U.S. patent application number 13/056606 was filed with the patent office on 2011-08-18 for assays for detection of phenylalanine ammonia-lyase and antibodies to phenylalanine ammonia-lyase.
This patent application is currently assigned to BioMarin Pharmaceutical Inc.. Invention is credited to Erik Damien Foehr, Carlos Fabricio Santamaria, Bin Zhao.
Application Number | 20110201022 13/056606 |
Document ID | / |
Family ID | 41100766 |
Filed Date | 2011-08-18 |
United States Patent
Application |
20110201022 |
Kind Code |
A1 |
Foehr; Erik Damien ; et
al. |
August 18, 2011 |
ASSAYS FOR DETECTION OF PHENYLALANINE AMMONIA-LYASE AND ANTIBODIES
TO PHENYLALANINE AMMONIA-LYASE
Abstract
Provided herein are methods of detecting the presence of a
pegylated enzyme, an enzyme-specific antibody (e.g., a neutralizing
antibody or of a particular isotype), or a polyethylene glycol
(PEG)-specific antibody in a sample, such as a bodily fluid or
tissue of a patient. In certain embodiments, the enzyme is
phenylalanine ammonia-lyase (PAL), such as Anabaena variabilis (Av)
PAL administered to the patient as part of an enzyme substitution
therapy for diseases or disorders, such as phenylketonuria (PKU),
or cancer therapy.
Inventors: |
Foehr; Erik Damien; (San
Rafael, CA) ; Zhao; Bin; (San Ramon, CA) ;
Santamaria; Carlos Fabricio; (San Francisco, CA) |
Assignee: |
BioMarin Pharmaceutical
Inc.
Novato
CA
|
Family ID: |
41100766 |
Appl. No.: |
13/056606 |
Filed: |
July 30, 2009 |
PCT Filed: |
July 30, 2009 |
PCT NO: |
PCT/US09/04386 |
371 Date: |
April 21, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61084961 |
Jul 30, 2008 |
|
|
|
Current U.S.
Class: |
435/7.4 |
Current CPC
Class: |
G01N 33/6854 20130101;
G01N 33/573 20130101; G01N 2333/988 20130101 |
Class at
Publication: |
435/7.4 |
International
Class: |
G01N 33/573 20060101
G01N033/573 |
Claims
1. A method of detecting the presence of a pegylated phenylalanine
ammonia-lyase (PAL-PEG) in a sample, comprising: (a) acidifying the
PAL-PEG in the sample by adding an acidification reagent; (b)
neutralizing the acidified PAL-PEG in the sample by adding a
neutralization buffer; (c) contacting the sample with an
immobilized first antibody that immunospecifically binds to
phenylalanine ammonia-lyase (PAL); (d) optionally removing unbound
sample; (e) contacting the sample bound to the immobilized first
antibody with a detectable second antibody, wherein the second
antibody immunospecifically binds to PEG; (f) optionally removing
unbound second antibody; and (g) detecting the presence of the
second antibody bound to the sample; wherein the PAL-PEG is a
pegylated recombinant Anabaena variabilis phenylalanine
ammonia-lyase (rAvPAL-PEG) or variant thereof, and wherein
detection above background of an amount of the second antibody
bound to the sample indicates the presence of PAL-PEG in the
sample.
2. (canceled)
3. (canceled)
4. The method according to claim 1, further comprising measuring
the amount of the PAL-PEG in the sample.
5-11. (canceled)
12. The method according to claim 1, wherein the rAvPAL-PEG variant
is rAvPAL-PEG_C503S, rAvPAL-PEG_C565S or rAvPAL-PEG_C565SC503S.
13. (canceled)
14. The method according to claim 1, wherein the sample comprises a
bodily fluid or tissue.
15. The method of claim 14, wherein the bodily fluid is blood,
serum or plasma.
16. The method according to claim 1, wherein the sample is from a
patient.
17. The method of claim 16, wherein the patient is a mammal.
18. The method of claim 17, wherein the mammal is a human.
19. The method according to claim 18, wherein the patient is a
patient having elevated phenylalanine levels.
20. The method according to claim 19, wherein the patient has
phenylketonuria.
21. The method according to claim 18, wherein the patient has
cancer.
22. The method according to claim 18, wherein the patient has been,
is, or will be administered a rAvPAL-PEG or variant thereof.
23. The method according to claim 22, wherein the rAvPAL-PEG
variant is rAvPAL-PEG_C503S, rAvPAL-PEG_C565S or
rAvPAL-PEG_C565SC503S.
24. A kit comprising: (a) an acidification reagent for acidifying a
PAL-PEG, wherein the PAL-PEG is a rAvPAL-PEG or variant thereof;
(b) a neutralization buffer for neutralizing the acidified PAL-PEG;
(c) a first antibody that immunospecifically binds to PAL that is
optionally immobilized on a solid support; and (d) a detectable
second antibody that immunospecifically binds to PEG.
25-31. (canceled)
32. The kit according to claim 24, wherein one or more components
of the kit are in a one or more containers, and wherein the kit
further comprises instructions for use.
33-37. (canceled)
38. The kit according to claim 24, wherein the rAvPAL-PEG variant
is rAvPAL-PEG_C503S, rAvPAL-PEG_C565S or rAvPAL-PEG_C565SC503S.
39. (canceled)
40. The method according to claim 1, wherein the acidification
reagent is 0.1 M glycine, pH 2.7.
41. The method according to claim 1, wherein the neutralization
buffer is 0.5 M Tris-HCl, pH 8.5.
42. The kit according to claim 24, wherein the acidification
reagent is 0.1 M glycine, pH 2.7.
43. The kit according to claim 24, wherein the neutralization
buffer is 0.5 M Tris-HCl, pH 8.5.
Description
FIELD
[0001] Provided herein are methods of detecting the presence of a
pegylated enzyme, an enzyme-specific antibody (e.g., a neutralizing
antibody or of a particular isotype), or a polyethylene glycol
(PEG)-specific antibody in a sample, such as a bodily fluid or
tissue of a patient.
BACKGROUND
[0002] Phenylalanine ammonia-lyase (PAL) is a non-mammalian enzyme
widely distributed in plants (Koukol et al., J Biol Chem
236:2692-2698, 1961; Hanson et al., The Enzymes 7:75-166, 1972;
Poppe et al., Curr Org Chem 7:1297-1315, 2003), some fungi (Rao et
al., Can J Biochem 45(12):1863-1872, 1967; Abell et al., Methods
Enzymol 142:242-253, 1987), and bacteria (Bezanson et al., Can J
Microbiol 16:147-151, 1970; Xiang et al., J Biol Chem
277:32505-32509, 2002; Hill et al., Chem Commun 1358-1359, 2003)
and can be recombinantly produced in Escherichia coli.
[0003] PAL from two cyanobacteria strains, Anabaena variabilis (Av)
and Nostoc punctiforme (Np), has been cloned and expressed in
bacteria (e.g., Escherichia coli (E. coli), and was shown to
display PAL enzyme activity in vitro and in vivo (see, e.g., U.S.
Pat. Nos. 7,531,341; 7,534,595; 7,537,923 and 7,560,263). A
pegylated recombinant Anabaena variabilis PAL (rAvPAL-PEG) has also
been produced, wherein the rAvPAL protein was derivatized by
covalent attachment of polyethylene glycol (PEG) to increase its
half-life and optimize its pharmacokinetic profile and/or reduce
its immunogenicity (Id.). rAvPAL-PEG has been shown to metabolize
phenylalanine and is being developed as an enzyme substitution
therapy (EST) for patients disorders or diseases associated with
elevated levels of phenylalanine, such as PKU, as well as in cancer
therapy (Id.).
[0004] A concern of administration of enzymes to patients, for
example, for PKU EST or cancer therapy, is whether sufficient
amounts of the enzyme are available in the body to exert a
therapeutic effect in the patient. Moreover, in the case of
pegylated enzymes, current methods used to determine the
concentration of PEG are relatively insensitive (see, e.g., U.S.
Pat. No. 6,596,849), and methods to detect the enzyme itself are
complicated by the presence of the PEG. To date, such methods have
not determined an effective way to expose the immunogenic epitopes
of the therapeutic enzyme (e.g., rAvPAL-PEG) for capture by an
immobilized antibody specific for the target therapeutic enzyme,
and maximize the number of epitopes recognized by the antibodies,
while at the same time minimizing the number of epitopes masked by
the PEG molecules.
[0005] A further concern of administration of enzymes to a patient,
e.g., for PKU EST or cancer therapy, is the possible development of
enzyme-specific antibodies in patients, for example, those
receiving multiple rounds of therapy. These enzyme-specific
antibodies may precipitate potential adverse events and lead to
changes in clinical efficacy, including, for example,
anaphylactoid-type reactions associated with antibodies of the IgE
isotype, changes in pharmacokinetic profile, neutralization of the
enzymatic activity, interference with receptor-mediated enzyme
uptake, and breaking of tolerance toward self proteins. Current
assays to measure the amount of anti-enzyme antibodies in body
fluids also suffer from a variety of technical issues, thereby
making their interpretation difficult (see Mire-Sluis et al., J.
Immunological Methods 289:1-16, 2004).
[0006] Given these concerns, as well as the shortcomings of
currently available assays, there remains a need for a reliable,
sensitive and specific assays to accurately detect or otherwise
measure (i) an enzyme (e.g., a rAvPAL) in body fluids, tissues or
other samples of a patient receiving the enzyme, e.g., for EST or
cancer therapy; (ii) a pegylated enzyme, such as extensively
pegylated enzyme (i.e., an enzyme having a sufficient number of PEG
molecules attached such that at least some of the immunogenic
epitopes of the enzyme are masked by the PEG molecules), (e.g., a
rAvPAL-PEG) in bodily fluids, tissues or other samples of patient
receiving the enzyme, e.g., for EST or cancer therapy; and/or (iii)
enzyme- or PEG-specific antibodies (e.g., anti-rAvPAL,
anti-rAvPAL-PEG antibodies, or anti-PEG antibodies) in body fluids,
tissues or other samples of patient receiving the enzyme, e.g., for
EST or cancer therapy. Such assays could enable assessment of the
treatment regimen in a patient receiving the enzyme, e.g., for EST
or cancer therapy and facilitate more efficient design of patient
therapy.
SUMMARY
[0007] Provided herein are methods of detecting the presence of a
pegylated enzyme, an enzyme-specific antibody (e.g., a neutralizing
antibody or of a particular isotype), or a PEG-specific antibody in
a sample, such as a bodily fluid or tissue of a patient. In certain
embodiments, the methods are for the detection and/or measurement
of therapeutic enzymes and antibodies associated with
administration of such therapeutic enzymes, including the use of a
PAL or a variant thereof, for example, in a patient receiving PAL
or a variant thereof (e.g., an AvPAL or rAvPAL-PEG) during the
course of EST for elevated phenylalanine levels (e.g.,
phenylketonuria) or cancer therapy. Also provided are kits for
carrying out said methods.
[0008] In a first aspect, provided herein is a method of detecting
the presence of a pegylated enzyme, e.g., rAvPAL-PEG or variant
thereof, in a sample (e.g., a body fluid, such as plasma, or a
tissue sample), said method comprising: (a) acidifying the
pegylated enzyme in the sample by adding an acidification reagent;
(b) neutralizing the acidified pegylated enzyme in the sample by
adding a neutralization buffer; (c) contacting the sample with an
immobilized first antibody that immunospecifically binds with the
pegylated enzyme; (d) optionally removing unbound sample; (e)
contacting the sample bound to the immobilized first antibody with
a detectable second antibody, wherein the second antibody
immunospecifically binds to PEG; (f) optionally removing unbound
second antibody; and (g) detecting the presence of the second
antibody bound to the sample; wherein detection above background of
an amount of the second antibody bound to the sample, e.g., an
increase in the amount of second antibody bound to the sample as
compared to a control sample having no pegylated enzyme, indicates
the presence of pegylated enzyme in the sample. In some
embodiments, the neutralized pegylated enzyme described in (b) is
introduced into a different assay buffer before contacting the
immobilized first antibody described in (c). In some embodiments,
the acidification reagent is 0.1 M glycine, pH 2.7 and/or the
neutralization buffer is 0.5 M Tris-HCl, pH 8.5.
[0009] In certain embodiments, the sample is a body fluid, such as
blood or plasma, or a tissue sample from a patient (e.g., a mammal,
such as a human). In an embodiment, the concentration of pegylated
enzyme in the sample is determined. In one embodiment, the limit of
detection is less than 5 ng/mL, or less than 3 ng/mL. In another
embodiment, the limit of detection is between 5 ng/mL and 2 ng/mL,
such as between 3 ng/mL and 2 ng/mL. In an embodiment, the sample
is plasma, which is equal to or less than 5% or equal to or less
than 2% of the volume as described in (a). In some embodiments, the
patient has elevated phenylalanine levels. In specific embodiments,
the patient has been (or will be) administered the pegylated
enzyme, for example, for EST (e.g., for PKU) or cancer therapy. In
an embodiment, the pegylated enzyme is rAvPAL-PEG or a variant
thereof. In one embodiment, the pegylated enzyme is rAvPAL-PEG
having its cysteine residue at position 503 substituted with serine
residues (rAvPAL-PEG_C503S). In another embodiment, the pegylated
enzyme is rAvPAL-PEG having its cysteine residues at position 565
substituted with serine residues (rAvPAL-PEG_C565S). In yet another
embodiment, the pegylated enzyme is rAvPAL-PEG having its cysteine
residues at positions 503 and 565 substituted with serine residues
(rAvPAL-PEG_C565SC503S). In other embodiments, the pegylated enzyme
is rAvPAL-PEG_C503S, rAvPAL-PEG_C565S, rAvPAL-PEG_C565SC503S, or
any combination thereof.
[0010] In a second aspect, provided herein is a method of detecting
the presence of an enzyme-specific antibody (e.g., an
anti-AvPAL-specific antibody) in a sample (e.g., a body fluid, such
as plasma, or a tissue sample), said method comprising: (a)
contacting the sample with an immobilized enzyme (e.g., AvPAL); (b)
optionally removing unbound sample; (c) contacting the sample bound
to the immobilized enzyme with a detectable antibody, wherein the
detectable antibody immunospecifically binds to Ig; (d) optionally
removing unbound detectable antibody; and (e) detecting the
presence of the detectable antibody bound to the sample; wherein
detection above background of an amount of the detectable antibody
bound to the sample, e.g., an increase in the amount of detectable
antibody bound to the sample as compared to a control sample having
no enzyme-specific antibody, indicates the presence of enzyme
specific antibody in the sample.
[0011] In certain embodiments, the sample is a body fluid, such as
blood or plasma, or a tissue sample from a patient (e.g., a mammal,
such as a human). In an embodiment, the concentration of
enzyme-specific antibody in the sample is determined. In the
methods provided herein, any isotype of enzyme-specific antibody
can be detected. In an embodiment, an enzyme-specific antibody of
IgG isotype can be detected, and, in certain embodiments, the limit
of detection is between 1.6 ng/mL and 8.6 ng/mL. In some
embodiments, the limit of detection is less than 8.6 ng/mL or less
than 4.2 ng/mL, such as between 2.1 ng/mL and 1.6 ng/mL. In other
embodiments, an enzyme-specific antibody of IgM isotype can be
detected, and, in certain embodiments, the limit of detection is
less than 5.9 ng/mL, such as between 5.9 ng/ml and 2.8 n/ml. In an
embodiment, an enzyme-specific antibody of IgE isotype can be
detected. In other embodiments, an enzyme-specific antibody of IgA
can be detected. In an embodiment, the sample is serum or plasma,
which is equal to or less than 5% or equal to or less than 2% of
the volume as described in (a).
[0012] In some embodiments, the patient has elevated phenylalanine
levels. In specific embodiments, the patient has been or will be
administered the enzyme or pegylated enzyme, for example, for EST
(e.g., for PKU) or cancer therapy.
[0013] In an embodiment, the enzyme is rAvPAL or a variant thereof.
In one embodiment, the enzyme is rAvPAL_C503S, rAvPAL_C565S,
rAvPAL_C565SC503S, or any combination thereof. In some embodiments,
the enzyme is a pegylated enzyme. In an embodiment, the pegylated
enzyme is rAvPAL-PEG. In other embodiments, the pegylated enzyme is
rAvPAL-PEG_C503S, rAvPAL-PEG_C565S, rAvPAL-PEG_C565SC503S, or any
combination thereof.
[0014] In a third aspect, provided herein is a method for detecting
the presence of neutralizing enzyme-specific antibodies (e.g., a
neutralizing anti-AvPAL-specific antibody) in a sample (e.g., a
body fluid, such as plasma, or a tissue sample), said method
comprising: (a) contacting the sample with the enzyme (e.g., AvPAL
or variant thereof) that is optionally immobilized on a solid
support; (b) optionally removing unbound sample; (c) adding a
substrate for the enzyme; (d) optionally removing unbound
substrate; and (e) detecting the presence of enzymatic reactivity
between the enzyme and substrate; wherein a reduction of enzymatic
activity, e.g., as compared to a sample having no neutralizing
anti-enzyme antibodies, indicates the presence of neutralizing
anti-enzyme antibodies in the sample. In certain embodiments, the
sample is a body fluid, such as blood or plasma, or a tissue sample
from a patient (e.g., a mammal, such as a human). In an embodiment,
the concentration of neutralizing enzyme-specific antibody in the
sample is determined. In certain embodiments, the limit of
detection is less than or equal to 10 .mu.g/mL. In an embodiment,
the sample is serum or plasma, which is equal to or less than 5% or
equal to or less than 2% of the volume as described in (a).
[0015] In some embodiments, the patient has elevated phenylalanine
levels. In specific embodiments, the patient has been (or will be)
administered the pegylated enzyme, for example, for EST (e.g., for
PKU) or cancer therapy.
[0016] In an embodiment, the enzyme is rAvPAL or a variant thereof.
In one embodiment, the enzyme is rAvPAL_C503S, rAvPAL_C565S,
rAvPAL_C565SC503S or any combination thereof. In some embodiments,
the enzyme is pegylated. In an embodiment, the pegylated enzyme is
rAvPAL-PEG. In other embodiments, the pegylated enzyme is
rAvPAL-PEG_C503S, rAvPAL-PEG_C565SC503S, or any combination
thereof.
[0017] In a fourth aspect, provided herein is a method for
detecting or otherwise measuring the amount of polyethylene glycol
(PEG)-specific antibodies (e.g., anti-PEG-specific antibodies) in a
sample (e.g., a body fluid, such as plasma, or a tissue sample),
said method comprising: (a) contacting the sample with an
immobilized PEG; (b) optionally removing unbound sample; (c)
contacting the sample bound to the immobilized PEG with a
detectable antibody, wherein the detectable antibody
immunospecifically binds to Ig; (d) optionally removing unbound
detectable antibody; and (e) detecting the presence of the
detectable antibody bound to the sample; wherein detection above
background of an amount of the detectable antibody bound to the
sample, e.g., an increase in the amount of detectable antibody
bound to the sample as compared to a control sample having no
enzyme-specific antibody, indicates the presence of PEG-specific
antibody in the sample.
[0018] In certain embodiments, the sample is a body fluid, such as
blood or plasma, or a tissue sample from a patient (e.g., a mammal,
such as a human). In an embodiment, the concentration of
PEG-specific antibody in the sample is determined. In the methods
provided herein, any isotype of PEG-specific antibody can be
detected (e.g., IgG, IgE, IgM or IgA). In an embodiment, the sample
is serum or plasma, which is equal to or less than 5% or equal to
or less than 2% of the volume as described in (a). In some
embodiments, the patient has elevated phenylalanine levels. In
specific embodiments, the patient has been (or will be)
administered the pegylated enzyme, for example, for EST (e.g., for
PKU) or cancer therapy.
[0019] In an embodiment, the PEG-specific antibodies are specific
for a pegylated enzyme. In some embodiments, the pegylated enzyme
is rAvPAL-PEG. In other embodiments, the pegylated enzyme is
rAvPAL-PEG_C503S, rAvPAL-PEG_C565S, rAvPAL-PEG_C565SC503S, or any
combination thereof.
DESCRIPTION OF THE FIGURES
[0020] FIGS. 1A-1B depict the (A) gene sequence (SEQ ID NO:1) and
(B) protein sequence of NpPAL (SEQ ID NO:2) of Nostoc punctiforme
PAL (NpPAL).
[0021] FIGS. 2A-2B depict the (A) gene sequence (SEQ ID NO:3) and
(B) protein sequence of AvPAL (SEQ ID NO:4) of Anabaena variabilis
PAL (AvPAL).
[0022] FIGS. 3A-3E depicts the protein sequences of AvPAL having a
cysteine to serine substitution at (A) position 64 (AvPAL_C64S, SEQ
ID NO:7); (B) position 318 (AvPAL_C318S, SEQ ID NO:8); (C) position
503 (AvPAL_C503S, SEQ ID NO:9); (D) position 565 (AvPAL_C565S, SEQ
ID NO:10); or (E) at positions 503 and 565 (AvPAL_C565SC503S, SEQ
ID NO:11). Cysteine to serine substitutions are underlined in
bold.
[0023] FIGS. 4A-4B depict the effect of cysteine to serine
substitutions at position 565 or both positions 565 and 503 of (A)
unpegylated AvPAL or (B) pegylated AvPAL on in vitro PAL specific
enzyme activity after incubation for various lengths of time at
37.degree. C.
[0024] FIGS. 5A-5B depict the effect of cysteine to serine
substitutions in AvPAL on formation of protein aggregates in
solution as analyzed by (A) gel electrophoresis under denaturing
conditions (left panel) or native conditions (right panel) or (B)
SEC-HPLC.
[0025] FIG. 6 depicts the effect of cysteine to serine
substitutions at positions 565 and 503 in AvPAL (AvPAL_C565SC503S)
on site-specific pegylation at various PEG concentrations.
[0026] FIGS. 7A-7B. FIG. 7A depicts the effect of a single
subcutaneous injection of a pegylated AvPAL with cysteine to serine
substitutions at positions 565 and 503 (AvPAL_C565SC503S) at 4
mg/kg (diamonds) and at 12 mg/kg (squares) into Cynomolgus monkeys
on the plasma AvPAL_C565SC503S levels over time (hours). FIG. 7B
depicts the effect of a single subcutaneous injection of
AvPAL_C565SC503S at 4 mg/kg into Cynomolgus monkeys on the plasma
AvPAL_C565SC503S (diamonds) and phenylalanine (squares) levels over
time (hours).
[0027] FIGS. 8A-8B. FIG. 8A depicts the effect of a single
intravenous injection of a pegylated AvPAL with cysteine to serine
substitutions at positions 565 and 503 (AvPAL_C565SC503S) at 1
mg/kg (diamonds), at 5 mg/kg (squares) and at 25 mg/kg (triangles)
into rats on the plasma AvPAL_C565SC503S levels over time (hours).
FIG. 8B depicts the effect of a single subcutaneous injection of
AvPAL_C565SC503S at 10 mg/kg (diamonds), at 25 mg/kg (squares) and
at 250 mg/kg (triangles) into rats on the plasma AvPAL_C565SC503S
levels over time (hours)
[0028] FIGS. 9A-9B. FIG. 9A depicts the effect of a pegylated AvPAL
with cysteine to serine substitutions at positions 565 and 503
(AvPAL_C565SC503S) at 0.01, 0.1, 1, 10 and 100 .mu.g/mL as
indicated on proliferation (as measured by propidium iodide
staining) of NOMO1 acute myeloid leukemia (AML) cells in vitro.
FIG. 9B depicts the effect of AvPAL_C565SC503S at 0.1, 1, 10 and
100 .mu.g/mL as indicated on proliferation of IM9 myeloma cells in
vitro.
[0029] FIGS. 10A-10D. FIG. 10A depicts the effect of a pegylated
AvPAL with cysteine to serine substitutions at positions 565 and
503 (AvPAL_C565SC503S) at 0.01, 0.1, 1, 10 and 100 .mu.g/mL as
indicated on proliferation (as measured by propidium iodide
staining) of SF268 (top) and 498L (bottom) brain/CNS tumor cells in
vitro. FIG. 10B depicts the effect of AvPAL_C565SC503S at 0.01,
0.1, 1, 10 and 100 .mu.g/mL as indicated on proliferation of HT29
(top) and HCT116 (bottom) colon tumor cells in vitro. FIG. 10C
depicts the effect of AvPAL_C565SC503S at 0.01, 0.1, 1, 10 and 100
.mu.g/mL as indicated on proliferation of H460 (top), 529L (middle)
and 629L (bottom) lung tumor cells in vitro. FIG. 10D depicts the
effect of AvPAL_C565SC503S at 0.01, 0.1, 1, 10 and 100 .mu.g/mL as
indicated on proliferation of LNCAP (top), PC3M (middle) and DU145
(bottom) prostate tumor cells in vitro.
TERMINOLOGY
[0030] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. The following exemplary references can
provide one of skill with a general definition of many of the terms
provided herein: Singleton et al., Dictionary of Microbiology and
Molecular Biology (2d ed. 1994); The Cambridge Dictionary of
Science and Technology (Walker ed., 1988); The Glossary of
Genetics, 5th Ed., Rieger et al. (eds.), Springer Verlag (1991);
and Hale & Marham, The Harper Collins Dictionary of Biology
(1991).
[0031] Each publication, patent application, patent, and other
reference cited herein is incorporated by reference in its entirety
to the extent that it is not inconsistent with the present
disclosure and to the same extent as if each individual
publication, patent application, patent, or other reference was
specifically and individually indicated to be incorporated by
reference.
[0032] It is noted here that as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural reference unless the context clearly dictates otherwise.
[0033] In the event there is a plurality of definitions for a term
herein, those in this section prevail unless stated otherwise.
[0034] The term "about" or "approximately" means an acceptable
error for a particular value as determined by one of ordinary skill
in the art, which depends in part on how the value is measured or
determined. In certain aspects, the term "about" or "approximately"
means within 1, 2, 3, or 4 standard deviations. In certain aspects,
the term "about" or "approximately" means within 50%, 20%, 15%,
10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given
value or range.
[0035] As used herein, "administer" or "administration" refers to
the act of injecting or otherwise physically delivering a substance
as it exists outside the body into a patient, such as by oral,
mucosal, intradermal, intravenous, intramuscular delivery and/or
any other method of physical delivery described herein or known in
the art. When a disease, or a symptom thereof, is being treated,
administration of the substance typically occurs after the onset of
the disease or symptoms thereof. When a disease, or a symptom
thereof, is being prevented, administration of the substance
typically occurs before the onset of the disease or symptoms
thereof.
[0036] The term "antibody" and "immunoglobulin" or "Ig" may be used
interchangeably herein. The term "antibody" refers to all types of
immunoglobulins and can be of any type (e.g., IgG, IgE, IgM, IgD,
IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin
molecule, or any antigen-recognition (or antigen-binding) fragments
thereof. The antibodies may be monoclonal or polyclonal and may be
of any species of origin, including, but not limited to, mouse,
rat, rabbit, horse, or human, or may be chimeric antibodies. See,
e.g., Walker et al., Molec. Immunol. 1989; 26: 403-411; Morrision
et al., Proc. Nat'l. Acad. Sci. 1984; 81: 6851; Neuberger et al.,
Nature 1984; 312: 604. The antibodies may be recombinant monoclonal
antibodies produced according to the methods disclosed in U.S. Pat.
No. 4,474,893 (Reading) or U.S. Pat. No. 4,816,567 (Cabilly et
al.). The antibodies may also be chemically constructed by specific
antibodies made according to the method disclosed in U.S. Pat. No.
4,676,980 (Segel et al.). Antibodies for use in the methods and
kits provided herein can include, but are not limited to, synthetic
antibodies, monoclonal antibodies, recombinantly produced
antibodies, multispecific antibodies (including bi-specific
antibodies), human antibodies, humanized antibodies, chimeric
antibodies, intrabodies, single-chain Fvs (scFv) (e.g., including
monospecific, bispecific, etc.), camelized antibodies, Fab
fragments, F(ab').sub.2 fragments, disulfide-linked Fvs (sdFv),
anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments
of any of the above. In particular, for use in the methods and kits
provided herein can include immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e.,
antigen binding domains or molecules that contain an
antigen-binding site that immunospecifically binds to AvPAL or
rAvPAL, or variants thereof (e.g., rAvPAL_C503S, rAvPAL_C5655,
rAvPAL_C565SC503S) and/or derivatives thereof (e.g., rAvPAL-PEG,
rAvPAL-PEG_C503S, rAvPAL-PEG_C565SC or rAvPAL-PEG_C565SC503S).
[0037] The term "body fluid" or "bodily fluid" as used herein
refers to a fluid that is obtained from a patient, such as a mammal
(e.g., human) patient. For example, a body fluid may be blood,
cerebral spinal fluid (CSF), breast milk or urine. The body fluid
can also be blood fractionated to remove cells (i.e., plasma) or
cells and clotting factors (i.e., serum).
[0038] The term "capture moiety" or "first antibody" as used herein
refers to a composition that is capable of being specifically bound
by another composition that is immobilized, e.g., attached or
otherwise linked, to a solid support. Many of the detection
moieties provided herein can also be used as capture moieties so
long as a binding event is involved. For example, useful capture
moieties include affinity labels for which specific and selective
ligands are available (e.g., biotin with avidin, glutathione with
GST), haptens and proteins for which antisera or monoclonal
antibodies are available (e.g., c-Myc), nucleic acid molecules with
a sequence complementary to a target, and peptides for which
specific and selective ligands are available (e.g., histidine tag
with Ni). Molecules that affect the binding characteristics to a
chromatographic resin are also envisioned. The solid support can
be, for example, a filter, a plate, a membrane, a chromatographic
resin, or a bead.
[0039] The term "cutpoint factor" or "threshold" as used herein
generally refers to a value that is used to mathematically
manipulate the signal from the naive pooled matrix (e.g., serum or
plasma) to set the minimum signal required from a sample to be
considered positive. In some embodiments, the cutpoint factor is
determined based on a confidence interval from a set of samples
from individuals that have not been previously exposed to the
therapeutic AvPAL enzyme. For example, the 95% confidence interval,
calculated as 1.645 multiplied by the standard deviation across the
individual samples, will lead to approximately a 5% false positive
rate.
[0040] The term "derivative" when used in connection with antibody
substances and polypeptides used in the methods provided herein
refers to polypeptides chemically modified by techniques including,
but not limited to, ubiquitination, conjugation to therapeutic or
diagnostic agents, labeling (e.g., with radionuclides or various
enzymes), covalent polymer attachment such as pegylation (i.e.,
derivatization with polyethylene glycol) and insertion or
substitution by chemical synthesis of amino acids such as
ornithine, which do not normally occur in human proteins.
Derivatives can retain the binding properties of underivatized
molecules. In certain embodiments, e.g., an AvPAL "derivative"
refers to an rAvPAL-PEG. In other embodiments, e.g., an AvPAL
variant "derivative" refers to an AvPAL-PEG_C503S, AvPAL-PEG_C565SC
or rAvPAL-PEG_C565SC503S.
[0041] The terms "detectable moiety," "detection moiety" or a
"label" as used herein refers to a composition (e.g., polypeptide
or antibody) detectable by means including, but not limited to,
spectroscopic, photochemical, biochemical, immunochemical,
chemical, or other physical means. For example, useful detectable
moieties or labels include Ruthenium (Ru)-based catalyst, Europium,
.sup.32P, .sup.35S, fluorescent dyes, electron-dense reagents,
enzymes (e.g., as commonly used in an ELISA), biotin-Streptavidin,
dioxigenin, haptens and proteins for which antisera or monoclonal
antibodies are available, and nucleic acid molecules with a
sequence complementary to a target. The detectable moiety or label
often generates a measurable signal, such as a radioactive,
chromogenic, luminescent, or fluorescent signal, which can be used
to quantitate the amount of bound detectable moiety or label in a
sample.
[0042] The term "detectable antibody" as used herein refers to any
antibody that can be detected. In some embodiments, the antibody is
directly labeled with a detectable moiety. In certain embodiments,
the antibody is a detectable anti-Ig antibody. The term "detectable
anti-Ig antibody" as used herein refers to an anti-Ig antibody that
can be detected. In some embodiments, the anti-Ig antibody is
directly labeled with a detectable moiety in addition to its
inherent binding to an Ig molecule. The Ig antibody can be of, for
example, the IgG, IgE, IgM, IgD, IgA or IgY isotype. In other
embodiments, the detectable antibody is a detectable anti-PEG
antibody. The term "detectable anti-PEG antibody" as used herein
refers to an anti-PEG antibody that can be detected. In certain
embodiments, the anti-PEG antibody is directly labeled with a
detectable moiety in addition to its inherent binding to a
pegylated molecule.
[0043] The term "detectable PAL" and similar terms used herein
refers to a PAL enzyme, e.g., AvPAL or a derivative or variant
thereof, that can be detected. In certain embodiments, the PAL is
directly labeled with a detectable moiety in addition to its
inherent binding to a PAL-specific antibody.
[0044] The term "detectable PAL-PEG" as used herein refers to a
pegylated (i.e., derivatized with polyethylene glycol) PAL enzyme
(e.g., rAvPAL-PEG) or variant thereof that can be detected. In
certain embodiments, the PAL-PEG reagent is directly labeled with a
detectable moiety in addition to its inherent binding to a
PAL-specific antibody or to a PEG-specific antibody.
[0045] In the context of a peptide or polypeptide, the term
"fragment" as used herein refers to a peptide or polypeptide that
comprises less than the full length amino acid sequence. Such a
fragment may arise, for example, from a truncation at the amino
terminus, a truncation at the carboxy terminus, and/or an internal
deletion of a residue(s) from the amino acid sequence. Fragments
may, for example, result from alternative RNA splicing or from in
vivo protease activity. In certain embodiments, fragments include
polypeptides comprising an amino acid sequence of at least 5
contiguous amino acid residues, at least 10 contiguous amino acid
residues, at least 15 contiguous amino acid residues, at least 20
contiguous amino acid residues, at least 25 contiguous amino acid
residues, at least 40 contiguous amino acid residues, at least 50
contiguous amino acid residues, at least 60 contiguous amino
residues, at least 70 contiguous amino acid residues, at least 80
contiguous amino acid residues, at least 90 contiguous amino acid
residues, at least contiguous 100 amino acid residues, at least 125
contiguous amino acid residues, at least 150 contiguous amino acid
residues, at least 175 contiguous amino acid residues, at least 200
contiguous amino acid residues, or at least 250 contiguous amino
acid residues of the amino acid sequence of an antibody that
immunospecifically binds to, e.g., an AvPAL or a variant or
derivative thereof. In a specific embodiment, the antibody fragment
that immunospecifically binds to, e.g., an AvPAL or a variant or
derivative thereof, retains at least 1, at least 2, or at least 3
functions of the antibody.
[0046] The terms "identical" or percent "identity," in the context
of two or more polynucleotide or polypeptide sequences, refer to
two or more sequences or subsequences that are the same or have a
specified percentage of nucleotides or amino acid residues that are
the same, when compared and aligned for maximum correspondence, as
measured using a sequence comparison algorithms or by visual
inspection.
[0047] The term "antibody that immunospecifically binds" with a
pegylated enzyme (e.g., rAvPAL-PEG) or "anti-pegylated enzyme
antibodies" (e.g., anti-AvPAL-PEG antibodies) and analogous terms
are used interchangeably herein and refer to antibodies and
fragments thereof, that specifically bind to only, e.g., the
pegylated enzyme, such as a rAvPAL-PEG antigen or epitope. For
example, an antibody or a fragment thereof that "immunospecifically
binds to" a rAvPAL-PEG antigen may be cross-reactive with related
antigens. In one embodiment, an antibody or a fragment thereof that
immunospecifically binds to a rAvPAL-PEG antigen does not
cross-react with other antigens. An antibody or a fragment thereof
that immunospecifically binds to a rAvPAL-PEG can be identified,
for example, by immunoassays, BIAcore, or other techniques known to
those of skill in the art. An antibody or a fragment thereof binds
specifically to a rAvPAL-PEG when it binds to a rAvPAL-PEG antigen
with higher affinity than to any cross-reactive antigen as
determined using experimental techniques, such as RIAs and ELISAs.
Typically, a specific or selective reaction will be at least twice
background signal or noise and more typically more than 10 times
background. Along the same lines, and in certain embodiments,
antibodies provided herein immunospecifically bind to AvPAL, a
variant thereof (e.g., rAvPAL_C503S, rAvPAL_C565S,
rAvPAL_C565SC503S) and/or a derivative thereof (e.g., rAvPAL-PEG,
rAvPAL-PEG_C503S, rAvPAL-PEG_C565SC or rAvPAL-PEG_C565SC503S). In
other embodiments, antibodies provided herein immunospecifically
bind to PEG. In yet other embodiments, antibodies provided herein
immunospecifically bind to an Ig, such as an IgG, IgE, IgM, IgD,
IgA isotype.
[0048] The term "interference" as used herein generally refers to
the presence of substances in body fluid (e.g., serum or plasma)
samples that prevent the target analyte from accurate detection and
measurement. As used herein, interference generally refers to the
effect of free drug or the effect of the matrix (e.g., serum or
plasma) on the concentration-response relationship. For example,
interference from matrix may be evaluated as the relative accuracy
to samples without the potential interference to target a range of
75-125% relative accuracy.
[0049] The term "in vivo," in the context of samples, refers to
samples obtained from a subject, e.g., a patient, such as a human
patient, including biological samples such as biological or body
fluids, e.g., blood, plasma, serum, bone marrow, spinal fluid,
brain fluid, or tissues, such as lymph tissue, a thin layer
cytological sample, a fresh frozen tissue sample or a tumor tissue.
The term "in vivo" is to be distinguished from the term "in vitro,"
which encompasses cells or cell lines or biomolecular components of
cells that have been cultured or propagated outside of a living
organism.
[0050] The term "limit of detection," "LOD" or "sensitivity" as
used herein generally refers to the lowest analyte concentration in
a body fluid (e.g., serum or plasma) sample that can be detected
but not necessarily quantitated as an exact value. For example, LOD
may be defined as the analyte concentration that consistently
generates a signal greater than the measured mean response of the
pooled naive matrix plus a cutpoint factor.
[0051] The term "matrix" or "matrices" as used herein generally
refers to the biological background in which the antibodies are
measured. Examples of matrices include, for example, body fluid and
tissue.
[0052] The term "monoclonal antibody" refers to an antibody
obtained from a population of homogenous or substantially
homogeneous antibodies, and each monoclonal antibody will typically
recognize a single epitope on the antigen. In certain embodiments,
a "monoclonal antibody," as used herein, is an antibody produced by
a single hybridoma or other cell, wherein the antibody
immunospecifically binds to only a enzyme, e.g., AvPAL or
rAvPAL-PEG as determined, e.g., by ELISA or other antigen-binding
or competitive binding assay known in the art. The term
"monoclonal" is not limited to any particular method for making the
antibody. For example, monoclonal antibodies used in the methods
provided herein may be made by the hybridoma method as described in
Kohler et al.; Nature, 256:495 (1975) or may be isolated from phage
libraries using the techniques known in the art. Other methods for
the preparation of clonal cell lines and of monoclonal antibodies
expressed thereby are well known in the art (see, for example,
Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th
Ed., Ausubel et al., eds., John Wiley and Sons, New York).
[0053] The term "naive" as used herein refers to individuals, e.g.,
humans, which have not been previously exposed to a PAL, e.g.,
AvPAL, enzyme.
[0054] "Polyclonal antibodies" as used herein refers to an antibody
population generated in an immunogenic response to a protein having
many epitopes and thus includes a variety of different antibodies
directed to the same and to different epitopes within the protein.
Methods for producing polyclonal antibodies are known in the art
(See, e.g., see, for example, Chapter 11 in: Short Protocols in
Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley
and Sons, New York).
[0055] The term "precision" as used herein generally refers to the
variability in signal between the analysts and days. For example,
precision may be evaluated as coefficient of variation, ranges of
values, or using ANOVA statistics.
[0056] As used herein, the terms "prevent," "preventing," and
"prevention" refer to the total or partial inhibition of the
development, recurrence, onset or spread of a disease and/or
symptom related thereto (e.g., a disease or symptom related thereto
that is associated with elevated phenylalanine levels, such as PKU
or cancer, in a patient), resulting from the administration of a
therapy or combination of therapies provided herein, e.g., AvPAL,
AvPAL variant, or any derivative thereof.
[0057] The term "reagent stability" as used herein generally refers
to the robustness of preparation and storage stability of the
reagents. For example, reagent stability may be established by the
conditions that still permit values to be measured within 75-125%
accuracy relative to freshly prepared reagents.
[0058] The term "robustness" as used herein generally refers to the
capacity of the assay to remain unaffected by small variations in
method parameters and indicates reliability of the assay during
normal run conditions. For example, robustness can be evaluated as
the percent change of reagent concentration, reagent volume, or
incubation time that still generates signal within 75-125% accuracy
relative to the nominal conditions.
[0059] The term "sample" as used herein generally refers to a test
fluid or tissue, e.g., taken from a patient, that can be used in
the methods provided herein. In some embodiments, the sample is an
in vivo sample, for example, bodily (or biological) fluid from a
subject, e.g., a patient, such as a human patient. Non-limiting
examples of such bodily fluids include blood (e.g., human
peripheral blood (HPB)), blood lysate, serum, blood plasma, fine
needle aspirate, ductal lavage, spinal fluid, brain fluid, bone
marrow, ascites fluid or any combination thereof. In other
embodiments, the sample is taken from a biopsy tissue such as a
tumor tissue from a subject or a thin layer cytological sample of
other body tissue or organ. In certain embodiments, the sample
comprises a peripheral blood sample, tumor tissue or suspected
tumor tissue, a thin layer cytological sample, a fine needle
aspirate sample, a bone marrow sample, a lymph node sample, a urine
sample, an ascites sample, a lavage sample, an esophageal brushing
sample, a bladder or lung wash sample, a spinal fluid sample, a
brain fluid sample, a ductal aspirate sample, a nipple discharge
sample, a pleural effusion sample, a fresh frozen tissue sample, a
paraffin embedded tissue sample. In other embodiments, the sample
is an extract or processed sample produced from any of a peripheral
blood sample, a tumor tissue or a suspected tumor tissue, a thin
layer cytological sample, a fine needle aspirate sample, a bone
marrow sample, a urine sample, an ascites sample, a lavage sample,
an esophageal brushing sample, a bladder or lung wash sample, a
spinal fluid sample, a brain fluid sample, a ductal aspirate
sample, a nipple discharge sample, a pleural effusion sample, a
fresh frozen tissue sample or a paraffin embedded tissue
sample.
[0060] The term "sample stability" as used herein generally refers
to the stability of the analyte in the biological fluid or tissue
sample to handling conditions that the collected samples are
anticipated to experience. Sample stability may be measured as the
conditions that still permit values to be measured within 75-125%
accuracy relative to freshly collected samples. For example, sample
stability may be evaluated at -20.degree. C. and -80.degree. C.
over time periods equal to a typical storage period, at RT or
4.degree. C. over a time period equal to the typical sample
preparation and analytical run times, at -20.degree. C., 4.degree.
