Test for predicting neutralization of asparaginase activity

Abstract

Method of in vitro measurement of the presence of factors that are able to neutralize asparaginase activity in a sample of blood, plasma, serum or derived medium that may contain asparaginase neutralizing factors, obtained from a patient, comprising mixing of said sample with asparaginase, incubation of said mixture, then measurement of the residual asparaginase activity in the mixture and determination or quantification of the presence of said neutralizing factors. Method for predicting the efficacy of a treatment with asparaginase.

Claims

1. A method of treatment of an asparaginase-sensitive pathology in a patient, said methods comprising: (a) obtaining a sample of blood, plasma, or serum from a patient; (b) incubating said sample with a known amount of asparaginase for a period of time sufficient to produce antibody-asparaginase immune complexes; (c) removing from the incubated sample any antibody-asparaginase immune complexes formed during incubation step (b) and recovering a sample free of antibody-asparaginase immune complexes; (d) incubating the sample obtained at step (c) with asparagine; (e) determining a residual asparaginase activity or the amount of asparaginase residual activity in the resultant mixture of step (d); and (f) treating said patient by means of the asparaginase and/or by means of another form of asparaginase.

2. The method according to claim 1, further comprises wherein the treating said patient of step (f) depends on step (e) determining the residual asparaginase activity or the amount of asparaginase residual activity in the resultant mixture of step (d).

3. The method according to claim 1, further comprises after step (e), determining or quantifying the presence of neutralizing factors.

4. The method according to claim 1, wherein the patient is currently being treated with asparaginase or has been treated with asparaginase.

5. The method according to claim 1, wherein measuring the activity of the asparaginase in the mixture is carried out by adding, to the mixture, asparagine and a reagent system suitable for assaying residual enzymatic activity.

6. The method according to claim 5, comprising the following stages: (a) incubating the mixture with the reagent system suitable for assaying the residual enzymatic activity; and (b) qualitatively or quantitatively evaluating a loss or retention of enzymatic activity, which is correlated with the presence or with the content of neutralizing factors of said asparaginase in the sample.

7. The method according to claim 1, comprising, before incubating the sample with said form of asparaginase, a step (a.sub.o) of removing or inactivating any asparaginase that may be present in the sample.

8. The method according to claim 1, comprising, before incubating the sample with said form of asparaginase, a step (a.sub.o) of measuring the baseline content of asparaginase in the sample.

9. The method according to claim 5, wherein the reagent system is sensitive to the appearance of ammonium ions resulting from enzymatic degradation of asparagine by asparaginase.

10. The method according to claim 9, which employs a reaction that consumes the ammoniums ions quantitatively.

11. The method according to claim 10, wherein the quantitative consumption of the ammonium ion is measured by measuring the decrease in optical density of the mixture.

12. The method according to claim 5, which employs the following reactions:
asparaginase+asparagine aspartic acid+NH.sub.4.sup.+(1)
-ketoglutarate+NH.sub.4.sup.++NADPH+glutamate dehydrogenase (catalyst) glutamate+NADP.sup.++H.sub.2O.(2)

13. The method according to claim 1, which comprises determining the patient's capacity to respond: (i) positively to treatment with the asparaginase; (ii) not respond to it; or (iii) only respond incompletely.

14. The method of claim 1, further comprising identifying the asparaginase is active in the patient sample and is able to be effective in the patient if the activity is present.

15. The method according to claim 13, which comprises, for cases (ii) and (iii), treating the patient using a different asparaginase.

16. The method according to claim 13, which comprises, for case (i), treating the patient using the asparaginase.

17. The method according to claim 15, wherein the different asparaginase is an asparaginase included in a biovector.

18. The method according to claim 15, wherein the different asparaginase is an asparaginase included in erythrocytes.

19. The method according to claim 2, wherein another form of asparaginase of step (f) is an asparaginase included in a biovector.

20. The method according to claim 2, wherein another form of asparaginase of step (f) is an asparaginase included in erythrocytes.

