Note: Descriptions are shown in the official language in which they were submitted.
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1 INDIVIDUALIZED ANTI-CANCER ANTIBODIES
2 Reference to Related Applications:
3 This application is a continuation-in-part of
4 application S.N. 09/415,278, filed October 8, 1999, now
U.S. Patent 6,180,357, the contents of which are herein
6 incorporated by reference.
7 Field of the Invention:
8 This invention relates to the production of anti-
9 cancer antibodies customized for the individual patient
which may be combined with chemotherapeutic agents that
11 can be used for therapeutic and diagnostic purposes. The
12 invention further relates to the process by which the
13 antibodies are made and to their methods of use.
14
Background of the Invention:
16 Each individual who presents with cancer is unique
17 and has a cancer that is as different from other cancers
18 as that person's identity. Despite this, current therapy
19 treats all patients with the same type of cancer, at the
same stage, in the same way. At least 30% of these
21 patients will fail the first line therapy, thus leading to
22 further rounds of treatment and the increased probability
23 of treatment failure, metastases, and ultimately, death.
24 A superior approach to treatment would be the
customization of therapy for the particular individual.
26 The only current therapy which lends itself to
27 customization is surgery. Chemotherapy and radiation
28 treatment can not be tailored to the patient, and surgery
29 by itself, in most cases is inadequate for producing
cures .
31 With the advent of monoclonal antibodies, the
32 possibility of developing methods for customized therapy
33 became more realistic since each antibody can be directed
34 to a single epitope. Furthermore, it is possible to
produce a combination of antibodies that are directed to
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1 the constellation of epitopes that uniquely define a
2 particular individual's tumor.
3 Having recognized that a significant difference
4 between cancerous and normal cells is that cancerous cells
contain antigens that are specific to transformed cells,
6 the scientific community has long held that monoclonal
7 antibodies can be designed to specifically target
8 transformed cells by binding specifically to these cancer
9 antigens; thus giving rise to the belief that monoclonal
antibodies can serve as "Magic Bullets" to eliminate
11 cancer cells.
12 At the present time, however, the cancer patient
13 usually has few options of treatment. The regimented
14 approach to cancer therapy has produced improvements in
global survival and morbidity rates. However, to the
16 particular individual, these improved statistics do not
17 necessarily correlate with an improvement in their
18 personal situation.
19 Thus, if a methodology was put forth which enabled
the practitioner to treat each tumor independently of
21 other patients in the same cohort, this would permit the
22 unique approach of tailoring therapy to just that one
23 person. Such a course of therapy would, ideally, increase
24 the rate of cures, and produce better outcomes, thereby
satisfying a long-felt need.
26 Historically, the use of polyclonal antibodies has
27 been used with limited success in the treatment of human
28 cancers. Lymphomas and leukemias have been treated with
29 human plasma, but there were few prolonged remission or
responses. Furthermore, there was a lack of
31 reproducibility and there was no additional benefit
32 compared to chemotherapy. Solid tumors such as breast
33 cancers, melanomas and renal cell carcinomas have also
34 been treated with human blood, chimpanzee serum, human
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1 plasma and horse serum with correspondingly unpredictable
2 and ineffective results.
3 There have been many clinical trials of monoclonal
4 antibodies for solid tumors. In the 1980s there were at
least four clinical trials for human breast cancer which
6 produced only one responder from at least 47 patients
7 using antibodies against specific antigens or based on
8 tissue selectivity. It was not until 1998 that there was
9 a successful clinical trial using a humanized anti-her 2
antibody in combination with Cisplatin. In this trial 37
11 patients were accessed for responses of which about a
12 quarter had a partial response rate and another half had
13 minor or stable disease progression.
14 The clinical trials investigating colorectal cancer
involve antibodies against both glycoprotein and
16 glycolipid targets. Antibodies such as 17-lA, which has
17 some specificity for adenocarcinomas, had undergone Phase
18 2 clinical trials in over 60 patients with only one
19 patient having a partial response. In other trials, use
of 17-lA produced only one complete response and two minor
21 responses among 52 patients in protocols using additional
22 cyclophosphamide. Other trials involving 17-lA yielded
23 results that were similar. The use of a humanized murine
24 monoclonal antibody initially approved for imaging also
did not produce tumor regression. To date there has not
26 been an antibody that has been effective for colorectal
27 cancer. Likewise there have been equally poor results for
28 lung cancer, brain cancers, ovarian cancers, pancreatic
29 cancer, prostate cancer, and stomach cancer. There has
been some limited success in the use of anti-GD3
31 monoclonal antibody for melanoma. Thus, it can be seen
32 that despite successful small animal studies that are a
33 prerequisite for human clinical trials, the antibodies
34 that have been tested have been for the most part
ineffective.
36
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1 Prior Patents:
2 U.S. Patent No. 5,750,102 discloses a process wherein
3 cells from a patient's tumor are transfected with MHC
4 genes which may be cloned from cells or tissue from the
patient. These transfected cells are then used to
6 vaccinate the patient.
7 U.S. Patent No. 4,861,581 discloses a process
8 comprising the steps of obtaining monoclonal antibodies
9 that are specific to an internal cellular component of
neoplastic and normal cells of the mammal but not to
11 external components, labeling the monoclonal antibody,
12 contacting the labeled antibody with tissue of a mammal
13 that has received therapy to kill neoplastic cells, and
14 determining the effectiveness of therapy by measuring the
binding of the labeled antibody to the internal cellular
16 component of the degenerating neoplastic cells. In
17 preparing antibodies directed to human intracellular
18 antigens, the patentee recognizes that malignant cells
19 represent a convenient source of such antigens.
U.S. Patent No. 5,171,665 provides a novel antibody
21 and method for its production. Specifically, the patent
22 teaches formation of a monoclonal antibody which has the
23 property of binding strongly to a protein antigen
24 associated with human tumors, e.g. those of the colon and
lung, while binding to normal cells to a much lesser
26 degree .
27 U.S. Patent No. 5,484,596 provides a method of cancer
28 therapy comprising surgically removing tumor tissue from a
29 human cancer patient, treating the tumor tissue to obtain
tumor cells, irradiating the tumor cells to be viable but
31 non-tumorigenic, and using these cells to prepare a
32 vaccine for the patient capable of inhibiting recurrence
33 of the primary tumor while simultaneously inhibiting
34 metastases. The patent teaches the development of
monoclonal antibodies which are reactive with surface
36 antigens of tumor cells. As set forth at col. 4, lines 45
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1 et seq., the patentees utilize autochthonous tumor cells
2 in the development of monoclonal antibodies expressing
3 active specific immunotherapy in human neoplasia.
4 U.S. Patent No. 5,693,763 teaches a glycoprotein
antigen characteristic of human carcinomas and not
6 dependent upon the epithelial tissue of origin.
7 U.S. Patent No. 5,783,186 is drawn to Anti-Her2
8 antibodies which induce apoptosis in Her2 expressing
9 cells, hybridoma cell lines producing the antibodies,
methods of treating cancer using the antibodies and
11 pharmaceutical compositions including said antibodies.
12 U.S. Patent No. 5,849,876 describes new hybridoma
13 cell lines for the production of monoclonal antibodies to
14 mucin antigens purified from tumor and non-tumor tissue
sources.
16 U.S. Patent No. 5,869,268 is drawn to a method for
17 producing a human lymphocyte producing an antibody
18 specific to a desired antigen, a method for producing a
19 monoclonal antibody, as well as monoclonal antibodies
produced by the method. The patent is particularly drawn
21 to the production of an anti-HD human monoclonal antibody
22 useful for the diagnosis and treatment of cancers.
