue
in the human antibody IgG1.
[0134] By "target antigen" as used herein is meant the molecule
that is bound specifically by the variable region of a given
antibody. A target antigen may be a protein, carbohydrate, lipid,
or other chemical compound.
[0135] By "target cell" as used herein is meant a cell that
expresses a target antigen.
[0136] By "variable region" as used herein is meant the region of
an immunoglobulin that comprises one or more Ig domains
substantially encoded by any of the V.kappa., V.lamda., and/or
V.sub.H genes that make up the .kappa., .lamda., and heavy chain
immunoglobulin genetic loci respectively.
[0137] By "variant polypeptide" as used herein is meant a
polypeptide sequence that differs from that of a parent polypeptide
sequence by virtue of at least one amino acid modification. Variant
polypeptide may refer to the polypeptide itself, a composition
comprising the polypeptide, or the amino sequence that encodes it.
Preferably, the variant polypeptide has at least one amino acid
modification compared to the parent polypeptide, e.g. from about
one to about ten amino acid modifications, and preferably from
about one to about five amino acid modifications compared to the
parent. The variant polypeptide sequence herein will preferably
possess at least about 80% homology with a parent polypeptide
sequence, and most preferably at least about 90% homology, more
preferably at least about 95% homology. Accordingly, by "Fc
protein" as used herein is meant an Fc sequence that differs from
that of a parent Fc sequence by virtue of at least one amino acid
modification. An Fc protein may only encompass an Fc region, or may
exist in the context of an antibody, Fc fusion, or other
polypeptide that is substantially encoded by Fc. Fc protein may
refer to the Fc polypeptide itself, compositions comprising the Fc
protein polypeptide, or the amino acid sequence that encodes it. In
an exemplary embodiment, the variant proteins described herein
comprise an Fc protein, as described herein, and as such, may
comprise an antibody (and the corresponding derivatives) with the
Fc protein, or an Fc fusion protein that comprises the Fc protein.
In addition, in some cases, the Fc is a variant as compared to a
wild-type Fc, or to a "parent" variant.
[0138] The term "Antibody-Dependent Cell-Mediated Cytotoxicity"
(ADCC) refers to a mechanism of cell-mediated immunity whereby an
effector cell of the immune system actively lyses a target cell
that has been bound by specific antibodies. The Fc region Variant
polypeptides and proteins described herein successfully modulate
ADCC as compared to the wild type Fc region antibodies. In certain
embodiments, the Variant polypeptide results in reduced ADCC as
compared to the wild-type Fc region. In certain other embodiments,
the Fc region Variant described herein results in increased ADCC as
compared to the corresponding wild-type Fc region.
[0139] The term geometric metrics describes the plurality of
measurable physical attributes of a structural unit or residue
(used interchangeably herein) of a biopolymer, an amino acid in the
case of a protein. Geometric metrics can be defined either by 1)
dihedral, plane or other measurable angles as defined by the atoms
of the respective residue, 2) distances between atoms of the same
residue or to atoms of another residue, and/or 3) distance between
atoms of the same residue and a reference atom or position in the
structure.
[0140] A residue population is the sum of all snapshots or samples
obtained for a single residue. In certain embodiments the
population is equal to the number of frames captured in a molecular
dynamics simulation or the number of samples taken from a Monte
Carlo simulation.
[0141] A residue conformation is defined in terms of the observed
combination of geometric metrics attributed to a particular residue
structure. A residue cluster refers to the plurality of snapshots,
which have been assigned the same conformation, based on clustering
all or a part of the defined geometric metrics. A conformational
frequency refers to the number of frames which have been assigned
the same residue conformation.
[0142] Simulation refers to the process of conformational sampling,
performed either by either molecular dynamics or a Monte Carlo
based sampling approach. A trajectory contains the conformational
frames produced by a simulation. Graph theory refers to the term as
used in mathematics and computer science (for example, see Reinhard
Diestel, Graph Theory; Edition 3, Springer 2005).
[0143] The term clustering tools refers to the plurality of
mathematical methods and algorithms and programs which implement
those, that can be used to identify clusters of similar data points
from data sets.
[0144] The term Dynamical Cross Correlation Method refers to a
graphical representation of the cross correlation matrix of atomic
displacements of a molecular structure in a molecular dynamics
trajectory with reference to another conformational state of that
structure.
[0145] Normal mode analysis is the study of characteristic harmonic
vibrations and frequencies about a local energy minimum of a
molecular system.
[0146] Elastic network model refers to the representation of a
protein structure as comprising of a network of harmonic springs
approximating the interaction between residue pairs.
[0147] Provided herein are polypeptides comprising a variant Fc
region, wherein said variant Fc region comprises at least three
amino acid modifications relative to a wild-type Fc region, and has
an altered effect relative to a polypeptide comprising a wild-type
Fc region or variant Fc region comprising only one or two amino
acid modifications; and wherein at least two of the modifications
provide a synergistic effect compared to single position
modifications thereby exhibiting a selected binding profile to
Fc.gamma. receptors. In certain embodiments, one or both amino acid
modifications are located between positions 234-330 according to
the EU index. In certain embodiments, the modifications do not
comprise simultaneous substitution at positions 317 and 353
according to the EU index. In some embodiments, the amino acid
modifications do not comprise a substitution at position 332
according to the EU index. In certain embodiments, the polypeptides
comprise the modifications L235A/S239E/D265E. In some embodiments,
the polypeptides comprise the modifications A327H/E269L/K236A. In
one embodiment, the polypeptides comprise the modifications
G237F/D270Q/S239E. In some other embodiments, the polypeptides
comprise the modifications A330V/I332L/K326. In one embodiment, the
polypeptides comprise the modifications G236S/A327H/A330I.
[0148] In certain embodiments of the polypeptides described herein,
the amino acid modifications produce amino acid interactions and
dynamics that result in enhanced binding affinity and/or
specificity to a first Fc.gamma. receptor while diminishing binding
affinity and/or specificity to a second Fc.gamma. receptor compared
to a polypeptide that lacks the three or more amino acid
modifications. In some of these embodiments, the first Fc.gamma.
receptor is Fc.gamma.RIIIa receptor and the second Fc.gamma.
receptor is Fc.gamma.RIIa or Fc.gamma.RIIb. In certain embodiments,
the amino acid modifications produce favorable
Fc.gamma.RIIIa-specific interactions and/or unfavorable
interactions with Fc.gamma.RIIa and/or Fc.gamma.RIIb receptors. In
some embodiments, the amino acid modifications have minimal impact
on the Fc.gamma.RIIIa receptor while producing detrimental effects
on binding of the polypeptide to Fc.gamma.RIIa and/or
Fc.gamma.RIIb.
[0149] Provided herein are polypeptides comprising a variant Fc
region, wherein said variant Fc region comprises at least three
amino acid modifications relative to a wild-type Fc region, wherein
the amino acid modifications produce amino acid interactions and
dynamics that result in enhanced binding affinity and/or
specificity to a Fc.gamma.RIIIa receptor while diminishing binding
affinity and/or specificity to Fc.gamma.RIIa or Fc.gamma.RIIb
receptor compared to a polypeptide that lacks the two amino acid
modifications. In some embodiments, the polypeptide comprises
modifications S239E/D265S/1332E. In certain embodiments, the
polypeptide comprises the modifications G237F/S239E/A327H. In
certain other embodiments, the polypeptide comprises modifications
H268D/E269L/S298A/K326A/A327H. In some embodiments, the polypeptide
comprises the modifications L235A/S239E/D265E/A327H. In an
embodiment, the polypeptide comprises the modifications
G237F/S239E/D270N. In some embodiments, the polypeptide comprises
the modifications G236E/G237F/S239E. In an embodiment, the
polypeptide comprises the modifications S239E/D265SI332E and
alternatively H268D. In certain embodiments, the polypeptide
comprises modifications selected from the group of
G237F/S239E/D265E, S239E/S298A/K326A/A327H, and
G236E/D270N/A327V/I332E. In certain embodiments, the polypeptide
comprises the modifications S298A/K326A/A327H wherein the
polypeptide has improved binding selectivity to Fc.gamma.RIIIa
receptor as compared to a polypeptide lacking the S298A/K326A/A327H
modifications.
[0150] In an aspect, provided herein are polypeptides comprising a
variant Fc region, wherein said variant Fe region comprises at
least three amino acid modifications relative to a wild-type Fc
region, wherein the amino acid modifications produce amino acid
interactions and dynamics that result in enhanced binding affinity
and/or specificity to a Fc.gamma.RIIa receptor while diminishing
binding affinity and/or specificity to Fc.gamma.RIIIa or
Fc.gamma.RIIb receptor compared to a polypeptide that lacks at
least one of the amino acid modifications. In some embodiments, the
polypeptide comprises modifications G237F, A327L and A330I. In
certain embodiments, the amino acid modifications produce favorable
Fc.gamma.RIIa-specific interactions and/or unfavorable interactions
with Fc.gamma.RIIIa and/or Fc.gamma.RIIb receptors. In certain
embodiments, the amino acid modifications have minimal impact on
the Fc.gamma.RIIa receptor while producing detrimental effects on
binding of the polypeptide to Fc.gamma.RIIIa and/or Fc.gamma.RIIb.
In certain embodiments, the polypeptide comprises modifications
G237F, S239E and H268D. In some embodiments, the polypeptide
comprises modifications D265E/S267D/A330S.
[0151] In a further aspect, provided herein are polypeptides
comprising a variant Fc region, wherein said variant Fc region
comprises at least three amino acid modifications relative to a
wild-type Fc region, wherein the amino acid modifications produce
amino acid interactions and dynamics that result in enhanced
binding affinity and/or specificity to a Fc.gamma.RIIb receptor
while diminishing binding affinity and/or specificity to
Fc.gamma.RIIIa or Fc.gamma.RIIa receptor compared to a polypeptide
that lacks at least one of the amino acid modifications. In certain
embodiments, the amino acid modifications produce favorable
Fc.gamma.RIIb-specific interactions and/or unfavorable interactions
with Fc.gamma.RIIIa and/or Fc.gamma.RIIa receptors. In certain
embodiments, the amino acid modifications have minimal impact on
the Fc.gamma.RIIb receptor while producing detrimental effects on
binding of the polypeptide to Fc.gamma.RIIIa and/or Fc.gamma.RIIa.
In certain embodiments, the polypeptide comprises modifications
S239D/D265S/S298A/I332E. In some other embodiments, the polypeptide
comprises the modifications G237F/S298A/A330L/I332E. In some
embodiments the polypeptide comprises the modifications H268D,
K326A, A327H and alternatively one or both of E269L and S298A. In
an embodiment, the polypeptide comprises the modifications
G237F/V266L/S267D. In some embodiments, the polypeptides comprise
the modifications L234F/S267G/N325L or L234F/S267E/N325L. In an
embodiment, the polypeptide comprises modifications
G236A/S239D/D270L/I332E.
[0152] In certain aspects described herein, the binding of the
polypeptide comprising a wild-type Fc region to Fc.gamma. receptors
is detectable by an in vitro assay.
[0153] In an aspect, provided herein are polypeptides comprising a
variant Fc region, wherein said variant Fc region comprises at
least three amino acid modifications relative to a wild-type Fc
region, wherein one of the modifications comprises the mutation
S239E wherein the polypeptide has higher selectivity in binding to
the Fc.gamma.RIIIa receptor compared to a polypeptide that lacks
the S239E mutation.
[0154] In an aspect, provided herein are polypeptides comprising a
variant Fc region, wherein said variant Fc region comprises at
least three amino acid modifications relative to a wild-type Fc
region, wherein one of the modifications comprises the mutation
S239E one of the modifications comprises the mutation S298A wherein
the polypeptide has reduced binding affinity to Fc.gamma.RIIa and
Fc.gamma.RIIb receptors compared to a polypeptide that lacks the
S298A mutation.
[0155] In an aspect provided herein are polypeptides comprising a
variant Fc region, wherein said variant Fc region comprises at
least three amino acid modifications relative to a wild-type Fc
region, said modifications selected from D270L/Y300L/A330K,
G237F/S267G/N325F, G237F/V266L/S267D, L234F/S267G/N325L,
L234F/S267E/N325L, G237F/S239E/A327H, G237F/A327L/A330I,
S239E/A327L/A330I, S239E/S267E/H268D, G237F/S239E/D270N,
G236E/G237F/S239E, S239E/D265S/I332E, G237F/S239E/D265E,
G237F/S239E/H268D, H268E/D270E/S267G, H268D/K326A/A327H,
D265E/S267D/A330S, L235A/S239E/D265E, A327H/E269L/K236A,
G237F/D270Q/S239E, A330V/I332L/K326, and G236S/A327H/A330I.
[0156] In an aspect provided herein are polypeptides comprising a
variant Fc region, wherein said variant Fc region comprises at
least four amino acid modifications relative to a wild-type Fc
region, said modifications selected from L235A/S239E/D265E/A327H,
S239E/D265S/H268D/I332E, S239D/D265S/S298A/I332E,
S239E/S298A/K326A/A327H, G237F/S298A/A330L/I332E, and
G236E/D270N/A327V/I332E, G236A/S239D/D270L/I332E and
H268D/E269L/S298A/K326A/A327H.
[0157] In an aspect provided herein are polypeptides comprising a
variant Fc region, wherein said variant Fc region comprises at
least three amino acid modifications relative to a wild-type Fc
region, wherein the at least three amino acid modifications are
selected from the group consisting of: L234Q, L234N, L235A, G236E,
E236L, E236D, G237F, G237N, S239E, S239D, D265E, D265S, S267E,
S267D, S267G, H268D, H268E, E269L, E269L, D270N, D270I, D270E,
S298A, K326A, K326D, A327H, A327V, A327L, A327T, A330V, A330L,
A330W, A330I, A330S, I332L, I332D, and 1332E.
