CHEMICAL COMPOUNDS
This invention relates to antibody directed enzyme prodrug therapy (ADEPT) using a
non-naturally occuring mutant form pf a host enzyme, especially a mutant form of
ribonuclease.
Targeting of drugs selectively to kill cancer cells in a patient has long been a problem for
medical research. ADEPT is one approach to overcome the problem. ADEPT uses a tumour
selective antibody conjugated to an enzyme. The conjugate is administered to the patient
(usually intravenously), allowed to localise at the tumour site(s) and clear from the general
circulation. Subsequently a prodrug is administered to the patient which is converted by the
enzyme (localised at the tumour sites) into a cytotoxic drug which kills tumour cells. Since
one molecule of enzyme can catalyse generation of many cytotoxic drug molecules an
amplification effect is produced. Furthermore tumour cells not displaying the antigen
recognised by the antibody (tumours usually display microheterogeneity) are also killed by
enzymically amplified generation of the cytotoxic drug. A known system uses the procaryotic
enzyme carboxypeptidase G2 (CPG2) as the enzyme component (see WO 88/07378). A
drawback of systems using procaryotic enzymes is that the normal gut flora may contain
procaryotic organisms capable of triggering non-selective cytotoxic drug generation.
A further problem with known systems is that repeated administration of the conjugate results
in a host immune response rendering the therapy less effective. The antibody component is
generally a mouse monoclonal which can be humanised using known techniques to reduce
immunogenicity. However reduction of the immunogenicity of the enzyme component has
proved more problematic. This is because the enzyme component must not be present
naturally in the human host circulation otherwise premature conversion of prodrug to cytotoxic
drug will occur and no selective toxicity to tumours will be observed. Akzo in WO90/02939
have proposed use of human enzymes for ADEPT with selectivity being maintained by choice
of a human enzyme not normally present in the circulation such as lysozyme. Akzo have
chosen human lysozyme as their enzyme and because of the nature of the substrate
requirements [being an endoglycosidase it requires pi_4linked polymers of N-acetylglucosamine
(NAG-chitin) for cleavage] they are forced into producing prodrugs containing such
functionalities. To prevent cell entry they further elaborate the oligomer with taurine residues -
relying on the sulphonic acids to prevent cell entry and hence cyiotoxicity - 20 fold less -
Figure 13 in WO90/02939.
Use of a mammalian enzyme such as alkaline phosphatase (Senter et al; US 4,975,278) or a
human enzyme such as beta-glucuronidase (Behringwerke DE 42336237) or lysozyme (Akzo;
WO 90/07929) for ADEPT has the advantage that such enzymes should have reduced, or
lack, immunogenicity compared with non-mammalian enzymes. A disadvantage of using a
mammalian or human enzyme is that it is present endogenously in patients and there will thus
be the potential for turnover of prodrug to drug which is not due to the administered
antibody-enzyme conjugate. This is likely to lead to enhanced toxicity with this type of ADEPT
approach. Prodrugs for alkaline phosphatase are rapidly converted to drugs both in mice
(Doyle, T.W. and Vyas, D.M., Cancer Treatment Reviews 17, 127-131, 1990) and in man
(Hande et al, Clinical Pharmacology and Therapeutics 53, 233, 1993) in the absence of any
administered conjugate due to the widespread distribution of endogenous alkaline phosphatase,
thus confirming this is a critical problem for this enzyme. Human data on prodrugs for
beta-glucuronidase or lysozyme are not available. Glucuronidase and lysozyme are present in
the plasma and in other tissue sites. Akzo report lysozyme is present in milk, tears, saliva,
spleen, leukocytes and monocytes. Behringwerke in DE4236237 report activated
macrophages, granulocytes and platelets secrete glucuronidase. Since these cells are widely
distributed throughout the body this could lead to undesirable prodrug activation. Indeed
Behringwerke have shown in mice that after administration of a Doxorubicin prodrug relatively
high levels of free drug accumulate in the spleen which is a rich source of these cells (see table
3 in DE4236237).
Use of human enzymes in this ADEPT approach is limited by the fact that only enzymes with a
predominant intracellular distribution can be used and the prodrugs that are used with them
must be kept out of cells to minimise toxicity. This severely limits the number of options to
produce an ADEPT system. Lysozyme although being a small enzyme has disadvantages for
ADEPT. Lyso/yme does not release the active drug but releases a derivative of unknown
pharmacological activity. In the example given by Akzo, Dox-(GlcNAc)i or Dox-(GlcNAc)s is
released rather than free Doxorubicin. Glucuronidase can release the active drug e.g.
adriamycin from a glucuronide prodrug and anti-tumour activities have been reported (Bosslet,
K et al Cancer Research 54, 2151-59, 1994). However, human glucuronidase is a high
molecular weight enzyme (150-300 KDa) and consequently the resulting targeting conjugate is
likely to be very large. This is likely to cause problems with penetration into tissues such as a
tumour since it is well documented that smaller proteins penetrate more rapidly into solid
tumours. In addition glucuronidase is glycosylated and this glycosylation leads to the rapid
blood clearance of the antibody-glucuronidase conjugate used in ADEPT. The rapid blood
clearance results in little conjugate localising to tumour xenografts. The combination of high
molecular weight and rapid blood clearance is likely to lead to poor tumour localisation in
patients. Thus glucuronidase is not an ideal enzyme for ADEPT.
The present invention is based on the discovery that a host enzyme (for example human
ribonuclease, an enzyme naturally present in the general circulation) can be engineered such
that it will recognise a prodrug for ADEPT therapy that is not significantly recognised by
natural host enzyme. Since the engineered enzyme is highly similar in terms of amino acid
composition to the native host enzyme it advantageously exhibits markedly reduced
immunogenicity compared with bacterial enzymes such as CPG2. The engineered enzyme
does not occur naturally and thus non-selective triggering of prodrug activation, by natural
flora or human enzymes, is advantageously reduced. The approach has the additional
advantages that it is applicable to a wide range of human or mammalian enzymes since it is not
limited by the natural distribution of the enzyme and prodrugs can be employed that get into
cells.
These problems have been addressed in part by International Patent application WO 95/13095
(Wellcome Foundataion) which was published after the earliest priority date of the present
invention. This application proposed ADEPT using mutant mammalian enzymes to activate
prodrugs which are not activated by the corresponding native enzyme but did not disclose the
presently claimed invention.
It is very surprising that the replacement of a charged residue, one located at or close to the
substrate binding or catalytic site of an enzyme, by a residue of opposite charge, produces a
mutant enzyme with an intact catalytic centre, and this mutant enzyme differs from the native
enzyme solely in possessing a related, complementary but charge inverted substate specificity
requirement.
Furthermore the prodrug/ drug combinations disclosed in Wellcome (based on methotrexate
and melphalan) rely on blockage of active transport mechanisms to prevent cell penetration of
the prodrug. This limits the range of prodrug/ drug possibilities to those possessing such
active transport menhanisms. In contrast the reversed polarity approach disclosed herein
allows choice of charge properties of prodrugs (which may or may not also possess active
transport properties) to block cell entry of the prodrug and thus enable application of the
invention to a wider range prodrug/ drug options.
According to one aspect of the present invention there is provided a matched two component
system designed for use in a host in which the components comprise:
(i) a first component that is a targeting moiety capable of binding with a tumour associated
antigen, the targeting moiety being linked to a mutated enzyme capable of converting a
prodrug into an antineoplastic drug and;
(ii) a second component that is a prodrug convertible under the influence of the enzyme to
the antineoplastic drug;
wherein:
the mutated enzyme is a mutated form of a host enzyme in which the natural host enzyme
recognises its natural substrate by an ion pair interaction and this interaction is reversed
("reversed polarity") in the design of mutated enzyme and complementary prodrug;
the first component is substantially non-immunogenic in the host and;
the prodrug second component is not significantly convertible into antineoplastic drug in the
host by natural unmutated host enzyme.
Preferably the system described above is one in which the first component comprises a mutated
enzyme based on an enzyme from the same species as the host for which the system is intended
for use.
Preferably the system described above is one in which the targeting moiety is an antibody or a
fragment thereof. Preferably the system described above is one in which the antibody fragment
is an F(ab')2 fragment.
Preferably the system described above is one in which the mutated enzyme is mutated
ribonuclease. Preferably the system described above is one in which the mutated enzyme is
human ribonuclease comprising a negatively charged amino acid at position 66. Preferably the
system described above is one in which the negatively charged amino acid at position 66 of
ribonuclease is Glu.
Another preferred embodiment for the system described above is one in which the mutated
enzyme is mutated glucuronidase.
According to another aspect of the present invention there is provided a matched two
component system designed for use in a host in which the components comprise:
(i) a first component that is a targeting moiety capable of binding with a tumour associated
antigen, the targeting moiety being linked to an enzyme capable of converting a prodrug
into an antineoplastic drug and;
(ii) a second component that is a prodrug convertible under the influence of the enzyme to
the antineoplastic drug;
wherein:
the enzyme is a mutated form of a host enzyme;
the first component is substantially non-immunogenic in the host and;
the prodrug is not significantly convertible into antineoplastic drug in the host by natural
unmutated host enzyme.
The term "the prodrug is not significantly convertible into antineoplastic drug in the host by
natural unmutated host enzyme" means that the prodrug does not give undue untargeted
toxicity problems on administration to the host.
The term "substantially non-immunogenic" means that the first component can be administered
to the host on more than one occasion without causing significant host immune response as
would be seen with for example the use of a mouse antibody linked to a bacterial enzyme in a
human host.
Preferably the mutated enzyme is based on an enzyme from the same species as the host for
which the system is intended for use but the mutated enzyme may be based on a host enzyme
from a different species as long as the structure of the enzyme is sufficiently conserved
between species so as not to create undue immunogenicity problems.
Preferably the targeting moiety is an antibody, especially an antibody fragment such as for
example F(ab')2. Linkage to enzyme may be effected by known methods such as use of
heterobifunctional reagents as cross-linkers or by gene fusion or any other suitable method.
Antibody may be from the same host (eg use of mouse antibody in mice) or the antibody may
be manipulated such that it is not significantly recognised as foreign in the chosen host (eg use
of chimeric, CDR grafted or veneered mouse antibodies in humans).
Transplantation of the variable domains of rodent antibodies into the constant domains of
human antibodies (Chimeric antibodies) or building the antigen binding loops (CDRs) of rodent
antibodies into a human antibody (CDR grafting) have both been shown to greatly decrease the
immunogenicity of the rodent antibody in preclinical studies in monkeys and in patients. Even
CDR grafted antibodies incorporate a large number (>50) of amino acids from the rodent
antibody sequence into the human framework. Despite this in monkeys and patients greatly
reduced immunogenicity has been reported. This provides evidence that mutating a very
limited number of amino acids in the catalytic site of a host enzyme is likely to result in an
enzyme with minimal immunogenicity and certainly lower immunogenicity than a non-host
enzyme. The reader is directed to the following references: A. Mountain and J. R. Adair,
Biotechnology and Genetic Engineering Reviews JjQ, 1-142, 1992; G. Winter and W. J. Harris,
Trends in Pharmacological Sciences, 14, 139-143, 1993; I.I. Singer et al, J. Immunol, 150.
2844-57, 1993; J. Hakimi et al, J. Immunol, 147. 11352-59, 1991 and; J. D. Isacs et al, The
Lancet, 340. 748-752, 1992. The constant region domains may be for example human IgA,
IgE, IgG or IgM domains. Human IgG2 and 3 (especially IgG2) are preferred but IgG 1 and 4
isotypes may also be used. Human antibodies per se may also be used such as those generated
in mice engineered to produce human antibodies.
The host enzyme is mutated (by any suitable method such as for example chemical or
biotechnological gene synthesis or targeted mutation) to give a change in mode of interaction
between enzyme active site and prodrug compared with the native host enzyme.
Preferably the enzyme mutation is a polarity change in its active site such that it turns over a
prodrug with a complementary polarity; the prodrug not being significantly turned over by the
unmutated host enzyme. Preferably the natural host enzyme recognises its natural substrate by
an ion pair interaction and this interaction is reversed in the design of mutated enzyme and
complementary prodrug. Preferably
the enzyme is mutated ribonuclease, especially human ribonuclease with reversed polarity (see
Figures 12-15).
Lysine 66 in human ribonuclease is a positively charged residue which interacts with negatively
charged phosphate groups on the natural RNA substrate for the enzyme. The polarity of this
residue is reversed for example by genetic engineering (but chemical synthesis is also
contemplated) to give a negatively charged residue such as glutamic acid. The resulting
'reversed polarity' enzyme recognises the prodrugs of the present invention which are not
significantly recognised by the unmutated host enzyme. Further alterations to residues in the
native site region are contemplated to optimise substrate binding and turnover characteristics.
Engineered forms of the ribonuclease enzyme represent a further aspect of the present
invention. Ribonuclease is an advantageous enzyme due to its low molecular weight (approx.
13600 Da; allowing good tumour penetration after administration) and good stability to heat
stress and proteolysis. Preferably the prodrug is a mustard-ribonucleotide of Formula 1 as set
out in Figure 11 wherein:
Q is O or NH (especially NH);
A is a group of formula -X-Y- wherein
Y is SOa, CO or a single bond (preferably CO) with the proviso that
when Q is oxygen then Y is not SO2;
X is -(CH2)n where n=l-4 (preferably n=l except when Y is a single bond then n is preferably
2) optionally substituted by
CM alkyl on any carbon atom (R and/or S configurations are contemplated at any chiral atom)
or
when Y is CO and n=l then X is optionally substituted on carbon with the side chain of
alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, serine, threonine,
cysteine, asparagine, glutamine, lysine, arginine or histidine (R and/or S configurations are
contemplated at any chiral atom);
Rl is uracil or cytosine as shown in Figure 11;
R2 and R3 independently represent H or Ci_4alkyl (preferably methyl and especially R2=R3=H);
R5 and R6 independently represent Cl, mesyl or tosyl (preferably R5=R6=C1);
R7, R8, R9 and RIO independently represent H, CM alkyl(preferably methyl),
Ci.4alkoxy (preferably methoxy), F or Cl (preferably Cl) and the preferred positions for
representing radicals other than H are R8 & R9 but R7=R8=R9=R10=H is especially preferred.
In a preferred embodiment the mustard ribonucleotide is one in which:
Q is NH;
X is -(CH2)n- where n is 1-4;
Y is -C(O)-;
Rl is uracil or cytosine;
R2 and R3 are H;
R5 and R6 are Cl; and
R7, R8, R9 and RIO are H;
or a salt thereof.
The following individualised compound is especially preferred;
Q-[(2R,3S)4R,5R)-2-(2-aminoacetamidomethyl)-5-(2,4-dioxo-l,2,3,4-tetrahydropyrimidin-
1 -yl)-4-hydroxy-2,3,4,5-tetrahydrofuran-3-yl] Q-[4-(bis[2-chloroethyl]
amino)phenoxy] hydrogen phosphate which is shown as the end product in Figure 7.
Another preferred compound is the cytosine analogue of the end product in Figure 7.
In this specification the generic term "alkyl" includes both straight-chain and branched-chain
alkyl groups. However references to individual alkyl groups such as "propyl" are specific for
the straight-chain version only and references to individual branched-chain alkyl groups such as
"isopropyl" are specific for the branched-chain version only. An analogous convention applies
to other generic terms.
It is to be understood that, insofar as certain of the compounds of Formula 1 may exist in
optically active or racemic forms by virtue of one or more asymmetric carbon atoms, the
invention includes in its definition any such optically active or racemic form which possesses
the property of being a substrate for mutant enzymes of the invention.
The synthesis of optically active forms may be carried out by standard techniques of organic
chemistry well known in the art, for example by synthesis from optically active starting
materials or by resolution of a racemic form. Similarly, substrate properties against mutant
enzymes may be evaluated using standard laboratory techniques.
Point mutations will be referred to as follows: natural amino acid (using the 1 letter
nomenclature), position, new amino acid. For example "D253K" means that at position 253
an aspartic acid (D) has been changed to lysine (K). Multiple mutations in one enzyme will be
shown between square brackets.
In this specification the term CPB includes the following:
i) mature, pro and prepro forms of the enzyme with or without "tags" (eg c-myc);
ii) any carboxypeptidase with specificity for peptidic substrates having Lys or Arg at the C
terminus;
pancreatic and plasma CPB enzymes (the pancreatic enzyme is preferred);
unless indicated otherwise or self evident from the context.
Mutant CPBs of the invention are mutants of any of the above CPBs having the desired
property required for the invention. The following mutants of pancreatic HCPB are preferred:
D253K, D253R and; especially [G251N,D253R]; corresponding mutations in other CPBs are
also contemplated. A mutant CPB of the invention may also comprise other "conservative"
mutations (insertions, substitutions and/or deletions) that do not significantly alter the
properties of the key mutation. For the purposes of this document a conservative amino acid
substitution is a substitution whose probability of occurring in nature is greater than ten times
the probability of that substitution occurring by chance (as defined by the computational
methods described by Dayhoff et al, Atlas of Protein Sequence and Structure, 1971, page
95-96 and figure 9-10).
References on CPBs include the following: Folk JE in The Enzymes Vol III, Academic Press
(1971), pg 57; Coll M et al (1991) EMBO Journal 1Q, 1-9; Eaton DL et al (1991) J Biol Chem
266. 21833-21838; Yamamoto K et al (1992) J Biol Chem 267. 2575-2581; US Patent
5364934 (Genentech) and; International Patent Application WO 95/14096 (Eli Lilly).
The compounds of this invention may form salts with various inorganic and organic acids and
bases which are also within the scope of the invention. Such salts include ammonium salts,
alkali metal salts like sodium and potassium salts, alkaline earth metal salts like the calcium and
magnesium salts, salts with organic bases; e.g. dicyclohexylamine salts, N-methyl-D-glucamine,
salts with amino acids like arginine, lysine, and the like. Also, salts with organic and inorganic
acids may be prepared; e.g., HC1, HBr, H2SO4, H3PC«4, methanesulfonic, toluenesulfonic,
maleic, fumaric and camphorsulfonic. Non-toxic physiologically acceptable salts are preferred,
although other salts are also useful; e.g., in isolating or purifying the product.
The salts can be formed by conventional means such as by reacting the free acid or free base
forms of the product with one or more equivalents of the appropriate base or acid in a solvent
or medium in which the salt is insoluble, or in a solvent such as water which is then removed in
vacuo or by freeze-drying or by exchanging the cations of an existing salt for another cation on
a suitable ion exchange resin.
The compounds of this invention may be utilized in compositions such as tablets, capsules or
elixirs for oral administration, suppositories for rectal administration, sterile solutions or
suspensions for parenteral or intramuscular administration, and the like. The compounds of
this invention can be adminstered to patients (animals and human) in need of such treatment in
dosages that will provide optimal pharmaceutical efficacy. Although the dose will vary from
patient to patient depending upon the nature and severity of disease, the patient's weight,
special diets then being followed by a patient, concurrent medication, and other factors which
those skilled in the art will recognize, the dosage range will generally be about 1 to 4000mg.
per patient per day which can be administered in single or multiple doses. Preferably, the
dosage range will be about 100 to 4000mg. per patient per day; more preferably about 500 to
3000mg. per patient per day.
The most effective mode of administration and dosage regimen for the conjugates and
prodrugs of this invention in cancer therapy depend on a number of factors such as the severity
of disease, the patient's health and response to treatment and the judgement of the treating
physician. Accordingly the dosages of the conjugates and prodrugs should be titred to the
individual patients. Nevertheless, an effective dose of conjugate is likely to be in the range of
20 to about 200 mg/m2. The effective dose of the prodrug will depend on the particular drug
used and the toxicity of the parent drug. Since the prodrug is less cytotoxic than the parent
drug the MTD of the parent drug, if known, would provide a starting point. For phenol
mustard based prodrugs where clinical data is not available on the parent drug the therapeutic
dose range is less certain and would need to be defined by standard animal toxicology studies
and dose escalation studies in patients starting at a low dose. However the therapeutic dose is
generally in the range 500-2000 mg/m2.
Naturally, these dose ranges can be adjusted on a unit basis as necessary to permit divided daily
dosage and, as noted above, the dose will vary depending on the nature and severity of the
disease, weight of patient, special diets and other factors.
Typically, these combinations can be formulated into pharmaceutical compositions as discussed
below.
About 1 to lOOmg. of compound or mixture of compounds of Formula 1 or a physiologically
acceptable salt thereof is compounded with a physiologically acceptable vehicle, carrier,
excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by
accepted pharmaceutically practice. The amount of active substance in these compositions or
preparations is such that a suitable dosage in the range indicated is obtained.
Illustrative of the adjuvants which can be incorporated in tablets, capsules and the like are the
following: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as
microcrystalline cellulose; a disintegrating agent such as corn starch, pregelatinized starch,
alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as
sucrose, lactose or saccharin; a flavoring agent such as peppermint, oil of wintergreen or
cherry. When the dosage unit form is a capsule, it may contain, in addition to materials of the
above type, a liquid carrier such as fatty oil. Various other materials may be present as
coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets may
be coated with shellac, sugar or both. A syrup or elixir may contain the active compound,
sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and a
flavoring such as cherry or orange flavor.
Sterile compositions for injection can be formulated according to conventional pharmaceutical
practice by dissolving or suspending the active substance in a vehicle such as water for
injection, a naturally occuring vegetable oil like sesame oil, coconut oil, peanut oil, cottonseed
oil, etc., or a synthetic fatty vehicle like ethyl oleate or the like. Buffers, preservatives,
antioxidants and the like can be incorporated as required.
According to another aspect of the present invention there is provided a system as herein
defined for use in a method of controlling the growth of neoplastic cells in a host in which the
method comprises administration to said host an effective amount of a first component,
allowing the first component to clear substantially from the general circulation, and
administering an effective amount of a second component. Preferably the components are
administered intravenously.
According to another aspect of the present invention there is provided a method of controlling
the growth of neoplastic cells in a host in which the method comprises administration to said
host an effective amount of a first component as defined above, allowing the first component
to clear substantially from general circulation in the host, and administering an effective
amount of a second component as defined above.
According to another aspect of the present invention there is provided a pharmaceutical
composition comprising an effective tumour localising amount of a first component as herein
defined and a pharmaceutically acceptable carrier or diluent. Preferably the composition is
suitable for intravenous administration. Preferably the first component is supplied as a dry
solid which is reconstituted before use with a suitable diluent.
According to another aspect of the present invention there is provided a pharmaceutical
composition comprising an effective antitumour amount of a second component as defined
herein and a pharmaceutically acceptable carrier or diluent. Preferably the composition is
suitable for intravenous administration. Preferably the second component is supplied as a dry
solid which is reconstituted before use with a suitable diluent.
According to another aspect of the present invention there is provided a pharmaceutical
composition comprising a first component as defined above.
According to another aspect of the present invention there is provided a pharmaceutical
composition comprising a second component as defined above.
Preferred pharmaceutical compositions are sterile (for intravenous administration).
According to another aspect of the present invention there is provided a first component as
defined above.
According to another aspect of the present invention there is provided a mutated enzyme as
defined above.
According to another aspect of the present invention there is provided plasmid pQR162.
Plasmid pQR162 was deposited as deposit reference NCIMB 40678 at NCIMB Limited, 23 St
Machar Drive, Aberdeen AB2 1RY, Scotland, UK under the Budapest Treaty on 16th August
1994.
E.coli MSD 1646 containing pCG330 (also known as pICI1698) was deposited under the
Budapest Treaty on 23rd November 1994 with the National Collection of Industrial and Marine
Bacteria (NCIMB), 23 St Machar Drive, Aberdeen, Scotland, United Kingdom AB2 1RY; the
accession number is NCIMB 40694. NCIMB 40694 is another aspect of the present
invention.
Antibody A5B7 was deposited as hybridoma deposit reference 93071411 under the Budapest
Treaty on 14th July 1993 at ECACC, PHLS centre for Applied Microbiology & Research,
Porton Down, Salisbury, Wiltshire SP4 OJG, UK. A humanised antibody A5B7 in the form of
a F(ab')2 is preferred.
Further antibodies useful in ADEPT have been described as follows. Antibody BW 431/26
was described in Haisma, H.J. et aj., Cancer Immunol. Immunother., 34: 343-348 (1992).
Antibodies L6,96.5, and 1F5 were described in European Patent 302 473. Antibody 16.88
was described in International Patent Application WO90/07929. Antibody B72.3 was
described in European Patent No. 392 745. Antibody CEM231 was described in European
Patent No. 382 411. Antibodies HMFG-1 and HMFG-11 (Unipath Ltd, Basingstoke, Hants,
United Kingdom) react with a mucin-like glycoprotein molecule on milk fat globule
membranes and may be used to target breast and ovarian cancers. Antibody SM3 (Chemicon
International Ltd, London, United Kingdom) reacts with core protein of mucin and may be
used to target breast and ovarian cancer. Antibodies 85A12 (Unipath Ltd, Basingstoke, Hants,
United Kingdom) and ZCEA1 (Pierce Chemical Company, Chester, United Kingdom) react
with tumour antigen CEA. Antibody PR4D1 (Serotec, Oxford, United Kingdom) reacts with a
colon tumour associated antigen. Antibody E29 (Dako Ltd, High Wycombe, United
Kingdom) reacts with epithelial membrane antigen. Antibody C242 is available from CANAG
Diagnostics, Gothenberg, Sweden. The reader is also referred to Table 3 on page 208 in
International patent application WO 95/13095 (Wellcome) which includes data on various
antibodies.
Generally, antibodies useful in ADEPT are poorly internalised by the tumour cells they
recognise. This allows the targeted prodrug-activating enzyme to be resident on the cell
surface and thus generate active drug at the tumour site from circulating prodrug.
Internalisation of antibody may be assayed by known techniques, for example as set out in
Jafrezou et ah, Cancer Research 52: 1352 (1992) and in Press et ah, Cancer Research, 48:
2249(1988).
Another utility of the present invention is in the use of the first and second components in in
vitro diagnostics. For example detection of a particular antigen may be achieved by exposing
a diagnostic sample to a first component of the invention comprising a targeting moiety such
as an antibody capable of binding with the antigen. Thereafter unbound first component can be
removed, for example by washing, then the amount of bound first component can be
quantitated by its ability to catalyse turnover of a second component prodrug. Turnover of
prodrug can be quantitated by any suitable means such as HPLC. The reader is referred to A
Practical Guide to ELISA by D.M. Kemeny, Pergamon Press 1991.
According to another aspect of the present invention there is provided a recombinant murine
F(ab')a fragment of antibody A5B7 wherein the fragment contains 3 inter-chain disulphide
bonds between heavy chains at the hinge region.
According to another aspect of the present invention there is provided a recombinant murine
F(ab')2 fragment of antibody A5B7 having the sequence set out in SEQ ID 25 & 26 for heavy
and light chain respectively. Sutler et al in Gene 113 (1992) 223-230 teaches that it is
necessary to introduce additional cysteines in the hinge region of the antibody to obtain good
dimer formation in recombinant production. Recombinantly produced fragment is
distinguished from proteolytically produced material by the absence of whole antibody
contaminants. Recombinantly produced material may also have a higher binding affinity for
CEA antigen as determined by a Pharmacia Biacore™ instrument.
According to another aspect of the present invention there is provided a method of making a
first component as herein described by linking:
a targeting moiety capable of binding with a tumour associated antigen and
an enzyme capable of converting a prodrug into an antineoplastic drug wherein the enzyme is a
mutated form of a host enzyme. The mutated enzyme and targeting moiety may be linked by
conventional methods known in the art such as for example by heterobifunctional reagents.
Gene fusion is also contemplated.
The mutated enzyme and targeting moiety may b^ n.ay be prepared by expression technologies
known in the art. Some expression systems involve transforming a host cell with a vector;
such systems are well known such as for example in E. coli. yeast and mammalian hosts (see
Methods in Enzymology 185. Academic Press 1990). Other systems of expression are also
contemplated such as for example transgenic non-human mammals in which the gene of
interest, preferably cut out from a vector but with a mammary promoter to direct expressed
protein into the animal's milk, is introduced into the pronucleus of a mammalian zygote
(usually by microinjection into one of the two nuclei (usually the male nucleus) in the
pronucleus) and thereafter implanted into a foster mother. A proportion of the animals
produced by the foster mother will carry and express the introduced gene which has integrated
into a chromosome. Usually the integrated gene is passed on to offspring by conventional
breeding thus allowing ready expansion, of stock. Preferably the protein of interest is simply
harvested from the milk of female transgenic animals. The reader is directed to the following
publications: Simons et al. (1988), Bio/Technology 6:179-183; Wright etal. (1991)
Bio/Technology 9:830-834; US 4,873,191 and; US 5,322,775. Manipulation of mouse
embryos is described in Hogan etal. "Manipulating the Mouse Embryo; A Laboratory
Manual", Cold Spring Harbor Laboratory 1986.
Transgenic plant technology is also contemplated such as for example described in the
following publications: Swain W.F. (1991) TIBTECH 9: 107-109; Ma J.K.C. etal (1994)
Eur. J. Immunology 24: 131-138; Hiatt A. etal (1992) FEES Letters 3.07:71-75; Hein M.B. et
a] (1991) Biotechnology Progress 7: 455-461; Duering K. (1990) Plant Molecular Biology 15:
281-294.
If desired, host genes can be inactivated or modified using standard procedures as outlined
briefly below and as described for example in "Gene Targeting; A Practical Approach", IRL
Press 1993. The target gene (or portion thereof) is preferably cloned into a vector with a
selection marker (such as Neo) inserted into the gene to disrupt its function. The vector is
linearised then transformed (usually by electroporation) into embryonic stem (ES) cells (eg
derived from a 129/Ola strain of mouse) and thereafter homologous recombination events take
place in a proportion of the stem cells. The stem cells containing the gene disruption are
expanded and injected into a blastocyst (such as for example from a C57BL/6J mouse) and
implanted into a foster mother for development. Chimeric offspring can be identified by coat
colour markers. Chimeras are bred to ascertain the contribution of the ES cells to the germ
line by mating to mice with genetic markers which allow a distinction to be made between ES
derived and host blastocyst derived gametes. Half of the ES cell derived gametes will carry the
gene modification. Offspring are screened (eg by Southern blotting) to identify those with a
gene disruption (about 50% of progeny). These selected offspring will be heterozygous and
therefore can be bred with another heterozygote and homozygous offspring selected thereafter
(about 25% of progeny). Transgenic animals with a gene knockout can be crossed with
transgenic animals produced by known techniques such as microinjection of DNA into
pronuclei, sphaeroplast fusion (Jakobovits etal. (1993) Nature 362:255-258) or lipid mediated
transfection (Lamb etal. (1993) Nature Genetics 5 22-29) of ES cells to yield transgenic
animals with an endogenous gene knockout and foreign gene replacement.
ES cells containing a targeted gene disruption can be further modified by transforming with the
target gene sequence containing a specific alteration, which is preferably cloned into a vector
and linearised prior to transformation. Following homologous recombination the altered gene
is introduced into the genome. These embryonic stem cells can subsequently be used to create
transgenics as described above.
The term "host cell" in this context includes any procaryotic or eucaryotic cell suitable for
expression technology such as for example bacteria, yeasts, plant cells and non-human
mammalian zygotes, oocytes, blastocysts, embryonic stem cells and any other suitable cells for
transgenic technology. Tf fhe context so permits the term "host cell" also includes a transgenic
plant or non-human mammal developed from transformed non-human mammalian zygotes,
oocytes, blastocysts, embryonic stem cells, plant cells and any other suitable cells for
transgenic technology.
