CA2471206A1 - Individualized anti-cancer antibodies - Google Patents

Abstract

The present invention relates to a method for producing patient specific ant i- cancer antibodies using a novel paradigm of screening. By segregating the an ti- cancer antibodies using cancer cell cytotoxicity as end point, the process makes possible the production of anti-cancer antibodies customized for the individual patient that can be used for therapeutic and diagnostic purposes.

Description

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.

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 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 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.

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 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, 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 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.

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 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.

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 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.

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 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 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 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 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 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 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 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 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 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 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 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

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 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 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:

19 Antibody ATCC Accession Number 24 ~ 2LN-21 2$ 2LN-28 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 12 2LN-31 100% 7.8%

13 2LN-33 94.2% <1 /o 14 2LN-34 56.6% 11.2%

2LN-35 66.5% 6.6%

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.

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, 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.

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 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 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 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-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 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.

17 Table 3. Anti-Breast Cancer Antibodies 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

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 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 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 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 29 LAC20N, .
K

23 8 57.6 25 5 0.6 0.6 0.6 0.6 0.6 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.

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 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.

_ CA 02471206 2004-06-21 P'~in~ed ~7 08~~fl03~~ s~3 zaFz os42:'~ ~E~CPAMi~ ~~~sT
.,._. . . f..r _.. . . .: ._a .. t, . .. _. .

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 .

lfi H460-5 1s x46o-lg H4fi0-16-2 23 H'460-22-1 .. -. _ .. ,.r ~, ar? '? :~ :.i.~~' ,.
~ a Empf eGeit.~ AMENDED SHEET (ARTICLE 19) ~ ;~1 fl~~2~03f 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 .

yl, a 1~ 46o-i6-i 33 ND ND 8 1. 0 . 0 . 0. ND

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 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 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.

Claims (25)

What is claimed is:
1. Claim 1. A method for treating a patient suffering from a cancerous disease comprising:
   administering to said patient anti-cancer antibodies or fragments thereof produced in accordance with a method for the production of individually customized anti-cancer antibodies which are useful in treating a cancerous disease, said antibodies including a subset of antibodies or fragments thereof characterised as being cytotoxic against cells of a cancerous tissue, said subset being essentially benign, to non-cancerous cells;
   wherein one or more antibodies or fragments thereof selected from said subset are placed in admixture with a pharmaceutically acceptable adjuvant sad are administered in an amount effective to mediate treatment of said cancerous disease;
   said one or more antibodies or fragments thereof being selected from the group consisting of a 1LN-8, 4BD-1, a 4BD-3, a 4BD-6, a 4BD-9, a 48D-13, a, 4BD-18, a 4BD-20, a 4BD-25, a 4BD-26, a 4BD-27, a 4BD-28, a 4BD-32, a 4BD-37, a 48D-50, a 6BD-1, a 68D-3, a 6BD-5, a 6BD-11, a 6BD-25, a 7BD-7, a 78D-12-1, a 7BD-12-2, a 7BD-13, a 7BD-14, a 7BD-39, a 7BD-21, a 7BD-24, a 7BD-29, a 7BD-30, a 7BD-31, a 7BDI-17, a 7BDI-58, a 7BDI-60, a 7BDI-62, a 5LAC2, a 5LAC4, a 5LAC20, a 5LAC23, a H460-1, a H450-4, a H460-5, a H460-10, a H460-14, a H460-16-1, a H460-16-2, a H460-22-1, a H460-23 and a H460-27 monoclonal antibody er combinations thereof.
2. Claim 2. The method for treating a patient suffering from a cancerous disease in accordance with claim 1, wherein said one or more antibodies or fragments thereof selected from said subset are humanized.
3. Claim 3. The method for treating a patient suffering from a cancerous disease in accordance with claim 1 comprising:
   conjugating said subset of antibodies or fragments thereof with a member selected from the group consisting of toxins, enzymes, radioactive compounds, and hematogenous cells; and administering conjugated antibodies or fragments thereof to said patient;
   wherein said conjugated antibodies are placed in admixture with a pharmaceutically acceptable adjuvant and are administered in as amount effective to mediate treatment of said cancerous disease.
4. Claim 4. The method of claim 3, wherein said one or more antibodies or fragments thereof selected from said subset are humanized.
5. Claim 5. The method for treating a patient suffering from a cancerous disease in accordance with claim 1 wherein:
   the cytotoxicity of said antibodies or fragments thereof is mediated through antibody dependent cellular toxicity.
6. Claim 6. The method for treating a patient suffering from a cancerous disease in accordance with claim 1 wherein:
   the cytotoxicity of said antibodies or fragments thereof is mediated through complement dependent cellular toxicity.
7. Claim 7. The method for treating a patient suffering from a cancerous disease in accordance with claim 1 wherein:
   the cytotoxicity of said antibodies or fragments thereof is mediated through catalyzing of the hydrolysis of cellular chemical bonds.
8. Claim 8. The method for treating a patient suffering from a cancerous disease in accordance with claim 1 wherein:
   the cytotoxicity of said antibodies or fragments thereof is mediated through producing an immune response against putative cancer antigens residing on tumor cells.
9. Claim 9. The method for treating a patient suffering from a cancerous disease in accordance with claim 1 wherein:
   the cytotoxicity of said antibodies or fragments thereof is mediated through targeting of cell membrane proteins to interfere with their function.
10. Claim 10. The method for treating a patient suffering from a cancerous disease in accordance with claim 1 wherein:
    the cytotoxicity of said antibodies or fragments thereof is mediated through production of a conformational change in a cellular protein effective to produce a signal to initiate cell-killing.
11. Claim 11. The method for treating a patient suffering from a cancerous disease in accordance with claim 1 wherein:
    said method of production utilizes a tissue sample containing cancerous and non-cancerous cells obtained from a particular individual.
12. Claim 12. A method for treating a patient suffering from a cancerous disease comprising:
    administering to said patient anti-cancer antibodies or fragments thereof produced in accordance with a method for the production of individually customized anti-cancer antibodies which are useful in treating a cancerous disease, said antibodies including a subset of antibodies or fragments thereof characterized as being cytotoxic against cells of a cancerous tissue, said subset being essentially benign to non-cancerous cells;
    wherein one or more antibodies or fragments thereof selected from said subset are placed in admixture with a pharmaceutically acceptable adjuvant and are administered in an amount effective to mediate treatment of said cancerous disease;
    said one or more antibodies or fragments thereof produced by a hybridoma cell line having an ATCC Accession Number selected from the group consisting of (to be provided before publication) or combinations thereof.
13. Claim 13. The method for treating a patient suffering from a cancerous disease in accordance with claim 12, wherein said one or more antibodies or fragments thereof selected from said subset are humanized.
14. Claim 14. The method for treating a patient suffering from a cancerous disease in accordance with claim 12 comprising:
    conjugating said subset of antibodies or fragments thereof with a member selected from the group consisting of toxins, enzymes, radioactive compounds, and hematogenous cells; and administering conjugated antibodies or fragments thereof to said patient;
    
