CZ276398A3 - Immunotoxic preparation for liquidation of undesired target cells - Google Patents

Abstract

An immunotoxic agent for destroying unwanted target cells in particular breast cancer, other cancer cells, expressing the same target antigens in a cell population containing nucleated cells derived from peripheral blood or CD-34 + cells collected from the above nucleated cells, or otherwise immature / early progenitor cells from blood containing the immuno-stem cell of the invention comprises two immunotoxins targeting antigens occurring in malignant cells, each immunotoxin consisting of a conjugate between an antibody, the toxin, fragments of atoxin antibodies or recombinantly produced antibodies, irrunotoxins or fragments thereof, wherein the antibodies are directed to epitopes on the EGP2 antigen expressed by the gG733 ana epitopes on antigen expressed by genes MUC1, MUC2 or MUC3 or their combination of atoxin is Pseuctomonas exotoxinA

Description

The invention relates to an immunotoxic composition for killing unwanted target cells, in particular breast cancer or other cancer cells expressing the same target antigens in a population of cells comprising nucleated cells derived from peripheral blood, or CD-34 + cells harvested from the above nucleated cells, or other immature / early blood progenitor cells containing multifunctional stem cells.

BACKGROUND OF THE INVENTION

The so-called autologous stem cell transplants contain isolated cells from blood or bone marrow from cancer patients, which, after pretreatment with patients, contain adequate amounts of immature progenitor blood and immune cells to restore inoperative bone marrow function in the short term or after prolonged periods after reintroduction cells into the blood of the patients from which the cells are derived. The separation of autologous hematopoietic transplants using antibodies is known in the art when the transplants represent unselected bone marrow samples. Such separation has been disclosed, inter alia, by J.R. Myklebust, A.T., Godal, A., Juell, S. and Fodstad, Comparison of two antibody-based methods for eliminating breast cancer cells from human bone marrow. Cancer Res. (USA) 1994, 54/1 (209-214) and Myklebust, A.T., Godal, A., Pharo, A. Juell, S. and Fodstat, 0. Eradication of small cell lung cancer cells from human bone marrow by immunotoxins, Cancer Res. (USA) 1993, 53 (16), 3784-88. Both • ·

The publications use immunotoxins, wherein the antibody is conjugated to a toxin. The principle is to destroy malignant cells from the acquired bone marrow cells before injecting the cell suspension into the patient's body. In recent years, methods have been developed in which the principle is actually the opposite.

Using so-called stem cell transplantation, it is possible to try to reliably separate a subset of normal cells from the blood or bone marrow that are capable of restoring normal bone marrow function after reintroducing the cells into the patient's body. These stem cells consist of a mixture of the least mature blood precursors and immune cells as well as more differentiated cells. Obtaining such cells can be accomplished either by so-called peripheral blood aparesis, a procedure lasting one or several days, or by immuno-absorbing / selecting CD34 + cells (immature progenitor cells) from blood or bone marrow using various techniques known in the art. Stray, K.M. et al., Separation of multiform cells from bone marrow or peripheral blood using avidin, biotin, immunoadsorption in: P.G. Adrian, G. Samuel, and A.W.-W. Diana (Eds), Advances in Purification and Bone Marrow Purification Procedures p.97-103, Orlando: Willelis Inc., 1994, describes the separation of bone marrow cells or afaresis product from peripheral blood. This procedure is used to perform cleansing in lymphoma and breast cancer patients. This method includes an enrichment procedure for CD34 + cells prior to purification of B cells or breast cancer cells with a so-called avidin column. In this case, purification is performed indirectly in that the cell suspension is initially incubated with primary antibodies that bind to breast cancer cells; the cell suspension is washed and incubated once more with an antibody that binds to the primary antibodies. This rat antibody is biotinylated, ie, attached to a molecule that provides a strong bond to •

-3 avidin. When this cell suspension is finally loaded onto a column of avidin-conjugated beads, tumor cells are captured by binding between cells with a primary antibody biotinylated with secondary antibody avidin. Purification using such a system resulted in at least 3.2 logs of malignant cell depletion. This principle is very time-consuming and laborious, since the cell suspension has to be handled in several procedures, including incubation with antibody and double wash before loading onto the column. Therefore, it is difficult to prevent stem cell damage or to prevent unwanted stem cell capture in the column, which unfortunately would mean the loss of cells very important for restoring normal bone marrow function. Tyer, C.L. et al.,

Breast cancer cells are effectively separated from peripheral blood progenitor cells using an immunomagnetic procedure. A summary of the first meeting of the International Society for Hematotherapy and Fission Technique, Orlando, Florida, 1993, describes an immunomagnetic method similar to that used in Myklebust's publication above. However, this method applies to peripheral blood cells. This principle is also quite different from the use of immunotoxins and the purification efficiency varying from 3.3 to 4.8 logs of malignant cell depletion in model experiments. This resume does not mention any further use of the indirect primary antibody incubation system followed by washing and re-incubation with the beads attached to the antibodies that provide binding to the primary antibody, but this is an acceptable assumption. This procedure also includes the additional and perhaps traumatic treatment of normal cells and the procedure is time consuming. The efficiency is limited and the resume does not mention separation of CD34 + cell populations at all, which will be a considerable problem in this method, since selection of CD34 + cells is itself time consuming and laborious. That is why I

Most cases use the immunomagnetic principle to select SD34 + cells, which in this case is followed by one or two immunomagnetic procedures for purification purposes. Therefore, there is a significant risk of cell destruction and cell loss with a method requiring long-lasting process and high cost. Lemoli, R.M. et al. (1994) in Bone Marrow Transplantation 13: 465-471 describes the purification of human hematopoietic cells and the use of an avidin-biotin immunoabsorption technique. Purification of neoplastic cells has been increased using several immunotoxins containing the ribosome inactivating protein saporin and targeting lymphoid related antigens CD30 and CD2. Tecce, R. et al. (1991), p

Int, J.Cancer, 49: 310-316 describes the purification of autologous bone marrow prior to transplantation in patients suffering from monocyte leukemia with 2 monocytic cell-specific immunotoxins produced with saporin and 2 MoAbs of high specificity for circulating monocytes and M5b acute non-lymphoid leukemia ( ANLL). WO91 / 00050 describes immunotoxins containing myelomonocytic specific MoAB 195 useful for clearing ANLL from bone marrow. Tonevitsky, A.G. et al. (1986) in Int. J. Cancer, 37: 263-373 describes the purification of mouse erythroleukemic stem cells from bone marrow using an immunotoxin containing a ricin-A-chain conjugate and MoAbMAE15 that confers binding to the surface of normal and neoplastic mouse erythroid cells: a model and examination of bone transplant therapy marrow. In the isolation of stem cells for transplantation, one of the main objectives was that the cells being considered for reintroduction into patients were selectively isolated in such a way that the transplants did not contain any malignant cells. Recently, it has been shown that in the prior art such stem cell preparations, in anticipation, contained malignant cells in a significant number in the investigated cases. To date, limited efforts have been made to remove or destroy selected malignant cells in such transplants. This is partly because a person skilled in the art has not seen the necessity of this, and also because current known methods were expected to be non-specific and thereby also destroy or remove vulnerable stem cells. Further, obtaining bone marrow or mobilized peripheral blood from a patient is not easy and unrestricted, and such a method of purifying stem cell transplants must be carried out within a short period of time and be without complications in order to avoid loss or damage to normal cells. In view of the above, the reason for using stem cell transplants is that the transplant is completely free of cancer cells and that the recovery of bone marrow function is faster than after a transplant with bone marrow without selection. Accordingly, it is imperative to find an immunotoxic agent for a method of destroying unwanted target cells that leaves fragile normal stem cells intact and which is practical to perform in combination with stem cell isolation procedures. It is an object of the present invention to provide a means for a method of purifying stem cell transplants that do not have the above drawbacks.

