METHODS OF DETERMINING PHARMACOKINETICS
OF TARGETED THERAPIES
RELATED APPLICATIONS
Priority is claimed to U.S. Provisional Patent Application No. 60/695,419, filed
on July 1, 2005, which is incorporated by reference in its entirety herein.
FIELD OF THE INVENTION
The present invention generally relates to methods for determining
pharmacokinetic properties of targeted therapies {e.g., immunoconjugates) using
mass-sensing techniques.
BACKGROUND OF THE INVENTION
Synthetic and natural macromolecules have become established therapeutics
in cancer treatment. Antibodies have proven clinical efficacy when administered as
a naked or unconjugated antibody or as an antibody/drug conjugate. According to
the latter approach, a therapeutic agent is coupled to an antibody with binding
specificity for a defined target cell population. Therapeutic agents that have been
conjugated to monoclonal antibodies include cytotoxins, biological response
modifiers, enzymes (e.g., ribonucleases), apoptosis-inducing proteins and peptides,
and radioisotopes.
Antibody-mediated drug delivery to tumor cells augments drug efficacy by
minimizing its uptake in normal tissues. See e.g., Reff et al. (2002) Cancer Control
9:152-66; Sievers (2000) Cancer Chemother. Pharmacol. 46 Suppl:S18-22;
Goldenberg (2001) Crit. Rev. Oncol. Hematol. 39:195-201. MYLOTARG®
(gemtuzumab ozogamicin) is a commercially available targeted immunotherapy that
works according to this principle and which is approved for the treatment of acute
myeloid leukemia in elderly patients. See Sievers et al. (1999) Blood 93: 3678-3684.
In this case, the targeting molecule is an anti-CD33 monoclonal antibody that is
conjugated to calicheamicin. Additional examples include ibritumomab tiuxetan
(ZEVALIN®) and tositumomab (BEXXAR®), which are radiolabeled anti-CD20
antibodies. See Dillman, Clin. Exp. Med., 2006, 6(1):1-12.
Despite progress in developing new antibody-targeted therapies, the
physiological characteristics conferring a favorable therapeutic index in the clinic are
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not well understood. Simple biochemical assays (e.g., the affinity of antibody for its
antigen) do not necessarily predict efficacy. See Graff & Wittrup, Cancer Res., 2003,
63(6): 1288-1296. Biological parameters in vivo such as circulation half-life, tissue
distribution rates, and rate of conjugate degradation may be more helpful in
comparing the potential therapeutic efficacy of these molecules. However,
preclinical experiments designed to assess these parameters are difficult because
they typically require large numbers of experimental animals and radiolabeling of the
conjugate.
To address the need for methods of predicting clinical efficacy, the present
invention provides plasmon resonance assays for pharmacokinetic characterization
of targeted therapies following their administration to a subject. The assays
disclosed herein accurately and reproducibly detect amounts of targeting molecule
and targeting molecule/drug conjugate in a single, minimal volume sample. Based
upon this determination, the circulation half-life of targeting molecule/drug conjugate,
rates of conjugate degradation, and linker stability can be monitored in a subject.
SUMMARY OF THE INVENTION
The present invention provides methods of determining an amount of
targeting molecule and an amount of targeting molecule/drug conjugate in a sample.
In a representative embodiment of the invention, the method comprises the steps of:
(a) providing a solid support comprising a surface to which a target is immobilized;
(b) providing a sample comprising a plurality of targeting molecule/drug conjugates;
(c) contacting the sample with the target immobilized to the surface of the solid
support; (d) detecting formation at the surface of the solid support of a first binding
complex of (i) the targeting molecule and (ii) the target at the surface of the solid
support, wherein formation of the first binding complex causes a first measurable
change in mass property of the solid support indicating an amount of targeting
molecule in the sample; (e) contacting the first binding complex with a drug binding
agent that specifically binds the drug of the targeting molecule/drug conjugate; and
(f) detecting formation at the surface of the solid support of a second binding
complex of (i) the drug binding agent and (ii) the first binding complex, wherein
formation of the second binding complex causes a second measurable change in
mass property of the solid support indicating an amount of targeting molecule/drug
conjugate in the sample.
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Methods of determining an amount of targeting molecule/drug conjugate in a
sample can also comprise the steps of: (a) providing a solid support comprising a
surface to which a first binding complex is immobilized, wherein the binding complex
comprises (i) a target and (ii) a targeting molecule/drug conjugate bound to the
target; (b) contacting a drug binding agent that specifically binds the drug of the
targeting molecule/drug conjugate with the first binding complex immobilized at the
surface of the solid support; and (c) detecting formation of a second binding complex
of (i) the drug binding agent and (ii) the first binding complex at the surface of the
solid support, wherein formation of the complex causes a measurable change in
mass property of the solid support indicating an amount of targeting molecule/drug
conjugate in the sample.
In another aspect of the invention, methods of determining an average
amount of drug loading per targeting molecule are provided. For example, a method
of determining drug loading of targeting molecule/drug conjugates in a sample can
comprise the steps of: (a) providing a solid support to which targeting molecule/drug
conjugates of a sample are bound; (b) determining an amount of drug in the sample
by measuring a change in mass property of a solid support upon binding of a drug
binding agent, which specifically binds the drug of the targeting molecule/drug
conjugate, to the targeting molecule/drug conjugates at the surface of the solid
support; and (c) calculating an average amount of drug per targeting molecule/drug
conjugate by dividing the amount of drug of (b) by an amount of targeting molecule in
the sample.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a sensorgram of a sandwich detection method. In the first
phase of the curve (between arrow 1 and 2), the sample of antibody/calicheamicin
conjugate was run over the immobilized antigen. A second phase (between arrow 3
and 4) was initiated by adding an anti-calicheamicin antibody. Response 1 indicates
mass addition proportionate to the concentration of antibody in the sample, and
response 2 is proportionate to the amount of calicheamicin in the
antibody/calicheamicin conjugate. RU, resonance units; gray circles, washing
period.
Figures 2A-2B show the correlation between the amount of antibody or
antibody/drug conjugate and the concentration of standard samples. Figure 2A is a
sensorgram showing resonance units as a function of time for each of the indicated
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concentrations (ng/ml) of hP67.6-AcBut-CalichDMH. Figure 2B is a line graph
showing resonance units as a function of concentration of hP67.6-AcBut-CalichDMH
+ anti-calicheamicin antibody (black filled circle), hP67.6-AcBut-CalichDMH (gray
filled circle), and anti-calicheamicin antibody (open circle).
Figures 3A-3C show plasma concentrations of hP67.6-AcBut-CalichDMH
determined using a sandwich detection method as described in Examples 3 and 4.
Each animal received antibody/drug conjugate for a total dose of 3 p,g of
calicheamicin. The dose of antibody/drug conjugate expressed in mg/kg is indicated.
Solid lines, animals bearing CD22-positive Ramos tumors; dotted lines, tumor-free
mice.
Figure 3A shows response 1, i.e., binding of hP67.6 and hP67.6-AcBut-
CalichDMH, to CD33 antigen immobilized on a CM5 chip.
Figure 3B shows response 2, i.e., binding of anti-calicheamicin to hP67.6-
AcBut-CalichDMH already bound to CD33 immobilized on a CM5 chip. The kinetics
of hP67.6-AcBut-CalichDMH in plasma are similar in tumor-bearing and tumor-free
animals.
Figure 3C shows the ratio of response 2 relative to response 1. The declining
concentration of antibody/drug conjugate as a fraction of the concentration of the
antibody moiety of the antibody/drug conjugate indicates the preferential clearance
of conjugated versus unconjugated antibody.
Figure 4 is a line graph showing resonance units as a function of
concentration of G5/44-AcBut-CalichDMH (inotuzumab ozogamicin) + anti-
calicheamicin antibody (black filled circle), G5/44-AcBut-CaIichDMH (gray filled
circle), and anti-calicheamicin antibody (open circle).
Figures 5A-5C show plasma concentrations of G5/44-AcBut-CalichDMH
determined using a sandwich detection method as described in Examples 3 and 5.
G5/44 anti-CD22 antibody was loaded with 72 jug calicheamicin per mg antibody,
and each animal received antibody/drug conjugate for a total dose of 3 (.ig of
calicheamicin. Solid lines, animals bearing CD22-positive Ramos tumors; dotted
lines, tumor-free mice.
Figure 5A shows response 1, i.e., binding of G5/44 and G5/44-AcBut-
CalichDMH to CD22 antigen immobilized on a CM5 chip.
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Figure 5B shows response 2, i.e., binding of anti-calicheamicin to G5/44-
AcBut-CalichDMH already bound to CD22 immobilized on a CM5 chip. The
presence of the CD22-positive Ramos tumor (solid lines) decreases the average
concentration of G5/44 antibody and G5/44-AcBut-CalichDMH conjugates in plasma.
Figure 5C shows the ratio of response 2 relative to response 1. The declining
concentration of antibody/drug conjugate as a fraction of the concentration of the
antibody moiety of the antibody/drug conjugate indicates the preferential clearance
of conjugated versus unconjugated antibody. Removal of calicheamicin from the
antibody was not influenced by the presence of the CD22-positive Ramos tumor.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods of characterizing samples comprising
compositions for targeted therapy, i.e., a targeting molecule conjugated either
directly or indirectly to a drug. Samples containing targeting molecule/drug
conjugates may include some proportion of the constituent parts (i.e., unconjugated
targeting molecule and free drug), for example, as a result of incomplete conjugation,
degradation of the conjugate, etc. In general, the unconjugated targeting molecule
and free drug each have limited efficacy and may contribute to patient toxicity.
Accordingly, for monitoring progress in patients receiving targeted therapies, drug
loading and the concentration of targeting molecule/drug conjugate (rather than the
constituent parts) is important. The disclosed methods provide for such
determination, which can be used to assess pharmacokinetic parameters of a
targeting molecule/drug conjugate, such as absorption, distribution, metabolism, and
excretion, following administration to a subject.
As compared to prior methods, the present disclosure describes use of a
mass-sensing technique to detect targeting molecule/drug conjugates, wherein such
conjugates are labile. The concentration of targeting molecule/drug conjugates can
be accurately determined in serum and/or at the targeting site to assess circulation
half-life, linker stability, and an amount of drug that is delivered to the targeting site.
A single, low-volume sample may be used to sequentially perform multiple detecting
steps in a same sample, which enables calculation of drug loading on the targeting
molecule/drug conjugate.
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I. Targeting Molecule/Drug Conjugates
Targeting molecules that may be used in the disclosed methods include any
molecule that shows specific binding to a target. Specific binding refers to an affinity
between two molecules which results in preferential binding in a heterogeneous
sample. Binding is generally characterized by an affinity of at least about 10'7 M or
higher, such as at least about 10"8 M or higher, or at least about 10'9 M or higher, or
at least about 10"11 M or higher, or at least about 10"12 M or higher.
Targeting molecules also include any molecule that, following administration
to a subject, selectively binds to cells expressing the target. The term targeting
refers to the preferential movement and/or accumulation in vivo of a molecule at a
target site (e.g., cells or tissues) as compared to a control site. A target site
comprises cells expressing a target, i.e., an intended site for accumulation of the
targeting molecule or targeting molecule/drug conjugate. A control site comprises
cells that substantially lack expression of the target and which therefore substantially
lack binding and/or accumulation of an administered targeting molecule or targeting
molecule/drug conjugate. Selective binding generally refers to a preferential
localization of a targeting molecule/drug conjugate such that an amount of targeting
molecule at a target site is about 2-fold greater than an amount of targeting molecule
at a control site, or about 5-fold greater, or about 10-fold greater or more.
Representative targeting molecules include antibodies, proteins, peptides,
peptide mimetics, peptide nucleic acids (PNAs), oligonucleotides, ligands, lectins,
and any other molecules that specifically and/or selectively bind to a target.
Targets bound by targeting molecules are generally associated with a disease
state, a disease susceptible state, or a condition requiring treatment. Representative
targets include antigens, haptens, proteins, peptides, receptors, oligonucleotides,
carbohydrates, and any other molecules expressed at elevated levels by cells of a
target site. A target is preferably present at the cellular surface or otherwise
accessible to targeting molecules. A target site may be localized, such as in a solid
tumor, or non-localized as in hematological malignancies. For example, a target site
can comprise cells expressing tumor-associated antigens (TAA), antigens expressed
on other malignant cells, immune cells contributing to inflammation, allergy,
autoimmunity, etc.
In one aspect of the invention, the targeting molecule is an antibody and the
invention relates to characterizing samples comprising immunoconjugates, i.e.,
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antibody/drug conjugates. The antibody moiety of an antibody/drug conjugate can
comprise any type of antibody, including for example, antibodies having tetrameric
structure (e.g., similar to naturally occurring antibodies), or any other structure
having at least one immunoglobulin light chain variable region or at least one
immunoglobulin heavy chain region, or antigen-binding fragments thereof (e.g., Fab,
modified Fab, Fab', F(ab')2 or Fv fragments). The disclosed methods may also be
used to characterize conjugates prepared using chimeric antibodies, humanized
antibodies, diabodies, single chain antibodies, tretravalent antibodies, and/or
multispecific antibodies (e.g., bispecific antibodies).
For preparation of targeted anti-cancer therapies, tumor-associated antigens
have been identified that specifically bind to cancer cells from solid tumors, such as
squamous/adenomatous lung carcinoma (non-small-cell lung carcinoma), invasive
breast carcinoma, colorectal carcinoma, gastric carcinoma, squamous cervical
carcinoma, invasive endometrial adenocarcinoma, invasive pancreas carcinoma,
ovarian carcinoma, squamous vesical carcinoma, and choriocarcinoma. Antigens for
targeted therapy of hematologic malignancies may also be useful drug targets, for
example, for the treatment of lymphomas and leukemias, such as including but not
limited to low grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL)
NHL, intermediate grade/ follicular NHL, intermediate grade diffuse NHL, high grade
immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved
cell NHL, bulky disease NHL and Waldenstrom's Macroglobulinemia, chronic
leukocytic leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia,
chronic lymphocytic leukemia, chronic myelogenous leukemia, lymphoblastic
leukemia, lymphocytic leukemia, monocytic leukemia, myelogenous leukemia, and
promyelocytic leukemia.
Representative antibodies that may be used to prepare antibody/drug
conjugates for targeted therapy include anti-5T4 antibodies, anti-CD19 antibodies,
anti-CD20 antibodies (e.g., RITUXAN®, ZEVALIN®, BEXXAR®), anti-CD22
antibodies, anti-CD33 antibodies (e.g., MYLOTARG®), anti-Lewis Y antibodies (e.g.,
Hu3S193, Mthu3S193, AGmthu3S193), anti-HER-2 antibodies (e.g., HERCEPTIN®
(trastuzumab), MDX-210, OMNITARG® (pertuzumab, rhuMAb 2C4)), anti-CD52
antibodies (e.g., CAMPATH®), anti-EGFR antibodies (e.g., ERBITUX® (cetuximab),
ABX-EGF (panitumumab)), anti-VEGF antibodies (e.g., AVASTIN® (bevacizumab)),
anti-DNA/histone complex antibodies (e.g., ch-TNT-1/b), anti-CEA antibodies (e.g.,
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CEA-Cide, YMB-1003), hLM609, anti-CD47 antibodies (e.g., 6H9), anti-VEGFR2 (or
kinase insert domain-containing receptor, KDR) antibodies (e.g., IMC-1C11), anti-
Ep-CAM antibodies (e.g., ING-1), anti-FAP antibodies (e.g., sibrotuzumab), anti-DR4
antibodies (e.g., TRAIL-R), anti-progesterone receptor antibodies (e.g., 2C5), anti-
CA19.9 antibodies (e.g., GIVAREX®), and anti-fibrin antibodies (e.g., MH-1).
As used herein, a drug refers to refers to any substance having biological or
detectable activity, for example, therapeutic agents, binding agents, etc., as well as
prodrugs, which are metabolized to an active agent in vivo. The term drug also
includes drug derivates, wherein a drug has been functionalized to enable
conjugation with a targeting molecule.
The drug may be bound to the targeting molecule either directly or indirectly,
but the linkage is such that it is compatible with preserving the therapeutic effect of
the drug moiety. The linker may be stable or hydrolyzable, and any suitable
technique for linking the drug to the antibody may be used. For example, hydrazides
and other nucleophiles may be conjugated to the aldehydes generated by oxidation
of the carbohydrates that naturally occur on antibodies. Hydrazone-containing
conjugates can be made with introduced carbonyl groups that provide the desired
drug-release properties. Conjugates can also be made with a linker that has a
disulfide at one end, an alkyl chain in the middle, and a hydrazine derivative at the
other end. Other representative linkers are thiol-reactive linkers such as esters,
amides, and acetals/ketals, and pH sensitive linkers, such as cis-aconitates, which
have a carboxylic acid juxtaposed to an amide bond. Linkers may also include
solubilizing agents such as PEG to limit aggregation of the targeting molecule/drug
conjugates. Peptdie linkers may also be used.
Representative drugs include anti-cancer agents, such as cytotoxic agents,
chemotherapeutic agents, immunomodulatory agents, anti-angiogenic agents, anti-
proliferative agents, pro-apoptotic agents, enzymes, and bioactive proteins. A drug
may also comprise a therapeutic nucleic acid, such as a gene encoding an
immunomodulatory agent, an anti-angiogenic agent, an anti-proliferative agent, or a
pro-apoptotic agent. These drug descriptors are not mutually exclusive, and thus a
therapeutic agent may be described using one or more of the above-noted terms.
Therapeutic agents may be prepared as pharmaceutically acceptable salts, acids or
derivatives of any of the above. In addition, conjugates can be made using
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secondary carriers as the cytotoxic agent, such as liposomes or polymers, for
example.
The term cytotoxic agent generally refers to an agent that inhibits or prevents
the function of cells and/or results in destruction of cells. Representative cytotoxic
agents include antibiotics, inhibitors of tubulin polymerization, alkylating agents that
bind to and disrupt DNA, and agents that disrupt protein synthesis or the function of
essential cellular proteins such as protein kinases, phosphatases, topoisomerases,
enzymes, and cyclins. For example, cytotoxic agents include, but are not limited to,
doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin,
carubicin, nogalamycin, rnenogaril, pitarubicin, valrubicin, cytarabine, gemcitabine,
trifluridine, ancitabine, enocitabine, azacitidine, doxifluridine, pentostatin, broxuridine,
capecitabine, cladribine, decitabine, floxuridine, fludarabine, gougerotin, puromycin,
tegafur, tiazofurin, adriamycin, cisplatin, carboplatin, cyclophosphamide,
dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine,
prednisone, procarbazine, methotrexate, flurouracils, etoposide, taxol, taxol analogs,
platins such as cis-platin and carbo-platin, mitomycin, thiotepa, taxanes, vincristine,
daunorubicin, epirubicin, actinomycin, authramycin, azaserines, bleomycins,
tamoxifen, idarubicin, dolastatins/auristatins, hemiasterlins, esperamicins and
maytansinoids.
In particular aspects of the invention, the targeting molecule/drug conjugates
characterized using the disclosed methods comprise an antibiotic drug moiety such
as a calicheamicin, also called the LL-E33288 complex, for example, gamma-
calicheamicin or a less potent derivative, N-acetyl gamma calicheamicin. See U.S.
Patent No. 4,970,198. Additional examples of calicheamicins suitable for use in
targeting molecule/drug candidates are disclosed in U.S. Patent Nos. 4,671,958;
5,053,394; 5,037,651; 5,079,233; and 5,108,912; which are each incorporated herein
in their entirety. Disulfide analogs of calicheamicin can also be used, for example,
analogs described in U.S. Patent Nos. 5,606,040 and 5,770,710, which are each
incorporated herein in their entirety. Representative techniques for preparation of
antibody/calicheamicin conjugates as set forth in Example 1 are described in U.S.
Patent Nos. 5,712,374; 5,714,586; 5,773,001; and 5,877,296; U.S. Publication Nos.
2004-0082764-A1 and 2006-0002942-A1; and PCT Publication No. WP
2005/089809; which are each incorporated herein in their entirety.
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Immunomodulatory agents that may be used to prepare targeting
molecule/drug conjugates include anti-hormones that block hormone action on
tumors and immunosuppressive agents that suppress cytokine production,
downregulate self-antigen expression, or mask MHC antigens. Representative anti-
hormones include anti-estrogens including for example tamoxifen, raloxifene,
aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY
117018, onapnstone, and toremifene; and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; and anti-adrenal agents.
Representative immunosuppressive agents include 2-amino-6-aryl-5-substituted
pyrimidines, azathioprine, cyclophosphamide, bromocryptine, danazol, dapsone,
glutaraldehyde, anti-idiotypic antibodies for MHC antigens and MHC fragments,
cyclosporin A, steroids such as glucocorticosteroids, cytokine or cytokine receptor
antagonists (e.g., anti-interferon antibodies, anti-IL10 antibodies, anti-TNFoc
antibodies, anti-IL2 antibodies), streptokinase, TGFp, rapamycin, T-cell receptor, T-
cell receptor fragments, and T cell receptor antibodies.
Representative anti-angiogenic agents include inhibitors of blood vessel
formation, for example, farnesyltransferase inhibitors, COX-2 inhibitors, VEGF
inhibitors, bFGF inhibitors, steroid sulphatase inhibitors (e.g., 2-methoxyoestradiol
bis-sulphamate (2-MeOE2bisMATE)), interleukin-24, thrombospondin,
metallospondin proteins, class I interferons, interleukin 12, protamine, angiostatin,
laminin, endostatin, and prolactin fragments.
Anti-proliferative agents and pro-apoptotic agents include activators of PPAR-
gamma (e.g., cyclopentenone prostaglandins (cyPGs)), retinoids, triterpinoids (e.g.,
cycloartane, lupane, ursane, oleanane, friedelane, dammarane, cucurbitacin, and
limonoid triterpenoids), inhibitors of EGF receptor (e.g., HER4), rampamycin,
CALCITRIOL® (1,25-dihydroxycholecalciferol (vitamin D)), aromatase inhibitors
(FEMARA® (letrozone)), telomerase inhibitors, iron chelators (e.g., 3-aminopyridine-
2-carboxaldehyde thiosemicarbazone (Triapine)), apoptin (viral protein 3 - VP3 from
chicken aneamia virus), inhibitors of Bcl-2 and Bcl-X(L), TNF-alpha, FAS ligand,
TNF-related apoptosis-inducing ligand (TRAIL/Apo2L), activators of TNF-alpha/FAS
ligand/TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) signaling, and inhibitors
of PI3K-Akt survival pathway signaling (e.g., UCN-01 and geldanamycin).
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Representative chemotherapeutic agents include alkylating agents such as
thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziidines such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine;
nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide,
estramustine, ifosfamide, mechiorethamine, mechiorethamine oxide hydrochloride,
melphalan, novembichin, phenesterine, prednimustine, trofosfarnide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,
ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, calicheamicins, carabicin, carminomycin,
carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-
oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin,
mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,
puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-
fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate,
pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-
azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine, 5-EU; androgens such as calusterone, dromostanolone propionate,
epitiostanol, mepitiostane, testolactone; anti-adrenal such as arninoglutethimide,
mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone;
aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene;
edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;
mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;
podophyilinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran;
spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2' -trichlorotriethylamine;
urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids,
e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology of Princeton, New Jersey)
and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer of Antony, France);
chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
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analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16);
ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine;
novantrone; teniposide; daunomycin; aininopterin; xeloda; ibandronate; CPT-11;
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid;
esperamicins; and capecitabine.
Additional therapeutic agents that may be conjugated to targeting molecules
and characterized using the methods disclosed herein include photosensitizing
agents (U.S. Patent Publication No. 2002/0197262 and U.S. Patent No. 5,952,329)
for photodynamic therapy; magnetic particles for thermotherapy (U.S. Patent
Publication No. 2003/0032995); binding agents, such as peptides, ligands, cell
adhesion ligands, etc., and prodrugs such as phosphate-containing prodrugs,
thiophosphate-containing prodrugs, sulfate containing prodrugs, peptide containing
prodrugs, (3-lactam-containing prodrugs, substituted phenoxyacetamide-containing
prodrugs or substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and
other 5-fluorouridine prodrugs that may be converted to the more active cytotoxic
free drug.
II Pharmacokinetics of Targeting Molecule/Drug Conjugates
The present invention provides methods of determining drug loading of a
targeting molecule, for example, to determine whether the conjugation reaction
achieved a level of drug loading which comprises an effective dose, i.e., an amount
of targeting molecule/drug conjugate sufficient to elicit a desired biological response,
and to maintain batch-to-batch consistency of commercially manufactured targeting
molecule/drug conjugates. To assess drug release or stability of targeting
molecule/drug conjugates, drug loading may also be assessed following
administration to a patient, for example, using a blood sample from the patient.
As disclosed herein, an amount of targeting molecule/drug conjugate may be
calculated from the separate determinations of (i) an amount of targeting molecule
and (ii) an amount of targeting molecule/drug conjugate in the same sample. Steps
(i) and (ii) are described herein below more fully under subheadings II.A and II.B,
respectively. See also Figure 1. Briefly, the method includes measurement of two
consecutive responses. A first response determines the number of resonance units
after contacting a sample that contains the targeting molecule/drug conjugates over
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a mass sensing device, such as a BIACORE® chip, with immobilized target
recognized by the targeting molecule of the conjugate. This response is proportional
to the sum of the free (unconjugated) and conjugated targeting molecule in the
sample. A second response is obtained after sequentially contacting a drug binding
agent with the conjugated and unconjugated targeting molecules bound to the
immobilized target on the same mass sensing device. This second response is
proportional to the amount of drug present as targeting molecule/drug conjugates in
the sample.
In accordance with the disclosed methods, any suitable mass-sensing
technique may be used. Representative techniques known in the art include
piezoelectric, optical, thermo-optical, surface acoustic wave (SAW) methods, as well
as electrochemical methods, such as potentiometric, voltametric, conductometric,
amperometric and capacitance methods.
Optical methods that may be used include methods for detecting mass
surface concentration (or refractive index), such as reflection-optical methods,
including both internal and external reflection methods, e.g., eliipsometry and
evanescent wave spectroscopy (EWS), the latter including surface plasmon
resonance (SPR), Brewster angle refractometry, critical angle refractometry,
frustrated total reflection (FTR), evanescent wave eliipsometry, scattered total
internal reflection (STIR), optical wave guide sensors, evanescent wave based
imaging, such as critical angle resolved imaging, Brewster angle resolved imaging,
SPR angle resolved imaging, etc., as well as methods based on evanescent
fluorescence (TIRF) and phosphorescence.
For example, to estimate the equilibrium constant of a targeting molecule in a
sample, the following mass-sensing technique may be used. First, a concentration
series (e.g., 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 ng/ml) of the
targeting molecule is prepared and sequentially injected into a biosensor having a
sensor chip operatively associated therewith, wherein the sensor chip has a
reference sensing surface and at least one sensing surface with immobilized target.
The relative responses at steady-state binding levels for each targeting molecule
concentration are measured. Because of bulk-refractive index contributions from
solvent additives in the biosensor's running buffer, a correction factor may be
calculated (via known calibration procedures) and applied to give corrected relative
responses. The corrected relative responses for each targeting molecule
-13-