According to another aspect of the present invention there is provided a polynucleotide
sequence selected from a polynucleotide sequence encoding any of the following:
any first component as defined above; and
any mutated enzyme as defined above.
According to another aspect of the present invention there is provided a vector comprising a
polynucleotide as defined above.
According to another aspect of the present invention there is provided a cell comprising a
polynucleotide as defined above.
The invention will now be illustrated in the following examples in which:
Figure 1 depicts construction of plasmid pQR177
Figure 2 depicts purification of bovine ribonuclease;
Figure 3 depicts PCR strategies leading to plasmid pATF4
Figure 4 depicts purity assessment by PAGE of expressed R4A,K6A human pancreatic RNase
Figure 5 depicts recombinant circle PCR generation of pATFZ44
Figure 6 depicts a comparison of toxicity against LoVo cells between a prodrug and
corresponding drug
Figure 7 depicts a scheme for synthesis of uracil based prodrug
Figure 8 depicts oligonucleotide primers
Figure 9 depicts a scheme for synthesis of a uracil based prodrug analogue
Figure 10 depicts a scheme for synthesis of a cytidine based prodrug analogue
Figure 11 depicts chemical formulas
Figure 12 is a schematic diagram of the active site of ribonuclease A - substrate complex
wherein B, R, and P indicate binding subsites for base, ribose and phosphate, respectively, B is specific for pyrimidines and 62 "prefers" purines. 3'-Pyrimidine mononucleotides bind to
S^RIPI. 5'-Purine mononucleotides bind to B2R2P1- 3'-AMP binds to B2R2P2. The
phosphate group of the phosphodiester bond hydrolysed by the enzyme binds to pj. The
residues known to be involved in each site are indicated.
Figure 13 is a schematic diagram of a prodrug in the active site of a reversed polarity mutant
enzyme wherein:
represents a reversed polarity residue (Lys 66 in native ribonuclease) and;
X is a positively charged group (attached by reversed polarity residue)
Figure 14 illustrates cleavage of prodrug by reversed polarity mutant enzyme
Figure 15 demonstrates the mechanism of action of native human RNase
Figure 16 depicts the structure of CpA & C>p RNase substrates
Figure 17 depicts a scheme for synthesis of a Cytosine based prodrug
Figure 18 illustrates pancreatic HCPB cloning.
Figure 19 illustrates pancreatic HCPB sequencing.
Figure 20 illustrates vector pICI1266.
Figure 21 illustrates pICI1266 expression vector gene cloning.
Figure 22 illustrates cytotoxicity of a prodrug and corresponding drug.
Figure 23 lists the composition of a growth medium.
Figure 24 is a diagram representing the key amino acid interactions between native
ribonuclease and a fragment of ribonucleic acid. The positively charged Lys66 at position Po
is shown making an ionic interaction with the negatively charged phospho-diester bond while
residues at Pj are important in the catalytic process.
Figure 25 depicts an interaction between a mustard prodrug and mutant RNAse. In order to
avoid turnover by native RNAse the key amino acid at postion 66 has been changed to a
negatively charged glutamic acid. This Glu-66 makes an ionic interaction with the positively
charged "X" moiety in the prodrug thus completing a reverse polarity interaction. It is
envisaged that further mutations at positions R2 and 82 would lead to enhanced interaction
with the prodrug.
Figure 26 shows two possible options for the positively charged moiety at position 5' of ribose
to affect an interaction with Glu-66 at P0.
Schemes 1-7 illustrate chemical synthetic procedures.
Abbreviations
Ac acetyl
ADEPT antibody directed enzyme prodrug therapy
BOC tert-butoxycarbonyl
BP-RNase bovine pancreatic ribonuclease
CPB carboxypeptidase B
DCCI 1,3-dicyclohexylcarbodiimide
DMAP 4-dimethylaminopyridine
DMF N.Nrdimethyl-formamide
DMSO dimethylsulfoxide
Et ethyl
EDCI 1 -(3-dimethylaminopropyl)-3-ethyl-carbodiirnide
HCPB human CPB
HOB T 1 -hydroxybenzotriazole
HP-RNase human pancreatic ribonuclease
PCR polymerase chain reaction
TFA trifluoroacetic acid
THF tetrahydrofuran
Reference Example 1
Preparation of recombinant mature bovine pancreatic ribonuclease
Recombinant bovine pancreatic ribonuclease was prepared from the coding sequence for the
bovine pancreatic ribonuclease (BP-RNase) precursor as described by Tarragona-Fiol et al. in
Gene (1992) 118. 239-245. The protein was expressed from E. coli under control of the tac
promoter from a two cistron expression fragment in pQR163. A plasmid containing the two
cistron fragment was designated pQR162 (NCIMB 40678).
Reference Example 2
Preparation of Arg4Ala,Lys6Ala human pancreatic ribonuclease
The coding sequence for the human pancreatic ribonuclease (HP-RNase) gene was obtained
from genomic DNA extracted from human buccal epithelial cells utilising the PCR technique as
described by Tarragona-Fiol et al. in Protein and Peptide Letters (1994) 1, 76-83. For the
preparation of HP-RNase expression of an engineered HP-RNase in E. coli was described. In
order to direct the expression of the recombinant human pancreatic enzyme to the periplasmic
space of E. coli. the bovine pancreatic RNase signal was fused 5' to the human gene. Initial
attempts to express the recombinant enzyme were not successful. Consequently site-directed
mutagenesis techniques were used to genetically engineer the HP-RNase gene to enable
expression in E. coli. The resultant engineered enzyme shows similar kinetic characteristics to
the homologous bovine enzyme.
(a) Cloning of the mature coding sequence for Arg4Ala,Lys6Ala HP-RNase
Restriction enzyme digestions, dephosphorylations, ligations, transformation and small scale
plasmid DNA purification was carried out as described by Maniatis et al.,(1982) Molecular
Cloning. A Laboratory Manual. Cold Spring Harbour, Laboratory, Cold Spring harbour. New
York. Oligonucleotides were synthesised using a Cyclone™ DNA synthesiser.
The mature sequence of the HP-RNase gene was obtained from genomic DNA extracted from
buccal epithelial cells using the PCR technique. Briefly, epithelial cells were obtained by
agitating vigorously 10 ml of 0.9% saline in the mouth for 20 seconds. The suspension of
buccal epithelial cells (1.5 ml) was pelleted by centrifugation and resuspended in 100 ul of 10
mM NaCl, 10 mM EDTA. After a further centrifugation the cell pellet was resuspended in 75
ul of 20 mM NaOH and incubated at 100°C for 30 minutes. The cell debris were pelleted and
the supernatant was stored at -20°C. An aliquot (2-3 ul) was normally used as template in PCR
incubations. Two primers (SEQ ID NO: 5 and SEQ ID NO: 6; see Figure 8, primers 1 & 2)
complementary to the 5'- and 3'-ends of the mature sequence of HP-RNase were used in a
PCR incubation (5 pmol/each), which also contained; human genomic DNA, 0.2 mM dNTPs,
Stratagene™ buffer (Ix) [10X buffer is 200 mM Tris-HCl (pH 8.2), 100 mM KC1, 60 mM
(NH4)2SO4,20 mM MgC^, 1% Triton™ X-100 and lOOug/ml nuclease-free BSA] and 2.5
units of pfu polymerase (Stratagene). The PCR incubation was carried out using 30 cycles of
denaturation at 92°C for 30 sec., annealing at 55°C for 30 sec and extension at 75°C for 1 min.
The resulting PCR products were analysed and separated by agarose gel electrophoresis. The
DNA fragment of interest was excised from the agarose gel and the DNA extracted using
centrifugal units (Spin-X™, Costar). In order to direct the expression of a complete
recombinant enzyme into the periplasmic space of Escherichia coli JM107 cells, the signal
sequence of bovine pancreatic RNase was fused to the 5'-end of the human gene, and the
coding sequence for the last seven amino acids of HP-RNase plus a termination codon was
attached to the 3'-end using the PCR technique. A PCR incubation was then set up containing
this PCR derived mature sequence of the HP-RNase gene which lacks the coding sequence for
the last 7 amino acids as template, a set of overlapping primers (SEQ ID NOS: 7 to 10; see
primers 3-6 in Figure 8) at different concentrations (0.1,0.5 and 50 pmol from the inner to the
outermost primer), 0.2 mM nucleotides, Stratagene buffer (Ix, see above) and 2.5 units of pfu
polymerase (Stratagene). The incubation was carried out using the same conditions as
described above. The PCR products were treated as described above and the fragment of
interest excised and extracted from the agarose gel. This fragment was cleaved with EcoRI and
ligated into previously digested and dephosphorylated pUCIS to enable double stranded DNA
sequencing by the dideoxy 3). The fused gene was then ligated into the expression vector
pKK223.3 3; see Example 1). The bovine signal sequence has incorporated a DNA sequence
coding for a hexapeptide 5'- to the open reading frame. This is utilised to disrupt the secondary
structure of the mRNA produced upon initiation of transcription of the promoter. Induction,
expression and purification of the recombinant enzyme was carried out as described above.
The analysis of periplasmic proteins obtained after this procedure revealed no product which
exhibited recombinant RNase activity.
The lack of expression of the human enzyme in these experiments was unexpected since the
bovine signal sequence has been used successfully to direct translocation of the recombinant
bovine enzyme to the periplasmic space. Comparison of the N-terminal sequence of the native
human and bovine enzymes show differences at positions 4 and 6 where alanine residues in the
bovine enzyme are replaced by arginine and lysine residues respectively in the human
counterpart. It is known that the presence of positive charged amino acids early in the mature
sequence can act as stop transfer signals preventing further translocation. To overcome this
problem a strategy was developed to replace the arginine and the lysine at positions 4 and 6 in
the human enzyme with alanine residues. Thus the technique of RCPCR (primers used for the
introduction of the desired mutation are SEQ ID NOS: 11 to 14; see primers E-H in Figure 8)
was used to generate a recombinant clone pATF3 containing the required replacements (see
Figure 3). This plasmid chimera was used as template in double stranded DNA sequencing to
verify the incorporation of the coding sequences for alanine residues at positions 4 and 6.
Excision with EcoRI and ligation with pKK223.3 produced the chimeric expression vector
pATF4 (Figure 3) which was used to express the engineered human enzyme.
(b) Expression and purification of the recombinant Arg4Ala,Lys6Ala HP-RNase from
E. coli
Transformation of Escherichia coli cells with pATF4 and IPTG induction results in the
expression of the engineered recombinant human enzyme which is isolated from the
periplasmic contents using protocols described above for the production of the homologous
bovine enzyme. Engineered recombinant HP-RNase was isolated from the periplasmic
contents and purified to homogeneity (see Figure 4). N-terminus sequencing of the
recombinant enzyme has been carried out and indicates that the bovine signal sequence has
been cleaved correctly. This also verifies the replacement of Arg-4 and Lys-6 with alanines.
The kinetic characterisation was carried out using CpA and C>p as substrates (Figure 16). The
kinetic parameters Km, kcat, and kcat/Km were compared with the values obtained for
commercial and recombinant bovine pancreatic RNase under the same assay conditions ( see
Tables ). The data indicate that the kinetic properties of the engineered HP-RNase enzyme are
not significantly different for the homologous bovine counterpart.
Kinetic parameters of the different enzymes for CpA as substrate at pH 7.0
kcat/Km
Rec. HP-RNase R4A:K6A 1700 (480)
Rec.BP-RNase 2800 (370)
BP-RNase 2300(600)
Kinetic parameters of the different enzymes for C>p as substrate at pH 7.0
kcat/Km (mM-Vs-1)
Rec. HP-RNase R4A:K6A 4.2 (0.8)
Rec.BP-RNase 3.9 (0.9)
BP-RNase 2.3 (0.5)
(n) indicates standard error.
Reference Example 3
Synthesis and isolation of murine A5B7-bovine pancreatic ribonuclease conjugate
A particular antibody capable of binding with a tumour associated antigen is mouse
monoclonal antibody A5B7. Antibody A5B7 binds to human carcinoembryonic antigen (CEA)
and is particularly suitable for targeting colorectal carcinoma. A5B7 is available from DAKO
Ltd., 16 Manor Courtyard, Hughenden Avenue, High Wycombe, Bucks HP 13 5RE, England,
United Kingdom. Antibody fragments can be prepared from whole IgG antibody by
conventional means such as for example F(ab')2 fragments as described by Mariani, M. etal
(1991), Molecular Immunology 28, 69 - 77. In general the antibody (or antibody fragment) -
enzyme conjugate should be at least divalent, that is to say capable of binding to at least 2
tumour associated antigens (which may be the same or different). Antibody molecules may be
humanised by known methods such as for example by "CDR grafting" as disclosed in
EP239400 or by grafting complete variable regions onto human constant regions as disclosed
in US 4816567. Humanised antibodies may be useful for reducing immunogenicity of an
antibody (or antibody fragment). A humanised version of antibody A5B7 has been disclosed in
PCTWO92/01059.
The hybridoma which produces monoclonal antioouy A5B7 was deposited with the European
Collection of Animal Cell Cultures, Division of Biologies, PHLS Centre for Applied
Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom.
The date of deposit was 14th July 1993 and the accession number is No. 93071411. Antibody
A5B7 may be obtained from the deposited hybridoma using standard techniques known in the
art such as documented in Fenge C, Fraune E & Schuegerl K in "Production of Biologicals
from Animal Cells in Culture" (Spier RE, Griffiths JR & Meignier B, eds)
Butterworth-Heinemann, 1991,262-265 and Anderson BL & Gruenberg ML in "Commercial
Production of Monoclonal Antibodies" (Seaver S, ed), Marcel Dekker, 1987, 175-195. The
cells may require re-cloning from time to time by limiting dilution in order to maintain good
levels of antibody production.
The linker used for derivitisation of murine A5B7 is S ATA™ (S-acetyl thioglycollic acid N
hydroxy succinimide ester), Sigma (product code A9043).
The linker used for bovine pancreatic ribonuclease (BP-RNase) derivatisation is SMPB
(4-(p-maleimidophenyl)butyric acid N-hydroxysuccinimide ester), Sigma (product code
M6139).
SATA (Sigma) was dissolved in DMSO (Fisons) at a concentration of lOmg/ml. To a solution
of 50mg of A5B7 at 5.4mg/ml in lOOmM phosphate/lOOmM NaCl/ ImM EDTA pH7.2 (buffer
A) was added 309|ig (30.9^,1) SATA solution (representing a 4 molar excess over A5B7),
mixed and allowed to stand at room temperature for 40 mins. The resulting solution was
passed down a Sephadex™ G25 column (Pharmacia) (210ml 2.6 x 38cm) to remove excess
reagents at room temperature yielding a final concentration of 2.09mg/ml of derivatised A5B7
(23.5ml total volume). The SATA derivatised A5B7 was mixed with 1.0ml 10%v/v 500mM
hydroxylamine HC1/ 500mM sodium phosphate/ 30mM EDTA pHS.O to deacetylate the
derivatised A5B7, the reaction proceeding for 40 mins at room temperature. The protein
concentration was determined by UV absorbance at 280nm assuming e= 1.4 (or by Bradford
Protein assay). The linker loading was determined by Ellmans -SH assay and found to be 1.2
linkers/mole A5B7.
BP-RNase (Sigma), was resuspended in 6.0 ml of lOOmM sodium phosphate /lOOmM NaCl
pH 7.2 (buffer B) to give a concentration of 8.33mg/ml.
SMPB (Sigma) was dissolved in DMSO (Fisons) at a concentration of lOmg/ml. A solution of
50mg BP-RNase was mixed with 6500mg (650ml) of the SMPB solution (representing a 5
molar excess over BP-RNase) and allowed to stand at room temperature for 120 mins. Excess
reagents were removed by gel permeation chromatography (Sephadex G25 210ml 2.6 x 30cm).
The derivatised protein concentration was determined by UV A280 assuming e = 0.6. The
linker loading was determined by a 'reverse' Ellmans assay, by adding a known amount of
2-mercaptoethanol to the maleimido derivatised BP-RNase and assaying unreacted SH groups.
The conjugation reaction preceded by the addition of equal weights of the deacetylated
derivatised A5B7 and derivatised BP-RNase and was diluted with deionised water to a
concentration of l.Omg/ml and mixed under nitrogen. The reaction was allowed to proceed
for 20hrs at room temperature followed by termination by the addition of Img/ml aqueous
glycine.
The crude conjugation was buffer exchanged by dialysis into 50mM Phosphate pH 8.0 (Buffer
C) and the resulting solution applied to a Q Sepharose™ (Pharmacia) column (30ml 1.6 x
15cm) equilibrated in Buffer C. The column was washed in buffer C to remove excess A5B7
and BRNase followed by elution of the conjugate in 0.5M NaCl wash at a flow rate of
Iml/min.
Purity of the resultant conjugate was determined by SDS-Page and contained a total of 5.75mg
conjugate with the corrnosition 88.4% conjugate and 11.6% free derivatised A5B7 by laser
densitometry.
Reference Example 4
Synthesis and isolation of murine A5B7 F(ab')2-bovine pancreatic ribonuclease
conjugate
The linker used for A5B7 F(ab')2 derivitisation is SATA (S-acetyl thioglycollic acid N hydroxy
succinimide ester), Sigma (product code A9043).
The linker used for bovine pancreatic ribonuclease (BP-RNase) derivitisation is SMPB
(4-(p-maleimidophenyl)butyric acid N-hydroxysuccinimide ester), Sigma (product code
M6139).
SATA (Sigma) was dissolved in DMSO (Fisons) at a concentration of lOmg/ml. To a solution
of 18.20mg of the F(ab')2 fragment at 2.14mg/ml in lOOmM phosphate/lOOmM NaCl/ ImM
EDTA pH7.2 (buffer A) was added 167|ng (16.7^1) SATA solution [representing a 4 molar
excess over A5B7 F(ab')2], mixed and allowed to stand at room temperature for 40 mins. The
resulting solution concentrated to 2.0 ml (9mg/ml) via an Amicon YM10™ (100,000 MW
cutoff) membrane followed by removal of excess reagents through a Sephadex G25™ column
(Pharmacia) (50ml 1.6 x 16cm) at room temperature yielding a final concentration of
1.04mg/ml of derivatised A5B7 F(ab')2 (10ml total volume). The SATA derivatised A5B7
F(ab')2 was mixed with 1.0ml 10%v/v SOOmM hydroxylamine HC1/ 500mM sodium phosphate/
30mM EDTA pHS.O to deacetylate the derivatised A5B7 F(ab')2, the reaction proceeding for
40 mins at room temperature. The protein concentration was determined by UV absorbance at
280nm assuming e= 1.4 (or by Bradford Protein assay). The linker loading was determined by
Ellmans -SH assay and found to be 1.2 linkers/mole Fab2.
BP-RNase (Sigma), was resuspended in 2.0 ml of lOOmM sodium phosphate /lOOmM NaCl
pH 7.2 (buffer B) to give a concentration of 7.50mg/ml.
SMPB (Sigma) was dissolved in DMSO (Fisons) at a concentration of lOmg/ml. A solution of
15mg BP-RNase was mixed with 1949mg (1.95ml) of the SMPB solution (representing a 5
molar excess over BP-RNase) and allowed to stand at room temperature for 120 mins. Excess
reagents were removed by gel permeation chromatography (Sephadex G25 50ml 1.6 x 16cm).
The derivatised protein concentration was determined by UV A280 assuming e = 0.6. The
linker loading was determined by a 'reverse' Ellmans assay, by adding a known amount of
2-mercaptoethanol to the maleimido derivatised BRNase and assaying unreacted SH groups.
The conjugation reaction preceded by the addition of equal weights of the deacetylated
derivatised A5B7 F(ab')2 and derivatised A5B7 F(ab')2 was diluted with deionised water to a
concentration of 1 .Omg/ml and mixed under nitrogen. The reaction was allowed to proceed for
20hrs at room temperature followed by termination by the addition of 1 mg/ml aqueous glycine.
The crude conjugation was buffer exchanged by dialysis into 50mM Tris pH 8.0 (Buffer C)and
5ml (6.5mg) of the resulting solution applied to a Mono Q ™ (HR5/5) (Pharmacia) column
equilibrated in Buffer C. The column was washed in buffer C to remove excess A5B7 F(ab')2
followed by elution of the conjugate and remaining BP-RNase in a salt gradient (0-1.OM over
20 column volumes) at a flow rate of Iml/min. Isolation of conjugate from residual enzyme
was achieved by applying pooled fractions containing conjugate on to a S200™ GPC column
(Pharmacia) (60ml 1.6 x 30cm) and running in PBS at a flow rate of Iml/min.
Purity of the resultant conjugate was determined by SDS-Page and contained a total of 0.70mg
conjugate with the composition 95.5% conjugate and 4.5% free derivatised A5B7 F(ab')2 by
laser densitometry.
Murine A5B7 F(ab')2 was made as described in Reference Example 5, or by the
following procedure:
The A5B7 antibody, described in Reference Example 3, (780ml at 5.4 mg/ml) was prepared for
digestion by diafiltration versus 7 volumes of 0.1 M sodium phosphate, 3mM EDTA (pH6.4),
using an Amicon™ CH2 spiral cartridge apparatus containing 1 30KDa membrane. The
material recovered (3682 mg estimated by ABS@280nm) was 0.22uM filtered and stored at
4°C until use. Crystalline papain suspension (9ml at lOmg/ml; Boehringer Mannheim, product
code 1080140) was mixed with 0.1M sodium phosphate, 3mM EDTA (pH6.4) containing
lOOmM L-cysteine and left for 30 minutes at 37°C. The excess cysteine was then removed by
size exclusion chromatography (Pharmacia G25M™ column size 2.6cm diameter, 30cm length
total volume approx 160ml) using 0.1M sodium phosphate, 3mM EDTA (pH6.4) run at 3
ml/min flow rate. Fractions (1 minute) were collected and monitered by OD280 and a simple
DTNB spot test to ensure clearance from free cysteine prior to pooling of reduced papain
pool. The concentration of the reduced papain pool was determined (by OD280 assuming
E=2.5) as 1.65mg/ml, volume 32.8ml, total protein available 54mg. The digestion was carried
out using a 1/60 w/w ratio of reduced papain to A5B7 at 37°C using all the available papain
and 655ml of the antibody (warmed to 37°C prior to commencement of the digestion) and at
an estimated protein concentration of 4.9 mg/ml. The reaction was quenched with O.lx total
reaction volume of lOOmM N-ethylmaleimide in 50% ethanol after 20 hours. The F(ab')2 was
purified from the Fc and trace undigested antibody using a 400ml Protein A Sepharose FF™
(Pharmacia) column (dimensions 5cm x 20cm) equilibrated with 25mM sodium phosphate,
150mM sodium chloride (pH7.33) until pH and conductivity matched that of the equilibration
buffer (19.7mS at 15oC). The crude digest was diluted 1:1 with column buffer and split into 2
batches (660 ml and 840 ml) and each loaded at 6.5ml/min (linear flow rate of
0.33ml/cm2/min) onto a protein A column. 10ml fractions were collected. Once loaded the
column was washed with equilibration buffer until the absorbance @ 280nm approached
baseline. The initial wash consisted of 50mM sodium acetate (pH 4.5) followed by an extended
50mM sodium acetate (pH 4.0) wash then a 50mM citric acid pH3.5 followed by a final 50mM
citric acid (pH2.8) wash. During the washes the OD280 values were measured and pools taken
then neutralised within 30 minutes using disodium hydrogen orthophosphate solution (0.4M).
Samples of the pools were analysed by SDS Page (Pharmacia Excel™ gel, coomassie stained).
F(ab')2 was eluted by the pH4.0 buffer and undigested A5B7 was eluted in the lowest pH
washes. The F(ab')2 pooled samples were diafiltered into lOOmM sodium phosphate, lOOmM
sodium chloride, ImM EDTA (pH7.2). (Amicon™ CH2 30KDa membrane, 7 volume
diafiltration) and yielded a total of 845mg F(ab')2 (@ 2mg/ml).
Reference Example 5
Preparation of recombinant murine A5B7 F(ab')2 in myeloma cells.
This example describes the preparation of cDNA from the A5B7 hybridoma, the isolation of
specific Fd and L chain fragments by PCR, determination of the complete DNA sequence of
these fragments, the subsequent co-expression in myeloma cells to generate a recombinant
F(ab')2 fragment, fermentation of the myeloma cells and purification of the recombinant F(ab')2
protein.
Several methods for production of genetically engineered antibodies in myeloma cells are
described in the literature, including Neuberger et al. (1984) Nature 312. 604-608, Williams
and Neuberger (1986) Gene 43, 319-324, Wright and Shin (1991) Methods 2, 125-135,
Traunecker (1991) Trends in Biotechnology 9, 109-113 and Bebbington et al. (1992)
Bio/Technology 10, 169-175. For convenience, this example will use essentially the procedure
described by Bebbington et al. based on glutamine synthetase (GS) gene as a selective marker.
a) Preparation ofmRNAfrom hybridoma cells
There are several procedures for the isolation of polyA+ mRNA from eukaryotic cells
(Sambrook J., Fritsch E.F., Maniatis T., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Second Edition, 1989, Chapter 8 p3 hereinafter referred to as
Maniatis). One such method is provided in kit form by Pharmacia and relies on the lysis of a
relatively small number of cells (10^ or less) followed by binding of polyA+ mRNA to an oligo
dT column. Unwanted cell components are removed by washing with a low salt concentration
before eluting the mRNA in high salt solution at elevated temperature.
mRNA was prepared from 10^ A5B7 hybridoma cells using the Quickprep™ mRNA kit
(Pharmacia Biotechnology Ltd.). The concentration of the mRNA was estimated by scanning
a sample from 300-220nm in a Uvikon 930 spectrophotometer (Kontron™ Instruments) and
using a factor of 40ug/ml/unit OD at 260nm. The mRNA was stored as 2.5ug aliqouts
precipitated in ethanol.
b) cDNA synthesis.
The method used for cDNA synthesis was based on that of Gubler and Hofman which relies on
reverse transcription from primed mRNA followed by RNAse H treatment to provide priming
and synthesis of the second strand by DNA polymerase I. Other methods for the synthesis of
cDNA are reviewed in Maniatis (Chapter 8).
A 5ug sample of mRNA was primed with oligo dT (12-18mer mixture, Pharmacia
Biotechnology Ltd., O.Sug) in a lOul solution containing 2.5u placental RNAse inhibitor (Life
Technologies Ltd.) made up with RNAse-free water by incubating at 70°C followed by cooling
on ice. First strand cDNA synthesis was then performed by adding 4ul 5x H-RT buffer
(250mM Tris, pH8.3, 200mM KC1, 30mM MgCl2 and 0.5mg/ml BSA), 2ul 0.1M DTT
(dithiothreitol), lul dNTP mix (dATP,dCTP,dGTP and dTTP at 20mM), 4ul Superscript™
Reverse transcriptase (Life Technologies Ltd.) and incubating at 42°C for 1 hour. For the
second strand reaction, l.Sul dNTP mix (as above), 92.5ul RNAse-free water, 30ul 5x
reaction buffer (125mM Tris, pH7.5, SOOmM KC1, 25mM MgCl2> 50mM (NH4)2SO4 and 0.5
mg/ml p-NAD), lul T4 DNA ligase (lOu, Life Technologies Ltd.), 4ul DNA polymerase I
(40u, Life Technologies Ltd.) and lul RNAse H (2.7u, Life Technologies Ltd.) were added
and incubation continued at 16°C for a further 2 hours. To ensure that blunt-ended cDNA was
prepared a final incubation at 16°C for 5 minutes after adding 2ul T4 DNA polymerase (10u,
Life Technologies Ltd.) was performed. Enzyme activity was then stopped by incubation at
70°C for 10 minutes.
c) Isolation of antibody gene fragments by PCR
Isolation of A5B7 Fd and L chain fragments was performed using the cDNA as template. The
Fd fragment was terminated immediately after the hinge sequence (c-terminal threonine)
hereinafter referred to as proteolytic type Fd. By proteolytic Fd we mean in this example it is a
recombinant Fd equivalent to a proteolytically produced Fd, described in Reference Example 4
Material from the first-strand cDNA reaction or after completion of the second strand reaction
is suitable as template. The material could be used neat from the completed reaction or as a
dilution (up to 1 in 100) in double-distilled water. Oligonucleotides (SEQ ID numbers 17-24)
were used in the generation of the Fd and L chain fragments. For each antibody fragment, the
5' region oligonucleotide (SEQ ID 17 for Fd fragment and SEQ ID 18 for the L chain)
encoded a restriction enzyme site (Hindlll for Fd and EcoRI for L chain) a consensus Kozak
sequence (GCCGCCACC) to maximise translation initiation and a portion of the natural
murine signal sequence. The 3' region oligonucleotide for the proteolytic type Fd fragment
(SEQ ID 19 was complementary to the 3' end of the antibody hinge region, encoded mutations
to introduce tandem translation termination codons (TAG and TAA) immediately after the
hinge and contained an EcoRI restriction enzyme site beyond this sequence. The 3' region of
the L chain was determined by an oligonucleotide (SEQ ID 20) complementary to the end of
the coding region, introduced an additional translation termination codon (TAA) and an EcoRI
restriction site. In addition pairs of partially overlapping and complementary oligonucleotides
for each fragment (SEQ IDS 21 and 22 for the Fd and SEQ IDS 23 and 24 for the L chain)
were used to introduce silent mutations into each DNA strand resulting in the removal of a
BamHI from the CHI of the Fd fragment and the VL of the L chain without altering the
encoded amino-acid sequence. Each 5' and 3' oligonucleotide was used with the appropriate
mutagenic oligonucleotide to generate 2 mutated fragments of each antibody chain. After
purification the two fragments were mixed in equal proportions and used as the templates for a
second PCR reaction using the relevant 5' and 3' region oligonucleotides. The products of
these reactions were the full-length Fd and L chain fragments without internal BamHI sites.
In general, 5ul of cDNA was added to a lOOul reaction containing lOmM Tris-HCl,pH 8.3,
50mM KC1, 0.1% gelatin, 1.5mM MgCl2) 1.25 mM each of dATP, dCTP, dGTP and dTTP,
luM each of an appropriate oligo pair and 2.5u Taq DNA polymerase (Amplitaq,
Perkin-Elmer Cetus). Each reaction was overlaid with lOOul mineral oil and incubated at 94°C
for 1.5 minutes, 50 or 55°C for 1.0 minute and 72°C for 2.0 minutes for 25 cycles plus 10
minutes at 72°C. Control reactions with no DNA were also set up.
The PCR reactions were analysed by running a 5ul sample of each on a 0.8% agarose (Sigma
Chemical Company Ltd.) gel which was subsequently stained in lug/ml Ethidium Bromide
(BDH Laboratory Supplies) solution and the DNA visualised on a UV transilluminator. Bands
of the appropriate size were visible in all PCRs with A5B7 cDNA present indicating successful
amplification of the fragments of the Fd and L chains. The absence of a DNA band in the
control reactions indicated that the reagents used did not contain contaminating DNA.
Each PCR product was purified by use of a Centricon 100™ microconcentrator (Amicon Ltd.).
Each reaction was added to a concentrator and the volume increased to 2ml by addition of
double distilled water. The unit was then centrifuged at 500xg (Sorval RT6000B™ benchtop
centrifuge with H1000B rotor) for 5 minutes and the "flow-through" discarded. The retentate
was diluted to 2ml again and the unit re-centrifuged. The process was repeated for a third
time. This procedure results in the removal of excess oligos and buffer components from the
amplified DNA. These purified DNAs were then used directly in subsequent PCR reactions.
