US007019 193B2

(12) United States Patent (10) Patent No.: US 7,019,193 B2

Ditullio et al. (45) Date of Patent: *Mar. 28, 2006
(54) TREATMENTS USING TRANSGENC GOAT FOREIGN PATENT DOCUMENTS
PRODUCED ANTITHROMBIN II
EP O 475 354 A2 10, 1991
(75) Inventors: Paul Ditullio, Framingham, MA (US); OTHER PUBLICATIONS
Harry M. Meade, Newton, MA (US); Houdebine, L.M. Transgenic Animal Bioreactors. Trans
Edward S. Cole, Bolton, MA (US) genic Research. 2000, vol. 9, pp. 305-320.*
(73) Assignee: GTC Biotherapeutics, Inc., Denman et al., “Transgenic expression of a variant of human
Framingham, MA (US) tissue-type Plasminogen activator in goat milk: purification
and characterization of the recombinant...’ Biotechnology,
(*) Notice: Subject to any disclaimer, the term of this 9:839-843, 1991.
patent is extended or adjusted under 35 Ebert et al., “Transgenic production of a variant of human
tissue-type Plasminogen activator in goat milk: generation
U.S.C. 154(b) by 238 days. of transgenic goats and analysis . . . . Biotechnology,
This patent is Subject to a terminal dis 9:836-838, 1991.
claimer. Bock, S. et al., “Cloning and Expression of the cDNA for
Human Antithrombin III, Nucleic Acids Research, Vol. 10
(21) Appl. No.: 10/188,658 (24), pp. 8113-8125 (1982).
Cole, E. et al., “Glycosylation Patterns of Human Proteins
Expressed in Transgenic Goat Milk'.), pp. 1 of Cellular
(22) Filed: Jul. 2, 2002 Biochemistry, Supp. 0 (180D), pp. 265 (1994).
Edmonds, T. et al., “Tissue Specific and Species Differences
(65) Prior Publication Data
in the Glycosylation Pattern of Antithrombin III”, Journal of
US 2003/0096974 A1 May 22, 2003 Cellular Biochemistry, Supp. 0 (180D), pp. 265 (1994).
Fan, B. et al., “Heterogeneity of Recombinant Human
Related U.S. Application Data Antithrombin III Expressed in Baby Hamster Kidney Cells'.
The Journal of Biological Chemistry, Vol. 268 (23), pp.
(63) Continuation of application No. 09/143,155, filed on 17588-17596 (1993).
Aug. 28, 1998, now Pat. No. 6,441,145, which is a Wall, R., “Transgenic Livestock: Progress and Prospects for
continuation of application No. 08/391743, filed on the Future'. Theriogenology, vol. 45, pp. 57-68 (1996).
Feb. 21, 1995, now Pat. No. 5,843,705.
* cited by examiner
(51) Int. C.
Primary Examiner Deborah Crouch
CI2P 2L/00 (2006.01) (74) Attorney, Agent, or Firm Byron V. Olsen; GTC
AIK 67/027 (2006.01) Biotherapeutics, Inc.
A6 IK 38/00 (2006.01)
(52) U.S. Cl. ............................... 800/7: 514/12: 800/14 (57) ABSTRACT
(58) Field of Classification Search ................ 530/393;
800/7, 14-18; 514/12 This invention relates to transgenically produced human
See application file for complete search history. Antithrombin III (tgATIII). The human ATIII produced by
the transgenic process of the present invention has a
(56) References Cited monosaccharide composition which comprises N-acetylga
lactosamine (GalNAc) along with fucose, N-acetylglu
U.S. PATENT DOCUMENTS cosamine, galactose, mannose, and N-acetylneuraminic
4,517,294 A 5/1985 Bock et al. ................... 435/70 acid/N-glycolyneuraminic acid. The monosaccharide com
4,632,981 A 12, 1986 Bock et al. ................. 530,393 position differs with that of plasma derived ATIII (phaTIII).
4,873,316 A 10, 1989 Meade et al. ............... 530/412 It has been found that tgATIII has an increased clearance rate
5,366,894 A 1 1/1994 Clark et al. ......... 435/320.1 when compared to phaTIII.
5,843,705 A * 12/1998 DiTullio et al. .... 800.7
6,441,145 B1* 8/2002 DiTullio et al. ............ 530,393 10 Claims, 11 Drawing Sheets
U.S. Patent Mar. 28, 2006 Sheet 1 of 11 US 7,019,193 B2
U.S. Patent Mar. 28, 2006 Sheet 2 of 11 US 7,019,193 B2

1 1 O KC
84 Kd

47 KC
1 MW markers
33 KC 2 hp AT
3 to ATIII
24 Kd

16 KC

SSSSSR;
g

FIG 2
U.S. Patent Mar. 28, 2006 Sheet 3 of 11 US 7,019,193 B2

plasma ATI

CyS 21-95 CyS 247-430

Cys 8-128 y

A
b
S
O
r

b
al

transgenic ATI
C cys 247-430 - Y
e Y
Cys 21-95
2 Cys 8-128

2O 40 60 8O 1 OO

Time (min)

FIG 3
U.S. Patent Mar. 28, 2006 Sheet 4 of 11 US 7,019,193 B2

plasma ATI
ASn 192 N

ASn 55
A ASn 96 \
b Y
S
O
r

b
3.

C transgenic ATII
e Y
2
1

5 AS 96 AS. 155
f y ?
n ASn 192
y
Asn 135

2O 40 6O 80 OO

Time (min)
FG. 4
U.S. Patent Mar. 28, 2006 Sheet 6 of 11 US 7,019,193 B2

:s

co
S.
co
N
O

co
s
-S
e
t-' is
S
g
r C
O) y wn
co
vs.
c)
vus
co w
e
O t
coCN No - SistsO
vm va
'n'
rt
S. 2 r
O
re

N O
O vo o

cdS : 35
C
O
— O
OO
,
O
CO
,
C-
vs.
O
CN
F
U.S. Patent Mar. 28, 2006 Sheet 7 of 11

