U.S. patent application number 10/658521 was filed with the patent office on 2005-03-10 for human antibodies derived from immunized xenomice.
Invention is credited to Brenner, Daniel G., Capon, Daniel J., Jakobovits, Aya, Klapholz, Sue, Kucherlapati, Raju.
Application Number | 20050054055 10/658521 |
Document ID | / |
Family ID | 31999910 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050054055 |
Kind Code |
A1 |
Kucherlapati, Raju ; et
al. |
March 10, 2005 |
Human antibodies derived from immunized xenomice
Abstract
Fully human antibodies against a specific antigen can be
prepared by administering the antigen to a transgenic animal which
has been modified to produce such antibodies in response to
antigenic challenge, but whose endogenous loci have been disabled.
Various subsequent manipulations can be performed to obtain either
antibodies per se or analogs thereof.
Inventors: |
Kucherlapati, Raju; (Darien,
CT) ; Jakobovits, Aya; (Menlo Park, CA) ;
Brenner, Daniel G.; (Redwood City, CA) ; Capon,
Daniel J.; (Hillsborough, CA) ; Klapholz, Sue;
(Stanford, CA) |
Correspondence
Address: |
FISH & NEAVE LLP
1251 AVENUE OF THE AMERICAS
50TH FLOOR
NEW YORK
NY
10020-1105
US
|
Family ID: |
31999910 |
Appl. No.: |
10/658521 |
Filed: |
September 8, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10658521 |
Sep 8, 2003 |
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09614092 |
Jul 11, 2000 |
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6713610 |
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09614092 |
Jul 11, 2000 |
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08724752 |
Oct 2, 1996 |
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6150584 |
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08724752 |
Oct 2, 1996 |
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08430938 |
Apr 27, 1995 |
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08430938 |
Apr 27, 1995 |
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08234145 |
Apr 28, 1994 |
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08234145 |
Apr 28, 1994 |
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08112848 |
Aug 27, 1993 |
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08112848 |
Aug 27, 1993 |
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08031801 |
Mar 15, 1993 |
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6673986 |
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08031801 |
Mar 15, 1993 |
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07919297 |
Jul 24, 1992 |
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07919297 |
Jul 24, 1992 |
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07610515 |
Nov 8, 1990 |
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07610515 |
Nov 8, 1990 |
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07466008 |
Jan 12, 1990 |
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Current U.S.
Class: |
435/70.21 |
Current CPC
Class: |
C12N 15/87 20130101;
C07K 16/2875 20130101; C07K 2317/24 20130101; A61K 38/00 20130101;
C12N 15/90 20130101; C07K 16/00 20130101; C07K 16/1282 20130101;
C07K 16/2812 20130101; C07K 16/248 20130101; A01K 2217/00 20130101;
C12N 15/8509 20130101; C12N 2800/206 20130101; A01K 2207/15
20130101; C07K 16/22 20130101; C07K 16/244 20130101; A01K 2217/30
20130101; C07K 16/241 20130101; C07K 16/2854 20130101; A01K
2217/075 20130101; C07K 16/462 20130101; C07K 2317/21 20130101;
C12N 2840/203 20130101; A01K 2267/01 20130101; A01K 2217/05
20130101 |
Class at
Publication: |
435/070.21 |
International
Class: |
C12P 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 29, 1996 |
WO |
PCT/US96/05928 |
Claims
1. A method to produce a human immunoglobulin or an analog thereof,
specific for a desired antigen, which method comprises:
administering said antigen or an immunogenic portion thereof to a
nonhuman animal under conditions to stimulate an immune response,
whereby said animal produces B cells that secrete immunoglobulin
specific for said antigen; wherein said nonhuman animal is
characterized by being substantially incapable of producing
endogenous heavy and light immunoglobulin chains, but capable of
producing human immunoglobulin; and recovering said immunoglobulin
or analog.
2. Canceled.
3. The method of claim 1 wherein said recovering step comprises
immortalizing B cells from said animal immunized with said antigen,
screening the resulting immortalized cells for the secretion of
said immunoglobulin specific for said antigen, and a) recovering
immunoglobulin secreted by said immortalized B cells, or b)
recovering the genes encoding at least the immunoglobulin from the
immortalized B cells, and optionally modifying said genes;
expressing said genes or modified forms thereof to produce
immunoglobulin or analog; and recovering said immunoglobulin or
analog.
4. The method of claim 1 wherein said recovering step comprises:
recovering genes encoding the immunoglobulins from the primary B
cells of the animal; generating a library of said genes expressing
the immunoglobulins; screening the library for an immunoglobulin
with the desired affinity for the antigen; recovering the genes
encoding the immunoglobulin; expressing said recovered genes to
produce an immunoglobulin or analog recovering said immunoglobulin
or analog.
5. A recombinant DNA molecule comprising a nucleotide sequence
encoding the immunoglobulin or analog produced by the method of
claim 1.
6. Canceled.
7. A cell or cell line modified to contain the DNA molecule of
claim 5.
8. A method to produce a fully human immunoglobulin or an analog
thereof which method comprises culturing the cells of claim 7 under
conditions whereby said encoding nucleotide sequence is expressed
to produce said immunoglobulin or analog; and recovering said
immunoglobulin or analog.
9. A DNA molecule comprising a nucleotide sequence corresponding to
the gene or modified gene prepared by the method of claim 3.
10. Canceled.
11. A cell or cell line modified to contain the DNA molecule of
claim 9.
12. A method to produce a fully human immunoglobulin or an analog
thereof which method comprises culturing the cells of claim 11
under conditions whereby said encoding nucleotide sequence is
expressed to produce said immunoglobulin or analog; and recovering
said immunoglobulin or analog.
13. A DNA molecule which comprises a nucleotide sequence encoding a
human immunoglobulin with desired affinity prepared according to
the method of claim 4.
14. Canceled.
15. A cell or cell line modified to contain the DNA molecule of
claim 13.
16. A method to produce a fully human immunoglobulin or an analog
thereof which method comprises culturing the cells of claim 15
under conditions whereby said encoding nucleotide sequence is
expressed to produce said immunoglobulin or analog; and recovering
said immunoglobulin or analog.
17. An immortalized B cell which secretes a fully human
immunoglobulin to a desired antigen prepared according to claim
3.
18. A method to produce an immunoglobulin or analog which comprises
culturing the cells of claim 17 and recovering said immunoglobulin
or analog.
19. A fully human immunoglobulin or analog produced by the method
of claim 1.
20. The immunoglobulin or analog of claim 19 wherein the desired
antigen is selected from the group consisting of the leukocyte
markers, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a,b,c, CD13, CD14,
CD18, CD19, CD20, CD22, CD23, CD27 and its ligand, CD28 and its
ligands B7.1, B7.2, B7.3, CD29 and its ligand, CD30 and its ligand,
CD40 and its ligand gp39, CD44, CD45 and isoforms, CDw52 (Campath
antigen), CD56, CD58, CD69, CD72, CTLA-4, LFA-1 and TCR; the
histocompatibility antigens, MHC class I or II, the Lewis Y
antigens, SLex, SLey, SLea, and SLeb; the integrins, VLA-1, VLA-2,
VLA-3, VLA-4, VLA-5, VLA-6, .alpha.V.beta.3, and LFA-1, Mac-1, and
pl50,95, .alpha..sub.V.beta..sub.1, gpIIbIIIa,
.alpha..sub.R.beta..sub.3, .alpha..sub.6.beta..sub.4,
.alpha..sub.V.beta..sub.5, .alpha..sub.V.beta..sub.6, and
.alpha..sub.V62 .sub.7; the selectins, L-selectin, P-selectin, and
E-selectin and their counterreceptors VCAM-1, ICAM-1, ICAM-2, and
LFA-3; the interleukins, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15; the
interleukin receptor is selected from the group consisting of
IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R,
IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, and IL-15R; the chemokine
is selected from the group consisting of PF4, RANTES, MIP1.alpha.,
MCP1, NAP-2, Gro.alpha., Gro.beta., and IL-8; the growth factor is
selected from the group consisting of TNFalpha, TGFbeta, TSH,
VEGF/VPF, PTHrP, EGF family, FGF, PDGF family, endothelin, Fibrosin
(F.sub.SF.sub.-1), human Laminin, and gastrin releasing peptide
(GRP); the growth factor receptor is selected from the group
consisting of TNFalphaR, RGFbetaR, TSHR, VEGFR/VPFR, FGFR, EGFR,
PTHrPR, PDGFR family, EPO-R, GCSF-R and other hematopoietic
receptors; the interferon receptor is selected from the group
consisting of IFNC.alpha.R, IFN.beta.R, and IFN.lambda.R; the Ig
and its receptor is selected from the group consisting of IgE,
FceRI, and FCERII; the tumor antigen is selected from the group
consisting of her2-neu, mucin, CEA and endosialin; the allergen is
selected from the group consisting of house dust mite antigen, lol
p1 (grass) antigens, and urushiol; the viral protein is selected
from the group consisting of CMV glycoproteins B, H, and gCIII,
HIV-1 envelope glycoproteins, RSV envelope glycoproteins, HSV
envelope glycoproteins, HPV envelope glycoproteins, Hepatitis
family surface antigens; the toxin is selected from the group
consisting of pseudomonas endotoxin and osteopontin/uropontin,
snake venom, spider venom, and bee venom conotoxin; the blood
factor is selected from the group consisting of complement C3b,
complement C4a, complement C4b-9, Rh factor, fibrinogen, fibrin,
and myelin associated growth inhibitor; and the enzyme is selected
from the group consisting of cholesterol ester transfer protein,
membrane bound matrix metalloproteases, and glutamic acid
decarboxylase (GAD).
21. Canceled.
22. A recombinant DNA molecule comprising a nucleotide sequence
that encodes the immunoglobulin or analog of claim 19.
23. Canceled.
24. A cell or cell line modified to contain the DNA molecule of
claim 22.
25. A method to produce an immunoglobulin or analog specific for a
desired antigen which method comprises culturing the cell or cell
line of claim 24 under conditions wherein said nucleotide sequence
is expressed to produce said immunoglobulin or analog; and
recovering the immunoglobulin or analog.
26. An human antibody or analog thereof which is specifically
immunoreactive with an antigen selected from the group consisting
of transition state mimics; leukocyte markers; histocompatibility
antigens; adhesion molecules; interleukins; interleukin receptors;
chemokines; growth factors; growth factor receptors; interferon
receptors; Igs and their receptors, tumor antigens; allergens;
viral proteins; toxins; blood factors; enzymes; and the
miscellaneous antigens ganglioside GD3, ganglioside GB2, LMP1,
LMP2, eosinophil major basic protein, eosinophil cationic protein,
PANCA, Amadori protein, Type IV collagen, glycated lipids,
.lambda.-interferon, A7, P-glycoprotein, Fas (AFO-1) and
oxidized-LDL.
27. The antibody or analog of claim 26 wherein the leukocyte marker
is selected from the group consisting of CD2, CD3, CD4, CD5, CD6,
CD7, CD8, CD11a,b,c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27
and its ligand, CD28 and its ligands B7.1, B7.2, B7.3, CD29 and its
ligand, CD30 and its ligand, CD40 and its ligand gp39, CD44, CD45
and isoforms, CDw52 (Campath antigen), CD56, CD58, CD69, CD72,
CTLA-4, LFA-1 and TCR; the histocompatibility antigen is selected
from the group consisting of MHC class I or II, the Lewis y
antigens, SLex, SLey, Slea, and SLeb; the adhesion molecule is
selected from the group consisting of VLA-1, VLA-2, VLA-3, VLA-4,
VLA-5, VLA-6, .alpha.V.beta.3, and LFA-1, Mac-1, p150,95,
.alpha..sub.V.beta..sub.1, gpIIbIIIa, .alpha..sub.R.beta..sub.3,
.alpha..sub.6.beta..sub.4, .alpha..sub.V.beta..sub.5,
.alpha..sub.V.beta..sub.6, and .alpha..sub.V.beta..sub.7,
L-selectin, P-selectin, and E-selectin and their counterreceptors
VCAM-1, ICAM-1, ICAM-2, and LFA-3; the interleukin is selected from
the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15; the
interleukin receptor is selected from the group consisting of
IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R,
IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, and IL-15R, the chemokine
is selected from the group consisting of PF4, RANTES, MIP1.alpha.,
MCP1, NAP-2, Gro.alpha., Gro.beta., and IL-8; the growth factor is
selected from the group consisting of TNFalpha, TGFbeta, TSH,
VEGF/VPF, PthrP, EGF family, FGF, PDGF family, endothelia, Fibrosin
(F.sub.SF.sub.-1), human Laminin, and gastrin releasing peptide
(GRP); the growth factor receptor is selected from the group
consisting of TNFalphaR, RGFbetaR, TSHR, VEGFR/VPFR, FGFR, EGFR,
PTHrPR, PDGFR family, EPO-R, GCSF-R and other hematopoietic
receptors; the interferon receptor is selected from the group
consisting of IFN.alpha.R, IFN.beta.R, and IFN.gamma.R; the Ig and
its receptor is selected from the group IgE, FceRI, and FCeRII;
tumor antigen is selected from the group her2-neu, mucin, CEA and
endosialin; the allergen is selected from the group consisting of
house dust mite antigen, lol p1 (grass) antigens, and urushiol; the
viral protein is selected from the group consisting of CVM
glycoproteins B, H, and GCIII, HIV-1 envelope glycoproteins, RSV
envelope glycoproteins, HSV envelope glycoproteins, EBV envelope
glycoproteins, VZV envelope glycoproteins, HPV envelope
glycoproteins, Hepatitis family surface antigens; the toxin is
selected from the group consisting of pseudomonas endotoxin and
osteopontin/uropontin, snake venom, and bee venom; the blood factor
is selected from the group consisting of complement C3b, complement
C5a, complement C5b-9, RH factor, fibrinogen, fibrin, and myelin
associated growth inhibitor; and the enzyme is selected from the
group consisting of cholesterol ester transfer protein, membrane
bound matrix metalloproteases, and glutamic acid decarboxylase
(GAD).
