WO1993006233A1 - Tumor immunotherapy utilizing a method of overcoming the resistance of a progressively growing tumor - Google Patents

Abstract

A method of overcoming the resistance of a progressively growing tumor in a host to lymphocytotoxic effector cells conferred by an anti-tumor polyclonal antibody response induced in the host. Themethod comprises the steps of obtaining a sample of the progressively growing tumor from the host, generating at least one human monoclonal antibody directed against at least one oncotope of the progressively growing tumor, subjecting the human monoclonal antibody to an analysis to make a determination of the therapeutic value of the human monoclonal antibody, generating a blocking human antibody which blocks anti-oncotope monoclonal antibodies; and delivering a therapeutic amount of the blocking human antibody to the host to permit the tumor to expose uncovered oncotopes thereby becoming sensitive to lymphocytotoxic effector cells.

Description

TUMOR IMMUNOTHERAPY UTILIZING A METHOD OF OVERCOMING THE RESISTANCE OF A PROGRESSIVELY GROWING TUMOR

Field of the Invention

Tumor immunotherapy utilizing a method of overcoming the resistance of a progressively growing tumor.

Background Art

Ever since the discovery of the technique of making monoclonal antibodies (MAbs) , published initially by Kohler & Milstein in Nature, Vol. 256, 495-497 (1975), many attempts have been made to use this procedure to derive MAbs directed against tumor-specific antigens in the hope that these would destroy the tumor when injected into the tumor-bearing host. During the last 10 years and after many such attempts, most recently reviewed by DeVita & al, ed. "Biologic Therapy of Cancer", published by Lippincott, Philadelphia, PA (1991) and Waldman, Science, Vol. 252, 1657-1662 (1991) a consensus has developed and articulated by Klausner in Biotechnology, Vol. 4, 185-194, (1986) that "naked" MAb therapy "would not do the trick".

There are two major reasons why this type of therapy has failed:

1) there is an absence of understanding that progressively-growing tumors become resistant to the lymphocytotoxic effector cells ("killer cells") they induce in the tumor-bearer directed against the specific "emergence- associated tumor immunogen" (EATI) present on the growing tumor cells, that this resistance is conferred on the growing tumor by an anti-tumor polyclonal antibody response simultaneously induced in the host, which coats the tumor cells in vivo, protects them from "killer cell" action and is itself harmless to the tumor; and

2) the MAbs that have been used clinically with human patients were generated by immunizing mice with human tumor tissue and choosing those mouse MAbs that preferentially reacted with human tumor tissue compared to their reactivity with normal human tissue, and as a consequence not likely to be directed against the molecules expressed on the surface of the tumor cells that the immune systems of the autochthonous host recognize as tumor-specific during progressive tumor growth (EATI) , each such antigenic determinant is referred to in this application as an "oncotope", as described in detail herein.

The discoveries made in this laboratory demonstrate that some virus-induced and carcinogen-induced progressively- growing tumors trigger in the tumor-bearing host both a B- cell and a T-cell immune response. The B-cell response resulted in the production of anti-tumor antibodies that coated the tumor cells without harming them or interfering with their rate of growth, but made them resistant to the "killer cells" (T-cell response) that coexisted in the host.

Objects of the Invention Accordingly, it is a general object of this invention to provide a tumor immunotherapy which overcomes the disadvantages of the prior art.

It is a further object of this invention to provide a method of overcoming the resistance of a progressively growing tumor.

It is yet another object of this invention to provide human monoclonal antibodies directed against the oncotopes of various tumors which will be useful for classifying human tumors, will provide standardized reagents for diagnostics tests and be the primary reagents for biochemically describing EATI.

Summary Of The Invention A method of overcoming the resistance of a progressively growing tumor in a host to lymphocytotoxic effector cells conferred by an anti-tumor polyclonal antibody response induced in the host. The method comprises the steps of obtaining a sample of the progressively growing tumor from the host, generating at least one human monoclonal antibody directed against at least one oncotope of the progressively growing tumor, subjecting the human monoclonal antibody to an analysis to make a determination of the therapeutic value of the human monoclonal antibody, generating a blocking human antibody which blocks anti-oncotope monoclonal antibodies; and delivering a therapeutic amount of the blocking human antibody to the host to permit the tumor to expose uncovered oncotopes thereby becoming sensitive to lymphocytotoxic effector cells.

Brief Description of the Drawings

Other objects and many attendant features of this invention will become readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

Fig. 1 shows the prophylactic effect of anti-EATI.

A hypertonic salt eluate of 14-day old EL4 tumors (grown in C57BL/6 mice from a subcutaneous inoculation of 5 x 10 4 cultured EL4) was made, centrifuged at 100,000 x g. , dialyzed overnight against PBS-azide, centrifuged after dialysis and concentrated to a volume of 1 ml/gm of original tumor. It contained 7.4 μg/ml of specific binding Ig when assayed on

P815Y discs and 15.4 μg specific binding Ig when assayed on

EL4 discs (for the method, see Fig. 2) . To each of three

DBA/2 mice, 0.1 ml. of the eluate was administered i.p. Three

DBA/2 mice, injected with PBS, served as controls. Six days after immunization, all animals received a challenge dose of 1 x 10 4 P815Y cultured cells inoculated i.p. All ascites were removed on day 15 after tumor challenge and the total number obtained plotted in the graph;