[0158] In an aspect provided herein are polypeptides comprising a
variant Fc region, said variant Fc region comprises a combination
of amino acid modifications wherein said combination is selected
from the group consisting of: L235A/S239E/D265E; L235A/G237F/D265E;
S239E/E269D/A327H; S239E/G237N/A327H; S239E/G237F/A327V;
G237F/D270I/S239E; G237F/A327L/S239E; A327H/E269L/K326A;
A330V/I332L/S239E; A327T/E269L/K326A; D270N/A327T/K326A;
A330V/I332L/S239E; A330W/I332D/S239E; G236E/D265E/A327H/A330I;
D270N/S298A/A327V; G236E/D265E/D270N/A327H/A330I;
G236E/D270N/A327H/A330I; G236E/D270N/A327V/I332E;
G236E/D270N/A327V/G237F; L234N/S239E/A330I/I332E;
L234Q/S239E/A330I/I332E; L234Q/S239E/A330I/I332E/S298A;
G237F/S239D/D265E/D270N/S298A; G237F/S239E/D270N/A330L/I332E;
G237F/S239E/D270N/A330L/I332E/S298A; S239E/G237F/A327H;
G237F/A327L/A330I; S239E/A330I/A327L; D265E/S239E/L235A/A327H;
S267E/S239E/H268D; G237F/D270N/S239E; S239E/G237F/G236E;
I332E/D265S/S239E/H268D; I332E/D265S/S239E; D265E/S239E/G237F;
S239E/H268D/G237F; S298A/D265S/S239D/I332E;
S298A/K326A/A327H/S239E; S298A/G237F/A3330L/I332E;
H268E/D270E/S267G; H268D/K326A/A327H;
H268D/K326A/A327H/E269L/S298A; A330S/D265E/S267D;
S239E/S267E/H268D; S237F/S239E/D265E and H268E/D270/E/S267G
[0159] In an aspect, provided herein are polypeptides that comprise
a variant Fc region, wherein said variant Fc region comprises at
least three amino acid modifications relative to a wild-type Fc
region wherein when said variant Fc region comprises amino acid
modification H268D, said variant does not comprise the modification
S267E.
[0160] In certain embodiments of the polypeptides described herein,
the Fc region of the parent polypeptide is a human IgG Fc region.
In some of these embodiments, the human IgG Fc region is a human
IgG1, IgG2, IgG3, or IgG4 Fc region.
[0161] In certain embodiments of the polypeptides described herein,
the polypeptide is an antibody. In some embodiments, the antibody
is a monoclonal antibody, a humanized antibody, or a human
antibody.
[0162] In an aspect is a nucleic acid comprising: a nucleotide
sequence encoding a polypeptide described herein. In certain
embodiments is a vector, comprising the nucleic acid.
[0163] In an aspect is a method for producing a polypeptide or
protein described herein, said method comprising: (i) culturing in
a medium a host cell comprising a nucleic acid encoding said
polypeptide, under conditions suitable for the expression of said
polypeptide; and (ii) recovering the polypeptide from said
medium.
[0164] In an aspect described herein is a therapeutic antibody that
specifically binds a cancer target antigen, said therapeutic
antibody comprising a variant Fc region polypeptide described
herein. In certain embodiments, the therapeutic antibody is
selected from the group consisting of abagovomab, adalimumab,
alemtuzumab, aurograb, bapineuzumab, basiliximab, belimumab,
bevacizumab, briakinumab, canakinumab, catumaxomab, certolizumab
pegol, cetuximab, daclizumab, denosumab, efalizumab, galiximab,
gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, infliximab,
ipilimumab, lumiliximab, mepolizumab, motavizumab, muromonab,
mycograb, natalizumab, nimotuzumab, ocrelizumab, ofatumumab,
omalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab,
reslizumab, rituximab, teplizumab, tocilizumab/atlizumab,
tositumomab, trastuzumab, Proxinium.TM., Rencarex.TM., ustekinumab,
zalutumumab, and any other antibodies. In certain embodiments, the
target antigen is selected from the group consisting of a-chain
(CD25) of IL-2R, Amyloid beta, anti-EpCAM.times.anti-CD3, BLyS (or
BAFF), CD11a, CD20, CD22, CD23, CD3, CD4, CD52, CD80, CTLA-4, EGFR,
EpCAM, F protein of RSV, G250, glycoprotein IIb/IIIa R, HER2,
HER2/neu R, Hsp90, IgE antibody, IL-12/IL-23, IL-1b, IL-5, IL-6
receptor, Integrin alpha-4/beta-1, Mucin 16/CA-125, RANKL, TNF
alpha, VEGF-A, and other therapeutically advantageous targets.
[0165] In an aspect described herein is a method of treating cancer
in a patient having a cancer characterized by a cancer antigen,
said method comprising administering to said patient a
therapeutically effective amount of a therapeutic antibody
described herein. In certain embodiments, the patient is human.
[0166] In an aspect described herein is a method of treating immune
disorders in a patient having an immune disorder characterized by
an immune antigen, said method comprising administering to said
patient a therapeutically effective polypeptide, antibody or
protein described herein.
[0167] In an aspect is a pharmaceutical composition, said
composition comprising a therapeutically effective amount of a
polypeptide described herein, and a pharmaceutically acceptable
carrier.
[0168] In an aspect described herein are polypeptides, said
polypeptides comprising a variant Fc region with at least three
amino acid substitutions, and wherein said polypeptides are more
effective at mediating antibody-dependent cellular cytotoxicity
(ADCC) relative to wild type. In certain embodiments, the
polypeptide comprising a variant Fc region is about 1.5 to about
100 fold more effective in mediating ADCC relative to wild type. In
certain embodiments, the polypeptide comprising a variant Fc region
is about 2 to about 50 fold more effective in mediating ADCC
relative to wild type.
[0169] In an aspect described herein are polypeptides, said
polypeptides comprising a variant Fc region with at least three
amino acid substitutions, wherein the polypeptide is more effective
at mediating inhibition of inflammatory immune responses relative
to wild type. In certain embodiments, the polypeptide comprising a
variant Fc region is about 2 fold more effective in mediating
inhibition of inflammatory immune responses relative to wild type.
In some embodiments, the polypeptide comprising a variant Fc region
is about 10 fold more effective in mediating inhibition of
inflammatory immune responses relative to wild type. In certain
embodiments, the polypeptide comprising a variant Fc region is
about 50 fold more effective in mediating inhibition of
inflammatory immune responses relative to wild type. In certain
embodiments, the polypeptide comprising a variant Fc region is
about 100 fold more effective in mediating inhibition of
inflammatory immune responses relative to wild type.
[0170] Also provided herein is a method for identifying Fc variant
polypeptides in silico based on calculated binding affinities to
Fc.gamma.RIIa, Fc.gamma.RIIb and/or Fc.gamma.RIIIa. In certain
embodiments, the method of identifying Fc variant polypeptides in
silico further calculates in silico electrostatics, solvation,
packing, packing density, hydrogen binding, and entropic effects of
said Fc variant polypeptides. In certain embodiments, the method of
identifying Fc variant polypeptides in silico further comprises
constructing the identified Fc variant polypeptides and expressing
said polypeptides in the context of an antibody in mammalian
cells.
[0171] The Fc polypeptides and proteins described herein may be
optimized for a variety of properties. Properties that may be
optimized include but are not limited to enhanced or reduced
affinity for an Fc.gamma.R. In a preferred embodiment, the Fc
proteins described herein are optimized to possess enhanced
affinity for a human activating Fc.gamma.RI, preferably
Fc.gamma.RII, Fc.gamma.RIIa, Fc.gamma.RIIc, Fc.gamma.RIIIa, and
Fc.gamma.RIIIb, most preferably Fc.gamma.RIIIa. In an alternately
preferred embodiment, the Fc proteins are optimized to possess
reduced affinity for the human inhibitory receptor F.gamma.RIIb.
These preferred embodiments are anticipated to provide antibodies
and Fc fusions with enhanced therapeutic properties in humans, for
example enhanced effector function and greater anti-cancer potency.
In an alternate embodiment, the Fc proteins described herein are
optimized to have reduced or ablated affinity for a human
Fc.gamma.RI, including but not limited to Fc.gamma.RII,
Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIc, Fc.gamma.RIIIa, and
Fc.gamma.RIIIb. These embodiments are anticipated to provide
antibodies and Fc fusions with enhanced therapeutic properties in
humans, for example reduced effector function and reduced toxicity.
Preferred embodiments comprise optimization of Fc binding to a
human Fc.gamma.R, however in alternate embodiments the Fc
polypeptides and proteins of the present invention possess enhanced
or reduced affinity for Fc.gamma.Rs from nonhuman organisms,
including but not limited to mice, rats, rabbits, and monkeys. Fc
proteins that are optimized for binding to a nonhuman Fc.gamma.R
may find use in experimentation. For example, mouse models are
available for a variety of diseases that enable testing of
properties such as efficacy, toxicity, and pharmacokinetics for a
given drug candidate. As is known in the art, cancer cells can be
grafted or injected into mice to mimic a human cancer, a process
referred to as xenografting. Testing of antibodies or Fc fusions
that comprise Fc proteins that are optimized for one or more mouse
Fc.gamma.Rs, may provide valuable information with regard to the
efficacy of the antibody or Fc fusion, its mechanism of action, and
the like. In certain embodiments, the Fc polypeptides and proteins
described herein are optimized for enhanced functionality and/or
solution properties in aglycosylated form. In an exemplary
embodiment, the aglycosylated Fc proteins described herein bind an
Fc ligand with greater affinity than the aglycosylated form of the
parent Fc polypeptide. Said Fc ligands include but are not limited
to Fc.gamma.Rs, C1q, FcRn, and proteins A and G, and may be from
any source including but not limited to human, mouse, rat, rabbit,
or monkey, preferably human. In an alternately preferred
embodiment, the Fc proteins are optimized to be more stable and/or
more soluble than the aglycosylated form of the parent Fc
polypeptide. An Fc protein that is engineered or predicted to
display any of the aforementioned optimized properties is herein
referred to as an "optimized Fc protein".
[0172] In certain embodiments, the Fc polypeptides and proteins
described herein are derived from parent Fc polypeptides that are
themselves from a wide range of sources. In some embodiments the
parent Fc polypeptide is substantially encoded by one or more Fc
genes from any organism, including but not limited to humans, mice,
rats, rabbits, camels, llamas, dromedaries, monkeys, preferably
mammals and most preferably humans and mice. In an embodiment, the
parent Fc polypeptide comprises an antibody, referred to as the
parent antibody. In certain embodiments, the parent antibody is
fully human, obtained for example using transgenic mice (Bruggemann
et al., 1997, Curr Opin Biotechnol 8:455-458) or human antibody
libraries coupled with selection methods (Griffiths et al., 1998,
Curr Opin Biotechnol 9:102-108). The parent antibody need not be
naturally occurring. In certain embodiments the parent antibody is
an engineered antibody, including but not limited to chimeric
antibodies and humanized antibodies (Clark, 2000, Immunol Today
21:397-402). In certain embodiments, the parent antibody is an
engineered variant of an antibody that is substantially encoded by
one or more natural antibody genes. In one embodiment, the parent
antibody has been affinity matured, as is known in the art.
Alternatively, the antibody has been modified in some other way,
for example as described in U.S. Ser. No. 10/339,788, filed on Mar.
3, 2003.
[0173] In certain embodiments, the Fc proteins described herein is
substantially encoded by immunoglobulin genes belonging to any of
the antibody classes. In an exemplary embodiment, the Fc proteins
described herein find use in antibodies or Fc fusions that comprise
sequences belonging to the IgG class of antibodies, including IgG1,
IgG2, IgG3, or IgG4. In an alternate embodiment the Fc proteins
described herein find use in antibodies or Fc fusions that comprise
sequences belonging to the IgA (including subclasses IgA1 and
IgA2), IgD, IgE, IgG, or IgM classes of antibodies. In some
embodiments, the Fc proteins described herein comprise more than
one protein chain. That is, in some embodiments, the polypeptides
described herein find use in an antibody or Fc fusion that is a
monomer or an oligomer, including a homo- or hetero-oligomer.
[0174] In some embodiments, the antibodies of the invention are
based on human sequences, and thus human sequences are used as the
"base" sequences, against which other sequences, such as rat,
mouse, and monkey sequences are compared. In order to establish
homology to primary sequence or structure, the amino acid sequence
of a precursor or parent Fc polypeptide is directly compared to the
human Fc sequence outlined herein. After aligning the sequences,
using one or more of the homology alignment programs known in the
art (for example using conserved residues as between species),
allowing for necessary insertions and deletions in order to
maintain alignment (i.e., avoiding the elimination of conserved
residues through arbitrary deletion and insertion), the residues
equivalent to particular amino acids in the primary sequence of
human Fc are defined. Alignment of conserved residues preferably
should conserve 100% of such residues. However, alignment of
greater than 75% or as little as 50% of conserved residues is also
adequate to define equivalent residues (sometimes referred to as
"corresponding residues"). Equivalent residues may also be defined
by determining homology at the level of tertiary structure for an
Fc polypeptide whose tertiary structure has been determined.