28. Canceled.
29. Canceled.
30. Canceled.
31. Canceled.
32. Canceled.
33. Canceled.
34. Canceled.
35. Canceled.
36. Canceled.
37. Canceled.
38. Canceled.
39. Canceled.
40. Canceled.
41. A recombinant DNA molecule encoding the antibody of any of
claim 26.
42. Canceled.
43. A recombinant host cell which is modified to contain the DNA
molecule of claim 41.
44. A method to produce an antibody or analog which method
comprises culturing cells of claim 43 under conditions wherein said
coding sequence is expressed; and recovery the antibody of analog
produced.
45. An isolated human antibody or an antigen-binding fragment
thereof that specifically binds a leukocyte marker selected from
the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a,
b, c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27, CD28, CD29,
CD30, CD40, CD44, CD45 and isoforms, Cdw52 (Campath antigen), CD56,
CD58, CD69, CD72, CTLA-4, LFA-1, and TCR, wherein said antibody or
fragment modulates the activity of said leukocyte marker.
46. The antibody or fragment according to claim 45 wherein the
leukocyte marker is CD4.
47. The antibody or fragment according to claim 45 herein the
leukocyte marker is CD8.
48. The antibody or fragment according to claim 45 wherein the
leukocyte marker is CD28.
49. The antibody or fragment according to claim 45 wherein the
leukocyte marker is CD40.
50. The antibody or fragment according to claim 45 wherein the
leukocyte marker is CD45.
51. The antibody or fragment according to claim 45 wherein the
leukocyte marker is TCR.
52. The antibody according to any one of claims 45-51, wherein the
antibody is monoclonal.
53. The fragment according to any one of claims 45-51, wherein the
fragment comprises an scFv, Fab, Fab', or F(ab').sub.2
fragment.
54. The antibody according to any one of claims 45-51, wherein the
antibody is detectably labeled.
55. The antibody according to any one of claims 45-51, wherein the
leukocyte marker is human.
56. The antibody according to any one of claims 45-51, wherein the
antibody decreases activity of the leukocyte marker.
57. The antibody according to any one of claims 45-51, wherein the
antibody comprises lambda light chain sequence.
58. The antibody according to any one of claims 45-51, wherein the
antibody increases an activity of the leukocyte marker.
59. The antibody according to any one of claims 45-51, further
comprising a pharmaceutical formulation.
60. A host cell that expresses the antibody according to any one of
claims 45-51.
61. A nucleic acid that encodes the antibody according to any one
of claims 45-51.
62. A host cell comprising the nucleic acid of claim 61.
63. A method of producing an isolated human antibody that
specifically binds and modulates the activity of a leukocyte marker
selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7,
CD8, CD11a, b, c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27,
CD28, CD29, CD30, CD40, CD44, CD45 and isoforms, Cdw52 (Campath
antigen), CD56, CD58, CD69, CD72, CTLA-4, LFA-1, and TCR
comprising: (a) administering the leukocyte marker or an
immunogenic fragment thereof to a mouse capable of expressing human
immunoglobulin; (b) screening the administered mouse for expression
of a human antibody that specifically binds to the leukocyte
marker; (c) selecting a mouse that produces a human antibody that
specifically binds to the leukocyte marker; (d) isolating an
antibody from the mouse that produces a human antibody that
specifically binds to the leukocyte marker; and (e) determining
whether the antibody modulates an activity of the leukocyte marker
thereby producing a human antibody that specifically binds to the
leukocyte marker and modulates an activity of the leukocyte
marker.
64. A method of producing an isolated human antibody that
specifically binds to and modulates the activity of a leukocyte
marker selected from the group consisting of CD2, CD3, CD4, CD5,
CD6, CD7, CD8, CD11a, b, c, CD13, CD14, CD18, CD19, CD20, CD22,
CD23, CD27, CD28, CD29, CD30, CD40, CD44, CD45 and isoforms, Cdw52
(Campath antigen), CD56, CD58, CD69, CD72, CTLA-4, LFA-1, and TCR
comprising: (a) administering the leukocyte marker or an
immunogenic fragment thereof to a mouse capable of expressing human
immunoglobulin; (b) isolating spleen cells from the mouse that
produces a human antibody that specifically binds to the leukocyte
marker; (c) fusing the spleen cells with a myeloma cell to produce
a hybridoma; and (d) screening the hybridoma for expression of a
human antibody that specifically binds to and modulates an activity
of the leukocyte marker thereby producing a human monoclonal
antibody that specifically binds to and modulates an activity of
the leukocyte marker.
65. The method according to claim 63 or 64 wherein the leukocyte
marker is CD4.
66. The method according to claim 63 or 64 wherein the leukocyte
marker is CD8.
67. The method according to claim 63 or 64 wherein the leukocyte
marker is CD28.
68. The method according to claim 63 or 64 wherein the leukocyte
marker is CD40.
69. The method according to claim 63 or 64 wherein the leukocyte
marker is CD45.
70. The method according to claim 63 or 64 wherein the leukocyte
marker is TCR.
71. A monoclonal antibody isolated from a hybridoma produced by the
method of claim 64.
72. A method for modulating an activity of a leukocyte marker
selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7,
CD8, CD11a, b, c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27,
CD28, CD29, CD30, CD40, CD44, CD45 and isoforms, Cdw52 (Campath
antigen), CD56, CD58, CD69, CD72, CTLA-4, LFA-1, and TCR comprising
contacting a cell that expresses the leukocyte marker with a
modulating amount of the antibody of claim 45.
73. The method according to claim 72 wherein the leukocyte marker
is CD4.
74. The method according to claim 72 wherein the leukocyte marker
is CD8.
75. The method according to claim 72 wherein the leukocyte marker
is CD28.
76. The method according to claim 72 wherein the leukocyte marker
is CD40.
77. The method according to claim 72 wherein the leukocyte marker
is CD45.
78. The method according to claim 72 wherein the leukocyte marker
is TCR.
79. The method of claim 72, wherein the leukocyte marker is
human.
80. The method of claim 72, wherein the activity is increased.
81. The method of claim 72, wherein the activity is decreased.
82. A method of increasing an activity of a leukocyte marker
selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7,
CD8, CD11a, b, c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27,
CD28, CD29, CD30, CD40, CD44, CD45 and isoforms, Cdw52 (Campath
antigen), CD56, CD58, CD69, CD72, CTLA-4, LFA-1, and TCR in a
subject comprising administering to the subject an amount of a
human antibody that increases an activity of the leukocyte
marker.
83. A method of decreasing an activity of a leukocyte marker
selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7,
CD8, CD11a, b, c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27,
CD28, CD29, CD30, CD40, CD44, CD45 and isoforms, Cdw52 (Campath
antigen), CD56, CD58, CD69, CD72, CTLA-4, LFA-1, and TCR in a
subject comprising administering to the subject an amount of a
human antibody that decreases an activity of the leukocyte
marker.
84. A method of detecting the presence of a leukocyte marker
selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7,
CD8, CD11a, b, c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27,
CD28, CD29, CD30, CD40, CD44, CD45 and isoforms, Cdw52 (Campath
antigen), CD56, CD58, CD69, CD72, CTLA-4, LFA-1, and TCR in a
sample or a cell, comprising contacting a sample having or
suspected of having the leukocyte marker, or a cell expressing or
suspected of expressing the leukocyte marker, with the antibody of
claim 1, and detecting the presence of the leukocyte marker in the
sample or cell.
85. The method according to claim 82, 83 or 84, wherein the
leukocyte marker is CD4.
86. The method according to claim 82, 83 or 84 wherein the
leukocyte marker is CD8.
87. The method according to claim 82, 83 or 84, wherein the
leukocyte marker is CD28.
88. The method according to claim 82, 83 or 84, wherein the
leukocyte marker is CD40.
89. The method according to claim 82, 83 or 84, wherein the
leukocyte marker is CD45.
90. The method according to claim 82, 83 or 84, wherein the
leukocyte marker is TCR.
91. The method of claim 84, wherein the cell is in a subject.
92. A method of detecting the presence of a disorder associated
with increased or decreased expression of a a leukocyte marker
selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7,
CD8, CD11a, b, c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27,
CD28, CD29, CD30, CD40, CD44, CD45 and isoforms, Cdw52 (Campath
antigen), CD56, CD58, CD69, CD72, CTLA-4, LFA-1, and TCR in a
human, comprising contacting a sample having or suspected of having
the leukocyte marker or a cell expressing or suspected of
expressing the leukocyte marker, wherein the sample or cell is from
or present in the human, with the human antibody of claim 45 and
detecting the presence of increased or decreased expression of the
leukocte marker in the sample or cell relative to a control thereby
detecting the presence of a disorder associated with increased or
decreased expression of the leukocyte marker in the human.
93. The method according to claim 92, wherein the leukocyte marker
is CD4.
94. The method according to claim 92, wherein the leukocyte marker
is CD8.
95. The method according to claim 92, wherein the leukocyte marker
is CD28.
96. The method according to claim 92, wherein the leukocyte marker
is CD40.
97. The method according to claim 92, wherein the leukocyte marker
is CD45.
98. The method according to claim 92, wherein the leukocyte marker
is TCR.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims benefit under 35 U.S.C.
.sctn. 120 as a continuation-in-part of U.S. patent application
Ser. No. 08/430,938, filed Apr. 27, 1995, which is a
continuation-in-part of U.S. patent application Ser. Nos.
08/234,143, 08/112,848, 08/031,801, 07/919,297, 07/610,515, and
07/466,008 (filed Apr. 28, 1994, Aug. 27, 1993, Mar. 15, 1993, Jul.
24, 1992, Nov. 8, 1990 and Jan. 12, 1990, respectively). The
present application also claims benefit under 35 U.S.C. .sctn. 119
to PCT/US96/05928, filed Apr. 29, 1996. The disclosures of each of
the aforementioned applications are hereby incorporated by
reference in their entirety.
TECHNICAL FIELD
[0002] The invention relates to the field of immunology, and in
particular to the production of antibodies. More specifically, it
concerns producing such antibodies by a process which includes the
step of immunizing a transgenic animal with an antigen to which
antibodies are desired. The transgenic animal has been modified so
as to produce human, as opposed to endogenous, antibodies.
BACKGROUND ART
[0003] PCT application WO 94/02602, published 3 Feb. 1994 and
incorporated herein by reference, describes in detail the
production of transgenic nonhuman animals which are modified so as
to produce fully human antibodies rather than endogenous antibodies
in response to antigenic challenge. Briefly, the endogenous loci
encoding the heavy and light immunoglobulin chains are
incapacitated in the transgenic hosts and loci encoding human heavy
and light chain proteins are inserted into the genome. In general,
the animal which provides all the desired modifications is obtained
by cross breeding intermediate animals containing fewer than the
full complement of modifications. The preferred embodiment of
nonhuman animal described in the specification is a mouse. Thus,
mice, specifically, are described which, when administered
immunogens, produce antibodies with human variable regions,
including fully human antibodies, rather than murine antibodies
that are immunospecific for these antigens.
[0004] The availability of such transgenic animals makes possible
new approaches to the production of fully human antibodies.
Antibodies with various immunospecificities are desirable for
therapeutic and diagnostic use. Those antibodies intended for human
therapeutic and in vivo diagnostic use, in particular, have been
problematic because prior art sources for such antibodies resulted
in immunoglobulins bearing the characteristic structures of
antibodies produced by nonhuman hosts. Such antibodies tend to be
immunogenic when used in humans.
[0005] The availability of the nonhuman, immunogen responsive
transgenic animals described in the above-referenced WO 94/02602
make possible convenient production of human antibodies without the
necessity of employing human hosts.
DISCLOSURE OF THE INVENTION
[0006] The invention is directed to methods to produce human
antibodies by a process wherein at least one step of the process
includes immunizing a transgenic nonhuman animal with the desired
antigen. The modified animal fails to produce endogenous
antibodies, but instead produces B-cells which secrete fully human
immunoglobulins. The antibodies produced can be obtained from the
animal directly or from immortalized B-cells derived from the
animal. Alternatively, the genes encoding the immunoglobulins with
human variable regions can be recovered and expressed to obtain the
antibodies directly or modified to obtain analogs of antibodies
such as, for example, single chain F.sub.v molecules.
[0007] Thus, in one aspect, the invention is directed to a method
to produce a fully human immunoglobulin to a specific antigen or to
produce an analog of said immunoglobulin by a process which
comprises immunizing a nonhuman animal with the antigen under
conditions that stimulate an immune response. The nonhuman animal
is characterized by being substantially incapable of producing
endogenous heavy or light immunoglobulin chain, but capable of
producing immunoglobulins with both human variable and constant
regions. In the resulting immune response, the animal produces B
cells which secrete immunoglobulins that are fully human and
specific for the antigen. The human immunoglobulin of desired
specificity can be directly recovered from the animal, for example,
from the serum, or primary B cells can be obtained from the animal
and immortalized. The immortalized B cells can be used directly as
the source of human antibodies or, alternatively, the genes
encoding the antibodies can be prepared from the immortalized B
cells or from primary B cells of the blood or lymphoid tissue
(spleen, tonsils, lymph nodes, bone marrow) of the immunized animal
and expressed in recombinant hosts, with or without modifications,
to produce the immunoglobulin or its analogs. In addition, the
genes encoding the repertoire of immunoglobulins produced by the
immunized animal can be used to generate a library of
immunoglobulins to permit screening for those variable regions
which provide the desired affinity. Clones from the library which
have the desired characteristics can then be used as a source of
nucleotide sequences encoding the desired variable regions for
further manipulation to generate antibodies or analogs with these
characteristics using standard recombinant techniques.
