PREDICTION OF CANCER TREATMENT BASED ON DETERMINATION OF ENZYMES OR METABOLITES OF THE KYNURENINE PATHWAY

Abstract

The present invention relates to a method of predicting the therapeutic efficacy of at least one therapy approach in the treatment of a neoplastic disease in a patient. The method comprises the following steps: a) Determining the presence or concentration of at least one enzyme or metabolite of the Kynurenine pathway in a patient sample, and b) Concluding, from step a), whether the at least one therapy approach will be therapeutically effective in the treatment of the neoplastic disease.

Claims

1-21. (canceled)

22. A method of predicting the therapeutic efficacy of at least one therapy approach involving an agent that targets an immune-checkpoint pathway in the treatment of a neoplastic disease in a patient by means of an immunoassay, which method comprises the following steps: a) determining the presence or concentration of at least one metabolite of the Kynurenine pathway in a patient sample, and b) concluding, from step a), whether the at least one therapy approach will be therapeutically effective in the treatment of the neoplastic disease.

23. A method of treating a neoplastic disease in a patient, which method comprises the following steps: a) determining the presence or concentration of at least one metabolite of the Kynurenine pathway in a patient sample by means of an immunoassay, and b) dependent on the result of step a), applying, in the patient, one or more therapy approaches involving an agent that targets an immune-checkpoint pathway.

24. The method of claim 22, wherein the presence, absence or concentration of at least one metabolite of the Kynurenine pathway is predictive for the efficacy of said therapy approach.

25. The method of claim 22, wherein the method avoids side effects in patients not responding on treatment with said agent that targets an immune-checkpoint pathway.

26. The method of claim 22, wherein a) the presence or a high concentration of at least one metabolite of the Kynurenine pathway is predictive of good efficacy of said therapy approach, and/or b) the absence or low concentration of at least one metabolite of the Kynurenine pathway is predictive of poor efficacy of said therapy approach.

27. The method of claim 26, wherein the high concentration of at least one metabolite of the Kynurenine pathway means a higher concentration thereof compared to a normal concentration of a healthy subject.

28. The method of claim 22, wherein the agent that targets the immune-checkpoint pathway is a modulator, inhibitor, antagonist and/or binder of CTLA4, OX40, PD1, PDL1, Lag3, B7-H3, B7-H4, IDO1, IDO2, TDO2 and/or TIM3.

29. The method of claim 22, wherein the agent that targets the immune-checkpoint pathway is at least one selected from the group consisting of: a monoclonal antibody (murine, chimeric, humanized, human) a fragment or derivative thereof (e.g., Fab, Fab2, scFv) a new antibody format a fusion peptide comprising at least one domain capable of binding an enzyme and/or a metabolite of the kynurenine pathway an antibody mimetic, an aptamer, and/or a small molecule antagonist.

30. The method of claim 22, wherein the method comprises in step a), the determination of the concentration of two or more metabolites of the Kynurenine pathway, whereupon a logical or arithmetical operation is made based on the determined concentrations, the result of which is then taken as a basis for the decision made in step b).

31. The method of claim 22, wherein the presence or concentration of at least one metabolite in the patient sample is determined by immunohistochemistry, ELISA, EIA and/or immunofluorescence.

32. The method of claim 22, wherein the metabolite of the kynurenine pathway in the sample is derivatized, prior to detection, by conjugating it to a carrier molecule.

33. The method of claim 32, wherein the carrier molecules added to the sample.

34. The method of claim 32, wherein the carrier molecule is a naturally occurring carrier molecule different from the serum proteins that are part of the sample.

35. The method of claim 22, wherein a detection immunoligand is used in the immunoassay, said immunoligand being created against a complex consisting of a metabolite of the Kynurenine pathway and a carrier.

36. The method of claim 35, wherein the detection immunoligand specifically binds to the metabolite-carrier complex and has been created by a method comprising the steps of: a) conjugating the Kynurenine pathway metabolite, in isolated form, to a carrier molecule to obtain an immunogenic conjugate, b) carrying out an immunization experiment with said immunogenic conjugate, and c) obtaining, directly or indirectly, detection antibodies from said experiment that specifically bind to the metabolite-carrier complex and/or to the metabolite.