Equivalent residues are defined as those for which the atomic
coordinates of two or more of the main chain atoms of a particular
amino acid residue of the parent or precursor (N on N, CA on CA, C
on C and 0 on 0) are within 0.13 nm and preferably 0.1 nm after
alignment. Alignment is achieved after the best model has been
oriented and positioned to give the maximum overlap of atomic
coordinates of non-hydrogen protein atoms of the Fc
polypeptide.
[0175] In some embodiments, the Fc polypeptides described herein
are combined with other Fc modifications, including but not limited
to modifications that alter effector function or interaction with
one or more Fc ligands. In certain embodiments, these combinations
provide additive, synergistic, or properties in antibodies or Fc
fusions. In one embodiment, the Fc proteins described herein is
combined with other known Fc proteins (Duncan et al., 1988, Nature
332:563-564; Lund et al., 1991, J Immunol 147:2657-2662; Lund et
al., 1992, Mol Immunol 29:53-59; Alegre et al., 1994,
Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl Acad
Sci USA 92:11980-11984; Jefferis et al., 1995, Immunol Left
44:111-117; Lund et al., 1995, Faseb J9:115-119; Jefferis et al.,
1996, Immunol Left 54:101-104; Lund et al., 1996, J Immunol
157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624;
Idusogie et al., 2000, J Immunol 164:4178-4184; Reddy et al., 2000,
J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26;
Idusogie et al., 2001, J Immunol 166:2571-2575; Shields et al.,
2001, J Biol Chem 276:6591-6604; Jefferis et al., 2002, Immunol
Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490;
Hinton et al., 2004, J Biol Chem 279:6213-6216) (U.S. Pat. No.
5,624,821; U.S. Pat. No. 5,885,573; U.S. Pat. No. 6,194,551; PCT WO
00/42072; PCT WO 99/58572; US 2004/0002587 A1). In an alternate
embodiment, the Fc proteins described herein are incorporated into
an antibody or Fc fusion that comprises one or more engineered
glycoforms. By "engineered glycoform" as used herein is meant a
carbohydrate composition that is covalently attached to an Fc
polypeptide, wherein said carbohydrate composition differs
chemically from that of a parent Fc polypeptide. Engineered
glycoforms may be useful for a variety of purposes, including but
not limited to enhancing or reducing effector function. Engineered
glycoforms may be generated by a variety of methods known in the
art (Umana et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,
2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol
Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem
278:3466-3473); (U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370;
U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/29246A1; PCT
WO 02/31140A1; PCT WO 02/30954A1); (Potelligent.RTM. technology
[Biowa, Inc., Princeton, N.J.]; GlycoMAb.RTM. glycosylation
engineering technology [GLYCART biotechnology AG, Zurich,
Switzerland]). Many of these techniques are based on controlling
the level of fucosylated and/or bisecting oligosaccharides that are
covalently attached to the Fc region, for example by expressing an
Fc polypeptide in various organisms or cell lines, engineered or
otherwise (for example Lec-13 CHO cells or rat hybridoma YB2/0
cells), by regulating enzymes involved in the glycosylation pathway
(for example FUT8 [.alpha.1,6-fucosyltranserase] and/or
.beta.1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by
modifying carbohydrate(s) after the Fc polypeptide has been
expressed. Engineered glycoform typically refers to the different
carbohydrate or oligosaccharide; thus an Fc polypeptide, for
example an antibody or Fc fusion, may comprise an engineered
glycoform. Alternatively, engineered glycoform may refer to the Fc
polypeptide that comprises the different carbohydrate or
oligosaccharide. Thus combinations of the Fc proteins described
herein with other Fc modifications, as well as undiscovered Fc
modifications, are contemplated with the goal of generating
antibodies or Fc fusions with optimized properties.
[0176] In certain embodiments, the variant Fc proteins and
polypeptides described herein find use in an antibody. By "antibody
described herein" as used herein is meant an antibody that
comprises an Fc protein described herein. The present invention
may, in fact, find use in any protein that comprises Fc, and thus
application of the Fc polypeptide and proteins described herein is
not limited to antibodies. The Fc proteins described herein may
find use in an Fc fusion. By "Fc fusion described herein" as used
herein refers to an Fc fusion that comprises an Fc protein
described herein. Fc fusions may comprise an Fc protein described
herein operably linked to a cytokine, soluble receptor domain,
adhesion molecule, ligand, enzyme, peptide, or other protein or
protein domain, and include but are not limited to Fc fusions
described in U.S. Pat. No. 5,843,725; U.S. Pat. No. 6,018,026; U.S.
Pat. No. 6,291,212; U.S. Pat. No. 6,291,646; U.S. Pat. No.
6,300,099; U.S. Pat. No. 6,323,323; PCT WO 00/24782; and in (Chamow
et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997,
Curr Opin Immunol 9:195-200).
[0177] Virtually any antigen may be targeted by the proteins and
polypeptides described herein, including but not limited to the
following list of proteins, subunits, domains, motifs, and epitopes
belonging to the following list of proteins: CD2; CD3, CD3E, CD4,
CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28,
CD29, CD30, CD32, CD33 (p67 protein), CD38, CD40, CD40L, CD52,
CD54, CD56, CD80, CD147, GD3, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5,
IL-6, IL-6R, IL-8, IL-12, IL-15, IL-18, IL-23, interferon alpha,
interferon beta, interferon gamma; TNF-alpha, TNFbeta2, TNFc,
TNFalphabeta, TNF-RI, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL,
RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, OX40L, TRAIL Receptor-1, A1
Adenosine Receptor, Lymphotoxin Beta Receptor, TACI, BAFF-R, EPO;
LFA-3, ICAM-1, ICAM-3, EpCAM, integrin beta1, integrin beta2,
integrin alpha4/beta7, integrin alpha2, integrin alpha3, integrin
alpha4, integrin alpha5, integrin alpha6, integrin alphav,
alphaVbeta3 integrin, FGFR-3, Keratinocyte Growth Factor, VLA-1,
VLA-4, L-selectin, anti-Id, E-selectin, HLA, HLA-DR, CTLA-4, T cell
receptor, B7-1, B7-2, VNRintegrin, TGFbeta1, TGFbeta2, eotaxin1,
BLyS (B-lymphocyte Stimulator), complement C5, IgE, factor VII,
CD64, CBL, NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3),
Her4 (ErbB-4), Tissue Factor, VEGF, VEGFR, endothelin receptor,
VLA-4, Hapten NP-cap or NIP-cap, T cell receptor alpha/beta,
E-selectin, digoxin, placental alkaline phosphatase (PLAP) and
testicular PLAP-like alkaline phosphatase, transferrin receptor,
Carcinoembryonic antigen (CEA), CEACAM5, HMFG PEM, mucin MUC1,
MUC18, Heparanase 1, human cardiac myosin, tumor-associated
glycoprotein-72 (TAG-72), tumor-associated antigen CA 125, Prostate
specific membrane antigen (PSMA), High molecular weight
melanoma-associated antigen (HMW-MAA), carcinoma-associated
antigen, Gcoprotein IIb/IIIa (GPIIb/IIIa), tumor-associated antigen
expressing Lewis Y related carbohydrate, human cytomegalovirus
(HCMV) gH envelope glycoprotein, HIV gp120, HCMV, respiratory
syncital virus RSV F, RSVF Fgp, VNRintegrin, IL-8, cytokeratin
tumor-associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV HIB
gp120 V3 loop, respiratory syncytial virus (RSV) Fgp, Herpes
simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB
envelope glycoprotein, and Clostridium perfringens toxin.
[0178] One skilled in the art will appreciate that the
aforementioned list of targets refers not only to specific proteins
and biomolecules, but the biochemical pathway or pathways that
comprise them. For example, reference to CTLA-4 as a target antigen
implies that the ligands and receptors that make up the T cell
co-stimulatory pathway, including CTLA-4, B7-1, B7-2, CD28, and any
other undiscovered ligands or receptors that bind these proteins,
are also targets. Thus target as used herein refers not only to a
specific biomolecule, but the set of proteins that interact with
said target and the members of the biochemical pathway to which
said target belongs. One skilled in the art will further appreciate
that any of the aforementioned target antigens, the ligands or
receptors that bind them, or other members of their corresponding
biochemical pathway, may be operably linked to the Fc proteins
described herein in order to generate an Fc fusion. Thus for
example, an Fc fusion that targets EGFR could be constructed by
operably linking an Fc protein to EGF, TGF.alpha., or any other
ligand, discovered or undiscovered, that binds EGFR. Accordingly,
an Fc protein described herein could be operably linked to EGFR in
order to generate an Fc fusion that binds EGF, TGF.alpha., or any
other ligand, discovered or undiscovered, that binds EGFR. Thus
virtually any polypeptide, whether a ligand, receptor, or some
other protein or protein domain, including but not limited to the
aforementioned targets and the proteins that compose their
corresponding biochemical pathways, may be operably linked to the
Fc proteins described herein to develop an Fc fusion.
[0179] A number of antibodies and Fc fusions that are approved for
use, in clinical trials, or in development benefit from the Fc
proteins described herein. Said antibodies and Fc fusions are
herein referred to as "clinical products and candidates". Thus in a
preferred embodiment, the Fc proteins described herein may find use
in a range of clinical products and candidates. For example, a
number of antibodies that target CD20 may benefit from the Fc
proteins described herein. For example the Fc proteins described
herein may find use in an antibody that is substantially similar to
rituximab (Rituxan.RTM., IDEC/Genentech/Roche) (see for example
U.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody approved to
treat Non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently
being developed by Genmab, an anti-CD20 antibody described in U.S.
Pat. No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20
(Immunomedics, Inc.), and HumaLYM (Intracel). A number of
antibodies that target members of the family of epidermal growth
factor receptors, including EGFR (ErbB-1), Her2/neu (ErbB-2), Her3
(ErbB-3), Her4 (ErbB-4), may benefit from the Fc proteins described
herein. For example the Fc proteins described herein may find use
in an antibody that is substantially similar to trastuzumab
(Herceptin.RTM., Genentech) (see for example U.S. Pat. No.
5,677,171), a humanized anti-Her2/neu antibody approved to treat
breast cancer; pertuzumab (rhuMab-2C4, Omnitarg.RTM.), currently
being developed by Genentech; an anti-Her2 antibody described in
U.S. Pat. No. 4,753,894; cetuximab (Erbitux.RTM.), Imclone) (U.S.
Pat. No. 4,943,533; PCT WO 96/40210), a chimeric anti-EGFR antibody
in clinical trials for a variety of cancers; ABX-EGF (U.S. Pat. No.
6,235,883), currently being developed by Abgenix/Immunex/Amgen;
HuMax-EGFr (U.S. Ser. No. 10/172,317), currently being developed by
Genmab; 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S.
Pat. No. 5,558,864; Murthy et al. 1987, Arch Biochem Biophys.
252(2):549-60; Rodeck et al., 1987, J Cell Biochem. 35(4):315-20;
Kettleborough et al., 1991, Protein Eng. 4(7):773-83); ICR62
(Institute of Cancer Research) (PCT WO 95/20045; Modjtahedi et al.,
1993, J. Cell Biophys. 1993, 22(1-3):129-46; Modjtahedi et al.,
1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi et al., 1996, Br
J Cancer, 73(2):228-35; Modjtahedi et al., 2003, Int J Cancer,
105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de
Immunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No.
6,506,883; Mateo et al., 1997, Immunotechnology, 3(1):71-81);
mAb-806 (Ludwig Institue for Cancer Research, Memorial
Sloan-Kettering) (Jungbluth et al. 2003, Proc Natl Acad Sci USA.
100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer
Institute) (PCT WO 0162931A2); and SC 100 (Scancell) (PCT WO
01/88138). In another preferred embodiment, the Fc proteins
described herein may find use in alemtuzumab (Campath.RTM.,
Millenium), a humanized monoclonal antibody currently approved for
treatment of B-cell chronic lymphocytic leukemia. The Fc proteins
described herein may find use in a variety of antibodies or Fc
fusions that are substantially similar to other clinical products
and candidates, including but not limited to muromonab-CD3
(Orthoclone OKT3.RTM.), an anti-CD3 antibody developed by Ortho
Biotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin.RTM.),
an anti-CD20 antibody developed by IDEC/Schering AG, gemtuzumab
ozogamicin (Mylotarg.RTM.), an anti-CD33 (p67 protein) antibody
developed by Celltech/Wyeth, alefacept (Amevive.RTM.), an
anti-LFA-3 Fc fusion developed by Biogen), abciximab (ReoPro.RTM.),
developed by Centocor/Lilly, basiliximab (Simulect.RTM.), developed
by Novartis, palivizumab (Synagis.RTM.), developed by Medlmmune,
infliximab (Remicade.RTM.), an anti-TNFalpha antibody developed by
Centocor, adalimumab (Humira.RTM.), an anti-TNFalpha antibody
developed by Abbott, Humicade.RTM., an anti-TNFalpha antibody
developed by Celltech, etanercept (Enbrel.RTM.), an anti-TNFalpha
Fc fusion developed by Immunex/Amgen, ABX-CBL, an anti-CD147
antibody being developed by Abgenix, ABX-IL8, an anti-IL8 antibody
being developed by Abgenix, ABX-MA1, an anti-MUC18 antibody being
developed by Abgenix, Pemtumomab (R1549, .sup.90Y-muHMFG1), an
anti-MUC1 In development by Antisoma, Therex (R1550), an anti-MUC1
antibody being developed by Antisoma, AngioMab (AS1405), being
developed by Antisoma, HuBC-1, being developed by Antisoma,
Thioplatin (AS1407) being developed by Antisoma, Antegren.RTM.)