concentration are then mathematically evaluated as is appreciated by those skilled in
the art to estimate the equilibrium constant of the targeting molecule.
In a particular aspect of the invention, the mass sensing technique is surface
plasmon resonance, which may be performed using a BIACORE® instrument
(Biacore AB of Uppsala, Sweden). The apparatus and theoretical background are
described in Jonsson et a!., BioTechniques, 1991, 11:620-627. This technique
involves immobilizing a first binding partner of a binding pair to a sensor chip,
contacting the sensor chip with a sample containing a second binding partner of the
binding pair, and then measuring a resultant change in the surface optical
characteristics of the sensor chip.
In general, a solid support comprises a hydrogel matrix coating coupled to the
top surface of the solid support, wherein the hydrogel matrix coating has a plurality of
functional groups. For use with a BIACORE® instrument, the solid support is
preferably in the form a sensor chip, wherein the sensor chip has a free electron
metal interposed between the hydrogel matrix and the top surface. Suitable free
electron metals for this purpose include copper, silver, aluminum and gold.
In a particular aspect of the invention, the method may comprise the steps of:
(a) providing a solid support comprising a surface to which a target is immobilized;
(b) providing a sample comprising a plurality of targeting molecule/drug conjugates;
(c) contacting the sample with the target immobilized to the surface of the solid
support; (d) detecting formation at the surface of the solid support of a first binding
complex of (i) the targeting molecule and (ii) the target at the surface of the solid
support, wherein formation of the first binding complex causes a first measurable
change in mass property of the solid support indicating an amount of targeting
molecule in the sample; (e) contacting the first binding complex with an anti-drug
antibody or drug-binding fragment thereof; and (f) detecting formation at the surface
of the solid support of a second binding complex of (i) the anti-drug antibody or drug-
binding fragment thereof and (ii) the first binding complex, wherein formation of the
second binding complex causes a second measurable change in mass property of
the solid support indicating an amount of targeting molecule/drug conjugate in the
sample.
In another aspect of the invention, a method of determining an average
amount of drug loading per antibody in a sample of targeting molecule/drug
conjugates can comprise the steps of: (a) providing a solid support to which targeting
-14-