FG 7
U.S. Patent Mar. 28, 2006 Sheet 8 of 11 US 7,019,193 B2

O
CO

G
St- O
C
l
C2
C
S O.
is
ave

O
C
r

O vu
C
ram
s
SS
U.S. Patent Mar. 28, 2006 Sheet 9 of 11 US 7,019,193 B2

G
la cN

9
c
E
CD
cu is O
- P. s

O
Cf)
- --

O
O
CD

O
c O
O
C
O
y
e s
vas

SQ
U.S. Patent Mar. 28, 2006 Sheet 10 of 11 US 7,019,193 B2

Sequence Listing

aga atttacccCaagatctoaaagaccoactgaatactaaagagacct cattgttggittac -263

aataatttggggactgggccaaaact tcc.gtgcatcccagoCaagatctgtagctactgg -2O3
acaattit cattt cotttatcagattgtgagttattoctgttaaaatgctccccagaattit -143
ctgggga cagaaaaataggaagaattcatttcctaatcatgcagatttctagga attcaa -83
atcCactgttggittittatttcaaaccacaaaattagcatgccattaaatactatatataa -23
acagccactaaatcagatcattaTCCATTCAGCTTCTCCTTCACTTCTTCTCCTCTACTT 37
TGGAAAAAAGgtaagaatctoagatataatttcagtgtatatgctacticatctittattitt 97
gga Ctaggittaaaatgtagaaagaacataattgct taaaatagat.cttaaaaataagggit lS7
gtttalagataaggtttacactattitt.ca.gcagatatgttaaaaaatagaagttgactataa 27
agaCttgataaaaattataggtgactgcaa. . . . . . . . . . . . . . . . . . . . tttittgcCat 277
gaggitttgcaggatcttggttccct gaccagggatcaaacctgcactCccCtggaag cat 337
ggag tottggacatttgtattatacactatotttggttccttittaaagggaagtaattitt 397
act taaataagaaaatagattgacaagtaatacgctgtttcctcatct tccCattcacag 457
GAATCGAGAGCCATGAAGGTCCTCATCCTTGCCTGTCTGGTGGCTCTGGCCATTGCAAGA 57
vir vir vir vir M K W L I L A C L V A. L. A. I. A R l6
GAGqtaaatacagaaaaaatgttgaaataatagactag tactgtctgcctatgtgtagaa 577
E. 7
atca Cattaccaacatcataaatgtataaataatgcacaatctagatttattttittaat 637
gCtaagaaagt catttatgttcatccactatotcaa.cagtaticcitataggaccacaactic 697
tgggtcaagtgctttctatag tattgtaccatctgtaccatcaatticcitaaagaaaaagg 757
aaaaga aaccalataagcaa.ca.gaccaacaagaaggaacacagacaagaacaaaaaatgag 817
taatattgtacaaatacaattgcacg.. . . . . . . . . . . . . . . . . . . . CtgCaggalactaala 877
gtgtttittttittctcitctictttittagoAGGAAGAACTCAATGTAGTCGGTGAGgtaagat 937
Q E E L N V V G E 26
atttittatacaaagaaaaaaattaatttaactgtaaaatagta acagactctgatgatct 997
agcagaaaacticagotaattgttcaatttittatttittcctittatagaCTGTGGAAAGCCTT lO57
T W E S L. 3.
TCAAGCAGTGAGgtaagataatgttcattcagaggcaattitcccagatttagagcaataa
S S S E 35
aacact g tattatctttttgttgttacattaattggcaa.cccacticcagtact.ctt. . . . . l177
as o a us as a . . . . . gacaaatatgaattitt totttaaagctaa acctgattittatttitt 237
atttitt coaaagGAATCTATTACACACATCAATAAGgtaaaacct tca tatttaaacgta 297
E S T H I N K - 43
Cattitt Caaaaattitcatgtttgatttittataaacagoatttctt tatgtgttgattittitt l357
tttt taccagAAAATTGAGAAGTTTCAAAGTGAGGAACAACAGCAAACAGAGgtaatttg 47
K I E. K. F Q S E E Q Q Q T E 57
Catatgagtatattittgagaagitat tatgaaacataacacataaaagatttataata l477
tatgttcagtctaagaatggtaatataagtgtcagtgtaagaaatgaaaactittgaca lS37

FIG 10A
U.S. Patent Mar. 28, 2006 Sheet 11 of 11 US 7,019,193 B2

aaatgaaaatattittaaagatagaaacacatttittaaacacataatcaaattitcagagta 59
tagaataaatacccaagaataactactggtatattoattittactaatggtatacctggct 657
ttaataaatgcatattagtaggaacaattcoagactagggactggatcCCCttattgta 1717
atgatggatatgctgatgaaagacagtagggtgacagtgttggCactaatcCtaat - - - - - li"
. . . . . . . . . . . . . . . aaatggaagattittctittctotCtcttcactgaattatgttittaa l837
aaagaggaggataattcatcatgaataacaattatalactggattatggactgcaaaggca 1897
ttggtttitccttctitt.ccaggATGAACTCCAGGATAAAATCCACCCCTTTGCCCAGGCAC 957
D E L Q D K I H P F A Q A Q
AGTCTCTAGTCTATCCCTTCACTGGGCCCATCCCTAACAGCCTCCCACAAAACATCCTGC 2017
S L. v Y P E T G P I P N S L. P. Q N I L. P 9.
CTCTTACTCAAACCCCTGTGGTGGTGCCGCCTTTCCTTCAGCCTGAAATAATGGGAGTCC 2O77
I. T. O. T P v v v P P E L Q. P E I M G W P lll
CCAAAGTGAAGGAGACTATGGTTCCTAAGCACAAAGAAATGCCCTTCCCTAAATATCGAG 237
K V K E T M W P K H K E M P F P K Y P V lig
TTGAGCCCTTACTGAAAGCCAGAGCCTGACTCTCACTGATGTGAAAAGCTGCACCTTC 297
E P F T E S Q S L T L T D V E K L. H. L. P. lSl
CTCTGCCTCTGGTCCAGTCTTGGATGCACCAGCCTCCCCAGCCTCTTTCTCCAACCGTCA 2257
P L. v Q S W M H Q P P Q P L S P T V M l71.
TGTTTCCTCCTCAGTCCGTGCTGTCCCTTCTCAGCCCAAAGTTCTGCCTGTTCCCCAGA 23.
F P P or s v I. s L S Q. P K V L. P V P Q K 191
AAGCAGTGCCCCAGAGAGATATGCCCATCCAGGCCTCTGCTGTACCAGGAGCCTGTAC 2377
A W P O. R D M P L Q A F L. L. Y Q E P W L 21
TTGGTCCTGTCCGGGGACCCTTCCCTATTCTTgtaagtctaaatttactaactgttgctgt 2437
G P W R G P F P I L. 22
ttaacttctgatgtttgtatgatatttgagtaattaagagccotacaaaaaaatcaataa 2497
tgaatggttcCaaaataagcatagotgagattaatgattctoagcattagttataaatag 2557
aataa.gctggaaaacCttCaCCtcCCCtcCaCCaCCagatcCC- - - - - - - - - - - - - - - - - 267
- - - aaaaacaaaatactgaagatgcttatttcaatactCagggaaaattittCttgcCaaa 2677
aaggcaagaattgtataattcattcacttattittatttitttittaatttittaaggTCTAAG 2737
W . 222
AGGATTTCAAAGTGAATGCCCCCTCCTCACTTTTGgt. . . . . . . . . . . . . -- - - - - - Cala 297
attggaaatgggggtgagatgaagagttataacatatalactaaatggacattgttcticta 2857
ttccacagAATTGACTGcGACTGGAAATATGGCAACTTTTCAATCCTTGCATCATGCTAC 297
TAAGATAATTTTAAATGAGTATACATGGAACAAAAAAGAAACTATTCCTTATA 2977
TTTTATGCTTTTTCATCTTAATTTGAATTTGAGTCATAAACCATATACTTTCAAAATGTT 3037
AATTCAACATTAGCATAAAAGTTCAATTTTAACTTGGAAATATCATGAACAATCAAAT 3097
ATGTATAAAAATAATTTCTGgaattgttgattattatttctttaagaatctatttcctaac 357
cagtcatttcaataaattaa.ccct taggcatatttaagttttcttgttctitt attatattt 32.
ttaaaaatgaaattgg totctittattgtta acttaaatttatctittgatgttaaaaatag 3.277
ctgttggaaaa 3.287