(natalizumab), an anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7
antibody being developed by Biogen, VLA-1 mAb, an anti-VLA-1
integrin antibody being developed by Biogen, LTBR mAb, an
anti-lymphotoxin beta receptor (LTBR) antibody being developed by
Biogen, CAT-152, an anti-TGF.beta.2 antibody being developed by
Cambridge Antibody Technology, J695, an anti-IL-12 antibody being
developed by Cambridge Antibody Technology and Abbott, CAT-192, an
anti-TGF.beta.1 antibody being developed by Cambridge Antibody
Technology and Genzyme, CAT-213, an anti-Eotaxin1 antibody being
developed by Cambridge Antibody Technology, LymphoStat-B.RTM. an
anti-Blys antibody being developed by Cambridge Antibody Technology
and Human Genome Sciences Inc., TRAIL-R1mAb, an anti-TRAIL-R1
antibody being developed by Cambridge Antibody Technology and Human
Genome Sciences, Inc., Avastin.RTM. (bevacizumab, rhuMAb-VEGF), an
anti-VEGF antibody being developed by Genentech, an anti-HER
receptor family antibody being developed by Genentech, Anti-Tissue
Factor (ATF), an anti-Tissue Factor antibody being developed by
Genentech, Xolair.RTM. (Omalizumab), an anti-IgE antibody being
developed by Genentech, Raptiva.RTM. (Efalizumab), an anti-CD1a
antibody being developed by Genentech and Xoma, MLN-02 Antibody
(formerly LDP-02), being developed by Genentech and Millenium
Pharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed by
Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab
and Amgen, HuMax-Inflam, being developed by Genmab and Medarex,
HuMax-Cancer, an anti-Heparanase 1 antibody being developed by
Genmab and Medarex and Oxford GcoSciences, HuMax-Lymphoma, being
developed by Genmab and Amgen, HuMax-TAC, being developed by
Genmab, IDEC-131, and anti-CD40L antibody being developed by IDEC
Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody
being developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80
antibody being developed by IDEC Pharmaceuticals, IDEC-152, an
anti-CD23 being developed by IDEC Pharmaceuticals, anti-macrophage
migration factor (MIF) antibodies being developed by IDEC
Pharmaceuticals, BEC2, an anti-idiotypic antibody being developed
by Imclone, IMC-1C11, an anti-KDR antibody being developed by
Imclone, DC101, an anti-flk-1 antibody being developed by Imclone,
anti-VE cadherin antibodies being developed by Imclone,
CEA-Cide.RTM. (labetuzumab), an anti-carcinoembryonic antigen (CEA)
antibody being developed by Immunomedics, LymphoCide.RTM.
(Epratuzumab), an anti-CD22 antibody being developed by
Immunomedics, AFP-Cide, being developed by Immunomedics,
MyelomaCide, being developed by Immunomedics, LkoCide, being
developed by Immunomedics, ProstaCide, being developed by
Immunomedics, MDX-010, an anti-CTLA4 antibody being developed by
Medarex, MDX-060, an anti-CD30 antibody being developed by Medarex,
MDX-070 being developed by Medarex, MDX-018 being developed by
Medarex, Osidem.RTM. (IDM-1), and anti-Her2 antibody being
developed by Medarex and Immuno-Designed Molecules, HuMax.RTM.-CD4,
an anti-CD4 antibody being developed by Medarex and Genmab,
HuMax-IL15, an anti-IL15 antibody being developed by Medarex and
Genmab, CNTO 148, an anti-TNF.alpha. antibody being developed by
Medarex and Centocor/J&J, CNTO 1275, an anti-cytokine antibody
being developed by Centocor/J&J, MOR101 and MOR102,
anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies
being developed by MorphoSys, MOR201, an anti-fibroblast growth
factor receptor 3 (FGFR-3) antibody being developed by MorphoSys,
Nuvion.RTM. (visilizumab), an anti-CD3 antibody being developed by
Protein Design Labs, HuZAF.RTM., an anti-gamma interferon antibody
being developed by Protein Design Labs,
Anti-.quadrature.5.quadrature.1 Integrin, being developed by
Protein Design Labs, anti-IL-12, being developed by Protein Design
Labs, ING-1, an anti-Ep-CAM antibody being developed by Xoma, and
MLN01, an anti-Beta2 integrin antibody being developed by Xoma.
[0180] Application of the Fc polypeptides, proteins to the
aforementioned antibody and Fc fusion clinical products and
candidates is not meant to be constrained to their precise
composition. In some embodiments, the Fc proteins described herein
are incorporated into the aforementioned clinical candidates and
products, or into antibodies and Fc fusions that are substantially
similar to them. In certain embodiments, the Fc proteins described
herein are incorporated into versions of the aforementioned
clinical candidates and products that are humanized, affinity
matured, engineered, or modified in some other way. Furthermore,
the entire polypeptide of the aforementioned clinical products and
candidates need not be used to construct a new antibody or Fc
fusion that incorporates the Fc proteins described herein; for
example only the variable region of a clinical product or candidate
antibody, a substantially similar variable region, or a humanized,
affinity matured, engineered, or modified version of the variable
region may be used. In another embodiment, the Fc proteins and
polypeptides described herein are used in an antibody or Fc fusion
that binds to the same epitope, antigen, ligand, or receptor as one
of the aforementioned clinical products and candidates.
[0181] In an aspect the Fc polypeptides described herein are used
in a wide range of antibody and Fc fusion products. In one
embodiment the antibody or Fc fusion comprising the Fc polypeptide
or protein described herein is a therapeutic, a diagnostic, or a
research reagent, preferably a therapeutic. In certain other
embodiments, the antibodies and Fc fusions comprise the Fc based
polypeptides described herein and are used for agricultural or
industrial uses. In an alternate embodiment, the Fc proteins and
polypeptides described herein compose a library that is screened
experimentally. In certain embodiments, this library comprises a
list of nucleic acid or amino acid sequences. In certain other
embodiments, the library is a physical composition of nucleic acids
or polypeptides that encode the library sequences. In some
embodiments, the Fc proteins find use in an antibody composition
that is monoclonal or polyclonal. The antibodies and Fe fusions
described herein encompass, but are not restricted to agonists,
antagonists, neutralizing, inhibitory, or stimulatory. In an
exemplary embodiment, the antibodies and Fc fusions described
herein are used to kill target cells that bear the target antigen,
for example cancer cells. In an alternate embodiment, the
antibodies and Fc fusions described herein are used to block,
antagonize, or agonize the target antigen, for example for
antagonizing a cytokine or cytokine receptor. In an alternate
embodiment, the antibodies and Fc fusions described herein are used
to block, antagonize, or agonize the target antigen and kill the
target cells that bear the target antigen.
[0182] In some embodiments, the polypeptides disclosed herein are
useful in regulating the immune response, e.g., in inhibiting the
immune response in connection with autoimmune diseases or
inflammatory diseases. Such polypeptides have therapeutic utility
in treating and/or preventing an autoimmune disorder. Examples of
autoimmune disorders that may be treated by administering the
polypeptides of the disclosed herein include, but are not limited
to, alopecia areata, ankylosing spondylitis, antiphospholipid
syndrome, autoimmune Addison's disease, autoimmune diseases of the
adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis,
autoimmune oophoritis and orchitis, autoimmune thrombocytopenia,
Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac
sprue-dermatitis, chronic fatigue immune dysfunction syndrome
(CFIDS), chronic inflammatory demyelinating polyneuropathy,
Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold
agglutinin disease, Crohn's disease, discoid lupus, essential mixed
cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis,
Graves' disease, Guillain-Barre, Hashimoto's thyroiditis,
idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura
(ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus
erthematosus, Meniere's disease, mixed connective tissue disease,
multiple sclerosis, type 1 or immune-mediated diabetes mellitus,
myasthenia gravis, pemphigus vulgaris, pernicious anemia,
polyarteritis nodosa, polychrondritis, polyglandular syndromes,
polymyalgia rheumatica, polymyositis and dermatomyositis, primary
agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic
arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid
arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man
syndrome, systemic lupus erythematosus, lupus erythematosus,
takayasu arteritis, temporal arteristis/giant cell arteritis,
ulcerative colitis, uveitis, vasculitides such as dermatitis
herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.
Examples of inflammatory disorders include, but are not limited to,
asthma, encephilitis, inflammatory bowel disease, chronic
obstructive pulmonary disease (COPD), allergic disorders, septic
shock, pulmonary fibrosis, undifferentitated spondyloarthropathy,
undifferentiated arthropathy, arthritis, inflammatory osteolysis,
and chronic inflammation resulting from chronic viral or bacteria
infections. Examples of inflammatory disorders which can be
prevented, treated or managed in accordance with the methods of the
invention include, but are not limited to, asthma, encephilitis,
inflammatory bowel disease, chronic obstructive pulmonary disease
(COPD), allergic disorders, septic shock, pulmonary fibrosis,
undifferentitated spondyloarthropathy, undifferentiated
arthropathy, arthritis, inflammatory osteolysis, and chronic
inflammation resulting from chronic viral or bacteria
infections.
[0183] The Fc polypeptides described herein are used for various
therapeutic purposes. In some embodiments, the Fc polypeptides and
proteins are administered to a patient to treat an antibody-related
disorder. A "patient" for the purposes described herein includes
both humans and other animals, preferably mammals and most
preferably humans. Thus the antibodies and Fc fusions described
herein have both human therapy and veterinary applications. In the
preferred embodiment the patient is a mammal, and in the most
preferred embodiment the patient is human. The term "treatment" in
the present invention is meant to include therapeutic treatment, as
well as prophylactic, or suppressive measures for a disease or
disorder. Thus, for example, successful administration of an
antibody or Fc fusion prior to onset of the disease results in
treatment of the disease. As another example, successful
administration of an optimized antibody or Fc fusion after clinical
manifestation of the disease to combat the symptoms of the disease
comprises treatment of the disease. "Treatment" also encompasses
administration of an optimized antibody or Fc fusion protein after
the appearance of the disease in order to eradicate the disease.
Successful administration of an agent after onset and after
clinical symptoms have developed, with possible abatement of
clinical symptoms and perhaps amelioration of the disease,
comprises treatment of the disease. Those "in need of treatment"
include mammals already having the disease or disorder, as well as
those prone to having the disease or disorder, including those in
which the disease or disorder is to be prevented. By "antibody
related disorder" or "antibody responsive disorder" or "condition"
or "disease" herein are meant a disorder that may be ameliorated by
the administration of a pharmaceutical composition comprising an
antibody or Fc fusion described herein. Antibody related disorders
include but are not limited to autoimmune diseases, immunological
diseases, infectious diseases, inflammatory diseases, neurological
diseases, and oncological and neoplastic diseases including cancer.
By "cancer" and "cancerous" herein 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 (including
liposarcoma), neuroendocrine tumors, mesothelioma, schwanoma,
meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid
malignancies. More particular examples of such cancers include
squamous cell cancer (e.g. epithelial squamous cell cancer), lung
cancer including small-cell lung cancer, non-small cell lung
cancer, adenocarcinoma of the lung and squamous carcinoma of the
lung, cancer of the peritoneum, hepatocellular cancer, gastric or
stomach cancer including gastrointestinal cancer, pancreatic
cancer, glioblastoma, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, hepatoma, breast cancer, colon cancer,
rectal cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney or renal cancer, prostate cancer,
vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma,
penile carcinoma, testicular cancer, esophageal cancer, tumors of
the biliary tract, as well as head and neck cancer. Furthermore,
the Fc proteins described herein may be used to treat conditions
including but not limited to congestive heart failure (CHF),
vasculitis, rosacea, acne, eczema, myocarditis and other conditions
of the myocardium, systemic lupus erythematosus, diabetes,
spondylopathies, synovial fibroblasts, and bone marrow stroma; bone
loss; Paget's disease, osteoclastoma; multiple myeloma; breast
cancer; disuse osteopenia; malnutrition, periodontal disease,
Gaucher's disease, Langerhans' cell histiocytosis, spinal cord
injury, acute septic arthritis, osteomalacia, Cushing's syndrome,
monoostotic fibrous dysplasia, polyostotic fibrous dysplasia,
periodontal reconstruction, and bone fractures; sarcoidosis;
multiple myeloma; osteolytic bone cancers, breast cancer, lung
cancer, kidney cancer and rectal cancer; bone metastasis, bone pain
management, and humoral malignant hypercalcemia, ankylosing
spondylitisa and other spondyloarthropathies; transplantation
rejection, viral infections, hematologic neoplasisas and
neoplastic-like conditions for example, Hodgkin's lymphoma;
non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic
lymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle
cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma,
marginal zone lymphoma, hairy cell leukemia and lymphoplamacytic
leukemia), tumors of lymphocyte precursor cells, including B-cell
acute lymphoblastic leukemia/lymphoma, and T-cell acute
lymphoblastic leukemia/lymphoma, thymoma, tumors of the mature T
and NK cells, including peripheral T-cell leukemias, adult T-cell
leukemia/T-cell lymphomas and large granular lymphocytic leukemia,
Langerhans cell histocytosis, myeloid neoplasias such as acute
myelogenous leukemias, including AML with maturation, AML without
differentiation, acute promyelocytic leukemia, acute myelomonocytic
leukemia, and acute monocytic leukemias, myelodysplastic syndromes,
and chronic myeloproliferative disorders, including chronic
myelogenous leukemia, tumors of the central nervous system, e.g.,
brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma,
ependymoma, and retinoblastoma), solid tumors (nasopharyngeal
cancer, basal cell carcinoma, pancreatic cancer, cancer of the bile
duct, Kaposi's sarcoma, testicular cancer, uterine, vaginal or
cervical cancers, ovarian cancer, primary liver cancer or
endometrial cancer, and tumors of the vascular system (angiosarcoma
and hemangiopericytoma), osteoporosis, hepatitis, HIV, AIDS,
spondylarthritis, rheumatoid arthritis, inflammatory bowel diseases
(IBD), sepsis and septic shock, Crohn's Disease, psoriasis,
schleraderma, graft versus host disease (GVHD), allogenic islet
graft rejection, hematologic malignancies, such as multiple myeloma
(MM), myelodysplastic syndrome (MDS) and acute myelogenous leukemia
(AML), inflammation associated with tumors, peripheral nerve injury
or demyelinating diseases.