C. and RT over a time period equal to the typical shipping period,
or through freeze-thaw cycles that may be experienced.
[0061] The term "specificity" as used herein generally refers to
the ability of the assay to detect antibodies that react with a
specific protein. For example, specificity may refer to a
proportional detection response with the specific analyte (e.g.,
AvPAL or variant thereof), while response to a non-specific protein
(e.g., not AvPAL or variant thereof) should be below the LOD. The
proportional response may be evaluated against a correlation
coefficient R value greater than or equal to 0.98. When used in
connection with an enzyme-linked immunosorbant assay (ELISA) method
to, e.g., detect anti-AvPAL antibodies, specificity refers to the
ability to detect antibodies that react with a specific protein
(e.g., rAvPAL-PEG or AvPAL, or variants thereof), and when used in
connection with an ELISA method to detect rAvPAL-PEG or variants
thereof, specificity refers to the ability to detect specific
analyte (e.g., rAvPAL-PEG or AvPAL, or variants thereof).
[0062] As used herein, the terms "subject" and "patient" are used
interchangeably. As used herein, a subject is preferably a mammal
such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats,
etc.) or a primate (e.g., monkey and human), most preferably a
human. In one embodiment, the subject is a mammal, preferably a
human, having been administered a PAL enzyme, such as AvPAL or
rAvPAL, or variants thereof (e.g., rAvPAL_C503S, rAvPAL_C565S,
rAvPAL_C565SC503S) and/or any derivatives thereof (e.g.,
rAvPAL-PEG, rAvPAL-PEG_C503S, rAvPAL-PEG_C565SC or
rAvPAL-PEG_C565SC503S). In some embodiments of the methods and kits
provided herein, the patient has a disease or symptom related
thereto that is associated with elevated phenylalanine levels, such
as HPA or PKU (e.g., classic PKU, severe PKU, moderate PKU or any
subpopulation thereof), or cancer. In some embodiments, the patient
is a patient receiving EST (e.g., rAvPAL or rAvPAL-PEG) for
elevated phenylalanine levels (e.g., a patient with PKU). In other
embodiments of the methods and kits provided herein, the patient is
a patient undergoing cancer therapy (e.g., using rAvPAL or
rAvPAL-PEG). In yet other embodiments of the methods and kits
provided herein, the patient is a pregnant female or an infant
(e.g., age 0 to about 36 months). In another embodiment of the
methods and kits provided herein, the patient is administered a low
or modified protein diet, or a low or modified phenylalanine diet
in combination with an AvPAL or rAvPAL variant thereof, such that
plasma phenylalanine are decreased, e.g., by at least about 25%.
See, e.g., U.S. Pat. Nos. 7,531,341 and 7,534,595 for further
information on the management of patient populations with elevated
phenylalanine levels (e.g., HPA and PKU) with a PAL or PAL-PEG
(e.g., AvPAL or rAvPAL-PEG, or any variant thereof), which, in
certain embodiments, can be used in conjunction with the methods
and kits provided herein. See also U.S. Pat. Nos. 7,560,263 and
7,537,923 for further information on the management of patient
populations with cancer (e.g., HPA and PKU) with a PAL or PAL-PEG
(e.g., AvPAL or rAvPAL-PEG, or any variant thereof), which, in
certain embodiments, can be used in conjunction with the methods
and kits provided herein.
[0063] As used herein, the term "tag" and "label" are used
interchangeably and refer to any type of moiety that is attached to
an antibody or antigen binding fragment thereof, or other
polypeptide used in the methods provided herein. The term
"detectable" or "detection" with reference to an antibody or tag
refers to any antibody or tag that is capable of being visualized
or wherein the presence of the antibody or tag is otherwise able to
be determined and/or measured (e.g., by quantitation). Non-limiting
examples of a detectable tag include fluorescent or other
chemiluminescent tags, and tags that can be amplified and
quantitated using PCR. In certain embodiments, the secondary
antibody used in the methods provided herein is a biotinylated
secondary antibody that is used in combination with a labeled
streptavidin.
[0064] As used herein, the term "therapy" refers to any protocol,
method and/or agent that can be used in the prevention, management,
treatment and/or amelioration of disease (or symptom related
thereto) associated with elevated phenylalanine levels (e.g., PKU)
or cancer. In certain embodiments, the terms "therapies" and
"therapy" refer to a biological therapy, supportive therapy, and/or
other therapies useful in the prevention, management, treatment
and/or amelioration of a disease associated with elevated
phenylalanine levels (e.g., PKU) or cancer known to one of skill in
the art such as medical personnel.
[0065] The term "tissue" as used herein refers to tissues that are
obtained from a mammal, e.g., human. For example, a tissue may be
from a biopsy sample, surgically removed tissue, or postmortem
collection. Furthermore, the tissue may be homogenized and
extracted to isolate the enzyme or antibodies from the tissue.
[0066] As used herein, the terms "treat," "treatment" and
"treating" refer to the reduction or amelioration of the
progression, severity, and/or duration of a disease (or symptom
related thereto) associated with elevated phenylalanine levels
(e.g., PKU) or cancer resulting from the administration of one or
more therapies, including, but not limited to, the administration
of a PAL, such as an AvPAL (or variant thereof) and derivatives
thereof (e.g., a pegylated AvPAL or a pegylated AvPAL variant
thereof).
[0067] The term "variant" as used herein refers to a polypeptide
sequence that contains at least one amino acid substitution,
deletion, or insertion in the coding region relative to the
original polypeptide coding domains. Variants retain the biological
activity of the naturally occurring polypeptide. For example, it is
contemplated that an AvPAL enzyme used in the methods provided
herein may be the naturally occurring enzyme or may comprise one or
more amino acid changes from the naturally occurring enzyme, but
retains the biological activity of the enzyme (i.e., conversion of
phenylalanine to ammonia and trans-cinnamic acid). In certain
embodiments, the enzyme comprises ten or less, five or less, four
or less, three or less, two or less or one amino acid change(s)
from the naturally occurring enzyme. Specific AvPAL variants that
can be used in the methods and kits provided herein, include
rAvPAL_C503S, rAvPAL_C565S, rAvPAL_C565SC503S (or derivatives, such
as pegylated forms, thereof), and are described elsewhere herein,
as well as in U.S. Pat. Nos. 7,531,341; 7,534,595; 7,537,923 and
7,560,263.
DETAILED DESCRIPTION
[0068] Provided herein are methods of detecting the presence of a
pegylated enzyme, an enzyme-specific antibody (e.g., a neutralizing
antibody or of a particular isotype), or a polyethylene glycol
(PEG)-specific antibody in a sample, such as a bodily fluid or
tissue of a patient. In certain embodiments, the methods provided
herein are useful for the detection and/or measurement of
therapeutic enzymes and antibodies associated with administration
of such therapeutic enzymes, including the use of a PAL (or any
variant or derivative thereof), for example, in a patient receiving
the PAL (e.g., an AvPAL or rAvPAL-PEG) during the course of EST for
elevated phenylalanine levels (e.g., phenylketonuria) or cancer
therapy. Also provided are kits for carrying our said methods. In
some embodiments, the assays and methods provided herein are used
to detect or otherwise measure the amount of a rAvPAL-PEG and
AvPAL-specific antibodies in a body fluid from a mammal, such as a
human. In some embodiments of the methods and kits provided herein,
the terms AvPAL and rAvPAL can be used interchangeably.
Production and Purification of Prokaryotic PAL
[0069] In certain embodiments, prokaryotic PAL or biologically
active fragment, mutant variant or analog thereof are used in the
methods described herein. Such prokaryotic PALs can be expressed
using any method known in the art.
[0070] For example, in certain embodiments, recombinant prokaryotic
PAL or a biologically active fragment, mutant, variant or analog
thereof is over-expressed, with or without an N-terminal tag (e.g.,
octahistidyl-tag), in a vector, such as pIBX1 (Su, et al., Appl.
Environ. Microbiol. 62:2723-2734 (1996)) or pET28a (Invitrogen)
with an inducible promoter such as with IPTG
(isopropyl-beta-D-thiogalactopyranoside), in E. coli BLR(DE3)/pLysS
(Novagen) or E. coli BL21(DE3)/pLysS (Invitrogen) cells. Seed
culture for a bioreactor/fermenter is grown from a glycerol stock
in shake flasks. Such seed culture is then used to spike into a
controlled bioreactor in fed-batch mode. Glucose is supplemented
and pH is controlled with base (NH4OH) and agitation is up to 1200
rpm. O.sub.2 feed keeps dissolved oxygen to greater than 20%. The
cells are grown at a temperature of 37.degree. C. until reaching an
OD.sub.600 of 70-100 (.about.22-25 hrs) and then induced with 0.4
mM IPTG. The temperature is reduced to 30.degree. C. and grown
until activity change is <0.1 IU/mL (approximately 40-48 hrs and
an OD.sub.600 typically of 200). Cell culture media is typically
defined and composed of yeast extract protein, peptone-tryptone,
glucose, glycerol, casamino acids, trace salts and phosphate
buffering salts. The recombinant prokaryotic PAL product or
biologically active fragment, mutant, variant or analog thereof is
produced intra-cellularly and not secreted. The bacteria are
harvested by continuous centrifugation (Alfa-Laval, Carr, Ceba, or
equivalent).
[0071] Purification of prokaryotic PAL or a biologically active
fragment, mutant, variant or analog thereof may also be
accomplished using any method known in the art. In one embodiment,
a transformed cell mass is grown and ruptured leaving crude
recombinant enzyme. Exogenous materials are normally separated from
the crude bulk to prevent fouling of the columns. Chromatographic
purification is conducted using one or several chromatographic
resins. Subsequently, the purified protein is formulated into a
buffer designed to provide stable activity over an extended period
of time. In another embodiment, the method to purify the
prokaryotic PAL or biologically active fragment, mutant, variant or
analog thereof comprises: (a) lysis of the bacteria containing
recombinant prokaryotic PAL or biologically active fragment,
mutant, variant or analog thereof using a pressure homogenizer (but
potentially by other physical means such as glass bead lysis); (b)
heat treatment; (c) clarification of this lysate using a second
continuous centrifugation step and/or depth filtration (as with
Cuono Zeta Plus or Maximizer, Pall Filtron, or Millipore Millistak
or Opticao filters); (d) passage through a charcoal filtration step
(as with Millipore Millistak 40AC); (e) passage through a final
filtration step (as with a Sartorious Sartopore 0.2 .mu.m filter);
(f) passage over a butyl hydrophobic interaction chromatography (as
in Toyopearl Butyl 650M from Tosoh Biosciences); (g) passage over a
Q ion exchange column (as in a Macroprep High Q from BioRad); and
(h) recovery of final product by buffer exchange with tangential
flow filtration (as with a Sartorious Hydrosart or PES 100 kDa
membrane). Those skilled in the art readily appreciate that one or
more of the chromatography steps may be omitted or substituted, or
that the order of the chromatography steps may be changed within
the scope of methods provided herein. Finally, appropriate
sterilizing steps may be performed as desired.
Variants
[0072] In certain embodiments, the prokaryotic PAL is AvPAL (or a
biologically active fragment, mutant, variant or analog thereof,
including derivatives thereof). The AvPAL may be prepared for use
in a variety of applications, including EST for elevated
phenylalanine levels (e.g., PKU) or for cancer therapy. AvPAL or
AvPAL fragments include the full-length enzyme, or any variant,
fragment or modification thereof that retains phenylalanine
convertase activity (i.e., conversion of phenylalanine to ammonia
and trans-cinnamic acid). Pegylated derivatives thereof may also be
used (e.g., a pegylated AvPAL or a biologically active fragment,
mutant, variant or analog thereof).
[0073] Full-length AvPAL can be isolated from natural sources
(e.g., purified from Anabaena variabilis infected cells) or
prepared using recombinant techniques, such as those described
elsewhere herein. For example, a general strategy to generate
recombinant full-length AvPAL or variants, fragments or
modifications thereof that retain phenylalanine convertase activity
is to amplify the coding region of interest from the AvPAL genomic
DNA or cDNA by PCR, clone the PCR product into a prokaryotic
expression vector, transfect the prokaryotic expression vector into
bacterial cells, e.g., E. coli cells, and purify the AvPAL proteins
using standard procedures (see, e.g., Examples 1 and 2 herein, as
well as U.S. Pat. Nos. 7,531,341; 7,534,595; 7,537,923 and
7,560,263). In certain embodiments, the AvPAL has the amino acid
sequence SEQ ID NO:4 (FIG. 2B).
[0074] Amino acid sequence variants of the AvPAL polypeptide can
be, for example, substitutional, insertional or deletion variants.
Deletion variants lack one or more residues of the native AvPAL,
which are not essential for function and/or immunogenic activity.
Insertional mutants typically involve the addition of material at a
non-terminal point in the polypeptide. This may include the
insertion of an immunoreactive epitope or simply a single residue.
Terminal additions are additions of amino acid sequences at either
the N- or C-terminus of the AvPAL. Terminal additions can be used
to improve the biophysical characteristics of AvPAL and/or simplify
its purification. Peptide additions include, for example and not
for limitation, HIS (e.g., 6 or 12), TAT, FLAG.TM., HA, c-Myc,
VSV-G, V5, S-peptide, and HSV. Protein additions include, for
example, GFP, MBP, and GST.
[0075] Substitutional variants typically exchange one amino acid of
the naturally occurring AvPAL for another at one or more sites, and
may be designed to modulate one or more properties of the AvPAL,
such as stability against proteolytic cleavage, without the loss of
other functions or properties. Substitutions of this kind
preferably are conservative, i.e., one amino acid is replaced with
one of similar shape and charge. In certain embodiments, the enzyme
comprises ten or less, five or less, four or less, three or less,
two or less or one amino acid change(s) from the naturally
occurring enzyme.
[0076] Variants may be substantially homologous or substantially
identical to the naturally occurring AvPAL. In the context of two
nucleic acids or polypeptides, substantially homologous generally
refers to two or more sequences or subsequences that have at least
40%, 60%, 80%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide or amino
acid residue identity, when compared and aligned for maximum
correspondence, as measured using sequence comparison algorithms
well known in the art or by visual inspection.
[0077] Preferred variants are those variants of AvPAL that retain
at least some of the biological activity of the full-length enzyme,
i.e., phenylalanine convertase activity. Skilled artisans may
easily design polypeptides comprising biologically active variants,
fragments, or modifications of the naturally occurring AvPAL, which
possess the same or similar biological activity to the naturally
occurring full-length enzyme.
[0078] Particularly preferred variants are those variants of AvPAL
in which one or more cysteine residues, e.g., at positions 64, 235,
318, 424, 503 and 565 of AvPAL, are replaced by serine residues, as
described elsewhere herein (see, e.g., U.S. Pat. Nos. 7,531,341;
7,534,595; 7,537,923 and 7,560,263). In certain embodiments, the
AvPAL variant has the amino acid sequence SEQ ID NO:7 (FIG. 3A),
SEQ ID NO:8 (FIG. 3B), SEQ ID NO:9 (FIG. 3C) or SEQ ID NO:10 (FIG.
3D). In specific embodiments, the variant is the AvPAL double
cysteine mutant, AvPAL_S565SC503S, in which the cysteine residues
at positions 503 and 565 of AvPAL are replaced by serine residues
(SEQ ID NO:11; FIG. 3E).
[0079] In any of the methods and kits provided herein, an AvPAL or
an AvPAL variant thereof (including pegylated forms) can be
used.
Pegylated Prokaryotic PAL Variants
[0080] Macromolecule chemical modification can be performed in a
non-specific fashion (leading to mixtures of derivatized species)
or in a site-specific fashion (based on wild-type macromolecule
reactivity-directed derivatization and/or site-selective
modification using a combination of site-directed mutagenesis and
chemical modification) or, alternatively, using expressed protein
ligation methods (Hofmann, et al., Curr. Opin. Biotechnol.
13(4):297-303 (2002)). In certain embodiments, chemical
modification is used to reduce immunogenicity. In certain
embodiments, the prokaryotic PAL variant comprises a water-soluble
polymer (i.e., polyethylene glycol (PEG)). Pegylation is a
demonstrated method to reduce immunogenicity of proteins (Bhadra,
et al., Pharmazie 57(1):5-29 (2002)), but glycosylation and other
chemical derivatization procedures, using modification with
phosphorylation, amidation, carboxylation, acetylation,
methylation, creation of acid-addition salts, amides, esters, and
N-acyl derivatives are also possible (Davis, Science 303:480-482
(2004)). Methods for pegylating PAL proteins and for determining
the optimal degree of pegylation are described in U.S. Pat. No.
7,553,653).
[0081] A series of different pegylation reactions on PAL, using a
range of PEG chemical reagent to PAL protein ratios, can provide
PEG-PAL derivatives for each modification method. The optimal
degree of pegylation can be determined based upon the residual
activity obtained for each derivatized PAL species using the
absorbance assay in combination with PAGE and native gel analysis
to determine the extent of PEG derivatization. After initial ranges
of optimal modification are determined, comparative kinetic
analysis (including Vmax and Km determinations, binding constants
of substrates, proteolytic stability, pH dependence of activity,
temperature-dependence of activity) and immunoreactivity of optimal
PEG-PAL species can be determined by ELISA, immunoprecipitation,
and Western blot. Protein engineering can also be used to generate
the most favorable PAL mutant for pegylation using the optimal
derivatization conditions; by minimizing the size of the PAL
protein and only modifying the most antigenic regions of the PAL
surface, cost of PEG modification will be reduced while at the same
time retaining the maximum amount of enzymatic activity and minimum
amount of immunogenicity. Similarly, site-specific pegylation can
be used to provide enzyme derivatives.
[0082] Other chemical modifications such as phosphorylation or
other chemical modification of lysine, arginine, and/or cysteine
residues can be used to mask immunogenic regions and/or proteolytic
sensitive regions. Such chemical modifications include the polymer
addition method of Bednarsaki and the Altus Corporation
cross-linking method for improving PAL stability, reducing
immunogenicity, and improving protease resistance are
representative examples. Bednarsaki demonstrated that polymer
addition improves protein temperature stability (Wang, et al., J.
Am. Chem. Soc. 114(1):378-380 (1992)), and Altus Corporation has
found that glutaraldehyde cross-linking improves enzyme
stability.
[0083] To discover if the in vivo therapeutic half-life of a
protein such as PAL would benefit from pegylation, a variety of
different PEG:PAL conjugates are synthesized, characterized in
vitro and tested in vivo for phenylalanine reduction. In order to
both optimize the potential effects of pegylation and to identify
the preferred sites of PEG attachment, a design strategy is
employed wherein polymer length, conformation, and the degree of
PEG attachment is varied.
[0084] Methods for preparing the pegylated PAL for use in the
therapies or other methods provided herein generally comprise the
steps of (a) reacting PAL with PEG under conditions whereby PAL
becomes attached to one or more PEG groups, and (b) obtaining the
reaction product(s). Because the specific sites of PAL modification
might significantly alter the intrinsic activity of the conjugate,
different types and amounts of PEG were explored. The chemistry
used for pegylation of PAL was the acylation of the primary amines
of PAL using the NHS-ester of methoxy-PEG
(O--[(N-Succinimidyloxycarbonyl)-methyl]-O'-methylpolyethylene
glycol). Acylation with methoxy-PEG-NHS or methoxy-PEG-SPA results
in an amide linkage that eliminates the charge from the original
primary amine.
[0085] The present methods provide for a substantially homogenous
mixture of polymer:protein conjugate. "Substantially homogenous" as
used herein means that only polymer:protein conjugate molecules are
observed. The polymer:protein conjugate has biological activity and
the present "substantially homogenous" pegylated PAL preparations
provided herein are those which are homogenous enough to display
the advantages of a homogenous preparation, e.g., ease in clinical
application in predictability of lot to lot pharmacokinetics.
[0086] The polymer molecules contemplated for use in the pegylation
approaches described herein may be selected from among
water-soluble polymers or a mixture thereof. The water-soluble
polymer may be selected from the group consisting of, for example,
polyethylene glycol, monomethoxy-polyethylene glycol, dextran,
poly-(N-vinyl pyrrolidone), propylene glycol homopolymers, a
polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated
polyols (e.g., glycerol), HPMA, Fleximer.TM., and polyvinyl
alcohol, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, tresyl monomethoxy
PEG, PEG propionaldehyde, bis-succinimidyl carbonate PEG,
cellulose, or other carbohydrate-based polymers. The polymer
selected should be water-soluble so that the protein to which it is
attached does not precipitate in an aqueous environment, such as a
physiological environment. The polymer may be branched or
unbranched. Preferably, for therapeutic use of the end-product
preparation, the polymer will be pharmaceutically acceptable.
[0087] In specific embodiments, the water-soluble polymer is PEG.
As used herein, PEG is meant to encompass any of the forms of PEG
that have been used to derivatize other proteins, such as
mono-(C1-C10)alkoxy- or aryloxy-polyethylene glycol.
[0088] The proportion of PEG molecules to protein molecules will
vary, as will their concentrations in the reaction mixture. In
general, the optimum ratio (in terms of efficiency of reaction in
that there is no excess unreacted protein or polymer) will be
determined by the molecular weight of the PEG selected and on the
number of available reactive groups (typically .epsilon. amino
groups) present. As relates to molecular weight, in general, the
higher the molecular weight of the polymer used, the fewer number
of polymer molecules which may be attached to the protein.
Similarly, branching of the polymer should be taken into account
when optimizing these parameters. Generally, the higher the
molecular weight (or the more branches) the higher the
polymer:protein ratio. Several different linear PEG polymer lengths
including, but not limited to, 5 kDa and 20 kDa, conjugates of
two-armed branched PEG polymers, including, but not limited to, 10
kDa and 40 kDa. In general, for the pegylation reactions
contemplated herein, the preferred average molecular weight is
about 2 kDa to about 100 kDa (the term "about" indicating +/-1
kDa). More preferably, the average molecular weight is about 5 kDa
to about 40 kDa. The ratio of water-soluble polymer to PAL will
generally range from 1:1 for monoPEG, 2:1 for diPEG, etc.
[0089] One or more lysine residues at or near the active site of a
prokaryotic PAL variant can be introduced to enhance catalytic
activity, reduce immunogenicity and/or improve biochemical
stability, in part by blocking potential pegylation of other amino
acid residues (e.g., tyrosine) at/near the active site of the
enzyme or by blocking potential pegylation of a lysine residue
important for enzyme activity. Without being bound to a particular
theory, it is hypothesized that a tyrosine residue at/near the
active site of a prokaryotic PAL (i.e., position 78 or 314 in
AvPAL) can be a site for pegylation, which reduces enzyme activity.
In some embodiments, one or more amino acid residues at/near the
active site of the prokaryotic PAL, which are not required for
enzyme activity, are substituted by a lysine residue.
[0090] In one embodiment, the prokaryotic PAL is AvPAL. In some
embodiments, the AvPAL tyrosine residue at position 78, 314 or 419
is not accessible for pegylation. Again without being bound to a
particular theory, it is hypothesized that a lysine residue of a
prokaryotic PAL (i.e., position 419 in AvPAL), which is normally
blocked from pegylation due to pegylation of a neighboring lysine
residue PAL (i.e., position 413 in AvPAL), can be a site for
pegylation, which reduces substrate binding and/or catalytic
activity. In some embodiments, one or more amino acid residues of
the prokaryotic PAL are substituted by a lysine residue, such that
a lysine residue important for the substrate binding and/or
catalytic activity of the enzyme is not accessible for
pegylation.
[0091] Pegylated PAL enzymes are effective for decreasing in vivo
phenylalanine concentrations in subjects. For instance, Examples
7-9 of U.S. Pat. No. 7,531,341, describe the effects of pegylated
and nonpegylated forms of lysine mutant R91K PAL from
Rhodosporidium toruloides (RtPAL), PAL produced by the
cyanobacterium Nostoc punctiforme (NpPAL), and PAL produced by the
cyanobacterium Anabaena variabilis (AvPAL) on phenylalanine levels
in the ENU2 or BTBR.sup.enu2 mouse. This animal model is a
homozygous mutant at the phenylalanine hydroxylase gene (PAH) locus
resulting in an animal with severe hyperphenylalanemia. The high
plasma phenylalanine levels make this animal the appropriate model
for evaluating the ability of PAL to reduce plasma phenylalanine.
Administration of pegylated forms of NpPAL and AvPAL resulted in
greater reduction in phenylalanine in the ENU2 mice as compared to
unpegylated NpPAL and AvPAL, respectively. Such effects were
maintained for NpPAL upon weekly injections over a ten-week period.
These results show that pegylation of PAL from the cyanobacteria,
Nostoc punctiforme and Anabaena variabilis, is essential in
reducing phenylalanine levels in PKU affected mice.
[0092] Similarly, certain pegylated AvPAL variants also effective
for decreasing in vivo phenylalanine concentrations in subjects and
also results in lower concentrations of anti-AvPAL antibodies. For
instance, Example 14 of U.S. Pat. No. 7,534,595 describes the
effect of serine substitution of the cysteine residues (e.g., at
positions 503 and 565) in the AvPAL polypeptide on phenylalanine
levels in ENU2 mice. The administration of the pegylated AvPAL
double cysteine mutant AvPAL_C565SC503S resulted in a reduction in
plasma phenylalanine that was comparable to that achieved with
pegylated wild-type AvPAL. In addition, the anti-PAL antibody
titers were lower in animals injected with pegylated AvPAL variant
as compared to pegylated wild-type AvPAL. These results show that a
pegylated AvPAL variant has (1) in vivo PAL enzyme activity that is
comparable to the pegylated wild-type AvPAL, and (2) has reduced
immunogenicity compared to the pegylated wild-type AvPAL.
[0093] In certain embodiments of the methods provided herein, the
AvPAL is an AvPAL variant, wherein the cysteine residue at position
503 of AvPAL has been substituted with a serine residue
(rAvPAL-PEG_C503S; SEQ ID NO:9). In some embodiments of the methods
provided herein, the AvPAL is an AvPAL variant, wherein the
cysteine residue at position 565 of AvPAL has been substituted with
a serine residue (rAvPAL-PEG_C565S; SEQ ID NO:10). In yet other
embodiments of the methods provided herein, the AvPAL is an AvPAL
variant, wherein the cysteine residue at positions 503 and 565 of
AvPAL have been substituted with serine residues
(rAvPAL_C565SC503S; SEQ ID NO:11)).
[0094] In some embodiments, the rAvPAL_C503S, rAvPALC565S or
rAvPAL_C565SC503S is pegylated (rAvPAL-PEG_C503S, rAvPAL-PEG_C565S,
or rAvPAL-PEG_C565SC503S, respectively).
[0095] In one embodiment, the rAvPAL-PEG_C565SC503S pegylation is
achieved by reacting the AvPAL variant with NHS-activated
polyethylene glycol at a ratio of at least 1.6 polyethylene glycol
per lysine residue of AvPAL variant. In one embodiment, at least 28
percent of lysine residues at positions 2, 10, 32, 145, 195, 301,
413, 493, and 522 of the rAvPAL-PEG_C565SC503S are pegylated. In
another embodiment, the rAvPAL-PEG_C565SC503S pegylation is
achieved by reacting the AvPAL variant with NHS-activated
polyethylene glycol at a ratio of at least 2.4 polyethylene glycol
per lysine residue of AvPAL variant. In some embodiments, at least
51 percent of lysine residues at positions 2, 10, 195, 413, 493,
and 522 of the rAvPAL-PEG_C565SC503S are pegylated. In other
embodiments, the rAvPAL-PEG_C565SC503S pegylation is achieved by
reacting the AvPAL variant with NHS-activated polyethylene glycol
at a ratio of 3 polyethylene glycol per lysine residue of AvPAL
variant. In certain embodiments, at least 75 percent of lysine
residues at positions 2, 10, 195, 493, and 522 of the
rAvPAL-PEG_C565SC503S are pegylated.
Labels
[0096] In some embodiments, an assay reagent is labeled to
facilitate its detection. Labels can be a substance used to
directly or indirectly detect the antibody or other protein (e.g.,
streptavidin) that the label is attached to. In certain
embodiments, the label is the antibody or other protein itself or,
alternatively, the label may be covalently or non-covalently linked
to the antibody or other protein. In certain embodiments, labels
are used in order to follow or track the given antibody or other
protein, for example, to determine its presence or amount.
[0097] A label or a detectable moiety is a composition detectable
by spectroscopic, photochemical, biochemical, immunochemical,
chemical, or other physical means. Antibodies can be detectably
labeled through the use of radioisotopes, affinity labels (such as
biotin, avidin, etc.), enzymatic labels (such as horseradish
peroxidase, alkaline phosphatase, etc.), fluorescent labels (such
as FITC or rhodamine, etc.), or luminescent or bioluminescent
labels (such as Europium, Vanadium, etc.), paramagnetic atoms,
electrochemiluminescent labels (such as Ru-based labels in
conjunction with substrates, etc.), and the like.
[0098] In some embodiments, an assay reagent is labeled to
facilitate its capture. A capture moiety is a composition that is
capable of being specifically bound by another composition that is
attached or linked to a solid support. An assay reagent can be
labeled through the use of affinity labels (such as biotin, avidin,
etc.) for which specific and selective ligands are available,
haptens and proteins for which antisera or monoclonal antibodies
are available, and nucleic acid molecules with a sequence
complementary to a target. Procedures for accomplishing such
labeling are described in Sternberger et al., J. Histochem.
Cytochem. 18:315 (1970); Bayer et al., Meth. Enzym. 62:308 (1979);
Engval et al., Immunol. 109:129 (1972); Goding, J. Immunol. Meth.
13:215 (1976)). The solid support can be a filter, plate, membrane
or bead, and the like.
[0099] In some embodiments of the methods and kits provided herein,
labels are used which may be detected directly, e.g., on the basis
of a physical or chemical property of the label (e.g., optical
absorbance, fluorescence, phosphorescence, chemiluminescence,
electrochemiluminescence, refractive index, light scattering,
radioactivity, magnetism, catalytic activity, or chemical
reactivity). Examples of directly detectable labels include, but
are not limited to, radioactive labels, fluorescent labels,
luminescent labels, enzyme labels, chemiluminescent labels,
electrochemiluminescent labels, phosphorescent labels, light
scattering or adsorbing particles (e.g., metal particles, gold
colloids, silver colloids), magnetic labels and the like.
[0100] Examples of labels suitable for use in the methods provided
herein include, but are not limited to, radioactive labels (e.g.,
.sup.32P), fluorophores (e.g., fluorescein), electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA), biotin,
digoxigenin, or haptens as well as proteins which can be made
detectable, e.g., by incorporating a radiolabel into the hapten or
peptide, or used to detect antibodies specifically reactive with
the hapten or peptide. Also contemplated are a nanotag, a molecular
mass bead, a magnetic agent, a nano- or micro-bead containing a
fluorescent dye, a quantum dot, a quantum bead, a fluorescent
protein, dendrimers with a fluorescent label, a micro-transponder,
an electron donor molecule or molecular structure, or a light
reflecting particle.
[0101] For example, labels contemplated for use in the methods
provided herein include, but are not limited to, fluorescent dyes
(e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the
like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C,
or .sup.32P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), biotin, and
colorimetric labels such as colloidal gold, colored glass or
plastic beads (e.g., polystyrene, polypropylene, latex, etc.), and
luminescent or chemiluminescent labels.
[0102] The label may be coupled directly or indirectly to the
desired component of the assay. In specific embodiments, the label
is covalently bound to the biopolymer (e.g., antibody or protein)
using an isocyanate or N-hydroxysuccinimide ester reagent for
conjugation of an active agent. In some embodiments, the
bifunctional isocyanate reagents provided herein can be used to
conjugate a label to a biopolymer to form a label biopolymer
conjugate without an active agent attached thereto. The label
biopolymer conjugate may be used as an intermediate for the
synthesis of a labeled conjugate or can be used to detect the
biopolymer conjugate. As indicated above, a wide variety of labels
can be used, with the choice of label depending on sensitivity
required, ease of conjugation with the desired component of the
assay, stability requirements, available instrumentation, and
disposal provisions. Non-radioactive labels are often attached by
indirect means. Generally, a ligand molecule (e.g., biotin) is
covalently bound to the molecule. The ligand then binds to another
molecule (e.g., Streptavidin) molecule, which is either inherently
detectable or covalently bound to a signal system, such as a
detectable enzyme, a fluorescent compound, or a chemiluminescent
compound, or to a solid support, such as a filter, a plate, a
membrane or a bead, and the like.
[0103] The compounds useful in the method provided herein can also
be conjugated directly to signal-generating compounds, e.g., by
conjugation with an enzyme or fluorophore. Enzymes suitable for use
as labels include, but are not limited to, hydrolases, particularly
phosphatases, esterases and glycosidases, or oxidotases,
particularly peroxidases. Fluorescent compounds, i.e.,
fluorophores, suitable for use as labels include, but are not
limited to, fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, etc. Further examples of
suitable fluorophores include, but are not limited to, eosin,
TRITC-amine, quinine, fluorescein W, acridine yellow, lissamine
rhodamine, B sulfonyl chloride erythroscein, ruthenium (tris,
bipyridinium), europium, Texas Red, nicotinamide adenine
dinucleotide, flavin adenine dinucleotide, etc. Chemiluminescent
compounds suitable for use as labels include, but are not limited
to, Europium (Eu), Samarium (Sm), luciferin and
2,3-dihydrophthalazinediones, e.g., luminol.
Electrochemiluminescent compounds suitable for use as labels
include, but are not limited to, MSD TAG, MSD Sulfo-TAG, BV-TAG,
and BV-TAG Plus. For a review of various labeling or signal
producing systems that can be used in the methods provided herein,
see U.S. Pat. No. 4,391,904.
Detection Methods and Kits
[0104] Where the label is radioactive, means for detection include
a scintillation counter or photographic film, as in
autoradiography. Where the label is a fluorescent label, exciting
the fluorochrome with the appropriate wavelength of light and
detecting the resulting fluorescence may detect it. The
fluorescence may be detected visually, by the use of electronic
detectors such as charge coupled devices (CCDs) or photomultipliers
and the like. Similarly, enzymatic labels may be detected by
providing the appropriate substrates for the enzyme and detecting
the resulting reaction product. Colorimetric labels may be detected
simply by observing the color associated with the label. Other
labeling and detection systems suitable for use in the methods
provided herein will be readily apparent to those of skill in the
art. Such labeled modulators and ligands can be used in the
diagnosis of a disease or health condition.
[0105] Chemiluminescent labels may be detected by observing the
light emitted upon reaction of the label with substrate.
Electrochemiluminescent labels may be detected by observing the
light emitted upon reaction of the label with substrate in an
electrical field.
[0106] In some embodiments, the labeled compositions that can be
used in the methods provided herein are linked to a solid support,
including, but not limited to, filters, plates or membranes. It is
further contemplated that the labeled compounds may be labeled and
interact in solution. For example, the capture antibody may be
labeled with a fluorescent resonance energy transfer (FRET) donor
molecule and the target molecule is labeled with a FRET acceptor
molecule such that the molecules are in proximity when binding
occurs. Alternatively, the target molecule may be labeled with the
FRET donor and the antibody molecule the FRET acceptor. Another
possibility is to separate quenching and fluorescent molecule both
present on the antibody or target when target and antibody
hybridize. The target molecule is only close enough for its label
to emit if it is interacting with the reagent. This produces a
system where the molecule only emits when it interacts with the
reagent (direct monitoring). A narrow band pass filter can be used
to block all wavelengths except that of the molecule's label. FRET
molecule pairs are commercially available in the art (e.g., from
Invitrogen), and may be used according to the manufacturer's
protocol. FRET emissions are detected using optical imaging
techniques, such as a CCD camera.
[0107] Another method of detecting the antibody-antigen
interactions is to label it with an electron donor. This donor
label would give electrons to an electrical contact to which the
reagent is bound. See, for example, Ghindilis, Biochem Soc Trans.
28:84-9, (2000) and Dai et al., Cancer Detect Prev. 29:233-40
(2005), which describe enzymes useful in and methods for electro
immunoassays. The electron contact would then be read by an A to D
(analog to digital) converter and quantified. The higher the
electron count the more interactions took place.
[0108] One embodiment of a label capable of single molecule
detection is the use of plasmon-resonant particles (PRPs) as
optical reporters, as described in Schultz et al., Proc. Natl.
Acad. Sci. USA 97:996-1001 (2000), incorporated herein by
reference. PRPs are metallic nanoparticles, typically 40-100 nm in
diameter, which scatter light elastically with remarkable
efficiency because of a collective resonance of the conduction
electrons in the metal (i.e., the surface plasmon resonance). The
magnitude, peak wavelength, and spectral bandwidth of the plasmon
resonance associated with a nanoparticle are dependent on the
particle's size, shape, and material composition, as well as the
local environment. By influencing these parameters during
preparation, PRPs can be formed that have scattering peak anywhere
in the visible range of the spectrum. For spherical PRPs, both the
peak scattering wavelength and scattering efficiency increase with
larger radius, providing a means for producing differently colored
labels. Populations of silver spheres, for example, can be
reproducibly prepared for which the peak scattering wavelength is
within a few nanometers of the targeted wavelength, by adjusting
the final radius of the spheres during preparation. Because PRPs
are bright, yet nanosized, they are used as indicators for
single-molecule detection; that is, the presence of a bound PRP in
a field of view can indicate a single binding event.
[0109] In one exemplary embodiment, the assay device is a lateral
flow test strip, optionally encased in a housing. A first labeled
antibody to an AvPAL is in solution, while a second antibody to the
AvPAL is immobilized on the test strip. When a patient sample
containing an AvPAL is contacted with both antibodies, an
antibody-target-antibody sandwich complex is formed, and the
resulting complex, which is immobilized on the solid support, is
detectable by virtue of the label. The test strip is then inserted
into a reader, where the signal from the label in the complex is
measured. The outcome may be either a positive or negative result,
or a quantitative determination of the concentration of an AvPAL in
the sample, which is correlated with a result indicative of a risk
or presence of a disease or disorder. The entire procedure may be
automated and/or computer-controlled. Alternatively, the test strip
may be read visually by comparison to a visual standard of the
appropriate color. This test provides similar clinically relevant
information as an AvPAL ELISA, but in significantly less time and
at the point of care.
[0110] Antigen-antibody complexes may also be detected using
nanoparticle-derived techniques. See, for example, Ao et al., Anal
Chem. 78:1104-6 (2006), which describes gold nanoparticle
quenching, Tang et al., Biosens Bioelectron. 2005 Nov. 30, which
describes SiO(2)/Au nanoparticle surfaces in antibody detection,
and Lieu et al., J Immunol Methods. 307:34-40 (2005), which
describes silicon dioxide nanoparticles containing
dibromofluorescein for use in solid substrate-room temperature
phosphorescence immunoassay (SS-RTP-IA).
[0111] Kits are also provided herein. The kit can contain one or
more reagents identified in the various methods provided herein.
For example, in one embodiment, the kit comprises an AvPAL,
optionally linked to a detectable label or a capture moiety, and/or
an antibody standard that specifically binds to the AvPAL, and/or
an AvPAL standard containing a known quantity of an AvPAL. In
another embodiment, the kit comprises an AvPAL substrate, and/or an
AvPAL standard containing a known quantity of an AvPAL, and/or an
antibody standard that specifically binds to, and neutralizes the
enzymatic activity of, the AvPAL. In another embodiment, the kit
comprises an AvPAL, optionally linked to a detectable label, and/or
an antibody that specifically binds to antibodies of IgG, IgA, IgE,
IgM or IgD isotype, and/or an IgG, IgA, IgE, IgM or IgD antibody
standard that specifically binds to the AvPAL, and/or an AvPAL
standard containing a known quantity of an AvPAL. Other components
of the kits may optionally include reagents and/or instructions for
carrying out the any of the methods provided herein, e.g., reagents
used to acidify or to neutralize rAvPAL-PEG or variants thereof, as
described elsewhere herein.
[0112] Spectral absorption labels may also be used. A possible
methodology for detection would be to mix into the bead polymer
different materials that absorb and pass different spectra of
light. Each different type of bead could be detected by passing a
multi-spectral light though the bead and detecting which spectra
are absorbed.
Antibody Production
[0113] Antibodies for use in the methods and kits provided herein
include, but are not limited to, synthetic antibodies, monoclonal
antibodies, recombinantly produced antibodies, multispecific
antibodies (including bi-specific antibodies), human antibodies,
humanized antibodies, chimeric antibodies, intrabodies,
single-chain Fvs (scFv) (e.g., including monospecific, bispecific,
etc.), camelized antibodies, Fab fragments, F(ab').sub.2 fragments,
disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies,
and epitope-binding fragments of any of the above. In specific
embodiments, the first and/or second antibody of the methods
provided herein is a monoclonal antibody.
[0114] In particular, antibodies for use in the methods and kits
provided herein include immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e.,
molecules that contain an antigen binding site that
immunospecifically binds to a target (such as AvPAL or rAvPAL-PEG
or a variant thereof). The immunoglobulin molecules provided herein
can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class
(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of
immunoglobulin molecule. In specific embodiments, the first and/or
second antibody of the methods provided herein is an IgG
antibody.
[0115] Variants and derivatives of antibodies include antibody
fragments that retain the ability to specifically bind to an
epitope. Preferred fragments include Fab fragments (an antibody
fragment that contains the antigen-binding domain and comprises a
light chain and part of a heavy chain bridged by a disulfide bond);
Fab' (an antibody fragment containing a single anti-binding domain
comprising an Fab and an additional portion of the heavy chain
through the hinge region); F(ab').sub.2 (two Fab' molecules joined
by interchain disulfide bonds in the hinge regions of the heavy
chains; the Fab' molecules may be directed toward the same or
different epitopes); a bispecific Fab (a Fab molecule having two
antigen binding domains, each of which may be directed to a
different epitope); a single chain Fab chain comprising a variable
region, also known as, a sFv (the variable, antigen-binding
determinative region of a single light and heavy chain of an
antibody linked together by a chain of 10-25 amino acids); a
disulfide-linked Fv, or dsFv (the variable, antigen-binding
determinative region of a single light and heavy chain of an
antibody linked together by a disulfide bond); a camelized VH (the
variable, antigen-binding determinative region of a single heavy
chain of an antibody in which some amino acids at the VH interface
are those found in the heavy chain of naturally occurring camel
antibodies); a bispecific sFv (a sFv or a dsFv molecule having two
antigen-binding domains, each of which may be directed to a
different epitope); a diabody (a dimerized sFv formed when the VH
domain of a first sFv assembles with the VL domain of a second sFv
and the VL domain of the first sFv assembles with the VH domain of
the second sFv; the two antigen-binding regions of the diabody may
be directed towards the same or different epitopes); and a triabody
(a trimerized sFv, formed in a manner similar to a diabody, but in
which three antigen-binding domains are created in a single
complex; the three antigen binding domains may be directed towards
the same or different epitopes). Derivatives of antibodies also
include one or more CDR sequences of an antibody combining site.