SUMMARY OF THE INVENTION

The deficiencies of the prior art are substantially eliminated and the stated object of the invention fulfills an immunotoxic means for destroying unwanted target cells, particularly breast cancer or other cancer cells expressing the same target antigens in a population of cells containing nucleated peripheral blood derived cells or CD-34 + cells harvested. from the above nucleated cells, or other immature / early blood progenitor cells, containing multifunctional stem cells, according to the invention, the essence of which is comprising two immunotoxins directed to antigens occurring in malignant cells, each immunotoxin consisting of a conjugate between the antibody and the cell toxin, antibody and toxin fragments or recombinantly produced antibodies, immunotoxins or fragments thereof, wherein the antibodies are directed to epi the tops on the EGP2 antigen expressed by the GA733 gene and the epitopes on the antigen expressed by the MUC1, MUC2 or MUC3 genes, or a combination thereof, and the toxin is Pseuctomonas exotoxin A. Preferably, the MOC31 antibodies and a particular antibody directed to the MUC1, MUC2, MUC3 their combination. According to a preferred embodiment, the antibodies used may be MoC31 and BM7 or fragments thereof or MoC31 and BM2 or 12H12 or fragments thereof or MoC31 and 595A6 or fragments thereof. According to a preferred embodiment, the antibodies may be MOC31 and optionally BM7, 595, RM2, 12H12, or combinations thereof, or fragments thereof, and the toxin is natural or recombinant Pseudomonas exotoxin A, or fragments thereof. Accordingly, the present invention provides an immunotoxic agent for selectively separating a population of cells. The present invention further provides a mixture of two or more antibodies linked to bacterial toxins for use in the purification of stem cell populations obtained in solid tumor cases. The antibodies used are directed to antigens related to the target cells.

DETAILED DESCRIPTION OF THE INVENTION

In the following, the invention is described in more detail using an example of purification of stem cell transplants.

-7 obtained from peripheral blood to remove breast cancer cells. Known cell retrieval procedures consist of immunoadsorption / selection of peripheral blood stem cells (PBSC) or CD-34 + cells from blood or bone marrow. However, these are known to be innocuous and do not have sufficiently effective procedures to purify these cell populations for tumor cells. It appears that even among these immature cells there are malignant cells which, to date, should not have CD34 receptors. Importantly, the invention described below surprisingly also purified cancer cells without toxicity to normal progenitors. Prior to obtaining stem cell transplants from peripheral blood, bone marrow stem cells must be mobilized using chemotherapy or growth factor treatment using methods known in the art. Stem cell recovery can be carried out in one or more ways, depending on the kind of cells desired. In one method, peripheral blood stem cells are collected. This can be done by scheduling patients to undergo leukopheresis on days 10 and 11 of G-CSF administration (10 μ / kg / day) after receiving high doses of chemotherapy and general body irradiation. Blood flow can be determined, for example, at 70 ml / min. using a CS-300 Plus blood cell separator (Baxter Healthcare Corporation, Fenwal Division, Deerfield, IL). The average volume of blood treated during such a procedure may be about 10 L for 2 hours on a two-pass central venous catheter. It can be collected with 50 ml PBSC and washed with physiological phosphate solution (PBS), 1% human serum albumin (HSA) in Cobe Processor 2991 to remove platelets. For use of the invention, the cell concentration (2-4 x 10 ¹⁰ / afaresis) can be regulated to 1 x 10 ⁸ / ml for negative selection (purification) by immunotoxins. If CD-34 + cells are desired, this can be obtained by positive selection with ISOLEX 50 ^R or ISOLEX 300 ^R (Baxter). In this method, the product of aparesis • · • Λ

or from bone marrow, which may be about 4 x 10 ¹⁰ cells, mixed together and incubated with, for example, anti-CD34 + - monoclonal antibody 9C5 at 0.5 µg / 1 x 10 ⁶ cells at 4 ° C for 30 minutes on a fine rotator. The treated cells are washed with PBS with 1% HSA on a Cobe Processor to remove unbound antibody. DYNAL ^R M-450 beads are added to the CD34 ⁺ fraction at 0.5 beads per nucleated cell at 4 ° C for 30 minutes. Magnetic separation of rosettes from non-target cells can be performed by washing unbound cells two or three times with PBS, 1% HSA. CD34 + cells can then be released from the dynal beads by adding, for example, ChymoCell-R (Chymopapain) at a final concentration of 200 pKat / ml at 15 minutes at room temperature. Thus, CD34 + cells can be obtained by washing with PBS in 5% sodium citrate. Other procedures for selecting stem cells / initial progenitor cells are also known. The binding profile of several antibodies in breast cancer cell lines and tumor materials has been investigated by other experts and partly confirmed by us. Antibodies that bind to a large percentage of breast cancer cells and not important immature normal cells in blood and bone marrow were conjugated to Pseudomonas exotoxin A (PE) and the ability to destroy breast cancer cells in culture was mainly investigated in colony-producing assays. Based on the antibody binding profile, these inventors produced a total of five different immunotoxins:

1. MOC31-PE: This conjugate binds to a very high percentage of all breast cancer cells and was highly effective in model experiments and at actual concentration with marginal toxicity to normal hematopoietic progenitor cells.

2. NrLul0-PE: This binds to the same antigen as above, but to another epitope. It is somewhat less active than MOC31-PE.

3. BM7-PE: This binds to the protein portion of the mucin antigen, which is mainly found on breast cancer cells.

·························; ; . , · · * «« * «« «« «« «« «« «·

Antigen occurs in a large percentage of breast cancer cells, but not all. This immunotoxin showed high specific activity against cancer cells but was not as effective as the two previous immunotoxins.

4. RM2-PE: This binds to a sugar containing an epitope on the same antigen as RMT-PE. This immunotoxin showed approximately the same efficacy as RM7-PE and, as a concomitant phenomenon, had a very low toxicity to normal cells.

5. MLuCl-PE: This binds to a completely different antigen, the Lewis ^Y antigen. This immunotoxin was somewhat less active than the previous one and also showed moderate toxicity to normal cells.

The immunotoxins of items 1, 2, 5 were tested individually and in combination in model experiments to purify regular bone marrow samples for cancer cells (Myklebust et al.,

Cancer Research, 1994). However, as mentioned earlier, it is a great advantage to use stem cell transplants because of the shorter interval to regain normal bone marrow function (i.e., a safer procedure). However, contrary to what was expected, such transplants are contaminated with tumor cells and it has been necessary to administer immunotoxins capable of destroying all cancer cells in the transplant without significantly affecting normal cells. Research has been initiated for immunotoxins more specific for breast cancer than MLuCl-PE and better suited for breast cancer purification. During this study, it was unexpectedly found that the combination of MoC31-PE and BM7-PE was more effective than the sum of each of the foregoing immunotoxins used alone. This is demonstrated further in Table 1. Further investigation of binding between antibody and cell lines revealed that the combination gave stronger binding than individual antibodies. MOC31 binds to most breast cancer cells, even those that are less differentiated. RM7 detects mucin antigen that ·· ·

10 is expressed on a significant fraction of breast cancer cells, also on cells that are more differentiated. NrLulO and RM2 bind to the antigen recognized by MOC31 and RM7, and in view of this, they were not very likely to add anything to the combination of MOC31 and RM7 immunotoxins. MLuCl-PE was theoretically interesting in that it binds to a different antigen than the immunotoxins mentioned above. However, MLuCl-PE was toxic to normal cells and the model experiments showed no clear preference for its inclusion in the combination. In the example below, purification of peripheral stem cell transplants (afaresis products) is described. In addition, the inventors conducted several experiments using a combination of MOC31-PE and BM7-PE in experiments where tumor cells were added to the obtained peripheral stem cells or bone marrow prior to immunomagnetic positive selection of CD-34 + cells. The results from one such experiment are shown in Table 2. With two different cell lines, it is shown that positive selection of CD34 ⁺ cells by itself (without any other form of purification) removes up to 3.8 log tumor cells from the original cell population obtained. In other experiments, the purification effect of CD34 selection differs from 2-3 logs, which is also reported in the literature. When immunotoxin treatment was applied to a positively selected CD34 population, the overall purifying effect was greater than 4.7 logs (Table 2) for both cell lines. More than 4.7 logs in this case means that all detectable tumor cells have been removed. In other experiments, we cultured tumor cells and normal progenitor cells taken from the CD34 + population after 1 hour immunotoxin treatment in separate assays. In these experiments, we observed that the tumor cells are destroyed or die shortly after treatment, whereas the tumor cells in unpurified control populations grew and formed colonies and / or exuberated with the susceptibility typical of cell cultures. Normal stem cells are not affected by this action, so in three different assay systems, the survival of normal progenitor cells shortens only insignificantly. Table 3 shows a similar experiment in which CD34 + cells were incubated with immunotoxins for two hours at 37 ° C and it was shown that the stem cells essentially survived the action of immunotoxins.

