U.S. patent application number 13/997069 was filed with the patent office on 2013-10-24 for method to optimize the treatment of patients with biological drugs.
This patent application is currently assigned to Proteomika, S.L.. The applicant listed for this patent is Antonio Martinez Martinez, Daniel Nagore Casas, Ainhoa Ruiz Del Agua, Laureano Simon Buela. Invention is credited to Antonio Martinez Martinez, Daniel Nagore Casas, Ainhoa Ruiz Del Agua, Laureano Simon Buela.
Application Number | 20130280171 13/997069 |
Document ID | / |
Family ID | 43569372 |
Filed Date | 2013-10-24 |
United States Patent
Application |
20130280171 |
Kind Code |
A1 |
Ruiz Del Agua; Ainhoa ; et
al. |
October 24, 2013 |
METHOD TO OPTIMIZE THE TREATMENT OF PATIENTS WITH BIOLOGICAL
DRUGS
Abstract
The invention relates to the field of personalized therapy and,
in particular, to a method for classifying a patient suffering from
rheumatoid arthritis as a responder or as a non-responder patient
to a treatment based on a biological drug.
Inventors: |
Ruiz Del Agua; Ainhoa;
(Derio (Vizcaya), ES) ; Martinez Martinez; Antonio;
(Derio (Vizcaya), ES) ; Nagore Casas; Daniel;
(Derio (Vizcaya), ES) ; Simon Buela; Laureano;
(Derio (Vizcaya), ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ruiz Del Agua; Ainhoa
Martinez Martinez; Antonio
Nagore Casas; Daniel
Simon Buela; Laureano |
Derio (Vizcaya)
Derio (Vizcaya)
Derio (Vizcaya)
Derio (Vizcaya) |
|
ES
ES
ES
ES |
|
|
Assignee: |
Proteomika, S.L.
Derio (Vizcaya)
ES
|
Family ID: |
43569372 |
Appl. No.: |
13/997069 |
Filed: |
December 22, 2011 |
PCT Filed: |
December 22, 2011 |
PCT NO: |
PCT/EP11/73836 |
371 Date: |
June 21, 2013 |
Current U.S.
Class: |
424/9.2 ;
424/133.1; 424/142.1 |
Current CPC
Class: |
A61K 39/3955 20130101;
C07K 2317/21 20130101; C07K 2317/24 20130101; G01N 2800/52
20130101; G01N 33/9493 20130101; G16H 40/20 20180101; G01N 2333/525
20130101; Y02A 90/10 20180101; C07K 16/241 20130101; G16H 20/17
20180101; G01N 33/564 20130101; G01N 33/6854 20130101; A61K 49/00
20130101; G01N 2800/24 20130101; G16H 50/70 20180101; G01N 33/6893
20130101; G01N 2800/102 20130101 |
Class at
Publication: |
424/9.2 ;
424/133.1; 424/142.1 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61K 39/395 20060101 A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2010 |
EP |
10382346.4 |
Claims
1. A method for classifying a patient suffering from rheumatoid
arthritis as a responder or as a non-responder patient to a
treatment, said treatment comprising the administration to said
patient of a biological drug selected from the group consisting of
infliximab and adalimumab that is periodically administered by
repetitive administrations, and wherein said patient has received
at least one dose from said biological drug, said method comprising
the steps of: a) determining the concentration of the circulating
biological drug in a sample from said patient at a time t1 wherein
said t1 corresponds to a time point within the period of time
between two successive administrations of said biological drug; b)
determining the concentration of antibodies against said biological
drug in a sample from said patient at a time t1; and c) comparing
the concentration of the circulating biological drug at said t1
with a Reference Value 1 (RV1) and the concentration of antibodies
against said biological drug at said time t1 with a Reference Value
2 (RV2), wherein the sample is selected from a sample of blood, a
sample of plasma and a sample of serum; RV1 is a therapeutic
efficiency cut-off value of the concentration of the circulating
biological drug; and RV2 is the cut-off value of the concentration
of said antibody against the biological drug as determined in a
group of treatment-naive individuals by the same assay as that used
for determining the concentration of the antibody against the
biological drug in step b), and wherein if the concentration of the
circulating biological drug is lower than RV1 and the concentration
of antibodies against said biological drug is higher than RV2, then
said patient is classified as a non-responder patient to said
treatment, and wherein if the concentration of the circulating
biological drug is equal to, or higher than RV1 and the
concentration of antibodies against said biological drug is equal
to, or lower than, RV2, then said patient is classified as a
responder patient to said treatment, and wherein if the biological
drug is infliximab, then RV1 is 1.5 .mu.g/ml; and wherein if the
biological drug is adalimumab, then RV1 is 0.8 .mu.g/ml.
2. The method according to claim 1, wherein said t1 is a time point
within two weeks of the administration of the biological drug.
3. The method according to claim 1, wherein the concentration of
the circulating biological drug is determined by an
immunoassay.
4. The method according to claim 1, wherein the concentration of
antibodies against said biological drug is determined by an
immunoassay.
5. The method according to claim 3, wherein the immunoassay is an
ELISA.
6. The method according to claim 4, wherein the immunoassay is an
ELISA.
7. The method according to claim 1, wherein said t1 is a time point
within one week of the administration of the biological drug.
8. The method according to claim 1, wherein if the biological drug
is infliximab, then RV2 is 150 ng/ml, and wherein if the biological
drug is adalimumab, then RV2 is 32 ng/ml.
9. A method for treating a patient suffering from rheumatoid
arthritis, comprising: a) administering to said patient a
biological drug at a time, ti, wherein the biological drug is
selected from the group consisting of infliximab and adalimumab; b)
determining the concentration of the circulating biological drug in
a sample from said patient at a time, ti, wherein t1 is a time
point after ti; c) determining the concentration of antibodies
against said biological drug in a sample from said patient at t1;
and d) comparing the concentration of the circulating biological
drug at ti with a Reference Value 1 (RV1) and the concentration of
antibodies against said biological drug at t1 with a Reference
Value 2 (RV2), wherein: the sample is selected from a sample of
blood, a sample of plasma and a sample of serum; RV1 is a
therapeutic efficiency cut-off value of the concentration of the
circulating biological drug; RV2 is the cut-off value of the
concentration of said antibody against the biological drug as
determined in a group of treatment-naive individuals by the same
assay as that used for determining the concentration of the
antibody against the biological drug in step c); wherein if the
concentration of the circulating biological drug is equal to, or
higher than RV1 and the concentration of antibodies against said
biological drug is equal to, or lower than, RV2, then classifying
the patient as a responder and repeating step a); and wherein if
the concentration of the circulating biological drug is equal to,
or higher than RV1 and the concentration of antibodies against said
biological drug is equal to, or lower than, RV2, then classifying
the patient as a non-responder and revising treatment.
10. The method according to claim 9, wherein t1 is a time point
within two weeks of ti.
11. The method according to claim 9, wherein t1 is a time point
within one week of ti.
12. The method according to claim 9, wherein the concentration of
the circulating biological drug is determined by an
immunoassay.
13. The method according to claim 12, wherein the immunoassay is an
ELISA.
14. The method according to claim 9, wherein the concentration of
antibodies against said biological drug is determined by an
immunoassay.
15. The method according to claim 14, wherein the immunoassay is an
ELISA.
16. The method according to claim 1, wherein if the biological drug
is infliximab, then RV1 is 1.5 .mu.g/ml, and wherein if the
biological drug is adalimumab, then RV1 is 0.8 .mu.g/ml.
17. The method according to claim 16, wherein if the biological
drug is infliximab, then RV2 is 150 ng/ml, and wherein if the
biological drug is adalimumab, then RV2 is 32 ng/ml.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is the U.S. National Phase application of
PCT International Application No. PCT/EP2011/073836, filed Dec. 22,
2011, and claims priority to European Patent Application No. EP
10382346.4, filed Dec. 22, 2010, the disclosures of which are
incorporated by reference in their entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of personalized
therapy and, in particular, to a method for classifying a patient
suffering from rheumatoid arthritis as a responder or as a
non-responder patient to a treatment based on a biological drug
selected from infliximab and adalimumab.
BACKGROUND OF THE INVENTION
[0003] The use of biological drugs has increased during the last
years. For example, the TNF-alpha neutralizing antibodies are
increasingly being used to treat diseases with a strong
inflammatory background like rheumatoid arthritis (RA) and
Inflammatory Bowel Disease (IBD).
[0004] Chronic inflammatory diseases represent a group of
heterogeneous conditions characterized by an elevated production of
cytokines, molecules which are essential for an organism's immune
response, that play a critical role in the pathology of these
diseases. Among the many cytokines Tumor Necrosis Factor alpha
(TNF-alpha) is a key player due to its role in initiating the
cascade of inflammatory processes and thus for triggering the
development of disease symptoms in individuals suffering from an
inflammatory disease.
[0005] The latest generation of biological drugs for the treatment
of inflammatory diseases is based on antibody constructs that exert
their effect by binding to TNF-alpha thus blocking initiation of
the inflammatory cascade. While potentially very effective the use
of these drugs requires the patient's response to the treatment to
be closely monitored in order, if necessary, to guide the treatment
regime.
[0006] In practice the response to treatment of patients with
autoimmune and inflammatory diseases is monitored based on a number
of clinical variables that reflect aspects of the disease process.
Increasing the uniformity and consistency of methods used to
measure patient response to treatment with anti-TNF-alpha
biological drugs will help in optimizing dosing and contribute to a
better use of expensive therapeutics by health care providers for
the benefit of their patients.
[0007] Due to their structure and nature anti-TNF-alpha biological
drugs are highly immunogenic. Unfortunately an immune response in
the patient against biological drugs can dramatically reduce the
efficacy of treatment.