21. The method of claim 1, wherein a medium is derived from the sample obtained in step (a) and used in the incubation step (b).

22. The method of claim 14, which comprises modifying the dosage regimen based on identifying the asparaginase is active in the patient sample and is able to be effective in the patient if the activity is present.

23. The method of claim 1, wherein the patient has leukemia.

24. The method of claim 1, wherein the patient has acute lymphoblastic leukemia.

25. The method of claim 1, wherein the patient has a solid tumor.

26. The method of claim 1, wherein the patient has pancreatic cancer.

27. The method of claim 1, wherein the patient has ovarian cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will be described with regard to examples using the Figures as follows:

(2) FIG. 1 is a graph showing effect of patient serum matrix on L-asparaginase activity in which seven fixed L-asparaginase concentrations (ranging from 0 to 800 IU/L were measured in three human sera and buffer controls.

(3) FIG. 2 is a graph showing effect of patient serum matrix on L-asparaginase activity measurement with distribution of activity measured for 52 human sera mixed with L-asparaginase at a final concentration of 500 IU/L.

(4) FIG. 3 is a graph showing L-asparaginase activity inhibition versus anti-asparaginase IgG concentration.

(5) FIG. 4 is a graph showing L-asparaginase activity inhibition versus anti-asparaginase IgG low concentration.

(6) FIG. 5 is a graph showing L-asparaginase activity inhibition versus L-asparaginase concentration.

(7) FIG. 6 is a graph showing % of inhibition of L-asparaginase activity in 57 human sera from 17 patients treated with L-asparaginase.

EXAMPLES

Example 1

Immunization of a Rabbit with L-Asparaginase

(8) A few milliliters of serum are taken from the rabbit before the first immunization so as to have a pre-immune serum. Then the rabbit is injected 4 times with 500 g of L-asparaginase (Kidrolase, OPI-EUSA Limonest France). Sera are taken between the first and second immunization and between the second and third immunization. Finally, after the last immunization, the total serum is recovered and stored at 20 C. The final serum is characterized according to its total protein concentration (Biuret method) and its total immunoglobulin concentration.

Example 2

Purification of Rabbit Total IgGs

(9) Purification of the rabbit total IgGs from the final serum, containing anti-asparaginase IgGs, is carried out with the Kit pure 1A from Sigma (# PURE1A). Briefly, 2 ml of serum is clarified by centrifugation or filtration on a 0.45 m filter before purification of the IgGs. Then 4 ml of binding buffer is added to the 2 ml of clarified serum. The mixture is passed, then eluted from the column following the protocol recommended by Sigma. So that they can be injected in the animal, the total IgGs are centrifuged in a filtration column with a threshold of 10 000 Dalton in order to replace the elution buffer with PBS.

Example 3a

Measurement of the Inhibition of an Intermediate Serum on the Enzymatic Activity of L-Asparaqinase (Assay of the Mixture)

(10) Assay of the L-asparaginase was carried out according to the protocol published in: Orsonneau et al., Automatic kinetic assay of plasma L-asparaginase activity in therapeutic monitoring of acute lymphoblastic leukaemias, Ann Biol Clin, 62: 568-572.

(11) An intermediate serum (obtained between the first and second immunization) was used first, for elaborating measurement of the inhibition of enzymatic activity. A concentration of 2 IU/ml of L-asparaginase is used. The enzyme is pre-incubated for 15 minutes at 37 C. with several dilutions of serum, then the enzymatic activity is measured in the mixture. The results are presented in Table 1:

(12) TABLE-US-00001 TABLE 1 L- L-asparaginase Tube asparaginase Serum in the mixture Residual No. added (IU/ml) dilution (IU/ml) activity, % 1 2 1.96 100 2 2 1.11 0.72 36.43 3 2 0.78 39.63 4 2 0.85 43.04 5 2 1/16 1.18 60.16 6 2 1/32 1.29 65.91 7 2 1/128 1.64 83.74

(13) Table 1 summarizes the measurements of residual enzymatic activities of L-asparaginase in the mixtures (tubes 2 to 7). The rabbit serum inhibits the enzymatic activity of L-asparaginase: the greater the dilution of the serum, the weaker the inhibition.

