WO1999025375A2

WO1999025375A2 - DIRECT CYTOTOXIC ACTIVITY BY ANTI-hCG MONOCLONAL ANTIBODIES

Info

Publication number: WO1999025375A2
Application number: PCT/US1998/024722
Authority: WO
Inventors: Hernan F. Acevedo; Gavreel Kalantarov
Original assignee: Allegheny-Singer Research Institute
Priority date: 1997-05-22
Filing date: 1998-11-19
Publication date: 1999-05-27
Also published as: WO1999025375A3; WO1999025375A8

Abstract

An investigation using cultured human cancer cells and 12 monoclonal antibodies (MAbs) directed against different determinants of human chorionic gonadotropin (hCG) was done to determine the presence or absence of direct dose dependent cytotoxic activity. Adenocarcinoma of uterine cervix cells (HeLa CCL 2.0) were grown in defined media. The cells were incubated with selected doses of each MAb for 2 to 3 days. There was a direct dose dependent effect by a MAb directed against the natural hCGβ carboxy terminal peptide (CTP) by a MAb directed against hCGα and by one of the three MAbs produced by the synthetic hCGβ-CTP, CTP103.

Description

DIRECT CYTOTOXIC ACTIVITY BY ANTI-hCG MONOCLONAL ANTIBODIES

Previous studies using a quantitative (analytical) flow cytometry technique and a battery of monoclonal antibodies (MAbs) directed to epitopes located in different sites of the human chorionic gonadotropin (hCG) molecule demonstrated the expression of membrane-associated hCG, its subunits and fragments, by live human cancer cells of different types and origins, and by human embryonic and fetal cells (1 -8). hCG expression by embryonic and fetal cells in vitro and in vivo has also been demonstrated (9J0). All of these findings were corroborated, in vivo and in vitro, by the presence of translatable levels of hCGβ and human luteinizing hormone β (hLHβ) mRNA's, not only in human embryonic and fetal cells, but also by cancer cells, indicating that the expression of the hCG β-hLHβ gene cluster constitutes a biochemical characteristic of all cancers. An important precept is the recognition that activation of the hCGβ-hLHβ gene cluster in cancer cells is a product of the process of malignant transformation and not an etiologic factor (1 1 -13).

The basic requirement for successful immunotherapy or immunoprotection of cancer is the preferential expression by the malignant cells of a membrane -associated antigen. Membrane-associated hCG, its subunits and fragments, expressed by all cancers irrespective of type or origin, fulfills this requirement. Taking advantage of the existence of a World Health Organization (WHO) synthetic vaccine created by Stevens and his associates for fertility control, arrangements were made to use the same vaccine for cancer treatment and prevention. The vaccine is based on a synthetic peptide corresponding to the 37 amino acid sequence (109-145) of the carboxy-terminal peptide (CTP) of the β chain of hCG (hCGβ) conjugated to diphtheria toxoid to make an immunogenic hapten -carrier complex. Synthetic nor-muramyl dipeptide is used as adjuvant and squalene/mannide monooleate is the vehicle (14-16).

The phase I clinical trials for fertility control were performed in Australia (17). Cytotoxicity studies were conducted with four samples of sera from the vaccinated women who developed a high titer of antibodies using cultured normal trophoblasts. The results of these experiments, shown in Table 1, demonstrated lysis of the trophoblast cells by the sera in the presence and absence of complement. TABLE 1

Lysis of Trophoblast Cells by Serum of Vaccinated Women

The controls, complement alone and sera of untreated non -pregnant women, were negative. In 1994, Triozzi and his associates published the results of a phase I clinical trial of the WHO vaccine in cancer patients (18). These investigators reported that in the patients who demonstrated measurable antitumor activity, peripheral blood mononuclear cytotoxicity was not observed using standard Cr-release assays and there was no increase in N cell acti\ ιt . At the same time, three mouse monoclonal antibodies (MAbs) raised by the synthetic vaccine, designated CTPl Ol , CTP102 and CTP103, were produced and characterized. Two other MAbs, CTP104 and CTP105, were generated by an immunogen derh ed from a carbohydrate-containing peptide isolated from hCG-holo (whole hCG) conjugated to bovine thyroglobulin (19).