[0008] In another aspect, the invention relates to an immortalized
nonhuman B cell line derived from the above described animal. In
still another aspect, the invention is directed to a recombinant
host cell which is modified to contain the gene encoding either the
human immunoglobulin with the desired specificity, or an analog
thereof which exhibits the same specificity.
[0009] In still other aspects, the invention is directed to
antibodies or antibody analogs prepared by the above-described
methods and to recombinant materials for their production.
[0010] In still other aspects, the invention is directed to
antibodies which are immunospecific with respect to particular
antigens set forth herein and to analogs which are similarly
immunospecific, as well as to the recombinant materials useful to
production of these antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a schematic of the construction of the yH1C human
heavy chain YAC.
[0012] FIG. 2 is a schematic of the construction of the yK2 human
kappa light chain YAC.
[0013] FIG. 3 shows the serum titers of anti-IL-6 antibodies from a
XenoMouse.TM. immunized with human IL-6 and which antibodies
contain human .kappa.n light chains and/or human .mu. heavy
chains.
[0014] FIG. 4 show the serum titers of anti-TNF.alpha. antibodies
from a XenoMouse.TM. immunized with human TNF-.alpha. and which
antibodies contain human .kappa. light chains and/or human .mu.
heavy chains.
[0015] FIG. 5 shows serum titers of anti-CD4 antibodies from a
XenoMouse.TM. immunized with human CD4 and which antibodies contain
human .kappa. light chains and/or human .mu. heavy chains.
[0016] FIG. 6 shows the serum titers of a XenoMouse.TM. immunized
with 300.19 cells expressing L-selectin at their surface. In the
ELISA assay used, these antibodies are detectable if they carry
human .mu. constant region heavy chains.
[0017] FIG. 7 shows the serum titers of a XenoMouse.TM. immunized
with 300.19 cells expressing L-selectin at their surface. In the
ELISA assay used, these antibodies are detectable only if they
carry human .kappa. light chains.
[0018] FIG. 8 shows a FACS Analysis of human neutrophils incubated
with serum from a XenoMouse.TM. immunized with human L-selectin and
labeled with an antibody immunoreactive with human light chain
.kappa. region.
[0019] FIG. 9 shows a diagram of a plasmid used to transfect
mammalian cells to effect the production of the human protein
gp39.
[0020] FIG. 10 represents the serum titration curve of mice
immunized with CHO cells expressing human gp39. The antibodies
detected in this ELISA must be immunoreactive with gp39 and contain
human heavy chain .mu. constant regions of human .kappa. light
chains.
[0021] FIG. 11 is a titration curve with respect to monoclonal
antibodies secreted by the hybridoma clone D5.1. This clone is
obtained from a XenoMouse.TM. immunized with tetanus toxin C (TTC)
and contains human .kappa. light chain and human .mu. constant
region in the heavy chain.
[0022] FIG. 12 DNA sequence of the heavy chain of anti tetanus
toxin monoclonal antibody D5.1.4 (a subclone of D5.1). Mutations
form germline are boxed.
[0023] FIG. 13 DNA sequence of the kappa light chain of
anti-tetanus toxin monoclonal antibody D5.1.4. Mutations form
germline are boxed.
[0024] FIG. 14 shows the serum titers of anti-IL-8 antibodies of
XenoMouse.TM. immunized with human IL-8 and which antibodies
contain human .kappa. light chains and/or human .mu. heavy
chains.
[0025] FIG. 15 Inhibition of IL-8 binding to human neutrophils by
monoclonal anti-human-IL-8 antibodies.
[0026] FIG. 16(A-H) DNA sequences of the heavy chain and kappa
light chain of the anti-IL-8 antibodies D1.1 (16A-B), K2.2 (16C-D),
K4.2 (16E-F), and K4.3 (16G-H).
MODES OF CARRYING OUT THE INVENTION
[0027] In general, the methods of the invention include
administering an antigen for which human forms of immunospecific
reagents are desired to a transgenic nonhuman animal which has been
modified genetically so as to be capable of producing human, but
not endogenous, antibodies. Typically, the animal has been modified
to disable the endogenous heavy and/or kappa light chain loci in
its genome, so that these endogenous loci are incapable of the
rearrangement required to generate genes encoding immunoglobulins
in response to an antigen. In addition, the animal will have been
provided, stably, in its genome, at least one human heavy chain
locus and at least one human light chain locus so that in response
to an administered antigen, the human loci can rearrange to provide
genes encoding human variable regions immunospecific for the
antigen.
[0028] The details for constructing such an animal useful in the
method of the invention are provided in the PCT application WO
94/02602 referenced above. Examples of YACs for the present
invention can be found in, for example, Green et al. Nature
Genetics 7:13-21 (1994). In a preferred embodiment of the
XenoMouse.TM., the human heavy chain YAC, yH1C (1020 kb), and human
light chain YAC, yK2 (880 kb) are used. yH1C is comprised of 870 kb
of the human variable region, the entire D and J.sub.H region,
human .mu., .delta., and .gamma.2 constant regions and the mouse 3'
enhancer. yK2 is comprised of 650 kb of the human kappa chain
proximal variable region (V.sub..kappa.), the entire J.kappa.
region, and C.kappa. with its flanking sequences that contain the
Kappa deleting element (.kappa.de). Both YACs also contain a human
HPRT selectable marker on their YAC vector arm. Construction of
yH1C and yK2 was accomplished by methods well known in the art. In
brief, YAC clones bearing segments of the human immunoglobulin loci
were identified by screening a YAC library (Calbertsen et al, PNAS
87:4256 (1990)) Overlapping clones were joined by recombination
using standard techniques (Mendez et al. Genomics 26:294-307
(1995)). Details of the schemes for assembling yH1C and yK2 are
shown in FIG. 1 and FIG. 2 respectively.
[0029] yK2 was constructed from the clones A80-C7, A210-F10 and
A203-C6 from the Olson library, disclosed in, for example, Burke et
al., Science 236:806-812 (1987), Brownstein et al., Science
244:1348-1351 (1989), and Burke et al ., Methods in Enzymology
194:251-270 (1991).
[0030] For production of the desired antibodies, the first step is
administration of the antigen. Techniques for such administration
are conventional and involve suitable immunization protocols and
formulations which will depend on the nature of the antigen per se.
It may be necessary to provide the antigen with a carrier to
enhance its immunogenicity and/or to include formulations which
contain adjuvants and/or to administer multiple injections and/or
to vary the route of the immunization, and the like. Such
techniques are standard and optimization of them will depend on the
characteristics of the particular antigen for which immunospecific
reagents are desired.
[0031] As used herein, the term "immunospecific reagents" includes
immunoglobulins and their analogs. The term "analogs" has a
specific meaning in this context. It refers to moieties that
contain the fully human portions of the immunoglobulin which
account for its immunospecificity. In particular, complementarity
determining regions (CDRs) are required, along with sufficient
portions of the framework (Frs) to result in the appropriate three
dimensional conformation. Typical immunospecific analogs of
antibodies include F(ab").sub.2, Fab', and Fab regions. Modified
forms of the variable regions to obtain, for example, single chain
F.sub.v analogs with the appropriate immunospecificity are known. A
review of such F.sub.V construction is found, for example, in
Huston et al., Methods in Enzymology 203:46-63 (1991). The
construction of antibody analogs with multiple immunospecificities
is also possible by coupling the variable regions from one antibody
to those of second antibody.
[0032] The variable regions with fully human characteristics can
also be coupled to a variety of additional substances which can
provide toxicity, biological functionality, alternative binding
specificities and the like. The moieties including the fully human
variable regions produced by the methods of the invention include
single-chain fusion proteins., molecules coupled by covalent
methods other than those involving peptide linkages, and aggregated
molecules. Examples of analogs which include variable regions
coupled to additional molecules covalently or noncovalently include
those in the following nonlimiting illustrative list. Traunecker,
A. et al. Int. J. Cancer Supp (1992) Supp 7:51-52 describe the
bispecific reagent janusin in which the F.sub.v region directed to
CD3 is coupled to soluble CD4 or to other ligands such as OVCA and
IL-7. Similarly, the fully human variable regions produced by the
method of the invention can be constructed into F.sub.v molecules
and coupled to alternative ligands such as those illustrated in the
cited article. Higgins, P. J. et al J. Infect Disease (1992)
166:198-202 described a heteroconjugate antibody composed of OKT3
cross-linked to an antibody directed to a specific sequence in the
V3 region of GP120. Such heteroconjugate antibodies can also be
constructed using at least the human variable regions contained in
the immunoglobulins produced by the invention methods. Additional
examples of bispecific antibodies include those described by
Fanger, M. W. et al. Cancer Treat Res (1993) 68:181-194 and by
Fanger M. W. et al. Crit Rev Immunol (1992) 12:101-124. Conjugates
that are immunotoxins including conventional antibodies have been
widely described in the art. The toxins may be coupled to the
antibodies by conventional coupling techniques or immunotoxins
containing protein toxin portions can be produced as fusion
proteins. The analogs of the present invention can be used in a
corresponding way to obtain such immunotoxins. Illustrative of such
immunotoxins are those described by Byers, B. S. et al. Seminars
Cell Biol (1991) 2:59-70 and by Fanger, M. W. et al. Immunol Today
(1991) 12:51-54.
[0033] It will also be noted that some of the immunoglobulins and
analogs of the invention will have agonist activity with respect to
antigens for which they are immunospecific in the cases wherein the
antigens perform signal transducing functions. Thus, a subset of
antibodies or analogs prepared according to the methods of the
invention which are immunospecific for, for example, a cell surface
receptor, will be capable of eliciting a response from cells
bearing this receptor corresponding to that elicited by the native
ligand. Furthermore, antibodies or analogs which are immunospecific
for substances mimicking transition states of chemical reactions
will have catalytic activity. Hence, a subset of the antibodies and
analogs of the invention will function as catalytic antibodies.
[0034] In short, the genes encoding the immunoglobulins produced by
the transgenic animals of the invention can be retrieved and the
nucleotide sequences encoding the fully human variable region can
be manipulated according to known techniques to provide a variety
of analogs such as those described above. In addition, the
immunoglobulins themselves containing the human variable regions
can be modified using standard coupling techniques to provide
conjugates retaining ittimunospecific regions.
[0035] Thus, immunoglobulin "analogs" refers to the moieties which
contain those portions of the antibodies of the invention which
retain their human characteristics and their immunospecificity.
These will retain sufficient human variable regions to provide the
desired specificity.
[0036] It is predicted that the specificity of antibodies (i.e.,
the ability to generate antibodies to a wide spectrum of antigens
and indeed to a wide spectrum of independent epitopes thereon) is
dependent upon the variable region genes on the heavy chain
(V.sub.H) and kappa light chain (V.sub.78 ) genome. The human heavy
chain genome includes approximately 82 genes which encode variable
regions of the human heavy chain of immunoglobulin molecules. In
addition, the human light chain genome includes approximately 40
genes on its proximal end which encode variable regions of the
human kappa light chain of immunoglobulin molecules. We have
demonstrated that the specificity of antibodies can be enhanced
through the inclusion of a plurality of genes encoding variable
light and heavy chains.
[0037] In preferred embodiments, therefore, greater than 10% of
V.sub.H and V.sub.78 genes are utilized. More preferably, greater
than 20%, 30%, 40%, 50%, 60% or even 70% or greater of V.sub.H and
V.sub.78 genes are utilized. In a preferred embodiment, constructs
including 32 genes on the proximal region of the V.sub..kappa.
light chain genome are utilized and 66 genes on the V.sub.H portion
of the genome are utilized. As will be appreciated, genes may be
included either sequentially, i.e., in the order found in the human
genome, or out , i.e., in an order other than that found in the
human genome, or a combination thereof. Thus, by way of example, an
entirely sequential portion of either the V.sub.H or V.sub..kappa.,
genome can be utilized, or various V genes in either the V.sub.H H
or V.sub..kappa., genome can be skipped while maintaining an
overall sequential arrangement, or V genes within either the
V.sub.H or V.sub..kappa. genome can be reordered, and the like. In
any case, it is expected and the results described herein
demonstrate that the inclusion of a diverse array of genes from the
V.sub.H and V.sub..kappa. genome leads to enhanced antibody
specificity and ultimately to enhanced antibody affinities.
[0038] With respect to affinities, antibody affinity rates and
constants derived through utilization of plural V.sub.H and
V.sub..kappa. genes (i.e., the use of 32 genes on the proximal
region of the V.sub..kappa. light chain genome and 66 genes on the
V.sub.H portion of the genome) results in association rates (Ka in
M.sup.-1S.sup.-1) of greater than about 0.50.times.10.sup.-6,
preferably greater than 2.00.times.10.sup.-6, and more preferably
greater than about 4.00.times.10.sup.-6; dissociation rates (kd in
S.sup.-1) of greater than about 1.00.times.10.sup.-4, preferably
greater than about 2.00.times.10.sup.-4, and more preferably
greater than about 4.00.times.10.sup.-4; and dissociation constant
(in M) of greater than about 1.00.times.10.sup.-10, preferably
greater than about 2.00.times.10.sup.-10, and more preferably
greater than about 4.00.times.10.sup.-10.
[0039] As stated above, all of the methods of the invention include
administering the appropriate antigen to the transgenic animal. The
recovery or production of the antibodies themselves can be achieved
in various ways.