(14) Tube 1 constitutes a control, showing that incubation of the enzyme alone at 37 C. for 15 minutes does not affect its enzymatic activity.

Example 3b

Measurement of the Inhibition of an Intermediate Serum on the Enzymatic Activity of L-Asparaqinase (Assay of a Supernatant)

(15) An intermediate serum (obtained between the first and second immunization) was used.

(16) In order to simulate phagocytosis of the antigen-antibody complexes by the reticuloendothelial system, the L-asparaginase/serum mixture is incubated for 15 minutes at 37 C., then centrifuged for 10 minutes at 17500 g at 4 C. in order to remove the immune complexes. The enzymatic activity is assayed in the supernatant. The assay results are presented in Table 2:

(17) TABLE-US-00002 TABLE 2 L- L-asparaginase Residual Tube asparaginase Serum in supernatant activity in No. added (IU/ml) dilution (IU/ml) supernatant (%) 1 2 1.91 95.63 2 2 1.11 0.21 10.39 3 2 0.14 6.77 4 2 0.05 2.72 5 2 1/16 0.03 1.53 6 2 1/32 0.02 0.98 7 2 1/128 0.02 1.00 control 2 pre-immune 1.82 90.81

(18) A control, replacing the serum with the pre-immune serum, was added so as to test the specificity of the interaction between L-asparaginase and the anti-asparaginase antibodies present in the serum. This demonstrates that more than 90% of the enzyme is not involved in interaction with nonspecific antibodies.

(19) The antibodies present in the serum interacted with all of the enzyme for the dilutions , 1/16, 1/32 and 1/128.

Example 4

Measurement of Inhibition of the Rabbit Total IgGs from the Enzymatic Activity of L-Asparaginase

(20) The same experiment as that presented in Example 3b was carried out with the rabbit total IgGs and a concentration of 1.25 IU/ml of L-asparaginase. The results are presented in Table 3:

(21) TABLE-US-00003 TABLE 3 L- L-asparaginase Residual Tube asparaginase Dilution of in supernatant activity in No. added (IU/ml) the IgGs (IU/ml) supernatant (%) control 1.25 1.19 94.99 1 1.25 1.26 100.00 2 0.02 1.38 3 1.25 0.02 1.52 4 1.25 1/16 0.01 0.40 5 1.25 1/32 0.01 0.62 6 1.25 1/64 0.01 0.98

(22) The rabbit total IgGs, containing anti-asparaginase IgGs, interact with L-asparaginase starting from the dilution 1/64 and cause total inhibition of the enzymatic activity (99.02% of enzyme precipitated).

Example 5

Inactivation, by the Rabbit Total IgGs, of Free L-Asparaqinase Injected in the Mouse

(23) An experiment was set up for the mouse (16 mice) to investigate the inhibition of L-asparaginase by the anti-asparaginase IgGs in vivo.

(24) The experimental conditions were as follows: dose of 100 IU/kg of L-asparaginase, equivalent to 1.25 IU/ml circulating in a 25 g mouse injection of 7.5 g of rabbit total IgGs.

(25) Injection of the IgGs or of PBS is carried out 20 minutes before injection of L-asparaginase, free or encapsulated in mouse red blood cells (Asp-RBC). Then 6 hours after this last-mentioned injection, the mice are sacrificed and the blood is collected.

