WO2007079953A2

WO2007079953A2 - In vitro method to predict the tumor sensitivity to pharmacotherapy and endoradiotherapy

Info

Publication number: WO2007079953A2
Application number: PCT/EP2006/012424
Authority: WO
Inventors: Samuel Samnick; Andreas Kluge
Original assignee: Samuel Samnick; Andreas Kluge
Priority date: 2005-12-21
Filing date: 2006-12-21
Publication date: 2007-07-19
Also published as: WO2007079953A3

Abstract

The present invention provides a method for the prediction of the sensitivity of malignant cells or malignant tissues to a cancer therapy which comprises the administration of a L-amino acid, conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 , wherein the L-amino acid is selected from the group consisting of phenylalanine, alanine, serine and cysteine. Moreover, the invention provides a pharmaceutical composition for an individual treatment of a malignant neoplasia and a kit for the prediction of the sensitivity of malignant cells or malignant tissues of an individual patient for such treatment. The invention also provides a use of one or more L-amino acid(s) selected from the group consisting of phenylalanine, alanine, serine and cysteine, conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 , for the preparation of a pharmaceutical composition for an individual patient for the treatment of malignant neoplasia, wherein for the selected one or more L-amino acid(s) a sensitivity of the malignant cells or malignant tissues from the individual patient is predicted by the method of the invention and a method for the individual treatment of a patient suffering from malignant neoplasia.

Description

In vitro method to predict the tumor sensitivity to pharmacotherapy and endoradiotherapy

The present invention provides a method for the prediction of the sensitivity of malignant cells or malignant tissue(s) to a cancer therapy which comprises the administration of a L-amino acid, conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 , wherein the L-amino acid is selected from the group consisting of phenylalanine, alanine, serine and cysteine. Moreover, the invention provides a pharmaceutical composition for an individual treatment of a malignant neoplasia and a kit for the prediction of the sensitivity of malignant cells or malignant tissues of an individual patient for such treatment. The invention also provides a use of one or more L-amino acids selected from the group consisting of phenylalanine, alanine, serine and cysteine, conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 , for the preparation of a pharmaceutical composition for an individual patient for the treatment of malignant neoplasia, wherein for the selected one or more L-amino acid(s) a sensitivity of the malignant cells or malignant tissues from the individual patient is predicted by the method of the invention and a method for the individual treatment of a patient suffering from malignant neoplasia.

A variety of documents is cited throughout this specification. The disclosure content of said documents including manufacturer's manuals is herewith incorporated by reference in its entirety.

Advanced malignant neoplasias are characterized by a locally infiltrative disease and the formation of one or multiple microscopic or macroscopic local or distant metastases. It is known in the art for such neoplasias that a concomitant phenomenon is the development of resistance to conventional chemotherapy regimens. Examples are recurrent malignant glioma, advanced breast cancer, advanced ovarian cancer, advanced prostate cancer, advanced malignant melanoma, or multiple myeloma. These neoplasias are typically not amenable to curative local treatment, such as e.g. surgery or local radiation therapy, but instead require the systemic administration of therapeutic agents, typically chemotherapeutic regimens. Even the use of chemotherapeutics, containing a combination of different agents in order to impede the development of chemoresistance to single agents and to optimize the tolerability of the usually highly toxic regimens to patients fails in a significant number of cases.

Established first line chemotherapy regimens for advanced neoplasias induce in some tumor entities complete remission rates of up to 10 - 20 % [1-2]. In contrast, the response rates of repeatedly recurrent disease are much lower due to the development of inducible chemoresistance or selection of chemoresistant mutants. Other tumors, such as malignant gliomas are primarily resistant to most chemotherapeutic agents, due to either pharmacokinetic (e.g. no penetrance of the blood brain barrier) or intrinsic chemoresistance. Diminished chemosensitivity may be mediated by inducible cellular detoxification mechanisms, such as PgP, MDR gene products and others.

Chemotherapy regimens used to treat advanced stage cancers include second line alkylating agents such as melphalan, platinum-containing compounds, topoisomerase inhibitors, or antimetabolites, are associated with extremely toxic effects on bone marrow and other organs, limiting therapeutic or palliative administration [3-5].

A series of amino acid derivatives have been tested for their antineoplastic activities in antitumor screens [6-11]. They include the halobenzoyl-DL-phenylalanines, N- chloroacetyl derivatives of para-substituted phenylalanines, N-benzoyl- fluorophenylalanine, p-chloro-DL-phenylalanine, α-methyl-phenylalanine, N-ethylcarb- aminomethyl-L-isoleicine and N-propionyl-L-valine, to name only some. However, the administration of all compounds from said group is also known to be associated with extremely toxic side effects on bone marrow and other organs, limiting therapeutic or palliative administration. For example it has been known in the art since 1974 that p- chlorophenylalanine interferes with the growth of developing rats [12] which disqualifies this class of compounds as a potential compound for the treatment of malignant diseases with an acceptable toxicity profile. The effective doses of the compounds described in corresponding document was relatively high. For example, in [11] it was described that a dose of 2.5 - 10 mmol/l of p-chloro-phenylalanine (4- chloro-phenylalanine) was necessary to demonstrate a cytotoxic effect of the compound on murine neuroblasts. Moreover, for some of the tested compounds the efficiency of an elimination of malignant cells or a reduction of the cell proliferation differed between samples derived from different subjects. Accordingly, apart from the limitations of the effectiveness of the tested compound it could not be predicted whether such compound may be effective in the treatment of an individual patient prior to such treatment which may be accompanied with serious side effects resulting in a significant reduction of the quality of life of the patient.

Thus, the technical problem underlying the present invention is to provide means and methods for an improved treatment of malignant neoplasias of individual patients. The solution to this technical problem is achieved by the embodiments characterized in the claims.

Accordingly, the present invention relates to a method for the prediction of the sensitivity of malignant cells or malignant tissue(s) to a cancer therapy comprising the steps (a) administration of a labeled L-amino acid, wherein the L-amino acid is selected from the group consisting of phenylalanine, alanine, serine and cysteine to a sample of the malignant cells or malignant tissue(s) from a patient and to a sample of control cells or control tissue(s) from a healthy donor and/or to a sample of diseased control cells or tissue(s), or alternatively to a positive and/or negative reference standard cell system, and (b) analyzing the functional activity level of an amino acid transporter protein in said samples, wherein the transporter is the L-type amino acid transporter 1 (LAT1) or the alanine-serine-cysteine transporter 2 (ASCT2), and wherein an increased functional activity level of at least one of the transporter proteins in the sample of the malignant cells or malignant tissue(s) compared to the one in the control cells or control tissue(s) is indicative for a sensitivity of the malignant cells or malignant tissue(s) for the therapy.

In a preferred embodiment the labeled L-amino acid is an L-amino acid conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine- 131 and astatine-211.

The present invention also relates to a method for the prediction of the sensitivity of malignant cells or malignant tissue(s) to a cancer therapy which comprises the administration of a L-amino acid, conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 , wherein the L-amino acid is selected from the group consisting of phenylalanine, alanine, serine and cysteine, the method comprising the step (a) analyzing the level of functional activity (i.e. the functional activity level) of an amino acid transporter protein in a sample of the malignant cells or malignant tissue(s) from a patient and in a sample of control cells or control tissue(s) from a healthy donor and/or in a sample of diseased control cells or tissue(s), or alternatively in a positive and/or negative reference standard cell system, wherein the transporter protein is preferably the L-type amino acid transporter 1 (LAT1) or the alanine-serine-cysteine transporter 2 (ASCT2), and wherein an increased functional activity level of at least one of the transporter proteins in the sample of the malignant cells or malignant tissue(s) compared to the one in the control cells or control tissue(s) is indicative for a sensitivity of the malignant cells or malignant tissue(s) for the therapy.