[0040] First, and most straightforward, the polyclonal antibodies
produced by the animal and secreted into the bloodstream can be
recovered using known techniques. Purified forms of these
antibodies can, of course, be readily prepared by standard
purification techniques, preferably including affinity
chromatography with Protein A, anti-immunoglobulin, or the antigen
itself. In any case, in order to monitor the success of
immunization, the antibody levels with respect to the antigen in
serum will be monitored using standard techniques such as ELISA,
RIA and the like.
[0041] For some applications only the variable regions of the
antibodies are required. Treating the polyclonal antiserum with
suitable reagents so as to generate Fab', Fab, or F(ab").sub.2
portions results in compositions retaining fully human
characteristics. Such fragments are sufficient for use, for
example, in immunodiagnostic procedures involving coupling the
immunospecific portions of immunoglobulins to detecting reagents
such as radioisotopes.
[0042] Alternatively, immunoglobulins and analogs with desired
characteristics can be generated from immortalized B cells derived
from the transgenic animals used in the method of the invention or
from the rearranged genes provided by these animals in response to
immunization.
[0043] Thus, as an alternative to harvesting the antibodies
directly from the animal, the B cells can be obtained, typically
from the spleen, but also, if desired, from the peripheral blood
lymphocytes or lymph nodes and immortalized using any of a variety
of techniques, most commonly using the fusion methods described by
Kohler and Milstein Nature 245:495 (1975) The resulting hybridomas
(or otherwise immortalized B cells) can then be cultured as single
colonies and screened for secretion of antibodies of the desired
specificity. As described above, the screen can also include a
confirmation of the fully human character of the antibody. For
example, as described in the examples below, a sandwich ELISA
wherein the monoclonal in the hybridoma supernatant is bound both
to antigen and to an antihuman constant region can be employed.
After the appropriate hybridomas are selected, the desired
antibodies can be recovered, again using conventional techniques.
They can be prepared in quantity by culturing the immortalized B
cells using conventional methods, either in vitro or in vivo to
produce ascites fluid. Purification of the resulting monoclonal
antibody preparations is less burdensome that in the case of serum
since each immortalized colony will secrete only a single type of
antibody. In any event, standard purification techniques to isolate
the antibody from other proteins in the culture medium can be
employed.
[0044] As an alternative to obtaining human immunoglobulins
directly from the culture of immortalized B cells derived from the
animal, the immortalized cells can be used as a source of
rearranged heavy chain and light chain loci for subsequent
expression and/or genetic manipulation. Rearranged antibody genes
can be reverse transcribed from appropriate mRNAs to produce cDNA.
If desired, the heavy chain constant region can be exchanged for
that of a different isotype or eliminated altogether. The variable
regions can be linked to encode single chain F.sub.v regions.
Multiple F.sub.v regions can be linked to confer binding ability to
more than one target or chimeric heavy and light chain combinations
can be employed. Once the genetic material is available, design of
analogs as described above which retain both their ability to bind
the desired target, and their human characteristics, is
straightforward.
[0045] Once the appropriate genetic material is obtained and, if
desired, modified to encode an analog, the coding sequences,
including those that encode, at a minimum, the variable regions of
the human heavy and light chain, can be inserted into expression
systems contained on vectors which can be transfected into standard
recombinant host cells. As described below, a variety of such host
cells may be used; for efficient processing, however, mammalian
cells are preferred. Typical mammalian cell lines useful for this
purpose include CHO cells, 293 cells, or NSO cells.
[0046] The production of the antibody or analog is then undertaken
by culturing the modified recombinant host under culture conditions
appropriate for the growth of the host cells and the expression of
the coding sequences. The antibodies are then recovered from the
culture. The expression systems are preferably designed to include
signal peptides so that the resulting antibodies are secreted into
the medium; however, intracellular production is also possible.
[0047] In addition to deliberate design of modified forms of the
immunoglobulin genes to produce analogs, advantage can be taken of
phage display techniques to provide libraries containing a
repertoire of antibodies with varying affinities for the desired
antigen. For production of such repertoires, it is unnecessary to
immortalize the B cells from the immunized animal; rather, the
primary B cells can be used directly as a source of DNA. The
mixture of cDNAs obtained from B cells, e.g., derived from spleens,
is used to prepare an expression library, for example, a phage
display library transfected into E. coli. The resulting cells are
tested for immunoreactivity to the desired antigen. Techniques for
the identification of high affinity human antibodies from such
libraries are described by Griffiths, A. D., et al., EMBO J (1994)
13:3245-3260; by Nissim, A., et al. ibid, 692-698, and by
Griffiths, A. D., et al., ibid, 12:725-734. Ultimately, clones from
the library are identified which produce binding affinities of a
desired magnitude for the antigen, and the DNA encoding the product
responsible for such binding is recovered and manipulated for
standard recombinant expression. Phage display libraries may also
be constructed using previously manipulated nucleotide sequences
and screened in similar fashion. In general, the cDNAs encoding
heavy and light chain are independently supplied or are linked to
form F.sub.v analogs for production in the phage library.
[0048] The phage library is then screened for the antibodies with
highest affinity for the antigen and the genetic material recovered
from the appropriate clone. Further rounds of screening can
increase the affinity of the original antibody isolated. The
manipulations described above for recombinant production of the
antibody or modification to form a desired analog can then be
employed.
[0049] Combination of phage display technology with the
XenoMouse.TM. offers a significant advantage over previous
applications of phage display. Typically, to generate a highly
human antibody by phage display, a combinatorial antibody library
is prepared either from human bone marrow or from peripheral blood
lymphocytes as described by Burton, D. R., et al., Proc. Natl.
Acad. Sci. USA (1991) 88:10134-10137. Using this approach, it has
been possible to isolate high affinity antibodies to human
pathogens from infected individuals, i.e. from individuals who have
been "immunized" as described in Burton, D. R., et al., Proc. Natl.
Acad. Sci. USA (1991) 88:10134-10137, Zebedee, S. L., et al. Proc.
Natl. Acad. Sci. USA (1992) 89:3175-3179, and Barbas III, C. F., et
al., Proc. Natl. Acad. Sci. USA (1991) 89:10164-20168. However, to
generate antibodies reactive with human antigens, it has been
necessary to generate synthetic libraries (Barbas III C. F., et
al., Proc. Natl. Acad. Sci. USA (1991) 89:4457-4461, Crameri, A.
et. al., BioTechniques (1995) 88:194-196) or to prepare libraries
from either autoimmune patients (Rapoport, B., et al., Immunol.
Today (1995) 16:43-49, Portolano, S., et al., J. Immunol. (1993)
151:2839-2851, and Vogel, M., et al., Eur J. Immunol. (1994)
24:1200-1207) or normal individuals, i.e. naive libraries
(Griffiths, A. D., et al., EMBO J. (1994) 13:3245-3260, Griffiths,
A. D., et al., EMBO J. (1993) 12:725-734, Persson, M. A. A., et
al., Proc. Natl. Acad. Sci. USA (1991) 88:2432-2436, Griffiths, A.
D., Curr. Opin. Immunol. (1993) 5:263-267, Hoogenboom, H. R., et
al., J. Mol. Biol. (1992) 227:381-388, Lerner, R. A., et al.,
Science (1992) 258:1313-1314, and Nissim A., et al., EMBO J. (1994)
13:692-698. Typically, high affinity antibodies to human proteins
have proven very difficult to isolate in this way. As is well
known, affinity maturation requires somatic mutation and somatic
mutation, in turn, is antigen driven. In the XenoMouse, repeated
immunization with human proteins will lead to somatic mutation and,
consequently, high affinity antibodies. The genes encoding these
antibodies can be readily amplified by PCR as described in Marks,
J. D., et al., J. Mol. Biol. (1991) 581-596 and immunospecific
antibodies isolated by standard panning techniques, Winter, G., et
al., Annu. Rev. Immunol. (1994) 12:433-55 and Barbas III, C. F., et
al., Proc. Natl. Acad. Sci. USA (1991) 88:7978-7982.
[0050] As above, the modified or unmodified rearranged loci are
manipulated using standard recombinant techniques by constructing
expression systems operable in a desired host cell, such as,
typically, a Chinese hamster ovary cell, and the desired
immunoglobulin or analog is produced using standard recombinant
expression techniques, and recovered and purified using
conventional methods.
[0051] The application of the foregoing processes to antibody
production has enabled the preparation of human immunospecific
reagents with respect to antigens for which human antibodies have
not heretofore been available. The immunoglobulins that result from
the above-described methods and the analogs made possible thereby
provide novel compositions for use in analysis, diagnosis,
research, and therapy. The particular use will, of course, depend
on the immunoglobulin or analog prepared. In general, the
compositions of the invention will have utilities similar to those
ascribable to nonhuman antibodies directed against the same
antigen. Such utilities include, for example, use as affinity
ligands for purification, as reagents in immunoassays, as
components of immunoconjugates, and as therapeutic agents for
appropriate indications.
[0052] Particularly in the case of therapeutic agents or diagnostic
agents for use in vivo, it is highly advantageous to employ
antibodies or their analogs with fully human characteristics. These
reagents avoid the undesired immune responses engendered by
antibodies or analogs which have characteristics marking them as
originating from nonhuman species. Other attempts to "humanize"
antibodies do not result in reagents with fully human
characteristics. For example, chimeric antibodies with murine
variable regions and human constant regions are easily prepared,
but, of course, retain murine characteristics in the variable
regions. Even the much more difficult procedure of "humanizing" the
variable regions by manipulating the genes encoding the amino acid
sequences that form the framework regions does not provide the
desired result since the CDRs, typically of nonhuman origin, cannot
be manipulated without destroying immunospecificity.
[0053] Thus, the methods of the present invention provide, for the
first time, immunoglobulins that are fully human or analogs which
contain immunospecific regions with fully human
characteristics.
[0054] There are large numbers of antigens for which human
antibodies and their human analogs would be made available by the
methods of the invention. These include, but are not limited to,
the following nonlimiting set:
[0055] leukocyte markers, such as CD2, CD3, CD4, CD5, CD6, CD7,
CD8, CD11a,b,c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27 and
its ligand, CD28 and its ligands B7.1, B7.2, B7.3, CD29 and its
ligand, CD30 and its ligand, CD40 and its ligand gp39, CD44, CD45
and isoforms, Cdw52 (Campath antigen), CD56, CD58, CD69, CD72,
CTLA-4, LFA-1 and TCR
[0056] histocompatibility antigens, such as MHC class I or II, the
Lewis Y antigens, Slex, Sley, Slea, and Selb;
[0057] adhesion molecules, including the integrins, such as VLA-1,
VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, LFA-1, Mac-1, .alpha.V.beta.3,
and p150, 95; and
[0058] the selectins, such as L-selectin, E-selectin, and
P-selectin and their counterreceptors VCAM-1, ICAM-1, ICAM-2, and
LFA-3;
[0059] interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15;
[0060] interleukin receptors, such as IL-1R, IL-2R, IL-3R, IL-4R,
IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R,
IL-14R and IL-15R;
[0061] chemokines, such as PF4, RANTES, MIP1a, MCP1, IP-10, ENA-78,
NAP-2, Gro.alpha., Gro.beta., and IL-8;
[0062] growth factors, such as TNFalpha, TGFbeta, TSH, VEGF/VPF,
PTHrP, EGF family, FGF, PDGF family, endothelin, Fibrosin
(F.sub.sF.sub.-1), Laminin, and gastrin releasing peptide
(GRP);
[0063] growth factor receptors, such as TNFalphaR, RGFbetaR, TSHR,
VEGFR/VPFR, FGFR, EGFR, PTHrPR, PDGFR family, EPO-R, GCSF-R and
other hematopoietic receptors;
[0064] interferon receptors, such as IFN.alpha.R, IFN.beta.R, and
IFN.sub.YR;
[0065] Igs and their receptors, such as IGE, FceRI, and FceRII;
[0066] tumor antigens, such as her2-neu, mucin, CEA and
endosialin;
[0067] allergens, such as house dust mite antigen, lol p1 (grass)
antigens, and urushiol;
[0068] viral proteins, such as CMV glycoproteins B, H, and gCIII,
HIV-1 envelope glycoproteins, RSV envelope glycoproteins, HSV
envelope glycoproteins, EBV envelope glycoproteins, VZV, envelope
glycoproteins, HPV envelope glycoproteins, Hepatitis family surface
antigens;
[0069] toxins, such as pseudomonas endotoxin and
osteopontin/uropontin, snake venom, spider venom, and bee
venom;
[0070] blood factors, such as complement C3b, complement C5a,
complement C5b-9, Rh factor, fibrinogen, fibrin, and myelin
associated growth inhibitor;
[0071] enzymes, such as cholesterol ester transfer protein,
membrane bound matrix metalloproteases, and glutamic acid
decarboxylase (GAD); and
[0072] miscellaneous antigens including ganglioside GD3,
ganglioside GM2, LMP1, LMP2, eosinophil major basic protein, PTHrp,
eosinophil cationic protein, pANCA, Amadori protein, Type IV
collagen, glycated lipids, .nu.-interferon, A7, P-glycoprotein and
Fas (AFO-1) and oxidized-LDL.