The appropriate pairs of fragments were mixed in equal proportions and aliquots used in the
second PCRs with the respective 5' and 3' oligonucleotides.
d) Subcloning the PCR generated fragments into pBluescript™
The products of the second PCR reactions showed bands of approximately 775bp and 730bp
consistent with the full-length Fd and L chains respectively. These products were also purified
using Centricon 100™ microconcentrators as above. Each DNA product was then precipitated
in a 1.5ml solution containing 50ul 3M sodium acetate, distilled water to 500ul and 1ml of
absolute ethanol. The solution was incubated on ice for at least 10 minutes before
centrifugation at 1 l,600xg for 10 minutes (MSB MicroCentaur™). The supernatant was
discarded and the pellet washed in 1ml 70% ethanol (v/v in distilled water) by centrifugation
for a further 5 minutes. The supernatant was discarded and the DNA pellet dried under
vacuum. Each DNA pellet was resuspended in distilled water. The Fd PCR product was then
digested with EcoRI and Hindlll in a 200ul reaction containing 20mM Tris-acetate, pH 7.9,
lOmM magnesium acetate, 50mM potassium acetate, ImM dithiothreitol (DTT), and 25u each
of Hindlll and EcoRI (Promega Corporation). The L chain product was digested with EcoRI
in a 30ul reaction containing 90mM Tris-HCl, pH7.5, lOmM magnesium chloride, 50mM
sodium chloride and lOu EcoRI. Digests were incubated at 37°C for 1 hr.
The digested fragments were then purified by electrophoresis on a 0.75% SeaPlaque™ GTG
agarose gel (FMC BioProducts Ltd) followed by excision of the appropriate bands from the
gel. The agarose gel slice was redissolved by incubation at 65°C for 2 minutes, diluted to a
final volume of 450ul with distilled water and 50ul 3M sodium acetate added. This solution
was extracted with an equal volume of liquified phenol, equilibrated with Tris buffer pH7.6
(Fisons Scientific Equipment) using cetrigugation at 1 l,600xg for 2 minutes (MSB
MicroCentaur™) to separate the aqueous and phenoiic phases. The subsequent aqueous phase
was re-extracted with a phenol:chloroform mixture (50:50 v:v) and again with chloroform
prior to ethanol precipitation as described above. Each purified pellet was resuspended in lOul
distilled water and a lul sample visualised by electrophoresis on a 0.8% agarose gel to estimate
quality and concentration.
pBluescript™ (Stratagene Cloning Systems) was used for initial cloning of Fd and L chain
cDNAs. This phagemid vector has unique EcoRI and Hindlll cloning sites, Ampicillin
resistance gene, and both ColEI and fl replication origins for isolation of either double- or
single stranded DNA. 5ug pBluescript™ KS- DNA was digested to completion with 30u
EcoRI (Promega Corporation) in a lOOul reaction containing 90mM Tris-HCl, pH7.5, lOmM
MgC12, 50mM NaCl or with EcoRI and Hindlll in a lOOul reaction containing 20mM
Tris-acetate, pH 7.9, lOmM magnesium acetate, 50mM potassium acetate, ImM dithiothreitol
(DTT), and 25u each of EcoRI and Hindlll (Promega Corporation) at 37°C for 1 hour.
calf-intestinal alkaline phosphatase (2u, Bohringer Mannheim) was the added to the EcoRI
digested plasmid to remove 5' phosphate groups and incubation continued at 37°C for a
further 30 minutes. Phosphatase activity was destroyed by incubation at 70°C for 10 minutes.
The EcoRI-HindlH cut plasmid was purified from a SeaPlaque GTG agarose gel as described
above.
25 - 50ng of digested Fd or L chain PCR product was ligated with 50ng of EcoRI-Hindll or
EcoRI/CIP treated pBluescript respectively in lOul of a solution containing 30mM Tris-HCl,
pH7.8, lOmM MgC12, lOmM DTT, ImM ATP and 1.5u T4 DNA ligase (Promega
Corporation) at 16°C for 2.5 hours. A lul aliquot of each reaction was used to transform
20ul of competent E.coli DHScx cells (Life Technologies Ltd.) using the protocol provided
with the cells. Transformed cells were plated onto L-agar plus lOOug/ml Ampicillin, ImM
IPTG and 0.2% X-gal and incubated overnight at 37°C. Clones containing cloned inserts were
selected on the basis of producing white colonies on the above medium compared to the blue
colour generated by cells containing the parental plasmid.
e) DNA sequence analysis ofcDNA clones
The potential Fd and L chain cDNA clones identified by colour selection were picked from the
agar plates and used for large scale plasmid DNA preparation. Each clone was used to
inoculate 200ml of L-broth plus lOOug/ml ampicillin in a 500ml conical flask. The cultures
were incubated, shaking at 37°C overnight. After growth the cells from each culture were
pelleted by centrifugation at SOOOxg for 10 minutes in a Sorvall RC5C centrifuge and GS3
rotor at 4°C. The cell pellet from each culture was resuspended in 20ml TE buffer and
re-centrifuged at 2000xg for 10 minutes in a Sorvall RC5C centrifuge and SS-34 rotor in an
oak-ridge tube at 4°C. Each washed cell pellet was resuspended in 3ml ice cold 25% sucrose,
50mM Tris, pHS.O, and left on ice. Fresh lysozyme solution (1.0ml at lOmg/ml) was added,
the contents mixed by rolling the tube and incubation on ice continued for 5 minutes. Sodium
ethylene diamine tetracetate (EDTA) solution (1.0ml at 0.5mM, pH8.5) was added and the
contents gently mixed. Finally, 5.0ml of iced Triton X™ solution (0.1% Triton X-100,
62.5mM EDTA, 50mM Tris, pHS.O) was added, the contents gently mixed and incubation on
ice continued for a further 10 minutes. The cell debris was then pelleted by centrifugation at
39,000xg for 30 minutes in a Sorvall RC5C centrifuge and SS-34 rotor at 4°C. The
supernatant containing plasmid DNA was added to 16g caesium chloride (Boehringer
Mannheim) and 150ul ethidium bromide solution (lOmg/ml) and the volume increased to
18.5ml by addition of TE buffer. This solution was transferred to an 18.5ml crimp top,
polypropylene centrifuge tube (Sorvall Instruments). The tube was sealed and centrifuged at
180,000xg for 16 hours in a Sorvall TV865B (titanium, vertical) rotor and OTD65B centrifuge
at 18°C.
After centrifugation, plasmid DNA was visible as a distinct orange band in the CsCl/EtBR
density gradient which had formed. The plasmid DNA was removed from the gradient using a
hypodermic syringe to pierce the tube wall. The sample taken from the gradient was diluted
3-4 fold with TE buffer and the DNA precipitated by addition of an equal volume of isopropyl
alcohol and incubation on ice for 10 minutes. The precipitated DNA was pelleted by
centrifugation at 17,000xg in a Sorvall RC5C centrifuge and SS-34 rotor at 4°C and the
supernatant discarded. The resulting pellet was washed in 70% ethanol (v/v) and
re-centrifuged for 5 minutes. The pellet was then dried under vacuum, resuspended in 1.8ml
TE buffer and 200ul 3M sodium acetate solution and extracted with an equal volume of phenol
using centrifugation at 17,000xg for 2 minutes to separate the phases. The aqueous phase was
re-extracted against an equal volume of chloroform befoie precipitating the DNA by addition
of an equal volume of ethanol at -20°C and incubating on ice for 10 minutes. The purified
DNA was pelleted as above, washed in 5ml 70% ethanol and the pellet vacuum dried. The
dried pellet was resuspended in 500ul double-distilled water and DNA concentration estimated
by scanning a diluted sample from 300 to 220nm in a UV spectrophotometer using and
extinction coefficient of 50ug/ml/OD260. A number of proprietary kits, e.g. Qiagen™ (Hybaid
Ltd), are also available for plasmid DNA purification.
This purified plasmid DNA was then used for DNA sequence analysis. Double stranded DNA
can be used for DNA sequence analysis by the dideoxy chain termination method of Sanger
(Proc.Nat.Acad.Sci. USA 74. 1977, p5463) using a proprietary sequencing kit such as the
Sequenase™ kit supplied by United States Biochemical Company and used in accordance with
the protocols provided.
Aliquots (2-4ug) of Fd and L chain cDNA clone plasmid DNA were used for DNA sequence
analysis. Each aliquot was initially denatured by incubation with 0.2M NaOH, 0.2mM EDTA
in a final volume of lOOul at room temperature for 10 minutes. The denatured DNA was then
precipitated by addition of lOul 3M sodium acetate (pHS.O) and 275ul ethanol and incubation
on ice for 10 minutes. The precipitated DNA was recovered as described for plasmid DNA
above. The denatured DNA was then primed for sequencing by incubation of each with
O.Spmoles of an appropriate primer in lOul of Sequenase™ reaction buffer (40mM Tris,
pH7.5, 25mM MgCl2, 50mM Nad) containing 10% di-methyl sulphoxide (DMSO) at 65°C
for 2 minutes followed by gradual cooling to below 30°C. These primed templates were then
used in sequencing reactions according to the protocols provided with 10% DMSO added to
labelling and termination mixtures.
The sequencing reactions were analysed by autoradiography after high resolution
electrophoresis on a 6% polyacrylamide: 8M urea denaturing gel (Sanger and Coulson, 1978,
FEES lett.87, p!07).
The complete Fd and L chain sequences of the cloned cDNAs are given below (SEQ ID 25 for
the proteolytic type Fd chain and SEQ ID 26 for L chain). The plasmid containing the
proteolytic type Fd was named pAFl and the L chain pAF3. The presence of the silent
mutation in each fragment for removal of the BamHI site was also confirmed. The DNA
sequence indicates that the antibody is an IgGlK isotype when compared to published constant
region DNA sequence data (in Kabat, E.A., Wu, T.T., Bilofsky, H., Reid-Milner, M, Perry,
H., 1987, Sequences of Proteins of Immunological Interest, Fourth Edition, Public Health
Service N.I.H. Washington DC).
f) Subcloning into myeloma expression vectors
To generate vectors capable of Fd and L chain coexpression in myeloma cells, the
OS-System™ system (Celltech Biologies) was used (WO 87/04462, WO 89/01036, WO
86/05807 and WO 89/10404).
The procedure requires cloning the Fd chain into the Hindlll-EcoRI region of vector pEE6
[this is a derivative of pEE6.hCMV - Stephens and Cockett (1989) Nucleic Acids Research 17,
7110 - in which a Hindni site upstream of the hCMV promoter has been converted to a Bglll
site] and the L chain into the EcoRI site of pEE12 [this vector is similar to pSV2.GS described
in Bebbington et al. (1992) Bio/Technology IQ, 169-175, with a number of restriction sites
originally present in pSV2.GS removed by site-directed mutagenesis to provide unique sites in
the multi-linker region]. Subsequently, a BglH-BamHI Fd expression cassette from pEE6 is
inserted into the BamHI region of pEE12. Alternatively, a Bglll-Sall fragment containing the
Fd expression cassette can be inserted into the BamHI-Sall region of the pEE12 plasmid
containing the L chain.
To construct the individual vectors (proteolytic Fd in pEE6 and L chain in pEE12), plasmids
pAFl and pEE6 were digested with EcoRI and Hindlll and pAF3 and pEE12 were digested
with EcoRI as described above. The appropriate vector and insert fragments from each digest
were then isolated from Seaplaque™ GTG agarose and ligated together and used to transform
competent DH5a cells also as described earlier. The transformed cells were plated onto L
agar plus lOOug/ml ampicillin. Screening of colonies from the transformation was by a PCR
method. Colonies were transfered into 200ul distilled water and mixed by vortexing. The
suspended cells were then heated to 100°C for 1 minute and centrifuged at 1 l,600xg for 2
minutes prior to using the supernatant in a PCR reaction. In each PCR reaction, an oligo
which primes within the CMV promoter (SEQ ID 27) was used with the oligo complementary
to the 3' region of either Fd (SEQ ID 19) or L chain (SEQ ID 20) as appropriate. Only clones
with the antibody fragment gene inserted in expressing orientation downstream from the CMV
promoter will produce specific PCR products of approximately 2.0kbp in each case. PCR
reactions of 20ul were set up containing 20pmoles of each oligo (SEQ ID NO 27 with either
SEQ ID NO 19 or 20) lOmM Tris-HCl,pH 8.3, 50mM KC1, 0.1% gelatin, 1.5mM MgC12,
1.25 mM each of dATP, dCTP, dGTP and dTTP and 0.5u Taq DNA polymerase (Amplitaq™,
Perkin-Elmer Cetus). Each reaction was overlaid with 20ul mineral oil and incubated at 94°C
for 1.5 minutes, 50°C for 1.0 minute and 72°C for 2.0 minutes for 25 cycles plus 10 minutes at
72°C. Control reactions with clones containing the parent plasmids and with no DNA were
also set up. The PCR reactions were analysed by agarose gel electrophoresis and potential
clones identified by the presence of a 2.0kbp PCR product. These possible clones were used
for large scale plasmid DNA preparation, were characterised by restriction enzyme digestion
with EcoRI-Hindlll or EcoRI and the sequence of the insertion confirmed by DNA sequence
analysis as described above. The isolates were named pAF4 (proteolytic type Fd in pEE6) and
pAF6 (L chain in pEE12).
To create the co-expressing vectors, 5-7.5fig of Fd plasmid pAF4 was digested with 30u each
of Bglll (Pharmacia) and BamHI (New England Biolabs) in a solution containing 50mM
Tris-HCl, pH7.9, lOmM magnesium chloride, ISOmM sodium chloride and ImM DTT for 1
hour at 37°C. Digestion was confirmed by agarose gel electrophoresis. 5ug of L chain plasmid
pAF6 was digested with 25 units of BamHI (New England Biolabs) in the solution described
above by incubating for 1 hour at 37°C. The DNA was then dephosphorylated by the addition
of 2u CIP and incubation at 37 °C for 40 minutes followed by three extractions with lOul of
Strataclean™ resin (Stratagene Ltd). The Fd expression cassette fragment and major pAF6
plasmid band was then purified from SeaPlaque™ GTG agarose gels, the appropriate
combination ligated together and the ligation used to transform competent DH5cc cells all as
described previously.
-35-
g) Identification of co-expressing vectors.
One hundred colonies from the above transformation were picked in duplicate in batches of 50
onto 9cm nitrocellulose discs (Schleicher and Schull) laid onto L-agar plus lOOug/ml ampicillin
plates. A third set of plates without filters was streaked to form a master stock of the selected
colonies. After overnight incubation at 37°C, the nitrocellulose filters were removed and
processed according to the method of Grunstein and Hogness (Maniatis, Chapter 1, pi02) to
lyse the bacterial cells in situ. The filters were overlaid on 3MM paper (Whatman) soaked in
the various reagents - 10% SDS for 2 minutes, 3M NaOH, 1M NaCl for 5 minutes and 1M
Tris, p6.8 for 2x 2 minutes. The filters containing lysed cells were transfered to 3MM paper
moistened with 20x SSC (3M NaCl, 0.3M sodium citrate) and the DNA cross-linked to the
filters by exposure to UV light in a Spectrolinker™ XL 1500 (Spectronics Corporation) set on
optional crosslink (120,OOOuJoules). The filters were air dried before being used in probing
(see below). The master stock plates were stored at 4°C until required.
Oligonucleotides specific for Fd and L chains (SEQ IDS 22 and 24 respectively) were used to
generate specific hybridisation probes for Fd and L chain containing clones. A hybridisation
probe can be generated from a synthetic oligonucleotide by the addition of a radio-active 5'
phospate group from y 32p ATP by the action of T4 polynucleotide kinase. 20 pmoles of the
oligonucleotide were added to a 20ul reaction containing lOOmM Tris, pH7.5 , lOmM
MgCl2,0.lmM Spermidine, 20mM DTT, 7.5uM ATP, 0.5uM 7 32P ATP and 2.5u T4
polynucleotide kinase (Pharmacia Biotechnology Ltd). The reactions were incubated for 30
minutes at 37°C and then for 10 minutes at 70°C prior to use in hybridisation. Methods for the
generation of hybridisation probes from oligonucleotides are provided in Maniatis (chapter 11).
A lOul aliquot of the radio-labelled oligo was added to 10ml of 6xSSC (1M NaCl, 0.1M
sodium citrate), 0.1% SDS (sodium dodecyl sulphate) and 0.25% Marvel™ (fat-reduced dried
milk powder) which was then used as a probe solution.
The processed filters containing the selected clones (see above) were pre-hybridised in
duplicate batches each in 90ml 6xSSC, 0.1%SDS, 0.25% Marvel™ at 65°C for 3 hours in a
Techne HB-1 hybridisation oven using rotating glass tubes. Each duplicate set was then
probed in 10ml of probe solution (one set with the VH probe and the other with VL) at 65 °C
overnight in the same apparatus. After incubation, each set of filters was washed in 100ml
6xSSC, 0,1% SDS at 65°C for 15 minutes, 100ml 3xSSC, 0.1%SDS at 65°C for 30 minutes
and 100ml IxSSC, 0.1%SDS at 65°C for 30 minutes in the same apparatus. The washed
filters were then air dried and autoradiographed using Hyperfilm™ MP (Amersham
International) in conjunction with a fast tungstate intensifying screen at -70°C. After
developing the film in a Kodak automatic film processor, potential F(ab')2 expression clones
were identified by hybridisation of both probes. The frequency of clones showing hybridisation
with both Fd and L chain specific probes was very low (approximately 2%).
The potential co-expressing clones were picked from the master plates and used for large-scale
plasmid DNA preparation. Restriction digestion analysis with the enzymes EcoRI and Hindin
was used to confirm the orientation of each expression cassette. Clones with the L and Fd
expresion cassettes in tandem orientation (rather than convergent) only were identified. The
generation of the co-expressing vector pAF8 (proteolytic Fd and L in pEE12).
h) Transfection of myeloma cells
Several methods exist for the introduction of DNA into eukaryotic cells (Bebbington, C., 1991,
Methods, vol 2, pi36-145). Electroporation has become a routinely used method more
recently, replacing the calcium phosphate-DNA co-precipitation method. NSO myeloma cells
(Methods in Enzymology, 1981,73B, p3-46. ECACC cat no. 85110503) are a suitable host
cell for this work due to the absence of any endogenous secreted antibody protein. It is
expected that a proportion of colonies arising in glutamine-free medium after transfection of
the Fd and L chain co-expressing plasmids will express functional A5B7 antibody fragments.
Prior to transfection 40ug of the pAF8 plasmid DNA was linearised by digestion with 200u
Sail (New England Biolabs) in a 400ul reaction containing lOmM Tris-HCl, pH7.9, lOmM
magnesium chloride, 150mM sodium chloride, ImM DTT and lOOug/ml acetylated BSA at
37°C for 1.75 hour. After digestion each DNA was precipitated in ethanol and resuspended in
50ul distilled water.
NSO cells were grown to near confluence in 160cm^ tissue culture flasks (Nunc or Costar)
containing 50ml non-selective growth medium (Dulbecco's Modified Eagle Medium, Life
Technologies Ltd., plus 10% foetal calf serum from an accredited source) incubated at 37°C in
an atmosphere of 5% CC«2. Prior to transfection the NSO cells were resuspended by knocking
the flask against a hand or bench and transferred to a 50ml conical centrifuge tube (Falcon). A
sample (40ul) was taken an used to estimate the cell concentration using a Coulter counter set
to count between 10 and 20um. The cells were pelleted by centrifugation at SOOxg for 5
minutes (Sorval RT6000C benchtop centrifuge) then washed with 45ml of ice-cold phosphate
buffered saline (PBS) and re-centrifuged. The washed cells were resuspended in ice-cold PBS
to a concentration of 1.3x10^ cells per ml and stored on ice. Each 50ul sample of Sail
digested plasmid DNA was mixed with SOOul (10^) NSO cells in a 0.4cm pathlength
electroporation cuvette (Bio-Rad Laboratories Ltd) avoiding bubbles and the cuvette incubated
on ice for 5 minutes. The cuvette was then wiped dry with a tissue and placed in a Gene
Pulser™ electroporation equipment (Bio-Rad Laboratories Ltd) and 2 consecutive pulses of
1500 volts at SuFarads delivered according to the manufacturer's instructions. After
electroporation the cuvettes were returned to ice for 5 minutes before mixing with 30ml
pre-warmed non-selective medium. Approximately 20ml of this cell suspension was distributed
into 4x flat-bottomed 96 well tissue culture plates (Nunc) at 50ul per well. A further 10ml was
diluted with 30ml non-selective medium and plated into 5x 96 well plates. The diluted
suspension was diluted further (10ml to 40ml) with non-selective medium and plated in a
further 5x 96-well plates. The cells were then incubated at 37°C in 5% CC>2 overnight.
Glutamine-free selective medium (150ul, Bebbington et al., (1992) Bio/Technology 1Q,
169-175) was added to each well of the 96 well plates and the plates returned to the incubator
to allow the gradual depletion of glutamine and until colonies were visible using the naked eye.
i) Expansion of cell lines.
Colonies were selected from the 96-well plates where 1 colony per well was present. The cells
were resuspended by pipetting up and down and lOOul transferred to a well of a 24-well plate
and 1ml selective medium added to each well. A further lOOul selective medium was added
back to each of the wells in the 96-well plaates from which colonies had been removed to
provide a back-up source of the cell lines. The 24-well plates were incubated at 37°C in 5%
CC2 until approx 50% confluent with cell growth. At this stage, lOOul culture supernatant was
removed and tested for anti-CEA binding activity in an ELISA assay (see below). Cell lines
showing binding activity were expanded further by pipetting up and down and transfering 1ml
to a 25cm2 tissue culture flask. A further 1ml of selective medium was added to each flask
and the flasks incubated sloping to concentrate the cells towards the bottom of the flask. After
several days incubation, 3ml of selective medium was added to each flask which was then
incubated horizontally until the cells achieved 50-75% confluence. At this stage the medium
was removed from the cells and the cells washed carefully with 5ml selective medium which
was then discarded and replaced with a further 5 ml of selective medium. The flasks were
returned to the incubator for 24hr. The cells were then harvested by knocking the flask, the
cell density counted either using a Coulter counter at 10-20um detection limits or using a
haemocytometer after staining with trypan blue solution (Life Technologies) and counting
viable (unstained) cells under a microscope. The cells were pelleted by centrifugation (~300xg
for 5 minutes) and the supernatant removed and stored at 4°C for use in analysis of antibody
fragment expression (see below). The cells were resuspended in 50% dialysed foetal calf
medium, 40% glutamine-free DMEM and 10% DMSO to a concentration of l-2x 10^ cell per
ml. The cells were then transfered in 1ml aliquots to screw cap cryotubes (Nunc), frozen at
-70°C overnight and then transferred to liquid nitrogen for long term storage.
Western blot analysis
Western blot analysis was performed as described below.
Aliquots (15ul) of each supernatant sample were mixed with an equal volume of sample buffer
(62.5mM Tris, pH6.8, 1% SDS, 10% sucrose and 0.05% bromophenol blue) with and without
reductant (50mM DTT). The samples were incubated at 100°C for 15 minutes before
electrophoresis on a 8-18% acrylamide gradient gel (Excel™ gel system from Pharmacia
Biotechnology Products) in a Multiphor™ II apparatus (LKB Produkter AB) according to the
manufacturer's instructions. After electrophoresis, the separated proteins were transfered to a
Hybond C-Super™ membrane (Amersham International) using a Novablot™ apparatus (LKB
Produkter AB) according to protocols provided by the manufacturer. After blotting, the
membrane was air dried.
The presence of antibody fragments was detected by the use of an anti-murine F(ab')2
antibody-peroxidase conjugate (ICN Biomedicals, product no. 67-430-1) Whilst this primary
antibody is raised against murine F(ab')2 it has been shown to bind primarily to the kappa L
chain. The presence of murine A5B7 antibody fragments was visualised using the ECL
detection system (Amersham International) according to the protocol provided.
This showed that about 90% of the material present in the cell supernatants was F(ab')2
protein.
k) ELISA analysis
Standard procedures for ELISA assay are available in "Laboratory Techniques in Biochemistry
-39-
and Molecular Biology" eds. Burdon, R.H. and van Kippenberg, P.H., volume 15, "Practice
and Theory of Enzyme Immunoassays", Tijssen, P., 1985, Elsevier Science Publishers B.V..
Another source of information is "Antibodies - A Laboratory Manual" Harlow, E. and Lane,
D.P. 1988, published by Cold Spring Harbor Laboratory.
The cell supernatants (see above) were used to detect the presence of anti-CEA binding
material according to the protocol given below:
/) ANTI-CEA ELISA
1. Prepare coating buffer (1 capsule of Carbonate-Bicarbonate buffer - Sigma C-3041 - in
100ml double distilled water).
2. Add 5ul of CEA stock solution (0.2mg/ml, Dako) to 10ml of coating buffer for each 96
well plate required.
3. Add lOOul of diluted CEA to each well of a Nunc "Maxisorp™" microtitre plate.
4. Incubate plates at 4°C overnight (or room temp, for 2 hours).
5. Wash plates 4 times for 5 minutes each with Phosphate buffered saline + 0.01% Sodium
azide (PBSA).
6. Block plates (after banging dry) with 1% BSA (Sigma A-7888) in PBSA at 150ul per well.
Incubate at room temp, for 2 hours.
7. Wash plates 4 times for 5 minutes each with PBSA.
8. Load samples (culture supernatants) and standards (doubling dilutions of proteolytic A5B7
F(ab')2) as appropriate. Dilute samples in growth medium (or PBS). Include PBSA + 1%
BSA and diluent as blanks.
9. Incubate at 4°C overnight.
10. Wash plates 6 times for 5 minutes each with PBSA + 0.5% Tween 20.
11. Prepare secondary antibody solution (anti-mouse IgG F(ab')2> from goat, peroxidase
conjugated - ICN 67 430-1 - at 20ul in 40ml-PBSA + 1% BSA + 0.5% Tween 20) and add
lOOul per well.
12. Incubate at room temp, for 2 hours.
13. Wash plates 6 times for 5 minutes each with PBSA + 0.5% Tween 20.
14. Prepare developing solution by dissolving 1 capsule of Phosphate-Citrate Perborate
buffer (Sigma P-4922) in 100ml double distilled water. Add 30mg o-Phenylenediamine
Dihydrochloride (OPD, Sigma P-8287) per 100ml buffer. Add lOOul per well.
15. Incubate at room temp, in darkness for 15 minutes.
16. Stop reaction by addition of 50ul per well of 2M Sulphuric acid.
17. Read OD 490nm in plate reader.
m) Calculation of Specific Production Rate (SPR)
The amount of anti-CEA binding activity in each sample was determined using the Softmax
data handling package. This figure was assumed to give an approximate figure for the amount
of A5B7 F(ab')2 fragment present in the cell supernatant taking into account the Western blot
analysis data which indicates that the majority of the antibody L chain (>90%) is present as
F(ab')2. This figure was then used to calculate a specific production rate in terms of ug/lO
cells/24 hours which was used to rank the cell lines according to productivity. SPR
calculations for the best cell lines isolated ranged typically from 4ug to lOug/lO^ cells/24
hours.
Purification of Recombinant A5B7 F(ab)i
The recombinant A5B7 F(ab)2 material was purified from myeloma medium supernatant using
a r-Protein A SOOmg cartridge such as for example manufactured by NyGene.
The cartridge was first washed in a citrate buffer at lOOmM citric acid pH2.8 and then
equilibrated with ISOmM sodium chloride lOmM sodium phosphate pH7.4 until the pH of the
wash matched that of the equilibration buffer. Both buffers were pre-filtered at 0.45um using a
Millipore filter.
The myeloma medium (1.8 litres) containing the recombinant A5B7 F(ab)2 was also
pre-filtered and diluted 1:1 with the equilibration buffer. This diluted medium was then loaded
onto the Protein A cartridge, collecting all the unbound wash. Once loaded the cartridge was
washed through with the equilibration buffer until the absorbance at 280 nm returned to
baseline.
The buffer was then changed to lOOmM sodium acetate pH4.0, also pre-filtered. This elution
buffer was collected as 45ml fractions. Once the absorbance at 280 nm had again returned to
baseline the buffer was changed to lOOmM citric acid pH2.8 in order to wash the column.
Optical density at 280 nm was determined on the fractions and those containing significant
absorbance were titrated to pH7.0. and analysed by SDS PAGE.
The fractions containing the recombinant A5B7 F(ab)2 were pooled. This volume was
concentrated (Amicon YM10™ membrane) and dialysed into 150mM sodium chloride, lOmM
sodium phosphate and 3mM EDTA disodium salt, pH7.4. and stored at 4°C. A total of 73 mg
F(ab)2 was obtained at a purity of > 90 % as judged by non-reducing SDS PAGE.
The myeloma cell supernatant used in the above purification was obtained essentially as
described Bebbington et al. (1992) in Bio/Technology 1Q, 169-175. The GS media (Cat. No.
51435) and supplement (Cat. No. 58672) is available from JRH Biosciences (JRH Biosciences
Europe, Hophurst Lane, Crawley Down, W. Sussex, U.K., RH10 4FF). At the end of the
fermentation procedure, the supernatant was filtered through a 0.45m filter to remove any
particulate matter and stored at 4°C until purification, typically no longer than 24 hours.
Reference Example 6
Synthesis of Uracil-based Prodrug analogue (see Scheme in FIGURE 9)
Compound 7 (5mg) was dissolved in 0.5ml hydrochloric acid (0.1N) to give the desired end
product (compound 9). After O.Shr at 25°C in the dark the stock solution was kept on ice and
aliquots diluted with buffer for test with mutant RNase.
Compound (7) was prepared from undine as follows:
Z'.S'-O-Methoxvethvlideneuridine (Compound 1)
Uridine (5g), p-toluenesulphonic acid monohydrate (Ig) and trimethylorthoacetate (15ml)
were stirred together at 20°C for 16hr. The reaction mixture was made slightly basic with
methanolic sodium methoxide and then concentrated to a gum. The required product was
purified by column chromatography on silica gel (Merck 9385) using chloroform/methanol
mixtures as eluant and in the proportion 96:4 (by volume) at first, followed by 92:8.
NMR (DMSOd6): (6) 11.38 (s,lH); 7.75 (d,lH); 5.95(d) and 5.80(d, total 1H); 5.62(d,lH);
4.70-5.10(m,3H); 4.18(q) and4.04(q, total 1H); 3.60(m,2H); 3.15(s) and 3.28(s, total 3H);
1.57(s)and 1.49 (s, total 3H).
S'-Azido-S'-deoxv-Z'.S'-O-methoxvethvlideneuridine (Compound 2)
To a solution of 2',3'-O-methoxyethylideneuridine (7.0g, 23.3 mmol) in dry pyridine (80ml) at
0° C was added methanesulphonyl chloride (1.9ml, 24 mmol). After stirring for 16hr at 4°C
the solvent was evaporated in vacuo and the residue dissolved in chloroform was washed with
water. The organic layer was separated, dried and concentrated fo give the crude mesylate.
The crude reaction product was dissolved in dry dimethylformamide (100ml) and sodium azide
(3.25g, 50 mmol) added. The mixture was stirred at 85°C for 7hr and then worked up by
evaporation of the solvent in vacuo to give a gum which was dissolved in chloroform and
washed with sodium bicarbonate solution. The chloroform extract was separated, dried over
anhydrous sodium sulphate and concentrated to give the crude 5'-azido product. The crude
azide intermediate was used as starting material for the next step.