The term "malignant neoplasia" describes in the context of the present invention a cancer, carcinoma, sarcoma, or other tumor, characterised by progressive, uncontrolled, invasive and/or metastatic growth. A malignant neoplasia leads invariably to death if not treated. Malignant cells and malignant tissues are understood in the context of the present invention as the cells and tissues characteristic for a malignant neoplasia.

It is preferred that the malignant neoplasia is selected from a group consisting of malignant glioma, multiple myeloma, malignant melanoma, prostatic and breast cancer. More preferably, the glioma is selected from the group consisting of glioblastoma multiforme, anaplastic astrozytoma, astrooligodendroglioma and oligoastrozytoma.

The term "alpha-, beta- or Auger-electron emitting isotope" defines in the context of the present invention radioactive isotopes, characterized by the emission of different particles (rays) formed during radioactive decay or by nuclear transition processes. An alpha emitting isotope is defined as a radioactive nuclide emitting alpha particles, corresponding to a helium nucleus consisting of two protons and two neutrons. A beta emitting isotope is defined as a nuclide emitting fast nuclear electrons (negatrons) formed during radioactive decay. An Auger-electron emitting isotope is defined as a nuclide emitting low energy nuclear electrons, formed by nuclear electron capture or internal transition processes. The maximum path lengths of these particles are in a range from 10 nm to 12 mm. The term "functional activity of an amino acid transporter protein" is defined in the context of this invention as the ability of said amino acid transporter protein to transport amino acids from the extracellular space into the cell, resulting into an increased intracellular concentration, and a decreased extracellular concentration, and consequently leading to an intracellular enrichment of said amino acid. The term " functional activity level of amino acid transporter protein" is defined in the context of this invention as the actual transport rate per unit of time of said amino acid transporter protein to transport amino acids from the extracellular space into the cell, resulting into an increased intracellular concentration.

The term "control cells or control tissues from a healthy donor" is defined in the context of this invention as cell or tissue samples derived from the same organ, from which the cancer test sample is derived, but stemming from a subject who has no cancer (e.g. for a prostatic cancer test sample, control cells or tissue will consist of healthy, non-diseased prostate epithelial cells or a cell preparation obtained by digestion of a prostate tissue sample comprising various other cell types, present in the prostate, in addition to prostate epithelial cells, from which prostate cancer is derived histologically).

The term "sample of diseased control cells or tissues" is defined in the context of this invention as cell or tissue samples derived from a patient with confirmed diagnosis of cancer of the same organ, from which the cancer test sample is derived, and for which the presence of an increased functional activity level of amino acid transporter protein, when compared to normal control tissue, has been characterized beforehand. The term "positive and/or negative reference standard cell system " is defined in the context of this invention as a cell line, which has been characterised with regard to the functional activity level of amino acid transporter protein beforehand, and whereas a negative reference standard cell system is a cell line, having low or minimal functional activity levels of amino acid transporter protein, as is found in healthy tissue, and whereas a positive reference standard cell system is a cell line, having increased functional activity levels of amino acid transporter protein, as found in tumor cells, but not in healthy cells. Reference standard cell systems, in contrast to control cells or control tissues form healthy donors and/or patient donors, do not necessarily need to be derived from the same organ as the test specimen. Instead, absolute values of functional activity levels of amino acid transporter protein, in test specimen and reference standards are compared.

The term labeled L-amino acid describes in the context of the present invention an L- amino acid conjugated for example to a radioactive isotope, a fluorescent dye or a chemoluminescent dye. A fluorescent dye or chemoluminescent dye may be conjugated to the L-amino acids according to standard protocols known in the art.

An example for a conjugated amino acid is represented for a phenylalanine derivative as presented in the general formula I:

General formula I in which,

X is bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 or astatine-211 linked to L-phenylalanine at the 3- (meta-) or 4- (para-) position within the aromatic ring. Ri is H, alkyl group, amino acid, peptide, protein or an other residue known to facilitate or improve tumor targeting. R₂ is OH₁ amino acid, or an other residue known to facilitate or improve tumor targeting. Preferred conjugates according to the formula I are those in which X is a bromine-77, bromine-82, iodine-125, iodine-131 , or astatine-211 linked to L-phenylalanine at the para- position of the aryl group, while Ri is H and R2 is OH.

According to the physical half life of the radionuclide conjugated to the L- phenylalanine, also the conjugates have a corresponding half life of 16.2 h for bromine-76, 57.04 h for bromine-77, 35.3 h for bromine-82, 13.27 h for iodine-123, 4.17 d for iodine-124, 59.41 d for iodine-125, 8.02 d for iodine-131 or 7.21 h astatine- 211 labelled L-phenylalanine, respectively. As described in more detail in the appended examples, the halogen isotope may e.g. be conjugated following a protocol for a "non-carrier-added" (n.c.a.) conjugation as well as following a protocol for a "carrier-added" (c.a.) conjugation; see e.g. appended example 1.

Conjugates of alanine, serine and cysteine with an alpha-, beta- or Auger-electron emitting isotope may be prepared in analogy to the exemplified protocol.

The identified amino acid transporter genes encode transmembrane proteins. The L- type amino acid transporter 1 (LAT1) is also known in the art as CD98 light chain, 4F2 light chain or Integral membrane protein E16; see GeneBank accession number: NM_003486. The nucleic acid sequence of a LAT1 is depicted in SEQ ID NO:1 , the amino acid sequence of the transporter protein encoded by the LAT1 gene is depicted in SEQ ID NO: 2. The alanine-serine-cysteine transporter 2 (ASCT2) is also known in the art as neutral amino acid transporter 2, Sodium-dependent neutral amino acid transporter type 2 or ATBO; see GeneBank accession number: NM_005628. The nucleic acid sequence of a ASCT2 is depicted in SEQ ID NO:3, the amino acid sequence of the transporter protein encoded by the ASCT2 gene is depicted in SEQ ID NO: 4. Methods for the analysis of the expression level of a gene in a cell are known in the art. Some embodiments of corresponding methods are exemplified herein below. It is known in the art, however, that protein function can be regulated and modulated at the post-translational level (i.e. in mature fully functional proteins, localized in the physiological cellular location for such a protein, e.g. a fully functional mature amino acid transporter protein localized in a transmembranal position) by a variety of physiological stimuli, such as intracellular pH (Anderson CM, et al.: Indirect regulation of the intestinal H+-coupled amino acid transporter hPAT1 (SLC36A1); J. Cell Physiol. 2005; 204:604-13), or ICAM 1 (Liu X, et al. : CD98 and intracellular adhesion molecule I regulate the activity of amino acid transporter LAT-2 in polarised intestinal epithelia: J. Biol. Chem. 2003; 278: 23672-7). For the present invention, therefore, the functional activity of amino acid transport via the specified transporter proteins is quantitatively or qualitatively measured in order to predict tumor cell sensitivity to treatment with amino acid derivatives specified in this invention, rather than measuring the expression level of amino acid transporter protein or RNA transcripts, using standard methodologies.

Functional activity of the LAT-1 amino acid transporter has been studied in an array of various cancer cell lines, by measuring internalisation of radiolabeled L-phenylalanine derivatives as described herein. Cell lines, actively taking up said amino acid derivatives, but not cell lines, not taking up said amino acid derivatives were susceptible to cellular killing by endoradiotherapy using the same radiolabeled amino acid both in vitro, and in vivo, as demonstrated by experiments using human glioblastoma cell lines in RNU rats.