23 U.S. Patent No. 5,869,045 relates to antibodies,
24 antibody fragments, antibody conjugates and single chain
immunotoxins reactive with human carcinoma cells. The
26 mechanism by which these antibodies function is two-fold,
27 in that the molecules are reactive with cell membrane
28 antigens present on the surface of human carcinomas, and
29 further in that the antibodies have the ability to
internalize within the carcinoma cells, subsequent to
31 binding, making them especially useful for forming
32 antibody-drug and antibody-toxin conjugates. In their
33 unmodified form the antibodies also manifest cytotoxic
34 properties at specific concentrations.
U.S. Patent No. 5,780,033 discloses the use of
36 autoantibodies for tumor therapy and prophylaxis. However,
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1 this antibody is an antinuclear autoantibody from an aged
2 mammal. In this case, the autoantibody is said to be one
3 type of natural antibody found in the immune system.
4 Because the autoantibody comes from "an aged mammal",
there is no requirement that the autoantibody actually
6 comes from the patient being treated. In addition the
7 patent discloses natural and monoclonal antinuclear
8 autoantibody from an aged mammal, and a hybridoma cell
9 line producing a monoclonal antinuclear autoantibody.
11 Summary of the Invention:
12 This application teaches a method for producing
13 patient specific anti-cancer antibodies using a novel
14 paradigm of screening. These antibodies can be made
specifically for one tumor and thus make possible the
16 customization of cancer therapy. Within the context of
17 this application, anti-cancer antibodies having either
18 cell-killing (cytotoxic) or cell-growth inhibiting
19 (cytostatic) properties will hereafter be referred to as
cytotoxic. These antibodies can be used in aid of staging
21 and diagnosis of a cancer, and can be used to treat tumor
22 metastases.
23 The prospect of individualized anti-cancer treatment
24 will bring about a change in the way a patient is managed.
A likely clinical scenario is that a tumor sample is
26 obtained at the time of presentation, and banked. From
27 this sample, the tumor can be typed from a panel of pre-
28 existing anti-cancer antibodies. The patient will be
29 conventionally staged but the available antibodies can be
of use in further staging the patient. The patient can be
31 treated immediately with the existing antibodies, and a
32 panel of antibodies specific to the tumor can be produced
33 either using the methods outlined herein or through the
34 use of phage display libraries in conjunction with the
screening methods herein disclosed. All the antibodies
36 generated will be added to the library of anti-cancer
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1 antibodies since there is a possibility that other tumors
2 can bear some of the same epitopes as the one that is
3 being treated.
4 In addition to anti-cancer antibodies, the patient
can elect to receive the currently recommended therapies
6 as part of a multi-modal regimen of treatment. The fact
7 that the antibodies isolated via the present methodology
8 are relatively non-toxic to non-cancerous cells allow
9 combinations of antibodies at high doses to be used,
either alone, or in conjunction with conventional therapy.
11 The high therapeutic index will also permit re-treatment
12 on a short time scale that should decrease the likelihood
13 of emergence of treatment resistant cells.
14 If the patient is refractory to the initial course of
therapy or metastases develop, the process of generating
16 specific antibodies to the tumor can be repeated for re-
17 treatment. Furthermore, the anti-cancer antibodies can be
18 conjugated to red blood cells obtained from that patient
19 and re-infused for treatment of metastases. There have
been few effective treatments for metastatic cancer and
21 metastases usually portend a poor outcome resulting in
22 death. However, metastatic cancers are usually well
23 vascularized and the delivery of anti-cancer antibodies by
24 red blood cells can have the effect of concentrating the
antibodies at the site of the tumor. Even prior to
26 metastases, most cancer cells are dependent on the host's
27 blood supply for their survival and anti-cancer antibody
28 conjugated red blood cells can be effective against in
29 situ tumors, too. Alternatively, the antibodies may be
conjugated to other hematogenous cells, e.g. lymphocytes,
31 macrophages, monocytes, natural killer cells, etc.
32
33 There are five classes of antibodies and each is
34 associated with a function that is conferred by its heavy
chain. It is generally thought that cancer cell killing
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1 by naked antibodies are mediated either through antibody
2 dependent cellular cytotoxicity or complement dependent
3 cytotoxicity. For example murine IgM and IgG2a antibodies
4 can activate human complement by binding the C-1 component
of the complement system thereby activating the classical
6 pathway of complement activation which can lead to tumor
7 lysis. For human antibodies the most effective complement
8 activating antibodies are generally IgM and IgGl. Murine
9 antibodies of the IgG2a and IgG3 isotype are effective at
recruiting cytotoxic cells that have Fc receptors which
11 will lead to cell killing by monocytes, macrophages,
12 granulocytes and certain lymphocytes. Human antibodies of
13 both the IgGl and IgG3 isotype mediate ADCC.
14 Another possible mechanism of antibody mediated
cancer killing may be through the use of antibodies that
16 function to catalyze the hydrolysis of various chemical
17 bonds in the cell membrane and its associated
18 glycoproteins or glycolipids, so-called catalytic
19 antibodies.
There are two additional mechanisms of antibody
21 mediated cancer cell killing which are more widely
22 accepted. The first is the use of antibodies as a vaccine
23 to induce the body to produce an immune response against
24 the putative cancer antigen that resides on the tumor
cell. The second is the use of antibodies to target
26 growth receptors and interfere with their function or to
27 down regulate that receptor so that effectively its
28 function is lost.
29 Accordingly, it is an objective of the invention to
teach a method for producing anti-cancer antibodies from
31 cells derived from a particular individual which are
32 cytotoxic with respect to cancer cells while
33 simultaneously being relatively non-toxic to non-cancerous
34 cells.
It is an additional objective of the invention to
36 produce novel anti-cancer antibodies.
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1 It is a further objective of the instant invention to
2 produce anti-cancer antibodies whose cytotoxicity is
3 mediated through antibody dependent cellular toxicity.
4 It is yet an additional objective of the instant
invention to produce anti-cancer antibodies whose
6 cytotoxicity is mediated through complement dependent
7 cellular toxicity.
8 It is still a further objective of the instant
9 invention to produce anti-cancer antibodies whose
cytotoxicity is a function of their ability to catalyze
11 hydrolysis of cellular chemical bonds.
12 Still an additional objective of the instant
13 invention is to produce anti-cancer antibodies useful as a
14 vaccine to produce an immune response against putative
cancer antigen residing on tumor cells.
16 A further objective of the instant invention is the
17 use of antibodies to target cell membrane proteins, such
18 as growth receptors, cell membrane pumps and cell
19 anchoring proteins, thereby interfering with or down
regulating their function.
21 Yet an additional objective of the instant invention
22 is the production of anti-cancer antibodies whose cell-
23 killing utility is concomitant with their ability to
24 effect a conformational change in cellular proteins such
that a signal will be transduced to initiate cell-killing.
26 A still further objective of the instant invention is
27 to produce anti-cancer antibodies which are useful for
28 diagnosis, prognosis, and monitoring of cancer, e.g.
29 production of a panel of therapeutic anti-cancer
antibodies to test patient samples to determine if they
31 contain any suitable antibodies for therapeutic use.
32 Yet another objective of the instant invention is to
33 produce novel antigens, associated with cancer processes,
34 which can be discovered by using anti-cancer antibodies
derived by the process of the instant invention. These
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1 antigens are not limited to proteins, as is generally the
2 case with genomic data; they may also be derived from
3 carbohydrates or lipids or combinations thereof.
4 Other objects and advantages of this invention will
5 become apparent from the following description wherein are
6 set forth, by way of illustration and example, certain
7 embodiments of this invention.
