WO2002017970A2 - Metal radionuclide labelled cd-30 antibodies and uses thereof - Google Patents

Abstract

The invention relates to metallic radionuclide containing immunoconjugates. The immunoconjugates bind specifically to the CD30 molecule. Both diagnostic and therapeutic uses of the immunoconjugates are described. The preferred immunoconjugates are monoclonal antibodies to which ?111In or 90¿Y is bound via a chelator.

Description

METAL RADIONUCLIDE LABELLED CD-30 ANTIBODIES AND USES

THEREOF

RELATED APPLICATIONS This application is a continuation in part of Serial No. 09/679,849, filed

August 28, 2000, and incorporated by reference in its entirety. FIELD OF THE INVENTION

The invention relates to labelled antibodies and their use. More specifically, it relates to the manufacture and use of metallic radionuclides as labels for antibodies specific to the molecule referred to as CD30. Expression of this molecule is associated with Hodgkin's Disease. Hence, in addition to the immunoconjugates, the invention relates to diagnostic and therapeutic uses of the immunoconjugates. BACKGROUND AND PRIOR ART

Conventional therapeutic approaches to Hodgkin's Disease include chemotherapy and radiotherapy. These approaches have been successful, but there is a significant proportion of patients who relapse following treatment, and for whom the disease becomes increasingly unresponsive to standard treatments. See, e.g., Engert, et al, Cancer Res 50:84-88 (1990). Hence, a need exists for improved, therapeutic approaches to Hodgkin's Disease. One proposed approach has been the use of radiolabelled rabbit antifeπitin polyclonal antibodies as a treatment for patients with recurrent Hodgkin's Disease. See Lai, et al, Clin. Cane. Res 5:3315s-3323s (1999). While the results of Lai's study were encouraging, several factors indicate that improvement to such approaches is needed. For example, ferritin is a ubiquitious component in humans, so precise targeting is not possible. Also, rabbit polyclonal antibodies are heterogeneous and immunogenic. Thus, their specificity is limited.

Interest has focused on the CD30 molecule as a target for therapy. The molecule is overexpressed in Hodgkins Reed-Steinberg cells, but in only a small portion of normal lymphoid cells. See Winkler, et al, Blood 83:466-475 (1994). Some have utilized this observation in combination with monoclonal antibodies as a possible therapy. For example, Hartmann, et al, Leukemia & Lymphoma 31(3-4):385-92 (1998), utilize bispecific monoclonal antibodies, to redirect CD 16 expressing, cytotoxic T lymphocytes to Hodgkin's Disease cells. Also see Renner, et al, Cancer Immunol Immunother 45 : 184- 186 (1997). Terenzi, et al, Brit. J. Hematol 92(4):872-9 (1996) have proposed conjugating biotoxins to CD30 specific antibodies to deliver them to Hodgkin's Disease cells. Further, US Patent No. 5,753,203 to Goodwin, et al, suggests delivery of a ligand to CD30 molecules to inhibit their action.

Work on the CD30 molecule has led to some understanding of it, although it is far from complete. Martinez, et al, Transplantation 65(9):1240-47 (1998), describe the cloning of the CD30 molecule and suggest that it is a growth factor receptor. Radiolabelled antibodies are well known to the artisan, and are useful both diagnostically and therapeutically. Exemplary radiolabels include radioactive isotopes of iodine, such as ^I251 and ¹³¹ 1, as well as metallic radiolabels, such as ⁹⁰ Y, ^ιπ In, and so forth. Radiolabels can be attached to antibodies in various ways. For example, radiolabelled iodine is attached to antibodies via tyrosine residues, catalyzed via iodogen or chloramine - T. See, e.g., da Costa, et al, Ann. Oncol 3(Supp 4):53-7 (1992), teaching radio labelling anti-CD-30 antibody HRS-3 with iodine. Methods for labelling antibodies with radioactive iodine include the use of ligands such as N-succinimidyl 3-iodo-5- pyridinecarboxylate. When metallic radiolabels are used, bifunctional metal ion chelators, such as CHX-A"-DTPA can be used. Such methods are well known, and are used in the invention described herein.