9 9 9 • 9

999 99 9 9 9

9 99 99

9 9 ·

9

99

CM ω

μ &

£ • Η>

ο β

Μ> <υ β

β Λ ι — I

WITH

CM 'Ή β

φ> ο

Ή β

• Η> β a

ω

ÍX φ

ω

Λ

AT

Ή υ

Ή • ΓΊ β

'> ι

4-) • β β

Ή

X ο

4J ο

β β

rH £ • Η fd

44 ι — I (1) β β

Λ -Η β> Φ Η '£>

CN ti x:

rH tí

Λ tí

H «· φ φ φ φ φ · ΦΦΦ ΦΦΦ φ φ φ φ φ · · · · φ φ φ φ φ φ φ φ φ φ · · · · ε

Ο) tí

Ή

X!

Ο

4J

I th i

* r-i

H>

O x:

tí tí

Ή three r a a

o \ ° * rd

x:

> three three three

X!

tí

4-)> o o

and

Ή

tí 4-1 0 'tí 'tí x: > tí oj 0 Xi Ό x: -H > N > u > tí tí O 4-1 x: • H tí 4-1 tí ra > tí ι — H 0 Ό ε tí 'tí tí > and • H 0 and > U tí 'tí and r ~ t x; '> < Ή Ό at ε > N tí Ή tí tí 4-1 x: 4-1 tí tí tí tí T3 X tí 'tí at tí i — 1 tí 0 X! ra x: • H tí > tí Xi > and 0 and Í> 1 ra 4-> rtí Ή with, AT > N Ή tí Ή í> 0 tí Ή and 0 i — 1 i — 1 4-> !> ί 0 0 1-1 x: tí τ) X! tí tí 4-1 W > tí and 0 > 1 and > -ti with 0 AT m x: oj tí tí tí tí tí £ XÍ w > O ra Ή and 1 tí 0 £ > 0 N i — 1 what N • tí at 0 X4 O and N at tí tí and T3 three r-1 ra with ít tí 0 N '> 1 • H tí tí XÍ at '> 1 tí tí > X 0 4-1 > u tí > tí 4-> 0 0 ε tí and • tí tí > u tí ε > -X tí and H (0 N • Q at

• fc · fcfc • fcfcfc fc · fcfc fc

Fcfc for survival of colonies in CD34 ⁺ cells selected from mobilized peririum (0 pts)

'(those>

O> U

O (t • 3 a

• H

X o

4-) a

Ή <0 r - I (L) o b Λ -H (t> 0 H '>>)

at 0 0 b \ m ω and -H 0 > b and and φ 34 - | -> ΓΊ 34 (those 0 and b 0 b 34 • H N X 0 0 b 4J 0 b b O b Ό ε <D • H > 0 ω b and (those b i — 1 '(those í> 34 0 h (those b 34 .—. b b • H 34 ω rd • H 1—1 ε h (0 b sT m m Q AT O — — 1 X 34 tn > (D b b Λ £ £ ω in O O 1 i — i O and X at Φ (0 • H 4J WITH b O (those 1 and h> and (ΰ at b Ό b <D b JJ i — 1 b 0) 34 > b O 34 T3

b Ό (0 i — I

B> b a

>

Ό

O

U (t

I - I '(t H b (D P (0 £

Ft ft ft ft ft ft ft

··

ft ft

It was a great surprise that by using two antibodies directed to the antigens expressed by epithelial cells according to the invention, each combined with Pseudomonas exotoxin A bacterial toxin destroyed the malignant cells without causing any damage to normal stem cells in obtaining them from peripheral blood and bone marrow . It is known in the art that cells can be destroyed by bacterial exotoxins, and that the destroying effect is enhanced by attaching the toxin to antibodies directed to epitopes expressed by the target cells. However, it is also known that if immature cells are exposed to one or more immunotoxins, there is an absolute possibility that this action destroys normal stem cells in the cell population. Furthermore, these normal stem cells are susceptible to ex vivo action accompanying mechanical injury and temperature changes. In the present invention, a population of cells, for example, a stem cell transplant derived from peripheral blood, is exposed to a mixture of two antibodies, each conjugated to PE. Since one of the immunotoxins was overactive, it is surprisingly demonstrated that by using a mixture of two antibodies linked to a bacterial toxin, the purifying effect appeared to be greater than the sum of the effects when the immunotoxins were used separately. This interacting effect is shown in Table 4 of this disclosure using BM7 and MoC31 antibodies coupled to Pseudomonas exotoxin A in killing human breast PM1 cells. These immunotoxins are both monoclonal antibodies directed against tumor-associated antigens linked to a bacterial toxin

Pseudomonas exotoxin A. One of the antibodies recognizes the epithelial antigen encoded by the GA 733-2 gene, which is expressed by most cancer cells and can therefore be used in all cases involving cancers (eg breast cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer and pancreatic cancer). Another antibody is directed to mucin, a mucilage protein that is somewhat different from

-16 ft ft ft ft ft ft ft ft ft one cancer to another ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft. . In general, an antigen can be described as proteins encoded by the MUC-1, MUC-2, and MUC-3 genes. Examples of the above monoclonal antibodies are MoC31 and BM7. Conjugation of the antibody and the toxin can be accomplished by various known methods. The selection of two or more antibodies in the composition for binding to bacterial exotoxins was performed in such a way that antibody binding was directed to epitopes expressed on most target cells and not normal cells. The problem with the former method was that both malignant cells and normal blood cells express common antigens on the cell surface. In this case, two monoclonal antibodies MOCC31 and BM7 are used in this paper. The first of these antibodies are directed to epithelial cells which, if they occur in peripheral blood, are malignant. The antigen (whole protein) is encoded by the GA 733-2 gene. However, this antigen has several epitopes and it is important to target the epitopes expressed most abundantly. The BM7 antibody is one of the antibodies directed to the epitope of the antigen expressed by the MUC1 gene. Several genes have encoded similar antigens, for example (MUC2, MUC3). The bacterial toxin Pseudomonas exotoxin A has a relatively mild toxic effect on normal stem cells and malignant cells. However, when coupled to antibodies directed to antigens expressed on target cells, the toxic effect on these is pronounced. Table 5 shows that the immunotoxin composition of the invention, even after an incubation period of only 60 minutes, destroys T-47 D cells, MCF7 cells and PM1 cells at a much higher degree of efficacy than known in the art with other methods. The combination of these two immunotoxins thus gives surprising results relative to what would be expected due to the selective efficacy, simplicity and only marginal toxicity to normal progenitor cells. It can be said that the use of several immunotoxins consisting of Pseudomonas exotoxin A conjugated to three different antibodies is known, see Myklebust et al. 1993 and 1994. One of them

99

4 4 ·

4 · 9

999 999

9

-174 9 9 9 9 ♦ ··· 4 9

of these antibodies (MoC31) was used in a similar manner as in the present invention to purify bone marrow cells without selection.

However, the other antibodies used do not appear to be optimal, inter alia because one of them is directed to the same antigen as M0C31 and because the other (MLuCl) cross-reacts with normal cells and thus the immunotoxin bound to the antibody can be easily toxic for most immature stem cells.

In the present invention regarding stem cell purification preparations, we have prepared another monoclonal antibody that enhances the effect of MOCC31 and we observe that the combination of the two antibodies used as immunotoxins gives considerably surprising results. Due to the highly specific activity of said immunotoxins, it appears possible to administer the composition for the treatment of patients suffering from various cancers in vivo. If the cancer disease is limited, it will be possible to inject or infuse or mix it intravenously, for example, when it appears to spread the disease to the bone marrow. Furthermore, it is possible to inject these immunotoxins alone or in combination in patients with a further spread of the disease with abdominal watery or pleural effusion. The third option is to treat patients with cancer spreading to the central nervous system. In this case, immunotoxins can be injected directly into tumor tissue, into the spinal fluid, or into an artery supplying blood to the brain. It is not known how to use these immunotoxins in vivo except when MOC31-PE has been used in a leptomeningeal tumor model for small cell breast cancer. BM7PE is not described in the literature at all. An important problem with the use of immunotoxins in vivo is that their half-lives are often very short, i.e., the immunotoxins disintegrate and are removed from the blood before their concentration in the tumor is reasonably high. In U.S. Pat. No. 5,322,678, Morgan et al., Patented some modification of a portion of an antibody of certain immunotoxins to reduce the problem of short half-life in vivo.