[0008] Therefore, there is a need in the art for methods suitable
for the determination of the patient response to a treatment based
in a biological drug that are more specific than the conventional
methods, and particularly for methods suitable for determining the
response of a patient suffering from rheumatoid arthritis to a
treatment based in an anti-TNF-alpha biological drug selected from
infliximab and adalimumab.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1. Infliximab bioavailability and immunogenicity
profiles over one year of treatment (top). The average values are
shown for the group of patients showing no response to infliximab.
.DELTA.DAS28 is indicated for each infusion time (bottom).
.DELTA.DAS28 does not increase over the year of treatment (bottom).
The horizontal line in the bottom panel indicates the
.DELTA.DAS28=1.2 threshold.
[0010] FIG. 2. Infliximab bioavailability and immunogenicity
profiles over one year of treatment (top). The average values are
shown for the group of patients showing response to infliximab.
.DELTA.DAS28 is indicated for each infusion time (bottom).
.DELTA.DAS28 does not increase over the year of treatment (bottom).
The horizontal line in the bottom panel indicates the
.DELTA.DAS28=1.2 threshold.
[0011] FIG. 3. DAS28 as a function of bioavailability (infliximab
concentration) and immunogenicity (antibody against infliximab)
variables.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0012] The term "antibody" (also known as immunoglobulins,
abbreviated Ig), as used herein is intended to gamma-globulin
proteins that are found in blood or other bodily fluids of
vertebrates, and that can bind to a target in an specific way. They
are typically made of basic structural units, each with two large
heavy chains and two small light chains, to form, for example,
monomers with one unit, dimers with two units or pentamers with
five units. The antibodies that can be used in the present
invention as biological drugs, directed to different targets, are
commercial products or can be obtained by conventional methods
known by the person skilled in the art. Also antibody fragments can
be used. An antibody fragment is a fragment of an antibody such as,
for example, Fab, F(ab')2, Fab', scFv, diabodies, etc. Various
techniques have been developed for the production of antibody
fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies but more recently these
fragments can be produced directly by recombinant host cells.
Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment. Pepsin
treatment yields an F(ab')2 fragment that has two antigen-binding
sites and is still capable of cross-linking antigen. "Fv" is the
minimum antibody fragment which contains a complete
antigen-recognition and antigen-binding site. Fab' fragments differ
from Fab fragments by the addition of a few residues at the carboxy
terminus of the heavy chain CHI domain including one or more
cysteines from the antibody hinge region. Other chemical couplings
of antibody fragments are also known. A single chain Fv (scFv)
fragment comprises the VH and VL domains of an antibody, wherein
these domains are present in a single polypeptide chain, and may be
monospecific or bispecific. The term "diabodies" refers to small
antibody fragments with two antigen-binding sites, which fragments
comprise a heavy-chain variable domain (VH) connected to a light
chain variable domain (VL) in the same polypeptide chain
(VH-VL).
[0013] By "anti-TNF-alpha for "anti-TNF-.alpha.", or "anti-TNF-a"
or simply "anti-TNF") therapy (or treatment)" is meant the
administration to a patient of a biological drug or biological
molecule (biopharmaceutical) capable of blocking, inhibiting,
neutralizing, preventing receptor binding, or preventing TNFR
activation by TNF-alpha. TNFR ("tumor necrosis factor receptor"),
or death receptor, is a cytokine receptor that binds tumor necrosis
factors (e.g., TNF-alpha) (Locksley R M, et al. 2001. Cell 104 (4):
487-501). Illustrative, non-limitative examples of such biological
drugs include inhibitory antibodies against TNF-alpha as well as
compounds, other than antibodies, capable of binding to TNF-alpha,
e.g., proteins, peptides, small chemical molecules, etc.
[0014] The term "biological drug", as used herein, refers to any
substance made or obtained from a living organism or its products
that is used in the prevention, diagnosis or treatment of a
pathology, e.g., a human pathology, like antibodies such as
IgG-like antibodies, Fab fragments, etc.; thus, a biological drug
or biopharmaceutical is a medical drug produced using
biotechnology, for example, a protein (including antibodies), a
nucleic acid (DNA, RNA or antisense oligonucleotides), used for
therapeutic or in vivo diagnostic purposes, and, generally are
produced by means other than direct extraction from a native
(non-engineered) biological source.
[0015] The term "dose" or "therapeutically effective amount" is
used herein to mean an amount sufficient to prevent, and preferably
reduce by at least about 25 percent, more preferably by at least 50
percent, most preferably by at least 90 percent, a clinically
significant change in a feature of pathology. As related to the
present invention, the term may also mean an amount sufficient to
ameliorate or reverse one or more symptoms associated with a
disease.
[0016] By "rheumatoid arthritis" or "RA" is meant a chronic,
systemic inflammatory disorder that may affect many tissues and
organs, but principally attacks synovial joints. The process
produces an inflammatory response of the synovium (synovitis)
secondary to hyperplasia of synovial cells, excess synovial fluid,
and the development of pannus in the synovium. The pathology of the
disease process often leads to the destruction of articular
cartilage and ankylosis of the joints. Rheumatoid arthritis can
also produce diffuse inflammation in lungs, pericardium, pleura,
and sclera, and also nodular lesions, most common in subcutaneous
tissue under the skin. Although the cause of RA is unknown,
autoimmunity plays a pivotal role in both its chronicity and
progression, and RA is considered as a systemic autoimmune
disease.
[0017] The term "patient", as used herein, refers to all animals
classified as mammals and includes, but is not restricted to,
domestic and farm animals, primates and humans, e.g., human beings,
non-human primates, cows, horses, pigs, sheep, goats, dogs, cats,
or rodents. Preferably, the patient is a male or female human of
any age or race.
[0018] The term "sample", as used herein, relates to any sample
which can be obtained from the patient, namely, a sample
susceptible of containing antibodies. Thus, the present method can
be applied to practically any type of biological sample from a
patient, such as a biopsy sample, tissue, cell or fluid, e.g.,
blood, brain extracts, cerebral spinal fluid (CSF), milk, mucus,
plasma, saliva, semen, serum, sputum, sweat, tears, and the like.
In a particular embodiment, said sample is selected from blood,
plasma or serum.
[0019] The term "TNF-alpha" (abbreviated herein as "TNF-.alpha.",
"TNFa" or simply "TNF"), as used herein, is intended to refer to a
human cytokine that exists as a 17 kD secreted form and a 26 kD
membrane associated form, the biologically active form of which is
composed of a trimer of noncovalently bound 17 kD molecules. The
sequence of human TNF-alpha is shown in CAA26669.1 (SEQ ID NO: 1).
The term "TNF-alpha" as used herein not only includes the human
gene and protein but also their orthologues from other species such
as dogs, mice, rats, etc., as well as functionally equivalent
variants thereof.
Method of the Invention
[0020] The authors of the present invention have now found that a
specific concentration of a circulating biological drug (e.g.,
infliximab, adalimumab, etc.) together with, optionally, a specific
concentration of antibodies to said biological drug
(anti-biological drug antibodies), particularly as determined in a
blood sample from a patient, said patient suffering from rheumatoid
arthritis and being treated with said biological drug, is
associated with the patient's response to the treatment with said
biological drug.
[0021] This information would allow physicians (medical doctors) to
follow more closely their patients' response to treatment and to
make informed decisions over treatment. These biological variables
have been clinically validated to show utility in correlating both
the levels of circulating biological drugs and the levels of
antibodies against said biological drugs with the efficacy of
treatment based on the patients Disease Activity Score (DAS28) used
to follow up the disease in patients.
[0022] Thus, in an aspect, the present invention refers to a
method, hereinafter referred to as the "method of the invention",
for classifying a patient suffering from rheumatoid arthritis as a
responder or as a non-responder patient to a treatment, said
treatment comprising the administration to said patient of a
biological drug selected from the group consisting of infliximab
and adalimumab that is periodically administered by repetitive
administrations, and wherein said patient has received at least one
dose from said biological drug, said method comprising the steps
of: [0023] a) determining the concentration of the circulating
biological drug in a sample from said patient at a time t1 wherein
said t1 corresponds to a time point within the period of time
between two successive administrations of said biological drug; and
[0024] b) comparing the concentration of the circulating biological
drug at said t1 with a Reference Value 1 (RV1), wherein [0025] RV1
is a therapeutic efficiency cut-off value of the concentration of
the circulating biological drug; and wherein if the concentration
of the circulating biological drug is lower than RV1, then said
patient is classified as a non-responder patient to said treatment,
and wherein if the concentration of the circulating biological drug
is equal to, or higher than, RV1, then said patient is classified
as a responder patient to said treatment, and wherein if the
biological drug is infliximab, then RV1 is 1.5 .mu.g/ml; and
wherein if the biological drug is adalimumab, then RV1 is 0.8
.mu.g/ml.
[0026] In the method of the invention, the pathology which is
treatable with a biological drug is a pathology wherein TNF-alpha
is involved, specifically rheumatoid arthritis. The patient is
subjected to an anti-TNF-alpha treatment comprising the
administration to said patient of a biological drug selected from
the group consisting of infliximab and adalimumab that is
periodically administered by repetitive administrations, said
biological drug being capable of blocking, inhibiting,
neutralizing, preventing receptor binding, or preventing TNFR
activation by TNF-alpha.
[0027] Said biological drug is an antibody or a fragment thereof;
particularly, an inhibitory anti-TNF-alpha antibody. An "inhibitory
anti-TNF-alpha antibody" or "inhibitory antibody against
TNF-alpha", as used herein, refers to an antibody which is capable
of preventing TNFR activation by TNF-alpha and thus the initiation
of the inflammation cascade. A "tumor necrosis factor receptor
(TNFR)", or death receptor, is a cytokine receptor that binds tumor
necrosis factors (e.g., TNF-alpha) (Locksley R M, et al. 2001. Cell
104 (4): 487-501). The determination of the inhibiting capacity on
the TNFR activation by TNF-alpha can be detected using standard
assays to measure the activation of TNFR such as the ones described
by Solorzano et al. (Solorzano C. C. et al. 1998. J Appl Physiol
84: 1119-1130) or by Hyunil et al. (Hyunil Ha et al. 2009. Current
Protocols in Immunology Chapter 11 Unit11.9D).