The recited cancer therapy, therefore, comprises the administration of one or more selected amino acid conjugates, e.g. in form of a pharmaceutical composition. The one or more selected amino acid conjugates are an active ingredient of the pharmaceutical composition which act as an endoradiotherapeutic agent and, optionally, also as a pharmacotherapeutic agent.

The term "endoradiotherapeutic agent" defines in the context of the present invention an agent which comprises at least one type of radioactive isotope. Such agent is to be administered to a subject in the need thereof and is effective in the therapy of the above described malignant neoplasia due to an endogenic irradiation, i.e. an irradiation with the radioactive compound within the body of the subject to be treated by the endoradiotherapy. The efficiency of a treatment with an endoradiotherapeutic agent depends inter alia on the specific incorporation and the enrichment of the agent in the malignant cells or tissues of an individual patient. The term "pharmacotherapeutic agent" defines, in contrast to an endoradiotherapeutic agent, in the context of the present invention an agent which exerts a pharmacological action in cells or tissues, by virtue of its chemical properties. The chemical properties of the substance may exert pharmacological effects by interfering with cell metabolism (e.g. antimetabolites), cellular signal transduction (e.g. ion channel blockers, receptor ligands inducing or antagonising the effect of a physiological endogenous receptor ligand) or intercellular signalling (e.g. hormone antagonists). The efficiency of a treatment with an pharmacotherapeutic agent also depends inter alia on the specific incorporation and the enrichment of the agent in the malignant cells or tissues of an individual patient. Preferably, the pharmacotherapeutic agent is the portion of nonradioactive conjugates comprised in a preparation of conjugates prepared according to a protocol for carrier added conjugation.

It is further preferred that the one or more amino acid conjugate(s) have on the malignant cells or malignant tissues of the neoplasia a radiosensitizing effect, a cytostatic effect and/or an effect to revert an acquired or constitutive state of cellular resistance to chemotherapy or radiotherapy.

The term "cytostatic effect" describes in the context of the present invention the capacity of a compound to slow down or to arrest the cell proliferation of malignant cells. The term "radiosensitizing effect" describes in the context of the present invention the capacity of a compound to enhance the therapeutic response to concomitantly administered radiation therapy, corresponding to the induction of an increased response to a given radiation dose administered in the presence of the radiosensitizing compound, compared to the response induced by the same radiation dose in the absence of the radiosensitizing compound, or alternatively the selective induction of the sensitivity of neoplastic cells for a radiotherapy, not present in the absence of the compound.

The term "an effect to revert an acquired or constitutive state of cellular resistance to chemotherapy or radiotherapy" describes in the context of the present invention the capacity of a compound to convert or to reconvert the cellular sensitivity for a chemotherapy or a radiotherapy.

The sample of the patient may be obtained by removal of tumor during a surgical operation, or by bioptic procedures. The same holds true for the control samples obtained from healthy cells or tissues of the patient or a healthy donor. These samples may be used as a negative control. The term "diseased control cells or tissues" defines in the context of the present invention cells or tissues isolated from a patient suffering from a malignant neoplasia identified as being sensitive for the treatment with one ore more of the recited conjugated L-amino acids. These samples may be used as a positive control for the sensitivity for said selected L-amino acids.

It has been surprisingly found that certain amino acids, but not others, are taken up by tumor cells or tissue and cells with high avidity, as compared to normal tissue of the same organ. Such uptake of the specific amino acids results in an enrichment of the amino acids in the tumor cells or tissue. Further analysis revealed that this observation is based on a pathological increase of the expression of amino acid transporter genes. More importantly, however, amino acid transporter proteins, can be regulated at the posttranslational level by various physiological and pharmacological stimuli, as has been shown for hPAT and LAT-2, which are referenced in this invention. Therefore, it was concluded that the in vitro measurement of increased transport of certain amino acids into tumor cells can be used to predict the uptake of amino acid based antineoplastic compounds into tumor cells. Moreover, the actual functional state of amino acid transporter gene products (i.e. the functional proteins), rather than measuring expression of amino acid transporter genes at the RNA or protein level, may be measured according to alternative approaches as described in more detail herein below. The prediction of the efficiency of a specific compound in the treatment of a malignant neoplasia of an individual patient allows an individual therapy optimisation (individualised medicine) and thereby minimising any side effects of a therapy approach, and moreover to recognise inefficient therapies for individual patients before such a therapy is initiated, thereby preventing any side effects of a therapy approach. Accordingly, it can be prevented that after a phase of a therapy with significant side effects it turns out that such therapy was not effective or less effective than a possible alternative approach.

At least some of the above identified amino acid conjugates are known in the art as being useful in the treatment or the diagnosis of cancer for some individual patients. However, there was no suggestion in the art for a general explanation of the success of such treatment of one patient (sensitivity of the malignant cells or malignant tissues of the patient for the therapy) and the failure or reduced success in the treatment of an other patient (lack of a sensitivity of the malignant cells or malignant tissues of the patient for the therapy). The sensitivity of malignant cells or malignant tissues of an individual patient is determined by the characteristics of said cells and tissues which comprise e.g. the upregulation of the expression of specific genes, whereas the expression of other genes is suppressed or downregulated. More importantly, however, the functional activity state of the specified amino acid transporter proteins has been found to be crucial for the prediction of therapeutic sensitivity. The specific functional activity pattern of said amino acid transporter proteins may be different for malignant cells or malignant tissues of individual patients. Thus, the present invention provides a method which allows a prediction of the efficiency of an individual therapy for an individual patient.

Generally, it is preferred that the L-amino acid employed in the method of the invention for the prediction of the sensitivity of malignant cells or malignant tissue(s) to a cancer therapy is the same as the L-amino acid used in the subsequent cancer therapy. It is further preferred that the same radioactive isotope conjugated to the L- amino acid is used in the prediction of the sensitivity of malignant cells or malignant tissue(s) to a cancer therapy and in the subsequent cancer therapy. Also preferred is the use of an L-amino acid conjugated to a radioactive isotope in the prediction of the sensitivity of malignant cells or malignant tissue(s) to a cancer therapy and the use of the same L-amino acid conjugated to an alternative radioactive isotope for the use in the subsequent cancer therapy.

In a preferred embodiment of the method of the invention the conjugated L-amino acid is a 4-[¹³¹l]iodo-L-phenylalanine (IPA-131), 4-[¹²⁴l]iodo-L-phenylalanine (IPA-124) and/or 4-[²¹¹At]astatine-L-phenylalanine (AtPA-211).

In a further preferred embodiment of the method of the invention the cancer therapy has to comprise the administration of a conjugated phenylalanine which is 4-[¹³¹l]iodo- L-phenylalanine (IPA-131), 4-[¹²⁴l]iodo-L-phenylalanine (IPA-124) and/or 4- [²¹¹At]astatine-L-phenylalanine (AtPA-211) to a patient. It is also preferred that the functional activity level of the amino acid transporter gene in the samples is analyzed using a whole cell assay in which the tumor specimen is exposed to a radiolabeled or fluorescent-labelled amino acid.

The term "whole cell assay" describes in accordance with the invention a group of assays which allow the analysis of the functional activity state of a protein, in a sample wherein said functional activity state is analyzed in a sample of whole cells. Such method may comprise the separation of single cells from a tissue. In accordance with the invention such whole cell assay may comprise the incubation of cells with a compound, which is specifically transported into the cells by the amino acid transporters or which specifically detects the transport activity using surrogate physiological parameters.