molecule/drug conjugates of a sample are bound; (b) determining an amount of drug
in the sample by measuring a change in mass property of a solid support upon
binding of an anti-drug antibody or drug-binding fragment thereof to the targeting
molecule/drug conjugates at the surface of the solid support; and (c) calculating an
average amount of drug per targeting molecule/drug conjugate by dividing the
amount of drug of (b) by an amount of targeting molecule in the sample. When
considered as a function of time following administration to a subject, this method is
useful for assessing circulation half-life of a targeting molecule/drug conjugate and
linker stability.
Using the disclosed methods, targeting molecule/drug conjugates were
detected in serum samples at a level of 100 to 1,000 ng/ml targeting molecule. As
described in Examples 4 and 5, PK values of targeting molecule/drug conjugates
were reproducibly determined in individual samples. The presence of a tumor
expressing a target reduced the circulation half-life of a targeting molecule/drug
conjugate with specificity for the target, but had no effect on the circulation half-life of
a targeting molecule/drug conjugate having different specificity. Compare Figures
5B and 3B, respectively. The reduction of circulation half-life may be attributable to
retention of the targeting molecule/drug conjugate in the presence of an appropriate
target.
II A. Methods of Determining an Amount of Targeting Molecule in a Sample
Comprising Targeting Molecule /Drug Conjugates
The present invention provides methods of determining an amount of
targeting molecule in a sample comprising a plurality of targeting molecule/drug
conjugates. In a particular aspect of the invention, the method comprises the steps
of (a) providing a solid support comprising a surface to which a target is immobilized;
(b) providing a sample comprising a plurality of targeting molecule/drug conjugates;
(c) contacting the sample with the target immobilized to the surface of the solid
support; and (d) detecting formation of a binding complex of (i) targeting molecules in
the sample and (ii) the target at the surface of the solid support, wherein formation of
the binding complex causes a measurable change in mass property of the solid
support.
Representative samples that may be used in accordance with the disclosed
methods include targeting molecule/drug conjugate preparations, i.e., a sample
comprising a conjugation reaction between a targeting molecule and a drug, which
-15-