• · · ···

- 18 • · · · ····

The applicant proposes a similar adaptation of the toxin moiety, a process which has not been carried out or known.

Example 1:

Effective purification of breast cancer cells from peripheral blood stem cells is obtained with immunotoxins. High-dose chemoradiotherapy with the support of autologous hematopoietic stem cells is increasingly used to treat patients with multiple malignancies (1,2). In cases where this procedure is unsuccessful, the most common cause of recurrence of the disease, rather than toxicity, is an infection and a transplant deficiency (3). Importantly, there is solid evidence that in patients receiving high-dose treatment, reinfusion of auto-transplants containing clonogenic tumor cells can contribute to recurrence and influence outcome (4). Gene-tagging studies of auto-transplanted cells suggest that tumor cells remaining in reinfused bone marrow (BM) contribute to disease recurrence (5). This conclusion is further corroborated by results in patients with follicular lymphomas suggesting that effective bone marrow cleansing improves the prospects of disease-free survival (6). Using sensitive immunocytochemical procedures, tumor cell contamination in histologically normal bone marrow autoantrants in the range of 37-62% can be observed in breast cancer patients undergoing high dose treatments (7). Peripheral blood stem cell (PMSC) auto-transplants collected by aphesis after pre-treatment with hematopoietic growth factors and chemotherapy are increasingly used with the belief that these products will barely contain tumor cells. However, it has recently been found that although the incidence of tumor cells is less widespread in PBSC autoantibodies against BM acquisition, malignant cells still occur frequently in mobilized PBSC collections in breast cancer patients (4,7). In addition, recent findings show that chemotherapy and / or

Growth factors can mobilize tumor cells into peripheral blood in patients with and without detectable bone marrow cancer cells (7.8), results that further increase the risk of tumor cell contamination in PBSC grafts. Purification of PBSC breast cancer auto-transplants may be necessary to prevent re-infusion of malignant cells in vitro. Here we report on a practical and rapid purification method, demonstrating that an incubation procedure of 60 minutes with immunotoxins directly added to the affinity product selectively destroys more than 5 logs of tumor cells. Materials and methods:

Cell line: The PM1 breast cancer cell line was taken from our laboratory from ascites fluid collected in a patient with advanced disease. MCF7 and T-47D cell lines were obtained from the American Type Culture Collection (Rockwille, MD) (ATCC HTB 22 and ATCC HTB 133). Cells were grown in culture at 37 ° C in an atmosphere of 5% CO 2 in RPMI 1640 medium supplemented with 10% heat activated fetal calf serum (FCS) and antibiotics (100 µg / ml penicillin, 100 µg / ml streptomycin). Medium and formulations were purchased from GIBCO in Paisley, UK. Human bone marrow and peripheral blood progenitor cells:

BM cells are obtained from healthy volunteer donors. BM mononuclear cells of the MNC fraction were obtained from Limphoprep ^R (Nycomed Pharma, Oslo, Norway) and washed twice in phosphate buffered saline (PBS) before being used in the experiments. PBSC were prepared from patients with non-Hodgkin's lymphoma. To mobilize PBSC, patients were pre-prepared with chemotherapy plus hematopoietic growth factors (G-CSF, Neupogen, Amgen / Hoffman-La Roche, Basel, Switzerland). Eleven to twelve days after chemotherapy, when CD34 + cells in peripheral blood are high, stem cells were collected using CS-3000 plus a blood cell separator (Baxter Healthcare Corp., Fenwal Division, Deerfield, IL).

9 ·

9 ·

9 9

999 ···

-209 9 9 9 9

9 9 9

9

Toxin, antibodies and immunotoxin assembly: Anti-MUC1 (9) BM7 antibody (IgG1) was donated by S. Kaul (Women's Clinic at Heidelberg University, Germany) and anti-EGP2 (10) antibody MO31 IgG2a was readily provided by L. de Leij (Groninngen University, The Netherlands) and MCA Development (Groninngen). PE was obtained from the Swiss Institute for Serums and Vaccines in Bern, Switzerland. Each antibody was conjugated to PE via a trioether bond formed by sulfosuccinimidyl-4- (N maleimidomethyl) cyclohexane-1-carboxylate (Pierce, Rockford, IL, USA) as described previously (11).

Immunotoxin treatment: The effect of immunotoxin treatment on the survival of clonogenic breast cancer cells was tested by incubating 2x10 ⁶ exponentially growing neoplasms of cells in RPMI with FCS at the indicated concentrations of immunotoxins at 37 ° C with gentle agitation (100 rpm in an orbital incubator Gallenkamp, Leicestershire, UK) ) to change the time slots as indicated for each experiment. Cells are washed twice in PBS with 1% FCS prior to inoculation in the clonogenic assay. In some experiments, 10% of neoplasm cells were admixed with BM mononuclear cells or PBSC, incubated with immunotoxins, washed and the effect assessed for survival of neoplasm cells or hematopietic progenitor clonogenic cells. Colon tests for neoplasm and hematopoietic progenitor cells. The fine agar clonogenic assay for neoplasm cells used herein has been previously described (12). Triple cultures were incubated for 14 days at 37 ° C in 5% CO 2, 5% O 2 and 90% N 2 and colonies of more than 50 cells were counted in a Zeiss stereomicroscope. Clonogenic capacity of conditioned and untreated normal progenitor cells was evaluated in CFU-GEMM assays (13) in which 5x10 ⁴ PBSC per ml were grown individually in standard methylcellulose cultures (HCC-4433 Methocult, Terry Fox Labs, Vancouver, Canada) in IMDM medium. (GIMBCO). After 19 days of incubation, the colonies

ΦΦ φ • φ φ

BFU-E and CFU-GM were counted in an inverse phase contrast microscope. Each test was performed with triplicate cultures in 1 ml with 35 mm dishes at 37 ° C in an atmosphere of 5% CO 2 and 100% humidity. Results: Growth of human breast cancer cells in some agar.

In several experiments, a linear relationship was observed between the number of inoculated cells and the number of tumor colonies formed. With the PM1 cell line, cloning efficiency ranged from 20 to 30% (not shown). In the T-47D and MCF7 cell line experiments, the previously reported linear relationships (14) with PE 27% and 22% were confirmed. These data were used to calculate the depletion efficiency of breast cancer cells obtained by this action.

Efficacy of single and mixed immunotoxins in killing breast cancer cells: Three different concentrations of each immunotoxin were used in model experiments. As shown in Table 4, only marginal effects with BM7 conjugate were obtained at two lower concentrations, whereas 2.5 log cell killing was achieved at 1.0 µg / ml. With MUC-31 at 0.1 µg / ml up to 3 log cell destruction was seen and at the highest concentration (1) / xg / ml, the efficacy was at least 5 log, which is the maximum effect possible for evaluation in this experiment (14). With a mixture of both immunotoxins, each at the indicated concentration, all neoplasm cells were destroyed as early as 0.1 gg / ml (Table 4). The results show that a mixture of the two immunotoxins can destroy breast cancer cells quite efficiently and the data also indicate that the enhanced effect can be achieved by combining the two conjugates. Similar results were obtained when the efficacy of immunotoxins was tested against two other breast cancer cell lines (not shown). Due to the expected diversity in the expression of antigens on target cells, it seemed logical to use a combination of immunotoxins in the further development of a method suitable for clinical use.