It is also preferred that prior to conducting the assay for functional activity of the specified amino acid transporter proteins, the expression level of the amino acid transporter gene in the samples is analyzed in a RNA assay, a protein assay or in a whole cell assay. The term "RNA assay" describes in accordance with the invention a group of assays which allow the analysis of the expression of a gene in a sample on RNA level. Accordingly, such method may comprise a preparation of RNA from the sample of the cells or tissues. Such assay may comprise e.g. a RT-PCR, RNase protection assay or a hybridization assay. Examples for a hybridization assay comprise an expression analysis by Northern-blot. The term "protein assay" describes in accordance with the invention a group of assays which allow the analysis of the expression of a gene in a sample on protein level (gene product level). Such method may comprise the lysis of the cells or tissues. Moreover, such assay may comprise a quantitative and/or qualitative analysis of the presence of the transporter protein encoded by the recited transporter genes in a sample. Such assays may comprise the use of antibodies or antibody fragments or derivatives thereof which specifically bind to/detect said transporter proteins. The specific binding to/detection of said transporter proteins by an antibody, antibody fragment or derivative thereof is understood to define a "specific recognition". The term "specifically recognizing" means in accordance with this invention that the antibody molecule is capable of specifically detecting and/or binding to at least two amino acids of each of the transporter proteins as defined herein. Said binding may be exemplified by the specificity of a "key-lock-principle". Thus, specific motifs in the amino acid sequence of the antigen-interaction-site of the antibody, antibody fragment or derivative thereof and the antigen (transporter proteins) bind to each other as a result of their primary, secondary or tertiary structure as well as the result of secondary modifications of said structure. The specific interaction of the antigen- interaction-site with its specific antigen may result as well in a simple binding of said site to the antigen.

The term "binding to/detecting" may also relate to a conformational epitope, a structural epitope or a discontinuous epitope consisting of two regions of the human target molecules or parts thereof. In context of this invention, a conformational epitope is defined by two or more discrete amino acid sequences separated in the primary sequence which come together on the surface of the molecule when the polypeptide folds to the native protein (SeIa, (1969) Science 166, 1365 and Laver, (1990) Cell 61 , 553-6).

The term "discontinuous epitope" means in context of the invention non-linear epitopes that are assembled from residues from distant portions of the polypeptide chain. These residues come together on the surface of the molecule when the polypeptide chain folds into a three-dimensional structure to constitute a conformational/structural epitope.

The term "antibody fragments and derivatives" is defined in detail herein below. The person skilled in the art is aware of methods for the production of antibodies with specificity of a known protein (antigen) and methods for the detection of said proteins; see e.g. Harlow et al. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988) or Wild, The Immunoassay Handbook, Elsevier Science Publishing Company (2005) .

In a preferred embodiment of the method of the invention the RNA assay comprises a RT-PCR or a hybridization assay. More preferably, the RT-PCR or the hybridization assay comprises the use of primers or probes derived from the nucleic acid sequence of the LAT1 as depicted in SEQ ID NO: 1 or the nucleic acid sequence of the ASCT2 as depicted in SEQ ID NO: 3 or the complementary strands thereof. Methods for the determination of suitable sequences for a primer or probe derived from a known nucleic acid sequence represent standard procedures for the person skilled in the art; see e.g. Sambrook et al. Molecular Cloning: A Laboratory Manual, 3 Vol., Cold Spring Harbor Laboratory Press (2001) or Ausubel et al. Short Protocols in Molecular Biology, 2 VoIs., John Wiley & Sons lnc (2003). Specific conditions for a RT-PCR or a hybridization assay are known to the person skilled in the art. The same holds true for modifications of such routine procedures since the parameters which may be modified and the potential effects of such modifications are known in the art. In line with the invention it is preferred that the hybridization conditions in a hybridization assay are high stringent; see Sambrook et al., loc cit. or Ausubel et al., loc cit.

In an also preferred embodiment of the method of the invention the protein assay or the whole cell assay comprises the use of an antibody specifically binding to/detecting LAT1 having an amino acid sequence as depicted in SEQ ID NO: 2 or the ASCT2 having an amino acid sequence as depicted in SEQ ID NO: 4. As described herein above, methods for the preparation and/or election of an antibody, antibody fragment or a derivative thereof, which specifically binds to/detect a protein are known to the person skilled in the art. Preferably, the whole cell assay according to the invention comprises a flowcytometric analysis. Protocols for a flowcytometric analysis of the expression of a gene product are also known to the person skilled in the art.

In an also preferred embodiment of the method of the invention the whole cell assay comprises an incubation of the malignant cells or malignant tissue and of the control cells or control with a labeled phenylalanine for an analysis of the LAT1 expression or a labeled alanine, serine or cysteine for an analysis of the ASCT2. Preferably, the phenylalanine, alanine, serine and/or cysteine is/are labeled with a radioactive isotope, a fluorescent dye or chemoluminescent dye. The amino acids may be radioactively labeled following a procedure as exemplified herein above for L- phenylalanine. A possible experimental approach for such whole cell assay is described in the appended example 2. A fluorescent dye or chemoluminescent dye may be conjugated to the amino acids according to standard protocols known in the art. Moreover, methods and means for a read out of an uptake of the amino acid(s) into the cells are known in the art and comprise e.g. fluorescence microscopy or flowcytometry.

It is preferred that in a whole cell assay according to the invention the cells or tissues are incubated with the labeled compound prior to the step of analyzing the expression for 1 to 48 h.

In an alternative embodiment the invention provides a pharmaceutical composition for an individual treatment of a malignant neoplasia comprising one or more L-amino acid(s), selected from the group consisting of phenylalanine, alanine, serine and cysteine, conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine- 124, iodine-125, iodine-131 and astatine-211 , for which a sensitivity of the malignant cells or malignant tissues from the individual patient is demonstrated by the use of a method for the prediction of the sensitivity of malignant cells or malignant tissues to a cancer therapy according to the invention.

In accordance with this invention, the term "pharmaceutical composition" relates to a composition for administration to a subject, preferably a human patient. The pharmaceutical composition is preferably administered orally, parenterally, transdermal^, intraluminal^, intra-arterially, intrathecal^ or intravenously. Also preferred is a direct injection of the pharmaceutical composition into malignant tissue. It is in particular envisaged that said pharmaceutical composition is administered to a patient via infusion or injection, or as a tablet or capsule. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, subcutaneous, intraperitoneal, intramuscular, topical or intradermal administration. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The individual selection of one or more amino acid derivative(s) for a treatment of a specific patient will depend on the results of an analysis of the sensitivity of the malignant cells or malignant tissues of the specific patient. Said sensitivity may be tested with a corresponding method of the invention. Preferred dosages for the administration of the identified amino acid derivatives are described herein below. The compositions may be administered locally or systemically. Administration will generally be parenteral, e.g., intravenous, or oral. In an preferred embodiment, the pharmaceutical composition is administered subcutaneously and in an even more preferred embodiment intravenously. In another preferred embodiment, the pharmaceutical composition is administered orally. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin. It is envisaged that the pharmaceutical composition might comprise, in addition to one or more amino acid derivative(s) further biologically active agents, depending on the intended use of the pharmaceutical composition in a treatment comprising the administration of additional agents for a concomitant therapy. Examples for such further biologically active agents are described herein below in the context of uses and methods comprising a concomitant therapy.