    wherein said conjugated antibodies are placed in admixture with a pharmaceutically acceptable adjuvant and are administered in an amount effective to mediate treatment of said cancerous disease.
15. Claim 15. The method of claim 14, wherein said one or more antibodies or fragments thereof selected from said subset are humanized.
16. Claim 16. The method for treating a patient suffering from a cancerous disease in accordance with claim 12 wherein:
    the cytotoxicity of said antibodies or fragments thereof is mediated through antibody dependent cellular toxicity.
17. Claim 17. The method for treating a patient suffering from a cancerous disease in accordance with claim 12 wherein:
    the cytotoxicity of said antibodies or fragments thereof is mediated through complement dependent cellular toxicity.
18. Claim 18. The method for treating a patient suffering from a cancerous disease in accordance with claim 12 wherein:
    
    the cytotoxicity of said antibodies or fragments thereof is mediated through catalyzing of the hydrolysis of cellular chemical bonds.
19. Claim 19. The method for treating a patient suffering from a cancerous disease in accordance with claim 12 wherein:
    the cytotoxicity of said antibodies or fragments thereof is mediated through producing an immune response against putative cancer antigens residing on tumor cells.
20. Claim 20. The method for treating a patient suffering from a cancerous disease in accordance with claim 12 wherein:
    the cytotoxicity of said antibodies or fragments thereof is mediated through targeting of cell membrane proteins to interfere with their function.
21. Claim 21. The method for treating a patient suffering from a cancerous disease in accordance with claim 12 wherein:
    the cytotoxicity of said antibodies or fragments thereof is mediated through production of a conformational change in a cellular protein effective to produce a signal to initiate cell-killing.
    
    Claim 22. The method for treating a patient suffering from a cancerous disease in accordance with claim 12 wherein:
    said method of production utilizes a tissue sample containing cancerous and non-cancerous cells obtained from a particular individual.
    
    Claim 23. Anti-cancer antibodies or fragments thereof selected from the group consisting of a 1LN-8, 4BD-1, a 4BD-3, a 4BD-6, a 4BD-9, a 4BD-13, a 4BD-18, a 4BD-20, a 4BD-25, a 4BD-26, a 4BD-27, a 4BD-28, a 4BD-32, a 4BD-37, a 4BD-50, a 6BD-1, a 6BD-3, a 6BD-5, a 6BD-11, a 6BD-25, a 7BD-7, a 7BD-12-1, a 7BD-12-2, a 7BD-13, a 7BD-14, a 7BD-19, a 7BD-21, a 7BD-24, a 7BD-29, a 7BD-30, a 7BD-31, a 7BDI-17, a 7BDI-58, a 7BDI-60, a 7BDI-62, a 5LAC2, a 5LAC4, a 5LAC20, a 5LAC23, a H460-1, a H460-4, a H460-5, a H460-10, a H460-14, a H460-16-1, a H460-16-2, a H460-22-1, a H460-23 and a H460-27 monoclonal antibody or combinations thereof.
    