8
9 Detailed Description of the Invention:
10 It is to be understood that while a certain form of
11 the invention is illustrated, it is not to be limited to
12 the specific form or arrangement herein described and
13 shown. It will be apparent to those skilled in the art
14 that various changes may be made without departing from
the scope of the invention and the invention is not to be
16 considered limited to what is shown and described in the
17 specification.
18 One of the potential benefits of monoclonal
19 antibodies with respect to the treatment of cancer is
their ability to specifically recognize single antigens.
21 It was thought that in some instances cancer cells possess
22 antigens that were specific to that kind of transformed
23 cell. It is now more frequently believed that cancer
24 cells have few unique antigens, rather, they tend to over-
express a normal antigen or express fetal antigens.
26 Nevertheless, the use of monoclonal antibodies provided a
27 method of delivering reproducible doses of antibodies to
28 the patient with the expectation of better response rates
29 than with polyclonal antibodies.
Traditionally, monoclonal antibodies have been made
31 according to fundamental principles laid.down by Kohler
32 and Milstein. Mice are immunized with antigens, with or
33 without, adjuvants. The splenocytes are harvested from
34 the spleen for fusion with immortalized hybridoma
partners. These are seeded into microtitre plates where
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1 they can secrete antibodies into the supernatant that is
2 used for cell culture. To select from the hybridomas that
3 have been plated for the ones that produce antibodies of
4 interest the hybridoma supernatants are usually tested for
antibody binding to antigens in an ELISA (enzyme linked
6 immunosorbent assay) assay. The idea is that the wells
7 that contain the hybridoma of interest will contain
8 antibodies that will bind most avidly to the test antigen,
9 usually the immunizing antigen. These wells are then
subcloned in limiting dilution fashion to produce
11 monoclonal hybridomas. The selection for the clones of
12 interest is repeated using an ELISA assay to test for
13 antibody binding. Therefore, the principle that has been
14 propagated is that in the production of monoclonal
antibodies the hybridomas that produce the most avidly
16 binding antibodies are the ones that are selected from
17 among all the hybridomas that were initially produced.
18 That is to say, the preferred antibody is the one with
19 highest affinity for the antigen of interest.
There have been many modifications of this procedure
21 such as using whole cells for immunization. In this
22 method, instead of using purified antigens, entire cells
23 are used for immunization. Another modification is the
24 use of cellular ELISA for screening. In this method
instead of using purified antigens as the target in the
26 ELISA, fixed cells are used. In addition to ELISA tests,
27 complement mediated cytotoxicity assays have also been
28 used in the screening process. However, antibody-binding
29 assays were used in conjunction with cytotoxicity tests.
Thus, despite many modifications, the process of producing
31 monoclonal antibodies relies on antibody binding to the
32 test antigen as an endpoint.
33 Most antibodies directed against cancer cells have
34 been produced using the traditional methods outlined
above. These antibodies have been used both
36 therapeutically and diagnostically. In general, for both
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1 these applications, the antibody has been used as the
2 targeting agent that delivers a payload to the site of the
3 cancer. These antibody conjugates can either be
4 radioactive, toxic, or serve as an intermediary for
further delivery of a drug to the body, such as an enzyme
6 or biotin. Furthermore, it was widely held, until
7 recently, that naked antibodies had little effect in vivo.
8 Both HERCEPTIN and RITUXIMAB are humanized murine
9 monoclonal antibodies that have recently been approved for
human use by the FDA. However, both these antibodies were
11 initially made by assaying for antibody binding and their
12 direct cytotoxicity was not the primary goal during the
13 production of hybridomas. Any tendency for these
14 antibodies to produce tumor cell killing is thus through
chance, not by design.
16 Although the production of monoclonal antibodies have
17 been carried out using whole cell immunization for various
18 applications the screening of these hybridomas have relied
19 on either putative or identified target antigens or on the
selectivity of these hybridomas for specific tissues. It
21 is axiomatic that the best antibodies are the ones with
22 the highest binding constants. This concept originated
23 from the basic biochemical principle that enzymes with the
24 highest binding constants were the ones that were the most
effective for catalyzing a reaction. This concept is
26 applicable to receptor ligand binding where the drug
27 molecule binding to the receptor with the greatest
28 affinity usually has the highest probability for
29 initiating or inhibiting a signal. However, this may not
always be the case since it is possible that in certain
31 situations there may be cases where the initiation or
32 inhibition of a signal may be mediated through non-
33 receptor binding. The information conveyed by a
34 conformational change induced by ligand binding can have
many consequences such as a signal transduction,
36 endocytosis, among the others. The ability to produce a
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1 conformational change in a receptor molecule may not
2 necessarily be due to the filling of a ligand receptor
3 pocket but may occur through the binding of another extra
4 cellular domain or due to receptor clustering induced by a
multivalent ligand.
6 The production of antibodies to produce cell killing
7 need not be predicated upon screening of the hybridomas
8 for the best binding antibodies. Rather, although not
9 advocated by those who produce monoclonal antibodies, the
screening of the hybridoma supernatants for cell killing
11 or alternatively for cessation of growth of the cancerous
12 cells may be selected as a desirable endpoint for the
13 production of cytotoxic or cytostatic antibodies. It is
14 well understood that the in-vivo antibodies mediate their
function through the Fc portions and that the utility of
16 the therapeutic antibody is determined by the
17 functionality of the constant region or attached moieties.
18 In this case the FAb portion of the antibody, the antigen-
19 combining portion, will confer to the antibody its
specificity and the Fc portion its functionality. The
21 antigen combining site of the antibody can be considered
22 to be the product of a natural combinatorial library. The
23 result of the rearrangement of the variable region of the
24 antibody can be considered a molecular combinatorial
library where the output is a peptide. Therefore, the
26 sampling of this combinatorial library can be based on any
27 parameter. Like sampling a natural compound library for
28 antibiotics, it is possible to sample an antibody library
29 for cytotoxic or cytostatic compounds.
The various endpoints in a screen must be
31 differentiated from each other. For example, the
32 difference between antibody binding to the cell is
33 distinct from cell killing. Cell killing (cytotoxicity) is
34 distinct from the mechanisms of cell death such as oncosis
or apoptosis. There can be many processes by which~cell
36 death is achieved and some of these can lead either to
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1 oncosis or apoptosis. There is speculation that there are
2 other cell death mechanisms other than oncosis or
3 apoptosis but regardless of how the cell arrives at death
4 there are some commonalities of cell death. One of these
is the absence of metabolism and another is the
6 denaturation of enzymes. In either case vital stains will
7 fail to stain these cells. These endpoints of cell death
8 have been long understood and predate the current
9 understanding of the mechanisms of cell death.
Furthermore, there is the distinction between cytotoxic
11 effects where cells are killed and cytostatic effects
12 where the proliferation of cells are inhibited.
13 In a preferred embodiment of the present invention,
14 the assay is conducted by focusing on cytotoxic activity
toward cancerous cells as an end point. In a preferred
16 embodiment, a live /dead assay kit , for example the
17 LIVE/DEAD~ Viability/Cytotoxicity Assay Kit (L-3224) by
18 Molecular Probes, is utilized. The Molecular Probes kit
19 provides a two-color fluorescence cell viability assay
that is based on the simultaneous determination of live
21 and dead cells with two probes that measure two recognized
22 parameters of cell viability - intracellular esterase
23 activity and plasma membrane integrity. The assay
24 principles are general and applicable to most eukaryotic
cell types, including adherent cells and certain tissues,
26 but not to bacteria or yeast. This fluorescence-based
27 method of assessing cell viability is preferred in place
28 of such assays as trypan blue exclusion, Cr release and
29 similar methods for determining cell viability and
cytotoxicity.