Currently, oncological disease entities are treated according to therapeutic guidelines, which are based on cohort comparisons. For example, a given therapy A would be generally recommended to be superior over a therapy B, if 60% of patients of a study cohort respond to therapy A, while 40% of the study cohort respond to therapy B. It is well known in the art, that the tumor biology of individual tumor strongly depends on the specific mutation inducing the uncontrolled tumor growth, and that considerable heterogeneity may exist with regard to the clinical behavior (pathological dignity) of two tumors, of identical histological appearance. A treatment, which is superior in 60% of patients, therefore, may be inferior in the individual tumor case. Individualized medicine, therefore, aims at identifying the individually best treatment, based on evidence generated in tumor specimen of an individual patients, rather than assuming that a treatment which is statistically superior in a group comparison, will also be the best treatment in the individual case.

Due to the presence of a radionuclide in the described conjugated amino acid derivatives the effective compound in this embodiment of the pharmaceutical composition is an endoradiotherapeutic agent. The term "endoradiotherapeutic agent" defines in the context of the present invention an agent which comprises at least one type of radioactive isotopes.

Moreover, it is preferred that the pharmaceutical composition of the invention further comprises a chemotherapeutic agent, an immunotherapeutic agent, a gene therapeutic agent, a vaccine, an antisense nucleotide therapeutic agent, an siRNA therapeutic agent and/or a (further) endoradiotherapeutic agent.

The administration of a chemotherapeutic agent, an immunotherapeutic agent, a gene therapeutic agent, a vaccine, an antisense nucleotide therapeutic agent, an siRNA therapeutic agent and/or a(n) (further) endoradiotherapeutic agent is understood as a concomitant therapy. Methods and means for such concomitant therapies are well known in the art.

The one or more amino acid derivative(s) and the additional therapeutic agent may be formulated as a single pharmaceutical composition for simultaneous administration of the effective compounds or in separate pharmaceutical compositions for sequential administration. Accordingly, an administration of a composition comprising one or more amino acid derivative(s) prior to the administration of a composition comprising one or more therapeutics selected from the group of a chemotherapeutic, an immunotherapeutic, a gene therapeutic, a vaccine, an antisense nucleotide therapeutic, an siRNA therapeutic and an endoradiotherapeutic agent is envisaged as well as simultaneous or subsequent administration.

An example for a chemotherapeutic agent comprises bioactive agents known to be effective in retarding or arresting the malignant growth or to be effective in the regression or elimination of malignant tissues or cells. Such agents might be e.g. drugs acting as cytostatics. Accordingly, a chemotherapy comprises in line with the medical standards in any systemic or local treatment the administration of cytostatic or cytotoxic agents. Chemotherapeutic agents used in oncology include among others, nitroso urea compounds (ACNU [nimustin], BCNU [carmustin], CCNU [lomustin]), temozolomid, procarbacin, metothrexate, cytarabin, gemcitabine, fluorouracil, cyclophosphamide, mitoxantron, anthracyclins, estramustin, or taxanes. The chemotherapeutic agents are intended to be administered in appropriate dosing regimens according to medical practice. In line with the invention nitroso urea compounds, temozolomide, procarbacin, and methotrexate are preferred chemotherapeutic agents.

Examples for an immunotherapeutic agent comprise but are not limited to compounds such as antibodies, antibody fragments and/or derivatives thereof which specifically detect malignant tissue or cells and/or cell with the ability to eliminate the malignant tissue or cells. The term "antibody fragment or derivative thereof relates to single chain antibodies, or fragments thereof, synthetic antibodies, antibody fragments, such as Fab, a F(ab')₂, Fv or scFv fragments, single domain antibodies etc., or a chemically modified derivative of any of these. Antibodies to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified outside the motifs using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) (e.g. posttranslational and chemical modifications, such as glycosylation and phosphorylation) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook et al.; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition 1989 and 3rd edition 2001. The specific detection of malignant tissue or cells may be effected via the detection of tumor specific markers by the antibodies, antibody fragments and/or derivatives thereof. A tumor-specific marker is a tumor-associated cell surface antigen which is either found exclusively on tumor cells or is overexpressed on tumor cells as compared to non-malignant cells. Tumor-associated cell surface antigens can be expressed not only on tumor cells but also on cells/tissue which are/is not essential for survival or which can be replenished by stem cells not expressing tumor-associated cell surface antigen. Furthermore, a tumor-associated cell surface antigen can be expressed on malignant cells and non-malignant cells but is better accessible by a therapeutic agent of interest on malignant cells. Examples of over-expressed tumor- associated cell surface antigens are Her2/neu, EGF-Receptor, Her-3 and Her-4. An example of a tumor-associated cell surface antigen which is tumor specific is EGFRV- III. An example of a tumor-associated cell surface antigen which is presented on a cell which is non-essential for survival is PSMA. Examples of tumor-associated cell surface antigens which are presented on cells which are replenished are CD19, CD20 and CD33. An example of a tumor-associated cell surface antigen which is better accessible in a malignant state than in a non-malignant state is EpCAM. Moreover, the definition of "immunotherapeutics" may comprise agents such as T-cell co-stimulatory molecules or cytokines, agents activating B-cells, NK-cells or other cells of the immune system as well as drugs inhibiting immune reactions (e.g. corticosteroids).

The term "gene therapeutic agent" defines in the context of the invention means for a therapy comprising the administration of one or more nucleic acid constructs functionally encoding e.g. one or more antigens which are characteristic for malignant cells. Such antigens comprise tumor specific markers. The sequence encoding such antigen is operably linked to a nucleic acid sequence which is a regulatory sequence. Thus, a gene therapy comprises the functional expression of a heterologous gene in a patient according to standard medical protocols using appropriate vector systems known in the art; see e.g. Haberkorn et al., Curr Med Chem. 2005;12(7):779-94. The term "regulatory sequence" refers to DNA sequences which are necessary to effect the expression of coding sequences to which they are ligated. Control sequences in the context of the described gene therapy generally include promoters, terminators and, in some instances, enhancers, transactivators or transcription factors. The term "control sequence" is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components. The term "operably linked" refers to a arrangement/configuration wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

The administration of a vaccine aims in the context of the present invention at activating the innate or adaptive immune system of the patient to act against the tumor tissue or the malignant cells. Such therapy comprises e.g. administering one or more antigen preparations containing tumor substances, or cells selected to react against tumor tissue or the malignant cells.

An antisense therapeutic agent is e.g. a nucleotide sequence being complementary to tumor-specific gene sequences, aiming at functionally neutralising tumor gene expression, and consequently inducing tumor cell death.

An siRNA therapeutic agent is e.g. a small interfering RNA capable of sequence- specifically silencing the expression and activity of various tumor-specific target genes by triggering cleavage of specific unique sequences in the mRNA transcript of the target gene and disrupting translation of the target mRNA, consequently inducing tumor cell death.

A concomitant therapy which requires the administration of one or more additional bioactive agents which is/are effective in the treatment of the malignant neoplasia may be accompanied by the administration of one or more additional compounds which minimize potential side effects of said bioactive agent(s) such as drugs acting on the gastro-intestinal system, drugs preventing hyperuricemia, and/or drugs acting on the circulatory system, e.g. on the blood pressure, known in the art. Such additional bioactive agents may be formulated in the form of the same or a separate pharmaceutical composition.

It is also preferred that the pharmaceutical composition is to be administered as a single dose once, as fractionized doses in 2 to 60 fraction doses or as continuous doses given daily until the disease progresses again, or until death of the patient. The administration of the described pharmaceutical composition results in an interception or a deceleration of the cancer development. Nevertheless, the disease may further progress after the interception or deceleration and the patient may die. Thus, it is also preferred that the conjugate is to be administered as a chronic maintenance therapy. It is preferred that a continuous dose is given daily. Dose fractionation is an established procedure in radiation therapy. By fractionating a total administered dose, improved tolerability for healthy non-target tissue, as well as an increased cytotoxic effect to tumor tissue is achieved. Repeated fractionated irradiation allows to therapeutically impact a higher percentage of cells in radiation sensitive stages of the cell cycle, compared to a one time single high dose irradiation. Therapeutic irradiation induces single and double strand breaks of DNA, which is counteracted by nuclear repair mechanisms upregulated following irradiation. It is believed, that cells undergoing DNA repair, are more susceptible to a renewed irradiation than radiation-naive cells.