(26) The L-asparaginase activity is then assayed in the plasma and in the RBCs. Table 4 summarizes the values obtained:

(27) TABLE-US-00004 TABLE 4 L-asparaginase activity (IU/ml) IgGs PBS Asp-RBC Red blood cells 0.798 0.126 0.879 0.146 Plasma 0.013 0.002 0.126 0.029 Free L- Red blood cells 0.132 0.019 0.098 0.013 asparaginase Plasma 0.002 0.002 0.417 0.103
Assay of L-asparaginase in the RBCs of mice that received the Asp-RBCs detects 0.798 and 0.879 IU/ml in the presence of IgGs or of PBS respectively. Therefore the IgGs present in the plasma did not have an inhibitory effect on the encapsulated enzyme. In the plasma of these same mice, free L-asparaginase injected with the Asp-RBCs (there is still in fact a small amount of free enzyme outside of the RBCs, of the order of 10% of the dose) is inhibited in the presence of the IgGs (0.013 IU/ml) and remains active in the presence of PBS (0.126 IU/ml).

(28) When free L-asparaginase was injected, the IgGs inhibited its activity (0.002 IU/ml) whereas injection of PBS had no effect on the activity of the enzyme (0.417 IU/ml). Taking into account the half-life of free L-asparaginase (10 hours), the measurement of 0.417 IU/ml of plasma L-asparaginase corresponds to the residual activity of the free enzyme 6 hours after its injection.

(29) The plasma concentration of L-asparagine was measured in the plasma of 14 of the mice in the study (two could not be used as the volume was too small). The results are presented in Table 5.

(30) TABLE-US-00005 TABLE 5 Plasma L-asparagine (mol/litre) IgGs PBS Asp-RBC <2 <2 Free L-asparaginase 28.09 3.63 <2

(31) The depletion of L-asparagine was total when the mice were treated with Asp-RBCs or when they received free L-asparaginase in the presence of PBS.

(32) Only the mice treated with free L-asparaginase in the presence of IgG have a plasma concentration of L-Asparagine of 28.09 M. The enzyme was therefore inhibited in the plasma by the IgGs.

Example 6

Measurement of the Inhibition of Rabbit Total IgGs on the Enzymatic Activity of PEG-Asparaginase

(33) Rabbit total IgGs, containing anti-asparaginase IgGs, were tested on PEG-asparaginase (Sigma # A5336). The same experiment as that presented in Example 4 was carried out with the PEG-asparaginase. The results are presented in Table 6.

(34) TABLE-US-00006 TABLE 6 PEG-Aspa PEG-Aspa Residual Tube added Dilution assayed activity in No. (IU/ml) of the IgGs (IU/ml) supernatant (%) 1 0.40 0.360 90.00 2 0.79 0.023 2.97 3 0.79 1/16 0.026 3.32 4 0.79 1/32 0.023 2.90 5 0.79 1/64 0.029 3.67

(35) The rabbit IgGs containing anti-asparaginase IgGs interacted with the PEG-asparaginase. The activity detected in the supernatant is less than 4% of the initial mixed activity with the IgGs.

Example 7

Test Protocol

(36) This protocol applies to a patient who is being considered for treatment with a particular asparaginase. A small blood sample is taken from this patient and is treated conventionally to obtain a serum sample.

(37) Then the following procedure is followed: (a.sub.0) optionally inactivation or removal of any asparaginase present in the serum sample, or measurement of the residual enzymatic activity (a) incubation of the sample with 0.1 to 5 IU/ml of asparaginase for 1 to 60 min; (a.sub.1) optionally removal of the immune complexes, preferably by centrifugation at 3000-25000 g for 1 to 30 min at the specified speed; (b) incubation of the mixture from (a) or of the supernatant from (a.sub.1) for 2 to 60 min with a saturating amount of asparagine, notably from 10 to 50 mg/ml; (c) incubation, notably for 3 to 20 min, of the preceding mixture with the reagent system (-ketoglutarate, NADPH, glutamate dehydrogenase) that is able to detect the residual enzymatic activity; (d) qualitative or quantitative evaluation of the loss or retention of enzymatic activity, which correlates with the presence or the content of asparaginase neutralizing factors in the sample.

(38) It is possible to determine levels or thresholds of levels of residual enzymatic activity depending on whether or not stage (a.sub.1) is included.