FIG 1 OB
 US 7,019, 193 B2
1. 2
TREATMENTS USING TRANSGENC GOAT (ph ATIII). It has been found that tdATIII has an increased
PRODUCED ANTITHROMBIN III clearance rate when compared to phaTIII.
This application is a continuation of U.S. application Ser. BRIEF DESCRIPTION OF THE DRAWINGS
No. 09/143,155, filed Aug. 28, 1998 now U.S. Pat. No. 5
6,441,145; which is a continuation of U.S. application Ser. FIG. 1 is a chromatograph comparing the HPLC profile of
No. 08/391743, filed Feb. 21, 1995, which issued as U.S. the tgATIII of the present invention and plasma derived
Pat. No. 5,843,705, the contents of which are incorporated ATIII.
herein by reference. FIG. 2 is a photograph of a SDS page gel showing a
10 greater than 98% purity of tgATIII.
BACKGROUND OF THE INVENTION FIG. 3 illustrates that the disulfide cross-linking between
phaTIII and the tgATIII. The peaks labelled with the arrows
A growing number of recombinant proteins are being are the peaks containing disulfide cross-linked peptides.
developed for therapeutic and diagnostic applications; how FIG. 4 shows the individual equivalent peptides of
ever, many of these proteins may be difficult or expensive to 15 tgATIII and phaTIII by mass spectrometry.
produce in a functional form in the required quantities using FIG. 5 illustrates the mass spectrometry data for one of
conventional methods. Conventional methods involve the individual glycopeptide residues from plasma derived
inserting the gene responsible for the production of a par ATIII.
ticular protein into host cells such as bacteria, yeast, or FIG. 6 shows the mass spectrometry data for the same
mammalian cells, and then growing the cells in culture glycopeptide residue as shown for FIG. 5, but for tgATIII.
media. The cultured cells then synthesize the desired pro FIG. 7 illustrates the sialic acid composition of human
tein. Traditional bacteria or yeast systems may be unable to ATIII, goat ATIII, tgATIII, and transgenic mouse ATIII
produce many complex proteins in a functional form. While (tmATIII).
mammalian cells can reproduce complex proteins, they are FIG. 8 shows a graph comparing the clearance rate of
generally difficult and expensive to grow, and produce only 25 phaTIII and tgATIII in mice.
mg/L quantities of protein. FIG. 9 shows a graph comparing the clearance rate of
The application of transgenic technology to the commer phaTIII and tgATIII in monkey.
cial production of recombinant proteins in the milk of FIGS. 10a and 10B depict the nucleotide sequence and the
transgenic animals offers significant advantages over tradi corresponding amino acid sequence of human antithrombin
tional methods of protein production. These advantages 30 III. FIG. 10a shows residues -263 through 1537 of the
include a reduction in the total amount of required capital nucleic acid sequence. FIG. 10b shows the entire amino acid
expenditures, elimination of the need for capital commit sequence of 1597 through 3287.
ment to build facilities early in the product development life
cycle, and lower direct production cost per unit for complex DETAILED DESCRIPTION OF THE
proteins. Of key importance is the likelihood that, for certain 35 INVENTION
complex proteins, transgenic production may represent the
only technologically and economically feasible method of This invention is based upon the discovery that haTIII
commercial production. produced by the transgenic method of the present invention
Antithrombin III (ATIII) is a serine protease inhibitor is structurally different than ATIII extracted from plasma
which inhibits thrombin and the activated forms of factors 40 (ph ATIII). The monosaccharide composition of the trans
X, VII, IX, XI, and XII. It is normally present in serum at genically produced ATIII (tgATIII) differs in the composi
levels of 14–20 mg/dL. Decreased levels of ATIII may be tion and the quantity when compared to plasma derived
found in the serum of individuals who have either a heredi ATIII. In particular, the glycosylation sites on the tgATIII
tary deficiency of ATIII or an acquired deficiency, which can vary considerably in contrast to phaTIII which are all
result from a number of pathologic conditions. The conven 45 uniform.
tional treatment for hereditary ATIII deficiency is protein
replacement therapy, which may also be effective in treating Exemplification
Some acquired deficiencies.
Current methods of obtaining ATIII involves isolating the Generation of the Gene Construct
protease inhibitor from blood plasma. However, the use of 50
A mammary gland-specific transgene was constructed by
plasma-based ATIII presents various problems due to the inserting the human Antithrombin III (haTIII) cDNA into
many components in plasma, including: Variation between the caprine beta casein gene (CSN2). The caprine beta
lots; immunogenicity problems; and biohazardous risks due casein gene was cloned as an 18.5 Kb fragment in a lambda
to viral contamination.
A need exists to develope a method to produce ATIII 55
EMBL3 vector (Roberts, et al., Gene., 1992. 121: p.
without the inherent problems of the present process. 255–262). The 6.2 Kb promoter (including exon 1 and part
of exon 2) was fused to the haTIII cDNA to direct high level
SUMMARY OF THE INVENTION
mammary gland-specific expression. A 7.2 Kb 3' flanking
region (including part of exon 7, exon 8, and exon 9) was
This invention relates to transgenically produced human 60
added to the 3' end of the haTIII cDNA to help stabilize the
expression levels. The 14.95 Kb transgene was excised from
Antithrombin III (tgATIII). The human ATIII (haTIII) pro bacterial sequences and injected into goat embryos for the
duced by the transgenic process of the present invention has production of hATIII in goats milk.
a monosaccharide composition which comprises N-acetyl
galactosamine (GalNAc) along with fucose, N-acetylglu Identification of Gene Coding for the Protein of Interest
cosamine, galactose, mannose, and N-acetylneuraminic 65 The haTIII cDNA was received from Dr G. Zettimei?B1
acid/N-glycolyneuraminic acid. The monosaccharide com (Behringwerke A. G., Marburg, Germany) in the plasmid
position differs with that of human plasma derived ATIII pBAT6. The sequence of the cDNA is the same as that
 US 7,019, 193 B2
3 4
published by Bock, et al., Nucleic Acids Research, 1982. 10: linker ligated into place. The ligation mixture was extracted
p. 8113-8125, except for the silent nucleotide changes at bp with phenol-chloroform, chloroform-isoamyl alcohol, and
1096 (TC) and bp 1417 (A-G). ethanol precipitated as described in Maniatis, et al., 1989.
Identification of Regulatory Sequences of Interest The DNA was resuspended in digestion buffer and digested
