U.S. patent application number 13/385527 was filed with the patent office on 2013-04-25 for extended soluble ph20 polypeptides and uses thereof.
The applicant listed for this patent is Louis Bookbinder, Gregory I. Frost, Krishnasamy Panneer Selvam, Ge Wei. Invention is credited to Louis Bookbinder, Gregory I. Frost, Krishnasamy Panneer Selvam, Ge Wei.
Application Number | 20130101577 13/385527 |
Document ID | / |
Family ID | 42035671 |
Filed Date | 2013-04-25 |
United States Patent
Application |
20130101577 |
Kind Code |
A9 |
Wei; Ge ; et al. |
April 25, 2013 |
Extended Soluble PH20 Polypeptides and uses thereof
Abstract
Soluble PH20 polypeptides are provided, including extended
soluble PH20 polypeptides, and uses thereof. Also provided are
other C-terminally truncated PH20 polypeptides and partially
deglycosylated PH20 polypeptides and uses thereof.
Inventors: |
Wei; Ge; (San Diego, CA)
; Selvam; Krishnasamy Panneer; (Poway, CA) ;
Bookbinder; Louis; (San Diego, CA) ; Frost; Gregory
I.; (Del Mar, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wei; Ge
Selvam; Krishnasamy Panneer
Bookbinder; Louis
Frost; Gregory I. |
San Diego
Poway
San Diego
Del Mar |
CA
CA
CA
CA |
US
US
US
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20120213767 A1 |
August 23, 2012 |
|
|
Family ID: |
42035671 |
Appl. No.: |
13/385527 |
Filed: |
February 21, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12653245 |
Dec 9, 2009 |
|
|
|
13385527 |
|
|
|
|
61281240 |
Nov 13, 2009 |
|
|
|
61201384 |
Dec 9, 2008 |
|
|
|
Current U.S.
Class: |
424/130.1 ;
424/94.3; 424/94.62; 435/188; 435/201; 435/252.3; 435/254.11;
435/254.2; 435/320.1; 435/325; 435/348; 435/350; 435/358; 435/367;
435/369; 435/419; 536/23.2 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 33/04 20180101; A61P 9/00 20180101; A61P 31/00 20180101; A61K
47/6435 20170801; A61P 9/12 20180101; A61K 47/61 20170801; C12N
9/2474 20130101; A61P 25/28 20180101; A61P 25/16 20180101; A61K
47/60 20170801; A61P 43/00 20180101; A61P 11/00 20180101; A61K
38/00 20130101; A61P 31/10 20180101; A61P 17/00 20180101; A61P
29/00 20180101; C12N 9/2408 20130101; A61P 3/04 20180101; A61P
25/00 20180101; A61P 33/06 20180101; A61P 3/00 20180101; A61P 27/02
20180101; A61P 13/08 20180101 |
Class at
Publication: |
424/130.1 ;
435/201; 435/188; 435/320.1; 435/358; 435/419; 435/252.3;
435/254.2; 435/254.11; 435/348; 435/325; 435/367; 435/350;
536/23.2; 435/369; 424/94.62; 424/94.3 |
International
Class: |
A61K 38/47 20060101
A61K038/47; C12N 9/96 20060101 C12N009/96; C12N 15/63 20060101
C12N015/63; C12N 5/10 20060101 C12N005/10; C12N 1/21 20060101
C12N001/21; C12N 1/19 20060101 C12N001/19; C12N 1/15 20060101
C12N001/15; C12N 15/56 20060101 C12N015/56; A61P 25/00 20060101
A61P025/00; A61P 35/00 20060101 A61P035/00; A61P 27/02 20060101
A61P027/02; A61P 9/00 20060101 A61P009/00; A61P 29/00 20060101
A61P029/00; A61P 9/12 20060101 A61P009/12; A61P 31/10 20060101
A61P031/10; A61P 33/04 20060101 A61P033/04; A61P 31/00 20060101
A61P031/00; A61P 25/16 20060101 A61P025/16; A61K 39/395 20060101
A61K039/395; A61P 33/06 20060101 A61P033/06; C12N 9/26 20060101
C12N009/26 |
Claims
1. A truncated soluble PH20 hyaluronidase, wherein: the truncated
PH20 hyaluronidase is neutral active; is soluble; and is either: a)
a polypeptide encoded by a nucleic acid molecule encoding a
C-terminally truncated polypeptide of the polypeptide set forth in
SEQ ID NO:107, where the C-terminal amino acid residue is 469, 470
or 471 of SEQ ID NO:107, or b) a polypeptide that contains amino
acid substitutions in the sequence of amino acids of a) whereby the
amino acid-substituted PH20 hyaluronidase consists of a sequence of
amino acids that has at least 91% to the polypeptide of a).
2. A truncated soluble PH20 hyaluronidase that consists of the
sequence of amino acids set forth as amino acids 36-469, 36-470 or
36-471 of SEQ ID NO: 107, or contains amino acid substitutions in
the sequence of amino acids set forth as amino acids 36-469, 36-470
or 36-471 of SEQ ID NO: 107, whereby the amino acid-substituted
PH20 hyaluronidase consists of a sequence of amino acids that has
at least 91% or more sequence identity with the sequence of amino
acids set forth as amino acids 36-469, 36-470 or 36-471 of SEQ ID
NO: 107.
3. The truncated soluble PH20 hyaluronidase of claim 1 that is
N-glycosylated.
4. The truncated soluble PH20 hyaluronidase of claim 3, wherein the
N-glycosylated polypeptide comprises an N-acetylglucosamine moiety
linked to each of at least three asparagine (N) residues.
5. The truncated soluble PH20 hyaluronidase of claim 4, wherein the
three asparagine residues are amino acid residues 235, 368 and 393
of SEQ ID NO: 107.
6. The truncated soluble PH20 hyaluronidase of claim 1 that is
selected from among: a polypeptide that consists of the sequence of
amino acids set forth in any of SEQ ID NOS: 81-83; and a
polypeptide that contains amino acid substitutions in the sequence
of amino acids set forth in any of SEQ ID NOS: 81-83, whereby the
amino acid-substituted polypeptide consists of a sequence of amino
acids that has at least 91% or more sequence identity with the
corresponding sequence of amino acids set forth in any of SEQ ID
NOS: 81-83.
7. The truncated soluble PH20 hyaluronidase of claim 6 that
consists of the sequence of amino acids set forth in any of SEQ ID
NOS: 81-83.
8. The truncated soluble PH20 hyaluronidase of claim 1 that is
modified by modification selected from among sialation,
albumination, farnesylation, carboxylation, hydroxylation and
phosphorylation.
9. The truncated soluble PH20 hyaluronidase of claim 1 that is
modified by a polymer.
10. The truncated soluble PH20 hyaluronidase of claim 9, wherein
the polymer is dextran or PEG.
11. The truncated soluble PH20 hyaluronidase of claim 1 that is
substantially purified.
12. A conjugate, comprising a conjugated moiety linked directly or
via a linker to the C-terminus or N-terminus of the truncated
soluble PH20 hyaluronidase of claim 1.
13. The conjugate of claim 12, wherein the truncated soluble PH20
hyaluronidase is conjugated to a moiety selected from among a
multimerization domain, toxin, detectable label or drug.
14. The conjugate of claim 13, wherein the truncated soluble PH20
hyaluronidase is conjugated to an Fc domain.
15. A nucleic acid molecule encoding the truncated soluble PH20
hyaluronidase of claim 1, wherein the nucleic acid molecule does
not encode a full-length PH20 hyaluronidase but encodes the
truncated soluble PH20 hyaluronidase.
16. A vector, comprising the nucleic acid molecule of claim 15.
17. An expression vector, comprising a polynucleotide that consists
of the sequence of nucleotides encoding a truncated soluble PH20
hyaluronidase of claim 1, including or immediately followed by a
stop codon operatively inserted into the vector for expression of
the truncated soluble PH20 hyaluronidase.
18. A cell, comprising the vector of claim 16.
19. The cell of claim 18 that is a prokaryotic or eukaryotic
cell.
20. The cell of claim 18 that is a mammalian cell.
21. The cell of claim 20 that is a Chinese Hamster Ovary (CHO)
cell.
22. A method for producing a polypeptide of claim 1, comprising
culturing a cell that comprises nucleic acid encoding the
polypeptide; and isolating the polypeptide from the cell culture
medium.
23. A composition, comprising a truncated soluble PH20
hyaluronidase polypeptide of claim 1.
24. The composition of claim 23 that is a pharmaceutical
composition and comprises a pharmaceutically acceptable
excipient.
25. The composition of claim 24, comprising an additional
therapeutic agent.
26. The composition of claim 25, wherein the therapeutic agent is
formulated with the soluble truncated PH20 hyaluronidase or is in a
separate composition.
27. The composition of claim 25, wherein the therapeutic agent is
selected from among a chemotherapeutic agent, an analgesic agent,
an anti-inflammatory agent, an antimicrobial agent, an amoebicidal
agent, a trichomonocidal agent, an anti-Parkinson agent, an
anti-malarial agent, an anticonvulsant agent, an anti-depressant
agent, an antiarthritic agent, an anti-fungal agent, an
antihypertensive agent, an antipyretic agent, an anti-parasite
agent, an antihistamine agent, an alpha-adrenergic agonist agent,
an alpha blocker agent, an anesthetic agent, a bronchial dilator
agent, a biocide agent, a bactericide agent, a bacteriostat agent,
a beta adrenergic blocker agent, a calcium channel blocker agent, a
cardiovascular drug agent, a contraceptive agent, a decongestant
agent, a diuretic agent, a depressant agent, a diagnostic agent, an
electrolyte agent, a hypnotic agent, a hormone agent, a
hyperglycemic agent, a muscle relaxant agent, a muscle contractant
agent, an ophthalmic agent, a parasympathomimetic agent, a psychic
energizer agent, a sedative agent, a sympathomimetic agent, a
tranquilizer agent, a urinary agent, a vaginal agent, a viricide
agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an
angiotensin converting enzyme inhibitor agent, a polypeptide, a
protein, a nucleic acid, a drug, an organic molecule or a sleep
inducer.
28. The composition of claim 25, wherein the therapeutic agent is
selected from among an antibody, an immunoglobulin, a
bisphosphonate, a cytokine, a chemotherapeutic agent and an
insulin.
29. The composition of claim 25, wherein the additional therapeutic
agent is selected from among Acivicins; Aclarubicins; Acodazoles;
Acronines; Adozelesins; Aldesleukins; Alemtuzumabs; Alitretinoins
(9-Cis-Retinoic Acids); Allopurinols; Altretamines; Alvocidibs;
Ambazones; Ambomycins; Ametantrones; Amifostines;
Aminoglutethimides; Amsacrines; Anastrozoles; Anaxirones;
Ancitabines; Anthramycins; Apaziquones; Argimesnas; Arsenic
Trioxides; Asparaginases; Asperlins; Atrimustines; Azacitidines;
Azetepas; Azotomycins; Banoxantrones; Batabulins; Batimastats; BCG
Live; Benaxibines; Bendamustines; Benzodepas; Bexarotenes;
Bevacizumab; Bicalutamides; Bietaserpines; Biricodars; Bisantrenes;
Bisantrenes; Bisnafide Dimesylates; Bizelesins; Bleomycins;
Bortezomibs; Brequinars; Bropirimines; Budotitanes; Busulfans;
Cactinomycins; Calusterones; Canertinibs; Capecitabines;
Caracemides; Carbetimers; Carboplatins; Carboquones; Carmofurs;
Carmustines with Polifeprosans; Carmustines; Carubicins;
Carzelesins; Cedefingols; Celecoxibs; Cemadotins; Chlorambucils;
Cioteronels; Cirolemycins; Cisplatins; Cladribines; Clanfenurs;
Clofarabines; Crisnatols; Cyclophosphamides; Cytarabine liposomals;
Cytarabines; Dacarbazines; Dactinomycins; Darbepoetin Alfas;
Daunorubicin liposomals; Daunorubicins/Daunomycins; Daunorubicins;
Decitabines; Denileukin Diftitoxes; Dexniguldipines;
Dexonnaplatins; Dexrazoxanes; Dezaguanines; Diaziquones;
Dibrospidiums; Dienogests; Dinalins; Disermolides; Docetaxels;
Dofequidars; Doxifluridines; Doxorubicin liposomals; Doxorubicin
HCL; Docorubicin HCL liposome injection; Doxorubicins;
Droloxifenes; Dromostanolone Propionates; Duazomycins; Ecomustines;
Edatrexates; Edotecarins; Eflornithines; Elacridars; Elinafides;
Elliott's B Solutions; Elsamitrucins; Emitefurs; Enloplatins;
Enpromates; Enzastaurins; Epipropidines; Epirubicins; Epoetin
alfas; Eptaloprosts; Erbulozoles; Esorubicins; Estramustines;
Etanidazoles; Etoglucids; Etoposide phosphates; Etoposide VP-16s;
Etoposides; Etoprines; Exemestanes; Exisulinds; Fadrozoles;
Fazarabines; Fenretinides; Filgrastims; Floxuridines; Fludarabines;
Fluorouracils; 5-fluorouracils; Fluoxymesterones; Fluorocitabines;
Fosquidones; Fostriecins; Fotretamines; Fulvestrants; Galarubicins;
Galocitabines; Gemcitabines; Gemtuzumabs/Ozogamicins; Geroquinols;
Gimatecans; Gimeracils; Gloxazones; Glufosfamides; Goserelin
acetates; Hydroxyureas; Ibritumomabs/Tiuxetans; Idarubicins;
Ifosfamides; Ilmofosines; Ilomastats; Imatinib mesylates; Imexons;
Improsulfans; Indisulams; Inproquones; Interferon alfa-2as;
Interferon alfa-2bs; Interferon Alfas; Interferon Betas; Interferon
Gammas; Interferons; Interleukin-2s and other Interleukins
(including recombinant Interleukins); Intoplicines; Iobenguanes
[131-I]; Iproplatins; Irinotecans; Irsogladines; Ixabepilones;
Ketotrexates; L-Alanosines; Lanreotides; Lapatinibs; Ledoxantrones;
Letrozoles; Leucovorins; Leuprolides; Leuprorelins (Leuprorelides);
Levamisoles; Lexacalcitols; Liarozoles; Lobaplatins; Lometrexols;
Lomustines/CCNUs; Lomustines; Lonafarnibs; Losoxantrones;
Lurtotecans; Mafosfamides; Mannosulfans; Marimastats; Masoprocols;
Maytansines; Mechlorethamines; Mechlorethamines/Nitrogen mustards;
Megestrol acetates; Megestrols; Melengestrols; Melphalans;
MelphalanslL-PAMs; Menogarils; Mepitiostanes; Mercaptopurines;
6-Mercaptopurine; Mesnas; Metesinds; Methotrexates; Methoxsalens;
Metomidates; Metoprines; Meturedepas; Miboplatins; Miproxifenes;
Misonidazoles; Mitindomides; Mitocarcins; Mitocromins;
Mitoflaxones; Mitogillins; Mitoguazones; Mitomalcins; Mitomycin Cs;
Mitomycins; Mitonafides; Mitoquidones; Mitospers; Mitotanes;
Mitoxantrones; Mitozolomides; Mivobulins; Mizoribines; Mofarotenes;
Mopidamols; Mubritinibs; Mycophenolic Acids; Nandrolone
Phenpropionates; Nedaplatins; Nelzarabines; Nemorubicins;
Nitracrines; Nocodazoles; Nofetumomabs; Nogalamycins; Nolatrexeds;
Nortopixantrones; Octreotides; Oprelvekins; Ormaplatins;
Ortataxels; Oteracils; Oxaliplatins; Oxisurans; Oxophenarsines;
Paclitaxels; Pamidronates; Patubilones; Pegademases; Pegaspargases;
Pegfilgrastims; Peldesines; Peliomycins; Pelitrexols; Pemetrexeds;
Pentamustines; Pentostatins; Peplomycins; Perfosfamides;
Perifosines; Picoplatins; Pinafides; Pipobromans; Piposulfans;
Pirfenidones; Piroxantrones; Pixantrones; Plevitrexeds; Plicamycid
Mithramycins; Plicamycins; Plomestanes; Plomestanes; Porfimer
sodiums; Porfimers; Porfiromycins; Prednimustines; Procarbazines;
Propamidines; Prospidiums; Pumitepas; Puromycins; Pyrazofurins;
Quinacrines; Ranimustines; Rasburicases; Riboprines; Ritrosulfans;
Rituximabs; Rogletimides; Roquinimexs; Rufocromomycins;
Sabarubicins; Safingols; Sargramostims; Satraplatins; Sebriplatins;
Semustines; Simtrazenes; Sizofurans; Sobuzoxanes; Sorafenibs;
Sparfosates; Sparfosic Acids; Sparsomycins; Spirogermaniums;
Spiromustines; Spiroplatins; Spiroplatins; Squalamines;
Streptonigrins; Streptovarycins; Streptozocins; Sufosfamides;
Sulofenurs; Sunitinib Malate; 6-TG; Tacedinalines; Talcs;
Talisomycins; Tallimustines; Tamoxifens; Tariquidars;
Tauromustines; Tecogalans; Tegafurs; Teloxantrones; Temoporfins;
Temozolomides; Teniposides/VM-26s; Teniposides; Teroxirones;
Testolactones; Thiamiprines; Thioguanines; Thiotepas; Tiamiprines;
Tiazofurins; Tilomisoles; Tilorones; Timcodars; Timonacics;
Tirapazamines; Topixantrones; Topotecans; Toremifenes;
Tositumomabs; Trabectedins (Ecteinascidin 743); Trastuzumabs;
Trestolones; Tretinoins/ATRA; Triciribines; Trilostanes;
Trimetrexates; Triplatin Tetranitrates; Triptorelins;
Trofosfamides; Tubulozoles; Ubenimexs; Uracil Mustards; Uredepas;
Valrubicins; Valspodars; Vapreotides; Verteporfins; Vinblastines;
Vincristines; Vindesines; Vinepidines; Vinflunines; Vinformides;
Vinglycinates; Vinleucinols; Vinleurosines; Vinorelbines;
Vinrosidines; Vintriptols; Vinzolidines; Vorozoles; Xanthomycin A's
(Guamecyclines); Zeniplatins; Zilascorbs [2-H]; Zinostatins;
Zoledronate; Zorubicins; and Zosuquidars.
30. A composition, comprising a truncated soluble PH20
hyaluronidase polypeptide of claim 10.
31. The composition of claim 30 that is a pharmaceutical
composition and comprises a pharmaceutically acceptable
excipient.
32. The composition of claim 31, comprising an additional
therapeutic agent.
33. The composition of claim 32, wherein the therapeutic agent is
selected from among a chemotherapeutic agent, an analgesic agent,
an anti-inflammatory agent, an antimicrobial agent, an amoebicidal
agent, a trichomonocidal agent, an anti-Parkinson agent, an
anti-malarial agent, an anticonvulsant agent, an anti-depressant
agent, an antiarthritic agent, an anti-fungal agent, an
antihypertensive agent, an antipyretic agent, an anti-parasite
agent, an antihistamine agent, an alpha-adrenergic agonist agent,
an alpha blocker agent, an anesthetic agent, a bronchial dilator
agent, a biocide agent, a bactericide agent, a bacteriostat agent,
a beta adrenergic blocker agent, a calcium channel blocker agent, a
cardiovascular drug agent, a contraceptive agent, a decongestant
agent, a diuretic agent, a depressant agent, a diagnostic agent, an
electrolyte agent, a hypnotic agent, a hormone agent, a
hyperglycemic agent, a muscle relaxant agent, a muscle contractant
agent, an ophthalmic agent, a parasympathomimetic agent, a psychic
energizer agent, a sedative agent, a sympathomimetic agent, a
tranquilizer agent, a urinary agent, a vaginal agent, a viricide
agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an
angiotensin converting enzyme inhibitor agent, a polypeptide, a
protein, a nucleic acid, a drug, an organic molecule or a sleep
inducer.
34. The composition of claim 32, wherein the therapeutic agent is
selected from among an antibody, an immunoglobulin, a
bisphosphonate, a cytokine, a chemotherapeutic agent and an
insulin.
35. A method for treating a hyaluronan-associated disease or
condition, comprising administering to a subject the composition of
claim 23.
36. A method for treating a hyaluronan-associated disease or
condition, comprising administering to a subject the composition of
claim 30.
37. A method for treating an excess of glycosaminoglycans; for
treating a tumor; for treating glycosaminoglycan accumulation in
the brain; for treating a cardiovascular disorder; for treating an
ophthalmic disorder; for treating pulmonary disease; for increasing
penetration of chemotherapeutic agents into solid tumors; for
treating cellulite; for treating a proliferative disorder; or for
increasing bioavailability of drugs and other therapeutic agents,
comprising administering to a subject the composition of claim
23.
38. The method of claim 37, wherein the proliferative disorder is
benign prostatic hyperplasia.
39. The method of claim 38, wherein the truncated soluble PH20
hyaluronidase is PEGylated.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/653,245, entitled "EXTENDED SOLUBLE PH20 POLYPEPTIDES AND
USES THEREOF," filed Dec. 9, 2009, which claims priority under 35
U.S.C. .sctn.119(e) to U.S. Provisional Application Ser. No.
61/281,240 to Ge Wei, Krishnasamy Panneerselvam, Louis Bookbinder
and Gregory I. Frost, entitled "EXTENDED SOLUBLE PH20 POLYPEPTIDES
AND USES THEREOF," filed Nov. 13, 2009; and to U.S. Provisional
Application Ser. No. 61/201,384, to Ge Wei, Krishnasamy
Panneerselvam, Louis Bookbinder and Gregory I. Frost, entitled
"EXTENDED SOLUBLE PH20 POLYPEPTIDES AND USES THEREOF," filed Dec.
9, 2008. The subject matter of each of the above-referenced
applications is incorporated by reference in its entirety.
[0002] This application is related to International Application No.
PCT/US09/006,501, filed the same day herewith, entitled "EXTENDED
SOLUBLE PH20 POLYPEPTIDES AND USES THEREOF," which claims priority
to U.S. Provisional Application Ser. Nos. 61/281,240 and
61/201,384.
[0003] The subject matter of the above-noted related application is
incorporated by reference in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT
DISCS
[0004] An electronic version on compact disc (CD-R) of the Sequence
Listing is filed herewith in duplicate (labeled Copy # 1 and Copy
#2), the contents of which are incorporated by reference in their
entirety. The computer-readable file on each of the aforementioned
compact discs, created on Feb. 16, 2012, is identical, 684
kilobytes in size, and titled 3074SEQB.001.txt.
FIELD OF INVENTION
[0005] Soluble PH20 polypeptides are provided, including extended
soluble PH20 polypeptides, and uses thereof. Also provided are
other C-terminally truncated PH20 polypeptides and partially
deglycosylated PH20 polypeptides and uses thereof.
BACKGROUND
[0006] Hyaluronan (hyaluronic acid; HA) is a polypeptide that is
found in the extracellular matrix of many cells, especially in soft
connective tissues. HA also is found predominantly in skin,
cartilage, and in synovial fluid in mammals. Hyaluronan also is the
main constituent of the vitreous of the eye. HA has a role in
various physiological processes, such as in water and plasma
protein homeostasis (Laurent T C et al (1992) FASEB J 6:
2397-2404). Certain diseases are associated with expression and/or
production of hyaluronan. Hyaluronidases are enzymes that degrade
hyaluronan. By catalyzing HA, hyaluronidases can be used to treat
diseases or disorders associated with accumulation of HA or other
glycosaminoglycans. Also, since HA is a major component of the
interstitial barrier, hyaluronidase increases tissue permeability
and therefore can be used to increase the dispersion and delivery
of therapeutic agents. Various hyaluronidases have been used
therapeutically (e.g. Hydase.TM., Vitrase.TM. and Wydase.TM.),
typically as dispersing and spreading agents in combination with
other therapeutic agents. Many of these are ovine or bovine forms,
which can be immunogenic for treatment of humans. Improved
compositions of hyaluronidases that can be used for treatment are
needed.
SUMMARY
[0007] Provided herein are soluble PH20 polypeptides, including
extended soluble PH20 (esPH20) polypeptides, and compositions. The
PH20 polypeptides provided herein are soluble proteins that are
truncated at the C-terminus and include those that lack all of the
GPI-anchor attachment signal sequence (e.g. are truncated at amino
acid positions 450 to 490). Soluble PH20 polypeptides also include
extended soluble PH20 polypeptides that retain one or more residues
located in the GPI-anchor attachment signal sequence of the
corresponding full length wild-type PH20 polypeptide. Also provided
herein are other modified PH20 polypeptides that contain C-terminal
truncations. Partially deglycosylated forms of any of the
polypeptides also are provided. Also provided are methods of
treatment using the PH20 polypeptides provided herein.
[0008] Provided herein are isolated substantially purified extended
soluble PH20 (esPH20) hyaluronidases, that can be N-glycosylated or
N-partially glycosylated. In some examples, the N-partially
glycosylated esPH20 polypeptide contains at least an
N-acetylglucosamine moiety linked to each of at least two N-linked
moieties, such as, for example, amino acid residues 368 and 393 of
SEQ ID NO:107 or residues corresponding to amino acid residues 368
and 393 of SEQ ID NO:107. In some aspects, the N-partially
glycosylated esPH20 polypeptide contains at least two
N-acetylglucosamine moieties linked to each of at least two
N-linked moieties. The N-partially glycosylated esPH20 polypeptide
provided herein also can contain a branched sugar.
[0009] Provided herein are esPH20 polypeptides that have the
sequence of amino acids set forth in any of SEQ ID NOS: 60-63 and
102-104 or an allelic or species variant thereof. Also provided are
esPH20 polypeptide variants having at least 40%, 45%, 50%, 55%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% sequence identity with any of SEQ ID NOS: 60-63 and
102-104 and retaining at least 30% of the hyaluronidase activity of
the corresponding unmodified form or of a polypeptide that is
encoded by a nucleic acid that encodes a polypeptide with amino
acids 36-482 of SEQ ID NO:107. Such esPH20 polypeptides remain
soluble and neutral active. In one example, the esPH20 is a human
esPH20, such as one with a sequence of amino acids set forth in any
of SEQ ID NOS: 60-63 and 102-104, or a chimpanzee esPH20, such as
one with a sequence of amino acids set forth as amino acids 36-491,
36-492, 36-493, 36-494, 36-495, 36-496, 36-497 or 36-498 of SEQ ID
NO:197.
[0010] Also provided herein are substantially purified PH20
polypeptides. These PH20 polypeptides can have a sequence of amino
acids set forth in any of SEQ ID NOS: 55-63 and 64-95 or an allelic
or species variant thereof. In other examples, the PH20
polypeptides are variants having at least 40%, 45%, 50%, 55%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% sequence identity with any of SEQ ID NOS: 55-63 and 64-95, that
retain at least 30% of the hyaluronidase activity of the
corresponding unmodified form or of a polypeptide that is encoded
by a nucleic acid that encodes a polypeptide with amino acids
36-482 of SEQ ID NO:107. Such PH20 polypeptides are neutral
active.
[0011] The PH20 polypeptides provided herein can be N-glycosylated
or N-partially glycosylated. In some examples, the N-partially
glycosylated esPH20 polypeptide contains at least an
N-acetylglucosamine moiety linked to each of at least two N-linked
moieties, such as, for example, amino acid residues 368 and 393 of
SEQ ID NO:107 or residues corresponding to amino acid residues 368
and 393 of SEQ ID NO:107. In some aspects, the N-partially
glycosylated PH20 polypeptide contains at least two
N-acetylglucosamine moieties linked to each of at least two
N-linked moieties. The N-partially glycosylated PH20 polypeptides
provided herein also can contain a branched sugar. In some aspects,
the PH20 polypeptides provided herein are soluble, and can be
selected from among human, chimpanzee, rhesus monkey, cynomolgus
monkey, mouse, rabbit, guinea pig, cow or sheep PH20.
[0012] The esPH20 and PH20 polypeptides provided herein can be
modified by, for example, sialation, albumination, farnysylation,
carboxylation, hydroxylation or phosphorylation. In some aspects,
the esPH20 and PH20 polypeptides are modified by a polymer, such as
dextran or PEG. Also provided herein are conjugates containing the
esPH20 or PH20 polypeptides. Exemplary conjugates include those in
which the esPH20 or PH20 is conjugated to a multimerization domain
(such as an Fc domain), toxin, detectable label or drug.
[0013] Provided herein are nucleic acids encoding the esPH20 and
PH20 polypeptides described above and provided herein. These
nucleic acids include those that encode an esPH20 or PH20
polypeptide with amino acids corresponding to amino acids 36-450,
36-451, 36-452, 36-453, 36-454, 36-455, 36-456, 36-457, 36-458,
36-459, 36-460, 36-461, 36-462, 36-463, 36-464, 36-465, 36-484,
36-485, 36-486, 36-487, 36-489, 36-491, 36-492, 36-493, 36-494,
36-495, 36-496 or 36-497 of SEQ ID NO:107, and those that encode an
esPH20 with amino acids corresponding to amino acids 36-491,
36-492, 36-493, 36-494, 36-495, 36-496, 36-497 or 36-498 of SEQ ID
NO:197. Also provided herein are vectors that contain these nucleic
acids. Further provided herein are cells that contain the vectors,
such as prokaryotic or eukaryotic cells. For example, the cells can
be mammalian cells, such as CHO cells.
[0014] Provided herein are compositions containing any one or more
of the esPH20 or PH20 polypeptides described herein. In some
examples, the compositions contain a plurality of esPH20 or PH20
polypeptides. For example, the compositions can contain a plurality
of esPH20 polypeptides that are encoded by a nucleic acid molecule
encoding amino acids corresponding to amino acids 36-450, 36-451,
36-452, 36-453, 36-454, 36-455, 36-456, 36-457, 36-458, 36-459,
36-460, 36-461, 36-462, 36-463, 36-464, 36-465, 36-484, 36-485,
36-486, 36-487, 36-489, 36-491, 36-492, 36-493, 36-494, 36-495,
36-496 or 36-497 of SEQ ID NO:107, and those that encode an esPH20
with amino acids corresponding to amino acids 36-491, 36-492,
36-493, 36-494, 36-495, 36-496, 36-497 or 36-498 of SEQ ID NO:197.
In some examples, the compositions contain esPH20 or PH20
polypeptides that are secreted from CHO cells.
[0015] The compositions provided herein can be pharmaceutical
compositions. In some examples, the compositions contain an
additional therapeutic agent, which can be formulated with the
composition or in a separate composition. Exemplary of the
therapeutic agents that can be included in the compositions
provided herein are chemotherapeutic agents, analgesic agents,
anti-inflammatory agents, antimicrobial agents, amoebicidal agents,
trichomonocidal agents, anti-parkinson agents, anti-malarial
agents, anticonvulsant agents, anti-depressant agents,
anti-arthritics agents, anti-fungal agents, antihypertensive
agents, antipyretic agents, anti-parasite agents, antihistamine
agents, alpha-adrenergic agonist agents, alpha blocker agents,
anesthetic agents, bronchial dilator agents, biocide agents,
bactericide agents, bacteriostat agents, beta adrenergic blocker
agents, calcium channel blocker agents, cardiovascular drug agents,
contraceptive agents, decongestant agents, diuretic agents,
depressant agents, diagnostic agents, electrolyte agents, hypnotic
agents, hormone agents, hyperglycemic agents, muscle relaxant
agents, muscle contractant agents, ophthalmic agents,
parasympathomimetic agents, psychic energizer agents, sedative
agents, sympathomimetic agents, tranquilizer agents, urinary
agents, vaginal agents, viricide agents, vitamin agents,
non-steroidal anti-inflammatory agents, angiotensin converting
enzyme inhibitor agents, polypeptides, proteins, nucleic acids,
drugs, organic molecules and sleep inducers. In particular
examples, the therapeutic agent is an antibody, an immunoglobulin,
a bisphosphonate (such as zolentronic acid), a cytokine, a
chemotherapeutic agent or an insulin (such as a fast-acting
insulin).
[0016] Other therapeutic agents that can be included in the
compositions provided herein include, but are not limited to,
Acivicins; Aclarubicins; Acodazoles; Acronines; Adozelesins;
Aldesleukins; Alemtuzumabs; Alitretinoins (9-Cis-Retinoic Acids);
Allopurinols; Altretamines; Alvocidibs; Ambazones; Ambomycins;
Ametantrones; Amifostines; Aminoglutethimides; Amsacrines;
Anastrozoles; Anaxirones; Ancitabines; Anthramycins; Apaziquones;
Argimesnas; Arsenic Trioxides; Asparaginases; Asperlins;
Atrimustines; Azacitidines; Azetepas; Azotomycins; Banoxantrones;
Batabulins; Batimastats; BCG Live; Benaxibines; Bendamustines;
Benzodepas; Bexarotenes; Bevacizumab; Bicalutamides; Bietaserpines;
Biricodars; Bisantrenes; Bisantrenes; Bisnafide Dimesylates;
Bizelesins; Bleomycins; Bortezomibs; Brequinars; Bropirimines;
Budotitanes; Busulfans; Cactinomycins; Calusterones; Canertinibs;
Capecitabines; Caracemides; Carbetimers; Carboplatins; Carboquones;
Carmofurs; Carmustines with Polifeprosans; Carmustines; Carubicins;
Carzelesins; Cedefingols; Celecoxibs; Cemadotins; Chlorambucils;
Cioteronels; Cirolemycins; Cisplatins; Cladribines; Clanfenurs;
Clofarabines; Crisnatols; Cyclophosphamides; Cytarabine liposomals;
Cytarabines; Dacarbazines; Dactinomycins; Darbepoetin Alfas;
Daunorubicin liposomals; Daunorubicins/Daunomycins; Daunorubicins;
Decitabines; Denileukin Diftitoxes; Dexniguldipines;
Dexonnaplatins; Dexrazoxanes; Dezaguanines; Diaziquones;
Dibrospidiums; Dienogests; Dinalins; Disermolides; Docetaxels;
Dofequidars; Doxifluridines; Doxorubicin liposomals; Doxorubicin
HCL; Docorubicin HCL liposome injection; Doxorubicins;
Droloxifenes; Dromostanolone Propionates; Duazomycins; Ecomustines;
Edatrexates; Edotecarins; Eflornithines; Elacridars; Elinafides;
Elliott's B Solutions; Elsamitrucins; Emitefurs; Enloplatins;
Enpromates; Enzastaurins; Epipropidines; Epirubicins; Epoetin
alfas; Eptaloprosts; Erbulozoles; Esorubicins; Estramustines;
Etanidazoles; Etoglucids; Etoposide phosphates; Etoposide VP-16s;
Etoposides; Etoprines; Exemestanes; Exisulinds; Fadrozoles;
Fazarabines; Fenretinides; Filgrastims; Floxuridines; Fludarabines;
Fluorouracils; 5-fluorouracils; Fluoxymesterones; Fluorocitabines;
Fosquidones; Fostriecins; Fostriecins; Fotretamines; Fulvestrants;
Galarubicins; Galocitabines; Gemcitabines; Gemtuzumabs/Ozogamicins;
Geroquinols; Gimatecans; Gimeracils; Gloxazones; Glufosfamides;
Goserelin acetates; Hydroxyureas; Ibritumomabs/Tiuxetans;
Idarubicins; Ifosfamides; Ilmofosines; Ilomastats; Imatinib
mesylates; Imexons; Improsulfans; Indisulams; Inproquones;
Interferon alfa-2 as; Interferon alfa-2bs; Interferon Alfas;
Interferon Betas; Interferon Gammas; Interferons; Interleukin-2s
and other Interleukins (including recombinant Interleukins);
Intoplicines; Iobenguanes [131-I]; Iproplatins; Irinotecans;
Irsogladines; Ixabepilones; Ketotrexates; L-Alanosines;
Lanreotides; Lapatinibs; Ledoxantrones; Letrozoles; Leucovorins;
Leuprolides; Leuprorelins (Leuprorelides); Levamisoles;
Lexacalcitols; Liarozoles; Lobaplatins; Lometrexols;
Lomustines/CCNUs; Lomustines; Lonafarnibs; Losoxantrones;
Lurtotecans; Mafosfamides; Mannosulfans; Marimastats; Masoprocols;
Maytansines; Mechlorethamines; Mechlorethamines/Nitrogen mustards;
Megestrol acetates; Megestrols; Melengestrols; Melphalans;
MelphalanslL-PAMs; Menogarils; Mepitiostanes; Mercaptopurines;
6-Mercaptopurine; Mesnas; Metesinds; Methotrexates; Methoxsalens;
Metomidates; Metoprines; Meturedepas; Miboplatins; Miproxifenes;
Misonidazoles; Mitindomides; Mitocarcins; Mitocromins;
Mitoflaxones; Mitogillins; Mitoguazones; Mitomalcins; Mitomycin Cs;
Mitomycins; Mitonafides; Mitoquidones; Mitospers; Mitotanes;
Mitoxantrones; Mitozolomides; Mivobulins; Mizoribines; Mofarotenes;
Mopidamols; Mubritinibs; Mycophenolic Acids; Nandrolone
Phenpropionates; Nedaplatins; Nelzarabines; Nemorubicins;
Nitracrines; Nocodazoles; Nofetumomabs; Nogalamycins; Nolatrexeds;
Nortopixantrones; Octreotides; Oprelvekins; Ormaplatins;
Ortataxels; Oteracils; Oxaliplatins; Oxisurans; Oxophenarsines;
Paclitaxels; Pamidronates; Patubilones; Pegademases; Pegaspargases;
Pegfilgrastims; Peldesines; Peliomycins; Pelitrexols; Pemetrexeds;
Pentamustines; Pentostatins; Peplomycins; Perfosfamides;
Perifosines; Picoplatins; Pinafides; Pipobromans; Piposulfans;
Pirfenidones; Piroxantrones; Pixantrones; Plevitrexeds; Plicamycid
Mithramycins; Plicamycins; Plomestanes; Plomestanes; Porfimer
sodiums; Porfimers; Porfiromycins; Prednimustines; Procarbazines;
Propamidines; Prospidiums; Pumitepas; Puromycins; Pyrazofurins;
Quinacrines; Ranimustines; Rasburicases; Riboprines; Ritrosulfans;
Rituximabs; Rogletimides; Roquinimexs; Rufocromomycins;
Sabarubicins; Safingols; Sargramostims; Satraplatins; Sebriplatins;
Semustines; Simtrazenes; Sizofurans; Sobuzoxanes; Sorafenibs;
Sparfosates; Sparfosic Acids; Sparsomycins; Spirogermaniums;
Spiromustines; Spiroplatins; Spiroplatins; Squalamines;
Streptonigrins; Streptovarycins; Streptozocins; Sufosfamides;
Sulofenurs; Sunitinib Malate; 6-TG; Tacedinalines; Talcs;
Talisomycins; Tallimustines; Tamoxifens; Tariquidars;
Tauromustines; Tecogalans; Tegafurs; Teloxantrones; Temoporfins;
Temozolomides; Teniposides/VM-26s; Teniposides; Teroxirones;
Testolactones; Thiamiprines; Thioguanines; Thiotepas; Tiamiprines;
Tiazofurins; Tilomisoles; Tilorones; Timcodars; Timonacics;
Tirapazamines; Topixantrones; Topotecans; Toremifenes;
Tositumomabs; Trabectedins (Ecteinascidin 743); Trastuzumabs;
Trestolones; Tretinoins/ATRA; Triciribines; Trilostanes;
Trimetrexates; Triplatin Tetranitrates; Triptorelins;
Trofosfamides; Tubulozoles; Ubenimexs; Uracil Mustards; Uredepas;
Valrubicins; Valspodars; Vapreotides; Verteporfins; Vinblastines;
Vincristines; Vindesines; Vinepidines; Vinflunines; Vinformides;
Vinglycinates; Vinleucinols; Vinleurosines; Vinorelbines;
Vinrosidines; Vintriptols; Vinzolidines; Vorozoles; Xanthomycin A's
(Guamecyclines); Zeniplatins; Zilascorbs [2-H]; Zinostatins;
Zoledronate; Zorubicins; and Zosuquidars.
[0017] Provided herein are methods for treating a
hyaluronan-associated disease or condition, wherein a subject is
administered an esPH20 or PH20, or a composition containing an
esPH20 or PH20, provided and described herein. Also provided are
methods for treating an excess of glycosaminoglycans; for treating
a tumor; for treating glycosaminoglycan accumulation in the brain;
for treating a cardiovascular disorder; for treating an ophthalmic
disorder; for treating pulmonary disease; for increasing
penetration of chemotherapeutic agents into solid tumors; for
treating cellulite; or for increasing bioavailability of drugs and
other therapeutic agents. Such methods involve administering to a
subject any of the esPH20 or PH20 polypeptides or compositions
described herein.
[0018] The esPH20 and PH20 polypeptides provided herein can be used
in place of a PH20 hyaluronidase, alone or in combination, in any
method of treatment or combination therapy for which a PH20
hyaluronidase is used in U.S. Publication Nos. US20040268425,
US20050260186, and US20060104968; and U.S. application Ser. Nos.
12/381,844, 12/386,249, 12/387,225 and 12/386,222.
[0019] Any of the esPH20 or PH20 polypeptides provided herein can
be substantially purified or isolated.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 depicts an alignment of the amino acid sequence of
the human (SEQ ID NO:107) and chimpanzee (SEQ ID NO:197) PH20
polypeptides (performed using the ClustalW2 alignment program). The
amino acid residues of the human PH20 GPI-anchor attachment signal
sequence, and the corresponding amino acids in the chimpanzee PH20
sequence, are in bold and underlined. "*" indicates that the
residues above are identical in both sequences in the alignment.
":" indicates conserved substitutions, and "." indicates
semi-conserved substitutions.
[0021] FIG. 2 depicts the major types of N-glycans in vertebrates,
including high mannose glycans, hybrid glycans and complex
glycans.
[0022] FIG. 3 depicts the endoglycosidase cleavage sites. FIG. 3A
illustrates the cleavage sites for Endoglycosidase F1 and Peptide N
Glycosidase F (PNGaseF). FIG. 3B illustrates the cleavage sites for
Endoglycosidase F2 and PNGaseF. FIG. 3C illustrates the cleavage
sites for Endoglycosidase F3 and PNGaseF. FIG. 3D illustrates the
cleavage sites for Endoglycosidase F4 and PNGaseF.
DETAILED DESCRIPTION
Overview
[0023] A. Definitions [0024] B. Overview [0025] 1. PH20 [0026] a.
Glycosylation [0027] b. GPI-Anchoring [0028] C. Extended Soluble
PH20 Polypeptides [0029] 1. Human esPH20 polypeptides [0030] 2.
Other species esPH20 polypeptides [0031] D. N-Partially
glycosylated PH20 polypeptides [0032] 1. PH20 polypeptides [0033]
2. C-terminally truncated PH20 polypeptides [0034] 3. Additional
Modifications [0035] Conjugation to polymers [0036] E. Methods of
Producing Nucleic Acids Encoding Extended Soluble PH20 and other
Soluble PH20 Hyaluronidases, and Polypeptides Thereof [0037] 1.
Vectors and Cells [0038] 2. Expression [0039] a. Prokaryotic Cells
[0040] b. Yeast Cells [0041] c. Insect Cells [0042] d. Mammalian
Cells [0043] e. Plants [0044] 3. Purification Techniques [0045] F.
Preparation, Formulation and Administration of Extended Soluble
PH20 Polypeptides, and Other Soluble PH20 Polypeptides [0046] 1.
Injectables, solutions and emulsions [0047] Lyophilized Powders
[0048] 2. Topical Administration [0049] 3. Compositions for other
routes of administration [0050] 4. Dosage and Administration [0051]
5. Packaging, Articles of Manufacture and Kits [0052] G. Assays
[0053] 1. Hyaluronidase Activity [0054] 2. Solubility [0055] H.
Methods of Treatment and Uses of Extended Soluble PH20 and other
Soluble PH20 and Combination Therapy [0056] 1. Use as a Spreading
Agent and Combination Therapy [0057] 2. Use to Remove Excess
Glycosaminoglycanases [0058] a. Use in cancer treatment [0059] b.
Use in treatment of glycosaminoglycan accumulation in the brain
[0060] c. Use in treatment of glycosaminoglycan accumulation in
cardiovascular disease [0061] d. Use in vitrectomy and ophthalmic
disorders and conditions [0062] e. Use in hypodermoclysis [0063] f.
Use in gene therapy [0064] g. Cosmetic uses [0065] h. Use in organ
transplantation [0066] i. Use in pulmonary disease [0067] 3. Other
uses [0068] I. Examples
A. Definitions
[0069] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the invention(s) belong. All patents,
patent applications, published applications and publications,
Genbank sequences, databases, websites and other published
materials referred to throughout the entire disclosure herein,
unless noted otherwise, are incorporated by reference in their
entirety. In the event that there are a plurality of definitions
for terms herein, those in this section prevail. Where reference is
made to a URL or other such identifier or address, it understood
that such identifiers can change and particular information on the
interne can come and go, but equivalent information can be found by
searching the Internet. Reference thereto evidences the
availability and public dissemination of such information.
[0070] As used herein, hyaluronidase refers to a class of enzymes
that degrade hyaluronan. Hyaluronidases include, but are not
limited to, bacterial hyaluronidases (EC 4.2.2.1 or EC 4.2.99.1),
hyaluronidases from leeches, other parasites, and crustaceans (EC
3.2.1.36), and mammalian-type hyaluronidases (EC 3.2.1.35).
Hyaluronidases include any of non-human origin including, but not
limited to, murine, canine, feline, leporine, avian, bovine, ovine,
porcine, equine, piscine, ranine, bacterial, and any from leeches,
other parasites, and crustaceans. Exemplary human hyaluronidases
include HYAL1, HYAL2, HYAL3, HYAL4, and PH20 (SEQ ID NO:107). Also
included amongst hyaluronidases are soluble hyaluronidases,
including, ovine and bovine PH20, soluble human PH20 and rHuPH20.
Examples of commercially available bovine or ovine soluble
hyaluronidases are Vitrase.RTM. hyaluronidase (ovine hyaluronidase)
and Amphadase.RTM. hyaluronidase (bovine hyaluronidase).
[0071] As used herein, PH20 refers to a type of hyaluronidase that
occurs in sperm and is neutral-active. PH-20 occurs on the sperm
surface, and in the lysosome-derived acrosome, where it is bound to
the inner acrosomal membrane. PH20 includes those of any origin
including, but not limited to, human, chimpanzee, Cynomolgus
monkey, Rhesus monkey, murine, bovine, ovine, guinea pig, rabbit
and rat origin. Exemplary PH20 polypeptides include those from
human (SEQ ID NO:107), chimpanzee (SEQ ID NO:197), Rhesus monkey
(SEQ ID NO:198), Cynomolgus monkey (SEQ ID NO:114), cow (e.g., SEQ
ID NOS:111 and 119); mouse (SEQ ID NO:117); rat (SEQ ID NO:116);
rabbit (SEQ ID NO:112); sheep (SEQ ID NOS:113, 118 and 120) and
guinea pig (SEQ ID NO:115). Reference to PH20 includes precursor
PH20 polypeptides and mature PH20 polypeptides (such as those in
which a signal sequence has been removed), truncated forms thereof
that have activity, and includes allelic variants and species
variants, variants encoded by splice variants, and other variants,
including polypeptides that have at least 40%, 45%, 50%, 55%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence
identity to the precursor polypeptides set forth in SEQ ID NO:107
and 109, or the mature forms thereof. PH20 polypeptides also
include those that contain chemical or posttranslational
modifications and those that do not contain chemical or
posttranslational modifications. Such modifications include, but
are not limited to, pegylation, albumination, glycosylation,
farnysylation, carboxylation, hydroxylation, phosphorylation, and
other polypeptide modifications known in the art. A truncated PH20
hyaluronidase is any C-terminal shortened form thereof,
particularly forms that are truncated and neutral active when
N-glycosylated.
[0072] As used herein, a soluble PH20 refers to any form of PH20
that is soluble under physiologic conditions. A soluble PH20 can be
identified, for example, by its partitioning into the aqueous phase
of a Triton.RTM. X-114 solution at 37.degree. C. (Bordier et al.,
(1981) J. Biol. Chem., 256:1604-7). Membrane-anchored PH20, such as
lipid-anchored PH20, including GPI-anchored PH20, will partition
into the detergent-rich phase, but will partition into the
detergent-poor or aqueous phase following treatment with
Phospholipase-C. Included among soluble PH20 are membrane-anchored
PH20 in which one or more regions associated with anchoring of the
PH20 to the membrane has been removed or modified, where the
soluble form retains hyaluronidase activity. Soluble PH20 also
include recombinant soluble PH20 and those contained in or purified
from natural sources, such as, for example, testes extracts from
sheep or cows. Exemplary of such soluble PH20 is soluble human
PH20.