Different forms of metallic radiolabels are available. For example, alpha particle emitters release high energy helium nuclei, depositing energy over short ranges of several cell diameters thickness. The linear energy transfer disrupts DNA function even under hypoxic conditions and is quite efficacious in bringing about cell destructions. Radioconjugates employing alpha emitters attack cells where conjugates accumulate, as well as adjacent cells. Hence, alpha emitters are useful in small volume diseases, as well as those with minimal residual disease.

Beta emitters have longer range, lower density ionizations along their tracks, and relatively lower Linear Energy Transfer, or "LET" values than alpha emitters. Experience has shown that radioimmunotherapy using beta emitters results in remarkable clinical responses, even in those patients refractory to conventional treatment. See, e.g., Rain, et al, Pathologie Biologie 46:341-345 (1998), for a report on the use of labelled anti-CD20 monoclonal antibodies in the treatment of Non-Hodgkin's lymphoma. Since beta emitters have longer ranges, and longer decay half lives, there is more time for irradiation of the tumors. More distant cells can be treated, including those cells in a tumor which do not necessarily present the target molecule interest. Beta emitters should be suitable for tumors with larger volumes.

A further form of radiolabel is the low energy electron emitter. Low energy electron emitters are cytotoxic if they reach the nucleus of cells, the result of a confined volume of Auger and Coster-Kronig electrons. Such isotopes are less effective outside of the nucleus, but this can be compensated for by using longer delay half life isotopes. Exemplary of such radiolabels are ^πι In, ^99m Tc, and ¹²⁵ 1. Such labels are especially effective in treating minimal residual diseases.

Yet further forms of radiolabels are gamma emitters, and positron emitters. ^! ' 'In is a gramma emitter, and ⁶⁸Ga and ⁸⁹Zr are examples of positron emitters. Positron emitters are useful because, inter alia, they provide higher resolution images in positron emission tomography ("PET"), as compared to conventional gamma camera imaging.

While there is an extensive literature on radiolabelled antibodies, it will be understood that not every radiolabel has been used on every antibody. Nor have all potential uses of such conjugates been explored.

It has now been found that conjugates of metallic radionuclides and anti-CD30 antibodies show surprising efficacy, suggesting their use as diagnostic markers and therapeutic agents. These, as well as other features of the invention, will become clear from the disclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the results of assays carried out to determine the immunoreactivity of ^nιIn and ¹²⁵1 labelled, anti-CD30 antibodies

Figure 2 compares in vivo uptake and retention of the ^πl In and ¹²⁵1 labelled anti- CD30 antibodies DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

EXAMPLE 1

Murine antibody HRS-3, described by Engert, et al, Cane. Res 50: 84-88 (1990), incorporated by reference, was used in this example, and in the examples which follow. Samples of antibody were purified, conjugated with chelate CHX-A"- DTP A,

(described by Nikula, et al, Nucl. Med. Biol. 22: 387-390 (1995), incorporated by reference), at a 3 : 1 molar ratio, purified, and then stored at -80 ° C until ready to use. The acronym "CHX-A" -DTPA" stands for C-functionalized trans cyclohexyl diethylenetriaminepentaacetic acid. Its synthesis is described by Wu, et al, Bioorg. Med. Chem 5: 1925-1934 (1997), incorporated by reference.

In order to radiolabel the antibodies, ^m In in an HC1 solution was converted to the acetate form via addition of concentrated acetate buffer and was then added to the antibody chelates. The pH was adjusted to 5-6, and after an appropriate period of time, any unbound isotope was quenched with EDTA, followed by chromatographic purification.

In parallel, ¹²⁵I was used to label the antibody following standard methods.

EXAMPLE 2

In these experiments, retention of the immunoreactivity of both the ^!i!In and the ¹²⁵I labelled chelated antibodies was determined. The Lindmo assay, as described by Lindmo, et al, J. Immunol Meth. 72: 77-89 (1984) incorporated by reference, was used.