3 -O (and 2 -O)-Acetyl-5' -azido-5 -deoxyuridine (Compound 3)
The crude azide (compound 2, above) was dissolved in acetic acid (70%) (100ml) and after 15
minutes the solvent was removed under reduced pressure. The residue was repeatedly
dissolved in absolute ethanol and concentrated to remove last traces of acetic acid. This
procedure gave a crude sample of the required product as a mixture of 2' and 3' -regioisomers.
NMRof2:l ratio of 2'acetoxy to 3' acetoxy in DMSOd6: (6) 11.40 (s.lH); 7.70(d,lH);
5.60-5.95(m,3H); 5.22(t,0.33H); 5.03(dd, 0.66H); 4.41 (q,0.66H); 4.24(q,0.33H); 4.14
(q,0.66H); 3.94(m,0.33H); 3.62(m,2H); 2.08(s,0.66H); 2.06(s,0.33H).
3>-O(and2>-O)-Acetvl-5>-azido-5>-deoxv-2>-Ofand3>-O)-tetrahvropvranvluridine
The crude acetate from the previous reaction was dissolved in dry dichloromethane (80ml).
Dihydropyran (6ml) plus p-toluenesulphonic acid monohydrate (SOOmg) were added to the
reaction flask. The mixture was stirred at 25°C for 3hr, after which, tic indicated that starting
material had been consumed. The reaction mixture was diluted with dichloromethane, washed
with aqueous sodium bicarbonate and the organic layer dried before evaporating under reduced
pressure. The crude product (mixture of 2' and 3'-regioisomers) was purified on silica gel
column using chloroform/methanol mixtures as eluant (97:3 by volume at first, followed by
95:5).
5*-Amino-5*-deoxv-2'-O(and 3'-O)-tetrahydropyranvluridine (Compound 4)
The azide intermediate from the previous reaction was dissolved in tetrahydrofuran (100ml)
and triphenylphosphine (6.5g, 25 mmole) was added followed by water (0.45ml). After
stirring for 16hr at 25 °C concentrated ammonia was added and the reaction continued for a
further 24hr. The reaction mixture was concentrated to dryness and purified by column
chromatography (first chloroform/methanol 9:1, followed by chloroform/methanol 1:1) to give
the required product as a mixture of 2' and 3' regioisomers.
5 * -(N-BenzyloxvcarbonvIglvcyl)amino-5' -deoxv-2'-O(and
3>-O)-tetrahydropyranvluridine (Compound 5)
To a solution of 5'-amino-5'-deoxy-2'-Q(and 3'-Q)-tetrahydropyranyluridine (3g) in
anhydrous tetrahydrofuran is added N-benzyloxycarbonylglycine p-nitrophenyl ester (3.1g, 9.2
mmol). The solution was stirred for 16hr at 25°C, concentrated to a gum and purified by
column chromatography on silica using chloroform/methanol (96:4) as eluant. The required
product was obtained as a mixture of regioisomers (3.6g, 75% yield).
NMR in DMSOd6 : (5) 11.36 (s,lH); 8.02(b,lH); 7.70(two d,lH); 7.32(m,6H); 5.90(d) and
5.70(d, total 1H); 5.64 (d, 1H); 5.444(d) and 5.20(d,total 1H); 5.02(s,2H); 4.75(m,lH);
3.20-4.25 (m, 9H); 1.35-1.80 (m, 6H).
Mass Spectrum (FAB), m/e, 519 (M+H+). C24H30N4O9 requires M+, 518
3'-O(and Z'-OVPhosphoramidite derivative of above product
To a solution of the product (1.9g, 3.67 mmol) from the previous reaction and
diisopropylethylamine (1.5ml) in dry dichloromethane (30ml) was added
N,N-diisopropylmethylphosphonamidic chloride. After stirring for 5hr at 25 °C the reaction
was diluted with chloroform and washed with aqueous sodium bicarbonate solution. The
chloroform extract was separated, dried and concentrated to give a gum. The crude mixture
was purified by column chromatography using the following eluants (first
chloroform/triethylamine 98:2, followed by chloroform/triethylamine/methanol 96:2:2) to give
the required phosphorus containing intermediate (1.9g).
Fully protected phosphate intermediate (Compound 6)
To a solution of the phosphoramidite (1.9g, 2.4 mmol) from the above reaction and
4-dipropylaminophenol (0.7g, 3.6 mmol) in dry acetonitrile (40ml) was added tetrazole (0.5g,
7.2 mmole). After stirring for 16hr at 25°C in the dark, tertiarybutylhydroperoxide (70%;
0.4ml) was added. After 15min the reaction mixture was concentrated, dissolved in
chloroform and washed with aqueous sodium bicarbonate. The chloroform layer was
separated, dried and then concentrated to a gum. This was purified by column
chromatography on silica using ethyl acetate followed by ethyl acetate/methanol (93:7 by
volume). Evaporation of appropriate fractions gave the product (1.5g) as a mixture of the 2'
and 3' regioisomers .
NMR in DMSOd6: (6) 11.43 (s, 1H); 8.10(b, 1H); 7.75(m,lH); 7.45(m, 1H); 7.35(s, 5H);
7.00(m, 2H); 6.60(m, 2H); 5.90(m, IH); 5.69 (m,lH); 5.17(m) and 4.97(m, total 1H); 5.02 (s,
2H); 4.69(bs) and 4.57(bs, total IH); 4.53(m) and 4.23 (m, total IH); 4.08(m, IH); 3.15-3.85
(m,13H); 1.35-1.75 (m,10H); 0.88 (t, 6H).
Mass Spectrum (FAB), m/e, 787 (M+) and 788 (M+ + H), C37H50N5O12P requires M+,
787
THP protected prodrug analogue (Compound 7)
The fully protected intermediate from the preceding reaction (1 mmole) was dissolved in a
mixture of ethanol (20ml)/cyclohexene (10ml) before addition of 20 % palladium on charcoal
(150mg) .The mixture was refluxed for Ihr and then filtered before concentrating under
reduced pressure. The resultant gum was purified by column chromatography on silica using
chloroform/methanol (9:1) as eluant to give the free glycyl derivative.
The methyl protected phosphate (1 mmole) from the preceding reaction was next dissolved in
tertiary butylamine (30ml). The reaction mixture was refluxed for 16hr and concentrated
before purifying by column chromatography on silica using chloroform/methanol (9:1)
followed by chloroform/methanol (7:3) as eluants to give the required THP protected prodrug
analogue as a mixture of 2' & 3'regioisomers.
Separation of 2' and 3' regioisomers by High Pressure Liquid Chromatography
The separation was accomplished by HPLC on a Partisil ODS-2 column by isocratic elution
with 60:40 methanol/ammonium formate (0.1M). Appropriate fractions were pooled and
freeze dried to give the required 3'-linked intermediate (7); see structure in Figure 9.
NMR in DMSOd6 : (6) 8.85 (s,lH); 8.25 (s,lH); 7.75 (d,!H); 6.95 (d,2H); 6.5 (d,2H); 5.85
(d,!H); 5.6 (d,!H); 4.5 (m, 2H); 4.3 (m.lH); 4.07 (m,lH); 3.2-3.6 (m,6H); 3.1 (m, 4H);
1.2-1.6(m,10H);0.8(m,6H).
Reference Example 7
Synthesis of a cytidine prodrug analogue (see scheme, Figure 10)
The cytidine prodrug analogue (compound 13) was prepared by analogy with the uridine
compounds described in Reference Example 6. The procedure described in Reference
Example 6 was followed but with compound 7 (Figure 9) replaced by compound 12 (Figure
10).
Standard work-up: Concentration of reaction mixture in vacuo, dissolve residue in
wash the solution with aq NaHCO3, dry on Na2SC»4, filter and concentrate. Purification by
flash column chromatography with indicated solvent mixture.
Compound 12 was prepared as follows (see scheme in Figure 10).
N"Benzoyl-2',3'-Q-methoxyethylidenecytidine (compound 1) was prepared according to
D.P.L. Green, T Ravindranathan, C B Reese and R Saffhill, Tetrahedron 26, 1031 (1970)
N4-Benzoyl-5'-Q-methanesulfonyl-2',3'-Q-methoxyethylidenecytidine (Compound 2) was
prepared as follows.
To a stirred solution of N^"benzoyl-2',3'-Q-Methoxyethylidenecytidine (9.85g, 25.0mmole) in
pyridine (100ml) was added methanesulphonyl chloride (1.9ml, 25mmole) at 0°C. After
stirring for 16hr at 25 °C, the reaction mixture was worked up by concentrating the solution
under vacuo, redissolving in chloroform and washing the organic layer with aqueous sodium
bicarbonate. The chloroform layer was separated, dried over sodium sulphate and
concentrated to give the product.
3 '-O-Acetyl-S'-azido-N^'benzoyl-S'-deoxy cytidine (mixture with 2'-0-acetyl isomer)
(Compound 3) was prepared as follows.
The crude mesylate (compound 2) was dissolved in anhydrous DMF (100ml). Sodium azide
(3.25g, 50mM) was added and the reaction mixture stirred at 80°C for 7 hr. The reaction was
worked up by concentrating the solvent, redissolving in chloroform and washing the
chloroform extract with sodium bicarbonate solution. The residue obtained from concentrating
the dried chloroform layer was dissolved in 120ml 70% HO Ac. After 15 minutes the solvent
was removed in vacuo and the crude product purified by column chromatography (95:5
CHCl3/MeOH followed by 92:8 CHCl3/MeOH). Yield of required product was 6g.
3 ' -O-Acetyl-5 ' -azido-N4-benzoyl-2 ' -O-tetrahydropyranyl-5 ' -deoxycytidine (mixture with
3'-O-tetrahydropyranyl isomer) (Compound 4) was prepared as follows.
Compound 3 (6g) from the above example was dissolved in methylene chloride (100ml) and
dihydropyran (4ml). After addition of 0.5g p-toluene sulphonic acid mono hydrate the
mixture was stirred for 16hr at 25°C. A similar work up procedure to that described above
gave a crude product which was purified by column chromatography eluting with 98:2
CHCla/MeOH to give the required product.
5'-Azido-5'-deoxy-2'-O-tetrahydropyranylcytidine (mixture with 3'-O-tetrahydropyranyl
isomer) (Compound 5) was prepared as follows.
The acetate (compound 4, 9.0g, impure) was dissolved in methanol (60ml) and sodium
methoxide (3.5g) was added. After stirring at 25°C for Ihr the reaction mixture was
concentrated and purified by flash column chromatography. Yield 3.7g.
Note :It is possible to separate the 2' and 3' isomers at this stage (as well as at most of the
next steps) by chromatography.
5' -Azido-N^-benzyloxycarbonyl-5' -deoxy-2' -O-tetrahydropyranylcytidine (mixture with
3'-O-tetrahydropyranyl isomer) (Compound 6) was prepared as follows.
The cytidine compound (compound 5, 3.7g) was dissolved in anhydrous pyridine (80ml) and a
catalytic amount of dimethylamino-pyridine (DMAP) and 2ml Z-C1 were added. After stirring
for 16hr at 25°C the reaction was worked up and the product purified using column
chromatography on silica and (CHCls/MeOH, 95:5) as eluant. 2.3g of product was obtained.
5'-(N-Benzyloxycarbonyl)amino-5'-deoxy-2'-O-tetrahydropyranylcytidine (mixture with
3'-O-tetrahydropyranyl isomer) (Compound 7) was prepared as follows
The azide (compound 6, 3.06g) in THF (30ml) was stirred with triphenylphosphine (1.7g) for
24hr at 50°C. Water (5ml) was added and stirring continued for another 1 hour at 50°C.
Concentration of the reaction mixture and purification on silica column using (first
CHCl3/MeOH 9:1, then 1:1 and finally 100% MeOH) gave 0.9g of the product.
N4-Benzyloxycarbonyl-5' -(N-benzyloxycarbonylglycyl)amino-5' -deoxy-2' -Otetrahydropyranylcytidine
(mixture with 3'-Q-tetrahydropyranyl isomer) (Compound 8) was
prepared as follows.
The amine (compound 7,0.9g) was dissolved in anhydrous dichloromethane (30ml) and
p-nitrophenyl- N-carbobenzyloxy-glycinate (700mg) was added. After stirring for 16hr at
25°C, the reaction mixture was concentrated and purified by column chromatography using :
(CHCls/MeOH first in the proportion 97:3, then 95:5). Ig of the required material was
obtained.
N4-Benzyloxycarbonyl-5'-N-(benzyloxycarbonylglycyl)amino-5'-deoxy-2'-Otetrahydropyranylcytidyl-
3'-(N,N-diisopropylmethyl) phosphonamidate (mixture with 3'
isomer) (Compound 9) was prepared as follows.
The alcohol (compound 8, Ig) was dissolved in anhydrous dichloromethane (30ml) and
EtN(iPr)2 (1.7ml) was added, followed by Cl-P(OMe)N(iPr)2 (34ml). After stirring for 6hrs at
25 °C and work-up the mixture was purified by column chromatography on silica using (first
CHCl3/Et3N 98:2, the CHCl3/Et3N/MeOH 97:2:1). 1. Ig product was obtained.
(Methyl)(4-N)N-dipropylarninophenyl)[N4-benzyloxycarbonyl-5'-(N-benzyloxycarbonylglycyl)
amino-5'-deoxy-2'-O-tetrahydropyranylcytidyl-3']-phosphate (mixture with 3' isomer)
(Compound 10) was prepared as follows.
The phosphonamidate (compound 9, l.lg) was dissolved in anhydrous acetonitrile (30ml) and
4-Af,W-dipropylaniinophenol (200mg)was added followed by tetrazole (420mg). After stirring
at 25°C for 16hr, 70% t-butylhydroperoxide (0.3ml) was added. After 15 minutes the reaction
mixture was worked up and the crude product purified by column chromatography on silica
eluting with : (first EtOAc, then EtOAc/MeOH 97:3) to give 0.85g product
(Methyl)(4-N,//-dipropylarninophenyl)(5' -deoxy-5' -glycylaminocytidyl-3')- phosphate
(mixture with 3' isomer) (Compound 11) was prepared as follows.
The bis-carbobenzyloxy-protected compound (compound 10, 0.85g) was dissolved in ethanol
(30ml) and cyclohexene (15ml). Pd-C 20% (400mg) were added and the stirred mixture
heated to reflux for 4hrs. After filtration the solution was concentrated and the gum purified
by column chromatography on silica eluting with :(first CHCla/MeOH 95:5 then 5:1 and finally
100% MeOH). lOOmg product was obtained.
(4-N, Af-dipropylaminopheny 1)(5' -deoxy-5' -glycylamino-2' -Qtetrahydropyranylcytidyl-
3') hydrogenphosphate. (Compound 12) was prepared as follows.
The phosphate (compound 11, lOOmg) was dissolved in f-butylamine (25ml) and heated to
reflux for 8hr. After concentration the product was purified by HPLC (Magnum 20 reversed
phase column, eluent MeOH/O.lM ammonium formate in the ratio 60:40)
NMR (DMSOd6): (8) 9.1 (s, 1H), 8.19 (s, 1H), 7.6 (d, 1H), 7.2 (m, 3H), 6.95 (d, 2H), 6.45
(d, 2H), 5.8 (d, 1H), 5.72 (d, 1H), 4.71 (m, 1H), 4.45 (m, 1H), 4.22 (m, 1H), 4.1 (m, 1H),
3.8 (t, 1H), 3.7-3.15 (m,2H), 3.51 (s, 2H), 3.35-3.5 (m, 1H), 3.0 (m, 4H), 1.8-1.2 (m, 10H),
0.8 (m, 6H).
Mass Spectrum FABMS [MH+] 639
Reference Example 8
Localisation of A5B7 F(ab')2-BP-RNase conjugate to LoVo tumour xenografts
The murine A5B7 F(ab')2-BP-RNase conjugate prepared as described in Reference Example 4,
was radioiodinated with carrier-free 125I using the IODOGEN™ reagent (Pierce and Warriner
(UK) Ltd, Chester England) following the manufacturer's recommended method. In vitro
retention of >50% immunoreactivity after radioiodination was confirmed by binding to LoVo
tumour cells using the method of Lindmo et al, J. Immunol. Meth., 72, 77-89, 1984.
Approximately 10n,g of conjugate containing lOjxCi 125I was injected intravenously into
athymic nude mice (nu/nu:Alpk [outbred]) bearing established LoVo tumour xenografts ( 1 x
107 LoVo tumour cells injected subcutaneously 7 days previously). Following injection of
conjugate, groups of 3 mice were killed at various time periods later and the tumour, a sample
of blood and a range of other tissues were removed, weighed and counted in a gamma counter.
The tumour and tissue distribution of the conjugate is shown in below.
Tumour and tissue localisation of A5B7 F(ah')2-BP-RNase
Tissue
Tumour
Blood
Liver
Kidney
Lung
4hr
2.54
6.83
1.81
2.76
2.85
24hr
3.27
1.06
0.62
0.55
0.28
48hr
1.00
0.25
0.12
0.23
0.15
72hr
0.66
0.12
0.07
0.18
0.09
96hr
0.41
0.06
0.06
0.11
0.08
Units = % injected dose/g tissue; results are mean values from 3 mice.
The results clearly show that the A5B7 F(ab')2-RNase conjugate specifically localises to the
LoVo xenograft. From 24 hr onwards there was more conjugate/g tissue in the tumour
compared to any other tissue including the blood. The levels of conjugate in the tumour were
similar to those achieved with a A5B7 F(ab')2-CPG2 conjugate (Blakey et al, Br. J. Cancer,
69 supplement XXI, p 14, 1994). These levels with this CPG2 conjugate have been shown
sufficient in combination with mustard prodrugs to result in tumour regressions and prolonged
growth delays in the LoVo xenograft model (Blakey et al, Br. J. Cancer, 69 supplement XXI,
p!4, 1994; Blakey et al, Proceedings of the American Association for Cancer Research, 35,
p507, 1994).
Reference Example 9
Synthesis of Hippuryl-L-Glutamic Acid (see Scheme 2)
Hippuryl-L-glutamic acid dibenzyl ester (compound 3) (2.06g, 4.2xlO~3 moles) and 30%
Pd/Carbon (50% moist) (0.77g) in THF were stirred in an atmosphere of hydrogen for 1.5
hours. The mixture was filtered through Celite™ and the filtrate evaporated to dryness.
Trituration with diethyl ether gave the desired end product as a white crystalline solid 1.02g
(78%). Melting point 169-171°C.20D = -2.5°
NMR DMSO d6 12.3, 2H (broad); 8.7, 1H (t); 8.2 ,1H (t); 7.9, 2H (m); 7.5, 3H (m); 4.3, 1H
(m); 3.9, 2H (m); 2.3, 2H (t); 1.9, 2H (m)
The starting material compound 3 was prepared as follows. To a solution of hippuric acid
(0.90g, 5xlO~3 moles) and L-glutamic acid dibenzyl ester (2.50g, 5xlO-3 moles) in DMF
(35ml) was added 1-hydroxybenzotriazole (0.73g, 5.5xlO"3 moles), triethylamine (1.4ml,
9.7xlO"3 moles) and l(3-dimethyl-aminopropyl)-3-ethylcarbodiimide, HCI salt (l.OSg, 5.5x10"
3 moles). The mixture was stirred overnight at room temperature, poured into water (400ml)
and extracted twice with ethyl acetate (100ml). The combined extracts were washed with
saturated sodium bicarbonate solution, water, 2N HCI and water. The organic phase was dried
over MgSC4 and evaporated to obtain the desired starting material as a yellow oil. 2.06g
(84%).
NMR DMSO d6 8.7, 1H (t); 8.4, 1H (d); 7.9, 2H (m); 7.5, 3H (m); 7.35, 10H (m); 5.15, 2H
(s); 5.05, 2H (s); 4.4, 1H (m); 3.9, 2H (t); 2.0,4H (m)
Reference Example 10
Synthesis of Hippuryl-L-Aspartic acid
Hippuryl-L-aspartic acid dibenzyl ester (1.28g , 2.7x10"3 moles) and 30% Pd/Carbon (50%
moist) (0.5 Ig) in THF were stirred in an atmosphere of hydrogen for 3 hours. The mixture was
filtered through Celite™ and the filtrate evaporated to dryness. Trituration with diethyl ether
gave an off-white crystalline solid 0.62g (78%). Melting point 200-202°C. 20D = + 7.9°
NMR DMSO d6 12.5, 2H (broad); 8.7, 1H (t); 8.2, 1H (d); 7.7 ,2H (m); 7.5, 3H (m); 4.6,
1H (m); 3.9, 2H (d); 2.7, 2H (m)
The starting material was synthesised as follows. To a solution of hippuric acid (0.90g, 5x10"^
moles) and L-aspartic acid dibenzyl ester (2.3Ig, 5x10"^ moles) in DMF (35ml) was added
1-hydroxybenzotriazole (0.73g, 5.5xlO~3 moles), triethylamine (1.4ml, 9.7x10"^ moles) and
l-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide, HC1 salt (1.05g, 5.5x10"^ moles). The
mixture was stirred for 4 hours at room temperature then poured into water (450ml) and
extracted twice with ethyl acetate (100ml). The extract was washed with saturated sodium
bicarbonate solution, water, 2N HC1 and water. The organic phase was dried over MgSO4 and
evaporated to dryness to obtain the desired starting material as a yellow oil. 1.90g (80%)
NMR DMSO d6 8.7, 1H, (t); 8.45, 1H, (d); 7.9, 2H (m); 7.5, 3H (m); 7.3, 10H (m); 5.15, 2H
(s); 5.05, 2H (s); 4.8, 1H (m); 3.9, 2H (m); 2.9, 2H (m)
Reference Example 11
Enzymic activity of recombinant HCPB against Hipp-Arg.
Purified human CPB, produced as described in Reference Example 20, was assayed for its
ability to convert hippuryl-L-arginine (Hipp-Arg) to hippuric acid using a spectrophotometric
assay.
The Km and kcat for native HCPB were determined by measuring the initial rate of conversion
of Hipp-Arg to hippuric acid at 254 nM using a range of Hipp-Arg concentrations (0.75-0.125
mM) and a CPB enzyme concentration of lug/ml. Measurements were carried out at 37°C in
0.25 mM Tris HC1 buffer, pH 7.5 using 1 cm path length cuvettes in a total volume of 1.0 ml
using a Perkin Elmer Lambda 2 spectrophotometer. Km and Vmax values were calculated
using the ENZFITTER™ software programme (Biosoft™, Perkin Elmer). Kcat was calculated
from Vmax by dividing by the enzyme concentration in the reaction mixture.
The results for human CPB against Hipp-Arg were:
Km = 0.18mM
kcat = 65s~1
The results demonstrate that the recombinant HCPB is enzymatically active and can cleave the
amide bond in Hipp-Arg to release Hippuric acid.
Reference Example 12
Synthesis of an Arginine mustard prodrug (see Scheme 1)
(2S),2-(3- {4-[bis-(2-chloroethyl)-amino)-phenoxycarbonyl} -propionylamino)-
5-guanidino-pentoic acid (compound 5c, scheme 1)
A solution of
(2S),2-(3-{4-[bis-(2-chloroethyl)-amino)-phenoxycarbonyl}-propionyl-amino)-5-(2-nitro)-
guanidino-pentoic acid benzyl ester (compound 4c, Scheme 1) (275 mg; 0.44 mmol) in ethyl
acetate/MeOH (1/1: V/V) (8 ml) containing 10 % Pd/C (200 mg) was hydrogenated in a Paar
apparatus at 80 psi for 6 h. After filtration the organic layer was evaporated. The resulting oil
was recrystallised using Cl^Ctydiethyl ether to give the desired compound 5c as a white solid
(180 mg), yield 84%.
1HNMR (CD30D): 1.55-1.7 (m, 3H); 1.8-1.9 (m, 1H); 2.6-2.7 (m, 2H); 2.75-2.85 (m, 1H);
2.9-2.95 (m, 1H); 3.1-3.2 (m, 2H); 3.6-3.7 (m, 4H); 3.7-3.8 (m, 4H); 4.3 (dd, 1H); 6.75 (dd,
2H); 6.95 (dd, 2H).
MS(ESI):512-514(MNa)+
Anal (C2oH29N504Cl2 1.5 H2O)
Calc. C: 47.91 H: 6.43 N: 13.97
Found C: 47.7 H: 6.21 N: 14.26
Starting material compound 4c was prepared as follows. To a solution of
(2S),2-amino-5-(2-nitro)-guanidino-pentoic acid benzyl ester (compound 2c) (654 mg; 1
mmol) in CHC13 (10 ml) was added dihydro-furan-2,5-dione (compound 1) (120 mg; 2 mmol)
followed by triethylamine (202 mg; 2 mmol) dropwise. After stirring for 2h at room
temperature, the solvent was evaporated and the crude residue was dissolved in water. pH was
adjusted to 2.5 with 2N HC1. The aqueous layer was extracted with ethyl acetate. The organic
layer was washed with brine, dried (MgSC>4) and evaporated to give
(2S),2-(3-carboxy-propionylamino)-5-(2-nitro)-guanidino-pentoic acid benzyl ester (compound
3c). The resulting solid was triturated with diethylether and filtered off: 280 mg (68 %).
-52-
1HNMR (CD3OD): 1.52-1.68 (m, 2H); 1.7-1.8 (m, IH); 1.85-1.95 (m, IH); 2.45-2.7 (m, 4H);
3.15-3.3 (m, 2H); 4.5 (m, IH); 5.15 (dd, 2H); 7.25-7.4 (m, 5H)
MS (ESI): 432 [MNa]+
To a suspension of compound 3c (204 mg; 0.5 mmol) in CHC13 (5 ml) was added
4-[bis(2-chloroethyl)amino]-phenol (compound 6) (135 mg; 0.5 mmol), EDCI (19 mg; 0.5
mmol) followed by DMAP (18 mg; 0.75 mmol). After stirring at room temperature for 6h, the
solvent was evaporated. The residue was partitioned between ethyl acetate and water and the
aqueous phase acidifed to pH = 3 with 2N HC1. After extraction with ethyl acetate, the organic
layer was washed with brine, dried (MgSO4) and evaporated. The residue was purified by flash
chromatography using C^C^^6^ (95/5: V/V) as eluant to give the desired starting
material 4c as a white foam (281 mg) yield: 90 %.
4c: IHNMR (CD3OD): 1.55-1.7 (m, 2H); 1.7-1.8 (m, IH); 1.85-1.95 (m, IH); 2.55-2.75 (m,
2H); 2.8-2.9 (m, 2H); 3.15-3.25 (m, 2H); 3.6-3.7 (m, 4H); 3.7-3.8 (m, 4H); 4.5 (dd, IH); 5.15
(dd, 2H); 6.7 (d, 2H); 6.95 (d, 2H); 7.32 (m, 5H)
MS (ESI): 647-649 [MNa]+
Reference Example 13
Synthesis of succinic acid mono-{4-[N,N-bis(2-chloroethyl)arnino]-pheny!} ester (also
called "intermediate" herein)
To a suspension of succinic anhydride (225mg, 2.25mmol) in CHC13 (10ml) was added under
stirring, 4-[N,N-bis(2-chloroethyl)-amino]phenol (compound 6, Scheme 1; 203mg, 0.75 mmol)
followed by triethylamine (75mg, 0.75 mmol). The mixture was stirred overnight and the
solvent evaporated. The crude residue was dissolved in EtOAC/Et2O/H2O and under stirring
the pH was adjusted to 3. The organic layer was washed with water, brine, dried (MgSO4), and
evaporated. The resulting oil was crystallised from Et2O/hexane and the white solid was
filtered off and dried under vacuum to obtain the desired end product (210 mg; yield 83%).
Melting point 98-100°C.
-53-
MS (ESI): 356-358 [MNa]+
1H NMR (CDC13): 2.8 (dd, 2H); 2.9 (dd,2H); 3.65 (dd, 4H); 3.75 (dd, 4H); 6.65 (d, 2H); 7.0
(d, 2H)
Analysis (C^HjyC^C^N 0.2 H2O):
Calc. %C: 49.78 H: 5.19 N:4.15
Found %C: 49.9 H: 5.3 N: 4.2
Reference Example 14
Cloning of human pancreatic carboxypeptidase B (HCPB)
Standard molecular biology techniques, such as restriction enzyme digestion, ligation, kinase
reactions, dephosphorylation, polymerase chain reaction (PCR), bacterial transformations, gel
electrophoresis, buffer preparation and DNA generation, purification and isolation, were
carried out as described by Maniatis et al., (1989) Molecular Cloning, A Laboratory Manual;
Second edition: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, or following
the recommended procedures of manufacturers of specific products. In most cases enzymes
were purchased from New England BioLabs, but other suppliers, and equivalent procedures
may be used. Oligonucleotide sequences were prepared in an Applied Biosystems 380A DNA
synthesiser from 5'dimethoxytrityl base-protected nucleoside-2-cyanoethyl-N,N'-di-isopropylphosphoramidites
and protected nucleoside linked to controlled-pore glass supports on a 0.2
umol scale, according to the protocols supplied by Applied Biosystems Inc..
The coding sequence for human pancreatic carboxypeptidase B was obtained from a human
pancreatic cDNA library cloned in the XgtlO vector (Clontech, Human pancreas 5' STRETCH
cDNA, HL1163a) using PCR technology, and cloned into the plasmid vector pBluescript™ II
KS+ (Stratagene).
Typically, an aliquot of the cDNA library (5ul at a litre of >10^pfu/ml) was mixed with
lOOpMols of two Oligonucleotide primers, BPT1 and BPB1, (SEQ ID NO: 46 and SEQ ID
NO: 47), dNTPs to a final concentration of 200|oM, Taq polymerase reaction buffer, and 2.5U
of Taq polymerase in a final volume of lOOul. The mixture was heated at 94°C for 10 minutes
prior to addition to the Taq enzyme, and the PCR incubation was carried out using 30 cycles
of 94°C for 1.5 minutes, 50°C for 2 minutes, and 72°C for 2 minutes, followed by a single
incubation of 72°C for 9.9 minutes at the end of the reaction.
The two oligonucleotide primers were designed to allow PCR extension from the 5' of the
gene from BPT1 (SEQ ID NO: 46), between the start of the pre-sequence and the start of the
pro-sequence, and PCR extension back from the 3' end of the gene from BPB1(SEQ ID NO:
47), as shown in Figure 18. BPT1 and BPB1 are also designed to introduce unique restriction
sites, SacI and Xhol respectively, into the PCR product.
An aliquot of the PCR product was analysed for DNA of the correct size (about 1250 base
pairs) by agarose gel electrophoresis and found to contain predominantly a band of the correct
size. The remainder of the product from the reaction mix was purified and separated from
excess reagents using a Centricon™ 100 microconcentrator column (Amicon), followed by
DNA isolation by ethanol/sodium acetate precipitation, centrifugation, vacuum drying and
re-suspension in distilled water. The isolated DNA was restriction digested with enzymes SacI
and Xhol, and a band of the correct size (about 1250 base pairs) purified and isolated from
agarose gel electrophoresis using excision and glass-milk (Geneclean™, Stratec Scientific, or
other similar product).
pBluescript™ IIKS+ double stranded DNA (Stratagene) was restriction digested with SacI
enzyme, and the product dephosphorylation treated with calf intestinal alkaline phosphatase to
remove 5'phosphoryl groups and reduce re-ligation and vector background following
transformation. The DNA product was purified from enzyme reaction contaminants using
glass-milk, and then restriction digested with Xhol enzyme. DNA of the correct size (about
2850 base pairs) was purified and isolated by agarose gel electrophoresis using excision and
glass-milk (Geneclean™, Stratec Scientific, or other similar product).