[0073] As used herein, soluble human PH20 or sHuPH20 includes PH20
polypeptides lacking all or a portion of the
glycosylphospatidylinositol (GPI) anchor sequence at the C-terminus
such that upon expression, the polypeptides are soluble under
physiological conditions. Solubility can be assessed by any
suitable method that demonstrates solubility under physiologic
conditions. Exemplary of such methods is the Triton.RTM. X-114
assay, that assesses partitioning into the aqueous phase and that
is described above and in the examples. In addition, a soluble
human PH20 polypeptide is, if produced in CHO cells, such as CHO--S
cells, a polypeptide that is expressed and is secreted into the
cell culture medium. Soluble human PH20 polypeptides, however, are
not limited to those produced in CHO cells, but can be produced in
any cell or by any method, including recombinant expression and
polypeptide synthesis. Reference to secretion in CHO cells is
definitional. Hence, if a polypeptide could be expressed and
secreted in CHO cells and is soluble, i.e. partitions into the
aqueous phase when extracted with Triton.RTM. X-114, it is a
soluble PH20 polypeptide whether or not it is so-produced. The
precursor polypeptides for sHuPH20 polypeptides can include a
signal sequence, such as a heterologous or non-heterologous (i.e.
native) signal sequence. Exemplary of the precursors are those that
include a signal sequence, such as the native 35 amino acid signal
sequence at amino acid positions 1-35 (see, e.g., amino acids 1-35
of SEQ ID NO:107).
[0074] As used herein, an "extended soluble PH20" or "esPH20"
includes soluble PH20 polypeptides that contain residues up to the
GPI anchor-attachment signal sequence and one or more contiguous
residues from the GPI-anchor attachment signal sequence such that
the esPH20 is soluble under physiological conditions. Solubility
under physiological conditions can be determined by any method
known to those of skill in the art. For example, it can be assessed
by the Triton.RTM. X-114 assay described above and in the examples.
In addition, as discussed above, a soluble PH20 is, if produced in
CHO cells, such as CHO--S cells, a polypeptide that is expressed
and is secreted into the cell culture medium. Soluble human PH20
polypeptides, however, are not limited to those produced in CHO
cells, but can be produced in any cell or by any method, including
recombinant expression and polypeptide synthesis. Reference to
secretion in CHO cells is definitional. Hence, if a polypeptide
could be expressed and secreted in CHO cells and is soluble, i.e.
partitions into the aqueous phase when extracted with Triton.RTM.
X-114, it is a soluble PH20 polypeptide whether or not it is
so-produced. Human soluble esPH20 polypeptides include, in addition
to residues 36-490, one or more contiguous amino acids from amino
acid residue position 491 of SEQ ID NO:107, inclusive, such that
the resulting polypeptide is soluble. Exemplary human esPH20
soluble polypeptides are those that have amino acids residues
corresponding to amino acids 36-491, 36-492, 36-493, 36-494,
36-495, 36-496 and 36-497 of SEQ ID NO:107. Exemplary of these are
those with an amino acid sequence set forth in any of SEQ ID
NOS:60-63 and 102-104. Also included are allelic variants and other
variants, such as any with 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater
sequence identity with the corresponding polypeptides of SEQ ID
NOS: 60-63 and 102-104 that retain neutral activity and are
soluble. Reference to sequence identity refers to variants with
amino acid substitutions.
[0075] As used herein, reference to esPH20s includes precursor
esPH20 polypeptides and mature esPH20 polypeptides (such as those
in which a signal sequence has been removed), truncated forms
thereof that have enzymatic activity (retaining at least 1%, 10%,
20%, 30%, 40%, 50% or more of the full-length form) and are
soluble, and includes allelic variants and species variants,
variants encoded by splice variants, and other variants, including
polypeptides that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to the precursor polypeptides set forth in SEQ ID
NOS:107 and 109, or the mature forms thereof.
[0076] As used herein, reference to esPH20s also include those that
contain chemical or posttranslational modifications and those that
do not contain chemical or posttranslational modifications. Such
modifications include, but are not limited to, PEGylation,
albumination, glycosylation, farnesylation, carboxylation,
hydroxylation, phosphorylation, and other polypeptide modifications
known in the art.
[0077] As used herein, soluble recombinant human PH20 (rHuPH20)
refers to a soluble form of human PH20 that as recombinantly
expressed and secreted in Chinese Hamster Ovary (CHO) cells.
Soluble rHuPH20 is encoded by nucleic acid that includes the signal
sequence and is set forth in SEQ ID NO:109. Also included are DNA
molecules that are allelic variants thereof and other soluble
variants. The nucleic acid encoding soluble rHuPH20 is expressed in
CHO cells which secrete the mature polypeptide. As produced in the
culture medium, there is heterogeneity at the C-terminus so that
the product includes a mixture of species that can include any one
or more of SEQ ID NO:122 to SEQ ID NO:127 in various abundance.
[0078] Similarly, for other forms of PH20, such as the esPH20s,
recombinantly expressed polypeptides and compositions thereof can
include a plurality of species whose C-terminus exhibits
heterogeneity. For example, compositions of recombinantly expressed
esPH20 produced by expression of the polypeptide of SEQ ID NO:8,
which encodes an esPH20 that has amino acids 36-497, can include
forms with fewer amino acids, such as 36-496, 36-495.
[0079] As used herein, an N-linked moiety refers to an asparagine
(N) amino acid residue of a polypeptide that is capable of being
glycosylated by post-translational modification of a polypeptide.
Exemplary N-linked moieties of human PH20 include amino acids N82,
N166, N235, N254, N368 and N393 of human PH20 set forth in SEQ ID
NO:107.
[0080] As used herein, an N-glycosylated polypeptide refers to a
PH20 polypeptide or truncated form thereof containing
oligosaccharide linkage of at least three N-linked amino acid
residues, for example, N-linked moieties corresponding to amino
acid residues N235, N368 and N393 of SEQ ID NO:107. An
N-glycosylated polypeptide can include a polypeptide where three,
four, five and up to all of the N-linked moieties are linked to an
oligosaccharide. The N-linked oligosaccharides can include
oligomannose, complex, hybrid or sulfated oligosaccharides, or
other oligosaccharides and monosaccharides.
[0081] As used herein, an N-partially glycosylated polypeptide
refers to a polypeptide that minimally contains an
N-acetylglucosamine glycan linked to at least three N-linked
moieties. A partially glycosylated polypeptide can include various
glycan forms, including monosaccharides, oligosaccharides, and
branched sugar forms, including those formed by treatment of a
polypeptide with EndoH, EndoF1, EndoF2 and/or EndoF3. The cleavage
of glycans by such enzymes is depicted, for example, in FIG. 2.
[0082] As used herein, a deglycosylated PH20 polypeptide refers to
a PH20 polypeptide provided herein in which fewer than all possible
glycosylation sites are glycosylated. Deglycosylation can be
effected, for example, by removing glycosylation, by preventing it,
or by modifying the polypeptide to eliminate a glycosylation site.
As shown herein, particular N-glycosylation sites are not required
for activity, whereas others are.
[0083] As used herein, a hyaluronan-associated disease, disorder or
condition refers to any disease or condition in which hyaluronan
levels are elevated as cause, consequence or otherwise observed in
the disease or condition. Hyaluronan-associated diseases and
conditions are associated with elevated hyaluronan expression in a
tissue or cell, increased interstitial fluid pressure, decreased
vascular volume, and/or increased water content in a tissue.
Hyaluronan-associated diseases, disorders or conditions can be
treated by administration of a composition containing a hyaluronan
degrading enzyme, such as a hyaluronidase, for example, a soluble
hyaluronidase, either alone or in combination with or in addition
to another treatment and/or agent. Exemplary diseases and
conditions, include, but are not limited to, hyaluronan-rich
cancers, for example, tumors, including solid tumors such as
late-stage cancers, a metastatic cancers, undifferentiated cancers,
ovarian cancer, in situ carcinoma (ISC), squamous cell carcinoma
(SCC), prostate cancer, pancreatic cancer, non-small cell lung
cancer, breast cancer, colon cancer and other cancers. Also
exemplary of hyaluronan-associated diseases and conditions are
diseases that are associated with elevated interstitial fluid
pressure, such as diseases associated with disc pressure, and
edema, for example, edema caused by organ transplant, stroke, brain
trauma or other injury. Exemplary hyaluronan-associated diseases
and conditions include diseases and conditions associated with
elevated interstitial fluid pressure, decreased vascular volume,
and/or increased water content in a tissue, including cancers, disc
pressure and edema. In one example, treatment of the
hyaluronan-associated condition, disease or disorder includes
amelioration, reduction, or other beneficial effect on one or more
of increased interstitial fluid pressure (IFP), decreased vascular
volume, and increased water content in a tissue.
[0084] As used herein, a conjugate refers to soluble PH20
polypeptides linked directly or indirectly to one or more other
polypeptides or chemical moieties. Such conjugates include fusion
proteins, those produced by chemical conjugates and those produced
by any other method whereby at least one soluble PH20 polypeptide
is linked, directly or indirectly to another polypeptide or
chemical moiety so long as the conjugate retains hyaluronidase
activity. Exemplary of conjugates provided herein include PH20
polypeptides linked directly or indirectly to a multimerization
domain, such as an Fc moiety, a toxin, a label or a drug.
[0085] As used herein, a fusion protein refers to a polypeptide
encoded by a nucleic acid sequence containing a coding sequence
from one nucleic acid molecule and the coding sequence from another
nucleic acid molecule in which the coding sequences are in the same
reading frame such that when the fusion construct is transcribed
and translated in a host cell, the protein is produced containing
the two proteins. The two molecules can be adjacent in the
construct or separated by a linker polypeptide that contains, 1, 2,
3, or more, but typically fewer than 10, 9, 8, 7, or 6 amino acids.
The protein product encoded by a fusion construct is referred to as
a fusion polypeptide. Exemplary of fusion polypeptides include Fc
fusions.
[0086] As used herein, a polymer that is conjugated to a hyaluronan
degrading enzyme, such as a hyaluronidase, refers to any polymer
that is covalently or otherwise stably linked, directly or via a
linker, to a hyaluronan degrading enzyme. Such polymers, typically
increase serum half-life, and include, but are not limited to
sialic moieties, pegylation moieties, dextran, and sugar and other
moieties, such as for glycosylation.
[0087] As used herein, activity refers to a functional activity or
activities of a polypeptide or portion thereof associated with a
full-length (complete) protein. Functional activities include, but
are not limited to, biological activity, catalytic or enzymatic
activity, antigenicity (ability to bind or compete with a
polypeptide for binding to an anti-polypeptide antibody),
immunogenicity, ability to form multimers, and the ability to
specifically bind to a receptor or ligand for the polypeptide.
[0088] As used herein, hyaluronidase activity refers to the ability
to enzymatically catalyze the cleavage of hyaluronic acid. The
United States Pharmacopeia (USP) XXII assay for hyaluronidase
determines hyaluronidase activity indirectly by measuring the
amount of higher molecular weight hyaluronic acid, or hyaluronan,
(HA) substrate remaining after the enzyme is allowed to react with
the HA for 30 min at 37.degree. C. (USP XXII-NF XVII (1990) 644-645
United States Pharmacopeia Convention, Inc, Rockville, Md.). A
Reference Standard solution can be used in an assay to ascertain
the relative activity, in units, of any hyaluronidase. In vitro
assays to determine the hyaluronidase activity of hyaluronidases,
such as PH20, including soluble PH20 and esPH20, are known in the
art and described herein. Exemplary assays include the
microturbidity assay described below (see e.g. Example 12) that
measures cleavage of hyaluronic acid by hyaluronidase indirectly by
detecting the insoluble precipitate formed when the uncleaved
hyaluronic acid binds with serum albumin. Reference Standards can
be used, for example, to generate a standard curve to determine the
activity in Units of the hyaluronidase being tested.
[0089] As used herein, neutral active refers to the ability of a
PH20 polypeptide to enzymatically catalyze the cleavage of
hyaluronic acid at neutral pH. A neutral active C-terminally
truncated or N-partially glycosylated PH20 provided herein has or
has about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 200%,
300%, 400%, 500%, 1000% or more activity compared to the
hyaluronidase activity of a corresponding neutral active PH20 that
is not C-terminally truncated or N-partially glycosylated.
[0090] As used herein, a GPI-anchor attachment signal sequence is a
C-terminal sequence of amino acids that directs addition of a
preformed GPI-anchor to the polypeptide within the lumen of the ER.
GPI-anchor attachment signal sequences are present in the precursor
polypeptides of GPI-anchored polypeptides, such as GPI-anchored
PH20 polypeptides. The C-terminal GPI-anchor attachment signal
sequence typically contains a predominantly hydrophobic region of
8-20 amino acids, preceded by a hydrophilic spacer region of 8-12
amino acids, immediately downstream of the co-site, or site of
GPI-anchor attachment. GPI-anchor attachment signal sequences can
be identified using methods well known in the art. These include,
but are not limited to, in silico methods and algorithms (see, e.g.
Udenfriend et al. (1995) Methods Enzymol. 250:571-582, Eisenhaber
et al., (1999) J. Biol. Chem. 292: 741-758, Kronegg and Buloz,
(1999), "Detection/prediction of GPI cleavage site (GPI-anchor) in
a protein (DGPI)", e.g., the website 129.194.185.165/dgpi/,
Fankhauser et al., (2005) Bioinformatics 21:1846-1852,
Omaetxebarria et al., (2007) Proteomics 7:1951-1960, Pierleoni et
al., (2008) BMC Bioinformatics 9:392), including those that are
readily available on bioinformatic websites, such as the ExPASy
Proteomics tools site (e.g., the WorldWideWeb site
expasy.ch/tools/).
[0091] As used herein, a bifucosylated polypeptide refers to a
polypeptide that has two fucose residues, one with a
.alpha.-1,3-linkage and the other with .alpha.-1,6-linkage, linked
to the same core N-acetylglucosamine moiety, with the
N-acetylglucosamine moiety linked to the asparagine residue in the
polypeptide chain. Bifucosylated polypeptides generally are
produced in insect cells.
[0092] As used herein, nucleic acids include DNA, RNA and analogs
thereof, including peptide nucleic acids (PNA) and mixtures
thereof. Nucleic acids can be single or double-stranded. When
referring to probes or primers, which are optionally labeled, such
as with a detectable label, such as a fluorescent or radiolabel,
single-stranded molecules are contemplated. Such molecules are
typically of a length such that their target is statistically
unique or of low copy number (typically less than 5, generally less
than 3) for probing or priming a library. Generally a probe or
primer contains at least 14, 16 or 30 contiguous nucleotides of
sequence complementary to or identical to a gene of interest.
Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids
long.
[0093] As used herein, a peptide refers to a polypeptide that is
greater than or equal to 2 amino acids in length, and less than or
equal to 40 amino acids in length. As used herein, the amino acids
which occur in the various sequences of amino acids provided herein
are identified according to their known, three-letter or one-letter
abbreviations (Table 1). The nucleotides which occur in the various
nucleic acid fragments are designated with the standard
single-letter designations used routinely in the art.
[0094] As used herein, an "amino acid" is an organic compound
containing an amino group and a carboxylic acid group. A
polypeptide contains two or more amino acids. For purposes herein,
amino acids include the twenty naturally-occurring amino acids,
non-natural amino acids and amino acid analogs (i.e., amino acids
wherein the a-carbon has a side chain).
[0095] As used herein, "amino acid residue" refers to an amino acid
formed upon chemical digestion (hydrolysis) of a polypeptide at its
peptide linkages. The amino acid residues described herein are
presumed to be in the "L" isomeric form. Residues in the "D"
isomeric form, which are so designated, can be substituted for any
L-amino acid residue as long as the desired functional property is
retained by the polypeptide. NH2 refers to the free amino group
present at the amino terminus of a polypeptide. COOH refers to the
free carboxy group present at the carboxyl terminus of a
polypeptide. In keeping with standard polypeptide nomenclature
described in J. Biol. Chem., 243: 3557-3559 (1968), and adopted 37
C.F.R. .quadrature..sctn..sctn.1.821-1.822, abbreviations for amino
acid residues are shown in Table 1:
TABLE-US-00001 TABLE 1 Table of Correspondence SYMBOL 1-Letter
3-Letter AMINO ACID Y Tyr Tyrosine G Gly Glycine F Phe
Phenylalanine M Met Methionine A Ala Alanine S Ser Serine I Ile
Isoleucine L Leu Leucine T Thr Threonine V Val Valine P Pro Proline
K Lys Lysine H His Histidine Q Gln Glutamine E Glu Glutamic acid Z
Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine D Asp Aspartic
acid N Asn Asparagine B Asx Asn and/or Asp C Cys Cysteine X Xaa
Unknown or other
[0096] All amino acid residue sequences represented herein by
formulae have a left to right orientation in the conventional
direction of amino-terminus to carboxyl-terminus. In addition, the
phrase "amino acid residue" is defined to include the amino acids
listed in the Table of Correspondence (Table 1) and modified and
unusual amino acids, such as those referred to in 37 C.F.R.
.sctn..sctn.1.821-1.822, and incorporated herein by reference.
Furthermore, it should be noted that a dash at the beginning or end
of an amino acid residue sequence indicates a peptide bond to a
further sequence of one or more amino acid residues, to an
amino-terminal group such as NH.sub.2 or to a carboxyl-terminal
group such as COOH.
[0097] As used herein, the "naturally occurring .alpha.-amino
acids" are the residues of those 20 .alpha.-amino acids found in
nature which are incorporated into protein by the specific
recognition of the charged tRNA molecule with its cognate mRNA
codon in humans. Non-naturally occurring amino acids thus include,
for example, amino acids or analogs of amino acids other than the
20 naturally-occurring amino acids and include, but are not limited
to, the D-isostereomers of amino acids. Exemplary non-natural amino
acids are described herein and are known to those of skill in the
art.
[0098] As used herein, a DNA construct is a single- or
double-stranded, linear or circular DNA molecule that contains
segments of DNA combined and juxtaposed in a manner not found in
nature. DNA constructs exist as a result of human manipulation, and
include clones and other copies of manipulated molecules.
[0099] As used herein, a DNA segment is a portion of a larger DNA
molecule having specified attributes. For example, a DNA segment
encoding a specified polypeptide is a portion of a longer DNA
molecule, such as a plasmid or plasmid fragment, which, when read
from the 5' to 3' direction, encodes the sequence of amino acids of
the specified polypeptide.
[0100] As used herein, the term polynucleotide means a single- or
double-stranded polymer of deoxyribonucleotides or ribonucleotide
bases read from the 5' to the 3' end. Polynucleotides include RNA
and DNA, and can be isolated from natural sources, synthesized in
vitro, or prepared from a combination of natural and synthetic
molecules. The length of a polynucleotide molecule is given herein
in terms of nucleotides (abbreviated "nt") or base pairs
(abbreviated "bp"). The term nucleotides is used for single- and
double-stranded molecules where the context permits. When the term
is applied to double-stranded molecules it is used to denote
overall length and will be understood to be equivalent to the term
base pairs. It will be recognized by those skilled in the art that
the two strands of a double-stranded polynucleotide can differ
slightly in length and that the ends thereof can be staggered; thus
all nucleotides within a double-stranded polynucleotide molecule
may not be paired. Such unpaired ends will, in general, not exceed
20 nucleotides in length.
[0101] As used herein, "similarity" between two proteins or nucleic
acids refers to the relatedness between the sequence of amino acids
of the proteins or the nucleotide sequences of the nucleic acids.
Similarity can be based on the degree of identity and/or homology
of sequences of residues and the residues contained therein.
Methods for assessing the degree of similarity between proteins or
nucleic acids are known to those of skill in the art. For example,
in one method of assessing sequence similarity, two amino acid or
nucleotide sequences are aligned in a manner that yields a maximal
level of identity between the sequences. "Identity" refers to the
extent to which the amino acid or nucleotide sequences are
invariant. Alignment of amino acid sequences, and to some extent
nucleotide sequences, also can take into account conservative
differences and/or frequent substitutions in amino acids (or
nucleotides). Conservative differences are those that preserve the
physico-chemical properties of the residues involved. Alignments
can be global (alignment of the compared sequences over the entire
length of the sequences and including all residues) or local (the
alignment of a portion of the sequences that includes only the most
similar region or regions).
[0102] "Identity" per se has an art-recognized meaning and can be
calculated using published techniques. (See, e.g. Computational
Molecular Biology, Lesk, A. M., ed., Oxford University Press, New
York, 1988; Biocomputing: Informatics and Genome Projects, Smith,
D. W., ed., Academic Press, New York, 1993; Computer Analysis of
Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular
Biology, von Heinje, G., Academic Press, 1987; and Sequence
Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton
Press, New York, 1991). While there exists a number of methods to
measure identity between two polynucleotide or polypeptides, the
term "identity" is well known to skilled artisans (Carillo, H.
& Lipton, D., SIAM J Applied Math 48:1073 (1988)).
[0103] As used herein, homologous (with respect to nucleic acid
and/or amino acid sequences) means about greater than or equal to
25% sequence homology, typically greater than or equal to 25%, 40%,
50%, 60%, 70%, 80%, 85%, 90% or 95% sequence homology; the precise
percentage can be specified if necessary. For purposes herein the
terms "homology" and "identity" are often used interchangeably,
unless otherwise indicated. In general, for determination of the
percentage homology or identity, sequences are aligned so that the
highest order match is obtained (see, e.g.: Computational Molecular
Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991;
Carillo et al. (1988) SIAM J Applied Math 48:1073). By sequence
homology, the number of conserved amino acids is determined by
standard alignment algorithms programs, and can be used with
default gap penalties established by each supplier. Substantially
homologous nucleic acid molecules would hybridize typically at
moderate stringency or at high stringency all along the length of
the nucleic acid of interest. Also contemplated are nucleic acid
molecules that contain degenerate codons in place of codons in the
hybridizing nucleic acid molecule.
[0104] Whether any two molecules have nucleotide sequences or amino
acid sequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%,
97%, 98% or 99% "identical" or "homologous" can be determined using
known computer algorithms such as the "FASTA" program, using for
example, the default parameters as in Pearson et al. (1988) Proc.
Natl. Acad. Sci. USA 85:2444 (other programs include the GCG
program package (Devereux, J., et al., Nucleic Acids Research
12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J
Mol Biol 215:403 (1990)); Guide to Huge Computers, Martin J.
Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al.
(1988) SIAM J Applied Math 48:1073). For example, the BLAST
function of the National Center for Biotechnology Information
database can be used to determine identity. Other commercially or
publicly available programs include, DNAStar "MegAlign" program
(Madison, Wis.) and the University of Wisconsin Genetics Computer
Group (UWG) "Gap" program (Madison Wis.). Percent homology or
identity of proteins and/or nucleic acid molecules can be
determined, for example, by comparing sequence information using a
GAP computer program (e.g., Needleman et al. (1970) J. Mol. Biol.
48:443, as revised by Smith and Waterman ((1981) Adv. Appl. Math.
2:482). Briefly, the GAP program defines similarity as the number
of aligned symbols (i.e., nucleotides or amino acids), which are
similar, divided by the total number of symbols in the shorter of
the two sequences. Default parameters for the GAP program can
include: (1) a unary comparison matrix (containing a value of 1 for
identities and 0 for non-identities) and the weighted comparison
matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as
described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE
AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358
(1979); (2) a penalty of 3.0 for each gap and an additional 0.10
penalty for each symbol in each gap; and (3) no penalty for end
gaps.
[0105] Therefore, as used herein, the term "identity" or "homology"
represents a comparison between a test and a reference polypeptide
or polynucleotide. As used herein, the term at least "90% identical
to" refers to percent identities from 90 to 99.99 relative to the
reference nucleic acid or amino acid sequence of the polypeptide.
Identity at a level of 90% or more is indicative of the fact that,
assuming for exemplification purposes a test and reference
polypeptide length of 100 amino acids are compared. No more than
10% (i.e., 10 out of 100) of the amino acids in the test
polypeptide differs from that of the reference polypeptide. Similar
comparisons can be made between test and reference polynucleotides.
Such differences can be represented as point mutations randomly
distributed over the entire length of a polypeptide or they can be
clustered in one or more locations of varying length up to the
maximum allowable, e.g. 10/100 amino acid difference (approximately
90% identity). Differences are defined as nucleic acid or amino
acid substitutions, insertions or deletions. At the level of
homologies or identities above about 85-90%, the result should be
independent of the program and gap parameters set; such high levels
of identity can be assessed readily, often by manual alignment
without relying on software.
[0106] As used herein, an aligned sequence refers to the use of
homology (similarity and/or identity) to align corresponding
positions in a sequence of nucleotides or amino acids. Typically,
two or more sequences that are related by 50% or more identity are
aligned. An aligned set of sequences refers to 2 or more sequences
that are aligned at corresponding positions and can include
aligning sequences derived from RNAs, such as ESTs and other cDNAs,
aligned with genomic DNA sequence.
[0107] As used herein, "primer" refers to a nucleic acid molecule
that can act as a point of initiation of template-directed DNA
synthesis under appropriate conditions (e.g., in the presence of
four different nucleoside triphosphates and a polymerization agent,
such as DNA polymerase, RNA polymerase or reverse transcriptase) in
an appropriate buffer and at a suitable temperature. It will be
appreciated that a certain nucleic acid molecules can serve as a
"probe" and as a "primer." A primer, however, has a 3' hydroxyl
group for extension. A primer can be used in a variety of methods,
including, for example, polymerase chain reaction (PCR),
reverse-transcriptase (RT)-PCR, RNA PCR, LCR, multiplex PCR,
panhandle PCR, capture PCR, expression PCR, 3' and 5' RACE, in situ
PCR, ligation-mediated PCR and other amplification protocols.
[0108] As used herein, "primer pair" refers to a set of primers
that includes a 5' (upstream) primer that hybridizes with the 5'
end of a sequence to be amplified (e.g. by PCR) and a 3'
(downstream) primer that hybridizes with the complement of the 3'
end of the sequence to be amplified.
[0109] As used herein, "specifically hybridizes" refers to
annealing, by complementary base-pairing, of a nucleic acid
molecule (e.g. an oligonucleotide) to a target nucleic acid
molecule. Those of skill in the art are familiar with in vitro and
in vivo parameters that affect specific hybridization, such as
length and composition of the particular molecule. Parameters
particularly relevant to in vitro hybridization further include
annealing and washing temperature, buffer composition and salt
concentration. Exemplary washing conditions for removing
non-specifically bound nucleic acid molecules at high stringency
are 0.1.times.SSPE, 0.1% SDS, 65.degree. C., and at medium
stringency are 0.2.times.SSPE, 0.1% SDS, 50.degree. C. Equivalent
stringency conditions are known in the art. The skilled person can
readily adjust these parameters to achieve specific hybridization
of a nucleic acid molecule to a target nucleic acid molecule
appropriate for a particular application. Complementary, when
referring to two nucleotide sequences, means that the two sequences
of nucleotides are capable of hybridizing, typically with less than
25%, 15% or 5% mismatches between opposed nucleotides. If
necessary, the percentage of complementarity will be specified.
Typically the two molecules are selected such that they will
hybridize under conditions of high stringency.
[0110] As used herein, substantially identical to a product means
sufficiently similar so that the property of interest is
sufficiently unchanged so that the substantially identical product
can be used in place of the product.
[0111] As used herein, it also is understood that the terms
"substantially identical" or "similar" varies with the context as
understood by those skilled in the relevant art.
[0112] As used herein, an allelic variant or allelic variation
references any of two or more alternative forms of a gene occupying
the same chromosomal locus. Allelic variation arises naturally
through mutation, and can result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or can encode polypeptides having altered amino acid
sequence. The term "allelic variant" also is used herein to denote
a protein encoded by an allelic variant of a gene. Typically the
reference form of the gene encodes a wildtype form and/or
predominant form of a polypeptide from a population or single
reference member of a species. Typically, allelic variants, which
include variants between and among species typically have at least
80%, 90% or greater amino acid identity with a wildtype and/or
predominant form from the same species; the degree of identity
depends upon the gene and whether comparison is interspecies or
intraspecies. Generally, intraspecies allelic variants have at
least about 80%, 85%, 90% or 95% identity or greater with a
wildtype and/or predominant form, including 96%, 97%, 98%, 99% or
greater identity with a wildtype and/or predominant form of a
polypeptide. Reference to an allelic variant herein generally
refers to variations n proteins among members of the same
species.
[0113] As used herein, "allele," which is used interchangeably
herein with "allelic variant" refers to alternative forms of a gene
or portions thereof. Alleles occupy the same locus or position on
homologous chromosomes. When a subject has two identical alleles of
a gene, the subject is said to be homozygous for that gene or
allele. When a subject has two different alleles of a gene, the
subject is said to be heterozygous for the gene. Alleles of a
specific gene can differ from each other in a single nucleotide or
several nucleotides, and can include modifications such as
substitutions, deletions and insertions of nucleotides. An allele
of a gene also can be a form of a gene containing a mutation.
[0114] As used herein, species variants refer to variants in
polypeptides among different species, including different mammalian
species, such as mouse and human. Exemplary of species variants
provided herein are primate PH20, such as, but not limited to,
human, chimpanzee, macaque and cynomologus monkey. Generally,
species variants have 70%, 75%. 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or sequence identity. Corresponding residues
between and among species variants can be determined by comparing
and aligning sequences to maximize the number of matching
nucleotides or residues, for example, such that identity between
the sequences is equal to or greater than 95%, equal to or greater
than 96%, equal to or greater than 97%, equal to or greater than
98% or equal to greater than 99%. The position of interest is then
given the number assigned in the reference nucleic acid molecule.
Alignment can be effected manually or by eye, particularly, where
sequence identity is greater than 80%. For example, the alignment
in FIG. 1 shows that amino acid residue 491 of human PH20
corresponds to amino acid residue 491 of chimpanzee PH20 and amino
acid residue 497 of human PH20 corresponds to amino acid residue
498 of chimpanzee PH20.
[0115] As used herein, a splice variant refers to a variant
produced by differential processing of a primary transcript of
genomic DNA that results in more than one type of mRNA.
[0116] As used herein, modification is in reference to modification
of a sequence of amino acids of a polypeptide or a sequence of
nucleotides in a nucleic acid molecule and includes deletions,
insertions, and replacements of amino acids and nucleotides,
respectively. Methods of modifying a polypeptide are routine to
those of skill in the art, such as by using recombinant DNA
methodologies.
[0117] As used herein, the term promoter means a portion of a gene
containing DNA sequences that provide for the binding of RNA
polymerase and initiation of transcription. Promoter sequences are
commonly, but not always, found in the 5' non-coding region of
genes.
[0118] As used herein, isolated or purified polypeptide or protein
or biologically-active portion thereof is substantially free of
cellular material or other contaminating proteins from the cell or
tissue from which the protein is derived, or substantially free
from chemical precursors or other chemicals when chemically
synthesized. Preparations can be determined to be substantially
free if they appear free of readily detectable impurities as
determined by standard methods of analysis, such as thin layer
chromatography (TLC), gel electrophoresis and high performance
liquid chromatography (HPLC), used by those of skill in the art to
assess such purity, or sufficiently pure such that further
purification would not detectably alter the physical and chemical
properties, such as enzymatic and biological activities, of the
substance. Methods for purification of the compounds to produce
substantially chemically pure compounds are known to those of skill
in the art. A substantially chemically pure compound, however, can
be a mixture of stereoisomers. In such instances, further
purification might increase the specific activity of the
compound.
[0119] Hence, reference to a substantially purified polypeptide,
such as a substantially purified extended soluble PH20 refers to
preparations of PH20 proteins that are substantially free of
cellular material includes preparations of proteins in which the
protein is separated from cellular components of the cells from
which it is isolated or recombinantly-produced. In one embodiment,
the term substantially free of cellular material includes
preparations of enzyme proteins having less that about 30% (by dry
weight) of non-enzyme proteins (also referred to herein as a
contaminating protein), generally less than about 20% of non-enzyme
proteins or 10% of non-enzyme proteins or less that about 5% of
non-enzyme proteins. When the enzyme protein is recombinantly
produced, it also is substantially free of culture medium, i.e.,
culture medium represents less than about or at 20%, 10% or 5% of
the volume of the enzyme protein preparation.
[0120] As used herein, the term substantially free of chemical
precursors or other chemicals includes preparations of enzyme
proteins in which the protein is separated from chemical precursors
or other chemicals that are involved in the synthesis of the
protein. The term includes preparations of enzyme proteins having
less than about 30% (by dry weight), 20%, 10%, 5% or less of
chemical precursors or non-enzyme chemicals or components.
[0121] As used herein, synthetic, with reference to, for example, a
synthetic nucleic acid molecule or a synthetic gene or a synthetic
peptide refers to a nucleic acid molecule or polypeptide molecule
that is produced by recombinant methods and/or by chemical
synthesis methods.
[0122] As used herein, production by recombinant means or using
recombinant DNA methods means the use of the well known methods of
molecular biology for expressing proteins encoded by cloned
DNA.
[0123] As used herein, vector (or plasmid) refers to discrete
elements that are used to introduce a heterologous nucleic acid
into cells for either expression or replication thereof. The
vectors typically remain episomal, but can be designed to effect
integration of a gene or portion thereof into a chromosome of the
genome. Also contemplated are vectors that are artificial
chromosomes, such as yeast artificial chromosomes and mammalian
artificial chromosomes. Selection and use of such vehicles are well
known to those of skill in the art.
[0124] As used herein, an expression vector includes vectors
capable of expressing DNA that is operatively linked with
regulatory sequences, such as promoter regions, that are capable of
effecting expression of such DNA fragments. Such additional
segments can include promoter and terminator sequences, and
optionally can include one or more origins of replication, one or
more selectable markers, an enhancer, a polyadenylation signal, and
the like. Expression vectors are generally derived from plasmid or
viral DNA, or can contain elements of both. Thus, an expression
vector refers to a recombinant DNA or RNA construct, such as a
plasmid, a phage, recombinant virus or other vector that, upon
introduction into an appropriate host cell, results in expression
of the cloned DNA. Appropriate expression vectors are well known to
those of skill in the art and include those that are replicable in
eukaryotic cells and/or prokaryotic cells and those that remain
episomal or those which integrate into the host cell genome.
[0125] As used herein, vector also includes "virus vectors" or
"viral vectors." Viral vectors are engineered viruses that are
operatively linked to exogenous genes to transfer (as vehicles or
shuttles) the exogenous genes into cells.
[0126] As used herein, "operably" or "operatively linked" when
referring to DNA segments means that the segments are arranged so
that they function in concert for their intended purposes, e.g.,
transcription initiates downstream of the promoter and upstream of
any transcribed sequences. The promoter is usually the domain to
which the transcriptional machinery binds to initiate transcription
and proceeds through the coding segment to the terminator.
[0127] As used herein the term "assessing" is intended to include
quantitative and qualitative determination in the sense of
obtaining an absolute value for the activity of a protease, or a
domain thereof, present in the sample, and also of obtaining an
index, ratio, percentage, visual or other value indicative of the
level of the activity. Assessment can be direct or indirect and the
chemical species actually detected need not of course be the
proteolysis product itself but can for example be a derivative
thereof or some further substance. For example, detection of a
cleavage product of a complement protein, such as by SDS-PAGE and
protein staining with Coomasie blue.
[0128] As used herein, biological activity refers to the in vivo
activities of a compound or physiological responses that result
upon in vivo administration of a compound, composition or other
mixture. Biological activity, thus, encompasses therapeutic effects
and pharmaceutical activity of such compounds, compositions and
mixtures. Biological activities can be observed in in vitro systems
designed to test or use such activities. Thus, for purposes herein
a biological activity of a protease is its catalytic activity in
which a polypeptide is hydrolyzed.
[0129] As used herein equivalent, when referring to two sequences
of nucleic acids, means that the two sequences in question encode
the same sequence of amino acids or equivalent proteins. When
equivalent is used in referring to two proteins or peptides, it
means that the two proteins or peptides have substantially the same
amino acid sequence with only amino acid substitutions that do not
substantially alter the activity or function of the protein or
peptide. When equivalent refers to a property, the property does
not need to be present to the same extent (e.g., two peptides can
exhibit different rates of the same type of enzymatic activity),
but the activities are usually substantially the same.
[0130] As used herein, "modulate" and "modulation" or "alter" refer
to a change of an activity of a molecule, such as a protein.
Exemplary activities include, but are not limited to, biological
activities, such as signal transduction. Modulation can include an
increase in the activity (i.e., up-regulation or agonist activity),
a decrease in activity (i.e., down-regulation or inhibition) or any
other alteration in an activity (such as a change in periodicity,
frequency, duration, kinetics or other parameter). Modulation can
be context dependent and typically modulation is compared to a
designated state, for example, the wildtype protein, the protein in
a constitutive state, or the protein as expressed in a designated
cell type or condition.
[0131] As used herein, a composition refers to any mixture. It can
be a solution, suspension, liquid, powder, paste, aqueous,
non-aqueous or any combination thereof.
[0132] As used herein, a combination refers to any association
between or among two or more items. The combination can be two or
more separate items, such as two compositions or two collections,
can be a mixture thereof, such as a single mixture of the two or
more items, or any variation thereof. The elements of a combination
are generally functionally associated or related.
[0133] As used herein, "disease or disorder" refers to a
pathological condition in an organism resulting from cause or
condition including, but not limited to, infections, acquired
conditions, genetic conditions, and characterized by identifiable
symptoms. Diseases and disorders of interest herein are those
involving components of the ECM. As used herein, "treating" a
subject with a disease or condition means that the subject's
symptoms are partially or totally alleviated, or remain static
following treatment. Hence treatment encompasses prophylaxis,
therapy and/or cure. Prophylaxis refers to prevention of a
potential disease and/or a prevention of worsening of symptoms or
progression of a disease. Treatment also encompasses any
pharmaceutical use of a modified interferon and compositions
provided herein.
[0134] As used herein, a pharmaceutically effective agent, includes
any therapeutic agent or bioactive agent, including, but not
limited to, for example, anesthetics, vasoconstrictors, dispersing
agents, conventional therapeutic drugs, including small molecule
drugs, including, but not limited to, bisphosphonates, and
therapeutic proteins, including, but not limited to, insulin, IgG
molecules, and antibodies.
[0135] As used herein, a therapeutic agent, includes any
pharmaceutically effective agent or bioactive agent, including, but
not limited to, for example, anesthetics, vasoconstrictors,
dispersing agents, conventional therapeutic drugs, including small
molecule drugs, including, but not limited to, bisphosphonates, and
therapeutic proteins, including, but not limited to, insulin, IgG
molecules, and antibodies.
[0136] As used herein, treatment means any manner in which the
symptoms of a condition, disorder or disease or other indication,
are ameliorated or otherwise beneficially altered.
[0137] As used herein, therapeutic effect means an effect resulting
from treatment of a subject that alters, typically improves or
ameliorates the symptoms of a disease or condition or that cures a
disease or condition. A therapeutically effective amount refers to
the amount of a composition, molecule or compound which results in
a therapeutic effect following administration to a subject.
[0138] As used herein, the term "subject" refers to an animal,
including a mammal, such as a human being.
[0139] As used herein, a patient refers to a human subject
exhibiting symptoms of a disease or disorder.
[0140] As used herein, amelioration of the symptoms of a particular
disease or disorder by a treatment, such as by administration of a
pharmaceutical composition or other therapeutic, refers to any
lessening, whether permanent or temporary, lasting or transient, of
the symptoms that can be attributed to or associated with
administration of the composition or therapeutic.
[0141] As used herein, prevention or prophylaxis refers to methods
in which the risk of developing disease or condition is
reduced.
[0142] As used herein, a "therapeutically effective amount" or a
"therapeutically effective dose" refers to the quantity of an
agent, compound, material, or composition containing a compound
that is at least sufficient to produce a therapeutic effect. Hence,
it is the quantity necessary for preventing, curing, ameliorating,
arresting or partially arresting a symptom of a disease or
disorder.
[0143] As used herein, unit dose form refers to physically discrete
units suitable for human and animal subjects and packaged
individually as is known in the art.
[0144] As used herein, a single dosage formulation refers to a
formulation for direct administration.
[0145] As used herein, an "article of manufacture" is a product
that is made and sold. As used throughout this application, the
term is intended to encompass a therapeutic agent with a soluble
PH20, such as esPH20, or an esPH20 alone, contained in the same or
separate articles of packaging.
[0146] As used herein, fluid refers to any composition that can
flow. Fluids thus encompass compositions that are in the form of
semi-solids, pastes, solutions, aqueous mixtures, gels, lotions,
creams and other such compositions.
[0147] As used herein, a "kit" refers to a combination of
compositions provided herein and another item for a purpose
including, but not limited to, reconstitution, activation, and
instruments/devices for delivery, administration, diagnosis, and
assessment of a biological activity or property. Kits optionally
include instructions for use.
[0148] As used herein, a cellular extract or lysate refers to a
preparation or fraction which is made from a lysed or disrupted
cell.
[0149] As used herein, animal includes any animal, such as, but are
not limited to primates including humans, gorillas and monkeys;
rodents, such as mice and rats; fowl, such as chickens; ruminants,
such as goats, cows, deer, sheep; pigs and other animals. Non-human
animals exclude humans as the contemplated animal. The enzymes
provided herein are from any source, animal, plant, prokaryotic and
fungal. Most enzymes are of animal origin, including mammalian
origin.
[0150] As used herein, a control refers to a sample that is
substantially identical to the test sample, except that it is not
treated with a test parameter, or, if it is a plasma sample, it can
be from a normal volunteer not affected with the condition of
interest. A control also can be an internal control.
[0151] As used herein, the singular forms "a," "an" and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to a compound comprising
"an extracellular domain" includes compounds with one or a
plurality of extracellular domains.
[0152] As used herein, ranges and amounts can be expressed as
"about" a particular value or range. About also includes the exact
amount. Hence "about 5 bases" means "about 5 bases" and also "5
bases."
[0153] As used herein, "optional" or "optionally" means that the
subsequently described event or circumstance does or does not
occur, and that the description includes instances where said event
or circumstance occurs and instances where it does not. For
example, an optionally substituted group means that the group is
unsubstituted or is substituted.
[0154] As used herein, the abbreviations for any protective groups,
amino acids and other compounds, are, unless indicated otherwise,
in accord with their common usage, recognized abbreviations, or the
IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972)
Biochem. 11:1726).
B. Overview
[0155] Hyaluronidases are enzymes that catalyze the hydrolysis of
hyaluronic acid, thereby lowering the viscosity of hyaluronic acid
and increasing tissue permeability. PH20 is a neutral-active and
acid-active hyaluronidase that exhibits optimal activity when
glycosylated. Human PH20 is a GPI-anchored protein that is anchored
to the extracellular leaflet of the plasma membrane via a
glycosylphosphatidylinositol (GPI) anchor attached to the
C-terminus of the protein. The addition of the GPI anchor to all
GPI-anchored proteins occurs following cleavage at a specific amino
acid position, called the .omega.-site (typically located
approximately 20-30 amino acids from the C-terminus), and removal
of the C-terminal portion in the ER. This C-terminal portion is the
GPI-anchor attachment signal sequence. The GPI-anchor attachment
signal sequence of human PH20 is located at amino acid positions
491-509 of the precursor polypeptide set forth in SEQ ID NO:107,
and the .omega.-site is amino acid position 490. GPI-anchored PH20
polypeptides such as human PH20 are membrane-bound and, therefore,
insoluble. Insoluble forms of PH20 typically are not suitable for
therapeutic purposes.
[0156] PH20 polypeptides that lack a GPI anchor generally are
secreted by cells upon expression because they do not contain a
GPI-attachment signal sequence that locks the polypeptide to the
membrane. It is found herein that soluble forms of PH20 also
include those that contain residues within the GPI-anchor
attachment signal sequence. Extended soluble PH20 (esPH20)
polypeptides are soluble PH20 proteins that are truncated at the
C-terminus but retain one or more amino acid residues located in
the GPI-anchor attachment signal sequence of the corresponding
wild-type PH20 polypeptide. Such esPH20 polypeptides are soluble
and can be used as therapeutic polypeptides, such as to treat
hyaluronan-associated diseases or conditions and/or to serve as a
spreading or dispersing agent to promote, enhance or increase the
dispersion and delivery of other agents, drugs and proteins thereby
improving the pharmacokinetic and pharmacodynamic profile of the
co-administered agent, drug or protein.
[0157] 1. PH20
[0158] PH20, also known as sperm surface protein, sperm adhesion
molecule 1, SPAM1 or HYAL3, is a hyaluronidase. Hyaluronidases are
a family of enzymes that degrade hyaluronic acid (also known as
hyaluronan or hyaluronate or HA), an essential component of the
extracellular matrix and a major constituent of the interstitial
barrier. By catalyzing the hydrolysis of hyaluronic acid,
hyaluronidase lowers the viscosity of hyaluronic acid, thereby
increasing tissue permeability. As such, hyaluronidases have been
used, for example, as a spreading or dispersing agent in
conjunction with other agents, drugs and proteins to enhance their
dispersion and delivery, and to improve the pharmacokinetic and
pharmacodynamic profile of the co-administered agent, drug or
protein.
[0159] PH20, like other mammalian hyaluronidases, is an
endo-.beta.-N-acetyl-hexosaminidase that hydrolyzes the
.beta.1.fwdarw.4 glycosidic bond of hyaluronic acid into various
oligosaccharide lengths such as tetrasaccharides and
hexasaccharides. PH20 has both hydrolytic and transglycosidase
activities and can degrade hyaluronic acid and chondroitin
sulfates, such as C4-S and C6-S. PH20 is naturally involved in
sperm-egg adhesion and aids penetration by sperm of the layer of
cumulus cells by digesting hyaluronic acid. PH20 is located on the
sperm surface, and in the lysosome-derived acrosome, where it is
bound to the inner acrosomal membrane. Plasma membrane PH20 has
hyaluronidase activity only at neutral pH, while inner acrosomal
membrane PH20 has activity at both neutral and acidic pH. In
addition to being a hyaluronidase, PH20 also appears to be a
receptor for HA-induced cell signaling, and a receptor for the zona
pellucida surrounding the oocyte.
[0160] Exemplary PH20 proteins include, but are not limited to,
human (precursor polypeptide set forth in SEQ ID NO:107, mature
polypeptide set forth in SEQ ID NO:108), bovine (SEQ ID NOS:111 and
119), rabbit (SEQ ID NO:112), ovine (SEQ ID NOS:113, 118 and 120),
Cynomolgus monkey (SEQ ID NO:114), guinea pig (SEQ ID NO:115), rat
(SEQ ID NO:116), mouse (SEQ ID NO:117), chimpanzee (SEQ ID NO:197)
and Rhesus monkey (SEQ ID NO:198) PH20 polypeptides. The human PH20
mRNA transcript is normally translated to generate a 509 amino acid
precursor protein (SEQ ID NO:107) containing a 35 amino acid signal
sequence at the N-terminus (amino acid residue positions 1-35 of
SEQ ID NO:107). Thus, following transport to the ER and removal of
the signal peptide, a 474 amino acid mature polypeptide with an
amino acid sequence set forth in SEQ ID NO:108 is produced. As
discussed below, a C-terminal peptide is then cleaved in the ER to
facilitate covalent attachment of a GPI anchor to the newly-formed
C-terminal amino acid at the amino acid position corresponding to
position 490 of the precursor polypeptide set forth in SEQ ID
NO:107.
[0161] Human PH20 is the prototypical neutral-active hyaluronidase
that is generally locked to the plasma membrane via a
glycosylphosphatidylinositol (GPI) anchor. As noted above, PH20
also is expressed on the inner acrosomal membrane where it has
hyaluronidase activity at both neutral and acidic pH. Evidence
suggests that the Peptide 1 region of PH20, which corresponds to
amino acids 142-172 of the precursor polypeptide set forth in SEQ
ID NO:107, is required for enzyme activity at neutral pH. The
Peptide 3 region, which corresponds to amino acids 277-297 of the
precursor polypeptide set forth in SEQ ID NO:107, appears to be
important for enzyme activity at acidic pH (Chem et al., (2001)
Matrix Biology 20:515-525). Thus, it appears that PH20 contains two
catalytic sites. In addition to the catalytic sites, PH20 also
contains a hyaluronan-binding site. Experimental evidence suggests
that this site is located in the Peptide 2 region, which
corresponds to amino acid positions 205-235 of the precursor
polypeptide set forth in SEQ ID NO:107. This region is highly
conserved among hyaluronidases and is similar to the heparin
binding motif
[0162] a. Glycosylation
[0163] Glycosylation, including N- and O-linked glycosylation, of
some hyaluronan degrading enzymes, including hyaluronidases, can be
important for their catalytic activity and stability. N-linked
oligosaccharides fall into several major types (oligomannose,
complex, hybrid), all of which have (Man).sub.3-GlcNAc-GlcNAc-cores
attached via the amide nitrogen of Asn residues that fall within
-Asn-Xaa-Thr/Ser-sequences (where Xaa is not Pro). An additional
glycosylation site at -Asn-Xaa-Cys- has been reported for
coagulation protein C. In some instances, a hyaluronan degrading
enzyme, such as a hyaluronidase, can contain both N-glycosidic and
O-glycosidic linkages. For example, PH20 has one O-linked
oligosaccharide at amino acid T475 as well as six N-linked
oligosaccharides at amino acids N82, N166, N235, N254, N368, and
N393 of human PH20, exemplified in SEQ ID NO: 107. Amino acid
residues N82, N166 and N254 are occupied by complex type glycans
whereas amino acid residues N368 and N393 are occupied by high
mannose type glycans (see, e.g. Example 6 below). Amino acid
residue N235 is occupied by approximately 80% high mannose type
glycans and 20% complex type glycans.
[0164] While altering the type of glycan modifying a glycoprotein
can have dramatic affects on a protein's antigenicity, structural
folding, solubility, and stability, most enzymes are not thought to
require glycosylation for optimal enzyme activity. For some
hyaluronidases, removal of N-linked glycosylation can result in
near complete inactivation of the hyaluronidase activity. Thus, for
such hyaluronidases, the presence of N-linked glycans is required
for generating an active enzyme. The presence of N-linked glycans
in PH20 polypeptides is required for generating an active enzyme.