In brief, small amounts of labelled chelated antibody (about 30ng protein) were added to L540 cells, which are known to express CD-30. The combined samples were incubated and, after 30 minutes, samples were centrifuged, and the resulting cell pellets were washed, three times, with culture medium. Pellets were then counted for radioactivity, and compared to similar standards, in order to calculate the immunoreactivity. The results are presented in Figure 1, in which it can be seen that the two radiolabelled antibodies both show excellent binding to the CD30 expressing cells. EXAMPLE 3

In these experiments, the ability of the antibodies to bind in vivo to the CD30 molecule was tested.

B ALB/c nude mice which bore L540 xenografts that express CD30 antigen were used. BALB/c mice bearing xenografts of colon tumors which did not express CD30 were used as controls.

Approximately 5 microcuries of each of the labelled antibody-chelates (2 microgram protein)Hodgkin's were mixed together, and inj ected, intravenously, into the mice. At varying time intervals after the intravenous injection, mice were sacrificed, in groups of 3, and blood, tumors, and other tissues were collected and weighed. These samples were and counted for radioactivity, as were standards. Presence of radiolabelled antibody in samples was expressed as percentage injected dose per gram (% ID/g).

Figure 2 shows that the radiometallic labelled antibody was taken up much faster, and was retained for a far longer time than the ¹²⁵I labelled antibody.

EXAMPLE 4

In these experiments, the HRS-3 antibodies, referred to supra, were labelled with ⁹⁰Y, using essentially the same methodology as is set out in example 1, supra. The labelled antibodies were then tested for retention of immunoreactivity, as in example 2, and the results were similar. These antibodies were used in experiments which follow. L540 Hodgkin's Disease cells are described supra. Approximately 20 million cells were injected, intravenously, into female "SOD" mice, approximately 5-6 weeks of age, via tail veins. The weight of the mice varied, but was essentially from 15-17g. The dose used (30 Ci)., is approximately 1.8Ci/kg, which is comparable to other protocols using ⁹⁰Y labelled antibodies. See, e.g., Herpst, et al, J. Clin. Oncol. 13:2394- 2400 (1995); Vriesendrop, et al, Clin. Cane. Res. 5 :3324s-3329s (1999). The method of

Kapp, et al. Annals. Oncol 3 suppl 4:21 (1992), incorporated by reference, was used.

The mice were left alone following the injections, for 13 days. Renner, et al., Blood 87:2930-2937 (1996), describes how this length of time allows for proliferation and dissemination of tumor cells in the animal, in a manner which simulates advanced disease. Survival was monitored ab initio.

The mice were divided into groups of 8, and received doses of either 30μCi or 40μCi of ⁹⁰Y CHX-A"-DTPA labelled HRS-3 antibodies, parallel doses of A33 antibodies labelled in the same fashion (A33 does not bind CD30), unlabelled HRS-3, or phosphate buffered saline.

At the 30 iCi dose, it was evident that the survival of the mice was enhanced. The survival of mice which received controls ranged from 38-49 days, with median survival of 41.5 days. Mice which received a single dose of unlabelled anti-CD30 (18.6 ig), survived for from 34 to 50 days, with median survival of 39.5 days. The 50% survival rate for both buffer and single antibody groups was 41 days. Those mice which received the labelled antibodies survived for from 34 to more than 75 days (the endpoint).

Mice which received a 40μCi dose of control, radiolabelled antibody had shorter survival periods, ranging from 27-29 days, with median survival of 27.5 days. The myelotoxicity was more rapid.

With respect to the group of mice which received 40μCi of the ⁹⁰Y radiolabelled conjugates, these had a survival period of from 21 to 48 days, with a median of 40.5 days. One mouse survived for 61 days post treatment.

In contrast, mice which received the radioconjugate of A33 only survived for 27 to 34 days, with a median survival time of 30 days. These mice also experienced rapid weight loss and, upon dissection and examination, discoloration of the gastrointestinal tract indicated radiotoxicity.