may include conjugated targeting molecule, unconjugated targeting molecule, and
free drug. Samples obtained from a subject following administration of antibodies to
the subject may also be used, for example, blood, serum, or urine samples. The
sample can comprise a minimal liquid volume, such as a sample less than about 100
fjj, or less than about 50 \L\, or less than about 25 JJ.I, or less than about 10 \x\, or less
than about 5 JJ,I. Larger sample volumes may be used to increase sensitivity. A
sample may also comprise a liquid extract prepared from a tissue sample, such as a
tumor. For example, a sample may be prepared from a squamous/adenomatous
lung carcinoma (non-small-cell lung carcinoma), invasive breast carcinoma,
colorectal carcinoma, gastric carcinoma, squamous cervical carcinoma, invasive
endometrial adenocarcinoma, invasive pancreas carcinoma, ovarian carcinoma,
squamous vesical carcinoma, choriocarcinoma, or other carcinomas of bronchi,
breast, colon, rectum, stomach, cervix, endometrium, pancreas, ovaria, chorium, and
seminal vesicles.
IIB. Methods of Determining an Amount of Drug in a Sample Comprising
Targeting Molecule/Drug Conjugates
For determining an amount of drug in a sample, targeting molecule/drug
conjugates are bound to a mass-sensing chip, and a drug binding agent that
specifically binds to the drug moiety of the targeting molecule/drug conjugate is used
to detect the conjugates. A drug binding agent can comprise an anti-drug antibody,
or drug-binding fragment thereof. Additional representative binding agents include
proteins, peptides, peptide mimetics, peptide nucleic acids (PNAs), ligands, or any
other molecule that specifically binds to a drug moiety as described herein.
For example, the method may comprise the steps of (a) providing a solid
support comprising a surface to which a first binding complex is immobilized,
wherein the binding complex comprises (i) a target as described herein and (ii) a
targeting molecule/drug conjugate bound to the target; (b) contacting an anti-drug
antibody or drug-binding fragment thereof with the first binding complex immobilized
at the surface of the solid support; and (c) detecting formation of a second binding
complex of (i) the anti-drug antibody or drug-binding fragment thereof and (ii) the first
binding complex at the surface of the solid support, wherein formation of the complex
causes a measurable change in mass property of the solid support indicating an
amount of targeting molecule/drug conjugate in the sample.
-16-