37. The method of claim 35, wherein the detection immunoligand is created by a method comprising the steps of: a) exposing said analyte, or a metabolite-carrier complex to a library of immunoligands, and b) screening said library for detection immunoligands that specifically bind to the metabolite-carrier complex and/or to the analyte.

Description

EXPERIMENTS AND FIGURES

[0199] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0200] FIG. 1: Progression of tumor volume in CT26 tumor bearing mice challenged or not with anti-CTLA4. Data represents mean tumor volume (in mm.sup.3) from 6 mice in each experimental setting. According to the literature, mice treated with anti-CTLA4 displayed a decrease in tumor progression.

[0201] FIG. 2: Quantification of L-Kynurenine in plasma from CT26 tumor bearing mice challenged with anti-CTLA4 using EIA (Enzyme immunoassay). Derivatized plasma samples were incubated with 3D4-F2 mAb (at 0.01 mg/ml) and an HRP-Kynurenine conjugate (Tracer, at 1 μg/mL) for 1.5 hour at 37° C. on a maxisporp plate coated with anti mouse IgG. Reaction was revealed using TetraMethylBenzidine (TMB). L-Kynurenine levels are correlated with tumor volume (mm.sup.3) at the date of sacrifice.

[0202] FIG. 3a: Entry reaction which initiates the kynurenine pathway (L-Tryoptophan->L-Formylkynurenine, but is not part thereof. The step is catalyzed by either a) Indoleamine 2,3-dioxygenase (IDO1) or b) Tryptophan 2,3-dioxygenase (TDO2). If one of the two is blocked, the reaction can still take place, while blocking both may have severe side effects.

[0203] FIG. 3b: Overview of the kynurenine pathway with its enzymes and metabolites. The enzymes are as follows: i) Kynurenine formamidase, a) Kynurenine amino-transferase, b) Kynurenine 3-hydroxylase (also called Kynurenine mono-oxygenase), c) Kynureninase (also called L-Kynurenine hydrolase), d) Kynurenine amino-transferase, e) Kynureninase (also called L-Kynurenine hydrolase), and f) 3-Hydroxyanthranilic Acid oxygenase (also called 3-Hydroxanthranilate dioxygenase).

[0204] The metabolites are as follows: L-Formylkynurenine, Kynuramine, L-Kynurenine, Kynurenic Acid, 3-hydroxyL-kynurenine, Anthranilic Acid, 3-hydroxyanthranilic Acid, Xanthurenic Acid, Quinaldic Acid, Picolinioc Acid and/or Quinolinic Acid.

[0205] Please note that some metabolites and enzymes of the Kynurenine pathway are not shown. This applies for example, for Niacin, which is formed out of Quinolinic Acid.

[0206] FIG. 4: Crosslinking reaction between a carboxylic acid group of one moiety (R1, e.g., the analyte or the carrier protein) and an amine group of another moiety (R2, e.g., the carrier protein or the analyte), as catalyzed by a carbodiimide. In this case, the latter is EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide). See more explanations in the text.

[0207] FIG. 5: Kynurenine concentration in a mice colorectal cancer model according to the response to anti-CTLA4. See more explanations in the text.

EXAMPLE 1: KYNURENINE LEVEL IS ASSOCIATED WITH CLINICAL RESPONSE TO ANTI-CTLA4

1. Experimental Procedure

1.1. Tumor Experiments and In Vivo Blockade of Anti-CTLA4.

[0208] BALB/c mice were implanted subcutaneously (s.c.) on the right flank with 5×10.sup.4 of CT26 cells (purchased from ATCC). One hundred μg of α-mouse α-CTLA-4 (clone 4F10) were administered intraperitoneally (i.p.), either 3, 6, and 9 days following CT26 inoculation and tumour size was monitored using caliper.