Since the mechanism(s) of action of the WHO vaccine is still not well defined, it was an object of the present invention to detenriine whether the MAbs against hCG, its subunits or fragments, are cytotoxic to cancer cells. More specifically, it was an object of the present invention to perform a series of experiments using MAbs (IgGl 's) directed to different epitopes of hCG. its subunits, or fragments, and to test the cytotoxicity of those MAbs to cultured adenocarcinoma of the uterine cervix cells. The cultured adenocarcinoma of the uterine cervix cells were the CCL 2.0 cells obtained from the American Type Culture Collection (ATCC, Rockville, MD). These cancer cells were grown under established conditions (4,5) m RPMI 1640 medium (JRH Biosciences. Lenexa. S) with 10% defined iron supplemented bovine calf serum (HyClone Labs. Inc., Logan, UT), 2 mM L-glutamine (Fisher Scientific Co., Pittsburgh, PA), I mM sodium pyruvate (JRH Biosciences), and 20 mg/L gentamicin sulfate (Sigma Chemical Co., St. Louis, MO) in the absence of any other type of human cell.

Ten of the twelve MAbs studied were obtained from the Irving Center for Clinical Research. Columbia University College of Physicians & Surgeons, New York, NY (1 -3, 19), while MAb GK-I. a baboon MAb, was produced in our laboratory, and AS1 1 , a MAb directed to the natural carboxy terminal peptide of hCG β, was a gift from ImmunoTherapy Corporation, Santa Ana, CA. The characteristics of each of the MAbs are listed in Tables 2 and 3.

TABLE 2

In Vitro Effects of Baboon and Mouse Anti-hCG MAb on HeLa Cells at 37°C

HeLa cells: (American Type Culture Collection CCL 2.0) incubated in defined media. Plating density: 10 cells/ml. Cells were trypsinized after 3 day treatment and counted using trypan blue. Monoclonal Antibodies used:

CTP 103 : Mouse MAb against synthetic carboxy terminal peptide (CTP) of hCG β (CTP109- 145) reacts also with the natural CTP of hCG-holo and hCGβ-free. GK-I: Baboon MAb against whole hCGβ

Negative controls: Mouse IgG, Monkey IgG, and PBS (without antibodies)

TABLE 3

Characteristics of the 10 MAbs Used in the Experiment Shown in Table 4

A 105 Reacts with hCGα-free or as part of the hCG-holo (intact hCG) A501 Reacts with hCGα-free or as part of hCG-holo B 109 Binds only hCG-holo B204 Binds hCGβ-free and hCGβ-fragment B207 Binds only hCGβ-free B210 Binds only hCGβ-fragment. It does not bind hCG-holo or hCGβ-free. CTP l O l and The two other MAbs produced by the vaccine. Like MAb CTP 103 CTP 102 (synthetic peptide 109- 145), they react with the synthetic CTP and with the CTP of hCG-holo and hCGβ-free.

CTP 105 Reacts with the natural CTP of hCG-holo, but does not react with the synthetic CTP or the asialo hCG (hCG without the sialic acid).

AS H Reacts with the natural CTP of hCG-holo (containing the four carbohydrate chains), hCGβ-free and the synthetic CTPs

The results of the in vitro effects of the MAbs on HeLa cells after incubation at 37°C for 3 days and 2 days are shown in Tables 2 and 4, respectively.

TABLE 4

In Vitro Effects of Anti-hCG MAb on HeLa Cells at 37°C

Cells: HeLa cells, incubated in defined media. Plating density: 150,000 cells/ml.

Negative controls: PBS, Mouse IgG

Cells were trypsinized after 2 days of treatment and counted using trypan blue.

n/d: not done

Of all the MAbs tested, only three demonstrated dose dependent cytotoxic activity. The three cytolytic MAbs were CTP 103, directed against the synthetic carboxy terminal peptide (CTP 109-145), AS 1 1 , directed against the natural carboxy terminal peptide of hCG, and A 105. directed against an epitope of the hCG , free, or as part of the dimer.