[0028] Inhibitory antibodies, or fragments thereof, against
TNF-alpha may be readily available, or may be readily produced by
using conventional molecular biology techniques. By illustrative,
using immunogens derived from, for example, the TNF-alpha molecule
it is possible to obtain anti-protein/anti-peptide antisera or
monoclonal antibodies by using standard protocols (see, for
example, "Antibodies: A Laboratory Manual", ed. by Harlow and Lane
(Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, a
hamster or rabbit can be immunized with an immunogenic form of the
peptide (e.g., TNF-alpha or an antigenic fragment thereof, which is
capable of eliciting an antibody response). Techniques for
conferring immunogenicity on a protein or peptide, include
conjugation to carriers or other techniques, are well known in the
art. An immunogenic portion of a polypeptide can be administered in
the presence of adjuvant. The progress of immunization can be
monitored by detection of antibody titers in plasma or serum.
Standard ELISA or other immunoassays can be used with the immunogen
as antigen to assess the levels of antibodies. In a preferred
embodiment, the antibodies forming part of the compositions of the
invention are immuno-specific for antigenic determinants of
TNF-alpha (or a variant at least 80%, 85%, 90%, 95%, or 98%
identical thereto). In certain embodiment, the immunospecific
subject antibodies do not substantially cross react with a
non-vertebrate (such as yeast) TNF-alpha related protein. By "not
substantially cross react" it is meant that the antibody has a
binding affinity for a non-homologous protein which is at least one
order of magnitude, more preferably at least 2 orders of magnitude,
and even more preferably at least 3 orders of magnitude less than
the binding affinity of the antibody for a TNF-alpha.
[0029] Thus, the antibody which can be used for the purposes of the
instant invention as an inhibitory antibody against TNF-alpha is
capable of binding to an epitope of TNF-alpha; typically, at least
6, 8, 10, or 12, contiguous amino acids are required to form an
epitope, however, epitopes which involve non-contiguous amino acids
may require more, e.g., at least 15, 25, or 50 amino acid.
Illustrative TNF-alpha inhibitory antibodies include, for example,
polyclonal antibodies, monoclonal antibodies (mAbs), Fab and scFv
fragments thereof, bispecific antibodies, heteroconjugates, human
and humanized antibodies, etc. Such antibodies may be produced in a
variety of ways, including hybridoma cultures, recombinant
expression in bacteria or mammalian cell cultures, and recombinant
expression in transgenic animals. Also antibodies can be produced
by selecting a sequence from a library of sequences expressed in
display systems such as filamentous phage, bacterial, yeast or
ribosome. There is abundant guidance in the literature for
selecting a particular production methodology (see, e.g., Chadd and
Chamow, Curr. Opin. Biotechnol., 12:188-194 (2001).
[0030] The inhibitory antibody against TNF-alpha is an inhibitory
mAb to TNF-alpha including, but not limited to, the antibodies sold
under the generic names of Infliximab (Remicade.RTM., Johnson and
Johnson; described in U.S. Pat. No. 5,656,272), Adalimumab
(Humira.RTM., Abbott Laboratories, a human anti-TNF-alpha mAb
described in U.S. Pat. No. 6,090,382 as D2E7), etc., and antibodies
in clinical development such as Golimumab (or CNTO 148; WO
02/12502), etc. Further examples of anti-TNF-alpha antibodies, or
fragments thereof, include CDP571 (a humanized monoclonal
anti-TNF-alpha IgG4 antibody), CDP 870 (a humanized monoclonal
anti-TNF-alpha antibody fragment), an anti-TNF dAb (Peptech),
etc.
[0031] Additional antibodies to human TNF-alpha and fragments
thereof are disclosed in U.S. Pat. No. 6,593,458, U.S. Pat. No.
6,509,015, U.S. Pat. No. 6,498,237, U.S. Pat. No. 6,451,983, U.S.
Pat. No. 6,448,380, U.S. Pat. No. 6,258,562, U.S. Pat. No.
6,090,382, U.S. Pat. No. 7,223,394 and U.S. patent application US
2003/0219438 A1.
[0032] In a preferred embodiment, the biological drug is an
inhibitory mAb to TNF-alpha selected from the group consisting of
infliximab and adalimumab.
[0033] The method of the invention allows for classifying a patient
as a responder or as a non-responder patient to a treatment, said
treatment comprising the administration to said patient of a
biological drug selected from infliximab and adalimumab that is
periodically administered by repetitive administrations, wherein
said patient suffers from rheumatoid arthritis which is treatable
with said biological drug under said treatment and wherein said
patient has received at least one dose from said biological
drug.
[0034] According to the method of the invention, the concentration
of the circulating biological drug in a sample from the patient
under study is determined at a time t1, wherein said time t1
corresponds to a time point within the period of time between two
successive administrations of said biological drug [step a)]. This
step is intended to determine the bioavailability of the biological
drug administered to the patient under study.
[0035] The term "t1" corresponds to a time point within the period
of time between two successive administrations of said biological
drug to the patient. The period of time between two successive
administrations, hereinafter referred to as "period of time ti-tj",
may vary within a broad range, for example, said period of time may
comprise, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or even
more days; typically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even
more weeks, or 1, 2, 3, 4, 5, 6 or even more months. In a
particular embodiment, t1 is a time point in the first half of said
period of time ti-tj; in another particular embodiment, t1 is a
time point in the second half of said period of time ti-tj; and, in
another particular embodiment, t1 is a time point around the half
of said period of time ti-tj. By illustrative, the period of time
ti-tj may be 4 weeks and t1 may be a time point within the first
half of said period of time ti-tj (e.g., day 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13 or 14 after the day of the prior
administration (ti)); alternatively, the period of time ti-tj may
be 4 weeks and t1 may be a time point within the second half of
said period of time ti-tj (e.g., day 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27 or 28 after the day of the prior administration
(ti)); or alternatively, the period of time ti-tj may be 4 weeks
and t1 may be a time point around the half of said period of time
ti-tj (e.g., day 13, 14, 15, 16, or 17 after the day of the prior
administration (ti)). In a particular embodiment, said period of
time ti-tj is 4 weeks and t1 is a time point within the second half
of said period of time ti-tj, i.e., within the last two weeks of
said period of time ti-tj, preferably within the last week of said
period of time ti-tj, more preferably 6, 5, 4 or 3 days before the
day of the following administration (tj).
[0036] The precise dose to be administered to the patient will
depend, among other features, on the route of administration, and
the seriousness of the disease or disorder to be treated, and
should be decided according to the judgment of the physician and
the patient's needs. Generally, the biological drug is administered
to the patient in need of treatment at a dose of approximately
0.005 mg per kilogram of body weight to approximately 50 mg per
kilogram of body weight; typically the dose ranges from
approximately 0.5 mg per kilogram of body weight to approximately
15 mg per kilogram of body weight. In a preferred embodiment the
dose is between 3 and 5 mg/kg intravenously. In another preferred
embodiment the dose is approximately 0.7 mg/kg subcutaneously.
[0037] The term "circulating biological drug" as used herein,
relates to the biological drug that is present in a fluid of the
patient's body (e.g., blood, serum, milk, etc.) and can be detected
by using standard methods.
[0038] The biological drug of the method of the invention is an
antibody. The concentration of an antibody can be determined by
practically any method known by the person skilled in the art, such
as, an immunoassay, for example, an ELISA (Enzyme-Linked
Immunosorbent Assay), ELISA Using Slope Correction, RIA
(radioimmunoassay), competitive EIA (competitive enzyme
immunoassay), DAS-ELISA (double antibody sandwich-ELISA),
bridging-ELISA, techniques based on the use of protein or antibody
microarrays, technologies based on discrete microparticles, assays
based on the precipitation of colloidal gold, affinity
chromatography techniques, ligand binding assays, lectin binding
assays, biosensors, etc., preferably by an immunoassay. In a
particular embodiment, the concentration of the circulating
antibodies (biological drug) is measured by an ELISA, as it is
shown in the examples of the present invention.
[0039] In a second step [step b)], the method of the invention
comprises comparing the concentration of the circulating biological
drug at said t1 with a Reference Value 1 (RV1) wherein RV1 is a
therapeutic efficiency cut-off value of the concentration of the
circulating biological drug.
[0040] The term "therapeutic efficiency cut-off value of the
concentration of the circulating biological drug" (RV1), as used
herein, relates to the concentration of the biological drug that is
available in the circulating blood for which no positive titer of
antibody against the biological drug is measured in the same
sample. The term "no positive titer" as used herein is equivalent
to a value below RV2, the term "RV2" being defined below.
[0041] Further, according to the method of the invention, if the
concentration of the circulating biological drug is lower than RV1,
then said patient is classified as a non-responder patient to said
treatment/biological drug.
[0042] On the contrary, if a t1, the concentration of the
circulating biological drug is equal to, or higher than, RV1, then
said patient is classified as a responder patient to said
treatment/biological drug.
[0043] In a more specific embodiment, the biological drug is
infliximab and RV1 is 1.5 .mu.g/ml. In another more specific
embodiment, the biological drug is adalimumab and RV1 is 0.8
.mu.g/ml.