The CDR sequences may be linked together on a scaffold when two or
more CDR sequences are present. In certain embodiments, the
antibody used in the methods and kits provided herein comprises a
single-chain Fv ("scFv"). scFvs are antibody fragments comprising
the VH and VL domains of an antibody, wherein these domains are
present in a single polypeptide chain. Generally, the scFv
polypeptide further comprises a polypeptide linker between the VH
and VL domains which enables the scFv to form the desired structure
for antigen binding. For a review of scFvs see Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
[0116] The antibodies may be from any animal origin including birds
and mammals (e.g., human, murine, donkey, sheep, rabbit, goat,
guinea pig, camel, horse, or chicken). In certain embodiments, the
antibodies are human or humanized monoclonal antibodies. As used
herein, "human" antibodies include antibodies having the amino acid
sequence of a human immunoglobulin and include antibodies isolated
from human immunoglobulin libraries or from mice that express
antibodies from human genes.
[0117] In some embodiments of the methods provided herein, the
first antibody and/or the second antibody is a monoclonal
antibody.
[0118] Monoclonal or polyclonal antibodies for a given PAL (e.g.,
AvPAL), PAL variant thereof, or any pegylated PAL or PAL variant,
that can be used in the methods and kits provided herein can be
prepared using standard techniques known in the art, such as those
described below. The term "monoclonal antibody" refers to an
antibody obtained from a population of homogenous or substantially
homogeneous antibodies, wherein each monoclonal antibody will
typically recognize a single epitope on the antigen. In certain
embodiments, a "monoclonal antibody," as used herein, is an
antibody produced by a single hybridoma or other cell, wherein the
antibody immunospecifically binds to only a given epitope of a PAL
(e.g., AvPAL) or variant thereof as determined, e.g., by ELISA or
other antigen-binding or competitive binding assay known in the
art. The term "monoclonal" is not limited to any particular method
for making the antibody. For example, monoclonal antibodies may be
made by the hybridoma method as described in Kohler et al.; Nature,
256:495 (1975) or may be isolated from phage libraries using
techniques known in the art, for example. Other methods for the
preparation of clonal cell lines and of monoclonal antibodies
expressed thereby are well known in the art (see, for example,
Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th
Ed., Ausubel et al., eds., John Wiley and Sons, New York).
"Polyclonal antibodies" as used herein refers to an antibody
population generated in an immunogenic response to a protein having
many epitopes and thus includes a variety of different antibodies
directed to the same and to different epitopes within the protein.
Methods for producing polyclonal antibodies are known in the art
(See, e.g., see, for example, Chapter 11 in: Short Protocols in
Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley
and Sons, New York).
[0119] Antibodies that immunospecifically bind to, e.g., a PAL,
such as AvPAL, or variant thereof, can be produced by any method
known in the art for the synthesis of antibodies. The practice of
the methods provided herein employs, unless otherwise indicated,
conventional techniques in molecular biology, microbiology, genetic
analysis, recombinant DNA, organic chemistry, biochemistry, PCR,
oligonucleotide synthesis and modification, nucleic acid
hybridization, and related fields within the skill of the art.
These techniques are described in the references cited herein and
are fully explained in the literature. See, e.g., Maniatis et al.
(1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press; Sambrook et al. (2001) Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.; Ausubel et al., Current Protocols in Molecular Biology, John
Wiley & Sons (1987 and annual updates); Current Protocols in
Immunology, John Wiley & Sons (1987 and annual updates); Gait
(ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL
Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A
Practical Approach, IRL Press; Birren et al. (eds.) (1999) Genome
Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory
Press.
[0120] Polyclonal antibodies that immunospecifically bind to an
antigen can be produced by various procedures well-known in the
art. For example, a human antigen can be administered to various
host animals including, but not limited to, rabbits, mice, rats,
etc. to induce the production of sera containing polyclonal
antibodies specific for the human antigen. Various adjuvants may be
used to increase the immunological response, depending on the host
species, and include, but are not limited to, Freund's (complete
and incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and corynebacterium parvum. Such
adjuvants are also well known in the art.
[0121] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681
(Elsevier, N.Y., 1981).
[0122] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
Briefly, mice can be immunized with a given PAL (e.g., AvPAL) or
variant thereof antigen and once an immune response is detected,
e.g., antibodies specific for the PAL (e.g., AvPAL) antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well known
techniques to any suitable myeloma cells, for example cells from
cell line SP20 available from the ATCC. Hybridomas are selected and
cloned by limited dilution.
[0123] Additionally, a RIMMS (repetitive immunization multiple
sites) technique can be used to immunize an animal (Kilptrack et
al., 1997 Hybridoma 16:381-9). The hybridoma clones are then
assayed by methods known in the art for cells that secrete
antibodies capable of binding a polypeptide. Ascites fluid, which
generally contains high levels of antibodies, can be generated by
immunizing mice with positive hybridoma clones.
[0124] Antibody fragments which recognize a PAL (e.g., AvPAL)
antigen may be generated by any technique known to those of skill
in the art. For example, Fab and F(ab').sub.2 fragments may be
produced by proteolytic cleavage of immunoglobulin molecules, using
enzymes such as papain (to produce Fab fragments) or pepsin (to
produce F(ab').sub.2 fragments). F(ab').sub.2 fragments contain the
variable region, the light chain constant region and the CH1 domain
of the heavy chain.
[0125] For example, antibodies which recognize a PAL (e.g., AvPAL)
antigen can also be generated using various phage display methods.
In phage display methods, functional antibody domains are displayed
on the surface of phage particles which carry the polynucleotide
sequences encoding them. In particular, DNA sequences encoding
variable heavy (VH) and variable light (VL) domains are amplified
from animal cDNA libraries (e.g., human or murine cDNA libraries of
affected tissues). The DNA encoding the VH and VL domains are
recombined together with an scFv linker by PCR and cloned into a
phagemid vector. The vector is electroporated in E. coli and the E.
coli is infected with helper phage. Phage used in these methods are
typically filamentous phage including fd and M13 and the VH and VL
domains are usually recombinantly fused to either the phage gene
III or gene VIII. Phage expressing an antigen binding domain that
binds to a particular antigen can be selected or identified with
antigen, e.g., using labeled antigen or antigen bound or captured
to a solid surface or bead. Examples of phage display methods that
can be used to make the antibodies useful in the methods provided
herein include those disclosed in Brinkman et al., 1995, J.
Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods
184:177-186; Kettleborough et al., 1994, Eur. J. Immunol.
24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al.,
1994, Advances in Immunology 57:191-280; PCT Application No.
PCT/GB91/O1 134; International Publication Nos. WO 90/02809, WO
91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO
95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409,
5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698,
5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and
5,969,108.
[0126] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described below. Techniques to
recombinantly produce Fab, Fab' and F(ab').sub.2 fragments can also
be employed using methods known in the art such as those disclosed
in PCT publication No. WO 92/22324; Mullinax et al., 1992,
BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and
Better et al., 1988, Science 240:1041-1043.
[0127] To generate whole antibodies, PCR primers including VH or VL
nucleotide sequences, a restriction site, and a flanking sequence
to protect the restriction site can be used to amplify the VH or VL
sequences in scFv clones. Utilizing cloning techniques known to
those of skill in the art, the PCR amplified VH domains can be
cloned into vectors expressing a VH constant region, e.g., the
human gamma 4 constant region, and the PCR amplified VL domains can
be cloned into vectors expressing a VL constant region, e.g., human
kappa or lambda constant regions. The VH and VL domains may also
cloned into one vector expressing the necessary constant regions.
The heavy chain conversion vectors and light chain conversion
vectors are then co-transfected into cell lines to generate stable
or transient cell lines that express full-length antibodies, e.g.,
IgG, using techniques known to those of skill in the art.
Antibody Purification
[0128] Antibodies may be purified using techniques standard in the
art, including, but not limited, to protein A-Sepharose,
hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography. The antibody composition prepared from
microbial or mammalian cells or serum can be purified using, for
example, hydroxylapatite chromatography cation or anion exchange
chromatography, and affinity chromatography, with affinity
chromatography being the preferred purification technique. For
affinity chromatography, the affinity ligand is typically the
antigen, e.g., rAvPAL-PEG or AvPAL, or variant thereof, which is
specifically recognized by the antibody to be purified. The
suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:1567-75 (1986)). The matrix to which the affinity
ligand is attached is most often agarose or acrylamide, but other
matrices are available. A suitable matrix to which the affinity
ligand is attached is Sepharose beads. Mechanically stable matrices
such as controlled pore glass or poly(styrenedivinyl)benzene allow
for faster flow rates and shorter processing times than can be
achieved with agarose. Where the antibody comprises a C.sub.H3
domain, the Bakerbond ABX.TM. resin (J. T. Baker, Phillipsburg,
N.J.) is useful for purification. Other techniques for protein
purification such as fractionation on an ion-exchange column,
ethanol precipitation, Reverse Phase HPLC, chromatography on
silica, chromatography on heparin SEPHAROSE.TM., chromatography on
an anion or cation exchange resin (such as a polyaspartic acid
column), chromatofocusing, SDS-PAGE, and ammonium sulfate
precipitation are also available depending on the antibody to be
recovered.
Solid Support Materials
[0129] For the methods provided herein, the antibody, enzyme (e.g.,
AvPAL, rAvPAL-PEG, or any variant thereof) or PEG can be
immobilized or otherwise bound to a variety of solid supports,
including but not limited to filters, PVC membranes, PDVF
membranes, PVC plates and other plates which bind protein,
microcarriers, macro solid phase beads, magnetic beads, made out
of, for example, polystyrene, nanoparticles, such as bimetallic
silver-gold nanoparticles (Yan Cui et al., J. Phys. Chem. B 110
(9):4002-06 (2006), and polyamide membrane (PAM) sheets (Sun et
al., Analytical Letters 34:1627-37 (2001)).
[0130] For example, microspheres with multiple fluorescent
molecular fillings, different materials, surface texture, surface
patterns, etc., can be utilized as identification tags. It is
contemplated that either the capture antibody or the AvPAL is
covalently bound to the bead and reacted against the opposite
binding partner to assay the amount of AvPAL-specific antibody in
serum. See, for example, Current Protocols in Immunology, Unit
6.11, 2006). Fluorescently filled microspheres are currently
available from Molecular Probes, Inc. and other companies.
Microspheres as small as 20 nm diameter polystyrene beads are
currently available.
[0131] The antibody, enzyme (e.g., AvPAL, rAvPAL-PEG, or any
variant thereof) or PEG can attached to the solid support using
standard protocols in the art, e.g., as described by the
manufacturer of the support, or using standard chemical
crosslinking techniques known in the art. See e.g., Pierce
Biotechnology, Inc. (Rockford, Ill.) crosslinking kits.
[0132] In one embodiment, antibody, enzyme (e.g., AvPAL,
rAvPAL-PEG, or any variant thereof) or PEG are attached to
Sepharose beads and used to purify anti-PAL enzyme antibodies,
e.g., anti-AvPAL antibodies.
[0133] In other embodiments of the methods and kits provided
herein, the antibody, PEG or rAvPAL-PEG or AvPAL, or variant
thereof is immobilized on a solid surface. In certain embodiments,
the antibody, enzyme (e.g., AvPAL, rAvPAL-PEG, or any variant
thereof) or PEG is immobilized in a well of a plate with a
plurality of wells, such as a multi-well plate or a multi-domain
multi-well plate. The use of multi-well assay plates allows for the
parallel processing and analysis of multiple samples distributed in
multiple wells of a plate. Multi-well assay plates (also known as
microplates or microtiter plates) can take a variety of forms,
sizes and shapes (e.g., 96-, 384-, 1536-, or 9600-well plates;
round- or flat-bottom multi-well plates). The methods provided
herein, when carried out in standardized plate formats can take
advantage of readily available equipment for storing and moving
these plates as well as readily available equipment for rapidly
dispensing liquids in and out of the plates (e.g., multi-well
pipettes, plate washers and the like). Exemplary multi-well plate
formats that can be used in the methods provided herein include
those found on 96-well plates (12.times.8 array of wells), 384-well
plates (24.times.16 array of wells) and 1536-well plate
(48.times.32 array of well). Other formats that may be used in the
methods provided herein include, but are not limited to, single or
multi-well plates comprising a plurality of domains.
[0134] In specific embodiments of the methods and kits provided
herein, solid phase supports are used for purifying, immobilizing,
or otherwise carrying out assays provided herein. Examples of solid
phases suitable for carrying out the methods and kits provided
herein include beads, particles, colloids, single surfaces, tubes,
multiwell plates, microtitre plates, slides, membranes, gels and
electrodes. When the solid phase is a particulate material (e.g.,
beads) it is, preferably, distributed in the wells of multi-well
plates to allow for parallel processing of the solid phase
supports. In specific embodiments of the methods and kits provided
herein, the primary capture reagents are immobilized on the solid
phase supports, e.g., by non-specific adsorption, covalent
attachment, or specific capture using an immobilized capture
reagent that binds the primary antibody or other protein of
interest. Immobilization may be accomplished by using proteins or
assay reagents that are labeled with binding species that form
binding pairs with immobilized capture reagents. Optionally, the
antibody is immobilized on a solid phase, and contacted with a
sample, and the solid phase is washed. The wash step allows for the
rapid purification of the antibody (e.g., an anti-AvPAL antibody)
or protein (e.g., rAvPAL-PEG} being identified from other,
potentially interfering, components of the sample. Optionally, the
sample is treated, for example with a lysis buffer, prior to
analysis.
Assays
[0135] Sandwich-based immunoassay methods are well established in
the art. See, e.g., U.S. Pat. Nos. 4,376,110 (David et al.);
4,016,043 (Schuurs et al.). Other related immunoassay formats and
variations thereof which may be useful for carrying out the methods
provided herein are well known in this field. See generally, E.
Maggio (1980) Enzyme-Immunoassay (CRC Press, Inc., Boca Raton,
Fla.); see also, e.g., U.S. Pat. Nos. 4,727,022 (Skold et al.);
4,659,678 (Forrest et al.); 4,376,110 (David et al.).
[0136] In the various methods provided herein, removing unbound
sample or other reagent is optional. In other words, the unbound
sample and/or other reagent may, in some embodiments, or may not,
in other embodiments, be removed when performing the methods
provided herein.
[0137] In certain embodiments of the various methods provided
herein, the two or more of the steps are performed sequentially. In
other embodiments of the methods provided herein, two or more of
steps are performed in parallel (e.g., at the same time).
[0138] It is noted that any combination of the embodiments provided
herein, for example, with respect to sample, patient populations,
solid phase immobilization, first antibody (or other reagent),
second antibody (or other reagent), labels and the like, are also
contemplated in relation to any of the various methods and/or kits
provided herein.
Methods and Kits to Detect a Pegylated Enzyme in a Sample
[0139] Pharmacokinetic (PK) studies are performed in mammalian
species, including humans, to evaluate exposure to drug or
therapeutic enzyme. As part of the evaluation of PK and toxicology
studies, the exposure to drug or therapeutic enzyme (i.e.,
rAvPAL-PEG or variant thereof) must be evaluated.
[0140] In a first aspect, provided herein is a method of detecting
the presence of a pegylated enzyme, e.g., rAvPAL-PEG or variant
thereof, in a sample (e.g., a body fluid, such as plasma, or a
tissue sample), said method comprising: (a) acidifying the
pegylated enzyme in the sample by adding an acidification reagent;
(b) neutralizing the acidified pegylated enzyme in the sample by
adding a neutralization buffer; (c) contacting the sample with an
immobilized first antibody that immunospecifically binds with the
pegylated enzyme; (d) optionally removing unbound sample; (e)
contacting the sample bound to the immobilized first antibody with
a detectable second antibody, wherein the second antibody
immunospecifically binds to PEG; (f) optionally removing unbound
second antibody; and (g) detecting the presence of the second
antibody bound to the sample; wherein detection above background of
an amount of the second antibody bound to the sample, e.g., an
increase in the amount of second antibody bound to the sample as
compared to a control sample having no pegylated enzyme, indicates
the presence of pegylated enzyme in the sample. In some
embodiments, the neutralized pegylated enzyme described in (b) is
introduced into a different assay buffer before contacting the
immobilized first antibody described in (c).
[0141] In certain embodiments, the sample is a body fluid (e.g.,
blood, serum, plasma, CSF, urine or breast milk) or a tissue sample
from a patient. In some embodiments, the patient is a mammal, such
as a human, monkey, dog, rabbit, rat or mouse.
[0142] In an embodiment, the concentration of pegylated enzyme in
the sample is determined. In one embodiment, the limit of detection
is less than 5 ng/mL, or less than 3 ng/mL. In another embodiment,
the limit of detection is between 5 ng/mL and 2 ng/mL, such as
between 3 ng/mL and 2 ng/mL. In an embodiment, the sample is
plasma, which is equal to or less than 5% or equal to or less than
2% of the volume as described in (a).
[0143] In some embodiments, the patient has elevated phenylalanine
levels. In specific embodiments, the patient has been or will be
administered the pegylated enzyme, for example, for EST (e.g., for
PKU) or cancer therapy. In an embodiment, the pegylated enzyme is
rAvPAL-PEG or a variant thereof. In one embodiment, the pegylated
enzyme is rAvPAL-PEG having its cysteine residue at position 503
substituted with serine residues (rAvPAL-PEG_C503S). In another
embodiment, the pegylated enzyme is rAvPAL-PEG having its cysteine
residues at position 565 substituted with serine residues
(rAvPAL-PEG.sub.--C565S). In yet another embodiment, the pegylated
enzyme is rAvPAL-PEG having its cysteine residues at positions 503
and 565 substituted with serine residues (rAvPAL-PEG_C565SC503S).
In other embodiments, the pegylated enzyme is rAvPAL-PEG_C503S,
rAvPAL-PEG_C565S, rAvPAL-PEG_C565SC503S, or any combination
thereof.
[0144] In one embodiment, the immunogenic epitopes of the pegylated
therapeutic enzyme are exposed by acidifying the pegylated enzyme
in the sample by adding an acidification reagent, and subsequently
neutralizing the pegylated enzyme in the sample by adding a
neutralization buffer. In one embodiment, the acidification reagent
is HCl. In one embodiment, the neutralization reagent is NaOH. In
another embodiment, the acidification reagent is 0.1 M glycine, pH
2.7. In some embodiments, the neutralization buffer is 0.5 M
Tris-HCl, pH 8.5. In one embodiment, the acidification reagent is
0.1 M glycine, pH 2.7 and the neutralization buffer is 0.5 M
Tris-HCl, pH 8.5. In another embodiment, the acidification and
subsequent neutralization of the pegylated enzyme in the sample is
performed before contacting the sample with the immobilized first
antibody.
[0145] In another embodiment, the immunogenic epitopes of the
pegylated therapeutic enzyme are exposed by denaturing the enzyme
in the sample by exposure to heat, and subsequently renaturing the
enzyme by cooling the sample.
[0146] In a specific embodiment, provided herein is a method to
measure the amount of a pegylated therapeutic enzyme (i.e.,
rAvPAL-PEG or variant thereof) in a mammal (e.g., human)
comprising: (a) binding an AvPAL-specific antibody to a solid
support, (b) acidifying a pegylated (i.e., derivatized by
polyethylene glycol) AvPAL (rAvPAL-PEG) enzyme in a body fluid
(e.g., plasma) sample from the mammal by adding an acidification
reagent, (c) neutralizing the acidified rAvPAL-PEG enzyme in the
body fluid sample by adding a neutralization buffer, (d) capturing
the acidified and neutralized rAvPAL-PEG enzyme in the body fluid
sample from the mammal by contacting the body fluid sample with
AvPAL-specific antibodies bound to the solid support in (a), (e)
contacting the captured rAvPAL-PEG enzyme from (d) with an anti-PEG
specific antibody labeled with a detectable moiety, and (f)
determining the amount of rAvPAL-PEG enzyme in the body fluid
sample by detecting the presence of anti-PEG specific antibody
labeled with the detectable moiety. In a another embodiment, (a)
the enzyme-specific antibody is bound to a solid support, (b) the
pegylated enzyme is acidified in the body fluid sample by adding an
acidification reagent, (c) the acidified pegylated enzyme is
neutralized in the body fluid sample by adding a neutralization
buffer, (d) the neutralized pegylated enzyme in the body fluid
sample is captured with the enzyme-specific antibody bound to the
solid support in (a), (e) the captured pegylated enzyme is
contacted with an anti-PEG specific antibody labeled with a
detectable moiety, and (f) the amount of captured pegylated enzyme
from the body fluid sample is determined by detecting the presence
of anti-PEG specific antibody labeled with the detectable moiety.
In some embodiments, (b), (c) and (d) are performed in the order
(b), (c), (d). In some embodiments, the neutralized pegylated
enzyme in (c) is introduced into a different assay buffer
performing the enzyme capture in (d).
[0147] In some embodiments, a method is provided of classifying a
patient for eligibility for a pegylated enzyme therapy (e.g.,
initial or continued pegylated enzyme therapy) with, e.g., a
rAvPAL-PEG or variant thereof, comprising: (a) providing a sample
(e.g., a body fluid or tissue) from the patient; (b) detecting or
otherwise determining the amount of pegylated enzyme in the sample
using an assay method provided herein; and (c) classifying the
patient as eligible to receive the one or more pegylated enzyme
therapies based on the detection or amount of pegylated enzyme in
the sample. In some embodiments, the patient has previously been
administered a pegylated enzyme, such as a rAvPAL-PEG or variant
thereof, e.g., for EST for elevated phenylalanine levels, or cancer
therapy.
[0148] In other embodiments, methods provided herein may be used to
observe or otherwise monitor how a patient is responding to a
therapy with a pegylated enzyme. Such information can be used, for
example, to make better decisions about the optimal methods, doses,
or treatments for the patient. For example, these methods are
applicable where a subject has been previously diagnosed as having
elevated phenylalanine levels (e.g., PKU) or cancer and possibly
has undergone treatment for the disease (e.g., pegylated enzyme
therapy, such as rAv-PAL-PEG), and the methods provided herein are
employed to monitor the progression of the disease or the treatment
thereof. In addition, the information obtained by said methods may
be used for selecting a patient suitable for pegylated enzyme
therapy, or determining if a patient is suitable for continued
pegylated enzyme therapy. In certain embodiments, the methods
herein are used in conjunction with treatment of a patient with
pegylated enzyme having or suspected of having elevated
phenylalanine levels (e.g., a patient having one or more PAH
mutations) and/or a disease associated with elevated phenylalanine
levels (e.g., PKU) or cancer, or symptom thereof. That is, in
certain embodiments, the assay methods provided herein are used to
monitor or otherwise track pegylated enzyme levels in a patient
that has been or will be administered the pegylated enzyme, such as
a rAvPAL-PEG or a pegylated variant thereof.
[0149] In other embodiments, provided herein are methods of
preventing, treating, or otherwise managing a disease or symptom
thereof associated with elevated phenylalanine levels (e.g., PKU)
or cancer in a patient, said method comprising: (a) administering a
pegylated enzyme, e.g., rAvPAL-PEG or a pegylated variant thereof,
to the patient; (b) obtaining a sample, such as a bodily fluid or
tissue, from the patient, (c) acidifying the pegylated enzyme in
the sample by adding an acidification reagent, (d) neutralizing the
acidified pegylated enzyme in the sample by adding a neutralization
buffer, (e) contacting the sample with an immobilized first
antibody that immunospecifically binds with the pegylated enzyme;
(f) optionally removing unbound sample; (g) contacting the sample
bound to the immobilized first antibody with a detectable second
antibody, wherein the second antibody immunospecifically binds to
PEG; (h) optionally removing unbound second antibody; and (i)
detecting the presence of the second antibody bound to the sample;
wherein detection above background of an amount of the second
antibody bound to the sample, e.g., an increase in the amount of
second antibody bound to the sample as compared to a control sample
having no pegylated enzyme, indicates the presence of pegylated
enzyme in the sample.
[0150] In certain embodiments of any of the methods provided
herein, the method further comprise providing results from the
assay to personnel, e.g., at a medical facility, such as a doctor,
nurse or other medical professional. In other embodiments, the
method further comprises providing therapeutic options to
personnel, e.g., at a medical facility, such as a doctor, nurse or
other medical professional. In one embodiment, the patient has
elevated phenylalanine concentrations (e.g., in blood, plasma or
serum) and/or cancer. In other embodiments, the patient has a
disease or disorder resulting from elevated phenylalanine levels,
such as PKU, or cancer. In certain embodiments, the patient has
been, is or will be treated with a pegylated enzyme, such as
rAvPAL-PEG. In some embodiments, the methods provided herein are
used to monitor or otherwise track pegylated enzyme, e.g.,
rAvPAL-PEG, therapy by detecting or otherwise measuring pegylated
enzyme levels in a sample (such as blood, blood lysate, plasma,
spinal fluid, cerebral fluid or bone marrow aspirate) from the
patient over a period of time, such as before, during and/or after
the treatment with the therapy over the course of a 1 hour, 2 hour,
3 hour, 4 hour, 6 hour, 8 hour, 10 hour, 12 hour, 18 hour, 24 hour,
2 day, 3 day, 4 day, 5 day, 6 day, 7 day, 2 week, 3 week, 4 week, 2
month, 3 month, 4 month, 5 month, 6 month, 7 month, 8 month, 9
month, 10, month 11 month, 1 year or more period of time.
[0151] In embodiments of the various methods provided herein, two
or more of the steps are performed sequentially. In other
embodiments of the methods provided herein, two or more of steps
are performed in parallel (e.g., at the same time).
[0152] Also provided herein is a kit comprising: (a) an
acidification reagent for acidifying a pegylated enzyme, (b) a
neutralization buffer for neutralizing the acidified pegylated
enzyme, (c) a first antibody that immunospecifically binds with the
pegylated enzyme that is optionally immobilized on a solid support,
and (d) a detectable second antibody that immunospecifically binds
to PEG.
[0153] Any combination of the above-listed embodiments, for
example, with respect to sample, pegylated enzymes, capture and
detectable antibodies, patient populations, solid phase
immobilization, labels and the like, are also contemplated
embodiments in connection with the kits provided herein.
[0154] The kits provided herein can be used to perform methods
provided herein for detecting the presence of a pegylated enzyme,
e.g., rAvPAL-PEG or variant thereof, in a sample (e.g., a body
fluid, such as plasma, or a tissue sample). The kits provided
herein can also be used to perform the methods provided herein for
classifying a patient for eligibility for a pegylated enzyme
therapy (e.g., initial or continued pegylated enzyme therapy) with,
e.g., a rAvPAL-PEG or variant thereof. The kits provided herein can
further be used in the methods provided herein to monitor or
otherwise track pegylated enzyme levels in a patient that has been
or will be administered the pegylated enzyme, such as a rAvPAL-PEG
or a pegylated variant thereof. The kits provided herein can also
be used in the methods provided herein to prevent, treat, or
otherwise manage a disease or symptom thereof associated with
elevated phenylalanine levels (e.g., PKU) or cancer in a
patient.
[0155] The kits can be packaged in any suitable manner, typically
with the various parts, in a suitable container along with
instructions for use. In certain embodiments, the kits may further
comprise, where necessary, other agents for reducing the background
interference in a test, control reagents, apparatus for conducting
a test, and the like.
Methods and Kits to Detect Enzyme-Specific Antibodies
[0156] Most protein or enzyme therapeutics elicit some level of
antibody response. In some cases this antibody response may lead to
serious side effects or loss of efficacy of the therapeutic
enzyme.
[0157] In a second aspect, provided herein is a method of detecting
the presence of an enzyme-specific antibody (e.g., an
anti-AvPAL-specific antibody or an anti-AvPAL-PEG-specific
antibody), in a sample (e.g., a body fluid, such as blood, serum or
plasma, or a tissue sample), said method comprising: (a) contacting
the sample with an immobilized enzyme (e.g., AvPAL or rAvPAL-PEG);
(b) optionally removing unbound sample; (c) contacting the sample
bound to the immobilized enzyme with a detectable antibody, wherein
the detectable antibody immunospecifically binds to Ig; (d)
optionally removing unbound detectable antibody; and (e) detecting
the presence of the detectable antibody bound to the sample;
wherein detection above background of an amount of the detectable
antibody bound to the sample, e.g., an increase in the amount of
detectable antibody bound to the sample as compared to a control
sample having no enzyme-specific antibody, indicates the presence
of enzyme specific antibody in the sample.
[0158] In certain embodiments, provided is a method of detecting
the presence of an enzyme-specific antibody (e.g., an
anti-AvPAL-specific antibody or an anti-AvPAL-PEG-specific
antibody), in a sample (e.g., a body fluid, such as blood, serum or
plasma, or a tissue sample), said method comprising: (a) contacting
the sample with an immobilized antibody that immunospecifically
binds to an Ig (e.g., an anti-Ig, anti-IgG, anti-IgA, anti-IgM,
anti-IgD or anti-IgE antibody); (b) optionally removing unbound
sample; (c) contacting the sample bound to the immobilized antibody
with a detectable enzyme (e.g., AvPAL or rAvPAL-PEG labeled with a
detection moiety); (d) optionally removing unbound detectable
enzyme; and (e) detecting the presence of the detectable enzyme
bound to the sample; wherein detection above background of an
amount of the detectable enzyme bound to the sample, e.g., an
increase in the amount of detectable enzyme bound to the sample as
compared to a control sample having no enzyme-specific antibody,
indicates the presence of enzyme specific antibody in the
sample.
[0159] In yet another embodiment, provided herein is a method of
detecting the presence of an enzyme-specific antibody (e.g., an
anti-AvPAL-specific antibody or an anti-AvPAL-PEG-specific
antibody), in a sample (e.g., a body fluid, such as blood, serum or
plasma, or a tissue sample), said method comprising: (a) contacting
the sample with an immobilized enzyme (e.g., AvPAL or rAvPAL-PEG);
(b) optionally removing unbound sample; (c) contacting the sample
bound to the immobilized antibody with a detectable enzyme (e.g.,
AvPAL or rAvPAL-PEG labeled with a detection moiety); (d)
optionally removing unbound detectable enzyme; and (e) detecting
the presence of the detectable enzyme bound to the sample; wherein
detection above background of an amount of the detectable enzyme
bound to the sample, e.g., an increase in the amount of detectable
enzyme bound to the sample as compared to a control sample having
no enzyme-specific antibody, indicates the presence of enzyme
specific antibody in the sample.
[0160] In certain embodiments, the sample is a body fluid (e.g.,
blood, serum, plasma, CSF, urine or breast milk) or a tissue sample
from a patient. In some embodiments, the patient is a mammal, such
as a human, monkey, dog, rabbit, rat or mouse. In an embodiment,
the concentration of enzyme-specific antibody in the sample is
determined.
[0161] In the methods provided herein, any isotype of
enzyme-specific antibody can be detected. In certain embodiments,
the anti-Ig antibody is an isotype-specific antibody, e.g., an
anti-IgG, anti-IgA, anti-IgM, anti-IgD or anti-IgE antibody. In an
embodiment, an enzyme-specific antibody of IgG isotype can be
detected, and, in certain embodiments, the limit of detection is
between 1.6 ng/mL and 8.6 ng/mL. In some embodiments, the limit of
detection is less than 8.6 ng/mL or less than 4.2 ng/mL, such as
between 2.1 ng/mL and 1.6 ng/mL. In other embodiments, an
enzyme-specific antibody of IgM isotype can be detected, and, in
certain embodiments, the limit of detection is less than 5.9 ng/mL,
such as between 5.9 ng/ml and 2.8 n/ml. In an embodiment, an
enzyme-specific antibody of IgE isotype can be detected. In other
embodiments, an enzyme-specific antibody of IgA can be detected. In
an embodiment, the sample is serum or plasma, which is equal to or
less than 5% or equal to or less than 2% of the volume as described
in (a).
[0162] In some embodiments, the patient has elevated phenylalanine
levels. In specific embodiments, the patient has been or will be
administered the enzyme, for example, for EST (e.g., for PKU) or
cancer therapy.
[0163] In an embodiment, the enzyme is rAvPAL or a variant thereof.
In one embodiment, the enzyme is rAvPAL_C503S, rAvPAL_C565S,
rAvPAL_C565SC503S, or any combination thereof. In some embodiments,
the enzyme is a pegylated enzyme. In an embodiment, the pegylated
enzyme is rAvPAL-PEG. In other embodiments, the pegylated enzyme is
rAvPAL-PEG_C503S, rAvPAL-PEG_C565S, rAvPAL-PEG_C565SC503S, or any
combination thereof.
[0164] In specific embodiments, a method is provided to measure the
amount of antibodies specific for a therapeutic enzyme (i.e., AvPAL
or variant thereof) in a mammal (e.g., human) comprising: (a)
binding the AvPAL to a solid support, (b) capturing AvPAL-specific
antibodies in a body fluid (e.g., serum) sample from the mammal by
contacting the body fluid with the AvPAL bound to the solid
support, (c) contacting the captured AvPAL-specific antibodies in
step (b) with an anti-Ig antibody labeled with a detectable moiety,
and (d) determining the amount of the captured AvPAL-specific
antibodies from the body fluid sample by detecting the presence of
anti-Ig antibody labeled with the detectable moiety. In some
embodiments, (c) and (d) are performed simultaneously.
[0165] In one embodiment, (a) the enzyme is bound to a solid
support, (b) the enzyme-specific antibody in a body fluid sample
from the mammal is captured by contacting the body fluid sample
with the enzyme bound to the solid support, (c) the captured
enzyme-specific antibody from (b) is contacted with an anti-Ig
specific antibody (e.g., anti-IgG, anti-IgM, anti-IgA, anti-IgD or
anti-IgE) labeled with a detection moiety, and (d) the amount of
captured enzyme-specific antibody from the body fluid sample is
determined by detecting the presence of anti-Ig specific antibody
(e.g., anti-IgG, anti-IgM, anti-IgA, anti-IgD or anti-IgE) labeled
with the detection moiety. In an alternative embodiment, the
anti-Ig specific antibody is bound to the solid support, the
enzyme-specific antibody in the body fluid sample from the mammal
is captured by contacting the body fluid sample with the anti-Ig
specific antibody bound to the solid support, the captured
enzyme-specific antibody is contacted with the enzyme labeled with
a detection moiety, and the amount of captured enzyme-specific
antibody is determined by detecting the presence of enzyme labeled
with the detection moiety. In another alternative embodiment, a
total antibody assay is employed that takes advantage of the binary
character of antibodies. In this assay, AvPAL is bound to a solid
support, and the body fluid sample, which contains the
enzyme-specific antibodies, is incubated with AvPAL labeled with a
detection moiety. The enzyme-specific antibody in the body fluid
sample is captured by contacting the body fluid sample with AvPAL
bound to the solid support, and the amount of captured
enzyme-specific antibody is determined by detecting the presence of
AvPAL labeled with the detection moiety.
[0166] In some embodiments, a method is provided of classifying a
patient for eligibility for an enzyme therapy (e.g., initial or
continued enzyme therapy) with, e.g., an AvPAL or rAvPAL-PEG, or
variants thereof, comprising: (a) providing a sample (e.g., a body
fluid or tissue) from the patient; (b) detecting or otherwise
determining the amount of enzyme-specific antibody in the sample
using an assay method provided herein; and (c) classifying the
patient as eligible to receive the one or more further enzyme
therapies based on the detection or amount of anti-enzyme antibody
in the sample. In some embodiments, the patient has previously been
administered an enzyme, such as an AvPAL or rAv-PAL-PEG, or a
variant thereof, e.g., for EST for elevated phenylalanine levels,
or cancer therapy.
[0167] In other embodiments, methods provided herein may be used to
observe or otherwise monitor how a patient is responding to a
therapy with an enzyme, e.g., an AvPAL or rAv-PAL-PEG, or a variant
thereof. Such information can be used, for example, to make better
decisions about the optimal methods, doses, or treatments for the
patient. For example, these methods are applicable where a subject
has been previously diagnosed as having elevated phenylalanine
levels (e.g., PKU) or cancer and possibly has undergone treatment
for the disease, and the methods provided herein are employed to
monitor the progression of the disease or the treatment thereof. In
addition, the information obtained by said methods may be used for
selecting a patient suitable for enzyme therapy, or determining if
a patient is suitable for continued enzyme therapy. In certain
embodiments, the methods herein are used in conjunction with
treatment of a patient with enzyme having or suspected of having
elevated phenylalanine levels (e.g., a patient having one or more
PAH mutations) and/or a disease associated with elevated
phenylalanine levels (e.g., PKU) or cancer, or symptom thereof.
That is, in certain embodiments, the assay methods provided herein
are used to monitor or otherwise track anti-enzyme antibody levels
in a patient that has been or will be administered the enzyme, such
as a AvPAL, rAvPAL-PEG, or a variant thereof.
[0168] In other embodiments, provided herein are methods of
preventing, treating, or otherwise managing a disease or symptom
thereof associated with elevated phenylalanine levels (e.g., PKU)
or cancer in a patient, said method comprising: (a) administering
an enzyme (e.g., AvPAL or rAvPAL-PEG, or a variant thereof), to the
patient; (b) obtaining a sample, such as a bodily fluid or tissue,
from the patient, (c) contacting the sample with an immobilized
enzyme (e.g., AvPAL or rAvPAL-PEG); (d) optionally removing unbound
sample; (e) contacting the sample bound to the immobilized enzyme
with a detectable antibody, wherein the detectable antibody
immunospecifically binds to Ig; (f) optionally removing unbound
detectable antibody; and (g) detecting the presence of the
detectable antibody bound to the sample; wherein detection above
background of an amount of the detectable antibody bound to the
sample, e.g., an increase in the amount of detectable antibody
bound to the sample as compared to a control sample having no
enzyme-specific antibody, indicates the presence of enzyme specific
antibody in the sample.
[0169] In another embodiment, provided herein are methods of
preventing, treating, or otherwise managing a disease or symptom
thereof associated with elevated phenylalanine levels (e.g., PKU)
or cancer in a patient, said method comprising: (a) administering
an enzyme (e.g., AvPAL or rAvPAL-PEG, or a variant thereof), to the
patient; (b) obtaining a sample, such as a bodily fluid or tissue,
from the patient, (c) contacting the sample with an immobilized
antibody that immunospecifically binds to an Ig (e.g., an anti-Ig,
anti-IgG, anti-IgA, anti-IgM, anti-IgD or anti-IgE antibody); (d)
optionally removing unbound sample; (e) contacting the sample bound
to the immobilized antibody with a detectable enzyme (e.g., AvPAL
or rAvPAL-PEG labeled with a detection moiety); (f) optionally
removing unbound detectable enzyme; and (g) detecting the presence
of the detectable enzyme bound to the sample; wherein detection
above background of an amount of the detectable enzyme bound to the
sample, e.g., an increase in the amount of detectable enzyme bound
to the sample as compared to a control sample having no
enzyme-specific antibody, indicates the presence of enzyme specific
antibody in the sample.
[0170] In another embodiment, provided herein are methods of
preventing, treating, or otherwise managing a disease or symptom
thereof associated with elevated phenylalanine levels (e.g., PKU)
or cancer in a patient, said method comprising: (a) administering
an enzyme (e.g., AvPAL or rAvPAL-PEG, or a variant thereof), to the
patient; (b) obtaining a sample, such as a bodily fluid or tissue,
from the patient, (c) contacting the sample with an immobilized
enzyme (e.g., AvPAL or rAvPAL-PEG); (d) optionally removing unbound
sample; (e) contacting the sample bound to the immobilized antibody
with a detectable enzyme (e.g., AvPAL or rAvPAL-PEG labeled with a
detection moiety); (f) optionally removing unbound detectable
enzyme; and (e) detecting the presence of the detectable enzyme
bound to the sample; wherein detection above background of an
amount of the detectable enzyme bound to the sample, e.g., an
increase in the amount of detectable enzyme bound to the sample as
compared to a control sample having no enzyme-specific antibody,
indicates the presence of enzyme specific antibody in the
sample.
[0171] In certain embodiments of any of the methods provided
herein, the method further comprise providing results from the
assay to personnel, e.g., at a medical facility, such as a doctor,
nurse or other medical professional. In other embodiments, the
method further comprises providing therapeutic options to
personnel, e.g., at a medical facility, such as a doctor, nurse or
other medical professional. In one embodiment, the patient has
elevated phenylalanine concentrations (e.g., in blood, plasma or
serum) and/or cancer. In other embodiments, the patient has a
disease or disorder resulting from elevated phenylalanine levels,
such as PKU, or cancer. In certain embodiments, the patient has
been, is or will be treated with an enzyme, such as AvPAL or
rAvPAL-PEG, or any variant thereof. In some embodiments, the
methods provided herein are used to monitor or otherwise track
enzyme-specific antibodies during the course of enzyme (e.g.,
AvPAL, AvPAL-PEG, or any variant thereof) therapy by detecting or
otherwise measuring enzyme-specific antibody levels in a sample
(such as blood, blood lysate, plasma, spinal fluid, cerebral fluid
or bone marrow aspirate) from the patient over a period of time,
such as before, during and/or after the treatment with the therapy
over the course of a 1 hour, 2 hour, 3 hour, 4 hour, 6 hour, 8
hour, 10 hour, 12 hour, 18 hour, 24 hour, 2 day, 3 day, 4 day, 5
day, 6 day, 7 day, 2 week, 3 week, 4 week, 2 month, 3 month, 4
month, 5 month, 6 month, 7 month, 8 month, 9 month, 10, month 11
month, 1 year or more period of time.
[0172] In embodiments of the various methods provided herein, two
or more of the steps are performed sequentially. In other
embodiments of the methods provided herein, two or more of steps
are performed in parallel (e.g., at the same time).
[0173] Also provided herein is a kit comprising: (a) an enzyme
(e.g., AvPAL or rAvPAL-PEG), which is optionally immobilized on a
solid support, and (b) a detectable antibody, wherein the
detectable antibody immunospecifically binds to Ig. In certain
embodiments, the kit further comprises one or more components in
one or more containers and/or instructions for use.
[0174] Also provided herein is a kit comprising: (a) an antibody
that immunospecifically binds to an Ig (e.g., an anti-Ig, anti-IgG,
anti-IgA, anti-IgM, anti-IgD or anti-IgE antibody), wherein the
anti-Ig antibody is optionally immobilized to a solid support; and
(b) a detectable enzyme (e.g., AvPAL or rAvPAL-PEG labeled with a
detection moiety). In certain embodiments, the kit further
comprises one or more components of the kit in one or more
containers and/or instructions for use.
[0175] Further provided herein is a kit comprising: (a) an enzyme
(e.g., AvPAL or rAvPAL-PEG), which is optionally immobilized on a
solid support; and (b) a detectable enzyme (e.g., AvPAL or
rAvPAL-PEG labeled with a detection moiety). In certain
embodiments, the kit further comprises one or more components of
the kit in one or more containers and/or instructions for use.
[0176] Any combination of the above-listed embodiments, for
example, with respect to sample, enzymes, enzyme-specific
antibodies, detectable enzymes, detectable enzymes, detectable
antibodies, patient populations, solid phase immobilization, labels
and the like, are also contemplated embodiments in connection with
the kits provided herein.