    Claim 24. Anti-cancer antibodies or fragments thereof produced by a hybridoma cell line having an ATCC Accession Number selected from the group consisting of (to be provided before publication).
    
    claim 25. The use of a composition for treating a patient suffering from a cancerous disease by administration of an effective amount of said composition to a patient to mediate treatment of said cancerous disease, wherein said composition comprises one or more antibodies or fragments thereof selected from a subset of said antibodies or fragments in admixture with a pharmaceutically acceptable adjuvant, said anti-cancer antibodies or fragments thereof produced in accordance with a method for the production of individually customized anti-cancer antibodies which are useful in treating cancerous disease, said subset of antibodies or fragments thereof characterized as being cytotoxic against cells of a cancerous tissue, as being essentially benign to non-cancerous cells and being selected from the group consisting of a 1LN-8, 4BD-1, a 4BD-3, a 4BD-6, a 4BD-9, a 4BD-13, a 4BD-18, a 4BD-20, a 4BD-25, a 4BD-26, a 4BD-27, a 4BD-28, a 4BD-32, a 4BD-37, a 4BD-50, a 6BD-1, a 6BD-3, a 6BD-5, a 6BD-11, a 6BD-25, a 7BD-7, a 7BD-12-1, a 7BD-12-2, a 7BD-13, a 7BD-14, a 7BD-19, a 7BD-21, a 7BD-24, a 7BD-29, a 7BD-30, a 7BD-31, a 7BDI-19, a 7BDI-58, a 7BDI-60, a 7BDI-62, a 5LAC2, a 5LAC4, a 5LAC20, a 5LAC23, a H460-1, a H460-4, a H460-5, a H460-10, a H460-14, a H460-16-1, a H460-16-2, a H460-22-1, a H460-23 and a H460-27 monoclonal antibody or combinations thereof.
    
    Claim 26. The use of a composition for treating a patient suffering from a cancerous disease by administration of an effective amount of the composition to the patient to mediate treatment of said cancerous disease, wherein said composition comprises one or more antibodies or fragments thereof from a subset of antibodies or fragments thereof characterized as being cytotoxic against cells of a cancerous tissue and essentially benign to non-cancerous cells placed in admixture with a pharmaceutically acceptable adjuvant, said one or more antibodies or fragments thereof produced by a hybridoma cell line having an ATCC Accession Number selected from the group consisting of (to be provided before publication) or combinations thereof.
    