[0044] In a particular embodiment, the method of the invention
further comprises, in addition to the determination of the
concentration of said circulating biological drug (infliximab or
adalimumab), the determination of the concentration of antibodies
to said biological drug; thus in a particular embodiment, the
method of the invention comprises the steps of: [0045] 1)
determining the concentration of the circulating biological drug in
a sample from said patient at a time t1 wherein said t1 corresponds
to a time point within the period of time between two successive
administrations of said biological drug, [0046] 2) determining the
concentration of antibodies against said biological drug in a
sample from said patient at a time; and [0047] 3) comparing the
concentration of the circulating biological drug at said t1 with a
Reference Value 1 (RV1) and the concentration of antibodies against
said biological drug at said time t1 with a Reference Value 2
(RV2), wherein [0048] RV1 is a therapeutic efficiency cut-off value
of the concentration of the circulating biological drug; and [0049]
RV2 is the cut-off value of the concentration of said antibody
against the biological drug as determined in a group of
treatment-naive individuals by the same assay as that used for
determining the concentration of the antibody against the
biological drug in step 2), and wherein if the concentration of the
circulating biological drug is lower than RV1 and the concentration
of antibodies against said biological drug is higher than RV2, then
said patient is classified as a non-responder patient to said
treatment, and wherein if the concentration of the circulating
biological drug is equal to, or higher than, RV1 and the
concentration of antibodies against said biological drug is equal
to, or lower than, RV2, then said patient is classified as a
responder patient to said treatment; and wherein if the biological
drug is infliximab, then RV1 is 1.5 .mu.g/ml; and wherein if the
biological drug is adalimumab, then RV1 is 0.8 .mu.g/ml.
[0050] Said particular embodiment comprises a first step 1) which
is equivalent to the first step [step a)] previously described for
the method of the invention. In a second step [step 2) of this
particular embodiment], the method of the invention comprises
determining the concentration of antibodies against said biological
drug in a sample from said patient at said time t1. This step is
intended to determine the immunogenicity of the biological drug
administered to the patient under study. Examples 1 and 2 of the
present invention show a method including said step. This step is
not necessary in order to classify a patient as a responder or
non-responder to a biological drug since it is sufficient to
determine the levels of concentration of the circulating biological
drug in a sample of said patient to obtain a good correlation with
the clinical response. This is shown in Examples 3 and 4 of the
present invention.
[0051] The term "antibody against a biological drug" as used
herein, relates to any antibody that the immunosystem of the
patient treated with said biological drug produces that binds
specifically to said biological drug. Types of antibodies include
IgA, IgD, IgE, IgG and IgM. The concentration of antibodies against
a biological drug can be measured by any method known by the person
skilled in the art, for example, an immunoassay, e.g., ELISA, ELISA
Using Slope Correction, RIA, competitive ETA, DAS-ELISA,
bridging-ELISA, techniques based on the use of antibody
microarrays, etc., as it has been previously discussed. In a
particular embodiment, the concentration of the antibodies against
a biological drug is measured by an ELISA, as it is shown in the
Examples 1 and 2 of the present invention.
[0052] If low amount of antibodies against a biological drug are
present in the sample, these can be complexed with the biological
drug, and, therefore, they would not be detected in the
determination of the antibodies against the biological drug (i.e.,
immunogenicity determination). This is a phenomenon called "drug
interference". In order to measure the antibody titer in the
presence of drug-antibody complexes, complexes can be disaggregated
by using, for example, an acid dissociation protocol. In a
particular embodiment, the samples can be treated with an acid
(e.g., acetic acid) prior to perform the immunogenicity
determination, such as it is mentioned in the accompanying
examples.
[0053] In the third step [step 3) of this particular embodiment],
the method of the invention comprises comparing the concentration
of the circulating biological drug at said t1 with a Reference
Value 1 (RV1) and the concentration of antibodies against said
biological drug at said time t1 with a Reference Value 2 (RV2),
wherein RV1 is a therapeutic efficiency cut-off value of the
concentration of the circulating biological drug, and RV2 is the
cut-off value of the concentration of said antibody against the
biological drug as determined in a group of treatment-naive
individuals by the same assay as that used for determining the
concentration of the antibody against the biological drug in step
b) of said particular embodiment).
[0054] The term "therapeutic efficiency cut-off value of the
concentration of the circulating biological drug" (RV1), as defined
above, relates to the concentration of the biological drug that is
available in the circulating blood for which no positive titer of
antibody against the biological drug is measured in the same
sample. The term "no positive titer" as used herein is equivalent
to a value below RV2, the term "RV2" being defined below, i.e., the
cut-off value of the immunogenicity determination as determined in
a group of treatment-naive individuals by the same assay as that
used for determining the concentration of the antibody against the
biological drug in step 2) [i.e., if the concentration of
antibodies against said biological drug is measured by a
conventional ELISA assay in step 2), the concentration of
antibodies against said biological in the group of treatment-naive
individuals is also determined under the same conditions by using
the same conventional ELISA assay]. In order to generate RV1,
ideally the concentrations of the circulating biological drug in a
group of patients treated with the biological drug are used.
Preferably at least 2, more preferably 2, 3, 10, 20, 40, 100 or
even more patients are used.
[0055] In a more specific embodiment, the biological drug is
infliximab and RV1 is 1.5 .mu.g/ml. In another more specific
embodiment, the biological drug is adalimumab and RV1 is 0.8
.mu.g/ml.
[0056] The term "cut-off value of the concentration of said
antibody against the biological drug as determined in a group of
treatment-naive individuals by the same assay as that used for
determining the concentration of the antibody against the
biological drug in step 2)" (RV2) is understood in the present
invention the value that defines the background measurements of the
assay used for determining the concentration of the antibody
against the biological drug in step 2), what means, that above said
value, the measurements are true positive values since are above
the background or noise threshold of the method used.
[0057] The term "treatment-naive individuals" as used herein,
relates to subjects who are new (naive) to the biological drug
therapy, that is, that were never treated before with said therapy.
Thus, those subjects should not present antibodies against the
biological drug and thus the concentration measured in a group of
said subjects could be used to determine the cut-off value of the
immunogenicity determination. The group of naive subjects is
preferably formed by more than 1, preferably 2 or more, more
preferably 3 or more, most preferably 4, 10, 20, 30, 40, 50, 60,
70, 80, 90, 100, 500, or even more treatment-naive individuals. The
cut off-value is then calculated as the mean of the values obtained
from each of the treatment-naive individuals.
[0058] In a particular embodiment, the immunoassay is an ELISA. In
a more specific embodiment, the immunoassay is an ELISA, the
biological drug is infliximab and RV2 is 150 ng/ml. In another more
specific embodiment, the immunoassay is an ELISA, the biological
drug is adalimumab and RV2 is 32 ng/ml.
[0059] Further, according to said particular embodiment of the
method of the invention, if the concentration of the circulating
biological drug is lower than RV1 and the concentration of
antibodies against said biological drug is higher than RV2, then
said patient is classified as a non-responder patient to said
treatment/biological drug.
[0060] On the contrary, if a t1, the concentration of the
circulating biological drug is equal to, or higher than, RV1 and
the concentration of antibodies against said biological drug is
equal to, or lower than, RV2, then said patient is classified as a
responder patient to said treatment/biological drug.
[0061] The term "responder patient" as used herein, relates to
patients for which the predicted response to the
treatment/biological drug is positive. Similarly, the term
"non-responder patient" as used herein, relates to patients for
which the predicted response to the treatment/biological drug is
negative.
[0062] The term "predicted response" or similar, as used herein
refers to the determination of the likelihood that the patient will
respond either favorably or unfavorably to a given
therapy/biological drug. Especially, the term "prediction", as used
herein, relates to an individual assessment of any parameter that
can be useful in determining the evolution of a patient. As it will
be understood by those skilled in the art, the prediction of the
clinical response to the treatment with a biological drug, although
preferred to be, need not be correct for 100% of the subjects to be
diagnosed or evaluated. The term, however, requires that a
statistically significant portion of subjects can be identified as
having an increased probability of having a positive response.
Whether a subject is statistically significant can be determined
without further effort by the person skilled in the art using
various well known statistic evaluation tools, e.g., determination
of confidence intervals, p-value determination, Student's t-test,
Mann-Whitney test, etc. Details are found in Dowdy and Wearden,
Statistics for Research, John Wiley & Sons, New York 1983.
Preferred confidence intervals are at least 50%, at least 60%, at
least 70%, at least 80%, at least 90% at least 95%. The p-values
are, preferably, 0.2, 0.1 or 0.05.
[0063] The term "clinical response", as used herein, refers to the
response to a biological drug of the subject suffering from a
pathology which is treatable with said biological. Standard
criteria may vary from disease to disease.
[0064] Patients achieving complete or partial response are
considered "responders", and all other patients are considered
"non-responders".
[0065] The response in individual patients may be characterized as
a complete response, a partial response, stable disease, and
progressive disease, as these terms are understood in the art.
Thus, the method of the invention allows for classifying a patient
as a responder or as a non-responder patient to a treatment, said
treatment comprising the administration to said patient of a
biological drug selected from the group consisting of infliximab
and adalimumab that is periodically administered by repetitive
administrations, wherein said patient suffers from rheumatoid
arthritis which is treatable with said biological drug under said
treatment and wherein said patient has received at least one dose
from said biological drug.
[0066] In the case of rheumatoid arthritis, the standard used is
the Disease Activity Score determination (DAS28, see table 1).
DAS28 is determined according to the European League against
Rheumatism (EULAR) (Aletaha D, et al. 2010. Ann. Rheum. Dis. 69
(9): 1580-8).
[0067] In a more particular embodiment, the biological drug is
infliximab. Infliximab is a mAb anti-TNF-alpha which can be used
for the treatment of, for example, psoriasis, Crohn's disease,
ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis
and ulcerative colitis. In another particular embodiment the
biological drug is adalimumab. Adalimumab is a TNF-alpha inhibitor
which can be used for the treatment of, for example, rheumatoid
arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's
disease, moderate to severe chronic psoriasis and juvenile
idiopathic arthritis. In a specific embodiment, said pathology of
the patient to be treated by a biological drug is rheumatoid
arthritis and said biological drug is infliximab, RV1 is 1.5
.mu.g/ml and RV2 is 150 ng/ml (as determined by ELISA).
[0068] In another specific embodiment, said pathology of the
patient to be treated by a biological drug is rheumatoid arthritis
and said biological drug is adalimumab, RV1 is 0.8 .mu.g/ml and RV2
is 32 ng/ml (as determined by ELISA).