1.2. L-Kynurenine Quantification in Plasma Using Enzyme Immuno Assay

[0209] Twenty four (24) days after cells inoculation, mice were anesthetized using ketamine/xylazine and subjected to plasma collection by intracardiac puncture.

[0210] L-Kynurenine was then quantified in plasma using a novel enzyme immunoassay in which the analyte was conjugated to a carrier, in order to make it detectable by an antibody that has been made against the same conjugate. Like other small metabolites, L-Kynurenine alone is not immunogenic, which makes it difficult to create antibodies that are capable of binding isolated L-Kynurenine with sufficient specificity, e.g., to detect it. For this reason, in a first step, L-Kynurenine is coupled to a carrier protein, which confers immunogenicity to the latter, thus making it possible to raise antibodies against it.

[0211] In a second step, L-Kynurenine in the sample is also derivatized prior to exposure to the detection antibody created with the method above, to render it detectable by the latter.

[0212] Because in this example, the detection antibody was murine, all endogenous proteins in the sample had to be precipitated before L-Kynurenine was then coupled (“derivatized”) to a carrier. The carrier was in this case BSA, and the antibody used had been obtained by immunization of a mouse with a conjugate consisting of L-Kynurenine and BSA.

[0213] Briefly, 100 μl of plasma and standards solutions were precipitated using 25 μl of Trichloro acid acetic (TCA) 1N, vortexed and centrifuged (10,000 g, 10 minutes, 4° C.). 80 μl of supernatant were equilibrated with 20 μl of Tris buffer 1M, pH=9, supplemented with Bovine Serum Albumin (BSA) to achieve 5 g/L and subjected to derivatization therewith using 100 μl of carbodiimide (EDC) and N-HydroxySuccinimide solubilized in MES buffer (0.3M, pH=6.3) over a 1 hour period under agitation (400 rpm) at 37° C. In this process, L-Kynurenine and other metabolites are coupled to BSA. Details of the derivatization and the conjugation are disclosed in patent application No GB 13 22 538 the content of which is fully incorporated herein by reference. HRP-Kynurenine tracer and a murine anti-L-Kynurenine monoclonal antibody were added to the solution at a final concentration at 0.3 μg/ml and 1 μg/ml respectively.

[0214] The latter solution was applied by mean of 200 μl per well on a maxisorp ELISA plate previously coated with unconjugated anti-mouse IgG immunoglobulin that has been raised against a conjugate consisting of L-Kynurenine and BSA. The plate was incubated for 1.30 hour at 37° C. and reaction was revealed using Tetramethylbenzidine. Coloration was monitored at 450 nm with a spectrophotometer.

2. Results

[0215] FIG. 1 shows the average tumour volume in CT26 tumor bearing mice treated with anti-CTLA4 (n=6) or the vehicle (n=6). Generally, a difference can be seen between the mice treated with anti-CTLA4 and with the vehicle.

[0216] However, in accordance with literature (Duraiswamy et al, Cancer Research, 2013), anti-CTLA4 exerts a benefit in only a fraction of the mice.

[0217] FIG. 2 shows a correlation between the L-Kynurenine plasma level and the tumour size from CT26 tumour bearing mice challenged with anti-CTLA4. These results indicate that mice with a higher plasma titer of L-Kynurenine display a better clinical response towards anti-CTLA4 than those with lower plasma titer.

[0218] The concentration of L-Kynurenine (which is a metabolite of the Kynurenine pathway) is thus predictive for the therapeutic efficacy of the drug anti-CTLA4 in the treatment of tumours.

EXAMPLE 2: KYNURENINE/TRYPTOPHAN RATIO IS ASSOCIATED WITH CLINICAL RESPONSE TO ANTI-CTLA4

3. Experimental Procedure

3.1. Tumor Experiments and In Vivo Blockade of Anti-CTLA4.

[0219] BALB/c mice are implanted subcutaneously (s.c.) on the right flank with 5×10.sup.4 of CT26 cells (purchased from ATCC). One hundred μg of α-mouse α-CTLA-4 (clone 4F10) are administered intraperitoneally (i.p.), either 3, 6, and 9 days following CT26 inoculation and tumour size is monitored using caliper.