[0177] The kits provided herein can be used to perform methods
provided herein for detecting the presence of an enzyme-specific
antibody (e.g., an anti-AvPAL-specific antibody or an
anti-AvPAL-PEG-specific antibody), in a sample (e.g., a body fluid,
such as blood, serum or plasma, or a tissue sample). The kits
provided herein can also be used to perform the methods provided
herein for classifying a patient for eligibility for an enzyme
therapy (e.g., initial or continued enzyme therapy) with, e.g., an
AvPAL or rAvPAL-PEG, or variants thereof. The kits provided herein
can further be used in the methods provided herein to monitor or
otherwise track anti-enzyme antibody levels in a patient that has
been or will be administered the enzyme, such as a AvPAL,
rAvPAL-PEG, or a variant thereof. The kits provided herein can also
be used in the methods provided herein to prevent, treat, or
otherwise manage a disease or symptom thereof associated with
elevated phenylalanine levels (e.g., PKU) or cancer in a
patient.
[0178] The kits can be packaged in any suitable manner, typically
with the various parts, in a suitable container along with
instructions for use. In certain embodiments, the kits may further
comprise, where necessary, other agents for reducing the background
interference in a test, control reagents, apparatus for conducting
a test, and the like.
Methods and Kits to Detect Neutralizing Enzyme-Specific
Antibodies
[0179] Most protein or enzyme therapeutics elicit some level of
antibody response. In some cases this antibody response may lead to
serious side effects or loss of efficacy.
[0180] In a third aspect, provided herein is a method for detecting
the presence of neutralizing enzyme-specific antibodies (e.g., a
neutralizing antibody that immunospecifically binds to an enzyme,
such as AvPAL, rAvPAL-PEG and/or any derivative thereof) in a
sample (e.g., a body fluid, such as plasma, or a tissue sample),
said method comprising: (a) contacting the sample with the enzyme
(e.g., AvPAL, rAvPAL-PEG, and/or any variant thereof), which is
optionally immobilized on a solid support; (b) optionally removing
unbound sample; (c) adding a substrate for the enzyme; (d)
optionally removing unbound substrate; and (e) detecting the
presence of enzymatic activity between the enzyme and substrate;
wherein a reduction of enzymatic activity, e.g., as compared to a
sample having no neutralizing anti-enzyme antibodies, indicates the
presence of neutralizing enzyme-specific antibodies in the
sample.
[0181] In certain embodiments, the neutralizing antibodies that
immunospecifically bind to an enzyme, such as AvPAL, rAvPAL-PEG, or
any derivative thereof, are contacted with a pegylated form of the
enzyme, such as rAvPAL-PEG or variant thereof, wherein the
antibodies neutralize the pegylated enzyme activity by interacting
with either the enzyme (e.g., AvPAL or derivative thereof) and/or
the PEG moiety. In other embodiments, the neutralizing antibodies
that immunospecifically bind to an enzyme, such as AvPAL,
rAvPAL-PEG, or any derivative thereof, are contacted with an enzyme
that is not pegylated, such as AvPAL or variant thereof, wherein
the antibodies neutralize the enzyme activity by interacting with
the enzyme (e.g., AvPAL or derivative thereof).
[0182] In certain embodiments, the sample is a body fluid (e.g.,
blood, serum, plasma, CSF, urine or breast milk) or a tissue sample
from a patient. In some embodiments, the patient is a mammal, such
as a human, monkey, dog, rabbit, rat or mouse. In an embodiment,
the concentration of enzyme-specific antibody in the sample is
determined.
[0183] In an embodiment, the concentration of neutralizing
enzyme-specific antibody in the sample is determined. In certain
embodiments, the limit of detection is less than or equal to 10
.mu.g/mL. In an embodiment, the sample is serum or plasma, which is
equal to or less than 5% or equal to or less than 2% of the volume
as described in (a). In one embodiment, the neutralizing
enzyme-specific antibodies in a sample are detected in an assay
using 96-well plates. In other embodiments, the neutralizing
enzyme-specific antibodies in a sample are detected in an assay
using cuvettes.
[0184] In some embodiments, the patient has elevated phenylalanine
levels. In specific embodiments, the patient has been or will be
administered the enzyme, for example, for EST (e.g., for PKU) or
cancer therapy.
[0185] In an embodiment, the enzyme is rAvPAL or a variant thereof.
In one embodiment, the enzyme is rAvPAL_C503S, rAvPAL_C565S,
rAvPAL_C565SC503S, or any combination thereof. In some embodiments,
the enzyme is a pegylated enzyme. In an embodiment, the pegylated
enzyme is rAvPAL-PEG. In other embodiments, the pegylated enzyme is
rAvPAL-PEG_C503S, rAvPAL-PEG_C565S, rAvPAL-PEG_C565SC503S, or any
combination thereof.
[0186] In a specific embodiment, provided is a method to measure
the amount of neutralizing antibodies specific for a therapeutic
enzyme (i.e., AvPAL or variant thereof) in a mammal (e.g., human)
comprising: (a) contacting AvPAL-specific antibodies in a body
fluid (e.g., serum) sample from the mammal with an AvPAL or variant
thereof, (b) adding a substrate for the AvPAL or variant thereof to
the mixture of AvPAL-specific antibodies and AvPAL or variant
thereof in (a), and (c) determining the amount of the neutralizing
AvPAL-specific antibodies from the body fluid by detecting the
reduction of the enzymatic reactivity of AvPAL or variant thereof
with the substrate in (b). In one embodiment, the method comprises
(a) contacting AvPAL-specific antibodies in a body fluid (e.g.,
serum) sample from the mammal with an AvPAL or variant thereof, (b)
adding a substrate for the AvPAL or variant thereof to the mixture
of AvPAL-specific antibodies and AvPAL or variant thereof in (a),
and (c) determining the amount of the neutralizing AvPAL-specific
antibodies from the body fluid by detecting the reduction of the
enzymatic reactivity of AvPAL or variant thereof with the substrate
in (b). In one embodiment, the neutralizing AvPAL-specific
antibodies in a body fluid sample are detected in an assay using
96-well plates. In an alternative embodiment, the neutralizing
AvPAL-specific antibodies in a body fluid sample are detected in an
assay similar to that described above, except that the assays are
performed in cuvettes. This assay can be low throughput and require
a higher volume of material, but can be suitable for performing
enzyme activity assays, e.g., for product release. In yet another
alternative embodiment, the neutralizing AvPAL-specific antibodies
in a body fluid sample are detected in an assay similar to that
described above, except that in (a) the AvPAL-specific antibodies
in a body fluid (e.g., serum) sample from the mammal are contacted
with rAvPAL-PEG or variant thereof. This assay can detect
antibodies that neutralize rAvPAL-PEG enzyme activity by
interacting with the AvPAL or PEG moiety.
[0187] In some embodiments, a method is provided of classifying a
patient for eligibility for an enzyme therapy (e.g., initial or
continued enzyme therapy) with, e.g., a AvPAL or rAvPAL-PEG, or
variants thereof, comprising: (a) providing a sample (e.g., a body
fluid or tissue) from the patient; (b) detecting or otherwise
determining the amount of neutralizing enzyme-specific antibody in
the sample using an assay method provided herein; and (c)
classifying the patient as eligible to receive the one or more
further enzyme therapies based on the detection or amount of
neutralizing enzyme-specific antibody in the sample. In some
embodiments, the patient has previously been administered an
enzyme, such as an AvPAL or rAvPAL-PEG, or a variant thereof, e.g.,
for EST for elevated phenylalanine levels or cancer therapy.
[0188] In other embodiments, methods provided herein may be used to
observe or otherwise monitor how a patient is responding to a
therapy with an enzyme, e.g., an AvPAL or rAvPAL-PEG, or a variant
thereof. Such information can be used, for example, to make better
decisions about the optimal methods, doses, or treatments for the
patient. For example, these methods are applicable where a subject
has been previously diagnosed as having elevated phenylalanine
levels (e.g., PKU) or cancer and possibly has undergone treatment
for the disease, and the methods provided herein are employed to
monitor the progression of the disease or the treatment thereof. In
addition, the information obtained by said methods may be used for
selecting a patient suitable for enzyme therapy, or determining if
a patient is suitable for continued enzyme therapy. In certain
embodiments, the methods herein are used in conjunction with
treatment of a patient with enzyme having or suspected of having
elevated phenylalanine levels (e.g., a patient having one or more
PAH mutations) and/or a disease associated with elevated
phenylalanine levels (e.g., PKU) or cancer, or symptom thereof.
That is, in certain embodiments, the assay methods provided herein
are used to monitor or otherwise track neutralizing anti-enzyme
antibody levels in a patient that has been or will be administered
the enzyme, such as an AvPAL, rAvPAL-PEG, or a variant thereof.
[0189] In other embodiments, provided herein are methods of
preventing, treating, or otherwise managing a disease or symptom
thereof associated with elevated phenylalanine levels (e.g., PKU)
or cancer in a patient, said method comprising: (a) administering
an enzyme (e.g., AvPAL or rAvPAL-PEG, or a variant thereof), to the
patient; (b) obtaining a sample, such as a bodily fluid or tissue,
from the patient, (c) contacting the sample with the enzyme (e.g.,
AvPAL, rAvPAL-PEG, and/or any variant thereof), which is optionally
immobilized on a solid support; (d) optionally removing unbound
sample; (e) adding a substrate for the enzyme; (f) optionally
removing unbound substrate; and (g) detecting the presence of
enzymatic activity between the enzyme and substrate; wherein a
reduction of enzymatic activity, e.g., as compared to a sample
having no neutralizing anti-enzyme antibodies, indicates the
presence of neutralizing enzyme-specific antibodies in the
sample.
[0190] In certain embodiments of any of the methods provided
herein, the method further comprise providing results from the
assay to personnel, e.g., at a medical facility, such as a doctor,
nurse or other medical professional. In other embodiments, the
method further comprises providing therapeutic options to
personnel, e.g., at a medical facility, such as a doctor, nurse or
other medical professional. In one embodiment, the patient has
elevated phenylalanine concentrations (e.g., in blood, plasma or
serum) and/or cancer. In other embodiments, the patient has a
disease or disorder resulting from elevated phenylalanine levels,
such as PKU, or cancer. In certain embodiments, the patient has
been, is or will be treated with an enzyme, such as AvPAL,
rAvPAL-PEG, or any variant thereof. In some embodiments, the
methods provided herein are used to monitor or otherwise track
neutralizing enzyme-specific antibodies during the course of enzyme
(e.g., AvPAL, rAvPAL-PEG, or any variant thereof) therapy by
detecting or otherwise measuring neutralizing enzyme-specific
antibody levels in a sample (such as blood, blood lysate, plasma,
spinal fluid, cerebral fluid or bone marrow aspirate) from the
patient over a period of time, such as before, during and/or after
the treatment with the therapy over the course of a 1 hour, 2 hour,
3 hour, 4 hour, 6 hour, 8 hour, 10 hour, 12 hour, 18 hour, 24 hour,
2 day, 3 day, 4 day, 5 day, 6 day, 7 day, 2 week, 3 week, 4 week, 2
month, 3 month, 4 month, 5 month, 6 month, 7 month, 8 month, 9
month, 10, month 11 month, 1 year or more period of time.
[0191] In embodiments of the various methods provided herein, two
or more of the steps are performed sequentially. In other
embodiments of the methods provided herein, two or more of steps
are performed in parallel (e.g., at the same time).
[0192] Also provided herein is a kit comprising: (a) an enzyme
(e.g., AvPAL, rAvPAL-PEG, and/or any variant thereof), which is
optionally immobilized on a solid support; and (b) a substrate for
the enzyme. In certain embodiments, the kit further comprises one
or more components of the kit in one or more containers and/or
instructions for use.
[0193] Any combination of the above-listed embodiments, for
example, with respect to sample, enzymes, neutralizing
enzyme-specific antibodies, enzymes, substrates and the like, are
also contemplated embodiments in connection with the kits provided
herein.
[0194] The kits provided herein can be used to perform methods
provided herein for detecting the presence of neutralizing
enzyme-specific antibodies (e.g., a neutralizing antibody that
immunospecifically binds to an enzyme, such as AvPAL, rAvPAL-PEG
and/or any derivative thereof) in a sample (e.g., a body fluid,
such as plasma, or a tissue sample). The kits provided herein can
also be used to perform the methods provided herein for classifying
a patient for eligibility for an enzyme therapy (e.g., initial or
continued enzyme therapy) with, e.g., an AvPAL or rAvPAL-PEG, or
variants thereof. The kits provided herein can further be used in
the methods provided herein to monitor or otherwise track
neutralizing anti-enzyme antibody levels in a patient that has been
or will be administered the enzyme, such as an AvPAL, rAvPAL-PEG,
or a variant thereof. The kits provided herein can also be used in
the methods provided herein to prevent, treat, or otherwise manage
a disease or symptom thereof associated with elevated phenylalanine
levels (e.g., PKU) or cancer in a patient.
[0195] The kits can be packaged in any suitable manner, typically
with the various parts, in a suitable container along with
instructions for use. In certain embodiments, the kits may further
comprise, where necessary, other agents for reducing the background
interference in a test, control reagents, apparatus for conducting
a test, and the like.
Methods and Kits to Detect PEG-Specific Antibodies
[0196] PEG and derivatives thereof are typically considered
non-immunogenic due to their chemical structures and are used in
pharmaceutical and cosmetic products. However, protein or enzyme
therapeutics that are conjugated to PEG or a derivative thereof can
still elicit some level of antibody response to the PEG or
derivative thereof. In some cases the antibody response to PEG or
derivative thereof can lead to serious side effects or loss of
efficacy.
[0197] In a fourth aspect, provided herein is a method for
detecting the presence of PEG-specific antibodies (e.g., an
antibody that immunospecifically binds, e.g., a PEG moiety of
rAvPAL-PEG or a variant thereof) in a sample (e.g., a body fluid,
such as plasma, or a tissue sample), said method comprising: (a)
contacting the sample with immobilized PEG; (b) optionally removing
unbound sample; (c) contacting the sample bound to the immobilized
PEG with a detectable antibody, wherein the detectable antibody
immunospecifically binds to Ig; (d) optionally removing unbound
detectable antibody; and (e) detecting the presence of the
detectable antibody bound to the sample; wherein detection above
background of an amount of the detectable antibody bound to the
sample, e.g., an increase in the amount of detectable antibody
bound to the sample as compared to a control sample having no
enzyme-specific antibody, indicates the presence of PEG-specific
antibody in the sample.
[0198] In another embodiment, provided herein is a method for
detecting the presence of PEG-specific antibodies (e.g., an
antibody that immunospecifically binds, e.g., a PEG moiety of
rAvPAL-PEG or a variant thereof) in a sample (e.g., a body fluid,
such as plasma, or a tissue sample), said method comprising: (a)
contacting the sample with immobilized antibody that
immunospecifically binds to an Ig; (b) optionally removing unbound
sample; (c) contacting the sample bound to the immobilized anti-Ig
antibody with a detectable PEG (e.g., a PEG labeled with a
detection moiety); (d) optionally removing unbound detectable PEG;
and (e) detecting the presence of the detectable PEG bound to the
sample; wherein detection above background of an amount of the
detectable PEG bound to the sample, e.g., an increase in the amount
of detectable PEG bound to the sample as compared to a control
sample having no PEG-specific antibody, indicates the presence of
PEG-specific antibody in the sample.
[0199] In yet another embodiment, provided herein is a method for
detecting the presence of PEG-specific antibodies (e.g., an
antibody that immunospecifically binds, e.g., a PEG moiety of
rAvPAL-PEG or a variant thereof) in a sample (e.g., a body fluid,
such as plasma, or a tissue sample), said method comprising: (a)
contacting the sample with immobilized PEG; (b) optionally removing
unbound sample; (c) contacting the sample bound to the immobilized
PEG with a detectable PEG (e.g., a PEG labeled with a detection
moiety); (d) optionally removing unbound detectable PEG; and (e)
detecting the presence of the detectable PEG bound to the sample;
wherein detection above background of an amount of the detectable
PEG bound to the sample, e.g., an increase in the amount of
detectable PEG bound to the sample as compared to a control sample
having no PEG-specific antibody, indicates the presence of
PEG-specific antibody in the sample.
[0200] In certain embodiments, the sample is a body fluid (e.g.,
blood, serum, plasma, CSF, urine or breast milk) or a tissue sample
from a patient. In some embodiments, the patient is a mammal, such
as a human, monkey, dog, rabbit, rat or mouse. In an embodiment,
the concentration of PEG-specific antibody in the sample is
determined.
[0201] In the methods provided herein, any isotype of PEG-specific
antibody can be detected. In certain embodiments, the anti-Ig
antibody is an isotype-specific antibody, e.g., an anti-IgG,
anti-IgA, anti-IgM, anti-IgD or anti-IgE antibody. In an
embodiment, the sample is serum or plasma, which is equal to or
less than 5% or equal to or less than 2% of the volume as described
in (a).
[0202] In some embodiments, the PEG is methoxy PEG, such as a 20
kDa methoxy PEG. In one embodiment, the methoxy PEG is inactivated.
In an embodiment, the PEG is hydroxy PEG, such as a 6 kDa hydroxy
PEG. The PEG may or may not be inactivated.
[0203] In some embodiments, the patient has elevated phenylalanine
levels. In specific embodiments, the patient has been or will be
administered the enzyme, for example, for EST (e.g., for PKU) or
cancer therapy.
[0204] In an embodiment, the enzyme is rAvPAL or a variant thereof.
In one embodiment, the enzyme is rAvPAL_C503S, rAvPAL_C565S,
rAvPAL_C565SC503S, or any combination thereof. In some embodiments,
the enzyme is a pegylated enzyme. In an embodiment, the pegylated
enzyme is rAvPAL-PEG. In other embodiments, the pegylated enzyme is
rAvPAL-PEG_C503S, rAvPAL-PEG_C565S, rAvPAL-PEG_C565SC503S, or any
combination thereof.
[0205] In a specific embodiment, provided is a method to measure
the amount of antibodies specific for PEG or derivative thereof in
a mammal (e.g., human) comprising: (a) binding the PEG (e.g.,
inactivated methoxy PEG) to a solid support, (b) capturing
PEG-specific antibodies in a body fluid (e.g., serum) sample from
the mammal by contacting the body fluid with the PEG bound to the
solid support, (c) contacting the captured PEG-specific antibodies
in (b) with an anti-Ig antibody labeled with a detectable moiety,
and (d) determining the amount of the captured PEG-specific
antibodies from the body fluid sample by detecting the presence of
anti-Ig antibody labeled with the detectable moiety. In some
embodiments, (c) and (d) are performed simultaneously. In one
embodiment, (a) the PEG is bound to a solid support, (b) the
PEG-specific antibody in a body fluid sample from the mammal is
captured by contacting the body fluid sample with the PEG bound to
the solid support, (c) the captured PEG-specific antibody from (b)
is contacted with an anti-Ig specific antibody (e.g., anti-IgG,
anti-IgM, anti-IgA, anti-IgD or anti-IgE) labeled with a detection
moiety, and (d) the amount of captured PEG-specific antibody from
the body fluid sample is determined by detecting the presence of
anti-Ig specific antibody (e.g., anti-IgG, anti-IgM, anti-IgA,
anti-IgD or anti-IgE) labeled with the detection moiety. In an
alternative embodiment, the anti-Ig specific antibody is bound to
the solid support, the PEG-specific antibody in the body fluid
sample from the mammal is captured by contacting the body fluid
sample with the anti-Ig specific antibody bound to the solid
support, the captured PEG-specific antibody is contacted with the
PEG labeled with a detection moiety, and the amount of captured
PEG-specific antibody is determined by detecting the presence of
PEG labeled with the detection moiety. In another alternative
embodiment, a total antibody assay is employed that takes advantage
of the binary character of antibodies. In this assay, PEG is bound
to a solid support, and the body fluid sample, which contains the
PEG-specific antibodies, is incubated with PEG labeled with a
detection moiety. The PEG-specific antibody in the body fluid
sample is captured by contacting the body fluid sample with PEG
bound to the solid support, and the amount of captured PEG-specific
antibody is determined by detecting the presence of PEG labeled
with the detection moiety.
[0206] In some embodiments, a method is provided of classifying a
patient for eligibility for a pegylated enzyme therapy (e.g.,
initial or continued pegylated enzyme therapy) with, e.g., a
rAvPAL-PEG or variant thereof, comprising: (a) providing a sample
(e.g., a body fluid or tissue) from the patient; (b) detecting or
otherwise determining the amount of PEG-specific antibody in the
sample using an assay method provided herein; and (c) classifying
the patient as eligible to receive the one or more further
pegylated enzyme therapies based on the detection or amount of
anti-pegylated enzyme antibody in the sample. In some embodiments,
the patient has previously been administered a pegylated enzyme,
such as a rAvPAL-PEG or variant thereof, e.g., for EST for elevated
phenylalanine levels or cancer therapy.
[0207] In other embodiments, methods provided herein may be used to
observe or otherwise monitor how a patient is responding to a
therapy with a pegylated enzyme, e.g., a rAvPAL-PEG or a variant
thereof. Such information can be used, for example, to make better
decisions about the optimal methods, doses, or treatments for the
patient. For example, these methods are applicable where a subject
has been previously diagnosed as having elevated phenylalanine
levels (e.g., PKU) or cancer and possibly has undergone treatment
for the disease, and the methods provided herein are employed to
monitor the progression of the disease or the treatment thereof. In
addition, the information obtained by said methods may be used for
selecting a patient suitable for pegylated enzyme therapy, or
determining if a patient is suitable for continued pegylated enzyme
therapy. In certain embodiments, the methods herein are used in
conjunction with treatment of a patient with enzyme having or
suspected of having elevated phenylalanine levels (e.g., a patient
having one or more PAH mutations) and/or a disease associated with
elevated phenylalanine levels (e.g., PKU) or cancer, or symptom
thereof. That is, in certain embodiments, the assay methods
provided herein are used to monitor or otherwise track anti-PEG
antibody levels in a patient that has been or will be administered
a pegylated enzyme, such as a rAvPAL-PEG or variant thereof.
[0208] In other embodiments, provided herein are methods of
preventing, treating, or otherwise managing a disease or symptom
thereof associated with elevated phenylalanine levels (e.g., PKU)
or cancer in a patient, said method comprising: (a) administering a
pegylated enzyme (e.g., rAvPAL-PEG or variant thereof), to the
patient; (b) obtaining a sample, such as a bodily fluid or tissue,
from the patient, (c) contacting the sample with immobilized PEG;
(d) optionally removing unbound sample; (e) contacting the sample
bound to the immobilized PEG with a detectable antibody, wherein
the detectable antibody immunospecifically binds to Ig; (f)
optionally removing unbound detectable antibody; and (g) detecting
the presence of the detectable antibody bound to the sample;
wherein detection above background of an amount of the detectable
antibody bound to the sample, e.g., an increase in the amount of
detectable antibody bound to the sample as compared to a control
sample having no enzyme-specific antibody, indicates the presence
of PEG-specific antibody in the sample.
[0209] In another embodiment, provided herein are methods of
preventing, treating, or otherwise managing a disease or symptom
thereof associated with elevated phenylalanine levels (e.g., PKU)
or cancer in a patient, said method comprising: (a) administering
an enzyme (e.g., AvPAL or rAvPAL-PEG, or a variant thereof), to the
patient; (b) obtaining a sample, such as a bodily fluid or tissue,
from the patient, (c) contacting the sample with immobilized
antibody that immunospecifically binds to an Ig; (d) optionally
removing unbound sample; (e) contacting the sample bound to the
immobilized anti-Ig antibody with a detectable PEG (e.g., a PEG
labeled with a detection moiety); (f) optionally removing unbound
detectable PEG; and (g) detecting the presence of the detectable
PEG bound to the sample; wherein detection above background of an
amount of the detectable PEG bound to the sample, e.g., an increase
in the amount of detectable PEG bound to the sample as compared to
a control sample having no PEG-specific antibody, indicates the
presence of PEG-specific antibody in the sample.
[0210] In another embodiment, provided herein are methods of
preventing, treating, or otherwise managing a disease or symptom
thereof associated with elevated phenylalanine levels (e.g., PKU)
or cancer in a patient, said method comprising: (a) administering
an enzyme (e.g., AvPAL or rAvPAL-PEG, or a variant thereof), to the
patient; (b) obtaining a sample, such as a bodily fluid or tissue,
from the patient, (c) contacting the sample with immobilized PEG;
(d) optionally removing unbound sample; (e) contacting the sample
bound to the immobilized PEG with a detectable PEG (e.g., a PEG
labeled with a detection moiety); (f) optionally removing unbound
detectable PEG; and (g) detecting the presence of the detectable
PEG bound to the sample; wherein detection above background of an
amount of the detectable PEG bound to the sample, e.g., an increase
in the amount of detectable PEG bound to the sample as compared to
a control sample having no PEG-specific antibody, indicates the
presence of PEG-specific antibody in the sample.
[0211] In certain embodiments of any of the methods provided
herein, the method further comprise providing results from the
assay to personnel, e.g., at a medical facility, such as a doctor,
nurse or other medical professional. In other embodiments, the
method further comprises providing therapeutic options to
personnel, e.g., at a medical facility, such as a doctor, nurse or
other medical professional. In one embodiment, the patient has
elevated phenylalanine concentrations (e.g., in blood, plasma or
serum) and/or cancer. In other embodiments, the patient has a
disease or disorder resulting from elevated phenylalanine levels,
such as PKU, or cancer. In certain embodiments, the patient has
been, is or will be treated with a pegylated enzyme, such as
rAvPAL-PEG or a variant thereof. In some embodiments, the methods
provided herein are used to monitor or otherwise track PEG-specific
antibodies during the course of enzyme (e.g., rAvPAL-PEG or a
variant thereof) therapy by detecting or otherwise measuring
PEG-specific antibody levels in a sample (such as blood, blood
lysate, plasma, spinal fluid, cerebral fluid or bone marrow
aspirate) from the patient over a period of time, such as before,
during and/or after the treatment with the therapy over the course
of a 1 hour, 2 hour, 3 hour, 4 hour, 6 hour, 8 hour, 10 hour, 12
hour, 18 hour, 24 hour, 2 day, 3 day, 4 day, 5 day, 6 day, 7 day, 2
week, 3 week, 4 week, 2 month, 3 month, 4 month, 5 month, 6 month,
7 month, 8 month, 9 month, 10, month 11 month, 1 year or more
period of time.
[0212] In embodiments of the various methods provided herein, two
or more of the steps are performed sequentially. In other
embodiments of the methods provided herein, two or more of steps
are performed in parallel (e.g., at the same time).
[0213] Also provided herein is a kit comprising: (a) a PEG, which
is optionally immobilized on a solid support; and (b) a detectable
antibody immunospecifically binds to Ig. In certain embodiments,
the kit further comprises one or more components of the kit in one
or more containers and/or instructions for use.
[0214] Also provided herein is a kit comprising: (a) an antibody
that immunospecifically binds to an Ig, which is optionally
immobilized on a solid support; and (b) a detectable PEG (e.g., a
PEG labeled with a detection moiety). In certain embodiments, the
kit further comprises one or more components of the kit in one or
more containers and/or instructions for use.
[0215] Further provided herein is a kit comprising: (a) a PEG,
which is optionally immobilized on a solid support; and (b) a
detectable PEG (e.g., a PEG labeled with a detection moiety). In
certain embodiments, the kit further comprises one or more
components of the kit in one or more containers and/or instructions
for use.
[0216] Any combination of the above-listed embodiments, for
example, with respect to sample, pegylated enzymes, PEG-specific
antibodies, anti-Ig antibodies, PEGs, detectable PEGs, detectable
antibodies, patient populations, solid phase immobilization, labels
and the like, are also contemplated embodiments in connection with
the kits provided herein.
[0217] The kits provided herein can be used to perform methods
provided herein for detecting the presence of PEG-specific
antibodies (e.g., an antibody that immunospecifically binds, e.g.,
a PEG moiety of rAvPAL-PEG or a variant thereof) in a sample (e.g.,
a body fluid, such as plasma, or a tissue sample). The kits
provided herein can also be used to perform the methods provided
herein for classifying a patient for eligibility for a pegylated
enzyme therapy (e.g., initial or continued pegylated enzyme
therapy) with, e.g., a rAvPAL-PEG or variant thereof. The kits
provided herein can further be used in the methods provided herein
to monitor or otherwise track anti-PEG antibody levels in a patient
that has been or will be administered a pegylated enzyme, such as
an AvPAL-PEG or variant thereof. The kits provided herein can also
be used in the methods provided herein to prevent, treat, or
otherwise manage a disease or symptom thereof associated with
elevated phenylalanine levels (e.g., PKU) or cancer in a
patient.
[0218] The kits can be packaged in any suitable manner, typically
with the various parts, in a suitable container along with
instructions for use. In certain embodiments, the kits may further
comprise, where necessary, other agents for reducing the background
interference in a test, control reagents, apparatus for conducting
a test, and the like.
[0219] The following examples are offered by way of illustration,
and not by way of limitation.
EXAMPLES
Example 1
Cloning of Nostoc punctiforme and Anabaena variabilis PAL DNA
Manipulations
[0220] N. punctiforme genomic DNA was purchased from ATCC (29133D)
and the PAL gene (ZP.sub.--00105927) was PCR-amplified from primers
5'-CACTGTCATATGAATATAACATCTCTACAACAGAACAT-3' (SEQ ID NO:12) and
5'-GACAGTGGCGGCCGCTCACGTTGACTTTAAGCTCGAAAAAATATG-3' (SEQ ID NO:13).
The resulting PCR product was digested with NdeI and NotI and the
1.7 kb fragment was ligated into pET-28a(+) and pET-30a(+)
(Novagen) for N-His tagged and =tagged, respectively.
[0221] A. variabilis cells were purchased from ATCC (29413).
Genomic DNA was extracted (Qiagen) and the PAL gene
(YP.sub.--324488) was amplified by SOE-PCR to remove an NheI site.
Primer 1 (5'-CACTGTGCTAGCATGAAGACACTATCTCAAGCACAAAG-3') (SEQ ID
NO:14) and primer 2
(5'-GGAAATTTCCTCCATGATAGCTGGCTTGGTTATCAACATCAATTAGTGG-3') (SEQ ID
NO:15) were used to amplify nucleotides 1-1190 and primer 3
(5'-CCACTAATTGATGTTGATAACCAAGCCAGCTATCATGGAGGAAATTTCC-3') (SEQ ID
NO:16) and primer 4
(5'-CACTGTGCGGCCGCTTAATGCAAGCAGGGTAAGATATCTTG-3') (SEQ ID NO:17)
were used to amplify nucleotides 1142-1771. These two PCR products
were combined to amplify the full-length gene with primers 1 and 4.
The resulting PCR product was digested with NheI, blunted with
Klenow (NEB), then digested with NotI. The 1.7 kb fragment was
ligated into pET-28a(+) and pET-30a(+) (Novagen). This plasmid was
named 3p86-23. The A. variabilis PAL (AvPAL) gene was also cloned
into the vector pIBX7 (Tkalec, et al., Appl. Environ. Microbiol.
66:29-35 (2000)), which was derived from pIBX1 (Su, et al., Appl.
Environ. Microbiol. 62:2723-2734 (1996)) (see Example 5).
Bacterial Strains and Culture Conditions
[0222] For N. punctiforme PAL (NpPAL), E. coli BL21(DE3) cells
(Stratagene) were transformed with pGro7 (TaKaRa) and competent
BL21(DE3)pGro7 cells were prepared by the Inoue Method (Sambrook
and Russell, Molecular Cloning: A Laboratory Manual, 3.sup.rd
Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
2001)). These cells were transformed with pET-28-NpPAL and cultured
in 25 mL LB with 50 mg/L kanamycin and 20 mg/L chloramphenicol
overnight at 37.degree. C. Twenty milliliters of this culture was
seeded into 1 L of LB medium with kanamycin, chloramphenicol, and
500 mg/L L-arabinose and grown at 37.degree. C. At an OD.sub.600 of
0.6, the culture was chilled on ice. After 5 minutes, the culture
was induced with 0.3 mM IPTG and grown for 16 hours at 20.degree.
C. Cells were harvested by centrifugation.
[0223] BL21(DE3)pLysS cells (Stratagene) were transformed with
AvPAL and cultured identically to NpPAL without the arabinose
induction.
[0224] AvPAL cloned in the pIBX7 vector (see Example 7) was
introduced by transformation into BLR(DE3)/pLysS (Novagen) cells
and cultured in 25 mL LB with 50 mg/L kanamycin overnight at
37.degree. C. Twenty milliliters of this culture was seeded into 1
L of LB medium with kanamycin, and grown at 37.degree. C. At an
OD.sub.600 of 0.6, the culture was chilled on ice. After 5 minutes,
the culture was induced with 0.3 mM IPTG and grown for 16 hours at
30.degree. C. Cells were harvested by centrifugation.
Example 2
Purification of NpPAL and AvPAL
[0225] The cultures were centrifuged in a bench-top centrifuge at
5,000 g for 20 minutes and the supernatant discarded. The cell
pellets were typically frozen at -70.degree. C. prior to further
processing. Upon thawing, the cell pellets were suspended to
approximately 80 optical density units (600 nm) in TBS (25 mM Tris,
150 mM NaCl, pH 7.8). The cells were lysed by two passes through an
APV pressure homogenizer at 12-14,000 psi. The crude lysate was
then heat-treated at 55.degree. C. for 2 hours. The lysate is
centrifuged at 10,000 g for 30 minutes and the supernatant retained
and filtered with a 0.2 .mu.m vacuum filter (Corning).
[0226] The PAL was purified from the clarified lysate by passage
sequentially over a butyl 650M column (Tosoh BioSciences) and a
MacroPrep High Q column (BioRad). The eluted product showed a high
level of purity by both SDS PAGE and reverse phase HPLC.
Example 3
Generation of Pegylated PAL Variants
[0227] A method for pegylation of PAL from Rhodosporidium
toruloides (RtPAL) is described below. Similar methods are used for
pegylation of prokaryotic PAL (e.g., Nostoc punctiforme (NpPAL) or
Anabaena variabilis (AvPAL)) are described in Example 4.
Protein Pegylation
[0228] Pegylation uses modifications of literature methods
(Hershfield, et al., (1991), ibid.; U.S. Pat. No. 6,057,292; Lu, et
al., Biochemistry 40(44):13288-13301 (2001); Nektar Therapeutics,
2003 catalog). Activated PEGs include both the linear PEG
succinimidyl succinates (mPEG-SPA, MW 5 kDa or MW 20 kDa) and the
branched PEG hydrosuccinimides (mPEG.sub.2-NHS ester, MW 10 kDa or
MW 40 kDa), which are both capped on one end with a methoxy group
and available from Nektar Therapeutics; experimental determination
of optimal pegylated proteins is normally required (Veronese, et
al., J. Bioactive Compatible Polymers 12:196-207 (1997)). Optimal
pegylation conditions are determined using different ratios of
PAL:PEG (taking into account the molar ratio of protein along with
the number of lysines per protein monomer), different pHs,
different buffers, various temperatures and incubation times. High
PAL protein:PEG derivatization ratios are necessary since native
PAL has a large number of lysines (e.g., 29 per Rhodosporidium
toruloides (Rt) monomer, 18 per Anabaena viriabilis (Av) monomer
and 18 per Nostoc punctiforme (Np) monomer) and because un-modified
PAL displays immunoreactivity upon repeated injection in mice and
since naked (wild-type) PAL is quickly inactivated upon exposure to
proteases. Pegylation reactions are stopped by freezing at
-20.degree. C., and the samples will be analyzed by SDS-PAGE,
MALDI-TOF mass spectroscopy, activity assessment, proteolytic
sensitivity, and immunoreactivity.
[0229] Prior to activity, proteolysis, and immune assessment, and
in order to remove excess unreacted PEG, reactions are dialyzed
against pH 8.5, 0.05 M potassium phosphate buffer overnight at
4.degree. C. with stirring using Tube-O-Dialyzers (GenoTechnology).
After protein concentration is determined using the NI protein
assay kit (GenoTechnology), PAL activity measurements will be
performed on underivatized and PEG derivatized PAL samples using
standard reaction conditions, as previously described. Following in
vitro characterization, in vivo trials will be conducted with the
most promising pegylated therapeutic candidates using the PKU mouse
model.
Characterization
[0230] Protein concentration is determined using the PAL extinction
coefficient (0.5 and 0.75 mg mL.sup.-1cm.sup.-1 for RtPAL and
AvPAL, respectively) at 280 nm for non-modified protein samples and
for pegylated protein samples the concentration is calculated using
the NI Protein Assay (GenoTechnology) that includes sample
processing to remove non-protein contaminants that might interfere
with accurate protein concentration determination.
[0231] PEG-PAL products are characterized by peptide mapping
techniques to determine site-specific pegylation (LC/ESI-MSD), and
trinitrobenzene sulfonate (TNBS) to determine the free amine
titration before and after pegylation. Peptide mapping determines
the relative occupancy of pegylation at a majority of the tryptic
peptides that terminate with lysine, however, due to size and
multiple adjacent lysine tryptic peptides, not all sites are
visible using this technique. The TNBS assay more accurately
defines the average number of PEG molecules per mol of enzyme, but
gives no information about which sites get pegylated. For this
reason, both assays are used and are complementary to each other.
Rough estimates of percent derivatization of PAL products by PEG
can be determined by SDS-PAGE and native gel analyses. Enzymatic
assays are used to assess specific activity before and after
pegylation and to provide evidence that there is no loss of the
tetrameric PAL structure.
PAL Activity Assay
[0232] The PAL activity assay is conducted using a Cary UV
spectrophotometer (Cary 50) in the kinetics mode. The activity of
PAL with L-phenylalanine substrate is assayed at room temperature
(25.degree. C.) by measuring the production of trans-cinnamate
monitored by the absorbance increase at 290 nm (Hodgins, (1968),
ibid). The molar extinction coefficient of trans-cinnamic acid at
290 nm is 10,238 liter M.sup.-1cm.sup.-1. Reaction mixtures contain
22.5 mM phenylalanine in 100 mM Tris-HCl buffer, pH 8.5. For
standard measurements the final enzyme concentration is 0.0035
mg/mL, but for kinetic studies the enzyme concentration in the
assay is adjusted so that the slope at 290 nm per min is in the
range of 0.005 to 0.02. Activity data is expressed as specific
activity (.mu.mol.times.min.sup.-1mg.sup.-1). One unit of PAL is
defined as that amount of enzyme that produces 1 .mu.mol of
trans-cinnamic acid per minute at room temperature.
Example 4
Generation of Pegylated NpPAL and AvPAL
[0233] In general, pegylation for both NpPAL and AvPAL involves
mixing the protein with SUNBRIGHT ME-200HS 20 kDa NHS-activated PEG
(NOF).
[0234] Protocol for pegylation, standard "HC" method using
NHS-activated 20 kDa linear PEG:
[0235] 1) The protein was evaluated for the presence of endotoxin.
A protein solution (0.1 mL) was diluted in 0.9 mL fresh MQ water
and tested with a hand-held Charles River apparatus (EndoPTS) for
endotoxin at the 0.5 EU/mL sensitivity level. If endotoxin was
greater than 0.5 EU/mL, then endotoxin was reduced initially by
Mustang E filtration, followed by Sterogene Etox resin, and less
preferably by further chromatographic purification. Reduction was
limited but sufficiently useful by passage over DEAE FF (Amersham)
at pH 7.8.
[0236] 2) Concentration and buffer exchange of protein. The protein
was concentrated to greater than 25 mg/mL but less than or equal to
75 mg/mL and buffer exchanged to 50 mM KPO.sub.4, pH 8.5. If a spin
filter was used to prepare this concentration, the filter was first
tested for endotoxin by spinning at reduced speed and time (3000
rpm, 3 minutes) with buffer alone, then testing the retained buffer
for endotoxin in the same way as the protein in step 1. The buffer
batch record/recipe for 50 mM KPO4, pH 8.5 consisted of water (QS
to 1 L), potassium phosphate dibasic (8.4913 g/L of 48.75 mM), and
potassium phosphate monobasic (0.17011 g/L of 1.25 mM). The
solution was filtered through a 0.2 .mu.m filter and stored at room
temperature. The concentrated product was slowly filtered (1-2
mL/min) through a Mustang E filter acrodisc. A sample diluted and
blanked with sterile TBS, pH 7.5 was measured at A280 to determine
protein concentration. The extinction coefficient was 0.83 for
NpPAL and 0.75 for AvPAL.
[0237] 3) Pegylation of NpPAL and AvPAL. PEG normally stored at
-80.degree. C. was warmed to room temperature. KPO4 buffer was
added to PEG to resuspend by vortexing at maximum speed, and
shaking tube hard in hand to ensure all large chunks were
suspended. The protein was added to the well-suspended PEG solution
within one minute of having first wetted the PEG and mixed by very
gentle inversion. Tubes wrapped in aluminum foil were placed on the
axis of a rocker and rocked very gently at room temperature for 3
hours. The tubes were filled with TBS (pH 7.5) and sterile
filtered. The suspensions were either formulated immediately or
stored at 4.degree. C. until ready for formulation.
[0238] 4) Formulation. The formulation buffer recipe/batch record
consisted of water (QS to 1 L), Tris-Base (3.2 mM), Tris-HCl (16.8
mM), and sodium chloride; the buffer solution was filtered through
a 0.2 .mu.m filter and stored at room temperature. The buffer
solution was subjected to tangential flow filtration using a
Vivaflow 50 (smaller lots) or Vivaflow 200 (larger lots) with a 100
MWCO regenerated cellulose membrane. The solution was flushed with
MQ water, 0.1 N NaOH, and 200 mL water again. The solution was
equilibrated with TBS, pH 7.5 at 50 mL/min cross-flow. The pH of
the permeate was determined to ensure a pH of 7.5.
[0239] The solution was buffer exchanged by first diluting with TBS
approximately 3-fold and returning to original volume at least four
times. Cross-flow was typically 180-200 mL/min for both Vivaflow 50
and 200.
[0240] The final product was filtered through Mustang E. The
presence of endotoxin was evaluated after diluting 0.1 mL with 1.9
mL sterile fresh water. If endotoxin was greater than 1 EU/mL,
reduction was conducted with Sterogene Etox gel. Formulated,
sterile pegylated NpPAL or AvPAL were sealed in vials and placed at
-70.degree. C. until ready for in vivo studies.
Example 5
Generation of AvPAL Variants (Cysteine Mutants)
[0241] Amino acid substitutions were made in the AvPAL polypeptide
to reduce aggregation that occurs in bacterially expressed,
recombinant proteins. Protein aggregation may reduce enzyme
activity and/or increase immunogenicity in vivo. One such form of
aggregation occurs as a result of formation of inter-chain
disulfide bonds. To minimize this possibility, various AvPAL
cysteine residues, alone or in combination, were replaced with
serine residues.