[0069] The following examples illustrate the invention and should
not be considered as limitative of the scope thereof.
EXAMPLES
[0070] The following methods were common to all the Examples (as
applicable).
Methods
Sampling
[0071] Blood samples from patients (Examples 1 and 2) were
collected before each infusion, and serum was obtained, aliquoted
and stored at -20.degree. C.
Analysis
[0072] An aliquot of each serum was defrozen. All sera were tested
simultaneously for free biological drug concentration
(bioavailability) and anti-biological drug antibodies
(immunogenicity).
Bioavailability Analysis
[0073] An immunoassay was designed to specifically measure the
concentration of the free biological drug (antibodies to human
TNF-alpha infliximab or adalimumab) in the sera of patients.
Briefly, a 96-well ELISA plate was coated with human recombinant
TNF-alpha, whose amino acid sequence is shown in SEQ ID NO: 2, via
a murine anti-TNF-alpha monoclonal antibody. Recombinant human
TNF-alpha was obtained in Escherichia coli, by cloning into an
expression vector. The protein was expressed as a fusion protein
bearing a six histidine tag in its amino terminus and purified by
affinity chromatography.
[0074] Sera were incubated in the plate in different serial
dilutions and replicas. If the biological drug is present in the
serum of the patient, it will bind to the fixed TNF-alpha.
Detection of the bound biological drug takes place with a
biotinylated monoclonal antibody to said biological drug
(infliximab or adalimumab). After washing, the amount of bound
biotinylated antibody can be measured by incubating with
streptavidin-polyHRP (Fitzgerald Industries Limited), and after
another washing step incubating with 3,3',5,5'-tetramethylbenzidine
(TMB), the substrate for horseradish peroxidase (HRP). If the
result is positive a blue color will appear. The reaction is
stopped with HCl, which changes the blue color into yellow. This
yellow color can be measured in an ELISA reader at 450 nm. In
parallel, a calibration standard curve is constructed using pure
biological drug. This correlates the concentration of the
biological drug to a given absorbance intensity. The concentration
of free biological drug in the serum of each patient is obtained in
micrograms per milliliter (.mu.g/ml) of serum by extrapolation of
the absorbance intensity in the calibration curve.
Immunogenicity Standard Analysis
[0075] A bridging immunoassay was designed to specifically measure
the concentration of the immunoglobulins against the biological
drug in the sera of patients. Briefly, a 96-well ELISA plate is
coated with the biological drug. Sera are incubated in the plate in
different serial dilutions and replicas. If anti-biological drug
antibodies are present in the serum of the patient, they will bind
to the fixed biological drug. Finally, biotinylated biological drug
is added as a detection reagent. If anti-biological drug antibodies
are present in the serum of the patient, the biotinylated
biological drug will bind to them. After washing, the amount of
anti-biological drug antibodies can be measured by incubating with
streptavidin-polyHRP, and after another washing step incubating
with TMB, the substrate for HRP. If the result is positive a blue
color will appear. The reaction is stopped with HCl, which changes
the blue color into yellow. This yellow color can be measured in an
ELISA reader at 450 nm. In parallel, a calibration standard curve
is constructed using serial dilutions of a serum sample with known
concentration of anti-biological drug antibodies as a positive
control. This correlates the concentration of the antibodies to a
given absorbance intensity. The concentration of antibodies in the
serum of each patient is obtained in arbitrary units per milliliter
(AU/ml) of serum by extrapolation of the absorbance intensity in
the calibration curve. For quantification purposes, in the case of
infliximab, 1 AU/ml corresponds to 10 ng/ml of anti-infliximab
antibodies, and, in the case of adalimumab, 1 AU/ml corresponds to
4 ng/ml of anti-adalimumab antibodies.
Immunogenicity Analysis with Acid Dissociation Protocol
[0076] If low amount of anti-biological drug antibodies are present
in the sample, these can be complexed with the biological drug,
and, therefore, they would not be detected in the immunogenicity
analysis. This is a phenomenon called "drug interference". In order
to measure the antibody titer in the presence of biological
drug-antibody complexes, complexes were disaggregated using an acid
dissociation protocol. The samples were treated with acetic acid
prior to the analysis, and the immunogenicity analysis was carried
out as explained before.
Disease Activity Score determination (DAS28)
[0077] Disease Activity Score was determined by the rheumatologist
according to the European League against Rheumatism (EULAR)
criteria for every patient at each sampling point (Table 1). A high
value of DAS28 indicates the progression of the disease. A low
value of DAS28 indicates the remission of the disease.
TABLE-US-00001 TABLE 1 DAS28 cut-off values according to EULAR
Status of disease DAS28 ranges Remission <1.6 Low activity
<2.4 Moderate activity 2.4 .ltoreq. DAS28 .ltoreq. 3.7 High
activity >3.7 Classification of clinical response. In rheumatoid
arthritis patients, clinical response to the biological drug
treatment was assessed according to DAS28 index. Definition of
responder and non-responder was assessed one year after the
initiation of the treatment with the corresponding drug. According
to EULAR: patients were grouped as responders if: .DELTA.DAS28 =
DAS28.sub.predose - DAS28.sub.1 year .gtoreq. 1.2; and patients
were grouped as non-responders if: .DELTA.DAS28 = DAS28.sub.predose
- DAS28.sub.1 year < 1.2
Bioavailability and Immunogenicity Cut-Off Values Determination
[0078] Pre-dose samples of 52 patients (infliximab) and 10 patients
(adalimumab) were analysed in order to calculate the cut-off value
of both the concentration of the biological drug (bioavailability
cut-off) and the concentration of anti-biological drug antibodies
(immunogenicity cut-off), which were defined as the average of the
background signals of all the pre-dose sera plus 1.645 times the
standard deviation of all the values.
Bioavailability and Immunogenicity Reference Values
Determination
[0079] A reference value 1 (RV1) corresponds to the therapeutic
efficiency threshold for each of the circulating biological drugs
as detailed in the "Determination of the diagnostic cut-offs"
sections for infliximab and adalimumab as shown in Examples 1 and
2. A reference value 2 (RV2) corresponds to the lowest positive
titer of antibodies against each biological drug that can be
quantified (expressed in units (U) or ng (nanograms) per
milliliter) as detailed in the "Determination of the diagnostic
cut-offs" sections for infliximab and adalimumab as shown in
Examples 1 and 2.
Statistical Analysis
[0080] SigmaPlot v11.0 and SPSS v11.0 were used for data analysis.
Samples did not follow a normal distribution according to
Kolmogorov-Smirnov and Shapiro-Wilk tests. Therefore non-parametric
tests were used for the subsequent analysis. Kendall's Tau and
Spearman's Rho were used to study the correlation between
variables. P values less than 0.05 were considered as statistically
significant.
Example 1
Correlation of Bioavailability and Immunogenicity of Infliximab
with the Clinical Response of Patients Suffering from Rheumatoid
Arthritis
[0081] Subjects:
[0082] Rheumatoid arthritis (RA) patients, 75, fulfilling the
criteria of the American College of Rheumatology (ACR) of 1987
(1987 ACR criteria) [Arnett F. et al. (1988). "The American
Rheumatism Association 1987 revised criteria for the classification
of rheumatoid arthritis". Arthritis Rheum. 31 (3): 315-24] and
about to start treatment with infliximab, were enrolled
consecutively. A total of 612 serum samples from said 75 patients
suffering from RA were analysed.
[0083] Treatment:
[0084] Patients received doses of 3 mg/kg of infliximab
(Remicade.RTM.) intravenously. The frequency of drug administration
was as follows: first infusion on the first day of treatment
(baseline), second infusion two weeks later, third infusion one
month after the second, fourth infusion two months after the third,
and subsequent infusions at eight weeks interval.
[0085] Evaluation of the Patient Clinical Response:
[0086] Disease Activity Score 28 (DAS28) was used to evaluate the
clinical response of the patient according scores in Table 1. DAS28
is a validated method according to the European League against
Rheumatism (EULAR) criteria. One year clinical response was
determined as explained before.
[0087] Determination of Bioavailability and Immunogenicity of
Infliximab:
[0088] Table 2 summarizes the average values of bioavailability and
immunogenicity of infliximab.
TABLE-US-00002 TABLE 2 Average of infliximab bioavailability and
immunogenicity. Infliximab Immunogenicity bioavailability
(.mu.g/ml) (antibodies against infliximab) Sam- Av- (AU/ml) ples
erage SD Cmin Cmax Average SD Cmin Cmax 612 4.27 7.39 0.002 54.4
437.47 2490.69 1.000 45904.64 SD: standard deviation; Cmin: minimum
concentration; Cmax: maximum concentration.
[0089] Further, Table 3 summarizes the average values of infliximab
bioavailability and immunogenicity as a function of the
treatment.
TABLE-US-00003 TABLE 3 Average of infliximab bioavailability and
immunogenicity as a function of the treatment. Immunogenicity
(antibodies against Infusion Infliximab bioavailability (.mu.g/ml)
infliximab) (AU/ml) number Average SD Cmin Cmax N Average SD Cmin
Cmax N 0 0.002 0.000 0.002 0.002 35 1.00 0.00 1.00 1.00 35 1 23.17
9.67 5.05 48.59 36 1.00 0.00 1.00 1.00 36 2 11.31 9.34 0.002 38.99
37 1.91 3.30 1.00 15.00 37 3 2.87 3.95 0.002 14.87 34 42.50 130.26
1.00 718.92 34 4 1.58 2.57 0.002 11.88 29 200.68 477.53 1.00
1665.90 29 5 1.82 3.41 0.002 13.52 25 479.24 1705.36 1.00 8458.88
25 6 2.61 3.78 0.002 13.13 22 460.31 1226.83 1.00 4606.24 22 7 2.61
4.00 0.002 15.40 18 791.14 2324.35 1.00 9607.56 18 8 3.56 4.81
0.002 14.35 14 1516.52 5666.53 1.00 21204.28 14 SD: standard
deviation; Cmin: minimum concentration; Cmax: maximum
concentration; N, number of patients.