    What is claimed is:
    Claim 1. A method for treating a patient suffering from a cancerous disease comprising:
    administering to said patient anti-cancer antibodies or fragments thereof produced in accordance with a method for the production of individually customized anti-cancer antibodies which are useful in treating a cancerous disease, said antibodies including a subset of antibodies or fragments thereof characterized as being cytotoxic against cells of a cancerous tissue, said subset being essentially benign to non-cancerous cells;
    wherein one or more antibodies or fragments thereof selected from said subset are placed in admixture with a pharmaceutically acceptable adjuvant and are administered in an amount effective to mediate treatment of said cancerous disease;
    said one or more antibodies or fragments thereof being selected from the group consisting of a 1LN-8, 4BD-1, a 4BD-3, a 4BD-6, a 4BD-9, a 4BD-13, a 4BD-18, a 4BD-20, a 4BD-25, a 4BD-26, a 4BD-27, a 4BD-28, a 4BD-32, a 4BD-37, a 4BD-50, a 6BD-1, a 6BD-3, a 6BD-5, a 6BD-11, a 6BD-25, a 7BD-7, a 7BD-12-1, a 7BD-12-2, a 7BD-13, a 7BD-14, a 7BD-19, a 7BD-21, a 7BD-24, a 7BD-29, a 7BD-30, a 7BD-31, a 7BDI-17, a 7BDI-58, a 7BDI-60, a 7BDI-62, a 5LAC2, a 5LAC4, a 5LAC20, a 5LAC23, a H460-1, a H460-4, a H460-5, a H460-10, a H460-14, a H460-16-1, a H460-16-2, a H460-22-1, a H460-23 and a H460-27 monoclonal antibody or combinations thereof.
    Claim 2. The method for treating a patient suffering from a cancerous disease in accordance with claim 1, wherein said one or more antibodies or fragments thereof selected from said subset are humanized.
    Claim 3. The method for treating a patient suffering from a cancerous disease in accordance with claim 1 comprising:
    conjugating said subset of antibodies or fragments thereof with a member selected from the group consisting of toxins, enzymes, radioactive compounds, and hematogenous cells; and administering conjugated antibodies or fragments thereof to said patient;
    wherein said conjugated antibodies are placed in admixture with a pharmaceutically acceptable adjuvant and are administered in an amount effective to mediate treatment of said cancerous disease.
    Claim 4. The method of claim 3, wherein said one or more antibodies or fragments thereof selected from said subset are humanized.
22. Claim 22. The method for treating a patient suffering from a cancerous disease in accordance with claim 12 wherein:
    said method of production utilizes a tissue sample containing cancerous and non-cancerous cells obtained from a particular individual.
23. Claim 23. Anti-cancer antibodies or fragments thereof selected from the group consisting of a 1LN-8, 4BD-1, a 4BD-3, a 4BD-6, a 4BD-9, a 4BD-13, a 4BD-18, a 4BD-20, a 4BD-25, a 4BD-26, a 4BD-27, a 4BD-28, a 4BD-32, a 4BD-37, a 4BD-50, a 6BD-1, a 6BD-3, a 6BD-5, a 6BD-11, a 6BD-25, a 7BD-7, a 7BD-12-1, a 7BD-12-2, a 7BD-13, a 7BD-14, a 7BD-19, a 7BD-21, a 7BD-24, a 7BD-29, a 7BD-30, a 7BD-31, a 7BDI-17, a 7BDI-58, a 7BDI-60, a 7BDI-62, a 5LAC2, a 5LAC4, a 5LAC20, a 5LAC23, a H460-1, a H460-4, a H460-5, a H460-10, a H460-14, a H460-16-1, a H460-16-2, a H460-22-1, a H460-23 and a H460-27 monoclonal antibody or combinations thereof.
24. Claim 24. Anti-cancer antibodies or fragments thereof produced by a hybridoma cell line having an ATCC Accession Number selected from the group consisting of (to be provided before publication).
25. Claim 25. The use of a composition for treating a patient suffering from a cancerous disease by administration of an effective amount of said composition to a patient to mediate treatment of said cancerous disease, wherein said composition comprises one or more antibodies or fragments thereof selected from a subset of said antibodies or fragments in admixture with a pharmaceutically acceptable adjuvant, said anti-cancer antibodies or fragments thereof produced in accordance with a method for the production of individually customized anti-cancer antibodies which are useful in treating cancerous disease, said subset of antibodies or fragments thereof characterized as being cytotoxic against cells of a cancerous tissue, as being essentially benign to non-cancerous cells and being selected from the group consisting of a 1LN-8, 4BD-1, a 4BD-3, a 4BD-6, a 4BD-9, a 4BD-13, a 4BD-18, a 4BD-20, a 4BD-25, a 4BD-26, a 4BD-27, a 4BD-28, a 4BD-32, a 4BD-37, a 4BD-50, a 6BD-1, a 6BD-3, a 6BD-5, a 6BD-11, a 6BD-25, a 7BD-7, a 7BD-12-1, a 7BD-12-2, a 7BD-13, a 7BD-14, a 7BD-19, a 7BD-21, a 7BD-24, a 7BD-29, a 7BD-30, a 7BD-31, a 7BDI-17, a 7BDI-58, a 7BDI-60, a 7BDI-62, a 5LAC2, a 5LAC4, a 5LAC20, a 5LAC23, a H460-1, a H460-4, a H460-5, a H460-10, a H460-14, a H460-16-1, a H460-16-2, a H460-22-1, a H460-23 and a H460-27 monoclonal antibody or combinations thereof.
    
    The amendments include:
    (1) amendments to claim 1 to correct editorial errors.
    (2) amendments to claims 1, 23 and 25 to insert reference to "a H460-22-1," in the three claims, support for this appearing in Table 5 on page 37, line 13.
    To provide consistency with the above, a new disclosure page 36 is included with the amended claim pages.
    Finally, attached hereto are copies of submissions and acknowledgement of the submission of Deposited Material and certain ATCC Numbers in relation to a related and counterpart U.S. application number 09/727,361 as provided to us by the instructing U.S. Attorney. This material also makes reference to H460-22-1.
    The cell line referred to in the ATCC material is found in the disclosure at the noted pages hereinbelow:
    Cell Line Reference Pages 1LN-8 22(23) 5LAC20 33(33) 3BD-26 19(20) 3BD-8 19(20) 7BD-14 27(30) 3BD-27 19(20) H460-27 36(37) H460-23 36(37) H460-16-2 36(37) H460-22-1 36(37) (amended as above) 7BDI-60 27(31) The first reference page number being reference to the page where the cell line is listed, whereas the second bracketed number is the page of the table in which the cell line appears.
    Appropriate amendments to the disclosure to add the ATCC numbers will be effected in due course.