[0242] The AvPAL polypeptide has 6 cysteine residues, at positions
64, 235, 318, 424, 503 and 565 (SEQ ID NO:4). The following AvPAL
single cysteine mutants were generated: AvPAL_C64S (SEQ ID NO:7),
AvPAL_C318S (SEQ ID NO:8), AvPAL_C503S (SEQ ID NO:9), and
AvPAL_C565S (SEQ ID NO:10). An AvPAL double cysteine mutant,
AvPAL_S565SC503S (SEQ ID NO:11), was also generated. FIGS. 3A-3E
shows the amino acid sequences of these AvPAL cysteine mutants.
Cloning
[0243] The AvPAL gene was amplified from Anabaena variabilis
genomic DNA (ATCC 29413-U, Qiagen DNeasy Kit) with forward primer
AvarPALfor (5'-CACTGTCATATGAAGACACTATCTCAAGCACAAAG-3') (SEQ ID
NO:18) and reverse primer AvarPALrev
(5'-CACTGTCTCGAGATGCAAGCAGGGTAAGATATCTTG-3') (SEQ ID NO:19). The
resulting PCR product was treated with Taq and then ligated into
pCR2.1 TOPO TA (Invitrogen). The resulting plasmid was named
1p40.
[0244] A 5' NheI site was added and an internal NheI site was
removed by SOE-PCR. The upstream AvPAL fragment was amplified from
1p40 with forward primer N-Nhe-AvPAL
(5'-CACTGTGCTAGCATGAAGACACTATCTCAAGCACAAAG-3') (SEQ ID NO:20) and
reverse primer Nhe-AvPALrev
(5'-GGAAATTTCCTCCATGATAGCTGGCTTGGTTATCAACATCAATTAGTGG-3') (SEQ ID
NO:21), and the downstream AvPAL fragment was amplified from 1p40
with forward primer Nhe-AvPAL for
(5'-CCACTAATTGATGTTGATAACCAAGCCAGCTATCATGGAGGAAATTTCC-3') (SEQ ID
NO:22) and reverse primer AvPALrev-r
(5'-ACAGTGGCGGCCGCTTAATGCAAGCAGGGTAAGATATCTTG-3') (SEQ ID NO:23).
In a single PCR reaction, the two PCR products were annealed and
extended with DNA polymerase to produce the full-length AvPAL gene,
and then amplified with primers N-Nhe-AvPAL and AvPALrev-r. The
resulting PCR product was digested with NheI, blunted with Klenow,
digested with NotI, and ligated into the pET28a+ vector (prepared
by digestion with NdeI, blunting with Klenow, and digestion with
NotI). The resulting plasmid was named 3p86-23.
[0245] New restriction sites were added by PCR. AvPAL was amplified
from plasmid 3p86-23 with forward primer AvEcoRIfor
(5'-CACTGTGAATTCATGAAGACACTATCTCAAGCACAAAG-3') (SEQ ID NO:24) and
reverse primer AvSmaIrev
(5'-CACTGTCCCGGGTTAATGCAAGCAGGGTAAGATATCT-3') (SEQ ID NO:25). The
resulting PCR product was digested with EcoRI and SmaI and ligated
into EcoRI- and SmaI-digested pIBX7 vector. The resulting plasmid
was named 7p56 Av3.
Cysteine Mutants
[0246] Two cysteine codons in the AvPAL gene, corresponding to
positions 503 and 565 of the AvPAL polypeptide, were substituted
with serine codons by site-directed mutagenesis (QuickChange XL II,
Stratagene). The cysteine codon at position 503 was changed to a
serine codon in plasmid 7p56 Av3 by PCR with forward primer
Av_C503S (5'-GTCATTACGATGCACGCGCCTCTCTATCACCTGCAACTGAG-3') (SEQ ID
NO:26) and reverse primer Av_C503Srev
(5'-CTCAGTTGCAGGTGATAGAGAGGCGCGTGCATCGTAATGAC-3') (SEQ ID NO:27).
The serine codon is underlined and the G to C mutation in the
coding strand (C to G mutation in the non-coding strand) is
indicated in bold. The resulting plasmid was named j282. The
cysteine codon at position 565 was changed to a serine codon in
plasmid j282 with forward primer Av_C565S
(5'-CAGTTCAAGATATCTTACCCTCCTTGCATTAACCCGGGCTGC-3') (SEQ ID NO:28)
and reverse primer Av_C565Srev
(5'-GCAGCCCGGGTTAATGCAAGGAGGGTAAGATATCTTGAACTG-3') (SEQ ID NO:29).
The serine codon is underlined and the G to C mutation in the
coding strand (C to G mutation in the non-coding strand) is
indicated in bold. The resulting plasmid was named j298a.
[0247] Cysteine codons in the AvPAL gene at positions 64, 318 and
565 of the AvPAL polypeptide were similarly substituted with serine
codons using the following primer pairs: C64S, forward primer
Av_C64S (5'-GCAGGGTATTCAGGCATCTTCTGATTACATTAATAATGCTGTTG-3') (SEQ
ID NO:30) and reverse primer Av_C64Srev
(5'-CAACAGCATTATTAATGTAATCAGAAGATGCCTGAATACCCTGC-3') (SEQ ID
NO:31); C318S, forward primer Av_C318S
(5'-CAAGATCGTTACTCACTCCGATCCCTTCCCCAGTATTTGGGGC-3') (SEQ ID NO:5)
and reverse primer Av_C318Srev
(5'-GCCCCAAATACTGGGGAAGGGATCGGAGTGAGTAACGATCTTG-3') (SEQ ID NO:6);
and C565S, forward primer Av_C565S (SEQ ID NO:28) and reverse
primer Av_C565Srev (SEQ ID NO:29). The serine codons are
underlined, and the G to C mutations in the coding strands and the
C to G mutations in the non-coding strands are indicated in
bold.
Example 6
In Vitro Enzyme Activity of AvPAL Variants (Cysteine Mutants)
[0248] The purpose of this study was to determine the effect of
serine substitution of the various cysteine residues in the AvPAL
polypeptide on in vitro phenylalanine ammonia-lyase (PAL) enzyme
activity.
[0249] AvPAL variants (i.e., cysteine mutants) were cloned as
described in Example 5. The AvPAL cysteine mutant expression
plasmids were transformed into bacteria and the AvPAL cysteine
mutant polypeptides were expressed as described in Example 1 and
purified as described in Example 2.
[0250] The wild-type (WT) AvPAL and AvPAL cysteine mutants were
tested for in vitro PAL enzyme activity as described in Example 3.
Table 1 shows that compared to unpegylated WT AvPAL, the in vitro
PAL specific activity of the purified, unpegylated AvPAL cysteine
mutant proteins was reduced by serine substitution of the cysteine
residue at position 64 (AvPAL_C64S), but was not adversely affected
by serine substitution of the cysteine residues at either of
positions 503 or 565, or at both positions 503 and 565
(AvPAL_C503S, AvPAL_C565S, and AvPAL_C565SC503S, respectively).
TABLE-US-00001 TABLE 1 Specific Activity of AvPAL Cysteine Mutants
AvPAL Protein Pegylation Specific Activity (U/mg) WT AvPAL - 1.7
AvPAL_C503S - 1.9 AvPAL_C64S - 1.3 AvPAL_C565S E1 - 2.0 AvPAL_C565S
E2 - 2.1 AvPAL_C565SC503S - 2.2 WT AvPAL + 1.1 AvPAL_C565SC503S +
1.1
[0251] To determine whether the introduction of the serine residues
had any effect on enzymatic activity of pegylated AvPAL proteins,
the WT AvPAL and double cysteine mutant, AvPAL_C565SC503S, were
pegylated as described in Example 4. Table 1 shows that the in
vitro PAL specific activity of the pegylated AvPAL protein was not
adversely affected by serine substitution of the cysteine residues
at both positions 503 and 565.
Example 7
In Vitro Biochemical Characterization of AvPAL Variants (Cysteine
Mutants)
[0252] The purpose of this study was to determine the effect of
serine substitution of the various cysteine residues in the AvPAL
polypeptide on: (1) accelerated stability; (2) aggregate formation;
and (3) site-specific pegylation.
Accelerated Stability
[0253] The effect of serine substitution of cysteine residues in
AvPAL on in vitro stability was determined by storing the purified
AvPAL cysteine mutants, either pegylated or un-pegylated, for
various time periods at 37.degree. C., and then measuring the in
vitro PAL specific activity of these proteins as described in
Example 3.
[0254] Wild-type AvPAL and AvPAL cysteine mutants, either
upegylated or pegylated, were prepared as described in Example
4.
[0255] As shown in FIG. 4A, the specific activities of the
unpegylated AvPAL proteins were stable for at least 5 days at
37.degree. C., and were not adversely affected by serine
substitution of the cysteine residues at position 565, or at both
positions 503 and 565. Similarly, as shown in FIG. 4B, the specific
activities of the pegylated AvPAL proteins were stable for at least
6 days at 37.degree. C. The single cysteine AvPAL mutant,
AvPAL_C565S, showed somewhat reduced stability compared to
wild-type AvPAL and the double cysteine AvPAL mutant,
AvPAL_C565SC503S, after 6 days at 37.degree. C.
Aggregate Formation
[0256] The effect of serine substitution of cysteine residues in
AvPAL on formation of protein aggregates in solution was determined
by separating the purified, unpegylated wild-type AvPAL and AvPAL
cysteine mutants by either denaturing and native gel
electrophoresis or by SEC-HPLC.
[0257] The purified AvPAL preparations were separated by gel
electrophoresis under either denaturing conditions (4-12% NuPAGE
Bis-Tris) or native conditions (8% Tris-Gly, pH 8.3). The separated
AvPAL proteins were stained with Coomassie Blue.
[0258] The purified AvPAL preparations were separated by SEC-HPLC.
AvPAL proteins were loaded onto a TSK gel column (G3000SW.times.1,
7.8 mm.times.30 cm, 5 .mu.m (Tosoh Bioscience, LLC)) in 20 mM
Na-phosphate, 300 mM NaCl, pH 6.9, and eluted at a flow rate of 0.5
mL/min. The separated AvPAL proteins were analyzed on an Agilent
series 1100 spectrometer.
[0259] Aggregates were present in the wild-type AvPAL preparation
and in the AvPAL_C503S and AvPAL_C64S preparations, but not in the
AvPAL_C565S and AvPAL_C565SC503S preparations, as judged by either
gel electrophoresis (FIG. 5A) or SEC-HPLC (FIG. 5B).
Site-Specific Pegylation
[0260] The effect of serine substitution of cysteine residues in
AvPAL on site-specific pegylation was determined by pegylating the
wild-type AvPAL and double cysteine mutant AvPAL_C503SC565S as
described in Example 4, and then comparing the relative pegylation
at the AvPAL lysine residues: K2, K10, K32, K115, K145, K195, K301,
K335, K413, K419, K493, K494 and K522.
[0261] Approximately 100 .mu.g (10 .mu.L at 10 .mu.g/.mu.L) of
unpegylated or pegylated AvPAL proteins were denatured in 8 M urea.
The denatured proteins were then digested in a 100 .mu.L reaction
volume with trypsin in 0.8 M urea at pH 8.2 overnight (.about.20
hours) at 37.degree. C. The trypsin-digested proteins were reduced
by treatment with 1 .mu.L of 1 M DTT for 1 hour at 37.degree. C.,
followed by quenching with 3 .mu.L 15% TFA. Digested proteins were
separated on a C18 reverse-phase column. Percent pegylation of each
of the pegylated AvPAL peptides was calculated by subtractive
peptide mapping of the corresponding unpegylated peptide.
[0262] As shown in FIG. 6, at a ratio of AvPAL protein:PEG of 1:3,
there was no striking difference in the percent pegylation of any
of the lysine (K) residues with the possible exception of K419, in
which the percent pegylation of the double cysteine mutant
C565SC503S was lower compared to wild-type AvPAL. However, the
results obtained using the double cysteine mutant at increasing
AvPAL protein:PEG ratios, in which no dose-response relationship
was observed, taken together with the relatively small percent
pegylation, indicates that the observed differences at K1419 are
not likely to be meaningful. Thus, serine substitution of cysteine
residues at positions 503 and 565 does not appear to affect
site-specific pegylation of AvPAL.
Example 8
Detection of Pegylated Recombinant Anabaena variabilis
Phenylalanine Ammonia-Lyase (rAvPAL-PEG) in Plasma
[0263] The purpose of this assay development was to create a method
to determine the plasma concentration of pegylated recombinant
Anabaena variabilis phenylalanine ammonia-lyase (rAvPAL-PEG) or
variant thereof. In particular, an enzyme-linked immunosorbent
assay (ELISA) method was developed to detect rAvPAL-PEG in rat,
monkey, and human plasma.
Materials
[0264] The following antibodies and chemicals were used: AvPAL
(unpegylated enzyme) and rAvPAL-PEG (pegylated enzyme) in
formulation buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.5); see
Examples 1, 2 and 4 above, as well as U.S. Pat. No. 7,531,341;
capture reagent: affinity-purified rabbit anti-rAvPAL-PEG
polyclonal antibody BP79ex (prepared using standard methods known
to those in the art); competing antibody: Protein G-purified rabbit
anti-rAvPAL-PEG polyclonal antibody BP80 (prepared using standard
methods known to those in the art); detection reagent: anti-PEG
mouse monoclonal IgM antibody (Academia Sinica, Cat No. AGP3);
biotinylated anti-PEG mouse monoclonal IgM antibody; conjugate:
HRP-conjugated Streptavidin (Pierce, Cat No. 21127); Dulbecco's
phosphate buffered saline (DPBS), 1.times. (Cellgro, Cat No.
21-031-CV); sulfuric acid (H.sub.2SO.sub.4), 12 N (VWR, Cat No.
VW3481-1); glycine (Sigma, Cat No. 67126); BupH
Carbonate-Bicarbonate Buffer Pack (Pierce, Cat No. 28382); Tween 20
(Acros Organics, VWR, Cat No. 233360010); Trizma (Sigma, Cat No.
T6066-500G); TMB 2 Component (BioRad Component A, Cat No.
210002430; Component B, Cat No. 210002327); ProClin 300 (Supelco,
Cat No. 48912-V); bovine serum albumin (BSA), Fraction V, Omnipure
(EMD, VWR, Cat No. 2930); pooled rat plasma LiHeparin
(Bioreclamation, Inc. Cat No., RATPLIHP); pooled cyno plasma
LiHeparin (Bioreclamation, Inc. Cat No., CYNPLIHP); and pooled
human plasma LiHeparin (Bioreclamation, Inc., Cat No. HMPLIHP).
[0265] The particular antibodies used here are illustrative and not
meant to be limiting in any way. Different capture reagents and
competing antibodies can be used in this assay, for example,
affinity-purified rabbit anti-rAvPAL-PEG polyclonal antibody BP83ex
as capture reagent, and Protein G-purified rabbit anti-rAvPAL-PEG
polyclonal antibody BP84 as competing antibody. Different detection
reagents can be used in this assay, for example, rabbit monoclonal
anti-PEG IgG (Epitomics, Inc.).
[0266] The following plasticware and disposables were used: pH
paper, Baker-pHIX (JT Baker, VWR, Cat No. 4393-01); 1.2 mL cluster
tubes (Costar, Cat No. 11508); microcentrifuge tubes, 1.5 mL (USA
Scientific, VWR, Cat No. 1615-550); flat bottom MaxiSorp 96-well
plate (Nunc International, VWR, Cat No. 12-565-135); microplate
adhesive film sealer (USA Scientific, Cat No. 2920-0000); pipetman
tips: 20 .mu.L, 250 .mu.L, and 1000 .mu.L (Rainin Instruments, Cat
Nos. GPS-L1000, GPS-L250, GPS-L10); reagent reservoir, 50 mL
Capacity (Corning, VWR, Cat No. 07200127); and serological
pipettes, 10 mL and 25 mL (VWR, Cat Nos. 13-678-11D and
13-678-11E).
[0267] The following equipment were used: El.times.405 Select Plate
Washer (Bio-Tek); multichannel pipetman, 12-well, LTS 20-200 .mu.L
(Rainin Instruments); multichannel pipetman, 12-well, LTS 100-1200
.mu.L (Rainin Instruments); pipet-aid, multi-speed, Drummond (VWR,
Cat No. 13-681-15E); SOFTmax Pro v3.0 (Molecular Devices);
SpectraMax PLUS Microplate Spectrophotometer (Molecular Devices);
timer, three channel alarm (VWR, Cat No. 62344-600); and titer
plate shaker (Barnstead/Lab-Line Instruments).
Buffers
[0268] The following buffers were used: acidification reagent: 0.1
M glycine, pH 2.7; neutralization buffer: 0.5 M Tris-HCl, pH 8.5;
coating buffer (CB): carbonate-bicarbonate buffer, pH 9.2; blocking
buffer (BB): 1.times.DPBS/0.05% Tween 20/5% BSA or 1.times.DPBS/5%
BSA; wash buffer (WB): 1.times.DPBS/0.1% Tween 20/0.05% Proclin
300; TMB substrate solution: 10 mL component A and 1.2 mL component
B; and stop solution: 2N H.sub.2SO.sub.4.
ELISA Protocol
[0269] The following ELISA protocol was used. The capture antibody
BP79ex was prepared in CB to a final concentration of 1 .mu.g/mL,
and 100 .mu.L/well was dispensed in a flat bottom MaxiSorp 96-well
plate, followed by incubating the plate overnight at 4.degree. C.
The plate was washed 3.times. with 300 .mu.L/well WB. BB was added
at 300 .mu.L/well, and the plate was incubated with shaking for 2
hours at room temperature (RT). After removing BB, the plate was
washed 3.times. with 300 .mu.L/well WB. For acidification, 1 mg/mL
rAvPAL-PEG standard stock solutions were diluted 20-fold in the
appropriate plasma matrix to a final concentration of 50 .mu.g/mL.
Four (4) volumes of 0.1 M glycine, pH 2.7, were added to the
diluted plasma standards, followed by incubating the plate for 30
min at RT. Standards and samples were neutralized with 1 volume 0.5
M Tris-HCl, pH 8.5. The neutralized standards and samples were then
diluted to a final plasma concentration of 2% (v/v) by adding BB.
Subsequent samples and standards dilutions were made in BB
containing 2% rat plasma to match the plasma matrix. Final
dilutions of standards and samples were added at 100 mL/well to the
plate, followed by incubating the plate without shaking for 2 hours
at 37.degree. C. After incubation, the plate was washed 3.times.
with 300 .mu.L/well WB. The Detection Antibody (mouse anti-PEG
monoclonal IgM antibody conjugated to biotin) was diluted to 1
.mu.g/mL in BB and then dispensed at 100 .mu.L/well, followed by
incubating the plate with shaking for 1 hour at RT. After
incubation, the plate was washed 3.times. with 300 .mu.L/well WB.
The Streptavidin HRP was diluted to 1 .mu.g/mL in BB and then
dispensed at 100 .mu.L/well, followed by incubating the plate with
shaking for 1 hour at RT. After incubation, the plate was washed
3.times. with 300 .mu.L/well WB. TMB Substrate Solution was added
to the plate at 100 .mu.L/well, followed by incubating the plate
for 30 min at RT. Stop Solution was added to the plate at 100
.mu.L/well, followed by reading the plate for absorbance at 450 nm.
It should be understood that the concentrations of antibodies used
in this assay may differ in assays using alternative
antibodies.
Assay Design Parameters
[0270] To establish the assay format and determine the
concentrations of capture and detection antibodies and additional
reagents, multiple rabbit polyclonal anti-AvPAL antibodies were
tested to identify the optimal coating antibody (i.e., BP77, BP78,
BP79, BP80). For coating, Protein G-purified rabbit polyclonal
anti-AvPAL antibodies were tested at 5 .mu.g/mL upon dilution in
CB. Some of these antibodies were tested for antigens diluted in CB
and in 2% rat plasma to identify antibodies that would give
stronger signals with minimal matrix interference. The
affinity-purified rabbit polyclonal anti-AvPAL antibody BP79ex was
also tested as a replacement for the affinity-purified
antibodies.
[0271] The initial screening of BP77, BP78, BP79, and BP80
antibodies identified BP79 and BP80 as lead candidates for coating
antibody. Further testing of these antibodies at 2 .mu.g/mL in CB
and in 2% rat plasma identified BP79 as the best coating antibody.
Further testing demonstrated that the affinity-purified BP79
antibody, BP79ex, showed improved sensitivity and linearity, and
the coating concentration was reduced to 1 .mu.g/mL. Additional
experiments demonstrated equivalent linearity and range in the
ELISA when used to detect the double cysteine mutant, pegylated
AvPAL, rAvPAL-PEG_C565SC503S, having the cysteine residues at
positions 503 and 565 substituted with serine residues, as compared
to the wild-type rAvPAL-PEG. Further experiments identified BP83ex
as a suitable coating antibody and BP84 as a suitable competing
antibody.
Acidification
[0272] Development of a suitable assay to detect an extensively
pegylated therapeutic enzyme (i.e., an enzyme having a sufficient
number of polyethylene glycol (PEG) molecules attached such that at
least some of the enzyme's immunogenic epitopes are masked by PEG
molecules) in a body fluid or tissue sample from a mammal requires
finding a way to expose the immunogenic epitopes of the therapeutic
enzyme (i.e., AvPAL) for capture by an immobilized antibody
specific for the target therapeutic enzyme, maximizing the number
of epitopes recognized by the antibodies, while at the same time
minimizing the number of epitopes masked by the PEG molecules. To
accomplish this, various methods were evaluated, including
acidification, to denature and subsequently renature the target
pegylated therapeutic enzyme (i.e., rAvPAL-PEG).
[0273] Acidification/neutralization has been used to remove bound
binding proteins in blood from interfering in an ELISA used to
measure transforming growth factor beta 1 (TGF-.beta.1) (Knopf et
al., Clin. Chem. 43:1965-1974 (1997)). In this procedure, plasma or
serum samples were acidified to pH 2 to 3 with HCl, and
subsequently neutralized to pH 7 to 8 with NaOH.
Acidification/neutralization has also been used to dissociate
inhibitor complexes from interfering in an ELISA to measure human
tissue plasminigen activator (tPA) in plasma samples (Ranby et al.,
Thromb. Haemost. 61:409-414 (1989). In this procedure, samples were
acidified by adding an equal volume of 1 M potassium acetate
buffer, pH 3.9, followed by neutralization with an equal volume of
0.5 M Tris containing 0.2 M NaH.sub.2PO.sub.4. Heat denaturation
and acidification/neutralization have been used to dissociate
immune complexes that interfere with an ELISA to detect HIV-1 p24
antigen in serum or plasma (Schupbach et al., AIDS 10:1085-1090
(1996).
[0274] The effect of acidification on assay sensitivity and
robustness was evaluated.
[0275] Standards were incubated under different
acidification/neutralization conditions with the following
acidification reagents and neutralization buffers: (1) acidify with
0.1 M glycine, pH 2.7, neutralize with 0.5 M Tris-HCl, pH 8.5; (2)
acidify with 0.5 M acetic acid and 0.2 M NaCL, neutralize with 0.5
M Tris-HCl, pH 8.5; (3) acidify with 0.5 N HCl, neutralize with 0.5
M Tris-HCl, pH 8.5; and (4) acidify with 2 N HCl, neutralize with 2
N NaOH; or no acid treatment.
[0276] Pegylation of AvPAL decreased the accessibility of capture
antibodies (i.e., BP79ex) to potential epitopes. Therefore, it was
necessary to acidify the pegylated AvPAL to reliably detect these
epitopes. Treatments (1) and (3) gave better results than treatment
(4) or no treatment (i.e., buffer curve). Treatment (3), however,
often showed a viscous, white precipitate at the higher end
concentrations that drastically lowered the signal. Treatment (2)
also showed improvement over no treatment, but was not deemed
significant over treatment (1) in the presence of plasma.
Therefore, treatment (1) was deemed the most feasible and reliable
acidification method.
Quantitative Range
[0277] The quantitative range of the ELISA assay was determined by
running standard curves of rAvPAL-PEG ranging from 1000 to 0.244
ng/mL when diluted in 2% rat plasma. The quantitative range was
established by running Quality Controls (QCs) from a stock of
rAvPAL-PEG at various concentrations in 2% rat plasma
(LiHeparin).
[0278] The standard curve, for example, of 0, 0.244, 0.977, 3.906,
15.625, 62.5, 250, and 1000 ng/mL rAvPAL-PEG in 2% plasma was
evaluated. Curve fits were generated by plotting the absorbance at
450 nm for the average of each triplicate versus nominal
concentration and fitting with the four-parameter model. The
accuracy of back-calculation of each point on the standard curve
was evaluated along with the R.sup.2 value for the overall fit.
[0279] The four-parameter model in SoftMax Pro generated a high
quality fit for standard curves constructed in 2% rat, monkey, and
human plasma. The back-calculation accuracy of the expected
concentration for at least 6 of the 7 non-zero standards was
86-124%, 76-110%, and 92-104% for rat, monkey and human plasma,
respectively.
[0280] To establish the upper limit of quantitation (ULOQ),
rAvPAL-PEG was prepared at 350, 300, and 200 ng/mL. The lower limit
of quantitation (LLOQ) was prepared at 5, 3, and 2 ng/mL. For rat
plasma, the ULOQ and LLOQ were 350 ng/mL and 5 ng/mL respectively,
with accuracy between 71-74%. For monkey plasma, the ULOQ was 350
ng/mL and the LLOQ was 2 ng/mL, with accuracy between 75-94%. For
human plasma, the ULOQ was 350 ng/mL and the LLOQ was 2 ng/mL, with
accuracy between 73-97%.
Selectivity
[0281] The selectivity of the ELISA assay was determined by
analyzing samples of rAvPAL-PEG in 2% plasma from different lots of
rat, monkey or human plasma.
[0282] Selectivity is a measure of variation in accurately
determining analyte concentration between different lots of plasma
due to interfering substances. The accuracy of determining
rAvPAL-PEG concentration in plasma from multiple individuals was
evaluated by analyzing samples of 200, 20, and 6 ng/mL rAvPAL-PEG
in 2% plasma from three lots of rat, monkey, or human plasma. These
controls were compared against a standard curve constructed in a 2%
pooled plasma lot from each species. The relative accuracy for each
lot of rat plasma (in relation to the pooled plasma) was 46%-115%
for 6, 20, and 200 ng/mL. However, if the 20 ng/mL concentration is
not considered, the relative accuracy across different lots of rat
plasma is 84%-115%. The relative accuracy for monkey plasma was
84-153% for 6, 20, and 200 ng/mL. The relative accuracy for the
human selectivity experiment was 83-246% for 6, 20, and 200 ng/mL.
However, if one sample was excluded from the analysis, the accuracy
across human plasma lots was 83%-123%. In addition, the high
quality control had low back calculated concentration, which skewed
nominal and relative accuracy. Because the variable accuracy
appears random across the high, mid, and low concentrations,
selectivity is not likely to be a major factor.
Specificity
[0283] The specificity of the ELISA assay for the rAvPAL-PEG
molecule when challenged with AvPAL or an inactivated PEG molecule
was evaluated. A standard curve was prepared from 1000 to 0.244
ng/mL rAvPAL-PEG. AvPAL and inactivated PEG were spiked into plasma
separately and tested in the standard ELISA assay.
[0284] The absorbance for rAvPAL-PEG samples was proportional to
concentration across the quantitative range. The absorbance for 200
ng/mL inactivated PEG, unpegylated AvPAL, and an unrelated protein,
such as recombinant human arylsufatase B (rhASB), was below the
limit of detection (LOD), indicating that the assay is specific to
rAvPAL-PEG.
Interference from Plasma Matrix
[0285] The effect of different matrices (i.e., rat, monkey and
human plasma) on the concentration-response relationship was
evaluated. Standard curves in plasma at various percentages from
different species were prepared, and the concentration of standards
in the presence of plasma as compared to the concentration of
standards in 2% plasma was calculated to determine the accuracy
relative to the nominal concentration.
[0286] Samples of rAvPAL-PEG at 200, 20, and 6 ng/mL in 10%, 5%,
and 2% plasma were compared against standard curves constructed in
2% rat, monkey, and human plasma. The accuracy was 78%-122% for 6,
20, and 200 ng/mL in 2% rat, monkey, and human plasma. In 5% human
plasma, the accuracy was 79%-98% for 6, 20, and 200 ng/mL.
Therefore, the rat, monkey, and human plasma dilutions of 1:50 (2%)
are acceptable for sample analysis. Human plasma dilution of 1:25
(5%) was also found to be acceptable. Plasma dilutions of 1:10
(10%) were not acceptable because accuracies in these matrices were
too low for rat and monkey plasma. For 10% human plasma, the
accuracy was slightly better, but still not acceptable across the
entire range (6-200 ng/mL).
Interference from Antibodies
[0287] The interfering effect of anti-rAvPAL-PEG antibodies on the
concentration-response relationship was evaluated by pre-incubating
combinations of anti-rAvPAL-PEG antibodies (BP80 and BP79ex) at
various concentrations with rAvPAL-PEG at various concentrations
for 30 min, carrying out the standard acidification procedure, and
then examining the accuracy of the controls in the presence of
increasing amounts of antibody.
[0288] Samples of 200, 20 and 6 ng/mL rAvPAL-PEG in rat plasma
containing 0, 4, 40, and 40 ng/mL affinity-purified rabbit
anti-rAvPAL-PEG antibody (BP79ex) were analyzed relative to a
standard curve in 2% plasma. BP79ex is the capture antibody for
this ELISA. Accuracy of the 6 ng/mL rAvPAL-PEG concentrations
decreased as antibody concentrations increased. The relative
accuracy of 200 ng/mL rAvPAL-PEG was within 87%-95% for up to 400
ng/mL antibody versus 0 ng/mL antibody. However, nominal accuracy
for 200 ng/mL was poor even without interfering antibody. The
relative accuracy for 20 ng/mL was within 93%-96% for up to 400
ng/mL antibody versus 0 ng/mL antibody. The relative accuracy for 6
ng/mL was 76%-107% for up to 400 ng/mL antibody versus 0 ng/mL
antibody. Although it is difficult to predict the likelihood or
impact of antibody interference on accuracy prior to non-clinical
and clinical studies, we did not measure a clear effect of BP79ex
on this assay.
Dilution Linearity
[0289] To ensure that accurate results are reported independent of
the final dilution factors that fall within the quantitative range
of the assay, the dilution linearity was investigated for
rAvPAL-PEG. Samples of 1000, 200, and 20 ng/mL rAvPAL-PEG in 2% rat
plasma were analyzed at dilutions of 1:2, 1:4, 1:8, and 1:16 (in
addition to the initial 1:50 dilution). For results within the
quantitative range of the assay, the plasma concentration was
calculated as dilution factor multiplied by the concentration of
the dilution. Within the quantitative range of the assay, the
percent accuracy for individual dilutions was within 69%-116% of
the expected value. The 1000 ng/mL sample did not accurately
dilute.
Precision and Accuracy
[0290] To assess precision, samples of rAvPAL-PEG were prepared at
200, 20 and 6 ng/mL in 2% rat plasma and analyzed against standard
curves. Two analysts evaluated intra-assay precision and
inter-assay precision with three experiments over at least a 2-day
period. Intra-assay (within run) precision (CV %) ranged from
1.6%-11.9% across the quality control concentrations tested.
Inter-assay (between runs) precision (CV %) was 17.7%, 10.6% and
17.3% for 200 ng/mL, 20 ng/mL and 6 ng/mL, respectively. Accuracy
was 85.9%, 115.9% and 103.3% respectively for these three quality
controls.
Robustness of Reagent Concentrations and Incubation Times
[0291] The capacity of the ELISA assay to remain unaffected by
small, but deliberate variations in the method parameters and
provide an indication of its reliability during normal usage was
determined.
[0292] Standard curves of rAvPAL-PEG were analyzed using minimum
and maximum reagent concentrations of BP79ex, and anti-IgM at 90%
and 110% of the optimized values of 1 .mu.g/mL. The accuracy of the
back-calculated minimum reagent concentration (90%) standard curve
did not meet acceptance criteria and had accuracies of 25%-41%
across the standard curve range. The accuracy of the
back-calculated maximum reagent concentration (110%) plate ranged
from 82%-126% across the quantitative range. The low end of the
curve suffered the most from the deviation from optimal conditions.
Overall, the maximum concentration standard curve was acceptable
with 5 of 7 non-zero points demonstrating good accuracy. It is
recommended that the optimal conditions are adhered to, but some
modest increase in BP79ex and anti-IgM antibody concentration can
be tolerated.
[0293] A minimum and a maximum plate were tested with the 10%
variation away from the optimal incubation times of each critical
assay steps. The cumulative decrease in incubation times at each
critical step adversely impacted accuracy of quality controls. The
minimal incubation plate had nominal accuracies of 71%-138% for the
200, 20, and 6 ng/mL quality controls. In contrast, the maximum
incubation time-plate had excellent curve fit and acceptable
accuracy (99%-121%). Therefore, it is recommended that the optimal
incubation times are adhered to, but some modest (10% increase) in
time of incubation at each step is likely to be tolerated in the
assay.
Reagent Stability
[0294] The stability of the reagents (e.g., antibodies and quality
controls) over a time period equal to the typical sample
preparation, sample handling, and analytical run times using the
intended storage temperatures was characterized.
[0295] Samples of 200, 20 and 6 ng/mL rAvPAL-PEG were frozen at
-70.degree. C. Samples subjected to multiple freeze-thaw cycles (FT
1-FT 3), or storage overnight at 4.degree. C., or for 4 hours at
RT, were analyzed against a freshly prepared standard curve along
with freshly thawed samples (FT 0). The signal for the 6 ng/mL
samples was above the limit of quantitation for all stability tests
and will not be considered herein. However, the mid- and
high-quality controls (20 ng/mL and 200 ng/mL) were stable after 3
freeze thaw cycles with nominal accuracy of 114% and 85%,
respectively. The stability of the mid- and high-quality controls
(20 ng/mL and 200 ng/mL) stored overnight at 4.degree. C. had
nominal accuracies of 89% and 66%, and relative accuracies of 101%
and 107%, respectively. RT stability after 4 hours demonstrated
nominal accuracies of 65% for the high quality control, and 74% for
the mid quality control, and relative accuracies of 105% and 84%,
respectively. These values indicate that test samples could
tolerate up to three freeze-thaw cycles. Storage of test samples
overnight at 4.degree. C. or for greater than 4 hours at RT is
tolerated.
Example 9
Detection of IgG Antibodies Specific to Anabaena variabilis
Phenylalanine Ammonia-Lyase in Serum
[0296] The purpose of this assay development was to create a method
to assess the immunogenicity of recombinant Anabaena variabilis
phenylalanine ammonia-lyase (AvPAL) or variant thereof. In
particular, an enzyme-linked immunosorbent assay (ELISA) method was
developed to detect IgG antibodies to AvPAL in serum.
Materials
[0297] The following enzymes were used: AvPAL (unpegylated enzyme)
and rAvPAL-PEG (pegylated enzyme) in formulation buffer (10 mM
Tris-HCl, 150 mM NaCl, pH 7.5); see Examples 1, 2 and 4 above, as
well as U.S. Pat. No. 7,531,341.
[0298] The following antibodies were used: BP79: Protein G-purified
rabbit anti-rAvPAL-PEG IgG (Positive Control) in 1.times.DPBS, pH
7.0-7.2; BP79ex: affinity-purified rabbit anti-rAvPAL-PEG IgG
(Positive Control) in 1.times.DPBS, pH 7.0-7.2; BP80: Protein
G-purified rabbit anti-rAvPAL-PEG IgG (Positive Control) in
1.times.DPBS, pH 7.0-7.2; BP14: rabbit anti-recombinant human
arylsulfatase B (rhASB) antiserum; J3549: purified rabbit
anti-rhASB IgG; quality control (QC) high positive control (HP)
anti-rAvPAL-PEG neat rat serum, 042-371 (SNBL USA, Inc.); QC mid
positive control (MP) anti-rAvPAL-PEG neat rat serum, 042-372 (SNBL
USA, Inc.); QC low positive control (LP) anti-rAvPAL-PEG neat rat
serum, 042-372 (SNBL USA, Inc.); detection reagent 1:
HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat.
No. 715-065-150); detection reagent 2: HRP-conjugated goat anti-rat
IgG Fc (Jackson ImmunoResearch, Cat. No. 112-035-008); detection
reagent 3: HRP-conjugated goat anti-rat IgG+IgM (H+L) (Jackson
ImmunoResearch, Cat. No. 111-036-068); detection reagent 4:
HRP-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch,
Cat. No. 111-036-045); detection reagent 5: HRP-conjugated donkey
anti-rabbit IgG (H+L) (Abcam, Cat. No. ab6721-1); detection reagent
6: HRP-conjugated goat anti-human IgG Fc (Jackson ImmunoResearch,
Cat. No. 111-036-098); detection reagent 7: HRP-conjugated goat
anti-human IgA+IgG+IgM (H+L) (Jackson ImmunoResearch, Cat. No.
111-036-064); and detection reagent 8: goat anti-rabbit IgG Fc
(Jackson ImmunoResearch, Cat. No. 111-006-046).
[0299] The following matrices were used: C57 mouse serum
(Fitzgerald, Cat No. 88-NM35); C57BL6 mouse serum (Bioreclamation,
Inc., Cat No. MSESRM.C57); C57BL6 mouse serum (Rockland, Code No.
D208-00-0050); pooled and individual naive-normal rat serum
(Bioreclamation, Inc., Cat No. RATBREC); pooled naive-normal Beagle
dog serum (Bioreclamation, Inc., Cat No. BGLBREC); pooled
naive-normal rabbit serum (Bioreclamation, Inc., Cat No. RABBREC);
pooled and individual naive-normal Cynomolgous monkey serum,
Mauritius origin (Bioreclamation, Inc., Cat No. CYNBREC, CYNSRM);
and pooled and individual naive-normal human serum (Bioreclamation,
Inc., Cat No. HUMSRM; The Binding Site, Cat No. CUS057).
[0300] The following ELISA reagents were used: coating buffer:
1.times.DPBS (Mediatech, Fisher Cat No. MT-21-031-CV); wash buffer
1: 1.times.DPBS/0.1% Polysorbate-20/0.05% Proclin 300; wash buffer
2: 1.times.DPBS/0.25% Polysorbate-20/0.05% Proclin 300; bovine
serum albumin (BSA), fraction V, Omnipure (EMD, VWR Cat No. 2930);
blocking buffer 1: Blocker Casein in PBS (Pierce, Cat No. 37528);
blocking buffer 2: Blocker Casein in TBS (Pierce, Cat No. 37532);
blocking buffer 3: SuperBlock T20 TBS (Pierce, Cat No. 37536);
blocking buffer 4: StartingBlock T20 PBS (Pierce, Cat No. 37539);
development reagent: TMB Peroxidase EIA Substrate Kit, 250 mL
(BioRad, Cat No. 172-1066); and stop solution: 2N
H.sub.2SO.sub.4.
[0301] The following chemicals were used: de-ionized water (MilliQ
filtered); Dulbecco's phosphate buffered saline (DPBS), 1.times.
(Mediatech, Fisher Cat No. MT-21-031-CV); Dulbecco's phosphate
buffered saline (DPBS), 10.times. (Mediatech, Fisher Cat No.
MT-20-031-CV); sulfuric acid (H.sub.2SO.sub.4), 11 N (VWR, Cat No.
VW3481-1); polysorbate-20 (Acros Organics, VWR Cat No. 233360010);
and Proclin 300 (Supelco, Cat No. 4-8126).
[0302] The following plasticware and other materials were used:
assay dilution block: 96-well, Polypropylene, V-bottom,
non-sterile, 2 mL well (VWR, Cat No. 40002-012); ELISA plate:
96-well, Maxisorp, flat bottom "F" (Nalge/Nunc International, VWR
Cat No. 12-565-135); microcentrifuge tubes, 1.5 mL (USA Scientific,
VWR Cat No. 1615-550); microcentrifuge tubes, 2.0 mL (Eppendorf,
VWR Cat No. 62111-754); microplate adhesive film (USA Scientific,
Cat No. 2920-0000); pipetman tips: 20 .mu.L, 250 .mu.L, and 1000
.mu.l, (Rainin Instruments, Cat Nos. GPS-L1000, GPS-L250, GPS-L10);
reagent reservoir, 50 mL capacity (Corning, VWR Cat No. 82026-350);
and serological pipettes, 10 mL and 25 mL (VWR, Cat No. 13-678-11D
and 13-678-11E).
[0303] The following equipment and software were used: El.times.405
Select Plate Washer (Bio-Tek); multichannel pipetman, 12-well, LTS
20-200 .mu.L (Rainin Instruments); multichannel pipetman, 12-well,
LTS 100-1200 .mu.L (Rainin Instruments); pipet-aid, multi-speed,
Drummond (VWR, Cat No. 13-681-15E); SOFTmax Pro v 3.0 (Molecular
Devices); SpectraMax PLUS Microplate Spectrophotometer (Molecular
Devices); timer, three channel alarm (VWR, Cat No. 62344-600); and
titer plate shaker (Barnstead/Lab-Line Instruments).
ELISA Protocol
[0304] Preparation of Samples. Serum Samples for Screening and
Titering were Initially diluted 1:50. For titering, the samples
were then serially diluted three-fold in Blocking Buffer 1. A
four-dilution factor cluster was chosen based on the screening
assay signal where one dilution factor sample will fall above the
established cutpoint. For suspected high titers, samples were
diluted at a higher initial dilution, and then serially diluted
three-fold thereafter.
[0305] Preparation of Positive Controls. Positive Controls were
Prepared in Blocking Buffer 1 at 10, 100, and 500 ng/mL. The
positive control was BP80, a Protein G-purified rabbit polyclonal
antibody against rAvPAL-PEG. An analogous lot of BP79 and BP79ex
(affinity-purified anti-AvPAL antibodies from BP79) were also used
in specialized applications during development. The detection
reagent used for this control was Detection Reagent 5, an
HRP-conjugated goat anti-rabbit-IgG diluted to 1:5,000 in Blocking
Buffer 1.
[0306] ELISA Procedure. AvPAL was prepared at 1 .mu.g/mL in Coating
Buffer (1.times.DPBS), dispensed at 100 .mu.L per well into the
ELISA plate (Nalgen/Nunc MaxiSorp surface), and incubated 12 to 20
hours (overnight) at 4.degree. C. Blocking Buffer 1 (Blocker Casein
PBS) was added at 300 .mu.L/well and incubated on a shaker for 1
hour at room temperature (RT). Blocking Buffer 1 was removed by
pouring into a waste reservoir. Serum samples and controls were
aliquoted at 100 .mu.L/well according to a template plate map
configuration and incubated on a shaker for 1 hour at RT.
HRP-conjugated Detection Reagents 2, 5 and 6 were prepared in
Blocking Buffer 1 at a 1:5,000 dilution, dispensed at 100
.mu.L/well, and incubated on a shaker for 1 hour at RT. TMB
development substrate was added at 100 .mu.L/well and the plate was
incubated on a shaker for 15 minutes at RT. Stop Solution was added
at 100 .mu.L/well and the plate was read immediately at 450 nm.