[0090] Tables 4-6 provide individual values of infliximab
bioavailability and immunogenicity as a function of the treatment
for each patient.
TABLE-US-00004 TABLE 4 Individual values of circulating infliximab
(IFX) and immunogenicity (antibodies). Infusion numbers 0 to 3 are
shown. Infusion number 0 Infusion number 1 Infusion number 2
Infusion number 3 IFX Antibodies IFX Antibodies IFX Antibodies IFX
Antibodies Patient (.mu.g/ml) (AU/ml) Patient (.mu.g/ml) (AU/ml)
Patient (.mu.g/ml) (AU/ml) Patient (.mu.g/ml) (AU/ml) 1 0.002 1.000
1 21.514 1.000 1 3.966 1.000 4 0.002 15.000 5 0.002 1.000 4 24.828
1.000 4 6.414 1.000 7 1.529 1.000 6 0.002 1.000 5 30.143 1.000 5
19.442 1.000 8 4.278 1.000 7 0.002 1.000 6 5.331 1.000 6 3.317
1.000 8 0.002 1.000 7 29.050 1.000 7 8.713 1.000 14 0.002 718.918
12 0.002 1.000 8 30.607 1.000 8 13.349 1.000 17 0.002 1.000 16
0.002 1.000 12 48.589 1.000 12 27.184 1.000 18 3.830 1.000 17 0.002
1.000 14 22.375 1.000 14 0.002 1.000 19 0.002 209.533 18 0.002
1.000 16 19.727 1.000 16 15.343 1.000 20 0.002 1.000 19 0.002 1.000
17 30.149 1.000 17 0.461 1.000 21 14.059 1.000 20 0.002 1.000 19
5.046 1.000 18 9.170 1.000 23 0.002 15.000 21 0.002 1.000 20 20.684
1.000 19 0.002 6.585 24 3.187 1.000 22 0.002 1.000 23 30.993 1.000
20 4.987 1.000 27 2.192 1.000 23 0.002 1.000 24 26.668 1.000 21
38.988 1.000 29 3.331 1.000 24 0.002 1.000 27 37.440 1.000 23 6.196
1.000 31 0.002 15.000 27 0.002 1.000 29 37.448 1.000 24 17.797
1.000 33 4.742 1.000 29 0.002 1.000 31 23.707 1.000 27 13.983 1.000
35 0.002 99.345 31 0.002 1.000 33 34.567 1.000 29 26.635 1.000 44
0.002 15.000 33 0.002 1.000 35 11.389 1.000 31 0.310 1.000 60 0.002
15.000 35 0.002 1.000 37 15.527 1.000 33 15.530 1.000 66 1.084
1.000 37 0.002 1.000 44 14.028 1.000 35 0.002 15.000 68 10.586
1.000 44 0.002 1.000 66 33.058 1.000 37 15.689 1.000 71 3.045 1.000
66 0.002 1.000 71 34.340 1.000 44 1.702 1.000 87 1.064 1.000 71
0.002 1.000 87 27.373 1.000 66 15.629 1.000 91 0.002 209.960 87
0.002 1.000 91 12.255 1.000 71 28.048 1.000 95 0.212 1.000 91 0.002
1.000 95 16.452 1.000 87 11.475 1.000 110 14.867 1.000 110 0.002
1.000 110 26.161 1.000 91 0.002 15.000 125 4.984 1.000 125 0.002
1.000 125 17.282 1.000 95 8.843 1.000 137 0.002 33.923 137 0.002
1.000 137 30.077 1.000 125 18.549 1.000 138 8.768 1.000 138 0.002
1.000 138 16.817 1.000 137 12.262 1.000 151 5.334 1.000 151 0.002
1.000 151 14.821 1.000 138 13.075 1.000 157 4.634 1.000 157 0.002
1.000 157 18.985 1.000 151 18.129 1.000 168 0.002 11.185 172 0.002
1.000 168 10.657 1.000 157 19.052 1.000 172 3.128 1.000 177 0.002
1.000 172 17.258 1.000 168 2.105 1.000 177 0.002 65.310 179 0.002
1.000 177 15.448 1.000 172 10.360 1.000 179 2.775 1.000 179 23.144
1.000 177 1.433 1.000 179 10.228 1.000
TABLE-US-00005 TABLE 5 Individual values of circulating infliximab
(IFX) and immunogenicity (antibodies). Infusion numbers 4 to 7 are
shown. Infusion number 4 Infusion number 5 Infusion number 6
Infusion number 7 IFX Antibodies IFX Antibodies IFX Antibodies IFX
Antibodies Patient (.mu.g/ml) (AU/ml) Patient (.mu.g/ml) (AU/ml)
Patient (.mu.g/ml) (AU/ml) Patient (.mu.g/ml) (AU/ml) 4 0.002
112.763 7 0.002 13.645 7 0.002 14.555 8 1.457 1.000 6 0.002 31.175
8 6.421 1.000 8 0.384 1.000 19 0.002 9607.560 7 0.040 1.000 18
0.002 1.000 19 0.002 4606.240 27 0.002 1.000 8 1.233 1.000 19 0.002
1676.180 27 0.002 35.580 29 2.255 1.000 14 0.002 1559.520 27 0.930
1.000 31 0.002 935.160 31 0.002 1352.732 17 0.002 200.373 29 0.807
1.000 33 12.269 1.000 33 8.662 1.000 18 4.579 1.000 31 0.002
297.668 44 0.002 15.000 44 0.002 15.000 19 0.002 1450.960 33 13.520
1.000 60 2.688 1.000 60 0.040 1.000 21 6.254 1.000 35 0.002 15.000
65 1.879 1.000 65 2.664 1.000 23 0.002 1.000 37 0.002 992.080 66
2.160 1.000 66 3.117 1.000 24 2.191 1.000 44 0.002 281.540 68
13.129 1.000 68 15.403 1.000 27 1.022 1.000 60 0.040 1.000 71 6.121
1.000 91 0.002 2985.120 29 0.731 1.000 66 0.911 1.000 87 0.954
1.000 95 0.002 15.000 31 0.002 117.420 68 9.977 1.000 91 0.002
3675.520 110 3.451 1.000 33 2.567 1.000 71 3.388 1.000 95 0.002
1.000 125 2.805 1.000 37 0.002 264.788 87 0.002 1.000 110 5.709
1.000 139 6.230 1.000 44 0.002 1665.900 91 0.002 8458.880 125 3.564
1.000 168 0.002 253.072 45 0.040 1.000 95 0.002 82.568 137 0.002
15.000 172 1.037 1.000 66 1.122 1.000 110 3.515 1.000 139 2.533
1.000 68 11.877 1.000 125 2.875 1.000 157 3.114 1.000 71 3.647
1.000 137 0.002 15.655 168 0.002 815.853 87 1.229 1.000 139 0.332
1.000 172 2.956 1.000 91 0.002 306.800 157 2.047 1.000 95 0.119
1.000 168 0.002 132.660 110 2.735 1.000 172 0.807 1.000 125 1.485
1.000 157 3.796 1.000 168 0.002 91.153 172 1.085 1.000
TABLE-US-00006 TABLE 6 Individual values of circulating infliximab
(IFX) and immunogenicity (antibodies). Infusion number 8 is shown.
Infusion number 8 Patient IFX (.mu.g/ml) Antibodies (AU/ml) 19
0.002 21204.280 27 0.298 1.000 29 2.057 1.000 33 14.348 1.000 44
0.002 15.000 54 0.298 1.000 60 5.181 1.000 65 6.815 1.000 66 1.861
1.000 68 13.291 1.000 95 0.426 1.000 110 1.780 1.000 139 3.466
1.000 168 0.040 1.000
[0091] After the analysis of the samples the following distribution
was observed for infliximab (Table 7).
TABLE-US-00007 TABLE 7 Distribution of patients according to
different combinations of infliximab bioavailability and
immunogenicity. Bioavailability/Immunogenicity Patients* Samples
positive/negative 68 390 negative/positive 35 142 negative/negative
51 80 positive/positive 0 0 *Due to multiple number of samples per
patient, patients can be present in more than one group.
[0092] FIG. 1 shows the average bioavailability and immunogenicity
profiles over one year of treatment of all the patients that do not
respond to the treatment with infliximab. In this example it is
shown that .DELTA.DAS28 is lower than 1.2; therefore, the patients
are classified as non-responders to the treatment with infliximab.
Concomitantly with the presence of antibodies against infliximab,
the concentration of free infliximab drops below the cut-off
value.
[0093] FIG. 2 shows the average bioavailability and immunogenicity
profiles over one year of treatment of the patients that respond to
the treatment with infliximab. In this example it is shown that
.DELTA.DAS28 is higher than 1.2; therefore, the patient is
classified as responder to infliximab treatment. Concomitantly with
the presence of antibodies against infliximab, the concentration of
free infliximab drops below the cut-off value.
[0094] Bioavailability and Immunogenicity Correlation:
[0095] Bioavailability and immunogenicity of infliximab strongly
correlate (p<0.01). Statistical correlation analysis was
performed using Kendall's Tau-b and Sperman tests. Correlation
results are shown in Table 8.
TABLE-US-00008 TABLE 8 Correlation between bioavailability and
immunogenicity of infliximab. Kendall's Tau-b test Sperman test
Correlation Correlation Parameter coefficient p-value coefficient
p-value Bioavailability -0.491 1.00E-06 -0.606 1.00E-06
Immunogenicity
[0096] Clinical Correlations:
[0097] The correlations between bioavailability, immunogenicity and
the disease activity based on the DAS28 values were analyzed.