31 In carrying out the assay, live cells are
32 distinguished by the presence of ubiquitous intracellular
33 esterase activity, determined by the enzymatic conversion
34 of the virtually nonfluorescent cell-permeant CALCEIN AM
to the intensely fluorescent Calcein. The polyanionic dye
36 Calcein is well retained within live cells, producing an
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1 intense uniform green fluorescence in live cells (ex/em
2 495 nm/--515 nm). EthD-1 enters cells with damaged
3 membranes and undergoes a 40-fold enhancement of
4 fluorescence upon binding to nucleic acids, thereby
5 producing a bright red fluorescence in dead cells (ex/em
6 --495 nm/-635 nm). EthD-1 is excluded by the intact plasma
7 membrane of live cells. The determination of cell
8 viability depends on these physical and biochemical
9 properties of cells. Cytotoxic events that do not affect
10 these cell properties may not be accurately assessed using
11 this method. Background fluorescence levels are inherently
12 low with this assay technique because the dyes are
13 virtually nonfluorescent before interacting with cells.
14 In addition to the various endpoints for screening,
15 there are two other major characteristics of the screening
16 process. The library of antibody gene products is not a
17 random library but is the product of a biasing procedure.
18 In the examples below, the biasing is produced by
19 immunizing mice with fixed cells. This increases the
proportion of antibodies that have the potential to bind
21 the target antigen. Although immunization is thought of as
22 a way to produce higher affinity antibodies (affinity
23 maturation) in this case it is not. Rather, it can be
24 considered as a way to shift the set of antigen combining
sites towards the targets. This is also distinct from the
26 concept of isotype switching where the functionality, as
27 dictated by the constant portion of the heavy chain, is
28 altered from the initial IgM isotype to another isotype
29 such as IgG.
The third key feature that is crucial in the
31 screening process is the use of multitarget screening. To
32 a certain extent specificity is related to affinity. An
33 example of this is the situation where an antigen has very
34 limited tissue distribution and the affinity of the
antibody is a key determinant of the specificity of the
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1 antibody-the higher the affinity the more tissue specific
2 the antibody and likewise an antibody with low affinity
3 may bind to tissues other than the one of interest.
4 Therefore, to address the specificity issue the antibodies
are screened simultaneously against a variety of cells. In
6 the examples below the hybridoma supernatants
7 (representing the earliest stages of monoclonal antibody
8 development?, are tested against a number of cell lines to
9 establish specificity as well as activity.
The antibodies are designed for therapeutic treatment
11 of cancer in patients. Ideally the antibodies can be naked
12 antibodies. They can also be conjugated to toxins. They
13 can be used to target other molecules to the cancer. e.g.
14 biotin conjugated enzymes. Radioactive compounds can also
be used for conjugation.
16 The antibodies can be fragmented and rearranged
17 molecularly. For example Fv fragments can be made; sFv-
18 single chain Fv fragments; diabodies etc.
19 It is envisioned that these antibodies can be used
for diagnosis, prognosis, and monitoring of cancer. For
21 example the patients can have blood samples drawn for shed
22 tumor antigens which can be detected by these antibodies
23 in different formats such as ELISA assays, rapid test
24 panel formats etc. The antibodies can be used to stain
tumor biopsies for the purposes of diagnosis. In addition
26 a panel of therapeutic antibodies can be used to test
27 patient samples to determine if there are any suitable
28 antibodies for therapeutic use.
29 Example one
In order to produce monoclonal antibodies specific
31 for a tumor sample the method of selection of the
32 appropriate hybridoma wells is complicated by the
33 probability of selecting wells which will produce false
34 positive signals. That is to say that there is the
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1 likelihood of producing antibodies that can react against
2 normal cells as well as cancer cells. To obviate this
3 possibility one strategy is to mask the anti-normal
4 antigen antibodies from the selection process. This can
be accomplished by removing the anti-normal antibodies at
6 the first stage of screening thereby revealing the
7 presence of the desired antibodies. Subsequent limiting
8 dilution cloning can delineate the clones that will not
9 produce killing of control cells but will produce target
cancer cell killing.
11 Biopsy specimens of breast, melanoma, and lung tumors
12 were obtained and stored at -70°C until used. Single cell
13 suspensions were prepared and fixed with -30°C, 70%
14 ethanol, washed with PBS and reconstituted to an
appropriate volume for injection. Balb/c mice were
16 immunized with 2.5x105-1x106 cells and boosted every third
17 week until a final pre-fusion boost was performed three
18 days prior to the splenectomy. The hybridomas were
19 prepared by fusing the isolated splenocytes with Sp2/0 and
NS1 myeloma partners. The supernatants from the fusions
21 were tested for subcloning of the hybridomas.
22 Cells (including A2058 melanoma cells, CCD-l2CoN
23 fibroblasts, MCF-12A breast cells among others) were
24 obtained from ATCC and cultured according to enclosed
instructions. The HEY cell line was a gift from Dr. Inka
26 Brockhausen. The non-cancer cells, e.g. CCD-l2CoN
27 fibroblasts and MCF-12A breast cells, were plated into 96-
28 well microtitre plates (NUNC) 1 to 2 weeks prior to
29 screening. The cancer cells, e.g. HEY, A2058, BT 483, and
HS294t, were plated two or three days prior to screening.
31 The plated normal cells were fixed prior to use. The
32 plates were washed with 100 microliters of PBS for 10
33 minutes at room temperature and then aspirated dry. 75
34 microliters of 0.01 percent glutaraldehyde diluted in PBS
were added to each well for five minutes and then
36 aspirated. The plates were washed with 100 microliters of
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1 PBS three times at room temperature. The wells were
2 emptied and 100 microliters of one percent human serum
3 albumin in PBS was added to each well for one hour at room
4 temperature. The plates were then stored at four degrees
Celsius.
6 Prior to the transfer of the supernatant from the
7 hybridoma plates the fixed normal cells were washed three
8 times with 100 microliters of PBS at room temperature.
9 After aspiration to the microliters of the primary
hybridoma culture supernatants were transferred to the
11 fixed cell plates and incubated for two hours at 37
12 degrees Celsius in a 8 percent COZ incubator. The
13 hybridoma supernatants derived from melanoma was incubated
14 with CCD-12 CoN cells and those derived from breast cancer
were incubated with MCF-12a cells. After incubation
16 the absorbed supernatant was divided into two 75
17 microliter portions and transferred to target cancer cell
18 plates. Prior to the transfer the cancer cell plates were
19 washed three times with 100 microliters of PBS. The
supernatant from the CCD-12 CoN cells were transferred to
21 the A2058 and the HS294t cells, whereas the supernatant
22 from MCF-12A cells were transferred to the HEY and BT 483
23 cells. The cancer cells were incubated with the hybridoma
24 supernatants for 18 hours at 37 degrees Celsisu in an 8
percent COZ incubator .
26 The Live/Dead cytotoxicity assay was obtained from
27 Molecular Probes (Eu,OR). The assays were performed
28 according to the manufacturer's instructions with the
29 changes outlined below. The plates with the cells were
washed once with 100 microliters of PBS at 37°C. 75 to 100
31 microliters of supernatant from the hybridoma microtitre
32 plates were transferred to the cell plates and incubated
33 in a 8% COZ incubator for 18-24 hours. Then, the wells that
34 served as the all dead control were aspirated until empty
and 50 microliters of 70% ethanol was added. The plate was
36 then emptied by inverting and blotted dry. Room
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19
1 temperature PBS was dispensed into each well from a
2 multichannel squeeze bottle, tapped three times, emptied
3 by inversion and then blotted dry. 50 microliters of the
4 fluorescent Live/Dead dye diluted in PBS was added to each
well and incubated at 37°C in a 5°s COz incubator for one
6 hour. The plates were read in a Perkin-Elmer HTS7000
7 fluorescence plate reader and the data was analyzed in
8 Microsoft Excel.
9 Four rounds of screening were conducted to produce
single clone hybridoma cultures. For two rounds of
11 screening the hybridoma supernatants were tested only
12 against the cancer cells. In the last round of screening
13 the supernatant was tested against a number of non-cancer
14 cells as well as the target cells indicated in table 1.