[0184] In one embodiment, an antibody or polypeptide described
herein is administered to a patient having a disease involving
inappropriate expression of a protein. Within the scope described
herein this is meant to include diseases and disorders
characterized by aberrant proteins, due for example to alterations
in the amount of a protein present, the presence of a mutant
protein, or both. An overabundance may be due to any cause,
including but not limited to overexpression at the molecular level,
prolonged or accumulated appearance at the site of action, or
increased activity of a protein relative to normal. Included within
this definition are diseases and disorders characterized by a
reduction of a protein. This reduction may be due to any cause,
including but not limited to reduced expression at the molecular
level, shortened or reduced appearance at the site of action,
mutant forms of a protein, or decreased activity of a protein
relative to normal. Such an overabundance or reduction of a protein
can be measured relative to normal expression, appearance, or
activity of a protein, and said measurement may play an important
role in the development and/or clinical testing of the antibodies
and Fc fusions described herein.
[0185] In one embodiment, an antibody or polypeptide described
herein is the only therapeutically active agent administered to a
patient. Alternatively, the antibody or Fc fusion described herein
is administered in combination with one or more other therapeutic
agents, including but not limited to cytotoxic agents,
chemotherapeutic agents, cytokines, growth inhibitory agents,
anti-hormonal agents, kinase inhibitors, anti-angiogenic agents,
cardioprotectants, or other therapeutic agents. Such molecules are
suitably present in combination in amounts that are effective for
the purpose intended. The skilled medical practitioner can
determine empirically the appropriate dose or doses of other
therapeutic agents useful herein. The antibodies and polypeptides
described herein may be administered concomitantly with one or more
other therapeutic regimens. For example, an antibody or polypeptide
described herein may be administered to the patient along with
chemotherapy, radiation therapy, or both chemotherapy and radiation
therapy. In one embodiment, the antibody or Fc fusion described
herein may be administered in conjunction with one or more
antibodies or polypeptides, which may or may not comprise an Fc
protein described herein.
[0186] In one embodiment, the antibodies and polypeptides described
herein are administered with a chemotherapeutic agent. By
"chemotherapeutic agent" as used herein is meant a chemical
compound useful in the treatment of cancer. Examples of
chemotherapeutic agents include but are not limited to alkylating
agents such as thiotepa and cyclosphosphamide (CYTOXAN.RTM.); alkyl
sulfonates such as busulfan, improsulfan and piposulfan; aziridines
such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitrogen
mustards such as chlorambucil, chlornaphazine, cholophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;
antibiotics such as aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, calicheamicin, carabicin,
caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,
epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,
mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK.RTM.; razoxane; sizofuran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine;
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel
(TAXOTERE.RTM., Rhne-Poulenc Rorer, Antony, France); chlorambucil;
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;
vincristine; vinorelbine; navelbine; novantrone; teniposide;
daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid;
esperamicins; capecitabine; thymidylate synthase inhibitor (such as
Tomudex); cox-2 inhibitors, such as celicoxib (CELEBREX.RTM.) or
MK-0966 (VIOXX.RTM.); and pharmaceutically acceptable salts, acids
or derivatives of any of the above. Also included in this
definition are anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti estrogens including for
example tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY
117018, onapristone, and toremifene (Fareston); and anti-androgens
such as flutamide, nilutamide, bicalutamide, leuprolide, and
goserelin; and pharmaceutically acceptable salts, acids or
derivatives of any of the above.
[0187] In certain embodiments, a chemotherapeutic or other
cytotoxic agent is administered as a prodrug. By "prodrug" as used
herein is meant a precursor or derivative form of a
pharmaceutically active substance that is less cytotoxic to tumor
cells compared to the parent drug and is capable of being
enzymatically activated or converted into the more active parent
form. See, for example Wilman, 1986, Biochemical Society
Transactions, 615th Meeting Belfast, 14:375-382; and Stella et al.,
"Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed
Drug Delivery, Borchardt et al., (ed.): 247-267, Humana Press,
1985. The prodrugs that may find use with the present invention
include but are not limited to phosphate-containing prodrugs,
thiophosphate-containing prodrugs, sulfate-containing prodrugs,
peptide-containing prodrugs, D-amino acid-modified prodrugs,
glycosylated prodrugs, beta-lactam-containing prodrugs, optionally
substituted phenoxyacetamide-containing prodrugs or optionally
substituted phenylacetamide-containing prodrugs, 5-fluorocytosine
and other 5-fluorouridine prodrugs which can be converted into the
more active cytotoxic free drug. Examples of cytotoxic drugs that
can be derivatized into a prodrug form for use with the antibodies
and Fc fusions described herein include but are not limited to any
of the aforementioned chemotherapeutic agents.
[0188] In some embodiments, the proteins and polypeptides described
herein are combined with other therapeutic regimens. For example,
in one embodiment, the patient to be treated with the antibody or
Fc fusion also receives radiation therapy. Radiation therapy can be
administered according to protocols commonly employed in the art
and known to the skilled artisan. Such therapy includes but is not
limited to cesium, iridium, iodine, or cobalt radiation. In some
embodiments, the radiation therapy is whole body irradiation, or
directed locally to a specific site or tissue in or on the body,
such as the lung, bladder, or prostate. Typically, radiation
therapy is administered in pulses over a period of time from about
1 to 2 weeks. In some embodiments, the radiation therapy is
administered over longer periods of time. In one embodiment,
radiation therapy is administered to patients having head and neck
cancer for about 6 to about 7 weeks. Optionally, the radiation
therapy is administered as a single dose or as multiple, sequential
doses. The skilled medical practitioner can determine empirically
the appropriate dose or doses of radiation therapy useful herein.
In accordance with another embodiment of the invention, the
antibody or Fc fusion described herein and one or more other
anti-cancer therapies are employed to treat cancer cells ex vivo.
It is contemplated that such ex vivo treatment may be useful in
bone marrow transplantation and particularly, autologous bone
marrow transplantation. In certain embodiments, treatment of cells
or tissue(s) containing cancer cells with antibody or Fc fusion and
one or more other anti-cancer therapies, such as described above,
is employed to deplete or substantially deplete the cancer cells
prior to transplantation in a recipient patient. II certain
embodiments, the proteins and Fc fusions of the invention are
employed in combination with still other therapeutic techniques
such as surgery.
[0189] In an alternate embodiment, the antibodies and polypeptides
described herein are administered with a cytokine. By "cytokine" as
used herein is meant a generic term for proteins released by one
cell population that act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-alpha and -beta;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-beta; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I
and -II; erythropoietin (EPO); osteoinductive factors; interferons
such as interferon-alpha, beta, and -gamma; colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-lalpha, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor
necrosis factor such as TNF-alpha or TNF-beta; and other
polypeptide factors including LIF and kit ligand (KL). As used
herein, the term cytokine includes proteins from natural sources or
from recombinant cell culture, and biologically active equivalents
of the native sequence cytokines.
[0190] A variety of other therapeutic agents find use for
administration with the Fc variant proteins and polypeptides
described herein. In one embodiment, the protein is administered
with an anti-angiogenic agent. By "anti-angiogenic agent" as used
herein is meant a compound that blocks, or interferes to some
degree, the development of blood vessels. The anti-angiogenic
factor may, for instance, be a small molecule or a protein, for
example an antibody, Fc fusion, or cytokine, that binds to a growth
factor or growth factor receptor involved in promoting
angiogenesis. The preferred anti-angiogenic factor herein is an
antibody that binds to Vascular Endothelial Growth Factor (VEGF).
In an alternate embodiment, the antibody or Fc fusion is
administered with a therapeutic agent that induces or enhances
adaptive immune response, for example an antibody that targets
CTLA-4. In an alternate embodiment, the antibody or Fc fusion is
administered with a tyrosine kinase inhibitor. By "tyrosine kinase
inhibitor" as used herein is meant a molecule that inhibits to some
extent tyrosine kinase activity of a tyrosine kinase. Examples of
such inhibitors include but are not limited to quinazolines, such
as PD 153035, 4-(3-chloroanilino) quinazoline; pyridopyrimidines;
pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP
60261 and CGP 62706; pyrazolopyrimidines,
4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloyl
methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines
containing nitrothiophene moieties; PD-0183805 (Warner-Lambert);
antisense molecules (e.g. those that bind to ErbB-encoding nucleic
acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S.
Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787
(Novartis/Schering A G); pan-ErbB inhibitors such as C1-1033
(Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate
(ST1571, Gleevec.RTM.); Novartis); PKI 166 (Novartis); GW2016
(Glaxo SmithKline); C1-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib
(Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG);
INC-1-C11 (Imclone); or as described in any of the following patent
publications: U.S. Pat. No. 5,804,396; PCT WO 99/09016 (American
Cyanimid); PCT WO 98/43960 (American Cyanamid); PCT WO 97/38983
(Warner-Lambert); PCT WO 99/06378 (Warner-Lambert); PCT WO 99/06396
(Warner-Lambert); PCT WO 96/30347 (Pfizer, Inc); PCT WO 96/33978
(AstraZeneca); PCT WO96/3397 (AstraZeneca); PCT WO 96/33980
(AstraZeneca), gefitinib (IRESSA.RTM., ZD1839, AstraZeneca), and
OSI-774 (Tarceva.RTM., OSI Pharmaceuticals/Genentech).
[0191] A variety of linkers find use in the present invention to
generate polypeptides (see definition above) or antibody--or
polypeptides--conjugates (see definition below). By "linker",
"linker sequence", "spacer", "tethering sequence" or grammatical
equivalents thereof, herein is meant a molecule or group of
molecules (such as a monomer or polymer) that connects two
molecules and often serves to place the two molecules in a
preferred configuration. A number of strategies may be used to
covalently link molecules together. These include, but are not
limited to polypeptide linkages between N- and C-termini of
proteins or protein domains, linkage via disulfide bonds, and
linkage via chemical cross-linking reagents. In one aspect of this
embodiment, the linker is a peptide bond, generated by recombinant
techniques or peptide synthesis. Choosing a suitable linker for a
specific case where two polypeptide chains are to be connected
depends on various parameters, including but not limited to the
nature of the two polypeptide chains (e.g., whether they naturally
oligomerize), the distance between the N- and the C-termini to be
connected if known, and/or the stability of the linker towards
proteolysis and oxidation. Furthermore, the linker may contain
amino acid residues that provide flexibility. Thus, the linker
peptide may predominantly include the following amino acid
residues: Gly, Ser, Ala, or Thr. The linker peptide should have a
length that is adequate to link two molecules in such a way that
they assume the correct conformation relative to one another so
that they retain the desired activity. Suitable lengths for this
purpose include at least one and not more than 30 amino acid
residues. Preferably, the linker is from about 1 to 30 amino acids
in length, with linkers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18 19 and 20 amino acids in length being
preferred. In addition, the amino acid residues selected for
inclusion in the linker peptide should exhibit properties that do
not interfere significantly with the activity of the polypeptide.
Thus, the linker peptide on the whole should not exhibit a charge
that would be inconsistent with the activity of the polypeptide, or
interfere with internal folding, or form bonds or other
interactions with amino acid residues in one or more of the
monomers that would seriously impede the binding of receptor
monomer domains. Useful linkers include glycine-serine polymers
(including, for example, (GS)n, (GSGGS)n, (GGGGS)n and (GGGS)n,
where n is an integer of at least one), glycine-alanine polymers,
alanine-serine polymers, and other flexible linkers such as the
tether for the shaker potassium channel, and a large variety of
other flexible linkers, as will be appreciated by those in the art.
Glycine-serine polymers are preferred since both of these amino
acids are relatively unstructured, and therefore may be able to
serve as a neutral tether between components. Secondly, serine is
hydrophilic and therefore able to solubilize what could be a
globular glycine chain. Third, similar chains have been shown to be
effective in joining subunits of recombinant proteins such as
single chain antibodies. Suitable linkers may also be identified by
screening databases of known three-dimensional structures for
naturally occurring motifs that can bridge the gap between two
polypeptide chains. In a preferred embodiment, the linker is not
immunogenic when administered in a human patient. Thus linkers may
be chosen such that they have low immunogenicity or are thought to
have low immunogenicity. For example, a linker may be chosen that
exists naturally in a human. In a preferred embodiment the linker
has the sequence of the hinge region of an antibody, that is the
sequence that links the antibody Fab and Fc regions; alternatively
the linker has a sequence that comprises part of the hinge region,
or a sequence that is substantially similar to the hinge region of
an antibody. Another way of obtaining a suitable linker is by
optimizing a simple linker, e.g., (Gly4Ser)n, through random
mutagenesis. Alternatively, once a suitable polypeptide linker is
defined, additional linker polypeptides can be created to select
amino acids that more optimally interact with the domains being
linked. Other types of linkers that may be used in the present
invention include artificial polypeptide linkers and inteins. In
another embodiment, disulfide bonds are designed to link the two
molecules. In another embodiment, linkers are chemical
cross-linking agents. For example, a variety of bifunctional
protein coupling agents may be used, including but not limited to
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 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., 1971, Science 238:1098. Chemical linkers may enable
chelation of an isotope. For example, Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody (see PCT WO 94/11026). The linker
may be cleavable, facilitating release of the cytotoxic drug in the
cell. For example, an acid-labile linker, peptidase-sensitive
linker, dimethyl linker or disulfide-containing linker (Chari et
al., 1992, Cancer Research 52: 127-131) may be used. Alternatively,
a variety of nonproteinaceous polymers, including but not limited
to polyethylene glycol (PEG), polypropylene glycol,
polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol, may find use as linkers, that is may find use
to link the Fc variants described herein to a fusion partner to
generate an Fc fusion, or to link the antibodies and polypeptides
described herein to a conjugate.