(Ν (Ν

Μ * β

γΗ β

X) β

Η · • · · · · · · · · · · ·

I «· · ·

Ή β

ω β

and 'β to

Ti • Η> β β

β

Λ

5 “ί

Μ β

1)> υ • Η β

• Η> β

Ch <

β β

Η> 4

Ο

4J

Ο χ

β ω

β ο

τ β

<υ (Ο

CM

0) to τ

υ

Ή υ

Ή π

β

4-) • β β

• Η

X ο

4J

Ο β

and

Η

4->

[Q

Ο β

β • Η> υ '2

9ί β

Η>

Ο β

β

Ο β

β>

ο

β 4-) CQ β '> 1 τ > Ν β 44 Ή ε β β 'β β ί> β 0 Τ) 44 β > υ 0 £ '> Ί • Η Ο 4-> £ CQ β 0 β β ω -Η > υ Τ 'β υ β 44 ω β £ 44 0 4-> Ch β β Τ Τ υ 'β '> < ι — 1 > τ • Η -Η γ ~ Ί -γ4 > β 4-) υ Ch 0 β β β CQ έ Ή β 'β > ο- υ τ β -η 4J β β Μ • Η -Η υ β β > β 0 0 I — 1 Ch 44 Ο β β Ch Ν Τ 'β 44 ο β β β β Τ

4J> υ ο

Ch ο

τ τ

β β

>

ο β

ο

Ν ο

Ch

Ν τ

υ β

ω> υ • β β

Ν β

4-1> υ ο

Ch what ο

β 'β τ

Ή> υ ο

<ΰ

4-) • Η β

β σι ο

ο β

• ΓΊ β

CQ

Ή

Β ’β

Τ β

I-1

C0 '> Ί>

β

4-)

Ο β

Τ

Ο

Τ

Ή β

τ β

> β

4-)

4- β 4-) β β 44 • Η> β 4-) β β Τ β>

Ο β

Ch υ β

>

β>

Each immunotoxin used

-23 Influence of incubation time. In the above experiments, an incubation of 120 minutes was used for immunotoxins. In clinical settings, it would be advantageous to use an even shorter incubation period for practical reasons. To investigate whether the time of immunotoxin exposure can be shortened without sacrificing the specific killing of neoplasm cells, a mixture of two conjugates, used at a concentration of 1 µg / ml for each, was tested with different incubation times against the three breast cancer cell lines. In all cases, a 12 minute exposure to immunotoxins caused eradication of all tumor cells. Importantly, this treatment was equally effective when the incubation time was reduced to 90 minutes and even 60 minutes. (Table 5) and data show that at the concentrations of immunotoxins used, the shortest incubation time is sufficient to destroy all clonogenic neoplasm cells occurring in tumor cell cultures.

·· ··> · · 1 »· <

• · · · · <

ΓΊ

LT)

Φ

Ή β

ω β

and> <υ β

Λ

Ή β

α)> υ • β

Η> β a

Γ>

ffl γΊ η

AND

AT

0 <0 • β β

• Η

X ο

JJ ο

β β

ε • Η ω

> φ

JJ ω

ο β

α • Η> Φ 'β

Φ β

Λ

Ο

Τ β

> υ (0

Λ β

β • Η β> Ό · Η γ-ι Η>

β β

> 0) ε

ο a

'Φ α

φ ω

Ή ε

Ή> β a

ο

Λ φ

β χ

J 'Φ β JJ Ο ε φ ω

2 ΐ> β β Λ 'Φ> Ο β. Ο 34 Τ Φ 'Φ β

Ή>

Ο

σι = t ω

β ra β

ο a

ε ο β Τ Φ ι — ι

Φ

Ή ε

φ> β β

τ a

φ

Q [>

AND

Η ω

Τ υ

φ ω

β ο

and

• Η Π υ at φ '2l β β JJ JJ β φ Φ JJ Ο ra β 0 0 ε 34 φ ra > β JJ 0 Ή > > Ν Ti β 0 φ and > β '> Ί • Η β X Φ 0 34 JJ ra 0 Ή β Ν β ε Ή 34 Φ τ |> and υ 2 '> Ί φ T ι — 1 Ρ > Ν ω υ Φ '> · > α > 18 Λ λ

• · · · · · · · · · · · · ·

To investigate whether toxicity to breast cancer cells can change in the presence of a large number of normal hematopoietic cells, experiments have been conducted in which tumor cells were admixed at a ratio of 1:10 to aparesis-derived PBPC. As shown in Table 5, immunotoxins destroyed more than 5 logs of PM1 tumor cells also in the presence of normal cells, after an incubation period of only 60 minutes. While these results were the same for all three cell lines, the data suggest that the immunotoxin procedure can also be effectively used in a clinical setting. Influence of incubation conditions and cell concentration. To investigate the efficacy of the immunotoxin procedure under conditions similar to that used in clinical specimens, PM1 tumor cells were admixed at a ratio of 1:10 to PBPC in experiments in which unwashed cells taken directly from the affection bag prior to incubation with immunotoxins were resuspended in normal saline with ACD (stock solution bag, R2220, Baxter Healthcare Corp., Fenwal Division). The results were compared to those obtained in the initial experiments with cells that were washed and resuspended in RPMI with 10% FCS. It was found (Table 6) that in both cases, all PM1 cells were destroyed by immunotoxins for 60 minutes, suggesting that for clinical use immunotoxins can be injected directly into the affection bag and that low pH under such conditions does not affect the cytotoxicity of the immunotoxins.

• · · · ·

CN

ί> Ί 0 'Η β > φ X β \ β X Ό ίΐ) Η > Ν > ι > 0 Φ AT/ > Li β X υ > Φ > 1SI 0 β β m β and ε β β ε 0 and X φ β _j 0 Ν > X fw ι — I > 1 τ — 1 β Ν > 1 γ — 1 φ 2 X ο X X Ρ4 η ο X Ή [J-. X X Ή β 0 φ φ 0 β • ι— » X 1 > υ 'β Φ • Η β > β > υ β >. θ ε β φ Η Φ β θ β ε > υ β 4-1 Φ L0 X β β • Η > Ν Ν X β • Η β 0 Ν > β Φ | ——1 γΜ [_) φ X & rt > 0) Ν Ή υ ρ X 'β Ο ε ίβ β- ω 'Φ φ β 2 \ β γ-Η β > β • Η m X Α ’Ι 1 η X X and > β X ι-Μ π φ β β β X φ X X β i> > ω ι — 1 X • η £ β ι — 1 ίβ φ ι \ ζζ ϊ> ε X > υ ρ> .X > β Ο 0 0 > tí γΗ β • 2 β X i-l m 0 Ή r- ¹ Λ ε m • β X β φ • β • Η Ν Sn · | -> , -χ β Η β Φ I — I X β X φ β 0 • Η 0 σι β n β and 4-) 0 4-1 AX '> Ί β β 0 β β tí β • Η X β and A-H Q > ϋ and ω β Ν \ (TS φ β .— £ X 'lU Vj β X Η !> Ί I 1 with 0 ω ω X r * fd X —- AT Η ο > β £ and ω i — 1 β β • β > Φ ·· m AND β β ο \ ο ε τ — 1 N -Η Ο ω β X ι — 1 β X 3 β 0 X β φ t- * -β X ω ω > φ β \with with ε 0 0 ε Ή r * TÍ β β H β 0 X β 2 β and β G) 1 — ί ε and -Η X »> β • Η (X > υ 'β Ή 0 • Η ί> Ν X υ β X Φ > d) 'φ β γΗ β υ β rj X X ε β M ω β Φ β 4-) P- * Γ) X X ι — I υ φ β rM • Η β β ω WHAT ι — 1 ♦ Η β X Ή β O β β ε 0 rH ο \ ° 0 X Ή > X 1—1 β ίβ β > β 0 rd and β • Η and β β 'β 0 Ή β !> X ·. X at Ο ο φ 'β Ή υ \ ι ~ ί X β β β m β β Ή • Η β β ι — 1 φ ε > and X ο \ ° 0 and X 0 φ β ο β ω <0 0 X ω ω CN X β and β X β ω β β Ή 0 ω 0 X X ο X > Φ ι — I φ Ή β \ β W β β β β ι — I X m 0 X X • Η ω 0 ) Ν and X > φ β > υ β Ν β β and Η '01 & Η X > • η 0

ί> Ί β

'β>

χ> υ ο

β β

(0> <

χ &

ο β

β

Ή

X ο

X ο

β i

ο χ;