Fig. 2 exemplifies a direct radioimmunoassay for anti-membrane Ig. The solid phase in this assay were filter- paper discs, 3/16 in diameter, (542 Whatman paper activated with cyanogen bromide and to which had been coupled either L5178Y MLP or thoroughly washed membrane preparations, of lysed sheep red blood cells (SRBC) ) . The SRBC membrane discs and a highly affinity-purified anti-SRBC Ig preparation were used to standardize the 125I-anti FAB used in the assay. This assay was run in parallel with the L5178Y MLP discs and the

M24F antiserum. It was concluded that the M24F serum (C57BL/6 anti-L5178Y (H-2 ) serum) contained 3.9 mg. of specific anti-

L5178Y antibody per ml. Other tests showed that this polyclonal antibody preparation was mainly anti MHC class I gene product antibody;

Fig. 3 is an antigen inhibition assay. A 1:100 dilution of the M24F serum in PBS was incubated with the indicated amounts of either the syngeneic MLP (EL4) or the allogeneic MLP (L5178Y) . The MLP is particulate, thus easily pelleted by centrifugation at 100,000 x g. for 10 min. The supernatant fluid was then titered as indicated in Fig. 2.

Fig. 4 shows the immunoprecipitation studies with P815Y. Tumor cells were grown in medium containing ³H- glucocsa ine, 3μCi/ml for 3 days. At this time, greater than 70% of the isotope in the medium was incorporated into the growing cells. Cells were harvested, washed and lysed in o

0.125% NP40 at 1 x 10 cells per ml. Lysate was clarified at

100,000 x g for 30 min. To an aliquot of lysate obtained from

₇ 2-4 x 10 cells was added 5 μl of a P815 X2 eluate (this cell line is EATI^") , followed by a goat or rabbit anti-mouse IgG- coated Staphylococcus aureus

Cowan I organisms. After 2 hour incubation, the preparation was pelleted. The clear supernatant fluid was divided into 2 aliquots. One was precipitated with 2 μl. of D4 antiserum, the other with 2 μl. of a P815Y eluate. After overnight incubation, coated Staph. aureus pellets were added, and 2 hours later pelleted. To each supernatant fluid was added 2 μl. of D4 antiserum and the process repeated. To each of the four Staph. aureus pellets was added 0.2 ml. of a 2% sodium dodecyl sulfate-2% mercaptoethanol solution and the tubes placed in a boiling water bath for 3 minutes. The eluates were then chromatographed on a 12% SDS-polyacrylamide discontinuous gel containing molecular weight markers as indicated in the figure, A was bovine serum albumin (68,000 kDa) B was egg albumin (48,000 kDa) and C was trypsinogen

(24,500 kDa) .

Detailed Description of the Preferred Embodiment

Introduction

It is believed that a tumor-bearing person is fully immune to tumors and is therefore an immediate source of immune B cells and immune T cells that constitute the immune network generated against the EATI. It is proposed first to generate a panel of human monoclonal antibodies (HMAbs) directed against the epitopes of EATI of progressively- growing tumors (oncotopes) . This will permit us to obtain a panel of monoclonal antibodies directed against the paratopes and the idiotopes of HMAbs

(HMAbs-2, sometimes referred to herein as human blocking antibodies) . The source of immune B cells with these latter activities will again be B-cells of the tumor-bearing patient.

The HMAbs directed against the oncotopes of various tumors will be immediately useful for classifying human tumors, will provide standardized reagents for diagnostic tests and be the primary reagents for biochemically describing EATI. Since they are specific for the oncotopes and should show little or no cross-reactivity for normal tissue, they should be ideal carriers for agents lethal to tumors, such as radioactive atoms and toxins (immunotoxins) .

Experiments with laboratory animals have shown that immunization of animals with anti-tumor antibodies eluted from in vivo grown tumors resulted in animals capable of resisting tumor challenges. It is therefore proposed that treating tumor-bearing patients with a single appropriate HMAb-2 or a panel of more than one HMAb-2 derived from a patient will result in the uncoating of the tumor, thereby making the tumor sensitive to endogenous "killer cells" or in vitro generated populations of "killer cells" initially derived from the patient. In addition, the HMAb-2 populations will have idiotypes which mimic the three-dimensional structure of the oncotopes, thus acting as immunogen and thereby enhancing the rate of formation of endogenous "killer cells".

The major advantage of this form of therapeutic intervention is that all of the effector reagents proposed to be used will act in a specific way on the immune systems of the host and therefore not interfere with the ability of the host immune systems to react against any non-tumor immunogens, such as bacteria, viruses and other infectious agents, and therefore not interfere with the capacity of the host immune systems to continue to act as a major defense system for the host. Description

The state of understanding at the present time of the immunological relationships between a host and the autochthonous tumor it may be bearing is in a state of disarray. There is no consensus that any or all such tumors in humans have triggered an immune response in the host during the time that the tumor has expanded from a single cell until the time that the tumor can be detected clinically (more than 10 cells) . The same can be said of the immune responses to tumors of experimental animals, be they autochthonous or transplantable. There is a consensus that there may be "tumor-specific" or "tumor-associated" antigens expressed on tumors, however there is no consensus as to what constitutes immunogenicity, what defines a tumor-specific antigen, what immune responses have taken place in the tumor- bearing host and are these responses compatible with progressive tumor growth.