[0073] Particularly preferred immunoglobulins and analogs are those
immunospecific with respect to human IL-6, human IL-8, human
TNF.alpha., human CD4, human L-selectin, human PTHrp and human
gp39. Antibodies and analogs immunoreactive with human TNF.alpha.
and human IL-6 are useful in treating cachexia and septic shock as
well as autoimmune disease. Antibodies and analogs immunoreactive
with GP39 or with L-selectin are also effective in treating or
preventing autoimmune disease. In addition, anti-gp39 is helpful in
treating graft versus host disease, in preventing organ transplant
rejection, and in treating glomerulonephritis. Antibodies and
analogs against L-selectin are useful in treating ischemia
associated with reperfusion injury. Antibodies to PTHrp are useful
in treating bone disease and metastatic cancer. In a particular
embodiment, human antibodies against IL-8 may be used for the
treatment or prevention of a pathology or condition associated with
IL-8. Such conditions include, but are not limited to, tumor
metastasis, reperfusion injury, pulmonary edema, asthma, ischemic
disease such as myocardial infarction,inflammatory bowel disease
(such as Crohn's disease and ulcerative colitis), encephalitis,
uveitis, autoimmune diseases (such as rheumatoid arthritis,
Sjogren's syndrome, vasculitis), osteoarthritis, gouty arthritis,
nephritis, renal failure, dermatological conditions such as
inflammatory dermatitis, psoriasis, vasculitic urticaria and
allergic angiitis, retinal uveitis, conjunctivitis, neurological
disorders such as stroke, multiple sclerosis and meningitis, acute
lung injury, adult respiratory distress syndrome (ARDS), septic
shock, bacterial pneumonia, diseases involving leukocyte
diapedesis, CNS inflammatory disorder, multiple organ failure,
alcoholic hepatitis, antigen-antibody complex mediated diseases,
inflammation of the lung (such as pleurisy, aveolitis, vasculitis,
pneumonia, chronic bronchitis, bronchiectasis, cystic fibrosis) ,
Behcet disease, Wegener's granulomatosis, and vasculitic
syndrome.
[0074] Typical autoimmune diseases which can be treated using the
above-mentioned antibodies and analogs include systemic lupus
erythematosus, rheumatoid arthritis, psoriasis, Sjogren's
scleroderma, mixed connective tissue disease, dermatomyositis,
polymyositis, Reiter's syndrome, Behcet's disease, Type 1 diabetes,
Hashimoto's thyroiditis, Grave's disease, multiple sclerosis,
myasthenia gravis and pemphigus.
[0075] For therapeutic applications, the antibodies may be
administered in a pharmaceutically acceptable dosage form. They may
be administered by any means that enables the active agent to reach
the desired site of action, for example, intravenously as by bolus
or by continuous infusion over a period of time, by intramuscular,
subcutaneous, intraarticular, intrasynovial, intrathecal, oral,
topical or inhalation routes. The antibodies may be administered as
a single dose or a series of treatments.
[0076] For parenteral administration, the antibodies may be
formulated as a solution, suspension, emulsion or lyophilized
powder in association with a pharmaceutically acceptable parenteral
vehicle. If the antibody is suitable. for oral administration, the
formulation may contain suitable additives such as, for example,
starch, cellulose, silica, various sugars, magnesium carbonate, or
calcium phosphate. Suitable vehicles are described in the most
recent edition of Remington's Pharmaceutical Sciences, A. Osol, a
standard reference text in this field.
[0077] For prevention or treatment of disease, the appropriate
dosage of antibody will depend upon known factors such as the
pharmacodynamic characteristics of the particular antibody; its
mode and route of administration, the age, weight, and health of
the recipient, the type of condition to be treated and the severity
and course of the condition, frequency of treatment, concurrent
treatment and the physiological effect desired. The examples below
are intended to illustrate but not to limit the invention.
[0078] In these examples, mice, designated XenoMouse.TM., are used
for initial immunizations. A detailed description of the
XenoMouse.TM. is found in the above referenced PCT application WO
94/02602. Immunization protocols appropriate to each antigen are
described in the specific examples below. The sera of the immunized
XenoMouse.TM. (or the supernatants from immortalized B cells) were
titrated for antigen specific human antibodies in each case using a
standard ELISA format. In this format, the antigen used for
immunization was immobilized onto wells of microtiter plates. The
plates were washed and blocked and the sera (or supernatants) were
added as serial dilutions for 1-2 hours of incubation. After
washing, bound antibody having human characteristics was detected
by adding antihuman .kappa., .mu., or .gamma. chain antibody
conjugated to horseradish peroxidase (HRP) for one hour. After
again washing, the chromogenic reagent o-phenylene diamine (OPD)
substrate and hydrogen peroxide were added and the plates were read
30 minutes later at 492 nm using a microplate reader.
[0079] Unless otherwise noted, the antigen was coated using plate
coating buffer (0.1 M carbonate buffer, pH 9.6); the assay blocking
buffer used was 0.5% BSA, 0.1% Tween 20 and 0.01% thimerosal in
PBS; the substrate buffer used in color development was citric acid
7.14 g/l; dibasic sodium phosphate 17.96 g/l; the developing
solution (made immediately before use) was 10 ml substrate buffer;
10 mg OPD, plus 5 ml hydrogen peroxide; the stop solution (used to
stop color development) was 2 M sulfuric acid. The wash solution
was 0.05% Tween 20 in PBS.
EXAMPLE 1
Human Antibodies Against Human IL-6
[0080] Three to five XenoMouse.TM. aged 8-20 weeks were age-matched
and immunized intraperitoneally with 50 .mu.g human IL-6 emulsified
in incomplete Freund's adjuvant for primary immunization and in
complete Freund's adjuvant for subsequent injections. The mice
received 6 injections 2-3 weeks apart. Serum titers were determined
after the second dose and following each dose thereafter. Bleeds
were performed from the retrobulbar plexus 6-7 days after
injections. The blood was allowed to clot at room temperature for
about 2 hours and then incubated at 4.degree. C. for at least 2
hours before separating and collecting the sera.
[0081] ELISAs were conducted as described above by applying 100
.mu.l/well of recombinant human IL-6 at 2 .mu.g/ml in coating
buffer. Plates were then incubated at 4.degree. C. overnight or at
37.degree. C. for 2 hours and then washed three times in washing
buffer. Addition of 100 .mu.l/well blocking buffer was followed by
incubation at room temperature for 2 hours, and an additional 3
washes.
[0082] Then, 50 .mu.l/well of diluted serum samples (and positive
and negative controls) were added to the plates. Plates were then
incubated at room temperature for 2 hours and again washed 3
times.
[0083] After washing, 100 .mu.l/well of either mouse antihuman .mu.
chain antibody conjugated to HRP at {fraction (1/2,000)} or mouse
antihuman .kappa. chain antibody conjugated to HRP at {fraction
(1/2,000)}, diluted in blocking buffer was added. After a 1 hour
incubation at room temperature, the plates were washed 3 times and
developed with OPD substrate for 10-25 minutes. 50 .mu.l/well of
stop solution was then added and the results read on an ELISA plate
reader at 492 nm. The dilution curves resulting from the titration
of serum from XenoMouse.TM. after 6 injections are shown in FIG. 3.
The data in FIG. 3 show production of anti-IL-6 immunoreactive with
antihuman .kappa. and antihuman .mu. detectable at serum dilutions
above 1:1,000.
EXAMPLE 2
Human Antibodies Against Human TNF.alpha.
[0084] Immunization and serum preparation were conducted as
described in Example 1 except that human recombinant TNFA (at 5
.mu.g per injection) was substituted for human IL-6. ELISAs were
conducted as described in Example 1 except that the initial coating
of the ELISA plate employed 100 .mu.l/well recombinant human
TNF.alpha. at 1 .mu.g/ml in coating buffer.
[0085] The dilution curves for serum from XenoMouse.TM. after 6
inductions obtained are shown in FIG. 4. Again significant titers
of human anti-TNF.alpha. binding were shown.
[0086] Serum titers for h.gamma., h.mu., and h.kappa. after one and
two immunizations of the XenoMouse.TM. are shown in Table 1. When
challenged with TNF-.alpha., the XenoMouse.TM. switches isotypes
from a predominant IgM response in the first immunization to an
immune response with a large IgG component in the second
immunization.
1TABLE 2 Anti TNF-alpha serum titer responses of Xenomouse-2. Bleed
1: after 2 immunizations Bleed 2: after 3 immunizations ELISA Serum
titers Specific for TNF-alpha titer titer titer XM2 (via h.gamma.)
(via h.mu.) (via h.kappa.) 1 bleed 1 500 3,000 1,500 bleed 2 10,000
8,000 15,000 2 bleed 1 200 3,000 500 bleed 2 2,700 5,000 1,000 3
bleed 1 <500 2,000 1,500 bleed 2 15,000 24,000 25,000 4 bleed 1
500 2,500 1,500 bleed 2 70,000 4,000 72,000 5 bleed 1 <500 2,500
1,500 bleed 2 1,000 10,000 7,000 6 bleed 1 1,000 13,000 4,500 bleed
2 10,000 24,000 25,000 7 bleed 1 <500 2,500 1,500 bleed 2 5,000
4,000 9,000 8 bleed 1 <500 1,000 500 bleed 2 2,700 5,000 9,000 9
bleed 1 200 6,000 4,000 bleed 2 40,000 80,000 80,000 10 bleed 1 200
2,000 500 bleed 2 15,000 8,000 60,000 11 bleed 1 1,500 1,000 1,500
bleed 2 24,000 2,700 72,000 12 bleed 1 200 2,000 1,000 bleed 2
10,000 4,000 25,000 13 bleed 1 500 30,000 500 bleed 2 2,000 4,000
12,000
EXAMPLE 3
Human Antibodies Against Human CD4
[0087] The human CD4 antigen was prepared as a surface protein
using human CD4 .zeta. on transfected recombinant cells as follows.
Human CD4 .zeta. consists of the extracellular domain of CD4, the
transmembrane domain of CD4, and the cytoplasmic domain
corresponding to residues 31-142, of the mature .zeta. chain of the
CD3 complex. Human CD4 zeta (F15 LTR) as described in Roberts et
al., Blood (1994) 84:2878 was introduced into the rat basophil
leukemic cell line RBL-2H3, described by Callan, M., et al., Proc
Natl Acad Sci USA (1993) 90:10454 using the Kat high efficiency
transduction described by Finer et al., Blood (1994) 83:43.
Briefly, RBL-2H3 cells at 10.sup.6 cells per well were cultured in
750 .mu.l DMEM +20% FBS (Gibco) and 16 .mu.g/ml polybrene with an
equal volume of proviral supernatant for 2 hours at 37.degree. C.,
5% CO.sub.2. One ml of medium was removed and 750 .mu.l of
infection medium and retroviral supernatant were added to each well
and the cultures incubated overnight. The cells were washed and
expanded in DMEM +10% FBS until sufficient cells were available for
sorting. The CD4 zeta transduced RBL-2H3 cells were sorted using
the FACSTAR plus (Becton Dickinson). The cells were stained for
human CD4 with a mouse antihuman CD4 PE antibody and the top 2-3%
expressing cells were selected.
[0088] Immunizations were conducted as described in Example 1 using
1.times.10.sup.7 cells per mouse except that the primary injection
was subcutaneous at the base of the neck. The mice received 6
injections 2-3 weeks apart. Serum was prepared and analyzed by
ELISA as described in Example 1 except that the initial coating of
the ELISA plate utilized 100 .mu.l per well of recombinant soluble
CD4 at 2 .mu.g/ml of coating buffer. The titration curve for serum
from XenoMouse.TM. after 6 injections is shown in FIG. 5. Titers of
human anti-CD4 reactivity were shown at concentrations representing
greater than those of 1:1,000 dilution.
EXAMPLE 4
Human Antibodies Against Human L-selectin
[0089] The antigen was prepared as a surface displayed protein in
C51 cells, a high expressing clone derived by transfecting the
mouse pre-B cell 300.19 with LAM-1 cDNA (LAM-1 is the gene encoding
L.-selectin) (Tedder, et al., J. Immunol (1990) 144:532) or with
similarly transfected CHO cells. The transfected cells were sorted
using fluorescent activated cell sorting using anti-Leu-8 antibody
as label.
[0090] The C51 and the transfected CHO cells were grown in DME 4.5
g/l glucose with 10% FCS and 1 mg/ml G418 in 100 mm dishes.
Negative control cells, 3T3-P317 (transfected with gag/pol/env
genes of Moloney virus) were grown in the same medium without
G418.
[0091] Primary immunization was done by injection subcutaneously at
the base of the neck; subsequent injections were intraperitoneal.
70-100 million C51 or transfected CHO cells were used per injection
for a total of five injections 2-3 weeks apart.
[0092] Sera were collected as described in Example 1 and analyzed
by ELISA in a protocol similar to that set forth in Example 1.
[0093] For the ELISA, the transfected cells were plated into 96
well plates and cell monolayers grown for 1-2 days depending on
cell number and used for ELISA when confluent. The cells were fixed
by first washing with cold 1.times.PBS and then fixing solution (5%
glacial acetic acid, 95% ethanol) was added. The plates were
incubated at -25.degree. C. for 5 minutes and can be stored at this
temperature if sealed with plate sealers.
[0094] The ELISA is begun by bringing the plates to room
temperature, flicking to remove fixing solution and washing 5 times
with DMEM medium containing 10% FCS at 200 .mu.l per well.
[0095] The wells were treated with various serum dilutions or with
positive or negative controls. Positive control wells contained
murine IgGl monoclonal antibody to human L-selectin.
[0096] The wells were incubated for 45 minutes and monolayer
integrity was checked under a microscope. The wells were then
incubated with antihuman .kappa. chain antibody or antihuman .mu.
chain antibody conjugates with HRP described in Example 1. The
plates were then washed with 1% BSA/PBS and again with PBS and
monolayer integrity was checked. The plates were developed,
stopped, and read as described above. The results for serum from
XenoMouse.TM. are shown in FIGS. 6 and 7; human antibodies both to
L-selectin and control 3T3 cells were obtained. However, the serum
titers are higher for the L-selectin-expressing cells as compared
to parental 3T3 cells. These results show the XenoMouse.TM.
produces antibodies specific for L-selectin with human .mu. heavy
chain regions and human .kappa. light chains.