For example, it is found herein, that complete deglycosylation of
human PH20, by treatment with the endoglycosidase PNGaseF or the
GlcNAc phosphotransferase (GPT) inhibitor tunicamycin, results in
the total loss of hyaluronidase activity (see, e.g. Examples 7-8,
below). In contrast, partial deglycosylation of human PH20, by
treatment with endoglycosidase EndoF1, EndoF2, EndoF3 or EndoH,
does not affect the hyaluronidase activity of human PH20 (see,
e.g., Example 7, below).
[0165] b. GPI-anchoring
[0166] Human PH20 is a GPI-anchored protein. As such, the PH20
polypeptide is anchored to the extracellular leaflet of the plasma
membrane via a glycosyl-phosphatidylinositol (GPI) anchor attached
to the C-terminus of the protein. GPI-anchored proteins such as
human PH20 are translated with a cleavable N-terminal signal
peptide that directs the protein to the endoplasmic reticulum (ER).
At the C-terminus of these proteins is another signal sequence that
directs addition of a preformed GPI-anchor to the polypeptide
within the lumen of the ER. Addition of the GPI anchor occurs
following cleavage of the C-terminal portion at a specific amino
acid position, called the co-site (typically located approximately
20-30 amino acids from the C-terminus). Although there appears to
be no consensus sequence to identify the location of the co-site,
GPI anchored proteins contain a C-terminal GPI-anchor attachment
signal sequence or domain that typically contains a predominantly
hydrophobic region of 8-20 amino acids, preceded by a hydrophilic
spacer region of 8-12 amino acids immediately downstream of the
co-site. This hydrophilic spacer region often is rich in charged
amino acids and proline (White et al., (2000) J. Cell Sci.
113(Pt.4):721-727). More detailed analysis suggests that there is a
region of approximately 11 amino acids before the .omega.-1
position that is characterized by a low amount of predicted
secondary structure, a region around the cleavage site (co-site),
from .omega.-1 to .omega.+2 that is characterized by the presence
of small side chain residues, the spacer region between positions
.omega.+3 and .omega.+9, and a hydrophobic tail from .omega.+10 to
the C-terminal end (Pierleoni et al., (2008) BMC Bioinformatics
9:392).
[0167] Although there is no GPI-anchor attachment signal consensus
sequence, various in silico methods and algorithms have been
developed that can be used to identify such sequences in
polypeptides (see, e.g. Udenfriend et al. (1995) Methods Enzymol.
250:571-582; Eisenhaber et al., (1999) J. Biol. Chem. 292: 741-758;
Kronegg and Buloz, (1999), "Detection/prediction of GPI cleavage
site (GPI-anchor) in a protein (DGPI)," 129.194.185.165/dgpi/;
Fankhauser et al., (2005) Bioinformatics 21:1846-1852;
Omaetxebarria et al., (2007) Proteomics 7:1951-1960; Pierleoni et
al., (2008) BMC Bioinformatics 9:392), including those that are
readily available on bioinformatic websites, such as the ExPASy
Proteomics tools site (expasy.ch/tools/). Thus, one of skill in the
art can determine whether a PH20 polypeptide likely contains a
GPI-anchor attachment signal sequence, and, therefore, whether the
PH20 polypeptide is a GPI-anchored protein.
[0168] The GPI-anchor attachment signal sequence of human PH20 is
located at amino acid positions 491-509 of the precursor
polypeptide set forth in SEQ ID NO:107, and the co-site is amino
acid position 490. Thus, in this modeling of human PH20, amino
acids 491-509 are cleaved following transport to the ER and a GPI
anchor is covalently attached to the serine residue at position
490. The covalent attachment of a GPI-anchor to the C-terminus of
human PH20 and, therefore, the membrane-bound nature of PH20, has
been confirmed using phosphatidylinositol-specific phospholipase C
(PI-PLC) hydrolysis studies (see, e.g., Lin et al., (1994) J. Biol.
Chem. 125:1157-1163 and Example 3, below).
Phosphatidylinositol-specific phospholipase C (PI-PLC) and D
(PI-PLD) hydrolyze the GPI anchor, releasing the PH20 polypeptide
from the cell membrane. The resulting released PH20 polypeptide is,
therefore, soluble. Soluble PH20 can be detected and discriminated
from insoluble, membrane-bound PH20 using methods well known in the
art, including, but not limited to, those using a Triton.RTM. X-114
assay, as described below and in Example 4. In this assay, soluble
PH20 hyaluronidases partition into the aqueous phase of a
Triton.RTM. X-114 solution warmed to 37.degree. C. (Bordier et al.,
(1981) J. Biol. Chem., 256:1604-7) while membrane-anchored PH20
hyaluronidases partition into the detergent rich phase. Thus, in
addition to using algorithms to assess whether a P1120 polypeptide
is naturally GPI-anchored, solubility experiments also can be
performed.
C. Extended Soluble PH20 Polypeptides
[0169] Provided herein are extended soluble P1120 (esPH20)
polypeptides and compositions. Exemplary of the esPH20 polypeptides
provided herein are primate esPH20 polypeptides, including, but not
limited to, human and chimpanzee esPH20 polypeptides. The esPH20
polypeptides provided herein are soluble, i.e. secreted, PH20
proteins that are truncated at the C-terminus but retain at least
one or more amino acid residues located in the GPI-anchor
attachment signal sequence of the corresponding wild-type PH20
polypeptide (e.g. are truncated at amino acid positions 491-500).
EsPH20 polypeptides can be produced from any GPI-anchored PH20
polypeptide by modification of the GPI-anchored PH20 polypeptide,
that is by removal of a portion of the GPI-anchor attachment signal
sequence, provided that the resulting esPH20 polypeptide is
soluble. Solubility, or secretion into the cell culture medium, can
be determined by SDS-PAGE and western blot analysis upon
expression, or alternatively, in a Triton.RTM. X-114 assay, as
described below and in Example 4, when the PH20 polypeptide is
produced by any method known to one of skill in the art, including
recombinant expression and polypeptide synthesis. The esPH20
polypeptides provided herein can be used, for example, as
therapeutic polypeptides, such as a spreading or dispersing agent
in conjunction with other agents, drugs and proteins to enhance
their dispersion and delivery, and to improve the pharmacokinetic
and pharmacodynamic profile of the co-administered agent, drug or
protein.
[0170] The esPH20 polypeptides provided herein contain 1, 2, 3, 4,
5, 6, 7 or more amino acid residues from the GPI-anchor attachment
signal sequence, providing the esPH20 polypeptide is soluble, i.e.,
partitions into the aqueous phase of a Triton.RTM. X-114 solution,
as described below. The extended soluble PH20 polypeptides provided
herein can be produced by making C-terminal truncations to any
naturally GPI-anchored PH20 polypeptide, wherein the resulting
esPH20 polypeptide is soluble and contains 1 or more amino acid
residues from the GPI-anchor attachment signal sequence. One of
skill in the art can determine whether a PH20 polypeptide is
GPI-anchored using methods well known in the art. Such methods
include, but are not limited to, using known algorithms to predict
the presence and location of the GPI-anchor attachment signal
sequence and .omega.-site, and performing solubility analyses
before and after digestion with phosphatidylinositol-specific
phospholipase C (PI-PLC) or D (PI-PLD).
[0171] Exemplary esPH20 polypeptides include, but are not limited
to, esPH20 polypeptides of primates, such as, for example, human
and chimpanzee esPH20 polypeptides. For example, the esPH20
polypeptides provided herein can be made by C-terminal truncation
of any of the mature or precursor polypeptides set forth in SEQ ID
NOS:107, 108, or 197, or allelic or other variations thereof,
including active fragments thereof, wherein the resulting
polypeptide is soluble and retains 1 or more amino acid residues
from the GPI-anchor attachment signal sequence. Allelic variants
and other variants are known to one of skill in the art, and
include polypeptides having 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,
95% or more sequence identity to any of SEQ ID NOS: 107, 108 and
197. The esPH20 polypeptides provided herein can be C-terminally
truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids
compared to the wild type polypeptide, such as a polypeptide with a
sequence set forth in SEQ ID NOS: 107, 108 and 197, provided the
resulting esPH20 polypeptide is soluble and retains 1 or more amino
acid residues from the GPI-anchor attachment signal sequence.
[0172] The extended soluble PH20 polypeptides provided herein
retain hyaluronidase activity. Additionally, the esPH20
polypeptides are neutral active, that is, they retain hyaluronidase
activity at neutral pH. The hyaluronidase activity can be increased
or decreased compared to the wild-type GPI-anchored form of the
PH20. For example, the esPH20 polypeptides provided herein can
exhibit hyaluronidase activity that is 1%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 300%,
400%, 500%, 1000% or more of the hyaluronidase activity exhibited
by the wildtype GPI-anchored form.
[0173] 1. Human esPH20 Polypeptides
[0174] Exemplary of the esPH20 polypeptides provided herein are
human esPH20 polypeptides. The human esPH20 polypeptides provided
herein are soluble and contain 1 or more amino acid residues from
the GPI-anchor attachment signal sequence. Thus, provided herein
are soluble forms of human PH20 that GPI do not completely lack the
GPI-anchor attachment signal sequence.
[0175] Precursor human esPH20 polypeptides provided herein include,
but are not limited to, those having C-terminal truncations to
generate polypeptides containing amino acid 1 to amino acid 491,
492, 493, 494, 495, 496, 497, 498, 499 or 500 of the sequence of
amino acids set forth in SEQ ID NO: 107. When expressed in
mammalian cells, the 35 amino acid N-terminal signal sequence is
cleaved during processing, and the mature form of the protein is
secreted. Thus, the mature human esPH20 polypeptides contain amino
acids 36 to 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 of
SEQ ID NO:107. Hence, mature human esPH20 polypeptides provided
herein include those set forth in SEQ ID NOS: 59-63 and 100-104, or
allelic or other variants thereof.
[0176] The human esPH20 polypeptides provided herein can be
expressed in CHO cells, or alternatively produced in any cell or by
any method known to one of skill in the art, provided they are
soluble and contain at least one amino acid from the GPI-anchor
attachment signal sequence. Soluble human esPH20 polypeptides
produced in CHO cells are those that are secreted into the cell
culture medium. It is understood by one of skill in the art that a
human esPH20 can be partially secreted, that is, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more of the expressed polypeptide is secreted into the
culture medium, provided that the secreted esPH20 is soluble, i.e.,
partitions into the aqueous phase of a Triton.RTM. X-114 solution,
as described below. Human esPH20 polypeptides provided herein that
contain amino acids 1-500, or 36-500, are partially secreted.
Additionally, when expressed in CHO cells, the precursor human
esPH20 polypeptides containing amino acids 1 to 498, 499 or 500, or
the mature human esPH20 polypeptides containing amino acids 36 to
498, 499 or 500, are weakly expressed (see, e.g., Example 3
below).
[0177] Thus, exemplary precursor human esPH20 polypeptides include,
but are not limited to, any having C-terminal truncations to
generate polypeptides containing amino acid 1 to amino acid 491,
492, 493, 494, 495, 496 or 497 of the sequence of amino acids set
forth in SEQ ID NO: 107. When expressed in mammalian cells,
following cleavage of the N-terminal signal peptide during
processing, mature human esPH20 polypeptides contain amino acids 36
to 491, 492, 493, 494, 495, 496 or 497 of SEQ ID NO:107. Hence,
exemplary mature human esPH20 polypeptides provided herein include
those that are 456, 457, 458, 459, 460, 461 or 462 amino acids in
length, such as set forth in any of SEQ ID NOS: 60-63 and 102-104,
or allelic or other variants thereof. Allelic variants and other
variants are known to one of skill in the art, and include
polypeptides having 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95% or
more sequence identity to any of SEQ ID NOS: 107 or 108.
[0178] Also provided herein are amino acid-substituted human esPH20
polypeptides, Amino acid substituted esPH20 polypeptides are human
esPH20 polypeptides that are modified such that they contain amino
acid substitutions, as compared to the human esPH20 polypeptides
provided herein, for example, as set forth in SEQ ID NOS: 60-63 and
102-104. Thus, amino acid-substituted human esPH20 polypeptides are
those having C-terminal truncations. In some examples, the amino
acid substituted human esPH20 polypeptides provided herein have at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with
the sequence of amino acids set forth as amino acids 1 to 491, 492,
493, 494, 495, 496 or 497, or amino acids 36 to 491, 492, 493, 494,
495, 496 or 497, of the sequence of amino acids set forth in SEQ ID
NO: 107. In other examples, the amino acid substituted human esPH20
polypeptides have 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more sequence identity with the sequence of amino acids
set forth as in SEQ ID NOS: 60-63 and 102-104.
[0179] The human esPH20 polypeptides provided herein can exhibit
hyaluronidase activity that is increased or decreased compared to
the wild-type GPI-anchored form of PH20. For example, the human
esPH20 polypeptides provided herein can exhibit hyaluronidase
activity that is 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100%, 110%, 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500%, 1000%
or more of the hyaluronidase activity exhibited by the wildtype
GPI-anchored form. In some examples, human esPH20 polypeptides
exhibit increased hyaluronidase activity compared to the wildtype
GPI-anchored form. The hyaluronidase activity of human esPH20
polypeptides can be increased by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%,
200%, 300%, 400%, 500%, 1000% or more compared to the hyaluronidase
activity of the wildtype GPI-anchored form.
[0180] The human esPH20 polypeptides provided herein exhibit
neutral active hyaluronidase activity, or hyaluronidase activity
when measured at neutral pH, that is increased or decreased
compared to the compared to the wild-type GPI-anchored form of
PH20. For example, the human esPH20 polypeptides provided herein
can exhibit hyaluronidase activity that is 1%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%,
300%, 400%, 500%, 1000% or more of the hyaluronidase activity
exhibited by the wildtype GPI-anchored form. In some examples,
human esPH20 polypeptides exhibit decreased neutral active
hyaluronidase activity compared to the wildtype GPI-anchored form.
The neutral active hyaluronidase activity can be decreased by 1%,
2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or
more compared to the neutral active hyaluronidase activity of the
wildtype GPI-anchored form. In other examples, human esPH20
polypeptides exhibit increased neutral active hyaluronidase
activity compared to the wildtype GPI-anchored form. The neutral
active hyaluronidase activity can be increased by 1%, 2%, 3%, 4%,
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%,
130%, 140%, 150%, 200%, 300%, 400%, 500%, 1000% or more compared to
the neutral active hyaluronidase activity of the wildtype
GPI-anchored form.
[0181] Typically, human esPH20 polypeptides are produced using
protein expression systems that facilitate correct N-glycosylation
to ensure the polypeptide retains activity, since glycosylation is
important for the catalytic activity and stability of these
polypeptides. Exemplary cells useful for recombinant expression of
esPH20 polypeptides include, for example Chinese Hamster Ovary
(CHO) cells (e.g. DG44 CHO or CHO--S cells).
[0182] 2. Other Species esPH20 Polypeptides
[0183] Provided herein are non-human extended soluble PH20
polypeptides. One of skill in art can align the amino acid sequence
of human PH20 with any non-human PH20 polypeptide to identify
positions corresponding to positions 491-500 of the human PH20
polypeptide set forth in SEQ ID NO:107, and at which C-terminal
truncations can be made to produce extended soluble PH20
polypeptides. Additionally, algorithms, such as those described
elsewhere herein, can be used to predict the location of the
GPI-anchor attachment signal sequence. The solubility of the
C-terminally truncated polypeptides can be assessed using methods
well known in the art, including the Triton.RTM. X-114 assays
described below and in Example 4, to determine whether the produced
C-terminally truncated polypeptides are soluble and, therefore,
esPH20 polypeptides.
[0184] Provided herein are extended soluble PH20 polypeptides of
non-human primate species. Exemplary non-human primate GPI-anchored
PH20 polypeptides include, but are not limited to, chimpanzee PH20
(SEQ ID NO:197). Thus, provided herein are chimpanzee esPH20
polypeptides. The esPH20 polypeptides of chimpanzee provided herein
contain C-terminal truncations that correspond to the C-terminal
truncations described above for the human esPH20 polypeptides.
Thus, the chimpanzee esPH20 polypeptides provided herein contain
amino acids corresponding to amino acid residues 1 to 491, 492,
493, 494, 495, 496, 497, 498, 499, 500 or 501 of the sequence of
amino acids set forth in SEQ ID NO: 107.
[0185] The chimpanzee PH20 polypeptides can be aligned to the human
PH20 polypeptide by any method known to those of skill in the art.
Such methods typically maximize matches, and include methods such
as using manual alignments and by using the numerous alignment
programs available (for example, BLASTP) and others known to those
of skill in the art. FIG. 1 provides an alignment of the precursor
polypeptides of human and chimpanzee PH20. Amino acid residues 491
to 500 of the human PH20 (at which the human esPH20 polypeptides
provided herein are truncated compared to the wild-type human PH20
polypeptide) correspond to amino acid residues 491 to 501 of
chimpanzee PH20. Thus, provided herein are chimpanzee esPH20
polypeptides that contain amino acid residues 1 to 491, 492, 493,
494, 495, 496, 497, 498, 499, 500 or 501 of the sequence of amino
acids set forth in SEQ ID NO: 197. When expressed in a mammalian
expression system, the 35 amino acid signal peptide is cleaved
during processing, and the mature form of the protein is secreted.
Thus, the mature chimpanzee esPH20 polypeptides contain amino acids
36 to 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 or 501 of
SEQ ID NO:197.
[0186] Exemplary chimpanzee esPH20 polypeptide are those that
contain amino acids residues 1 to 491, 492, 493, 494, 495, 496, 497
or 498 of the sequence of amino acids set forth in SEQ ID NO:197.
When expressed in a mammalian expression system, the 35 amino acid
signal peptide is cleaved during processing, and the mature form of
the protein is secreted. Thus, the mature chimpanzee esPH20
polypeptides contain amino acids 36 to 491, 492, 493, 494, 495,
496, 497, or 498 of SEQ ID NO:197.
[0187] D. N-Partially Glycosylated PH20 Polypeptides
[0188] Provided herein are N-partially glycosylated hyaluronidases,
including partially deglycosylated PH20 polypeptides, that retain
all or a portion of the hyaluronidase activity of an N-glycosylated
hyaluronidase. Exemplary partially deglycosylated hyaluronidases
include partially deglycosylated PH20 polypeptides from any
species, such as any set forth in any of SEQ ID NOS:107-109,
111-120, 197 and 198, or allelic variants or other variants
thereof. Allelic variants and other variants are known to one of
skill in the art, and include polypeptides having 60%, 70%, 80%,
90%, 91%, 92%, 93%, 94%, 95% or more sequence identity to any of
SEQ ID NOS: NOS:107-109, 111-120, 197 and 198, or truncated forms
thereof. The partially deglycosylated hyaluronidases provided
herein also include hybrid, fusion and chimeric partially
deglycosylated hyaluronidases, and partially deglycosylated
hyaluronidase conjugates.
[0189] The N-partially glycosylated hyaluronidases provided herein
can be produced by digestion with one or more glycosidases. Thus,
although all N-linked glycosylation sites (such as, for example,
those at amino acids N82, N166, N235, N254, N368, and N393 of human
PH20, exemplified in SEQ ID NO:107) can be glycosylated, the extent
of glycosylation is reduced compared to a hyaluronidase that is not
digested with one or more glycosidases. The partially
deglycosylated hyaluronidase polypeptides provided herein,
including partially deglycosylated soluble PH20 polypeptides, can
have 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the level of
glycosylation of a fully glycosylated hyaluronidase. In one
example, 1, 2, 3, 4, 5 or 6 of the N-glycosylation sites
corresponding to amino acids N82, N166, N235, N254, N368, and N393
of SEQ ID NO: 107 are partially deglycosylated, such that they no
longer contain high mannose or complex type glycans, but rather
contain at least an N-acetylglucosamine moiety. In some examples,
1, 2 or 3 of the N-glycosylation sites corresponding to amino acids
N82, N166 and N254 of SEQ ID NO: 107 are deglycosylated, that is,
they do not contain a sugar moiety. In other examples, 3, 4, 5, or
6 of the N-glycosylation sites corresponding to amino acids
N82,
[0190] N166, N235, N254, N368, and N393 of SEQ ID NO: 107 are
glycosylated. Glycosylated amino acid residues minimally contain an
N-acetylglucosamine moiety.
[0191] Also provided herein are N-partially glycosylated
C-terminally truncated PH20 polypeptides. The partially
deglycosylated C-terminally truncated PH20 polypeptides provided
herein lack one or more amino acids from the C-terminus of a full
length PH20 polypeptide, such as any of those set forth in SEQ ID
NOS:107-109, 111-120, 197 and 198. Thus, the N-partially
glycosylated C-terminally truncated PH20 polypeptides provided
herein can be C-terminally truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,
55, 60 or more amino acids compared to the full length wild type
polypeptide, such as a full length wild type polypeptide with a
sequence set forth in SEQ ID NOS:107-109, 111-120, 197 and 198. In
some examples, 3, 4, 5, or 6 of the N-glycosylation sites
corresponding to amino acids N82, N166, N235, N254, N368, and N393
of SEQ ID NO: 107 are glycosylated. Glycosylated amino acid
residues minimally contain an N-acetylglucosamine moiety. In other
examples, 1, 2 or 3 of the N-glycosylation sites corresponding to
amino acids N82, N166, N235, N254, N368, and N393 of SEQ ID NO: 107
are not glycosylated. In further examples, the extent of
glycosylation can be reduced, such that, the partially glycosylated
C-terminally truncated PH20 polypeptides do not contain high
mannose and complex type glycans, rather they contain at least an
N-acetylglucosamine moiety, so long as they retain hyaluronidase
activity. Thus the partially deglycosylated C-terminally truncated
PH20 polypeptides provided herein can have 10%, 20%, 30%, 40%, 50%,
60%, 70% or 80% of the level of glycosylation of a fully
glycosylated C-terminally truncated PH20 polypeptide.
[0192] The partially deglycosylated PH20 polypeptides and
C-terminally truncated PH20 polypeptides provided herein retain
hyaluronidase activity. Additionally, the partially deglycosylated
PH20 polypeptides and C-terminally truncated PH20 polypeptides are
neutral active, that is, they retain hyaluronidase activity at
neutral pH. The hyaluronidase activity can be increased or
decreased compared to the glycosylated full length and C-terminally
truncated PH20 polypeptides. For example, the partially
deglycosylated PH20 polypeptides and C-terminally truncated PH20
polypeptides provided herein can exhibit hyaluronidase activity
that is 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
110%, 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500%, 1000% or more
of the hyaluronidase activity exhibited by the glycosylated full
length and C-terminally truncated PH20 polypeptides.
[0193] Thus, the PH20 polypeptides provided herein can be used as
therapeutic polypeptides, such as to treat hyaluronan-associated
diseases or conditions. The partially deglycosylated PH20
polypeptides and C-terminally truncated PH20 polypeptides also can
be used, for example, in combination therapy.
[0194] 1. PH20 Polypeptides
[0195] Exemplary N-partially glycosylated hyaluronidases provided
herein include partially deglycosylated PH20 polypeptides from any
species, such as any set forth in any of SEQ ID NOS:107-109,
111-120, 197 and 198, or allelic variants or other variants
thereof. Allelic variants and other variants are known to one of
skill in the art, and include polypeptides having 60%, 70%, 80%,
90%, 91%, 92%, 93%, 94%, 95% or more sequence identity to any of
SEQ ID NOS: NOS:107-109, 111-120, 197 and 198, or truncated forms
thereof. In some examples, 3, 4, 5, or 6 of the N-glycosylation
sites corresponding to amino acids N82, N166, N235, N254, N368, and
N393 of SEQ ID NO: 107 are glycosylated. In other examples, 1, 2,
or 3 of the N-glycosylation sites corresponding to amino acids N82,
N166 and N254 of SEQ ID NO: 107 are not glycosylated. In some
examples, 1, 2, 3, 4, 5, or 6 of the N-glycosylation sites
corresponding to amino acids N82, N166, N235, N254, N368, and N393
of SEQ ID NO: 107 minimally contain an N-acetylglucosamine
moiety.
[0196] The partially deglycosylated hyaluronidases provided herein
can be produced by digestion with one or more glycosidases. Thus,
although all N-linked glycosylation sites (such as, for example,
those at amino acids N82, N166, N235, N254, N368, and N393 of human
PH20, exemplified in SEQ ID NO:107) can be glycosylated, the extent
of glycosylation is reduced compared to a hyaluronidase that is not
digested with one or more glycosidases. In particular, partially
glycosylated hylaruonidases retain at least an N-acetylglucosamine
moiety at each of the N-linked glycosylation sites. Partially
glycosylated hyaluronidases can be glycosylated at 3, 4, 5, or 6 of
the N-glycosylation sites corresponding to amino acids N82, N166,
N235, N254, N368, and N393 of SEQ ID NO: 107. In some examples, the
hyaluronidases are deglycosylated at 1, 2, or 3 of the
N-glycosylation sites corresponding to amino acid residues N82,
N166, N235, N254, N368, and N393 of SEQ ID NO: 107. The partially
deglycosylated PH20 polypeptides provided herein can have 10%, 20%,
30%, 40%, 50%, 60%, 70% or 80% of the level of glycosylation of a
fully glycosylated hyaluronidase.
[0197] Glycosidases, or glycoside hydrolases, are enzymes that
catalyze the hydrolysis of the glycosidic linkage to generate two
smaller sugars. As shown in FIG. 2, the major types of N-glycans in
vertebrates include high mannose glycans, hybrid glycans and
complex glycans. There are several glycosidases that result in only
partial protein deglycosylation, including: EndoF1, which cleaves
high mannose and hybrid type glycans; EndoF2, which cleaves
biantennary complex type glycans; EndoF3, which cleaves biantennary
and more branched complex glycans; and EndoH, which cleaves high
mannose and hybrid type glycans (FIG. 3). Treatment of a
hyaluronidase, such as a soluble hyaluronidase, such as a soluble
PH20, with one or all of these glycosidases results can result in
only partial deglycosylation and, therefore, retention of
hyaluronidase activity.
[0198] For example, treatment of rHuPH20 with one or all of these
glycosidases results in partial deglycosylation. These partially
deglycosylated rHuPH20 polypeptides exhibit hyaluronidase enzymatic
activity that is comparable to the fully glycosylated polypeptides
(see e.g. Example 7). In contrast, treatment of rHuPH20 (SEQ ID
NO:122) with PNGaseF, a glycosidase that cleaves all N-glycans (see
FIG. 3), or treatment with the GlcNAc phosphotransferase (GPT)
inhibitor tunicamycin, results in complete deglcosylation of all
N-glycans and thereby renders PH20 enzymatically inactive (see
e.g., Examples 7-8, below).
[0199] The partially deglycosylated hyaluronidase polypeptides
provided herein, including partially deglycosylated soluble PH20
polypeptides, can have 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of
the level of glycosylation of a fully glycosylated hyaluronidase.
Typically, the partially deglyclosylated hyaluronidases, including
partially deglycosylated soluble PH20 polypeptides, provided herein
exhibit hyaluronidase activity that is 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 300%,
400%, 500%, 1000% or more of the hyaluronidase activity exhibited
by the fully glycosylated hyaluronidase.
[0200] The partially deglycosylated hyaluronidases provided herein
also include hybrid, fusion and chimeric partially deglycosylated
hyaluronidases, and partially deglycosylated hyaluronidase
conjugates.
[0201] 2. C-terminally Truncated PH20 Polypeptides
[0202] Exemplary of the N-partially glycosylated, or partially
deglycosylated, PH20 peptides provided herein are C-terminally
truncated PH20 polypeptides. The partially glycosylated
C-terminally truncated PH20 polypeptides provided herein lack one
or more amino acids from the C-terminus of the full length PH20
polypeptide as set forth in SEQ ID NOS:107-109, 111-120, 197 and
198. Thus, the partially glycosylated C-terminally truncated PH20
polypeptides provided herein can be C-terminally truncated by 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
25, 30, 35, 40, 45, 50, 55, 60 or more amino acids compared to the
full length wild type polypeptide, such as a full length wild type
polypeptide with a sequence set forth in SEQ ID NOS:107-109,
111-120, 197 and 198. In some examples, 3, 4, 5, or 6 of the
N-glycosylation sites, corresponding to amino acids N82, N166,
N235, N254, N368, and N393 of SEQ ID NO: 107, are glycosylated. In
other examples, 1, 2, or 3 of the N-- glycosylation sites,
corresponding to amino acids N82, N166 and N254 of SEQ ID NO: 107,
are not glycosylated.
[0203] The partially deglycosylated C-terminally truncated PH20
polypeptides provided herein can be produced by digestion with one
or more glycosidases. Although all N-linked glycosylation sites
(such as, for example, those at amino acids N82, N166, N235, N254,
N368, and N393 of human PH20, exemplified in SEQ ID NO:107) can be
glycosylated, the extent of glycosylation is reduced compared to a
hyaluronidase that is not digested with one or more glycosidases.
Thus, the partially deglycosylated C-terminally truncated PH20
polypeptides provided herein can have 10%, 20%, 30%, 40%, 50%, 60%,
70% or 80% of the level of glycosylation of a fully glycosylated
hyaluronidase. In particular, N-partially glycosylated
hylaruonidases retain at least an N-acetylglucosamine moiety at
each of the N-linked glycosylation sites. In some examples, 1, 2,
3, 4, 5, or 6 of the N-glycosylation sites corresponding to amino
acids N82, N166, N235, N254, N368, and N393 of SEQ ID NO: 107
minimally contain an N-acetylglucosamine moiety. In other examples,
3, 4, 5, or 6 of the N-glycosylation sites corresponding to amino
acids N82, N166, N235, N254, N368, and N393 of SEQ ID NO: 107 are
glycosylated at the level of glycosylation of a fully glycosylated
hyaluronidase at each of the 3, 4, 5, or 6 N-glycosylation sites.
In further examples, 1, 2, or 3 of the N-glycosylation sites
corresponding to amino acids N82, N166, N235, N254, N368, and N393
of SEQ ID NO: 107 are fully deglycosylated. In these examples,
typically, amino acids N82, N166 or N254 are fully
deglycosylated.
[0204] Exemplary N-partially glycosylated C-terminally truncated
PH20 polypeptides are from any species, such as any set forth in
any of SEQ ID NOS: 107-109, 111-120, 197 and 198, or allelic
variants or other variants thereof. Allelic variants and other
variants are known to one of skill in the art, and include
polypeptides having 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95% or
more sequence identity to any of SEQ ID NOS: NOS:107-120, 197 and
198, or truncated forms thereof. The N-partially glycosylated
C-terminally truncated PH20 polypeptides provided herein also
include hybrid, fusion and chimeric PH20 polypeptides, and PH20
conjugates. For example, the partially deglycosylated C-terminally
truncated PH20 polypeptides provided herein can be conjugated to a
polymer, such as dextran, a polyethylene glycol (pegylation (PEG))
or sialyl moiety, or other such polymers, such as natural or sugar
polymers. In other examples, the N-partially glycosylated
C-terminally truncated PH20 polypeptide is linked or fused to a
domain such as an Fc domain from an IgG immunoglobulin.
[0205] Included amongst the glycosylated or partially glycosylated
C-terminally truncated polypeptides provided herein are those that
are truncated at the C-terminus by 2 amino acids up to 44 amino
acids compared to the wild type PH20 set forth in SEQ ID NO:107
(precursor polypeptide) or 108 (mature polypeptide), or allelic or
species variants thereof. Thus, C-terminally truncated PH20
polypeptides include any having C-terminal truncations to generate
polypeptides containing amino acid 1 to amino acid 450, 451, 452,
453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,
466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,
479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491,
492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504,
505, 506 or 507 of the sequence of amino acids set forth in SEQ ID
NO: 107, or corresponding positions in an allelic or species
variant thereof, with 2, 3, 4, 5, or 6 of the N-glycosylation
sites, corresponding to amino acids N82, N166, N235, N254, N368,
and N393 of SEQ ID NO: 107, glycosylated. When expressed in
mammalian cells, the 35 amino acid N-terminal signal sequence is
cleaved during processing, and the mature form of the protein is
secreted. Thus, provided herein are mature C-terminally truncated
PH20 polypeptides that contain amino acids 36 to 450, 451, 452,
453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,
466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,
479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491,
492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504,
505, 506 or 507 of the sequence of amino acids set forth in SEQ ID
NO: 107 or corresponding positions in an allelic or species variant
thereof, with 3, 4, 5, or 6 of the N-glycosylation sites,
corresponding to amino acids N82, N166, N235, N254, N368, and N393
of SEQ ID NO: 107, glycosylated.
[0206] Table 2 provides non-limiting examples of exemplary
C-terminally truncated PH20 polypeptides that can be glycosylated
or partially deglycosylated. In Table 2 below, the length (in amino
acids) of the precursor and mature polypeptides, and the sequence
identifier (SEQ ID NO) in which exemplary amino acid sequences of
the precursor and mature polypeptides of the C-terminally truncated
PH20 proteins are set forth, are provided. The wild-type PH20
polypeptide also is included in Table 2 for comparison.
TABLE-US-00002 TABLE 2 Exemplary C-terminally truncated PH20
polypeptides Precursor Mature Precursor SEQ ID Mature SEQ ID
Polypeptide (amino acids) NO (amino acids) NO SPAM1-VASL 509 1 474
108 SPAM1-SSVA 507 3 472 55 SPAM1-ISSV 506 45 471 97 SPAM1-IISS 505
4 470 56 SPAM1-LIIS 504 46 469 98 SPAM1-FLII 503 5 468 57
SPAM1-LFLI 502 47 467 99 SPAM1-ILFL 501 6 466 58 SPAM1-SILF 500 48
465 100 SPAM1-VSIL 499 7 464 59 SPAM1-IVSI 498 49 463 101
SPAM1-FIVS 497 8 462 60 SPAM1-MFIV 496 50 461 102 SPAM1-TMFI 495 9
460 61 SPAM1-ATMF 494 51 459 103 SPAM1-SATM 493 10 458 62
SPAM1-LSAT 492 52 457 104 SPAM1-TLSA 491 11 456 63 SPAM1-PSTL 489
12 454 64 SPAM1-STLS 490 13 455 65 SPAM1-SPST 488 53 453 105
SPAM1-ASPS 487 14 452 66 SPAM1-NASP 486 54 451 106 SPAM1-YNAS 485
15 450 67 SPAM1-FYNA 484 16 449 68 SPAM1-IFYN 483 17 448 69
SPAM1-QIFY 482 18 447 70 SPAM1-PQIF 481 19 446 71 SPAM1-EPQI 480 20
445 72 SPAM1-EEPQ 479 21 444 73 SPAM1-TEEP 478 22 443 74 SPAM1-ETEE
477 23 442 75 SPAM1-METE 476 24 441 76 SPAM1-PMET 475 25 440 77
SPAM1-PPME 474 26 439 78 SPAM1-KPPM 473 27 438 79 SPAM1-LKPP 472 28
437 80 SPAM1-FLKP 471 29 436 81 SPAM1-AFLK 470 30 435 82 SPAM1-DAFL
469 31 434 83 SPAM1-IDAF 468 32 433 84 SPAM1-CIDA 467 33 432 85
SPAM1-VCID 466 34 431 86 SPAM1-GVCI 465 35 430 87 SPAM1-GDVC 464 36
429 88 SPAM1-IADG 462 37 427 89 SPAM1-VCIA 460 38 425 90 SPAM1-VDVC
458 39 423 91 SPAM1-DAVD 456 40 421 92 SPAM1-DTDA 454 41 419 93
SPAM1-VKDT 452 42 417 94 SPAM1-ADVK 450 43 415 95
[0207] The N-glycosylated and partially deglycosylated C-terminal
truncated PH20 polypeptides provided herein include those that are
soluble, i.e. partition into the aqueous phase of a Triton.RTM.
X-114 solution, and those that are insoluble, i.e. partition into
the detergent phase of a Triton.RTM. X-114 solution. The partially
deglycosylated C-terminally truncated PH20 polypeptides provided
herein can have 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the
level of glycosylation of a fully glycosylated hyaluronidase.
Alternatively, the partially deglycosylated C-terminal truncated
PH20 polypeptides can have 1, 2 or 3 of the N-glycosylation sites,
corresponding to amino acids N82, N166 and N254 of SEQ ID NO: 107,
that are not glycosylated. Minimally, to be glycosylated, an
N-glycosylation site contains at least an N-acetylglucosamine
moiety.
[0208] In some examples, the N-partially glycosylated C-terminally
truncated polypeptides provided herein are soluble, i.e., are not
GPI-anchored. This can be assessed, for example, using a
Triton.RTM. X-114 assay following incubation with PI-PLC or PI-PLD,
as described below and in Example 4. For example, PH20 polypeptides
that are C-terminally truncated at or 5' to the amino acid position
corresponding to amino acid residue position 490 of the PH20
polypeptide set forth in SEQ ID NO:107 typically are soluble when
expressed in a mammalian expression system (see, e.g. Example 3).
These polypeptides are soluble by virtue of the fact that they
completely lack the GPI-anchor attachment signal sequence. In other
examples, the partially glycosylated C-terminally truncated
polypeptides provided herein are insoluble and membrane-bound when
expressed in a mammalian expression system. For example, PH20
polypeptides that are C-terminally truncated at or 3' of the amino
acid position corresponding to amino acid position 500 of the PH20
polypeptide set forth in SEQ ID NO:107 typically are insoluble when
expressed in a mammalian expression system (see, e.g. Example 3).
The C-terminally truncated polypeptides provided herein can be
partially glycosylated in that 3, 4, 5, or 6 of the N-glycosylation
sites corresponding to amino acids N82, N166, N235, N254, N368, and
N393 of SEQ ID NO: 107 are glycosylated.
[0209] Soluble partially glycosylated C-terminal truncated PH20
polypeptides provided herein include those that are truncated but
retain at least one or more amino acid residues located in the
GPI-anchor attachment signal, and those that completely lack the
GPI-anchor attachment signal sequence and the co-site. Thus,
instead of having a GPI-anchor covalently attached to the
C-terminus of the protein in the ER and being anchored to the
extracellular leaflet of the plasma membrane, these polypeptides
are secreted. These C-terminal truncated soluble PH20 polypeptides
can be partially glycosylated such that 3, 4, 5, or 6 of the
N-glycosylation sites are glycosylated. Exemplary soluble
C-terminally truncated PH20 polypeptides that lack the GPI-anchor
attachment signal sequence are from any species, such as any set
forth in any of SEQ ID NOS: 107-109, 111-120, 197 and 198, or
allelic variants or other variants thereof. These partially
glycosylated soluble C-terminal truncated PH20 polypeptides have
C-terminal truncations to generate polypeptides containing amino
acids 1 to 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460,
461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473,
474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,
487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or
500 of the sequence of amino acids set forth in SEQ ID NO: 107.
Upon cleavage of the N-terminal signal sequence following
expression in mammalian cells, the mature partially glycosylated
soluble C-terminal truncated PH20 polypeptides polypeptides contain
amino acids 36 to 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,
460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,
473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,
486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,
499 or 500 of the sequence of amino acids set forth in SEQ ID NO:
107. In some examples, the C-terminally GPI-anchor signal sequence
truncated soluble PH20 polypeptides are partially glycosylated,
containing, for example, at least an N-acetylglucosamine at 3, 4,
5, or 6 of the N-glycosylation sites corresponding to amino acids
N82, N166, N235, N254, N368, and N393 of SEQ ID NO: 107. In other
examples, the C-terminally GPI-anchor signal sequence truncated
soluble PI-120 polypeptides have 10%, 20%, 30%, 40%, 50%, 60%, 70%
or 80% of the level of glycosylation of a fully glycosylated
hyaluronidase.
[0210] Partially deglycosylated C-terminal truncated PH20
polypeptides that retain at least one amino acid in the GPI-anchor
attachment signal sequence provided herein are partially
deglycosylated extended soluble PH20 polypeptides. In some
examples, the partially deglycosylated C-terminal truncated PH20
polypeptides are not glycosylated at 1, 2 or 3 of the
N-glycosylation sites corresponding to amino acids N82, N166 and
N254 of SEQ ID NO: 107. These partially deglycosylated extended
soluble PH20 polypeptides contain amino acids 1 to 491, 492, 493,
494, 495, 496, 497, 498, 499 or 500 of the sequence of amino acids
set forth in SEQ ID NO: 107. When expressed in mammalian cells, the
35 amino acid N-terminal signal sequence is cleaved during
processing, and the mature form of the protein is secreted. Thus,
the mature form of the partially deglycosylated esPH20 polypeptides
contain amino acids 36 to 491, 492, 493, 494, 495, 496, 497, 498,
499 or 500 of the sequence of amino acids set forth in SEQ ID NO:
107. Mature human forms of partially glycosylated esPH20
polypeptides provided herein include those set forth in SEQ ID NOS:
59-63 and 100-104 containing at least an N-acetylglucosamine at 3,
4, 5 or 6 of the N-glycosylation sites corresponding to amino acids
N82, N166, N235, N254, N368, and N393 of SEQ ID NO: 107. In some
examples, the extent of glycosylation is reduced by treatment with
a endoglycosidase. Thus, the partially deglycosylated C-terminally
truncated PH20 polypeptides that contain at least one amino acid in
the GPI-anchor attachment signal sequence provided herein can have
10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the level of
glycosylation of a fully glycosylated hyaluronidase.
[0211] Also provided herein are partially deglycosylated
C-terminally truncated PH20 polypeptides that are not soluble, that
is, they are attached to the cell membrane and therefore not
secreted into the media upon expression. The C-terminal truncated
PH20 polypeptides that are not soluble can be partially
deglycosylated as long as they retain hyaluronidase activity.
Exemplary partially glycosylated mature C-terminally truncated PH20
polypeptides that are not soluble are those that contain amino
acids corresponding to amino acid positions 36 to 501, 502, 503,
504, 505, 506 or 507 of SEQ ID NO:107. Hence, partially
glycosylated C-terminally truncated PH20 polypeptides provided
herein that are not soluble include those that are 466, 467, 468,
469, 470, 471 or 472 amino acids in length, such as those set forth
in SEQ ID NOS: 55-58 and 97-99, that retain at least 10%, 20%, 30%,
40%, 50%, 60%, 70% or 80% of the level of glycosylation of a fully
glycosylated hyaluronidase. In some examples, 3, 4, 5, or 6 of the
N-glycosylation sites corresponding to amino acids N82, N166, N235,
N254, N368, and N393 of SEQ ID NO: 107 are glycosylated. In other
examples, the 1, 2, 3, 4, 5 or 6 of the N-glycosylation sites,
corresponding to amino acids N82, N166, N235, N254, N368, and N393
of SEQ ID NO: 107, contain at least an N-acetylglucosamine
moiety.
[0212] The partially glycosylated C-terminally truncated
polypeptides provided herein can exhibit hyaluronidase activity
that is increased or decreased compared to the wild-type
GPI-anchored form of the PH20. Additionally, the partially
deglycosylated C-terminally truncated PH20 polypeptides are neutral
active, that is, they retain hyaluronidase activity at neutral pH.
For example, the C-terminal truncated PH20 polypeptides provided
herein can exhibit hyaluronidase activity that is 1%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%,
150%, 200%, 300%, 400%, 500%, 1000% or more of the hyaluronidase
activity exhibited by the wildtype GPI-anchored form. In some
examples, partially glycosylated C-terminal truncated PH20
polypeptides exhibit increased hyaluronidase activity compared to
the wildtype GPI-anchored form.
[0213] The C-terminal truncated PH20 polypeptides provided herein
may also be N-glycosylated. The N-glycosylated and N-partially
glycosylated hyaluronidases provided herein also include hybrid,
fusion and chimeric N-glycosyated and partially deglycosylated
hyaluronidases, and N-glycosylated and partially deglycosylated
hyaluronidase conjugates.
[0214] 3. Additional Modifications
[0215] The PH20 polypeptides included herein, including human
esPH20 polypeptides, N-glycosylated and N-partially glycosylated
C-terminally truncated PH20 polypeptides and partially glycosylated
PH20 polypeptides, also include those that contain chemical or
posttranslational modifications and those that do not contain
chemical or posttranslational modifications. Such modifications
include, but are not limited to, pegylation, sialation,
albumination, glycosylation, farnysylation, carboxylation,
hydroxylation, phosphorylation, and other polypeptide modifications
known in the art. Thus, C-terminally truncated PH20 polypeptides,
including esPH20 polypeptides, provided herein can contain other
modifications that are or are not in the primary sequence of the
polypeptide, including, but not limited to, of a carbohydrate
moiety, a polyethylene glycol (PEG) moiety, a silation moiety, an
Fc domain from immunoglobulin G, or any other domain or moiety. For
example, such additional modifications can be made to increase the
stability or serum half-life of the protein. The C-terminally
truncated PH20 polypeptides, including esPH20 polypeptides,
provided herein can be conjugated or fused to any moiety using any
method known in the art, including chemical and recombinant
methods, providing the resulting polypeptide retains hyaluronidase
activity.
[0216] Decreased Immunogenicity
[0217] The PH20 polypeptides provided herein, including the human
esPH20 polypeptides, can be made to have decreased immunogenicity.
Decreased immunogenicity can be effected by sequence changes that
elimiminate antigenic epitopes from the polypeptide or by altering
post-translational modifications. For example, altering the
glycosylation of the peptide is contemplated, so long as the
polypeptides minimally contain at least N-acetylglucosamine at
amino acid residues N235, N368 and N393 of SEQ ID NO:107.
[0218] For example, the PH20 polypeptides can be modified such that
they lack fucose, particularly bifucosylation. In particular, the
PH20 polypeptides provided herein are not bifucosylated. This can
be achieved by expressing and producing the PH20 polypeptide in a
host cells, typically insect host cells, that do not effect
bifucosylation. Fucose is a deoxyhexose that is present in a wide
variety of organisms, including mammals, insects and plants.
Fucosylated glycans are synthesized by fucosyl-tranferases. See,
e.g., Ma et al., Glycobiology, 15(2):158R-184R, (2006); Nakayama et
al., J. Biol. Chem., 276:16100-16106 (2001); and Sturla et al.,
Glycobiology, 15(10):924-935 (2005). In humans, fucose frequently
exists as a terminal modification to glycan structures, and the
presence of fucose .alpha.-1,6-linked to N-acetylglucosamine has
been shown to be important in glycoprotein processing and
recognition. In insects, N-glycan core structures exhibit
bifucosylation with .alpha.-1,6- and .alpha.-1,3-linkages. Insect
cell core fucosylation with .alpha.-1,3-linkages generates a
carbohydrate epitope that is immunogenic in humans (see, e.g., US
patent application No. 20070067855). For example, PH20 polypeptides
provided herein, including esPH20 polypeptides, can be generated in
host cells that are incapable of bifucosylating the polypeptide.
Thus, while insect cells or other cells that bifucosylate can be
used for expression of the polypeptides, typically mammalian cells,
such as CHO cells, are used.
[0219] In some examples, defucosylated, or fucose-deficient PH20
polypeptides can be generated in insect cells with modified
glycosylation pathways, through the use of baculovirus expression
vectors containing eukaryotic oligosaccharide processing genes,
thereby creating "mammalianized" insect cell expression systems
(see, e.g., U.S. Pat. No. 6,461,863). Alternatively, antigenicity
can be eliminated by expression of PH20 polypeptides in insect
cells lacking .alpha.1,3-fucosyltransferase (FT3) (see, e.g., US
patent application No. 20070067855). In other examples,
defucosylated or fucose-deficient PH20 polypeptides can be
generated, for example, in cell lines that produce defucosylated
proteins, including Lec 13 CHO cells deficient in protein
fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545
(1986); U.S. Pat. Appl. No. 2003/0157108; and WO 2004/056312), and
knockout cell lines, such as alpha-1,6-fucosyltransferase gene,
FUT8, knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87:
614 (2004)).
[0220] Conjugation to Polymers
[0221] In some examples, the esPH20 polypeptides and other
C-terminally truncated PH20 polypeptides, including partially
glycosylated PH20 polypeptides, provided herein are conjugated to
polymers. Exemplary polymers that can be conjugated to the PH20
polypeptides, include natural and synthetic homopolymers, such as
polyols (i.e. poly-OH), polyamines (i.e. poly-NH.sub.2) and
polycarboxylic acids (i.e. poly-COOH), and further heteropolymers
i.e. polymers comprising one or more different coupling groups e.g.
a hydroxyl group and amine groups. Examples of suitable polymeric
molecules include polymeric molecules selected from among
polyalkylene oxides (PAO), such as polyalkylene glycols (PAG),
including polyethylene glycols (PEG), methoxypolyethylene glycols
(mPEG) and polypropylene glycols, PEG-glycidyl ethers (Epox-PEG),
PEG-oxycarbonylimidazole (CDI-PEG), branched polyethylene glycols
(PEGS), polyvinyl alcohol (PVA), polycarboxylates,
polyvinylpyrrolidone, poly-D, L-amino acids, polyethylene-co-maleic
acid anhydride, polystyrene-co-maleic acid anhydride, dextrans
including carboxymethyl-dextrans, heparin, homologous albumin,
celluloses, including methylcellulose, carboxymethylcellulose,
ethylcellulose, hydroxyethylcellulose, carboxyethylcellulose and
hydroxypropylcellulose, hydrolysates of chitosan, starches such as
hydroxyethyl-starches and hydroxypropyl-starches, glycogen,
agaroses and derivatives thereof, guar gum, pullulan, inulin,
xanthan gum, carrageenan, pectin, alginic acid hydrolysates and
bio-polymers.