Studies carried out in my laboratory with several mouse tumor lines, have demonstrated that transplantable car¬ cinogen-induced and virus-induced tumors trigger early, during progressive tumor growth, both a specific B-cell response and a specific T-cell response. The B cells produce antibodies directed against tumor-unique structures not found on normal tissue cells (EATI) , and "killer cells" can be demonstrated in the tumor-bearing host, also directed against similar structures, which are capable of lysing the tumor cells and thereby destroying them (the T-cell response) . The first significant observation made in this laboratory with these systems, as described by Biddison & Palmer, Proc. Nat. Acad. Sci. USA, Vol. 74, 329-323, (1977), was the demonstration that progressively growing tumor cells in vivo become resistant to "killer cells". Tumor cells isolated from animals inoculated two weeks earlier with 1000 cells of a syngeneic transplantable tumor line, that were initially sensitive to anti-tumor "killer cells", were now completely resistant even though the tumor had increased one million fold in size from the initial inoculum. The resistant cells were found to be covered with antibodies the host had synthesized, directed against the tumor-unique structures. These anti-tumor antibodies were found only on the growing tumor cells in the tumor-bearing host, and not in its serum. Since the host has a limited capacity to synthesize these antibodies, the tumor cells bind all that is made as soon as it is elaborated and therefore none was found in the serum.

That these anti-tumor antibodies were responsible for progressive tumor growth was shown in a series of experiments in which normal mice were immunized with the antibody preparation eluted from in vivo grown tumor cells, as published by Manson, Transpl. Proc, Vol. 16, 524-527, (1984) . Three mice were pre-immunized with one injection of 1 μg. of the eluted antibody each, then challenged six days later with 10⁴ tumor cells. Fifteen days later, the number of tumor cells detectable in the ascites of these animals as well as three control animals was determined.

In Fig. 1 are shown the results, the three controls showed normal ascites growth of tumor, whereas two of the three animals given anti-tumor antibody showed no significant tumor growth. It was concluded that an anti-idiotype response was most probably responsible for the rejection of the tumor, i.e. the rate of formation of the blocking antibody was interfered with whereas the rate of formation of the "killer cells" was not affected by the treatment, thus leading to the elimination of the challenge tumor dose.

Similar immune responses can be induced in experimental animals in mice syngeneic to the transplantable tumor being studied, using X-ray treated tumor cells as the im unogen. The immune B cells so generated have been immortalized as hybridomas in this laboratory and these provide a plethora of specific monoclonal antibodies (MAbs) for future studies. The key discovery derived from the animal studies is that the tumor-bearing host is completely immunized to the tumor that the host is bearing at the time that the tumor becomes detectable clinically and therefore the tumor-bearing host is an immediate source of immune effector cells specifically directed against the EATI of the tumor, both B cells and T cells.

The understanding of the interactions of the immune response systems of the human tumor-bearing host to its autochthonous tumor may be analogous to that found with laboratory animals. Many attempts are being made to isolate cytolytic cells from patients, expand their number in tissue culture in appropriate growth media with IL-2, and then re- infiltrate the resultant lymphoid population into the patient from which the cells were initially isolated in the hope that the cytolytic cells will decrease the size of the tumor or eliminate it completely. Limited success has been achieved because of the lack of appreciation of the fact that the tumor cells may be resistant to cytolytic immune effectors due to host anti-tumor antibody coating the tumor cells and making them resistant, as was found with laboratory animal tumor systems.

There is a need for a framework around which one can describe the nature of the substances which are expressed uniquely in tumors and not found on normal cells and which are responsible for the tumor-specific immune responses of the host. I call these molecules that are uniquely found on tumor cells and which induce the specific immune responses

"emergence-associated tumor immunogens (EATI)". The importance of establishing this framework can be best appreciated in reviewing the knowledge of tissue transplantation antigens and how it evolved. It is now known that histocompatibility is controlled by a region of the genome called the Major Histocompatibility Complex (MHC) and tissue compatibility is coded for in the MHC Class I genes.

The gene product of Class I genes is a glycoprotein, 46-49 kDa in size, with multiple antigenic determinants (epitopes) in the molecule. Among inbred mice, the large polymorphism seen with respect to these epitopes (more than one hundred) have been organized into two types, those that are specific for a single strain of mouse ("private") and those that are found in several different mouse strains ("public") . A similar system has been found with human tissue-typing data, namely a large degree of polymorphism that can be observed as "public" and "private" epitopes, a combination of which constitute the Class 1 antigens of the human MHC (HLA) . The reagents which made this classification possible in humans and in mice are antibodies directed against these epitopes which describe this polymorphism.

The lack of a reproducible source of anti-tumor specific antibodies has prevented a biochemical analysis of the EATI of immunogenic tumors, in fact it has prevented the development of a consensus that there is an EATI on which are expressed multiple epitopes. This family of epitopes I am calling "oncotopes" to indicate that they are antigenic determinants found on molecules expressed only on tumor cells. If such molecules were also expressed on normal cells, then anti-EATI bound to in vivo growing tumor cells would not have been detected. Due to the overwhelming amount of normal tissue available in the animal relative to the amount of tumor tissue present, cross-reactive antibodies would have been bound by the normal tissues dispersed throughout the body and not have localized on the growing tumor cells as was found.