[0097] The antisera obtained from the immunized XenoMouse.TM. were
also tested for staining of human neutrophils which express
L-selectin. Human neutrophils were prepared as follows:
[0098] peripheral blood was collected from normal volunteers with
100 units/ml heparin. About 3.5 ml blood was layered over an equal
volume of One-step Polymorph Gradient (Accurate Chemical, Westbury,
N.Y.) and spun for 30 minutes at 450.times.g at 20.degree. C. The
neutrophil fraction was removed and washed twice in DPBS/2%
FBS.
[0099] The neutrophils were then stained with either;
[0100] (1) antiserum from XenoMouse.TM. immunized with C51 cells
(expressing L-selectin);
[0101] (2) as a negative control, antiserum from a XenoMouse.TM.
immunized with cells expressing human gp39.
[0102] The stained, washed neutrophils were analyzed by FACS. The
results for antiserum from XenoMouse.TM. are shown in FIG. 8.
[0103] These results show the presence of antibodies in immunized
XenoMouse.TM. serum which contain fully human light chains
immunoreactive with L-selectin. The negative control antiserum from
mice immunized with gp39 does not contain antibodies reactive
against human neutrophils.
EXAMPLE 5
Human Antibodies Against Human gp39
[0104] gp39 (the ligand for CD40) is expressed on activated human
CD4 T cells. The sera of XenoMouse.TM. immunized with recombinant
gp39 according to this example contained fully human antibodies
immunospecific for gp39.
[0105] The antigen consisted of stable transfectants of 300.19
cells or of CHO cells expressing gp39 cDNA cloned into the
mammalian expression vector P1K1.HUgp39/IRES NEO as shown in FIG.
9. CHO cells were split 1:10 prior to transfection in DMEM 4.5 g/l
glucose, 10% FBS, 2 mM glutamine, MEM, NEAA supplemented with
additional glycine, hypoxanthine and thymidine. The cells were
cotransfected with the gp39 vector at 9 .mu.g/10 cm plate
(6.times.10.sup.5 cells) and the DHFR expressing vector pSV2DHFRs
(Subranani et al., Mol Cell Biol (1981) 9:854) at 1 .mu.g/10 cm
plate using calcium phosphate transfection. 24 hours later the
cells were split 1:10 into the original medium containing G418 at
0.6 mg/ml. Cells producing gp39 were sorted by FACS using an
anti-gp39 antibody.
[0106] Mice grouped as described in Example 1 were immunized with
300.19 cells expressing gp39 using primary immunization
subcutaneously at the base of the neck and with secondary
intraperitoneal injections every 2-3 weeks. Sera were harvested as
described in Example 1 for the ELISA assay. The ELISA procedure was
conducted substantially as set forth in Example 1; the microtiter
plates were coated with CHO cells expressing gp39 grown in a 100 mm
dish in DMEM, 4.5 g/l glucose, 10% FCS, 4mM glutamine, and
nonessential amino acid (NEAA) solution for MEM (100 X). On the day
preceding the ELISA assay, the cells were trypsinized and plated
into well filtration plates at 10.sup.5 cells/200 .mu.l well and
incubated at 37.degree. C. overnight. The positive controls were
mouse antihuman gp39; negative controls were antisera from mice
immunized with an antigen other than gp39. 50 .mu.l of sample were
used for each assay. The remainder of the assay is as described in
Example 1.
[0107] The dilution curves for the sera obtained after 4 injections
from mice immunized with gp39 expressed on CHO cells are shown in
FIG. 10. As shown, the sera contained antihuman gp39
immunospecificity which is detectable with anti-human .kappa. and
anti-human .mu. chain antibodies coupled to HRP.
EXAMPLE 6
Preparation of Human Mabs Against Tetanus Toxin
[0108] The antibodies prepared in this example were secreted by
hybridomas obtained by immortalizing B cells from xenomice
immunized with tetanus toxin. The immunization protocol was similar
to that set forth in Example 1 using 50 .mu.g tetanus toxin
emulsified in complete Freund's adjuvant for intraperitoneal
primary immunization followed by subsequent intraperitoneal
injections with antigen incorporated into incomplete Freund's
adjuvant. The mice received a total of 4 injections 2-3 weeks
apart.
[0109] After acceptable serum titers of antitetanus toxin C
(anti-TTC) were obtained, a final immunization dose of antigen in
PBS was give 4 days before the animals were sacrificed and the
spleens were harvested for fusion.
[0110] The spleen cells were fused with myeloma cells P3X63-Ag8.653
as described by Galfre, G. and Milstein, C. Methods in Enzymology
(1981) 73:3-46.
[0111] After fusion the cells were resuspended in DMEM, 15% FCS,
containing HAT supplemented with glutamine, pen/strep for culture
at 37.degree. C. and 10% CO.sub.2. The cells were plated in
microtiter plates and maintained in HAT-supplemented medium for two
weeks before transfer to HAT-supplemented medium. Supernatants from
wells containing hybridomas were collected for a primary screen
using an ELISA.
[0112] The ELISA was conducted as described in Example 1 wherein
the antigen coating consisted of 100 .mu.l/well of tetanus toxin C
(TTC) protein at 2 .mu.g/ml in coating buffer, followed by
incubation at 4.degree. C. overnight or at 37.degree. C. for two
hours. In the primary ELISA, HRP-conjugated mouse antihuman IgM was
used as described in Example 1. Two hybridomas that secreted
anti-TTC according to the ELISA assay, clone D5.1 and clone K4.1
were used for further analysis.
[0113] As shown in FIG. 11, clone D5.1 secretes fully human
anti-TTC which is detectable using HRP-conjugated antihuman .mu.
chain antibody and HRP-conjugated antihuman .kappa. chain antibody.
This is confirmed in FIG. 11.
[0114] The antibody secreted by D5.1 did not immunoreact in ELISAs
using TNF.alpha., IL-6, or IL-8 as immobilized antigen under
conditions where positive controls (sera from xenomice immunized
with TNF.alpha., IL-6 and IL-8 respectively) showed positive ELISA
results.
[0115] The complete nucleotide sequence of the cDNAs encoding the
heavy and light chains of the monoclonal were determined as shown
in FIGS. 12 and 13. polyA mRNA was isolated from about 10.sup.6
hybridoma cells and used to generate cDNA using random hexamers as
primers. Portions of the product were amplified by PCR using the
appropriate primers.
[0116] The cell line was known to provide human .kappa. light
chains; for PCR amplification of light chain encoding cDNA, the
primers used were HKP1 (5'-CTCTGTGACACTCTCCTGGGAGTT-3') for priming
from the constant region terminus and two oligos, used in equal
amounts to prime from the variable segments; B3
(5'-GAAACGACACTCACGCAGTCTCCAGC-3').
[0117] For amplification of the heavy chain of the antibody derived
form D5.1 (which contains the human .mu. constant region), MG-24 VI
was used to prime from the variable and .mu.P1
(5'-TTTTCTTTGTTGCCGTTGGGGTGC-3') was used to prime from the
constant region terminus.
[0118] Referring to FIG. 12 which sets forth the sequence for the
heavy chain of the antibody secreted by clone D5.1, this shows the
heavy chain is comprised of the human variable fragment VH6, the
human diversity region DN1 and the human joining segment JH4 linked
to the human .mu. constant region. There were two base-pair
mutations from the germline sequence in the variable region, both
in the CDRs. Two additional mutations were in the D segment and six
nongermline nucleotide additions were present at the D -J
junction.
[0119] Finally, referring to FIG. 13 which presents the light chain
of the antibody secreted by D5.1, the human .kappa. variable region
B3 and human .kappa. joining region JK3 are shown. There are nine
base-pair differences from the germline sequences, three falling
with CDR1.
EXAMPLE 7
Human Antibodies Against PTHrp
[0120] Groups of XenoMouse.TM.-2 were immunized intraperitoneally
with either PTHrp (1-34) conjugated with BTG, as described by
Ratcliffe et al., J. Immunol. Methods 127:109 (1990), or with PTHrp
(1-34) synthesized as a 4 branched-MAP (multiple antigenic peptide
system). The antigens were emulsified in CFA (complete Freunds
adjuvant) and injected i.p. at a dose of 25 .mu.g per animal at 2
week intervals, and bled after two injections. The sera obtained
from this bleed were analyzed by ELISA as described supra.
[0121] Serum titers for h.gamma., h.mu., and h.kappa. after one
immunization of the XenoMouse.TM. are shown in Table 2. When
immunized with PTHrp, the XenoMouse.TM. showed low serum titers in
5 of 7 mice on the first bleed, but when PTHrp-MAP is used, 7 of 7
mice show high serum titers on the first bleed.
2TABLE 1 AntiPTHrp serum titer responses of Xenomouse-2. First
bleed after 2 immunizations with either PTHrp-BTG conjugate Human
Responses titer titer titer (via h.gamma.) (via h.mu.) (via
h.kappa.) XM2 PTHrp-BTG Conjugate 1 <30 850 100 2 <30 3,000
50 3 <30 7,000 1,000 4 <30 800 200 5 <30 400 90 6 <30
500 50 7 <30 300 50 XM2 PTHrp-MAP 1 <30 1,000 50 2 <30
2,500 300 3 <30 1,200 150 4 150 1,000 270 5 100 2,500 300 6
<30 1,000 150 7 <30 4,000 800
EXAMPLE 8
Human Antibodies Against Human IL-8
[0122] Immunization and serum preparation were as described in
Example 1 except that human recombinant IL-8 was used as an
immunogen.
[0123] ELISA assays were performed with respect to the recovered
serum, also exactly as described in Example 1, except that the
ELISA plates were initially coated using 100 .mu.l/well of
recombinant human IL-8 at 0.5 mg/ml in the coating buffer. The
results obtained for various serum dilutions from XenoMouse.TM.
after 6 injections are shown in FIG. 14. Human anti-IL-8 binding
was again shown at serum dilutions having concentrations higher
than that represented by a 1:1,000 dilution.
EXAMPLE 9
Preparation of High Affinity Human Monoclonal Antibodies Against
Human IL-8
[0124] Groups of 4 to 6 XenoMouse.TM. aged between 8 to 10 weeks
old were used for immunization and for hybridoma generation.
XenoMouse.TM. were immunized intraperitoneally with 25 .mu.g of
human recombinant-IL-8 (Biosource International, Calif., USA)
emulsified in complete Freund's adjuvant (CFA, Sigma) for the
primary immunization. All subsequent injections were done with the
antigen incorporated into incomplete Freund's adjuvant (IFA,
Sigma). For animals used as spleen donors for hybridoma generation
a final dose of antigen in phosphate buffer saline (PBS) was given
4 days before the fusion. Serum titers of immunized XenoMouse.TM.
were first analyzed after a secondary dose of antigens, and from
there after, following every antigen dose. Test bleeds were
performed 6 to 7 days after the injections, by bleeding from the
retro-bulbar plexus. Blood was allowed to clot at room temperature
for about 2 hours and then incubated at 4.degree. C. for at least 2
hours before separating and collecting the sera.
[0125] Generation of Hybridomas
[0126] Spleen cells obtained from XenoMouse.TM. previously
immunized with antigen, were fused with the non secretory NSO
myeloma cells transfected with bcl-2 (NSO-bcl2) as described in
Galfre G, et al., Methods in Enzymology 73, 3-46, (1981). Briefly,
the fusion was performed by mixing washed spleen cells and myeloma
cells at a ratio of 5:1 and gently pelleting them by centrifugation
at 800.times.g. After complete removal of the supernatant the cells
were treated with 1 ml of 50% PEG/DMSO (polyethylene glycol MW
1500, 10% DMSO Sigma) which was added over 1 min., the mixture was
further incubated for one minute, and gradually diluted with 2 ml
of DMEM over 2 minutes and diluted further with 8 ml of DMEM over 3
minutes. The process was performed at 37.degree. C. with continued
gentle stirring. After fusion the cells were resuspended in DMEM,
15% FCS, containing HAT, and supplemented with L glutamine,
pen/strep, for culture at 37.degree. C. and 10% CO2 in air. Cells
were plated in flat bottomed 96 well microtiter trays. Cultures
were maintained in HAT supplemented media for 2 weeks before
transfer to HT supplemented media. Cultures were regularly examined
for hybrid cell growth, and supernatants from those wells
containing hybridomas were collected for a primary screen analysis
for the presence of human .mu., human gamma 2, and human kappa
chains in an antigen specific ELISA as described above. Positive
cultures were transferred to 48 well plates and when reaching
confluence transferred to 24 well plates. Supernatants were tested
in an antigen specific ELISA for the presence of human .mu., human
gamma 2, and human kappa chains.
[0127] As shown in Table 3 several hybridomas secreting fully human
monoclonal antibodies with specificity for human IL-8 have been
generated from representative fusions. In all of these human
monoclonal antibodies the human gamma-2 heavy chain is associated
with the human kappa light chain.
3TABLE 3 ELISA determination of heavy and light chain composition
of anti-IL-8 human monoclonal antibodies generated in XenoMouse
.TM. reactivity to hIL8 H.sub..kappa. m.lambda. h.gamma. Total
Sample OD OD OD hlgG ID lg class titers (1:1) (1:1) (1:1) (ng/ml)
Bkgd 0.08 0.04 0.12 I8D1.1 hlgG2 500 4.12 0.04 4.09 1,159 I8K2.1
hlgG2 200 4.18 0.18 4.11 2,000 I8K2.2 hlgG2 1,000 4.00 0.04 4.00
4,583 I8K4.2 hlgG2 200 3.98 0.04 3.49 450 I8K4.3 hlgG2 200 3.80
0.05 4.09 1,715 I8K4.5 hlgG2 1,000 4.00 0.06 4.00 1,468
[0128] Evaluation of Kinetic Constants of XenoMouse.TM.