[0222] Typically, the polymers are polyalkylene oxides (PAO), such
as polyethylene oxides, such as PEG, typically mPEG, which, in
comparison to polysaccharides such as dextran, pullulan and the
like, have few reactive groups capable of cross-linking.
[0223] Typically, the polymers are non-toxic polymeric molecules
such as (methoxy)polyethylene glycol (mPEG) which can be covalently
conjugated to the esPH20 polypeptides and other C-terminally
truncated PH20 polypeptides (e.g., to attachment groups on the
protein surface) using a relatively simple chemistry.
[0224] Suitable polymeric molecules for attachment to the esPH20
polypeptides and other C-terminally truncated PH20 polypeptides
include, but are not limited to, polyethylene glycol (PEG) and PEG
derivatives such as methoxy-polyethylene glycols (mPEG),
PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG),
branched PEGs, and polyethylene oxide (PEO) (see e.g. Roberts et
al., Advanced Drug Delivery Review 2002, 54: 459-476; Harris and
Zalipsky (eds.) "Poly(ethylene glycol), Chemistry and Biological
Applications" ACS Symposium Series 680, 1997; Mehvar et al., J.
Pharm. Pharmaceut. Sci., 3(1):125-136, 2000; Harris and Chess
(2003) Nat Rev Drug Discov. 2(3):214-21; and Tsubery, J Biol. Chem.
279(37):38118-24, 2004). The polymeric molecule can be of a
molecular weight typically ranging from about 3 kDa to about 60
kDa. In some embodiments the polymeric molecule that is conjugated
to a PH20 polypeptide provided herein has a molecular weight of 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more than 60 kDa.
[0225] Various methods of modifying polypeptides by covalently
attaching (conjugating) a PEG or PEG derivative (i.e. "PEGylation")
are known in the art (see e.g., U.S. 2006/0104968; U.S. Pat. No.
5,672,662; U.S. Pat. No. 6,737,505; and U.S. 2004/0235734).
Techniques for PEGylation include, but are not limited to,
specialized linkers and coupling chemistries (see e.g., Harris,
Adv. Drug Deliv. Rev. 54:459-476, 2002), attachment of multiple PEG
moieties to a single conjugation site (such as via use of branched
PEGs; see e.g., Veronese et al., Bioorg. Med. Chem. Lett.
12:177-180, 2002), site-specific PEGylation and/or mono-PEGylation
(see e.g., Chapman et al., Nature Biotech. 17:780-783, 1999), and
site-directed enzymatic PEGylation (see e.g., Sato, Adv. Drug
Deliv. Rev., 54:487-504, 2002) (see, also, for example, Lu and
Felix (1994) Int. J. Peptide Protein Res. 43:127-138; Lu and Felix
(1993) Peptide Res.
[0226] 6:142-6, 1993; Felix et al. (1995) Int. J. Peptide Res.
46:253-64; Benhar et al. (1994) J. Biol. Chem. 269:13398-404;
Brumeanu et al. (1995) J Immunol. 154:3088-95; see also, Caliceti
et al. (2003) Adv. Drug Deliv. Rev. 55(10):1261-77 and Molineux
(2003) Pharmacotherapy 23 (8 Pt 2):3S-8S). Methods and techniques
described in the art can produce proteins having 1, 2, 3, 4, 5, 6,
7, 8, 9, 10 or more than 10 PEG or PEG derivatives attached to a
single protein molecule (see e.g., U.S. 2006/0104968).
[0227] Numerous reagents for PEGylation have been described in the
art. Such reagents include, but are not limited to,
N-hydroxysuccinimidyl (NHS) activated PEG, succinimidyl mPEG,
mPEG2-N-hydroxysuccinimide, mPEG succinimidyl
alpha-methylbutanoate, mPEG succinimidyl propionate, mPEG
succinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acid
succinimidyl ester, homobifunctional PEG-succinimidyl propionate,
homobifunctional PEG propionaldehyde, homobifunctional PEG
butyraldehyde, PEG maleimide, PEG hydrazide,
p-nitrophenyl-carbonate PEG, mPEG-benzotriazole carbonate,
propionaldehyde PEG, mPEG butryaldehyde, branched mPEG2
butyraldehyde, mPEG acetyl, mPEG piperidone, mPEG methylketone,
mPEG "linkerless" maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG
orthopyridylthioester, mPEG orthopyridyl disulfide, Fmoc-PEG-NHS,
Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylate PEG-NHS, fluorescein
PEG-NHS, and biotin PEG-NHS (see e.g., Monfardini et al.,
Bioconjugate Chem. 6:62-69, 1995; Veronese et al., J. Bioactive
Compatible Polymers 12:197-207, 1997; U.S. Pat. No. 5,672,662; U.S.
Pat. No. 5,932,462; U.S. Pat. No. 6,495,659; U.S. Pat. No.
6,737,505; U.S. Pat. No. 4,002,531; U.S. Pat. No. 4,179,337; U.S.
Pat. No. 5,122,614; U.S. Pat. No. 5,183,550; U.S. Pat. No.
5,324,844; U.S. Pat. No. 5,446,090; U.S. Pat. No. 5,612,460; U.S.
Pat. No. 5,643,575; U.S. Pat. No. 5,766,581; U.S. Pat. No.
5,795,569; U.S. Pat. No. 5,808,096; U.S. Pat. No. 5,900,461; U.S.
Pat. No. 5,919,455; U.S. Pat. No. 5,985,263; U.S. Pat. No.
5,990,237; U.S. Pat. No. 6,113,906; U.S. Pat. No. 6,214,966; U.S.
Pat. No. 6,258,351; U.S. Pat. No. 6,340,742; U.S. Pat. No.
6,413,507; U.S. Pat. No. 6,420,339; U.S. Pat. No. 6,437,025; U.S.
Pat. No. 6,448,369; U.S. Pat. No. 6,461,802; U.S. Pat. No.
6,828,401; U.S. Pat. No. 6,858,736; U.S. 2001/0021763; U.S.
2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430; U.S.
2002/0072573; U.S. 2002/0156047; U.S. 2003/0114647; U.S.
2003/0143596; U.S. 2003/0158333; U.S. 2003/0220447; U.S.
2004/0013637; US 2004/0235734; U.S. 2005/000360; U.S. 2005/0114037;
U.S. 2005/0171328; U.S. 2005/0209416; EP 01064951; EP 0822199; WO
00176640; WO 0002017; WO 0249673; WO 9428024; and WO 0187925).
[0228] Other Modifications
[0229] The esPH20 polypeptides and other C-terminally truncated
PH20 polypeptides provided herein also include fusions and
conjugates thereof
E. Methods of Producing Nucleic Acids Encoding Extended Soluble
PH20 and other Soluble PH20 Hyaluronidases, and Polypeptides
Thereof
[0230] Polypeptides of extended soluble PH20, C-terminal truncated
PH20 hyaluronidases, and partially glycosylated PH20 hyaluronidases
set forth herein, and nucleic acid molecules encoding such
polypeptides, can be obtained by methods well known in the art for
recombinant protein expression and protein purification. For
example, the DNA can be obtained from cloned DNA (e.g. from a DNA
library), by chemical synthesis, by cDNA cloning, or by the cloning
of genomic DNA or fragments thereof, purified from the desired
cell. When the polypeptides are produced by recombinant means, any
method known to those of skill in the art for identification of
nucleic acids that encode desired genes can be used. Any method
available in the art can be used to obtain a full length (i.e.
encompassing the entire coding region) cDNA or genomic DNA encoding
a desired PH20 enzyme, such as from a cell or tissue source.
Modified or variant, including truncated forms such as provided
herein, can be engineered from a wildtype polypeptide using
standard recombinant DNA methods.
[0231] Polypeptides can be cloned or isolated using any available
methods known in the art for cloning and isolating nucleic acid
molecules. Such methods include PCR amplification of nucleic acids
and screening of libraries, including nucleic acid hybridization
screening, antibody-based screening and activity-based
screening.
[0232] Methods for amplification of nucleic acids can be used to
isolate nucleic acid molecules encoding a desired polypeptide,
including for example, polymerase chain reaction (PCR) methods. PCR
can be carried out using any known methods or procedures in the
art. Exemplary of such methods include use of a Perkin-Elmer Cetus
thermal cycler and Taq polymerase (Gene Amp). A nucleic acid
containing material can be used as a starting material from which a
desired polypeptide-encoding nucleic acid molecule can be isolated.
For example, DNA and mRNA preparations, cell extracts, tissue
extracts from an appropriate source (e.g. testis, prostate,
breast), fluid samples (e.g. blood, serum, saliva), samples from
healthy and/or diseased subjects can be used in amplification
methods. The source can be from any eukaryotic species including,
but not limited to, vertebrate, mammalian, human, porcine, bovine,
feline, avian, equine, canine, and other primate sources. Nucleic
acid libraries also can be used as a source of starting material.
Primers can be designed to amplify a desired polypeptide. For
example, primers can be designed based on expressed sequences from
which a desired polypeptide is generated. Primers can be designed
based on back-translation of a polypeptide amino acid sequence. If
desired, degenerate primers can be used for amplification.
Oligonucleotide primers that hybridize to sequences at the 3' and
5' termini of the desired sequence can be uses as primers to
amplify by PCR sequences from a nucleic acid sample. Primers can be
used to amplify the entire full-length PH20, or a truncated
sequence thereof, such as a nucleic acid encoding any of the
soluble PI-120 polypeptides provided herein. Nucleic acid molecules
generated by amplification can be sequenced and confirmed to encode
a desired polypeptide.
[0233] Additional nucleotide sequences can be joined to a
polypeptide-encoding nucleic acid molecule, including linker
sequences containing restriction endonuclease sites for the purpose
of cloning the synthetic gene into a vector, for example, a protein
expression vector or a vector designed for the amplification of the
core protein coding DNA sequences. Furthermore, additional
nucleotide sequences specifying functional DNA elements can be
operatively linked to a polypeptide-encoding nucleic acid molecule.
Examples of such sequences include, but are not limited to,
promoter sequences designed to facilitate intracellular protein
expression, and secretion sequences, for example heterologous
signal sequences, designed to facilitate protein secretion. Such
sequences are known to those of skill in the art. For example,
exemplary heterologous signal sequences include, but are not
limited to, human and mouse kappa IgG heterologous signal sequences
set forth in SEQ ID NOS:144 and 145, respectively. Additional
nucleotide residues sequences such as sequences of bases specifying
protein binding regions also can be linked to enzyme-encoding
nucleic acid molecules. Such regions include, but are not limited
to, sequences of residues that facilitate or encode proteins that
facilitate uptake of an enzyme into specific target cells, or
otherwise alter pharmacokinetics of a product of a synthetic
gene.
[0234] In addition, tags or other moieties can be added, for
example, to aid in detection or affinity purification of the
polypeptide. For example, additional nucleotide residues sequences
such as sequences of bases specifying an epitope tag or other
detectable marker also can be linked to enzyme-encoding nucleic
acid molecules. Exemplary of such sequences include nucleic acid
sequences encoding a His tag (e.g., 6.times.His, HHHHHH; SEQ ID
NO:142) or Flag Tag (DYKDDDDK; SEQ ID NO:143).
[0235] The identified and isolated nucleic acids can then be
inserted into an appropriate cloning vector. A large number of
vector-host systems known in the art can be used. Possible vectors
include, but are not limited to, plasmids or modified viruses, but
the vector system must be compatible with the host cell used. Such
vectors include, but are not limited to, bacteriophage such as
lambda derivatives, or plasmids such as pCMV4, pBR322 or pUC
plasmid derivatives or the Bluescript vector (Stratagene, La Jolla,
Calif.). Other expression vectors include the HZ24 expression
vector exemplified herein (set forth in SEQ ID NO:140). The
insertion into a cloning vector can, for example, be accomplished
by ligating the DNA fragment into a cloning vector which has
complementary cohesive termini. If the complementary restriction
sites used to fragment the DNA are not present in the cloning
vector, the ends of the DNA molecules can be enzymatically
modified. Alternatively, any site desired can be produced by
ligating nucleotide sequences (linkers) onto the DNA termini; these
ligated linkers can include specific chemically synthesized
oligonucleotides encoding restriction endonuclease recognition
sequences. In an alternative method, the cleaved vector and protein
gene can be modified by homopolymeric tailing. Insertion can be
effected using TOPO cloning vectors (INVITROGEN, Carlsbad,
Calif.).
[0236] Recombinant molecules can be introduced into host cells via,
for example, transformation, transfection, infection,
electroporation and sonoporation, so that many copies of the gene
sequence are generated. In specific embodiments, transformation of
host cells with recombinant DNA molecules that incorporate the
isolated protein gene, cDNA, or synthesized DNA sequence enables
generation of multiple copies of the gene. Thus, the gene can be
obtained in large quantities by growing transformants, isolating
the recombinant DNA molecules from the transformants and, when
necessary, retrieving the inserted gene from the isolated
recombinant DNA.
[0237] In addition to recombinant production, soluble PH20,
including any esPH20 provided herein, can be produced by direct
peptide synthesis using solid-phase techniques (see e.g., Stewart
et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San
Francisco; Merrifield J (1963) J Am Chem. Soc., 85:2149-2154). In
vitro protein synthesis can be performed using manual techniques or
by automation. Automated synthesis can be achieved, for example,
using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer,
Foster City Calif.) in accordance with the instructions provided by
the manufacturer. Various fragments of a polypeptide can be
chemically synthesized separately and combined using chemical
methods.
[0238] 1. Vectors and Cells
[0239] For recombinant expression of one or more of the desired
proteins, such as any described herein, the nucleic acid containing
all or a portion of the nucleotide sequence encoding the protein
can be inserted into an appropriate expression vector, i.e., a
vector that contains the necessary elements for the transcription
and translation of the inserted protein coding sequence. The
necessary transcriptional and translational signals also can be
supplied by the native promoter for PH20 genes, and/or their
flanking regions.
[0240] Also provided are vectors that contain a nucleic acid
encoding the enzyme. Cells containing the vectors also are
provided. The cells include eukaryotic and prokaryotic cells, and
the vectors are any suitable for use therein.
[0241] Prokaryotic and eukaryotic cells, including endothelial
cells, containing the vectors are provided. Such cells include
bacterial cells, yeast cells, fungal cells, Archea, plant cells,
insect cells and animal cells. The cells are used to produce a
protein thereof by growing the above-described cells under
conditions whereby the encoded protein is expressed by the cell,
and recovering the expressed protein. For purposes herein, for
example, soluble PH20 polypeptides, including extended soluble PH20
polypeptides, can be secreted into the medium.
[0242] A host cells strain can be chosen for its ability to
modulate the expression of the inserted sequences or to process the
expressed protein in the desired fashion. Such modifications of the
polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation, phosphorylation, lipidation and
acylation. Post-translational processing can impact the folding
and/or function of the polypeptide. Different host cells, such as,
but not limited to, CHO (DG44, DXB11, CHO-K1), HeLa, MCDK, 293 and
WI38 have specific cellular machinery and characteristic mechanisms
for such post-translational activities and can be chosen to ensure
the correct modification and processing of the introduced protein.
Generally, the choice of cell is one that is capable of introducing
N-linked glycosylation into the expressed polypeptide. Hence,
eukaryotic cells containing the vectors are provided. Exemplary of
eukaryotic cells are mammalian Chinese Hamster Ovary (CHO) cells.
For example, CHO cells deficient in dihydrofolate reductase (e.g.
DG44 cells) are used to produce polypeptides provided herein. Note
that bacterial expression of an extended soluble PH20 or
C-terminally truncated PH20 provided herein will not result in a
catalytically active polypeptide, but when combined with proper
glycosylation machinery, the PH20 can be artificially
glycosylated.
[0243] Provided are vectors that contain a sequence of nucleotides
that encodes the hyaluronidase polypeptide, including extended
soluble PH20 polypeptides and other C-terminal truncated PH20
polypeptides, coupled to the native or heterologous signal
sequence, as well as multiple copies thereof. The vectors can be
selected for expression of the enzyme protein in the cell or such
that the enzyme protein is expressed as a secreted protein.
[0244] A variety of host-vector systems can be used to express the
protein coding sequence. These include but are not limited to
mammalian cell systems infected with virus (e.g. vaccinia virus,
adenovirus and other viruses); insect cell systems infected with
virus (e.g. baculovirus); microorganisms such as yeast containing
yeast vectors; or bacteria transformed with bacteriophage, DNA,
plasmid DNA, or cosmid DNA. The expression elements of vectors vary
in their strengths and specificities. Depending on the host-vector
system used, any one of a number of suitable transcription and
translation elements can be used.
[0245] Any methods known to those of skill in the art for the
insertion of DNA fragments into a vector can be used to construct
expression vectors containing a chimeric gene containing
appropriate transcriptional/translational control signals and
protein coding sequences. These methods can include in vitro
recombinant DNA and synthetic techniques and in vivo recombinants
(genetic recombination). Expression of nucleic acid sequences
encoding protein, or domains, derivatives, fragments or homologs
thereof, can be regulated by a second nucleic acid sequence so that
the genes or fragments thereof are expressed in a host transformed
with the recombinant DNA molecule(s). For example, expression of
the proteins can be controlled by any promoter/enhancer known in
the art. In a specific embodiment, the promoter is not native to
the genes for a desired protein. Promoters which can be used
include but are not limited to the SV40 early promoter (Bemoist and
Chambon, Nature 290:304-310 (1981)), the promoter contained in the
3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. Cell
22:787-797 (1980)), the herpes thymidine kinase promoter (Wagner et
al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), the
regulatory sequences of the metallothionein gene (Brinster et al.,
Nature 296:39-42 (1982)); prokaryotic expression vectors such as
the b-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci.
USA 78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad.
Sci. USA 80:21-25 (1983)); see also "Useful Proteins from
Recombinant Bacteria": in Scientific American 242:79-94 (1980));
plant expression vectors containing the nopaline synthase promoter
(Herrara-Estrella et al., Nature 303:209-213 (1984)) or the
cauliflower mosaic virus 35S RNA promoter (Gardner et al., Nucleic
Acids Res. 9:2871 (1981)), and the promoter of the photosynthetic
enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al.,
Nature 310:115-120 (1984)); promoter elements from yeast and other
fungi such as the Gal4 promoter, the alcohol dehydrogenase
promoter, the phosphoglycerol kinase promoter, the alkaline
phosphatase promoter, and the following animal transcriptional
control regions that exhibit tissue specificity and have been used
in transgenic animals: elastase I gene control region which is
active in pancreatic acinar cells (Swift et al., Cell 38:639-646
(1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol.
50:399-409 (1986); MacDonald, Hepatology 7:425-515 (1987)); insulin
gene control region which is active in pancreatic beta cells
(Hanahan et al., Nature 315:115-122 (1985)), immunoglobulin gene
control region which is active in lymphoid cells (Grosschedl et
al., Cell 38:647-658 (1984); Adams et al., Nature 318:533-538
(1985); Alexander et al., Mol. Cell. Biol. 7:1436-1444 (1987)),
mouse mammary tumor virus control region which is active in
testicular, breast, lymphoid and mast cells (Leder et al., Cell
45:485-495 (1986)), albumin gene control region which is active in
liver (Pinckert et al., Genes and Devel. 1:268-276 (1987)),
alpha-fetoprotein gene control region which is active in liver
(Krumlauf et al., Mol. Cell. Biol. 5:1639-1648 (1985); Hammer et
al., Science 235:53-58 1987)), alpha-1 antitrypsin gene control
region which is active in liver (Kelsey et al., Genes and Devel.
1:161-171 (1987)), beta globin gene control region which is active
in myeloid cells (Magram et al., Nature 315:338-340 (1985); Kollias
et al., Cell 46:89-94 (1986)), myelin basic protein gene control
region which is active in oligodendrocyte cells of the brain
(Readhead et al., Cell 48:703-712 (1987)), myosin light chain-2
gene control region which is active in skeletal muscle (Shani,
Nature 314:283-286 (1985)), and gonadotrophic releasing hormone
gene control region which is active in gonadotrophs of the
hypothalamus (Mason et al., Science 234:1372-1378 (1986)).
[0246] In a specific embodiment, a vector is used that contains a
promoter operably linked to nucleic acids encoding a PH20 protein,
or a domain, fragment, derivative or homolog, thereof, one or more
origins of replication, and optionally, one or more selectable
markers (e.g., an antibiotic resistance gene). Depending on the
expression system, specific initiation signals also are required
for efficient translation of a PH20 sequence. These signals include
the ATG initiation codon and adjacent sequences. In cases where the
initiation codon and upstream sequences of PH20 or soluble forms
thereof are inserted into the appropriate expression vector, no
additional translational control signals are needed. In cases where
only coding sequence, or a portion thereof, is inserted, exogenous
transcriptional control signals including the ATG initiation codon
must be provided. Furthermore, the initiation codon must be in the
correct reading frame to ensure transcription of the entire insert.
Exogenous transcriptional elements and initiation codons can be of
various origins, both natural and synthetic. The efficiency of
expression can be enhanced by the inclusion of enhancers
appropriate to the cell system in use (Scharf et al. (1994) Results
Probl Cell Differ 20:125-62; Bittner et al. (1987) Methods in
Enzymol, 153:516-544).
[0247] Exemplary plasmid vectors for transformation of E. coli
cells, include, for example, the pQE expression vectors (available
from Qiagen, Valencia, Calif.; see also literature published by
Qiagen describing the system). pQE vectors have a phage T5 promoter
(recognized by E. coli RNA polymerase) and a double lac operator
repression module to provide tightly regulated, high-level
expression of recombinant proteins in E. coli, a synthetic
ribosomal binding site (RBS II) for efficient translation, a
6.times.His tag coding sequence, t.sub.0 and T1 transcriptional
terminators, ColE1 origin of replication, and a beta-lactamase gene
for conferring ampicillin resistance. The pQE vectors enable
placement of a 6.times.His tag at either the N- or C-terminus of
the recombinant protein. Such plasmids include pQE 32, pQE 30, and
pQE 31 which provide multiple cloning sites for all three reading
frames and provide for the expression of N-terminally
6.times.His-tagged proteins. Other exemplary plasmid vectors for
transformation of E. coli cells, include, for example, the pET
expression vectors (see, U.S. Pat. No. 4,952,496; available from
NOVAGEN, Madison, Wis.; see, also literature published by Novagen
describing the system). Such plasmids include pET 11a, which
contains the T7lac promoter, T7 terminator, the inducible E. coli
lac operator, and the lac repressor gene; pET 12a-c, which contains
the T7 promoter, T7 terminator, and the E. coli ompT secretion
signal; and pET 15b and pET19b (NOVAGEN, Madison, Wis.), which
contain a His-Tag.TM. leader sequence for use in purification with
a H is column and a thrombin cleavage site that permits cleavage
following purification over the column, the T7-lac promoter region
and the T7 terminator.
[0248] Exemplary of a vector for mammalian cell expression is the
HZ24 expression vector. The HZ24 expression vector was derived from
the pCI vector backbone (Promega). It contains DNA encoding the
Beta-lactamase resistance gene (AmpR), an F1 origin of replication,
a Cytomegalovirus immediate-early enhancer/promoter region (CMV),
and an SV40 late polyadenylation signal (SV40). The expression
vector also has an internal ribosome entry site (IRES) from the
ECMV virus (Clontech) and the mouse dihydrofolate reductase (DHFR)
gene. Cells transfected with such a vector can be cultured in
chemically defined medium in the absence of hypoxanthine and
thymidine, followed by further gene amplification with increasing
concentrations of methotrexate. Such methods are described herein
in Examples 13 and 15.
[0249] 2. Expression
[0250] PH20 polypeptides, including esPH20 polypeptides and
C-terminally truncated PH20 polypeptides provided herein, can be
produced by any method known to those of skill in the art including
in vivo and in vitro methods. Desired proteins can be expressed in
any organism suitable to produce the required amounts and forms of
the proteins, such as for example, needed for administration and
treatment. Expression hosts include prokaryotic and eukaryotic
organisms such as E. coli, yeast, plants, insect cells, mammalian
cells, including human cell lines and transgenic animals.
Expression hosts can differ in their protein production levels as
well as the types of post-translational modification that are
present on the expressed proteins. The choice of expression host
can be made based on these and other factors, such as regulatory
and safety considerations, production costs and the need and
methods for purification.
[0251] Many expression vectors are available and known to those of
skill in the art and can be used for expression of proteins. The
choice of expression vector will be influenced by the choice of
host expression system. In general, expression vectors can include
transcriptional promoters and optionally enhancers, translational
signals, and transcriptional and translational termination signals.
Expression vectors that are used for stable transformation
typically have a selectable marker which allows selection and
maintenance of the transformed cells. In some cases, an origin of
replication can be used to amplify the copy number of the
vector.
[0252] Soluble hyaluronidase polypeptides, including esPH20, and
other C-terminally truncated PH20 polypeptides, also can be
utilized or expressed as protein fusions. For example, an enzyme
fusion can be generated to add additional functionality to an
enzyme. Examples of enzyme fusion proteins include, but are not
limited to, fusions of a signal sequence, a tag such as for
localization, e.g. a his.sub.6 tag or a myc tag, or a tag for
purification, for example, a GST fusion, a sequence for directing
protein secretion and/or membrane association and other sequences
used to increase half-life such as an Fc fusion.
[0253] For long-term, high-yield production of recombinant
proteins, stable expression is desired. For example, cell lines
that stably express a soluble PH20, such as an esPH20, or another
C-terminally truncated PH20 polypeptide, can be transformed using
expression vectors that contain viral origins of replication or
endogenous expression elements and a selectable marker gene.
Following the introduction of the vector, cells can be allowed to
grow for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells that successfully express the introduced
sequences. Resistant cells of stably transformed cells can be
proliferated using tissue culture techniques appropriate to the
cell types.
[0254] Any number of selection systems can be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler M et al. (1977) Cell,
11:223-32) and adenine phosphoribosyltransferase (Lowy I et al.
(1980) Cell, 22:817-23) genes, which can be employed in TK- or
APRT-cells, respectively. Also, antimetabolite, antibiotic or
herbicide resistance can be used as the basis for selection. For
example, DHFR, which confers resistance to methotrexate (Wigler M
et al. (1980) Proc. Natl. Acad. Sci, 77:3567-70); npt, which
confers resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin F et al. (1981) J. Mol. Biol., 150:1-14); and als
or pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively, can be used.
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of typtophan or hisD,
which allows cells to utilize histinol in place of histidine
(Hartman S C and RC Mulligan (1988) Proc. Natl. Acad. Sci,
85:8047-51). Visible markers, such as but not limited to,
anthocyanins, beta glucuronidase and its substrate, GUS, and
luciferase and its substrate luciferin, also can be used to
identify transformants and also to quantify the amount of transient
or stable protein expression attributable to a particular vector
system (Rhodes C A et al. (1995) Methods Mol. Biol.
55:121-131).
[0255] The presence and expression of soluble PH20 polypeptides,
including esPH20, and other C-terminal truncated PH20 polypetpides,
can be monitored. For example, detection of a functional
polypeptide can be determined by testing the conditioned media for
hyaluronidase enzyme activity under appropriate conditions. Section
G below provides exemplary assays to assess the solubility and
activity of expressed proteins.
[0256] a. Prokaryotic Cells
[0257] Prokaryotes, especially E. coli, provide a system for
producing large amounts of proteins. Transformation of E. coli is
simple and rapid technique well known to those of skill in the art.
Expression vectors for E. coli can contain inducible promoters,
such promoters are useful for inducing high levels of protein
expression and for expressing proteins that exhibit some toxicity
to the host cells. Examples of inducible promoters include the lac
promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6
RNA promoters and the temperature regulated XPL promoter.
[0258] Proteins, such as any provided herein, can be expressed in
the cytoplasmic environment of E. coli. The cytoplasm is a reducing
environment and for some molecules, this can result in the
formation of insoluble inclusion bodies. Reducing agents such as
dithiothreotol and .beta.-mercaptoethanol and denaturants, such as
guanidine-HCl and urea can be used to resolubilize the proteins. An
alternative approach is the expression of proteins in the
periplasmic space of bacteria which provides an oxidizing
environment and chaperonin-like and disulfide isomerases and can
lead to the production of soluble protein. Typically, a leader
sequence is fused to the protein to be expressed which directs the
protein to the periplasm. The leader is then removed by signal
peptidases inside the periplasm. Examples of periplasmic-targeting
leader sequences include the pelB leader from the pectate lyase
gene and the leader derived from the alkaline phosphatase gene. In
some cases, periplasmic expression allows leakage of the expressed
protein into the culture medium. The secretion of proteins allows
quick and simple purification from the culture supernatant.
Proteins that are not secreted can be obtained from the periplasm
by osmotic lysis. Similar to cytoplasmic expression, in some cases
proteins can become insoluble and denaturants and reducing agents
can be used to facilitate solubilization and refolding. Temperature
of induction and growth also can influence expression levels and
solubility, typically temperatures between 25.degree. C. and
37.degree. C. are used. Typically, bacteria produce aglycosylated
proteins. Thus, if proteins require glycosylation for function,
glycosylation can be added in vitro after purification from host
cells.
[0259] b. Yeast Cells
[0260] Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces
pombe, Yarrowia lipolytica, Kluyveromyces lactis and Pichia
pastoris are well known yeast expression hosts that can be used for
production of proteins, such as any described herein. Yeast can be
transformed with episomal replicating vectors or by stable
chromosomal integration by homologous recombination. Typically,
inducible promoters are used to regulate gene expression. Examples
of such promoters include GAL1, GALT and GALS and metallothionein
promoters, such as CUP1, AOX1 or other Pichia or other yeast
promoter. Expression vectors often include a selectable marker such
as LEU2, TRP1, HIS3 and URA3 for selection and maintenance of the
transformed DNA. Proteins expressed in yeast are often soluble.
Co-expression with chaperonins such as Bip and protein disulfide
isomerase can improve expression levels and solubility.
Additionally, proteins expressed in yeast can be directed for
secretion using secretion signal peptide fusions such as the yeast
mating type alpha-factor secretion signal from Saccharomyces
cerevisae and fusions with yeast cell surface proteins such as the
Aga2p mating adhesion receptor or the Arxula adeninivorans
glucoamylase. A protease cleavage site such as for the Kex-2
protease, can be engineered to remove the fused sequences from the
expressed polypeptides as they exit the secretion pathway. Yeast
also is capable of glycosylation at Asn-X-Ser/Thr motifs.
[0261] c. Insect Cells
[0262] Insect cells, particularly using baculovirus expression, are
useful for expressing polypeptides such as hyaluronidase
polypeptides. Insect cells express high levels of protein and are
capable of most of the post-translational modifications used by
higher eukaryotes. Baculovirus have a restrictive host range which
improves the safety and reduces regulatory concerns of eukaryotic
expression. Typical expression vectors use a promoter for high
level expression such as the polyhedrin promoter of baculovirus.
Commonly used baculovirus systems include the baculoviruses such as
Autographa califormica nuclear polyhedrosis virus (AcNPV), and the
bombyx mori nuclear polyhedrosis virus (BmNPV) and an insect cell
line such as Sf9 derived from Spodoptera frugiperda, Pseudaletia
unipuncta (A7S) and Danaus plexippus (DpN1). For high-level
expression, the nucleotide sequence of the molecule to be expressed
is fused immediately downstream of the polyhedrin initiation codon
of the virus. Mammalian secretion signals are accurately processed
in insect cells and can be used to secrete the expressed protein
into the culture medium. In addition, the cell lines Pseudaletia
unipuncta (A7S) and Danaus plexippus (DpN1) produce proteins with
glycosylation patterns similar to mammalian cell systems. Exemplary
insect cells are those that have been altered to reduce
immunogenicity, including those with "mammalianized" baculoviruse
expression vectors and those lacking the enzyme FT3.
[0263] An alternative expression system in insect cells is the use
of stably transformed cells. Cell lines such as the Schnieder 2
(S2) and Kc cells (Drosophila melanogaster) and C7 cells (Aedes
albopictus) can be used for expression. The Drosophila
metallothionein promoter can be used to induce high levels of
expression in the presence of heavy metal induction with cadmium or
copper. Expression vectors are typically maintained by the use of
selectable markers such as neomycin and hygromycin.
[0264] d. Mammalian Cells
[0265] Mammalian expression systems can be used to express proteins
including soluble hyaluronidase polypeptides. Expression constructs
can be transferred to mammalian cells by viral infection such as
adenovirus or by direct DNA transfer such as liposomes, calcium
phosphate, DEAE-dextran and by physical means such as
electroporation and microinjection. Expression vectors for
mammalian cells typically include an mRNA cap site, a TATA box, a
translational initiation sequence (Kozak consensus sequence) and
polyadenylation elements. IRES elements also can be added to permit
bicistronic expression with another gene, such as a selectable
marker. Such vectors often include transcriptional
promoter-enhancers for high-level expression, for example the SV40
promoter-enhancer, the human cytomegalovirus (CMV) promoter and the
long terminal repeat of Rous sarcoma virus (RSV). These
promoter-enhancers are active in many cell types. Tissue and
cell-type promoters and enhancer regions also can be used for
expression. Exemplary promoter/enhancer regions include, but are
not limited to, those from genes such as elastase I, insulin,
immunoglobulin, mouse mammary tumor virus, albumin, alpha
fetoprotein, alpha 1 antitrypsin, beta globin, myelin basic
protein, myosin light chain 2, and gonadotropic releasing hormone
gene control. Selectable markers can be used to select for and
maintain cells with the expression construct. Examples of
selectable marker genes include, but are not limited to, hygromycin
B phosphotransferase, adenosine deaminase, xanthine-guanine
phosphoribosyl transferase, aminoglycoside phosphotransferase,
dihydrofolate reductase (DHFR) and thymidine kinase. For example,
expression can be performed in the presence of methotrexate to
select for only those cells expressing the DHFR gene. Fusion with
cell surface signaling molecules such as TCR-.lamda. and
Fc.sub..epsilon.RI-.gamma. can direct expression of the proteins in
an active state on the cell surface.
[0266] Many cell lines are available for mammalian expression
including mouse, rat human, monkey, chicken and hamster cells.
Exemplary cell lines include but are not limited to CHO, Balb/3T3,
HeLa, MT2, mouse NSO (nonsecreting) and other myeloma cell lines,
hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts,
Sp2/0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines
also are available adapted to serum-free media which facilitates
purification of secreted proteins from the cell culture media.
Examples include CHO--S cells (Invitrogen, Carlsbad, Calif., cat
#11619-012) and the serum free EBNA-1 cell line (Pham et al.,
(2003) Biotechnol. Bioeng. 84:332-42.). Cell lines also are
available that are adapted to grow in special mediums optimized for
maximal expression. For example, DG44 CHO cells are adapted to grow
in suspension culture in a chemically defined, animal product-free
medium.
[0267] e. Plants
[0268] Transgenic plant cells and plants can be used to express
proteins such as any described herein. Expression constructs are
typically transferred to plants using direct DNA transfer such as
microprojectile bombardment and PEG-mediated transfer into
protoplasts, and with agrobacterium-mediated transformation.
Expression vectors can include promoter and enhancer sequences,
transcriptional termination elements and translational control
elements. Expression vectors and transformation techniques are
usually divided between dicot hosts, such as Arabidopsis and
tobacco, and monocot hosts, such as corn and rice. Examples of
plant promoters used for expression include the cauliflower mosaic
virus promoter, the nopaline syntase promoter, the ribose
bisphosphate carboxylase promoter and the ubiquitin and UBQ3
promoters. Selectable markers such as hygromycin, phosphomannose
isomerase and neomycin phosphotransferase are often used to
facilitate selection and maintenance of transformed cells.
Transformed plant cells can be maintained in culture as cells,
aggregates (callus tissue) or regenerated into whole plants.
Transgenic plant cells also can include algae engineered to produce
hyaluronidase polypeptides. Because plants have different
glycosylation patterns than mammalian cells, this can influence the
choice of protein produced in these hosts.
[0269] 3. Purification Techniques
[0270] Host cells transformed with a nucleic acid sequence encoding
a soluble PH20, including esPH20, and other C-terminal truncated
PH20 polypeptides, can be cultured under conditions suitable for
the expression and recovery of the encoded protein from cell
culture. The protein produced by a recombinant cell is generally
secreted, but may be contained intracellularly depending on the
sequence and/or the vector used. As will be understood by those of
skill in the art, expression vectors containing nucleic acid
encoding PH20 can be designed with signal sequences that facilitate
direct secretion of PH20 through prokaryotic or eukaryotic cell
membrane.
[0271] Thus, method for purification of polypeptides from host
cells will depend on the chosen host cells and expression systems.
For secreted molecules, proteins are generally purified from the
culture media after removing the cells. For intracellular
expression, cells can be lysed and the proteins purified from the
extract. When transgenic organisms such as transgenic plants and
animals are used for expression, tissues or organs can be used as
starting material to make a lysed cell extract. Additionally,
transgenic animal production can include the production of
polypeptides in milk or eggs, which can be collected, and if
necessary, the proteins can be extracted and further purified using
standard methods in the art.
[0272] Proteins, such as soluble PH20 polypeptides, including
esPH20 polypeptides, or other C-terminal truncated PH20
polypeptides, can be purified using standard protein purification
techniques known in the art including but not limited to, SDS-PAGE,
size fractionation and size exclusion chromatography, ammonium
sulfate precipitation and ionic exchange chromatography, such as
anion exchange. Affinity purification techniques also can be
utilized to improve the efficiency and purity of the preparations.
For example, antibodies, receptors and other molecules that bind
PH20 hyaluronidase enzymes can be used in affinity purification.
For example, soluble. PH20 can be purified from conditioned
media.
[0273] Expression constructs also can be engineered to add an
affinity tag to a protein such as a myc epitope, GST fusion or
His.sub.6 and affinity purified with myc antibody, glutathione
resin and Ni-resin, respectively. Such tags can be joined to the
nucleotide sequence encoding a soluble PH20 as described elsewhere
herein, which can facilitate purification of soluble proteins. For
example, soluble PH20 can be expressed as a recombinant protein
with one or more additional polypeptide domains added to facilitate
protein purification. Such purification facilitating domains
include, but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp., Seattle
Wash.). The inclusion of a cleavable linker sequence such as Factor
XA or enterokinase (Invitrogen, San Diego, Calif.) between the
purification domain and the expressed PH20 polypeptide is useful to
facilitate purification. One such expression vector provides for
expression of a fusion protein containing a soluble PH20 and
contains nucleic acid encoding 6 histidine residues followed by
thioredoxin and an enterokinase cleavage site. The histidine
residues facilitate purification on IMIAC (immobilized metal ion
affinity chromatography), while the enterokinase cleavage site
provides a means for purifying the polypeptide from the fusion
protein.
[0274] Purity can be assessed by any method known in the art
including gel electrophoresis, orthogonal HPLC methods, staining
and spectrophotometric techniques. The expressed and purified
protein can be analyzed using any assay or method known to one of
skill in the art, for example, any described in Section G. These
include assays based on the physical and/or functional properties
of the protein, including, but not limited to, analysis by gel
electrophoresis, immunoassay and assays of hyaluronidase
activity.
[0275] Depending on the expression system and host cells used, the
resulting polypeptide can be heterogeneous due to peptidases
present in the culture medium upon production and purification. For
example, culture of soluble PH20 in CHO cells can result in a
mixture of heterogeneous polypeptides. An exemplary protocol for
the generation, production and purification of a soluble PH20 (e.g.
rHuPH20) is described in Examples 13-15 below. Similarly, for
example, expression of a nucleic acid encoding a polypeptide having
a sequence of amino acids 36-497 set forth in SEQ ID NO:60, can
result in a heterogeneous mixture of polypeptides variably
including polypeptides that end at 497, 496, 495, 494, 493, 492,
491, 490, 489 or shorter.
F. Preparation, Formulation and Administration of Extended Soluble
PH20 Polypeptides, and Other Soluble PH20 Polypeptides
[0276] Pharmaceutical compositions of soluble PH20 polypeptides,
including esPH20, are provided herein for administration. The
soluble PH20 polypeptides can be formulated separately, or can be
co-formulated or co-administered with pharmaceutical formulations
of other therapeutic agents, for example, as described in Section
G. The compounds can be formulated into suitable pharmaceutical
preparations such as solutions, suspensions, tablets, dispersible
tablets, pills, capsules, powders, sustained release formulations
or elixirs, for oral administration, as well as transdermal patch
preparation and dry powder inhalers. Typically, the compounds are
formulated into pharmaceutical compositions using techniques and
procedures well known in the art (see e.g., Ansel Introduction to
Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126).
[0277] Typically, a therapeutically effective dosage is
contemplated. The amount of a selected soluble PH20 to be
administered for the treatment of a disease or condition can be
determined by standard clinical techniques. In addition, in vitro
assays and animal models can be employed to help identify optimal
dosage ranges. The precise dosage, which can be determined
empirically, can depend on the particular enzyme, the route of
administration, the type of disease to be treated and the
seriousness of the disease.
[0278] Hence, it is understood that the precise dosage and duration
of treatment is a function of the disease being treated and can be
determined empirically using known testing protocols or by
extrapolation from in vivo or in vitro test data. It is to be noted
that concentrations and dosage values also can vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that the
concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or use of compositions and
combinations containing them. The compositions can be administered
hourly, daily, weekly, monthly, yearly or once. Generally, dosage
regimens are chosen to limit toxicity. It should be noted that the
attending physician would know how to and when to terminate,
interrupt or adjust therapy to lower dosage due to toxicity, or
bone marrow, liver or kidney or other tissue dysfunctions.
Conversely, the attending physician would also know how to and when
to adjust treatment to higher levels if the clinical response is
not adequate (precluding toxic side effects).
[0279] Pharmaceutically acceptable compositions are prepared in
view of approvals for a regulatory agency or other agency prepared
in accordance with generally recognized pharmacopeia for use in
animals and in humans. Compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders, and
sustained release formulations. A composition can be formulated as
a suppository, with traditional binders and carriers such as
triglycerides. Oral formulation can include standard carriers such
as pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, and
other such agents. The formulation should suit the mode of
administration.
[0280] Pharmaceutical compositions can include carriers such as a
diluent, adjuvant, excipient, or vehicle with which an soluble PH20
is administered. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, generally in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. Such pharmaceutical carriers can be
sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, and sesame oil. Water is a typical
carrier when the pharmaceutical composition is administered
intravenously. Saline solutions and aqueous dextrose and glycerol
solutions also can be employed as liquid carriers, particularly for
injectable solutions. Compositions can contain along with an active
ingredient: a diluent such as lactose, sucrose, dicalcium
phosphate, or carboxymethylcellulose; a lubricant, such as
magnesium stearate, calcium stearate and talc; and a binder such as
starch, natural gums, such as gum acacia, gelatin, glucose,
molasses, polyvinylpyrrolidine, celluloses and derivatives thereof,
povidone, crospovidones and other such binders known to those of
skill in the art. Suitable pharmaceutical excipients include
starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,
chalk, silica gel, sodium stearate, glycerol monostearate, talc,
sodium chloride, dried skim milk, glycerol, propylene, glycol,
water, and ethanol. A composition, if desired, also can contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents, for example, acetate, sodium citrate, cyclodextrine
derivatives, sorbitan monolaurate, triethanolamine sodium acetate,
triethanolamine oleate, and other such agents.
[0281] Formulations of pharmaceutically therapeutically active
compounds and derivatives thereof are provided for administration
to humans and animals in unit dosage forms or multiple dosage
forms. For example compounds can be formulated as tablets,
capsules, pills, powders, granules, sterile parenteral solutions or
suspensions, and oral solutions or suspensions, and oil water
emulsions containing suitable quantities of the compounds or
pharmaceutically acceptable derivatives thereof. Each unit dose
contains a predetermined quantity of therapeutically active
compound sufficient to produce the desired therapeutic effect, in
association with the required pharmaceutical carrier, vehicle or
diluent. Examples of unit dose forms include ampoules and syringes
and individually packaged tablets or capsules. Unit dose forms can
be administered in fractions or multiples thereof. A multiple dose
form is a plurality of identical unit dosage forms packaged in a
single container to be administered in segregated unit dose form.
Examples of multiple dose forms include vials, bottles of tablets
or capsules or bottles of pints or gallons. Hence, multiple dose
form is a multiple of unit doses that are not segregated in
packaging. Generally, dosage forms or compositions containing
active ingredient in the range of 0.005% to 100% with the balance
made up from non-toxic carrier can be prepared.
[0282] Compositions provided herein typically are formulated for
administration by subcutaneous route, although other routes of
administration are contemplated, such as any route known to those
of skill in the art including intramuscular, intraperitineal,
intravenous, intradermal, intralesional, intraperitoneal injection,
epidural, vaginal, rectal, local, otic, transdermal administration
or any route. Formulations suited for such routes are known to one
of skill in the art. Administration can be local, topical or
systemic depending upon the locus of treatment. Local
administration to an area in need of treatment can be achieved by,
for example, but not limited to, local infusion during surgery,
topical application, e.g., in conjunction with a wound dressing
after surgery, by injection, by means of a catheter, by means of a
suppository, or by means of an implant. Compositions also can be
administered with other biologically active agents, either
sequentially, intermittently or in the same composition.
[0283] The most suitable route in any given case depends on a
variety of factors, such as the nature of the disease, the
tolerance of the subject to a particular administration route, the
severity of the disease, and the particular composition that is
used. Typically, the compositions provided herein are administered
parenterally. In some examples, soluble PH20 compositions are
administered so that they reach the interstitium of skin or
tissues, thereby degrading the interstitial space for subsequent
delivery of a therapeutic agent. Thus, in some examples, direct
administration under the skin, such as by subcutaneous
administration methods, is contemplated. Thus, in one example,
local administration can be achieved by injection, such as from a
syringe or other article of manufacture containing a injection
device such as a needle. In another example, local administration
can be achieved by infusion, which can be facilitated by the use of
a pump or other similar device. Other modes of administration also
are contemplated. Pharmaceutical compositions can be formulated in
dosage forms appropriate for each route of administration.
[0284] Administration methods can be employed to decrease the
exposure of selected soluble PH20 polypeptides to degradative
processes, such as proteolytic degradation and immunological
intervention via antigenic and immunogenic responses. Examples of
such methods include local administration at the site of treatment.
Pegylation of therapeutics has been reported to increase resistance
to proteolysis, increase plasma half-life, and decrease
antigenicity and immunogenicity. Examples of pegylation
methodologies are known in the art (see for example, Lu and Felix,
Int. J. Peptide Protein Res., 43: 127-138, 1994; Lu and Felix,
Peptide Res., 6: 142-6, 1993; Felix et al., Int. J. Peptide Res.,
46:253-64, 1995; Benhar et al., J. Biol. Chem., 269: 13398-404,
1994; Brumeanu et al., J Immunol., 154: 3088-95, 1995; see also,
Caliceti et al. (2003) Adv. Drug Deliv. Rev. 55(10):1261-77 and
Molineux (2003) Pharmacotherapy 23 (8 Pt 2):3S-8S). Pegylation also
can be used in the delivery of nucleic acid molecules in vivo. For
example, pegylation of adenovirus can increase stability and gene
transfer (see, e.g., Cheng et al. (2003) Pharm. Res. 20(9):
1444-51).
[0285] 1. Injectables, Solutions and Emulsions
[0286] Parenteral administration, generally characterized by
injection or infusion, either subcutaneously, intramuscularly,
intravenous or intradermally is contemplated herein. Injectables
can be prepared in conventional forms, either as liquid solutions
or suspensions, solid forms suitable for solution or suspension in
liquid prior to injection, or as emulsions. Suitable excipients
are, for example, water, saline, dextrose, glycerol or ethanol. The
pharmaceutical compositions may contain other minor amounts of
non-toxic auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, stabilizers, solubility enhancers, and
other such agents, such as for example, sodium acetate, sodium
phosphate, sorbitan monolaurate, triethanolamine oleate and
cyclodextrins. Implantation of a slow-release or sustained-release
system, such that a constant level of dosage is maintained (see,
e.g., U.S. Pat. No. 3,710,795) is also contemplated herein. The
percentage of active compound contained in such parenteral
compositions is highly dependent on the specific nature thereof, as
well as the activity of the compound and the needs of the
subject.
[0287] Injectables are designed for local and systemic
administration. For purposes herein, local administration is
desired for direct administration to the affected interstitium.
Preparations for parenteral administration include sterile
solutions ready for injection, sterile dry soluble products, such
as lyophilized powders, ready to be combined with a solvent just
prior to use, including hypodermic tablets, sterile suspensions
ready for injection, sterile dry insoluble products ready to be
combined with a vehicle just prior to use and sterile emulsions.
The solutions may be either aqueous or nonaqueous. If administered
intravenously, suitable carriers include physiological saline or
phosphate buffered saline (PBS), and solutions containing
thickening and solubilizing agents, such as glucose, polyethylene
glycol, and polypropylene glycol and mixtures thereof.