Materials and Methods

It is anticipated that there may be as many as three classes of oncotopes among human tumors:

Private oncotopes - each tumor may have a unique oncotope, characteristic of that tumor, and different from that found in every other tumor;

Semi-public oncotopes - there may be a class of oncotopes found on all members of a similar type of tumor, e.g. mammary, colon carcinoma, leukemia, sarcoma, etc. Public oncotopes - these are tumor-specific structures found on all tumors and which are not expressed on normal cells.

The major effect will be to obtain human hybridomas secreting monoclonal antibodies (HMAbs) directed against such oncotopes with a variety of malignant tumors. The objective will be to develop a panel of such HMAbs for diagnostic studies, classification of tumors and ultimately for therapeutic purposes as will be described in this application.

It is first necessary to obtain samples of individual tumors and establish them in tissue culture. Methods for growing malignant cells in tissue culture are quite varied, but there is good reason to expect that this will be accomplishable. The individual whole tumor line cells will be used as the solid-phase immunoadsorbent in the enzyme-linked immunoadsorbent assay (ELISA) and as a source of cell-free membrane particulates ( icrosomal lipoproteins, MLP) which are bound to filter paper discs that become the solid phase immunoadsorbent in the radioimmunoassay (RIA) to detect, assay and quantify a polyclonal anti-tumor antibody as well as HMAbs. Only small amounts of tissue will be required to initiate such cultures. It may be possible to use a portion of the sample removed from the patient at the initial biopsy for diagnostic purposes as a source for tissue culture-grown cells.

Lymphoid and ascitic tumors grow easily in standard tissue culture media after a short adaptation period of several days. Successful cultures have also been developed by dissociating solid tumors, carcinomas and sarcomas with a mixture of enzymes (deoxyribonuclease, hyaluronidase and a bacterial collagenase) . A goodly proportion of the tumor cells survive the dissociating protocols and will then grow as adherent cultures on plastic surfaces with normal tissue culture media. If enzymes have been used to dissociate the tumor cells, it is essential that the cells undergo several days growth in culture in order to re-express the oncotopes on the surface membranes of the cells. It is also essential that the cells be removed from the plastic surfaces by scraping, enzymes should not be used. Radioimmunoassay (RIA)

An RIA was developed to measure quantitatively specific antibody directed against oncotopes and against other epitopes found on cell surfaces. The details have been described by Manson & al, in Curr. Topics Microbiol. Immunol., Vol. 81, 232-234 (1978). MLP particles are prepared from tumor cells grown in culture or normal tissues such as spleen, liver, kidneys as described by Manson & al, J. Cell Comp. Physiol. Vol. 61, 109-118 (1963). These MLP particles, microsomal in size, were shown to be complete histocompatibility and tumor immunogens by Manson & al, Transpl. Proc, Vol. 7, 161-164 (1975). The cell-free particles are covalently bound to cyanogen-treated filter paper discs, 3/16 in, diameter (Whatman No. 52) , 2μg protein per disc.

To carry out the RIA, MLP discs are placed in flat- bottom wells of a 96-well Microtiter plate. A dilution containing 0.5-2 ng. of specific antibody is added to the well (diluted in PBS buffer which contains 0.2% bovine serum albumin and 2% horse serum) and incubated at room temperature overnight. The wells are washed and an 125I-labelled anti- immunoglobulin is added and the plates incubated again overnight. The discs are washed, dried and counted in a gamma-spectrometer. As indicated in Fig. 2, a linear relationship was observed between the 125I bound to the disc and the original anti-membrane antibody added to the discs.

Control experiments showed that under these conditions, all of the antibody added to the well is bound, thus the assay can be used to quantitate an antibody preparation.

A further refinement of this assay permitted us to measure dispersed, cell-free antigen, using an inhibition protocol. In this case the aliquot containing putative antigen is pre-incubated with an appropriate antibody aliquot before the MLP-disc is added to the well. The assay is then carried out in a normal way. Fig. 3 is an example of such an assay. An assay such as this was used to detect the presence of materials in sera obtained from women with metastatic mammary cancer that might be cross-reactive to epitopes found on mouse mammary tumor virus, using a panel of MAbs developed against mouse mammary tumor virus, as published by Manson & al, in Current Controversies in Breast Cancer, ed. by Ames & al. p.425-431, University of Texas Press, Austin (1984).

Eight of eleven sera of a group of patients with metastatic breast cancer blocked MAb VE7 and IIIA1 to a significantly higher degree than did sera from normal blood donors.

Enzyme-Linked Immunoadsorbent Assay (ELISA)

The procedure used has been described by Kennett in

Monoclonal Antibodies ed. by Kennett & al, pp. 376-377,

Plenum Press, New York (1980). Tumor cells (0.5-1.0 x 10 per well) are attached to the bottom of flat-bottom 96-well polyvinyl chloride Microtiter plates, treated with poly-1- lysine. The cells are lightly fixed and covalently-bound to the PLL layer with glutaraldehyde. Antibody dilutions are then assayed by being adsorbed onto the cells in the well, which are then reacted with a purified anti-immunoglobulin reagent that has been conjugated to an enzyme, such as horseradish peroxidase, alkaline phosphatase or β- galactosidase. After an appropriate period of time, the conjugate is washed out and substrate for the enzyme added.

By following the rate of color development, one can determine qualitatively the presence of an anti-cell antibody with the first two enzymes or quantitated with 3-galactosidase. The

ELISA test is much more rapid than the RIA, but not as sensitive. A sensitivity approaching that seen in the RIA has been obtained using 0-galactosidase and a fluorescent substrate, methylumbelliferyl galactoside and a fluorescence plate reader.