Hybridomas
[0129] In order to determine the kinetic parameters of these
antibodies, specifically their on and off rates and their
dissociation constants (KD), they were analyzed on the BIAcore
instrument (Pharmacia). The BIAcore instrument uses plasmon
resonance to measure the binding of an antibody to an
antigen-coated gold chip.
[0130] BIAcore Reagents and Instrumentation:
[0131] The BIAcore instrument, CM5 sensor chips, surfactant P20,
and the amine coupling kit containing N-hydroxysuccinimide (NHS),
N-ethyl-N.sup.1-(3-diethylaminopropyl)-carbodimide (EDC), and
ethanolamine were purchased from Pharmaicia Biosensor.
Immobilization of human recombinant IL-8 onto the sensor surface
was carried out at low levels of antigen density immobilized on the
surface and was performed according to the general procedures
outlined by the manufacturers. Briefly, after washing and
equilibrating the instrument with HEPES buffer (HBS; 10 mM HEPES,
150 mM NaCl, 0.05% surfactant P20, pH 7.4) the surface was
activated and IL-8 immobilized for the subsequent binding and
kinetic studies. The sensor surface was activated with 5 .mu.l of a
mixture of equal volumes of NHS (0.1 M) and EDC (0.1 M) injected at
10 .mu.l/min across the surface for activation, then 5 .mu.l of the
ligand (human recombinant IL-8) at 12 .mu.g/ml in 5 mM maleate
buffer, pH 6.0 was injected across the activated surface, and
finally non-conjugated active sites were blocked with an injection
of 35 .mu.l of 1 M ethanolamine. The surface was washed to remove
non-covalently bound ligand by injection of 5 .mu.l 0.1 M HCI. All
the immobilization procedure was carried out with a continuous flow
of HBS of 10 .mu.l/min. About 100 resonance units (RU) of ligand
(82 and 139 RU, in separate experiments) were immobilized on the
sensorship, (according to the manufacturers 1,000 RU corresponds to
about 1 ng/mm.sup.2 of immobilized protein).
[0132] These ligand coated surfaces were used to analyze hybridoma
supernatants for their specific binding to ligand and for kinetic
studies. The best regenerating condition for the analyte
dissociation from the ligand in these sensorships was an injection
of 10 .mu.l 100 mM HCl with no significant losses of binding
observed after many cycles of binding and regeneration.
[0133] Determination of the Dissociation, and Association Rates and
the Apparent Affinity Constants of Fully Human Monoclonal
Antibodies Specific for IL-8
[0134] The determination of kinetic measurements using the BIAcore
in which one of the reactants is immobilized on the sensor surface
was done following procedures suggested by the manufacturers and
described in Karlsson et al. "Kinetic analysis of monoclonal
antibody-antigen interaction with a new biosensor based analytical
system." J. Immunol. Methods (19910 145, 229. Briefly the single
site interaction between two molecules A and B is described by the
following equation.
d[AB]/dt=ka[A][B]-kd[AB]
[0135] In which B is immobilized on the surface and A is injected
at a constant concentration C. The response is a measure of the
concentration of the complex [AB] and all concentration terms can
be expressed as Response Units (RU) of the BIAcore:
dR/dt-kaC(Rmax-R)-kdR
[0136] where dR/dt is the rate of change of the signal, C is the
concentration of the analyte, Rmax is the maximum analyte binding
capacity in RU and R is the signal in RU at time t. In this
analysis the values of ka and kd are independent of the
concentration of immobilized ligand on the surface of the sensor.
The dissociation rates (kd) and association rates (ka) were
determined using the software provided by the manufacturers, BIA
evaluation 2.1. The dissociation rate constant was measured during
the dissociation phase that extended for 10 minutes at a constant
buffer flow rate of 45 ul/min, after the completion of the
injection of the hybridoma supernatants onto the surface containing
immobilized IL-8. The association phase extended over 1.25 minutes
at a flow rate of 45 ul/min and the data was fitted into the model
using the previously determined kd values. At least two surfaces
with different levels of immobilized ligand were used in which
different concentrations of anti IL-8 hybridoma supernatants were
tested for binding and analyzed for kinetic data. The kinetic
constants determined on these two surfaces are presented in Table
4. The affinities were determined to be very, ranging from
7.times.10.sup.-11 to 2.times.10.sup.-9 M. This compares vary
favorably with the affinities of murine monoclonal antibodies
derived from normal mice.
4TABLE 4 Kinetic constants of fully human rnonoclonal antibodies
(lgG2, kappa) derived from XenoMouse .TM. II-a with specificity to
human IL-8, determined by BIAcore. BIAcore association dissociation
Dissociation surface Hybri- rate rate Constant h-IL-8 doma ka
(M.sup.-1.sub.s.sup.-1) kd (.sub.s.sup.-1) KD (M) = kd/ka [RU]
I8D1-1 1 3.36 .times. 106 2.80 .times. 106 2 2.58 .times. 10 - 4
1.73 .times. 10 - 4 3 7.70 .times. 10 - 11 6.20 .times. 10 - 11 4
81 134 I8K2-1 5 4.38 .times. 105 3.83 .times. 105 6 6.73 .times. 10
- 4 6.85 .times. 10 - 4 7 1.54 .times. 10 - 9 1.79 .times. 10 - 9 8
81 134 I8K2-2 9 5.24 .times. 105 4.35 .times. 105 10 2.26 .times.
10 - 4 2.30 .times. 10 - 4 11 4.30 .times. 10 - 10 5.30 .times. 10
- 10 12 81 134 I8K4-2 13 5.76 .times. 106 1.95 .times. 106 14 8.17
.times. 10 - 4 3.84 .times. 10 - 4 15 1.42 .times. 10 - 10 1.96
.times. 10 - 10 16 81 134 I8K4-3 17 2.66 .times. 106 1.46 .times.
106 18 7.53 .times. 10 - 4 5.72 .times. 10 - 4 19 2.83 .times. 10 -
10 3.90 .times. 10 - 10 20 81 134 I8K4-5 21 4.00 .times. 105 1.70
.times. 105 22 9.04 .times. 10 - 4 4.55 .times. 10 - 4 23 2.26
.times. 10 - 9 2.68 .times. 10 - 9 24 81 134
[0137] Methods for Isolation of Human Neutrophils and Assays for
Antibody Activity
[0138] The primary in vivo function of IL-8 is to attract and
activate neutrophils. Neutrophils express on their surface two
distinct receptors for IL-8, designated the A receptor and the B
receptor. In order to determine whether the fully human antibodies
could neutralize the activity of IL-8, two different in vitro
assays were performed with human neutrophils. In one assay, the
ability of the antibodies to block binding or radiolabelled IL-8 to
neutrophil IL-8 receptors was tested. In a second assay, the
antibodies were tested for their ability to block an IL-8-induced
neutrophil response, namely the upregulation of the integrin Mac-1
on the neutrophil surface. Mac-1 is composed of two polypeptide
chains, CD11b and CD18. Typically, anti-CD11b antibodies are used
for its detection.
[0139] Isolation of Neutrophils
[0140] Human neutrophils are isolated from either freshly drawn
blood or buffy coat. Human blood is collected by venipuncture into
sterile tubes containing EDTA. Buffy coats are obtained from
Stanford Blood Bank. They are prepared by centrifuging
anticoagulated blood (up to 400 ml) in plastic bags at 2600.times.g
for 10 min at 20.degree. C. with the brake off. The plasma
supernatant is aspirated out of the bag and the buffy coat, i.e.,
the upper cell layer (40-50 ml/bag) is collected. One unit of buffy
coat (40-50 ml) is diluted to final volume of 120 ml with
Ca.sup.2+, Mg.sup.2+-free PBS. 30 milliliters of blood or diluted
buffy coat are transferred into 50-ml centrifuge tubes on top of a
20-ml layer of Ficoll-Paque Plus (Pharmacia Biotech). The tubes are
centrifuged at 500 .times.g for 20 min at 20.degree. C. with brake
off. The supernatant, the mononuclear cells at the interface, and
the layer above the pellet are carefully withdrawn. To completely
remove the mononuclear cells, the cell pellet containing
neutrophils and erythrocytes is resuspended with 5 ml of PBS and
transferred into clean 50-ml tubes. The cells are washed in
Ca.sup.2+, Mg.sup.2+-free PBS (300 .times.g for 5 min at 4.degree.
C.). The erythrocytes are then lysed with ammonium chloride. The
cells are resuspended in 40 ml of an ice-cold solution containing
155 mM NH.sub.4Cl and 10 nM EDTA, pH 7.2-7.4. The tubes are kept on
ice for 10 min with occasional mixing and then centrifuged at 300
.times.g for 5 min at 4.degree. C. The pellet is resuspended in PBS
and washed once (300 .times.g for 5 min at 4.degree. C.). If
erythrocyte lysis appears incomplete, the treatment with ammonium
chloride is repeated. The neutrophils are again washed and finally
suspended either in assay medium (RPMI-1640 supplemented with 10%
fetal calf serum, 2 mM L-glutamine, 5.times.10.sup.-5
2-mercapthoethanol, 1.times. non-essential amino acids, 1 mM sodium
pyruvate and 10 mM Hepes) at a density of 3.times.10.sup.7 cells/ml
or in a binding buffer (PBS containing 0.1% bovine serum albumin
and 0.02% NaN.sub.3), at a density of 6.times.10.sup.6
cells/ml.
[0141] IL-8 Receptor Binding Assay
[0142] Multiscreen filter plates (96-well, Millipore, MADV N6550)
were pretreated with a PBS binding buffer containing 0.1% bovine
serum albumin and 0.02% NaN.sub.3 at 25.degree. C. for 2 hours. A
final volume of 150 .mu.l, containing 4.times.10.sup.5 neutrophils,
0.23 nM [.sup.125I]-human-IL-8 (Amersham, IM-249) and varying
concentrations of antibodies made up in PBS binding buffer, was
added to each well, and plates were incubated for 90 min at
4.degree. C. Cells were washed 5 times with 200 .mu.l of ice-cold
PBS, which was removed by aspiration. The filters were air-dried,
3.5 ml of scintillation fluid was added (Beckman Ready Safe) and
filters were counted on a Beckman LS6000IC counter. The data
obtained is presented as % specific bound [I.sup.125]-IL-8, which
is calculated as the cpm in the presence of antibody divided by the
cpm in the presence of PBS binding buffer only and multiplied by
100 (FIG. 15). All six of the human anti-IL-8 monoclonals tested
blocked IL-8 binding to human neutrophils.
[0143] Neutrophil CD11b (Mac-1) Expression Assay
[0144] Human IL-8 at a final concentration of 10 nM was
preincubated with varying concentrations of monoclonal antibodies
at 4.degree. C. for 30 minutes and at 37.degree. C. for an
additional 30 min. Neutrophils (4.times.10.sup.5/well) were exposed
to IL-8 in the presence or absence of antibodies at 4.degree. C.
for 90 min, and incubated with PE-conjugated mouse-anti-human-CD11b
(Becton Dickinson) for 45 min at 4.degree. C. The cells were washed
with ice-cold PBS containing 2% fetal calf serum. Fluorescence was
measured on a Becton Dickinson FACscan cell analyzer. A mouse
monoclonal antibody against human CD11b obtained from R&D
System, Inc. was used as a positive control while the purified
myeloma human IgG2 (Calbiochem) was used as a negative control in
the experiments. The expression levels of CD11b on neutrophils were
measured and expressed as the mean fluorescence channel. The mean
fluorescence channel derived form the negative control antibody was
subtracted from those of experimental samples. 25 % inhibition =
mean fluorescence in presence of IL - 8 only - mean fluorescence in
the presence of antibodies mean fluorescence in the presence of IL
- 8 only - mean fluorescence in the presence of human IgG2 .times.
100
[0145] As shown in Table 5, five of the six antibodies blocked
upregulation of CD11b to some degree, with three of the five giving
complete blocking.
5TABLE 5 Inhibition of CD11b expression on human neutrophils by
monoclonal antibodies against IL-8. Inhibition of CD11b Antibody
Concentration (nM) expression (%) R&D anti-IL8 333 100 I8K1.1 6
100 I8K2.1 10 60 I8K2.2 32 100 I8K4.2 3 10 I8K4.3 8 100 I8K4.5 5 0
Human IgG2 33 0
[0146] Background of CD11b expression is 670 (mean fluorescence)
while CD11b expression in the presence of 10 nM of human IL-8 is
771.
[0147] Sequence Analysis of Immunoglobulin Transcripts Derived from
Anti-hIL-8 Hybridomas.
[0148] All sequences were derived by direct sequencing of PCR
fragments generated form RT-PCR reactions of RNA prepared from
hybridomas D1.1, K2.2, K4.2 and K4.3, using human V.sub.H and human
V.sub..kappa. family specific primers (Marks et. al. 1991; Euro J.
Immunol 21;985-991) and a primer specific for either the human
gamma 2 constant region (MG-40d; 5'GCTGAGGGAGTAGAGTCCTGAGGACTGT-3')
or human kappa constant region (HKP2; Green et al 1994; Nature
Genetics 7: 13-21)). In FIG. 16 A-H, both strands of the four
clones were sequenced and analyzed to generate the complete
sequence. All sequences were analyzed by alignments to the "V BASE
sequence directory", Tomlinson et al., MRC Centre for Protein
Engineering, Cambridge, UK. The variable and joining regions are
indicated by brackets []. Nucleotides containing an "N" indicate
uncertainty in the generated sequence.