[0192] In one embodiment, an antibody or polypeptide described
herein is conjugated or operably linked to another therapeutic
compound, referred to herein as a conjugate. The conjugate may be a
cytotoxic agent, a chemotherapeutic agent, a cytokine, an
anti-angiogenic agent, a tyrosine kinase inhibitor, a toxin, a
radioisotope, or other therapeutically active agent.
Chemotherapeutic agents, cytokines, anti-angiogenic agents,
tyrosine kinase inhibitors, and other therapeutic agents have been
described above, and all of these aforemention therapeutic agents
may find use as antibody or Fc fusion conjugates. In an alternate
embodiment, the antibody or Fc fusion is conjugated or operably
linked to a toxin, including but not limited to small molecule
toxins and enzymatically active toxins of bacterial, fungal, plant
or animal origin, including fragments and/or variants thereof.
Small molecule toxins include but are not limited to calicheamicin,
maytansine (U.S. Pat. No. 5,208,020), trichothene, and CC1065. In
one embodiment of the invention, the antibody or polypeptide is
conjugated to one or more maytansine molecules (e.g. about 1 to
about 10 maytansine molecules per antibody molecule). Maytansine
may, for example, be converted to May-SS-Me which may be reduced to
May-SH3 and reacted with modified antibody or Fc fusion (Chari et
al., 1992, Cancer Research 52: 127-131) to generate a
maytansinoid-antibody or maytansinoid-Fc fusion conjugate. Another
conjugate of interest comprises an antibody or Fc fusion conjugated
to one or more calicheamicin molecules. The calicheamicin family of
antibiotics are capable of producing double-stranded DNA breaks at
sub-picomolar concentrations. Dolastatin 10 analogs such as
auristatin E (AE) and monomethylauristatin E (MMAE) may find use as
conjugates for the Fc variants described herein (Doronina et al.,
2003, Nat Biotechnol 21(7):778-84; Francisco et al., 2003 Blood
102(4):1458-65). Useful enzymatically active toxins include but are
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. See, for example, PCT WO 93/21232. The present
invention further contemplates a conjugate or fusion formed between
an antibody or Fc fusion described herein and a compound with
nucleolytic activity, for example a ribonuclease or DNA
endonuclease such as a deoxyribonuclease (DNase).
[0193] In an alternate embodiment, an Fc variant protein described
herein is conjugated or operably linked to a radioisotope to form a
radioconjugate. A variety of radioactive isotopes are available for
the production of radioconjugate antibodies and Fc fusions.
Examples include, but are not limited to, 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, and radioactive isotopes of Lu.
[0194] In yet another embodiment, an Fc varaint described herein is
conjugated to a "receptor" (such streptavidin) for utilization in
tumor pretargeting wherein the antibody-receptor or Fc
fusion-receptor conjugate is administered to the patient, followed
by removal of unbound conjugate from the circulation using a
clearing agent and then administration of a "ligand" (e.g. avidin)
which is conjugated to a cytotoxic agent (e.g. a radionucleotide).
In an alternate embodiment, the antibody or Fc fusion is conjugated
or operably linked to an enzyme in order to employ Antibody
Dependent Enzyme Mediated Prodrug Therapy (ADEPT). ADEPT may be
used by conjugating or operably linking the antibody or Fc fusion
to a prodrug-activating enzyme that converts a prodrug (e.g. a
peptidyl chemotherapeutic agent, see PCT WO 81/01145) to an active
anti-cancer drug. See, for example, PCT WO 88/07378 and U.S. Pat.
No. 4,975,278. The enzyme component of the immunoconjugate useful
for ADEPT includes any enzyme capable of acting on a prodrug in
such a way so as to covert it into its more active, cytotoxic form.
Enzymes that are useful in the method of this invention include but
are not limited to alkaline phosphatase useful for converting
phosphate-containing prodrugs into free drugs; arylsulfatase useful
for converting sulfate-containing prodrugs into free drugs;
cytosine deaminase useful for converting non-toxic 5-fluorocytosine
into the anti-cancer drug, 5-fluorouracil; proteases, such as
serratia protease, thermolysin, subtilisin, carboxypeptidases and
cathepsins (such as cathepsins B and L), that are useful for
converting peptide-containing prodrugs into free drugs;
D-alanylcarboxypeptidases, useful for converting prodrugs that
contain D-amino acid substituents; carbohydrate-cleaving enzymes
such as .beta.-galactosidase and neuramimidase useful for
converting glycosylated prodrugs into free drugs; beta-lactamase
useful for converting drugs derivatized with .alpha.-lactams into
free drugs; and penicillin amidases, such as penicillin V amidase
or penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used
to convert the prodrugs of the invention into free active drugs
(see, for example, Massey, 1987, Nature 328: 457-458).
Antibody-abzyme and Fc fusion-abzyme conjugates can be prepared for
delivery of the abzyme to a tumor cell population.
[0195] Other modifications of the Fc Variants comprise linking said
protein or poly-peptide to one of a variety of nonproteinaceous
polymers, e.g., polyethylene glycol (PEG), polypropylene glycol,
polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol.
[0196] Pharmaceutical compositions are encompassed wherein a Fc
variant protein or polypeptide or Fc fusion described herein and
one or more therapeutically active agents are formulated.
Formulations of the antibodies and Fc fusions described herein are
prepared for storage by mixing said antibody or Fc fusion having
the desired degree of purity with optional pharmaceutically
acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980), in the
form of lyophilized formulations or aqueous solutions. Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at
the dosages and concentrations employed, and include buffers such
as phosphate, citrate, acetate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl orbenzyl 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; sweeteners and other flavoring agents; fillers such as
microcrystalline cellulose, lactose, corn and other starches;
binding agents; additives; coloring agents; salt-forming
counter-ions such as sodium; metal complexes (e.g. Zn-protein
complexes); and/or non-ionic surfactants such as TWEEN.RTM.,
PLURONICS.RTM. or polyethylene glycol (PEG). In a preferred
embodiment, the pharmaceutical composition that comprises the
antibody or Fc fusion described herein is in a water-soluble form,
such as being present as pharmaceutically acceptable salts, which
is meant to include both acid and base addition salts.
"Pharmaceutically acceptable acid addition salt" refers to those
salts that retain the biological effectiveness of the free bases
and that are not biologically or otherwise undesirable, formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid and the like, and
organic acids such as acetic acid, propionic acid, glycolic acid,
pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic
acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic
acid, p-toluenesulfonic acid, salicylic acid and the like.
"Pharmaceutically acceptable base addition salts" include those
derived from inorganic bases such as sodium, potassium, lithium,
ammonium, calcium, magnesium, iron, zinc, copper, manganese,
aluminum salts and the like. Particularly preferred are the
ammonium, potassium, sodium, calcium, and magnesium salts. Salts
derived from pharmaceutically acceptable organic non-toxic bases
include salts of primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted
amines, cyclic amines and basic ion exchange resins, such as
isopropylamine, trimethylamine, diethylamine, triethylamine,
tripropylamine, and ethanolamine. The formulations to be used for
in vivo administration are preferrably sterile. This is readily
accomplished by filtration through sterile filtration membranes or
other methods.
[0197] In certain embodiments, the proteins and polypeptides
disclosed herein are formulated as immunoliposomes. A liposome is a
small vesicle comprising various types of lipids, phospholipids
and/or surfactant that is useful for delivery of a therapeutic
agent to a mammal. Liposomes containing the antibody or Fc fusion
are prepared by methods known in the art, such as described in
Epstein et al., 1985, Proc Natl Acad Sci USA, 82:3688; Hwang et
al., 1980, Proc Natl Acad Sci USA, 77:4030; U.S. Pat. No.
4,485,045; U.S. Pat. No. 4,544,545; and PCT WO 97/38731. Liposomes
with enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556. The components of the liposome are commonly arranged in
a bilayer formation, similar to the lipid arrangement of biological
membranes. Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. A chemotherapeutic agent or other therapeutically active
agent is optionally contained within the liposome (Gabizon et al.,
1989, J National Cancer Inst 81:1484).
[0198] In certain embodiments, the antibodies, polypeptides, and
other therapeutically active agents are entrapped in microcapsules
prepared by methods including but not limited to coacervation
techniques, interfacial polymerization (for example using
hydroxymethylcellulose or gelatin-microcapsules, or
poly-(methylmethacylate) microcapsules), colloidal drug delivery
systems (for example, liposomes, albumin microspheres,
microemulsions, nano-particles and nanocapsules), and
macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980.
Sustained-release preparations may be prepared. Suitable examples
of sustained-release preparations include semipermeable matrices of
solid hydrophobic polymer, which matrices are in the form of shaped
articles, e.g. films, or microcapsules. Examples of
sustained-release matrices include polyesters, hydrogels (for
example poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOTS (which are injectable microspheres composed of
lactic acid-glycolic acid copolymer and leuprolide acetate),
poly-D-(-)-3-hydroxybutyric acid, and ProLease.RTM.) (commercially
available from Alkermes), which is a microsphere-based delivery
system composed of the desired bioactive molecule incorporated into
a matrix of poly-DL-lactide-co-glycolide (PLG).
[0199] In embodiments, the concentration of the therapeutically
active antibody or polypeptide described herein in the formulation
varies from about 0.1 to 100 weight %. In one embodiment, the
concentration of the antibody or polypeptide is in the range of
0.003 to 1.0 molar. In order to treat a patient, a therapeutically
effective dose of the antibody or polypeptide described herein may
be administered. By "therapeutically effective dose" herein is
meant a dose that produces the effects for which it is
administered. The exact dose will depend on the purpose of the
treatment, and will be ascertainable by one skilled in the art
using known techniques. Dosages may range from 0.01 to 100 mg/kg of
body weight or greater, for example 0.1, 1, 10, or 50 mg/kg of body
weight, with 1 to 10 mg/kg being preferred. As is known in the art,
adjustments for antibody or polypeptide degradation, systemic
versus localized delivery, and rate of new protease synthesis, as
well as the age, body weight, general health, gender, diet, time of
administration, drug interaction and the severity of the condition
may be necessary, and will be ascertainable with routine
experimentation by those skilled in the art.
[0200] Administration of the pharmaceutical composition comprising
a protein or polypeptide described herein in the form of a sterile
aqueous solution, is done in a variety of ways, including, but not
limited to orally, subcutaneously, intravenously, intranasally,
intraotically, transdermally, topically (e.g., gels, salves,
lotions, creams, etc.), intraperitoneally, intramuscularly,
intrapulmonary (e.g., AERx.RTM. inhalable technology commercially
available from Aradigm, or Inhance.RTM. pulmonary delivery system
commercially available from Inhale Therapeutics), vaginally,
parenterally, rectally, or intraocularly. In some instances, for
example for the treatment of wounds, inflammation, etc., the
antibody or Polypeptide may be directly applied as a solution or
spray. As is known in the art, the pharmaceutical composition may
be formulated accordingly depending upon the manner of
introduction.
[0201] According to the teachings herein, a polypeptide can be
prepared with a variant Fc region which has improved, or
diminished, ADCC activity. Such molecules find applications in the
treatment of different disorders.
[0202] In certain embodiments, the polypeptide variant with
improved ADCC activity is employed in the treatment of diseases or
disorders where destruction or elimination of tissue or foreign
micro-organisms is desired. For example, the polypeptide may be
used to treat cancer; inflammatory disorders; infections (e.g.
bacterial, viral, fungal or yeast infections); and other conditions
(such as goiter) where removal of tissue is desired, etc.
[0203] Where the polypeptide variant has diminished ADCC activity,
such variants are used to treat diseases or disorders where a Fc
region-containing polypeptide with long half-life is desired, but
the polypeptide preferably does not have undesirable effector
function(s). For example, in an embodiment, the Fc
region-containing polypeptide is an anti-tissue factor (TF)
antibody; anti-IgE antibody; and anti-integrin antibody (e.g. an
anti-a.alpha.4.beta.7 antibody). The desired mechanism of action of
such Fc region-containing polypeptides may be to block
ligand-receptor binding pairs. Moreover, the Fc-region containing
polypeptide with diminished ADCC activity may be an agonist
antibody.
[0204] In an aspect are provided methods for generating Fc proteins
that are subsequently screened experimentally to single out
optimized Fc proteins. General methods for antibody molecular
biology, expression, purification, and screening are described in
Antibodies: A Laboratory Manual by Harlow & Lane, New York:
Cold Spring Harbor Laboratory Press, 1988.