EXAMPLE 5

Myelosuppression is a common occurrence following radio therapy. White cell and platelet counts, measured approximately two weeks after treatment to study whether or not this had occurred. With respect to the group which received the ⁹⁰Y HRS-3 conjugates, white cell and platelet levels recovered, approaching normal levels within two weeks. In contrast, the mice treated with ⁹⁰Y- A33 conjugates experienced more rapid myelosuppression, and did not survive. It is believed that disseminated tumor deposits take up the ⁹⁰Y-HR-S3 radioconjugates, inhibiting growth of tumor cells. This is reflected in a 25% survival rate after 70 days. The radiolabelled control antibody cannot bind to tumor cells, and as a result bone marrow is subjected to greater, lethal radiotoxicity.

EXAMPLE 6

The results set forth supra showed that a single dose of 18.4 μg of unlabelled HRS-3 antibody did not have a significant effect on the survival of the mice. A second set of experiments was carried out, in which a group of mice each received 13.6^g of unlabelled, anti-CD 30 antibody, at 1 week intervals.

The mice has a longer period of survival compared to buffer control, or mice given a single inj ection of antibody. Mice which received the two inj ections survived for anywhere from 19 to beyond 75 days, with median survival time of 49 days, as compared to 41 days for buffer control, and mice which received a single dose of antibody. When the log rank test was used to evaluate survival, the P value was 0.0369, which suggested a significant survival advantage over control mice.

EXAMPLE 7

In view of the results in the preceding examples, studies were carried out to determine the effect of the conjugates on cell morphology. L540 cells were treated with nutrient media only for 3 days, and were viable, as demonstrated by the lack of trypan blue uptake. These served as a control.

Cells were then treated with 0.2μg of HRS-3 antibody alone. These cells were viable, for the most part, with some dead cells taking up the dye.

Cells which were treated with ⁹⁰Y-conjugated HRS-3 were more affected, with pronounced profusions.

Cells treated with ⁹⁰Y labelled A33 antibodies were normal, i.e., they exhibited the same pattern as did the cells treated with medium only. Membrane profusions have been shown in, e.g., antibodies binding to Fas antigen

(Quirk, et al, Endocrinology 138:4558-66(1997), and the process itself, referred to as

"blebbing" or "zerosis," is considered to be a hallmark of apoptosis (Stevens, et al, Infect

& Immun.64:2687-94 (1996)), suggesting that these processes are involved in the action of the ⁹⁰ Y labelled antibody.

The foregoing examples describe various features of the invention, which include, inter alia, CD30 specific antibodies containing immunoconjugates, where the immunoconjugate is labelled with a metallic radionuclide. "Antibody" as used herein, not only refers to whole antibodies, such as murine antibodies and antibodies secured from species including humans, sheep, rabbits, etc., but also fragments of the antibodies, as long as these are binding fragments, i.e., portions of the antibodies which are able to bind with CD30. Such fragments include Fab, Fab, Fab(ab)_2j and scFv fragments. Also a part of "antibody" as used herein are structures such as diabodies, oligo - and multimeric antibodies, synthetic proteins designed to simulate CD30 specific antibodies and so forth. Polyclonal, monoclonal, humanized chimeric and other forms of antibody are all considered part of the invention.

The metallic radionuclides of the invention may be attached to the antibody directly or, more preferably, via a linker, such as a chelating molecule, i.e., CHX-A"- DTPA. Depending upon the nature of the chelator or the label, the site of attachment will vary. For example, in the case of CHX-A"-DTPA, attachment is via a lysine residue on the antibody molecule.

The metallic radionuclide itself may vary in its properties and may be, for example, an alpha emitter, a beta emitter, and/or a low energy electron emitter. Exemplary of the type of metallic radionuclides encompassed by the invention are ^ιuln, ⁹⁰Y, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²⁵Ac, ¹⁷⁷Lu, ¹⁸⁸Re, ⁶⁸Ga, and ^99mTc, although ^nlIn and ⁹⁰Y are preferred. Other examples of useful metallic radionuclides will be clear to the skilled artisan.

The immunoconjugates may be used in various diagnostic and therapeutic applications. For example, the uptake of the metallic radionuclide labelled antibodies suggests that imaging studies, or other forms of in vivo assays, can be used to determine if CD30 is being expressed, the degree to which it is being expressed, and whether the level of expression has changed with reference to a previous measurement. Of course, the antibodies can be used in standard in vitro immunoassays for determining presence, amount, and/or a change in status of CD30, expression, as discussed in connection with in vivo assays, supra.