These data demonstrate the existence of cytotoxic MAbs reacting with hCG, subunits or fragments. However, although the three MAbs produced by the synthetic carboxy terminal peptide have very similar immunoreactivity (19), only one, MAb CTP103, was cytotoxic. Mabs against hCGα have been shown to have similar characteristics (20). As in the case of the MAbs against the synthetic CTP, another MAb against hCGα, MAb A501 , was devoid of cytotoxic activity.

Results indicating that only one (CTP103) of the three MAbs directed against the synthetic carboxy terminal peptide of hCGβ and only one of the MAbs against hCGα exhibited cytotoxic activity were exceptional and unique. However, the present invention is not considered to be limited to that single CTP103 MAb. Those skilled in the art will recognize that additional MAbs to the carboxy-terminal peptide of the β chain of hCG can be made, using conventional hybridoma fusion methods, and screened for cytotoxic activity using the methods set out herein and that, although it may be necessary to screen a large number of such hybridomas, other MAbs will be identified which have similar binding and cytotoxicity characteristics, and the present invention is intended to include all such MAbs. Those skilled in the art will also recognize that MAbs having the desired cytotoxic characteristics may require the use of the same synthetic peptide, or a peptide having an amino acid sequence which is about 90% homologous to the sequence of the amino acid sequence of the carboxy-terminal peptide of the β chain of human chorionic gonadotropin, and in particular to the 37 amino acid sequence corresponding to CTP109-145.

The particular property of CTP103 which confers these unique characteristics is not clear from these data. However, because all the MAbs used in this study were IgGl's, a protein with a molecular weight around 150 KD having a tertiary structure, the possibility exists that the cytotoxic activity could be due to a change in the spacial configuration of these exceptional MAbs. In the event there is a difference between the spacial configuration of the cytotoxic MAbs and those MAbs which are not cytotoxic which confers this particular property of cytotoxicity upon those MAbs, the present invention is considered to be directed to all MAbs to the CTP portion of hCGβ which are characterized by the particular spacial configuration which confers this property upon those MAbs.

The unique characteristics of the MAbs of the present invention may explain the inability to produce a primate MAb directed against the synthetic hCG β-CTP using the classical hybridoma technique, which requires a human heteromyeloma as a fusion partner. Because the human heteromyeloma, a human cancer cell, has an abundance of membrane-associated hCG, its subunits and fragments, a cytotoxic hCG antibody produced by such a hybridoma will attack the heteromyeloma, causing the destruction of the hybridoma. Efforts to protect the hybridoma from this "suicidal effect" have failed. Only one baboon hybridoma producing the MAb GK-I directed against an epitope of hCGβ has been obtained and, as shown in the data presented herein, that MAb was devoid of cytotoxic acti\ ity. These results indicate that production of a primate cytotoxic MAb against hCG and/or its subunits using the hybridoma technique may be a biological impossibility.

Mouse hybridomas producing cytolytic MAbs such as MAb AS1 1 are unstable. It be for this reason that hybridomas producing MAbs against hCG β or its natural CTP, such as MAb AS1 1 , have been lost forever.

Because of the specificity of CTP103 for the CTP109-145 of hCGβ, CTP103 does not cross-react with the other hormones (LH and FSH) in vivo even though all have a common CX chain. It therefore represents an ideal candidate for immunotherapeutic treatment of cancer. The data set out herein indicate that CTP103 is cytotoxic to cancer cells in vitro and that the cytotoxicity is dose-dependent. Further, it is known to those skilled in the art that in vitro activity is indicative of an agent which is a candidate for in vivo activity against cancer. Consequently, these data indicate that Mabs against the carboxy-terminal peptide of the hCGβ, and particularly the CTP103 MAb, have utility as immunotherapeutic agents which, when administered to the carcinoma in anti-tumor effective amounts, is toxic to the cancer cells and not to other cells.