O

X> β β

S4 σι = t β

β • Η

X

Ο

X

Ο a

υ ο

>

η

Η> β> 1 β

'β>

Ο

X β

X β

• Η

Ϊ> Ί

X> β β

Λ ίΝ

Λ · · · · · · · · · · · · · · · · · · · · ·

«·

In the aparse bag, the total number of cells is usually very high and it was conceivable that at such a high cell concentration, the efficiency of the procedure may be reduced when compared to the conditions used in the model examination. However, in experiments where this option was tested, no difference in efficacy was observed where the total cell concentration was increased first from 1x10 ⁷ to 5x10 ⁷ and then to ΙχΙΟθ per ml (Table 6). Toxicity of immunotoxins against normal hematopoietic progenitor cells. The effect of immunotoxins on the survival of CFU-GM and BFU-E was examined under incubation conditions as described above. It was found (Table 7) that even a 20 minute incubation of nucleated PBPCs with a mixture of immunotoxins did not shorten the survival of progenitor cells, whether tested after washing and resuspending cells in RPMI with 10% FCS, or when unwashed cells resuspended in normal saline. ACD. Since the treated cells will be frozen and thawed in the clinical environment before being returned to the patient, the effect of such procedures on progenitor cells has also been investigated. It was found that freezing and thawing only slightly decreased the number of CFU-GM and BFU-E (Table 7). Remarkably, immunotoxin treatment alone did not significantly shorten the survival of progenitor cells, although a slight decrease in the mean number of cell colonies was observed in the group where cells were not treated under low pH conditions. These data demonstrate that the concentration of immunotoxins that efficiently eradicates neoplasm cells after a 60 minute incubation has only an insignificant effect on the survival of normal clonogenic cells that have been treated twice as long.

• · ► »» »»>> * * * * * * * *

ΓΟ>

β ι — I β

β

Η

X!

υ β

β ω

Ή

Ν

U a

£ Ώ a

U ω

Ό • Η σι

β.

'Φ β

'Η β \ β _k > ο

AT

Φ

-η β

β ω

β

Ο a

Ή

Ή β

Φ

Ν β

β ε

Ν

Ο a

what

Ολ

VD ιρ rd

Ο

ΓΟ +1 +1 <0

VD

C ΓΟ ι — I c + 1 a

rn

Q what β> β> and ο

β β

• Η β

ω • Η ε

4-) N γ — 1 what β ^ 1 -Η β 44 > υ ε Ν Ο > and β CQ and and and AND β and 4-) 'Φ and ω β m 0 β and ί> β 0 β φ £ ι — 1 O φ 44 AND AT β and β £ and 44 at β • 44 VD H β > U Ή Φ • β υ > 44 ι — 1 Φ β O β 44 £ Ή β PQ ε 4->

Ό ο

and> φ β

β

X

Lf) ε

Ή β

'β ί>

ο> 0

Ο β

β

Ό

Φ> β a

at

4-) ω

'β> υ

Ή β

I — ι ο

β

4-) β

Ο 'Φ β

β>

Ο

U β

β a

Ν φ

β ο \ ο πι

Μ • Η β

Ο ι — I Ο 44

4J

Φ> υ ο

and ε

Ή β

φ

Ν β

β ε

Ν

Ti φ

> β a

Ή β

φ

Ν β

β ε

Ν ο

and ε

Ή β

φ

Ν β

β ε

Ν

Ti

Φ> β a

Ο ο

γΟ

LP rd • 4 * t-d +1 +1 ο

Γ • Ψ a

ro r ~ + 1 ro a

Τ)

Φ ω

β ο

and υ

<ο what ΓΟ

LP a

+1 a

ο

1 ^- 1

C0

ΓΟ +1 νο what a

+1 a

l — i a

ο ο

rd γο

ΓΟ ι — I

VD

O a

vd +1

C0

Γ0

ΓΟ +1

LP

Μ ’

LP a

+1

LP co β

γΗ 0 • ΓΏ 44 44 m 1 Φ β ο β 1 Γ ~ 1 Η Ti ο • Η > β 0 £ > υ and 4-) β (a) «Β • Η 44 ε β • and • Η and β ΓΟ β X ω 44 0 AT it 0 > ί 4-) 1 and γ — 1 0 ο Ή \ β β β β • Η β β > β β ε 0 • Η φ Ή Ή υ X 4J β β 0 β • Η 'β β 4-) £ ω !> and 0 > φ 0 β ε Ν β β I.E ω • Η ε 0 rH Μ Ή and β • Η β 4-) 44 γΗ 0 0 Ή 0 0 44 β AT ε β 'Φ 'β • Η Ν 4J Τ5 C0 X β > Ν and 0 0 0 β 0 Η and and 44 and

φ υ

β β

β • Η ί> Ί β

Ή ε

Ό

Ο a

'Φ β

β>

υ β

β a

Ν α

u ο \ ο

Ο

C0 ω

β

4J

Ν β

and £

and a

'Φ β β>

U 'Φ> <

> β β

β β

and

Ν

Φ £

4-) ο

φ £

Ν a

ε

Ή β

1-1 'β ε

β ο

β 'Φ β

β ί>

ο υ

β β

and

Ν β

'Φ

4->

£> 1> β β

m ο \ ° Ο j — I ω

4-)

Ο β

Ti φ

γο β

>

Ti

Η> β a

β β

4J I — I β

AT

Ή

Ό ο

β

4->

Ν υ

'> 1 β

β ω

νβ

Ν to β 44 ι — I ί> 1 44 ζ ¹ Ti ^? ο

Q ω

+1

τ) φ

> β

4J

Ή β

τ β

β

Ti β

β

4-) ω

• · • ·

-29Discussion: Analogous transplantation of circulating hematopoietic stem cells has recently aroused interest because of their advantages over BM transplantation (15, 16). In addition to rapid bone marrow function reconstitution, it is believed that the use of PBSC could eliminate the risk of re-infusion of tumor cells contaminating the transplant. However, the problem of tumor cell contamination has been shown to be reduced but not excluded (4). It should also be noted that high-dose chemotherapy using colony-promoting factors can drain tumor cells into peripheral blood (7, 8). Therefore, a quick and practical procedure for the purification of afaresis products is highly justified.

Several ways of removing breast cancer cells from BM have been reported, including chemo-immunoseparation, immunomagnetic procedures and the use of immunotoxins (14, 17, 18, 19). In contrast, few studies on PBSC purification preparations have been described so far, but immunotoxins prepared with ribosome-inactivating protein (22, 23) have been used to kill lymphoid tumor cells added to collections of CD34 positive cells prepared from BM (24). At the last examination, a purification efficiency of 2 logs was obtained in addition to 3 logs of indirect purification achieved by the CD34 selection procedure. The aim of this examination was to develop a safe immunotoxin procedure for purifying breast cancer cells from PBSC. The results obtained in the model experiments show that a 60-minute incubation with 1 µg / ml of each of the 2 anti-carcinoma antibody and PE conjugates effectively destroyed all tumor cells admixed to PBSC without toxicity to normal progenitor cells. Importantly, this method permits the addition of immunotoxins directly to the affinity product and after incubation the cells are washed, centrifuged and ready to freeze. Particularly because of its simplicity and efficacy, this method should be attractive for use in the treatment of selected groups of breast cancer patients in conjunction with high-dose chemotherapy combined with PBSC transplantation.

-30 flfl flfl flfl flflfl flflfl flflfl

9 9 9 9 flflfl 9 9 · · flfl

The high selectivity of our procedure can be attributed to the following factors: First, the antigen recognized by MOC31 is known to be expressed on most cells in almost all breast cancer samples examined (10). Also, BM7, which recognizes the marrow protein expressed by the MUC-1 gene (9), binds to a high fraction of breast cancer cells (25). Taken together, these two monoclonals appear to include, to an acceptable extent, the diversity in antigen expression occurring in breast cancer. Secondly, we have previously shown that it is important to use a toxin that fits the antibodies used in the assembly of immunotoxins (11). We have found that PE conjugations with a number of monoclonals, including those used herein, are quite effective (14). In addition, immunotoxins with PE are always more toxic than equimolar concentrations of free PE (11, 18) demonstrating the specificity of such immunotoxins. The purification procedures must be effective and safe, and the method must also be practical and clinically applicable. In addition to the advantage that immunotoxins can be added directly to the affection bag, our method has only a 60-minute incubation period to destroy all clonogenic tumor cells. Furthermore, this procedure is not toxic to normal hematopoietic progenitor cells and much higher concentrations of immunotoxins were well tolerated in BM purification attempts (14). We have also shown that freezing and thawing of PBSCs treated with immunotoxins did not induce any further toxicity, and it is noteworthy that the immunotoxin procedure does not involve non-specific cell loss that could occur in procedures using immuno-spherical tumor cell removal or immunoadsorption. We have previously calculated that the amount of conjugate remaining in the bone marrow treated with immunotoxins after washing is about 0.75% of the total added amount (26). In a clinical setting it would then be expected that PBSC treatments containing approximately 2x10 ¹⁰ · ·

-31 ft ft ft ft ft mononuclear cells, with a recommended concentration of 2 aminotoxin per ml (1x10 ⁸ cells) yields at most 3 µg of immunotoxins in the final product. This represents 100-150 times less free toxin than the theoretically calculated maximum allowable dose (26). This would not be expected to re-infuse purified PBSC with any systemic toxicity. The success or failure of high-dose therapy combined with autologous hematopoietic progenitor cell transplantation may depend even more on the effectiveness of systemic action than on the efficiency of graft cleaning (1). However, it is logical to remove cancer cells that might be present in the auto transplant, and recent evidence from studies of other tumor types shows the importance of purification (16). For breast cancer, we suggest using a simple, safe and effective procedure such as that described here.