Generally, it is preferred that the alpha-, beta- or Auger-electron emitting isotope is administered in doses of 10^~5 to 10^~18 g / kg body weight. More preferably, the alpha-, beta- or Auger-electron emitting isotope is administered in doses of 10^'7 to 10^"15 g / kg body weight and more preferably in doses of 10^~8 to 10^~10 g / kg body weight. It is particularly preferred that such a dose is formulated or contained in 1 to 10, preferably 2 to 5 ml of sterile solution, such as phosphate buffered saline solutions, water for injection, etc.

It is additionally preferred that the irradiation dose of the alpha-, beta- or Auger- electron emitting isotope is in the range of 0.1 to 1000 MBq/kg body weight. More preferably, the irradiation dose of the alpha-, beta- or Auger-electron emitting isotope is in the range of 10 to 400 MBq/kg body weight and more preferably the irradiation dose of the alpha-, beta- or Auger-electron emitting isotope is in the range of 20 to

120MBq/kg body weight. The administered dose is determined using an appropriate dose meter, calibrated to quantitatively measure alpha, beta or gamma radiation.

An example for an embodiment of a pharmaceutical composition for an individual patient may comprise e.g. a combination of 3-iodo-L-phenylalanine or 4-iodo-L- phenylalanine as amino acid derivatives, wherein the iodine is the stable, nonradioactive [¹²⁷l]-iodine isotope and an endoradiotherapeutic agent which is a halogenated-L-phenylalanine, wherein the halogen isotope is selected from the group of alpha-, beta- or Auger-electron emitting isotopes bromine-76, bromine-77, bromine- 82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211.

In a further preferred embodiment of the pharmaceutical composition of the invention the radioactive halogen isotopes are 4-[¹³¹l]iodo-L-phenylalanine (IPA-131), 4- [¹²⁴l]iodo-L-phenylalanine (IPA-124) and/or 4-[²¹¹At]astatine-L-phenylalanine (AtPA- 211). Iodine-131 is widely available, has a favourable half life and can be handled by most institutions licensed to apply open radionuclides. Iodine-131 allows for the convenient extracorporal therapy monitoring using a gamma camera owing to a gamma ray component, emitted in a fixed ratio relative to the therapeutic beta particle emission, which is itself not detectable extracorporeal^. Iodine-124 has a positron emission component, allowing for PET imaging, in addition to the therapeutic beta- emission. Using quantitative PET imaging, internal dosimetry measurements at an ongoing basis can be conducted for therapy planning and therapy monitoring for a period of up to 15 days following a single injection. Astatine-211 is also preferred, as it emits high energy (6.8 MeV) alpha particles, with a short path length in tissue (65μm), allowing to administer a highly cytotoxic radiation to targeted tissue, while minimising undesirable radiation effects to non-target tissue.

It is preferred for the use of the invention that the pharmaceutical composition is to be administered to a patient and that this patient is subsequently irradiated percutaneously (percutaneous radiotherapy or external field radiation therapy). Such external field radiation therapy is understood in the context of the invention as a concomitant therapy.

External field radiation therapy is typically administered as an external beam radiation stemming from, among others, radioactive cobalt-60 sources, linear accelerators, proton, neutron, or hadron beam sources. Preferably, the irradiation is started in a period of 0 to 7 days subsequent to the administration of the selected one or more compounds. More preferably, the irradiation is started in a period of 0.5 to 24 hours subsequent to the administration of the selected one or more amino acid derivatives. The concomitant radiotherapy may comprise a cumulative external irradiation of a patient in a dose of 1 to 100 Gy. A preferred range of the irradiation dose is 1 to 60 Gy. It is preferred that the external irradiation dose is administered in 1 to 60 fractional doses, more preferably in 5 to 30 fractional doses. Preferably, the fractionized doses are administered over a period of 1 to 26 weeks, more preferably over a period of 6 to 12 weeks. In accordance with the present invention, the term 'fractional dose' is to be understood to mean that the overall activity of the fractional dose adds up or essentially adds up to the cumulative external irradiation otherwise also achievable by administering one single dose.

In a further alternative embodiment the invention provides a kit for the prediction of the sensitivity of malignant cells or malignant tissues of an individual patient for a therapy comprising the administration of a phenylalanine, serine or cysteine conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 to the patient, the kit comprising one or more of the primers or probes as described herein above, antibodies as described herein above or a labeled, preferably halogenated, amino acid as described herein above. Optionally, the kit may also comprise additional compounds, auxiliary substances and/or a description of the protocol for the execution of a method for the prediction of the sensitivity of malignant cells or malignant tissues to a cancer therapy according to the invention.

Moreover, the invention provides a use of one or more L-amino acid(s) selected from the group consisting of phenylalanine, alanine, serine and cysteine, conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 , for the preparation of a pharmaceutical composition for an individual patient for the treatment of malignant neoplasia, wherein for the selected one or more compound(s) a sensitivity of the malignant cells or malignant tissues from the individual patient is predicted by the method for the prediction of the sensitivity of malignant cells or malignant tissues to a cancer therapy according to the invention.

In a further alternative embodiment the invention provides a method for the individual treatment of a patient suffering from malignant neoplasia, the method comprising the steps of:

(a) selecting one or more L-amino acid(s) selected from the group consisting of phenylalanine, alanine, serine and cysteine, conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 , wherein the L-amino acid is and

• wherein for the selected one or more L-amino acid(s) a sensitivity of the malignant cells or malignant tissues from the individual patient is demonstrated by analyzing the functional activity level of an amino acid transporter protein in a sample of the malignant cells or malignant tissues from the patient and in a sample of control cells or control tissues from a healthy donor and/or a diseased control sample,

• wherein the transporter is the L-type amino acid transporter 1 (LAT1) or the alanine-serine-cysteine transporter 2 (ASCT2), and

• wherein an increased functional activity level of at least one of the transporter proteins in the sample of the malignant cells or malignant tissues compared to the one in the control cells or control tissues is indicative for a sensitivity of the malignant cells or malignant tissues for the therapy; and (b) administering the one or more selected L-amino acid(s) to the patient.

The figures show:

Figure 1 : Uptake of [¹²³I]IPA by 5 different human glioma cell lines

Figure 2:

Uptake of [¹²³I]IPA by 2 different human malignant melanoma cell lines

Figure 3:

Uptake of [¹²³I]IPA by normal brain homogenate (rat), and normal breast homogenate (rat). For ethical reasons, no human material could be obtained The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of scope of the present invention.