Alternatively, the method may comprise the steps of (a) providing a solid
support comprising a surface to which an anti-drug antibody or drug-binding
fragment thereof is immobilized; (b) contacting a sample comprising targeting
molecule/drug conjugates with the anti-drug antibody or drug-binding fragment
immobilized at the surface of the solid support; and (c) detecting a measurable
change in mass property of the solid support indicating an amount of targeting
molecule/drug conjugate in the sample.
An antibody that is used to detect the drug moiety of the targeting
molecule/drug conjugate may be any antibody that shows specific binding, i.e.,
preferential binding to the drug when the drug is presented in a sample containing
other antigens. The antibody may be polyclonal or monoclonal. Anti-drug antibodies
having low off rates provide the greatest sensitivity. When using anti-drug antibodies
having moderate off-rates, background corrections may be used to quantify targeting
molecule/drug conjugates at reduced sensitivity.
Methods for preparing and characterizing anti-drug antibodies are well known
in the art. See, e.g., Harlow & Lane, Antibodies: A Laboratory Manual. Cold Spring
Harbor Laboratory, 1988. Additional techniques and reagents useful for generating
and screening an antibody display library can be found in, for example, U.S. Patent
No. 5,223,409 and PCT International Application Publication Nos. WO 92/18619,
WO 91/17271, WO 92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO
92/09690, and WO 90/02809.
Briefly, a polyclonal antibody is prepared by immunizing an animal with an
immunogen comprising a drug as described herein, and collecting antisera from that
immunized animal. A wide range of animal species can be used for the production
of antisera, for example rabbits, mice, rats, hamsters, guinea pigs, goats, and
donkeys.
As is well known in the art, the immunogen may be coupled with a carrier,
such as keyhole limpet hemocyanin (KLH) and serum albumins (e.g., BSA), to
improve immunogenicity. Techniques for conjugating an immunogen to a carrier
polypeptide are well known in the art and include glutaraldehyde, m-
maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized
benzidine. Immunogenicity of an immunogen can also be enhanced by the use of
adjuvants, for example, complete Freund's adjuvant, incomplete Freund's adjuvants,
and aluminum hydroxide adjuvant.
-17-