To direct high level tissue-specific expression of haTIII to with EcoRI and BamHI. The resulting 1.5 Kb fragment was
the mammary gland of transgenic goats, the goat beta casein gel purified and ligated into puC to form the plasmid Bc 150.
gene was cloned from a lambda EMBL3 goat genomic The entire available 5' region of the beta casein gene was
library. The goat beta casein gene is a mammary gland constructed by sequential addition of fragments contained in
specific gene which directs expression of high levels of beta the subclones Bc 104, Bc147, and Bcl03. The orientation of
casein into the milk. In goats, beta casein is thought to
10 the Bc 147 clone was verified by restriction mapping. The
comprise 25–50% of the total milk proteins (~10–20 replacement of the 2.6 Kb Xbal fragment with the Xbal
mg/ml). The goat beta casein gene was cloned from a Saanen fragment from Bc147 was needed to replace a 31 bp deletion
goat genomic library and characterized in transgenic mice as in the promoter between adjacent EcoRI restriction sites. To
described in Roberts, et al., 1992. allow for the addition of the human antithrombin III cDNA,
15 the BamHI site on the 3' end of Bc 113 was converted to an
Cloning DNA fragments XhoI site to form Bc114.
High molecular weight goat DNA was isolated from a The 3' end of the goat beta casein gene was constructed in
Saanen goat blood sample according to the procedure a similar fashion to the 5' end. The 1.8Kb Bc107 clone was
described by Herrmann and Frischauf, Methods Enzymol restricted with PpuMI to allow for the addition of a BamHI
ogy, 1987. 152: p. 180-183. The genomic DNA was partially linker. The BamHI site was placed in exon 7 to include the
digested with Mbol and ligated into BamHI cleaved lambda intron between exons 7 and 8. The BamHI site is seven
EMBL3 phage arms (Stratagene, La Jolla, Calif.) by stan amino acids 5' to the termination of the goat beta casein
dard methods (Maniatis et al., Molecular Cloning, A Labo mRNA but will have no effect on the hATIII since the cDNA
ratory Manual. 2d, Cold Spring Harbor Laboratory Press, for ATIII contains a termination signal. The BamHI/HindIII
1989). The Saanen goat genomic library consisted of 1.2x 25 fragment spanning exon 7 from Bc 107 was ligated into the
10' recombinant phage and was used to isolate the goat beta vector Bc109 which contained the extreme 3' HindIII/Sa.
casein gene. The goat genomic library was screened using a fragment to form Bc165. The 3' end was completed by the
1.5 Kb HindIII/TthIII1 fragment encoding the entire mouse addition of the 4.4 Kb HindIII fragment from Bc 108 to form
CSN2 cDNA (Yoshimura, et al., Nucleic Acids Res., 1986. the vector Bc1 18. The orientation of HindIII fragment from
14: p. 8224) and three overlapping clones were identified. 30 Bc 108 was verified by restriction mapping. To allow for the
The three overlapping clones were designated EMBL 3-7, addition of the haTIII cDNA, the 5' BamHI site in the vector
3-8, and 3-11. Only clone 3-11 contained the entire goat beta Bc 118 was changed to XhoI and the 3' SalI was converted
casein gene and was used for all future analysis and vector to Not to form Bc 122.
construction (Roberts, et al., 1992). In order to clone the entire haTIII cDNA into the goat
35
Characterization of Cloned Material beta casein expression vector, the 5' EcoRI restriction site
The goat beta casein gene was isolated as an 18.5 Kb was converted to an XhoI site using site-directed mutagen
fragment in lambda EMBL3 phage clone designated esis. The new vector designated pAT7 was sequenced to
EMBL3-11 (Roberts, et al., 1992). The fragment contains confirm the addition of the SalI site and the absence of any
4.2 Kb of 5" flanking region, exons 1 through 9, and 5.3. Kb nucleotide changes in the 5' end. The haTIII clNA was
40 isolated as a 1.45 Kb XhoI/SalI fragment for cloning into the
of 3' flanking sequence. The gene was subcloned into puC19
or pGEM37 vectors. The subclones were sequenced by beta casein expression vector.
Sanger dideoxy sequencing to verify all intron/exon bound The 1.45KBXhoI/SalI haTIII cDNA was first ligated into
aries and the sequence is shown in SEQ. ID. No.: 1. All XhoI digested Bc 114 and Bc 122 to form Bc143 and Bc 144,
sequences are deposited with GenBank (accession Nos. 45
respectively. The orientation of the cDNA was confirmed by
M90556, M90557, M90558, M90559, M90560, M90561, sequencing and restriction analysis. The unique SacII
and M90562). restriction in the haTIII cDNA allows for the directional
cloning of the intact goat beta casein haTIII vector. The
Assembly and Characterization of the Gene Construct complete transgene was assembled by ligating the SalI/SacII
The beta casein haTIII transgene was constructed to fragment from Bc 143 into a SalI/SacII digested Bc 144. The
resemble as accurately as possible the intact goat beta casein 50
5' and 3' junction of the haTIII cDNA were confirmed by
gene. The vector was 14.95 Kb in length with 4.2 Kb of 5' sequencing to ensure no mutations had occurred. The final
flanking sequence and 5.3. Kb of 3' flanking sequence. The vector (Bc6) was 14.95 Kb in length and contained 4.2 Kb
vector was engineered with three introns between exons of 5' flanking region, exon 1, intron 1, part of exon 2, the
1–2, 7–8, and 8–9 to allow for splicing of the transcript. The 1.45 Kb human AT III clNA, part of exon 7, intron 7, exon
introns were added to increase expression of the transgene 55
8, intron 8, exon 9, and 5.3 Kb of 3' flanking sequence. The
(Brinster et al., Proc. Natl. Acad. Sci. USA., Devel. Biol. 85, transgene was excised from the puC backbone by digestion
pp.836-840, 1988.) and the 3' flanking sequence to decrease with SalI/NotI. The Bc6 transgene directs mammary specific
chromosomal position effects. expression of an approximately 1.5 Kb haTIII-goat beta
To allow for the cloning of various inserts, the beta casein casein hybrid mRNA containing the 5' and 3' untranslated
promoter and 3’ flanking region were reassembled from the 60
regions of the goat beta casein mRNA and coding region of
Subclones with unique restriction sites in exon 2 and exon 7. the hATIII mRNA.
To remove the initiator methionine of the goat beta casein,
the plasmid Bc106 was digested with the restriction enzyme Testing and Characterization of Gene Constructs
Taq I. The Taq.I site restricts the plasmid Bc 106 six nucle Transgene constructs are generally tested in a mouse
otides upstream of the initiator methionine (TaqI site marked 65 model system to assess their ability to direct high levels of
with ****, FIG.10). Following digestion with Taq, the ends expression and their ability to express in a tissue-specific