Example 1 :

4-Bromo-L-phenylalanine (4-BrPA), 3-bromo-L-phenylalanine (3-BrPA), 4-iodo-L- phenylalanine (4-IPA), 4-ter.butyltinn-L-phenylalanine (4-TBSnPA), 3-ter.butyltinn-L- phenylalanine (3-TBSnPA), 4-methylsilyl-L-phenylalanine (4-Me₃SiPA) and 3- methylsilyl-L-phenylalanine (3-Mβ₃SiPA) used as starting materials (precursor) for radiolabeling were either purchased commercially or prior synthesized in analogy to the literature. Unless stated otherwise, all other chemicals and solvent were of analytical grade and obtained commercially or via our local hospital pharmacy. Sodium [¹²⁴l]iodide, sodium [¹²⁵l]iodide, sodium [¹³¹l]iodide, sodium [⁷⁷Br]bromide, sodium [⁸²Br]bromide, and sodium [²¹¹At]astate for radiolabeling was obtained in the highest obtainable radiochemical purity, generally in 0.01 N NaOH or in phosphate buffered saline (PBS) from different suppliers. HPLC purification was performed on a Hewlett Packard HPLC system consisting of a binary gradient pump (HP 1100), a Valco 6-port valve with 2500 μl loop, a variable wavelength detector (HP 1100) with a UV detection at 254 nm and a sodium iodide scintillation detector (Berthold, Wildbad, Germany), using reversed-phased column (250 x 4 mm, Nucleosil-100). The column was eluted at different flow rates in with water/ethanol/acetic acid (89:10:1 ; v/v) or PBS / ethanol (90:10; v/v).

The proposed radiolabeled phenylalanines were obtained either by non-isotopic halogen exchange (carrier-added/c.a.) or by radio-demetalation of the corresponding precursor as described in the general scheme 1 , resulting to no-carrier-added (n. c. a) products after HPLC separation.

R = 124/125/1311 _or 77/82g_r

X = m-, p-Br or m-, p-l for 77/82Br n.c.a. (m, p)-IPA-124, -IPA-125, -IPA-131 or n.c.a. (m, p)-BrPA-77 and -BrPA-82

X = 1-Bu₃Sn or R = (124/125/13Di₁ 77/82_Br

(CH₃)₃Si Or ^{21 1}At n.c.a. (m, p)-BrPA-77, -BrPA-82, -IPA-124, -IPA-125, -IPA-131 and n.c.a. (m, p)-AtPA-211

Scheme 1 : scheme of the radiosyntheses of n.c.a. IPA-124, IPA-125, IPA-131 , BrPA- 77, BrPA-82 and AtPA-211

Example 2:

4-Bromo-L-phenylalanine (4-BrPA), 3-bromo-L-phenylalanine (3-BrPA)₁ 3-iodo-L- phenylalanine (3-IPA) and 4-iodo-L-phenylalanine (IPA) used in this work were purchased commercially or prior synthesized in analogy to the literature. Unless stated otherwise, all other chemicals and solvent were of analytical grade and obtained commercially or via our local hospital pharmacy.

Cell lines and cell cultures

Five human glioma cell lines, normal brain tissue homogenate (rat), two human prostate cancer cell lines, as well as one human breast cancer cell line, normal breast tissue homogenate (rat), and two human melanoma cell line were investigated. The human glioma cell lines Tx 3868 and T 5135 (from primary human glioblastoma multiforme), and the rat C6 glioma cells were provided by the Institute of Human Genetics, University of the Saarland (Homburg, Germany). The human high-grade glioma cells, designated as A1207, M059K and U373MG, the human prostate cancer cells PC3 and DU425, the pancreatic carcinoma cell line PanC1 , the human breast cancer cell line MCF-07 (American Type Culture Collection, Rockville, MD), and the pancreatic carcinoma cell line PaCa44 (established by Dr Bulow, Mainz, Germany) were purchased commercially or provided by the oncological research laboratory of the University Medical Center of Saarland (Homburg, Germany). Cells were cultivated in RPMI-1640 medium or in Dulbecco's modified Eagle medium (sodium pyruvate- free, supplemented with L-glucose and pyridoxine), respectively, supplemented with 10 % (v/v) heat-inactivated foetal calf serum (FCS), penicillin (50 U/ml), streptomycin (50 μg/ml), and insulin (50 μg/ml, PromoCell, Heidelberg, Germany). All cells lines were maintained in appropriate flasks in a humidified incubator (5% CO₂) at 37°C. Before the experiment, subconfluent cell cultures were trypsinized with a solution of 0.05% trypsin containing 0.02% EDTA but without Ca²⁺ and Mg²⁺, and resuspended in fresh medium to various cell concentrations after counting by vital staining on a hemocytometer, depending upon the study. Cells were free of mycoplasms. Viability of the cells was > 95 %.

Example of internalisation experiments

Uptake experiments were undertaken to evaluate the affinity of the proposed L- phenylalanine derivatives for the proposed human tumors, and to assess their therapeutic activity in vitro.

All experiments were performed fourfold, simultaneously with 250000, 500000 and 10⁶ freshly prepared human tumor cells, including human malignant glioma cells, pancreatic prostatic and breast cancer cells. Before experiments, subconfluent cells were trypsinized as described above. The suspension was mixed thoroughly, transferred to a 50-ml centrifuge tube (Falcon®, Becton Dickinson, USA). Cells were centrifuged for 5 min at 200 x g; the resulting supernatant was removed and the pellet resuspended in serum-free Dulbecco^'s Mod Eagle medium and then transferred to Eppendorf tubes at concentrations of 10⁶ cells/ml for the uptake investigations. Before incubation with the corresponding radiolabeled phenylalanine, the tumor cells were preincubated for 5 min in 500 μl_ medium at 37°C in 1.5-ml Eppendorf centrifuge tubes. Aliquots of 30-50 μl_ (10⁶ - 1.5 x 10⁶ cpm) freshly prepared radiopharmaceutical were added and cells incubated at 37°C / 5% CO₂ for 1 , 2, 5, 15, 30, 60, 90 and 120 min while shaking. Uptake was stopped with 500 μl_ ice-cold PBS (pH 7.4) and an additional 3-min in an ice bath, the cells were centrifuged for 2 min at 300 x g, the supernatant removed and the pellet washed three time with ice-cold PBS. Cell pellets were counted for radioactivity together with 3 aliquots of standards on a Berthold LB951 counter. The percentage of binding of the radiopharmaceutical was calculated by the formula: (cpm cell pellet/mean cpm radioactive standards) x 100. The results were expressed either as percent of the applied dose per 10⁶ cells or as cpm/1000 cells for better comparison.

In vitro studies Figures 1 - 3 show examples of uptake kinetic of a IPA in human tumor cells. We used the radiolabeled derivative 4-[¹²³l]iodo-L-phenylalanine to facilitate quantification, using a gamma counter. As shown, IPA exhibit high uptake in human tumor cells with a continuous increase over the investigation time. This result provides evidence of the high affinity of the proposed diagnostic tracers for human tumors, including the human malignant gliomas, malignant melanoma, prostate and breast cancer, which is not paralleled by uptake into normal tissue.

References:

1. Hortobagy G₁ Cancer Control 4 ; suppl., 1997

2. O^'Day S et al., Cancer Control 9 : 31 - 38, 2002

3. Nieto Y et al. Biology of blood and marrow transplantation 11 : 297-306, 2005 4. Carlson K et al. Bone marrow transplantation 35 : 985-90, 2005

5. Gruenhagen D et al. Annals of surgery 240 : 939-47, 2004

6. Fukushima K, Toyoshima S. Antitumor activity of amino acid derivatives in the primary screening. Gann. 66(1):29-36, 1975.

7. Otani TT, Briley MR. Structure-activity relationships among substituted N-benzoyl derivatives of phenylalanine and its analogues in a microbial antitumor prescreen

III: derivatives of p-fluoro-DL-phenylalanine. J Pharm Sci. 74(1):40-43, 1985.