Aliquots of both restricted and purified DNA samples were checked for purity and
concentration estimation using agarose gel electrophoresis compared with known standards.
From these estimates ligation mixes were prepared to clone the HCPB gene into the vector,
using a molar ratio of about 1 vector to 2.5 insert (1 pBluescript™ II KS+ to 2.5 HCPB PCR
product), and a final DNA concentration of about 2.5ng/ul, in the presence of T4 DNA ligase,
ImM ATP and enzyme buffer.
Following the ligation reaction the DNA mixture was used to transform E.coli strain DH5a
(Gibco-BRL, maximum efficiency competent cells). Cell aliquots were plated on L-agar
nutrient media containing lOOug/ml ampicillin as selection for plasmid vector, and incubated
over-night at 37°C. Colonies containing plasmids with inserts of interest were identified by
hybridisation.
About 200 colonies were picked and plated onto duplicate sterile nitro-cellulose filters
(Schleicher and Schull), pre-wet on plates of L-agar nutrient media containing lOOug/ml
ampicillin as selection for plasmid vector, and incubated over-night at 37°C. One duplicate
plate is stored at 4°C, and acts as a source of live cells for the colonies, the other plate is
treated to denature and fix the DNA from the individual colonies to the nitro-cellulose. The
nitro-cellulose filter is removed from the agar plate and placed in succession onto Whatman™
filter papers soaked in :
1. 10% SDS for 2 minutes
2. 0.5M NaOH, 1.5M NaCl for 7 minutes
3. 0.5M NaOH, 1.5M NaCl for 4 minutes
4. 0.5M NaOH, 1.5M NaCl for 2 minutes
5. 0.5M Tris pH7.4,1.5M NaCl for 2 minutes
6. 2xSSC (standard saline citrate) for 2 minutes.
The filter is then placed on a Whatman™ filter paper soaked in lOxSSC and the denatured
DNA is crossed linked to the nitro-cellulose by ultra violet light treatment (Spectrolinker™
XL-1500 UV crosslinker). The filters are then allowed to air dry at room temperature, and are
then pre-hybridised at 60°C for one hour in a solution of 6xSSC with gentle agitation (for
example using a Techne HB-1D hybridizer). Pre-hybridization blocks non-specific DNA
binding sites on the filters.
In order to determine which colonies contain DNA inserts of interest the DNA crosslinked to
the nitro-cellulose filter is hybridised with a radio-labelled 32P-DNA probe prepared from
HCPB PCR product of the pancreatic cDNA library (see above). About 50ng of DNA was
labelled with 50uCi of 32P-dCTP (~3000Ci/mMol) using T7 DNA polymerase in a total
volume of 50ul (Pharmacia T7 Quickprime kit), and the reaction allowed to proceed for
minutes at 37°C. The labelled probe is then heated to 95°C for 2 minutes, to denature the
double stranded DNA, immediately added to 10ml of 6xSSC at 60°C, and this solution used to
replace the pre-hybridisation solution on the filters. Incubation with gentle agitation is
continued for about 3 hours at 60°C. After this time the hybridisation solution is drained off,
and the filters washed twice at 60°C in 2xSSC for 15 minutes each time. Filters were then
gently blotted dry, covered with cling film (Saran™ wrap or similar), and exposed against
X-ray film (for example Kodak Xomat™-AR5) over-night at room temperature. Following
development of the film, colonies containing inserts of interest were identified as those which
gave the strongest exposure (darkest spots) on the X-ray film. In this series of experiments
about 15% of the colonies gave positive hybridisation. From this 12 colonies were chosen for
further screening. These colonies were picked from the duplicate filter, streaked and
maintained on L-agar nutrient media containing lOOug/ml ampicillin, and grown in L-broth
nutrient media containing lOOug/ml ampicillin.
The selected isolates were checked by PCR for inserts of the correct size, using primers BPT1
and BPB1, (SEQ ID NO: 46 and SEQ ID NO: 47), and for priming with an internal primer
BPT2 (SEQ ID NO: 48) and BPB1. BPT2 is designed to prime at the end of the
pro-sequence, prior to the start of the mature gene and to introduce an Xbal restriction site.
For PCR screening colonies of the selected isolates were picked and dispersed into 200ul of
distilled water and heated at 100°C for 10 minutes in a sealed Eppendorf™ tube. The
suspensions were then centrifuged for 10 minutes in a microfuge to pellet cell debris, and Ijol
of the supernatant used as the DNA template in PCR screening. Typically, lul of supernatant
was mixed with 20pmols of two oligonucleotide primers, BPT1 and BPB1, or BPT2 and
BPB1, dNTPs to a final concentration of 200uM, Taq polymerase reaction buffer, and 0.5U of
Taq polymerase in a final volume of 20ul. The PCR incubation was carried out using 25 cycles
of 94°C for 1.5 minutes, 50°C for 2 minutes, and 72°C for 2 minutes, followed by a single
incubation of 72°C for 9.9 minutes at the end of the reaction.
The PCR products were analysed for DNA of the correct size (about 1250 base pairs from
primers BPT1 to BPB1, and about 900 base pairs from primers BPT2 to BPB1, see Figure 18)
by agarose gel electrophoresis. Ten of the twelve clones gave PCR DNA products of the
correct size. Six of the ten clones were then taken for plasmid DNA preparation (using Qiagen
Maxi™ kits, from 100ml of over-night culture at 37°C in L-broth with lOOug/ml ampicillin).
These plasmid DNA preparations were then sequenced over the region of PCR product insert
using an USB Sequenase™ DNA sequencing kit, which incorporates bacteriophage T7 DNA
polymerase. Each clone was sequenced using eight separate oligonucleotide primers, known
as 676, 336, 337, 679, 677, 1280, 1279 and 1281 (SEQ ID NOs: 48 to 55). The positioning
of the sequencing primers within the HCPB sequence is shown diagramatically in Figure 19,
primers 336, 1279, 676, 1280, 677 and 1281 being 'forward', and 337 and 679 'backwards'.
Five of the six clones were found to have identical sequence (SEQ ID NO: 56) of 1263 base
pairs between and including the SacI and Xhol restriction sites, and this sequence was used in
further experiments. The translation of the DNA sequence into its polypeptide sequence is
shown in SEQ ID NO: 57, and is numbered 1 from the start of the mature protein sequence.
Amino acid numbered -95 marks the start of the putative pro-enzyme sequence. Only part of
the enzyme secretion leader sequence (pre-sequence) is present in the cloned PCR generated
DNA. The polypeptide sequence shows an aspartate residue at position 253, which when the
whole sequence is aligned with other mammalian carboxypeptidase A and B sequences
indicates a B type specificity (see amino acids numbered 255 by Catasus L, et al, Biochem J.,
287. 299-303, 1992, and discussion). However, the cysteine residue at position 135 in the
cloned sequence is not observed in other published human pancreatic carboxypeptidase B
sequences, as highlighted by Yamamoto et al, in the Journal of Biological Chemistry, 267.
2575-2581, 1992, where she shows a gap in her sequence following the position numbered
244, when aligned with other mammalian pancreatic carboxypeptidase B amino acid
sequences. Also shown on Figure 19 are the approximate sites of the aspartate amino acid
residue in the enzyme recognition site, and the cysteine residue at position 135 of the mature
enzyme.
One of the clones was deposited on 23-November-1995 with the National Collection of
Industrial and Marine Bacteria Limited (23 St. Machar Drive, Aberdeen AB2 IRY, Scotland)
and has the designation NCIMB 40694. The plasmid from this clone is known as pICI1698.
Reference Example 15
Expression of mature HCPB-(His)5'C-Myc from E. coli
In order to achieve the expression of mature HCPB from E.coli the mature gene from
pICI1698 was transferred into a plasmid vector which allows controlled secretion of protein
products into the periplasm of the bacteria. This secretion vector, known as pICI266, in a
bacterial host MSD522 suitable for controlled expression, has been deposited on 11 October
1993 with the National Collection of Industrial and Marine Bacteria Limited (Aberdeen AB2
IRY, Scotland) and has the designation NCIMB 40589. A plasmid map of pICI266 is shown
in Figure 20. The plasmid has genes for tetracycline resistance and induction (TetA and
TetR), an AraB operator and promoter sequence for inserted gene expression, and an AraC
gene for expression control. The promoter sequence is followed by the PelB translation leader
sequence which directs the polypeptide sequence following it to the periplasm. The site of
gene cloning has several unique restriction sites and is followed by a phage T4 transcription
terminator sequence. The DNA sequence in this region and the features for gene cloning are
shown diagramatically in Figure 21.
For the cloning of the mature HCPB sequence into pICI266 it was decided to generate HCPB
DNA by PCR, and to make some alterations to the codon usage at the start of the mature gene
to introduce E.coli preferred codons. Also, to help with detection and purification of the
expression construct a C-term peptide tag, known as (His)6'C-myc was added to the enzyme.
The tag consists of 6 histidines, a tri-peptide linker (EPE) and a peptide sequence
(EQKLISEEDL) from c-myc which is recognised by the antibody 9E10 (as published by Evan
et al, Mol Cell Biol, 5, 129-136, 1985, and available from Cambridge Research Biochemicals
and other antibody suppliers). The C-term is completed by the addition of an Asparagine. The
6 histidine residues should allow the purification of the expressed protein on a metal chelate
column (for example Ni-NTA Agarose from Qiagen). In addition the PCR primers are used to
introduce unique restriction sites at the 5' (Fspl) and 3' (EcoRI) of the gene to facilitate the
introduction of the PCR product into the expression vector. The sequence of the two primers,
known as FSPTS1 and 6HIS9E10R1BS1, are shown in SEQ ID NOs: 58 and 59.
To generate a modified gene for cloning into pICI266, PCRs were set up using lOOpMols of
primers FSPTS1 and 6HIS9E10R1BS1 in the presence of approximately 5ng of pICI1698
DNA, dNTPs to a final concentration of 200uM, Taq polymerase reaction buffer, and 2.5U of
Taq polymerase in a final volume of lOOul. The mixture was heated at 94°C for 10 minutes
prior to addition to the Taq enzyme, and the PCR incubation was carried out using 30 cycles
of 94°C for 1.5 minutes, 50°C for 2 minutes, and 72°C for 2 minutes, followed by a single
incubation of 72°C for 9.9 minutes at the end of the reaction. An aliquot of the PCR product
was analysed for DNA of the correct size (about 1000 base pairs) by agarose gel
electrophoresis and found to contain predominantly a band of the correct size. The remainder
of the product from the reaction mix was purified and separated from excess reagents using a
Centricon™ 100 microconcentrator column (Amicon), followed by DNA isolation by
ethanol/sodium acetate precipitation, centrifugation, vacuum drying and re-suspension in
distilled water. The isolated DNA was restriction digested with enzymes Fspl and EcoRI, and
a band of the correct size (about 1000 base pairs) purified and isolated from agarose gel
electrophoresis using excision and glass-milk (Geneclean™, Stratec Scientific, or other similar
product).
pICI266 double stranded DNA, prepared using standard DNA technology (Qiagen plasmid kits
or similar), was restriction digested with Kpnl enzyme, being very careful to ensure complete
digestion. The enzyme was then inactivated by heating at 65°C for 10 minutes, and then
cooling on ice. The 3' over-hang generated by the Kpnl was then enzymatically digested by
the addition of T4 DNA polymerase as recommended by the supplier (New England BioLabs),
in the presence of dNTPs and incubation at 16°C for 15 minutes. The reaction was stopped by
inactivating the enzyme by heating at 70°C for 15 minutes. The DNA product was purified
from enzyme reaction contaminants using glass-milk, an aliquot checked for yield by agarose
gel electrophoresis, and the remainder restriction digested with EcoRI enzyme. Again care
was taken to ensure complete restriction digest. DNA of the correct size (about 5600 base
pairs) was purified and isolated by agarose gel electrophoresis using excision and glass-milk
(Geneclean™, Stratec Scientific, or other similar product).
Aliquots of both restricted and purified DNA samples were checked for purity and
concentration estimation using agarose gel electrophoresis compared with known standards.
From these estimates ligation mixes were prepared to clone the HCPB gene into the vector,
using a molar ratio of about 1 vector to 2.5 insert (1 pICI266 to 2.5 HCPB PCR product), and
a final DNA concentration of about 2.5ng/ul, in the presence of T4 DNA ligase, ImM ATP
and enzyme buffer, using conditions suitable for the ligation of blunt ended DNA (Fspl to T4
DNA polymerase treated Kpnl).
Following the ligation reaction the DNA mixture was used to transform E.coli strain DHSoc
(Gibco-BRL, maximum efficiency competent cells). Cell aliquots were plated on L-agar
nutrient media containing lOug/ml tetracycline as selection for plasmid vector, and incubated
over-night at 37°C. Colonies containing plasmids with inserts of interest were identified by
hybridisation.
About 350 colonies were picked and plated onto duplicate sterile nitro-cellulose filters
(Schleicker and Schull), pre-wet on plates of L-agar nutrient media containing lOug/ml
tetracycline as selection for plasmid vector, and incubated over-night at 37°C. One duplicate
plate is stored at 4°C, and acts as a source of live cells for the colonies, the other plate is
treated to denature and fix the DNA from the individual colonies to the nitro-cellulose. The
nitro-cellulose filter is removed from the agar plate and placed in succession onto Whatman™
filter papers soaked in :
1. 10% SDS for 2 minutes
2. 0.5M NaOH, 1.5M NaCl for 7 minutes
3. 0.5M NaOH, 1.5M NaCl for 4 minutes
4. 0.5M NaOH, 1.5M NaCl for 2 minutes
5. 0.5M Tris pH7.4,1.5M NaCl for 2 minutes
6. 2xSSC (standard saline citrate) for 2 minutes.
The filter is then placed on a Whatman filter paper soaked in lOxSSC and the denatured DNA
is crossed linked to the nitro-cellulose by ultra violet light treatment (Spectrolinker XL-1500
UV crosslinker). The filters are then allowed to air dry at room temperature, and are then
pre-hybridised at 60°C for one hour in a solution of 6xSSC with gentle agitation (for example
using a Techne HB-1D hybridizer™). Pre-hybridization blocks non-specific DNA binding sites
on the filters.
In order to determine which colonies contain DNA inserts of interest, the DNA crosslinked to
the nitro-cellulose filter is hybridised with a radio-labelled 32P-DNA probe prepared from
HCPB PCR product of the pancreatic cDNA library (see above). About 50ng of DNA was
labelled with 50uCi of 32P-dCTP (~3000Ci/mMol) using TV DNA polymerase in a total
volume of 50ul (Pharmacia T7 Quickprime™ kit), and the reaction allowed to proceed for 15
minutes at 37°C. The labelled probe is then heated to 95°C for 2 minutes, to denature the
double stranded DNA, immediately added to 10ml of 6xSSC at 60°C, and this solution used to
replace the pre-hybridisation solution on the filters. Incubation with gentle agitation is
continued for about 3 hours at 60°C. After this time the hybridisation solution is drained off,
and the filters washed twice at 60°C in 2xSSC for 15 minutes each time. Filters were then
gently blotted dry, covered with cling film (Saran™ wrap or similar), and exposed against
X-ray film (for example Kodak Xomat™-ARS) over-night at room temperature. Following
development of the film, colonies containing inserts of interest were identified as those which
gave the strongest exposure (darkest spots) on the X-ray film. In this series of experiments
about 50% of the colonies gave positive hybridisation. From this 12 colonies were chosen for
further screening. These colonies were picked from the duplicate filter, streaked and
maintained on L-agar nutrient media containing lOug/ml tetracycline, and grown in L-broth
nutrient media containing lOug/ml tetracycline.
The selected isolates were checked by PCR for inserts of the correct size, using primers
FSPTS1 and 6HIS9E10R1BS1, (SEQ ID NO: 58 and SEQ ID NO: 59), and for priming with
an internal primer BPB2 (SEQ ID NO: 51) and FSPT1. BPB2 is designed to prime within the
mature gene and generate a fragment of about 430 base pairs.
For PCR screening colonies of the selected isolates were picked and dispersed into 200ul of
distilled water and heated at 100°C for 10 minutes in a sealed Ependorph tube. The
suspensions were then centrifuged for 10 minutes in a microfuge to pellet cell debris, and lul
of the supernatant used as the DNA template in PCR screening. Typically, lul of supernatant
was mixed with 20pMols of two oligonucleotide primers, FSPT1 and 6HIS9E10R1BS1, or
FSPT1 and BPB2, dNTPs to a final concentration of 200uM, Taq polymerase reaction buffer,
and 0.5U of Taq polymerase in a final volume of 20ul. The PCR incubation was carried out
using 25 cycles of 94°C for 1.5 minutes, 50°C for 2 minutes, and 72°C for 2 minutes, followed
by a single incubation of 72°C for 9.9 minutes at the end of the reaction.
The PCR products were analysed for DNA of the correct size (about 1000 base pairs from
primers FSPTS1 to 6HIS9E10R1BS1, and about 430 base pairs from primers FSPTS1 to
BPB2) by agarose gel electrophoresis. All twelve clones gave PCR DNA products of the
correct size. Six of the clones were then taken for plasmid DNA preparation (using Qiagen
Maxi™ kits, from 100ml of over-night culture at 37°C in L-broth with lOug/ml tetracycline).
These plasmid DNA preparations were then sequenced over the region of PCR product insert
using an USB Sequenase™ DNA sequencing kit, which incorporates bacteriophage T7 DNA
polymerase. Alternatively the DNA was sequenced using an automated DNA sequencing
service (using ABI sequencing equipment). The clones were sequenced using several separate
oligonucleotide primers. Three of the primers, known as 1504, 1590 and 1731, were used to
check the cloning junctions between the expression vector and the inserted gene (SEQ ID
NOs: 60, 61 and 62), as well as giving sequence data from the start and end of the inserted
gene. Other primers, including those known as 679, 677, 1802, and 1280 (SEQ ID NOs: 51,
52, 63 and 53) were used to confirm the remainder of the inserted gene sequence. This
plasmid containing the modified mature HCPB gene is known as pICI1712. The confirmed
sequence of the cloned gene, showing amino acid translation, from the start of the PelB
sequence to the end of the (His)6-c-myc tag is shown as SEQ ID NO: 64 with DNA numbering
starting from 1 in the first codon of PelB, and peptide numbering starting from 1 in the mature
HCPB.
To obtain controlled expression of the modified HCPB the pICI1712 plasmid DNA was
transformed into calcium chloride transformation competent E.coli expression strains.
Included amongst these strains were a number which were incapable of growing with arabinose
as the major carbon source, and were chromosome deleted for the arabinose (Ara) operon. A
preferred strain is known as MSD213 (strain MC1000 of Casadaban et al, Journal of
Molecular Biology, v!38, 179-208, 1980), and has the partial genotype, F- Ara A(Ara-Leu) A
LacX74 GalV GalK StrR. Another preferred strain is known as MSD525 (strain MCI061)
and has the genotype, AraD139 A(Ara Leu)7697 ALac74 GalU HsdR RpsL. E.coli strains of
similar genotype, suitable for controlled expression of genes from the AraB promoter in
plasmid pICI266, may be obtained from The E.coli Genetic Stock Centre, Department of
Biology, Yale University, CT, USA. Selection for transformation was on L-agar nutrient
media containing lOug/ml tetracycline, over night at 37°C. Single colonies were picked from
the transformation plates, purified by streaking and maintained on L-agar nutrient media
containing lOug/ml tetracycline, and grown in L-broth nutrient media containing 10ng/ml
tetracycline.
All pICI1712 transformed expression strains were treated in the same manner to test for
expression of the cloned HCPB gene.
1. A single colony was used to inoculate 10ml of L-broth nutrient media containing
lOug/ml tetracycline in a 25ml Universal container, and incubated over night at 37°C
with shaking.
2. 75ml of L-broth nutrient media containing lOug/ml tetracycline pre-warmed to 37°C in
a 250ml conical flask was inoculated with 0.75 ml (l%v/v) of the over-night culture.
Incubation was continued at 37°C with shaking, and growth monitored by light
absorbance at 540nm. Induction of cloned protein expression was required during
exponential growth of the culture, and this was taken as between 0.4 and 0.6 O.D. at
540nm, and generally took 90 to 150 minutes from inoculation.
3. When the cells had reached the required optical density the cultures were allowed to cool
to approximately 30°C by placing the flasks at room temperature for 30 minutes.
Arabinose was then added to a final concentration of 1% (w/v), and incubation continued
at 30°C with shaking for 4 to 6 hours.
4. After incubation a final optical density measurement is taken, and the cells were
harvested by centrifugation. The final O.D. measurement is used to calculate the the
volume of protein acrylamide gel (Laemmli) loading buffer that is used to resuspend the
cell pellet. For O.D. less than 1 a volume of lOul is used for each 0.1 O.D. unit, and for
-63-
an O.D. greater than 1 a volume of 15ul is used for each 0.1 O.D. unit. The Laemmli
loading buffer consists of 0.125M Tris-HCl pH 6.8, containing 2% SDS, 2% (5
-mercaptoethanol, 10% glycerol and 0.1% Bromophenol blue.
5. Following re-suspension the samples were denatured by heating at 100°C for 10 minutes,
and then centrifuged to separate the viscous cell debris from the supernatant. Expression
samples, usually 20ul of the supernatant, typically were loaded onto 17% SDS
acrylamide gels for electrophoretic separation of the proteins. Duplicate gels were
generally prepared so that one could be stained for total protein (using Coomassie or
similar stain and standard conditions), and the other could be processed to indicate
specific products using Western analysis.
For Western analysis proteins in the run gel were transferred to nylon membrane (Problot™,
Applied Biosystems for example), using a semi-dry electrophoresis blotting apparatus (Bio-rad
or similar). Before and during processing care was taken to ensure that the membrane
remained damp. After transfer of the proteins from the gel, further binding was blocked with a
solution of 5% low fat milk powder (Marvel™ or similar) in phosphate buffered saline (PBS)
at room temperature with gentle agitation for 5 hours. The membrane was then washed 3
times at room temperature with gentle agitation for 5 minutes each time in PBS containing
0.05% Tween 20. The washed membrane was then incubated with the primary antibody,
monoclonal 9E10 mouse anti-c-myc peptide (see above), at a suitable dilution (typically 1 in
10,000 for ascites or 1 in 40 for hybridoma culture supernatant) in PBS containing 0.05%
Tween 20 and 0.5% low fat milk powder, at room temperature with gentle agitation over
night. The membrane was then washed 3 times at room temperature with gentle agitation for
at least 5 minutes each time in PBS containing 0.05% Tween 20. The washed membrane was
then incubated with the secondary antibody, horseradish peroxidase labelled anti-mouse IgG
(typically raised in goat, such as A4416 from Sigma), at a suitable dilution (typically 1 in
10,000) in PBS containing 0.05% Tween 20 and 0.5% low fat milk powder, at room
temperature with gentle agitation for at least three hours. The membrane was then washed
times at room temperature with gentle agitation for at least 10 minutes each time in PBS
containing 0.05% Tween 20. The membrane was then processed using the Amersham ECL™
Western detection kit methodology, and exposed against Amersham Hyperfilm™ ECL for 30
seconds in the first instance, and then for appropriate times to give a clear image of the
expressed protein bands. Other methods of similar sensitivity for the detection of peroxidase
labelled proteins on membranes may be used.
Good expression of the cloned tagged HCPB in pICI266 (pICI1712) was demonstrated in
E.coli strains MSD213 and MSD525 by the Coomassie stained gels showing an additional
strong protein band at about 35,000 Daltons when compared to vector (pICI266) alone clones,
and a band of the same size giving a strong signal by Western analysis detection of the c-myc
peptide tag.
Reference Example 16
Expression of mature HCPB from E. coli
The method of cloning and expressing the mature HCPB in E.coli was very similar to the
method described in Reference Example 15. Again pICI266 was used as the cloning vector,
but in this case the starting material for PCR of the mature HCPB gene was plasmid pICI1712,
the tagged gene in the expression vector. Two oligonucleotides, known as 2264 and 2265
(SEQ ID NOs: 65 and 66) were used in the PCR reactions (instead of primers FSPTS1 and
6HIS9E10R1BS1), using similar conditions to Reference Example 15. but using pICI1712
DNA instead of pICI1698. The first, top strand, oligonucleotide, 2264, was designed to prime
on pICI1712 and to include the Ncol restriction enzyme site in the PelB leader sequence, and
to continue to the start of the inserted mature HCPB gene (DNA bases 36 to 66 inclusive in
SEQ ID NO: 64). The second, bottom strand, oligonucleotide, 2265, was designed to prime at
the end of the mature HCPB gene, prior to the start of the (His)6-c-myc tag sequence
(complementary to DNA bases 965 to 987 inclusive in SEQ ID NO: 64), and to introduce
translation termination codons (complementary to TAA TAA) at the end of the gene followed
by an EcoRI (GAATTC) restriction enzyme site and fill-in bases. This oligo primes back into
the gene in the PCR to isolate the mature gene sequence.
An aliquot of the PCR product was analysed for DNA of the correct size (about-970 base
pairs) by agarose gel electrophoresis and found to contain predominantly a band of the correct
size. The remainder of the product from the reaction mix was purified in a similar manner to
Reference Example 15. The isolated DNA was restriction digested with enzymes Ncol and
EcoRI, and a band of the correct size (about 940 base pairs) purified in a similar manner to
Reference Example 15.
pICI266 double stranded DNA, prepared in a similar manner to Reference Example 15. was
restriction digested with Ncol and EcoRI enzymes, being very careful to ensure complete
digestion. DNA of the correct size (about 5600 base pairs) was purified in a similar manner to
Reference Example 15.
Aliquots of both restricted and purified DNA samples were checked for purity and
concentration estimation using agarose gel electrophoresis compared with known standards.
From these estimates ligation mixes were prepared to clone the HCPB gene into the pICI266
vector in a similar manner to Reference Example 15.
Following the ligation reaction the DNA mixture was used to transform E.coli strain DH5a,
colonies were picked and tested by hybridisation, in a similar manner to Reference Example 15.
Six of the clones were then taken for plasmid DNA preparation, which were then sequenced
over the region of PCR product in a similar manner to Reference Example^ The clones were
sequenced using six separate oligonucleotide primers known as 1504, 1802, 679, 1280,677
and 1731 (SEQ ID NOs: 60, 63, 51, 53, 52 and 62). From the sequencing results a clone
containing a plasmid with the required mature HCPB gene sequence was selected, and is
known as pICI 1736.
The confirmed sequence of the cloned gene, showing amino acid translation, from the start of
the PelB sequence to the EcoRI restriction site is shown as SEQ ID NO: 67 with DNA
numbering starting from 1 in the first codon of PelB, and peptide numbering starting from 1 in
the mature HCPB.
To obtain controlled expression of the mature HCPB, the pICI1736 plasmid DNA was
transformed into calcium chloride transformation competent E.coli expression strains in a
similar manner to Reference Example 15. All pICI1736 transformed expression strains were
treated in a similar manner to Reference Example 15 to test for expression of the cloned HCPB
gene. However, in this case the 9E10 monoclonal antibody specific for the c-myc peptide tag
cannot be used in the Western analysis, as the mature HCPB has no C-terminal tag. Therefore,
the primary antibody was an anti-bovine carboxypeptidase. A raised in rabbit (from
Biogenesis) which had previously been shown to cross-react with purified human pancreatic
carboxypeptidase B. The secondary antibody was an anti-rabbit IgG antibody labelled with
horseradish peroxidase and raised in goat (Sigma A9169 or similar).
Expression of the cloned mature HCPB in pICI266 (pICI1736) was demonstrated in E.coli
strains MSD213 and MSD525 by the Coomassie stained gels showing an additional protein
band at about 34,000 daltons when compared to vector (pICI266) alone clones. A band of the
same size gave a signal by Western analysis detection using the anti-bovine carboxypeptidase
A.
Reference Example 17
Expression of mature HCPB from COS cells
A gene encoding preHCPB was generated by PCR from pICI1698 (Reference Example ty.
The PCR was set up with template pICI1689 (lOug) and oligos SEQ ID NO 34 and SEQ ID
NO 35 (lOOpMoles of each) in buffer (lOOul) containing lOmM Tris-HCl (pH8.3), 50mM
KCL, 1.5mM MgCl2,0.125mM each of dATP, dCTP, dGTP and dTTP and 2.5u Taq DNA
polymerase (Amplitaq, Perkin-Elmer Cetus). The reaction was overlaid with mineral oil
(lOOul) and incubated at 94°C for 1 min, 53°C for 1 min and 72°C for 2.5 min for 25 cycles,
plus 10 min at 72°C. The PCR product of 985bp was isolated by electrophoresis on a 1%
agarose (Agarose type I, Sigma A-6013) gel followed by excision of the band from the gel and
isolation of the DNA fragment by use of Geneclean™ (Geneclean II kit, Stratech Scientific
Ltd. or Bio 101 Inc.). The Geneclean kit contains 1) 6M sodium iodide 2) a concentrated
solution of sodium chloride, Tris and EDTA for making a sodium chloride/ethanol/water wash;
3) Glassmilk™- a 1.5 ml vial containing 1.25 ml of a suspension of a specially formulated silica
matrix in water.
This is a technique for DNA purification based on the method of Vogelstein and Gillespie
published in Proceedings of the National Academy of Sciences USA (1979) Vol 76, p 615.
Alternatively any of the methods described in "Molecular Cloning - a laboratory manual"
Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory, 1989) can
be used. Briefly, the Geneclean procedure is as follows. To 1 volume of gel slice is added 3
volumes of sodium iodide solution from the kit. The agarose is melted by heating the mix at
55°C for 10 min then Glassmilk (5-10ul) is added, mixed well and left to stand for 10 min at
ambient temperature. The glassmilk is spun down and washed 3 times with NEW WASH
(SOOul) from the kit. The wash buffer is removed from the Glassmilk which is to dry in air.
The DNA is eluted by incubating the dried Glassmilk with water (5-10ul) at 55°C for 5-10
min. The aqueous supernatant containing the eluted DNA is recovered by centrifugation. The
elution step can be repeated and supernatants pooled.
The preHCPB gene was digested for Ih at 37°C with EcoRI and Hindffl in a 100|il reaction
containing lOOmM Tris-HCl (pH 7.5), lOmM magnesium chloride, 50mM NaCl, 0.025%
triton X-100, and 25u each of Hindlll and EcoRI (New England Biolabs). The digested
fragment was purified by agarose gel electrophoresis and GeneClean as described above for the
uncut fragment and cloned into pBluescript™ (Stratagene Cloning Systems).
pBluescript™ KS+ DNA (5ug) was digested to completion with EcoRI and Hindlll (25u each)
in a lOOul reaction as described above. Calf-intestinal alkaline phosphatase (lul; New England
Biolabs, lOu/ul) was the added to the digested plasmid to remove 5' phosphate groups and
incubation continued at 37°C for a further 30 minutes. Phosphatase activity was destroyed by
incubation at 70°C for 10 minutes. The EcoRI-HindlH cut plasmid was purified from an
agarose gel as described above. The EcoRI-Hindlll digested preHCPB gene (50ng) was
ligated with the above cut plasmid DNA in 20ul of a solution containing 30mM Tris-Hcl
(pH7.8), lOmM MgCl2, lOmM DTT, ImM ATP, 50 ug/ml BSA and 400u T4 DNA ligase
(New England Biolabs, Inc) at 25°C for 4h. A lul aliquot of the reaction was used to
transform 20ul of competent E. coli DH5a cells (MAX efficiency DH5a competent cells, Life
Technologies Ltd) using the protocol provided with the cells. Transformed cells were plated
onto L-agar plus lOOug/ml Ampicillin. Potential preHCPB clones were identified by PCR.