The amount of immunogen used for the production of polyclonal antibodies
varies upon the nature of the immunogen, the animal used for immunization, and the
administration route (e.g., subcutaneous, intramuscular, intradermal, intravenous, or
intraperitoneal). The production of polyclonal antibodies is monitored by sampling
blood of the immunized animal at various points following immunization. When a
desired titer of antibody is obtained, the immunized animal is bled and the serum
isolated and stored.
An anti-drug monoclonal antibody for use in the disclosed methods can be
readily prepared through use of well-known techniques such as those exemplified in
U.S. Pat. No. 4,196,265. For example, mice or rats are immunized with a drug for a
sufficient period to obtain an immune response, and then spleen cells from the
immunized animal are then fused with immortal myeloma cells. Fused cells are
separated from the mixture of non-fused parental cells, for example, by the addition
of agents to the culture media that block de novo nucleotide synthesis (e.g.,
aminopterin, methotrexate, and azaserine). Individual hybridomas are cultured and
supematants are tested for reactivity with the drug immunogen. The selected clones
can be propagated indefinitely as a source of the monoclonal antibody.
By way of specific example, to produce an anti-drug antibody as described
herein, mice are injected intraperitoneally with between about 1 -200 |ag of an antigen
comprising a drug of a targeting molecule/drug conjugate. B lymphocyte cells are
stimulated to grow by injecting the drug in association with an adjuvant such as
complete Freund's adjuvant. As needed, mice are boosted by injection with a
second dose of the drug mixed with incomplete Freund's adjuvant. A few weeks
after the second injection, mice are tail bled and the sera titered by
immunoprecipitation. The steps of boosting and titering are repeated until a suitable
titer is achieved. The spleen of the mouse is removed, spleen lymphocytes are
isolated, and myeloma cells are combined with the spleen lymphocytes under
conditions appropriate for cell fusion. Fusion conditions include, for example, the
presence of polyethylene glycol. Fused cells are separated from unfused myeloma
cells by culturing in a selection medium such as HAT media (hypoxanthine,
aminopterin, thymidine). The resultant hybridomas are screened for the production
of anti-drug antibodies. Selected clones are cultured in high volumes to achieve
-18-

suitable amounts of antibody. The antibodies may be purified by affinity
chromatography or other methods, as is known in the art,
EXAMPLES
The following examples have been included to illustrate modes of the
invention. Certain aspects of the following examples are described in terms of
techniques and procedures found or contemplated by the present co-inventors to
work well in the practice of the invention. These examples illustrate standard
laboratory practices of the co-inventors. In light of the present disclosure and the
general level of skill in the art, those of skill will appreciate that the following
examples are intended to be exemplary only and that numerous changes,
modifications, and alterations may be employed without departing from the scope of
the invention.
EXAMPLE 1
Preparation of Antibodv/Calicheamicin Conjugates
Gemtuzumab ozogamicin and inotuzumab ozogamicin are calicheamicin
conjugates of the anti-CD33 and anti-CD22 antibodies, hP67.6 and G5/44,
respectively. Gemtuzumab ozogamicin is the generic name for the marketed drug
MYLOTARG® and is also referred to as hP67.6-AcBut-CalichDMH. The anti-
CD22/calicheamicin conjugate, inotuzomab ozogamicin, also known as G5/44-
AcBut-CalichDMH, is currently in phase I clinical trials. To obtain these conjugates,
hP67.6 and G5/44 were linked to N-acetyl gamma calichemicin dimethyl hydrazide
with the acid labile (4-(4' acetylphenoxy)butanoic acid (AcBut) linker. Antibodies
were loaded at a density of approximately 35 ng calicheamicin per mg hP67.6 and
approximately 73 ng calicheamicin per mg G5/44. Anti-Lewis Y/calicheamicin and
anti-5T4/calicheamicin conjugates were similarly prepared and used in the disclosed
assays.
EXAMPLE 2
Administration of Antibodv/Calicheamicin Conjugates
The Ramos cell line (CRL-1923) was obtained from the American Type
Culture Collection (ATCC). Ramos is a CD22+, CD33" cell line derived from a human
B-cell lymphoma. The cells were maintained in suspension cultures in RPMI1640
supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 0.2 % (w/v) glucose, 100
-19-

U/ml penicillin G sodium, 100 (xg/ml streptomycin sulphate and 10 % (v/v) fetal
bovine serum.
Balb/c nude mice of 16 weeks old (Charles River Laboratories, Wilmington,
Massachusetts) were irradiated with 400 rad gamma rays. Ramos cells (107/200 \i\)
were injected in the right flank of each mouse. After 8 days, 10 mice with a tumor
size of approximately 0.5 cm3 (± s=0.16) were selected. Four treatment groups were
created: (1) tumor-bearing mice treated with hP67.6-AcBut-CalichDMH, (2) tumor-
free mice treated with hP67.6-AcBut-CalichDMH, (3) tumor-bearing mice treated with
G5/44-AcBut-CalichDMH, and (4) tumor-free mice treated with G5/44-AcBut-
CalichDMH. Two days prior to administration of antibody/calicheamicin conjugates,
a 5 JLXI blood sample was taken from each mouse. A single dose of 150 \xantibody/calicheamicin conjugate (3 |ug calicheamicin per mouse) was injected into
the lateral tail vein. Blood samples of exactly 5 p.l were taken at 24, 48, 72, and 96
hours thereafter. To obtain reproducible small volume samples, the mice were kept
under a heating lamp until tail veins became visible. The tail was disinfected with
70% isopropyl alcohol, and the lateral tail vein was ruptured with a needle. The
resultant blood droplet was then aspirated with a capillary mounted to a micropipettor
(Drummond of Broomall, Pennsylvania) preset to an aspiration volume of 5 nl. This
blood sample was immediately transferred to a test tube containing 195 jal of the
following mixture: 0.01 M HEPES (pH 7.4), 0.15 M NaCI, 3 mM EDTA, 0.005%
Surfactant P20 (HBS-EP buffer, available from Biacore of Uppsala, Sweden).
EXAMPLE 3
Plasmon Resonance Sandwich Detection Assay
A plasmon resonance sandwich detection assay was developed to determine
in a serum sample (1) an amount of targeting molecule and targeting molecule/drug
conjugate in a sample, and (2) an amount of drug present in targeting molecule/drug
conjugates of the same sample. The principle of this method is illustrated in Figure
1. The assay allows for an evaluation of the clearance of targeting molecule/drug
conjugate. The method does not discriminate between a reduction of drug on all the
conjugate molecules and the generation of a fraction of unconjugated antibody.
The analyses described herein were performed on a BIACORE® instrument
(Biacore International AB of Uppsala, Sweden) using antibody/calicheamicin
conjugates. The detection system of this instrument relies upon the measurement
-20-