[0288] Pharmaceutically acceptable carriers used in parenteral
preparations include aqueous vehicles, nonaqueous vehicles,
antimicrobial agents, isotonic agents, buffers, antioxidants, local
anesthetics, suspending and dispersing agents, emulsifying agents,
sequestering or chelating agents and other pharmaceutically
acceptable substances. Examples of aqueous vehicles include Sodium
Chloride Injection, Ringers Injection, Isotonic Dextrose Injection,
Sterile Water Injection, Dextrose and Lactated Ringers Injection.
Nonaqueous parenteral vehicles include fixed oils of vegetable
origin, cottonseed oil, corn oil, sesame oil and peanut oil.
Antimicrobial agents in bacteriostatic or fungistatic
concentrations can be added to parenteral preparations packaged in
multiple-dose containers, which include phenols or cresols,
mercurials, benzyl alcohol, chlorobutanol, methyl and propyl
p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and
benzethonium chloride. Isotonic agents include sodium chloride and
dextrose. Buffers include phosphate and citrate. Antioxidants
include sodium bisulfate. Local anesthetics include procaine
hydrochloride. Suspending and dispersing agents include sodium
carboxymethylcelluose, hydroxypropyl methylcellulose and
polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80
(TWEEN 80). A sequestering or chelating agent of metal ions include
EDTA. Pharmaceutical carriers also include ethyl alcohol,
polyethylene glycol and propylene glycol for water miscible
vehicles and sodium hydroxide, hydrochloric acid, citric acid or
lactic acid for pH adjustment.
[0289] The concentration of the pharmaceutically active compound is
adjusted so that an injection or infusion provides an effective
amount to produce the desired pharmacological effect, such as
glycemic control. The exact dose depends on the age, weight and
condition of the patient or animal as is known in the art. The
unit-dose parenteral preparations can be packaged in, for example,
an ampoule, a cartridge, a vial or a syringe with a needle. The
volume of liquid solution or reconstituted powder preparation,
containing the pharmaceutically active compound, is a function of
the disease to be treated and the particular article of manufacture
chosen for package. All preparations for parenteral administration
must be sterile, as is known and practiced in the art.
[0290] In one example, pharmaceutical preparation can be in liquid
form, for example, solutions, syrups or suspensions. If provided in
liquid form, the pharmaceutical preparations can be provided as a
concentrated preparation to be diluted to a therapeutically
effective concentration before use. Such liquid preparations can be
prepared by conventional means with pharmaceutically acceptable
additives such as suspending agents (e.g., sorbitol syrup,
cellulose derivatives or hydrogenated edible fats); emulsifying
agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g.,
almond oil, oily esters, or fractionated vegetable oils); and
preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic
acid). In another example, pharmaceutical preparations can be
presented in lyophilized form for reconstitution with water or
other suitable vehicle before use.
[0291] Lyophilized Powders
[0292] Of interest herein are lyophilized powders, which can be
reconstituted for administration as solutions, emulsions and other
mixtures. They may also be reconstituted and formulated as solids
or gels.
[0293] The sterile, lyophilized powder is prepared by dissolving a
compound of inactive enzyme in a buffer solution. The buffer
solution may contain an excipient which improves the stability or
other pharmacological component of the powder or reconstituted
solution, prepared from the powder. Subsequent sterile filtration
of the solution followed by lyophilization under standard
conditions known to those of skill in the art provides the desired
formulation. Briefly, the lyophilized powder is prepared by
dissolving an excipient, such as dextrose, sorbital, fructose, corn
syrup, xylitol, glycerin, glucose, sucrose or other suitable agent,
in a suitable buffer, such as citrate, sodium or potassium
phosphate or other such buffer known to those of skill in the art.
Then, a selected enzyme is added to the resulting mixture, and
stirred until it dissolves. The resulting mixture is sterile
filtered or treated to remove particulates and to insure sterility,
and apportioned into vials for lyophilization. Each vial will
contain a single dosage or multiple dosages of the compound. The
lyophilized powder can be stored under appropriate conditions, such
as at about 4.degree. C. to room temperature. Reconstitution of
this lyophilized powder with an appropriate buffer solution
provides a formulation for use in parenteral administration.
[0294] 2. Topical Administration
[0295] Topical mixtures are prepared as described for the local and
systemic administration. The resulting mixture can be a solution,
suspension, emulsions or the like and are formulated as creams,
gels, ointments, emulsions, solutions, elixirs, lotions,
suspensions, tinctures, pastes, foams, aerosols, irrigations,
sprays, suppositories, bandages, dermal patches or any other
formulations suitable for topical administration.
[0296] The compounds or pharmaceutically acceptable derivatives
thereof may be formulated as aerosols for topical application, such
as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209,
and 4,364,923, which describe aerosols for delivery of a steroid
useful for treatment inflammatory diseases, particularly asthma).
These formulations for administration to the respiratory tract can
be in the form of an aerosol or solution for a nebulizer, or as a
microfine powder for insufflation, alone or in combination with an
inert carrier such as lactose. In such a case, the particles of the
formulation will typically have diameters of less than 50 microns,
or less than 10 microns.
[0297] The compounds can be formulated for local or topical
application, such as for topical application to the skin and mucous
membranes, such as in the eye, in the form of gels, creams, and
lotions and for application to the eye or for intracisternal or
intraspinal application. Topical administration is contemplated for
transdermal delivery and also for administration to the eyes or
mucosa, or for inhalation therapies. Nasal solutions of the active
compound alone or in combination with other pharmaceutically
acceptable excipients also can be administered.
[0298] Formulations suitable for transdermal administration are
provided. They can be provided in any suitable format, such as
discrete patches adapted to remain in intimate contact with the
epidermis of the recipient for a prolonged period of time. Such
patches contain the active compound in optionally buffered aqueous
solution of, for example, 0.1 to 0.2 M concentration with respect
to the active compound. Formulations suitable for transdermal
administration also can be delivered by iontophoresis (see, e.g.,
Pharmaceutical Research 3(6):318 (1986)) and typically take the
form of an optionally buffered aqueous solution of the active
compound.
[0299] 3. Compositions for Other Routes of Administration
[0300] Depending upon the condition treated other routes of
administration, such as topical application, transdermal patches,
oral and rectal administration are also contemplated herein. For
example, pharmaceutical dosage forms for rectal administration are
rectal suppositories, capsules and tablets for systemic effect.
Rectal suppositories include solid bodies for insertion into the
rectum which melt or soften at body temperature releasing one or
more pharmacologically or therapeutically active ingredients.
Pharmaceutically acceptable substances utilized in rectal
suppositories are bases or vehicles and agents to raise the melting
point. Examples of bases include cocoa butter (theobroma oil),
glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate
mixtures of mono-, di- and triglycerides of fatty acids.
Combinations of the various bases may be used. Agents to raise the
melting point of suppositories include spermaceti and wax. Rectal
suppositories may be prepared either by the compressed method or by
molding. The typical weight of a rectal suppository is about 2 to 3
gm. Tablets and capsules for rectal administration are manufactured
using the same pharmaceutically acceptable substance and by the
same methods as for formulations for oral administration.
[0301] Formulations suitable for rectal administration can be
provided as unit dose suppositories. These can be prepared by
admixing the active compound with one or more conventional solid
carriers, for example, cocoa butter, and then shaping the resulting
mixture.
[0302] For oral administration, pharmaceutical compositions can
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinized maize starch, polyvinyl
pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,
lactose, microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets can be
coated by methods well-known in the art.
[0303] Formulations suitable for buccal (sublingual) administration
include, for example, lozenges containing the active compound in a
flavored base, usually sucrose and acacia or tragacanth; and
pastilles containing the compound in an inert base such as gelatin
and glycerin or sucrose and acacia.
[0304] Pharmaceutical compositions also can be administered by
controlled release formulations and/or delivery devices (see, e.g.,
in U.S. Pat. Nos. 3,536,809; 3,598,123; 3,630,200; 3,845,770;
3,847,770; 3,916,899; 4,008,719; 4,687,610; 4,769,027; 5,059,595;
5,073,543; 5,120,548; 5,354,566; 5,591,767; 5,639,476; 5,674,533
and 5,733,566).
[0305] Various delivery systems are known and can be used to
administer selected soluble PH20 polypeptides, such as but not
limited to, encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the
compound, receptor mediated endocytosis, and delivery of nucleic
acid molecules encoding selected soluble PH20 polypeptides such as
retrovirus delivery systems.
[0306] Hence, in certain embodiments, liposomes and/or
nanoparticles also can be employed with administration of soluble
PH20 polypeptides. Liposomes are formed from phospholipids that are
dispersed in an aqueous medium and spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles
(MLVs)). MLVs generally have diameters of from 25 nm to 4 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 angstroms
containing an aqueous solution in the core.
[0307] Phospholipids can form a variety of structures other than
liposomes when dispersed in water, depending on the molar ratio of
lipid to water. At low ratios, the liposomes form. Physical
characteristics of liposomes depend on pH, ionic strength and the
presence of divalent cations. Liposomes can show low permeability
to ionic and polar substances, but at elevated temperatures undergo
a phase transition which markedly alters their permeability. The
phase transition involves a change from a closely packed, ordered
structure, known as the gel state, to a loosely packed,
less-ordered structure, known as the fluid state. This occurs at a
characteristic phase-transition temperature and results in an
increase in permeability to ions, sugars and drugs.
[0308] Liposomes interact with cells via different mechanisms:
endocytosis by phagocytic cells of the reticuloendothelial system
such as macrophages and neutrophils; adsorption to the cell
surface, either by nonspecific weak hydrophobic or electrostatic
forces, or by specific interactions with cell-surface components;
fusion with the plasma cell membrane by insertion of the lipid
bilayer of the liposome into the plasma membrane, with simultaneous
release of liposomal contents into the cytoplasm; and by transfer
of liposomal lipids to cellular or subcellular membranes, or vice
versa, without any association of the liposome contents. Varying
the liposome formulation can alter which mechanism is operative,
although more than one can operate at the same time. Nanocapsules
can generally entrap compounds in a stable and reproducible way. To
avoid side effects due to intracellular polymeric overloading, such
ultrafine particles (sized around 0.1 .mu.m) should be designed
using polymers able to be degraded in vivo. Biodegradable
polyalkyl-cyanoacrylate nanoparticles that meet these requirements
are contemplated for use herein, and such particles can be easily
made.
[0309] 4. Dosage and Administration
[0310] The soluble PH20 polypeptides, including esPH20, provided
herein can be formulated as pharmaceutical compositions for single
dosage or multiple dosage administration. The selected hyaluronan
degrading enzyme is included in an amount sufficient to exert a
therapeutically useful effect in the absence of undesirable side
effects on the patient treated. The therapeutically effective
concentration can be determined empirically by testing the
polypeptides in known in vitro and in vivo systems such as by using
the assays provided herein or known in the art (see e.g., Taliani
et al. (1996) Anal. Biochem., 240: 60-67; Filocamo et al. (1997) J
Virology, 71: 1417-1427; Sudo et al. (1996) Antiviral Res. 32:
9-18; Buffard et al. (1995) Virology, 209:52-59; Bianchi et al.
(1996) Anal. Biochem., 237: 239-244; Hamatake et al. (1996)
Intervirology 39:249-258; Steinkuhler et al. (1998) Biochem.,
37:8899-8905; D'Souza et al. (1995) J. Gen. Virol., 76:1729-1736;
Takeshita et al. (1997) Anal. Biochem., 247:242-246; see also e.g.,
Shimizu et al. (1994) J. Virol. 68:8406-8408; Mizutani et al.
(1996) J. Virol. 70:7219-7223; Mizutani et al. (1996) Biochem.
Biophys. Res. Commun., 227:822-826; Lu et al. (1996) Proc. Natl.
Acad. Sci. (USA), 93:1412-1417; Hahm et al., (1996) Virology,
226:318-326; Ito et al. (1996) J. Gen. Virol., 77:1043-1054;
Mizutani et al. (1995) Biochem. Biophys. Res. Commun., 212:906-911;
Cho et al. (1997) J. Virol. Meth. 65:201-207 and then extrapolated
therefrom for dosages for humans.
[0311] Typically, a therapeutically effective dose of a soluble
PH20 enzyme is at or about 10 Unit (U) to 500,000 Units, 100 Units
to 100,000 Units, 500 Units to 50,000 Units, 1000 Units to 10,000
Units, 5000 Units to 7500 Units, 5000 Units to 50,000 Units, or
1,000 Units to 10,000 Units, generally 1,000 to 50,000 Units, in a
stabilized solution or suspension or a lyophilized from. The
formulations can be provided in unit-dose forms such as, but not
limited to, ampoules, syringes and individually packaged tablets or
capsules. The dispersing agent can be administered alone, or with
other pharmacologically effective agent or therapeutic agent in a
total volume of 0.1-100 ml, 1-50 ml, 10-50 ml, 10-30 ml, 1-20 ml,
or 1-10 ml, typically 10-50 ml.
[0312] For example, a soluble PH20, including esPH20, can be
administered subcutaneously at or about 10 U, 20 U, 30 U, 40 U, 50
U, 100 U, 150 U, 200 U, 250 U, 300 U, 350 U, 400 U, 450 U, 500 U,
600 U, 700 U, 800 U, 900 U, 1000 U, 2,000 U, 3,000 U, 4,000 Units,
5,000 U or more. In some examples, dosages can be provided as a
ratio of amount of a soluble PH20 to therapeutic agent
administered. For example, a soluble PH20 polypeptide can be
administered at 1 hyaluronidase U/therapeutic agent U (1:1) to 50:1
or more, for example, at or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,
7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 20:1, 25:1,
30:1, 35:1, 40:1, 45:1, 50:1 or more. Typically, volumes of
injections or infusions of a soluble PH20 contemplated herein are
from at or about 0.01 mL, 0.05 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL,
0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 ml, 10
ml, 20 ml, 30 ml, 40 ml, 50 ml or more. The soluble PH20 can be
provided as a stock solution at or about 100 U/ml, 150 U/ml, 200
U/ml, 300 U/ml, 400 U/ml, 500 U/mL, 600 U/mL, 800 U/mL or 1000
U/mL, or can be provided in a more concentrated form, for example
at or about 2000 U/ml, 3000 Units/ml, 4000 U/ml, 5000 U/ml, 8000
U/ml, 10,000 U/mL or 20,000 U/mL for use directly or for dilution
to the effective concentration prior to use. The soluble PH20 can
be provided as a liquid or lyophilized formulation.
[0313] 5. Packaging, Articles of Manufacture and Kits
[0314] Pharmaceutical compounds of soluble PH20 polypeptides,
including esPH20, or nucleic acids encoding such polypeptides, or a
derivative or variant thereof can be packaged as articles of
manufacture containing packaging material, a pharmaceutical
composition which is effective for treating a disease or disorder,
and a label that indicates that the soluble PH20 or nucleic acid
molecule is to be used for treating the disease or disorder.
Combinations of a selected soluble PH20 hyaluronidase, or
derivative or variant thereof and an therapeutic agent also can be
packaged in an article of manufacture.
[0315] The articles of manufacture provided herein contain
packaging materials. Packaging materials for use in packaging
pharmaceutical products are well known to those of skill in the
art. See, for example, U.S. Pat. Nos. 5,323,907, 5,052,558 and
5,033,252, each of which is incorporated herein in its entirety.
Examples of pharmaceutical packaging materials include, but are not
limited to, blister packs, bottles, tubes, inhalers, pumps, bags,
vials, containers, syringes, bottles, and any packaging material
suitable for a selected formulation and intended mode of
administration and treatment. The articles of manufacture can
include a needle or other injection device so as to facilitate
administration (e.g. sub-epidermal administration) for local
injection purposes. A wide array of formulations of the compounds
and compositions provided herein are contemplated including a
soluble PH20, such as an esPH20, and a therapeutic agent known to
treat a particular disease or disorder. The choice of package
depends on the soluble PH20 and/or therapeutic agent, and whether
such compositions will be packaged together or separately. In one
example, the soluble PH20 can be packaged as a mixture with the
therapeutic agent. In another example, the components can be
packaged as separate compositions
[0316] Selected soluble PH20 polypeptides, such as esPH20
polypeptides, therapeutic agents and/or articles of manufacture
thereof also can be provided as kits. Kits can include a
pharmaceutical composition described herein and an item for
administration provided as an article of manufacture. For example a
soluble PH20 polypeptide can be supplied with a device for
administration, such as a syringe, an inhaler, a dosage cup, a
dropper, or an applicator. The compositions can be contained in the
item for administration or can be provided separately to be added
later. The kit can, optionally, include instructions for
application including dosages, dosing regimens and instructions for
modes of administration. Kits also can include a pharmaceutical
composition described herein and an item for diagnosis. For
example, such kits can include an item for measuring the
concentration, amount or activity of the selected protease in a
subject.
G. Assays
[0317] Soluble PH20 polypeptides provided herein, including esPH20
polypeptides, are soluble and retain a hyaluronidase enzymatic
activity. N-glycosylated or N-partially glycosylated PH20
polypeptides provided herein retain a hyaluronidase enzymatic
activity. The activity of a PH20 provided herein is or is about
30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more compared to the activity of a corresponding
PH20 that is not C-terminally truncated or N-partially
glycosylated. The activity of a soluble PH20 hyaluronidase
polypeptide, such as an esPH20, can be assessed using methods well
known in the art. These methods include, for example, a
microturbidity assay and a microtiter assay using biotinylated
hyaluronic acid. Activity and assessments can be performed on
conditioned medium or supernatants or on purified protein. The
solublity of a protein also can be determined, for example, by a
Triton.RTM. X-114 partition assay. In all assays, the activity or
solubility of a soluble PH20 can be compared to a control, for
example, a full length PH20 lacking C-terminal truncations.
[0318] 1. Hyaluronidase Activity
[0319] The activity of a soluble PH20 polypeptide can be assessed
using methods well known in the art. For example, the USP XXII
assay for hyaluronidase determines activity indirectly by measuring
the amount of undegraded hyaluronic acid, or hyaluronan, (HA)
substrate remaining after the enzyme is allowed to react with the
HA for 30 min at 37.degree. C. (USP XXII-NF XVII (1990) 644-645
United States Pharmacopeia Convention, Inc, Rockville, Md.). A
Hyaluronidase Reference Standard (USP) or National Formulary (NF)
Standard Hyaluronidase solution can be used in an assay to
ascertain the activity, in units, of any hyaluronidase.
[0320] In one example, activity is measured using a microturbidity
assay, as described in Example 12. This is based on the formation
of an insoluble precipitate when hyaluronic acid binds with serum
albumin. The activity is measured by incubating hyaluronidase with
sodium hyaluronate (hyaluronic acid) for a set period of time (e.g.
10 minutes) and then precipitating the undigested sodium
hyaluronate with the addition of acidified serum albumin. The
turbidity of the resulting sample is measured at 640 nm after an
additional development period. The decrease in turbidity resulting
from hyaluronidase activity on the sodium hyaluronate substrate is
a measure of hyaluronidase enzymatic activity.
[0321] In another example, hyaluronidase activity is measured using
a microtiter assay in which residual biotinylated hyaluronic acid
is measured following incubation with hyaluronidase (see e.g. Frost
and Stern (1997) Anal. Biochem. 251:263-269, U.S. Patent
Publication No. 20050260186). In Example 4, the hyaluronidase
activity of truncated human PH20 hyaluronidase is determined using
biotinylated hyaluronic acid. The free carboxyl groups on the
glucuronic acid residues of hyaluronic acid are biotinylated, and
the biotinylated hyaluronic acid substrate is covalently coupled to
a microtiter plate. Following incubation with hyaluronidase, the
residual biotinylated hyaluronic acid substrate is detected using
an avidin-peroxidase reaction, and compared to that obtained
following reaction with hyaluronidase standards of known activity.
As the substrate is covalently bound to the microtiter plate,
artifacts such as pH-dependent displacement of the biotinylated
substrate does not occur. The sensitivity permits rapid measurement
of hyaluronidase activity from cultured cells and biological
samples with an inter-assay variation of less than 10%.
[0322] Other assays to measure hyaluronidase activity also are
known in the art and can be used in the methods herein (see e.g.
Delpech et al., (1995) Anal. Biochem. 229:35-41; Takahashi et al.,
(2003) Anal. Biochem. 322:257-263).
[0323] Many hyaluronidase assays have been based upon the
measurement of the generation of new reducing N-acetylamino groups
(Bonner and Cantey, Clin. Chim. Acta 13:746-752, 1966), or loss of
viscosity (De Salegui et al., Arch. Biochem. Biophys.121:548-554,
1967) or turbidity (Dorfman and Ott, J. Biol. Chem. 172:367, 1948).
With purified substrates all of these methods suffice for
determination of the presence or absence of endoglucosamidic
activity.
[0324] Substantially purified glycosaminoglycan substrates can also
be used in a Gel Shift Assay. Glycosaminoglycans are mixed with
recombinant PH20, such as a soluble PH20, to test for
endoglucosidase activity that results in a shift in substrate
mobility within the gel. Exemplary of such substrates include, but
are not limited to, chondroitin-4 and 6 sulfate, dermatan sulfate,
heparan-sulfate, which can be obtained from Sigma Chemical. Human
umbilical cord Hyaluronan can be obtained from ICN. For example,
each test substrate can be diluted to at or about 0.1 mg/ml in a
buffer range from pH 3.5-7.5. In such an exemplary assay, at or
about 10 .mu.l samples of purified soluble PH20 or conditioned
media from PH20 expressing cells can be mixed with at or about 90
.mu.l of test substrate in desired buffer and incubated for 3 hours
at 37.degree. C. Following incubation, samples are neutralized with
sample buffer (Tris EDTA pH 8.0, Bromophenol Blue and glycerol)
followed by electrophoresis. Glycosaminoglycans can be detected
using any method known in the art, for example, glycosaminoglycans
can be detected by staining the gels using 0.5% Alcian Blue in 3%
Glacial Acetic Acid overnight followed by destaining in 7% Glacial
Acetic Acid. Degradation is determined by comparison of substrate
mobility in the presence and absence of enzyme.
[0325] Hyaluronidase activity can also be detected by substrate gel
zymography (Guentenhoner et al., 1992, Matrix 388-396). In this
assay a sample is applied to a SDS-PAGE gel containing hyaluronic
acid and the proteins in the sample separated by electrophoresis.
The gel is then incubated in an enzyme assay buffer and
subsequently stained to detect the hyaluronic acid in the gel.
Hyaluronidase activity is visualized as a cleared zone in the
substrate gel.
[0326] The ability of a soluble PH20 polypeptide to act as a
spreading or diffusing agent also can be assessed. For example,
trypan blue dye can be injected subcutaneously with or without a
soluble PH20 into the lateral skin on each side of nude mice. The
dye area is then measured, such as with a microcaliper, to
determine the ability of the hyaluronan degrading enzyme to act as
a spreading agent (U.S. patent No. 20060104968). The effect of
co-administration of hyaluronidase with another agent, for example
a therapeutic agent, on the pharmacokinetic and pharmacodynamic
properties of that agent also can be assessed in vivo using animal
model and/or human subjects, such as in the setting of a clinical
trial.
[0327] The functional activity of a soluble PH20, such as esPH20
can be compared and/or normalized to a reference standared using
any of these assays. This can be done to determine what a
functionally equivalent amount of a soluble PH20 is. For example,
the ability of a soluble PH20 to act as a spreading or diffusing
agent can be assessed by injecting it into the lateral skin of mice
with trypan blue, and the amount required to achieve the same
amount of diffusion as, for example, 100 units of a Hyaluronidase
Reference Standard, can be determined. The amount of soluble PH20
required is, therefore, functionally equivalent to 100 hyauronidase
units.
[0328] 2. Solubility
[0329] Solubility of a hyaluronidase can be determined by any
method known to one of the skill in the art. One method for
determining solubility is by detergent partitioning. For example, a
soluble PI-120 polypeptide can be distinguished, for example, by
its partitioning into the aqueous phase of a Triton.RTM. X-114
solution at 37.degree. C. (Bordier et al., (1981) J. Biol. Chem.,
256:1604-1607). For example, the solubility of the PH20
polypeptides described herein is assessed as described in Example
4. Membrane-anchored hyaluronidases, such as lipid-anchored
hyaluronidases, including GPI-anchored hyaluronidases, will
partition into the detergent-rich phase, but will partition into
the detergent-poor or aqueous phase following treatment with
Phospholipase C. Phospholipase C is an enzyme that cleaves the
phospho-glycerol bond found in GPI-anchored proteins. Treatment
with PLC will cause release of GPI-linked proteins from the outer
cell membrane.
[0330] Another method for assessing solubility is to determine
whether a PI-120 polypeptide is GPI-anchored. A GPI-anchored PH20
polypeptide is bound to the cell membrane and therefore insoluble.
To determine whether a PH20 polypeptide is GPI-anchored, one can
assess solubility before and after PLC/PLD hydrolysis, and also use
predictive algorithms to identify a GPI-anchor attachment signal
sequence. GPI-anchored proteins can be identified by their
solubilization after specific enzymatic or chemical cleavage, in
conjunction with detergent partitioning (e.g., in Triton.RTM.
X-114), antibody recognition, and metabolic radioactive
labeling.
[0331] A common method used to demonstrate that a protein has a GPI
anchor is its release from the cell surface or its solubilization
by treating with bacterial PI-PLC or trypanosome-derived
GPI-specific phospholipase C (GPI-PLC). These enzymes cleave a
diacylglycerol in the membrane and produce the immunoreactive
glycan epitope (CRD) on the protein, which can be detected by
Western blotting with antibodies produced against the GPI of
trypanosomes. One common problem with this approach especially
encountered in mammalian cells is that the lipases cannot cleave a
GPI anchor in which the inositol is acylated. These require prior
treatment with mild alkali to remove the fatty acid on the inositol
ring. Alternatively, serum-derived GPI-specific phospholipase D can
be used to cleave GPI anchors. This enzyme cleaves between the
inositol ring and the phosphatidic acid moiety and is not inhibited
by inositol acylation. Hydrofluoric acid cleaves GPI anchors
between the inositol ring and phosphatidic acid and also cleaves
the phosphodiester linkages between any phosphoethanolamines and
mannosyl residues. Dilute nitrous acid is particularly useful in
the study of GPI anchors because it cleaves specifically between
the nonacetylated glucosamine and the inositol ring, releasing the
protein-bound glycan (now containing a diagnostic anhydromannose
moiety) and phosphatidylinositol. In combination with CRD
antibodies, composition analyses, radioactive labeling with
myo-inositol, ethanolamine, glucosamine, mannose, or fatty acids
and chromatographic or detergent partitioning methods, these
degradation methods represent a powerful set of tools to study GPI
anchors on proteins.
[0332] Various in silico methods and algorithms have been developed
that can be used to identify GPI-anchor attachment signal consensus
sequences in polypeptides (see, e.g. Udenfriend et al. (1995)
Methods Enzymol. 250:571-582; Eisenhaber et al., (1999) J. Biol.
Chem. 292: 741-758, Kronegg and Buloz, (1999);
"Detection/prediction of GPI cleavage site (GPI-anchor) in a
protein (DGPI)", 129.194.185.165/dgpi/; Fankhauser et al., (2005)
Bioinformatics 21:1846-1852; Omaetxebarria et al., (2007)
Proteomics 7:1951-1960; Pierleoni et al., (2008) BMC Bioinformatics
9:392); including those that are readily available on bioinformatic
websites, such as the ExPASy Proteomics tools site
(expasy.ch/tools/). Thus, one of skill in the art can determine
whether a PH20 polypeptide contains a GPI-anchor attachment signal
sequence, and, therefore, whether the PH20 polypeptide is a
GPI-anchored protein.
H. Methods of Treatment and Uses of Extended Soluble PH20 and other
Soluble PH20 and Combination Therapy
[0333] Various forms of PH20 hyaluronidases have been prepared and
approved for therapeutic use in humans. For example, animal-derived
hyaluronidase preparations include Vitrase.RTM. (ISTA
Pharmaceuticals), a purified ovine testicular hyaluronidase, and
Amphadase.RTM. (Amphastar Pharmaceuticals), a bovine testicular
hyaluronidase. Hylenex.RTM. (Halozyme Therapeutics) is a human
recombinant hyaluronidase produced by genetically engineered
Chinese Hamster Ovary (CHO) cells containing nucleic acid encoding
for soluble rHuPH20. Approved therapeutic uses for hyaluronidase
include use as an adjuvant to increase the absorption and
dispersion of other therapeutic agents, for hypodermoclysis
(subcutaneous fluid administration), and as an adjunct in
subcutaneous urography for improving resorption of radiopaque
agents. In addition to these indications hyaluronidases can be used
as a therapeutic or cosmetic agent for the treatment of additional
diseases and conditions.
[0334] Hyaluronidases have also been used to enhance the activity
of chemotherapeutics and/or the accessibility of tumors to
chemotherapeutics (Schuller et al., 1991, Proc. Amer. Assoc. Cancer
Res. 32:173, abstract no. 1034; Czejka et al., 1990, Pharmazie
45:H.9). Combination chemotherapy with hyaluronidase is effective
in the treatment of a variety of cancers including urinary bladder
cancer (Horn et al., 1985, J. Surg. Oncol. 28:304-307), squamous
cell carcinoma (Kohno et al., 94, J. Cancer Res. Oncol.
120:293-297), breast cancer (Beckenlehner et al., 1992, J. Cancer
Res. Oncol. 118:591-596), and gastrointestinal cancer (Scheithauer
et al., 1988, Anticancer Res. 8:391-396). Hyaluronidase is
effective as the sole therapeutic agent in the treatment of brain
cancer (gliomas) (PCT published application no. WO88/02261,
published Apr. 7, 1988). Administration of hyaluronidase also
induces responsiveness of previously chemotherapy-resistant tumors
of the pancreas, stomach, colon, ovaries, and breast (Baumgartner
et al., 1988, Reg. Cancer Treat. 1:55-58; Zanker et al., 1986,
Proc. Amer. Assoc. Cancer Res. 27:390). Unfortunately, the
contaminants and non human nature of such hyaluronidases result in
anaphylactic reactions.
[0335] In addition to its indirect anticancer effects,
hyaluronidases also have direct anticarcinogenic effects.
Hyaluronidase prevents growth of tumors transplanted into mice (De
Maeyer et al., 1992, Int. J. Cancer 51:657-660) and inhibits tumor
formation upon exposure to carcinogens (Pawlowski et al., 1979,
Int. J. Cancer 23:105-109; Haberman et al., 1981, Proceedings of
the 17th Annual Meeting of the American Society of Clinical
Oncology, Washington, D.C., 22:105, abstract no. 415).
[0336] In particular, PH20 hyaluronidase can be used to treat
hyaluronan-associated diseases or conditions associated with high
interstitial fluid pressure, such as disc pressure, proliferative
disorders, such as cancer and benign prostatic hyperplasia, and
edema. Edema can result from or be manifested in, for example, from
organ transplant, stroke or brain trauma. Proliferative disorders
include, but are not limited to, cancer, smooth muscle cell
proliferation, systemic sclerosis, cirrhosis of the liver, adult
respiratory distress syndrome, idiopathic cardiomyopathy, lupus
erythematosus, retinopathy, e.g., diabetic retinopathy or other
retinopathies, cardiac hyperplasia, reproductive system associated
disorders, such as benign prostatic hyperplasia (BPH) and ovarian
cysts, pulmonary fibrosis, endometriosis, fibromatosis, harmatomas,
lymphangiomatosis, sarcoidosis, desmoid tumors. Cancers include
solid and lymphatic/blood tumors and metastatic disease, and
undifferentiated tumors. The tumors amenable to treatment typically
exhibit cellular and/or stromal expression of a hyaluronan,
compared to a non-cancerous tissue of the same tissue type or
compared to a non-metastatic tumor of the same tumor-type. Cancers
include any one or more of ovarian cancer, in situ carcinoma (ISC),
squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer,
other gastric cancers, non-small cell lung cancer, breast cancer,
brain cancer and colon cancer.
[0337] Hence, PH20 hyaluronidases have multiple uses, including and
in addition to their use as a spreading agent. Hyaluronidase is
commonly used, for example, for peribulbar block in local
anesthesia prior ophthalmic surgery. The presence of the enzyme
prevents the need for additional blocks and speeds the time to the
onset of akinesia (loss of eye movement). Peribulbar and
sub-Tenon's block are the most common applications of hyaluronidase
for ophthalmic procedures. Hyaluronidase also can promote akinesia
in cosmetic surgery, such as blepharoplasties and face lifts. It is
understood that soluble PH20 hyaluronidases provided herein,
including esPH20 hyaluronidases, can be used in any method of
treatment or combination therapy for which a PH20 hyaluronidase is
used (see e.g., U.S. Publication Nos. US20040268425; US20050260186;
US20060104968; and U.S. application Ser. Nos. 12/381,844,
12/386,249, 12/387,225 and 12/386,222, incorporated by reference in
their entirety). Exemplary therapeutic and cosmetic uses for
hyaluronidase are described below.
[0338] 1. Use as a Spreading Agent and Combination Therapy
[0339] As noted above, hyaluronidase is a spreading or diffusing
substance which modifies the permeability of connective tissue
through the hydrolysis of hyaluronic acid, a polysaccharide found
in the intercellular ground substance of connective tissue, and of
certain specialized tissues, such as the umbilical cord and
vitreous humor. When no spreading factor is present, materials
injected subcutaneously, such as drugs, proteins, peptides and
nucleic acid, spread very slowly. Co-injection with hyaluronidase,
however, can cause rapid spreading. The rate of diffusion is
proportional to the amount of enzyme, and the extent of diffusion
is proportional to the volume of solution.
[0340] PH20, including soluble PH20 such as esPH20 provided herein,
can be used to promote or enhance the delivery agents and molecules
to any of a variety of mammalian tissues in vivo. It can be used to
facilitate the diffusion and, therefore, promote the delivery, of
small molecule pharmacologic agents as well as larger molecule
pharmacologic agents, such as proteins, nucleic acids and
ribonucleic acids, and macromolecular compositions than can contain
a combination of components including, but not limited to, nucleic
acids, proteins, carbohydrates, lipids, lipid-based molecules and
drugs (see e.g. U.S. Publication Nos. US20040268425; US20050260186;
and US20060104968). PH20, including soluble PH20 such as esPH20 can
be co-administered and/or co-formulated with a therapeutic agent to
improve the bioavailability as well as pharmacokinetic (PK) and/or
pharmacodynamic (PD) characteristics of co-formulated or
co-administered agents. PK/PD parameters that can be improved by
using soluble PH20, such as esPH20, include such measures as
C.sub.max (the maximal concentration of agent achieved following
absorption in, e.g., the bloodstream), T.sub.max (the time required
to achieve maximal concentration), T.sub.1/2 (the time required for
the concentration to fall by half), C.sub.min (the minimal
concentration of agent following metabolism and excretion), AUC
(area under the curve of concentration versus time, a measure of
the overall amount of bioavailability), concentrations in various
tissues of interest (including, e.g., the rate of achieving desired
concentrations, the overall levels, and the duration of maintaining
desired levels), and E.sub.max (the maximal effect achieved).
[0341] The methods of treatment provided herein include combination
therapies with a therapeutic agent for the treatment of a disease
or disorder for which the therapeutic agent threats. Any
therapeutic agent that ameliorates and or otherwise lessens the
severity of a disease or condition can be combined with a soluble
PH20 provided herein in order to increase the bioavailability of
such therapeutic agent. In particular, soluble PH20 polypeptides
provided herein, such as esPH20s, can be used in each and all of
the combinations described in applications see e.g., U.S.
Publication Nos. US20040268425; US20050260186; US20060104968 and
U.S. Appl. Serial Nos. 12/381,844, 12/386,249, 12/387,225 and
12/386,222 in place of the disclosed hyaluronidase or hyaluronidase
degrading enzyme.
[0342] Soluble PH20 polypeptides provided herein, in particular
esPH20 polypeptides, can be administered prior, subsequently,
intermittently or simultaneously to the therapeutic agent
preparation. Generally, the soluble PH20 is administered prior to
or simultaneously with administration of the therapeutic agent
preparation to permit the soluble PH20 to degrade the hyaluronic
acid in the interstitial space. The soluble PH20 can be
administered at a site different from the site of administration of
the therapeutic molecule or the soluble PH20 can be administered at
a site the same as the site of administration of the therapeutic
molecule.
[0343] Examples of pharmaceutical, therapeutic and cosmetic agents
and molecules that can be administered with hyaluronidase include,
but are not limited to, a chemotherapeutic or anticancer agent, an
analgesic agent, an antibiotic agent, an anti-inflammatory agent,
an antimicrobial agent, an amoebicidal agent, a trichomonocidal
agent, an anti-parkinson agent, an anti-malarial agent, an
anticonvulsant agent, an anti-depressant agent, an anti-arthritics
agent, an anti-fungal agent, an antihypertensive agent, an
antipyretic agent, an anti-parasitic agent, an antihistamine agent,
an alpha-adrenergic agonist agent, an alpha blocker agent, an
anesthetic agent, a bronchial dilator agent, a biocide agent, a
bactericide agent, a bacteriostatic agent, a beta adrenergic
blocker agent, a calcium channel blocker agent, a cardiovascular
drug agent, a contraceptive agent, a cosmetic or esthetic agent, a
decongestant agent, a diuretic agent, a depressant agent, a
diagnostic agent, an electrolyte agent, a hypnotic agent, a hormone
agent, a hyperglycemic agent, a muscle relaxant agent, a muscle
contractant agent, an ophthalmic agent, a parasympathomimetic
agent, a psychic energizer agent, a sedative agent, a sleep
inducer, a sympathomimetic agent, a tranquilizer agent, a urinary
agent, a vaginal agent, a viricide agent, a vitamin agent, a
non-steroidal anti-inflammatory agent, or an angiotensin converting
enzyme inhibitor agent, and any combination thereof. In particular,
therapeutic agents include antibodies, including monoclonal
antibodies, bisphosphonates, insulins and immunoglobulins.
[0344] For example, exemplary antibiotic agents include, but are
not limited to, Aminoglycosides; Amphenicols; Ansamycins;
Carbacephems; Carbapenems; Cephalosporins or Cephems; Cephamycins;
Clavams; Cyclic lipopeptides; Diaminopyrimidines; Ketolides;
Lincosamides; Macrolides; Monobactams; Nitrofurans; Oxacephems;
Oxazolidinones; Penems, thienamycins and miscellaneous
beta-lactams; Penicillins; Polypeptides antibiotics; Quinolones;
Sulfonamides; Sulfones; Tetracyclines; and other antibiotics (such
as Clofoctols, Fusidic acids, Hexedines, Methenamines,
Nitrofurantoins Nitroxolines, Ritipenems, Taurolidines,
Xibomols).
[0345] Also included among exemplary therapeutic agents are blood
modifiers such as antihemophilic factors, anti-inhibitor coagulent
complexes, antithrombin IIIs, coagulations Factor Vhs, coagulation
Factor VIIIs, coagulation Factor IXs, plasma protein fractions, von
Willebrand factors; antiplatelet agents (including, for example,
abciximabs, anagrelides, cilostazols, clopidogrel bisulfates,
dipyridamoles, epoprostenols, eptifibatides, tirofibans; colony
stimulating factors (CSFs) (including, for example, Granulocyte
CSFs and Granulocyte Macrophage CSFs); erythropoiesis stimulators
(including, for example, erythropoietins such as darbepoetin alfas)
and epoetin alfas; hemostatics and albumins (including, for
example, aprotinins, combinations of antihemophilic factors and
plasma, Desmopressin Acetates, and albumins); immune globulins, as
well as hepatitis B immune globulins; thrombin inhibitors
(including for example direct thrombin inhibitors and lepirudin),
and drotecogin alfas; anticoagulants (including, for example,
dalteparins, enoxaperins and other heparins, and warfarins).
[0346] Other exemplary therapeutic agents that can be combined by
co-administration and/or co-formulation with a soluble PH20, such
as an esPH20, include, but are not limited to, Adalimumabs,
Agalsidase Betas, Alefacepts, Ampicillins, Anakinras,
Antipoliomyelitic Vaccines, Anti-Thymocytes, Azithromycins,
Becaplermins, Caspofungins, Cefazolins, Cefepimes, Cefotetans,
Ceftazidimes, Ceftriaxones, Cetuximabs, Cilastatins, Clavulanic
Acids, Clindamycins, Darbepoetin Alfas, Deaclizumabs, Diphtheria,
Diphtheria antitoxins, Diphtheria Toxoids, Efalizumabs,
Epinephrines, Erythropoietin Alphas, Etanercepts, Filgrastims,
Fluconazoles, Follicle-Stimulating Hormones, Follitropin Alphas,
Follitropin Betas, Fosphenyloins, Gadodiamides, Gadopentetates,
Gatifloxacins, Glatiramers, GM-CSF's, Goserelins, Goserelin
acetates, Granisetrons, Haemophilus Influenza B's, Haloperidols,
Hepatitis vaccines, Hepatitis A Vaccines, Hepatitis B Vaccines,
Ibritumomab Tiuxetans, Ibritumomabs, Tiuxetans, Immunoglobulins,
Hemophilus influenza vaccines, Influenza Virus Vaccines,
Infliximabs, Insulins, Insulin Glargines, Interferons, Interferon
alphas, Interferon Betas, Interferon Gammas, Interferon alpha-2a's,
Interferon alpha-2b's, Interferon alpha-1's, Interferon alpha-n3's,
Interferon Betas, Interferon Beta-1a's, Interferon Gammas,
Interferon alpha-consensus, Iodixanols, Iohexyls, Iopamidols,
Ioversols, Ketorolacs, Laronidases, Levofloxacins, Lidocaines,
Linezolids, Lorazepams, Measles Vaccines, Measles virus, Mumps
viruses, Measles-Mumps-Rubella Virus Vaccines, Rubella vaccines,
Medroxyprogesterones, Meropenems, Methylprednisolones, Midazolams,
Morphines, Octreotides, Omalizumabs, Ondansetrons, Palivizumabs,
Pantoprazoles, Pegaspargases, Pegfilgrastims, Peg-Interferon
Alfa-2a's, Peg-Interferon Alfa-2b's, Pegvisomants, Pertussis
vaccines, Piperacillins, Pneumococcal Vaccines and Pneumococcal
Conjugate Vaccines, Promethazines, Reteplases, Somatropins,
Sulbactams, Sumatriptans, Tazobactams, Tenecteplases, Tetanus
Purified Toxoids, Ticarcillins, Tositumomabs, Triamcinolones,
Triamcinolone Acetonides, Triamcinolone hexacetonides, Vancomycins,
Varicella Zoster immunoglobulins, Varicella vaccines, other
vaccines, Alemtuzumabs, Alitretinoins, Allopurinols, Altretamines,
Amifostines, Anastrozoles, Arsenics, Arsenic Trioxides,
Asparaginases, Bacillus Calmette-Guerin (BCG) vaccines, BCG Live,
Bexarotenes, Bleomycins, Busulfans, Busulfan intravenous, Busulfan
orals, Calusterones, Capecitabines, Carboplatins, Carmustines,
Carmustines with Polifeprosans, Celecoxibs, Chlorambucils,
Cisplatins, Cladribines, Cyclophosphamides, Cytarabines, Cytarabine
liposomals, Dacarbazines, Dactinomycins, Daunorubicin liposomals,
Daunorubicins, Daunomycins, Denileukin Diftitoxes, Dexrazoxanes,
Docetaxels, Doxorubicins, Doxorubicin liposomals, Dromostanolone
propionates, Elliott's B Solutions, Epirubicins, Epoetin alfas,
Estramustines, Etoposides, Etoposide phosphates, Etoposide VP-16s,
Exemestanes, Floxuridines, Fludarabines, Fluorouracils,
5-Fluorouracils, Fulvestrants, Gemcitabines, Gemtuzumabs,
Ozogamicins, Gemtuzumab ozogamicins, Hydroxyureas, Idarubicins,
Ifosfamides, Imatinib mesylates, Irinotecans, Letrozoles,
Leucovorins, Levamisoles, Lomustines, CCNUs, Mechlorethamines,
Nitrogen mustards, Megestrols, Megestrol acetates, Melphalans,
L-PAMs, Mercaptopurines, 6-Mercaptopurines, Mesnas, Methotrexates,
Methoxsalens, Mitomycins, Mitomycin C's, Mitotanes, Mitoxantrones,
Nandrolones, Nandrolone Phenpropionates, Nofetumomabs, Oprelvekins,
Oxaliplatins, Paclitaxels, Pamidronates, Pegademases, Pentostatins,
Pipobromans, Plicamycins, Mithramycins, Porfimers, Porfimer
sodiums, Procarbazines, Quinacrines, Rasburicases, Rituximabs,
Sargramostims, Streptozocins, Talcs, Tamoxifens, Temozolomides,
Teniposides, Testolactones, Thioguanines, 6-Thioguanines,
Triethylenethiophosphoramides (Thiotepas), Topotecans, Toremifenes,
Trastuzumabs, Tretinoins, Uracil Mustards, Valrubicins,
Vinblastines, Vincristines, Vinorelbines, Zoledronates, Acivicins,
Aclarubicins, Acodazoles, Acronines, Adozelesins, Aldesleukins,
Retinoic Acids, Alitretinoins, 9-Cis-Retinoic Acids, Alvocidibs,
Ambazones, Ambomycins, Ametantrones, Aminoglutethimides,
Amsacrines, Anaxirones, Ancitabines, Anthramycins, Apaziquones,
Argimesnas, Asperlins, Atrimustines, Azacitidines, Azetepas,
Azotomycins, Banoxantrones, Batabulins, Batimastats, Benaxibines,
Bendamustines, Benzodepas, Bicalutamides, Bietaserpines,
Biricodars, Bisantrenes, Bisnafide Dimesylates, Bizelesins,
Bortezomibs, Brequinars, Bropirimines, Budotitanes, Cactinomycins,
Canertinibs, Caracemides, Carbetimers, Carboquones, Carmofurs,
Carubicins, Carzelesins, Cedefingols, Cemadotins, Chiorambucils,
Cioteronels, Cirolemycins, Clanfenurs, Clofarabines, Crisnatols,
Decitabines, Dexniguldipines, Dexormaplatins,
[0347] Dezaguanines, Diaziquones, Dibrospidiums, Dienogests,
Dinalins, Disermolides, Dofequidars, Doxifluridines, Droloxifenes,
Duazomycins, Ecomustines, Edatrexates, Edotecarins, Eflomithines,
Elacridars, Elinafides, Elsamitrucins, Emitefurs, Enloplatins,
Enpromates, Enzastaurins, Epipropidines, Eptaloprosts, Erbulozoles,
Esorubicins, Etanidazoles, Etoglucids, Etoprines, Exisulinds,
Fadrozoles, Fazarabines, Fenretinides, Fluoxymesterones,
Fluorocitabines, Fosquidones, Fostriecins, Fotretamines,
Galarubicins, Galocitabines, Geroquinols, Gimatecans, Gimeracils,
Gloxazones, Glufosfamides, Ilmofosines, Ilomastats, Imexons,
Improsulfans, Indisulams, Inproquones, Interleukins,
Interleukin-2s, recombinant Interleukins, Intoplicines,
lobenguanes, Iproplatins, Irsogladines, Ixabepilones, Ketotrexates,
L-Alanosines, Lanreotides, Lapatinibs, Ledoxantrones, Leuprolides,
Leuprorelins, Lexacalcitols, Liarozoles, Lobaplatins, Lometrexols,
Lonafarnibs, Losoxantrones, Lurtotecans, Mafosfamides,
Mannosulfans, Marimastats, Masoprocols, Maytansines,
Mechiorethamines, Melengestrols, Meiphalans, Menogarils,
Mepitiostanes, Metesinds, Metomidates, Metoprines, Meturedepas,
Miboplatins, Miproxifenes, Misonidazoles, Mitindomides,
Mitocarcins, Mitocromins, Mitoflaxones, Mitogillins, Mitoguazones,
Mitomalcins, Mitonafides, Mitoquidones, Mitospers, Mitozolomides,
Mivobulins, Mizoribines, Mofarotenes, Mopidamols, Mubritinibs,
Mycophenolic Acids, Nedaplatins, Neizarabines, Nemorubicins,
Nitracrines, Nocodazoles, Nogalamycins, Nolatrexeds,
Nortopixantrones, Ormaplatins, Ortataxels, Oteracils, Oxisurans,
Oxophenarsines, Patubilones, Peldesines, Peliomycins, Pelitrexols,
Pemetrexeds, Pentamustines, Peplomycins, Perfosfamides,
Perifosines, Picoplatins, Pinafides, Piposulfans, Pirfenidones,
Piroxantrones, Pixantrones, Plevitrexeds, Plomestanes,
Porfiromycins, Prednimustines, Propamidines, Prospidiums,
Pumitepas, Puromycins, Pyrazofurins, Ranimustines, Riboprines,
Ritrosulfans, Rogletimides, Roquinimexs, Rufocromomycins,
Sabarubicins, Safingols, Satraplatins, Sebriplatins, Semustines,
Simtrazenes, Sizofurans, Sobuzoxanes, Sorafenibs, Sparfosates,
Sparfosic Acids, Sparsomycins, Spirogermaniums, Spiromustines,
Spiroplatins, Squalamines, Streptonigrins, Streptovarycins,
Sufosfamides, Sulofenurs, Tacedinalines, Talisomycins,
Tallimustines, Tariquidars, Tauromustines, Tecogalans, Tegafurs,
Teloxantrones, Temoporfins, Teroxirones, Thiamiprines, Tiamiprines,
Tiazofurins, Tilomisoles, Tilorones, Timcodars, Timonacics,
Tirapazamines, Topixantrones, Trabectedins, Ecteinascidin 743,
Trestolones, Triciribines, Trilostanes, Trimetrexates, Triplatin
Tetranitrates, Triptorelins, Trofosfamides, Tubulozoles, Ubenimexs,
Uredepas, Vaispodars, Vapreotides, Verteporfins, Vinbiastines,
Vindesines, Vinepidines, Vinflunines, Vinformides, Vinglycinates,
Vinleucinols, Vinleurosines, Vinrosidines, Vintriptols,
Vinzolidines, Vorozoles, Xanthomycin A's, Guamecyclines,
Zeniplatins, Zilascorbs [2-H], Zinostatins, Zorubicins,
Zosuquidars, Acetazolamides, Acyclovirs, Adipiodones,
Alatrofloxacins, Alfentanils, Allergenic extracts, Alpha
1-proteinase inhibitors, Aiprostadils, Amikacins, Amino acids,
Aminocaproic acids, Aminophyllines, Amitriptylines, Amobarbitals,
Amrinones, Analgesics, Anti-poliomyelitic vaccines, Anti-rabic
serums, Anti-tetanus immunoglobulins, tetanus vaccines,
Antithrombin III's, Antivenom serums, Argatrobans, Arginines,
Ascorbic acids, Atenolols, Atracuriums, Atropines,
Aurothioglucoses, Azathioprines, Aztreonams, Bacitracins,
Baclofens, Basiliximabs, Benzoic acids, Benztropines,
Betamethasones, Biotins, Bivalirudins, Botulism antitoxins,
Bretyliums, Bumetanides, Bupivacaines, Buprenorphines,
Butorphanols, Calcitonins, Calcitriols, Calciums, Capreomycins,
Carboprosts, Carnitines, Cefaniandoles, Cefoperazones, Cefotaximes,
Cefoxitins, Ceftizoximes, Cefuroximes, Chioramphenicols,
Chloroprocaines, Chloroquines, Chlorothiazides, Chiorpromazines,
Chondroitinsulfuric acids, Choriogonadotropin alfas, Chromiums,
Cidofovirs, Cimetidines, Ciprofloxacins, Cisatracuriums,
Clonidines, Codeines, Coichicines, Colistins, Collagens,
Corticorelin ovine triflutates, Corticotrophins, Cosyntropins,
Cyanocobalamins, Cyclosporines, Cysteines, Dacliximabs,
Dalfopristins, Dalteparins, Danaparoids, Dantrolenes,
Deferoxamines, Desmopressins, Dexamethasones, Dexmedetomidines,
Dexpanthenols, Dextrans, Iron dextrans, Diatrizoic acids,
Diazepams, Diazoxides, Dicyclomines, Digibinds, Digoxins,
Dihydroergotamines, Diltiazems, Diphenhydramines, Dipyridamoles,
Dobutamines, Dopamines, Doxacuriums, Doxaprams, Doxercalciferols,
Doxycyclines, properidols, Dyphyllines, Edetic acids, Edrophoniums,
Enalaprilats, Ephedrines, Epoprostenols, Ergocalciferols,
Ergonovines, Ertapenems, Erythromycins, Esmolols, Estradiols,
Estrogenics, Ethacrynic acids, Ethanolamines, Ethanols, Ethiodized
oils, Etidronic acids, Etomidates, Factor VIII's, Famotidines,
Fenoldopams, Fentanyls, Flumazenils, Fluoresceins, Fluphenazines,
Folic acids, Fomepizoles, Fomivirsens, Fondaparinuxs, Foscarnets,
Fosphenyloins, Furosemides, Gadoteridols, Gadoversetamides,
Ganciclovirs, Gentamicins, Glucagons, Glucoses, Glycines,
Glycopyrrolates, Gonadorelins, Gonadotropin chorionics, Haemophilus
B polysaccarides, Hemins, Herbals, Histamines, Hydralazines,
Hydrocortisones, Hydromorphones, Hydroxocobalamins, Hydroxyzines,
Hyoscyamines, Ibutilides, Imiglucerases, Indigo carmines,
Indomethacins, Iodides, Iopromides, Iothalamic acids, loxaglic
acids, Ioxilans, Isoniazids, Isoproterenols, Japanese encephalitis
vaccines, Kanamycins, Ketamines, Labetalols, Lepirudins,
Levobupivacaines, Levothyroxines, Lincomycins, Liothyronines,
Luteinising hormones, Lyme disease vaccines, Mangafodipirs,
Manthtols, Meningococcal polysaccharide vaccines, Meperidines,
Mepivacaines, Mesoridazines, Metaraminols, Methadones,
Methocarbamols, Methohexitals, Methyldopates, Methylergonovines,
Metoclopramides, Metoprolols, Metronidazoles, Minocyclines,
Mivacuriums, Morrhuic acids, Moxifloxacins, Muromonab-CD3s,
Mycophenolate mofetils, Nafcillins, Nalbuphines, Nalmefenes,
Naloxones, Neostigmines, Niacinamides, Nicardipines,
Nitroglycerins, Nitroprussides, Norepinephrines, Orphenadrines,
Oxacillins, Oxymorphones, Oxytetracyclines, Oxytocins,
Pancuroniums, Panthenols, Pantothenic acids, Papaverines,
Peginterferon-alpha (e.g. interferon alpha 2a or 2b), Penicillin
Gs, Pentamidines, Pentazocines, Pentobarbitals, Perfiutrens,
Perphenazines, Phenobarbitals, Phentolamines,
[0348] Phenylephrines, Phenyloins, Physostigmines, Phytonadiones,
Polymyxin bs, Pralidoximes, Prilocalnes, Procainamides, Procaines,
Prochiorperazines, Progesterones, Propranolols, Pyridostigmine
hydroxides, Pyridoxines, Quinidines, Quinupristins, Rabies
immunoglobulins, Rabies vaccines, Ranitidines, Remifentanils,
Riboflavins, Rifampins, Ropivacaines, Samariums, Scopolamines,
Seleniums, Sermorelins, Sincalides, Somatrems, Spectinomycins,
Streptokinases, Streptomycins, Succinylcholines, Sufentanils,
Sulfamethoxazoles, Tacrolirnuss, Terbutalines, Teriparatides,
Testosterones, Tetanus antitoxins, Tetracaines, Tetradecyl
sulfates, Theophyllines, Thiamines, Thiethylperazines, Thiopentals,
Thyroid stimulating hormones, Tinzaparins, Tirofibans, Tobramycins,
Tolazolines, Tolbutamides, Torsemides, Tranexamic acids,
Treprostinils, Trifluoperazines, Trimethobenzamides, Trimethoprims,
Tromethamines, Tuberculins, Typhoid vaccines, Urofollitropins,
Urokinases, Vaiproic acids, Vasopressins, Vecuroniums, Verapamils,
Voriconazoles, Warfarins, Yellow fever vaccines, Zidovudines,
Zincs, Ziprasidone hydrochlorides, Aclacinomycins, Actinomycins,
Adriamycins, Azaserines, 6-Azauridines, Carzinophilins,
Chromomycins, Denopterins, 6-Diazo-5-Oxo-L-Norleucines,
Enocitabines, Loxuridines, Olivomycines, Pirarubicins, Piritrexims,
Pteropterins, Tagafurs, Tubercidins, Alteplases, Arcitumomabs,
bevacizumabs, Botulinum Toxin Type A's, Botulinum Toxin Type B's,
Capromab Pendetides, Daclizumabs, Dornase alfas, Drotrecogin alfas,
Imciromab Pentetates, and Iodine-131's.