Methods For Obtaining Membrane Fractions Rich In EATI (MLP) For In Vitro Stimulation Of Lymphocytes Obtained From Normal Individuals Or Tumor-Bearing Patients And For Use In The RIA The detailed procedures for isolating MLP from cells grown in culture have been described by Manson & al, J.

Cell, Comp. Physiol., Vol. 61, 109-118, (1963). Washed cultured cells are suspended in a hypotonic medium containing a variable amount of sucrose (0-0.18 M, depending on the particular cell that is being used) but always 0.01 M in Mg⁺.

The cells are homogenized in a nitrogen decompression apparatus, which results in complete breakage of the cells but very little breakage of nuclei. The homogenate is then fractionated by differential centrifugation, the fraction sedimenting after 10' at 5000 x g is discarded, whereas the pellet obtained after 1 hour at 100,000 x g is retained

(microsomes) . This pellet is suspended in a ground-glass homogenizer in 2 M sucrose, and centrifuged in a swinging bucket rotor overnight. The pellicle floating at the top of each tube is suspended in distilled water and preserved at either 0"C or -20°C as is required. The usual yield of MLP per gram of tissue used is 2-4 mg. of protein as assayed in a fluorescent protein assay using BSA as a standard.

Methods for Immortalizing Anti-EATI Human B Cells Secreting Polyclonal Antibodies Directed Against Oncotopes

A. Methods for Obtaining Immune B Cells From The Peripheral Circulation of the Tumor-bearing Patient

A significant percentage of the lymphoid cells found in the peripheral circulation are B cells (15-30%) .

This is the most readily available source of immune B cells from the tumor-bearing patient. Heparinized peripheral blood is fractionated with Ficoll-Hypaque and the buffy coat interface cells are used. The mononuclear cells are infected with a supernatant fluid containing an adequate amount of tissue-culture-grown Epstein-Barr virus (EBV) . To the cultures are then added 1 μg per ml. cyclosporin A. The cultures are then incubated for approximately 3 weeks in a humidified 5% CO incubator. Culture is continued until anti- EATI can be detected by RIA or ELISA in the culture fluid.

The amount of anti-EATI that will be produced in such cultures is expected to be small. It is intended to cryopreserve these cultures at this stage to permit these to be used as starting preparations for developing anti-tumor HMAbs.

B. From Surgically-excised Samples of Tumor Tissue Immune B cells may be isolated from the tumor itself taken from the tumor bearing patient during a biopsy or surgery. Solid tumors are relatively easily dissociated with enzymes as has already been described. The tumor cells will most likely be adherent to plastic, whereas lymphoid cells are not, and therefore easily removed from such cultures. These lymphoid cultures will then be immortalized with EBV as described above and cryopreserved.

C. Immunized or Further Stimulated During Tissue Culture With Tumor MLP As Source of Antigen

It has been shown that MLP can act as a complete immunogen in vitro and in vivo as reviewed by Manson &

Palmer, In Vitro, Vol. 11, 186-204, (1975). It will be attempted to increase the number of immune B cells in the lymphocyte preparations removed from patients by incubating them with syngeneic tumor MLP prior to immortalization. The

EBV immortalization procedure works best with resting B cells, thus cultures stimulated with MLP will be allowed to rest before EBV treatment.

Methods for Developing Human Hybridomas Secreting HMAbs Directed Against Oncotopes

The first step will be to obtain immune B cells from the patient and immortalize them with EBV as described above. The EBV transformed cultures are known to be polyclonal. Procedures will be used to enhance antibody production to permit isolating hybridomas secreting HMAbs.

Some success has been achieved by fusing EBV- immortalized lines with mouse myeloma line P3X63Ag8.Clone653. The resultant hybridomas, using standard fusion protocols, should yield relatively stable mixed mouse-human hybrids secreting HMAbs in reasonable amounts.

Excellent success has been obtained using the

₇ following protocol to generate mouse hybridomas. To 10 lymphoid cells are added an equal number of log phase growing SP2/0-Agl4 myeloma cells (drug-marked, nonsecretory; ATCC #CRL 1581) . The cell mixture is centrifuged. To the well- drained pellet, warmed to 37°C is added drop-wise 1 ml. of 50% PEG, also warmed to 37°C. All subsequent reactions are carried out at 37°C. One milliliter of serum-free medium is added slowly over a period of 1 min. This step is then repeated. Next is added 7 ml. of serum-free medium over 2-3 min., after which time the cells are pelleted. To the pellet is added 30 ml. of complete selection medium to which has been added azaserine and hypoxathine. The cell suspension is distributed, 2 drops per well, into six 96-well flat-bottom Microtest plates and incubated in a C0₂-incubator for 10 days to 2 weeks. Hybrids begin to appear by day 10 and are complete by day 20. The usual yield of hybrids with DBA/2 spleens are 50 per 10 7 ori.gi.nal spleen cells, whereas with

C57BL/6 or B6D2F1 spleen cells, the average yield has been

₇ 150 hybrids per 10 spleen cells.