The immunoconjugates of the invention have therapeutic efficacy as well. Volkert, et al, J. Nucl. Med 32:174-185 (1991), the disclosure of which is incorporated by reference, teaches that there are various therapeutic isotopes which release, e.g., alpha, beta, or gamma emissions, Auger electrons, or Coster-Kroning electrons. As the CD30 molecule is associated with Hodgkin's Disease, as described supra, the assays and therapeutic methodologies described herein are especially useful for the diagnosis, monitoring, and treatment of Hodgkin's Disease.

The dose used will vary; however as was seen supra, a dose range from about 1.0 to about 2.5 Ci kg, more preferably about 1.5 to about 2.0 Ci/kg, and most preferably, from about 1.7 to about 1.δ Ci kg is preferred.

The mode of administration may vary. Systemic administration such as via intravenous administration is preferred, but other forms of administration such as in situ administration are also a part of the invention.

Bolus administration, as well as multiple administration, etc, can all be used in the therapeutic modalities associated with the invention.

Other features of the invention will be clear to the skilled artisan and need not be set forth here.

It will be understood that the specification and examples are illustrative but not limitative of the present invention and that other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.

Claims

WE CLAIM:

1. An immunoconjugate comprising an antibody which specifically binds to a CD30 molecule, or a binding fragment of said antibody, and a metallic radionuclide.

2. The immunoconjugate of claim 1, wherein said antibody is a monoclonal antibody.

3. The immunoconjugate of claim 1, wherein said metallic radionuclide is conjugated to said antibody or binding fragment via a lysine residue of said antibody or binding fragment.

4. The immunoconjugate of claim 1, wherein said metallic radionuclide is an alpha emitter, a beta emitter, a gamma emitter, a positron emitter or a low energy electron emitter.

5. The immunoconjugate of claim 1, wherein said metallic radionuclide is conjugated to said antibody or binding fragment via a linker.

6. The immunoconjugate of claim 5, wherein said linker is a metal ion chelator.

7. The immunoconjugate of claim 6, wherein said metal ion chelator is c- fimctionalized trans cyclohexyl diethylene triamine pentaacetic acid (CHX-A"- DTPA).

8. The immunoconjugate of claim 1, wherein said metallic radionuclide is ^nιIn,⁹⁰Y, ²¹²Bi, ²¹³Bi, ²ⁿAt, ²²⁵Ac, ¹⁷⁷Lu, ¹⁸⁸Re, ⁶⁷Ga, ^99mTc, ⁶⁸ Ga, or ⁸⁹ Zr.

9. The immunoconjugate of claim 8, wherein said metallic radionuclide is ^mIn.

10. The immunoconjugate of claim 8, wherein said metallic radionuclide is ⁹⁰Y .

11. A method for determining a tumor which expresses CD30 on surface of its cells, comprising contacting said tumor with an amount of the immunoconjugate of claim 1, and determining radio emission from said immunoconjugate as a determination of said tumor.

12. The method of claim 11 , wherein said immunoconjugate comprises a monoclonal antibody.

13. The method of claim 11 , wherein said immunoconjugate comprises ^π 'In.

14. The method of claim 11 , wherein said immunoconjugate comprises ⁹⁰Y.

15. A method for treating a subj ect with a tumor that expresses CD30 on surface of its cells, comprising administering to a subject in need thereof a therapeutically effective amount of the immunoconjugate of claim 1.

16. The method of claim 15, wherein said immunoconjugate is administered systemically.

17. The method of claim 15, wherein said immunoconjugate is administered intravenously.

18. The method of claim 15, wherein said metallic radionuclide is an alpha emitter, a beta emitter, a gamma emitter, a positron emitter, or a low energy electron ion emmiter.

19. The method of claim 15, wherein said metallic radionuclide is ' ' 'In.

20. The method of claim 15, wherein said metallic radionuclide is ⁹⁰Y.