[0149] Based on sequence alignments with sequences found in the
V-base database the heavy chain transcript from hybridoma D1.1 has
a human V.sub.H4-21(DP-63) variable region (7 point mutations were
observed compared to the germline sequence), a human 21-10rc D
segment, a human J.sub.H3 joining region and a human gamma 2
constant region. See FIG. 16A.
[0150] The kappa light chain transcript from hybridoma D1.1 is
comprised of a human kappa variable region with homology to
V.sub..kappa. 08/018 (DPK1) (16 point mutations were observed when
compared to the germline sequence) a human J.sub..kappa.3 joining
region, and a human kappa constant region. See FIG. 16B.
[0151] Based on sequence alignments with sequences found in the
V-base database the heavy-chain transcript from hybridoma K2.2 has
a human V.sub.H3-30 variable region (3 point mutations were
observed compared to the germline sequence), a human IR3rc D
segment, a human J.sub.H4 joining region and a human gamma 2
constant region. See FIG. 16C.
[0152] The kappa light chain transcript from hybridoma K2.2 is
comprised of a human kappa variable region with homology to
V.sub.kIV (B3; DPK24) (9 point mutations were observed when
compared to the germline sequence), a human J.sub.K3 joining
region, and a human kappa constant region. See FIG. 16D.
[0153] Based on sequence alignments with sequences found in the
V-base database the heavy chain transcript from hybridoma K4.2 has
a human V.sub.H4-34 variable region (8 point mutations were
observed compared to the germline sequence), a human K1 D segment,
a human J.sub.H4 joining region and a human gamma 2 constant
region. See FIG. 16E.
[0154] The kappa light chain transcript from hybridoma K4.2 is
comprised of a human kappa variable region with homology to
V.sub..kappa. 08/018 (DPK1) (6 point mutations were observed when
compared to the germline sequence), a human J.sub..kappa.4 joining
region, and a human kappa constant region. See FIG. 16F.
[0155] Based on sequence alignments with sequences found in the
V-base database the heavy chain transcript from hybridoma K4.3 has
a human V.sub.H5-51 (DP-73) variable region, a human M5-a/M5-b D
segment, a human J.sub.H4 joining region and a human gamma 2
constant region. See FIG. 16G.
[0156] The kappa light chain transcript from hybridoma K4.3 is
comprised of a human kappa variable region with homology to
V.sub..kappa. 02/012 (DPK9) (9 point mutations were observed when
compared to the germline sequence), a human J.sub..kappa.4 joining
region, and a human kappa constant region. See FIG. 16H.
[0157] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0158] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
[0159] Biological Deposits
[0160] yH1C contained in S. cerivisiae was deposited with the
American Type Culture Collection ("ATCC") , 12301 Parklawn Drive,
Rockville Md. 20852, USA, on Apr. 26, 1996, and given ATCC
accession no. 74367. The deposit of this YAC is for exemplary
purposes only, and should not be taken as an admission by the
Applicant that such deposit is necessary for enablement of the
claimed subject matter.
Sequence CWU 1
1
21 1 259 DNA Homo sapiens 1 agaccctctc actcacctgt gccatctccg
gggacagtgt ctctagcaac agtgctgctt 60 ggaactggat caggcagtcc
ccatcgagag gccttgagtg gctgggaagg acatactaca 120 ggtccaagtg
gtataatgat tatgcagtat ctgtgaaaag tcgaataacc atcaacccag 180
acacatccaa gaaccagttc tccctgcagc tgaactctgt gactcccgag gacacggctg
240 tgtattactg tgcaagaga 259 2 400 DNA Artificial Sequence
Description of Artificial Sequence Heavy chain of the antibody
secreted by clone D5.1 2 agaccctctc actcacctgt gccatctccg
gggacagtgt ctctagcgac agtgctgctt 60 ggaactggat caggcagtcc
ccatcgagag gccttgagtg gctgggaagg acatactaca 120 ggtccaagtg
gtataatgat tatgcagttt ctgtgaaaag tcgaataacc atcaacccag 180
acacatccaa gaaccagttc tccctgcagc tgaactctgt gactcccgag gacacggctg
240 tgtattactg tgcaagagat atagcagtgg ctggcgtcct ctttgactgc
tggggccagg 300 gaaccctggt caccgtctcc tcagggagtg catccgcccc
aacccttttc cccctcgtct 360 cctgtgagaa ttccccgtcg gatacgagca
gcgtggccgt 400 3 43 DNA Homo sapiens 3 cttgactagc tggggccaag
gaaccctggt caccgtctcc tca 43 4 15 DNA Homo sapiens 4 tatagcagca
gctgg 15 5 77 DNA Homo sapiens 5 gggagtgcat ccgccccaac ccttttcccc
ctcgtctcct gtgagaattc cccgtcggat 60 acgagcagcg tggccgt 77 6 302 DNA
Homo sapiens 6 gacatcgtga tgacccagtc tccagactcc ctggctgtgt
ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta
tacagctcca acaataagaa ctacttagct 120 tggtaccagc agaaaccagg
acagcctcct aagctgctca tttactgggc atctacccgg 180 gaatccgggg
tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240
atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatagtact
300 cc 302 7 442 DNA Artificial Sequence Description of Artificial
Sequence Light chain of the antibody secreted by clone D5.1 7
accatcaagt gcaagtccag ccagagtgtt ttgtacactt ccagcaataa gaactactta
60 gcttggtacc agcagaaacc aggacagcct cctaaactac tcatttactg
ggcatctacc 120 cgggaatccg gggtccctga ccgattcagt ggcagcgggt
ctgggacaga tttcactctc 180 accatccgca gcctgcaggc tgaagatgtg
gcagtttatt actgtcagca atattatact 240 attccattca atttcggccc
tgggaccaga gtggatatca aacgaactgt ggctgcacca 300 tctgtcttca
tcttcccgcc atctgatgag cagttgaaat ctggaactgc ctctgttgtg 360
tgcctgctga ataacttcta tcccagagag gccaaagtac agtggaaggt ggataacgcc
420 ctccaatcgg gttggggaaa aa 442 8 38 DNA Homo sapiens 8 attcactttc
ggccctggga ccaaagtgga tatcaaac 38 9 149 DNA Homo sapiens 9
gaactgtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg
60 gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc
aaagtacagt 120 ggaaggtgga taacgccctc caatcgggt 149 10 399 DNA
Artificial Sequence Description of Artificial Sequence Heavy chain
anti-IL-8 antibody D1.1 10 cctgtccctc acctgcgctg tctatggtgg
gtccttcagt ggttactact ggagctggat 60 ccgccagccc ccagggaagg
gactggagtg gattggggaa atcaatcaaa gtggaagcac 120 caattacaac
ccgtccctca agagtcgagt catcatatca atagacacgt ccaagaccca 180
gttctccctg aagttgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag
240 agagactccc catgcttttg atatctgggg ccaagggaca atggtcaccg
tctcttcagc 300 ctccaccaag ggcccatcgg tcttccccct ggcgccctgc
tccaggagca cctccgagag 360 cacagcgcgc cctgggctgc ctggtcaagg
actacttcc 399 11 444 DNA Artificial Sequence Description of
Artificial Sequence Kappa light chain anti-IL-8 antibody D1.1 11
cagtctccat cctccctgtc tgcatctgta ggcgacagag tcaccatcac ttgccaggcg
60 agtcaggaca ttagtaagtt tttaagttgg tttcaacaga aaccagggaa
agcccctaaa 120 ctcctgatct acggtacatc ctatttggaa accggggtcc
catcaagttt cagtggaagt 180 ggatctggga cagattttac tctcaccatc
agcagcctgc agcctgaaga tgttgcaaca 240 tatttctgta acagnatgat
gatctcccat acactttcgg ccctgggacc aaagtggata 300 tcaaacgaac
tgtggctgca ccatctgtct tcatcttccc gccatctgat gagcagttga 360
aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga gaggccaaag
420 tacagtggaa ggtggataac gccc 444 12 453 DNA Artificial Sequence
Description of Artificial Sequence Heavy chain anti-IL-8 antibody
K2.2 12 aggtccctga gactctcctg tgcagcctct ggattcacct tcagtagcta
tggcatgcac 60 tggntccgcc aggctccagg caaggggctg gagtgggtgg
cagaaatatc atatgatgga 120 agtaataaat actatgtaga ctccgtgaag
ggccgactca ccatctccag agacaattcc 180 aagaacacgc tgtatctgca
aatgaacagc ctgagagctg aggacacggc tgtgtattac 240 tgtgcgagag
accgactggg gatctttgac tactggggcc agggaaccct ggtcaccgtc 300
tcctcagcct ccaccaaggg cccatcggtc ttccccctgg cgccctgctc caggagcacc
360 tccgagagca cagcgcggcc ctgggctgcc tggtccaagg actacttccc
ccgaaccggt 420 gacggtgtcg tggaactcag gcgctctgac cag 453 13 470 DNA
Artificial Sequence Description of Artificial Sequence Kappa light
chain anti-IL-8 antibody K2.2 13 ctgacncagt ctccagactc cctggctgtg
tctctgggcg agagggccac catcaactgc 60 aagtccagcc agagtgtttt
atacatctcc aacaataaaa ctacttagct tggtaccagc 120 agaaaccagg
acagtctcct aaactgctca tttactgggc atctacccgg aaatccgggg 180
tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc atcagcagcc
240 tgcaggctga agatgtggca gtttattact gtcaacagta ttatgatact
ccattcactt 300 tcggccctgg gaccaaagtg gatatcaaac gaactgtggc
tgcaccatct gtcttcatct 360 tcccgccatc tgatgagcag ttgaaatctg
gaactgcctc tgttgtgtgc ctgctgaata 420 acttctatcc cagagaggcc
aaagtacagt ggaaggtggn taacgcccca 470 14 462 DNA Artificial Sequence
Description of Artificial Sequence Heavy chain anti-IL-8 antibody
K4.2 14 tccctcacct gcgctgtcta tggtgggtcc ttcagtggtt actactggac
ctggatccgc 60 cagcccccag ggaaggggct ggagtggatt ggggaaatca
ttcatcatgg aaacaccaac 120 tacaacccgt ccctcaagag tcgagtctcc
atatcagttg acacgtccaa gaaccagttc 180 tccctgacac tgagctctgt
gaccgccgcg gacacggctg tgtattactg tgcgagaggg 240 ggagcagtgg
ctgcgtttga ctactggggc cagggaaccc tggtcaccgt ctcctcagcc 300
tccaccaagg gcccatcggt cttccccctg gcgccctgct ccaggagcac ctccgagagc
360 acagcgcggc cctgggctgc ctggtcaagg actacttccc ccgaaccggt
gacggtgtcg 420 tggaactcag gcgctctgac cagcggcgtg cacaccttcc ca 462
15 437 DNA Artificial Sequence Description of Artificial Sequence
Kappa light chain anti-IL-8 antibody K4.2 15 tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc atcacttgcc 60 aggcgagtca
ggacattagt aactatttaa attggtatca acagaaagca gggaaagccc 120
ctaaggtcct gatctacgct gcatccaatt tggaagcagg ggtcccatca aggttcagtg
180 gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct
gaagatattg 240 caacatatta ttgtcaacac tatgataatc tactcacttt
cggcggaggg accaaggtag 300 agatcaaacg aactgtggct gcaccatctg
tcttcatctt cccgccatct gatgagcagt 360 tgaaatctgg actgcctctg
ttgtgtgcct gctgaataac ttctatccca gagaggccaa 420 agtacagtgg aaggtgg
437 16 477 DNA Artificial Sequence Description of Artificial
Sequence Heavy chain anti-IL-8 antibody K4.3 16 agtctctgaa
gatctcctgt aagggttctg gatacagctt taccagctac tggatcggct 60
gggtgcgcca gatgcccggg aaaggcctgg agtggatggg gatcatctat cctggtgact
120 ctgataccag atacagcccg tccttccaag gccaggtcac catctcagcc
gacaagtcca 180 tcagcaccgc ctacctgcag tggagcagcc tgaaggcctc
ggacaccgcc atgtattact 240 gtgcgagaca ggacggtgac tcctttgact
actggggcca gggaaccctg gtcaccgtct 300 cctcagcctc caccaagggc
ccatcggtct tccccctggc gccctgctcc aggagcacct 360 ccgagagcac
agcgcggccc tgggctgcct ggtccaagga ctacttcccc cgaaccggtg 420
acggtgtcgt ggaactcagg cgctctgacc agcggcgtgc acaccttccc actgcca 477
17 410 DNA Artificial Sequence Description of Artificial Sequence
Kappa light chain anti-IL-8 antibody K4.3 17 tgtctgcatc tattggagac
agagtcacca tcacttgccg ggcaagtcag agcattagca 60 actatttaaa
ttggtatcag cagaaaccag ggcaaagccc ctaagttcct gatctatggt 120
gcatccagtt tggaaagtgg ggtcccatca nggttcagtg gcagtggatc tgggacagat
180 ttcactctca ccatcagcag cctgcaacct gnggattttg caacttacta
ctgtcaacag 240 agttacagta accctctcac tttcggcggn gggaccaang
tggagatcaa acgaactgtg 300 gctgcaccat ctgtcttcat cttcccgcca
tctgatgagc agttgaaatc tggaactgcc 360 tctgttgtgt gcctgctgaa
taacttctat cccagagagg ccaaagtaca 410 18 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 18 ctctgtgaca ctctcctggg
agtt 24 19 26 DNA Artificial Sequence Description of Artificial
Sequence Primer 19 gaaacgacac tcacgcagtc tccagc 26 20 24 DNA
Artificial Sequence Description of Artificial Sequence Primer 20
ttttctttgt tgccgttggg gtgc 24 21 28 DNA Artificial Sequence
Description of Artificial Sequence Primer 21 gctgagggag tagagtcctg
aggactgt 28
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