After growth in a well has covered at least 1/3 of the surface of the bottom of the well, samples of the supernatant fluid are tested in either the RIA or ELISA for reactivity against cell surface oncotopes. Those that test positively are then tested for lack of reactivity against a cell line, also grown in culture, that does not express the oncotope. As a potentially negative control cell strain for each patient, fibroblast cultures will be derived from scrapings taken from inside the check or surgically removed from the patient's skin, as described by Sly and Grubb,

Methods Enzymol., Vol. 58, 444-450, (1970). Human fibroblast cultures grow rapidly and rarely transform spontaneously and therefore are not likely to express EATI, but do have a limited life-time in culture, as has been summarized by

Hayflick in Exptl. Cell Res., Vol. 37, 614-636 (1965). The limited life-span seen with human fibroblast cultures (50-100 doublings) will be more than adequate for these tests. A first objective will be to find a continuously-growing cell line that may act as a negative control cell for RIA and

ELISA tests. Whether such mixed mouse-human hybridomas will be stable enough to produce continuously an HMAb in large quantities is not known at the present time. It may be that the development of these human-mouse hybridomas will serve only as an interim step yielding a cell which produces the mRNA characteristic of the immunoglobulin it secretes in quantity.

Biochemical Studies of EATI

As soon as immortalized cultures of B cells obtained from patients are available, supernatant fluids that test positive for putative EATI, i.e. positive in ELISA with the patient's tumor cells growing in culture, and negative on fibroblast cultured cells obtained from the same patient, will be used directly to begin a biochemical characterization of EATI. The plan of the experiments will follow that which was successfully used initially to characterize the P815Y EATI, using, as a source of anti-EATI, eluates made with hypertonic salt of P815Y(H-2 ) grown in vivo as subcutaneous tumors in their syngeneic host DBA/2 mice. These experiments have been described by Manson, in Cancer Detection and Prevention Suppl., Vol.l, 111-120 (1987). An eluate obtained from P815Y that assayed 2.8 μg of specific antibody per ml. was used. Immunoprecipitation experiments were done using P815Y cells grown in 3H-glucosamine. The cells were lysed with NP40 and the supernatant fluid extract cleared with an eluate of P815-X2 tumor cells. This latter cell line is non- immunogenic and EATI-negative, it is however gp69-71 C virus positive, as is P815Y. This step removed from the cell lysate all extraneous materials. Equal aliquots of the treated cell extract were allowed to react with 2 μl each of the P815Y-eluate or D-4 antiserum (an antiserum obtained from the Transplantation Immunology Branch of the National Institute of Allergy and Infectious Diseases of the National Institutes of Health in Bethesda, MD) specifically directed against the #4 specificity of the Major Histocompatibility Complex Class I gene product, a private specificity for the H-2 haplotype. Both supernatant fluids obtained from this immunoprecipitation step were treated with the D4 antiserum. The four pellets that were obtained were subjected to sodium dodecyl sulfate gel electrophoresis with internal molecular weight standards. The results have been combined and are illustrated in Fig.4. What is apparent is that regardless of whether the first precipitation was with the D4 antiserum or the anti-EATI eluate, four glycoprotein peaks are seen, with migration rates of molecules approximately 35, 48, 55 and 65 kDa. Experiments w th cells labelled with 125I, an additional peak is seen on both chromatograms, of 12 kDa, which is

The second sequential precipitation with D4 antiserum on each supernatant fluid shows no specific peaks. In other tests, the anti-EATI is definitely not an anti-MHC Class I antibody. It has been concluded that the EATI and the Class I gene products are hydrophobically associated in the cell membrane, and the EATI is expressed on either the three non-Class I antigen peaks

(33, 55 and 65 kDa) or a complex containing all or some of these compounds. Similar results have been obtained with EL4 (H-2 ) , whose EATI is cross-reactive with several tumors studies in this laboratory, P815Y and L5178Y, both carcinogen-induced lymphomas, and MCA-105, a carcinogen- induced sarcoma.

Production Of HMAb-2

As soon as HMAb is obtained, it will be possible to screen the immortalized and cryopreserved polyclonal B cell populations for their capacity to elaborate anti-HMAb activity. Since the original immortalized populations will be secreting polyclonal anti-EATI, it may not be possible to detect the presence of a clone that has the capacity to synthesize HMAb-2 and secrete it into the supernatant fluid. Such molecules will be neutralized by the polyclonal anti- EATI and their presence would not be detectable in an ELISA.

However, a B cell which is secreting anti-EATI should also have a surface im unoglobulin with the same anti- EATI idiotype. Two procedures will be used to take advantage of this fact. In one, it should be possible to allow the immortalized B cells to adsorb onto live cultures of the tumor line, expecting that they would attach themselves to the EATI molecules of the tumor cells. If the immortalized B cells obtained from the patient contain B cell secreting anti-idiotype immunoglobulin, they would have an idiotype that mimics the three-dimensional structure of EATI and therefore would not bind to the tumor cell monolayer. Thus, after a suitable adsorption period at 37°C, the non-adsorbed lymphoid population will be removed and cultured separately. The supernatant fluid elaborated by such cultures will be assayed in an ELISA or RIA. In this case, the solid phase will be a plastic surface to which will first be attached a mixture of rabbit anti-human Fc purified IgG, rabbit anti- human IgM and rabbit anti-human IgA. The rabbit immunoglobulin will bind all the human immunoglobulin secreted by the purified population of immortalized B cells.