[0205] One embodiment described herein relates to a rational design
process to identify Fc proteins with improved selectivity and
binding affinity to the Fc receptors. In one aspect, the invention
includes understanding the structural features associated with
dynamic properties which drive the interaction between the Fc and
its receptors. Another embodiment described herein provides a
method for identifying Fc protein polypeptides based on their
binding affinities to Fc.gamma.RIIa, Fc.gamma.RIIb, and
Fc.gamma.RIIIa, electrostatics, solvation, packing, packing
density, hydrogen binding networks and entropic effects associated
with the dynamic nature of the protein molecule. The method of the
current invention further provides for constructing the in silico
identified mutants by, for example site-directed mutagenesis and/or
de novo synthesis, and expression in mammalian cells for in vitro
validation.
[0206] In one embodiment described herein, the Fc protein
polypeptides identified in silico are reverse engineered to create
nucleic acids that encode the member sequences, that may then be
cloned into host cells, expressed and assayed, if desired. These
practices are carried out using well-known procedures. For example,
a variety of methods that may find use in the present invention are
described in Molecular Cloning--A Laboratory Manual, 3rd Ed.
(Maniatis, Cold Spring Harbor Laboratory Press, New York,
2001).
[0207] Fc proteins are screened using a variety of methods,
including but not limited to those that use in vitro assays, in
vivo and cell-based assays, and selection technologies. Automation
and high-throughput screening technologies may be utilized in the
screening procedures. Screening may employ the use of a fusion
partner or label.
[0208] Design Strategies
[0209] A rational design process was undertaken to design Fc
proteins with multiple amino acid substitutions that
synergistically provide enhanced selectivity and binding affinity
to a target Fc receptor. Central to this process is the
understanding of structural features and associated dynamic
properties which drive the interaction between the Fc and its
receptors. Starting with the known co-complex structure between the
Fc and the Fc.gamma.RIIIb, homology models were constructed for the
receptors of question: Fc.gamma.RIIa, Fc.gamma.RIIb, and
Fc.gamma.RIIIa. Applying a proprietary suite of in silico structure
and dynamics-based technologies (including but not limited to
ZymeCAD.TM. and ResidueNetworks.TM. (as described in WO
2009/098596, US20070276791 and US 20080147360)), unique insights
into the structure-function relationships of these protein-protein
interactions were identified and led to the proposal of unique
combinations of mutations which were predicted to preferentially
bind to various Fc receptors, and in certain examples, with
improved binding strength. The primary quantitative metric to
evaluate the Fc proteins is the binding energies. However, a number
of other parameters such as changes in electrostatics, solvation,
packing and packing density, hydrogen binding networks and entropic
effects associated with the dynamic nature of the protein molecule
are also employed in selecting attractive Fc proteins.
[0210] WO 2009/098596 provides methods and systems of determining
biopolymer profiles and correlations between structural units
(residues) of a biopolymer based on sampling of the conformational
space available to the molecule. The correlations between these
structural units are further used to find coupled residue networks
in the protein.
[0211] In one embodiment, the functional and/or biophysical
properties of Fc proteins are screened in an in vitro assay. Assays
may employ a variety of detection methods including but not limited
to chromogenic, fluorescent, luminescent, or isotopic labels. Assay
to detect ADCC include but are not limited to, ADCC reporter
assays, cytotoxicity assays, chromium release assay, europium
release assay, sulfur release assay, and flow cytometry. Examples
of well known assays can be found in the art, for example in
Stavenhagen et al. (2007) "Fc Optimization of Therapeutic
Antibodies Enhances Their Ability to Kill Tumor Cells In vitro and
Controls Tumor Expansion In vivo via low-affinity Activating
Fc.gamma. Receptors," Cancer Res. 67:882 and Stavenhagen et al.
(2008) "Enhancing the potency of therapeutic monoclonal antibodies
via Fc optimization," Adv. Enzyme Regul. 48:152.
[0212] The biological properties of the antibodies and polypeptides
that comprise the Fc proteins described herein may be characterized
in cell, tissue, and whole organism experiments. Drugs are often
tested in animals, including but not limited to mice, rats,
rabbits, dogs, cats, pigs, and monkeys, in order to measure a
drug's efficacy for treatment against a disease or disease model,
or to measure a drug's pharmacokinetics, toxicity, and other
properties. Therapeutics are often tested in mice, including but
not limited to nude mice, SCID mice, xenograft mice, and transgenic
mice (including knock-ins and knockouts).
[0213] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of this invention. All literature and patent
citations mentioned herein are expressly incorporated by
reference.
Example 1
Illustration of Rational Design of Polypeptides Having a
Combination of Mutations Selected for Producing Selected Fc.gamma.R
Binding Profile
[0214] Antibodies Targeting the HER2/Neu Receptor:
[0215] The Human Epidermal growth factor Receptors (HERs) are
proteins embedded in the cell membrane and communicate molecular
signals from outside the cell to inside the cell, and turn genes on
and off. The HER proteins regulate cell growth, survival, adhesion,
migration, and differentiation--functions that are amplified or
weakened in cancer cells. Human Epidermal growth factor Receptor
2''--a member of the epidermal growth factor receptor family, is a
protein giving higher aggressiveness in breast cancers. HER2/neu
has also been designated as CD340. Approximately 15-20 percent of
breast cancers have an amplification of the HER2/neu gene or
overexpression of its protein product. Overexpression of this
receptor in breast cancer is associated with increased disease
recurrence and worse prognosis. Because of its prognostic role as
well as its ability to predict response to trastuzumab, breast
tumors are routinely checked for overexpression of HER2/neu.
Overexpression also occurs in other cancer such as ovarian cancer,
stomach cancer, and biologically aggressive forms of uterine
cancer, such as uterine serous endometrial carcinoma.
[0216] Trastuzumab is a humanized monoclonal antibody that binds to
the domain IV of the extracellular segment of the HER2/neu
receptor. Cells treated with trastuzumab undergo arrest during the
G1 phase of the cell cycle so there is reduced proliferation. It
has been suggested that trastuzumab induces some of its effect by
downregulation of HER2/neu leading to disruption of receptor
dimerization and signaling through the downstream PI3K cascade.
P27Kip1 is then not phosphorylated and is able to enter the nucleus
and inhibit cdk2 activity, causing cell cycle arrest. Also,
trastuzumab suppresses angiogenesis by both induction of
antiangiogenic factors and repression of proangiogenic factors. It
is thought that a contribution to the unregulated growth observed
in cancer could be due to proteolytic cleavage of HER2/neu that
results in the release of the extracellular domain. Trastuzumab has
been shown to inhibit HER2/neu ectodomain cleavage in breast cancer
cells.
Design of a Polypeptide Comprising a Combination of Modifications
that Produce a Selected Fc.gamma.R Binding Profile:
[0217] Some of the proteins and polypeptides disclosed herein are
designed based on the impact of selected amino acid substitutions
to the protein interactions and dynamics, which are indicative of
enthalpic and entropic changes, respectively. Relative changes to
protein binding characteristics, especially compared to the
wild-type system, can be optimized.
[0218] In this example, a polypeptide is designed with the goal of
selecting amino acid mutations that increase binding selectivity to
the Fc.gamma.RIIIa receptor when compared to a wild-type antibody
(Trastuzumab/Herceptin.RTM.). The rational design performed
resulted in a quadruple variant (S239E/S298A/K326A/A327H). In the
following example, the main strategies of introducing
Fc.gamma.RIIIa-specific electrostatic interactions and adding
steric repulsion to both Fc.gamma.RIIa and Fc.gamma.RIIb receptors
are highlighted such that the selectivity design goal was achieved.
In some cases, the mutations' contribution to binding on one chain
of the Fc is different than the other chain and these
chain-specific effects are also demonstrated. The specific effects
to binding as a result of each of the four mutations are discussed
with the binding profile of the Fc variant being an overall
synergistic combination of the structural and dynamic changes.
A327H as a Steric Selectivity Driver:
[0219] His327 is a much bulkier group compared to the wild-type
alanine and this substitution is a main selectivity driver that has
minimal impact on the Fc.gamma.RIIIa receptor while being
detrimental for Fc.gamma.RIIa and Fc.gamma.RIIb. As shown in FIG.
1, the binding interface and the protein-protein interactions with
the Fc.gamma.RIIIa are preserved with the addition of His327 when
compared to the wild-type Fc, which suggests that binding will not
be affected between these two partners. The primary factor for the
interface stabilization is due to the presence of the sidechain
hydroxyl group from Tyr132 in Fc.gamma.RIIIa. This non-conserved
moiety is not present in Fc.gamma.RIIa and hence the addition of
His327 resulted in a major destabilization of the binding
interface, most notably with the rapid dynamics observed for the
receptor's neighboring His134 residue (FIG. 1). It is anticipated
that binding to the IIa receptor will be severely disrupted.
[0220] While Fc.gamma.RIIIa and Fc.gamma.RIIa share a conserved
His134 residue that is differentially affected with the addition of
His327 to the Fc, Fc.gamma.RIIb contains of an even bulkier Arg134.
While this residue is also perturbed with the His134 mutation, it
is however able to adopt a pseudo-favorable conformation that is
stabilized with a serine sidechain on the Fc (FIG. 1). It is
expected that binding of the Fc to the IIb receptor will be reduced
but not abolished because of the stabilized Arg rotomer.
S239E as an Electrostatic General Stabilizer and a Selectivity
Driver:
[0221] The mutation S239E displays Fc chain-specific interactions
to the receptors thereby facilitating both of its roles as a
general stabilizing mutation as well as a selectivity driver for
the Fc.gamma.RIIIa receptor.Glu239 on chain-B of the Fc (FIG. 1) is
a general stabilizing residue across all three receptors as it is
able to form a hydrogen bond with conserved Lys120 residues.
General stabilizing residues are beneficial in protein variant
design as they ensure that the integrity of the binding interfaces
is maintained when numerous mutations are introduced within
confined areas.
[0222] The S239E mutation, when introduced on chain-A of the Fc
(FIG. 2) is a selectivity driver for improved Fc.gamma.RIIIa
binding as it is able to form a bifurcated electrostatic
interaction with the non-conserved Lys161 while the enhanced
binding interaction is not possible with the Thr161 in the
Fc.gamma.RIIa and IIb receptors.
S298A as Both a Steric and Electrostatic Selectivity Driver
[0223] The Ser298 residue in the wild-type antibody Fc interacts
with Ser129 in the Fc.gamma.RIIa and IIb receptors via hydrogen
bonding while the receptors' Lys128 is stabilized intramolecularly
and is not involved in any binding interactions (FIG. 3). The S298A
mutation removes one of the key electrostatic binding partners
between the Fc and the receptors and is therefore expected to
drastically reduce binding to Fc.gamma.RIIa and IIb receptors.
[0224] The wild-type Fc binds to the Fc.gamma.RIIIa receptor using
a different mechanism, with the interaction mediated by the
backbone carbonyl group of Ser298 on the Fc with Lys128 from the
receptor, which is able to partially occupy the binding interface.
The sidechain flexibility and motions required for this interaction
was demonstrated using molecular dynamics simulations (FIG. 3). The
Fc.gamma.RIIIa's Lys128 is `locked` and pre-positioned in the
binding conformation when the S298A mutation is introduced in the
Fc.
Synergistic Combination of Mutations
[0225] By combining the above computational and structural insights
in the Fc design process; the present disclosure provides a new
variant that has, relative to the wild-type Fc, improved
Fc.gamma.RIIIa binding while having reduced binding to
Fc.gamma.RIIb and substantially abolished binding to Fc.gamma.RIIa.
As illustrated by in vitro binding assays with the resulting
binding profile shown in FIG. 4.
[0226] These results also demonstrate the unexpected synergistic
nature of these mutations (FIG. 4). With the single S239E mutation,
it is shown that the variants cannot clearly differentiate binding
between the highly homologous receptors. With the addition of the
S298A/K326A/A327H mutation combination, Fc.gamma.RIIIa selectivity
was improved.
Example 2
In Vitro and Ex Vivo Validation of Designed Antibodies
[0227] Once designed in silico individual antibodies are tested
according to the methods described in Stavenhagen et al. (2007)
Cancer Res. 67:882 and Stavenhagen et al. (2008), Adv. Enzyme
Regal., 48:152.
[0228] Briefly, the gene for each mutant is constructed by standard
chemical synthesis using, for example, Trastuzumab IgG1 as the
wild-type framework. After cloning into a suitable vector, the
mutant Fc polypeptides are expressed in mammalian HEK293 cells. The
Fc.gamma.RIIa, Fc.gamma.RIIb and Fc.gamma.RIIIa are also cloned and
expressed in HEK293 cells. The binding affinities of the antibodies
to each of the three receptors is then determined by surface
plasmon resonance.
[0229] Surface Plasmon Resonance Analysis:
[0230] Affinity of Fc.gamma. receptors to antibody Fc was measure
by SPR (surface Plasmon resonance) using a ProteOn XPR36 system
from BIO-RAD. HER-2 in buffer (10 mM Hepes pH 6.8) was immobilized
on CM5 chip through amine coupling until 3000 RU. Fc variants in an
antibody format containing anti HER2 F(ab)2 were immobilized to the
HER-2 surface to 300 RU. Running buffer and the surfactant was
maintained at pH 6.8. Purified analyte FcR was diluted in its
running buffer and injected at a flow rate of 20-30 mul/min for 2
minutes, followed by dissociation for another 4 minutes. Five
twofold dilutions of each antibody beginning at 20 nM were analyzed
in triplicate. Sensograms were fit globally to a 1:1 Langmuir
binding model. All experiments were conducted at room temperature.
The in vitro binding Kd determined by SPR for each variant is shown
in Table 1.