Example 2:

Since breast cancer cells may have different sensitivity to immunotoxins, so that BM7 and MOC31 purification activity may be different on breast cancer cells in different patients, the same experiment was repeated as previously performed with PM1 breast cancer cells (Example 1) with a different cell line , MA11. The effect of the immunotoxin effect was found to be equally good or even better against MA11 cells over PM1 cells (Table 4). The results confirm the high specific activity of the purification action with immunotoxins. In separate experiments, kinetics of immunotoxin cell killing activity was examined in an experiment where PM1 breast cancer cells were added to peripheral blood progenitor cells (1: 100 ratio). After incubation for two hours, the mixed cell suspension was frozen and then thawed before cell inoculation and viability of cancer cells and normal progenitor cells in

999-99-9-9-9 9-9999-9999-9999 9-99-9999-999-99-99-9 9 cultures of cells that were or were not treated with immunotoxin for the same time period.

To Examples 3-4:

Cancer cells that spread to the bone or bone marrow, pleural and abdominal cavities, brain and spinal tissues, and the urogenital tract can be destroyed selectively by the immunotoxins injected into the tumor, into the body fluids or systematically, e.g., to target metastatic tumor cells. in tissues such as blood, bone, and bone marrow.

Example 3:

Human breast cancer MA-11 cells were injected into the left ventricle of immune-deficient rats. Control animals without intervention developed symptoms of spinal cord compression and had to be sacrificed 24-37 days after cell injection. Animals treated intravenously with a single dose of MOC31-PE (20 per rat) achieved extended symptom-free survival and some of these animals lived for more than 50 days. Another experiment in the same model confirmed the results and in this case some of the animals survived for 110 days. In these experiments, one group of rats was treated with a certain immunotoxin consisting of antibody 425.3 directed against the EGF receptor conjugated to PE. All animals in this group survived. In a third experiment in this model, control rats showed symptoms of spinal cord compression and had to be sacrificed between day 40 and day 60 after cell injection. Three treatment groups were enrolled in this experiment, one with 20 µg 423.5-PE and one receiving 10 mg of each of the two immunotoxins.

Significant prolongation of disease-free survival was achieved with both immunotoxins used individually, giving a 6% long survival time with MOC31-PE and 60% with 425.3-PE. In the combination trials, all animals survived without disease. In the fourth experiment in this model, the effect of MOC31-PE was compared to that of cis • φ · · · φφφ

-33platin and doxorubicin. In this experiment, all MOC31-PE-treated animals survived for more than 70 days, whereas doxorubicin showed only a marginal effect and cisplatin-treated rats did not live longer than saline-treated control animals. These data convincingly show that the immunotoxins used are of significantly superior quality in the control of breast cancer metastases than doxorubicin and cisplatin, two of the most commonly used drugs in the clinic.

Example 4:

The human breast cancer cell line MT-1 was used in two different experiments. In the first case, cells were injected into the left heart cavity and control animals had to be sacrificed due to spinal cord compression symptoms after an average of 19 days. Animals treated with 425.3-PE intravenously one day after cell injection survived all. In other experiments, MT-1 tumor cells were injected directly into the bone marrow cavity in the tibia of rats. All untreated animals had to be sacrificed 20 days later due to increasing tibial tumors, whereas rats treated with 20 gg 425.3-PE intravenously one day after cell injection survived all for more than 100 days. Furthermore, in a model where MT-1 tumor cells were injected directly into the bone marrow of the concomitant shaving, the effect of BM7-PE administered either on days 1 or 7 was compared with the effects of 425.3-OE in groups of animals treated on the same days as BM7-PE. . Furthermore, the effect of doxorubicin (Adriamycin) administered intravenously on days 7 and 14 was also investigated. Both immunotoxins were found to cure 80% of the rats, whether administered on day 1 or 7. When half concentrations of each immunotoxin were combined, all animals survived. In contrast, doxorubicin was clearly less effective, leaving only 35% of the animals alive after 90 days. Control animals had to be sacrificed 20 days after cell injection as in previous experiments. The data confirm the effect of 425.3-PE

-34 ftft ftft ftft ftft ftftftft ftft ftftftft ftft ftft ftft ftft ftft ftft ftft ftft ftft ftft ftft ftft as before shown. Importantly, RM7-PE was found to be as effective as 425.3-PE. Both were clearly superior to doxorubicin, one of the clinical drugs most commonly used against breast cancer. In addition, the combination of these two immunotoxins cured all animals from their disease.

Example 5:

In two sets of experiments, the effect of recombinantly produced immunotoxins directed against the erbB2 gene product and with a recombinantly produced PE modification was tested. In the model described in Example 4, the highest concentration of recombinant immunotoxins significantly prolonged the life span of the animals and 35% of the rats survived. In a model where MT-1 breast cancer cells were injected intrathecally in immune-deficient rats, recombinant immunotoxin treatment was also administered intrathecally (days 1, 2, and 3), resulting in a significant increase in the life span of the animals. This effect was dose-dependent and the life span of the animals increased from 10.6 days (saline-treated control animals) to 23.4 days and 32.8 days with two different doses of immunotoxins with two different doses. At the highest dose, 20% of rats survived. Since toxicity was not observed even at the highest dose of immunotoxins, it is expected that the effect at optimal doses may be even better.

·· ·· »♦ · · · • · φ · φ ·

-35 • · φ • ···· *

References to literature

1. Peters, W.P., Ross,,, Vredenburgh, J.J., Meisenberg, B., Marks, L.B., Winer, E., Kurtzberg, J., Bast, R.C.J., Jones,

R., Shpall, E., Wu, K., Rosner, G., Gilbert, C., Mathias, B., Coniglio, D., Petros, W., Henderson, IC, Norton, L., Weiss, RB. , Budman, D. and Hurd, D .: High Dose Chemotherapy and Autologous Bone Marrow Support as Consolidation after Adjuvant Treatment at Standard Dose for High Risk Primary Breast Cancer. J. Clin. Oncol., 11: 1132-1143, 1993.

2. Armitage, J.O .: Bone marrow transplantation. N. Engl. J.

Med., 330: 827-838 (1994).

3. Moss, T.J., Sanders, D.G., Lasky, L.C., and Bostrom, B .: Contamination in peripheral blood stem cell acquisition by circulating neurobastastic cells. Blood, 76: 1879-1883 (1990).

4. Ross, AA, Cooper, BW, Lazarus, HM, Mackay, W., Moss, TJ, Cioban, B., Tallman, MS, Kennedy, MJ, Davidson, NE, Sweet, D., et al. Detection and viability of tumor cells in peripheral blood stem cell collection in breast cancer patients using immunocytochemical and clonogenic assay procedures. Blood, 82: 2605-2610, 1993.

5. Brenner, M.K., Rill, D.R., Moen, R.C., Krance, R.A., Mirro,

J., Jr., Anderson, W. F., and Ihle, J.N .: Gene Tagging for Monitoring the Origin of Relapses after Anal Bone Marrow Transplantation. Lancet, 341: 85-86 (1993).