3.2. L-Kynurenine and Tryptophan Quantification in Plasma Using Immunoassay

[0220] Twenty four (24) days after cells inoculation, mice are anesthetized using ketamine/xylazine and subjected to plasma collection by intracardiac puncture.

[0221] L-Kynurenine is then quantified in plasma using a novel enzyme immuno assay in which the analyte is conjugated to a carrier, in order to make it detectable by an antibody that has been made against the same conjugate. Briefly, 100 μl of plasma and standards solutions are precipitated using 25 μl of Trichloro acid acetic (TCA) 1N, vortexed and centrifuged (10,000 g, 10 minutes, 4° C.). 80 μl of supernatant are equilibrated with 20 μl of Tris buffer 1M, pH=9 supplemented with Bovine Serum Albumin (BSA) to achieve 5 g/L and subjected to derivatization using 100 μl of carbodiimide (EDC) and N-HydroxySuccinimide solubilized in MES buffer (0.3M, pH=6.3) over a 1 hour period under agitation (400 rpm) at 37° C. Details of the derivatization and the conjugation are disclosed in patent application no GB 13 22 538, the content of which is fully incorporated herein by reference. HRP-Kynurenine tracer and a murine anti-L-Kynurenine monoclonal antibody are added to the solution at a final concentration at 0.3 μg/ml and 1 μg/ml respectively. The latter solution is applied by mean of 200 μl per well on a maxisorp ELISA plate previously coated with unconjugated anti-mouse IgG immunoglobulin. The plate is incubated for 1.30 hour at 37° C. and reaction is revealed using Tetramethylbenzidine. Coloration is monitored at 450 nm with a spectrophotometer.

[0222] Tryptophan is measured using commercially available ELISA kit—purchased from LDN, Nordhorn, Germany—according to the provider procedure.

4. Results

[0223] Experiments demonstrate that anti-CTLA4 therapy induced an increase in L-Kynurenine/Tryptophan ratio (as an indicator of IDO1, IDO2, TDO2 dependent tryptophan degradation) level in plasma compared to mice who received only vehicle.

[0224] Also, we show a correlation between the Kynurenine to tryptophan ratio in plasma and the tumour size from CT26 tumour bearing mice challenged with anti-CTLA4. These results indicate that mice with a higher plasma Kynurenine to Tryptophan ratio display a better clinical response towards anti-CTLA4 than those with lower ratio value.

[0225] The L-Kynurenine/Tryptophan ratio is thus predictive for the therapeutic efficacy of the drug anti-CTLA4 in the treatment of tumours.

EXAMPLE 3: KYNURENINE LEVEL IS ASSOCIATED WITH CLINICAL RESPONSE TO ANTI-PD1

5. Experimental Procedure

5.1. Tumor Experiments and In Vivo Blockade of Anti-PD1.

[0226] BALB/c mice are implanted subcutaneously (s.c.) on the right flank with 5×10.sup.4 of CT26 cells (purchased from ATCC). Two hundred μg of α-mouse α-PD1 (clone RMP1-14) are administered intraperitoneally (i.p.), either 3, 6, and 9 days following CT26 inoculation and tumour size is monitored using caliper.

5.2. L-Kynurenine Quantification in Plasma Using Enzyme Immuno Assay

[0227] Twenty four (24) days after cells inoculation, mice are anesthetized using ketamine/xylazine and subjected to plasma collection by intracardiac puncture.