[0231] Antibody-Dependent Cellular Cytotoxicity Analysis:
[0232] The SKBR-3 cells were used as target cells in these
experiments. A fresh vial of cryopreserved SKBR3 cells was thawed
and a robust culture established. The SKBR3 cells were maintained
in McKoy's medium enriched with 10% Fetal Bovine Serum and 1%
PenStrep. The cells were passaged regularly upon attaining
.about.75% confluence (approximately every 3-4 days). To verify the
binding of the variant antibodies to the target cells (SKBR3),
binding curves for all variants and positive control (Herceptin)
were obtained. SKBR-3 cells were washed with PBS and resuspended in
FACS (Fluorescence-activated cell sorting) tubes at
2.times.10.sup.5 cells per tube in 100 .mu.l volume of PBS/1% BSA.
Antibodies were added to the tubes to achieve 0.1, 1 and 10
.mu.g/ml final concentration, cells incubated on ice for 1 hour,
washed with 1% BSA/PBS and resuspended in 1:200 dilution of
FITC-conjugated anti-human IgG. Cells were incubated on ice for
another 40 min, washed again and resuspended in 200 .mu.l of PBS.
10 .mu.l of 10 mg/ml Propidium Iodide were added to each tube, and
samples were analyzed by FACS. FACS gates were set to exclude dead
cells (PI+). The Mean Fluorescence Intensity ("MFI") for each
sample was measured. Cells stained with secondary but not primary
antibody were used as negative control. As measured from the
fold-difference in mean fluorescence intensity ("MFI"), all
variants demonstrated levels of binding comparable to
Herceptin.
[0233] To establish an effector:target cell (E:T) ratio and
duration of incubation to achieve suitable cell killing, a
preliminary ADCC experiment was performed using Herceptin as a
positive control. SKBR-3 cells were seeded in flat bottom 96 well
plates a day prior to experiment, at 2.times.10.sup.4 cells per
well in 200 .mu.l of culture media. PBMCs were purified from fresh
buffy coats from five different donors using Ficoll gradient
centrifugation, washed 3 times with PBS and resuspended in RPMI
containing 10% Heat Inactivated FBS and 10 ng/ml IL-2. Twenty four
hours later, three wells containing SKBR-3 cells were trypsinized
to verify cell count and to determine the exact number of PBMCs
required to achieve the desired E:T ratio. Target cells were
labeled with 10 .mu.g/ml CFSE immediately prior of assay. Antibody
(Herceptin) was added to cells at 1 and 10 .mu.g/ml and incubated
for 15 min. PBMCs at 10:1, 50:1 and 100:1 effector:target (E:T)
ratio were then added to corresponding wells, and plates were
briefly spun down at low RPM to concentrate cells in the bottom of
the wells. The plates were then incubated in a standard tissue
culture incubator for four, eight and twenty four hours. Following
treatment, cells were harvested and added to 400 .mu.l PBS
containing propidium iodide ("PI"), a viability stain, and
immediately analyzed by FACS. The extent of ADCC activity was
determined by measuring the frequency of PI.sup.+ green cells
(killed targets) as the fraction of total target cells (PI.sup.-
and PI.sup.+ green cells). The pilot ADCC experiment demonstrated
significant ADCC activity of Herceptin in SKBR3 cells. At 24 hours,
higher E:T ratios (50:1 and 100:1) appeared to result in some
antibody independent cell death. Because the killing of the cells
at antibody concentrations of 1 and 10 .mu.g/ml is
indistinguishable, ADCC activity appears to be saturating above 1
.mu.g/ml. Optimal results were observed at 24 hours with an E:T
ratio of 10:1.
[0234] For the subsequent ADCC experiment, PBMCs from five buffy
coats were purified using Ficoll gradient centrifugation. Following
centrifugation and washes, cells were resuspended in 100 ml of
pre-warmed RPMI. The cells were counted, and viability was
determined by the Trypan Blue exclusion. An aliquot of cells from
each donor was cryopreseved for future use. An additional aliquot
of cells was used for FACS-based genotyping for the 158V/F CD16
polymorphism using a two-antibody staining protocol that exploits
the fact that the G38 anti-CD16 antibody binds with equal affinity
to both V and F alleles and comparing that with staining using the
MEM-154 anti-CD16 antibody that has a lower affinity for the V
allele than for the F allele (S. Bottcher et al., 2005, Journal of
Immunological Methods, Volume 306, Issues 1-2, pp 128-136). Based
on the CD16 genotyping results, samples from three donors
heterozygous in the F/V, high cell viability and no endogenous cell
killing were selected. All data points were obtained in triplicate
wells and the viability for each donor was normalized to the
viability of SKBR-3 cells incubated with PBMCs of the same donor,
but without test antibody. The average ADCC results for these
samples are summarized in Table 1.
[0235] As seen from table 1, the ex vivo ADCC assay revealed that a
designed variants had significantly lowered ADCC than the wild-type
antibody. This shows that the variations described herein are
useful in modulating ADCC.
TABLE-US-00001 TABLE 1 Results from SPR and ADCC assay for
different designed antibodies. ex vivo in vitro Binding Kd [M] in
silico .DELTA..DELTA.G solv [kcal/mol] ADCC assay IIIa (F) IIa (H)
IIb IIIa (F) IIa (H) IIb Relative EC50 Mutations Compared to
Wildtype Trastuzumab variant variant variant variant variant
variant [ug/mL] Wildtype Trastuzumab 1.00E-06 4.62E-07 5.41E-07 +++
+++ +++ 1.0 G237F S239E A327H -- -- 1.58E-06 8.19E-06 1.16E-06 ++++
+++ ++ 0.7 G237F A327L A330I -- -- NB NB 1.46E-06 ++ ++++ ++++ 0.0
S239E A327L A330I -- -- 1.47E-06 NB 6.15E-06 +++++ ++ + ND L235A
S239E D265E A327H -- NB NB NB +++++ +++++ + 0.0 S239E S267E H268D
-- -- 1.75E-07 6.42E-07 1.61E-08 +++++ +++++ +++++ 2.1 G237F S239E
D270N -- -- NB NB NB ++ + + 1.0 G236E G237F S239E -- -- ND NB
8.56E-07 +++ + + ND S239E D265S H268D I332E -- ND 4.44E-06 4.16E-06
++++ + + ND S239E D265S I332E -- -- 2.46E-06 NB NB ++++ +++ + ND
G237F S239E D265E -- -- NB NB 2.95E-06 +++++ +++++ + ND G237F S239E
H268D -- -- 5.94E-07 7.40E-07 6.84E-08 +++ +++++ +++++ 0.7 S239D
D265S S298A I332E 1.04E-06 NB 4.01E-06 +++ ++ +++++ ND S239E S298A
K326A A327H -- 3.33E-07 NB 2.71E-06 +++++ ++ ++ ND G237F S298A
A330L I332E -- 4.29E-07 NB 7.63E-06 +++ +++ ++++ 5.3 G236E D270N
A327V I332E -- NB NB NB +++++ + + ND H268E D270E S267G -- --
1.27E-06 5.31E-07 5.83E-07 +++++ +++++ +++++ ND H268D K326A A327H
-- -- 1.33E-06 1.10E-06 2.71E-07 ++++ ++++ +++++ ND H268D E269L
S298A K326A A327H 2.43E-06 NB 6.17E-06 + ++ +++++ ND D265E S267D
A330S -- -- NB NB 1.59E-06 + +++++ ++++ 0.0 +++++ <-5 kcal/mol;
++++ -5 to -2 kcal/mol; +++ Between -2 and 2 kcal/mol; ++ 2 to 5
kcal/mol; + >5 kcal/mol NB: no binding; ND: Not Determined.
Relative EC50: >1 = Enhanced ADCC; <1 = Supressed ADCC; ND =
Not Determined
Example 3
[0236] Additional antibodies comprising modifications based on the
in silico methods described above are summarized in Table 2.
TABLE-US-00002 TABLE 2 in silico .DELTA..DELTA.G solv [kcal/mol]
IIIa (F) IIIa (V) IIa (H) IIa (R) IIb (F) IIb (Y) Mutations variant
variant variant variant variant variant WT Herceptin -- -- -- -- --
+++ +++ +++ +++ +++ +++ D270L Y300L A330K -- -- + + +++++ ++++ ++++
+++ G236A S239D D270L I332E -- +++++ +++++ ++++ ++ +++++ +++++
G237F S267G N325F -- -- + +++ +++ + ++ + G237F V266L S267D -- -- ++
++ +++ ++++ +++++ ++++ L234F S267G N325L -- -- ++ + + + +++ +++
L234F S267E N325L -- -- +++ + + + +++++ ++++ +++++ <-5 kcal/mol;
++++ -5 to -2 kcal/mol; +++ Between -2 and 2 kcal/mol; ++ 2 to 5
kcal/mol; + >5 kcal/mol NB: no binding; ND: Not Determined
Example 4
Treatment of Non-Hodgkins Lymphoma Using Anti-CD20 Antibodies
[0237] An antibody comprising a combination of modifications as
described herein is administered to a patient having low-grade or
follicular NHL. The recommended dosage for patients with low-grade
or follicular NHL is 375 mg/m 2 infused i.v. at weekly intervals
for a total of four doses. In a majority of patients, this can be
accomplished in an outpatient clinic over a 22-day period.
[0238] Just before administration, the antibody preparation is
diluted with 5% dextrose in water or 0.9% sodium chloride injection
to a final concentration of 1-4 mg/mL. A 1-mg/mL dilution is
preferable to facilitate adjustments in the infusion and to avoid
adverse effects potentially caused by inadvertently rapid
administration. Prepared infusions are stable in polyvinyl chloride
or polyethylene bags at 2-8.degree. C. (36-46.degree. F.) for 24
hours and at room temperature for an additional 12 hours. Unused
portions of undiluted drug must be discarded because of the absence
of a preservative.
[0239] Thirty to 60 minutes before each infusion, acetaminophen
650-1000 mg and diphenhydramine hydrochloride 50-100 mg can be
administered to help prevent infusion-related effects. Infusion may
be through either a central or peripheral i.v. catheter but should
never be given by i.v. push or bolus injection, owing to the risk
of potentially serious infusion-related adverse effects. Before
beginning each infusion, the administering clinician must prime the
i.v. tubing with drug-containing solution to guarantee that active
drug rather than other solution is being infused from the
beginning. The first infusion should be initiated at 50 mg/hr with
the rate increased by 50 mg/hr every 30 minutes as tolerated until
a maximum rate of 400 mg/hr is reached. Subsequent infusions may be
started at 100 mg/hr, with 100-mg/hr increases every 30 minutes as
tolerated until the maximum rate (400 mg/hr) is attained. In
patients with a large tumor burden (white blood cell count,
>25,000/mm 3), an initial infusion rate of 25 mg/hr should be
considered.
Example 5
Treatment of Systemic Lupus Erythematosus in Serologically-Active
Patients
[0240] All subjects receive anti-CD22 antibodies incorporating the
Fc polypeptide modifications described herein monthly, with loading
doses on days 8 and 15 of month one, until disease progression or
subject discontinuation. The drug is prepared in a 10 mg/ml
prepared in 17.5 vials. Administration is carried out by slow
intravenous infusion using PBS as a vehicle/buffer for the infusion
procedures. All patients were given 1200 mg antibody given in 2
doses every other week in 12 week treatment cycles. Assessments of
the patients treated were accomplished through a combined response
index analysis evaluating BILAG, SLEDAI, and a physician's global
assessment and treatment failure status.
Example 6
Treatment of BLL Using Anti-CD22 Antibodies
[0241] The dosage used is 120-1000-mg/m2 by infusion with diffuse
large B-cell lymphoma.
[0242] Sub-Cutaneous Administration of Antibodies comprising the
amino acid modifications described herein
[0243] The subcutaneous immunoglobulin is infused in the
subcutaneous tissue on the abdomen twice or thrice a week, with a
maximal speed of 2 mL/h. Every time 20 mL is infused, the needle is
removed to a new place.
Example 7
Administration of Immunoglobulin to Control Chronic Disease Such as
Type B Hepatitis
[0244] Continuous monthly administration of anti-hepatitis B
antibodies incorporating the Fc polypeptides described herein at
5000 IU a month as a treatment results in reduced serum levels of
the patient's own anti-hepatitis B antibodies and decreased
symptoms of liver disease.
Example 8
Treatment of Organ Rejection Using Anti-CD20 Antibodies
[0245] Treatment of organ transplant patients is done either
weekly, biweekly, bi-monthly, or monthly, by infusion of anti-CD20
antibodies comprising the amino acid modifications provided herein
from 100 to 1000 mg/m2, according the the above protocol for
infusion and pretreatment.
[0246] The polypeptides and methods disclosed herein are used,
without limitation to develop antibodies and polypeptides based on
synergistic improvements to the Fc region of the following
antibodies: Bevacizumab (Avastin); Abciximab (ReoPro); Adalimumab
(Humira); Alemtuzumab (Campath); Cetuximab (Erbitux); Efalizumab
(Raptiva); Etanercept (Enbrel); Gemtuzumab oxogamicin (Mylotarg);
Infliximab (Remicade); Natalizumab (Tysabri, aka Antegren);
Omalizumab (Xolair); Palivizumab (Syangis); Rituximab (Rituxan);
Trastuzumab (Herceptin); Golimumab (Simponi); Panitumumab
(Vectibix); Canakinumab (ILARIS); Ustekinumab (Stelara); Denosumab
(Prolia); Ofatumumab (Arzerra)
[0247] While specific embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. It should be understood that various alternatives to
the embodiments of the invention described herein may be employed
in practicing the invention. It is intended that the following
claims define the scope of the invention and that methods and
structures within the scope of these claims and their equivalents
be covered thereby.
* * * * *