Clinical responses strongly correlate with the levels of free
infliximab (drug bioavailability) and the formation of
anti-infliximab antibodies (drug immunogenicity) (Table 9). In
addition, infliximab concentration and antibodies against the drug
strongly correlate (p<-0.001). The concentration of infliximab
inversely correlates with DAS28 (p<-0.001), whereas the
concentration of anti-infliximab antibodies directly correlates
with DAS28 (p<0.001).
TABLE-US-00009 TABLE 9 Correlation between the infliximab
bioavailability, anti-infliximab antibodies (immunogenicity) and
DAS28. Concentration Concentration of of anti-infliximab infliximab
antibodies (.mu.g/ml) (AU/ml) (Bioavailability) (Immunogenicity)
DAS28 Concentration of -- p < -0.001 p < -0.001 infliximab
(.mu.g/ml) Concentration of p < -0.001 -- p < 0.001
anti-infliximab antibodies (AU/ml) DAS28 p < -0.001 p < 0.001
-- A positive p value indicates a direct correlation. A negative p
value indicates an inverse correlation. p values <0.05 or
<-0.05 are statistically significant.
[0098] Determination of Diagnostic Cut-Offs:
[0099] According to the standard protocol to determine
immunogenicity, there is never a positive titer of anti-infliximab
antibodies above 1.5 .mu.g/ml of free infliximab. Therefore,
diagnostic cut-off was determined by measuring the anti-infliximab
antibodies concentration with the acid dissociation protocol in
those samples with concentrations of free infliximab below 1.5
.mu.g/ml. 45 patients (183 samples) fulfilling this criteria were
analysed. 51% of the patients (33% of the samples) showed a
positive immunogenicity titer (Table 10).
TABLE-US-00010 TABLE 10 Detection of antibodies anti-infliximab
using the acid dissociation protocol in samples with free
infliximab below 1.5 .mu.g/ml. Positive samples after acid Samples
# Patients # dissociation protocol/patients 183 45 60 (33%)/23
[0100] It was further studied if there is a relationship between
the level of free infliximab below 1.5 .mu.g/ml and the presence of
antibodies anti-infliximab above 15 AU/ml (equivalent to 150 ng/ml
of antibodies) (limit of detection) with DAS28 (Table 11) in the
whole cohort of patients.
TABLE-US-00011 TABLE 11 Disease activity as a function of
bioavailability and immunogenicity cut-offs. Grouping Average DAS28
SD p-value Infliximab >1.5 .mu.g/ml AND 3.35 0.97 <0.01
antibodies anti-infliximab <15 AU/ml Infliximab <1.5 .mu.g/ml
AND 3.89 1.41 antibodies anti-infliximab >15 AU/ml SD, standard
deviation. p value <0.05 is statistically significant.
[0101] Inventors studied the same relationship between responder
and non-responder patients (Table 12).
TABLE-US-00012 TABLE 12 Correlation of infliximab bioavailability
and immunogenicity cut-offs with the clinical response. Average
Population Cut-offs combination DAS28 (N) SD p-value With Free
infliximab >1.5 .mu.g/ml 3.30 (107) 0.98 0.546 clinical AND
antibodies anti-infliximab <15 AU/ml response Free infliximab
<1.5 .mu.g/ml 3.51 (41) 1.28 AND antibodies anti-infliximab
>15 AU/ml With no Free infliximab >1.5 .mu.g/ml 3.52 (33)
0.97 <0.01 clinical AND antibodies anti-infliximab <15 AU/ml
response Free infliximab <1.5 .mu.g/ml 4.25 (42) 1.46 AND
antibodies anti-infliximab >15 AU/ml SD, standard deviation.
Different combinations of cut-offs are considered. p values
<0.05 are statistically significant.
[0102] It is demonstrated that patients with an infliximab
bioavailability lower than 1.5 .mu.g/ml and antibodies
anti-infliximab higher than 15 AU/ml show a higher DAS28 than those
patients with infliximab bioavailability higher than 1.5
.mu.g/ml.
[0103] Therefore, if a patient shows a combination of free
infliximab lower than (<) 1.5 .mu.g/ml AND antibodies
anti-infliximab higher than (>) 15 AU/ml, there is a higher
probability that the individual is a non-responder to the
infliximab treatment, showing a high activity of the disease (see
EULAR guidelines on Table 1).
[0104] FIG. 3 shows how the bioavailability of infliximab
(circulating concentration of infliximab) and immunogenicity of
infliximab (anti-infliximab antibodies) variables are associated to
the DAS28 index, and therefore can be used to classify patients and
predict the status of a patient according to the EULAR DAS
guidelines.
[0105] Accordingly, a patient showing a combination of free
infliximab equal to or higher than (.gtoreq.) 1.5 .mu.g/ml AND
antibodies anti-infliximab equal to or lower than (.ltoreq.) 15
AU/ml has a higher probability of being a responder patient to the
infliximab treatment; however, patients showing bioavailability
lower than 1.5 .mu.g/ml and antibodies anti-infliximab higher than
15 AU/ml have a higher probability of being non-responder patients
to the infliximab treatment.
[0106] Infliximab Decision Algorithm:
[0107] It is demonstrated that the clinical response to infliximab
closely follows the drug levels and the presence of antibodies
directed against the drug.
[0108] It is thus demonstrated that if a patient has a
bioavailability value below 1.5 .mu.g/ml AND an immunogenicity
value above 15 AU/ml, this will correlate with no clinical response
to the treatment, therefore an increased DAS28 value (moderate or
high disease activity) compared to those with bioavailability
values above 1.5 .mu.g/ml (p<0.001).
[0109] None of the analysed patients showed anti-infliximab
antibodies if the concentration of free infliximab was above 1.5
.mu.g/ml.
[0110] The following decision algorithm can be constructed from the
data:
TABLE-US-00013 1. Analysis Free infliximab bioavailability
determination AND anti-infliximab antibodies analysis 2. Result
Free infliximab Free infliximab concentration .gtoreq.1.5 .mu.g/ml
concentration <1.5 .mu.g/ml AND anti-infliximab AND
anti-infliximab antibodies .ltoreq.15 AU/ml antibodies >15 AU/ml
3. Patient Responder Non-responder classification 4. Action None.
Adequate treatment Inefficient treatment. Follow up required. The
treatment should be considered.
Example 2
Correlation of Bioavailability and Immunogenicity of Adalimumab
with the Clinical Response of Patients Suffering from Rheumatoid
Arthritis
[0111] Subjects:
[0112] Rheumatoid arthritis patients, 49, fulfilling the 1987 ACR
criteria and about to start treatment with adalimumab, were
enrolled consecutively. 171 serum samples were analysed.
[0113] Treatment:
[0114] Patients received doses of 40 mg of adalimumab
(Humira.RTM.), subcutaneously, every two weeks.
[0115] Evaluation of the Patient Clinical Response:
[0116] DAS28 was used to evaluate the clinical response of the
patient according scores in Table 1 (Example 1).
[0117] Bioavailability and Immunogenicity Correlation:
[0118] Bioavailability and immunogenicity of adalimumab strongly
correlate (p<0.05). Statistical correlation analysis was
performed using Kendall's Tau-b and Sperman tests. Correlation
results are shown in Table 13.
TABLE-US-00014 TABLE 13 Correlation between bioavailability and
immunogenicity of adalimumab. Kendall's Tau-b test Sperman test
Correlation Correlation Parameter coefficient p-value coefficient
p-value Bioavailability -0.363 1.00E-06 -0.444 1.00E-06
Immunogenicity
[0119] Clinical Correlations:
[0120] The correlation between bioavailability, immunogenicity and
the disease activity based on the DAS28 values was analyzed.
Clinical response strongly correlates with the level of free
adalimumab (drug bioavailability) and antibodies against adalimumab
(immunogenicity) (Table 14).
TABLE-US-00015 TABLE 14 Correlation between the adalimumab
bioavailability, anti-adalimumab antibodies (immunogenicity) and
DAS28. Concentration of anti- Concentration of adalimumab
adalimumab antibodies (.mu.g/ml) (AU/ml) (Bioavailability)
(Immunogenicity) DAS28 Concentration of -- -0.001 (171) -0.001
(102) adalimumab (.mu.g/ml) Concentration of -0.001 (171) -- 0.09
(102) anti-adalimumab antibodies (AU/ml) DAS28 -0.001 (102) 0.09
(102) -- A positive p value indicates a direct correlation. A
negative p value indicates an inverse correlation. p values
<0.05 or <-0.05 are statistically significant. *p value of
0.06 is considered statistically significant. The value is very
close to the significance criteria of p < 0.05, and therefore
clinically relevant.
[0121] Determination of Diagnostic Cut-Offs:
[0122] According to the standard protocol to determine
immunogenicity, there is never a positive titer of anti-adalimumab
antibodies above 0.8 .mu.g/ml of free adalimumab. Therefore,
diagnostic cut-off was determined by measuring the anti-adalimumab
antibodies concentration with the acid dissociation protocol in
those samples with concentrations of free adalimumab below 0.8
.mu.g/ml. 54 samples fulfilling this criteria were analysed. 22% of
the patients with free adalimumab below 0.8 .mu.g/ml showed a
positive immunogenicity titer, and 77% of the patients with free
adalimumab below the limit of detection (2 ng/ml) showed a positive
immunogenicity titer (Table 15).
TABLE-US-00016 TABLE 15 Detection of antibodies anti-adalimumab
using the acid dissociation protocol in samples with free
adalimumab below 0.8 ug/ml and no antibodies against adalimumab.
Free adalimumab Free adalimumab <2 ng/ml, no antibodies <0.8
.mu.g/ml, no antibodies Samples # Patients # Samples # Patients #
28 18 26 13 Positive after acid 5 (18%) 4 (22%) 10 (38%) 10 (77%)
dissociation
[0123] It was studied if there is a relationship between the level
of free adalimumab below 0.8 .mu.g/ml and the presence of
antibodies anti-adalimumab above 8 AU/ml (equivalent to 32 ng/ml of
antibodies) (limit of detection) with DAS28 (Table 16) in the whole
cohort of patients.