The antibodies were isotyped using a commercial isotyping
16 kit .
17 A number of monoclonal antibodies were produced in
18 accordance with the method of the present invention.
19 These antibodies, whose characteristics are summarized in
Table 1, are identified as 3BD-3, 3BD-6, 3BD-8, 3BD-9,
21 3BD-15, 3BD-25, 3BD-26 and 3BD-27. Each of the designated
22 antibodies is produced by a hybridoma cell line deposited
23 with the American Type Culture Collection at 10801
24 University Boulevard, Manassas, Va. having an ATCC
Accession Number as follows:
26 Antibody ATCC Accession Number
27 3BD-3
28 3BD-6
29 3BD-8
3BD-9
31 3BD-15
32 3BD-25
33 3BD-26
34 3BD-27
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1 These antibodies are considered monoclonal after four
2 rounds of limiting dilution cloning. The anti-melanoma
3 antibodies did not produce significant cancer cell
4 killing. The panel of anti-breast cancer antibodies killed
5 32-87% of the target cells and <1-3% of the control cells.
6 The predominant isotype was IgGl even though it was
7 expected that the majority of anti-tumor antibodies would
8 be directed against carbohydrate antigens, and thus, be of
9 the IgM type. There is a high therapeutic index since most
10 antibodies spare the control cells from cell death.
11 Table 1. Anti-Breast Cancer Antibodies
12
13 % Celi Death
14 Clones Target for Normal FibroblastFibrocystic Isotype
Anti-Breast Cells Breast Cells
Cancer Antibodies(CCD-l2CoN) (MCF-12A)
(HEY & A2058)
15 3BD-3 74.9% 3.7% <1% vl, ~
16 3BD-6 68.5% 5.6% <1 % vl, ~
17 3BD-8 81.9% 4.5% 2.6% v~, K
18 3BD-9 77.2% 7.9% <1% vl,
19 3BD-15 87.1 % <1 % <1 % v~ , ~
20 3BD-26 54.8% 3.3% <1 % N,K
21 3BD-25 32.4% 3.6% <1 % vl.x
22 3BD-27 60.1 % 8.3% 1.3% y~, K
23
24 Example 2
In this example customized anti-cancer antibodies are
26 produced by first obtaining samples of the patient's
27 tumor. Usually this is from a biopsy specimen from a
28 solid tumor or a blood sample from hematogenous tumors.
29 The samples are prepared into single cell suspensions and
fixed for injection into mice. After the completion of the
31 immunization schedule the hybridomas are produced from the
32 splenocytes. The hybridomas are screened against a variety
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21
1 of cancer cell lines and normal cells in standard
2 cytotoxicity assays. Those hybridomas that are reactive
3 against cancer cell lines but are not reactive against
4 normal non-transformed cells are selected for further
propagation. Clones that were considered positive were
ones that selectively killed the cancer cells but did not
7 kill the non-transformed cells. The antibodies are
8 characterized for a large number of biochemical parameters
9 and then humanized for therapeutic use.
The melanoma tumor cells isolated and cell lines were
11 cultured as described in Example 1. Balb/c mice were
12 immunized according to the following schedule: 200,000
13 cells s.c. and i.p. on day 0, then 200,000 cells were
14 injected i.p. on day 21, then 1,000,000 cells were
injected on day 49, then 1,250,000 cells in Freund's
16 Complete Adjuvant were injected i.p. on day 107, and then
17 200,000 cells were injected on day 120 i.p. and then the
18 mice were sacrificed on day 123. The spleens were
19 harvested and the splenocytes were divided into two
aliquots for fusion with Sp2/0 (1LN) or NS-1 (2LN) myeloma
21 partners using the methods outlined in example 1.
22 The screening was carried out 11 days after the
23 fusion against A2058 melanoma cells and CCD-l2CoN
24 fibroblasts. Each pair of plates were washed with 100
microliters of room temperature PBS and then aspirated to
26 near dryness. Then 50 microliters of hybridoma supernatant
27 was added to the same wells on each of the two plates. The
28 spent Sp2/0 supernatant was added to the control wells at
29 the same volume and the plates were incubated for around
18 hours at 37 degrees Celsius at a 8°sC02, 98°s relative
31 humidity incubator. Then each pair of plates were removed
32 and in the positive control wells 50 microliters of 70°s
33 ethanol was substituted for the media for 4 seconds. The
34 plates were then inverted and washed with room temperature
PBS once and dried. Then 50uL of fluorescent live/dead dye
36 diluted in PBS (Molecular Probes Live/Dead Kit) was added
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22
1 for one hour and incubated at 37 degrees Celsius. The
2 plates were then read in a Perkin-Elmer fluorescent plate
3 reader and the data analyzed using Microsoft Excel. The
4 wells that were considered positive were subcloned and the
same screening process was repeated 13 days later and then
6 33 days later. The results of the last screening is
7 outlined in Table 2 below. A number of monoclonal
8 antibodies were produced in accordance with the method of
9 the present invention. These antibodies, whose
characteristics are summarized in Table 2, are identified
11 as 1LN-1, 1LN-8, 1LN-12, 1LN-14, 2LN-21, 2LN-28, 2LN-29,
12 2LN-31, 2LN-33, 2LN-34 and 2LN-35. Each of the designated
13 antibodies is produced by a hybridoma cell line deposited
14 with the American Type Culture Collection at 10801
University Boulevard, Manassas, Va. having an ATCC
16 Accession Number as follows:
17
18
19 Antibody ATCC Accession Number
1LN-1
21 1LN- 8
22 1LN-12
23 1LN-14
24 ~ 2LN-21
2$ 2LN-28
26 2LN-29
27 2LN-31
28 2LN-33
29 2LN-34
2LN-35
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1
2 Table 2, Anti-Melanoma Antibodies
Cell Death
3 Target for
Anti- Normal Fibroblast
4 Clones
Melanoma Cells
Antibodies (CCD-1 2CoN)
(A2058)
1LN-1 59.4% <1
6 1 LN-8 11.0% 5.0%
7 1 LN-12 55.2% 1.4%
$ 1LN-14 51.4% <1%
2LN-21 72.0% 15.9%
2LN-28 66.6% 12.4%
1 2LN-29 78.2% 6.1
1
12 2LN-31 100% 7.8%
0
13 2LN-33 94.2% <1 /o
14 2LN-34 56.6% 11.2%
2LN-35 66.5% 6.6%
16
17 The table illustrates that clones from both the Sp2/0
18 and NS-1 fusions were able to produce antibodies that had
19 a greater than 50% killing rate for cancerous cells and at
the same time some of the clones were able to produce less
21 than one percent killing of normal control fibroblasts.
22
23 Example 3
24 In this example antibodies were produced to several
different breast tumor samples following the method of
26 Example 2 in order to demonstrate the generality of
27 producing customized antibodies. Biopsy specimens of
28 breast tumors were obtained and stored at -70°C until used
29 as noted in Example 1. Single cell suspensions were
prepared for each specimen and fixed with -30°C, 70%
31 ethanol, washed with PBS and reconstituted to an
32 appropriate volume for injection. Female, 7-8 week old, A
33 strain, H-2d haplotype Balb/c mice (Charles River Canada,
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24
1 St. Constant, QC, Can), were immunized with 2.5x105-1x106
2 cells and boosted every third week until a final pre-
3 fusion boost was performed three days prior to the
4 splenectomy. The hybridomas were prepared by fusing the
isolated splenocytes with Sp2/0 myeloma partners. The
6 supernatants from the fusions were tested for subcloning
7 of the hybridomas.