Each clone was subjected to PCR as described above for preparation of the preHCPB gene
except that the mix with the cells was incubated at 94°C (hot start procedure) for 5 min prior
to 25 cycles of PCR and oligos SEQ ID NOs 36 and 37 replace oligos SEQ ID NOs 34 and
35. A sample (lOul) of the PCR reaction was analysed by electrophoresis on a 1% agarose
gel. Clones containing the preHCPB gene were identified by the presence of a 1.2kb PCR
product. Clones producing the 1.2kb were used for large scale plsamid DNA preparation and
the sequence of the insert confirmed by DNA sequnece analysis. The plasmid containing the
preHCPB gene in pBluescript™ was named pMF15.
To generate vectors capable of expressing HCPB in eukaryotic cells the GS-System™
(Celltech Biologies) was used (WO 87/04462, WO 89/01036, WO 86/05807 and WO
89/10404). The procedure requires cloning the preHCPB gene into the Hindlll-EcoRI region
of vector pEE12 [this vector is similar to pSV2.GS described in Bebbington et al. (1992)
Bio/Technology 10, 169-175, with a number of restriction sites originally present in pSV2.GS
removed by site-directed mutagenesis to provide unique sites in the multi-linker region]. To
construct the expression vector, plasmids pEE12 and pMFIS were digested with EcoRI and
-68-
Hindlll as described above. The appropriate vector (from pEE12) and insert (from pMF15)
from each digest were isloated from a 1% agarose gel and ligated together and used to
transform competent DH5a cells. The transformed cells were were plated onto L agar plus
ampicillin (lOOug/ml). Colonies were screened by PCR as described above, with oligos which
prime within the CMV promoter (SEQ ID NO 38) and in the HCPB gene (SEQ ID NO 39).
Clones producing a 1.365kb PCR product were used for large scale plasmid DNA preparation
and the sequence of the insert confirmed by DNA sequence analysis. The plasmid containing
the preHCPB sequence in pEE12 was named pMF48.
A second eukaryotic expression plasmid, pEE12 containing the prepro sequence of
preproHCPB was prepared as described above. Oligos SEQ ID NOs 40 and 41 were used in
the initial PCR to isolate a gene for the prepro sequence from pMFIS (described in Reference
Example 19). In this case the PCR was performed with a hot start procedure by first incubating
the mix without Taq DNA polymerase for 5 min at 94°C. Taq DNA polymerase (2.5u) was
then added and the PCR continued through the 25 cycles as described above. The 360bp
fragment was clone into pBluescript to give pMF66 and subsequently into pEE12 (screening
by PCR with SEQ ID NOS 40 and 41 to give pMF67.
For expression in eukaryotic cells, vectors containing genes capable of expressing preHCPB
and the prepro sequence were cotransfected into COS-7 cells. COS cells are an African green
monkey kidney cell line, CV-1, transformed with an origin-defective SV40 virus and have been
widely used for short-term transient expression of a variety of proteins because of their
capacity to replicate circular plasmids containing an S V40 origin of replication to very high
copy number. There are two widely available COS cell clones, COS-1 and COS-7. The basic
methodology for transfection of COS cells is described by Bebbington in Methods: A
Companion to Methods in Enzymology (1991) 2, p. 141. For expression of HCPB, the
plasmid vectors pMF48 and pMF67 (4ug of each) were used to transfect the COS-7 cells (2 X
10e5) in a six-well culture plate in 2ml Dulbecco's Modified Eagle's Medium (DMEM)
containing 10% heat inactivated foetal calf serum (PCS) by a method known as lipofection -
cationic lipid-mediated delivery of polynucleotides [Feigner et al. in Methods: A Companion to
Methods in Enzymology (1993) 5, 67-75]. The cells were incubated at 37°C in a CO2
incubator for 20h. The mix of plasmid DNA in serum-free medium (200ul; OPTI-MEM
Reduced Serum Medium; GibcoBRL Cat. No. 31985) was mixed gently with LIPOFECTIN
reagent (12ul; GibcoBRL Cat. No. 18292-011) and incubated at ambient temperature for
15min. The cells were washed with serum-free medium (2ml; OPTI-MEM). Serum-free
medium (600ul; OPTI-MEM) was added to the DNA/LIPOFECTIN and the mix overlaid onto
the cells which were incubated at 37°C for 6h in a CO2 incubator. The DNA containing
medium was replaced with normal DMEM containing 10% PCS and the cells incubated as
before for 72h. Cell supernatants (250ul) were analysed for HCPB activity against Hipp-Arg
(5h assay) as described in Reference Example 11. COS cell supernatants which had been
treated with LIPOFECTIN reagent, but without plasmid DNA, hydrolysed 1.2% of the
substrate, whereas the COS cell supernatants transfected with the mix of plasmids expressing
preHCPB and prepro sequence hydrolysed 61% of the Hipp-Arg substrate. COS cells
transfected with only the preHCPB plasmid hydrolysed Hipp-Arg at the level seen for COS
cells which had been treated with LIPOFECTIN reagent alone.
LIPOFECTIN Reagent is a 1:1 (w/w) liposome formulation of the cationic lipid
N-[l-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl
phosphatidylethanolamine (DOPE) in membrane filtered water. It binds sponaneously with
DNA to form a lipid-DNA complex - see Feigner et al. in Proc. Natl. Acad. Sci. USA (1987)
84,7431.
Reference Example 18
Expression of proHCPB from E. coli
The method of cloning and expressing the pro-HCPB in E.coli was very similar to the method
described in Reference Example 15. Again pICI266 was used as the cloning vector, and the
starting material for PCR of the pro-HCPB gene was plasmid pICI1698 (as described in
Reference Example 14). Two oligonucleotides, known as 2310 and 2265 (SEQ ID NOs: 68
and 66) were used in the PCR reactions (instead of primers FSPTS1 and 6HIS9E10R1BS1),
using similar conditions to Reference Example 15.
The first, top strand, oligonucleotide, 2310, was designed to prime on pICI1698, and to add
the Ncol restriction enzyme site from the PelB leader sequence (DNA bases 51 to 66 inclusive
in SEQ ID NO: 64) to the start of the inserted pro-HCPB gene (DNA bases 40 to 57 inclusive
in SEQ ID NO: 56). The second, bottom strand, oligonucleotide, 2265, was designed to prime
at the end of the mature HCPB gene, prior to the start of the (His)6_c-myc tag sequence
(complementary to DNA bases 965 to 987 inclusive in SEQ ID NO: 64), and to introduce
translation termination codons (complementary to TAA TAA) at the end of the gene followed
by an EcoRI (GAATTC) restriction enzyme site and fill-in bases. This oligo primes back into
the gene in the PCR to isolate the pro-gene sequence.
An aliquot of the PCR product was analysed for DNA of the correct size (about 1240 base
pairs) by agarose gel electrophoresis and found to contain predominantly a band of the correct
size. The remainder of the product from the reaction mix was purified in a similar manner to
Reference Example 15. The isolated DNA was restriction digested with enzymes Ncol and
EcoRI, and a band of the correct size (about 1210 base pairs) purified in a similar manner to
Reference Example 15.
pICI266 double stranded DNA, prepared in a similar manner to Reference Example 15. was
restriction digested with Ncol and EcoRI enzymes, being very careful to ensure complete
digestion. DNA of the correct size (about 5600 base pairs) was purified in a similar manner to
Reference Example 15.
Aliquots of both restricted and purified DNA samples were checked for purity and
concentration estimation using agarose gel electrophoresis compared with known standards.
From these estimates ligation mixes were prepared to clone the pro-HCPB gene into the
pICI266 vector in a similar manner to Reference Example 15.
Following the ligation reaction the DNA mixture was used to transform E.coli strain DHSoc,
colonies were picked and tested by hybridisation, in a similar manner to Reference Example
15.
Four positive hybridisation isolates were checked by PCR for inserts of the correct size, using
primers 2310 and 2265, (SEQ ID NOs: 68 and 66), and for priming with a pair of internal
primers 1279 (SEQ ID NO: 54) and 679 (SEQ ID NO: 51) in a similar manner to Reference
Example 15. The PCR products were analysed for DNA of the correct size (about 1200 base
pairs from primers 2310 to 2265, and about 580 base pairs from primers 1279 to 679) by
agarose gel electrophoresis. All clones gave PCR DNA products of the correct size.
All four of the clones were then taken for plasmid DNA preparation, and were then sequenced
over the region of PCR product in a similar manner to Reference Example 15. The clones
were sequenced using six separate oligonucleotide primers known as 1504, 1802,679, 1281,
1590 and 1592 (SEQ ID NOs: 60, 63, 51, 55, 69 and 70). From the sequencing results a clone
containing a plasmid with the required pro-HCPB gene sequence was selected, and is known
as pICI1738.
The confirmed sequence of the cloned pro-HCPB gene in pICI1738, showing amino acid
translation, from the start of the PelB sequence to the EcoRI restriction site is shown as SEQ
ID NO: 71 with DNA numbering starting from 1 in the first codon of PelB, and peptide
numbering starting from 1 in the mature HCPB.
To obtain controlled expression of the pro-HCPB the pICI1738 plasmid DNA was
transformed into calcium chloride transformation competent E.coli expression strains in a
similar manner to Reference Example 15. All pICI1738 transformed expression strains were
treated in a similar manner to Reference Example 15 to test for expression of the cloned HCPB
gene. However, in this case the 9E10 monoclonal antibody specific for the c-myc peptide tag
cannot be used in the Western analysis, as the pro-HCPB has no C-terminal tag. Therefore,
the primary antibody was an anti-bovine carboxypeptidase A raised in rabbit (from Biogenesis)
which had previously been shown to cross-react with purified human pancreatic
carboxypeptidase B. The secondary antibody was an anti-rabbit IgG antibody labelled with
horseradish peroxidase and raised in goat (Sigma A0545 or similar).
Expression of the cloned pro-HCPB in pICI266 (pICI1738) was demonstrated from E.coli by
the Coomassie stained gels showing an additional protein band at about 40,000 Daltons when
compared to vector (pICI266) alone clones, and clones producing the tagged HCPB
(Reference Example 15). A band of the same size gave a signal by Western analysis detection
using the anti-bovine carboxypeptidase A.
Reference Example 19
Expression of proHCPB from COS cells
A gene for preproHCPB was prepared by PCR as described in Reference Example 17 using as
template pICI1689 and oligos SEQ ID NOS 34 and 40 to give a 1270bp PCR product. The
gene was digested with EcoRI and Hindlll and cloned initially into pBluescript KS+ (to give
pMFIS) then into pEE12 in DH5a (to give pMF49) as described in Reference Example 17.
Plasmid pEE12 was transfected into COS-7 cells by use of LIPOFECTIN reagent as described
in Reference Example 17 and cell supernatants (250ul) assayed for HCPB activity against
Hipp-Arg (5h assay), as described in Reference Example 11, following activation with trypsin
(700ug/ml) in 50mM Tris-Hcl (pH7.6), 150mM NaCl at 4°C for Ih. Under these condition,
complete hydrolysis of the Hipp-Arg substrate was achieved, whereas supernatant from COS
cells which had been treated with LIPOFECTEN reagent alone (without plasmid DNA) when
activated with trypsin hydrolysed 30% of the Hipp-Arg substrate.
Reference Example 20
Purification of native HCPB
A system has been determined for the initial purification of the native and the different mutant
enzymes via two routes. The preferred route is described first.
Recombinant E.coli cell paste containing the recombinant enzyme was taken from storage at
-70°C and allowed to thaw. The weight of cell paste was measured in grams and the paste
resuspended with the addition of buffer A (200mM Tris (hydroxymethyl)aminomethane
hydrochloride (TRIS-HC1), 20% sucrose (C12H22O11), pH 8.0 ) to a volume equal to the
initial weight of the cell paste. The cell suspension was incubated at room temperature for
minutes with occasional gentle mixing before an equal volume of distilled water was added and
thoroughly mixed in. The cell suspension was again incubated at room temperature for 20
minutes with occassional gentle mixing. The resulting crude osmotic shockate was clarified by
centrifugation at 98000 x g for 90 minutes at 4°C after which the supernatant was decanted off
from the pelleted insoluble fraction. Deoxyribonuclease 1 was added to the supernatant to a
final concentration of 0.1 mg/ml The mixture was incubated at room temperature, with
continuous shaking, until the vicosity was reduced enough for it to be loaded on to a
Carboxypeptidase Inhibitor CNBr activated sepharose affinity column.prepared according to
instructions with the CNBr activated Sepharose 4B from Pharmacia and carboxypeptidase
inhibitor from potato tuber (c-0279,Sigma). The supernatant was adjusted to pHS.O and
loaded on to the affinity column, pre-equilibrated with lOmM TRIS-HC1, SOOmM sodium
chloride, pH 8.0. After loading the supernatant the column was washed until the absorbance of
the flow through was back to baseline before the bound material was eluted from the column
by elution buffer (lOOmM sodium carbonate, SOOmM sodium chloride, pH 11.4). The eluted
fractions were frozen at -20°C whilst those containing the recombinant carboxypeptidase were
determined by western blot analysis using an anti- c-myc tag antibody ( 9E10), followed by an
anti-mouse -horse raddish peroxidase conjugate (a-9044, sigma) that gave a colour reaction
with exposure to 4-chloro-naphthol and hydrogen peroxide.
Fractions containing the rscombinant carboxypeptidase B were pooled, concentrated and the
pH adjusted to pH 7.5 before being snap-frozen and stored at -20°C. Further purification of
the pooled sample, utilising known methods such as ion exchange and gel permeation
chromatography may performed if required.
The second route involves the total lysis of the E.coli cells as opposed to a periplasmic shock,
as used in the preferred route.
Recombinant E.coli cell paste containing the recombinant enzyme was taken and resuspended
in lysis buffer (50mM TRIS-HC1, 15% Sucrose, pH 8.0). Lysozyme was added to a
concentration of Img/ml and at the same time lithium dodecyl sulphate (LDS) was added (80ul
of a 25% solution per 25ml of suspension). The suspension was incubated on ice for SOminutes
with occasional shaking, followed by the addition deoxyribonuclease 1 to a concentration of
Img/ml and again the suspension was incubated on ice for 30 minutes with occasion shaking.
The suspension was subsequently divided in to 200ml volumes and sonicated to complete the
disruption of the cells for 10 x 30 sec bursts with 30sec intervals between bursts. Sonicated
suspensions were centrifuged at 98,000x g for 90 minutes at 4°C after which the supernatant
was decanted off from the pelleted insoluble fraction. The supernatant was adjusted to pH 8.0
and loaded on to the affinity column, pre-equilibrated with lOmM TRIS-HC1, SOOmM sodium
chloride, pH 8.0. After loading the supernatant the column was washed until the absorbance of
the flow through was back to baseline before the bound material was eluted from the column
by elution buffer (lOOmM sodium carbonate, 500mM sodium chloride, pH 11.4). The eluted
fractions were frozen at -20oC whilst those containing the recombinant carboxypeptidase were
determined by western blot analysis using an anti- c-myc tag antibody (9E10), followed by an
anti-mouse -horse raddish peroxidase conjugate (a-9044, sigma) that gave a colour reaction
with exposure to 4-chloronaphthol and hydrogen peroxide. Fractions containing the
recombinant carboxypeptidase B were pooled, concentrated and the pH adjusted to pH 7.5
before being snap-frozen and stored at -20°C. Further purification of the pooled sample,
utilising known methods such as ion exchange and gel permeation chromatography may
performed if required.
Samples of the pooled material from both routes, analysed by SDS-PAGE and Coomassie
stained nitrocellulose blot provided Coomassie stained bands at the correct molecular weight
for the recombinant carboxypeptidase B's. These bands sequenced by an automated
protein/peptide sequencer using the Edman degradation technique gave positive matches for
the particular recombinant carboxypeptidase B being purified.
Reference Example 21
Expression of murine A5B7 F(ab')2'HCPB fusion protein from COS cells
This example describes the preparation of cDNA from the A5B7 hybridoma, the isolation of
specific Fd and light chain fragments by PCR, determination of the complete DNA sequence of
these fragments, the subsequent preparation of an Fd-HCPB fusion gene and a co-expression
vector capable of producing both light chain and Fd-HCPB fusion protein in eukaryotic cells,
expression of the F(ab')2-HCPB from COS cells by co-transfection with a prepro sequence
fromHCPB.
The procedure described in Reference Example 5 is repeated as far as item (e).
f) Preparation of Fd-HCPB fusion DNA sequence
A gene encoding the C-terminal region of the Fd sequence, from the Ncol site in SEQ ID NO
25 (position 497) was joined to the HCPB sequence by PCR. In this process DNA for an 8
amino-acid linker sequence (VPEVSSVF) was introduced. Plasmid pAFl (described in
Reference Example 5) was subjected to PCR (hot start procedure) as described in Reference
Example 17 with oligos SEQ ID NOS 42 and 43 to give a 338bp product. Similarly, pICI1698
was subjected to PCR with oligos SEQ ID NOS 44 and 34 to give a 998bp product. Both
products were isolated by agarose gel electrophoresis and Geneclean™ as described in
Reference Example 17 and used (0.2ng each in 50ul total volume) in a second hot start PCR
with 10 cycles for 1 min at 94°C and 4 min at 63°C followed by 2 min at 94°C. Flanking
oligos (SEQ ID NOS 42 and 34; lOOpM each) were added in 50ul buffer with Amplitaq
(2.5u). After heating to 94°C for 3 min, the mix was subjected to 25 cycles of 1.5 min at
94°C, 2 min at 55°C and 2 min at 72°C followed by 10 min at 72°C. The product was a band
at 1336bp, isolated as described previously, then cut with EcoRI and Hindlll and cloned into
pBluescript™ in DH5oc (clones were screened by PCR with oligos SEQ ID NOS 36 and 37) to
give pMF35 To make the complete Fd-HCPB fusion sequence, plasmids pAFl and pMF35
were cut (lOug of each) with Ncol and EcoRI for 2h in buffer (lOOul) containing 50mM
potassium acetate, 20mM Tris-acetate (pH 7.9), lOmM MgCl2, ImM DTT, EcoRI (40u) and
Ncol (20u). The vector fragment (3.4kb) from pAFl was isolated and treated with calf
intestinal alkaline phosphatase as described in Reference Example 17 and ligated to the purified
1.2kb fragment from pMF35. The resulting vector was cloned in DH5a (screened by PCR
with oligos SEQ ID NOS 36 and 37 for a l,922bp insert) and named pMF39. The
EcoRI-Hindlll fragment from pMF39 was cloned into pEE6 [this is a derivative of
pEE6.hCMV - Stephens and Cockett (1989) Nucleic Acids Research JJ, 7110 - in which a
Hindlll site upstream of the hCMV promoter has been converted to a Bglll site] in DH5a
(screened by PCR with oligos SEQ ID NOS 38 and 39 for a 2,200bp, approximately, insert) to
givepMF43.
To make the co-expression vector, pMF43 (lOug) was cut with Bglll (20u) and Sail (40U) in
buffer (100|il) containing lOmM Tris-HCl (pH 7.9), 150mM NaCl, lOmM MgCl2, ImM DTT
and BSA (lOOug/ml) and the 4348bp fragment isolated by agarose gel electrophoresis and
purified with Geneclean™ as described previously. Similarly, pAF6 (described in e) in
Reference Example 5) was cut with BamHI (40u) and Sail (40u) and the 7.8kb vector
fragment isolated and ligated to the BglH-Sall fragment from pMF43 and cloned into DH5a.
Colonies were screened by PCR with 2 sets of oligos (SEQ ID NOS 18 and 45, and SEQ ID
NOS 17 and 39). Clones giving PCR products of 360bp and 1.3kp respectively were
characterised by DNA sequencing. A clone with correct sequence was named pMF53 - light
chain/Fd-HCPB co-expression vector in DH5a.
g) Expression ofA5B7 F(ab %-HCPB in COS cells
The procedure described in Reference Example 17 for co-transfection of COS-7 cells with the
plasmid encoding the prepro sequence (pMF67) was repeated with pMF48 replaced by
pMF53. COS cell supernatants were examined for HCPB activity as described in Reference
Examples 11 and 17. COS cell supernatants which had been treated with LIPOFECTIN
reagent, but without plasmid DNA, hydrolysed 1.2% of the substrate, whereas the COS cell
supernatants transfected with the mix of plasmids expressing light chain/Fd-HCPB and prepro
sequence hydrolysed 34% of the Hipp-Arg substrate. COS cells transfected with only pMF53
plasmid hydrolysed Hipp-Arg at the level seen for COS cells which had been treated with
LIPOFECTIN reagent alone. By Western analysis (see h below) bands of approximatey
SOkDa and 160kDa were visible, corresponding to Fab'-HCPB and F(ab')2-(HCPB)2
respectively. In a CEA ELISA assay (see i and j below) cell supernatants (see above) were
used to detect the presence of CEA binding material according to the protocol given in j.
h) Western blot analysis
Western blot analysis was performed as described below.
Aliquots (20ul) of each supernatant sample were mixed with an equal volume of sample buffer
(62.5mM Tris, pH6.8, 1% SDS, 10% sucrose and 0.05% bromophenol blue) with and without
reductant. The samples were incubated at 65°C for 10 minutes before electrophoresis on a
8-18% acrylamide gradient gel (Excel™ gel system from Pharmacia Biotechnology Products)
in a Multiplier™ II apparatus (LKB Produkter AB) according to the manufacturer's
instructions. After electrophoresis, the separated proteins were transfered to a Hybond™
C-Super membrane (Amersham International) using a Novablot™ apparatus (LKB Produkter
AB) according to protocols provided by the manufacturer. After blotting, the membrane was
air dried.
The presence of antibody fragments was detected by the use of an anti-murine F(ab')2
antibody-peroxidase conjugate (ICN Biomedicals, product no. 67-430-1). The presence of
murine A5B7 antibody fragments was visualised using the ECL™ detection system (Amersham
International) according to the protocol provided.
i) ELISA analysis
Standard procedures for ELISA assay are available in "Laboratory Techniques in Biochemistry
and Molecular Biology" eds. Burdon, R.H. and van Kippenberg, P.H., volume 15, "Practice
and Theory of Enzyme Immunoassays", Tijssen, P., 1985, Elsevier Science Publishers B.V..
Another source of information is "Antibodies - A Laboratory Manual" Harlow, E. and Lane,
D.P. 1988, published by Cold Spring Harbor Laboratory.
j) ANTI-CEA ELISA
1. Prepare coating buffer (1 capsule of Carbonate-Bicarbonate buffer - Sigma C-3041 - in
100ml double distilled water).
2. Add 5ul of CEA stock solution (Img/ml, Dako) to 10ml of coating buffer for each 96 well
plate required.
3. Add lOOul of diluted CEA to each well of a Nunc "Maxisorp™" microtitre plate -
50ng/well/100ul.
4. Incubate plates at 4°C overnight (or room temp, for 2 hours).
-77-
5. Wash plates 4 times for 5 minutes each with Phosphate buffered saline + 0.01% Sodium
azide (PBSA) + 0.05% Tween 20.
6. Block plates (after banging dry) with 1% BSA (Sigma A-7888) in PBSA containing 0.05%
Tween 20 at 200ul per well. Incubate at room temp, for 2 hours.
7. Wash plates 4 times for 5 minutes each with PBSA containing 0.05% Tween 20.
8. Load samples (culture supernatants) and standards (doubling dilutions of proteolytic A5B7
F(ab')2) as appropriate. Dilute samples in growth medium (or PBS). Include PBSA +1%
BSA and diluent as blanks.
9. Incubate at ambient temperature for 3h.
10. Wash plates 6 times for 5 minutes each with PBSA + 0.5% Tween 20.
11. Prepare secondary antibody solution (anti-mouse IgG F(ab')2, from goat, peroxidase
conjugated - ICN 67-430-1 - at 20ul in 40ml PBSA + 1% BSA + 0.5% Tween 20) and add
lOOul per well.
12. Incubate at room temp, for Ih.
13. Wash plates 6 times for 5 minutes each with PBSA + 0.5% Tween 20.
14. Prepare developing solution by dissolving 1 capsule of Phosphate-Citrate Perborate
buffer (Sigma P-4922) in 100ml double distilled water. Add 30mg o-Phenylenediamine
Dihydrochloride (OPD, Sigma P-8287) per 100ml buffer. Add 150ul per well.
15. Incubate at room temp, in darkness for 15 minutes.
16. Stop reaction by addition of 50ul per well of 2M Sulphuric acid.
17. Read OD 490nm in plate reader.
Example 1
Preparation of bovine Lys66Glu pancreatic ribonuclease
(a) Construction of a RNase gene sequence containing the substitution in the codon 66
(Lys ->Glu) via recombinant circle polymerase chain reaction (RCPCR).
A plasmid containing the pre-sequence coding for bovine pancreatic RNase (pQR162:
NCIMB No 40678 and described in Tarragona-Fiol et al., Gene (1992) 118. 239-245) was
used in a PCR incubation as template. Primers for PCR incubations were synthesised by the
phosphite-triester method using cyanoethyl phosphoramidites on a Cyclone™ DNA synthesiser
(Milligen/Millipore). The primers were designed such that when they are used in PCR
incubations, the products generated are double-stranded, linear DNA molecules which upon
combination, denaturation and re-annealing form double-stranded DNA with discrete,
cohesive, single stranded ends in addition to the original blunt ended products. These ends will
anneal to form recombinant circles of DNA. These molecules are then ready for
transformation into competent E. coli cells.
Two PCR incubations, one with oligonucleotides SEQ ID NO: 1 and SEQ ID NO: 2 (see
primers A & B in Figure 8) and the other with oligonucleotides SEQ ID NO: 3 and SEQ ID
NO: 4 (see primers C & D in Figure 8), were allowed to undergo 25-30 cycles of 1.5 min at
92°C, 1.5 min at 55°C and 6 min at 75°C, with a final 10 min at 75°C in a Techne PHC-1
thermal cycler. The reaction contained pQR162 as template (10 ng), 50 pmol/primer, 5 ul of
10 x Buffer 1 [200 mM Tris-HCl (pH 8.2), 100 mM KC1, 60 mM (NH4)2SO4, 20 mM MgCl2,
1% Triton X-100 and lOOug/ml nuclease-free BSA] and 2.5 U of pfu polymerase (a
thermostable polymerase from Pyrococcus furiosus, Stratagene) in a total volume of 50 ul,
overlaid with the same volume of paraffin oil to prevent evaporation.
PCR products were analysed on a 1% agarose gel. The DNA fragment generated from each
PCR incubation (approx. 3.1 kb) was removed from the gel and the DNA separated from the
agarose by centrifugation (Spin-X™, Costar). The two extracted DNA fragments were
precipitated with ethanol and resuspended in 20|il of water. Aliquots from each (lOul) were
combined in a total volume of 50ul containing 10 mM Tris/HCl pH 8.8, 10 mM NaCl and
mM Na2EDTA. The combined DNA fragments were denatured for 5 min at 92°C and
re-annealed for 2 hours at 55-57°C. The recombinant circles thus formed were used to
transform an aliquot of competent cells.
Mini-preps for the isolation of plasmids were carried out [Maniatis et al. (1982) Molecular
Cloning. A Laboratory Manual. Cold Spring Harbour, Laboratory, Cold Spring harbour. New
York] and used as templates for double stranded DNA sequencing using the dideoxy chain
termination method [Sanger et al.,(1977) Proc. Natl. Acad. Sci. USA 14, 5463-5467]. A
plasmid containing the altered coding sequence without any mis-incorporation was designated
pQR176. This was digested in a total volume of 20ul containing 20 U of EcoRI and reaction
buffer. The DNA fragment having the altered coding sequence was obtained from an agarose
gel as described above and ligated to previously digested and dephosphorylated pKK223.3
[Pharmacia Biotech; this vector contains the tac promoter, regulated by the lac represser and
induced by the addition of isopropyl-p-D-thiogalactoside (IPTG)); Brosius and Holy, Proc.
Natl. Acad. Sci. USA (1984) 81, 6929] in a total volume of 20ul containing 20 U of T4 DNA
ligase and reaction buffer. The ligated products were used to transform an aliquot of E. coli
competent cells. Restriction enzyme analysis of plasmids obtained from the different
recombinant colonies were carried out to ascertain size and orientation of the inserts with
respect to the tac promoter. The correct construct was named pQR177 (Figure 1).
(b) Production and purification ofLys66Glu bovine pancreatic RNase
The strategy for the production and purification of the engineered sequence follows protocols
developed for the expression of bovine pancreatic ribonuclease A in E. coli (Tarragona-Fiol et
al., Gene 1992). This system utilises the natural signal sequence of bovine pancreatic
ribonuclease to direct the production of ribonuclease or its engineered mutants to the periplasm
of E. coli. The oxidative environment of the periplasm facilitates the correct folding of the
protein resulting in the expression of fully active recombinant RNase. The high net positive
charge of the recombinant or engineered mutants facilitates the rapid purification from
endogenous periplasmic proteins. Expression and subsequent purification to homogeneity of
mutant proteins takes place in 48 hours from inoculation of the medium.
The plasmid pQR177 contains two Ribosome Binding Sites (RBS), one provided by the tac
promoter of the vector and the other, for translation of the second cistron, is contained within
the coding sequence of the first cistron. The mRNA produced upon IPTG induction of
Escherichia coli cells harbouring pQR177 is bicistronic and starts from the tac promoter. The
first cistron encodes a 6-aa peptide (Met-Phe-Leu-Glu-Asp-Asp). The stop codon of the first
cistron and the start codon of the second cistron overlap such that ribosomes will continue
translation of the mRNA and produce pre-RNase. The synthesised precursor form of RNase is
translocated to the periplasm and N-terminal sequencing has shown that the signal sequence is
correctly cleaved. The oxidative environment of the periplasm allows the correct folding of
RNase to form the native enzyme as evidenced by the recovery of fully active enzyme.
Escherichia coli [pQR177] cells were grown in 5 litres of media containing lOOug Ap/ml for
8 hrs at 28°C. When cells were in the exponential phase of growth, IPTG was added to a final
concentration of 0.5 mM and growth of cells was continued overnight at 28°C with shaking.
The release of the periplasmic proteins was carried out using a modified spheroplast/osmotic
shock procedure. Cells from an overnight culture (5 litres) were pelleted by centrifugation at
8300 xg (average) for 10 min at 10°C. The cell pellet was resuspended in 60 ml of 200 mM
Tris-HCl pH 7.5 / 20% (w/v) sucrose (RNase free) / 1 mM Na2EDTA. The suspension was
left for 30 min at room temperature. An osmotic shock was obtained by adding an equal
volume of sterile water and mixing thoroughly. The mixture was left for a further 30 min at
room temperature. Spheroplasts were pelleted by centrifugation at 100000 xg (average) for 90
min at 10°C.