[0228] L-Kynurenine is then quantified in plasma using a novel enzyme immuno assay in which the analyte is conjugated to a carrier, in order to make it detectable by an antibody that has been made against the same conjugate. Briefly, 100 μl of plasma and standards solutions are precipitated using 25 μl of Trichloro acid acetic (TCA) 1N, vortexed and centrifuged (10,000 g, 10 minutes, 4° C.). 80 μl of supernatant are equilibrated with 20 μl of Tris buffer 1M, pH=9 supplemented with Bovine Serum Albumin (BSA) to achieve 5 g/L and subjected to derivatization using 100 μl of carbodiimide (EDC) and N-HydroxySuccinimide solubilized in MES buffer (0.3M, pH=6.3) over a 1 hour period under agitation (400 rpm) at 37° C. Details of the derivatization and the conjugation are disclosed in patent application No GB 13 22 538, the content of which is fully incorporated herein by reference. HRP-Kynurenine tracer and a murine anti-L-Kynurenine monoclonal antibody are added to the solution at a final concentration at 0.3 μg/ml and 1 μg/ml respectively.

[0229] The latter solution is applied by mean of 200 μl per well on a maxisorp ELISA plate previously coated with unconjugated anti-mouse IgG immunoglobulin. The plate is incubated for 1.30 hour at 37° C. and reaction is revealed using Tetramethylbenzidine. Coloration is monitored at 450 nm with a spectrophotometer.

6. Results

[0230] In accordance with literature (Duraiswamy et al, Cancer Research, 2013), our experiment shown a difference of the average tumor size between the mice treated with anti-PD1 and mice exposed only to the vehicle. However anti-PD1 exerts a benefit in only a fraction of the mice.

[0231] Also, we show a correlation between the L-Kynurenine plasma level and the tumour size from CT26 tumour bearing mice challenged with anti-PD1. These results indicate that mice with a higher plasma titer of L-Kynurenine display a better clinical response towards anti-PD1 than those with lower plasma titer.

[0232] The concentration of L-Kynurenine (which is a metabolite of the Kynurenine pathway) is thus predictive for the therapeutic efficacy of the drug anti-PD1 in the treatment of tumours.

EXAMPLE 4: KYNURENINE LEVEL IN HUMAN SAMPLES IS ASSOCIATED WITH CLINICAL RESPONSE TO ANTI-CTLA4

7. Experimental Procedure

7.1. Clinical Settings and In Vivo Blockade of CTLA4.

[0233] Patients suffering from advanced metastatic melanoma are treated with Ipilimumab (fully human anti CTLA4, Bristol Myers Squibb, 3 mg/kg, i.v, over 90 mins, triweekly, 4 doses in total). Before initiating the therapy, biopsy and plasma samples are taken from patients. For longitudinal study, plasma is then taken at 3, 12 and 24 weeks after treatment initiation.

7.2. L-Kynurenine Quantification in Plasma Using Enzyme Immuno Assay

[0234] L-Kynurenine is then quantified in plasma using an enzyme immuno assay in which the analyte is conjugated to proteins present in the plasma. Because the murine antibody used is raised against L-Kynurenine conjugated to BSA, the proteins in the sample (which is human) do not have to be precipitated (unlike in a murine sample, where endogenous murine immunoglobulins would lead to false positives).

[0235] It is in this context surprising that a murine antibody raised against a conjugate consisting of L-Kynurenine and BSA is capable of detecting, in a human sample, different conjugates consisting of L-Kynurenine and different respective proteins present in the plasma, with a sufficient degree of specificity.

[0236] Antibody targeting L-Kynurenine-BSA conjugate is affine and specific enough to recognize L-Kynurenine bound to a plasma protein. Briefly, 100 μl of plasma and standards solutions are subjected to derivatization using 100 μl of carbodiimide (EDC) and N-HydroxySuccinimide solubilized in MES buffer (0.3M, pH=6.3) over a 1 hour period under agitation (400 rpm) at 37° C. Details of the derivatization and the conjugation are disclosed in patent application No GB 13 22 538, the content of which is fully incorporated herein by reference. HRP-Kynurenine tracer and a murine anti-L-Kynurenine monoclonal antibody are added to the solution at a final concentration at 0.3 μg/ml and 1 μg/ml respectively.

[0237] The latter solution is applied by mean of 200 μl per well on a maxisorp ELISA plate previously coated with unconjugated anti-mouse IgG immunoglobulin. The plate is incubated for 1.30 hour at 37° C. and reaction is revealed using Tetramethylbenzidine. Coloration is monitored at 450 nm with a spectrophotometer.