were filled in with Klenow DNA polymerase and a BamHI a.
 US 7,019, 193 B2
5 6
The Bc6 transgene was prepared for microinjection by prevent adhesions. The linea alba was closed with simple
digestion of the Bc6 plasmid with SalI/NotI, removal of the interrupted sutures of 2.0 Polydioxanone or Supramid and
bacterial sequences by TAE agarose gel electrophoresis the skin closed with sterile wound clips.
followed by banding of the transgene on a cesium chloride Fertilized goat eggs were collected from the PBS oviduc
gradient according to Lacy, et al., A Laboratory Manual, tal flushings on a stereomicroscope, and were then washed
1986. Cold Springs Harbor Laboratory, New York. The in Ham's F12 medium (Sigma, St. Louis, Mo.) containing
14.95 Kb transgene was then microinjected into mouse 10% fetal bovine serum (FBS) purchased from Sigma. In
embryos to test the ability of the goat beta casein promoter cases where the pronuclei were visible, the embryos were
to direct high level expression of recombinant human Anti immediately microinjected. If pronuclei were not visible, the
thrombin III (rhATIII) into the milk. Seventeen transgenic 10 embryos were placed in Hams F12 containing 10% FBS for
mouse lines were generated as determined by Southern short term culture at 37° C. in a humidified gas chamber
analysis. One mouse line showed a level of expression of containing 5% CO in air until the pronuclei became visible
rhAT III of 0.7–1.0 mg/ml as determined by Western blot (Selgrath, et al...Theriogenology, 1990. p. 1195–1205).
analysis and a thrombin inhibition assay. Based on expres Microinjection Procedure
sion results and data collected from other ongoing mouse 15
One-cell goat embryos were placed in a microdrop of
and goat experiments, the Bc6 transgene was microinjected
into goat embryos for production of transgenic goats. medium under oil on a glass depression slide. Fertilized eggs
having two visible pronuclei were immobilized on a flame
Generation and Characterization of Transgenic Animals polished holding micropipet on a Zeiss upright microscope
A founder (Fo) transgenic goat is defined as a viable with a fixed stage using Normarski optics. A pronucleus was
transgenic animal resulting from embryo transfer of fertil microinjected with the Bc6 DNA construct in injection
ized goat eggs that have been microinjected with a specified buffer (Tris-EDTA) using a fine glass microneedle (Selgrath,
construct (Bc6). Four founder BcG goats were produced. The et al., Theriogenology, 1990. p. 1195–1205).
general methodologies that follow in this section were used Embryo Development
to generate all transgenic goats. 25 After microinjection, the Surviving embryos were placed
Goat Species and Breeds in a culture of Ham's F12 containing 10% FBS and then
The transgenic goats produced for ATIII production are of incubated in a humidified gas chamber containing 5% CO
Swiss origin, and are the Alpine, Saanen, and Toggenburg in air at 37°C. until the recipient animals were prepared for
breeds. embryo transfer (Selgrath, et al. Theriogenology, 1990. p.
30
Goat Superovulation 1195-1205).
The timing of estrus in the donors was synchronized on Preparation of Recipients
Day 0 by 6 mg Subcutaneous norgestomet ear implants Estrus synchronization in recipient animals was induced
(Syncromate-B, CEVA Laboratories, Inc., Overland Park, by 6 mg norgestomet ear implants (Syncromate-B). On Day
Kans.). Prostaglandin was administered after the first seven 35 13 after insertion of the implant, the animals were given a
to nine days to shut down the endogenous synthesis of single non-Superovulatory injection (400 I.U.) of pregnant
progesterone. Starting on Day 13 after insertion of the mares serum gonadotropin (PMSG) obtained from Sigma.
implant, a total of 18 mg of follicle-stimulating hormone Recipient females were mated to vasectomized males to
(FSH Schering Corp., Kenilworth, N.J.) was given intra ensure estrus synchrony (Selgrath, et al., Theriogenology,
muscularly over three days in twice-daily injections. The 1990. p. 1195-1205).
implant was removed on Day 14. Twenty-four hours fol
lowing implant removal the donor animals were mated Embryo Transfer
several times to fertile males over a two-day period (Sel All embryos from one donor female were kept together
grath, et al., Theriogenology, 1990. pp. 1195–1205). and transferred to a single recipient when possible. The
Embryo Collection 45
Surgical procedure was identical to that outlined for embryo
Surgery for embryo collection occurred on the second day collection outlined above, except that the oviduct was not
following breeding (or 72 hours following implant removal). cannulated, and the embryos were transferred in a minimal
Superovulated does were removed from food and water 36 volume of Hams F12 containing 10% FBS into the oviduc
hours prior to Surgery. Does were administered 0.8 mg/kg tal lumen via the fimbria using a glass micropipet. Animals
Diazepam (Valium(R), IV, followed immediately by 5.0 50
having more than six to eight ovulation points on the ovary
mg/kg Ketamine (Keteset), IV. Halothane (2.5%) was were deemed unsuitable as recipients. Incision closure and
administered during Surgery in 2 L/min oxygen via an post-operative care were the same as for donor animals
(Selgrath, et al., Theriogenology, 1990. p. 1195–1205).
endotracheal tube. The reproductive tract was exteriorized
through a midline laparotomy incision. Corpora lutea, Monitoring of Pregnancy and Parturition
unruptured follicles greater than 6 mm in diameter, and 55 Pregnancy was determined by ultrasonography 45 days
ovarian cysts were counted to evaluate Superovulation after the first day of standing estrus. At Day 110 a second
results and to predict the number of embryos that should be ultrasound exam was conducted to confirm pregnancy and
collected by oviductal flushing. A cannula was placed in the assess fetal stress. At Day 130 the pregnant recipient doe
ostium of the oviduct and held in place with a single was vaccinated with tetanus toxoid and Clostridium C&D.
temporary ligature of 3.0 Prolene. A 20 gauge needle was 60 Selenium and vitamin E (Bo-Se) were given IM and Iver
placed in the uterus approximately 0.5 cm from the utero mectin was given SC. The does were moved to a clean stall
tubal junction. Ten to twenty ml of sterile phosphate buffered on Day 145 and allowed to acclimatize to this environment
saline (PBS) was flushed through the cannulated oviduct and prior to inducing labor on about Day 147. Parturition was
collected in a Petri dish. This procedure was repeated on the induced at Day 147 with 40 mg of PGFa (LutalyseR)
opposite side and then the reproductive tract was replaced in 65 purchased from Upjohn Company, Kalamazoo, Mich. This