Assay Design Parameters
[0307] Development of a suitable assay to detect AvPAL-specific
antibodies of a particular isotype in a body fluid or tissue sample
from a mammal requires identifying an immunoassay and reaction
conditions that are sensitive to detect small amounts
AvPAL-specific antibodies of a particular isotype in a sample
containing AvPAL-specific antibodies of different isotypes, as well
as large amounts of non-specific antibodies.
[0308] To identify the parameters for assay reagents and format to
detect AvPAL-specific antibodies of the IgG isotype, the
concentrations for capture and detection reagents, as well as the
incubation times and methods to reduce background signal, were
determined.
[0309] Conditions optimal for assay development included coating
with 1 .mu.g/mL PAL, blocking with Blocker Casein PBS (Buffer 1),
and then testing samples and controls. Detection of anti-AvPAL
antibodies was achieved by using HRP-conjugated species-specific
anti-IgG antibodies at 1:5,000 dilution. Additional experiments
were performed to evaluate the blocking buffer selection, detection
antibody evaluation, and PAL coating condition. The Casein PBS
block decreased background as compared to StartingBlock and
SuperBlock. Different anti-rabbit antibodies detecting rabbit BP80
positive control in 2% naive pooled rat, monkey, and human serum
were compared. Jackson Labs HRP-conjugated anti-rabbit (Detection
Reagent 4) worked best with our BP79, BP79ex, and BP80 positive
controls. During sample testing, species-specific HRP conjugated
secondary antibodies were used (Detection Reagent 2 for rat and
Detection Reagent 6 for monkey/human). One (1) .mu.g/mL PAL was
found to be a superior coating concentration for good
signal-to-noise ratio (i.e., positive signal versus
negatives/background).
Cutpoint Determination
[0310] To determine the threshold for a positive result (i.e.,
cutpoint), the signal distribution for individual naive sera and a
pooled serum lot was tested. The cutpoint was set at a 95%
confidence interval to provide a 5% false positive rate.
[0311] The standard ELISA protocol described above was used. Twenty
naive samples (10 males and 10 females) were examined for signal
distribution in 2% serum (i.e., 50-fold dilution). The average
assay signal for multiple naive individuals was calculated from two
replicates in the same manner as unknown samples. The percent
difference relative to a pooled lot was calculated. The average and
standard deviation were also calculated. To obtain the cutpoint
factor, the standard deviation was multiplied by 1.65 (i.e., the
student's t-factor for a one-tailed 95% confidence interval). The
cutpoint factor was then added to the absorbance obtained for the
pooled sample to obtain the plate cutpoint for each species.
[0312] Cutpoints were established by: assaying 20 lots of
individual naive sera per species (e.g., rat, monkey, and human).
Two replicates were used for each serum sample to match the
samples, which were also analyzed in duplicate. The average signal
(A450) for the samples in 2% sera was 0.109 for rat, 0.169 for
monkey, and 0.193 for human. This was lower than the normal pooled
serum at 0.215 for rat and 0.221 for human, and similar to the
signal for pooled serum at 0.163 for monkey. It was also notable
that the A450 varied among individual sera from 0.065 to 0.227 in
rat, 0.060 to 0.597 in monkey, and 0.083 to 0.401 in human. The CV
% between samples was 35.2% in rat, 88.1% in monkey, and 44.9% in
human. There was little difference between male and female rat
samples, but human female sera had a higher signal than male sera
(0.227 versus 0.177). Monkey male sera had a higher signal than
female (0.220 vs. 0.163). The cutpoint factors for rat, monkey and
human were 0.0635, 0.279, and 0.143, respectively. Based on the
determination of the cutpoint factors, the cutpoint for rat, monkey
and human were 0.279, 0.442, and 0.364, respectively.
Free Drug Interference
[0313] To evaluate the effect of free drug (e.g., rAvPAL-PEG) on
the concentration-response relationship, Protein G-purified rabbit
anti-rAvPAL-PEG (BP80) was prepared at 10, 100, and 500 ng/mL in
naive serum containing 0, 10, 100, 1,000, and 10,000 ng/mL
rAvPAL-PEG. The signal accuracy with and without free drug was
compared following the standard ELISA protocol described above.
[0314] The signal accuracy using 2% rat and human sera was 80-100%
for all QCs, regardless of free drug concentration. For 500 ng/mL
BP80 QC in 2% monkey serum, the accuracy fell below 80% at 100
ng/ml rAVPAL-PEG and higher. The low and mid QCs in monkey serum
were within .+-.15%.
Matrix Interference
[0315] To evaluate the effect of matrix (e.g., serum) on the
concentration-response relationship, the accuracy was determined
from the signal of samples diluted into increasing amounts of
serum. Protein G-purified anti-rAvPAL-PEG rabbit polyclonal
antibody (BP80) was prepared at 10, 100, 250, 500, 750, and 1000
ng/mL in naive-pooled serum from rat, monkey and human at 2%, 5%,
and 10%.
[0316] The concentration-response relationship of samples diluted
in Casein Blocking Buffer versus 2% rat, monkey, and human serum
was determined. The 1,000 ng/mL QC was omitted from the analysis
due to signal saturation in some samples (OD=4.0). Accuracy in 2%
serum was within .+-.25% for all QCs for all three species
(90.7-113.0% for rat, 74.5-112.2% for monkey, and 86.0-102.0% for
human). Serum increased the signal in 10 ng/mL QC, and attenuated
the signal in the 100-750 ng/mL QCs. The accuracy remained within
.+-.25% for 5% and 10% human serum, but was unacceptable for some
rat and monkey QCs at these serum concentrations. Therefore, matrix
interference is acceptable if rat, monkey, and human sera
concentrations remain at 2% in the samples.
Limit of Detection
[0317] To determine the lowest amount of analyte that can be
detected but not necessarily quantitated (i.e., the limit of
detection or LOD, which is the analyte concentration for which the
measured mean signal is higher than the cutpoint), a known sample
of AvPAL-seropositive rat individual sera positive QC (study 042,
animal 371, pooled days 32-59) was prepared at dilutions of
1:3,000, 1:9,000, 1:27,000, and 1:81,000 in 2% rat serum. Using a
reference range of anti-AvPAL affinity-purified rabbit IgGs
(BP79ex), these dilutions corresponded to approximately 8.0, 3.1,
1.4, and 0.9 ng/mL, respectively. A known sample of
AvPAL-seropositive human individual sera positive QC(CUS057 adult
male lot 50) was prepared at dilutions of 1:250, 1:750, 1:2,250,
1:6,750, and 1:20,250 in 2% human serum. Using a reference range of
BP79ex, these dilutions corresponded to 8.6, 4.2, 2.1, 1.6, and 1.1
ng/mL, respectively. The signal from the dilutions of prepared
concentrations was compared to the signal from a 1:50 dilution of
naive pooled serum. Duplicates for each concentration were analyzed
and reported as positive or negative versus the rat or human
cutpoints. LOD in monkey serum was not calculated at present due to
lack of monkey positive control.
[0318] The lowest concentration that was consistently above the
cutpoint (i.e., 4 out of 5 replicates) determined the LOD. The LOD
was associated with 1.4 ng/mL IgG concentration for the rat
positive control, and 1.6 ng/mL IgG concentration for the human
positive control.
Reagent Stability
[0319] To characterize the robustness of preparation and storage
stability of the reagents, reagent quality and integrity was
observed throughout assay development.
[0320] The reagents should be stable over a time period equal to
the typical sample preparation, sample handling, and analytical run
times using the intended storage temperatures. The quality controls
should remain above the pooled serum and the signal proportionality
from freshly prepared quality controls must be maintained from the
beginning through the end of assay development. No significant
changes in control antibody (BP80), PAL coating, Casein block, or
detection antibody were observed.
Robustness
[0321] To determine its robustness, the capacity of the assay to
remain unaffected by small, but deliberate variations in method
parameters, and to provide an indication of its reliability during
normal usage, was determined. Although not formally tested, most
assay parameters and characteristics behaved robustly throughout
assay development.
[0322] The assay was somewhat sensitive to changes in coating
concentration and coating time. The detection antibody seemed to
tolerate a range of dilutions (1:2,000-1:10,000). Other assay
parameters appeared to be robust.
Selectivity
[0323] To determine selectivity (i.e., the ability of the assay to
accurately detect the analyte in the presence of different matrix
lots), the accuracy of detecting anti-AvPAL control spiked into 5-6
different lots of 2% individual serum at 0, 10, 100, 250, 500, 750,
and 1,000 ng/mL was tested.
[0324] The 1,000 ng/mL QC was omitted from the analysis due to
signal saturation in some samples (A450=4.0). At the 500-750 ng/mL
concentrations of anti-PAL BP80, the accuracy was between 77.8 and
105.3%, between 74.3 and 94.2%, and between 82.2 and 102.2% for the
rat, monkey and human serum lots. At and below 250 ng/mL BP80, the
reproducibility of the signal was even tighter (i.e., all
accuracies were within 80-120% and most were very close to 100%).
This indicates that lot-to-lot variation will probably not be a
serious concern during assay validation.
Specificity
[0325] To determine specificity of the assay for anti-AvPAL
antibodies and for species cross-reactivity of detection
antibodies, rabbit anti-rhASB antibodies (1,000 ng/mL) were tested
for cross-reactivity to immobilized PAL. The signals to BP79ex and
BP80 rabbit anti-PAL antibodies at high, mid, and low QCs were
compared. The ability of excess PAL to compete away positive signal
(i.e., confirmatory assay) was determined.
[0326] Recombinant human arylsufatase B (rhASB) has no significant
homology and identity with AvPAL. The detection response for the
positive control was proportional to analyte concentration, and all
QCs had mean signals above the purified rabbit anti-rhASB
antibodies (BP14 and J3549) and buffer signals. BP14, J3549, and
buffer signals all fell below the cutpoints of the three species.
Therefore, the assay is specific for the detection of AvPAL
antibodies. Rabbit polyclonal antibodies to other PAL variants
(BP60ex for Rhodosporidium toruloides PAL ((RtPAL); BP64 for Nostoc
punctiforme PAL (NpPAL)) also demonstrated varying levels of
cross-reactivity with AvPAL at equivalent concentrations, but
should not be present in samples from pre-clinical and clinical
AvPAL studies.
[0327] The confirmatory assay also demonstrated specificity for
AvPAL antigen in some mammal's samples. Adding excess rAvPAL-PEG
(10 .mu.g/mL) deceased the positive control signal by a percentage
greater than (cutpoint factor)/(cutpoint). A sample with
.gtoreq.22.8% decrease in signal in the confirmatory assay was
considered a true positive for rat serum. The 042-study rat
positive control satisfied this criterion. We also noted
seropositive samples from so-called naive human and monkey samples.
A sample with .gtoreq.63.1% decrease in signal in the confirmatory
assay was considered a true positive for monkey serum. A sample
with .gtoreq.39.3% decrease in signal in the confirmatory assay was
considered a true positive for human serum.
Example 10
Detection of IgM Antibodies Specific to Anabaena variabilis
Phenylalanine Ammonia-Lyase (AvPAL) in Serum
[0328] The purpose of this assay development was to create a method
to assess the immunogenicity of recombinant Anabaena variabilis
phenylalanine ammonia-lyase (AvPAL) or variant thereof. In
particular, an enzyme-linked immunosorbent assay (ELISA) method was
developed to detect IgM antibodies to AvPAL in serum.
Materials
[0329] The following therapeutic enzymes were used: AvPAL
(unpegylated enzyme) and rAvPAL-PEG (pegylated enzyme) in
formulation buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.5); see
Examples 1, 2 and 4 above, as well as U.S. Pat. No. 7,531,341.
[0330] The following antibodies were used: BP79: Protein G-purified
rabbit anti-rAvPAL-PEG IgG (Positive Control) in 1.times.DPBS, pH
7.0-7.2; BP79ex: affinity-purified rabbit anti-rAvPAL-PEG IgG
(Positive Control) in 1.times.DPBS, pH 7.0-7.2; BP80: Protein
G-purified rabbit anti-rAvPAL-PEG IgG (Positive Control) in
1.times.DPBS, pH 7.0-7.2; BP14: rabbit anti-recombinant human
arylsulfatase B (rhASB) purified IgG; rat quality control (QC) at
high, mid and low dilutions (HP=1:50, MP=1:150, LP=1:450) of
anti-AvPAL IgM in neat rat serum (unpurified individual rat sera
confirmed positive against AvPAL from Bioreclamation, Inc., Cat No.
RATBREC.34982F and RATBREC.26135M); monkey quality control (QC) at
high, mid and low dilutions (HP=1:50, MP=1:150, LP=1:450) of
anti-AvPAL IgM in neat monkey serum (unpurified individual monkey
sera confirmed positive against AvPAL from Bioreclamation, Inc.,
Cat No. CYNSRM.28918F); human quality control (QC) at high, mid and
low dilutions (HP=1:50, MP=1:150, LP=1:450) of anti-AvPAL IgM in
neat human serum (unpurified individual human sera confirmed
positive against AvPAL from The Binding Site, Cat No. CUS057 lot 3
and Bioreclamation, Inc., Cat No. HUMSRM.BRH73661); detection
reagent 2: HRP-conjugated goat anti-rat IgM Fc (Jackson
ImmunoResearch, Cat. No. 112-036-075); detection reagent 4:
HRP-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch,
Cat. No. 111-036-045); detection reagent 5: HRP-conjugated donkey
anti-human IgM Fc (Jackson ImmunoResearch, Cat. No.
709-036-064).
[0331] The following matrices were used: pooled and individual
naive-normal Sprague Dawley rat serum (Bioreclamation, Inc., Cat
No. RATSRM.RATBREC); pooled and individual naive-normal Cynomolgous
monkey serum, Mauritius origin (Bioreclamation, Inc., Cat No.
CYNSRM.CYNBREC); and pooled and individual naive-normal human serum
(Bioreclamation, Inc., Cat No. HUMSRM; The Binding Site, Cat No.
CUS057).
[0332] The following ELISA reagents were used: coating buffer:
1.times.DPBS (Mediatech, Fisher Cat No. MT-21-031-CV); wash buffer
1: 1.times.DPBS/0.1% Tween-20/0.05% Proclin 300; wash buffer 2:
1.times.DPBS/0.25% Tween-20/0.05% Proclin 300; bovine serum albumin
(BSA), fraction V, Omnipure (EMD, VWR Cat No. 2930); blocking
buffer 1: Blocker Casein in PBS (Pierce, Cat No. 37528); blocking
buffer 2: Blocker Casein in TBS (Pierce, Cat No. 37532); blocking
buffer 3: SuperBlock T20 TBS (Pierce, Cat No. 37536); blocking
buffer 4: StartingBlock T20 PBS (Pierce, Cat No. 37539);
development reagent: TMB Peroxidase EIA Substrate Kit, 250 mL
(BioRad, Cat No. 172-1066); and stop solution: 2 N
H.sub.2SO.sub.4.
[0333] The following chemicals were used: de-ionized water (MilliQ
filtered); Dulbecco's phosphate buffered saline (DPBS), 1.times.
(Mediatech, Fisher Cat No. MT-21-031-CV); Dulbecco's phosphate
buffered saline (DPBS), 10.times. (Mediatech, Fisher Cat No.
MT-20-031-CV); sulfuric acid (H.sub.2SO.sub.4), 11 N (VWR, Cat No.
VW3481-1); Tween-20 (Acros Organics, VWR Cat No. 233360010); and
Proclin 300 (Supelco, Cat No. 4-8126).
[0334] The following plasticware and other materials were used:
recombinant human arylsulfatase B (rhASB) in 1.times.DPBS; assay
dilution block: 96-well, polypropylene, V-bottom, non-sterile, 2 mL
well (VWR, Cat No. 40002-012); ELISA plate: 96-well, Maxisorp, flat
bottom "F" (Nalge/Nunc International, VWR Cat No. 12-565-135);
microcentrifuge tubes, 1.5 mL (USA Scientific, VWR Cat No.
1615-550); microcentrifuge tubes, 2.0 mL (Eppendorf, VWR Cat No.
62111-754); microplate adhesive film (USA Scientific, Cat No.
2920-0000); pipetman tips: 20 .mu.L, 250 .mu.L, and 1000 .mu.L
(Rainin Instruments, Cat Nos. GPS-L1000, GPS-L250, GPS-L10);
reagent reservoir, 50 mL capacity (Corning, VWR Cat No. 82026-350);
and serological pipettes, 10 mL and 25 mL (VWR, Cat No. 13-678-11D
and 13-678-11E)
[0335] The following equipment and software were used: El.times.405
Select Plate Washer (Bio-Tek); multichannel pipetman, 12-well, LTS
20-200 .mu.L (Rainin Instruments); multichannel pipetman, 12-well,
LTS 100-1200 .mu.L (Rainin Instruments); pipet-aid, multi-speed,
Drummond (VWR, Cat No. 13-681-15E); SOFTmax Pro v 3.0 (Molecular
Devices); SpectraMax PLUS Microplate Spectrophotometer (Molecular
Devices); timer, three channel alarm (VWR, Cat No. 62344-600); and
titer plate shaker (Barnstead/Lab-Line Instruments).
ELISA Protocol
[0336] Preparation of Samples: Serum samples for screening were
initially diluted 1:50. For suspected highly positive samples,
dilutions were higher. Screened samples were compared to the
species-specific assay cutpoint. For positive samples above the
cutpoint, the confirmation step was performed to determine whether
the sample was a true positive (i.e., observed signal loss due to
spiked AvPAL in sample).
[0337] Preparation of Positive Controls: Rabbit Positive Controls
were Prepared in Blocking Buffer 1 at 10, 100, and 500 ng/mL BP80.
BP80 is a Protein G-purified rabbit polyclonal antibody against
rAvPAL-PEG. An analogous lot of BP79 and BP79ex (affinity-purified
anti-AvPAL antibodies from BP79) were also used in specialized
applications. These rabbit antibodies were used as surrogate QCs in
some experiments due to scarcity of supply of anti-AvPAL IgM. The
detection reagent used for these controls was Detection Reagent 4,
an HRP-conjugated goat anti-rabbit-IgG diluted to 1:5,000 in
Blocking Buffer 1. Rat IgM positive controls were prepared in
Blocking Buffer 1 at 1:50 dilution (approximately 2.1 ng/mL IgM).
The rat positive controls were RATBREC.34982F and RATBREC.26135M,
individual rat serum lots seropositive against AvPAL. The detection
reagent used for these controls was Detection Reagent 2, an
HRP-conjugated goat anti-rat-IgM diluted to 1:5,000 in Blocking
Buffer 1. Monkey IgM positive controls were prepared in Blocking
Buffer 1 at 1:150 dilution (approximately 2.8 ng/mL IgM). The
monkey positive control was CYNSRM.28918F, an individual monkey
serum lot seropositive against AvPAL. The detection reagent used
for this control was Detection Reagent 5, an HRP-conjugated donkey
anti-human-IgM diluted to 1:5,000 in Blocking Buffer 1. Human IgM
positive controls were prepared in Blocking Buffer 1 at 1:150
dilution (approximately 2.8 ng/mL IgM). The human/monkey positive
controls were CUS057 lot 3 and HUMSRM.BRH73661, individual human
serum lots seropositive against AvPAL. The detection reagent used
for these controls was Detection Reagent 5, an HRP-conjugated
donkey anti-human-IgM diluted to 1:5,000 in Blocking Buffer 1.
[0338] ELISA Procedure: AvPAL was prepared at 1 .mu.g/mL in Coating
Buffer (1.times.DPBS), dispensed at 1004 per well into the ELISA
plate (Nalge/Nunc MaxiSorp surface), and incubated 12 to 20 hours
(overnight) at 4.degree. C. Blocking Buffer 1 (Blocker Casein PBS)
was added at 300 .mu.L/well and incubated on a shaker for 1 hour at
room temperature (RT). Blocking Buffer 1 was removed by pouring
into a waste reservoir. Serum samples and controls were aliquoted
at 100 .mu.L/well according to a template plate map configuration
and incubated on a shaker for 1 hour at RT. HRP-conjugated
Detection Reagents were prepared in Blocking Buffer 1 at a 1:5,000
dilution, dispensed at 100 .mu.L/well, and incubated on a shaker
for 1 hour at RT. TMB development substrate was added at 100
.mu.L/well and the plate was incubated on a shaker for 15 minutes
at RT. Stop Solution was added at 100 .mu.L/well and the plate was
read immediately at 450 nm.
Assay Design Parameters
[0339] To identify the parameters for assay reagents and format to
identify AvPAL-specific antibodies of the IgM isotype, the
concentrations for capture and detection reagents, as well as the
incubation times and methods to reduce background, were
determined.
[0340] Conditions optimal for assay development included coating
with 1 .mu.g/mL AvPAL, blocking with Blocker Casein PBS (Buffer 1),
and then testing samples and controls. Detection of anti-AvPAL
antibodies was achieved by using HRP-conjugated species-specific
anti-IgM antibodies at 1:5,000 dilution. Casein PBS was the
superior blocker to decrease background when compared to other
Pierce blockers.
Cutpoint Determination
[0341] To determine the threshold for a positive result (i.e.,
cutpoint), the signal distribution for individual naive sera and a
pooled serum lot was tested. The cutpoint was set at a 95%
confidence interval to provide a 5% false positive rate.
[0342] Signal distribution in 2% serum was tested for 20 naive rat,
monkey, and human serum samples (approximately 50% male and 50%
female). The average assay signal for multiple naive individuals
was calculated from two replicates in the same manner as unknown
samples. The percent difference relative to a pooled lot was
calculated. The average and standard deviation were also
calculated. To obtain the cutpoint factor, the standard deviation
was multiplied by 1.65 (i.e., the student's t-factor for a
one-tailed 95% confidence interval). The cutpoint factor was then
added to the pool average to obtain the plate cutpoint for each
species.
[0343] Cutpoints were established by assaying 10-20 lots of
individual naive sera per species (i.e., rat, monkey, and human).
Two replicates were used for each serum sample to match the
samples, which were also analyzed in duplicate. The average signal
(A450) for the samples in 2% sera was 0.385 for rat, 0.341 for
monkey, and 0.167 for human. The naive pooled serum was 0.314 for
rat, 0.187 for monkey, and 0.300 for human. It was also notable
that the A450 varies among individual sera from 0.248 to 0.596 in
rat, 0.152 to 0.627 in monkey, and 0.090 to 0.321 in human. The CV
% between samples was 27.2% in rat, 50.6% in monkey, and 37.2% in
human. There was little difference between male and female rat
samples, but monkey and human female sera had higher signals than
male sera (0.411 versus 0.262 and 0.204 versus 0.130,
respectively). The cutpoint factors for rat, monkey, and human were
0.0635, 0.285, and 0.102, respectively. Based on the determination
of the cutpoint factors, the cutpoints for rat, monkey, and human
were 0.475, 0.472, and 0.402, respectively. The number of false
positives of individual lots for rat, monkey and human were 3/19
(16%), 4/16 (25%), and 0/20 (0%), respectively. Since the desired
rate of false positives in the assay is 5%, the IgM ELISA for rat
and monkey may over-estimate the number of positive samples above
the cutpoint. However, the confirmation step in the assay is
designed to identify true positives versus samples with unusually
high background or nonspecific IgM binding.
Free Drug Interference
[0344] The effect of free drug on the concentration-response
relationship was evaluated. Seropositive QCs against AvPAL from
each species were prepared at low (LP=1:450), mid (MP=1:150), and
high (HP=1:50) QC dilutions in naive serum containing 0, 10, 100,
1,000, and 10,000 ng/mL AvPAL or rAvPAL-PEG. The signal accuracies
with and without free drug were compared.
[0345] The accuracy results for the different rAvPAL-PEG drug
concentrations showed that the accuracy in 2% rat, monkey, and
human sera was 80-110% for all QCs, regardless of free drug
concentration. The QCs that had signals above the cutpoints in
buffer did not fall below the cutpoints in the presence of drug. In
general, the LP had better accuracies than MP or HP. The accuracy
results for the different unpegylated AvPAL concentrations showed
that, similar to the confirmatory data, the accuracy for most of
the QCs at HP and LP dilutions decreased with increasing AvPAL
spike. In many cases, the reduction in accuracy was greater with
unpegylated AvPAL spike versus rAvPAL-PEG
Matrix Interference
[0346] To evaluate the effect of matrix (e.g., serum) on the
concentration-response relationship, the effect of matrix on
accuracy from the signal of QC samples diluted into increasing
amounts of serum was determined. Seropositive QCs against AvPAL
from each species were prepared at low, mid, and high QC dilutions
(1:540, 1:180, and 1:60) in naive pooled serum from rat, monkey and
human each at 1%, 2%, and 10%.
[0347] The accuracy of the QCs at the different serum
concentrations showed that serum served to increase the signal in
most QCs of each species, but attenuated the signal at the 1:60 QC
in human. The accuracy remained within .+-.25% for the mid and low
dilutions in 1%, 2%, and 10% human serum, but was unacceptable for
most rat and monkey QCs. Since the standard 2% naive pooled serum
background for all three species was approximately 0.3 (A450), the
mean signal seemed to be additive with QCs in rat and monkey (i.e.,
the QC signal in 2% serum was approximately equal to the QC signal
in buffer plus 2% serum background signal). Therefore, when the 2%
serum background signal was subtracted from the rat and monkey QCs,
most of the QCs were within .+-.25% accuracy. But the low absolute
signal strength of the rat and monkey QCs (.about.0.8 A450 at HP
1:50 dilution) may have contributed to the significant matrix
interference. This problem is probably not an attribute of the
assay itself, because the human QCs in naive matrix passed
properly.
Relative Limit of Detection (LOD)
[0348] The approximate lowest amount of analyte that can be
detected, but not necessarily quantitated, was determined. The
relative limit of detection or LOD is the analyte concentration for
which the measured mean signal is higher than the cutpoint in 4 out
of 5 replicates. Known samples of AvPAL-seropositive rat
(RATBREC.34982F), monkey (CYNSRM.28918F), and human (CUS057 adult
female lot 3) individual sera positive QCs were prepared at
dilutions of 1:50, 1:150, 1:450, and 1:1,350 in 2% naive pooled
serum. Using a 50-0 ng/mL reference range of
AvPAL-affinity-purified rabbit IgGs (BP79ex), these dilutions
corresponded to approximations of specific anti-AvPAL IgMs. The
signal from the dilutions of prepared concentrations was compared
to the signal from a 1:50 dilution of naive pooled serum.
Duplicates for each concentration were analyzed and reported as
positive or negative versus the rat, monkey, or human
cutpoints.
[0349] To estimate the relative LOD, we compared the signal from
true positive rat, monkey, and human individual serum lots to a
reference curve of BP79ex anti-rAvPAL-PEG affinity purified rabbit
IgGs of known concentration. We therefore made three assumptions:
(1) a comparable concentration of rat, monkey, and human anti-AvPAL
IgMs bound similarly to immobilized AvPAL as the anti-rAvPAL-PEG
rabbit IgG reference, (2) said antibodies are similarly detected
with species-and-chain-specific HRP-conjugated secondaries, and (3)
the presence of .ltoreq.2% naive serum did not greatly affect the
QC signals versus BP79ex. These assumptions allowed for a ballpark
estimate of the relative LOD. The lowest concentration that is
consistently above the cutpoint (4 out of 5 replicates) determines
the relative LOD. A known sample of AvPAL-seropositive rat
individual sera QC(RATBREC.34982F) was prepared at dilutions of
1:50, 1:150, 1:450, and 1:1,350 in 2% rat serum. Using a reference
range of AvPAL-affinity-purified rabbit IgGs (BP79ex), these
dilutions corresponded to approximately 2.1, 0.6, <0.6, and
<<0.6 ng/mL, respectively. A known sample of
AvPAL-seropositive monkey individual sera QC (CYNSRM.28918F) was
prepared at dilutions of 1:50, 1:150, 1:450, and 1:1,350 in 2%
monkey serum. Using a reference range of AvPAL-affinity-purified
rabbit IgGs (BP79ex), these dilutions corresponded to approximately
5.0, 2.6, 0.9, and 0.2 ng/mL, respectively. A known sample of
AvPAL-seropositive human individual sera QC (CUS057 adult female
lot 3) was prepared at dilutions of 1:50, 1:150, 1:450, and 1:1,350
in 2% human serum. Using a reference range of BP79ex, these
dilutions corresponded to 5.9, 2.8, 0.95, and <0.95 ng/mL,
respectively. The LOD was associated with 2.1 ng/mL IgM
concentration for rat positive control, 2.6 ng/mL IgM concentration
for monkey positive control, and 2.8 ng/mL IgM concentration for
human positive control. The true LODs may be higher or lower than
2-3 ng/mL, but cannot be determined without purified,
well-characterized anti-AvPAL IgM antibodies from each of the three
species.
Reagent Stability
[0350] To characterize the robustness of preparation and storage
stability of the reagents, reagent quality and integrity was
observed throughout assay development.
[0351] This assay parameter will be formally tested during
validation. The reagents should be stable over a time period equal
to the typical sample preparation, sample handling, and analytical
run times using the intended storage temperatures. The quality
controls should remain above the pooled serum and the signal
proportionality from freshly prepared quality controls must be
maintained from the beginning through the end of assay development.
No significant changes in control antibody, AvPAL coating, Casein
Block, or detection antibody were observed.
Robustness
[0352] To determine its robustness, the capacity of the assay to
remain unaffected by small, but deliberate variations in method
parameters, and to provide an indication of its reliability during
normal usage, was determined. Although not formally tested, most
assay parameters and characteristics behaved robustly throughout
assay development.
[0353] The assay was somewhat sensitive to changes in coating
concentration and coating time. The detection antibody seemed to
tolerate a range of dilutions (1:2,000-1:10,000). Other assay
parameters appear to be robust.
Selectivity
[0354] The selectivity (i.e., the ability of the assay to
accurately detect analyte in the presence of different matrix lots)
was determined by testing the accuracy of detecting anti-AvPAL QCs
spiked into at least 3 different lots of 2% individual serum at low
(LP=1:450), mid (MP=1:150), and high (HP=1:50) QC dilutions.
[0355] In general, the HP and MP QCs had better accuracies than LP.
For the lots of rat serum, the accuracy varied from 68.7-119.4%,
with all but one QC within .+-.25%. For the lots of monkey serum,
the accuracy varied from 64.5-93.2%, with all but two QCs within
.+-.25%. For the lots of human serum, the accuracy varied from
77.1-110.8%, with all QCs within .+-.25%. This indicates that
lot-to-lot variation will probably not be a serious concern during
assay validation.
Specificity
[0356] To determine the specificity of the assay for anti-AvPAL
IgMs versus species cross-reactivity and foreign
antibodies/antigens, anti-rhASB and anti-AvPAL QCs were tested for
cross-reactivity to immobilized AvPAL and recombinant human ASB
(rhASB). The ability of excess AvPAL to compete away positive
signal (i.e., confirmatory assay) was determined.
[0357] Recombinant human arylsulfatase B (rhASB) has no significant
homology and identity with AvPAL. The determination of specificity
showed that the species-specific anti-AvPAL IgM QCs had a
concentration-dependent A450 response, and the signal detected with
anti-IgM-HRP was always higher than the anti-IgG-HRP signal under
the same conditions. Therefore, the QCs are specific to anti-AvPAL
IgMs versus IgGs. The anti-AvPAL IgM QCs preferentially bound to
immobilized AvPAL versus immobilized rhASB. The anti-AvPAL QC
signals were above the species cutpoints, and the anti-rhASB
negative control was below the cutpoint. Therefore, the assay is
specific for the detection of AvPAL antibodies. The rhASB and AvPAL
coated properly because they were readily detectable by specific
rabbit IgGs with no observed cross-reactivity (i.e., BP14 against
rhASB and BP79ex against AvPAL). Therefore, the anti-AvPAL IgM QCs
are specific for AvPAL, and the detected IgM signal is not heavily
contaminated with IgG.
[0358] The confirmatory assay also demonstrated specificity for
AvPAL antigen. By adding excess AvPAL (0.1 mg/mL), the positive
control signal was decreased by a percentage greater than (cutpoint
factor)/(cutpoint). A sample with .gtoreq.34.0% decrease in signal
in the confirmatory assay was considered a true positive for rat
serum. A sample with .gtoreq.60.4% decrease in signal in the
confirmatory assay was considered a true positive for monkey serum.
A sample with .gtoreq.25.4% decrease in signal in the confirmatory
assay was considered a true positive for human serum. We observed
true seropositives from several so-called naive individual samples.
Such samples will be omitted from future cutpoint determinations
during assay validation.
Example 11
Detection of Neutralizing Antibodies Specific to Anabaena
variabilis Phenylalanine Ammonia-Lyase (AvPAL) in Serum
[0359] The purpose of this assay development was to create a method
to detect the neutralizing antibodies to recombinant Anabaena
variabilis phenylalanine ammonia-lyase (AvPAL) or variant thereof.
In particular, an enzyme activity assay method was developed to
detect antibodies in serum that are able to inhibit the AvPAL
enzymatic activity.
Materials
[0360] The following materials were used: rAvPAL-PEG (pegylated
enzyme) (1.4 mg/mL); see Examples 1, 2 and 4 above, as well as U.S.
Pat. No. 7,531,341.
[0361] The following antibodies were used: KK84: positive control
antibody (Cynomolgous anti-rAvPAL-PEG, affinity purified); and
G192: (Sheep anti-recombinant human arylsulfatase B (rhASB)
antiserum, affinity purified). KK84 was generated by immunizing
Cynomolgous monkeys with rAvPAL-PEG with adjuvant. Sera from the
immunized Cynomolgous monkeys were collected and purified for
anti-AvPAL antibodies; IgG from three Cynomolgous monkeys were
tested, and one was positive.
[0362] The following matrices were used: normal Cynomolgous monkey
serum, Mauritius origin (Bioreclamation, Inc., lot #16895A); and
normal human serum (Bioreclamation, Inc., lot #22234).
[0363] The following chemicals were used: Trizma (Sigma
T1503-500G); 6N HCL (J. T. Baker 5619-02); BSA Fraction V (Sigma
A3059-500G); phenylalanine (Sigma P-5482); sodium azide (Sigma
58032-25G); sodium chloride (VWR BDH0286-500G); Tween 80 (EM
Science OmniPur 9490); and sulfuric acid (H.sub.2SO.sub.4) 12N (VWR
Cat No. VW3481-1).
[0364] The following plasticware and other materials were used: UV
plate: 96-well flat bottom, Costar 3635; microcentrifuge tubes, 1.5
mL (USA Scientific, VWR Cat No. 1615-550); microcentrifuge tubes,
2.0 mL (Eppendorf, VWR Cat No. 62111-754); microplate adhesive film
(USA Scientific, Cat No. 2920-0000); pipetman tips: 20 .mu.L, 250
.mu.L, and 1000 .mu.L (Rainin Instruments, Cat Nos. GPS-L1000,
GPS-L250, GPS-L10); reagent reservoir, 50 mL capacity (Corning, VWR
Cat No. 82026-350); and serological pipettes, 10 mL and 25 mL (VWR,
Cat No. 13-678-11D and 13-678-11E).
[0365] The following equipment and software were used: Jitterbug
incubator/shaker; multichannel pipetman, 12-well, LTS 20-200 .mu.L
(Rainin Instruments); multichannel pipetman, 12-well, LTS 100-1200
.mu.L (Rainin Instruments); pipet-aid, multi-speed, Drummond (VWR,
Cat No. 13-681-15E); SOFTmax Pro v 5.0 (Molecular Devices);
SpectraMax M2E Microplate Spectrophotometer (Molecular Devices);
timer, three channel alarm (VWR, Cat No. 62344-600); and pipetman
LTS-20, LTS-100, LTS-200, and LTS-1000 (Rainin Instruments).
[0366] The following buffers were used: dilution buffer: 1.6 mM
Tris-base, 8.4 mM Tris-HCl, 140 mM NaCl, 2 mg/mL BSA, 1 mM Phe,
0.05% Tween 80 and 0.01% sodium azide, pH 7.3; and substrate
buffer: 0.1 M Tris, pH 8.5, 22.5 mM L-Phe.
Activity Assay Protocol
[0367] Preparation of rAvPAL-PEG working solution: The 1.4 mg/mL
rAvPAL-PEG stock solution was diluted 1:140 in Dilution Buffer to
give a working solution at 10 .mu.g/mL.
[0368] Preparation of Samples: a Baseline Sample, Test Samples and
a Positive Neutralizing antibody control were prepared in cluster
tubes as follows. For the baseline sample, 75 .mu.L, of rAvPAL-PEG
was combined with 7.5 .mu.L of pooled cyno serum, then 67.5 .mu.L
of Dilution Buffer was added to test sample for a final volume of
150 .mu.L. For the test samples, 75 .mu.L of rAvPAL-PEG was
combined with 7.5 .mu.L of serum, then 67.5 .mu.L of Dilution
Buffer was added to test sample for a final volume of 150 .mu.L.
For the positive neutralizing antibody control, 75 .mu.L of
rAVPAL-PEG was combined with 7.5 .mu.L of pooled cyno serum, then
29 .mu.L of KK84 positive control antibody (520 .mu.g/mL stock) was
added. 38.5 .mu.L of Dilution Buffer was added for final volume of
150 .mu.L.
[0369] Activity Assay: 100 .mu.L of mixed samples were transferred
to a Costar UV flat bottom plate. Rows 1, 5 and 9 on the plate were
used to run up to 22 test samples, baseline sample and positive
control samples. 200 .mu.L of Substrate Buffer was added to rows 2,
3, 4, 6, 7, 8, 10, 11 and 12. The plate was preincubated for 30
minutes at 30.degree. C. Following preincubation, the activity
assay was started by transferring in triplicate, 20 .mu.L of test
samples, baseline sample and positive control sample from row 1 to
substrate containing rows 2, 3 and 4; this transfer step was
repeated from row 5 to substrate containing rows 6, 7 and 8, and
from row 9 to substrate containing rows 10, 11 and 12. Reactions
containing mixed samples and substrate were incubated for an
additional 30 minutes at 30.degree. C., then the reactions were
stopped by adding 50 .mu.L of 2N H.sub.2SO.sub.4. In end point
mode, the plate was read after 30 minutes using 290 nm absorbance.
The plate cutpoint was determined by subtracting the cutpoint
factor (0.048 for cyno serum and 0.065 for human serum) from the
baseline sample. A test sample was scored as positive for
neutralizing anti-rAvPAL-PEG antibody if the OD value was found to
be below plate cutpoint.
Assay Design Parameters
[0370] Development of a suitable assay to detect neutralizing
AvPAL-specific antibodies in a body fluid or tissue sample from a
mammal requires identifying an enzyme assay (i.e., phenylalanine
converting activity) and reaction conditions that are sensitive to
detect small amounts of neutralizing AvPAL-specific antibodies in a
sample containing large amounts of non-neutralizing AvPAL-specific
antibodies and non-specific antibodies. In addition, the enzyme
assay has to take into consideration the baseline level of AvPAL
activity, which can easily be affected by active AvPAL enzyme in
the serum or plasma samples, thereby causing potential false
negative results.
[0371] To identify the parameters for assay reagents and format to
detect neutralizing AvPAL-specific antibodies, the reagent
concentration, incubation buffers, and controls for the activity
assay, as well as the performance characteristics of the kinetic
method and endpoint method, were determined.
[0372] The optimal concentrations of rAvPAL-PEG were established by
examining a range of concentrations via kinetic and endpoint modes.
Serial 2-fold dilutions of rAvPAL-PEG from 50 .mu.g/mL to 3 ng/mL
in dilution buffer were prepared, and the concentration-response
relationship was examined in the activity assay. The
substrate-enzyme concentration response relationship was measured
using both endpoint and kinetic measurements in 2% matrix and
buffer. Measurements at 290 nm (trans-cinnamic acid absorbance)
were taken every 30 seconds using the kinetic read mode, and the
Vmax (mOD/min) was determined. For endpoint analysis, the reactions
were stopped at 20, 30, and 60 minutes, the OD at 290 nm was
measured, and results were plotted on SoftMaxPro using a
4-parameter fit.
[0373] Increasing the rAvPAL-PEG concentration in the presence of
excess Phe substrate resulted in increased trans-cinnamic acid
using the kinetic read. The concentration of rAvPAL-PEG spanned 50
.mu.g/mL to 3 ng/mL and provided good curve fit and back-calculated
accuracy to approximately 1 mg/mL. This dose response information
can be used to estimate the optimal amount of rAvPAL-PEG to use to
assess the presence of neutralizing antibodies. For the endpoint
assay, the same reaction was performed and at the end the reaction
was stopped by addition of 504 of 2N H.sub.2SO.sub.4 and read at
290 nm after 20 minutes or 60 minutes. In both of the kinetic and
endpoint assays, the interference from 2% rat and 2% cyno serum was
minimal when compared to reaction buffer. Both the endpoint and
kinetic assays gave similar concentration response curves. The
kinetic assay measures the rate of change of the trans-cinnamic
acid production and is more prone to deviations. The endpoint assay
measures the trans-cinnamic acid production after a finite time
point and is less susceptible to variability. Evaluation of both
endpoint and kinetic assays showed that a rAvPAL-PEG concentration
of between 5-10 .mu.g/mL would give an OD signal that was robust
yet could be inhibited by an antibody control. Further tests
established that 5 .mu.g/mL is an appropriate amount of rAvPAL-PEG
to use for the activity assay. The endpoint assay is preferred over
the kinetic assay because the latter is more variable. A 30 minute
endpoint using 5% serum with rAvPAL-PEG at 5 .mu.g/mL was
determined to be a feasible approach for the activity assay.
Baseline Activity in Normal Serum
[0374] Naive sera were tested to establish a 95% confidence
interval using the endpoint method with increasing concentrations
of rAvPAL-PEG.
[0375] Pooled cyno serum was compared to 20 naive individual
samples (10 male and 10 female) for signal variability. Similarly,
pooled human serum was compared test 10 individual lots of naive
human serum. 5% serum (1:20 dilution) was used in the activity
assay. Following 30 minutes of incubation, enzyme activity was
neutralized with 50 .mu.L 2N H.sub.2SO.sub.4 and read via endpoint
measurement. Each sample was examined using a range of rAvPAL-PEG
concentrations (312.5-20,000 ng/mL), and the average signal,
standard deviation, cutpoint factor and plate cutpoint using 95%
confidence interval were determined.