8
9 Hs574.T breast ductal carcinoma cells, A2058
melanoma cells, NCI-H460 human lung large cell carcinoma,
11 NCI-H661 human lung large cell carcinoma, CCD-112CoN human
12 colon fibroblasts, CCD-27sk human skin fibroblasts, MCF-
T3 12A human mammary epithelial cells, Hs574.mg human breast
14 cells and other cell lines, were obtained from ATCC and
cultured according to enclosed instructions. Both cancer
16 and non-cancer cells were plated three to four days prior
17 to screening.
18 The hybridomas were cultured for ten to twelve days
19 after fusion and observed under the microscope. When 20 to
25°s of the wells were over 80°s confluent, the hybridoma
21 supernatants were screened in a cytotoxicity assay. The
22 hybridoma supernatants were divided into two 75 microliter
23 portions; one portion was added to a target cancer cell
24 plate and another to a non-cancer cell plate. Prior to
transfer of hybridoma supernatants, the cell plates were
26 washed three times with 100 microliters of PBS. The
27 supernatant from the anti-breast cancer hybridomas were
28 transferred to the Hs574.T and the Hs574.mg cells, whereas
29 the supernatant from the anti-lung cancer hybridoma were
transferred to the NCI-H460 and CCD-27SK cells. The
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1 cancer cells were incubated with the hybridoma
2 supernatants for 18 hours at 37 degrees Celsius in an 8
3 percent COZ incubator.
4 The Live/Dead cytotoxicity assay was obtained from
5 Molecular Probes (Eugene,OR). The assays were performed
6 according to the manufacturer's instructions with the
7 changes outlined below. The plates with the cells were
8 washed once with 100 microliters of PBS at 37°C. 75 to 100
9 microliters of supernatant from the hybridoma microtitre
10 plates were transferred to the cell plates and incubated
11 in a 8% COZ incubator for 18-24 hours. Then, the wells that
12 served as the dead control cells were aspirated until
13 empty and 50 microliters of 70% ethanol was added. The
14 plate was then emptied by inverting and blotted dry. Room
15 temperature PBS was dispensed into each well from a
16 multichannel squeeze bottle, tapped three times, emptied
17 by inversion and then blotted dry. 50 microliters of the
18 fluorescent Live/Dead dye diluted in PBS was added to each
19 well and incubated at 37°C in a 5% C02 incubator for one
20 hour. The plates were read in a Perkin-Elmer HTS7000
21 fluorescence plate reader and the data was analyzed in
22 Microsoft Excel (Microsoft, Redmond, WA).
23 Four rounds of screening were conducted to
24 produce single clone hybridoma cultures. For two rounds of
25 screening the hybridoma supernatants were tested only
26 against the cancer cells. In the last round of screening
27 the supernatant was tested against a number of non-cancer
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26
1 cells as well as the target cells indicated in Table 3.
2 The antibodies were isotyped using a commercial isotyping
3 kit (Roche, Indianapolis, IN).
4 A number of monoclonal antibodies were produced in
accordance with the method of the present invention.
6 These antibodies, whose characteristics are summarized in
7 Table 3, are identified as 4BD-1, 4BD-3, 4BD-6, 4BD-9,
$ 4BD-13, 4BD-18, 4BD-20, 4BD-25, 4BD-37, 4BD-32, 4BD-26,
9 4BD-27, 4BD-28, 4BD-50, 6BD-1, 6BD-3, 6BD-5, 6BD-11, 6BD-
25, 7BD-7, 7BD-12-1, 7BD-12-2, 7BD-13, 7BD-14, 7BD-19,
11 7BD-21, 7BD-24, 7BD-29, 7BD-30, 7BD-31, 7BDI-17, 7BDI-58,
12 7BDI-60 and 7BDI-62. Each of the designated antibodies is
13 produced by a hybridoma cell line deposited with the
14 American Type Culture Collection at 10801 University
1$ Boulevard, Manassas, Va. having an ATCC Accession Number
16 as follows:
17 Antibody ATCC Accession Number
18 4BD-1
19 4BD-3
4BD-6
21 4BD-9
22 4BD-13
23 4BD-18
24 4BD-20
4BD-25
26 4BD-37
27 4BD-32
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1 4BD-26
2 4BD-27
3 4BD-28
4 4BD-50
S 6BD-1
6 6BD-3
7 6BD-5
8 6BD-11
9 6BD-25
7BD-7
11 7BD-12-1
12 7BD-12-2
13 7BD-13
14 7BD-14
7BD-19
16 7BD-21
17 7BD-24
18 7BD-29
19 7BD-30
7BD-31
21 7BDI-17
22 7BDI-58
23 7BDI-60
24 7BDI-62
These antibodies are considered monoclonal after four
26 rounds of limiting dilution cloning. The panel of anti-
27 breast cancer antibodies killed 15-79s of the target cells
28 and <1-31~ of the control cells. The majority of anti-
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28
1 tumor antibodies were IgM type, suggesting they could be
2 directed against carbohydrate antigens on the surface of
3 tumor cells. There is a high therapeutic index since most
4 of the antibodies do not cause the normal cells to undergo
cell death.
6 These monoclonal antibodies are characterized for a
7 number of immunological and biochemical parameters. A
8 cell based enzyme linked immunosorbent assay (ELISA) was
9 established for measuring the binding of the antibodies
derived of each clones to different cell lines. Cells were
11 seeded and grown on 96-well tissue culture plates. The
12 plates were washed with 100 microliters of PBS. 100
13 microliters of cold 4 percent paraformaldehyde in PBS were
14 added to each well for ten minutes and then aspirated. The
plates were washed with PBS using a multichannel squeeze
16 bottle . The wells were emptied and 100 microliters of
17 blocking buffer (1 percent hydrocasein, 0.1 percent
18 geletin in 50mM Tris-HC1 buffer, pH 9.3) was added to each
19 well for one hour at room temperature. The plates were
washed three times with a buffer (0.05 percent Tween 20 in
21 10 mM PBS) at room temperature and then stored at -30
22 degrees Celsius with 100 microliters of the buffer. Prior
23 to use the plates were thawed and the buffer was aspirated
24 from each well. 75 microliters of hybridoma supernatant
were added to each well and incubated for 60 minutes at
26 room temperature. After the plates were washed with PBS
27 using a multichannel squeeze bottle, 50 microliters of a
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29
1 combination of peroxidase conjugated goat anti-mouse IgG
2 and peroxidase conjugated donkey anti-mouse IgM (Jackson
3 ImmunoResearch Lab, Inc., West Grove, PA.) was added and
4 incubated for 30 minutes at room temperature. After the
last wash, 50 microliters of orthophenylene diamine (OPD)
6 (Sigma, St. Louis, MO) was added to each well and the
7 optical density was read at 492 nm on the HTS7000 plate
8 reader after adding equal volume of 1 N sulfuric acid.
9 Different clones show different profiles in binding to
different cells (Table 3). This indicates that they may
11 target different cell surface antigen and further suggests
12 the variable distribution of these antigen on the surface
13 of cancer cells. Those binding only to cancer cells but
14 not to normal cells could identify certain tumor-
associated antigen.