the abdomen. Before closure, 10–20 ml of a sterile saline injection was given IM in two doses, one 20 mg dose
glycerol Solution was poured into the abdominal cavity to followed by a 20 mg dose four hours later. The doe was
 US 7,019, 193 B2
7
under periodic observation during the day and evening AT III Activity Assay
following the first injection of Lutalyse?R) on Day 147. Materials
Observations were increased to every 30 minutes beginning 1. 12x75 mm Glass Tubes;
on the morning of the second day. Parturition occurred 2. Behring hATIII dilute to 0.5 mg/ml with diHO
between 30 and 40 hours after the first injection. Following 5
(E9%280–0.72);
delivery the doe was milked to collect the colostrum and 3. Thrombin (100 units/vial diluted to 1.82 mls with 0.1
passage of the placenta was confirmed. mg/ml BSA), purchased from Calbiochem, San Diego
Verification of the Transgenic Nature of F Animals Calif.). Final Concentration 20 g/ml;
To screen for transgenic Fo animals, genomic DNA was 4. Heparin (Sigma) from porcine intestinal mucosa 50 mg
10
isolated from two different cell lines to avoid missing any dissolved 5.0 mls diHO. Final Concentration 10 mg/ml;
mosaic transgenics. A mosaic animal is defined as any goat 5. Substrate (Kabi) S2238 25 mg diluted to 10 mls with
that does not have at least one copy of the transgene in every diHO:
cell. Therefore, an ear tissue sample (mesoderm) and blood 6. Dilution Buffer: 50 mM Tris, 150 mM NaCl, 0.1 mg/ml
sample were taken from a two day old Fo animal for the 15 BSA pH 8.1;
isolation of genomic DNA (Lacy, et al., A Laboratory 7. Glacial Acetic Acid;
Manual, 1986, Cold Springs Harbor, N.Y.; and Herrmann 8. Repeat Pipettor; and
and Frischauf, Methods Enzymology, 1987. 152: pp. 9. 37° C. circulating HO bath.
180–183). The DNA samples were analyzed by the poly
merase chain reaction (Gould, et al., Proc. Natl. Acad. Sci. ASSAY SET-UP
1989. 86.p. 1934–1938) using primers specific for hATIII
and by Southern blot analysis (Thomas, Proc Natl. Acad. Dilution of Stocks
Sci., 1980. 77:5201–5205) using a random primed hATIII
cDNA probe (Feinberg and Vogelstein, Anal. Bioc., 1983. 1. ATIII stock to 1 ug/ml: 20 ul ATIII+980 uls dilution buffer
132: pp. 6-13). Assay sensitivity was estimated to be the then 100 ul--900 ul dilution buffer;
detection of one copy of the transgene in 10% of the somatic 25 2. Heparin/Buffer: 12.5 ul Heparin-i-25 mls dilution buffer;
cells. and
Generation and Selection of Production Herd 3. Thrombin stock: 1:10 dilution into dilution buffer (2.0 mls
The procedures described above were utilized for produc for a 30 tube assay).
tion of the transgenic founder (Fo) goats, as well as other 30 Standard Curve Set-Up
transgenic goats in our herd. The transgenic Fo founder
goats, for example, were bred to produce milk, if female, or
to produce a transgenic female offspring if it was a male
founder. Final Concentration Diluted AT Heparin
AT III III Stock Dil Buffer Buffer Thrombin
This transgenic founder male, was bred to non-transgenic 35
females, and produced transgenic female offspring. Ongfml Oil 100 ul 750 pil 50 ul
Transmission of Transgene and Pertinent Characteristics 5 ng/ml 5 ul 95 ul 750 pil 50 ul
10 ng/ml 10 ul 90 ul 750 pil 50 ul
Transmission of the Bc6 transgene in our goat line was 20 ng/ml 20 ul 80 ul 750 pil 50 ul
analyzed in ear tissue and blood by PCR and Southern blot 30 ng/ml 30 ul 70 ul 750 pil 50 ul
analysis. For example, Southern blot analysis of the founder 40 40 ng/ml 40 ul 60 ul 750 pil 50 ul
male and the three transgenic offspring showed no rear 50 ng/ml 50 ul 50 ul 750 pil 50 ul
60 ng/ml 60 ul 40 ul 750 pil 50 ul
rangement or change in the copy number between genera
tions. The Southern blots were probed with the 1.45 Kb
ATIII cDNA and a 0.38 Kb probe corresponding to goat beta 1. Incubate the above at 37° C. for 15 minutes;
casein exon 7. The blots were analyzed on a Betascope 603 45 2. Then add 100 ul of substrate to each tube using a repeat
and copy number determined by comparison of the trans pipettor and incubate for 15 minutes at 37° C.;
gene to the goat beta casein endogenous gene. 3. Stop with 50 ul of glacial acetic acid to each tube; and
Evaluation of Expression Levels 4. Read at 405 nm. Blank with 150 ul Buffer, 750 ul Heparin
The expression level of rhaTIII in the milk of transgenic Buffer, 100 ul substrate and 50 ul Acetic acid stopped at
animals is determined using a thrombin inhibition assay, 50 time zero (NOT INCUBATED).
which measures the inhibition of thrombin’s ability to Calculations
remove a small peptide from an artificial substrate (S2238, Use a second order polynomial curve fit for the standard
Kabi, Franklin Ohio). The basis for this assay is described as curve, plug A 405 nm readings into the equation. Divide by
follows. The interaction between ATIII and thrombin
amounts to rapid irreversible inhibition of the protease by 55
the Volume of sample used in the assay tube in mls and
multiply by the appropriate inverse dilution factor for the
ATIII in the presence of heparin. However, the interaction is sample.
very slow in the absence of heparin. Attempts to extend the
range of ATIII detectable on a single standard curve reveal Characterization of Human ATIII Produced in the Milk of a
that ATIII can only be determined accurately in stoichio Transgenic Goat
metric titration across the linear range of standard curves. At 60 TgATIII was purified from goat milk by adaptation of
low total thrombin concentration (0.7x10M), the effective methods previously developed for extracting ATIII from
measuring range for ATIII is 0.15-0.75x10M (-7.3–36.8 mouse milk and human blood. Other more novel methods
ng/ml). At high total thrombin, the effective measuring range for separating proteins from milk are also in development.
for AT III is 0.25 to 1.25x10M (-12-60 ng/ml) if the data The method described is the earliest method that we utilized.
are fit with a first degree polynomial, and 0.25 to 2.5x10M 65 Milk from one BC6 goat contained tgATIII at a concentra
(~12–120 ng/ml) if the data are fit with a second degree tion estimated to be 0.08 mg/ml by ELISA. Other goats with
polynomial. much higher expression levels (4 to 6 mg/ml) were also
 US 7,019, 193 B2
10
developed and milk from those goats was purified using methionines on this ATIII may be a function of the process
similar methods. Small scale purification was performed by ing of the molecule following removal of the milk from the
the following method. goat.
Monosaccharide compositional analysis indicating the
ATIII Purification Process amounts of fucose (Fuc), GalNac, N-Acetylglucosamine
ATIII Extraction/Clarification