[0376] The mean signals for cyno serum samples using 2.5, 5, and 10
.mu.g/mL rAvPAL-PEG were 0.307, 0.436, and 0.666, respectively. The
variability was minimal (CV % less than 10%) within conditions and
between individuals. By applying a 95% confidence interval to the
data set, the plate cutpoint was determined. The cutpoint factor is
calculated as [(95% CI)=1.645.times. standard deviation]. The plate
cutpoint is calculated as [pooled normal cyno or human serum sample
OD minus the cutpoint factor]. A sample is deemed as having
positive neutralizing anti-rAvPAL-PEG antibody activity if the OD
is below the plate cutpoint. For 5% cyno serum, the cutpoint factor
using 5 .mu.g/mL rAvPAL-PEG was 0.048 and the plate cutpoint was
0.431. The cutpoint factor accounted for an 11% reduction in signal
(0.048/0.431.times.100=11%). Although slightly more variable (CV %
of 4.7%-10.3%), a 95% confidence interval and plate cutpoint were
calculated for 5% human serum using 5 .mu.g/mL rAvPAL-PEG. The
cutpoint factor was 0.065 and the plate cutpoint was 0.405. The
cutpoint corresponded to a modest decrease in signal that equated
to approximately 10%-20% reduction that could be relied upon to
determine antibody mediated neutralization of rAvPAL-PEG.
Specificity
[0377] The specificity of antibody mediated neutralization of
rAvPAL-PEG activity was established. The response to 0, 10, 100,
and 300 .mu.g/mL neutralizing rAvPAL-PEG antibody (KK84,
affinity-purified cyno anti-rAvPAL-PEG antibody) was compared to
300 .mu.g/mL non-neutralizing rhASB antibody (G192), using 2.5, 5,
and 10 .mu.g/mL of rAvPAL-PEG in the endpoint assay in both human
and cyno serum.
[0378] Increasing amounts of anti-rAvPAL-PEG antibody (KK84)
reduced the activity of rAvPAL-PEG. The 100 and 300 .mu.g/mL
concentrations of KK84 worked best. The reduction was more marked
in human serum than in cyno serum. The 300 .mu.g/mL anti-rhASB
antibody (G192) did not significantly reduce enzymatic activity.
These results demonstrate a concentration response relationship and
specific nature of the antibody-enzyme interaction.
Sensitivity/Limit of Detection
[0379] The lowest concentration of neutralizing antibody in a
sample which can be detected (i.e., decrease rAvPAL-PEG activity)
was determined.
[0380] Positive control antibodies were prepared in cyno and human
serum in a dilution series, and the lowest concentration that gave
a signal below the assay cutpoint was identified. 2.5, 5, and 10
.mu.g/mL rAvPAL-PEG was tested in combination with 0, 10, 100, and
300 .mu.g/mL positive control rAvPAL-PEG antibody (KK84).
[0381] The KK84 antibody at 10 .mu.g/mL reduced the 2.5, 5 and 10
.mu.g/mL rAvPAL-PEG signal by less than 10% in both cyno and human
serum. This reduction was to a level that was not less than the
cutpoint and therefore unlikely to be consistently positive.
However, the KK84 antibody at 100 .mu.g/mL reduced the 2.5, 5, and
10 .mu.g/mL rAvPAL-PEG signal by 12%-21% in both cyno and human
serum, and at 300 .mu.g/mL up to 32% reduction in cyno serum and up
to 38% in human serum, depending on the level of rAvPAL-PEG.
Therefore, the limit of detection was approximately 100 .mu.g/mL
using the KK84 antibody. The 5 .mu.g/mL concentration of rAvPAL-PEG
seemed most responsive using either cyno or human serum. The limit
of detection is an approximation of the sensitivity of the assay
and is dependent on the specific character of the positive control
antibody used in the assay (i.e., affinity and epitope). Several
anti-rAvPAL-PEG antibodies were examined, but only the KK84
antibody had measurable neutralizing activity response. The nature
of the rAvPAL-PEG enzyme (i.e., pegylated AvPAL tetramer) supports
the notion that activity neutralization via antibody interaction
may be difficult to measure. A neutralizing antibody may need to
block or sterically modify the catalytic pocket to achieve the
desired effect.
Interference by Free Drug
[0382] The effect of free drug on the antibody-activity response
relationship was evaluated. The samples obtained for antibody
analysis are typically acquired several days post injection, and
immediately before the next injection. Prior pharmacokinetic
analysis has demonstrated that the half-life of rAvPAL-PEG is
several days (.about.5 days). Therefore, measurable concentrations
of free drug may be present and cause interference.
[0383] Increasing amounts of rAvPAL-PEG (0, 1, 10, and 100
.mu.g/mL) were added into the serum along with 0, 1, 10, and 100
.mu.g/mL positive control antibody (KK84), and the drug-antibody
mixtures were pre-incubated for 30 minutes. The level of
interference at different amounts of free drug using 5 .mu.g/mL
rAvPAL-PEG in the activity assay was determined.
[0384] The neutralizing antibody activity assay was modestly
sensitive to free drug in serum. Addition of 100 .mu.g/mL of
rAvPAL-PEG eliminated the neutralizing effect of the 10 and 100
.mu.g/mL KK84 positive control antibody.
Interference by Matrix
[0385] The effect of the matrix (i.e., serum) on the
antibody-activity response relationship was evaluated by increasing
the amounts of cyno serum on the activity assay. Specifically, the
level of inhibition obtained with the KK84 control antibody on
rAvPAL-PEG in buffer, 2.5%, 5%, 10%, 20% cyno serum was
measured.
[0386] Increasing serum concentrations had a minimal effect on the
activity assay at 2% serum as compared to buffer. At 5% serum,
modest matrix interference was observed, and at 10% serum, there
was significant matrix interference. 2.5% and 5% serum did not
prevent antibody neutralization obtained with KK84 at 100 .mu.g/ml,
however, 10% and 20% cyno serum caused enough matrix interference
to mask the neutralization effect of KK84. Thus, dilution of a test
sample by 1:20 (5%) is recommended.
Precision
[0387] The intra-assay and inter-assay variability of the activity
neutralizing antibody assay over a two day period was examined.
Replicate 5 .mu.g/mL rAvPAL-PEG samples in 5% cyno serum were
prepared and enzyme activity was measured in the absence and
presence of KK84 (100 .mu.g/mL). The intra-day variability and
inter-day variability as measured by CV %.
[0388] The intra-assay and inter-assay replicates showed less than
5% variability as measured by CV % when tested by one analyst over
two days. The KK84 was able to inhibit to below the cutpoint on
both days.
Robustness
[0389] The capacity of the assay to remain unaffected by small, but
deliberate variations in method parameters was determined to
provide an indication of its reliability during normal usage. The
reduction in activity when using KK84 at 80, 100, and 120 .mu.g/mL
was examined. Pre-incubation times and assay incubation times at
24, 30, 36 minutes, which represent 80%, 100%, and 120% of the
optimized times, were examined. The stability of reagents used in
the activity assay was evaluated.
[0390] The KK84 antibody, when used at 100 .mu.g/mL, was able to
inhibit the reaction to below cutpoint at increased incubation
times up to 36 minutes. It is expected that decreased incubation
times within 10% of the 30 minute time may not affect the assay.
The substrate and dilution solutions have been used after
preparation and storage at 4.degree. C. for up to 6 months without
significant loss in activity. The KK84 antibody is typically stored
at 4.degree. C. after being thawed; after storage for 1 month, the
KK84 antibody was able to inhibit activity to below cutpoint and
compared similarly to freshly thawed material.
Selectivity
[0391] The variability between individual matrices using pooled and
individual serum samples was examined. Pooled and individual serum
samples with KK84 control and rAvPAL-PEG at 5 .mu.g/ml in 5% serum
was tested in the endpoint assay.
[0392] KK84 was able to neutralize activity of 5 .mu.g/mL
rAvPAL-PEG to below cutpoint for pooled and individual serum
samples. The percent reduction ranged from 14-18%. Thus, the
activity assay demonstrates selectivity.
Example 12
Detection of IgG Antibodies Specific to Polyethylene Glycol (PEG)
in Serum
[0393] An assay was developed to detect the presence of antibodies
specific for polyethylene glycol (PEG) or derivatives thereof. In
particular, an enzyme-linked immunosorbent assay (ELISA) method was
developed to detect IgG antibodies to PEG in serum.
Materials
[0394] The following forms of PEG were used: 20 kDa methoxy PEG
(NOF Corporation, Cat No. ME-200HS) inactivated by dissolution in
TBS buffer, pH 7.5, and reconstituted to 2 mg/mL in TBS buffer, pH
7.5; and 6 kDa hydroxy PEG (Fluka, Cat No. 53770) (1.8 mg/mL in 0.1
M Tris-HCl, pH 8.0, 30% PEG 6000).
[0395] The following antibodies were used: rabbit anti-PEG IgG
(Epitomics); and detection reagent: HRP-conjugated goat anti-human
IgG (H+L) (KPL, Cat No. 474-1006).
[0396] The following matrices were used: Human serum (pooled); and
individual human serum (Bioreclamation, Inc.).
[0397] The following ELISA reagents were used: coating buffer and
wash buffer: 1.times.DPBS (Cellgro, Cat No. 21-031-CV); blocking
buffer: Blocker Casein in PBS (Thermo, Cat No. 37528); development
reagent: TMB Peroxidase EIA Substrate Kit, 250 mL (BioRad, Cat No.
172-1066); and stop solution: 2 N H.sub.2SO.sub.4.
[0398] The following chemicals were used: de-ionized water (MilliQ
filtered); Dulbecco's phosphate buffered saline (DPBS), 1.times.
(Cellgro, Cat No. 21-031-CV); sulfuric acid (H.sub.2SO.sub.4), 11 N
(VWR, Cat No. VW3481-1); and Proclin 300 (Supelco, Cat No.
4-8126).
[0399] The following plasticware and other materials were used:
ELISA plate: 96-well, Maxisorp, flat bottom "F" (Nalge/Nunc
International, VWR Cat No. 12-565-135); microcentrifuge tubes, 2.0
mL (Eppendorf, VWR Cat No. 62111-754); microplate adhesive film
(USA Scientific, Cat No. 2920-0000); pipetman tips: 20 .mu.L, 250
.mu.L, and 1000 .mu.L, (Rainin Instruments, Cat Nos. GPS-L1000,
GPS-L250, GPS-L10); reagent reservoir, 50 mL capacity (Corning, VWR
Cat No. 82026-350); and serological pipettes, 10 mL and 25 mL (VWR,
Cat No. 13-678-11D and 13-678-11E).
[0400] The following equipment and software were used: El.times.405
Select Plate Washer (Bio-Tek); multichannel pipetman, 12-well, LTS
20-200 .mu.L (Rainin Instruments); multichannel pipetman, 12-well,
LTS 100-1200 .mu.L (Rainin Instruments); pipet-aid, multi-speed,
Drummond (VWR, Cat No. 13-681-15E); SOFTmax Pro v 3.0 (Molecular
Devices); SpectraMax PLUS Microplate Spectrophotometer (Molecular
Devices); timer, three channel alarm (VWR, Cat No. 62344-600); and
titer plate shaker (Barnstead/Lab-Line Instruments).
ELISA Protocol
[0401] Preparation of Samples. Serum samples for screening were
initially diluted 1:50 in Blocking Buffer. Samples with positive
results were serially diluted three-fold in Blocking Buffer.
[0402] Preparation of Positive Controls. Positive Controls were a
Cyno Anti-Peg polyclonal antibody and a rabbit anti-PEG monoclonal
antibody. The Cyno positive control used a Protein G purified total
IgG from day 84 serum from a Cynomolgous monkey that had been
immunized with rAvPAL-PEG. The Cyno positive control could be
replaced by other Cyno or human sera identified as positive for
anti-PEG IgG antibodies in this assay. Fluka 6 kDa hydroxyl PEG and
inactivated 20 kDa methoxy PEG showed similar reactivity with the
Cyno and rabbit anti-PEG antibody positive controls in this
assay.
[0403] ELISA Procedure. Inactivated methoxy PEG or hydroxy PEG was
diluted to 1 .mu.g/mL with Coating Buffer and dispensed at 100
.mu.L per well into the ELISA plate. After incubation for 1 hour at
4.degree. C., plates were washed 3.times. with 300 .mu.L/well of
Wash Buffer, and then 300 .mu.L/well of Blocking Buffer was added
to each well and incubated on a shaker for 1 hour at room
temperature (RT). Plates were washed 3.times. with 300 .mu.L/well
of Wash Buffer. Serum samples and controls, diluted 1:50 in
Blocking Buffer, were aliquoted at 100 .mu.L/well according to a
template plate map and incubated on a shaker for 1 hour at RT.
Plates were washed 3.times. with 300 .mu.L/well of Wash Buffer.
HRP-conjugated Detection Reagent was prepared in Blocking Buffer at
a 1:1,000 dilution, dispensed at 100 .mu.L/well, and incubated on a
shaker for 1 hour at RT. Plates were washed 3.times. with 300
.mu.L/well of Wash Buffer. Development Reagent was added at 100
.mu.L/well and the plate was incubated on a shaker for 15 minutes
at RT. Stop Solution was added at 100 .mu.L/well and the plate was
read immediately at 450 nm.
[0404] Using this assay, anti-PEG antibodies of the IgG isotype can
be specifically detected in clinical (e.g., serum) samples from
humans,
Example 13
Detection of IgM Antibodies Specific to Polyethylene Glycol (PEG)
in Serum
[0405] An assay was developed to detect the presence of antibodies
specific for polyethylene glycol (PEG) or derivatives thereof. In
particular, an enzyme-linked immunosorbent assay (ELISA) method was
developed to detect IgM antibodies to PEG in serum.
Materials
[0406] The following forms of PEG were used: 20 kDa methoxy PEG
(NOF Corporation, Cat No. ME-200HS) inactivated by dissolution in
TBS buffer, pH 7.5, and reconstituted to 2 mg/mL in TBS buffer, pH
7.5; and 6 kDa hydroxy PEG (Fluka, Cat No. 53770) (1.8 mg/mL in 0.1
M Tris-HCl, pH 8.0, 30% PEG 6000).
[0407] The following antibodies were used: detection reagent:
HRP-conjugated goat anti-human IgM (Fc specific) (BioMarin).
[0408] The following matrices were used: Human serum (pooled); and
individual human serum (Bioreclamation, Inc.).
[0409] The following ELISA reagents were used: coating buffer and
wash buffer: 1.times.DPBS (Cellgro, Cat No. 21-031-CV); blocking
buffer: Blocker Casein in PBS (Thermo, Cat No. 37528); development
reagent: TMB Peroxidase EIA Substrate Kit, 250 mL (BioRad, Cat No.
172-1066); and stop solution: 2 N H.sub.2SO.sub.4.
[0410] The following chemicals were used: de-ionized water (MilliQ
filtered); Dulbecco's phosphate buffered saline (DPBS), 1.times.
(Cellgro, Cat No. 21-031-CV); sulfuric acid (H.sub.2SO.sub.4), 11 N
(VWR, Cat No. VW3481-1); and Proclin 300 (Supelco, Cat No.
4-8126).
[0411] The following plasticware and other materials were used:
ELISA plate: 96-well, Maxisorp, flat bottom "F" (Nalge/Nunc
International, VWR Cat No. 12-565-135); microcentrifuge tubes, 2.0
mL (Eppendorf, VWR Cat No. 62111-754); microplate adhesive film
(USA Scientific, Cat No. 2920-0000); pipetman tips: 20 .mu.L, 250
.mu.L, and 1000 .mu.L (Rainin Instruments, Cat Nos. GPS-L1000,
GPS-L250, GPS-L10); reagent reservoir, 50 mL capacity (Corning, VWR
Cat No. 82026-350); and serological pipettes, 10 mL and 25 mL (VWR,
Cat No. 13-678-11D and 13-678-11E).
[0412] The following equipment and software were used: El.times.405
Select Plate Washer (Bio-Tek); multichannel pipetman, 12-well, LTS
20-200 .mu.L (Rainin Instruments); multichannel pipetman, 12-well,
LTS 100-1200 .mu.L (Rainin Instruments); pipet-aid, multi-speed,
Drummond (VWR, Cat No. 13-681-15E); SOFTmax Pro v 3.0 (Molecular
Devices); SpectraMax PLUS Microplate Spectrophotometer (Molecular
Devices); timer, three channel alarm (VWR, Cat No. 62344-600); and
titer plate shaker (Barnstead/Lab-Line Instruments).
ELISA Protocol
[0413] Preparation of Samples. Serum samples for screening were
initially diluted 1:50 in Blocking Buffer. Samples with positive
results were serially diluted in Blocking Buffer as needed.
[0414] Preparation of Positive Controls. Positive Controls were
Cyno Anti-Peg Polyclonal antibodies. The Cyno positive control used
a Protein G purified total IgG from day 84 serum from a Cynomolgous
monkey that had been immunized with rAvPAL-PEG. This sera elicited
a strong anti-PEG IgG and an even stronger IgM antibody response.
The Cyno positive control could be replaced by other Cyno or human
sera identified as positive for anti-PEG IgM antibodies in this
assay. Fluka 6 kDa hydroxy PEG and inactivated 20 kDa methoxy PEG
showed similar reactivity with the Cyno anti-PEG antibody positive
control in this assay.
[0415] ELISA Procedure. Inactivated methoxy PEG or hydroxy PEG was
diluted to 1 .mu.g/mL with Coating Buffer and dispensed at 100
.mu.L per well into the ELISA plate. After incubation for 1 hour at
4.degree. C., plates were washed 3.times. with 300 .mu.L/well of
Wash Buffer, and then 300 .mu.L/well of Blocking Buffer was added
to each well and incubated on a shaker for 1 hour at room
temperature (RT). Plates were washed 3.times. with 300 .mu.L/well
of Wash Buffer. Serum samples and controls, diluted 1:50 in
Blocking Buffer, were aliquoted at 100 .mu.L/well according to a
template plate map and incubated on a shaker for 1 hour at RT.
Plates were washed 3.times. with 300 .mu.L/well of Wash Buffer.
HRP-conjugated Detection Reagent was prepared in Blocking Buffer at
a 1:5,000 dilution, dispensed at 100 .mu.L/well, and incubated on a
shaker for 1 hour at RT. Plates were washed 3.times. with 300
.mu.L/well of Wash Buffer. Development Reagent was added at 100
.mu.L/well and the plate was incubated on a shaker for 15 minutes
at RT. Stop Solution was added at 100 .mu.L/well and the plate was
read immediately at 450 nm.
[0416] Using this assay, anti-PEG antibodies of the IgM isotype can
be specifically detected in clinical (e.g., serum) samples from
humans.
Example 14
Toxicity/Pharmacokinetic Studies of Pegylated Forms of AvPAL
Variants (Cysteine Mutants) in Cynomolgus Monkeys and Rats
[0417] Toxicity/pharmacokinetic studies were performed to determine
the effect of administration of a single dose of a pegylated form
of an AvPAL polypeptide variant (e.g., with serine substitution of
the cysteine residues at positions 503 and 565) in Cynomolgus
monkeys and in rats.
[0418] The pegylated AvPAL double cysteine mutant AvPAL_C565SC503S
was prepared as described in Example 5.
Cynomolgus Monkey Toxicity/Pharmacokinetic Study
[0419] This study used four (4) groups of monkeys, each with three
males and three females. Group 1 received placebo (mL/kg); and
Groups 2, 3 and 4 received a single subcutaneous injection of
pegylated AvPAL double cysteine mutant AvPAL_C565SC503S in solution
at 4, 12 and 60 mg/kg, respectively. Plasma samples were collected
from the monkeys pre-dose, and at various times post-dose, from 3
to 504 hours. The 60 mg/kg dose was found to be toxic to the
monkeys, so the Group 4 portion of this study was terminated.
[0420] FIG. 7A shows the concentration of pegylated AvPAL double
cysteine mutant AvPAL_C565SC503S in the plasma at various times
after a single subcutaneous injection at 4 and 12 mg/kg. The data
shows monophasic elimination of the pegylated AvPAL double cysteine
mutant AvPAL_C565SC503S. A single compartment model with 1.sup.st
order absorption appears to describe the plasma profile of the
pegylated AvPAL double cysteine mutant AvPAL_C565SC503S after a
single subcutaneous injection.
[0421] FIG. 87B shows the concentrations of phenylalanine (Phe) and
pegylated AvPAL double cysteine mutant AvPAL_C565SC503S in the
plasma at various times after a single subcutaneous injection at 4
mg/kg. At this dose, the plasma Phe concentration was reduced to
below the limit of quantitation in the GC/MS assay within 24 hours,
and the drop in plasma Phe was sustained over 10 days.
Rat Toxicity/Pharmacokinetic Study
[0422] This study used eight (8) groups of rats, with 3 males and 3
females in the placebo groups, and 6 males and 6 females in the
test groups. Groups 1 and 5 received single intravenous and
subcutaneous injections of placebo. Groups 2, 3 and 4 received
single intravenous injections of pegylated AvPAL double cysteine
mutant AvPAL_C565SC503S at 1, 5 and 25 mg/kg, respectively. Groups
6, 7 and 8 received single subcutaneous injections of pegylated
AvPAL double cysteine mutant AvPAL_C565SC503S at 10, 25 and 250
mg/kg, respectively. Blood samples were collected from the rats
pre-dose, and at various times post-dose, from 1 to 360 hours. At
each collection time, blood was collected from 3 rats in each
group. No toxicity was observed in the rats in this study.
[0423] FIG. 8A shows the concentration of pegylated AvPAL double
cysteine mutant AvPAL_C565SC503S in the plasma at various times
after a single intravenous injection at 1, 5 and 25 mg/kg. The data
shows monophasic elimination of the pegylated AvPAL double cysteine
mutant AvPAL_C565SC503S from the plasma after a single intravenous
injection.
[0424] FIG. 8B shows the concentration of pegylated AvPAL double
cysteine mutant AvPAL_C565SC503SC65SC503S in the plasma at various
times after a single subcutaneous injection at 10, 25 and 250
mg/kg. A single compartment model with first order absorption
appears to describe the plasma profile of the pegylated AvPAL
double cysteine mutant AvPAL_C565SC503S after a single subcutaneous
injection.
[0425] Table 2 shows pharmacokinetic parameters of the pegylated
AvPAL double cysteine mutant AvPAL_C565SC503S after a single
intravenous or subcutaneous injection.
TABLE-US-00002 TABLE 2 Pharmacokinetic Parameters of Pegylated
Double Cysteine Mutant AvPAL C565SC503S After a Single Intravenous
or Subcutaneous Dose Dose AUC.sub.0-.infin. C.sub.max T.sub.max
t.sub.1/2* F Route (mg/kg) (ng-hr/mL) (ng/mL) (hr) (hr) (%)
Intravenous 1 657131 12600 4.5 27.9 -- 5 3579327 87667 2 39.1 -- 25
10860907 202238 9.0 30.4 -- Subcutaneous 10 1304016 16674 18.0 46.9
19.7 25 2290754 29260 42.0 21.0 12.5.sup.# 250 37254683 225200 72.0
62.8 34.0 *For the subcutaneous route of administration, terminal
t.sub.1/2 is longer than intravenous; this may be due to a slower
rate of absorption from subcutaneous tissues than the rate of
elimination (so that the t.sub.1/2 observed is actually
absorption). .sup.#Bioavailability using intravenous AUC data at 25
mg/kg is 21.5%.
[0426] There appeared to be no gender difference in this
pharmacokinetic study. The AUC.sub.inf and C.sub.max were roughly
proportional with dose for both the intravenous and subcutaneous
routes of administration.
Multiple Dose Toxicity Studies in Rats and Cynomolgus Monkeys
[0427] The safety of pegylated AvPAL double cysteine mutant
AvPAL_C565SC503S was evaluated in repeat-dose toxicity studies in
rats and Cynomolgus monkeys.
[0428] Rats administered up to 25 mg/kg pegylated AvPAL double
cysteine mutant AvPAL_C565SC503S twice weekly, subcutaneously over
28 days exhibited no toxicity.
[0429] Cynomolgus monkeys administered up to doses of 1 mg/kg
pegylated AvPAL double cysteine mutant AvPAL_C565SC503S twice
weekly, subcutaneously over 28 days exhibited no significant
toxicity. A dose dependent decrease in plasma Phe levels was
observed after the first dose; however, after the seventh dose,
plasma Phe levels returned to baseline in all dose groups,
indicating a possible antibody response toward the administered
enzyme. Minimal anti-AvPAL_C565SC503S IgG titers were observed in
most 1 mg/kg treated animals at day 28. No IgM titers were observed
in any animal in the study at day 28.
Example 15
Effects of AvPAL Variants (Cysteine Mutants) on Tumor Cells in
Culture
[0430] Studies were performed to investigate the effect a pegylated
form of an AvPAL polypeptide variant (e.g., with serine
substitution of the cysteine residues at positions 503 and 565) on
the proliferation of tumor cells grown in culture in vitro.
[0431] The pegylated AvPAL double cysteine mutant AvPAL_C565SC503S
was prepared as described in Example 7.
[0432] The proliferation of tumor cells in vitro was measured using
a propidium iodide fluorescence assay as described in Dengler, et
al., Anti-Cancer Drugs 6:522-532 (1995).
Hematological Tumors
[0433] A panel of twenty-four (24) hematological tumor cell lines,
including 14 leukemias, 5 lymphomas and 5 myelomas, were evaluated
for the effect of pegylated double cysteine mutant AvPAL_C565SC503S
on cell proliferation in vitro.
[0434] The hematological tumor cell lines were seeded into culture
plates at 5,000 cells/well on Day 0. On Day 1, pegylated double
cysteine mutant AvPAL_C565SC503S was added to the cultures at
various concentrations, from 0.01 to 100 .mu.g/mL. On Day 5, cells
were harvested and DNA content was measured by propidium iodide
staining. The IC.sub.50, IC.sub.70 and IC.sub.90 were determined.
These experiments were performed twice or three times for each
hematological tumor cell line.
[0435] Table 3 shows that pegylated double cysteine mutant
AvPAL_C565SC503S was effective in inhibiting in vitro
proliferation, as measured by propidium iodide staining, of several
hematological tumor cell lines.
TABLE-US-00003 TABLE 3 Inhibition of Propidium Iodide Staining of
Hematological Tumor Lines In Vitro by Pegylated Double Cysteine
Mutant AvPAL_C565SC503S Tumor IC.sub.50 IC.sub.70 Line Cell Type
.mu.g/mL .mu.g/mL CCRF CEM ALL - T Cell Lymphoma 1 >100 >100
>100 >100 >100 EM2 CML >100 >100 >100 >100
>100 >100 HL-60 APL 0.904 >100 >100 >100 46.41
>100 JURKAT Human T Cell Leukemia 0.38 2.928 14.125 >100
10.000 >100 JURLMK1 CML 0.766 >100 10 >100 3.162 >100
K562 CML 0.701 59.948 >100 >100 11.659 >100 KCL22 CML 0.9
15.399 >100 >100 1 >100 KG1 AML 43.287 >100 >100
>100 >100 >100 MEG01 CML 1.258 >100 >100 >100
0.926 >100 MOLT4 ALL - T cell lymphoma 0.326 1.873 1.082 5.298
1.096 6.918 Mv411 AML 5.994 74.989 >100 >100 >100 >100
NOMO1 AML 0.304 2.511 0.732 8.659 0.863 6.449 OCIAML2 AML 0.261
0.938 >100 >100 7.305 >100 PL21 AML >100 >100
>100 >100 >100 >100 HUT78 Lym CTL 6.105 18.276 17.782
>100 0.096 >100 L5178Y Mouse T cell Leukemia 6.683 41.595
3.981 10 3.019 7.585 MYLA Lym CTL 4.436 >100 5.379 >100 8.171
>100 RAJI Burkitt Lymphoma 0.261 0.938 21.544 >100 2.154
>100 U937 Histio Lymphoma 0.803 >100 >100 >100 >100
>100 8226 Myeloma 0.229 0.825 >100 >100 0.691 7.742 IM9
Human Lymphoblastic Cells 0.271 1.467 0.295 1.311 0.063 0.188 L363
Human Plasma Cell Leukemia 7.943 >100 1.73 15.505 LP1 Human
Multiple Myeloma 0.774 100 0.71 6.309 NCIH929 Human Multiple
Myeloma >100 >100 11.288 >100 2.154 >100
[0436] Dose-dependent inhibition of cell proliferation, as
determined by a reduction in propidium iodide staining, by the
pegylated double cysteine mutant AvPAL_C565SC503S in two sensitive
hematological tumor cells, NOMO1 and IM9, are shown in FIGS. 9A and
9B, respectively. These tumor cell lines had IC.sub.50 and
IC.sub.70 of less than 1.0 and 10.0 .mu.g/mL, respectively. For
comparison, asparaginase has an IC.sub.50 of 1-10 .mu.g/mL in human
leukemia cell lines. In general, however, the hematological tumor
cell lines were more resistant, as judged by IC.sub.70 values, than
the solid tumor lines (see below).
Solid Tumors
[0437] A panel of thirty-six (36) solid tumor cell lines, including
tumors derived from bladder, brain, colon, stomach, head and neck,
lung, breast, ovary, pancreas, prostate, kidney and uterus, were
evaluated for the effect of pegylated double cysteine mutant
AvPAL_C565SC503S on cell proliferation in vitro. The solid tumor
cell lines were seeded into culture plates at 5,000 cells/well on
Day 0. On Day 1, pegylated double cysteine mutant AvPAL_C565SC503S
was added to the cultures at various concentrations, from 0.01 to
100 .mu.g/mL. On Day 5, DNA content was measured by propidium
iodide staining. The IC.sub.50, IC.sub.70 and IC.sub.90 were
determined.
[0438] Table 4 shows that pegylated double cysteine mutant
AvPAL_C565SC503S was effective in inhibiting in vitro
proliferation, as measured by propidium iodide staining, of several
solid tumor cell lines.
TABLE-US-00004 TABLE 4 Inhibition of Propidium Iodide Staining of
Solid Tumor Lines In Vitro by Pegylated Double Cysteine Mutant
AvPAL_C565SC503S Tumor IC.sub.50 IC.sub.70 Line Organ/Cell Type
.mu.g/mL .mu.g/mL Bladder 1218L ATCC, Freiburg; Urothelial 1.1
7.498 Adenocarcinoma T24 Xenograft 0.617 2.154 Brain/CNS 498NL
Xenograft, Freiburg 0.691 2.154 SF268 NCI 0.59 1.492 Colon HCT116
NCI; Adenocarcinoma, pd 0.316 0.9 HT29 NCI; Adenocarcinoma, pd
0.508 0.94 Gastric 251L Xenograft, Freiburg; Adenocarcinoma, pd
2.682 37.275 Head and Neck 536L Xenograft, Freiburg; Hypopharynx
0.606 1.887 Carcinoma Lung 1121L Xenograft 0.715 3.548 289L
Xenograft, Freiburg; Adenocarcinoma, pd 2.807 23.101 529L
Xenograft, Freiburg; Large Cell, du 0.539 1.73 629L Xenograft,
Freiburg; Adenocarcinoma, pd 0.457 1.467 H460 NCI; Large Cell
Carcinoma 0.215 0.644 Breast 401NL Xenograft, Freiburg; Pap 1.873
7.564 Adenocarcinoma, wd MCF7 NCI; Mammary Carcinoma 0.599 1.623
Melanoma 276L Xenograft 4.124 268.269 394NL Xenograft 0.887 3.856
462NL Xenograft 0.954 6.189 514L Xenograft 0.828 4.216 520L
Xenograft 1.359 6.309 Ovarian 1619L Xenograft, Freiburg;
Adenocarcinoma, md 0.322 0.688 899L Xenograft, Freiburg; Pap Serous
1.279 6.628 Carcinoma, md OVCAR3 NCI; Adenocarcinoma, md 1.185
6.528 Pancreatic 1657L Xenograft, Freiburg; Adenocarcinoma, md
1.951 8.619 PANC1 ATCC 0.825 5.179 Prostate 22RV1 ATCC;
Adenocarcinoma, md 0.87 7.079 DU145 NCI; Adenocarcinoma, md 0.622
1.873 LNCAP DSMZ; Adenocarcinoma, md 0.584 0.974 PC3M NCI;
Adenocarcinoma, md 0.501 1.274 Pleuramesothelioma 1752L Xenograft,
Freiburg; Pleuramesothelioma 1.637 8.483 Renal 1781L Xenograft,
Freiburg; Renal Carcinoma 2.371 10 393NL Xenograft, Freiburg;
Hypernephroma, wd 0.55 1.995 486L Xenograft 0.859 5.336 944L
Xenograft 0.71 3.727 Uterine 1138L Xenograft, Freiburg;
Carcinosarcoma, wd 0.621 1.258
[0439] Dose-dependent inhibition of cell proliferation, as
determined by a reduction of propidium iodide staining, by the
pegylated double cysteine mutant AvPAL_C565SC503S in tumor cell
lines derived from brain/CNS, colon, lung and prostate cancer is
shown in FIGS. 10A-10D, respectively.
[0440] The AvPAL_C565SC503S displayed a selective
anti-proliferative activity in this broad panel of solid tumor cell
lines, and was particularly potent (i.e., IC.sub.50 between 0.2 and
0.7 .mu.g/mL) in tumor cell lines derived from lung, brain/CNS,
colon, prostate and kidney. At least on tumor cell line derived
from bladder, head and neck, breast, ovary and uterus were also
sensitive to cell killing by AvPAL_C565SC503S. Several melanomas
were also sensitive to cell killing by AvPAL_C565SC503S.
Example 16
Antitumor Activity of AvPAL Variants (Cysteine Mutants) in Nude
Mice
[0441] Studies are performed to investigate the effect of a
pegylated form of an AvPAL polypeptide variant (e.g., with serine
substitution of the cysteine residues at positions 503 and 565) on
the proliferation of tumor cells grown in nude mice in vivo.
[0442] The pegylated AvPAL double cysteine mutant AvPAL_C565SC503S
is prepared as described in Example 7.
[0443] Subcutaneous xenografts of human tumor cells in
immunodeficient nude or SCID mice have been successfully used as
models for human cancers to test the in vivo efficacy of cancer
therapeutic agents as well as targeted cancer therapeutic agents,
such as antibodies and toxin conjugates (for review, see Kerbel,
Cancer Biol. Ther. 2(4):Suppl. 1:S134-S139 (2003)).
[0444] The in vivo antitumor activity of the pegylated AvPAL double
cysteine mutant AvPAL_C565SC503S can be tested alone or in
combination with cancer therapeutic agents or targeted cancer
therapeutic agents, or in combination with a
phenylalanine-restricted diet, using xenografts of human tumor
cells in nude mice.
[0445] To establish human tumor xenografts, nude mice are injected
subcutaneously with about 5.times.10.sup.6 human tumor cells in 0.2
mL PBS. The average tumor size increases over time. Human xenograft
tumors are excised from the tumor bearing nude mice and tumor
tissue blocks of approximately 30 mm.sup.3 are prepared. Naive nude
mice to be used for evaluating in vivo antitumor activity of
pegylated AvPAL double cysteine mutant AvPAL_C565SC503S are each
implanted subcutaneously with one tumor tissue block. Therapeutic
treatment is initiated before tumor initiation or when the average
tumor size within a group of nude mice is approximately 100-150
mm.sup.3 (prevention model), and/or after the establishment of
tumors when the average tumor size within a group of nude mice is
above 500 mm.sup.3 (treatment model).
[0446] In a first step, the dose of pegylated AvPAL double cysteine
mutant AvPAL_C565SC503S that will lower plasma phenylalanine (Phe)
levels to near zero is determined. Experiments are performed such
as those described in Examples 7 to 9 and 14 in prior co-pending
U.S. patent application Ser. No. 11/451,999 filed on Jun. 12, 2006,
except nude mice rather than ENU2 mice are used. The PAL enzyme
dose and the frequency of administration are determined in this
initial step.
[0447] In a second step, the anti-tumor activity of pegylated AvPAL
double cysteine mutant AvPAL_C565SC503S is assessed in various
human tumor xenografts derived from patients or cell lines. Tumor
models include different cancer types, for example and not for
limitation, central nervous system (CNS), colon, lung, prostate,
metastatic melanoma and renal cancer. Non-comprehensive lists of
tumors and tumor cell lines that can be tested are provided in
Tables 3 (hematological tumors) and 4 (solid tumors).
[0448] To assess the antitumor activity of pegylated AvPAL double
cysteine mutant AvPAL_C565SC503S, nude mice bearing different human
tumor xenografts subcutaneously are treated with AvPAL_C565SC503S
given subcutaneously at, e.g., three different dose levels, ranging
from about 5 to 500 mg/kg. This dose may result in a human dose of
about 0.1 to 10 mg/kg. Antitumor activity is analyzed as tumor
volume inhibition and/or absolute growth delay. The tolerability of
the AvPAL_C565SC503S is also evaluated as mortality and/or body
weight changes.
[0449] Each in vivo antitumor study consists of at least four
groups, one vehicle control group and at least three prokaryotic
PAL enzyme-treated groups. The group size will be at least 8 mice,
resulting in a total of 32 mice receiving subcutaneous tumor
implantations. Mice with similar sized tumors (100-150 mm.sup.3)
will be used for randomization (Day 0).
[0450] In the case of an antitumor effect, mice may be monitored
for additional 2 weeks after termination of prokaryotic PAL enzyme
treatment to detect a possible reinitiation of tumor growth.
According to regulations for animal experiments, mice are
sacrificed if the tumor diameters exceed 1.6 cm.
[0451] Tumor diameters are measured twice weekly together with body
weight. Tumor volume is calculated according to the formula
a*b.sup.2/2 (where `a` is the largest diameter of the tumor and `b`
is the perpendicular axis). Relative tumor volumes and body weights
are calculated for each individual tumor based on the value on Day
0 (the first day of dosing). Treatment starts when the tumors have
reached a volume of approximately 100-150 mm.sup.3. Mice are
sacrificed if the tumor volume exceeds 1600 mm.sup.3, per
regulations for animal studies.
[0452] Patient-derived tumors established in serial passage in nude
mice can also be used as test tumors. Typically, these tumors
retain important characteristics of the original patient tumor,
including histology and drug sensitivity. For certain tumors, e.g.,
one CNS and both prostate cancers, cancer cell line-derived tumors
are used.
Example 17
Clinical Evaluation with Prokaryotic PAL Compositions for Treatment
of Cancer
[0453] The following example provides guidance on the parameters to
be used for the clinical evaluation of compositions comprising
prokaryotic PAL or biologically active fragments, mutant, variants
or analogs thereof in the therapeutic methods and kits provided
herein. As discussed herein throughout, prokaryotic PAL
compositions can be used, e.g., in the treatment of cancer.
Clinical trials will be conducted which will provide an assessment
of oral or subcutaneous doses of prokaryotic PAL for safety,
pharmacokinetics, and initial response of both surrogate and
defined clinical endpoints. The trial will be conducted for a
minimum, but not necessarily limited to, 24 weeks to collect
sufficient safety information for 100 evaluable patients. The
initial dose for the trials will vary from about 0.001 to about 1.0
mg/kg/week. In the event that this dose does not produce a
reduction in plasma phenylalanine (Phe) levels in a patient, e.g.,
a reduction from the normal range about 50 .mu.M to about 70 .mu.M
to a range from below the level of detection to less than about 30
.mu.M, preferably less than about 20 .mu.M, and even more
preferably less than about 10 .mu.M, the dose should be increased
as necessary, and maintained for an additional minimal period of,
but necessarily limited to, 24 weeks to establish safety and to
evaluate further efficacy.
[0454] Measurements of safety will include adverse events, allergic
reactions, complete clinical chemistry panel (kidney and liver
function), urinalysis, and CBC with differential. In addition,
other parameters including the reduction in levels of blood Phe
levels, neuropsychological and cognitive testing, and global
assessments also will be monitored. The present example also
contemplates the determination of pharmacokinetic parameters of the
drug in the circulation, and general distribution and half-life of
PAL in blood. It is anticipated that these measures will help
relate dose to clinical response.
Methods
[0455] Cancer-free control patients and patients who have been
diagnosed with a form of cancer will undergo a baseline a medical
history and physical exam, neuropsychological and cognitive
testing, a standard set of clinical laboratory tests (CBC, Panel
20, CH50, UA), levels of urinary pterins, dihydropteridine
reductase (DHPR) levels, and a fasting blood (plasma) panel of
serum amino acids. Baseline blood, serum or plasma Phe levels will
be measured. The patient will be followed closely with weekly
visits to the clinic. Patients will return to the clinic for a
complete evaluation one week after completing the treatment period.
Should dose escalation be required, the patients will follow the
same schedule outlined above. Safety will be monitored throughout
the trial.
Diagnosis and Inclusion/Exclusion Criteria
[0456] The patient may be male or female, with a documented
diagnosis of a form of cancer. The study will include cancer
patients who have previously undergone surgery, chemotherapy,
radiation therapy and/or other anti-cancer therapy and are in
remission (e.g., disease-free for at least 5 years). A patient will
be excluded from this initial study if the patient has been
diagnosed with a form of cancer, but has not undergone some form of
anti-cancer therapy.
Prokaryotic PAL Safety
[0457] Prokaryotic PAL therapy will be determined to be safe if no
significant acute or chronic drug reactions occur during the course
of the study. The longer-term administration of the drug will be
determined to be safe if no significant abnormalities are observed
in the clinical examinations, clinical labs, or other appropriate
studies.
Prokaryotic PAL Efficacy
[0458] Once prokaryotic PAL therapy has been determined to be safe
and effective to reduce the plasma phenylalanine (Phe) levels in a
patient, e.g., a reduction from the normal range about 50 .mu.M to
about 70 .mu.M to a range from below the level of detection to less
than about 30 .mu.M, preferably less than about 20 .mu.M, and even
more preferably less than about 10 .mu.M, the prokaryotic PAL
compositions provided herein can be tested in cancer patients who
have previously undergone surgery, chemotherapy, radiation therapy
and/or other anti-cancer therapy and are in remission (e.g.,
disease-free for at least 5 years), as well as in patients who have
been diagnosed with a form of cancer, but have not as yet undergone
any form of anti-cancer therapy.
[0459] For cancer patients in remission, prokaryotic PAL is
administered, alone or in combination with standard cancer therapy
for the particular form of cancer, to determine whether patients
given the PAL therapy remain in remission (i.e., disease-free) for
a longer period of time than patients not given prokaryotic PAL
compositions provided herein.
[0460] For cancer patients with an active form of cancer,
prokaryotic PAL is administered, alone or in combination with
standard cancer therapy for the particular form of cancer, to
determine whether patients given the PAL therapy have a better
response to the cancer therapy (e.g., remain disease-free longer,
have longer survival time, or have lower tumor growth, tumor size
or tumor burden) than patients not given prokaryotic PAL
compositions provided herein.
[0461] Prokaryotic PAL therapy can be administered alone, or in
combination with a cancer therapeutic agent or targeted cancer
therapeutic agent, or with a protein-restricted diet (i.e.,
phenylalanine-free), or both.
SEQUENCE LISTING
[0462] The present specification is being filed with a computer
readable form (CRF) copy of the Sequence Listing. The CRF entitled
11808-029-228_SEQLIST.txt, which was created on Jul. 22, 2009 and
is 44,912 bytes in size, is identical to the paper copy of the
Sequence Listing and is incorporated herein by reference in its
entirety.