Once the human immunoglobulin are bound, the surface will then be reacted with an 125I-culture supernatant fluid from the original immortalized B cell cultures. If any anti- idiotype immunoglobulins are being produced by the frac¬ tionated B cell populations, they should bind ¹²⁵I-anti-EATI present in the culture fluids. A positive result will be an indication to continue to process this population of B cells into the human-mouse hybrid stage to be a source of mRNA for HMAb-2 production, and for significant quantities of HMAb-2.

A recently marketed apparatus made by CellPro Incorporated, 22322-20th Avenue, S.W., Bothell, WA 98021, sold under the trademark CEPRATE SC Stem Cell, uses the avidin-biotin system to isolate unique cells from heterogene¬ ous cell populations for various purposes. The affinity binding constant of avidin with biotin is several orders of magnitude higher than that of antibodies combining with their antigens. Thus the developers propose to biotinylate an antibody which will bind uniquely with a subpopulation of cells, then pass the mixture of cells, some coated with biotmylated antibody, the majority free, through a solid phase bead column to which has been bound avidin covalently. Those cells which have biotinylated antibody bound to them will remain fixed to the column, the rest will pass through and be collected in the effluent fluid. The bound cells can then easily be released by physical agitation. In my system, the polyclonal antibody preparation released by the immortalized B cells into the supernatant culture fluid will be biotinylated. This preparation will then be allowed to bind to a fresh preparation of immortalized B cells obtained from the patient initially, or with a later fresh preparation of lymphoid cells obtained from the patient. An appropriate time to obtain such a fresh preparation of cells from the peripheral circulation .of the patient will be when anti- oncotope antibody can be detected in the circulation, perhaps one to two weeks after surgery/biopsy. The cell preparation will then be passed through the apparatus Those B cells elaborating anti-oncotope antibody (HMAb) will not bind to the avidin column, however any B cell elaborating antibody directed against an anti-oncotope idiotype (HMAb-2) will be bound to the biotinylated antibody and thus remain in the avidin column. This may prove to be an efficient way of obtaining cells secreting HMAb-2 antibodies. According to the manufacturer, the apparatus is most efficient for obtaining sub-populations that are relatively rare in the normal population and therefore it may be well suited for the present application.

Large scale production of HMAb and HMAb-2 Some experience has been obtained with commercially-developed methods for production of milligram quantities of mouse Mabs that may prove useful in obtaining larger quantities of HMAbs or HMAbs-2. One such procedure, developed by Damon Corp. involved encapsulating hybridoma cells in Ca-alginate "microcapsules". In a trial experiment, the anti-MMTV gp-52 Mab VE7 was encapsulated and the capsules incubated in an appropriate medium for 7 days. At the end of this time, the capsules were harvested and their contents liberated into 5 ml of saline. The yield of material, as assayed by RIA, was 5.6 mg. of VE7 per ml starting with 2 x

7 10 hybridoma cells.

Another apparatus that used in the laboratory to obtain milligram amounts of Mab was the Amicon Vitafiber artificial capillary unit. In an experiment with the 2 ml. chamber, a culture was initiated with 1 x 10 7 cells of VE7 and culture continued in the apparatus for 14 days. At the end of that time the contents of the chamber were removed.

7 The total yield of cells was 6.5 x 10 and the supernatant fluid, 1.9 ml. assayed 3.7 mg. of VE7 MAb activity in RIA.

Production of rHMAb and rHMAb-2

For all uses projected for anti-oncotope HMAb and possible therapeutic use of oncotope HMAb-2, large commercial quantities of the materials will be required that can be purified and are stable for clinical use. It may be that the mouse-human hybridomas may not be useful in this respect. However, it should be possible, using modern recombinant DNA technology to transfect cells that have the potential of growing on a large scale and therefore yielding commercial quantities. Two procedures appear to have the possibilities just described. Each will require isolating mRNA for the light chain and for the heavy chain of a HMAb and HMAb-2. Many attempts during the last few years have been made to induce Escherichia coli cells to produce a recombinant immunoglobulin but these have failed. It is believed that bacteria do not correctly process and/or glycosylate eukaryotic proteins (Smith et al, Proc. Nat. Acad. Sci. USA Vol. 82, 8404-8408, (1985).

In a recent publication, Hasemann and Capra (Proc. Nat. Acad. Sci. USA, Vol. 87, 3942-3946, (1990) describe the successful production of a mouse Mab by a baculovirus expression system using, as a vector, a double-recombinant virus containing both the immunoglobulin heavy and light chain cDNA. A major advantage of using this insect system is that late in virus infection as much as 50-75% of total cellular protein is claimed to be the foreign protein. The authors claim that the rig produced in culture is identical to that produced by the parent Mab grown in mouse ascites using a variety of functional tests.

A more recent publication describes a different system for the production of a recombinant molecule which, in this case, is a disulfide-bonded heterodimeric lymphokine, cytotoxic lymphocyte maturation factor (CLMF) as published by Gubler et al, Proc. Nat. Acad. Sci. USA Vol. 88, 4143-4147 (1991) . In this case, the cDNA for each of the two chains of CLMF was prepared from a human lymphoblastoid cell NC-37 and incorporated separately into vectors containing simian virus 40 early promoter. When COS cells were double transfected with both vectors mixed together, fully active CLMF was produced. It would be desirable to repeat this study and attempt to use as a host cell, one of the lymphoid lines used in my laboratory, such as P815Y, P815-X2, L5178Y, EL4 or L1210. For example, L5178Y have been grown under chemostat conditions in 8L stirred vessels and achieved higher than 6 xlO cells per ml. over a number of days. All of the cell lines are immunoglobulin-negative and therefore may prove useful as hosts for recombinant immunoglobulin production.