6. Gribben, J. G., Frredman, A. S., Neuberg, D., Roy, D.C.,

Blake, K. W., Woo, S. D., Grossbard, M. L., Rabinowe, S.N.,

Coral, F., Freeman, G.J., et al .: Immunologic purification of pulp evaluated by PVR prior to autologous bone marrow transplantation for B-cell lymphons. N. Engl. J. Med., 325: 1525-1533 (1991).

7.Sphall, E.J. and Jones, R.B .: Release of neoplasm cells from bone marrow. Blood, 83: 623-625 (1994).

-36φ φ φ · · · φ φ φ φ · φ φ φ φ φ φ φ · · · φ φ φ φ φ φ φ Φ Φ Φ Φ Φ

ΦΦ ΦΦ

8. Bruger, W., Bross, K.J., Glatt,,., Weber, F., Mertelsmann, R., and Kanz, L .: Mobilization of Neoplasmic Cells and Hematopoietic Progenitor Cells into the Peripheral Blood of Patients with Tissue Tumors. Blood, 83: 636-640, 1994. and Dekker, J.: Mucionic Glycoproteins. Crit. Roar. Biochem. Molecu. Biol., 27: 57-92,1992.

10. Leij, L.D., Postmus, P.E., Poppema, S., Elema, J.D., and

The, T.H .: Use of Monoclonal Antibodies for Pathological Diagnosis of Lung Cancer. V: H.H. Hansen (ed.), Lung Cancer: Basic and Clinical Aspects 31-48. Boston:

Martinus Niijhoff Publishers (1986).

11. Godal, A., Kumle, B., Pihl, A., Juell, S., and Fodstad, O .: Immunotoxins directed against high molecular weight melanoma-related antigens. Identification of effective combinations of antibody toxins. Int. J. Cancer, 52: 631-635 (1992).

ftft ft ftftft ft ft ft

-37 ft ft ft ft ft 9 ft 9 ft ft

12. Courtenay, V.D. and Mills, J .: In vitro colony analysis for human tumor grown in immunosuppressed mice and processed in vivo with cytotoxic agents. Br. J. Cancer, 37: 261-268 (1978).

13. Wang, MY, Kvalheim, G., Kvaloy, S., Beiske, K., Jakobsen, E., Wijdens, J., Pihl, A., & Fodstad, 0: An Effective Immunomagnetic Procedure for Bone Marrow Purification in T-cell malignancies. Bone Marrow Transplant., 9: 319-323,1992.

14. Myklebust, A.T., Godal, A., Juell, S., Pharo, A., and Fodstad, 0 .: Comparison of two antibody-based methods for removing breast cancer cells from human bone marrow.

Cancer Res., 54: 209-214,1994.

15. Eaves, C.J .: Peripheral blood stem cells reach new heights. Blood, 82: 1957-1959 (1993).

Sphall, E.J., Jones, R.B., Bearman, S.I., Franklin, W.A., Archer, P.G., Curiel, T., Bitter, Μ., Claman, H.N., Stemmer,

SM, Purdy,,, Myers, SE, Hami, L., Taffs, S., Heimfeld, S., Hallagan, J. and Berenson, J. Transplantation Enriched with CD34 - Positive Autologous Marrow of Breast Cancer Patients After High-Dose Chemotherapy : Influence of CD34 - positive peripheral blood progenitors and growth factor on vaccinated transplantation. J. Clin. Oncol., 12: 28-36, 1994.

-38 φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ

17. Bjorn, M.J., Groetsema, G., and Scalapino, L .: Antibody Pseudomonad Exotoxin A Cytotoxic Conjugates to Human Breast Cancer Cells in vitro. Cancer Res., 46: 3262-3267 (1986).

18. Anderson ,. I.C., Shpall, E.J., Leslie, D.S., Nustad, K., Ugelstad, J., Peters, W.P., and Bast, R.C.Jr .: Exclusion of Malignant Breast Cancer Clonogenic Cells from Human Bone Marrow. Cancer Res., 49: 4659-4664,1989.

19.0'Briant, KC, Shpall, EJ, Houston, LL, Peters, WP, Bast, RCJr .: Exclusion of Breast Cancer Clonogenic Cells from Human Bone Marrow: Comparison of Immunotoxin Treatment with Chemoimmune Separation Using 4-Hydroperoxy-cyclophosphamide, Monoclonal Antibodies and Magnetic microspheres.

Cancer 68: 1272-1278, 1991.

20O. Stray, K.M. , Corpuz, D., Colter, K. M., Berenson, R., and Heimfeld, S .: Purification of Neoplasm Cells from Bone Marrow or Peripheral Blood Using Avidin Biotin Immunoadsorption. Listed in: P.G. Adrian. G. Samuel, and A. W. W. Diana (eds.), Advances in Bone Marrow Purification and Processing, pp. 97-103. Orlando: Wiley-Liss, Inc., 1994.

21. Tyer, C.L., Vredenburgh, J.J., Heimer, M., Bast, R.C.Jr., and Peters, W.P .: Breast cancer cells were effectively separated from peripheral blood progenitor cells using an immunomagnetic procedure. Summary of the first session

International Society of Hematology and Grafting Technology, Orlando, FL, 1993.

22. Stirpe, F., Barbieri, L., Battelli, M.G., Soria, M., and Lappi, D.A .: Ribosome Inactivation Protein from Plants: Present State and Future Prospects. Bio / Technology 10: 405-412 (1992).

23.Barbieri, L., Battelli, M.G., Stirpe, F .: The Rubosone Inactivation Protein from Plants. Biochem. Biophys. Acta.

1154: 237-282 (1993).

Ft. Ft

-39ftft · ftft · ftft ftftftft ftft ftft

24. Lemoli, RM, Tazzari, PL, Fortuna, A., Bolognesi, A., Gulati, SC, Stirpe, F., and Tura, S .: Positive selection of hematopoietic CD34 + stem cells ensures indirect purification of CD34 - lymphoid cells and segregation the effect is enhanced by anti-CD2 and CD30 immunotoxins. Bone Marrow Transplant., 13: 465-471,1994.

25. Diel, I.J., Kaufmann,,., Goerner, R., Costa, S.D., Kaul, S., and Bastert, G .: Detection of neoplasm cells in bone marrow in patients with primary breast cancer: prognostic factor for distant metastasis. J.Clin. Oncol., 10: 1534-1539,1992.

26. Myclebust, A.T., Godal, A., Pharo, A., Juell, S., and Fodstad, O .: Eradication of small cell lung cancer cells from human bone marrow by immunotoxins. Cancer Res., 53: 37843788 (1993).

ft v

9 9 9

9 9 9

999 999

9

99 • 9

9 9

9999

9

Claims (6)

Patent claims Immunotoxic means for killing unwanted target cells, in particular breast cancer or other cancer cells, expressing the same target antigens in a population of cells comprising nucleated cells derived from peripheral blood, or CD-34 + cells harvested from the above nucleated cells, or other immature / early blood progenitor cells containing multifunctional stem cells, characterized in that it comprises two immunotoxins directed to antigens occurring in malignant cells, each immunotoxin consisting of a conjugate between an antibody and a cell toxin, antibody and toxin fragments or recombinantly produced antibodies, immunotoxins or fragments thereof, wherein the antibodies are directed to epitopes on the EGP2 antigen expressed by the GA733 gene and epitopes on the antigen expressed by the MUC1, MUC2 or MUC3 genes, or a combination thereof, and a toxin is Pseuctomonas exotoxin A. The immunotoxic composition of claim 1, wherein the antibodies used are MOC31 and an antibody directed to the antigen-encoded MUC1, MUC2, MUC3 genes, or combinations thereof. The immunotoxic composition of claim 2, wherein the antibodies used are MOC31 and BM7 or fragments thereof. The immunotoxic composition of claim 2, wherein the antibodies used are MOC31 and BM2 or 12H12 or fragments thereof. • Β ΒΒ -41 ΒΒ Β · · · · · · · ΒΒΒ ΒΒ Β Β · ΒΒ The immunotoxic composition of claim 2, wherein the antibodies used are MoC31 and 595A6 or fragments thereof. t The immunotoxic composition according to claim 2, wherein the immunotoxic agent is as follows characterized in that the antibodies are MoC31 and optionally BM7, 595, RM2, 12H12 or combinations thereof, or fragments thereof, and the toxin is natural or recombinant Pseudomonas exotoxin A, or fragments thereof.