Cation exchange chromatography (S-Sepharose™ FF) was used to obtain all the positively
charged proteins from the periplasmic extract. Buffer A was 50 mM MES, pH 6.5 and Buffer
B, 50 mM MES, 1 M NaCl, pH 6.5. Recombinant RNase was purified from the pool of
positively charged proteins by cation exchange chromatography (Mono-S™, Pharmacia-LKB)
on a gradient of 17.5 mM NaCl/min. Assessment of the purity of recombinant RNase by
PAGE-SDS electrophoresis and silver staining clearly shows that this combination of
techniques results in purification of the protein to homogeneity (see Figure 2). RNase activity
of the recombinant enzyme was estimated on the hydrolysis of cytidylyl-3':5'-adenosine (CpA)
and cytidine-2':3'-cyclic monophosphate (C>p) showing an equal specific activity to that of
the commercial enzyme (see Table). Protein concentration was determined measuring the OD
at 278 (OD278nm= 0.71 is equivalent to 1 mg/ml of RNase). Kinetic measurements were
carried out by monitoring the increase (C>p hydrolysis) in absorbance with time at 286 nm
[Witzel and Barnard (1962) Biochem. Biophys. Res. Commun. 7, 295-299]. The initial
velocity and substrate concentration values were used to determine the parameters Km and
kcat and their standard errors by a computational method based on the analysis described by
Wilkinson (1961) Biochem. J. 80, 324-332. Differences between these parameters obtained
using different ribonucleases were assessed using the Student t-test. The rate of hydrolysis of
Cp was measured at room temperature in cuvettes of 0.1 cm path length (Hellma) in a total
volume of 250ul. Reactions contained varying concentrations of C>p in 0.1 M
(l,3-bis[tris(hydroxymethyl)-methylamino] propane) pH 7.0, 50 mM NaCl (1=0.1) and were
initiated by the addition of the enzyme (see Table). The data indicates that the kinetic
properties of the engineered Lys66Glu RNase enzyme are not significantly different to the
commercial bovine enzyme.
Example 2
Preparation Arg4AIa,Lys6Ala,Lys66Glu human pancreatic ribonuclease
Plasmid pATF3 (described in Reference Example 2, contains the Arg4Ala,Lys6Ala HP-RNase
gene) and was used as template (2 ng) in a PCR incubation containing the primer SEQ ID
NOS 15 and 16 (5 pmol/each), nucleotides (0.2 mM), PCR buffer and 2.5 units of pfu
polymerase. After 5 min of initial denaturation at 92°C, 30 cycles were carried out of
denaturation (92°C, 1 min), annealing (55°C, Imin) and extension (75°C, Imin). The PCR
fragment was gel extracted as described in Example 1 and digested with EcoRI (10-15 units) at
37°C for 1 hour. After heat inactivation of the enzyme, the EcoRI fragment was ligated into
EcoRI digested and dephosphorylated pUC18. The resulting plasmid was named pATFZl.
Plasmid pATFZl was used to confirm the DNA sequence of the mutated HP-RNase gene.
To generate the Arg4Ala,Lys6Ala,Lys66Glu HP-RNase, pATFZl was used as template in
RCPCR incubations as described in Example 1 but with oligonucleotide primers SEQ ID NO:
30 to 33 replacing SEQ ID NO 1 to 4 respectively). The resulting plasmid was called
pATFZ3. The gene for Arg4Ala,Lys6Ala,Lys66Glu HP-RNase was excised from pATFZ3 by
digestion with EcoRI and Ncol (10-15 units of each) and ligated to previously digested (EcoRI
and Ncol) and dephosphorylated pICI266 (NCIMB 40589) for expression studies. Ligations,
expression and purification, were carried out as the example described in Example 1, except
that a double digestion with Ncol and EcoRI was used to excised the fragment from pATFZ3,
as described above, and was ligated to previously dephosphorylated and digested (with EcoRI
and Ncol) pICI266 and, the induction was carried out with 1% arabinose (instead of IPTG).
The resulting construct was called pATFZ44 (see Figure 5). Expression and purification of
the mutant enzyme was as described in Example 1, but with induction with 1% arabinose
instead of IPTG.
Example 3
Preparation of
Q-[(2R,3S,4R,5R)-2-(2-aminoacetamidomethyl)-5-(2,4-dioxo-l,2,3,4-tetrahydropyrimidin-
l-yl)-4-hydroxy-2,3,4,5-tetrahydrofuran-3-yl]O-[4-(bis[2-chloroethyl]
amino)phenoxy] hydrogen phosphate (which is shown as the end product in Figure
7).
Compound 4 (Figure 7; 31mg, 0.034mM) was dissolved in 0.01M HC1 in
N,N-dimethylformamide (DMF) and 30% palladium on carbon catalyst (60mg) added as a
suspension in dimethylformarnide. The mixture was stirred under an atmosphere of hydrogen
for 2hrs 45mins. After filtration through Celite™ the filtrate was evaporated to dryness at
30°C. The crude product was suspended in dry dichloromethane and the mixture centrifuged.
The supernatant dichloromethane layer was discarded. The process was repeated and finally
the solid residue dried to give the desired product 9.4mg (compound 5, Figure 7).
NMR data DMSO d6, d4 Acetic (8) 3,3 (1H, m); 3.5 (3H, m); 3.62 (8H,s); 4.05 (1H, m); 4.25
(1H, m); 4.53 (1H, m); 5.62 (1H, d); 5.72 (IH.d); 6.63 (2H,d); 7.05 (2H,d); 7.63 (lH,d).
Compound 4 was made by the following procedure.
T -O-Benzyl-5' -bromo-5' -deoxyuridine (compound 1, Figure 7)
To a mixture of 2'-O-benzyluridine [Wagner et al. (1974), J. Org. Chem. 39, 24-30] (334mg
ImM) carbon tetrabromide (SOOmg) and DMF (4ml) at 20°C under Argon was added over 5
mins a solution of triphenylphosphine (340mg) in DMF (2ml). The mixture was stirred at
20°C for 2hrs, poured into water (60ml) and extracted twice with ethyl acetate. The combined
organic extracts were washed with water, dried, and evaporated to an oil. This oil was
chromatographed on 20g of Merck silica gel (Art. 9385). Elution with 5% methanol in
toluene gave 2'-O-benzyl-5'-bromo-5'-deoxyuridine (160mg, 40%).
NMR (DMSO d6) 8 11.4 (slH); 7.6 (dlH); 7.3(m5H); 5.95(dlH); 5.6(ddlH); 4.6(q2H);
4.0-4.2(m3H); 3.6-3.8(m2H)
5' -Azido-2' -O-benzyl-5' -deoxyuridine (compound 2, Figure 7)
2'-O-Benzyl-5'-bromo-5'-deoxyuridine (4.3g) was dissolved in DMF (86ml) and sodium azide
(7g) added. The mixture was stirred and heated at 60°C for 45 mins. After cooling and
decanting from unreacted sodium azide the DMF was evaporated to dryness. The residue was
dissolved in ethyl acetate and washed twice with water, dried and evaporated to dryness. The
residue was chromatographed on Merck silica gel (Art. 9385). Elution with 10% methanol in
toluene gave 1.5g of pure 5'-azido-2'-O-benzyl-5'-deoxyuridine.
NMR (DMSO d6) 8 11.4(slH); 7.6(dlH); 7.3(m5H); 5.9(dlH); 5.6(dlH); 5.4(dlH);
4.65(q2H); 3.9-4.2(m3H); 3.6(d2H).
2' -O-Benzyl-5'-carbobenzoxyglycylamino-5'-deoxyuridine (compound 3, Figure 7)
To a mixture of 5'-azido-2'-O-benzyl-5'deoxyuridine (1.5g), tetrahydrofuran (25ml) and
benzyloxycarbonyl glycine N-hydroxysuccinyl ester (1.3g) was added 10% platinum on carbon
(50% moist with water) (1.5g). The mixture was stirred under an atmosphere of hydrogen for
4 hours. After filtration through Celite the filtrate was evaporated to dryness. The residue was
dissolved in ethyl acetate and washed with 5% citric acid soln. (x2) water, sodium bicarbonate
soln. (x2) dried and evaporated to dryness. The residue was triturated with 1:1 ether/ethyl
acetate to give a solid (960mg) (42%).
NMR (DMSO d6) 8 11.3 (slH); S.O(tlH); 7.6(dlH); 7.4(mlOH); 5.9(dlH); 5.6(dlH);
5.4(dlH); 5.0(s2H); 4.6(q.2H); 4.0(m2H); 3.9(mlH); 3.6(d2H); 3.5(m2H)
Preparation of compound 4 (Figure 7)
a) Benzyloxydichlorophosphine [Scott et al. (1990), J. Org. Chem. 55,4904-4911] (135mg,
0.64mM) was dissolved in dry dichloromethane (4.0mls). the solution was cooled to -20°C
and a mixture of diisopropylamine (0.091 ml, 0.64mM) and diisopropylethylamine (0.11ml,
0.64mM) dissolved in dry dichloromethane (2.0ml) was added. The solution was stirred at
-20°C for 45min and then allowed to warm to room temperature over 30 min and then
stirred at room temp, for a further 30min. This solution was then added dropwise to a
solution of 2'-O-benzyl-5'-carbobenzoxyglycylamino-5'-deoxyuridine (280mg, 0.53mM)
and diisopropylethylamine (0.336ml, 2.14mM) in dichloromethane (3.0ml) cooled to 0°C.
The solution was stirred 10 mins at 0°C and at room temperature for 2 hours. The reaction
mixture was then diluted with dichloromethane washed with saturated sodium bicarbonate
(x2), dried and evaporated to an oil. The oil was azeotroped with toluene (2x) ready for the
next reaction.
b) The crude product from the previous stage was dissolved in dry dichloromethane (2.5ml)
and a solution of 4-N,N-bis-(2-chloroethyl)aminophenol (125mg, 0.534mM) in dry
dichloromethane (3.0ml) was added. A solution of 0.46M tetrazole in dry acetonitrile
(3.2ml) was then added and the solution stirred at room tenp for 2hrs. After this time 70%
t-butyl hydroperoxide solution in water (0.11ml, O.SOlmM) was added and the solution
stirred a further 1 hour at room temperature. The reaction mixture was diluted with
dichloromethane and washed with saturated sodium bicarbonate (Ix), dilute sodium
bisulphite (Ix), saturated sodium chloride (Ix), dried and evaporated to dryness. The crude
product was chromatographed on Merck silica gel (ART 9385) elution with 2% methanol.
in dichloromethane and then 3.5% methanol in dichloromethane gave the pure product
118mg.
NMR data. DMSOd6 (6) Mixture of diastereoisomers 3.37 1H (m); 3.42 2H (d); 3.67 8H (d);
4.12 1H (m); 4.33 1H (m); 4.56 2H (m); 5.0 2H (s); 5.14 3H (m); 5.59 1H (d); 5.91 1H (d);
6.64 2H (dd); 7.05 2H (t); 7.28 15H (m); 7.45 1H (t); 7.62 1H (dd); 8.13 1H (brs); 11.35 1H
(s).
Example 4
Synthesis and isolation of murine A5B7 F(ab')2-Lys66Glu bovine pancreatic
ribonuclease conjugate
The procedure described in Reference Example 4 is repeated but with bovine pancreatic
ribonuclease replaced by Lys66Glu bovine pancreatic ribonuclease (described in Example 1).
Example 5
Synthesis and isolation of murine A5B7 F(ab')2-Arg4Ala,Lys6Ala,Lys66Glu human
pancreatic ribonuclease conjugate
The procedure described in Reference Example 4 is repeated but with bovine pancreatic
ribonuclease replaced by Arg4Ala,Lys6Ala,Lys66Glu human pancreatic ribonuclease
(described in example 2).
Example 6
Synthesis and isolation of humanised A5B7 F(ab')2-Arg4Ala,Lys6Ala,Lys66Glu human
pancreatic ribonuclease conjugate
The procedure described in Example 5 is repeated but with murine A5B7 F(ab')2 replaced by
humanised A5B7 F(ab')2.
The humanised A5B7 F(a^'); is made by the following procedure. The procedure described in
Reference Example 5 is followed from step f) therein but the murine sequences for Fd and light
chain, as shown in SEQ ID NOs 25 and 26 respectively, are replaced by the humanised
sequences shown in SEQ ID NOs 28 and 29 respectively.
The humanised sequences shown in SEQ ID NOs 28 and 29 may be prepared by a variety of
methods including those described by Edwards (1987) Am. Biotech. Lab. 5, 38-44, Jayaraman
et al. (1991) Proc. Natl. Acad. Sci. USA 88,4084-4088, Foguet and Lubbert (1992)
Biotechniques 13, 674-675 and Pierce (1994) Biotechniques 16, 708.
Example 7
In vitro cytotoxicity of uracil based prodrug of Example 3, corresponding drug and,
prodrug plus mutant enzyme Arg4Ala,Lys6Ala,Lys66Glu Human Pancreatic-RNase
(HP-RNase).
The differential cytotoxicity to tumour cells of the RNase prodrug and corresponding drug has
been demonstrated by the following means. LoVo colorectal tumour cells were incubated with
prodrug or drug over a final concentration range of 5 X 10"4 to 5 X 10"8M in 96 well (2,500
cells/well) microtitre plates for Ihr at 37°C. The cells were then washed and incubated for a
further three days at 37°C. TCA was then added to the wells and, after washing to remove
dead cells, the amount of cellular protein adhering to the plates was assessed by addition of
SRB dye as described by P. Skehan et al, J. Natl. Cancer Inst. 82, 1107 (1990). Potency of the
compounds was assessed by the concentration required to inhibit cell growth by 50% (IC50).
Upon treatent of LoVo cells with the drug an IC50 of approximately luM was seen. In
contrast the prodrug was much less cytotoxic with an IC50 of approximately 30uM (Figure
6). Thus the RNase prodrug is approximately 30 fold less cytotoxic to tumour cells than the
drug generated by cleavage with the mutant RNase.
If either free Arg4Ala,Lys6Ala,Lys66Glu HP-RNase (lOug enzyme) or A5B7
F(ab')2-Arg4Ala,Lys6Ala,Lys66Glu HP-RNase conjugate (lOug enzyme) is added to the assay
wells containing the prodrug cytotoxicity can be seen which is comparable to that of the active
drug thus demonstrating conversion of the prodrug by the mutant enzyme to release the more
potent drug.
These studies demonstrate the activity of a conjugate of mutant human RNase to convert a
relatively inactive prodrug into a potent cytotoxic drug capable of killing tumour cells in an
ADEPT system.
Example 8
Anti-tumour activity of RNase prodrug and antibody-mutant RNase conjugate in
xenografted mice
The anti-tumour efficacy of the RNase prodrug and Arg4Ala,Lys6Ala,Lys66Glu HP-RNase
conjugate (or Lys66Glu bovine pancreatic RNase) can be demonstrated in the following
model. LoVo tumour cells (107) are injected subcutaneously into athymic nude mice. When the
tumours are 4-5mm in diameter the conjugate is administered iv at doses between 10-100
mg/kg. Following localisation of the conjugate to the tumours and allowing a suitable time
interval for residual conjugate to clear from the bloodstream and normal tissues (1-4 days) the
prodrug is administered either iv or ip to the mice in dose ranging between 100-1000 mg/kg.
The combination of conjugate and prodrug cause the tumours to grow significantly slower than
untreated control tumours or tumours treated with either the same dose of conjugate or
prodrug alone. These studies demonstrate that the Arg4Ala,Lys6Ala,Lys66Glu HP-RNase
conjugate in combination with the prodrug result in anti-tumour activity .
Example 9
Clinical dosing in patients
The most effective mode of administration and dosage regimen for the conjugates and
prodrugs of this invention in cancer therapy depend on a number of factors such as the severity
of disease, the patient's health and response to treatment and the judgement of the treating
physician. Accordingly the dosages of the conjugates and prodrugs should be titred to the
individual patients. Nevertheless, an effective dose of conjugate is likely to be in the range of
20 to about 200 mg/m2. The effective dose of the prodrug will depend on the particular drug
used and the toxicity of the parent drug. Since the prodrug is less cytotoxic than the parent
drug the MTD of the parent drug, if known, would provide a starting point. For phenol
mustard based prodrugs where clinical data is not available on the parent drug the therapeutic
dose range is less certain and would need to be defined by standard animal toxicology studies
and dose escalation studies in patients starting at a low dose. However the therapeutic dose
may be in range 500-2000 mg/m2.
Example 10
Enzyme kinetics of the uracil based prodrug of Example 3 (RNase prodrug) versus
native and mutant Lys66Glu bovine pancreatic RNase
The absorbancies of RNase prodrug and corresponding phenol mustard drug were scanned
from 200 nm to 350 nm using a spectrophotometer (Perkin Elmer Lambda 2) and the
wavelength was selected were the absorbance difference (due to cleavage of the phosphate
linkage) between prodrug and drug was maximal. This absorbance was 256 nm. The km and
Vmax were then determined by measuring the initial rate of conversion of prodrug to drug at
this wavelenghth using a range of prodrug concentrations (0.2-2 mM) and RNase enzyme
concentrations (5-80p,g/ml). Measurements were carried out at 37°C in 0.025M Tris-HCL plus
0.01% Brij-35 buffer pH7.5 in cuvettes of 0.1 cm path length (Hellma) in a total volume of
250uL. Kcat was calculated from the Vmax by dividing by the amount of RNase in the
reaction mixture. The enzymic activity of both enzymes against the standard substrate Cytidine
2'3' Cyclic monophosphate (C>p) was measured by determining the absorbance change at 284
nm and using a range of C>p concentrations (0.5-6 mM) and RNase enzyme concentrations
(5-35ug/ml). The results are shown in below.
Kcat/Km enzyme kinetics for RNase prodrug and C>p with bovine native and mutant
Lys66Glu RNase.
Substrate BP-RNase Lys66GluBP-RNase
(kcat/Km mM-V1)
RNase Prodrug
(Example 3) 0.37 18
Cp 3.0 3.0
The results show that both native and mutant bovine RNase turn over the standard substrate
Op at a similar rate. In contrast, the mutant RNase hydrolyses the prodrug much faster than
the native enzyme does. Thus, introducing the mutation of Lys66Glu in RNase has not
compromised the ability of the bovine enzyme to cleave the phosphate bond but has produced
an enzyme which can specifically cleave the RNase Prodrug (Example 3) to release active
drug.
Example 11
Enzyme kinetics of uracil based prodrug of Example 3 (RNase prodrug) versus native
and Arg4Ala,Lys6AIa,Lys66Glu human pancreatic RNase
The enzyme kinetic measurements with native HP-RNase and Arg4Ala,Lys6Ala,Lys66Glu
HP-RNase were carried out as described in example 10 except that the the buffer used was 0.1
M l,3-bis[tris(hydroxymethyl)-methylamino]-propane, pH 7.0, 50 mM NaCl. The results are
shown below.
Kcat/Km enzyme kinetics for RNase prodrug and C>p with native HP-RNase and
Arg4Ala,Lys6AIa,Lys66GIu HP-RNase.
Substrate HP-RNase Arg4AIa,Lys6Ala,Lys66Glu
HP-RNase
RNase prodrug
(Example 3) 0.2 3.6
Cp 1.2 1.2
Units = kcat/Km mM-V1
The results show that both the native and mutant human enzymes turn over the standard
substrate Cp at a similar rate. In contrast, the mutant human RNase hydrolyses the RNase
prodrug much faster than the native enzyme. Thus, introducing the mutation Lys66Glu into
human pancreatic RNase has also not compromised the ability of the human enzyme to cleave
the phosphate bond but has produced an enzyme which can specificaly cleave the RNase
prodrug to release active drug.
Example 12
Synthesis of cytosine based prodrug (see Scheme in Figure 17)
The procedure described in Example 3 is followed but with compound 6 (Figure 17) replacing
compound 4 (Figure 7). Compound 6 (Figure 17) is prepared as described for compound 4
(Figure 7) but with N4"benzyloxycarbonyl-2'-O-benzylcytidine replacing 2'-O-benzyluridine.
N"Benzyloxycarbonyl-2'-O-benzylcytidine (compound 2, Figure 17) is prepared from
2'-O-benzylcytidine [Christensen and Broom (1972), J. Org. Chem. 37, 3398-3401] by the
procedure used to prepare compound 6 in Reference Example 7.
Example 13
Enzyme activity of bovine Lys66Glu pancreatic RNase on Uridine and Cytidine based
prodrug analogues of Reference Examples 6 and 7 respectively
The experiment was performed in a manner analagous to that described in Example 10 but the
assays were performed at 25°C. The results are shown below.
Kcat/Km enzyme kinetics for RNase prodrug analogues and C>p with bovine native
and mutant Lys66Glu RNase.
Substrate BP-RNase Lys66GIuBP-RNase
(kcat/Km mM-is-1)
RNase Prodrug 1(0.2) 25(6)
analogue
(RefEx6)
RNase Prodrug
analogue
(RefEx) 5.5(0.3) 109(11)
Cp 3.0 3.0
The results show that both native and mutant bovine RNase turn over the standard substrate
C p at a similar rate. In contrast, the mutant RNase hydrolyses the prodrug analogues much
faster than the native enzyme does. Thus, introducing the mutation of Lys66Glu in RNase has
not compromised the ability of the bovine enzyme to cleave the phosphate bond but has
produced an enzyme which can specifically cleave the RNase prodrug analogues (Reference
Examples 6 & 7) to indicate release of active drugs with appropriate prodrugs.
Example 14
Typical pharmaceutical compositions containing a prodrug compound of the invention
A: Dry Filled Capsules Containing SOmg of Active Ingredient Per Capsule
Ingredient Amount per
capsule (mg)
Compound 50
Lactose 149
Magnesium stearate _1
Capsule (size No 1) 200
The compound can be reduced to a No. 60 powder and the lactose and magnesium stearate
can then be passed through a No. 60 blotting cloth onto the powder. The combined
ingredients can then be mixed for about 10 minutes and filled into a No. 1 dry gelatin capsule.
B: Tablet
A typical tablet would contain compound (25mg), pregelatinized starch USP
(82mg), microcrystaline cellulose (82mg) and magnesium stearate (Img).
C: Suppository
Typical suppository formulations for rectal administration can contain compound
(0.08-l.Omg), disodium calcium edetate (0.25-0.5mg). and polyethylene glycol (775-1600mg).
Other suppository formulations can be made by substituting, for example butylated
hydroxytoluene (0.04-0.08mg) for the disodium calcium edetate and a hydrogenated vegetable
oil (675-1400mg) such as Suppocire L, Wecobee FS, Wecobee M, Witepsols, and the like, for
the polyethylene glycol.
D: Injection
A typical injectible formulation would contain compound (lOmg) benzylalcohol
(0.01ml) and water for injection (1.0ml).
Example 15
Cloning and expression of D253K HCPB-(His)6 c-Myc from E. coli
The method of cloning and expressing the D253K-HCPB in E.coli was very similar to the
method described in Reference Example 15. Again pICI266 was used as the cloning vector,
and the starting material for PCR of the pro-HCPB gene was plasmid pICI1698 (as described
in Reference Example 14). However, in this case site directed mutagenesis was used during
the PCR amplification of the gene to change the codon at amino acid position 253 in the
mature gene from Aspartate to Lysine (GAC to AAA), the D253K change. Two PCR
mixtures were prepared, in a manner similar to that described in Reference Example 15. In the
first reaction primers were FSPTS1 (SEQ ID NO: 58) and 1398 (SEQ ID NO: 72). In the
second reaction primers were 6HIS9E10R1BS1 (SEQ ID NO: 59) and 1397 (SEQ ID NO:
73). In both reactions the starting DNA was pICI1698. Primers 1398 and 1397 (SEQ ID
NOs: 72 and 73) are designed to anneal around amino acid codon 253, introduce the GAC to
AAA change in the DNA sequence, and produce complementary sequence at the ends of the
two PCR products. The other two primers, FSPTS1 and 6HIS9E10R1BS1 (SEQ ID NOs: 58
and 59) are described in Reference Example 15. Aliquots of the two PCR reactions were
analysed for DNA of the correct size (about 750 and 250 base pairs) and estimation of
concentration by Agarose gel electrophoresis, and found to contain predominantly bands of the
correct size. Another PCR was then set up using approximately 4ng of each of the first two
PCR products, in the presence of dNTPs to a final concentration of 200uM, Taq polymerase
reaction buffer, 2U of Taq polymerase in a final volume of 80ul. The mixture was heated at
94°C for 10 minutes prior to the addition of the Taq enzyme, and PCR incubation was carried
out using 10 cycles of 94°C for 1 minute and 63°C for 4 minutes. On completion of these
cycles the reaction mix was made up to 120ul by the addition of 120pmols of each end primer,
FSPTS1 and 6HIS9E10R1BS1 (SEQ ID NOs: 58 and 59), additional dNTPs (approximately
an extra lOOuM), Taq polymerase reaction buffer, and 4U of Taq polymerase. The mixture
was heated at 94°C for 10 minutes prior to addition of Taq enzyme, and the PCR incubation
was carried out using 30 cycles of 94°C for 1.5 minutes, 50°C for 2 minutes, and 72°C for 2
minutes, followed by a single incubation of 72°C for 9.9 minutes at the end of the reaction.
An aliquot of the PCR product was analysed for DNA of the correct size (about 1000 base
pairs) by agarose gel electrophoresis and found to contain predominantly a band of the correct
size. The remainder of the product from the reaction mix was purified in a similar manner to
Reference Example 15. The isolated DNA was restriction digested with enzymes Fspl and
EcoRI, and a band of the correct size (about 1000 base pairs) purified in a similar manner to
Reference Example 15.
pICI266 double stranded DNA, prepared in a similar manner to Reference Example 15. was
restriction digested with Kpnl enzyme, and blunt-end treated with T4 DNA polymerase being
very careful to ensure complete digestion. The purified DNA was then digested with
restriction enzyme EcoRI. DNA of the correct size (about 5600 base pairs) was purified in a
similar manner to Reference Example 15.
Aliquots of both restricted and purified DNA samples were checked for purity and
concentration estimation using agarose gel electrophoresis compared with known standards.
From these estimates ligation mixes were prepared to clone the HCPB gene into the pICI266
vector in a similar manner to Reference Example 15.
Following the ligation reaction the DNA mixture was used to transform E.coli strain DHSoc,
colonies were picked and tested by hybridisation, in a similar manner to Reference Example 15.
Six positive hybridisation isolates were checked by PCR for inserts of the correct size, using
primers FSP1TS1 and 6HIS9E10R1BS1 (SEQ ID NOs: 58 and 59), and for priming with an
internal primer FSPTS1 (SEQ ID NO: 58) and 679 (SEQ ID NO: 51) in a similar manner to
Reference Example 15. The PCR products were analysed for DNA of the correct size (about
1000 base pairs from primers FSPTS1 to 6HIS9E10R1BS1, and about 430 base pairs from
primers FSPTS1 to 679) by agarose gel electrophoresis. All clones gave PCR DNA products
of the correct size.
All six of the clones were then taken for plasmid DNA preparation, and two were sequenced
over the region of PCR product in a similar manner to Reference Example 15. The clones
were sequenced using eight separate oligonucleotfde primers known as 1281, 677, 1504, 679,
1802, 1590, 1280 and 1731 (SEQ ID NOs: 55, 52, 60, 51, 63, 61, 53 and 62). From the
sequencing results a clone containing a plasmid with the required D253K-HCPB gene
sequence was selected, and is known as pICI1713.
The confirmed sequence of the cloned D253K-HCPB gene in pICI1713, showing amino acid
translation, from the start of the PelB sequence to the EcoRI restriction site is shown as SEQ
ID NO: 74 with DNA numbering starting from 1 in the first codon of PelB, and peptide
numbering starting from 1 ^ the mature HCPB.
To obtain controlled expression of the D253K-HCPB, the pICI1713 plasmid DNA was
transformed into calcium chloride transformation competent E.coli expression strains in a
similar manner to Reference Example 15. All pICI1713 transformed expression strains were
treated in a similar manner to Reference Example 15 to test for expression of the cloned
D253K-HCPB gene. In this case the 9E10 monoclonal antibody specific for the C-myc
peptide tag was used in the Western analysis, as the D253K-HCPB has the C-terminal
(His)6_c-myc tag in a similar manner to Reference Example 15.
Expression of the cloned tagged D253K-HCPB in pICI266 (pICI1713) was demonstrated
from E.coli by the Coomassie stained gels showing a strong protein band at about 35,000
Daltons when compared to vector (pICI266) alone clones, and clones producing the tagged
HCPB (Reference Example 15). A band of the same size gave a strong signal by Western
analysis detection of the c-myc tag.
Example 16
Cloning and expression of D253R HCPB-(His)g-c-Myc from E. coli
The method of cloning and expressing the D253R-HCPB in E.coli was very similar to the
method described in Reference Example 16. Again pICI266 was used as the cloning vector,
and the starting material for PCR of the pro-HCPB gene was plasmid pICI1712 (as described
in Reference Example 15. However, in this case site directed mutagenesis was used during the
PCR amplification of the gene to change the codon at amino acid position 253 in the mature
gene from Aspartate to Arginine (GAG to CGC), the D253R change. Two PCR mixtures
were prepared, in a manner similar to that described in Reference Examples 15 and 16. In the
first reaction primers were 2264 (SEQ ID NO: 65) and 2058 (SEQ ID NO: 75). In the second
reaction primers were 6HIS9E10R1BS1 (SEQ ID NO: 59) and 2054 (SEQ ID NO: 76). In
both reactions the starting DNA was pICI1712.
Primers 2058 and 2054 (SEQ ID NOs: 75 and 76) are designed to anneal around amino acid
codon 253, introduce the GAC to CGC change in the DNA sequence, and produce
complementary sequence at the ends of the two PCR products. The other two primers, 2264
and 6HIS9E10R1BS1 (SEQ ID NOs: 65 and 59) are described in Reference Examples 15 and
16. Aliquots of the two PCR reactions were analysed for DNA of the correct size (about 750
and 250 base pairs) and estimation of concentration by Agarose gel electrophoresis, and found
to contain predominantly bands of the correct size. Another PCR was then set up using
approximately 4ng of each of the first two PCR products, in the presence of dNTPs to a final
concentration of 200uM, Taq polymerase reaction buffer, 2U of Taq polymerase in a final
volume of 80ul. The mixture was heated at 94°C for 10 minutes prior to the addition of the
Taq enzyme, and PCR incubation was carried out using 10 cycles of 94°C for 1 minute and
63°C for 4 minutes. On completion of these cycles the reaction mix was made up to 120ul by
the addition of 120pmols of each end primer, 2264 and 6HIS9E10R1BS1 (SEQ ID NOs:
and 59), additional dNTPs (approximately an extra lOOuM), Taq polymerase reaction buffer,
and 4U of Taq polymerase. The mixture was heated at 94°C for 10 minutes prior to addition
of Taq enzyme, and the PCR incubation was carried out using 30 cycles of 94°C for l.Smin,
50°C for 2min, and 72°C for 2min, followed by a single incubation of 72°C for 9.9min at the
end of the reaction.
An aliquot of the PCR product was analysed for DNA of the correct size (about 1000 base
pairs) by agarose gel electrophoresis and found to contain predominantly a band of the correct
size. The remainder of the product from the reaction mix was purified in a similar manner to
Reference Example 15. The isolated DNA was restriction digested with enzymes Ncol and
EcoRI, and a band of the correct size (about 1000 base pairs) purified in a similar manner to
Reference Example 15.
pICI266 double stranded DNA, prepared in a similar manner to Reference Example 15. was
restriction digested with Ncol and EcoRI enzymes, being very careful to ensure complete
digestion. DNA of the correct size (about 5600 base pairs) was purified in a similar manner to
Reference Example 15.
Aliquots of both restricted and purified DNA samples were checked for purity and
concentration estimation using agarose gel electrophoresis compared with known standards.