Such immunotherapy can be accomplished in accordance with a variety of modalities as known to those skilled in the pharmacological and immunological arts. For instance, for certain types of tumors, the MAb may be administered by direct injection into the tumor and/or by infusion into the tumor. In a tumor such as a lung cancer, the MAbs may be given by inhalation or aerosol izatio . In the case of certain diffuse tumors, the cytotoxic MAbs of the present may be given by IV or IM injection, for instance, in buffered serum albumin containing dilute NaCI. The MAbs of the present invention may be formulated for administration in accordance with known techniques for administering therapeutically effective doses of exogenous antibodies to a patient, for instance, in buffered, preserved saline solutions. The formulation may or may not include one or more ingredients also know n in the art for stimulating the effects of the immune system. As a general rule, it is preferred that the MAbs are given in either repeated doses or over an extended period of time to maximize their efficacy, but it is not necessary that they ge administered in this manner. Administering the MAbs of the present invention over an extended period of time is particularly contemplated for immunotherapy against diffuse tumors in which the MAbs are administered by IV so as to increase antibody titer in the blood, thereby increasing the likelihood of the binding of the MAbs to the cancer cells. The MAbs of the present invention may also be coupled with toxins, chemicals, enz> ιnes to tumor cell surfaces, and/or radioisotopes or radionuclides for the purpose of increasing their cytotoxicity. A wide range of labeling techniques are disclosed in Feteanu, "Labeled Antibodies in Biology and Medicine," pages 214-309 (New York: McGraw-Hill lnt'l. Book Co.), 1978. The introduction of various metal radioisotopes may be accomplished according to the procedures of Wagner, et al., 20 J. Nucl. Med. 428 (1979), Sundberg, et a]., 17 J. Med. Chem. 1304 (1974), and Saha, et al., 6 J. Nucl. Med. 542 (1976), but such procedures are illustrative and there are many other methods of radiolabeling known to the art. For instance, a useful method of labeling antibodies with radiometals is described in Khaw. et al.. 209 Science 295 (1980). Toxins such as diptheria toxin, IL-2R-directed Pseυdomonas exotoxin (PE) (Pastan, et al., Al Cell 641 (1986)), and other toxins known to those skilled in the art may be used to advantage in this manner. A variety of methods are utilized to couple these various toxin(s) to the MAbs, as described in, for instance, Jansen, et al., 62 Immunol. Rev. 185 (1982), and Vitetta, et al., 37 Transplant. 535 (1984).

In a further aspect of the present invention, administration of therapeutically effective doses of cytotoxic Mabs are combined with other treatment modalities such as surgery, conjugated antibodies, and/or chemotherapy in the manner described, for instance, in Mellstedt, et al., 18 Semin. Oncol. 462 (1991) and Frodin, et al, 1 Hybridoma 309 (1988). Similarly, therapy may include administration of the cytotoxic MAbs of the present invention with such agents as interferon in the manner described in Weiner, et al., 48 Cancer Res. 2568 ( 1988) and Caulfield. et al, 9 J. Biol. Response Modif. 319 (1990). It is also envisioned that therapy may require co-administration of agents for controlling toxicity and/or the side effects of MAb administration, for instance, prophylactic use of diphenhydramine or corticosteroids . to control allergic or hypersensitivity reactions and epinephrine for anaphylaxis. Although described in terms of specific embodiments, those skilled in the art who have the benefit of this disclosure will recognize that certain changes can be made to that embodiment without changing the manner in which the component parts thereof function to achieve their intended results. All such changes which do not depart from the spirit of the present invention are intended to fall within the scope of the following claims.

REFERENCES CITED

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Claims

WHAT IS CLAIMED IS:

1. Use of a monoclonal antibody which binds to the carboxy-terminal peptide of the ╬▓ chain of human chorionic gonadotropin for the preparation of a therapeutic composition for treating cancer.

2. The use of claim 1 wherein the monoclonal antibody is CTP-103.

3. The use of claim 1 wherein the monoclonal antibody is a monoclonal antibody made from a synthetic peptide having a sequence which is about 90% homologous with the sequence of amino acids 109-145 of the carboxy-terminal peptide of the ╬▓ chain of human chorionic gonadotropin.