7.3. L-Kynurenine Detection in Tumour Specimens by Immunohistochemistry

[0238] Tumours samples are taken from patients suffering from advanced metastatic melanoma before initiation of Ipilimumab therapy. Experimentally, paraffin embedded sections are deparafinized using successive bath of Xylene and Ethanol. Sections are then subjected to antigen retrieval with citrate buffer pH=6 (Dako) for 20 minutes at 95° C. Sections are washed in TBS before incubation with methanol containing 0.03% of hydrogen peroxide to block endogenous peroxydase. After two washes, sections are saturated in antibody diluent (Dako) plus 5% of BSA (Sigma-Aldrich) for 30 minutes at room temperature.

[0239] Anti L-Kynurenine mAb (3D4-F2) is then added at 0.01 mg/ml, in the presence of 2% of normal goat serum, and incubated overnight at 4° C. Sections are washed three times in TBS, and incubated for 30 minutes with envision system (dextran polymer grafted with anti mouse IgG conjugated with HRP, Dako) at room temperature. Sections are washed three times before revelation with DAB (Dako) for 10 minutes at room temperature.

[0240] Sections are rinsed, subjected to hematoxylin, dehydrated and mounted in DPX mountant media (Sigma-Aldrich). Pictures are obtained after a systematic scan of all cores (Hamamatsu, Nanozzomer). Quantification is performed according to the following grades:

0: No staining
1: Weak staining
2: Intermediate staining
3: Strong staining

8. Results

[0241] Experiments demonstrate that anti-CTLA4 therapy induced an increase in L-Kynurenine level in plasma compared to patients who received only chemotherapy. Increase is observed at early time points after treatment initiation and is maintained over the time of observation.

[0242] Also, patients harboring better clinical response (assessed by radiological imaging) following Ipilimumab based regimen displayed higher amount of L-Kynurenine in plasma.

[0243] The concentration of L-Kynurenine (which is a metabolite of the Kynurenine pathway) is thus predictive for the therapeutic efficacy of the drug anti-CTLA4 in the treatment of tumours. Experiments demonstrate that only a fraction of melanoma patients displayed Kynurenine positivity

[0244] Also, patients harboring better clinical response (assessed by radiological imaging) following Ipilimumab based regimen are in most of the case highly positive for L-Kynurenine.

[0245] The L-Kynurenine positivity in tumour biopsy (which is a metabolite of the Kynurenine pathway) is thus predictive for the therapeutic efficacy of the drug anti-CTLA4 in the treatment of tumours.

9. Kynurenine Concentration in a Mice Colorectal Cancer Model According to the Response to Anti-CTLA4

[0246] Mice were exposed to anti-CTLA4 (clone UC10-4F10, 100 μg/mouse, Day 3, 6 and 9, ip) and bleedings from the tail vein were performed at different time. Kynurenine measurements from plasma were performed by means of ELISA and revealed that mice rejecting the tumor upon anti-CTLA4 mAb treatment display higher production of Kynurenine seven (7) days after tumor cells inoculation when compared to responding (strong delay in tumor growth when compared to vehicle treated mice) and not responding mice (weak delay in tumor growth when compared to vehicle treated mice).

[0247] These results show that Kynurenine overproduction upon anti-CTLA4 antibody exposure is a good predictor of response to anti-CTLA4, strongly suggesting that Kynurenine itself could be critical at tuning the host immune response against the tumor. Results are shown in FIG. 5.

REFERENCES

[0248] Duraiswamy et al, Cancer Res; 73(23) Dec. 1, 2013 [0249] Ott et al, Clin Cancer Res Oct. 1, 2013, 19:5300-5309 [0250] Topalian et al., N Engl J Med 2012; 366:2443-2454 [0251] Bessede et al, Nature 511, 184-190 (10 Jul. 2014) [0252] Opitz et al, Nature 2011; 478:197-203.