8. Loeffler LJ, Sajadi Z, Hall IH. Antineoplastic agents. 2. Structure-activity studies on N-protected vinyl, 1 ,2-dibromoethyl, and cyanomethyl esters of several amino acids. J Med Chem 20: 1584-1588, 1977. 9. Otani TT, Briley MR. m- And p-halobenzoyl derivatives of p-halo-DL- phenylalanine as inhibitors in a microbial antitumor prescreen. Res Commun Chem Pathol Pharmacol. 40(2): 325-8, 1983

10. Otani TT, Briley MR. The study of structure-activity relationships among substituted N-benzoyl derivatives of phenylalanine and its analogs in a microbial antitumor prescreen: II. Derivatives of m-fluoro-DL-phenylalanine. Res Commun

Chem Pathol Pharmacol 40: 321- 324, 1983.

11. Kelly CJ, Johnson TC: Effects of p-chlorophenylalanine and alpha- methylphenylalanine on amino acid uptake and protein synthesis in mouse neuroblastoma cells. Biochem J. 1978 15;174(3):931-8. 12. Prohaska JR, Wells WW, Luecke RW.: Effect of phenylalanine and p- chlorophenylalanine administration on the development of rat brain 2',3'-cyclic nucleotide 3'-phosphohydrolase. Proc Soc Exp Biol Med. 1974;147(2):566-71.

Claims

1. A method for the prediction of the sensitivity of malignant cells or malignant tissue(s) to a cancer therapy comprising the steps

(a) administration of a labeled L-amino acid, wherein the L-amino acid is selected from the group consisting of phenylalanine, alanine, serine and cysteine to a sample of the malignant cells or malignant tissue(s) from a patient and to a sample of control cells or control tissue(s) from a healthy donor and/or to a sample of diseased control cells or tissue(s), or alternatively to a positive and/or negative reference standard cell system, and

(b) analyzing the functional activity level of an amino acid transporter protein in said sample, wherein the transporter is the L-type amino acid transporter 1 (LAT1) or the alanine-serine-cysteine transporter 2 (ASCT2), and wherein an increased functional activity level of at least one of the transporter proteins in the sample of the malignant cells or malignant tissue(s) compared to the one in the control cells or control tissue(s) is indicative for a sensitivity of the malignant cells or malignant tissues for the therapy.

2. The method according to claim 1 , wherein the labeled L-amino acid is an L- amino acid conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211.

3. A method for the prediction of the sensitivity of malignant cells or malignant tissue(s) to a cancer therapy which comprises the administration of a L-amino acid, conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 , wherein the L-amino acid is selected from the group consisting of phenylalanine, alanine, serine and cysteine, the method comprising the step

(a) analyzing the functional activity level of an amino acid transporter protein in a sample of the malignant cells or malignant tissue(s) from a patient and in a sample of control cells or control tissue(s) from a healthy donor and/or in a sample of diseased control cells or tissue(s), or alternatively in a positive and/or negative reference standard cell system, wherein the transporter is the L-type amino acid transporter 1 (LAT1) or the alanine-serine-cysteine transporter 2 (ASCT2), and wherein an increased functional activity level of at least one of the transporter proteins in the sample of the malignant cells or malignant tissue(s) compared to the one in the control cells or control tissue(s) is indicative for a sensitivity of the malignant cells or malignant tissues for the therapy.

4. The method according to claims 1 to 3, wherein the conjugated L-amino acid is 4-[¹³¹l]iodo-L-phenylalanine (IPA-131), 4-[¹²⁴l]iodo-L-phenylalanine (IPA-124) and/or 4- [²¹¹At]astatine-L-phenylalanine (AtPA-211).

5. The method according to claim 1 to 3, wherein the cancer therapy has to comprise the administration of a conjugated phenylalanine which is 4-[¹³¹l]iodo-L- phenylalanine (IPA-131), 4-[¹²⁴l]iodo-L-phenylalanine (IPA-124) and/or 4- [²¹¹At]astatine-L-phenylalanine (AtPA-211) to a patient.

6. The method according to claim 1 to 5, wherein the functional activity level of the amino acid transporter protein in the samples is analyzed in a whole cell assay, and wherein the expression level of the amino acid transporter gene in the samples is analysed in a RNA assay, a protein assay, or a whole cell assay.

7. The method according to claim 6, wherein the RNA assay comprises a RT-PCR or a hybridization assay.

8. The method according to claim 7, wherein the RT-PCR or hybridization assay comprises the use of primers or probes derived from the nucleic acid sequence of the LAT1 as depicted in SEQ ID NO: 1 or the nucleic acid sequence of the ASCT2 as depicted in SEQ ID NO: 3 or the complementary strands thereof.

9. The method according to claim 6, wherein the protein assay or the whole cell assay comprises the use of an antibody specifically binding to/detecting LAT1 having an amino acid sequence as depicted in SEQ ID NO: 2 or the ASCT2 having an amino acid sequence as depicted in SEQ ID NO: 4.

10. The method according to claim 9, wherein the whole cell assay comprises a flowcytometric analysis.

11. The method according to claim 6, wherein the whole cell assay comprises an incubation of the malignant cells or malignant tissue and of the control cells or control with a labeled phenylalanine for an analysis of the LAT1 functional activity level or a labeled alanine, serine or cysteine for an analysis of the ASCT2.

12. The method according to claim 11 , wherein the phenylalanine, alanine, serine and/or cysteine is/are labeled with a radioactive isotope, a fluorescent dye or chemoluminescent dye.

13. The method according to claim 11 or 12, wherein the cells or tissues are incubated with the labeled compound prior to the step of analyzing the expression for 1 to 48 h.

14. A pharmaceutical composition for an individual treatment of a malignant neoplasia comprising one or more L-amino acid(s) selected from the group consisting of phenylalanine, alanine, serine and cysteine, conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 , for which a sensitivity of the malignant cells or malignant tissue(s) from the individual patient is demonstrated by the use of a method according to claims 1 to 12.

15. A kit for the prediction of the sensitivity of malignant cells or malignant tissue(s) of an individual patient for a therapy comprising the administration of a phenylalanine, serine or cysteine conjugated to an alpha-, beta- or Auger- electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 to the patient, the kit comprising one or more of the primers or probes described in claim 8, antibodies described in claim 9 or a labeled, preferably halogenated, amino acid described in claim 11 or 12.

16. Use of one or more L-amino acid(s) selected from the group consisting of phenylalanine, alanine, serine and cysteine, conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine- 76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 , for the preparation of a pharmaceutical composition for an individual patient for the treatment of malignant neoplasia, wherein for the selected one or more L-amino acid(s) a sensitivity of the malignant cells or malignant tissues from the individual patient is predicted by the method according to claims 1 to 13.

17. A method for the individual treatment of a patient suffering from malignant neoplasia, the method comprising the steps of:

(a) selecting one or more L-amino acid(s) selected from the group consisting of phenylalanine, alanine, serine and cysteine, conjugated to an alpha-, beta- or Auger-electron emitting isotope selected from the group consisting of bromine-76, bromine-77, bromine-82, iodine-123, iodine-124, iodine-125, iodine-131 and astatine-211 , and

• wherein for the selected one or more L-amino acid(s) a sensitivity of the malignant cells or malignant tissue(s) from the individual patient is demonstrated by analyzing the functional activity level of an amino acid transporter protein in a sample of the malignant cells or malignant tissue(s) from the patient and in a sample of control cells or control tissue(s) from a healthy donor and/or a diseased control sample, • wherein the transporter is the L-type amino acid transporter 1 (LAT1) or the alanine-serine-cysteine transporter 2 (ASCT2), and

• wherein an increased functional activity level of at least one of the transporter proteins in the sample of the malignant cells or malignant tissues compared to the one in the control cells or control tissues is indicative for a sensitivity of the malignant cells or malignant tissues for the therapy; and

(b) administering the one or more selected L-amino acid(s) to the patient.