[0349] In particular, therapeutic agents include, but are not
limited to, immunoglobulins, Interferon beta, Interferon alpha-2
as, Interferon alpha-1s, Interferon alpha-n3s, Interferon beta-1,
Interferon beta-1 as, Interferon gamma-lbs, Peg-interferon alpha-2
and Peginterferon alpha-2bs, insulin, a bisphosphate (e.g.
Pamidronates or Zoledronates), Docetaxels, Doxorubincins,
Doxorubicin liposomals and bevacizumabs.
[0350] 2. Use to Remove Excess Glycosaminoglycanases
[0351] Provided herein are methods for treating
hyaluronan-associated diseases and conditions by administration of
a composition containing a soluble PH20, typically a soluble
hyaluronidase either alone or in combination with or in addition to
another treatment and/or agent. Hyaluronan-associated conditions
and diseases are diseases and conditions in which hyaluronan levels
are elevated as cause, consequence or otherwise observed in the
disease or condition, and can be treated by administration of a
composition hyaluronidases, such as a soluble PH20, either alone or
in combination with or in addition to another treatment and/or
agent.
[0352] Typically, hyaluronan-associated diseases and conditions are
associated with elevated hyaluronan expression in a tissue, cell,
or body fluid (e.g. tumor tissue or tumor-associated tissue, blood,
or interstitial space) compared to a control, e.g. another tissue,
cell or body fluid. The elevated hyaluronan expression can be
elevated compared to a normal tissue, cell or body fluid, for
example, a tissue, cell or body fluid that is analogous to the
sample being tested, but isolated from a different subject, such as
a subject that is normal (i.e. does not have a disease or
condition, or does not have the type of disease or condition that
the subject being tested has), for example, a subject that does not
have a hyaluronan-associated disease or condition. The elevated
hyaluronan expression can be elevated compared to an analogous
tissue from another subject that has a similar disease or
condition, but whose disease is not as severe and/or is not
hyaluronan-associated or expresses relatively less hyaluronan and
thus is hyaluronan-associated to a lesser degree. For example, the
subject being tested can be a subject with a hyaluronan-associated
cancer, where the HA amounts in the tissue, cell or fluid are
relatively elevated compared to a subject having a less severe
cancer, such as an early stage, differentiated or other type of
cancer. In another example, the cell, tissue or fluid contains
elevated levels of hyaluronan compared to a control sample, such as
a fluid, tissue, extract (e.g. cellular or nuclear extract),
nucleic acid or peptide preparation, cell line, biopsy, standard or
other sample, with a known amount or relative amount of HA, such as
a sample, for example a tumor cell line, known to express
relatively low levels of HA, such as exemplary tumor cell lines
described herein that express low levels of HA, for example, the
HCT 116 cell line, the HT29 cell line, the NCl H460 cell line, the
DU145 cell line, the Capan-1 cell line, and tumors from tumor
models generated using such cell lines.
[0353] In some cases, hyaluronan-associated diseases and conditions
are associated with increased interstitial fluid pressure,
decreased vascular volume, and/or increased water content in a
tissue, such as a tumor. In one example, treatment with the
compositions and compounds provided herein ameliorates one or more
of these symptoms or other symptoms associated with the disease or
condition, for example, improves survival or quality of life of the
subject over time, or inhibits tumor growth.
[0354] Exemplary hyaluronan-associated diseases and conditions that
can be treated using the provided enzymes, compositions and
methods, include, but are not limited to, hyaluronan-rich cancers,
for example, tumors, including solid tumors such as late-stage
cancers, a metastatic cancers, undifferentiated cancers, ovarian
cancer, in situ carcinoma (ISC), squamous cell carcinoma (SCC),
prostate cancer, pancreatic cancer, non-small cell lung cancer,
breast cancer, colon cancer and other cancers.
[0355] Also exemplary of hyaluronan-associated diseases and
conditions are diseases that are associated with elevated
interstitial fluid pressure, such as diseases associated with disc
pressure, and edema, for example, edema caused by organ transplant,
stroke, brain trauma or other injury. Exemplary
hyaluronan-associated diseases and conditions include diseases and
conditions associated with elevated interstitial fluid pressure,
decreased vascular volume, and/or increased water content in a
tissue, including cancers, disc pressure and edema. In one example,
treatment of the hyaluronan-associated condition, disease or
disorder includes amelioration, reduction, or other beneficial
effect on one or more of increased interstitial fluid pressure
(IFP), decreased vascular volume, and increased water content in a
tissue.
[0356] Typically, the hyaluronan-associated disease or condition is
associated with increased HA expression, for example, in a diseased
tissue, for example, a tumor. In one example, HALOs (pericellular
matrix regions that are rich in proteoglycans, including
hyaluronan) form in a tissue of the subject, for example, in a
diseased tissue. In another example, the presence of HALOs is
detected in an in vitro culture of cells from a tissue of the
subject, for example, a diseased tissue.
[0357] a. Use in Cancer Treatment
[0358] Hyaluronidase has direct anticarcinogenic effects by
degradation of hyaluronic acid in tumors. Thus, soluble PH20
hyaluronidases, such as esPH20, can be used to treat tumors, in
particular, tumors that are hyaluronan rich. The hyaluronan-rich
cancer can be a cancer in which the cancer cells produce HALOs,
cancers that have elevated expression of hyaluronan (as determined
by immunostaining, e.g. histological staining of sections from the
tumor), cancers that have elevated HAS2 (Hyaluronan synthase 2),
cancers that do not produce hyaluronidase (HYAL1) in vitro.
Hyaluronan-rich cancers can be identified by any method for
assessing hyaluronan expression, and other known methods for
assaying protein/mRNA expression.
[0359] Several hyaluronan-rich cancers have been identified. In
some cases, hyaluronan expression correlates with poor prognosis,
for example, decreased survival rate and/or recurrence-free
survival rate, metastases, angiogenesis, cancer cell invasion into
other tissues/areas, and other indicators of poor prognosis. Such
correlation has been observed, for example, in hyaluronan-rich
tumors including ovarian cancer, SCC, ISC, prostate cancer, lung
cancer, including non-small-cell lung cancer (NSCLC), breast
cancer, colon cancer and pancreatic cancer (see, for example,
Maarit et al., Cancer Research, 60:150-155 (2000); Karvinen et al.,
British Journal of Dermatology, 148:86-94 (2003); Lipponen et al.,
Eur. Journal of Cancer, 849-856 (2001); Pirinen et al., Int. J.
Cancer: 95: 12-17 (2001); Auvinen et al., American Journal of
Pathology, 156(2):529-536 (2000); Ropponen et al., Cancer Research,
58: 342-347 (1998)). Thus, hyaluronan-rich cancers can be treated
by administration of a hyaluronidase, such as a soluble PH20, to
treat one or more symptoms of the cancer. Hyaluronan-rich tumors
include, but are not limited to, prostate, breast, colon, ovarian,
stomach, head and neck and other tumors and cancers.
[0360] Hyaluronidases can also be used to increase the sensitivity
of tumors that are resistant to conventional chemotherapy. For
example, a hyaluronidase, such as soluble PH20, can be administered
to a patient having a tumor associated with a HYAL1 defect in an
amount effective to increase diffusion around the tumor site (e.g.,
to facilitate circulation and/or concentrations of chemotherapeutic
agents in and around the tumor site), inhibit tumor cell motility,
such as by hyaluronic acid degradation, and/or to lower the tumor
cell apoptosis threshold. This can bring the tumor cell(s) to a
state of anoikis, which renders the tumor cell more susceptible to
the action of chemotherapeutic agents. Administration of a
hyaluronidase can induce responsiveness of previously
chemotherapy-resistant tumors of the pancreas, stomach, colon,
ovaries, and breast (Baumgartner et al. (1988) Reg. Cancer Treat.
1:55-58; Zanker et al. (1986) Proc. Amer. Assoc. Cancer Res.
27:390). Thus, in addition to treatment of a cancer with a soluble
PH20 alone, the compositions and methods provided herein also can
be used to treat hyaluronan-associated cancers by administration of
a soluble PH20 in combination with, for example, simultaneously or
prior to, a chemotherapeutic or other anti-cancer agent or
treatment. In this example, the hyaluronidase, such as a soluble
PH20, typically enhances penetration of chemotherapeutic or other
anti-cancer agents into solid tumors, thereby treating the
disease.
[0361] Compositions containing soluble PH20 can be injected
intratumorally with anti-cancer agents or intravenously for
disseminated cancers or hard to reach tumors. The anticancer agent
can be a chemotherapeutic, an antibody, a peptide, or a gene
therapy vector, virus or DNA. Additionally, hyaluronidase can be
used to recruit tumor cells into the cycling pool for sensitization
in previously chemorefractory tumors that have acquired multiple
drug resistance (St Croix et al., (1998) Cancer Lett September
131(1): 35-44).
[0362] Exemplary anti-cancer agents that can be administered after,
coincident with or before administration of a soluble PH20, such as
an esPH20, include, but are not limited to Acivicins; Aclarubicins;
Acodazoles; Acronines; Adozelesins; Aldesleukins; Alemtuzumabs;
Alitretinoins (9-Cis-Retinoic Acids); Allopurinols; Altretamines;
Alvocidibs; Ambazones; Ambomycins; Ametantrones; Amifostines;
Aminoglutethimides; Amsacrines; Anastrozoles; Anaxirones;
Ancitabines; Anthramycins; Apaziquones; Argimesnas; Arsenic
Trioxides; Asparaginases; Asperlins; Atrimustines; Azacitidines;
Azetepas; Azotomycins; Banoxantrones; Batabulins; Batimastats; BCG
Live; Benaxibines; Bendamustines; Benzodepas; Bexarotenes;
Bevacizumab; Bicalutamides; Bietaserpines; Biricodars; Bisantrenes;
Bisantrenes; Bisnafide Dimesylates; Bizelesins; Bleomycins;
Bortezomibs; Brequinars; Bropirimines; Budotitanes; Busulfans;
Cactinomycins; Calusterones; Canertinibs; Capecitabines;
Caracemides; Carbetimers; Carboplatins; Carboquones; Carmofurs;
Carmustines with Polifeprosans; Carmustines; Carubicins;
Carzelesins; Cedefingols; Celecoxibs; Cemadotins; Chlorambucils;
Cioteronels; Cirolemycins; Cisplatins; Cladribines; Clanfenurs;
Clofarabines; Crisnatols; Cyclophosphamides; Cytarabine liposomals;
Cytarabines; Dacarbazines; Dactinomycins; Darbepoetin Alfas;
Daunorubicin liposomals; Daunorubicins/Daunomycins; Daunorubicins;
Decitabines; Denileukin Diftitoxes; Dexniguldipines;
Dexonnaplatins; Dexrazoxanes; Dezaguanines; Diaziquones;
Dibrospidiums; Dienogests; Dinalins; Disermolides; Docetaxels;
Dofequidars; Doxifluridines; Doxorubicin liposomals; Doxorubicin
HCL; Docorubicin HCL liposome injection; Doxorubicins;
Droloxifenes; Dromostanolone Propionates; Duazomycins; Ecomustines;
Edatrexates; Edotecarins; Eflornithines; Elacridars; Elinafides;
Elliott's B Solutions; Elsamitrucins; Emitefurs; Enloplatins;
Enpromates; Enzastaurins; Epipropidines; Epirubicins; Epoetin
alfas; Eptaloprosts; Erbulozoles; Esorubicins; Estramustines;
Etanidazoles; Etoglucids; Etoposide phosphates; Etoposide VP-16s;
Etoposides; Etoprines; Exemestanes; Exisulinds; Fadrozoles;
Fazarabines; Fenretinides; Filgrastims; Floxuridines; Fludarabines;
Fluorouracils; 5-fluorouracils; Fluoxymesterones; Fluorocitabines;
Fosquidones; Fostriecins; Fostriecins; Fotretamines; Fulvestrants;
Galarubicins; Galocitabines; Gemcitabines; Gemtuzumabs/Ozogamicins;
Geroquinols; Gimatecans; Gimeracils; Gloxazones; Glufosfamides;
Goserelin acetates; Hydroxyureas; Ibritumomabs/Tiuxetans;
Idarubicins; Ifosfamides; Ilmofosines; Ilomastats; Imatinib
mesylates; Imexons; Improsulfans; Indisulams; Inproquones;
Interferon alfa-gas; Interferon alfa-lbs; Interferon Alfas;
Interferon Betas; Interferon Gammas; Interferons; Interleukin-2s
and other Interleukins (including recombinant Interleukins);
Intoplicines; Iobenguanes [131-I]; Iproplatins; Irinotecans;
Irsogladines; Ixabepilones; Ketotrexates; L-Alanosines;
Lanreotides; Lapatinibs; Ledoxantrones; Letrozoles; Leucovorins;
Leuprolides; Leuprorelins (Leuprorelides); Levamisoles;
Lexacalcitols; Liarozoles; Lobaplatins; Lometrexols;
Lomustines/CCNUs; Lomustines; Lonafarnibs; Losoxantrones;
Lurtotecans; Mafosfamides; Mannosulfans; Marimastats; Masoprocols;
Maytansines; Mechlorethamines; Mechlorethamines/Nitrogen mustards;
Megestrol acetates; Megestrols; Melengestrols; Melphalans;
MelphalanslL-PAMs; Menogarils; Mepitiostanes; Mercaptopurines;
6-Mercaptopurine; Mesnas; Metesinds; Methotrexates; Methoxsalens;
Metomidates; Metoprines; Meturedepas; Miboplatins; Miproxifenes;
Misonidazoles; Mitindomides; Mitocarcins; Mitocromins;
Mitoflaxones; Mitogillins; Mitoguazones; Mitomalcins; Mitomycin Cs;
Mitomycins; Mitonafides; Mitoquidones; Mitospers; Mitotanes;
Mitoxantrones; Mitozolomides; Mivobulins; Mizoribines; Mofarotenes;
Mopidamols; Mubritinibs; Mycophenolic Acids; Nandrolone
Phenpropionates; Nedaplatins; Nelzarabines; Nemorubicins;
Nitracrines; Nocodazoles; Nofetumomabs; Nogalamycins; Nolatrexeds;
Nortopixantrones; Octreotides; Oprelvekins; Ormaplatins;
Ortataxels; Oteracils; Oxaliplatins; Oxisurans; Oxophenarsines;
Paclitaxels; Pamidronates; Patubilones; Pegademases; Pegaspargases;
Pegfilgrastims; Peldesines; Peliomycins; Pelitrexols; Pemetrexeds;
Pentamustines; Pentostatins; Peplomycins; Perfosfamides;
Perifosines; Picoplatins; Pinafides; Pipobromans; Piposulfans;
Pirfenidones; Piroxantrones; Pixantrones; Plevitrexeds; Plicamycid
Mithramycins; Plicamycins; Plomestanes; Plomestanes; Porfimer
sodiums; Porfimers; Porfiromycins; Prednimustines; Procarbazines;
Propamidines; Prospidiums; Pumitepas; Puromycins; Pyrazofurins;
Quinacrines; Ranimustines; Rasburicases; Riboprines; Ritrosulfans;
Rituximabs; Rogletimides; Roquinimexs; Rufocromomycins;
Sabarubicins; Safingols; Sargramostims; Satraplatins; Sebriplatins;
Semustines; Simtrazenes; Sizofurans; Sobuzoxanes; Sorafenibs;
Sparfosates; Sparfosic Acids; Sparsomycins; Spirogermaniums;
Spiromustines; Spiroplatins; Spiroplatins; Squalamines;
Streptonigrins; Streptovarycins; Streptozocins; Sufosfamides;
Sulofenurs; Sunitinib Malate; 6-TG; Tacedinalines; Talcs;
Talisomycins; Tallimustines; Tamoxifens; Tariquidars;
Tauromustines; Tecogalans; Tegafurs; Teloxantrones; Temoporfins;
Temozolomides; Teniposides/VM-26s; Teniposides; Teroxirones;
Testolactones; Thiamiprines; Thioguanines; Thiotepas; Tiamiprines;
Tiazofurins; Tilomisoles; Tilorones; Timcodars; Timonacics;
Tirapazamines; Topixantrones; Topotecans; Toremifenes;
Tositumomabs; Trabectedins (Ecteinascidin 743); Trastuzumabs;
Trestolones; Tretinoins/ATRA; Triciribines; Trilostanes;
Trimetrexates; Triplatin Tetranitrates; Triptorelins;
Trofosfamides; Tubulozoles; Ubenimexs; Uracil Mustards; Uredepas;
Valrubicins; Valspodars; Vapreotides; Verteporfins; Vinblastines;
Vincristines; Vindesines; Vinepidines; Vinflunines; Vinformides;
Vinglycinates; Vinleucinols; Vinleurosines; Vinorelbines;
Vinrosidines; Vintriptols; Vinzolidines; Vorozoles; Xanthomycin A's
(Guamecyclines); Zeniplatins; Zilascorbs [2-H]; Zinostatins;
Zoledronate; Zorubicins; and Zosuquidars, for example:
[0363] Aldesleukins (e.g. PROLEUKIN.RTM.); Alemtuzumabs (e.g.
CAMPATH.RTM.); Alitretinoins (e.g. PANRETIN.RTM.); Allopurinols
(e.g. ZYLOPRIM.RTM.); Altretamines (e.g. HEXALEN.RTM.); Amifostines
(e.g. ETHYOL.RTM.); Anastrozoles (e.g. ARIMIDEX.RTM.); Arsenic
Trioxides (e.g. TRISENOX.RTM.); Asparaginases (e.g. ELSPAR.RTM.);
BCG Live (e.g. TICE.RTM. BCG); Bexarotenes (e.g. TARGRETIN.RTM.);
Bevacizumab (AVASTIN.RTM.); Bleomycins (e.g. BLENOXANE.RTM.);
Busulfan intravenous (e.g. BUSULFEX.RTM.); Busulfan orals (e.g.
MYLERAN.TM.); Calusterones (e.g. METHOSARB.RTM.); Capecitabines
(e.g. XELODA.RTM.); Carboplatins (e.g. PARAPLATIN.RTM.);
Carmustines (e.g. BCNU.RTM., BiCNU.RTM.); Carmustines with
Polifeprosans (e.g. GLIADEL.RTM. Wafer); Celecoxibs (e.g.
CELEBREX.RTM.); Chlorambucils (e.g. LEUKERAN.RTM.); Cisplatins
(e.g. PLATINOL.RTM.); Cladribines (e.g. LEUSTATIN.RTM.,
2-CdA.RTM.); Cyclophosphamides (e.g. CYTOXAN.RTM., NEOSAR.RTM.);
Cytarabines (e.g. CYTOSAR-U.RTM.); Cytarabine liposomals (e.g.
DepoCyt.RTM.); Dacarbazines (e.g. DTIC-Domeu): Dactinomycins (e.g.
COSMEGEN.RTM.); Darbepoetin Alfas (e.g. ARANESP.RTM.); Daunorubicin
liposomals (e.g. DANUOXOME.RTM.); Daunorubicins/Daunomycins (e.g.
CERUBIDINE.RTM.); Denileukin Diftitoxes (e.g. ONTAK.RTM.);
Dexrazoxanes (e.g. ZINECARD.RTM.); Docetaxels (e.g. TAXOTERE.RTM.);
Doxorubicins (e.g. ADRIAMYCIN.RTM., RUBEX.RTM.); Doxorubicin
liposomals, including Docorubicin HCL liposome injections (e.g.
DOXIL.RTM.); Dromostanolone propionates (e.g. DROMOSTANOLONE.RTM.
and MASTERONE.RTM. Injection); Elliott's B Solutions (e.g.
Elliott's B Solution.RTM.); Epirubicins (e.g. ELLENCE.RTM.);
Epoetin alfas (e.g. EPOGEN.RTM.); Estramustines (e.g. EMCYT.RTM.);
Etoposide phosphates (e.g. ETOPOPHOS.RTM.); Etoposide VP-16s (e.g.
VEPESID.RTM.); Exemestanes (e.g. AROMASIN.RTM.); Filgrastims (e.g.
NEUPOGEN.RTM.); Floxuridines (e.g. FUDR.RTM.); Fludarabines (e.g.
FLUDARA.RTM.); Fluorouracils incl. 5-FUs (e.g. ADRUCIL.RTM.);
Fulvestrants (e.g. FASLODEX.RTM.); Gemcitabines (e.g. GEMZAR.RTM.);
Gemtuzumabs/Ozogamicins (e.g. MYLOTARG.RTM.); Goserelin acetates
(e.g. ZOLADEX.RTM.); Hydroxyureas (e.g. HYDREA.RTM.);
Ibritumomabs/Tiuxetans (e.g. ZEVALIN.RTM.); Idarubicins (e.g.
IDAMYCIN.RTM.); Ifosfamides (e.g. IFEX.RTM.); Imatinib mesylates
(e.g. GLEEVEC.RTM.); Interferon alfa-2 as (e.g. ROFERON-A.RTM.);
Interferon alfa-2bs (e.g. INTRON A.RTM.); Irinotecans (e.g.
CAMPTOSAR.RTM.); Letrozoles (e.g. FEMARA.RTM.); Leucovorins (e.g.
WELLCOVORIN.RTM., LEUCOVORIN.RTM.); Levamisoles (e.g.
ERGAMISOL.RTM.); Lomustines/CCNUs (e.g. CeeBU.RTM.);
Mechlorethamines/Nitrogen mustards (e.g. MUSTARGEN.RTM.); Megestrol
acetates (e.g. MEGACE.RTM.); Melphalans/L-PAMs (e.g. ALKERAN.RTM.);
Mercaptopurine incl. 6-MPs (e.g. PURINETHOL.RTM.); Mesnas (e.g.
MESNEX.RTM.); Methotrexates; Methoxsalens (e.g. UVADEX.RTM.);
Mitomycin Cs (e.g. MUTAMYCIN.RTM., MITOZYTREX.RTM.); Mitotanes
(e.g. LYSODREN.RTM.); Mitoxantrones (e.g. NOVANTRONE.RTM.);
Nandrolone Phenpropionates (e.g. DURABOLIN-50.RTM.); Nofetumomabs
(e.g. VERLUMA.RTM.); Oprelvekins (e.g. NEUMEGA.RTM.); Oxaliplatins
(e.g. ELOXATIN.RTM.); Paclitaxels (e.g. PAXENE.RTM., TAXOL.RTM.);
Pamidronates (e.g. AREDIA.RTM.); Pegademases (e.g. ADAGEN.RTM.);
Pegaspargases (e.g. ONCASPAR.RTM.); Pegfilgrastims (e.g.
NEULASTA.RTM.); Pentostatins (e.g. NIPENT.RTM.); Pipobromans (e.g.
VERCYTE.RTM.); Plicamycin/Mithramycins (e.g. MITHRACIN.RTM.);
Porfimer sodiums (e.g. PHOTOFRIN.RTM.); Procarbazines (e.g.
MATULANE.RTM.); Quinacrines (e.g. ATABRINE.RTM.); Rasburicases
(e.g. ELITEK.RTM.); Rituximabs (e.g. RITUXAN.RTM.); Sargramostims
(e.g. PROKINE.RTM.); Streptozocins (e.g. ZANOSAR.RTM.); Sunitinib
Malates (e.g. SUTENT.RTM.); Talcs (e.g. SCLEROSOL.RTM.); Tamoxifens
(e.g. NOLVADEX.RTM.); Temozolomides (e.g. TEMODAR.RTM.);
Teniposides/VM-26s (e.g. VUMON.RTM.); Testolactones (e.g.
TESLAC.RTM.); Thioguanines incl. 6-TG; Thiotepas (e.g.
THIOPLEX.RTM.); Topotecans (e.g. HYCAMTIN.RTM.); Toremifenes (e.g.
FARESTON.RTM.); Tositumomabs (e.g. BEXXAR.RTM.); Trastuzumabs (e.g.
HERCEPTIN.RTM.); Tretinoins/ATRA (e.g. VESANOID.RTM.); Uracil
Mustards; Valrubicins (e.g. VALSTAR.RTM.); Vinblastines (e.g.
VELBAN.RTM.); Vincristines (e.g. ONCOVIN.RTM.); Vinorelbines (e.g.
NAVELBINE.RTM.); and Zoledronates (e.g. ZOMETA.RTM.).
[0364] In one example, a soluble PH20, such as an esPH20, for
example, PEGylated rHuPH20, is administered to a subject after,
coincident with or before administration of one or more of
docetaxel (e.g. TAXOTERE.RTM.), Doxorubicin liposomal (e.g.
DOXIL.RTM.), Sunitinib Malate (e.g. SUTENT.RTM.) or Bevacizumab
(AVASTIN.RTM.).
[0365] Hence, soluble PH20 polypeptides provided herein can be used
in the treatment of metastatic and non-metastatic cancers,
including those that have decreased endogenous hyaluronidase
activity relative to non-cancerous cells. Hyaluronidases can be
used as a chemotherapeutic agent alone or in combination with other
chemotherapeutics. Exemplary cancers include, but are not limited
to, small lung cell carcinoma, squamous lung cell carcinoma, and
cancers of the breast, ovaries, head and neck, or any other cancer
associated with depressed levels of hyaluronidase activity or
decreased hyaluronic acid catabolism.
[0366] b. Use in Treatment of Glycosaminoglycan Accumulation in the
Brain
[0367] Hyaluronic acid levels are elevated in a number of
cerebrospinal pathologic conditions. Levels of cerebrospinal
hyaluronic acid are normally less than 200 .mu.g/L in adults
(Laurent et al. (1996) Acta Neurol Scand September 94(3):194-206),
but can elevate to levels of over 8000 .mu.g/L in diseases such as
meningitis, spinal stenosis, head injury and cerebral infarction.
Hyaluronidases, such as, for example, soluble rHuPH20, can be
utilized to degrade critically elevated levels of substrate.
[0368] The lack of effective lymphatics in the brain also can lead
to life threatening edema following head trauma. Hyaluronic acid
accumulation is a result of increased synthesis by hyaluronic acid
synthases and decreased degradation. Accumulation of hyaluronic
acid can initially serve the beneficial purpose of increasing water
content in the damaged tissue to facilitate leukocyte
extravasation, but continued accumulation can be lethal.
Administration of hyaluronidase, such as intrathecally or
intravenously, to a patient suffering from head trauma can serve to
remove tissue hyaluronic acid accumulation and the water associated
with it.
[0369] Soluble PH20 also can be used in the treatment of edema
associated with brain tumors, particularly that associated with
glioblastoma multiform. The edema associated with brain tumors
results from the accumulation of hyaluronic acid in the
non-cancerous portions of the brain adjacent the tumor.
Administration of a soluble PH20 hyaluronidase to the sites of
hyaluronic acid accumulation (e.g., by intravenous injection or via
a shunt) can relieve the edema associated with such malignancies by
degrading the excess hyaluronic acid at these sites.
[0370] c. Use in Treatment of Glycosaminoglycan Accumulation in
Cardiovascular Disease
[0371] Soluble PH20 hyaluronidases can be used in the treatment of
some cardiovascular disease. Administration of hyaluronidase in
animal models following experimental myocardial infarct can reduce
infarct size (Maclean, et al (1976) Science 194(4261):199-200). One
proposed mechanism by which this can occur is by reducing
hyaluronic acid accumulation that occurs following ischemia
reperfusion. Reduction of infarct size is believed to occur from
increased lymph drainage and increased tissue oxygenation and
reduction of myocardial water content.
[0372] Soluble PH20 hyaluronidases also can be used to limit
coronary plaques from arteriosclerosis. Such plaques accumulate
glycosaminoglycans and mediate macrophage and foam cell adhesion
(Kolodgie et al. (2002) Arterioscler Thromb Vasc Biol.
22(10):1642-8).
[0373] d. Use in Vitrectomy and Ophthalmic Disorders and
Conditions
[0374] Hyaluronidase, such as a soluble PH20, can be used to
minimize the detachment or tearing of the retina during vitrectomy.
This could cause, for example, the vitreous body to become
uncoupled or "disinserted" from the retina, prior to removal of the
vitreous body. Such disinsertion or uncoupling of the vitreous body
can minimize the likelihood that further tearing or detachment of
the retina will occur as the vitreous body is removed.
[0375] Hyaluronidase, such as a soluble PH20, can be used for
various ophthalmic applications, including the vitrectomy adjunct
application described in U.S. Pat. No. 5,292,509. The use of a
highly purified hyaluronidase, such as, for example, soluble PH20
provided herein, is preferable for intraocular procedures to
minimize immunogenicity and toxicity.
[0376] Soluble PH20 hyaluronidases can be used to treat and/or
prevent ophthalmic disorders by, for example, preventing
neovascularization and increasing the rate of clearance from the
vitreous of materials toxic to the retina. A soluble PH20
hyaluronidase can be administered in an amount effective to liquefy
the vitreous humor of the eye without causing toxic damage to the
eye. Liquefaction of the vitreous humor increases the rate of
liquid exchange from the vitreal chamber. This increase in exchange
removes the contaminating materials whose presence can cause
ophthalmologic and retinal damage.
[0377] Soluble PH20 hyaluronidases also can be used to reduce
postoperative pressure. Hyaluronic acid has been used in eye
primarily as a spacer during cataract and intraocular lens surgical
procedures. It also is used in other ocular surgical procedures
such as glaucoma, vitreous and retina surgery and in corneal
transplantation. A common side effect occurring in postoperative
cataract patients is a significant early, and occasionally
prolonged, rise in intraocular pressure. Such a condition is
sometimes serious, especially in patients with glaucomatous optic
disc changes. Hyaluronidase, such as soluble PH20, can be
co-administered with hyaluronic acid to the eye prior to surgery to
reduce postoperative pressure in the eye. The hyaluronidase is
administered in an amount effective to reduce the intraocular
pressure to pre-operative levels by breaking down the hyaluronic
acid without decreasing its effectiveness during surgery nor
causing side effects in the patient (U.S. Pat. No. 6,745,776).
[0378] Soluble PH20 hyaluronidases also can be administered to
patients with glaucoma to remove glycosaminoglycans from the
trabecular meshwork and reduce intraocular pressure, and can be
applied to the vitreous to promote the resolution of vitreous
hemorrhages (i.e. extravasation of blood into the vitreous), which
can occur in connection with conditions such as diabetic
retinopathy, retinal neovascularization, retinal vein occlusion,
posterior vitreous detachment, retinal tears, ocular traumas and
the like. The presence of vitreous hemorrhages, which are typically
slow to resolve, can delay, complicate or prevent procedures that
require the retina to be visualized through the vitreous for
diagnosis and/or for treatment procedures such as laser
photocoagulation and the like which are often primary treatments
for conditions such as proliferative diabetic retinopathy.
[0379] e. Use in Hypodermoclysis
[0380] Hypodermoclysis, the infusion of fluids and electrolytes
into the hypodermis of the skin, is a useful and simple hydration
technique suitable for mildly to moderately dehydrated adult
patients, especially the elderly. Although considered safe and
effective, the most frequent adverse effect is mild subcutaneous
edema that can be treated by local massage or systemic diuretics.
Approximately 3 L can be given in a 24-hour period at two separate
sites. Common infusion sites include the chest, abdomen, thighs and
upper arms. Solutions used in hypodermoclysis include, for example,
normal saline, half-normal saline, glucose with saline and 5%
glucose. Potassium chloride also can be added to the solution. The
addition of a hyaluronidase, such as a soluble PH20, to the
solution can enhance fluid absorption and increase the overall rate
of administration.
[0381] f. Use in Gene Therapy
[0382] The efficacy of most gene delivery vehicles in vivo does not
correspond to the efficacy found observed in vitro.
Glycosaminoglycans can hinder the transfer and diffusion of DNA and
viral vectors into many cell types. The levels such extracellular
matrix material can hinder the process considerably. Administration
of hyaluronidase, such as a soluble PH20, can open channels in the
extracellular matrix, thus enhancing delivery of gene therapy. For
example, soluble PH20 can be administered with collagenase to
facilitate transduction of DNA in vivo (Dubensky et al. (1984) Proc
Natl Acad Sci USA 81(23):7529-33). Hyaluronidase also can enhance
gene therapy using adeno-associated virus (Favre et al, (2000) Gene
Therapy 7(16):1417-20). The channels opened following
administration of hyaluronidase are of a size that typically
enhance diffusion of smaller molecules such as retroviruses,
adenoviruses, adeno-associated viruses and DNA complexes (as well
as other therapeutic and pharmacological agents of interest). The
pores are not so large, however, as to promote the dislocation and
movement of cells.
[0383] In some examples, viruses can be engineered to express
hyaluronidase, such as a soluble PH20, to facilitate their
replication and spread within a target tissue. The target tissue
can be, for example, a cancerous tissue whereby the virus is
capable of selective replication within the tumor. The virus also
can be a non-lytic virus wherein the virus selectively replicates
under a tissue specific promoter. As the viruses replicate, the
co-expression of hyaluronidase with viral genes can facilitate the
spread of the virus in vivo.
[0384] g. Cosmetic Uses
[0385] Hyaluronidases, such as a soluble PH20, can be by
administered to remove glycosaminoglycans involved in the
accumulation of cellulite and to promote lymphatic flow. For
example, soluble PH20 can be used for the treatment of cellulite.
The hyaluronidase can be administered through repeated subcutaneous
injections, through transdermal delivery in the form of ointments
or creams or through the use of injectable slow release
formulations to promote the continual degradation of
glycosaminoglycans and prevent their return.
[0386] Hyaluronidase, such as a soluble PH20, also can be used to
treat conditions such as "pigskin" edema or "orange peel" edema.
Hyaluronidases can effect depolymerization of the long
mucopolysaccharide chains that can accumulate in the dermis and
which are responsible for the retention of bound water and of the
slowing, by capillary compression, of the diffusion of organic
liquids, which eliminate metabolic wastes. Such retention of water
and wastes associated with fat overloading of the lipocytes,
constitutes classical "pigskin" edema or "orange peel" edema.
Depolymerization can cut the long chains of mucopolysaccharides
into shorter chains, resulting in the elimination of the bound
water and wastes and restoration of the venous and lymphatic
circulation, culminating in the disappearance of local edema.
[0387] h. Use in Organ Transplantation
[0388] The content of hyaluronic acid in an organ can increase with
inflammation. An increased concentration of hyaluronic acid has
been observed in tissue from different organs characterized by
inflammatory-immunological injury such as alveolitis (Nettelbladt
et al. (1991) Am. Rev. Resp. Dis. 139: 759-762) and myocardial
infarction (Waldenstrom et al. (1991) J. Clin. Invest. 88(5):
1622-1628). Other examples include allograft rejection after a
renal (Hallgren et al. (1990) J. Exp. Med. 171: 2063-2076; Wells et
al. (1990) Transplantation 50: 240-243), small bowel (Wallander et
al. (1993) Transplant. Int 6: 133-137) or cardiac (Hallgren et al.
(1990) J Clin Invest 85:668-673) transplantation; or a myocardial
inflammation of viral origin (Waldenstrom et al. (1993) Eur. J.
Clin. Invest. 23: 277-282). The occurrence of interstitial edemas
in connection with the grafting of an organ constitutes a severe
problem in the field of transplantation surgery. Grafts with
interstitial edemas can swell to such a degree that the function is
temporarily be lost. In some instances, the swelling can cause
disruption of the kidney, resulting in a massive hemorrhage.
Hyaluronidases, such as a soluble PH20, can be used to degrade
accumulated glycosaminoglycans in an organ transplant. Removal of
such glycosaminoglycans promotes removal of water from the graft
and thus enhances organ function.
[0389] i. Use in Pulmonary Disease
[0390] Levels of hyaluronic acid in broncheoalveolar lavages (BAL)
from normal individuals are generally below 15 ng/ml. Hyaluronic
acid levels in BAL rise dramatically in conditions of respiratory
distress (Bjermer et al. (1987) Br Med J (Clin Res Ed)
295(6602):803-6). The increased hyaluronic acid in the lung can
prevent oxygen diffusion and gas exchange as well as activating
neutrophil and macrophage responses. Purified preparations of
soluble PH20, such as any provided herein, can be delivered by
either pulmonary or intravenous delivery to patients presenting
with such conditions to reduce hyaluronan levels. Hyaluronidases,
such as a soluble PH20, also can be administered to patients
suffering from other pulmonary complications that are associated
with elevated glycosaminoglycans or to enhance the delivery of
other co delivered molecules to the lung.
[0391] 3. Other Uses
[0392] In further examples of its therapeutic use, hyaluronidase,
such as a soluble PH20 including esPH20 provided herein, can be
used for such purposes as an antidote to local necrosis from
paravenous injection of necrotic substances such as vinka alkaloids
(Few et al. (1987) Amer. J. Matern. Child Nurs. 12, 23-26),
treatment of ganglion cysts (Paul et al. (1997) J Hand Surg. 22
(2): 219-21) and treatment of tissue necrosis due to venous
insufficiency (Elder et al. (1980) Lancet 648-649). Soluble PH20
also can be used to treat ganglion cysts (also known as a wrist
cyst, Bible cyst, or dorsal tendon cyst), which are the most common
soft tissue mass of the hand and are fluid filled sacs that can be
felt below the skin.
[0393] Hyaluronidases, such as soluble PH20, can be used in the
treatment of spinal cord injury by degrading chondroitin sulfate
proteoglycans (CSPGs). Following spinal cord injury, glial scars
containing CSPGs are produced by astrocytes. CSPGs play a crucial
role in the inhibition of axon growth. In addition, the expression
of CSPG has been shown to increase following injury of the central
nervous system (CNS). Soluble PH20 also can be utilized for the
treatment of herniated disks in a process known as
chemonucleolysis. Chondroitinase ABC, an enzyme cleaving similar
substrates as hyaluronidase, can induce the reduction of
intradiscal pressure in the lumbar spine. There are three types of
disk injuries. A protruded disk is one that is intact but bulging.
In an extruded disk, the fibrous wrapper has torn and the NP has
oozed out, but is still connected to the disk. In a sequestered
disk, a fragment of the NP has broken loose from the disk and is
free in the spinal canal. Chemonucleolysis is typically effective
on protruded and extruded disks, but not on sequestered disk
injuries.
I. Examples
[0394] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
Example 1
Generation of Human PH20 Hyaluronidase Carboxy-terminus Deletion
Mutants
[0395] In this example, a series of human PF120 hyaluronidase
carboxy-terminus deletion mutants were generated. Mature human
PI-120 hyaluronidase, or sperm adhesion molecule 1 (SPAM1),
contains 474 amino acids while the mature carboxy-terminus deletion
mutants generated in this example ranged in length from 472 amino
acids to 415 amino acids.
[0396] DNA oligonucleotides encoding truncated human PH20
hyaluronidase carboxy-terminus deletion mutants from amino acid
A507 to amino acid K450 were synthesized according to standard DNA
synthesis protocols. The parent DNA sequence was a codon-optimized
human PH20 hyaluronidase, the nucleotide sequence of which is set
forth in SEQ ID NO:2. This codon-optimized human PH20 hyaluronidase
contained an heterologous immunoglobulin kappa (IgK) signal
sequence, set forth in SEQ ID NO:144. Additionally, the sequences
contained a 5' NheI and a 3' BamHI restriction site to allow
cloning into the HZ24 plasmid (SEQ ID NO:140). The human PH20
hyaluronidase carboxy-terminus deletion mutant nucleotide sequences
are set forth in SEQ ID NOS:146-185 and 199-201. The synthetic DNA
sequences were digested with NheI and BamHI restriction enzymes and
cloned into a similarly digested HZ24 plasmid to generate a mutant
SPAM 1-HZ24 plasmid for each individual clone.
[0397] The human PH20 hyaluronidase carboxy-terminus deletion
mutants are set forth in Table 3. The SPAM1 mutants are identified
by the 4 amino acids at the C-terminal end of the proteins. Also
set forth are the length, in amino acids, of the precursor and
mature carboxy-terminus deletion mutants.
TABLE-US-00003 TABLE 3 Human PH20 hyaluronidase carboxy-terminus
deletion mutants. Precursor Mature Precursor SEQ ID Mature SEQ ID
Mutant (amino acids) NO (amino acids) NO SPAM1-VASL 509 1 474 108
SPAM1-SSVA 507 3 472 55 SPAM1-IISS 505 4 470 56 SPAM1-FLII 503 5
468 57 SPAM1-LFLI 502 47 467 99 SPAM1-ILFL 501 6 466 58 SPAM1-SILF
500 48 465 100 SPAM1-VSIL 499 7 464 59 SPAM1-IVSI 498 49 463 101
SPAM1-FIVS 497 8 462 60 SPAM1-TMFI 495 9 460 61 SPAM1-SATM 493 10
458 62 SPAM1-TLSA 491 11 456 63 SPAM1-PSTL 489 12 454 64 SPAM1-STLS
490 13 455 65 SPAM1-ASPS 487 14 452 66 SPAM1-YNAS 485 15 450 67
SPAM1-FYNA 484 16 449 68 SPAM1-IFYN 483 17 448 69 SPAM1-QIFY 482 18
447 70 SPAM1-PQIF 481 19 446 71 SPAM1-EPQI 480 20 445 72 SPAM1-EEPQ
479 21 444 73 SPAM1-TEEP 478 22 443 74 SPAM1-ETEE 477 23 442 75
SPAM1-METE 476 24 441 76 SPAM1-PMET 475 25 440 77 SPAM1-PPME 474 26
439 78 SPAM1-KPPM 473 27 438 79 SPAM1-LKPP 472 28 437 80 SPAM1-FLKP
471 29 436 81 SPAM1-AFLK 470 30 435 82 SPAM1-DAFL 469 31 434 83
SPAM1-IDAF 468 32 433 84 SPAM1-CIDA 467 33 432 85 SPAM1-VCID 466 34
431 86 SPAM1-GVCI 465 35 430 87 SPAM1-DGVC 464 36 429 88 SPAM1-IADG
462 37 427 89 SPAM1-VCIA 460 38 425 90 SPAM1-VDVC 458 39 423 91
SPAM1-DAVD 456 40 421 92 SPAM1-DTDA 454 41 419 93 SPAM1-VKDT 452 42
417 94 SPAM1-ADVK 450 43 415 95
Example 2
Expression of Human PH20 Hyaluronidase Carboxy-terminus Deletion
Mutants
[0398] In this example, the human PH20 hyaluronidase
carboxy-terminus deletion mutants generated in Example 1 were
expressed in CHO--S cells. Additionally, rHuPH20 and H1s-tagged
PH20 were expressed in each of four strains of lectin resistant CHO
mutants, including Lec1 (Cat No. CRL-1735, ATCC), Lec2 (Cat No.