16
17 Table 3. Anti-Breast Cancer Antibodies
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1 Binding
to
cell
lines
2 ClonesIsotype
3 Hs574.T Hs574.mgHs574.THs574,mgNCI-H460CCD-27skA2058
4 BD-1 38.2 5 0.8 0.5 0.6 0.3 ND*
N
K
5 BD-3 79 12 0.35 0.25 0.24 0.14 ND
~
K
6 BD-5 57.3 8 1.0 0.3 0.14 0.25 ND
a
K
7 BD-11 52.3 11 0.15 0.1 0.17 0.1 ND
~
K
8 BD-25 33.3 2 0.15 0.1 0.2 0.1 ND
N,
K
9 BD-26 27 1.8 0.5 ND ND <0.1 ND
N,
K
10 BD-27 19.6 <1 0.9 ND ND 0.5 ND
~
K
11 BD-28 26.4 <1 0.8 ND ND <0.1 ND
~
K
12 BD-32 41.7 4 0.8 ND ND <0.1 ND
~,
K
13 BD-50 20 <1 0.8 ND ND <0.1 ND
N,
K
14 BD-1 23 31 0.6 ND ND <0.1 ND
N,
K
15 BD-3 29.7 8.2 1.7 ND ND 0.1 ND
N
K
16 BD-6 17 <1 0.9 ND ND <0.1 ND
N,
K
17 BD-9 15 <1 0.6 ND ND <0.1 ND
~
K
18 BD-13 31 <1 1.2 ND ND <0.1 ND
N,
K
19 BD-18 23.3 2.4 0.7 ND ND 0.12 ND
~
K
20 BD-20 45 <1 0.95 ND ND <0.1 ND
~
K
21 BD-25 26 14.16 1.8 ND ND 0.1 ND
N,
K
22 BD-37 30 <1 0.8 ND ND
<0.1 ND
~
K
23 BD-7 24 3 0.8 0.3 1.4 0.26 ND
N,
K
24 BD-12-1 22 6 0.36 0.16 0.43 0.1 ND
N,
K
25 BD-12-2 31 2 0.2 0,2 0.2 0.2 0.2
~
K
26 BD-13 29 12 0.1 0.15 0.2 0.1 0.2
N,
K
27 BD-14 32 13 0.4 0.4 0.6 0.3 0.5
~,
K
28 BD-19 20 4 1.3 0.4 0.43 0.2 ND
~,
K
29 BD-21 21 13 0.4 0.5 0.25 0.3 ND
~J,
K
30 BD-24 32 15 0.3 0.1 0.14 0.15 ND
~,
K
31 BD-29 15 16 0.3 0.24 0.14 0.16 ND
N,
K
32 BD-30 23 13 0.34 0.24 0.2 0.16 ND
N,
K
33 BD-31 28 10 0.3 0.4 0.4 0.3 0.4
~,
K
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1 BDI-17 23 <1 0.75 ND ND ND ND
~,
K
2 8DI-58 17.5 <1 0.77 ND ND ND ND
Y1,
K
3 BDI-60 15 <1 0.73 ND ND ND ND
Y1,
K
4 BDI-62 15 5 0.55 ND ND ND ND
'ND: not done.
6 Example 4
7 In this example customized anti-cancer antibodies are
8 produced to a lung cancer sample by first obtaining
9 samples of the patient's tumor preparing single cell
suspensions which are then fixed for injection into mice
11 as noted in Example 1. After the completion of the
12 immunization schedule the hybridomas are produced from the
13 splenocytes. The hybridomas are screened against a variety
14 of cancer cell lines and normal cells in standard
cytotoxicity assays. Those hybridomas that are reactive
16 against cancer cell lines but are not reactive against
17 normal non-transformed cells are selected for further
18 propagation. Clones that were considered positive were
19 ones that selectively killed the cancer cells but did not
kill the non-transformed cells.
21 The lung cancer cells were isolated and cell lines
22 were cultured as described in Example 1. Female, 7-8 week
23 old, A strain, H-2d haplotype Balb/c mice (Charles River
24 Canada, St. Constant, QC, Can), were immunized with human
lung cancer cells. The lung cancer cell suspensions were
26 emulsified in an equal volume of Freund's complete
27 adjuvant (FCA) fox the first immunization and then in
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1 Freund's incomplete adjuvant (FIA) for subsequent
2 immunizations at 0, 21, 45 days. 5x105 cells were used to
3 immunize each mouse either through a subcutaneous or
4 intra-peritoneal route. Immunized mice were sacrificed 3-4
days after the final immunization with human lung cancer
6 cells at 148 days, given intra-peritoneally, in PBS at pH
7 7.4. The spleens were harvested and the splenocytes were
8 divided into two aliquots for fusion with Sp2/0 myeloma
9 partners using the methods outlined in Example 1.
The screening was carried out 10 days after the
11 fusion against NCI-H460 and/or NCI-H661 cells and CCD-27SK
12 fibroblasts. Each pair of plates were washed with 100
13 microliters of room temperature PBS and then aspirated to
14 near dryness. Then 75 microliters of hybridoma supernatant
was added per well on each of the two plates. The spent
16 Sp2/0 supernatant was added to the control wells at the
17 same volume and the plates were incubated for around 18
18 hours at 37 degrees Celsius at a 8%COz, 98% relative
19 humidity incubator. Then each pair of plates was removed
and in the positive control wells 50 microliters of 70%
21 ethanol was substituted for the media for 4 seconds. The
22 plates were then inverted and washed with room temperature
23 PBS once and dried. Then 50 microliters of fluorescent
24 live/dead dye diluted in PBS (Molecular Probes Live/Dead
Kit) was added for one hour and incubated at 37 degrees
26 Celsius. The plates were then read in a Perkin-Elmer
27 fluorescent plate reader and the data analyzed using
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33
1 Microsoft Excel. The wells that were considered positive
2 were subcloned and the same screening process was repeated
3 6 days later and then 13 days later. The result of the
4 last screening is outlined in Table 4 below. Antibodies
were characterized for binding to different cell lines
6 with a cellular ELISA according to the methods of Example
7 3. A number of monoclonal antibodies were produced in
8 accordance with the method of the present invention.
9 These antibodies, whose characteristics are summarized in
Table 4, are identified as 5LAC2, 5LAC4, 5LAC20, and
11 5LAC23. Each of the designated antibodies is produced by
12 a hybridoma cell line deposited with the American Type
13 Culture Collection at 10801 University Boulevard,
14 Manassas, Va. having an ATCC Accession Number as follows:
Antibody ATCC Accession Number
16 5LAC2
17 5LAC4
18 5LAC20
19 5LAC23.
Table 4. Anti-Lung Cancer Antibodies
21 ~ Binding
to
cell
lines
22 ClonesIsotype
23 Hs574.T NCI-H460NCI-H6612058 CCD-27skHs574.THs574.mgNCI-
H460CCD-27skA2058
24 30 7 45.3 23 <1 0.2 0.2 0.26 0.2 0.2
LAC2 N,
K
26 21 11 20.5 23 3 0.7 0.9 1.7 0.8 0.9
27 ~AC4 u,
K
28 23 7 66 24 3 0.5 0.2 0.6 0.2 0.2
4
29 LAC20N, .
K
23 8 57.6 25 5 0.6 0.6 0.6 0.6 0.6
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1 *ND: not done
2 The table illustrates that clones were able to
3 produce antibodies that had a greater than 7-67% killing
4 rate for cancerous cells and at the same time some of the
clones were able to produce less than five percent killing
6 of normal control fibroblasts.
7
8 Example 5
9 In this example customized anti-cancer antibodies are
produced to a patient's lung cancer cells, but cultured
11 cells were used in the antibody development process to
12 demonstrate the generality of the immunization process.