TABLE-US-00017 TABLE 16 Disease activity as a function of
bioavailability and immunogenicity cut-offs. p value <0.05 is
statistically significant. Average Grouping DAS28 p-value
Adalimumab >0.8 .mu.g/ml AND antibodies anti- 2.56 (53) 0.037
adalimumab <8 AU/ml Adalimumab <0.8 .mu.g/ml AND antibodies
anti- 3.27 (4) adalimumab >8 AU/ml
[0124] It is demonstrated that patients with an adalimumab
bioavailability lower than 0.8 .mu.g/ml and antibodies
anti-adalimumab higher than 8 AU/ml (equivalent to 32 ng/ml of
antibodies) show a higher DAS28 value compared to those with an
adalimumab bioavailability higher than 0.8 .mu.g/ml and antibodies
anti-adalimumab lower than 8 AU/ml. This DAS28 difference is
statistically significant.
[0125] Therefore, if a patient shows a combination of free
adalimumab lower than (<) 0.8 .mu.g/ml AND antibodies
anti-adalimumab higher than (>) 8 AU/ml, there is a higher
probability that the individual is a non-responder to the
adalimumab treatment, showing a high activity of the disease (see
EULAR guidelines on Table 1 and Table 16).
[0126] Accordingly, a patient showing a combination of free
adalimumab equal to or higher than (.gtoreq.) 0.8 .mu.g/ml AND
antibodies anti-adalimumab equal to or lower than (.ltoreq.) 8
AU/ml has a higher probability of being a responder patient to the
adalimumab treatment.
[0127] Adalimumab Decision Algorithm:
[0128] It is demonstrated that the clinical response to adalimumab
closely follows the drug levels and the presence of antibodies
directed against the drug.
[0129] It is demonstrated that if a patient has a bioavailability
value below 0.8 .mu.g/ml AND an immunogenicity value above 8 AU/ml
(equivalent to 32 ng/ml of antibodies), this will correlate with a
higher probability of showing no clinical response to the
treatment, therefore an increased DAS28 value (moderate or high
disease activity) compared to those with bioavailability values
above 0.8 .mu.g/ml (p<0.001).
[0130] None of the analysed patients showed anti-adalimumab
antibodies if the concentration of free adalimumab was above 0.8
.mu.g/ml.
[0131] The following decision algorithm can be constructed from the
data:
TABLE-US-00018 1. Analysis Free adalimumab bioavailability
determination AND anti-adalimumab antibodies analysis 2. Result
Free adalimumab Free adalimumab concentration .gtoreq.0.8 .mu.g/ml
concentration <0.8 .mu.g/ml AND anti-adalimumab AND
anti-adalimumab antibodies .ltoreq.8 AU/ml antibodies >8 AU/ml
3. Patient Responder Non-responder classification 4. Action None.
Adequate treatment Inefficient treatment. Follow up required. The
treatment should be considered.
Example 3
Correlation of Bioavailability of Infliximab with the Clinical
Response of Patients Suffering from Rheumatoid Arthritis
[0132] Experiments were performed as described in Example 1 except
that the anti-infliximab antibody levels were not analized. Thus,
patients were classified according to the infliximab therapeutical
cut-off.
[0133] Infliximab trough levels are significantly higher (2.73
.mu.g/ml) in patients suffering from rheumatoid arthritis that show
a good or moderate clinical response (responders) to the treatment
(p=1E-09) compared to non-responders (0.002 .mu.g/ml) after one
year of treatment (Table 17).
TABLE-US-00019 TABLE 17 Comparison between trough infliximab levels
according to response criteria. Patient classification Median
trough infliximab (.mu.g/ml) N p Responder 2.73 71 1E-09
Non-responder 0.002 54
[0134] To calculate the frequency of responders and non-responders
rheumatoid arthritis patients after one year of treatment, we
performed significance tests by Chi-squared analysis (Table 18).
The number of patients that are classified as responders is
significantly higher than non-responders when trough infliximab
concentration is .gtoreq.1.5 .mu.g/ml (p=3.55E-07). On the other
hand, the number of patients that are classified as responders is
significantly lower than non-responders when trough infliximab
concentration is <1.5 .mu.g/ml (p=0.028).
TABLE-US-00020 TABLE 18 Comparison of the frequency of responder
and non-responder rheumatoid arthritis patients depending on the
infliximab cut-off. Patient Infliximab concentration classification
N p (.chi..sup.2) .gtoreq.1.5 .mu.g/ml Responder 43 3.5E-07
Non-responder 7 <1.5 .mu.g/ml Responder 28 0.028 Non-responder
47
[0135] When trough infliximab concentration is .gtoreq.1.5
.mu.g/ml, the median drug level of responder patients is 3.64
.mu.g/ml, while is 2.68 .mu.g/ml in the non-responder population
(p=0.142). However, when trough infliximab concentration is <1.5
the median drug level of responder patients is significantly higher
(0.06 .mu.g/ml) than in the non-responder population (0.002
.mu.g/ml) (p=4.23E-03) (Table 19).
TABLE-US-00021 TABLE 19 Median infliximab concentration for
responder and non-responder populations depending on the infliximab
cut-off. Patient p (Kruskal- Infliximab concentration
classification N Median Wallis) .gtoreq.1.5 .mu.g/ml Responder 43
3.6472 0.142 Non-responder 7 2.688 <1.5 .mu.g/ml Responder 28
0.0607 4.2E-03 Non-responder 47 0.002
[0136] Therefore, if a patient has an infliximab trough level
<1.5 .mu.g/ml it is more likely to have lost drug efficacy and
be classified as a non-responder, therefore necessitating of a
treatment regime revision. On the other hand, if a patient has an
infliximab trough level .gtoreq.1.5 .mu.g/ml it is more likely that
the patient corresponds to a responder, since the patient is
exposed to effective drug levels.
Example 4
Correlation of Bioavailability of Adalimumab with the Clinical
Response of Patients Suffering from Rheumatoid Arthritis
[0137] Experiments were performed as described in Example 2 except
that the anti-adalimumab antibody levels were not analized. Thus,
patients were classified according to the adalimumab therapeutical
cut-off.
[0138] Adalimumab trough levels are significantly higher (12.25
.mu.g/ml) in patients suffering from rheumatoid arthritis that show
a good or moderate clinical response (responders) to the treatment
(p=0.0005) compared to non-responders (4.15 .mu.g/ml) after two
years of treatment (Table 20).
TABLE-US-00022 TABLE 20 Comparison between trough adalimumab levels
according to response criteria. Patient Median trough adalimumab
classification (.mu.g/ml) N p Responder 12.25 16 0.0005
Non-responder 4.15 45
[0139] To calculate the frequency of responder and non-responder
rheumatoid arthritis patients after two years of treatment, we
performed significance tests by Chi-squared analysis (Table 21).
All patients with an adalimumab trough level <0.8 .mu.g/ml are
non-responders. No patients were classified as responders with
adalimumab trough levels <0.8 .mu.g/ml.
TABLE-US-00023 TABLE 21 Comparison of the frequency of responder
and non-responder rheumatoid arthritis patients depending on the
adalimumab cut-off. Adalimumab concentration Patient N p
(.chi..sup.2) .gtoreq.0.8 .mu.g/ml Responder 16 0.093 Non-responder
27 <0.8 .mu.g/ml Responder 0 p < 0.05 Non-responder 16
[0140] When trough adalimumab concentration is .gtoreq.0.8
.mu.g/ml, the median drug level of responder rheumatoid arthritis
patients is 12.25 .mu.g/ml, while is 6.73 .mu.g/ml in the
non-responder population (Table 22).
TABLE-US-00024 TABLE 22 Median adalimumab concentration of
responder and non-responder populations depending on the adalimumab
cut-off. Adalimumab Patient p (Kruskal- concentration
classification N Median Wallis) .gtoreq.0.8 .mu.g/ml Responder 16
12.25 0.06 Non-responder 27 6.73 <0.8 .mu.g/ml Responder 0 0 p
< 0.05 Non-responder 16 0.00008
[0141] In conclusion, if a patient has an adalimumab trough level
<0.8 .mu.g/ml it is more likely to have lost drug efficacy and
be classified as a non-responder, therefore necessitating of a
treatment regime revision. On the other hand, if a patient has an
adalimumab trough level .gtoreq.0.8 .mu.g/ml there is an increased
probability that the patient could be responding to the therapy.
Sequence CWU 1
1
21233PRTHomo sapiens 1Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu
Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln
Gly Ser Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu
Ile Val Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe
Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro 50 55 60 Arg Asp Leu
Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser 65 70 75 80 Ser
Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 85 90
95 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu
100 105 110 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val
Pro Ser 115 120 125 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe
Lys Gly Gln Gly 130 135 140 Cys Pro Ser Thr His Val Leu Leu Thr His
Thr Ile Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys Val
Asn Leu Leu Ser Ala Ile Lys Ser Pro 165 170 175 Cys Gln Arg Glu Thr
Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 180 185 190 Pro Ile Tyr
Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205 Ser
Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215
220 Gln Val Tyr Phe Gly Ile Ile Ala Leu 225 230 2164PRTHomo sapiens
2Met His His His His His His Val Arg Ser Ser Ser Arg Thr Pro Ser 1
5 10 15 Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln Ala Glu Gly
Gln 20 25 30 Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala
Asn Gly Val 35 40 45 Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser
Glu Gly Leu Tyr Leu 50 55 60 Ile Tyr Ser Gln Val Leu Phe Lys Gly
Gln Gly Cys Pro Ser Thr His 65 70 75 80 Val Leu Leu Thr His Thr Ile
Ser Arg Ile Ala Val Ser Tyr Gln Thr 85 90 95 Lys Val Asn Leu Leu
Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr 100 105 110 Pro Glu Gly
Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly 115 120 125 Gly
Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn 130 135
140 Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly
145 150 155 160 Ile Leu Ala Leu
* * * * *