CRL-1736, ATCC), Lec8 (Cat No. CRL-1737, ATCC) and Pro-5 (Cat No.
CRL-1781). The expression of PH20 in Lec mutant cells is further
discussed in Example 9 below.
A. Transient Expression in CHO-S Cells in 6-well Plates
[0399] The mutant PH20-HZ24 plasmids generated in Example 1 were
transiently infected into CHO--S cells (derived from Chinese
Hamster Ovary CHO K1 cells) using GeneJuice.RTM. (Novagen)
according to the manufacturer's instructions. In short, the CHO--S
cells were grown in CD CHO medium supplemented with L-glutamine.
Prior to transfection, the CHO--S cells were plated in 6-well
plates, with approximately 5.times.10.sup.5 cells per well, and
grown overnight at 37.degree. C. with 5% CO.sub.2. The medium was
then removed and the CHO--S cells were washed 2 times with 1 mL
serum-free medium. GeneJuice.RTM. was mixed with serum-free media
followed by the addition of 2 .mu.g mutant-HZ24 DNA. After
incubating at room temperature for 5-15 minutes, the
GeneJuice.RTM./DNA mixture was added dropwise to an individual well
containing the washed CHO--S cells. After 4 hours, the medium was
replaced with 1 mL CD-CHO medium supplemented with L-glutamine and
the cells were incubated for 72 hours at 37.degree. C. with 5%
CO.sub.2. Following expression, the media and cells were harvested
separately.
B. Transient Expression in CHO Cells in 10 cm Cell Culture
Dishes
[0400] The mutant PH20-HZ24 plasmids generated in Example 1 were
transiently infected into CHO--S cells using GeneJuice.RTM.
(Novagen) according to the manufacturer's instructions.
Alternatively, HZ24-PH20, (SEQ ID NO:108, encoding rHuPH20),
PH20His, (SEQ ID NO:187, encoding his-tagged PH20) and
HZ24-mut(B/S) (SEQ ID NO:122, encoding PH20 truncated at amino acid
482), were transiently infected into four strains of lectin
resistant CHO mutants, including Lec 1 (Cat No. CRL-1735, ATCC),
Lec2 (Cat No. CRL-1736, ATCC), Lec8 (Cat No. CRL-1737, ATCC) and
Pro-5 (Cat No. CRL-1781), using GeneJuice.RTM. (Novagen) according
to the manufacturer's instructions.
[0401] In short, CHO--S cells were maintained in CD-CHO medium
supplemented with 8 mM GlutaMax. Lectin resistant CHO mutant cells
were grown in DMEM medium supplemented with 10% FBS. Prior to
transfection, the CHO cells were plated in 10 cm cell culture
dishes, with approximately 3.times.10.sup.6 cells per well and
grown overnight in DMEM medium supplemented with 10% FBS at
37.degree. C. with 5% CO.sub.2. The medium was then removed and the
monolayer of cells was washed 2 times with 10 mL serum-free medium.
36 .mu.L GeneJuice.RTM. was mixed with 1.2 mL DMEM and incubated at
room temperature for 5 minutes. Following incubation, 12 mg DNA was
added and mixed gently. After incubating at room temperature for 15
minutes, the GeneJuice.RTM./DNA mixture was added dropwise the
monolayer of CHO cells and the cell culture dish was shaken gently
to allow for mixing. The plate was incubated for 4 hours at
37.degree. C. with 5% CO.sub.2. After 4 hours, the medium was
replaced with 12 mL detergent free CD DG44 medium supplemented with
Glutamax-1 and the cells were incubated for 48 hours at 37.degree.
C. with 5% CO.sub.2. Following expression, the media and cells were
harvested separately.
Example 3
Solubility of Human PH20 Hyaluronidase Carboxy-terminus Deletion
Mutants
[0402] In this example, following transient expression, as
described in Example 2 above, the media and cells were harvested
separately and analyzed for PH20 expression and solublility by
Western blot analysis. Solubility of the C-terminus truncation
mutants was determined by examining whether the expressed protein
was present in the growth media or in the cells. C-terminus
deletion mutants from 455 to 472 amino acids in length,
corresponding to SEQ ID NOS:55-65 and 99-101, contain amino acid
residues from the GPI-anchor which serves to attach the protein to
the cell membrane. Cells expressing these mutants were treated with
phosphoinositol-phospholipase C (PI-PLC), which cleaves the
GPI-anchor allowing the release of soluble protein into the media,
and the presence of PH20 in the resulting media and cells was
determined by Western blot analysis.
A. Western Blot Analysis
[0403] Non-reduced samples were run on a 4-20% Tris-Glycine gel and
transferred to PVDF membrane using iBlot (Invitrogen). For the
Western blot, rabbit anti-PH20 IgG (0.5 .mu.g/mL) was used as the
primary antibody and HRP-conjugated goat anti-rabbit IgG (0.1
ng/mL, Cat#DC03L, EMD) was used as the secondary antibody. Evidence
of expression is determined by a band at approximately 66 kDa,
corresponding to recombinant human PH20 hyaluronidase.
B. PI-PLC Treatment
[0404] 1. Transient Expression in 6-well Plates
[0405] Following expression of rHuPH20 in CHO--S cells for 72
hours, as described in Example 2A above, the media and cells were
harvested separately. The cells were washed with serum-free media
followed by the addition of 2 mL serum-free media per well. PI-PLC
(0.5 units/well) was added to each well and the cells were
incubated in the PI-PLC for 2 hours. The resultant media and cells
were analyzed by Western blot analysis as described above.
[0406] 2. Transient Expression in 10 cm Tissue Culture Dishes
[0407] Two plates each of rHuPH20 expressing CHO--S cells, one for
treatment with PI-PLC and one without treatment with PI-PLC, were
prepared for each C-terminus mutant as described in Example 2B
above. Following expression for 48 hours, for cells untreated with
PI-PLC, the media and cells were harvested separately. The
harvested media was spun down, concentrated to a volume of 10 mL,
and buffer exchanged into PBS using an Amicon 30 kD MWCO
concentrator. The cells were rinsed with cold PBS and scraped and
resuspended in 1.2 mL PBS with protease inhibitor Set III (Cat No.
539134, Calbiochem). The resuspended cells were briefly sonicated
to prepare whole-cell extract. For PI-PLC treatment of cells,
following expression for 48 hours, the untreated media was
harvested, as described above. The cells were rinsed once with
fresh CD DG44 medium with Glutamax-1, and the media was replaced
with 12 mL fresh detergent free CD DG44 medium supplemented with
Glutamax-1 with 3.0 units PI-PLC per dish, and the cells were
incubated for 2 hours at 37.degree. C. with 5% CO.sub.2. After 2
hours, the PI-PLC media and cells were harvested separately, as
described above. The resultant untreated media and cells, and
PI-PLC treated media and cells, were analyzed by Western blot
analysis as described above.
C. Results
[0408] The results are described in Table 4 below. Four mutants,
ILFL (SEQ ID NO:58), SILF (SEQ ID NO:100), VSIL (SEQ ID NO:59) and
IVSI (SEQ ID NO:101), exhibited low expression of PH20. Western
blot analysis shows that human PH20 hyaluronidase carboxy-terminus
deletion mutants shorter than F500 (SEQ ID NOS:59-95 and 100-101)
are expressed in the media, as evidenced by a protein band at
approximately 66 kDa. Human PH20 hyaluronidase carboxy-terminus
deletion mutants with lengths between L501 and A507 (SEQ ID
NOS:55-58 and 99) are expressed in the cells. Upon treatment of
these cells with PI-PLC, human PH20 hyaluronidase is released into
the media, as evidenced by a protein band at approximately 66 kDa.
Treatment of cells from human PH20 hyaluronidase carboxy-terminus
deletion mutants, corresponding to SEQ ID NOS:59-65 and 100-101,
with PI-PLC had no effect since these proteins were initially
expressed into the media.
TABLE-US-00004 TABLE 4 Human PH20 hyaluronidase carboxy-terminus
deletion mutant expression. Expressed in Media Mature Protein
Expressed Following Addition Mutant (AA) Expression in Media of
PI-PLC SPAM1-VASL 474 YES NO YES (SEQ ID NO: 108) SPAM1-SSVA 472
YES NO YES (SEQ ID NO: 55) SPAM MISS 470 YES NO YES (SEQ ID NO: 56)
SPAM1-FLII 468 YES NO YES (SEQ ID NO: 57) SPAM1-LFLI 467 YES NO YES
(SEQ ID NO: 99) SPAM1-ILFL 466 WEAK NO YES (SEQ ID NO: 58)
SPAM1-SILF 465 WEAK WEAK/YES Initially in media (SEQ ID NO: 100)
SPAM1-VSIL 464 WEAK YES Initially in media (SEQ ID NO: 59)
SPAM1-IVSI 463 WEAK YES Initially in media (SEQ ID NO: 101)
SPAM1-FIVS 462 YES YES Initially in media (SEQ ID NO: 60)
SPAM1-TMFI 460 YES YES Initially in media (SEQ ID NO: 61)
SPAM1-SATM 458 YES YES Initially in media (SEQ ID NO: 62)
SPAM1-TLSA 456 YES YES Initially in media (SEQ ID NO: 63)
SPAM1-STLS 455 YES YES Initially in media (SEQ ID NO: 65)
SPAM1-PSTL 454 YES YES Initially in media (SEQ ID NO: 64)
SPAM1-ASPS 452 YES YES n/a (SEQ ID NO: 66) SPAM1-YNAS 450 YES YES
n/a (SEQ ID NO: 67) SPAM1-FYNA 449 YES YES n/a (SEQ ID NO: 68)
SPAM1-IFYN 448 YES YES n/a (SEQ ID NO: 69) SPAM1-QIFY 447 YES YES
n/a (SEQ ID NO: 70) SPAM1-PQIF 446 YES YES n/a (SEQ ID NO: 71)
SPAM1-EPQI 445 YES YES n/a (SEQ ID NO: 72) SPAM1-EEPQ 444 YES YES
n/a (SEQ ID NO: 73) SPAM1-TEEP 443 YES YES n/a (SEQ ID NO: 74)
SPAM1-ETEE 442 YES YES n/a (SEQ ID NO: 75) SPAM1-METE 441 YES YES
n/a (SEQ ID NO: 76) SPAM1-PMET 440 YES YES n/a (SEQ ID NO: 77)
SPAM1-PPME 439 YES YES n/a (SEQ ID NO: 78) SPAM1-KPPM 438 YES YES
n/a (SEQ ID NO: 79) SPAM1-LKPP 437 YES YES n/a (SEQ ID NO: 80)
SPAM1-FLKP 436 YES YES n/a (SEQ ID NO: 81) SPAM1-AFLK 435 YES YES
n/a (SEQ ID NO: 82) SPAM1-DAFL 434 YES YES n/a (SEQ ID NO: 83)
SPAM1-IDAF 433 YES YES n/a (SEQ ID NO: 84) SPAMI-CIDA 432 YES YES
n/a (SEQ ID NO: 85) SPAM1-VCID 431 YES YES n/a (SEQ ID NO: 86)
SPAM1-GVCI 430 YES YES n/a (SEQ ID NO: 87) SPAM1-DGVC 429 YES YES
n/a (SEQ ID NO: 88) SPAM1-IADG 427 YES YES n/a (SEQ ID NO: 89)
SPAM1-VCIA 425 YES YES n/a (SEQ ID NO: 90) SPAM1-VDVC 423 YES YES
n/a (SEQ ID NO: 91) SPAM1-DAVD 421 YES YES n/a (SEQ ID NO: 92)
SPAM1-DTDA 419 YES YES n/a (SEQ ID NO: 93) SPAM1-VKDT 417 YES YES
n/a (SEQ ID NO: 94) SPAM1-ADVK 415 YES YES n/a (SEQ ID NO: 95)
Example 4
Solubility of Human PH20 Hyaluronidase Carboxy-terminus Deletion
Mutants using Triton.RTM. X-114 Assay
[0409] In this example, the solubility of the human PH20
hyaluronidase carboxy-terminus deletion mutants was tested using a
Triton.RTM. X-114 assay. In this assay, soluble PH20 hyaluronidases
will partition into the aqueous phase of a Triton.RTM. X-114
solution warmed to 37.degree. C. (modification as described by
Bordier et al., (1981) J. Biol. Chem., 256:1604-7) while
membrane-anchored PH20 hyaluronidases will partition into the
detergent rich phase.
[0410] For this purpose, 2% (v/v) Triton.RTM. X-114 in PBS at
0.degree. C. was added to 200 .mu.L of tissue culture media or cell
extract, as prepared in Example 3B above, and the samples were
incubated on ice. For separation, the sample was overlaid on a 30
.mu.L sucrose cushion (6% w/v) containing 0.06% Triton.RTM. X-114
at 4.degree. C. in a microfuge tube. The samples were heated to
37.degree. C. for 3 minutes to induce phase separation and
centrifuged for 3 min at 4000 g at room temperature. Aqueous and
detergent phases were removed for SDS-PAGE analysis and Western
blotting. Rabbit anti-PH20 IgG (0.5 .mu.g/mL) was used as the
primary antibody and HRP-conjugated goat anti-Rabbit IgG (0.1
ng/mL, Cat#DC03L, EMD) was used as the secondary antibody. Full
length human PH20, which partitions strongly into the detergent
phase, was used as a control.
[0411] The results of the solubility of the carboxy-terminus
deletion mutants are shown in Table 5. Human PH20 hyaluronidase
carboxy-terminus deletion mutants up to F500 (precursor SEQ ID
NOS:7-13 and 48-49 or mature SEQ ID NOS:59-65 and 100-101)
partition into the aqueous phase and are therefore soluble. Human
PH20 hyaluronidase carboxy-terminus deletion mutants longer than
F500 (SEQ ID NOS:55-58 and 99) partition into the detergent phase
and are insoluble. Full length PH20 is also insoluble.
TABLE-US-00005 TABLE 5 Solubility of human PH20 hyaluronidase
carboxy-terminus deletion mutants Mutant SEQ ID NO Mature (AA)
Soluble SPAM1-VASL 108 474 NO SPAM1-SSVA 55 472 NO SPAM1-IISS 56
470 NO SPAM1-FLII 57 468 NO SPAM1-LFLI 99 467 NO SPAM1-ILFL 58 466
NO SPAM1-SILF 100 465 YES SPAM1-VSIL 59 464 YES SPAM1-IVSI 101 463
YES SPAM1-FIVS 60 462 YES SPAM1-TMFI 61 460 YES SPAM1-SATM 62 458
YES SPAM1-TLSA 63 456 YES SPAM1-PSTL 64 454 YES SPAM1-STLS 65 455
YES
Example 5
Hyaluronidase Activity of Human PH20 Hyaluronidase Carboxy-terminus
Deletion Mutants
[0412] In this example, the human PH20 hyaluronidase
carboxy-terminus deletion mutants were tested for their PH20
hyaluronidase activity using a microtiter assay with
biotinylated-hyaluronic acid (biotinylated-HA or bHA). The human
PH20 hyaluronidase carboxy-terminus deletion mutants were tested
for hyaluronidase activity at both pH 7.4 and pH 5.5.
[0413] In short, a 4xBH 96-well plate was coated with
biotinylated-HA (1.1 MDa). The 72 hour post transfection
supernatant from cells transfected with human PH20 hyaluronidase
carboxy-terminus deletion mutants was diluted in buffer at either
pH 7.4 or pH 5.5 and added to individual wells of the plate and
allowed to incubate at 37.degree. C. for 90 minutes. The reaction
was terminated by addition of 4M guanidine HCl. The wells were
washed 4.times. with Phosphate Buffered Saline with Tween20 (PBST)
to remove any digested biotinylated-HA followed by addition of
streptavidin-HRP for 1 hour at room temperature. The wells were
washed 4.times. with PBST and the plate was developed with TMB. The
plate was read at 450 nm using an ELISA plate reader. Hyaluronidase
activity (in Units/mL) was determined by interpolating the measured
absorbance at 450 nm with a hyaluronidase reference standard curve.
Full length mature human PH20 hyaluronidase and untransfected CHO
cells were used as positive and negative controls.
[0414] The results are shown in Tables 6 and 6A, below. Human PH20
hyaluronidase carboxy-terminus deletion mutants shorter than 1430,
corresponding to SPAM1-GDVC to SPAM1-ADVK (SEQ ID NOS:88-95), are
inactive. Human PH20 hyaluronidase carboxy-terminus deletion
mutants ending at 1498 (SEQ ID NO:101), L499 (SEQ ID NO:59), F500
(SEQ ID NO:100), L501 (SEQ ID NO:58) and 1502 (SEQ ID NO:99) have
little detectable activity due to low expression level. All other
human PH20 hyaluronidase carboxy-terminus deletion mutants (SEQ ID
NOS:55-57 and 60-87) are active hyaluronidases at both pH 7.4 and
pH 5.5.
TABLE-US-00006 TABLE 6 Hyaluronidase Activity pH 7.4 pH 5.5
Precursor Mature Activity Activity Deletion Mutant (AA) (AA)
(Units/ml) (Units/ml) SPAM1-SSVA 507 472 1.4715 1.125 (SEQ ID NO:
55) SPAM1-IISS 505 470 1.458 0.837 (SEQ ID NO: 56) SPAM1-FLII 503
468 0.9405 0.6345 (SEQ ID NO: 57) SPAM1-ILFL 501 466 0.0405 0.0405
(SEQ ID NO: 58) SPAM1-VSIL 499 464 0.02025 0.045 (SEQ ID NO: 59)
SPAM1-FIVS 497 462 0.1755 0.216 (SEQ ID NO: 60) SPAM1-TMFI 495 460
0.45 0.612 (SEQ ID NO: 61) SPAM1-SATM 493 458 0.5715 0.7335 (SEQ ID
NO: 62) SPAM1-TLSA 491 456 0.3645 0.5625 (SEQ ID NO: 63) SPAM1-STLS
490 455 0.819 1.2375 (SEQ ID NO: 65) SPAM1-PSTL 489 454 1.557 1.089
(SEQ ID NO: 64) SPAM1-ASPS 487 452 1.017 0.9225 (SEQ ID NO: 66)
SPAM1-YNAS 485 450 1.8765 1.74825 (SEQ ID NO: 67) SPAM1-FYNA 484
449 1.4985 1.26225 (SEQ ID NO: 68) SPAM1-IFYN 483 448 2.45025
2.3085 (SEQ ID NO: 69) SPAM1-QIFY 482 447 2.03175 1.647 (SEQ ID NO:
70) SPAM1-PQIF 481 446 1.818 1.701 (SEQ ID NO: 71) SPAM1-EPQI 480
445 2.1825 1.6425 (SEQ ID NO: 72) SPAM1-EEPQ 479 444 1.917 2.0745
(SEQ ID NO: 73) SPAM1-TEEP 478 443 1.764 1.584 (SEQ ID NO: 74)
SPAM1-ETEE 477 442 2.088 2.0475 (SEQ ID NO: 75) SPAM1-METE 476 441
1.332 1.278 (SEQ ID NO: 76) SPAM1-PMET 475 440 2.223 2.0925 (SEQ ID
NO: 77) SPAM1-PPME 474 439 1.2105 1.341 (SEQ ID NO: 78) SPAM1-KPPM
473 438 0.8595 0.91575 (SEQ ID NO: 79) SPAM1-LKPP 472 437 0.5445
0.9 (SEQ ID NO: 80) SPAM1-FLKP 471 436 3.321 2.79 (SEQ ID NO: 81)
SPAM1-AFLK 470 435 3.204 2.925 (SEQ ID NO: 82) SPAM1-DAFL 469 434
2.3895 2.2365 (SEQ ID NO: 83) SPAM1-IDAF 468 433 0.5625 0.62775
(SEQ ID NO: 84) SPAM1-CIDA 467 432 0.5535 0.4725 (SEQ ID NO: 85)
SPAM1-VCID 466 431 0 0.2115 (SEQ ID NO: 86) SPAM1-GVCI 465 430
0.441 0.468 (SEQ ID NO: 87) SPAM1 -DGVC 464 429 0 0.045 (SEQ ID NO:
88) SPAM1-IADG 462 427 0 0.00225 (SEQ ID NO: 89) SPAM1-VCIA 460 425
0 0.0135 (SEQ ID NO: 90) SPAM1-VDVC 458 423 0.0495 0.0585 (SEQ ID
NO: 91) SPAM1-DAVD 456 421 0 0.0675 (SEQ ID NO: 92) SPAM1-DTDA 454
419 0 0.054 (SEQ ID NO: 93) SPAM1-VKDT 452 417 0.054 0.0225 (SEQ ID
NO: 94) SPAM1-ADVK 450 415 0.063 0.0405 (SEQ ID NO: 95) VASL 509
474 1.8045 0.891 (SEQ ID NO: 108) VASL + PLC 509 474 3.96 2.313
(SEQ ID NO: 108) HZ24-PH20 482 447 0.499 0.726188 (SEQ ID NO: 109)
CHO-S n/a n/a 0 0.012375
TABLE-US-00007 TABLE 6A Hyaluronidase Activity pH 7.4 pH 5.5
Precursor Mature Activity Activity Deletion Mutant (AA) (AA)
(Units/ml) (Units/ml) SPAM1-SSVA 507 472 1.782 1.256 (SEQ ID NO:
55) SPAM1-IISS 505 470 1.863 0.932 (SEQ ID NO: 56) SPAMI-FLII 503
468 1.094 0.648 (SEQ ID NO: 57) SPAM1-LFLI 502 467 0.608 0.324 (SEQ
ID NO: 99) SPAM1-ILFL 501 466 0.446 0.122 (SEQ ID NO: 58)
SPAM1-SILF 500 465 0.365 0.162 (SEQ ID NO: 100) SPAM1-VSIL 499 464
0.486 0.122 (SEQ ID NO: 59) SPAM1-IVSI 498 463 0.527 0.203 (SEQ ID
NO: 101) SPAM1-FIVS 497 462 0.365 0.162 (SEQ ID NO: 60) SPAM1-TMFI
495 460 0.689 0.770 (SEQ ID NO: 61) SPAM1-SATM 493 458 0.689 0.851
(SEQ ID NO: 62) SPAM1-TLSA 491 456 0.851 0.729 (SEQ ID NO: 63)
SPAM1-PSTL 489 454 1.985 3.321 (SEQ ID NO: 64) SPAM1-ASPS 487 452
1.134 1.580 (SEQ ID NO: 66)
Example 6
Glycan Analysis of rHuPH20 by LC-MS
[0415] In this example, a glycan analysis study of rHuPH20 (SEQ ID
NO:122) was performed by mass spectral analysis of trypsin digested
PH20.
[0416] Briefly, rHuPH20 (as produced in Example 15C), was
lyophilized and resuspended in buffer containing 6M guanidine HCL,
0.002 M EDTA and 0.02 M Tris, pH 8.28 to a final concentration of
0.5 mg/mL. DTT (10 mM final concentration) was added and the
protein/DTT mixture was incubated for 1 hour at 37.degree. C.
Following reduction, iodoacetamide was added to a final
concentration of 20 mM. Finally, trypsin (1:25 w/w) was added and
the mixture was incubated for 20 hours at 37.degree. C.
[0417] The tryptic digests were analyzed by LC-MS. Briefly, the
tryptic digests were injected onto a C18 reverse phase column using
the conditions set forth in Table 7 below. MS data was collected on
a Q-TOF Ultima mass spectrometer using electrospray ionization
(ESI) in positive ion mode. Data was acquired from m/z 200-1950 in
MS mode. The glycopeptides were analyzed using GlycoMod software
(www.expasy.ch/tools/glycomod/) to determine the glycan type.
TABLE-US-00008 TABLE 7 LC-MS parameters and settings Parameter
Setting Column Phenomenex Synergi Hydro-RP Column Temperature
30.degree. C. Mobile Phase A Deionized water containing 0.2% formic
acid Mobile Phase B Acetonitrile containing 0.2% formic acid
Gradient Time (min) % A % B 0.0 97.0 3.0 5.0 97.0 3.0 144.0 60.0
40.0 150.0 10.0 90.0 160.0 10.0 90.0 161.0 97.0 3.0 180.0 97.0 3.0
Flow Rate 0.2 mL/min Injection Volume 5 .mu.L Run Time (total) 180
minutes
[0418] Human PH20 hyaluronidase has one O-glycosylation site at
T475. The site is occupied by a core type 1 glycan that has one or
two sialic acids. rHuPH20 is glycosylated at six different
asparagine residues, including N82, N166, N235, N254, N368, and
N393. The results show that N254 is approximately 75% occupied,
N393 is approximately 85% occupied, and the four remaining sites,
N82, N166, N235 and N368, are greater than 99% occupied. All of the
three types of N-glycans, high mannose, hybrid and complex types,
are present in rHuPH20. In general, rHuPH20 contains about 45% high
mannose glycans, 45% complex glycans and 10% hybrid glycans. About
35% of the total glycans are anionic, of which 25% contain a sialic
acid and the remaining 10% contain an unknown anionic group,
possibly a phosphate group. Most of the complex glycans are
fucosylated and the anionic complex glycans contain mostly one
sialic acid while a few of them contain two sialic acids. Each
asparagine residue has about 90% of one type of glycan and a small
proportion of the other two types of glycans, with the exception of
N235. The major glycan type for each residue is set forth in Table
8 below. Residues N82, N166 and N254 are occupied by complex
glycans. Residues N368 and N393 are occupied by high mannose
glycans. Residue N235 is occupied by approximately 80% high mannose
glycans, with approximately 20% complex glycans.
TABLE-US-00009 TABLE 8 Types of N-glycans at asparagine residues in
rHuPH20 Glycan Site High Mannose glycans Complex Glycans N82 X N166
X N235 ~80% ~20% N254 X N368 X N393 X
Example 7
Deglycosylation of Human PH20 Hyaluronidase by Treatment with
Endoglycosidases
[0419] In this example, human PH20 hyaluronidase was deglycosylated
by treatment of purified rHuPH20 (SEQ ID NO:122) with various
glycosidases and hyaluronidase activity was assessed. Human PI-120
hyaluronidase is glycosylated at six different asparagine residues,
including N82, N166, N235, N254, N368, and N393. Five glycosidases
were used to generate deglycosylated human PH20 hyaluronidase,
including: PNGaseF (New England Biolabs, Cat. No. P0704S, Lot #34),
which cleaves all N-glycans; EndoF1, which cleaves high mannose and
hybrid type glycans; EndoF2, which cleaves biantennary complex type
glycans; EndoF3, which cleaves biantennary and more branched
complex glycans; and EndoH (New England Biolabs, Cat. No. P0702S),
which cleaves high mannose and hybrid type glycans. Therefore,
treatment with PNGaseF results in complete deglycosylation whereas
treatment with endoglycosidases results in only partial
deglycosylation.
[0420] For complete deglycosylation, purified rHuPH20 (0.1 mg/mL
final concentration) was incubated with PNGaseF (50,000 units/mL)
in 50 mM phosphate buffer pH 7.2 overnight at 37.degree. C. For
partial deglycosylation, purified rHuPH20 (0.5 mg/mL final
concentration) was incubated with 0.3 units/mL of endoglycosidase
(either EndoF1, EndoF2, EndoF3 or EndoH) or a mixture all four
endoglycosidases in 50 mM sodium acetate buffer pH 5.0 overnight at
35.degree. C. Deglycosylation of rHuPH20 was analyzed by the shift
in the mobility of PH20 by SDS-PAGE. Hyaluronidase enzymatic
activity was determined as described in Example 5.
[0421] Human PH20 hyaluronidase has a molecular weight of
approximately 66 kDa. Treatment with EndoF1, EndoH or a mixture of
EndoF1, EndoF2, EndoF3 and EndoH resulted in partially
deglycosylated human PH20 hyaluronidase as determined by SDS-PAGE
mobility shift to a molecular weight of approximately 56 kDa.
Treatment with PNGaseF resulted in complete deglycosylation of
human PH20 hyaluronidase. Partial deglycosylation of rHuPH20 did
not result in inactivation of hyaluronidase enzymatic activity
whereas exhaustive digestion with PNGaseF to completely remove
N-glycans resulted in the total loss of hyaluronidase enzymatic
activity (see Table 9 below).
TABLE-US-00010 TABLE 9 Effect of glycosidase treatment on rHuPH20
activity rHuPH20 Control EndoF1, (U/ml) PH20 EndoF1 EndoF2 EndoF3
EndoH F2, F3, H PNGaseF 1.0000 0.3195 0.2983 0.2573 0.2965 0.2144
1.9315 0.2000 0.7910 0.7656 0.6048 0.5880 0.7435 0.5366 1.9173
0.0400 1.4299 1.3450 1.3117 1.2255 1.3584 1.3877 1.9926 0.0080
1.8397 1.7338 1.6900 1.6698 1.6998 1.8418 1.9172
Example 8
Treatment of Human PH20 Hyaluronidase with Glycosylation
Inhibitors
[0422] In this example, rHuPH20 (SEQ ID NO:122) was transiently
expressed in the presence of each of two glycosylation inhibitors
and hyaluronidase secretion and activity were assessed. Kifunensine
is a potent inhibitor mannosidase I, an enzyme involved in glycan
processing (see e.g., Elbein et al., J Biol Chem, 265:15599-15605
(1990)). Tunicamycin is a mixture of homologous nucleoside
antibiotics that inhibit the enzyme GlcNAc phospho-transferase
(GPT), thereby blocking the synthesis of all N-glycans (see e.g.,
Bohme et al., Eur. J. Biochem. 269:977-988 (2002)).
[0423] Briefly, 1.times.10.sup.6 HZ24-2B2 cells expressing rHuPH20
(see Example 14 below) were seeded in 24 mL complete CD-CHO medium
in two 125 mL flasks. Tunicamycin (dissolved in DMSO) or
Kifunensine (freshly dissolved in water) was added to a final
concentration of 5 .mu.g/mL (containing 12 .mu.L DMSO). As a
control, one flask was seeded with 1.times.10.sup.6 HZ24-2B2 cells
expressing rHuPH20 and 12 .mu.L DMSO was added as a vehicle
control. Following addition of either tunicamycin or kifunensine,
the cells were incubated for 4-6 hours at 37.degree. C. with 5%
CO.sub.2. Following expression, a 2 mL culture was removed and
centrifuged for 5 minutes at 500 g. The supernatant was stored at
4.degree. C. and the cell pellets were stored at -20.degree. C. The
remaining 22 mL cultures were centrifuged for 5 minutes at 500 g.
The supernatant was stored at 4.degree. C. The cells were
resuspended in 22 mL complete CD-CHO medium in the original two 125
mL flasks. Tunicamycin or Kifunensine was added to the culture at a
final concentration of 5 .mu.g/mL and the cells were incubated at
37.degree. C. with 5% CO.sub.2. Two mL (2 mL) cultures were removed
from each flask at approximately every 24 hours post changing
medium. For each time point, the supernatant was stored at
4.degree. C. and the cell pellets were stored at -20.degree. C. The
expression of rHuPH20 was analyzed by Western blot analysis and the
hyaluronidase activity was measured using the biotinylated HA
enzymatic assay (as described in Examples 3 and 5 above).
[0424] The results are shown in Tables 10-13 below, which set forth
the number of viable cells and the PH20 activity. As shown in
Tables 10-11, tunicamycin inhibits PH20 activity in both tissue
culture media and inside the cell and also results in a complete
loss of cell viability. Additionally, one hour of treatment with
tunicamycin resulted in the accumulation of deglycosylated human
PH20 hyaluronidase inside the cell, as determined by SDS-PAGE
mobility shift to a molecular weight of approximately 56 kDa in the
cell pellet fractions. As shown in Tables 12-13, kifunensine did
not affect the activity of PH20 while western blot analysis
revealed kifunensine inhibited the expression and secretion of
rHuPH20 in treated cells.
TABLE-US-00011 TABLE 10 Effect of Tunicamycin on Cell Viability and
PH20 activity in Tissue Culture Media With Tunicamycin Without
Tunicamycin Time Viable Cells PH20 activity Viable Cells PH20
activity (hours) (.times.10.sup.6) (U/mL) (.times.10.sup.6) (U/mL)
0 1.04 0.50 1.04 0.00 1 1.04 2.80 1.04 1.50 2 1.04 5.00 1.04 3.00 4
0.910 8.80 1.30 7.00 25 1.08 5.80 1.32 82.50 49 0.200 6.80 2.72
171.30 73 0.080 7.80 3.80 331.00 91 0 7.50 6.25 313.30
TABLE-US-00012 TABLE 11 Effect of Tunicamycin on Cell Viability and
PH20 activity in Cell Pellets With Tunicamycin Without Tunicamycin
Time Viable Cells PH20 activity Viable Cells PH20 activity (hours)
(.times.10.sup.6) (U/mL) (.times.10.sup.6) (U/mL) 0 1.04 34.50 1.04
35.00 1 1.04 38.00 1.04 38.10 2 1.04 34.00 1.04 36.60 4 0.910 18.00
1.30 31.90 25 1.08 1.00 1.32 14.40 49 0.200 0.80 2.72 33.10 73
0.080 0.30 3.80 67.50 91 0 0.30 6.25 79.40
TABLE-US-00013 TABLE 12 Effect of Kifunensine on Cell Viability and
PH20 activity in Tissue Culture Media With Kifunensine Without
Kifunensine Time Viable Cells PH20 activity Viable Cells PH20
activity (hours) (.times.10.sup.6) (U/mL) (.times.10.sup.6) (U/mL)
0 1 0.4 1 0.45 6 1 23.85 1 15.75 24 1.2 129.6 1.4 75.6 50 2.1 299.7
2.4 206.55 72 3 535.95 4.4 444.15 96 3.7 945 6.3 726.3 144 5.8
2968.65 8.5 2241
TABLE-US-00014 TABLE 13 Effect of Kifunensine on Cell Viability and
PH20 activity in Cell Pellets With Kifunensine Without Kifunensine
Time Viable Cells PH20 activity Viable Cells PH20 activity (hours)
(.times.10.sup.6) (U/mL) (.times.10.sup.6) (U/mL) 0 1 22.25 1 23 6
1 21.25 1 27 24 1.2 27.75 1.4 14.45 50 2.1 43 2.4 26 72 3 98.75 4.4
52.75 96 3.7 208.75 6.3 167.5 144 5.8 497.25 8.5 107
Example 9
Transient Expression of rHuPH20 in Lectin Resistant CHO Mutants
[0425] In this example, rHuPH20 was transiently expressed in four
Lectin resistant CHO mutants and hyaluronidase secretion and
activity were assessed. The Lectin resistant CHO mutants are
summarized in Table 14 below. Pro.sup.-5 cells lack the
galactosyltransferase .beta.4galT-6 causing a reduction in
galactosylated N-glycans (see, e.g., Lee et al. J. Biol. Chem.
276:13924-13934 (2001)). Lec1 cells lack
N-acetylglucosaminyltranferase I activity and therefore do not
synthesize complex or hybrid glycans (see, e.g., Chen and Stanley,
Glycobiology, 13:43-50 (2003)). Lec2 and Lec8 are deficient in
nucleotide-sugar transporters, which transport nucleotide-sugars
across the ER or golgi membrane. Lec2 cells are unable to
translocate CMP-sialic acid (namely CMP-NeuAc) therefore causing
the expression of asialo cell surfaces (see, e.g., Eckhardt et al.,
J. Biol. Chem. 273:20189-20195 (1998)). Lec8 cells are unable to
translocate UDP-galactose therefore causing glycans devoid of
galactose (see, e.g., Bakker et al., Glycobiology, 15:193-201
(2005)).
TABLE-US-00015 TABLE 14 Lectin resistant CHO mutants CHO line
Biochemical Change Genetic Change Pro.sup.-5 .dwnarw. Gal on
N-glycans No expression of .beta.4galt6 (parent) Lec1 .dwnarw.
GlcNAc-TI Insertion/deletion in Mgat1 ORF Lec2 .dwnarw. CMP-sialic
acid Golgi Mutation in Slc35a1 ORF transporter Lec8 .dwnarw.
UDP-Gal Golgi transporter Mutation in Slc35a2 ORF
[0426] In brief, PH20His (encoding his-tagged PH20, SEQ ID NO:187)
wa transiently expressed in each of four strains of lectin
resistant CHO mutants, including Lec1 (Cat No. CRL-1735, ATCC),
Lec2 (Cat No. CRL-1736, ATCC), Lec8 (Cat No. CRL-1737, ATCC) and
Pro.sup.-5 (Cat No. CRL-1781) as described in Example 2A above.
Additionally, HZ24-mut(B/S) (encoding PH20 truncated at amino acid
482, SEQ ID NO:122) was transiently expressed in Pro.sup.-5 cells
and as negative control, Pro.sup.-5 cells were subjected to a mock
transfection. The resulting cell culture media was analyzed by
Western blot analysis and hyaluronidase activity was measured using
the biotinylated HA enzymatic assay (as described in Examples 3 and
5 above).
[0427] The results show that rHuPH20 expressed in the Lec mutants
is secreted into the medium, as evidenced by a protein band at
approximately 66 kDa. The results of the bHA enzymatic assay are
set forth in Table 15 below, which sets for the lectin resistant
CHO mutant, the PH20 encoding plasmid used to transfect the cells,
and the PH20 activity at pH5.5 for both a 1:27 and 1:81 dilution.
rHuPH20 expressed by Lec mutant cells is enzymatically active.
TABLE-US-00016 TABLE 15 PH20 activity (U/mL) of rHuPH20 transiently
expressed in Lec mutant cells. Lectin Mutant Pro-5 Pro-5 Lec1 Lec2
Lec8 Pro-5 Plasmid HZ24- HZ24- HZ24- HZ24- HZ24- Mock mut(B/S)
PH20sHis PH20sHis PH20sHis PH20sHis transfection PH20 0.6615 0.297
0.54 0.675 0.2565 0.081 Activity (1:27) PH20 1.1745 0.6075 0.7695
1.053 0.567 0.1215 Activity (1:81)
Example 10
Site-Directed Mutagenesis of Human PH20 Hyaluronidase
N-glycosylation Sites
[0428] In this example, N-glycan site specific human PH20
hyaluronidase deglycosylation mutants were generated and their
secretion patterns and hyaluronidase enzymatic activity were
assessed. The N-glycan site specific deglycosylation mutants and
glycan types are set forth in Table 16 below.
[0429] PH20sHis (SEQ ID NO:210) was used as a template for
mutagenesis of each asparagine residue to alanine using
QuikChange.RTM. Site-Directed Mutagenesis Kit (Cat No. 200518,
Stratagene). The protein encoded by the template DNA corresponds to
PH20sHis (SEQ ID NO:187), a human PH20 clone that contains a
HexaHis tag (SEQ ID NO:142) after amino acid 5490. Wild type
PH20sHis and deglycosylated mutants are set forth in Table 16. Six
single mutants were generated, one for each of the N-glycosylation
sites. Additionally, three double mutants and a triple mutant were
generated for asparagines N82, N166 and N254, all of which are
occupied by complex type glycans. Finally, a double mutant
N368A/N393A was generated, lacking high mannose glycans. The
mutants were transfected into CHO--S cells and expression was
performed as described in Example 2A. Secretion into the media and
hyaluronidase activity were determined as described in Examples 3
and 5, above.
[0430] The results are shown in Table 16 below, which sets forth
the mutation, the glycan types, whether the protein was secreted
into the media and the hyaluronidase activity at both pH 5.5 and pH
7.4. Western blot analysis showed that mutation of residues N82,
N166, N235 and N254 had no effect on secretion of the rHuPH20
protein into the media. Alternatively, mutation of residues N368A
and N368A/N393A prevented PH20 expression and secretion, as
evidenced by a lack of protein at approximately 66 kDa in the
media. Mutation of residue N393A resulted in reduced protein
expression, but rHuPH20 was observed in the media, as evidenced by
a protein band at approximately 66 kDa. Mutation of a residues N82,
N 166 and/or N254 had no effect on rHuPH20 activity. These residues
are occupied by complex glycans. In contrast, mutation of residues
N235, N368 and/or N393, which contain high mannose glycans,
resulted in a complete loss of detectable activity in the media due
to a lack of secretion.
TABLE-US-00017 TABLE 16 Human PH20 Hyaluronidase Deglycosylation
Mutants SEQ Activity Activity Mutant ID NO Glycan Type Secretion pH
5.5 pH 7.4 PH20sHis 187 Both YES YES YES (parent) N82A 202 Complex
YES YES YES N166A 203 Complex YES YES YES N235A 204 High Mannose
YES NO NO (80%) Complex (20%) N254A 205 Complex YES YES YES N368A
188 High Mannose NO NO NO N393A 189 High Mannose YES NO NO (WEAK)
N82A/N166A 206 Complex YES YES YES N82A/N254A 207 Complex YES YES
YES N166A/N254A 208 Complex YES YES YES N82A/N166A/ 209 Complex YES
YES YES N254A N368A/N393A 190 High Mannose NO NO NO
[0431] Immunofluorescent analysis of CHO cells with an anti-PH20
antibody was used to visualize the expression of the N-glycan site
specific deglycosylation mutants N368A, N393A and N368A/N393A. CHO
cells were seeded for monolayer culture onto 8-well chamber slides
with 200 .mu.L of cells at 2.5.times.10.sup.4 cells per ml of
Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine
serum (FBS) and grown at 37.degree. C. in a humidified atmosphere
of 5% CO.sub.2. Cells were transfected 36 hours later at 80%
confluency using Lipofectamine.TM. 2000 (Invitrogen) as follows.
DNA (0.4 .mu.g in 50 .mu.L of DMEM without serum) and
Lipofectamine.TM. 2000 (1 .mu.L in DMEM without serum) were mixed
gently for 20 minutes at room temperature and then added to each
well containing cells and 100 .mu.L serum free medium. Mixing was
effected by gently rocking the plate back and forth. The cells were
then incubated at 37.degree. C. in a CO.sub.2 incubator for 4-6
hours after which the medium was replaced with medium containing
10% FBS. At 48 hours post-transfection, the cells on the chamber
slides were fixed with 4% paraformaldehyde for 15 minutes. The
cells were washed 3.times. with PBS and 200 .mu.l, of a 1%
NP-40/PBS solution was added and incubated for 30 minutes at room
temperature. The cells were washed 3.times. with PBS and stored at
4.degree. C. prior to immunolabeling.
[0432] To immunolabel the cells, the samples were blocked with 15%
normal goat serum for 30 minutes at room temperature. The cells
were incubated with a 1:20 solution of anti-PI-120 rabbit IgG
diluted in 5% normal goat serum in PBS for 2 hours. Finally, the
cells were washed 3.times. with PBS followed by incubation with a
FITC-conjugated goat anti-rabbit IgG for 1 hour followed by
visualization. In addition, the mounting solution contained DAPI
allowing for nuclei staining. Immunofluorescent analysis using the
anti-PH20 antibody showed that N368A and N393A mutations caused
PH20 to accumulate inside the cells.
Summary of N-glycosylation Studies
[0433] As exhibited in Examples 7-10 above, N-linked glycosylation
is essential for proper folding and enzymatic activity of rHuPH20.
Complete deglycosylation of rHuPH20, effected by exhaustive
digestion with PNGaseF or by inhibition of glycosylation during
biosynthesis by treatment with tunicamyicn, abolished all
detectible enzymatic activity. In addition, unglycosylated rHuPH20
was shown to accumulate in the cell. In contrast, partially
deglycosylated rHuPH20, effected by treatment with kifunensine or
by expression in Lec mutants, retained enzymatic activity. Finally,
detailed mutational analysis using site-directed mutagenesis
revealed that the presence of high mannose type glycans is
necessary for production of soluble, enzymatically active
rHuPH20.
Example 11
Generation of a Soluble rHuPH20-expressing Cell Line
[0434] The HZ24 plasmid (set forth in SEQ ID NO:140) was used to
transfect Chinese Hamster Ovary (CHO cells) (see e.g. U.S. patent
application Ser. Nos. 10,795,095, 11/065,716 and 11/238,171). The
HZ24 plasmid vector for expression of soluble rHuPH20 contains a
pCI vector backbone (Promega), DNA encoding amino acids 1-482 of
human PH20 hyaluronidase (SEQ ID NO:110), an internal ribosomal
entry site (IRES) from the ECMV virus (Clontech), and the mouse
dihydrofolate reductase (DHFR) gene. The pCI vector backbone also
includes DNA encoding the Beta-lactamase resistance gene (AmpR), an
f1 origin of replication, a Cytomegalovirus immediate-early
enhancer/promoter region (CMV), a chimeric intron, and an SV40 late
polyadenylation signal (SV40). The DNA encoding the soluble rHuPH20
construct contains an NheI site and a Kozak consensus sequence
prior to the DNA encoding the methionine at amino acid position 1
of the native 35 amino acid signal sequence of human PH20, and a
stop codon following the DNA encoding the tyrosine corresponding to
amino acid position 482 of the human PH20 hyaluronidase set forth
in SEQ ID NO:107, followed by a BamHI restriction site. The
construct pCI-P1-120-IRES-DHFR-SV40pa (HZ24), therefore, results in
a single mRNA species driven by the CMV promoter that encodes amino
acids 1-482 of human PH20 (set forth in SEQ ID NO:109 and amino
acids 1-186 of mouse dihydrofolate reductase (set forth in SEQ ID
NO:141), separated by the internal ribosomal entry site (IRES).
[0435] Non-transfected DG44 CHO cells growing in GIBCO Modified
CD-CHO media for DHFR(-) cells, supplemented with 4 mM Glutamine
and 18 ml/L Plurionic F68/L (Gibco), were seeded at
0.5.times.10.sup.6 cells/ml in a shaker flask in preparation for
transfection. Cells were grown at 37.degree. C. in 5% CO.sub.2 in a
humidified incubator, shaking at 120 rpm. Exponentially growing
non-transfected DG44 CHO cells were tested for viability prior to
transfection.
[0436] Sixty million viable cells of the non-transfected DG44 CHO
cell culture were pelleted and resuspended to a density of
2.times.10.sup.7 cells in 0.7 mL of 2.times. transfection buffer
(2.times. HeBS: 40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mM KCl, 1.4 mM
Na.sub.2HPO.sub.4, 12 mM dextrose). To each aliquot of resuspended
cells, 0.09 mL (250n) of the linear HZ24 plasmid (linearized by
overnight digestion with Cla I (New England Biolabs) was added, and
the cell/DNA solutions were transferred into 0.4 cm gap BTX
(Gentronics) electroporation cuvettes at room temperature. A
negative control electroporation was performed with no plasmid DNA
mixed with the cells. The cell/plasmid mixes were electroporated
with a capacitor discharge of 330 V and 960 .mu.F or at 350 V and
960 .mu.F.
[0437] The cells were removed from the cuvettes after
electroporation and transferred into 5 mL of Modified CD-CHO media
for DHFR(-) cells, supplemented with 4 mM Glutamine and 18 ml/L
Plurionic F68/L (Gibco), and allowed to grow in a well of a E-well
tissue culture plate without selection for 2 days at 37.degree. C.
in 5% CO.sub.2 in a humidified incubator.
[0438] Two days post-electroporation, 0.5 mL of tissue culture
media was removed from each well and tested for the presence of
hyaluronidase activity, using the microturbidity assay described in
Example 12. Results are shown in Table 17.
TABLE-US-00018 TABLE 17 Initial Hyaluronidase Activity of HZ24
Transfected DG44 CHO cells at 40 hours post- transfection Activity
Dilution Units/ml Transfection 1 330 V 1 to 10 0.25 Transfection 2
350 V 1 to 10 0.52 Negative Control 1 to 10 0.015
[0439] Cells from Transfection 2 (350V) were collected from the
tissue culture well, counted and diluted to 1.times.10.sup.4 to
2.times.10.sup.4 viable cells per mL. A 0.1 mL aliquot of the cell
suspension was transferred to each well of five, 96 well round
bottom tissue culture plates. One hundred microliters of CD-CHO
media (GIBCO) containing 4 mM GlutaMAX.TM.-1 supplement (GIBCO.TM.,
Invitrogen Corporation) and without hypoxanthine and thymidine
supplements were added to the wells containing cells (final volume
0.2 mL).
[0440] Ten clones were identified from the 5 plates grown without
methotrexate (Table 18).
TABLE-US-00019 TABLE 18 Hyaluronidase activity of identified clones
Plate/Well ID Relative Hyaluronidase 1C3 261 2C2 261 3D3 261 3E5
243 3C6 174 2G8 103 1B9 304 2D9 273 4D10 302
[0441] Six HZ24 clones were expanded in culture and transferred
into shaker flasks as single cell suspensions. Clones 3D3, 3E5,
2G8, 2D9, 1E11, and 4D10 were plated into 96-well round bottom
tissue culture plates using a two-dimensional infinite dilution
strategy in which cells were diluted 1:2 down the plate, and 1:3
across the plate, starting at 5000 cells in the top left hand well.