of refractive index changes caused by the interaction of macromolecules on
biosensor chips. See e.g., Johne et al., J. Immunol. Methods, 1993,160(2):191-198;
Karlson et al., J. Immunol. Methods, 1991,145(1-2):229-240.
Antigens were immobilized to the surface of a CM5 biosensor chip at a
density of 4000-9000 resonance units/flow cell. The chip was activated by the
coupling reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-HCI/N-
hydroxysuccinimide at a flow rate of 5 (il/minute for 6 minutes, followed by addition of
antigens. Lewis-BSA antigens were loaded by contacting the chip with 50 p,g/ml
protein in a solution of 10 mM sodium acetate (pH 4.0-4.5) at. a flow rate of 5
I/minute for 6 minutes. CD33 or CD22Fc were covalently linked to CM5 chips by
contacting the chip to 0.1 mg/ml protein in a solution of 10 mM sodium acetate (pH
5) at a flow rate of 2 JLX! per minute for 30 minutes. The chip was then washed with
HBS-EP containing 300 mM NaCI.
Following immobilization of the antigen on a CM5 chip, calibration curves
were established for each antigen. As a representative result, Figures 2A-2B show
the correlation between the concentration of standard samples and the number of
resonance units upon binding of the anti-CD33/calicheamicin conjugate hP67.6-
AcBut-CalichDMH. A correlation coefficient of approximately 1.0 allows for accurate
determination of the total amount of antibody and the amount of calicheamicin bound
to antibody. Using the calibration curves, the serum concentration of the antibody
moiety of an antibody/drug conjugate was determined.
By thereafter contacting the chip with an anti-calicheamicin antibody, the
amount of calicheamicin present in the serum sample was also determined. As
demonstrated by the absence of a second response in Figure 2B, unconjugated
antibody at the same concentrations does not react to the anti-calicheamicin
antibody. This result provides evidence for the specificity of the second response for
the presence of calicheamicin on the antibody.
The response after binding of the conjugate to CD33 by itself (hP67.6-AcBut-
CalichDMH) as well as followed by a secondary response (hP67.6-AcBut-
CalichDMH + anti-calicheamicin) was linear (1=0.9996 and r2=0.9994, respectively)
for a concentration range of conjugate between 0 and 500 ng/ml. The difference of
these responses (i.e., resonance units attributable to binding of anti-calicheamicin) is
also linear (=0.9947) within this range. The regression coefficients of the quadratic
-21 -

equations of these functions were larger than 0.99 when a concentration range of 0
to 1000 ng/ml was used. Interpolation using a quadratic equation of resonance units
plotted as a function of concentration allows for the accurate determination of
antibody/drug conjugate concentration in a sample containing between 0 and 1000
ng/ml of antibody/drug conjugate.
A similar strategy was used to establish the calibration curves depicting (1)
the relationship between resonance units and the concentration of G5/44 anti-CD22
antibody and G5/44-AcBut-CalichDMH (see Figure 4); and (2) the relationship
between resonance units and the concentration of G193 anti-Lewis Y antibody and
CMD193, a calicheamicin conjugate thereof. These relationships were also best
described (rO.99) by a quadratic equation for a concentration range between 0 and
1000 ng/ml.
EXAMPLE 4
Pharmacokinetic Properties of Anti-CD33/Calicheamicin Conjugates
The pharmacokinetic properties of hP67.6-AcBut-CalichDMH were
determined in tumor-bearing and tumor-free mice. Five animals were used for each
group. Tumor-bearing mice had an average body weight of 19 g (standard deviation
= 1 g) and had xenografted Ramos tumors with an average volume of 528 mm3
(standard deviation 102 mm3) Tumor-free mice had an average body weight of 20 g
(standard deviation = 1 g).
Figure 3A shows the concentration of hP67.6-AcBut-CalichDMH in plasma of
nude mice at various time points following intravenous injection of a single dose of
antibody/drug conjugate. A dose of 3 jxg calicheamicin was administered to each
mouse. The dose of antibody as ng/kg body mass is indicated. The concentration of
the antibody/drug conjugate in plasma was calculated by correcting for a normal
hematocrit of 45%, and it was assumed that no antibody/drug conjugate was bound
to the cell fraction. A 3 |ng calicheamicin dose, which is provided as 86 jug
antibody/drug conjugate having 35 jag calicheamicin per mg antibody, is
administered in a blood volume of 1.5 ml (approximate blood volume of a 20 g
mouse). Therefore, one would theoretically anticipate 105 (j.g/ml as a maximum
concentration. Based upon a blood sample volume of 5 p.l, the experimentally
determined concentration of antibody/drug conjugate after 20 minutes was
approximately 80 |ag/ml.
-22-

The amounts of antibody/drug conjugate that were administered to each
mouse varied depending on the actual body mass of the animal. Within a range of
4.1 to 4.5 mg antibody/drug conjugate per kg, the administered dose was not directly
proportional to the maximum concentration of the conjugate in plasma. In addition,
the data did not indicate that dose variation was responsible for variations in
circulation half-life. An exceptionally high circulation half-life was observed in a
single mouse that received a dose of 5 mg antibody/drug conjugate per kg.
The amount of hP67.6 conjugated to calicheamicin has a shorter circulation
half-life than the unconjugated antibody. This is illustrated in Figure 3C, which
shows a consistently declining concentration of conjugated calicheamicin (response
2) as a fraction of the antibody-moiety of hP67.6-AcBut-CalichDMH (response 1).
The reproducible reduction of total calicheamicin bound to antibody was not
influenced by the presence of the CD22+ Ramos tumor.
EXAMPLE 5
Pharmacokinetic Properties of Anti-CD22/Calicheamicin Conjugates
The pharmacokinetic properties of G5/44-AcBut-CalichDMH were determined
in tumor-bearing and tumor-free mice. Three tumor-bearing mice had an average
body weight of 19 g (standard deviation = 1 g) and had xenografted Ramos tumors
with an average volume of 1276 mm3 (standard deviation 398 mm3). Six tumor-free
mice had an average body weight of 20 g (standard deviation = 1 g). Administration
of anti-CD22/calicheamicin conjugates and surface plasmon resonance assay were
performed as described in Examples 2, 3, and 4.
Calibration curves depicting the relationship between resonance units and the
concentration of the G5/44 antibody and G5/44-AcBut-CalichDMH conjugate are
shown in Figure 4. The relationship was best described (r>0.99) by a quadratic
equation for a concentration range between 0 and 1000 ng/ml. See Figure 4. As for
unconjugated hP67.6, a response to free calicheamicin was not observed with
unconjugated G5/44.
Figure 5A shows the declining concentration of the antibody moiety of G5/44-
AcBut-CalichDMH in plasma of tumor bearing and non-tumor bearing mice.
Concentrations of the antibody moiety of G5/44-AcBut-CalichDMH (Figure 5A) and
of the amount of calicheamicin bound to G5/44 (Figure 5B) declined faster in tumor-
bearing mice. This was reflected in the decreased circulation half-life of G5/44-
AcBut-CalichDMH. See Table I. The presence of a tumor that expresses the CD22
-23-

target enhanced the removal of the conjugate from plasma. The decline of the
calicheamicin concentration as a function of time was identical in tumor bearing and
non-tumor bearing mice (Figure 5C), indicating that the presence of the tumor did not
influence the release of calicheamicin from the antibody moiety of the conjugate.
Table I