(GlcNAc), Galactose (Gal), Mannose (Man) and Sialic Acid
is shown below in Table 1.
Thaw Milk
Add an equal volume of 2M Arginine TABLE 1.
Centrifuge at 8000 rpm for 30 minutes and remove the fat 10
pad on the top mole Sugar mole protein
Filter through a 0.2 um nominal polypropylene filter Sample Fuc GalNAc GlcNAc Gal Man Sialic Acid
Dilute the conductivity to less than 20 mS/cm (approxi
mately a 1/7 dilution) phATIII 0.39 0.00 20.70 11.74 14.90 11.8O
15 tATIII 3.18 2.39 14.75 6.44 1818 S.21
Expected step yield of AT III is 2.95%
Heparin-Hyper DTM Chromatography
Equilibrate with 3 column volumes of 20 mM sodium Lysylendopeptidase maps of plasma derived hATIII and
phosphate, 50 mM NaCl, pH7.0 tgATIII have been analyzed by liquid chromatography/mass
Load, the target operational capacity is approximately 8 spectrometry (LC/MS) to compare the amino acid backbone
mg of ATIII/mL of resin structure, the glycosylation profile and to confirm the iden
Wash with 4 column volumes of Equilibration Buffer tity of the disulfide linked peptides. For non-reduced human
Step Elute with 4 column volumes of 20 mM sodium plasma ATIII and ty. ATIII, 32 of the 35 predicted peptides
phosphate, 3.0M NaCl, pH7.0 were identified, including the three pairs of disulfide linked
Dilute with 9 volumes of 1.5 M sodium citrate 25
peptides. The disulfide cross-linking was identical in the
Expected step yield of AT III is 2.95% plasma ATIII and the tgATIII as shown in the peptide maps
Ether-PorosTM 50 Chromatography of the non-reduced proteins (FIG. 3). The peaks labeled with
Equilibrate with 3 column volumes of 1.40M sodium the arrows are the peaks containing disulfide cross-linked
citrate, pH7.0 peptides. These peaks have identical retention times for each
Load, the target operational capacity is approximately 4 30
disulfide crosslinked peptide pair of each protein. The
mg of ATIII/mL of resin remaining peaks on the maps are identical with the excep
Wash with 4 column volumes of Equilibration Buffer tion of the glycosylated peptides. The maps of the reduced,
Step Elute with 4 column volumes of 1.10M sodium pyridylethylated ATIII digests detail this variability more
citrate, pH7.0 specifically.
Expected step yield of AT III is 2.75% 35 For reduced and pyridylethylated plasma and transgenic
Ultrafiltration with Amicon YM10 Spiral Crossflow hATIII, 33 of the 35 predicted peptides were identified by
Concentrate to approximately 20 mg/mL (160 units/mL) LC/MS. Chromatograms of the plasma ATIII and the indi
and buffer exchange into 10 mM vidual forms of the tgATIII can be seen in FIG. 4 with the
ammonium bicarbonate, pH 7.4. identification of the individual equivalent peptides by mass
Expected step yield of AT III is 2.95% 40 spectrometry shown in Table 2 below. Again, all of the
Lyophilize peptides are identical with the exception of the glycosylated
peptides.
Reconstitute into buffer of choice
Expected step yield of AT III is 2.95% TABLE 2
A sample of the concentrated pool was examined by SDS 45 LCMS ANALYSIS of ATIII PEPTIDE MAPS
PAGE (10% separating gel) with a human ATIII standard for
comparison and estimation of purity. The purity is estimated PEPTIDE RESIDUES
THEORETICAL
MASS ph ATIII tgATIII
to be >98% (see FIG. 2.). The UV/Vis spectrum of tgATIII
is indistinguishable (within experimental error) from that of K1 1–11 232.4 232.O 232.3
plasma derived hATIII. N-terminal sequence analysis gave 50 K2 12-28 2152.6 2152.6 2152.3
the predicted sequence for hATIII. Reversed phase HPLC K3 29-29 146.2 ID ID
K4 30-39 O93.1 O92.9 O92.8
analysis showed three peaks for goat tgATIII compared to KS 40-53 699.O 698.7 698.7
two peaks for plasma ATIII (FIG. 1). All three peaks were K6 S4 70 957.1 956.9 956.9
found to be ATIII by N-terminal sequence analysis. Peak #1 K7 71-91 2299.6 2299.3 2299.4
is miniscule in the tgATIII and not apparent in the plasma 55 K8 92-107 GP GP GP
K9 108-114 838.9 838.7 838.7
derived ATIII. Peak 2 is present in both samples and varies K2O 115-125 34O.S 340.3 340.2
with preparations. Peak three is the predominant peak. K11 126-133 170.5 170.1 170.1
Examination of specific peaks from FIG. 1 following diges K12 134 136 GP GP GP
tion with lysylendopeptidase and separation by reverse K13
K14
137 139
140-150
3204
219.4
ND
219.1
ND
219.4
phase HPLC to create peptide maps identified the presence 60 K15
151-169 GP GP GP
of oxidized methionine residues in the two early small peaks K16 170–176 860.O 860.2 860.O
of the tgATIII. The middle peak contained oxidized K17 177 188 3304 330.4 330.2
methionines mainly on a single peptide (K30 on table 2), K18 189-193 GP GP GP
K19 194-222 3248.8 3248.8 3248.8
whereas the earliest peak contained oxidized methionines on K2O 223-226 SO2.6 SO2.9 SO2.8
most of the peptides that contained methionine. The major 65 K21 227 228 233.3 ND ND
peak did not contain any oxidized methionines as measured K22 229-236 978.1 978.1 977.9
by the technique being used above. The presence of oxidized
 US 7,019, 193 B2
11 12
much more heterogeneous than phaTIII, with a higher
TABLE 2-continued degree of fucosylation and more varied sialylation (Table 3).
LCMS ANALYSIS of ATIII PEPTIDE MAPS
Several glycoforms with a mass difference of 41 were
observed by LC/MS which can be accounted for by the
THEORETICAL substitution of a Hexose residue by a HexNAc (Tables 4). In
PEPTIDE RESIDUES MASS ph ATIII tgATIII view of the monosaccharide composition (Table 1) and the
K23 237-241 698.8 698.5 698.6
lack of O-linked glycosylation (based on comparison of the
K24 242-257 1799.0 1798.8 1798.9 observed vs theoretical mass for all peptides other than those
K2S 258. 275 2209.6 2209.3 2209.6 containing an N-linked site) this could be accounted for by
K26 276-287 1314.6 ID ID 10 the Substitution of one or more galactose residues by Gal
K27 288-290 372.4 ID ID NAc. This result was confirmed by fragmentation analysis
K28 291-294 417.5 417.3 417.5
K29 295 297 3745 374.2 374.2 mass spectroscopy of isolated glycopeptides. FIG. 5 is an
K3O 298–332 4261.O 42611 4260.9 example of the mass spectrometry data for one of the
K31 333-348 1849.1 1849.O 1848.9 individual glycopeptide residues from phaTIII. It’s pattern
K32 349-350 233.3 ID ID 15 is fairly simple. In contrast, FIG. 6 is an example of the same
K33 351 370 22O2S 22O2S 22O2.2
K34 371-403 3448.9 3448.7 3448.7 glycopeptide from tgATIII. The increased complexity of the
K35 404-432 3421.2 3421.2 3421.2 pattern is obvious, and can be deciphered into specific
K3–4 29-39 1221.3 12214 1221.1 structures as described above.
K26-27 276-290 1669.0 1668.8 1668.9
K32-33 349-370 2417.7 2417.5 2417.6
Several glycoforms with mass differences of 16 mass
units were also identified. The difference is explained by the