Diluted clones were grown in a background of 500 non-transfected
DG44 CHO cells per well, to provide necessary growth factors for
the initial days in culture. Ten plates were made per subclone,
with 5 plates containing 50 nM methotrexate and 5 plates without
methotrexate.
[0442] Clone 3D3 produced 24 visual subclones (13 from the no
methotrexate treatment, and 11 from the 50 nM methotrexate
treatment. Significant hyaluronidase activity was measured in the
supernatants from 8 of the 24 subclones (>50 Units/mL), and
these 8 subclones were expanded into T-25 tissue culture flasks.
Clones isolated from the methotrexate treatment protocol were
expanded in the presence of 50 nM methotrexate. Clone 3D35M was
further expanded in 500 nM methotrexate giving rise to clones
producing in excess of 1,000 Units/ml in shaker flasks (clone
3D35M; or Gen1 3D35M). A master cell bank (MCB) of the 3D35M cells
was then prepared.
Example 12
Determination of Hyaluronidase Activity of Soluble rHuPH20
[0443] Hyaluronidase activity of soluble rHuPH20 in samples such as
cell cultures, purification fractions and purified solutions was
determined using a turbidometric assay, which is based on the
formation of an insoluble precipitate when hyaluronic acid binds
with serum albumin. The activity is measured by incubating soluble
rHuPH20 with sodium hyaluronate (hyaluronic acid) for a set period
of time (10 minutes) and then precipitating the undigested sodium
hyaluronate with the addition of acidified serum albumin. The
turbidity of the resulting sample is measured at 640 nm after a 30
minute development period. The decrease in turbidity resulting from
enzyme activity on the sodium hyaluronate substrate is a measure of
the soluble rHuPH20 hyaluronidase activity. The method is performed
using a calibration curve generated with dilutions of a soluble
rHuPH20 assay working reference standard, and sample activity
measurements are made relative to this calibration curve.
[0444] Dilutions of the sample were prepared in Enzyme Diluent
Solution. The Enzyme Diluent Solution was prepared by dissolving
33.0.+-.0.05 mg of hydrolyzed gelatin in 25.0 mL of the 50 mM PIPES
Reaction Buffer (140 mM NaCl, 50 mM PIPES, pH 5.5) and 25.0 mL of
SWFI, and diluting 0.2 mL of 25% Buminate solution into the mixture
and vortexing for 30 seconds. This was performed within 2 hours of
use and stored on ice until needed. The samples were diluted to an
estimated 1-2 U/mL. Generally, the maximum dilution per step did
not exceed 1:100 and the initial sample size for the first dilution
was not be less than 20 pt. The minimum sample volumes needed to
perform the assay were: In-process Samples, FPLC Fractions: 80
.mu.L; Tissue Culture Supernatants: 1 mL; Concentrated Material 80
.mu.L; Purified or Final Step Material: 80 .mu.l. The dilutions
were made in triplicate in a Low Protein Binding 96-well plate, and
30 .mu.L of each dilution was transferred to Optilux black/clear
bottom plates (BD BioSciences).
[0445] Dilutions of known soluble rHuPH20 with a concentration of
2.5 U/mL were prepared in Enzyme Diluent Solution to generate a
standard curve and added to the Optilux plate in triplicate. The
dilutions included 0 U/mL, 0.25 U/mL, 0.5 U/mL, 1.0 U/mL, 1.5 U/mL,
2.0 U/mL, and 2.5 U/mL. "Reagent blank" wells that contained 60
.mu.L of Enzyme Diluent Solution were included in the plate as a
negative control. The plate was then covered and warmed on a heat
block for 5 minutes at 37.degree. C. The cover was removed and the
plate was shaken for 10 seconds. After shaking, the plate was
returned to the heat block and the MULTIDROP 384 Liquid Handling
Device was primed with the warm 0.25 mg/mL sodium hyaluronate
solution (prepared by dissolving 100 mg of sodium hyaluronate
(LifeCore Biomedical) in 20.0 mL of SWFI. This was mixed by gently
rotating and/or rocking at 2-8.degree. C. for 2-4 hours, or until
completely dissolved). The reaction plate was transferred to the
MULTIDROP 384 and the reaction was initiated by pressing the start
key to dispense 30 .mu.L sodium hyaluronate into each well. The
plate was then removed from the MULTIDROP 384 and shaken for 10
seconds before being transferred to a heat block with the plate
cover replaced. The plate was incubated at 37.degree. C. for 10
minutes
[0446] The MULTIDROP 384 was prepared to stop the reaction by
priming the machine with Serum Working Solution and changing the
volume setting to 240 .mu.L. (25 mL of Serum Stock Solution [1
volume of Horse Serum (Sigma) was diluted with 9 volumes of 500 mM
Acetate Buffer Solution and the pH was adjusted to 3.1 with
hydrochloric acid] in 75 mL of 500 mM Acetate Buffer Solution). The
plate was removed from the heat block and placed onto the MULTIDROP
384 and 240 .mu.L of serum Working Solutions was dispensed into the
wells. The plate was removed and shaken on a plate reader for 10
seconds. After a further 15 minutes, the turbidity of the samples
was measured at 640 nm and the hyaluronidase activity (in U/mL) of
each sample was determined by fitting to the standard curve.
[0447] Specific activity (Units/mg) was calculated by dividing the
hyaluronidase activity (U/ml) by the protein concentration
(mg/mL).
Example 13
Production and Purification of Gen1 Human sPH20
A. 5 L Bioreactor Process
[0448] A vial of 3D35M was thawed and expanded from shaker flasks
through 1 L spinner flasks in CD-CHO media (Invitrogen, Carlsbad
Calif.) supplemented with 100 nM Methotrexate and GlutaMAX.TM.-1
(Invitrogen). Cells were transferred from spinner flasks to a 5 L
bioreactor (Braun) at an inoculation density of 4.times.10.sup.5
viable cells per ml. Parameters were temperature Setpoint
37.degree. C., pH 7.2 (starting Setpoint), with Dissolved Oxygen
Setpoint 25% and an air overlay of 0-100 cc/min. At 168 hrs, 250 ml
of Feed #1 Medium (CD CHO with 50 g/L Glucose) was added. At 216
hours, 250 ml of Feed #2 Medium (CD CHO with 50 g/L Glucose and 10
mM Sodium Butyrate) was added, and at 264 hours 250 ml of Feed #2
Medium was added. This process resulted in a final productivity of
1600 Units per ml with a maximal cell density of 6.times.10.sup.6
cells/ml. The addition of sodium butyrate was to dramatically
enhance the production of soluble rHuPH20 in the final stages of
production.
[0449] Conditioned media from the 3D35M clone was clarified by
depth filtration and tangential flow diafiltration into 10 mM Hepes
pH 7.0. Soluble rHuPH20 was then purified by sequential
chromatography on Q Sepharose (Pharmacia) ion exchange, Phenyl
Sepharose (Pharmacia) hydrophobic interaction chromatography,
phenyl boronate (Prometics) and Hydroxapatite Chromatography
(Biorad, Richmond, Calif.).
[0450] Soluble rHuPH20 bound to Q Sepharose and eluted at 400 mM
NaCl in the same buffer. The eluate was diluted with 2M ammonium
sulfate to a final concentration of 500 mM ammonium sulfate and
passed through a Phenyl Sepharose (low sub) column, followed by
binding under the same conditions to a phenyl boronate resin. The
soluble rHuPH20 was eluted from the phenyl sepharose resin in Hepes
pH 6.9 after washing at pH 9.0 in 50 mM bicine without ammonium
sulfate. The eluate was loaded onto a ceramic hydroxyapatite resin
at pH 6.9 in 5 mM potassium phosphate and 1 mM CaCl.sub.2 and
eluted with 80 mM potassium phosphate, pH 7.4 with 0.1 mM
CaCl.sub.2.
[0451] The resultant purified soluble rHuPH20 possessed a specific
activity in excess of 65,000 USP Units/mg protein by way of the
microturbidity assay (Example 12) using the USP reference standard.
Purified sPH20 eluted as a single peak from 24 to 26 minutes from a
Pharmacia 5RPC styrene divinylbenzene column with a gradient
between 0.1% TFA/H.sub.2O and 0.1% TFA/90% acetonitrile/10%
H.sub.2O and resolved as a single broad 61 kDa band by SDS
electrophoresis that reduced to a sharp 51 kDa band upon treatment
with PNGASE-F. N-terminal amino acid sequencing revealed that the
leader peptide had been efficiently removed.
B. Upstream Cell Culture Expansion Process into 100 L Bioreactor
Cell Culture
[0452] A scaled-up process was used to separately purify soluble
rHuPH20 from four different vials of 3D35M cell to produce 4
separate batches of sHuPH20; HUA0406C, HUA0410C, HUA0415C and
HUA0420C. Each vial was separately expanded and cultured through a
125 L bioreactor, then purified using column chromatography.
Samples were taken throughout the process to assess such parameters
as enzyme yield. The description of the process provided below sets
forth representative specifications for such things as bioreactor
starting and feed media volumes, transfer cell densities, and wash
and elution volumes. The exact numbers vary slightly with each
batch, and are detailed in Tables 24 to 30.
[0453] Four vials of 3D35M cells were thawed in a 37.degree. C.
water bath, CD CHO containing 100 nM methotrexate and 40 mL/L
GlutaMAX was added and the cells were centrifuged. The cells were
re-suspended in a 125 mL shake flask with 20 mL of fresh media and
placed in a 37.degree. C., 7% CO.sub.2 incubator. The cells were
expanded up to 40 mL in the 125 mL shake flask. When the cell
density reached 1.5-2.5.times.10.sup.6 cells/mL, the culture was
expanded into a 125 mL spinner flask in a 100 mL culture volume.
The flask was incubated at 37.degree. C., 7% CO.sub.2. When the
cell density reached 1.5-2.5.times.10.sup.6 cells/mL, the culture
was expanded into a 250 mL spinner flask in 200 mL culture volume,
and the flask was incubated at 37.degree. C., 7% CO.sub.2. When the
cell density reached 1.5-2.5.times.10.sup.6 cells/mL, the culture
was expanded into a 1 L spinner flask in 800 mL culture volume and
incubated at 37.degree. C., 7% CO.sub.2. When the cell density
reached 1.5-2.5.times.10.sup.6 cells/mL, the culture was expanded
into a 6 L spinner flask in 5 L culture volume and incubated at
37.degree. C., 7% CO.sub.2. When the cell density reached
1.5-2.5.times.10.sup.6 cells/mL, the culture was expanded into a 36
L spinner flask in 20 L culture volume and incubated at 37.degree.
C., 7% CO.sub.2.
[0454] A 125 L reactor was sterilized with steam at 121.degree. C.,
20 PSI and 65 L of CD CHO media was added. Before use, the reactor
was checked for contamination. When the cell density in the 36 L
spinner flasks reached 1.8-2.5.times.10.sup.6 cells/mL, 20 L cell
culture were transferred from the 36 L spinner flasks to the 125 L
bioreactor (Braun), resulting a final volume of 85 L and a seeding
density of approximately 4.times.10.sup.5 cells/mL. Parameters were
temperature setpoint, 37.degree. C.; pH: 7.2; Dissolved oxygen:
25%.+-.10%; Impeller Speed 50 rpm; Vessel Pressure 3 psi; Air
Sparge 1 L/min.; Air Overlay: 1 L/min. The reactor was sampled
daily for cell counts, pH verification, media analysis, protein
production and retention. Nutrient feeds were added during the run.
At Day 6, 3.4 L of Feed #1 Medium (CD CHO+50 g/L Glucose+40 mL/L
GlutaMAX.TM.-1) was added, and culture temperature was changed to
36.5.degree. C. At day 9, 3.5 L of Feed #2 (CD CHO+50 g/L
Glucose+40 mL/L GlutaMAX.TM.-1+1.1 g/L Sodium Butyrate) was added,
and culture temperature was changed to 36.degree. C. At day 11, 3.7
L of Feed #3 (CD CHO+50 g/L Glucose+40 mL/L GlutaMAX.TM.-1+1.1 g/L
Sodium Butyrate) was added, and the culture temperature was changed
to 35.5.degree. C. The reactor was harvested at 14 days or when the
viability of the cells dropped below 50%. The process resulted in
production of soluble rHuPH20 with an enzymatic activity of 1600
Units/ml with a maximal cell density of 8 million cells/mL. At
harvest, the culture was sampled for mycoplasma, bioburden,
endotoxin, and virus in vitro and in vivo, transmission electron
microscopy (TEM) for viral particles, and enzyme activity.
[0455] The one hundred liter bioreactor cell culture harvest was
filtered through a series of disposable capsule filters having a
polyethersulfone medium (Sartorius): first through a 8.0 .mu.m
depth capsule, a 0.65 .mu.m depth capsule, a 0.22 .mu.m capsule,
and finally through a 0.22 .mu.m Sartopore 2000 cm.sup.2 filter and
into a 100 L sterile storage bag. The culture was concentrated
10.times. using two TFF with Spiral Polyethersulfone 30 kDa MWCO
filters (Millipore), followed by a 6.times. buffer exchange with 10
mM HEPES, 25 mM Na.sub.2SO.sub.4, pH 7.0 into a 0.22 .mu.m final
filter into a 20 L sterile storage bag. Table 19 provides
monitoring data related to the cell culture, harvest, concentration
and buffer exchange steps.
TABLE-US-00020 TABLE 19 Monitoring data for cell culture, harvest,
concentration and buffer exchange steps. Parameter HUA0406C
HUA04010C HUA0415C HUA0420C Time from thaw to inoculate 100 L 21 19
17 18 bioreactor (days) 100 L inoculation density (.times.10.sup.6
cells/mL) 0.45 0.33 0.44 0.46 Doubling time in logarithmic 29.8
27.3 29.2 23.5 growth (hr) Max. cell density (.times.10.sup.6
cells/mL) 5.65 8.70 6.07 9.70 Harvest viability (%) 41 48 41 41
Harvest titer (U/ml) 1964 1670 991 1319 Time in 100-L bioreactor
(days) 13 13 12 13 Clarified harvest volume (mL) 81800 93300 91800
89100 Clarified harvest enzyme assay 2385 1768 1039 1425 (U/mL)
Concentrate enzyme assay 22954 17091 8561 17785 (U/mL) Buffer
exchanged concentrate 15829 11649 9915 8679 enzyme assay (U/mL)
Filtered buffer exchanged 21550 10882 9471 8527 concentrate enzyme
assay (U/mL) Buffer exchanged concentrate 10699 13578 12727 20500
volume(mL) Ratio enzyme units 0.87 0.96 1.32 1.4
concentration/harvest
[0456] A Q Sepharose (Pharmacia) ion exchange column (3 L resin,
Height=20 cm, Diameter=14 cm) was prepared. Wash samples were
collected for a determination of pH, conductivity and endotoxin
(LAL) assay. The column was equilibrated with 5 column volumes of
10 mM Tris, 20 mM Na.sub.2SO.sub.4, pH 7.5. The concentrated,
diafiltered harvest was loaded onto the Q column at a flow rate of
100 cm/hr. The column was washed with 5 column volumes of 10 mM
Tris, 20 mM Na.sub.2SO.sub.4, pH 7.5 and 10 mM Hepes, 50 mM NaCl,
pH 7.0. The protein was eluted with 10 mM Hepes, 400 mM NaCl, pH
7.0 and filtered through a 0.22 .mu.m final filter into a sterile
bag.
[0457] Phenyl-Sepharose (Pharmacia) hydrophobic interaction
chromatography was next performed. A Phenyl-Sepharose (PS) column
(9.1 L resin, Height=29 cm, Diameter=20 cm) was prepared. The
column was equilibrated with 5 column volumes of 5 mM potassium
phosphate, 0.5 M ammonium sulfate, 0.1 mM CaCl.sub.2, pH 7.0. The
protein eluate from above was supplemented with 2M ammonium
sulfate, 1 M potassium phosphate and 1 M CaCl.sub.2 stock solutions
to final concentrations of 5 mM, 0.5 M and 0.1 mM, respectively.
The protein was loaded onto the PS column at a flow rate of 100
cm/hr. 5 mM potassium phosphate, 0.5 M ammonium sulfate and 0.1 mM
CaCl.sub.2 pH 7.0 was added at 100 cm/hr. The flow through was
passed through a 0.22 .mu.M final filter into a sterile bag.
[0458] The PS-purified protein was the loaded onto an aminophenyl
boronate column (ProMedics) (6.3 L resin, Height=20 cm, Diameter=20
cm) that had been equilibrated with 5 column volumes of 5 mM
potassium phosphate, 0.5 M ammonium sulfate. The protein was passed
through the column at a flow rate of 100 cm/hr, and the column was
washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate, pH
7.0. The column was then washed with 20 mM bicine, 100 mM NaCl, pH
9.0 and the protein eluted with 50 mM Hepes, 100 mM NaCl pH 6.9
through a sterile filter and into a 20 L sterile bag. The eluate
was tested for bioburden, protein concentration and enzyme
activity.
[0459] A hydroxyapatite (HAP) column (BioRad) (1.6 L resin,
Height=10 cm, Diameter=14 cm) was equilibrated with 5 mM potassium
phosphate, 100 mM NaCl, 0.1 mM CaCl.sub.2 pH 7.0. Wash samples were
collected and tested for pH, conductivity and endotoxin (LAL assay.
The aminophenyl boronate purified protein was supplemented with
potassium phosphate and CaCl.sub.2 to yield final concentrations of
5 mM potassium phosphate and 0.1 mM CaCl.sub.2 and loaded onto the
HAP column at a flow rate of 100 cm/hr. The column was washed with
5 mM potassium phosphate pH 7.0, 100 mM NaCl, 0.1 mM CaCl.sub.2,
then 10 mM potassium phosphate pH 7.0, 100 mM NaCl, 0.1 mM
CaCl.sub.2 pH. The protein was eluted with 70 mM potassium
phosphate pH 7.0 and filtered through a 0.22 .mu.m filter into a 5
L sterile storage bag. The eluate was tested for bioburden, protein
concentration and enzyme activity.
[0460] The HAP-purified protein was then pumped through a 20 nM
viral removal filter via a pressure tank. The protein was added to
the DV20 pressure tank and filter (Pall Corporation), passing
through an Ultipor DV20 Filter with 20 nm pores (Pall Corporation)
into a sterile 20 L storage bag. The filtrate was tested for
protein concentration, enzyme activity, oligosaccharide,
monosaccharide and sialic acid profiling, and process-related
impurities. The protein in the filtrate was then concentrated to 1
mg/mL using a 10 kD molecular weight cut off (MWCO) Sartocon Slice
tangential flow filtration (TFF) system (Sartorius). The filter was
first prepared by washing with a Hepes/saline solution (10 mM
Hepes, 130 mM NaCl, pH 7.0) and the permeate was sampled for pH and
conductivity. Following concentration, the concentrated protein was
sampled and tested for protein concentration and enzyme activity. A
6.times. buffer exchange was performed on the concentrated protein
into the final buffer: 10 mM Hepes, 130 mM NaCl, pH 7.0. The
concentrated protein was passed though a 0.22 .mu.m filter into a
20 L sterile storage bag. The protein was sampled and tested for
protein concentration, enzyme activity, free sulfydryl groups,
oligosaccharide profiling and osmolarity.
[0461] Tables 20 to 26 provide monitoring data related to each of
the purification steps described above, for each 3D35M cell
lot.
TABLE-US-00021 TABLE 20 Q sepharose column data Parameter HUA0406C
HUA0410C HUA0415C HUA0420C Load volume 10647 13524 12852 20418 (mL)
Load Volume/ 3.1 4.9 4.5 7.3 Resin Volume ratio Column 2770 3840
2850 2880 Volume (mL) Eluate volume 6108 5923 5759 6284 (mL)
Protein Conc. 2.8 3.05 2.80 2.86 of Eluate (mg/mL) Eluate Enzyme
24493 26683 18321 21052 Assay (U/mL) Enzyme Yield 65 107 87 76
(%)
TABLE-US-00022 TABLE 21 Phenyl Sepharose column data Parameter
HUA0406C HUA0410C HUA0415C HUA0420C Volume Before 5670 5015 5694
6251 Stock Solution Addition (mL) Load Volume 7599 6693 7631 8360
(mL) Column 9106 9420 9340 9420 Volume (mL) Load Volume/ 0.8 0.71
0.82 0.89 Resin Volume ratio Eluate volume 16144 18010 16960 17328
(mL) Protein Cone 0.4 0.33 0.33 0.38 of Eluate (mg/mL) Eluate
Enzyme 8806 6585 4472 7509 Assay (U/mL) Protein Yield 41 40 36 37
(%) Enzyme Yield 102 88 82 96 (%)
TABLE-US-00023 TABLE 22 Amino Phenyl Boronate column data Parameter
HUA0406C HUA0410C HUA0415C HUA0420C Load Volume 16136 17958 16931
17884 (mL) Load Volume/ 2.99 3.15 3.08 2.98 Resin Volume ratio
Column 5400 5700 5500 5300 Volume (mL) Eluate volume 17595 22084
20686 19145 (mL) Protein Conc. 0.0 0.03 0.03 0.04 of Eluate (mg/mL)
Protein Conc. not tested 0.03 0.00 0.04 of Filtered Eluate (mg/mL)
Eluate Enzyme 4050 2410 1523 4721 Assay (U/mL) Protein Yield 0 11
11 12 (%) Enzyme Yield not 41 40 69 (%) determined
TABLE-US-00024 TABLE 23 Hydroxyapatite column data Parameter
HUA0406C HUA0410C HUA0415C HUA0420C Volume Before 16345 20799 20640
19103 Stock Solution Addition (mL) Load Volume/ 10.95 13.58 14.19
12.81 Resin Volume ratio Column 1500 1540 1462 1500 Volume (mL)
Load volume 16429 20917 20746 19213 (mL) Eluate volume 4100 2415
1936 2419 (mL) Protein Conc. not tested 0.24 0.17 0.23 of Eluate
(mg/mL) Protein Conc. NA NA 0.17 NA of Filtered Eluate (mg/mL)
Eluate 14051 29089 20424 29826 Enzyme Assay (U/mL) Protein Yield
Not tested 93 53 73 (%) Enzyme Yield 87 118 140 104 (%)
TABLE-US-00025 TABLE 24 DV20 filtration data Parameter HUA0406C
HUA0410C HUA0415C HUA0420C Start volume 4077 2233 1917 2419 (mL)
Filtrate Volume 4602 3334 2963 3504 (mL) Protein Conc. 0.1 NA 0.09
NA of Filtrate (mg/mL) Protein Conc. NA 0.15 0.09 0.16 of Filtered
Eluate (mg/mL) Protein Yield not tested 93 82 101 (%)
TABLE-US-00026 TABLE 25 Final concentration data Parameter HUA0406C
HUA0410C HUA0415C HUA0420C Start volume 4575 3298 2963 3492 (mL)
Concentrate 562 407 237 316 Volume (mL) Protein Conc. 0.9 1.24 1.16
1.73 of Concentrate (mg/mL) Protein Yield 111 102 103 98 (%)
TABLE-US-00027 TABLE 26 Buffer Exchange into Final Formulation data
Parameter HUA0406C HUA0410C HUA0415C HUA0420C Start Volume 562 407
237 316 (mL) Final Volume 594 516 310 554 Buffer Exchanged
Concentrate (mL) Protein Conc. 1.00 0.97 0.98 1.00 of Concentrate
(mg/mL) Protein Conc. 0.95 0.92 0.95 1.02 of Filtered Concentrate
(mg/mL) Protein Yield 118 99 110 101 (%)
[0462] The purified and concentrated soluble rHuPH20 protein was
aseptically filled into sterile vials with 5 mL and 1 mL fill
volumes. The protein was passed though a 0.22 .mu.m filter to an
operator controlled pump that was used to fill the vials using a
gravimetric readout. The vials were closed with stoppers and
secured with crimped caps. The closed vials were visually inspected
for foreign particles and then labeled. Following labeling, the
vials were flash-frozen by submersion in liquid nitrogen for no
longer than 1 minute and stored at <-15.degree. C.
(-20.+-.5.degree. C.).
Example 14
Production Gen2 Cells Containing Soluble Human PH20 (rHuPH20)
[0463] The Gen1 3D35M cell line described in Example 13 was adapted
to higher methotrexate levels to produce generation 2 (Gen2)
clones. 3D35M cells were seeded from established
methotrexate-containing cultures into CD CHO medium containing 4 mM
GlutaMAX-1.TM. and 1.0 .mu.M methotrexate. The cells were adapted
to a higher methotrexate level by growing and passaging them 9
times over a period of 46 days in a 37.degree. C., 7% CO.sub.2
humidified incubator. The amplified population of cells was cloned
out by limiting dilution in 96-well tissue culture plates
containing medium with 2.0 .mu.M methotrexate. After approximately
4 weeks, clones were identified and clone 3E10B was selected for
expansion. 3E10B cells were grown in CD CHO medium containing 4 mM
GlutaMAX-1.TM. and 2.0 .mu.M methotrexate for 20 passages. A master
cell bank (MCB) of the 3E10B cell line was created and frozen and
used for subsequent studies.
[0464] Amplification of the cell line continued by culturing 3E10B
cells in CD CHO medium containing 4 mM GlutaMAX-1.TM. and 4.0 .mu.M
methotrexate. After the 12.sup.th passage, cells were frozen in
vials as a research cell bank (RCB). One vial of the RCB was thawed
and cultured in medium containing 8.0 .mu.M methotrexate. After 5
days, the methotrexate concentration in the medium was increased to
16.0 .mu.M, then 20.0 .mu.M 18 days later. Cells from the 8.sup.th
passage in medium containing 20.0 .mu.M methotrexate were cloned
out by limiting dilution in 96-well tissue culture plates
containing CD CHO medium containing 4 mM GlutaMAX-1.TM. and 20.0
.mu.M methotrexate. Clones were identified 5-6 weeks later and
clone 2B2 was selected for expansion in medium containing 20.0
.mu.M methotrexate. After the 11th passage, 2B2 cells were frozen
in vials as a research cell bank (RCB).
[0465] The resultant 2B2 cells are dihydrofolate reductase
deficient (dhfr-) DG44 CHO cells that express soluble recombinant
human PH20 (rHuPH20). The soluble PH20 is present in 2B2 cells at a
copy number of approximately 206 copies/cell. Southern blot
analysis of Spe I-, Xba I- and BamH I/Hind III-digested genomic 2B2
cell DNA using a rHuPH20-specific probe revealed the following
restriction digest profile: one major hybridizing band of
.about.7.7 kb and four minor hybridizing bands (.about.13.9,
.about.6.6, .about.5.7 and .about.4.6 kb) with DNA digested with
Spe I; one major hybridizing band of .about.5.0 kb and two minor
hybridizing bands (.about.13.9 and .about.6.5 kb) with DNA digested
with Xba I; and one single hybridizing band of .about.1.4 kb
observed using 2B2 DNA digested with BamH I/Hind III. Sequence
analysis of the mRNA transcript indicated that the derived cDNA
(SEQ ID NO:139) was identical to the reference sequence (SEQ ID
NO:110) except for one base pair difference at position 1131, which
was observed to be a thymidine (T) instead of the expected cytosine
(C). This is a silent mutation, with no effect on the amino acid
sequence.
Example 15
A. Production of Gen2 soluble rHuPH20 in 300 L Bioreactor Cell
Culture
[0466] A vial of HZ24-2B2 was thawed and expanded from shaker
flasks through 36 L spinner flasks in CD-CHO media (Invitrogen,
Carlsbad, Calif.) supplemented with 20 .mu.M methotrexate and
GlutaMAX-1.TM. (Invitrogen). Briefly, the vial of cells was thawed
in a 37.degree. C. water bath, media was added and the cells were
centrifuged. The cells were re-suspended in a 125 mL shake flask
with 20 mL of fresh media and placed in a 37.degree. C., 7%
CO.sub.2 incubaor. The cells were expanded up to 40 mL in the 125
mL shake flask. When the cell density reached greater than
1.5.times.10.sup.6 cells/mL, the culture was expanded into a 125 mL
spinner flask in a 100 mL culture volume. The flask was incubated
at 37.degree. C., 7% CO.sub.2. When the cell density reached
greater than 1.5.times.10.sup.6 cells/mL, the culture was expanded
into a 250 mL spinner flask in 200 mL culture volume, and the flask
was incubated at 37.degree. C., 7% CO.sub.2. When the cell density
reached greater than 1.5.times.10.sup.6 cells/mL, the culture was
expanded into a 1 L spinner flask in 800 mL culture volume and
incubated at 37.degree. C., 7% CO.sub.2. When the cell density
reached greater than 1.5.times.10.sup.6 cells/mL the culture was
expanded into a 6 L spinner flask in 5000 mL culture volume and
incubated at 37.degree. C., 7% CO.sub.2. When the cell density
reached greater than 1.5.times.10.sup.6 cells/mL the culture was
expanded into a 36 L spinner flask in 32 L culture volume and
incubated at 37.degree. C., 7% CO.sub.2.
[0467] A 400 L reactor was sterilized and 230 mL of CD-CHO media
was added. Before use, the reactor was checked for contamination.
Approximately 30 L cells were transferred from the 36 L spinner
flasks to the 400 L bioreactor (Braun) at an inoculation density of
4.0.times.10.sup.5 viable cells per ml and a total volume of 260 L.
Parameters were temperature setpoint, 37.degree. C.; Impeller Speed
40-55 RPM; Vessel Pressure: 3 psi; Air Sparge 0.5-1.5 L/Min.; Air
Overlay: 3 L/min. The reactor was sampled daily for cell counts, pH
verification, media analysis, protein production and retention.
Also, during the run nutrient feeds were added. At 120 hrs (day 5),
10.4 L of Feed #1 Medium (4.times.CD-CHO+33 g/L Glucose+160 mL/L
Glutamax-1.TM.+83 mL/L Yeastolate+33 mg/L rHulnsulin) was added. At
168 hours (day 7), 10.8 L of Feed #2 (2.times.CD-CHO+33 g/L
Glucose+80 mL/L Glutamax-1.TM.+167 mL/L Yeastolate+0.92 g/L Sodium
Butyrate) was added, and culture temperature was changed to
36.5.degree. C. At 216 hours (day 9), 10.8 L of Feed #3 (1.times.
CD-CHO+50 g/L Glucose+50 mL/L Glutamax-1.TM.+250 mL/L
Yeastolate+1.80 g/L Sodium Butyrate) was added, and culture
temperature was changed to 36.degree. C. At 264 hours (day 11),
10.8 L of Feed #4 (1.times. CD-CHO+33 g/L Glucose+33 mL/L
Glutamax-1.TM.+250 mL/L Yeastolate+0.92 g/L Sodium Butyrate) was
added, and culture temperature was changed to 35.5.degree. C. The
addition of the feed media was observed to dramatically enhance the
production of soluble rHuPH20 in the final stages of production.
The reactor was harvested at 14 or 15 days or when the viability of
the cells dropped below 40%. The process resulted in a final
productivity of 17,000 Units per ml with a maximal cell density of
12 million cells/mL. At harvest, the culture was sampled for
mycoplasma, bioburden, endotoxin and viral in vitro and in vivo,
Transmission Electron Microscopy (TEM) and enzyme activity.
[0468] The culture was pumped by a peristaltic pump through four
Millistak filtration system modules (Millipore) in parallel, each
containing a layer of diatomaceous earth graded to 4-8 .mu.m and a
layer of diatomaceous earth graded to 1.4-1.1 .mu.m, followed by a
cellulose membrane, then through a second single Millistak
filtration system (Millipore) containing a layer of diatomaceous
earth graded to 0.4-0.11 .mu.m and a layer of diatomaceous earth
graded to <0.1 .mu.m, followed by a cellulose membrane, and then
through a 0.22 .mu.m final filter into a sterile single use
flexible bag with a 350 L capacity. The harvested cell culture
fluid was supplemented with 10 mM EDTA and 10 mM Tris to a pH of
7.5. The culture was concentrated 10.times. with a tangential flow
filtration (TFF) apparatus using four Sartoslice TFF 30 kDa
molecular weight cut-off (MWCO) polyether sulfone (PES) filter
(Sartorious), followed by a 10.times. buffer exchange with 10 mM
Tris, 20 mM Na.sub.2SO.sub.4, pH 7.5 into a 0.22 .mu.m final filter
into a 50 L sterile storage bag.
[0469] The concentrated, diafiltered harvest was inactivated for
virus. Prior to viral inactivation, a solution of 10% Triton.RTM.
X-100, 3% tri (n-butyl) phosphate (TNBP) was prepared. The
concentrated, diafiltered harvest was exposed to 1% Triton.RTM.
X-100, 0.3% TNBP for 1 hour in a 36 L glass reaction vessel
immediately prior to purification on the Q column.
B. Purification of Gen2 Soluble rHuPH20
[0470] A Q Sepharose (Pharmacia) ion exchange column (9 L resin,
H=29 cm, D=20 cm) was prepared. Wash samples were collected for a
determination of pH, conductivity and endotoxin (LAL) assay. The
column was equilibrated with 5 column volumes of 10 mM Tris, 20 mM
Na.sub.2SO.sub.4, pH 7.5. Following viral inactivation, the
concentrated, diafiltered harvest was loaded onto the Q column at a
flow rate of 100 cm/hr. The column was washed with 5 column volumes
of 10 mM Tris, 20 mM Na.sub.2SO.sub.4, pH 7.5 and 10 mM Hepes, 50
mM NaCl, pH7.0. The protein was eluted with 10 mM Hepes, 400 mM
NaCl, pH 7.0 into a 0.22 .mu.m final filter into sterile bag. The
eluate sample was tested for bioburden, protein concentration and
hyaluronidase activity. A.sub.280 absorbance reading were taken at
the beginning and end of the exchange.
[0471] Phenyl-Sepharose (Pharmacia) hydrophobic interaction
chromatography was next performed. A Phenyl-Speharose (PS) column
(19-21 L resin, H=29 cm, D=30 cm) was prepared. The wash was
collected and sampled for pH, conductivity and endotoxin (LAL
assay). The column was equilibrated with 5 column volumes of 5 mM
potassium phosphate, 0.5 M ammonium sulfate, 0.1 mM CaCl.sub.2, pH
7.0. The protein eluate from the Q sepharose column was
supplemented with 2M ammonium sulfate, 1 M potassium phosphate and
1 M CaCl.sub.2 stock solutions to yield final concentrations of 5
mM, 0.5 M and 0.1 mM, respectively. The protein was loaded onto the
PS column at a flow rate of 100 cm/hr and the column flow thru
collected. The column was washed with 5 mM potassium phosphate, 0.5
M ammonium sulfate and 0.1 mM CaCl.sub.2 pH 7.0 at 100 cm/hr and
the wash was added to the collected flow thru. Combined with the
column wash, the flow through was passed through a 0.22 pm final
filter into a sterile bag. The flow through was sampled for
bioburden, protein concentration and enzyme activity.
[0472] An aminophenyl boronate column (Prometics) was prepared. The
wash was collected and sampled for pH, conductivity and endotoxin
(LAL assay). The column was equilibrated with 5 column volumes of 5
mM potassium phosphate, 0.5 M ammonium sulfate. The PS flow through
containing purified protein was loaded onto the aminophenyl
boronate column at a flow rate of 100 cm/hr. The column was washed
with 5 mM potassium phosphate, 0.5 M ammonium sulfate, pH 7.0. The
column was washed with 20 mM bicine, 0.5 M ammonium sulfate, pH
9.0. The column was washed with 20 mM bicine, 100 mM sodium
chloride, pH 9.0. The protein was eluted with 50 mM Hepes, 100 mM
NaCl, pH 6.9 and passed through a sterile filter into a sterile
bag. The eluted sample was tested for bioburden, protein
concentration and enzyme activity.
[0473] The hydroxyapatite (HAP) column (Biorad) was prepared. The
wash was collected and tested for pH, conductivity and endotoxin
(LAL assay). The column was equilibrated with 5 mM potassium
phosphate, 100 mM NaCl, 0.1 mM CaCl.sub.2, pH 7.0. The aminophenyl
boronate purified protein was supplemented to final concentrations
of 5 mM potassium phosphate and 0.1 mM CaCl.sub.2 and loaded onto
the HAP column at a flow rate of 100 cm/hr. The column was washed
with 5 mM potassium phosphate, pH 7, 100 mM NaCl, 0.1 mM
CaCl.sub.2. The column was next washed with 10 mM potassium
phosphate, pH 7, 100 mM NaCl, 0.1 mM CaCl.sub.2. The protein was
eluted with 70 mM potassium phosphate, pH 7.0 and passed through a
0.22 .mu.m sterile filter into a sterile bag. The eluted sample was
tested for bioburden, protein concentration and enzyme
activity.
[0474] The HAP purified protein was then passed through a viral
removal filter. The sterilized Viosart filter (Sartorius) was first
prepared by washing with 2 L of 70 mM potassium phosphate, pH 7.0.
Before use, the filtered buffer was sampled for pH and
conductivity. The HAP purified protein was pumped via a peristaltic
pump through the 20 nM viral removal filter. The filtered protein
in 70 mM potassium phosphate, pH 7.0 was passed through a 0.22
.mu.m final filter into a sterile bag. The viral filtered sample
was tested for protein concentration, enzyme activity,
oligosaccharide, monosaccharide and sialic acid profiling. The
sample also was tested for process related impurities.
[0475] The protein in the filtrate was then concentrated to 10
mg/mL using a 10 kD molecular weight cut off (MWCO) Sartocon Slice
tangential flow filtration (TFF) system (Sartorius). The filter was
first prepared by washing with 10 mM histidine, 130 mM NaCl, pH 6.0
and the permeate was sampled for pH and conductivity. Following
concentration, the concentrated protein was sampled and tested for
protein concentration and enzyme activity. A 6.times. buffer
exchange was performed on the concentrated protein into the final
buffer: 10 mM histidine, 130 mM NaCl, pH 6.0. Following buffer
exchange, the concentrated protein was passed though a 0.22 .mu.m
filter into a 20 L sterile storage bag. The protein was sampled and
tested for protein concentration, enzyme activity, free sulfydryl
groups, oligosaccharide profiling and osmolarity.
[0476] The sterile filtered bulk protein was then asceptically
dispensed at 20 mL into 30 mL sterile Teflon vials (Nalgene). The
vials were then flash frozen and stored at -20.+-.5.degree. C.
C. Comparison of Production and Purification of Gen1 Soluble
rHuPH20 and Gen2 Soluble rHuPH20
[0477] The production and purification of Gen2 soluble rHuPH20 in a
300L bioreactor cell culture contained some changes in the
protocols compared to the production and purification Gen1 soluble
rHuPH20 in a 100L bioreactor cell culture (described in Example
13B). Table 27 sets forth exemplary differences, in addition to
simple scale up changes, between the methods.
TABLE-US-00028 TABLE 27 Process Difference Gen1 soluble rHuPH20
Gen2 soluble rHuPH20 Cell line 3D35M 2B2 Media used to expand cell
Contains 0.10 .mu.M Contains 20 .mu.M inoculum methotrexate (0.045
mg/L) methotrexate (9 mg/L) Media in 6 L cultures Contains 0.10
.mu.M Contains no methotrexate onwards methotrexate 36 L spinner
flask No instrumentation Equipped with 20 L operating volume.
instrumentation that monitors and controls pH, dissolved oxygen,
sparge and overlay gas flow rate. 32 L operating volume Final
operating volume in Approx. 100 L in a 125 L Approx. 300 L in a 400
L bioreactor bioreactor bioreactor (initial culture (initial
culture volume + volume + 260 L) 65 L) Culture media in final No
rHuInsulin 5.0 mg/L rHuInsulin bioreactor Media feed volume Scaled
at 4% of the Scaled at 4% of the bioreactor cell culture bioreactor
cell culture volume i.e. 3.4, 3.5 and 3.7 L, volume i.e. 10.4,
10.8, resulting in a target 11.2 and 11.7 L, resulting bioreactor
volume of ~92 L. in a target bioreactor volume of ~303 L. Media
feed Feed #1 Medium: CD Feed #1 Medium: 4x CD CHO + 50 g/L Glucose
+ CHO + 33 g/L Glucose + 8 mM GlutaMAX .TM.-1 32 mM Glutamax + 16.6
g/L Feed #2 (CD CHO + 50 g/L Yeastolate + 33 mg/L Glucose + 8 mM
rHuInsulin GlutaMAX + 1.1 g/L Feed #2: 2x CD CHO + 33 g/L Sodium
Butyrate Glucose + 16 mM Feed #3: CD CHO + 50 g/L Glutamax + 33.4
g/L Glucose + 8 mM Yeastolate + 0.92 g/L GlutaMAX + 1.1 g/L Sodium
Butyrate Sodium Butyrate Feed #3: 1x CD CHO + 50 g/L Glucose + 10
mM Glutamax + 50 g/L Yeastolate + 1.80 g/L Sodium Butyrate Feed #4:
1x CD CHO + 33 g/L Glucose + 6.6 mM Glutamax + 50 g/L Yeastolate +
0.92 g/L Sodium Butyrate Filtration of bioreactor cell Four
polyethersulfone 1.sup.st stage - Four modules in culture filters
(8.0 .mu.m, 0.65 .mu.m, parallel, each with a layer 0.22 .mu.m and
0.22 .mu.m) in of diatomaceous earth series graded to 4-8 .mu.m and
a 100 L storage bag layer of diatomaceous earth graded to 1.4-1.1
.mu.m, followed by a cellulose membrane. 2.sup.nd stage - single
module containing a layer of diatomaceous earth graded to 0.4-0.11
.mu.m and a layer of diatomaceous earth graded to <0.1 .mu.m,
followed by a cellulose membrane. 3.sup.rd stage - 0.22 .mu.m
polyethersulfone filter 300 L storage bag Harvested cell culture is
supplemented with 10 mM EDTA, 10 mM Tris to a pH of 7.5.
Concentration and buffer Concentrate with 2 TFF Concentrate using
four exchange prior to with Millipore Spiral Sartorius Sartoslice
TFF chromatography Polyethersulfone 30K 30K MWCO Filter MWCO Filter
Buffer Exchange the Buffer Exchange the Concentrate 10.times. with
10 mM Concentrate 6.times. with 10 mM Tris, 20 mM Na2SO4, Hepes, 25
mM NaCl, pH 7.5 pH 7.0 50 L sterile storage bag 20 L sterile
storage bag Viral inactivation prior to None Viral inactivation
chromatography performed with the addition of a 1% Triton .RTM.
X-100, 0.3% Tributyl Phosphate, pH 7.5, 1.sup.st purification step
(Q No absorbance reading A280 measurements at the sepharose)
beginning and end Viral filtration after Pall DV-20 filter (20 nm)
Sartorius Virosart filter (20 nm) chromatography Concentration and
buffer Hepes/saline pH 7.0 buffer Histidine/saline, pH 6.0 exchange
after Protein concentrated to 1 mg/ml buffer chromatography Protein
concentrated to 10 mg/ml
Example 16
Determination of Sialic Acid and Monosaccharide Content
[0478] The sialic acid and monosaccharide content of soluble
rHuPH20 can be assessed by reverse phase liquid chromatography
(RPLC) following hydrolysis with trifluoroacetic acid. In one
example, the sialic acid and monosaccharide content of purified
hyaluronidase lot #HUB0701E (1.2 mg/mL; produced and purified
essentially as described in Example 15) was determined. Briefly,
100 .mu.g sample was hydrolyzed with 40% (v/v) trifluoroacetic acid
at 100.degree. C. for 4 hours in duplicate. Following hydrolysis,
the samples were dried down and resuspended in 300 .mu.L water. A
45 .mu.L aliquot from each re-suspended sample was transferred to a
new tube and dried down, and 10 .mu.L of a 10 mg/mL sodium acetate
solution was added to each. The released monosaccharides were
fluorescently labeled by the addition of 50 .mu.L of a solution
containing 30 mg/mL 2-aminobenzoic acid, 20 mg/mL sodium
cyanoborohydride, approximately 40 mg/mL sodium acetate and 20
mg/mL boric acid in methanol. The mixture was incubated for 30
minutes at 80.degree. C. in the dark. The derivitization reaction
was quenched by the addition of 440 .mu.L of mobile phase A (0.2%
(v/v) n-butylamine, 0.5% (v/v) phosphoric acid, 1% (v/v)
tetrahydrofuran). A matrix blank of water also was hydrolyzed and
derivitized as described for the hyaluronidase sample as a negative
control. The released monosaccharides were separated by RPLC using
an Octadecyl (C.sub.18) reverse phase column (4.6.times.250 mm, 5
particle size; J. T. Baker) and monitored by fluorescence detection
(360 nm excitation, 425 nm emission). Quantitation of the
monosaccharide content was made by comparison of the chromatograms
from the hyaluronidase sample with chromatograms of monosaccharide
standards including N-D-glucosamine (GlcN), N-D-galactosamine
(GaiN), galactose, fucose and mannose. Table 28 presents the molar
ratio of each monosaccharide per hyaluronidase molecule.
TABLE-US-00029 TABLE 28 Monosaccharide content of soluble rHuPH20
Lot Replicate GlcN GalN Galactose Mannose Fucose HUB0701E 1 14.28
0.07* 6.19 25.28 2.69 2 13.66 0.08* 6.00 24.34 2.61 Average 13.97
0.08* 6.10 24.81 2.65 *GalN results were below the limit of
detection
Example 17
C-terminal Heterogeneity of Soluble rHuPH20 from 3D35M and 2B2
Cells
[0479] C-terminal sequencing was performed on two lots of sHuPH20
produced and purified from 3D35M cells in a 100 L bioreactor volume
(Lot HUA0505MA) and 2B2 cells in a 300L bioreactor volume (Lot
HUB0701EB). The lots were separately digested with endoproteinase
Asp-N, which specifically cleaves peptide bonds N-terminally at
aspartic and cysteic acid. This releases the C-terminal portion of
the soluble rHuPH20 at the aspartic acid at position 431 of SEQ ID
NO:122. The C-terminal fragments were separated and characterized
to determine the sequence and abundance of each population in Lot
HUA0505MA and Lot HUB0701EB.
[0480] It was observed that the soluble rHuPH20 preparations from
3D35M cells and 2B2 cells displayed heterogeneity, and contained
polyepeptides that differed from one another in their C-terminal
sequence (Tables 30 and 31). This heterogeneity is likely the
result of C-terminal cleavage of the expressed 447 amino acid
polypeptide (SEQ ID NO:122) by peptidases present in the cell
culture medium or other solutions during the production and
purification process. The polypeptides in the soluble rHuPH20
preparations have amino acid sequences corresponding to amino acids
1-447, 1-446, 1-445, 1-444 and 1-443 of the soluble rHuPH20
sequence set forth SEQ ID NO:122. The full amino acid sequence of
each of these polypeptides is forth in SEQ ID NOS: 122 to 126,
respectively. As noted in tables 29 and 30, the abundance of each
polypeptide in the soluble rHuPH20 preparations from 3D35M cells
and 2B2 cells differs.
TABLE-US-00030 TABLE 29 Analysis of C-terminal fragments from Lot
HUA0505MA Amino acid position (relative to Frag- SEQ ID NO: Theor.
Exp. Elution Abun- ment 122) Sequence mass Mass Error time dance
D28a 431-447 DAFKLPPMETEEPQIFY 2053.97 2054.42 0.45 99.87 0.2% (SEQ
ID NO: 191) D28b 431-446 DAFKLPPMETEEPQIF 1890.91 1891.28 0.37
97.02 18.4% (SEQ ID NO: 192) D28c 431-445 DAFKLPPMETEEPQI 1743.84
1744.17 0.33 86.4 11.8% (SEQ ID NO: 193) D28d 431-444
DAFKLPPMETEEPQ 1630.70 1631.07 0.32 74.15 56.1% (SEQ ID NO: 194)
D28e 431-443 DAFKLPPMETEEP 1502.70 1502.98 0.28 77.36 13.6% (SEQ ID
NO: 195) D28f 431-442 DAFKLPPMETEE 1405.64 ND N/A N/A 0.0% (SEQ ID
NO: 196)
TABLE-US-00031 TABLE 30 Analysis of C-terminal fragments from Lot
HUB0701EB Amino acid position (relative to Frag- SEQ ID NO: Theor.
Exp. Elution Abun- ment 122) Sequence Mass mass Error time dance
D28a 431-447 DAFKLPPMETEEPQIFY 2053.97 2054.42 0.45 99.89 1.9% (SEQ
ID NO: 191) D28b 431-446 DAFKLPPMETEEPQIF 1890.91 1891.36 0.45
96.92 46.7% (SEQ ID NO: 192) D28c 431-445 DAFKLPPMETEEPQI 1743.84
1744.24 0.40 85.98 16.7% (SEQ ID NO: 193) D28d 431-444
DAFKLPPMETEEPQ 1630.70 1631.14 0.39 73.9 27.8% (SEQ ID NO: 194)
D28e 431-443 DAFKLPPMETEEP 1502.70 1503.03 0.33 77.02 6.9% (SEQ ID
NO: 195) D28f 431-442 DAFKLPPMETEE 1405.64 ND N/A N/A 0.0% (SEQ ID
NO: 196)
[0481] Since modifications will be apparent to those of skill in
this art, it is intended that this invention be limited only by the
scope of the appended claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20130101577A9).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20130101577A9).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References