13 The samples were prepared into single cell suspensions and
14 fixed for injection into mice as noted in Example 1. After
the completion of three rounds of immunization with cells
16 derived directly from a patient's lung cancer, the mice
17 were immunized twice with a human lung large cell
18 carcinoma cell line (NCI-H460). Hybridomas were produced
19 from splenocytes and the supernatants were screened
against a variety of cancer cell lines and normal cells in
21 standard cytotoxicity assays. Those hybridomas that were
22 reactive against cancer cell lines but were not reactive
23 against normal non-transformed cells were selected for
24 further propagation. Clones that were considered positive
were ones that selectively killed the cancer cells but did
26 not kill the non-transformed cells. The antibodies are
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1 characterized for a large number of biochemical parameters
2 and then humanized for therapeutic use.
3 The lung tumor cells isolated and cell lines were
4 cultured as described''in Example'1. Balb/c mice, A strain
5 with H-2d haplotype from Charles River Canada, St.
6 Constant, Quebec, Canada, female, 7-8 week old, were
7 immunized with the human lung cancer cells emulsified in
8 an equal volume of either Freund's complete adjuvant (FCA)
9 for the first immunization and then in Freund's incomplete
10 adjuvant (FIA) for subsequent immunizations at 0, 21, 45
11 days with 5x105 cells. The mice were immunized with fixed
12 NCI H460 cells, which were prepared from NCI H460 cells
13 grown in T-75 cell culture flask by scraping mono-layer
14 cells into cell suspensions at 105, 150 and 170 days.
15 Immunized mice were sacrificed 3-4 days after the final
16 immunization with NCI H460 cells, given intra-
17 peritoneally, in phosphate buffered saline buffer (PBS),
18 pH 7.4. The spleens were harvested and the splenocytes
19 were divided into two aliquots for fusion with Sp2/0
20 myeloma partners using the methods outlined in Example 1.
21 The screening was carried out 10 days after the
22 fusion against NCI H460 cells and CCD-27SK fibroblasts as
23 described in Example 4. Antibodies were characterized for
24 binding to different cell lines with a cellular ELISA
25 according to the methods of Example 3.
26
_ CA 02471206 2004-06-21
P'~in~ed ~7 08~~fl03~~ s~3 zaFz os42:'~ ~E~CPAMi~ ~~~sT
.,._. . . f..r _.. . . .: ._a .. t, . .. _. .
36
1' The wel7.s that were considered positive were
.2 subelaned and the same screening process was repeated 9
3 days and,l8, days later. The results aze outlined in Table
4 ~ 5 below. A 'number of . monoclonal ~ ~atibodies ~rere produced
in accordance with the method of the present invention_
6 . These antibodies, whose characteristics are summarized in
7 Table 5, are identified as H460-1, H~60-4, H460-5, H~60-
8 lo, H460-lg, H46p-16-l, H450-16-2, H~60-23 and ~I~60-2?.
9 Each of the designated antibodies is produced by a
hybridoma cell line depo$ited with the American Type
11 Cultwre Collection at 10801 University Bvulevardr
12 Manassas, va _ having an .p,TCC ,pccessioz~. Number as follows n
13 Antzbodv ATCC Acaeasian N,msber .
14 H460-1
H460-4
lfi H460-5
17 H460-10
1s x46o-lg
19 H46Q-Z6-1
H4fi0-16-2
Z1 H460-23
H460-27
23 H'460-22-1
.. -. _ .. ,.r ~, ar? '? :~ :.i.~~'
,.
~ a Empf eGeit.~ AMENDED SHEET (ARTICLE 19) ~ ;~1 fl~~2~03f
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1 Table 5. Anti-Lung Cancer Antibodies
2 Binding
to
cell
lines
3 Clones
4 NCI-H46o Hs574.A2058 CCD- Hs574.Hs574.NCI- CCD- A2058
$ 460-1 16 30 23 <1 1.0 0.6 0.5 0.7 ND
yi,e
6 460-4 37 21 23 3 1.0 0.6 0.4 0.6 ND
7 460-5 22.5 23 24 3 1.0 0.3 0.3 0.2 ND
u,
K
$ 4so-io 8 23 25 5 0.3 0.14 0.2 0.1 ND
u,
K
460-i4 17 ND ND 4 1.1 0 . 0 . 0 ND
6 4 .
54
yl,
a
1~ 46o-i6-i 33 ND ND 8 1. 0 . 0 . 0. ND
0 6 3 5
yl,
a
11 46o-i6-z 22 ND ND 3 1.0 0.6 0.3 0.7 ND
yl,e
12 a6o-22-iy1, 21 ND ND 5 0.6 0.4 0.3 0.4 ND
a
13 460-z2-2 23 ND ND 3 0.4 0.1 0.1 0.1 ND
u,
K
14 4so-z3 36 36 18 1 0.4 1.1 0.54 0.53 0.58
~,1,
K
1$ 460-27u, 33 31 16 8 0.3 0.4 0.4 0.3 0.4
K
16 *ND: not done
17 The table illustrates that clones were able to
18 produce antibodies that had a greater than 15% killing
19 rate for cancerous cells and at the same time some of the
20 clones were able to produce less than eight percent
21 killing of normal control fibroblasts.
22 The anti-cancer antibodies of the invention are
23 useful for treating a patient with a cancerous disease
24 when administered in admixture with a pharmaceutically
25 acceptable adjuvant, for example normal saline, a lipid
26 emulsion, albumen, phosphate buffered saline or the like
27 and are administered in an amount effective to mediate
28 treatment of said cancerous disease, for example with a
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1 range of about 1 microgram per milliliter to about 1 gram
2 per milliliter.
3 The method for treating a patient suffering from a
4 cancerous disease may further include the use of
S conjugated anti-cancer antibodies and would this include
6 conjugating patient specific anti-cancer antibodies with a
7 member selected from the group consisting of toxins,
8 enzymes, radioactive compounds, and hematogenous cells;
9 and administering these conjugated antibodies to the
patient; wherein said anti-cancer antibodies are
11 administered in admixture with a pharmaceutically
12 acceptable adjuvant, for example normal saline, a lipid
13 emulsion, albumen, phosphate buffered saline or the like
14 and are administered in an amount effective to mediate
treatment of said cancerous disease, for example with a
16 range of about 1 microgram per mil to about 1 gram per
17 mil. In a particular embodiment, the anti-cancer
18 antibodies useful in either of the above outlined methods
19 may be a humanized antibody.
The anti-cancer antibodies of the invention are
21 useful for treating a patient with a cancerous disease
22 when administered in admixture with a pharmaceutically
23 acceptable adjuvant, for example normal saline, a lipid
24 emulsion, albumen, phosphate buffered saline or the like
and are administered in an amount effective to mediate
26 treatment of said cancerous disease, for example with a
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1 range of about 1 microgram per mil to about 1 gram per
2 mil.
3 The method for treating a patient suffering from a
4 cancerous disease may further include the use of
conjugated anti-cancer antibodies and would this include
6 conjugating patient specific anti-cancer antibodies with a
7 member selected from the group consisting of toxins,
8 enzymes, radioactive compounds, and hematogenous cells;
9 and
administering these conjugated antibodies to the patient;
11 wherein said anti-cancer antibodies are administered in
12 admixture with a pharmaceutically acceptable adjuvant, for
13 example normal saline, a lipid emulsion, albumen,
14 phosphate buffered saline or the like and are administered
in an amount effective to mediate treatment of said
16 cancerous disease, for example with a range of about 1
17 microgram per mil to about 1 gram per mil. In a
18 particular embodiment, the anti-cancer antibodies useful
19 in either of the above outlined methods may be a humanized
antibody.
21
22
23
24