The lymphoblastoid cell lines utilized are easily grown in large quantities also in other commercial apparatuses.

There are many newer procedures appearing in the literature almost daily for new expression systems to produce recombinant human molecules for a variety of purposes. It is intended to follow this field actively and chose an appropriate system to obtain recombinant molecules that are suitable for the intended purposes. For example, even though E. coli have been found not to produce complete human molecules due to their inability to glycosylate peptides appropriately, still E. coli have been found to produce the V- XI, and VJ-l region and Fab molecules of immunoglobulin using a variety of vectors and methodologies. Many recent approaches to such problems, as well as attempts to engineer better fitting antibody-combining sites as well as using plants and domestic animals to produce large quantities of human recombinant molecules cheaply have recently been reviewed by Borrebaeck, ed. Antibody Engineering, A Practical Guide, published by W. H. Freeman & Co. New York (1992) and in the September issue of Biotechnology. Those expression systems that produce only the antibody-combining site of the antibody molecule without the F portion of the human Ig may be most suitable for the present purposes. Since the approach of the present inventor has shown that the effector portions of the antibody molecule located in the F region do not appear to function in vivo, molecules lacking this function may act much more efficiently as blocking agents, either as HMAb to block lymphocytotoxic cells or HMAb-2 to block anti-oncotope HMAb.

It is not intended to overlook the possibility that having obtained polyclonal and monoclonal anti-oncotope anti¬ bodies, it may be useful to make an anti-oncotope idiotype antibody in a species other than human, mice, rats, etc. Having available the correct cDNA for the anti-oncotope antibody combining site, having a vector expression systems that produce only the V L_τi and/or V-ri. and/or Fab portions of the molecule, may also produce molecules much smaller than the complete Ig, lacking the F portion. Since this latter portion of the mouse Ig may be the major immunogen when one crosses species, it may be practical to develop anti-human EATI antibodies, polyclonal and monoclonal, in species other than man. As has been indicated above, the antibody combining site for EATI or for anti-EATI are the functional molecules aimed for, since they should have equal blocking and combining capacity to the entire Ig molecule.

Without further elaboration the foregoing will so fully illustrate my invention that others may, by applying current or future knowledge, adopt the same for use under various conditions of service.

Claims

Claims

1. A method of overcoming the resistance of a progressively growing tumor in a host to lymphocytotoxic effector cells conferred by an anti-tumor polyclonal antibody response induced in the host, the method comprising the steps of:

(a) obtaining a sample of the progressively growing tumor from the host;

(b) generating at least one human monoclonal antibody directed against at least one oncotope of the progressively growing tumor;

(c) subjecting the human monoclonal antibody to an analysis to make a determination of the therapeutic value of the human monoclonal antibody;

(d) generating a blocking human antibody which blocks anti-oncotope monoclonal antibodies; and

(e) delivering a therapeutic amount of the blocking human antibody to the host to permit the tumor to expose uncovered oncotopes thereby becoming sensitive to the lymphocytotoxic effector cells.

2. The method of Claim 1 wherein the determination involves the step of utilizing the human antibody to classify progressively growing tumors in a host.

3. The method of Claim 1 wherein the determination involves the step of utilizing the human antibody to provide standardized reagents for diagnostic test purposes.

4. The method of Claim 1 wherein the determination involves the step of utilizing the human antibody as a primary reagent for describing emergence- associated tumor immunogen.

5. The method of Claim 1 additionally comprising the step of utilizing the at least one human monoclonal antibody as a carrier for accessory molecules.

6. A method of overcoming the resistance of a progressively growing tumor in a host to lymphocytotoxic effector cells conferred by an anti-tumor polyclonal antibody response induced in the host, the method comprising the steps of:

(a) obtaining a sample of the progressively growing tumor from the host;

(b) generating at least one human monoclonal antibody which blocks the binding of the anti- oncotope antibody to the progressively growing tumor;

(c) subjecting the human blocking monoclonal antibody to an analysis to make a determination of the therapeutic value of the human blocking monoclonal antibody; and

(d) using the human blocking monoclonal antibody in therapeutic protocols.

7. The method of Claim 6 wherein the step of using the human blocking monoclonal antibody in therapeutic protocols comprises the step of delivering a therapeutic amount of the human blocking monoclonal antibody into a host to act as an immunogen and enhance the rate of formation of endogenous killer cells of the host.

8. The method of Claim 6 wherein the step of using the human blocking monoclonal antibody in therapeutic protocols comprises the step of delivering a therapeutic amount of the human blocking monoclonal antibody into a host to interfere with the endogenous ^• formation of antitumor antibody to permit the progressively growing tumor to uncoat and become more sensitive to endogenous or in vitro cultured killer cells.

9. A method of overcoming the resistance of a progressively growing tumor in a host comprising the steps of:

(a) generating at least one recombinant material containing the antibody combining site of human anti-EATI or of human anti-oncotope antibody;

(b) determining the therapeutic value of the at least one recombinant material;

(c) delivering a therapeutic amount of the at least one recombinant material to a person suspected of having a tumor.