AB = antibody moiety, CM = calicheamicin bound to antibody, 2T = plasma half-life
(h), AUC = area under the curve (h*ng/ml), CL = clearance (ml/min/kg), Vss =
volume distribution (ml/kg)
-24-

CLAIMS
1. A method of determining an amount of targeting molecule and an
amount of targeting molecule/drug conjugate in a sample comprising the steps of:
(a) providing a solid support comprising a surface to which a target is
immobilized;
(b) providing a sample comprising a plurality of targeting molecule/drug
conjugates;
(c) contacting the sample with the target immobilized to the surface of the
solid support;
(d) detecting formation at the surface of the solid support of a first binding
complex of (i) the targeting molecule and (ii) the target at the surface of the solid
support, wherein formation of the first binding complex causes a first measurable
change in mass property of the solid support indicating an amount of targeting
molecule in the sample;
(e) contacting the first binding complex with a drug binding agent that
specifically binds the drug of the targeting molecule/drug conjugate; and
(f) detecting formation at the surface of the solid support of a second
binding complex of (i) the drug binding agent and (ii) the first binding complex,
wherein formation of the second binding complex causes a second measurable
change in mass property of the solid support indicating an amount of targeting
molecule/drug conjugate in the sample.

2. The method of claim 1, wherein the target is expressed on cancer cells
or on cells involved in an autoimmune response.
3. The method of claim 2, wherein the target expressed on cancer cells is
5T4, CD19, CD20, CD22, CD33, Lewis Y, HER-2, type I Fc receptor for
immunoglobulin G (Fc gamma Rl), CD52, epidermal growth factor receptor (EGFR),
vascular endothelial growth factor (VEGF), DNA/histone complex, carcinoembryonic
antigen (CEA), CD47, VEGFR2 (vascular endothelial growth factor receptor 2 or
kinase insert domain-containing receptor, KDR), epithelial cell adhesion molecule
(Ep-CAM), fibroblast activation protein (FAP), Trail receptor-1 (DR4), progesterone
receptor, oncofetal antigen CA19.9, or fibrin.
4. The method of claim 1, wherein the targeting molecule is an antibody.
5. The method of claim 1, wherein the drug is calicheamicin.
6. The method of claim 1, wherein the drug binding agent is an antibody.
-25-

7. The method of claim 1, wherein the sample comprises a volume of
about 5 p.l or less.
8. The method of claim 1, wherein the sample is a blood sample.
9. A method of determining an amount of targeting molecule/drug
conjugate in a sample comprising the steps of:
(a) providing a solid support comprising a surface to which a first binding
complex is immobilized, wherein the binding complex comprises (i) a target and (ii) a
targeting molecule/drug conjugate bound to the target;
(b) contacting a drug binding agent that specifically binds the drug of the
targeting molecule/drug conjugate with the first binding complex immobilized at the
surface of the solid support; and
(c) detecting formation of a second binding complex of (i) the drug binding
agent and (ii) the first binding complex at the surface of the solid support, wherein
formation of the complex causes a measurable change in mass property of the solid
support indicating an amount of targeting molecule/drug conjugate in the sample.

10. The method of claim 9, wherein the target is expressed on cancer cells
or on cells involved in an autoimmune response.
11. The method of claim 9, wherein the target expressed on cancer cells is
5T4, CD19, CD20, CD22, CD33, Lewis Y, HER-2, type I Fc receptor for
immunoglobulin G (Fc gamma Rl), CD52, epidermal growth factor receptor (EGFR),
vascular endothelial growth factor (VEGF), DNA/histone complex, carcinoembryonic
antigen (CEA), CD47, VEGFR2 (vascular endothelial growth factor receptor 2 or
kinase insert domain-containing receptor, KDR), epithelial cell adhesion molecule
(Ep-CAM), fibroblast activation protein (FAP), Trail receptor-1 (DR4), progesterone
receptor, oncofetal antigen CA19.9, or fibrin.
12. The method of claim 9, wherein the targeting molecule is an antibody.
13. The method of claim 9, wherein the drug is calicheamicin.
14. The method of claim 9, wherein the drug binding agent is an antibody.
15. The method of claim 9, wherein the sample comprises a volume of
about 5 nl or less.
16. The method of claim 9, wherein the sample is a blood sample.
17. The method of claim 9, wherein the amount of targeting molecule in
the sample is determined by measuring a change in mass property of a solid support
-26-

upon binding of targeting molecule/drug conjugates to a target immobilized at a
surface of a solid support.
18. A method of determining an average amount of drug loading per
targeting molecule in a sample of targeting molecule/drug conjugates comprising the
steps of:
(a) providing a solid support to which targeting molecule/drug conjugates
of a sample are bound;
(b) determining an amount of drug in the sample by measuring a change in
mass property of a solid support upon binding of a drug binding agent that
specifically binds the drug of the targeting molecule/drug conjugate to the targeting
molecule/drug conjugates at the surface of the solid support; and
(c) calculating an average amount of drug per targeting molecule/drug
conjugate by dividing the amount of drug of (b) by an amount of targeting molecule in
the sample.

19. The method of claim 18, wherein the target is expressed on cancer
cells or on cells involved in an autoimmune response.
20. The method of claim 18, wherein the target expressed on cancer cells
is 5T4, CD19, CD20, CD22, CD33, Lewis Y, HER-2, type I Fc receptor for
immunoglobulin G (Fc gamma Rl), CD52, epidermal growth factor receptor (EGFR),
vascular endothelial growth factor (VEGF), DNA/histone complex, carcinoembryonic
antigen (CEA), CD47, VEGFR2 (vascular endothelial growth factor receptor 2 or
kinase insert domain-containing receptor, KDR), epithelial cell adhesion molecule
(Ep-CAM), fibroblast activation protein (FAP), Trail receptor-1 (DR4), progesterone
receptor, oncofetal antigen CA19.9, or fibrin.
21. The method of claim 18, wherein the targeting molecule is an antibody.
22. The method of claim 18, wherein the drug is calicheamicin.
23. The method of claim 18, wherein the drug binding agent is an antibody.
24. The method of claim 18, wherein the sample comprises a volume of
about 5 p.I or less.
25. The method of claim 18, wherein the sample is a blood sample.
26. The method of claim 18, wherein the amount of targeting molecule in
the sample is determined by measuring a change in mass property of a solid support
-27-

upon binding of targeting molecule/drug conjugates to a target immobilized at a
surface of a solid support.
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The present invention relates to methods for determining pharmacokinetics of targeted therapies using mass-sensing
techniques.