GP = Glycopeptide presence of an oxidized form of sialic acid, N-Glycoly
ND = Not detected (m/z 2+ below scan range) ineuraminic acid (NGNA) in place of N-Acetylineuraminic
D = Incomplete digestion product acid (NANA). NGNA is a common form of sialic acid found
phATIII = Human Plasma. ATIII
gATIII = Transgenic (goat) Human ATIII in goats. Approximately 25% of the sialic acids found in
25 tgATIII are NGNA. Approximately 25% of goat plasma
The glycopeptide peak retention times (glycopeptide ATIII sialic acid is NGNA (FIG. 7).
peaks are labeled with Kils on FIG. 4) did not vary Thus, we have determined that: (1) one of the four
significantly from those of the plasma derived protein, but glycosylation sites on tgATIII has mainly high mannose
the chromatograms show that peak widening and peak (oligomannose) and hybrid type oligosaccharide structures,
splitting did occur due to the variability in the oligosaccha 30 whereas the phaTIII has biantenarry, complex oligosaccha
ride residues attached to the glycopeptides. rides on each of the four sites; (2) the complex oligosac
Based on the LC/MS, peptide mapping, and carbohydrate charides of tgATIII are not fully sialylated, whereas the
composition data, the only differences observed between the phaTIII oligosaccharides are fully sialylated; (3) the tgATIII
tgATIII and the plasma derived hATIII arise due to the has a percentage of its sialic acid that is NGNA whereas the
different patterns of glycosylation. Despite the presence of 35 phaTIII has only NANA; and (4) tgATIII contains N-acetyl
N-Acetylgalactosamine (GalNAc) in the monosaccharide galactosamine on its N-linked oligosaccharides and the
analysis, no O-linked glycosylation was present on the phaTIII does not; and (5) the tgATIII has fucose on its
transgenic protein, Suggesting the presence of oligosaccha proximal GlcNAc on each of the three sites having complex
rides different than found on the plasma derived human oligosaccharides, whereas the phaTIII has only a very Small
ATIII. 40 amount of fucose on any site.
The major glycoform at each glycosylation site of the The tgATIII exhibits a faster clearance time in rabbits,
plasma derived (pATIII) is a complex type oligosaccharide mice and monkeys than does phaTIII. Twenty ug samples of
and has a mass corresponding to HexNAc4, Hex5, NANA2 test ATIII was injected via the tail vein and residual ATIII
which is in agreement with the reported structure GlcNAc4, determined using an ELISA assay which has little cross
Man3, Gal2, NANA2. With the exception of Asn'ss, located 45 reactivity with mouse ATIII. The pattern shown in FIG. 8 for
on K15, the major glycoforms on tgATIII contain oligosac clearance in mice mimics the pattern found for the same
charides of the complex type. The major glycoform at each materials in rabbits. The clearance appears to be bimodal
of the complex oligosaccharide containing sites on tgATIII and is approximately 10 times faster than for haTIII. In vivo
has a mass corresponding to HexNAc4, Hex6, NANA1 clearance was also examined in a monkey model system.
which is in agreement with the structure GlcNAc4, Fuc, 50
Both trace and high levels of radioiodinated ATIII were
Man3, Gal2, NANAL with the second most abundant form injected and detected in plasma samples by counting in a
being the disialylated form of the same structure, HexNAc4, gamma counter. The clearance pattern of tgATIII in mon
Hex6, NANA2 which is in agreement with the structure keys indicated only a 4 to 5-fold faster clearance (FIG.9)
GlcNAc4, Fuc1, Man3, Gal2, NANA2 from the circulation than the phaTIII and could also be
TgATIII contains a significant amount of oligomannose 55 defined by a biphasic mechanism.
type and hybrid forms at ASn55 and only a very low level Early experiments indicate that the tgATIII may have a
of hybrid structures at the other locations. Oligomannose stronger affinity for heparin than the phaTIII. This would be
type structures are more primitive structures that are remod important since ATIII inhibits thrombin at inflamation or
eled into the complex type oligosaccharides in the endo injury sites by binding to heparan Sulfate in the endothelial
plasmic reticulum. Oligomannose structures display masses 60
layer of the vasculature. Once bound its affinity for thrombin
ranging from Hex5 to HexNAc2, Hex9. These values agree is enhanced 1000 fold and it binds to and irreversibly
with structures comprised of 5 up to GlcNAc2, Man9, with inhibits thrombin.
only the number of mannose residues varying. Hybrid
oligosaccharides contain elements of complex oligosaccha Equivalents
rides on one antenna of an individual glycosylation site and 65 Those skilled in the art will recognize, or be able to
components of oligomannose type oligosaccharides on the ascertain, using no more than routine experimentation, many
other antenna. The N-linked glycosylation for tgATIII was equivalents to the specific embodiments of the invention
 US 7,019, 193 B2
13 14
described herein. Such equivalents are intended to be 6. The method of claim 1, wherein the ATIII deficiency is
encompassed by the following claims: acquired ATIII deficiency.
The invention claimed is: 7. A method of treating inflammation in a subject, com
1. A method of treating a subject having an ATIII defi prising:
ciency or inflammation, comprising: administering to the Subject a Moat mammary gland
administering to the Subject goat mammary gland pro produced antithrombin III having a monosaccharide
duced antithrombin III having a monosaccharide com composition which comprises a higher level of man
position which comprises a higher level of mannose nose than plasma derived antithrombin III, wherein
than plasma derived antithrombin III, wherein throm thrombin is inhibited.
bin is inhibited. 10
2. The method of claim 1, wherein the mammary gland 8. The method of claim 7, wherein the mammary gland
produced antithrombin III further has a monosaccharide produced antithrombin III further has a monosaccharide
composition which comprises fucose. composition which comprises fucose.
3. The method of claim 1, wherein the mammary gland 9. The method of claim 7, wherein the mammary gland
produced antithrombin III further has a monosaccharide 15 produced antithrombin III further has a monosaccharide
composition which comprises GalNAc. composition which comprises GalNAc.
4. The method of claim 1, wherein the mammary gland 10. The method of claim 7, wherein the mammary gland
produced antithrombin III is produced in the mammary produced antithrombin III is produced in the mammary
glands of a transgenic goat. glands of a transgenic goat.
5. The method of claim 1, wherein the ATIII deficiency is 20
hereditary ATIII deficiency.