Sequence CWU 1
1
3111710DNANostoc punctiforme 1atgaatataa catctctaca acagaacata
acgcgttctt ggcaaatacc tttcactaat 60agttcagatt caatcgtaac tgtaggcgat
cgcaatctga caatcgacga ggttgtaaat 120gttgctcgtc atggaacaca
ggtgcgctta actgataatg cagatgtcat tcggggtgtt 180caagcatctt
gtgattacat taacaatgca gtcgaaacag cacagccaat ttacggggtg
240acatctggct ttggcggtat ggcagatgtt gtcatctctc gcgaacaagc
agcggaactt 300cagactaatt taatttggtt tctgaaatcc ggcgcaggaa
acaaattatc gttagcagac 360gtgcgtgcag ctatgctctt acgtgcaaat
tcacatttgt atggtgcgtc tggtatacga 420ctcgaactta ttcagcggat
tgaaactttc ctcaacgctg gcgtgacacc ccatgtctat 480gagtttggct
ctatcggtgc tagcggcgat ttggtgccat tatcctacat tactggggca
540ctaatcggtc tagatcctag ctttacagtt gacttcgacg gtaaagaaat
ggatgccgtt 600acagccttgt ctcgtttggg tttgccaaag ttgcaattgc
aaccgaaaga aggtttagca 660atgatgaatg gcacctcagt catgacaggt
attgcagcta actgtgtgta cgatgcgaaa 720gttttgctcg ctctgacaat
gggtgtacac gccttagcca tccaaggttt atacggaacg 780aatcaatctt
tccacccgtt tattcatcag tgcaagccac atcccggtca actatggaca
840gcagatcaaa tgttttctct gctgaaagat tcatctttag ttcgtgaaga
gttggatggt 900aaacacgaat accgtggtaa agatctgata caggatcgtt
attctctccg ctgtctggca 960cagttcatag ggccaatcgt tgatggggta
tcagagatta ccaagcaaat cgaggtagaa 1020atgaactcag tcaccgataa
cccattgatt gatgtcgaga accaagttag ttatcacggc 1080ggcaattttc
tcggacagta tgtgggtgtg acaatggatc gcctacgtta ttacataggg
1140ctattggcca aacacatcga tgtgcagatt gcacttcttg tctcgccaga
gtttagcaac 1200ggcttaccac cctctttagt tggtaatagc gatcgcaaag
ttaatatggg actcaaaggt 1260ttgcaaatca gtggaaactc gattatgcca
ctgttgagct tctatggaaa ttccctagcc 1320gatcgctttc ctacccacgc
cgagcaattt aatcaaaata ttaacagcca aggctatatt 1380tccgcaaatt
tgacacgtcg ttccgtagac atatttcaga attatatggc gatcgcgttg
1440atgtttggag ttcaagctgt tgacctccgc acatataaga tgaaaggtca
ttatgatgca 1500cgtacatgcc tctcacccaa tactgtgcag ttatacacag
cagtctgcga ggtagttgga 1560aagccactaa cgtctgtgcg tccatacatt
tggaacgaca acgagcaatg tttagatgag 1620catattgccc ggatttcagc
tgatatcgct ggtggtggtt taattgtgca agcagttgag 1680catatttttt
cgagcttaaa gtcaacgtaa 17102569PRTNostoc punctiforme 2Met Asn Ile
Thr Ser Leu Gln Gln Asn Ile Thr Arg Ser Trp Gln Ile1 5 10 15Pro Phe
Thr Asn Ser Ser Asp Ser Ile Val Thr Val Gly Asp Arg Asn 20 25 30Leu
Thr Ile Asp Glu Val Val Asn Val Ala Arg His Gly Thr Gln Val 35 40
45Arg Leu Thr Asp Asn Ala Asp Val Ile Arg Gly Val Gln Ala Ser Cys
50 55 60Asp Tyr Ile Asn Asn Ala Val Glu Thr Ala Gln Pro Ile Tyr Gly
Val65 70 75 80Thr Ser Gly Phe Gly Gly Met Ala Asp Val Val Ile Ser
Arg Glu Gln 85 90 95Ala Ala Glu Leu Gln Thr Asn Leu Ile Trp Phe Leu
Lys Ser Gly Ala 100 105 110Gly Asn Lys Leu Ser Leu Ala Asp Val Arg
Ala Ala Met Leu Leu Arg 115 120 125Ala Asn Ser His Leu Tyr Gly Ala
Ser Gly Ile Arg Leu Glu Leu Ile 130 135 140Gln Arg Ile Glu Thr Phe
Leu Asn Ala Gly Val Thr Pro His Val Tyr145 150 155 160Glu Phe Gly
Ser Ile Gly Ala Ser Gly Asp Leu Val Pro Leu Ser Tyr 165 170 175Ile
Thr Gly Ala Leu Ile Gly Leu Asp Pro Ser Phe Thr Val Asp Phe 180 185
190Asp Gly Lys Glu Met Asp Ala Val Thr Ala Leu Ser Arg Leu Gly Leu
195 200 205Pro Lys Leu Gln Leu Gln Pro Lys Glu Gly Leu Ala Met Met
Asn Gly 210 215 220Thr Ser Val Met Thr Gly Ile Ala Ala Asn Cys Val
Tyr Asp Ala Lys225 230 235 240Val Leu Leu Ala Leu Thr Met Gly Val
His Ala Leu Ala Ile Gln Gly 245 250 255Leu Tyr Gly Thr Asn Gln Ser
Phe His Pro Phe Ile His Gln Cys Lys 260 265 270Pro His Pro Gly Gln
Leu Trp Thr Ala Asp Gln Met Phe Ser Leu Leu 275 280 285Lys Asp Ser
Ser Leu Val Arg Glu Glu Leu Asp Gly Lys His Glu Tyr 290 295 300Arg
Gly Lys Asp Leu Ile Gln Asp Arg Tyr Ser Leu Arg Cys Leu Ala305 310
315 320Gln Phe Ile Gly Pro Ile Val Asp Gly Val Ser Glu Ile Thr Lys
Gln 325 330 335Ile Glu Val Glu Met Asn Ser Val Thr Asp Asn Pro Leu
Ile Asp Val 340 345 350Glu Asn Gln Val Ser Tyr His Gly Gly Asn Phe
Leu Gly Gln Tyr Val 355 360 365Gly Val Thr Met Asp Arg Leu Arg Tyr
Tyr Ile Gly Leu Leu Ala Lys 370 375 380His Ile Asp Val Gln Ile Ala
Leu Leu Val Ser Pro Glu Phe Ser Asn385 390 395 400Gly Leu Pro Pro
Ser Leu Val Gly Asn Ser Asp Arg Lys Val Asn Met 405 410 415Gly Leu
Lys Gly Leu Gln Ile Ser Gly Asn Ser Ile Met Pro Leu Leu 420 425
430Ser Phe Tyr Gly Asn Ser Leu Ala Asp Arg Phe Pro Thr His Ala Glu
435 440 445Gln Phe Asn Gln Asn Ile Asn Ser Gln Gly Tyr Ile Ser Ala
Asn Leu 450 455 460Thr Arg Arg Ser Val Asp Ile Phe Gln Asn Tyr Met
Ala Ile Ala Leu465 470 475 480Met Phe Gly Val Gln Ala Val Asp Leu
Arg Thr Tyr Lys Met Lys Gly 485 490 495His Tyr Asp Ala Arg Thr Cys
Leu Ser Pro Asn Thr Val Gln Leu Tyr 500 505 510Thr Ala Val Cys Glu
Val Val Gly Lys Pro Leu Thr Ser Val Arg Pro 515 520 525Tyr Ile Trp
Asn Asp Asn Glu Gln Cys Leu Asp Glu His Ile Ala Arg 530 535 540Ile
Ser Ala Asp Ile Ala Gly Gly Gly Leu Ile Val Gln Ala Val Glu545 550
555 560His Ile Phe Ser Ser Leu Lys Ser Thr 56531704DNAAnabaena
variabilis 3atgaagacac tatctcaagc acaaagcaaa acctcatctc aacaattttc
ttttactgga 60aattcttctg ccaatgtaat tattggtaat cagaaactca caatcaatga
tgttgcaagg 120gtagcgcgta atggcacctt agtgtcttta accaataaca
ctgatatttt gcagggtatt 180caggcatctt gtgattacat taataatgct
gttgaatctg gggaaccaat ttatggagtg 240acatctggtt ttggcggtat
ggccaatgtt gccatatccc gtgaacaagc atctgaactc 300caaaccaact
tagtttggtt cctgaaaaca ggtgcaggga acaaattacc cttggcggat
360gtgcgcgcag ctatgctctt gcgtgcaaac tctcatatgc gcggtgcatc
tggcatcaga 420ttagaactta tcaagcgtat ggagattttc cttaacgctg
gtgtcacacc atatgtgtat 480gagtttggtt caattggtgc aagtggtgat
ttagtgccac tatcctacat tactggttca 540ctgataggct tagatcccag
ttttaaggtt gacttcaacg gtaaagaaat ggatgcgcca 600acagctctac
gtcaactgaa tttgtcaccc ttgacattgt tgccgaagga aggcttggcg
660atgatgaacg gcacttcagt catgacaggt attgcagcaa actgcgtcta
cgatactcaa 720attttaactg cgatcgctat gggcgttcac gctctagata
tccaagcttt aaacggaacc 780aatcaatcat tccatccatt tatccataat
tccaaaccac atcctggtca attatgggca 840gcagatcaga tgatttcttt
gttagccaat tcccagttag ttcgtgatga gttagatggt 900aaacacgatt
atcgtgatca cgagttgatt caagatcgtt actcactccg atgccttccc
960cagtatttgg ggccaatcgt tgatggaatt tcccagattg ccaaacaaat
tgaaatcgaa 1020atcaactcag tcaccgataa cccactaatt gatgttgata
accaagctag ctatcatgga 1080ggaaatttcc tcggacagta cgtgggtatg
ggaatggatc acctgcgtta ctatattggg 1140ttattggcta aacacctaga
tgtgcagatt gccctcctcg cctcaccaga gtttagcaat 1200ggactaccac
catctttatt aggcaaccga gaacgtaaag tcaatatggg actcaaaggt
1260ctgcaaatat gcggtaactc aattatgcca ctgttgacct tctatggaaa
ttccatcgcc 1320gatcgctttc ctacccatgc agaacaattt aatcagaaca
tcaacagtca aggatacact 1380tcagcgactc tagcccgccg ttctgtggat
atcttccaga attatgtggc gatcgctctg 1440atgtttggag tccaagctgt
tgacctccgc acatataaaa agactggtca ttacgatgca 1500cgcgcctgtc
tatcacctgc aactgagcgc ttatattcag cagtccgcca cgtagttgga
1560caaaaaccaa cttcagatcg cccatatatt tggaatgata atgagcaagg
actggatgag 1620catattgccc ggatttctgc tgatatcgct gctggtggtg
tgattgtgca agcagttcaa 1680gatatcttac cctgcttgca ttaa
17044567PRTAnabaena variabilis 4Met Lys Thr Leu Ser Gln Ala Gln Ser
Lys Thr Ser Ser Gln Gln Phe1 5 10 15Ser Phe Thr Gly Asn Ser Ser Ala
Asn Val Ile Ile Gly Asn Gln Lys 20 25 30Leu Thr Ile Asn Asp Val Ala
Arg Val Ala Arg Asn Gly Thr Leu Val 35 40 45Ser Leu Thr Asn Asn Thr
Asp Ile Leu Gln Gly Ile Gln Ala Ser Cys 50 55 60Asp Tyr Ile Asn Asn
Ala Val Glu Ser Gly Glu Pro Ile Tyr Gly Val65 70 75 80Thr Ser Gly
Phe Gly Gly Met Ala Asn Val Ala Ile Ser Arg Glu Gln 85 90 95Ala Ser
Glu Leu Gln Thr Asn Leu Val Trp Phe Leu Lys Thr Gly Ala 100 105
110Gly Asn Lys Leu Pro Leu Ala Asp Val Arg Ala Ala Met Leu Leu Arg
115 120 125Ala Asn Ser His Met Arg Gly Ala Ser Gly Ile Arg Leu Glu
Leu Ile 130 135 140Lys Arg Met Glu Ile Phe Leu Asn Ala Gly Val Thr
Pro Tyr Val Tyr145 150 155 160Glu Phe Gly Ser Ile Gly Ala Ser Gly
Asp Leu Val Pro Leu Ser Tyr 165 170 175Ile Thr Gly Ser Leu Ile Gly
Leu Asp Pro Ser Phe Lys Val Asp Phe 180 185 190Asn Gly Lys Glu Met
Asp Ala Pro Thr Ala Leu Arg Gln Leu Asn Leu 195 200 205Ser Pro Leu
Thr Leu Leu Pro Lys Glu Gly Leu Ala Met Met Asn Gly 210 215 220Thr
Ser Val Met Thr Gly Ile Ala Ala Asn Cys Val Tyr Asp Thr Gln225 230
235 240Ile Leu Thr Ala Ile Ala Met Gly Val His Ala Leu Asp Ile Gln
Ala 245 250 255Leu Asn Gly Thr Asn Gln Ser Phe His Pro Phe Ile His
Asn Ser Lys 260 265 270Pro His Pro Gly Gln Leu Trp Ala Ala Asp Gln
Met Ile Ser Leu Leu 275 280 285Ala Asn Ser Gln Leu Val Arg Asp Glu
Leu Asp Gly Lys His Asp Tyr 290 295 300Arg Asp His Glu Leu Ile Gln
Asp Arg Tyr Ser Leu Arg Cys Leu Pro305 310 315 320Gln Tyr Leu Gly
Pro Ile Val Asp Gly Ile Ser Gln Ile Ala Lys Gln 325 330 335Ile Glu
Ile Glu Ile Asn Ser Val Thr Asp Asn Pro Leu Ile Asp Val 340 345
350Asp Asn Gln Ala Ser Tyr His Gly Gly Asn Phe Leu Gly Gln Tyr Val
355 360 365Gly Met Gly Met Asp His Leu Arg Tyr Tyr Ile Gly Leu Leu
Ala Lys 370 375 380His Leu Asp Val Gln Ile Ala Leu Leu Ala Ser Pro
Glu Phe Ser Asn385 390 395 400Gly Leu Pro Pro Ser Leu Leu Gly Asn
Arg Glu Arg Lys Val Asn Met 405 410 415Gly Leu Lys Gly Leu Gln Ile
Cys Gly Asn Ser Ile Met Pro Leu Leu 420 425 430Thr Phe Tyr Gly Asn
Ser Ile Ala Asp Arg Phe Pro Thr His Ala Glu 435 440 445Gln Phe Asn
Gln Asn Ile Asn Ser Gln Gly Tyr Thr Ser Ala Thr Leu 450 455 460Ala
Arg Arg Ser Val Asp Ile Phe Gln Asn Tyr Val Ala Ile Ala Leu465 470
475 480Met Phe Gly Val Gln Ala Val Asp Leu Arg Thr Tyr Lys Lys Thr
Gly 485 490 495His Tyr Asp Ala Arg Ala Cys Leu Ser Pro Ala Thr Glu
Arg Leu Tyr 500 505 510Ser Ala Val Arg His Val Val Gly Gln Lys Pro
Thr Ser Asp Arg Pro 515 520 525Tyr Ile Trp Asn Asp Asn Glu Gln Gly
Leu Asp Glu His Ile Ala Arg 530 535 540Ile Ser Ala Asp Ile Ala Ala
Gly Gly Val Ile Val Gln Ala Val Gln545 550 555 560Asp Ile Leu Pro
Cys Leu His 565543DNAArtificial SequenceAnabaena variabilis PAL
primer to create cysteine to serine substitution at position 318
(forward) 5caagatcgtt actcactccg atcccttccc cagtatttgg ggc
43643DNAArtificial SequenceAnabaena variabilis PAL primer to create
cysteine to serine substitution at position 318 (reverse)
6gccccaaata ctggggaagg gatcggagtg agtaacgatc ttg
437567PRTArtificial SequenceCysteine to serine substitution at
position 64 in Anabaena variabilis PAL 7Met Lys Thr Leu Ser Gln Ala
Gln Ser Lys Thr Ser Ser Gln Gln Phe1 5 10 15Ser Phe Thr Gly Asn Ser
Ser Ala Asn Val Ile Ile Gly Asn Gln Lys 20 25 30Leu Thr Ile Asn Asp
Val Ala Arg Val Ala Arg Asn Gly Thr Leu Val 35 40 45Ser Leu Thr Asn
Asn Thr Asp Ile Leu Gln Gly Ile Gln Ala Ser Ser 50 55 60Asp Tyr Ile
Asn Asn Ala Val Glu Ser Gly Glu Pro Ile Tyr Gly Val65 70 75 80Thr
Ser Gly Phe Gly Gly Met Ala Asn Val Ala Ile Ser Arg Glu Gln 85 90
95Ala Ser Glu Leu Gln Thr Asn Leu Val Trp Phe Leu Lys Thr Gly Ala
100 105 110Gly Asn Lys Leu Pro Leu Ala Asp Val Arg Ala Ala Met Leu
Leu Arg 115 120 125Ala Asn Ser His Met Arg Gly Ala Ser Gly Ile Arg
Leu Glu Leu Ile 130 135 140Lys Arg Met Glu Ile Phe Leu Asn Ala Gly
Val Thr Pro Tyr Val Tyr145 150 155 160Glu Phe Gly Ser Ile Gly Ala
Ser Gly Asp Leu Val Pro Leu Ser Tyr 165 170 175Ile Thr Gly Ser Leu
Ile Gly Leu Asp Pro Ser Phe Lys Val Asp Phe 180 185 190Asn Gly Lys
Glu Met Asp Ala Pro Thr Ala Leu Arg Gln Leu Asn Leu 195 200 205Ser
Pro Leu Thr Leu Leu Pro Lys Glu Gly Leu Ala Met Met Asn Gly 210 215
220Thr Ser Val Met Thr Gly Ile Ala Ala Asn Cys Val Tyr Asp Thr
Gln225 230 235 240Ile Leu Thr Ala Ile Ala Met Gly Val His Ala Leu
Asp Ile Gln Ala 245 250 255Leu Asn Gly Thr Asn Gln Ser Phe His Pro
Phe Ile His Asn Ser Lys 260 265 270Pro His Pro Gly Gln Leu Trp Ala
Ala Asp Gln Met Ile Ser Leu Leu 275 280 285Ala Asn Ser Gln Leu Val
Arg Asp Glu Leu Asp Gly Lys His Asp Tyr 290 295 300Arg Asp His Glu
Leu Ile Gln Asp Arg Tyr Ser Leu Arg Cys Leu Pro305 310 315 320Gln
Tyr Leu Gly Pro Ile Val Asp Gly Ile Ser Gln Ile Ala Lys Gln 325 330
335Ile Glu Ile Glu Ile Asn Ser Val Thr Asp Asn Pro Leu Ile Asp Val
340 345 350Asp Asn Gln Ala Ser Tyr His Gly Gly Asn Phe Leu Gly Gln
Tyr Val 355 360 365Gly Met Gly Met Asp His Leu Arg Tyr Tyr Ile Gly
Leu Leu Ala Lys 370 375 380His Leu Asp Val Gln Ile Ala Leu Leu Ala
Ser Pro Glu Phe Ser Asn385 390 395 400Gly Leu Pro Pro Ser Leu Leu
Gly Asn Arg Glu Arg Lys Val Asn Met 405 410 415Gly Leu Lys Gly Leu
Gln Ile Cys Gly Asn Ser Ile Met Pro Leu Leu 420 425 430Thr Phe Tyr
Gly Asn Ser Ile Ala Asp Arg Phe Pro Thr His Ala Glu 435 440 445Gln
Phe Asn Gln Asn Ile Asn Ser Gln Gly Tyr Thr Ser Ala Thr Leu 450 455
460Ala Arg Arg Ser Val Asp Ile Phe Gln Asn Tyr Val Ala Ile Ala
Leu465 470 475 480Met Phe Gly Val Gln Ala Val Asp Leu Arg Thr Tyr
Lys Lys Thr Gly 485 490 495His Tyr Asp Ala Arg Ala Cys Leu Ser Pro
Ala Thr Glu Arg Leu Tyr 500 505 510Ser Ala Val Arg His Val Val Gly
Gln Lys Pro Thr Ser Asp Arg Pro 515 520 525Tyr Ile Trp Asn Asp Asn
Glu Gln Gly Leu Asp Glu His Ile Ala Arg 530 535 540Ile Ser Ala Asp
Ile Ala Ala Gly Gly Val Ile Val Gln Ala Val Gln545 550 555 560Asp
Ile Leu Pro Cys Leu His 5658567PRTArtificial SequenceCysteine to
serine substitution at position 318 in Anabaena variabilis PAL 8Met
Lys Thr Leu Ser Gln Ala Gln Ser Lys Thr Ser Ser Gln Gln Phe1 5 10
15Ser Phe Thr Gly Asn Ser Ser Ala Asn Val Ile Ile Gly Asn Gln Lys
20 25 30Leu Thr Ile Asn Asp Val Ala Arg Val Ala Arg Asn Gly Thr Leu
Val 35 40 45Ser Leu Thr Asn Asn Thr Asp Ile Leu Gln Gly Ile Gln Ala
Ser Cys 50 55 60Asp Tyr Ile Asn Asn Ala Val Glu Ser Gly Glu Pro Ile
Tyr Gly Val65 70 75 80Thr Ser Gly Phe Gly Gly Met Ala Asn Val Ala
Ile Ser Arg Glu Gln 85 90 95Ala Ser Glu Leu Gln Thr Asn Leu Val Trp
Phe Leu Lys Thr
Gly Ala 100 105 110Gly Asn Lys Leu Pro Leu Ala Asp Val Arg Ala Ala
Met Leu Leu Arg 115 120 125Ala Asn Ser His Met Arg Gly Ala Ser Gly
Ile Arg Leu Glu Leu Ile 130 135 140Lys Arg Met Glu Ile Phe Leu Asn
Ala Gly Val Thr Pro Tyr Val Tyr145 150 155 160Glu Phe Gly Ser Ile
Gly Ala Ser Gly Asp Leu Val Pro Leu Ser Tyr 165 170 175Ile Thr Gly
Ser Leu Ile Gly Leu Asp Pro Ser Phe Lys Val Asp Phe 180 185 190Asn
Gly Lys Glu Met Asp Ala Pro Thr Ala Leu Arg Gln Leu Asn Leu 195 200
205Ser Pro Leu Thr Leu Leu Pro Lys Glu Gly Leu Ala Met Met Asn Gly
210 215 220Thr Ser Val Met Thr Gly Ile Ala Ala Asn Cys Val Tyr Asp
Thr Gln225 230 235 240Ile Leu Thr Ala Ile Ala Met Gly Val His Ala
Leu Asp Ile Gln Ala 245 250 255Leu Asn Gly Thr Asn Gln Ser Phe His
Pro Phe Ile His Asn Ser Lys 260 265 270Pro His Pro Gly Gln Leu Trp
Ala Ala Asp Gln Met Ile Ser Leu Leu 275 280 285Ala Asn Ser Gln Leu
Val Arg Asp Glu Leu Asp Gly Lys His Asp Tyr 290 295 300Arg Asp His
Glu Leu Ile Gln Asp Arg Tyr Ser Leu Arg Ser Leu Pro305 310 315
320Gln Tyr Leu Gly Pro Ile Val Asp Gly Ile Ser Gln Ile Ala Lys Gln
325 330 335Ile Glu Ile Glu Ile Asn Ser Val Thr Asp Asn Pro Leu Ile
Asp Val 340 345 350Asp Asn Gln Ala Ser Tyr His Gly Gly Asn Phe Leu
Gly Gln Tyr Val 355 360 365Gly Met Gly Met Asp His Leu Arg Tyr Tyr
Ile Gly Leu Leu Ala Lys 370 375 380His Leu Asp Val Gln Ile Ala Leu
Leu Ala Ser Pro Glu Phe Ser Asn385 390 395 400Gly Leu Pro Pro Ser
Leu Leu Gly Asn Arg Glu Arg Lys Val Asn Met 405 410 415Gly Leu Lys
Gly Leu Gln Ile Cys Gly Asn Ser Ile Met Pro Leu Leu 420 425 430Thr
Phe Tyr Gly Asn Ser Ile Ala Asp Arg Phe Pro Thr His Ala Glu 435 440
445Gln Phe Asn Gln Asn Ile Asn Ser Gln Gly Tyr Thr Ser Ala Thr Leu
450 455 460Ala Arg Arg Ser Val Asp Ile Phe Gln Asn Tyr Val Ala Ile
Ala Leu465 470 475 480Met Phe Gly Val Gln Ala Val Asp Leu Arg Thr
Tyr Lys Lys Thr Gly 485 490 495His Tyr Asp Ala Arg Ala Cys Leu Ser
Pro Ala Thr Glu Arg Leu Tyr 500 505 510Ser Ala Val Arg His Val Val
Gly Gln Lys Pro Thr Ser Asp Arg Pro 515 520 525Tyr Ile Trp Asn Asp
Asn Glu Gln Gly Leu Asp Glu His Ile Ala Arg 530 535 540Ile Ser Ala
Asp Ile Ala Ala Gly Gly Val Ile Val Gln Ala Val Gln545 550 555
560Asp Ile Leu Pro Cys Leu His 5659567PRTArtificial
SequenceCysteine to serine substitution at position 503 in Anabaena
variabilis PAL 9Met Lys Thr Leu Ser Gln Ala Gln Ser Lys Thr Ser Ser
Gln Gln Phe1 5 10 15Ser Phe Thr Gly Asn Ser Ser Ala Asn Val Ile Ile
Gly Asn Gln Lys 20 25 30Leu Thr Ile Asn Asp Val Ala Arg Val Ala Arg
Asn Gly Thr Leu Val 35 40 45Ser Leu Thr Asn Asn Thr Asp Ile Leu Gln
Gly Ile Gln Ala Ser Cys 50 55 60Asp Tyr Ile Asn Asn Ala Val Glu Ser
Gly Glu Pro Ile Tyr Gly Val65 70 75 80Thr Ser Gly Phe Gly Gly Met
Ala Asn Val Ala Ile Ser Arg Glu Gln 85 90 95Ala Ser Glu Leu Gln Thr
Asn Leu Val Trp Phe Leu Lys Thr Gly Ala 100 105 110Gly Asn Lys Leu
Pro Leu Ala Asp Val Arg Ala Ala Met Leu Leu Arg 115 120 125Ala Asn
Ser His Met Arg Gly Ala Ser Gly Ile Arg Leu Glu Leu Ile 130 135
140Lys Arg Met Glu Ile Phe Leu Asn Ala Gly Val Thr Pro Tyr Val
Tyr145 150 155 160Glu Phe Gly Ser Ile Gly Ala Ser Gly Asp Leu Val
Pro Leu Ser Tyr 165 170 175Ile Thr Gly Ser Leu Ile Gly Leu Asp Pro
Ser Phe Lys Val Asp Phe 180 185 190Asn Gly Lys Glu Met Asp Ala Pro
Thr Ala Leu Arg Gln Leu Asn Leu 195 200 205Ser Pro Leu Thr Leu Leu
Pro Lys Glu Gly Leu Ala Met Met Asn Gly 210 215 220Thr Ser Val Met
Thr Gly Ile Ala Ala Asn Cys Val Tyr Asp Thr Gln225 230 235 240Ile
Leu Thr Ala Ile Ala Met Gly Val His Ala Leu Asp Ile Gln Ala 245 250
255Leu Asn Gly Thr Asn Gln Ser Phe His Pro Phe Ile His Asn Ser Lys
260 265 270Pro His Pro Gly Gln Leu Trp Ala Ala Asp Gln Met Ile Ser
Leu Leu 275 280 285Ala Asn Ser Gln Leu Val Arg Asp Glu Leu Asp Gly
Lys His Asp Tyr 290 295 300Arg Asp His Glu Leu Ile Gln Asp Arg Tyr
Ser Leu Arg Cys Leu Pro305 310 315 320Gln Tyr Leu Gly Pro Ile Val
Asp Gly Ile Ser Gln Ile Ala Lys Gln 325 330 335Ile Glu Ile Glu Ile
Asn Ser Val Thr Asp Asn Pro Leu Ile Asp Val 340 345 350Asp Asn Gln
Ala Ser Tyr His Gly Gly Asn Phe Leu Gly Gln Tyr Val 355 360 365Gly
Met Gly Met Asp His Leu Arg Tyr Tyr Ile Gly Leu Leu Ala Lys 370 375
380His Leu Asp Val Gln Ile Ala Leu Leu Ala Ser Pro Glu Phe Ser
Asn385 390 395 400Gly Leu Pro Pro Ser Leu Leu Gly Asn Arg Glu Arg
Lys Val Asn Met 405 410 415Gly Leu Lys Gly Leu Gln Ile Cys Gly Asn
Ser Ile Met Pro Leu Leu 420 425 430Thr Phe Tyr Gly Asn Ser Ile Ala
Asp Arg Phe Pro Thr His Ala Glu 435 440 445Gln Phe Asn Gln Asn Ile
Asn Ser Gln Gly Tyr Thr Ser Ala Thr Leu 450 455 460Ala Arg Arg Ser
Val Asp Ile Phe Gln Asn Tyr Val Ala Ile Ala Leu465 470 475 480Met
Phe Gly Val Gln Ala Val Asp Leu Arg Thr Tyr Lys Lys Thr Gly 485 490
495His Tyr Asp Ala Arg Ala Ser Leu Ser Pro Ala Thr Glu Arg Leu Tyr
500 505 510Ser Ala Val Arg His Val Val Gly Gln Lys Pro Thr Ser Asp
Arg Pro 515 520 525Tyr Ile Trp Asn Asp Asn Glu Gln Gly Leu Asp Glu
His Ile Ala Arg 530 535 540Ile Ser Ala Asp Ile Ala Ala Gly Gly Val
Ile Val Gln Ala Val Gln545 550 555 560Asp Ile Leu Pro Cys Leu His
56510567PRTArtificial SequenceCysteine to serine substition at
position 565 in Anabaena variabilis PAL 10Met Lys Thr Leu Ser Gln
Ala Gln Ser Lys Thr Ser Ser Gln Gln Phe1 5 10 15Ser Phe Thr Gly Asn
Ser Ser Ala Asn Val Ile Ile Gly Asn Gln Lys 20 25 30Leu Thr Ile Asn
Asp Val Ala Arg Val Ala Arg Asn Gly Thr Leu Val 35 40 45Ser Leu Thr
Asn Asn Thr Asp Ile Leu Gln Gly Ile Gln Ala Ser Cys 50 55 60Asp Tyr
Ile Asn Asn Ala Val Glu Ser Gly Glu Pro Ile Tyr Gly Val65 70 75
80Thr Ser Gly Phe Gly Gly Met Ala Asn Val Ala Ile Ser Arg Glu Gln
85 90 95Ala Ser Glu Leu Gln Thr Asn Leu Val Trp Phe Leu Lys Thr Gly
Ala 100 105 110Gly Asn Lys Leu Pro Leu Ala Asp Val Arg Ala Ala Met
Leu Leu Arg 115 120 125Ala Asn Ser His Met Arg Gly Ala Ser Gly Ile
Arg Leu Glu Leu Ile 130 135 140Lys Arg Met Glu Ile Phe Leu Asn Ala
Gly Val Thr Pro Tyr Val Tyr145 150 155 160Glu Phe Gly Ser Ile Gly
Ala Ser Gly Asp Leu Val Pro Leu Ser Tyr 165 170 175Ile Thr Gly Ser
Leu Ile Gly Leu Asp Pro Ser Phe Lys Val Asp Phe 180 185 190Asn Gly
Lys Glu Met Asp Ala Pro Thr Ala Leu Arg Gln Leu Asn Leu 195 200
205Ser Pro Leu Thr Leu Leu Pro Lys Glu Gly Leu Ala Met Met Asn Gly
210 215 220Thr Ser Val Met Thr Gly Ile Ala Ala Asn Cys Val Tyr Asp
Thr Gln225 230 235 240Ile Leu Thr Ala Ile Ala Met Gly Val His Ala
Leu Asp Ile Gln Ala 245 250 255Leu Asn Gly Thr Asn Gln Ser Phe His
Pro Phe Ile His Asn Ser Lys 260 265 270Pro His Pro Gly Gln Leu Trp
Ala Ala Asp Gln Met Ile Ser Leu Leu 275 280 285Ala Asn Ser Gln Leu
Val Arg Asp Glu Leu Asp Gly Lys His Asp Tyr 290 295 300Arg Asp His
Glu Leu Ile Gln Asp Arg Tyr Ser Leu Arg Cys Leu Pro305 310 315
320Gln Tyr Leu Gly Pro Ile Val Asp Gly Ile Ser Gln Ile Ala Lys Gln
325 330 335Ile Glu Ile Glu Ile Asn Ser Val Thr Asp Asn Pro Leu Ile
Asp Val 340 345 350Asp Asn Gln Ala Ser Tyr His Gly Gly Asn Phe Leu
Gly Gln Tyr Val 355 360 365Gly Met Gly Met Asp His Leu Arg Tyr Tyr
Ile Gly Leu Leu Ala Lys 370 375 380His Leu Asp Val Gln Ile Ala Leu
Leu Ala Ser Pro Glu Phe Ser Asn385 390 395 400Gly Leu Pro Pro Ser
Leu Leu Gly Asn Arg Glu Arg Lys Val Asn Met 405 410 415Gly Leu Lys
Gly Leu Gln Ile Cys Gly Asn Ser Ile Met Pro Leu Leu 420 425 430Thr
Phe Tyr Gly Asn Ser Ile Ala Asp Arg Phe Pro Thr His Ala Glu 435 440
445Gln Phe Asn Gln Asn Ile Asn Ser Gln Gly Tyr Thr Ser Ala Thr Leu
450 455 460Ala Arg Arg Ser Val Asp Ile Phe Gln Asn Tyr Val Ala Ile
Ala Leu465 470 475 480Met Phe Gly Val Gln Ala Val Asp Leu Arg Thr
Tyr Lys Lys Thr Gly 485 490 495His Tyr Asp Ala Arg Ala Cys Leu Ser
Pro Ala Thr Glu Arg Leu Tyr 500 505 510Ser Ala Val Arg His Val Val
Gly Gln Lys Pro Thr Ser Asp Arg Pro 515 520 525Tyr Ile Trp Asn Asp
Asn Glu Gln Gly Leu Asp Glu His Ile Ala Arg 530 535 540Ile Ser Ala
Asp Ile Ala Ala Gly Gly Val Ile Val Gln Ala Val Gln545 550 555
560Asp Ile Leu Pro Ser Leu His 56511567PRTArtificial
SequenceCysteine to serine substitutions at positions 565 and 503
in Anabaena variabilis PAL 11Met Lys Thr Leu Ser Gln Ala Gln Ser
Lys Thr Ser Ser Gln Gln Phe1 5 10 15Ser Phe Thr Gly Asn Ser Ser Ala
Asn Val Ile Ile Gly Asn Gln Lys 20 25 30Leu Thr Ile Asn Asp Val Ala
Arg Val Ala Arg Asn Gly Thr Leu Val 35 40 45Ser Leu Thr Asn Asn Thr
Asp Ile Leu Gln Gly Ile Gln Ala Ser Cys 50 55 60Asp Tyr Ile Asn Asn
Ala Val Glu Ser Gly Glu Pro Ile Tyr Gly Val65 70 75 80Thr Ser Gly
Phe Gly Gly Met Ala Asn Val Ala Ile Ser Arg Glu Gln 85 90 95Ala Ser
Glu Leu Gln Thr Asn Leu Val Trp Phe Leu Lys Thr Gly Ala 100 105
110Gly Asn Lys Leu Pro Leu Ala Asp Val Arg Ala Ala Met Leu Leu Arg
115 120 125Ala Asn Ser His Met Arg Gly Ala Ser Gly Ile Arg Leu Glu
Leu Ile 130 135 140Lys Arg Met Glu Ile Phe Leu Asn Ala Gly Val Thr
Pro Tyr Val Tyr145 150 155 160Glu Phe Gly Ser Ile Gly Ala Ser Gly
Asp Leu Val Pro Leu Ser Tyr 165 170 175Ile Thr Gly Ser Leu Ile Gly
Leu Asp Pro Ser Phe Lys Val Asp Phe 180 185 190Asn Gly Lys Glu Met
Asp Ala Pro Thr Ala Leu Arg Gln Leu Asn Leu 195 200 205Ser Pro Leu
Thr Leu Leu Pro Lys Glu Gly Leu Ala Met Met Asn Gly 210 215 220Thr
Ser Val Met Thr Gly Ile Ala Ala Asn Cys Val Tyr Asp Thr Gln225 230
235 240Ile Leu Thr Ala Ile Ala Met Gly Val His Ala Leu Asp Ile Gln
Ala 245 250 255Leu Asn Gly Thr Asn Gln Ser Phe His Pro Phe Ile His
Asn Ser Lys 260 265 270Pro His Pro Gly Gln Leu Trp Ala Ala Asp Gln
Met Ile Ser Leu Leu 275 280 285Ala Asn Ser Gln Leu Val Arg Asp Glu
Leu Asp Gly Lys His Asp Tyr 290 295 300Arg Asp His Glu Leu Ile Gln
Asp Arg Tyr Ser Leu Arg Cys Leu Pro305 310 315 320Gln Tyr Leu Gly
Pro Ile Val Asp Gly Ile Ser Gln Ile Ala Lys Gln 325 330 335Ile Glu
Ile Glu Ile Asn Ser Val Thr Asp Asn Pro Leu Ile Asp Val 340 345
350Asp Asn Gln Ala Ser Tyr His Gly Gly Asn Phe Leu Gly Gln Tyr Val
355 360 365Gly Met Gly Met Asp His Leu Arg Tyr Tyr Ile Gly Leu Leu
Ala Lys 370 375 380His Leu Asp Val Gln Ile Ala Leu Leu Ala Ser Pro
Glu Phe Ser Asn385 390 395 400Gly Leu Pro Pro Ser Leu Leu Gly Asn
Arg Glu Arg Lys Val Asn Met 405 410 415Gly Leu Lys Gly Leu Gln Ile
Cys Gly Asn Ser Ile Met Pro Leu Leu 420 425 430Thr Phe Tyr Gly Asn
Ser Ile Ala Asp Arg Phe Pro Thr His Ala Glu 435 440 445Gln Phe Asn
Gln Asn Ile Asn Ser Gln Gly Tyr Thr Ser Ala Thr Leu 450 455 460Ala
Arg Arg Ser Val Asp Ile Phe Gln Asn Tyr Val Ala Ile Ala Leu465 470
475 480Met Phe Gly Val Gln Ala Val Asp Leu Arg Thr Tyr Lys Lys Thr
Gly 485 490 495His Tyr Asp Ala Arg Ala Ser Leu Ser Pro Ala Thr Glu
Arg Leu Tyr 500 505 510Ser Ala Val Arg His Val Val Gly Gln Lys Pro
Thr Ser Asp Arg Pro 515 520 525Tyr Ile Trp Asn Asp Asn Glu Gln Gly
Leu Asp Glu His Ile Ala Arg 530 535 540Ile Ser Ala Asp Ile Ala Ala
Gly Gly Val Ile Val Gln Ala Val Gln545 550 555 560Asp Ile Leu Pro
Ser Leu His 5651238DNAArtificial SequenceNostoc punctiforme PAL
primer 1 (forward) 12cactgtcata tgaatataac atctctacaa cagaacat
381345DNAArtificial SequenceNostoc punctiforme PAL primer 2
(reverse) 13gacagtggcg gccgctcacg ttgactttaa gctcgaaaaa atatg
451438DNAArtificial SequenceAnabaena variabilis PAL primer 1
(forward, N-terminal fragment) 14cactgtgcta gcatgaagac actatctcaa
gcacaaag 381549DNAArtificial SequenceAnabaena variabilis PAL primer
2 (reverse, N-terminal fragment) 15ggaaatttcc tccatgatag ctggcttggt
tatcaacatc aattagtgg 491649DNAArtificial SequenceAnabaena
variabilis PAL primer 3 (forward, C-terminal fragment) 16ccactaattg
atgttgataa ccaagccagc tatcatggag gaaatttcc 491741DNAArtificial
SequenceAnabaena variabilis PAL primer 4 (reverse, C-terminal
fragment) 17cactgtgcgg ccgcttaatg caagcagggt aagatatctt g
411835DNAArtificial SequenceAnabaena variabilis PAL forward primer
18cactgtcata tgaagacact atctcaagca caaag 351936DNAArtificial
SequenceAnabaena variabilis PAL reverse primer 19cactgtctcg
agatgcaagc agggtaagat atcttg 362038DNAArtificial SequenceAnabaena
variabilis PAL primer to create 5' NheI site and delete internal
NheI site (forward, N-terminal) 20cactgtgcta gcatgaagac actatctcaa
gcacaaag 382149DNAArtificial SequenceAnabaena variabilis PAL primer
to create 5' NheI site and delete internal NheI site (reverse,
N-terminal ) 21ggaaatttcc tccatgatag ctggcttggt tatcaacatc
aattagtgg 492249DNAArtificial SequenceAnabaena variabilis PAL
primer to create 5' NheI site and delete internal NheI site
(forward, C-terminal fragment) 22ccactaattg atgttgataa ccaagccagc
tatcatggag gaaatttcc 492341DNAArtificial
SequenceAnabaena variabilis PAL primer to create 5' NheI site and
delete internal NheI site (reverse, C-terminal fragment)
23acagtggcgg ccgcttaatg caagcagggt aagatatctt g 412438DNAArtificial
SequenceAnabaena variabilis PAL primer to create 5' NheI site and
3' SmaI site (forward) 24cactgtgaat tcatgaagac actatctcaa gcacaaag
382537DNAArtificial SequenceAnabaena variabilis PAL primer to
create 5' NheI site and 3' SmaI site (reverse) 25cactgtcccg
ggttaatgca agcagggtaa gatatct 372641DNAArtificial SequenceAnabaena
variabilis PAL primer to create cysteine to serine substitution at
position 503 (forward) 26gtcattacga tgcacgcgcc tctctatcac
ctgcaactga g 412741DNAArtificial SequenceAnabaena variabilis PAL
primer to create cysteine to serine substitution at position 503
(reverse) 27ctcagttgca ggtgatagag aggcgcgtgc atcgtaatga c
412842DNAArtificial SequenceAnabaena variabilis PAL primer to
create cysteine to serine substitution at position 565 (forward)
28cagttcaaga tatcttaccc tccttgcatt aacccgggct gc
422942DNAArtificial SequenceAnabaena variabilis PAL primer to
create cysteine to serine substitution at position 565 (reverse)
29gcagcccggg ttaatgcaag gagggtaaga tatcttgaac tg
423044DNAArtificial SequenceAnabaena variabilis PAL primer to
create cysteine to serine substitution at position 64 (forward)
30gcagggtatt caggcatctt ctgattacat taataatgct gttg
443144DNAArtificial SequenceAnabaena variabilis PAL primer to
create cysteine to serine substitution at position 64 (reverse)
31caacagcatt attaatgtaa tcagaagatg cctgaatacc ctgc 44
* * * * *