From these estimates ligation mixes were prepared to clone the HCPB gene into the pICI266
vector in a similar manner to Reference Example 15.
Following the ligation reaction the DNA mixture was used to transform E.coli strain DH5cc,
colonies were picked and tested by hybridisation, in a similar manner to Reference Example 15.
Three of the clones were then taken for plasmid DNA preparation, and were sequenced over
the region of PCR product in a similar manner to Reference Example 15. The clones were
sequenced using nine separate oligonucleotide primers known as 1281, 677, 1504, 679, 1802,
1590, 1280, 1731 and 1592 (SEQ ID NOs: 55, 52, 60, 51, 63, 61, 53, 62 and 70). From the
sequencing results a clone containing a plasmid with the required D253R-HCPB gene
sequence was selected, and is known as pICI1746.
The confirmed sequence of the cloned D253R-HCPB gene cloned in pICI1746, showing amino
acid translation, from the start of the PelB sequence to the EcoRI restriction site is shown as
SEQ ID NO: 77 with DNA numbering starting from 1 in the first codon of PelB, and peptide
numbering starting from 1 in the mature HCPB.
To obtain controlled expression of the D253R-HCPB the pICI1746 plasmid DNA was
transformed into transformation competent E.coli expression strains in a similar manner to
Reference Example 15. All pICI1746 transformed expression strains were treated in a similar
manner to Reference Example 15 to test for expression of the cloned D253R-HCPB gene. In
this case the 9E10 monoclonal antibody specific for the C-myc peptide tag was used in the
Western analysis, as the D253R-HCPB has the C-terminal (His)6-C-myc tag in a similar
manner to Reference Example 15.
Expression of the cloned tagged D253R-HCPB in pICI266 (pICI1746) was demonstrated from
E.coli by the Coomassie stained gels showing a strong protein band at about 35,000 Daltons
when compared to vector (pICI266) alone clones, and clones producing the tagged HCPB
(Reference Example 15). A band of the same size gave a strong signal by Western analysis
detection of the c-myc tag.
Purification is achieved using methodology analogous to that set out below in Example 17.
Example 17
Purification of mutant D253K HCPB-(His)6 c-Myc proteins from E. coli
First a 20 litre fermentation process for carboxypeptidase B analogue D253K in a cell paste is
described. E. coli K12 strain MSD 1924 was transformed with plasmid pZen 1713 (pICI
1713; see Example 15 above) and the resultant strain MSD 2230 (MSD 1924 pZen 1713) was
stored in glycerol freezing mix at -80°C.
MSD 2230 was streaked onto agar plates containing L-tetracycline (10ugml-l) medium to
separate single colonies after overnight growth at 37°C. Six single colonies of MSD 2230
were removed from the surface of the L-tetracycline (lOugml-1) agar, re suspended in a 10ml
L-tetracycline (lOugml-1) broth and lOOul of this culture was immediately inoculated into each
of six 250ml Erlenmeyer flasks containing 75ml of L-tetracycline (lOugml- *) broth. After
growth for 15-16 hours at 37°C on a reciprocating shaker (300rpm) the contents of the flasks
were pooled and used to inoculate a single fermenter (U30D vessel, B. Braun, Melsungen,
Germany) containing 15 litres of the growth medium described in Figure 23.
The fermentation was performed at a temperature of 37°C and pH of 6.7 and pH of 6.7 which
was automatically controlled to the set point by the addition of 6M sodium hydroxide or 2M
sulphuric acid. The dissolved oxygen tension (dOT) set point was 50% air saturation and it
was maintained by the automatic adjustment of the fermenter stirrer speed between 200 and
1000 rpm. The air flow to the fermenter was maintained at 20 standard litres per minute which
corresponds to 1.3 vessel volumes per minute (vvm) by a Tylan mass flow controller.
4.5 Hours following inoculation, a solution of yeast extract (225gl-^) was fed into the
fermenter at a rate of 190-210mlh-l for 28.5 hours. 1.5 hours after the yeast extract feed was
started, the fermentation temperature set point was reduced to 25°C. When this temperature
was attained, approximately 1 hour later, expression of the carboxypeptidase analogue D253K
was induced with a single shot addition of 50% arabinose to give a final concentration in the
fermenter vessel of 0.5%. 1-2 hours following induction, a mixture of glycerol (714gl-1) and
ammonium sulphate (143gl-l) was fed into the fermenter at 45-55mlh-l until harvest. The
fermentation was continued under these conditions until ca. 75 hours post fermenter
inoculation when the culture was harvested by transferring aliquots of the fermenter contents
into 1 litre centrifuge bottles. The spent medium was separated from the bacterial cells by
centrifugation in a Sorvall RC-3B centrifuge (7,000x g, 4°C, 30min.). This process typically
yields a final dry weight of ca.20gl-1.
The cell paste was purified as follows. Recombinant E.coli cell paste containing the
recombinant enzyme, D253K HCPB, was taken from storage at -70°C and allowed to thaw.
The weight of cell paste was measured and found to be 309 grams.The paste was resuspended
with the addition of buffer A [200mM Tris (hydroxymethyl)aminomethane hydrochloride
(TRIS-HC1), 20% sucrose, pH 8.0] to give a resuspended volume of 320 ml. The cell
suspension was incubated at room temperature for 20 minutes with occasional gentle mixing
before an equal volume of distilled water, at room temperature, was added and thoroughly
mixed in. The cell suspension was again incubated at room temperature for 20 minutes with
occasional gentle mixing.
The resulting crude osmotic shockate was clarified by centrifugation at 98000 x g for 90
minutes at 4°C after which the supernatant was decanted off from the pelleted insoluble
fraction, giving a clarified volume of 240 ml. Deoxyribonuclease 1 (24mg) was dissolved in
distilled water (5ml) and added to the supernatant. The mixture was incubated at room
temperature, with continuous shaking for 30 minutes to reduce the vicosity of the supernatant
enough for it to be loaded on to a Carboxypeptidase Inhibitor CNBr activated Sepharose™
affinity column, prepared according to instructions with the CNBr activated Sepharose™ 4B
from Pharmacia and carboxypeptidase inhibitor from potato tuber (c-0279,Sigma). The
supernatant was diluted 1:1 with lOmM TRIS-HC1, 500mM sodium chloride, pH 8.0 (Buffer
B), adjusted to pH8.0 and loaded.over night, on to the Carboxypeptidase inhibitor affinity
column at 0.5 ml/min. The column was pre-equilibrated with buffer B at 4°C. After loading
the supernatant, the column was washed until the absorbance of the flow through was back to
baseline before the bound material was eluted from the column by elution buffer (lOOmM
sodium carbonate, SOOmM sodium chloride, pH 11.4) at 4°C,with 1ml fractions being
collected. The eluted fractions were frozen at -20°C after samples were taken to determine
those containing the recombinant carboxypeptidase. This was accomplished by Western blot
analysis using an anti- c-myc tag antibody (9E10), followed by an anti-mouse -horseradish
peroxidase conjugate (a-9044, sigma) that gave a colour reaction with exposure to
4-chloro-naphthol and hydrogen peroxide.
Fractions 11 to 44 were determined to contain the recombinant carboxypeptidase B. These
were pooled, the pH adjusted to pH7.5 and concentrated using a Millipore Centifugal
Ultrafree™ -20 (10,000 molecular weight cut off) before being snap-frozen and stored at
-20°C. The purification detailed here provided 4.7mg of D253K mutant carboxypeptidase at a
purity of 80%, in a volume of 0.95 ml.
Example 18
Synthesis of an aspartic acid phenol mustard prodrug (compound 5a, Scheme 1)
(2S),2-(3-{4-[bis-(2-chloroethyl)-amino)-phenoxycarbonyl}-propionyI-amino)-succinic
acid
Analagous methodology to that set out in Reference Example 12 was used.
(2S),2-(3- {4-[bis-(2-chloroethyl)-amino)-phenoxycarbonyl} -propionylamino)-
succinic acid dibenzyl ester (4a) was hydrogenated for 2h at 80 psi to give the desired end
product 5a (yield: 86%).
5a: 1HNMR (CD3OD): 2.65-2.75 (t, 2H); 2.8-2.9 (m, 4H); 3.7-3.75 (m, 4H); 3.8-3.85 (m,
4H); 4.75 (t, 1H); 6.7-6.8 (m, 2H); 7.0-7.1 (m, 2H).
MS (ESI): 471-473 (MNa)+
Anal. (Ci8H22N2O7Cl2 1.4 H2O)
Calc. %C: 45.56 H: 5.27 N: 5.90
Found %C: 45.79 H: 5.60 N: 5.91
Starting material compound 4a was prepared as follows.
(2S),2-amino-succinic acid dibenzyl ester (Compound 2a) was reacted with
compound 1 to give (2S),2-(3-carboxypropionylarnino)-succinic acid dibenzyl ester
(compound 3a) after recrystallisation with diethyl ether/hexane: (Yield: 80%).
3a: 1HNMR (CDC13): 2.42-2.6 (m, 2H); 2.6-2.75 (m, 2H); 2.85 (dd, 2H); 3.1 (dd, 1H); 4.9
(dd, 1H); 5.05 (dd, 2H); 5.15 (s, 2H); 6.7 (d, 1H); 7.25-7.5 (m, 10 H).
MS (ESI): 436 [MNa]+
Anal. (C22H23NO7 O.4H2O):
Calculated %C: 62.82 H: 5.70 N: 3.33
Found %C: 63.2 H: 5.75 N: 2.9
Compound 3a was reacted to give the desired starting material 4a (yield: 78 %)
stirring was maintained for 3h at room temperature and purification was achieved by flash
chromatography using diethyl ether/hexane (70/30 V/V as eluent).
4a: 1HNMR (CDC13): 2.55-2.65 (m, 2H); 2.8-2.9 (m, 2H); 2.9 (dd, 1H); 3.1 (dd, 1H); 3.6
(dd, 4H); 3.7 (dd, 4H); 4.9 (dd, 1H); 5.05 (dd, 2H); 5.15 (s, 2H); 6.58 (d, 1H); 6.65 (d, 2H);
6.95 (d, 2H); 7.25-7.4 (m, 10 H).
MS (ESI): 651-653 (MNa)+
Exanwle 19
Synthesis of a glutamic acid phenol mustard prodrug (5b; Scheme 1)
(2S),2-(3-{4-[bis-(2-chloroethyl)-amino)-phenoxycarbonyl}-propionyl-amino)-pentanedioic
acid
Analagous methodology to that set out in Reference Example 12 was used.
(2S),2-(3- {4-[bis-(2-chloroethyl)-amino)-phenoxycarbonyl} -propionylamino)-pentanedioic
acid dibenzyl ester (4b) was hydrogenated for 3 h at 60 psi to give the desired end product 5b
(yield: 93%).
5b: 1HNMR (CD3OD): 1.9-2.0 (m, 1H); 2.1-2.2 (m, 1H); 2.35-2.45 (m, 2H); 2.55-2.7 (m,
2H); 2.8-2.9 (m, 2H); 3.65-3.7 (m, 4H); 3.72-3.8 (m, 4H); 4.4-4.5 (m, 1H); 6.75 (d, 2H); 6.95
(d, 2H).
MS (ESI): 485-487 (MNa)+
Starting material compound 4b was prepared as follows.
(2S),2-amino-pentanedioic acid dibenzyl ester (2b) was reacted to give
(2S),2-(3-carboxypropionylamino)-pentanedioic acid dibenzyl ester (3b) (Yield: quantitative)
3b: 1HNMR (CDC13): 2.0-2.1 (m, 1H); 2.2-2.3 (m, 1H); 2.3-2.5 (m, 4H); 2.6-2.7 (m, 2H);
4.65 (dd, 1H); 5.05 (s, 2H); 5.15 (s, 2H); 6.5 (d, 1H); 7.3-7.4 (m, 10 H).
MS (ESI): 450 [MNa]+
3b was reacted to give the desired starting material 4b (yield: 82%).
4b: 1HNMR (CDC13): 1.95-2.05 (m, 1H); 2.2-2.3 (m, 1H); 2.3-2.5 (m, 2H); 2.6 (dt, 2H);
2.8-3.0 (m, 2H); 3.6 (dd, 4H); 3.7 (dd, 4H); 4.7 (dd, 1H); 5.1 (s, 2H); 5.2 (s, 2H); 6.3 (d, 1H);
6.6 (d, 2H); 6.95 (d, 2H); 7.3-7.4 (m, 10 H).
MS (ESI): 665-667 (MNa)+
Example 20
Assay of activity of mutant human CPB and native human CPB against Hipp-Asp and
Hipp-GIu prodrug analogues.
Purified mutants of human CPB (D253K and D253R; Examples 15-17) and native human
CPB, produced as described in Reference Example 20, were assayed for their ability to convert
either hippuryl-L-aspartic acid (Hipp-Asp - Reference Example 10), hippuryl-L-glutamic acid
(Hipp-Glu - Reference Example 9) or hippuryl-L-arginine (Sigma Chemical Company - cat no.
H6625) to hippuric acid using a HPLC based assay.
The reaction mixture (250 ul) contained 4 ug human CPB (native or mutant) and 0.5 mM
Hipp-Asp or Hipp-Glu in 0.025 M Tris-HCL, pH 7.5. Samples were incubated for 5 hr at
37°C. The reactions were terminated by the addition of 250 ul of 80% methanol, 20% distilled
water, 0.2% trifluoro acetic acid and the amount of hippuric acid generated was quantified by
HPLC.
HPLC analysis was carried out using a Hewlett Packard 1090 Series 11 (with diode array)
HPLC system. Samples (50 ul) were injected onto a Hichrom Hi-RPB column (25 cm) and
separated using a mobile phase of 40% methanol, 60% distilled water, 0.1% trifluoro acetic
acid at a flow rate of Iml/min. The amount of product (hippuric acid) produced was
determined from calibration curves generated with known amounts of hippuric acid
(Sigma-H6375). The results are shown in the Table and are expressed as the percentage
conversion of substrate into product in 5 hr at 37°C with 4 ug enzyme.
Conversion of Hipp-Asp and Hipp-Glu by mutant and native human CPB
Hipp-Asp Hipp-Glu Hipp-Arg
(% conversion to Hippuric acid)
Native CPB 0 0 100
D253K mutant CPB 78 91 2
D253R mutant CPB 72 52 3
The data show that introduction of either a lysine or arginine residue at position 253 in human
CPB instead of the aspartate residue present in the native enzyme changes the substrate
specificity of the enzyme so that it is capable of conversion of either Hipp-Asp or Hipp-Glu. In
contrast, the native enzyme is unable to convert either of these compounds into Hippuric acid
but does convert Hipp-Arg to hippuric acid. The best activity was seen with the D253K mutant
and the Hipp-Glu substrate.
Example 21
Determination of Km and kcat of D253K mutant HCPB with Hipp-Asp and Hipp-Glu.
Purified D253K HCPB, produced as described in Example 17, was assayed against Hipp-Asp
(Reference Example 10) and Hipp-Glu (Reference Example 9) to determine Km and kcat for
these substrates. Hipp-Glu and Hipp-Asp were diluted in range 0.25-8.0 mM and 0.25-5.0 mM
respectively in 0.025 M Tris-HCL buffer, pH 7.5. Where necessary substrate samples were
adjusted to pH 7.5 with 1M NaOH.
D253K HCPB (4ug/ml for Hipp-Asp and 0.5ug/ml for Hipp-Glu) was added to these
substrates (500ul reaction volume) to start the reaction. Samples were incubated for 5h at
37°C. Reactions were terminated by the addition of 500ul methanol/distilled water (80/20)
containing 0.2% TFA. The amount of hippuric acid produced was quantified by HPLC as
described in Example 20.
Km and Vmax values were calculated using the ENZFITTER software programme (Biosoft,
Perkin Elmer), kcat was calculated from Vmax by dividing by the enzyme concentration in the
reaction mixture (using a molecular weight for HCPB of 34 KDa). The results are shown in the
Table.
Km and kcat data for Hipp-Asp and Hipp-Glu with D253K mutant HCPB
Km(mM) kcat(s"1) kcat/Km
(rnM'V1)
Hipp-Asp 2.7 0.26 0.1
Hipp-Glu 5.3 3.8 0.7
The data confirm that replacing aspartate with a lysine residue at position 253 in human CPB
results in an enzyme which can convert both Hipp-Asp and Hipp-Glu into hippuric acid with
reasonable enzyme kinetics. The kcat/Km is approximately 7 fold greater with the Hipp-Glu
compared to the Hipp-Asp substrate.
Example 22
Assay of activity of mutant HCPB and native HCPB against glutamic acid prodrug
Purified D253K HCPB and native human CPB, produced as described in Example 17 and
Reference Example 20 respectively, were assayed for their ability to enzymatically cleave
glutamic acid from a glutamic acid prodrug (Example 18). Cleavage liberates an intermediate
(Reference Example 13) which self collapses non-enzymatically to release the active phenol
mustard drug. Conversion of the glutamic acid prodrug to intermediate was measured using a
HPLC based assay.
Prodrug was diluted in the range 0.25-5.0 mM in 0.025 M Tris-HCL buffer, pH 7.5. Where
necessary prodrug samples were adjusted to pH 7.5 with 1M NaOH. D253K mutant HCPB or
native HCPB, both at a final concentration of 0.25 mg/ml, were added to the these substrates
(250ul reaction volume prewarmed to 37°C for 2 min) to start the reaction. Samples were
incubated for 4 minutes at 37°C. The reaction was terminated by the addition of 250ul 98.8%
MeCN, 0.2% TFA and the samples placed on ice. The amount of intermediate produced was
then quantified by HPLC.
HPLC separation was carried out as described in Example 20 except that a mobile phase of
MeCN/distilled water (55/45 V/V) containing 0.1% TFA was used to achieve separation of
the prodrug (retention time 4.9 minutes) and intermediate (retention time 8.4 minutes). The
amount of intermediate produced was quantified from calibration curves generated with known
amounts of the intermediate.
The amount of intermediate formed at 5.0 mM and 0.25 mM prodrug with native and mutant
(D253K) HCPB in replicate samples is shown in the Table.
Conversion of prodrug to intermediate by native and mutant (D253K) HCPB.
Prodrug concentration Intermediate concentration(mM)
(mM) Native HCPB Mutant HCPB
5.0 0,0 0.023,0.022
0.25 0,0 0.005,0.005
Km, Vmax and kcat values for the mutant human enzyme (D253K) and the prodrug were
calculated from the amount of intermediate produced over a range of substrate concentrations
(0.25-5.0 mM) using the ENZFTTTER™ software described in Example 21.
The results for the D253K mutant HCPB were:
Km=1.25mM
Vmax = 1.17 X lO^mMsec'1
kcat = 0.016sec"1
The data show that introduction of a lysine residue at position 253 in human CPB instead of
the aspartate residue present in the native enzyme changes the substrate specificity of the
enzyme so that it is capable of conversion of the glutamic acid prodrug into its self-collapsing
intermediate. In contrast, the native enzyme is unable to convert the prodrug to its
intermediate. Since the prodrug is relatively non-cytotoxic (Example 23) and the intermediate
is non-enzymatically broken down to release free phenol mustard drug which kill tumour cells
(Example 23) these results demonstrate that mutation of active site residues of CPB can yield a
mutant human enzyme capable of converting a relatively non-cytotoxic prodrug into a potent
cytotoxic drug capable of killing tumour cells.
Example 23
Cytotoxicity of glutamic acid prodrug and phenol mustard drug in LoVo human
colorectal tumour cells.
The differential cytotoxicity to tumour cells of the glutamic acid prodrug and corresponding
phenol mustard drug has been demonstrated by the following means.
LoVo colorectal tumour cells were incubated with prodrug or drug over a final concentration
range of 5 X 10'4 to 5 X 10'8M in 96-well (2,500 cells/well) microtitre plates for Ih at 37°C.
The cells were then washed and incubated for a further three days at 37°C. TCA was then
added to the wells and, after washing to remove dead cells, the amount of cellular protein
adhering to the plates was assessed by addition of SRB dye as described by P. Skehan et al, J.
Natl. Cancer Inst. 82, 1107 (1990). Potency of the compounds was assessed by the
concentration required to inhibit cell growth by 50% (IC50).
Upon treatment of LoVo cells with the phenol mustard drug an IC50 of approximately luM
was seen. In contrast the glutamic acid prodrug was much less cytotoxic with an IC50 of
approximately 50uM (Figure 22). Thus the mutant CPB glutamic acid prodrug is
approximately 50 fold less cytotoxic to tumour cells than the phenol mustard drug.
If lOOug of mutant HCPB (D253K) produced as described in Example 17 is added to the assay
wells containing the glutamic acid prodrug cytotoxicity can be seen which is comparable to
that of the active drug thus demonstrating conversion of the prodrug by the mutant enzyme to
release the more potent drug. Addition of lOOug of native human CPB to each well does not
significantly enhance the cytotoxicity of the glutamic acid prodrug. These studies demonstrate
the potential of the mutant human CPB enzyme (D253K) to selectively convert a relatively
inactive prodrug into a potent cytotoxic drug capable of killing tumour cells.
Example 24
Preparation of humanised A5B7 F(ab')2-D253K HCPB fusion protein
The procedure described in Reference Example 21 is repeated but with murine A5B7 light
chain and Fd sequences replace by sequences for humanised A5B7, and with the HCPB
sequence replaced by D253K sequence. The fusion protein is expressed from COS cells by
co-transfection with the HCPB prepro sequence as described in Reference Example 21.
Large-scale expression of the fusion protein is performed by transiently introducing the plasmid
vectors (750ug of each) into COS-7 cells (11) essentially as described in Reference Example
21. The product is purified either by passing the supernatant containing the fusion protein over
immobilised protein A and elution of the bound fusion protein with high pH buffer or by
passing the supernatant containing the fusion protein over immobolised carboxypeptidase
inhibitor, following the route used for the purification of the recombinant carboxypeptidase
enzyme, and elution with the same high pH as used with the enzyme in Example 12. Both
these routes may involve further purification of the fusion protein by either gel permeation
chromatography, ion exchange chromatography, hydrophobic interaction chromatography
singly, or a combination of them.
The procedure described in Reference Example 21 is repeated but the murine sequences for Fd
and light chain, as shown in SEQ ID NOS 25 and 26 respectively, are replaced by the
humanised sequences shown in SEQ ID NOs 28 and 29 respectively. The HCPB sequence in
Reference Example 21 is replaced by the D253K sequence [described in Example 15, but
without the (His)6'C-Myc tags]. The template for PCR in Reference Example 21 (pICI1698) is
replaced by pICI1713 (described in Example 15).
The humanised sequences shown in SEQ ID NOs 28 and 29 are prepared by a variety of
methods including those described by Edwards (1987) Am. Biotech. Lab. 5, 38-44, Jayaraman
et al. (1991) Proc. Natl. Acad. Sci. USA 88,4084-4088, Foguet and Lubbert (1992)
Biotechniques 13, 674-675 and Pierce (1994) Biotechniques 16, 708.
Example 25
Shake flask fermentation for preparation of D253K HCPB
E.coli strain MSD 213 was transformed with plasmid pICI 1713 (see Example 15) and the
resultant strain MSD 213 pZen 1713 stored as a glycerol stock at -80°C. An aliquot of MSD
213 pZen 1713 was streaked onto agar plates of L-tetracycline to separate single colonies after
overnight growth at 37°C. A single colony of MSD 213 pZen 1713 was removed and
inoculated into a 250ml Erlenmeyer flask containing 75ml of L-tetracycline broth. After
growth for 16h at 37°C on a reciprocating shaker the contents of the flask were used to
inoculate to OD550 = 0.1 each of nine 2L Erlenmeyer flasks containing 600ml of
L-tetracycline broth. The flasks were then incubated at 20°C on a reciprocal shaker until
growth, estimated by measuring the optical density of the culture, reached OD550 = 0.5. At
this point heterologous protein production was induced by adding L-arabinose to the cultures
to a final concentration of 0.01%w/v and the incubation continued at 20°C as described above
for a further 42h. The spent medium was separated from the bacterial cells by centrifugation in
a Sorvall RC-3B centrifuge (7000x g, 4°C, 30min) and the cell paste stored at -70°C.
Example 26
Use of ADEPT in autologous bone marrow transplantation
Autologous bone marrow transplantation involves removal of a portion of the patient's own
marrow before giving the patient intensive radiochemotherapy. The bone marrow is returned to
the patient on completion of the treatment. In some cancers, such as leukaemias and
lymphomas of B- and T- cell lineage and carcinomas of breast, lung and colon, malignant cells
infiltrate the marrow and should be eliminated before reinfusing the marrow to optimise
survival. Antibody-toxin conjugates have been used previously to eliminate these tumour cells
for autologous bone marrow (Blakey, D. C. et al, Prog. Allergy vol 45, 50, 1988).
ADEPT could be used for this purpose especially if a short-lived reactive mustard alkylating
agent is used as the drug component. Thus autologous bone marrow containing tumour cells
could be incubated with an appropriate antibody-enzyme conjugate. Following binding of the
conjugate selectively to the tumour cells residual conjugate would be washed away. Prodrug
would then be added and drug would be generated adjacent to antigen positive tumour cells
resulting in selective tumour cell killing. Normal bone marrow cells could be protected by
optimising the dilution of the bone marrow to ensure that sufficient distance existed between
the site of generation of drug on tumour cells and the bone marrow cells so that the drug
became inactivated due to chemical decomposition before it reached the bone marrow cells.
Addition of protein to act as a nucleophile for the reactive mustard drug could also be used to
minimise normal bone marrow damage.
Example 27
Use of mutated Glucuronidase for reverse polarity ADEPT
Human glucuronidase is another enzyme where the* re verse polarity' concept can be used to
produce a specific human enzyme capable of cleaving a prodrug to release an active drug.
Bosslet et al (Cancer Res. 54, 2151, 1994) have already described an adriamycin-glucuronide
prodrug for native human glucuronidase and have described the synthesis of a range of
alternative prodrugs releasing a range of drugs (Bosslet in patent application AU -50225/93 ).
Endogenous native glucuronidase present in blood and tissues will potentially turn over these
prodrugs to release active drug in the absence of antibody-glucuronidase conjugate and thus
reduce the specificity of the approach. Cheng and Touster (J.B.C. 247. 2650, 1972) have
reported that there is a positively charged amino acid in the active site of glucuronidase that
reacts with the negatively charged carboxyl group on the glucuronide ring.
The linker could be either a direct linkage between the glucuronide and the cytotoxic agent or
a self imolating linker which for example could be of the type described by Bosslet et al
(Cancer Res. 54, 2151, 1994 and Patent Au-A-50225/93). If the negatively charged carboxyl
group on the glucuronide ring is replaced with a positive charged group R where, for example,
or other suitable
linkers) then the positive charged prodrug should no longer be a substrate for native
glucuronidase. If the positive charge residue in the active site of glucuronidase is then
converted to a negative charged amino acid e.g. glutamate or aspartate this mutant
glucuronidase will now turn over the positively charged prodrug selectively in a manner
analogous to the RNase and CPB examples. Thus the reverse polarity concept can be
extended to human glucuronidase and positively charged glucuronide based prodrugs.

CLAIMS
1 A matched two component system designed for use in a host in which the
components comprise:
(i) a first component that is a targeting moiety capable of binding with a tumour
associated antigen, the targeting moiety being linked to a mutated enzyme capable of
converting a prodrug into an antineoplastic drug and;
(ii) a second component that is a prodrug convertible under the influence of the
enzyme to the antineoplastic drug;
wherein:
the mutated enzyme is a mutated form of a host enzyme in which the natural host enzyme
recognises its natural substrate by an ion pair interaction and this interaction is reversed
("reversed polarity") in the design of mutated enzyme and complementary prodrug;
the first component is substantially non-immunogenic in the host and;
the prodrug second component is not significantly convertible into antineoplastic drug in the
host by natural unmutated host enzyme.
2 A system according to claim 1 in which the first component comprises a mutated
enzyme based on an enzyme from the same species as the host for which the system is intended
for use.
3 A system according to any one of claims 1-2 in which the targeting moiety is an
antibody or a fragment thereof.
4 A system according to claim 3 in which the antibody fragment is an F(ab')2
fragment.
5 A system according to any one of claims 1-4 in which the mutated enzyme is
mutated ribonuclease.
6 A system according to any one of claims 1-5 in which the mutated enzyme is
human ribonuclease comprising a negatively charged amino acid at position 66.
7 A system according to claim 6 in which the negatively charged amino acid at
position 66 is Glu.
8 A system according to any one of claims 1-4 in which the mutated enzyme is
mutated glucuronidase.
9 A second component as defined in claim 1 which is a mustard-ribonucleotide of
Formula 1
wherein:
Q is O or NH ;
A is a group of formula -X-Y- ,with Y next to Q, wherein
Y is SO2, CO or a single bond with the proviso that
when Q is oxygen then Y is not SO2i
X is -(CH2)n- where n=l-4 optionally substituted by
CM alkyl on any carbon atom or
when Y is CO and n=l then X is optionally substituted on carbon with the side chain of
alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, serine, threonine,
cysteine, asparagine, glutamine, lysine, arginine or histidine;
Rl is uracil or cytosine;
R2 and R3 independently represent H or Ci^alkyl;
R5 and R6 independently represent Cl, mesyl or tosyl;
R7, R8, R9 and RIO independently represent H, CM alkyl, Q.4alkoxy, F or Cl
or a salt thereof.
10 A mustard ribonucleotide" according to claim 9 in which:
X is -(CH2)n- where n is 1-4;
Y is -C(O)-;
Rl is uracil or cytosine;
R2 and R3 are H;
R5 and R6 are Cl; and
R7, R8, R9 and RIO are H;
or a salt thereof.
11 A second component as defined in claim 1 which is the compound
Q-[(2R,3S,4R,5R)-2-(2-aminoacetamidomethyl)-5-(2,4-dioxo-l,2)3,4-tetrahydropyrimidin-
1 -yl)-4-hydroxy-2,3,4,5-tetrahydrofuran-3-yl] O-[4-(bis[2-chloroethyl]
amino)phenoxy] hydrogen phosphate or a salt thereof.
12 A pharmaceutical composition comprising a first component as defined in any one
of claims 1-8.
13. A pharmaceutical composition comprising a second component as defined in any
one of claims 1 or 9-11.
14 A pharmaceutical composition according to any of claims 12-13 which is sterile.
15. A first component as defined in any one of claims 1-8.
16. A mutated enzyme as defined in any one of claims 1,2,5,6,7 or 8.
17. A method of controlling the growth of neoplastic cells in a host in which the
method comprises administration to said host an effective amount of a first component as
defined in any one of claims 1-8, allowing the first component to clear substantially from
general circulation in the host, and administering an effective amount of a second component
as defined in any one of claims 1 or 9-11.
18 PlasmidpQR 162 deposited as deposit reference NCIMB 40678.
19 A polynucleotide sequence selected from a polynucleotide sequence encoding any
of the following:
a first component as defined in any one of claims 1-8; and
a mutated enzyme as defined in any one of claims 1,2,5,6,7 and 8.
20 A vector comprising a polynucleotide as defined in claim 19.
21 A cell comprising a polynucleotide as defined in claim 19.
22. A matched two component system substantially as herein
described with reference to the accompanying drawings.
23. A mustard ribonucleotide substantially as herein described
with reference to the accompanying drawings.
24. A pharmaceutical composition substantially as herein
described with reference to the accompanying drawings.
25. A mutated enzyme substantially as herein described with
reference to the accompanying drawings.
26. A polynucleotide sequence substantially as herein described
with reference to the accompanying drawings.