(39) In a patient who has already been treated, if the method shows marked presence of neutralizing factors, a replacement treatment using a different form of asparaginase is recommended and applied. Notably, when the free or modified form is likely to be inhibited, the enzyme encapsulated in erythrocytes is recommended.

(40) The same protocol is applicable simply for testing the potential efficacy of an asparaginase in the patient.

Example 8

Effect of Patient Serum Matrix on L-Asparaginase Activity Measurement

(41) Three human sera, nave to L-asparaginase treatment, were mixed with 7 L-asparaginase concentrations (from 0 to 800 IU/L) to ensure that human serum has no interference on L-asparaginase activity measurement. Samples were incubated 15 minutes at 37 C. and were centrifuged 10 minutes at 17500 g at 4 C. L-asparaginase activity is checked in the supernatant. As a control, buffer 1PBS 4% BSA was mixed with the same L-asparaginase concentrations. Results are presented in Table 7.

(42) TABLE-US-00007 TABLE 7 Added L- asparaginase Measured L-asparaginase activity (IU/L) condition IU/L srum 246922 srum 25579 srum 255810 pure 800 685 783 788 serum 400 ND* 442 461 200 220 220 246 100 101 125 122 50 30 76 72 25 19 37 33 0 0 0 3 buffer 800 753 802 851 control 400 407 455 463 200 218 236 244 100 115 120 124 50 63 59 77 25 24 45 37 0 0 0 *Not Determined

(43) No interference of human serum was observed on L-asparaginase activity measurement (compared with buffer control: see Table 7 and FIG. 1).

Example 9

Effect of Patient Serum Matrix on L-Asparaginase Activity Measurement and Determination of Basal Activity of Human Serum

(44) Fifty-two human sera, nave to L-asparaginase treatment, were mixed with L-asparaginase at a final concentration of 500 IU/L to ensure that human serum has no interference on L-asparaginase activity measurement. Samples were incubated 15 minutes at 37 C. and were centrifuged 10 minutes at 17500 g at 4 C. L-asparaginase activity is checked in the supernatant. As a control, buffer 1PBS 4% BSA was mixed with the same L-asparaginase final concentration. Results are presented in Table 8 and in FIG. 2.

(45) TABLE-US-00008 TABLE 8 Measurement of l-asparaginase activity on 52 human sera with L-asparaginase added at a final concentration of 500 (IU/L) Serum Measured L-asparaginase activity (IU/L) 1 467 2 493 3 505 4 492 5 466 6 482 7 470 8 474 9 453 10 474 11 482 12 472 13 467 14 469 15 468 16 456 17 476 18 477 19 481 20 477 21 479 22 486 23 478 24 479 25 472 26 479 27 457 28 457 29 483 30 472 31 418 32 462 33 477 34 477 35 467 36 442 37 464 38 464 39 489 40 452 41 475 42 484 43 480 44 480 45 475 46 478 47 473 48 482 49 480 50 473 51 478 52 477 Control 480 Mean 473.02 Standard deviation 13.4 Mean 2SD 446.22 Mean + 2SD 499.82

(46) The mean activity measured for the 52 human sera is 473 IU/L the standard deviation (SD) is 13.4 IU/L. The mean activity measured for the sera is not significantly different from the control activity. All values are comprised within an acceptable range: on the 52 measurements only 3 are outside the confidence range of [mean2SD; mean+2SD]. The distribution of the activity measured according to the serum assayed indicate that L-asparaginase activity is not affected by the matrix serum (FIG. 2).

(47) To check the absence of enzymatic activity signal in human serum: 25 human sera nave to L-asparaginase treatment were assayed for l-asapraginase activity.

(48) TABLE-US-00009 TABLE 9 Measurement of l-asparaginase activity on 25 human sera nave to L-asparaginase treatment Serum Measured L-asparaginase activity (IU/L) 1 0 2 2 3 2 4 1 5 2 6 0 7 0 8 0 9 0 10 0 11 0 12 0 13 0 14 1 15 2 16 1 17 5 18 2 19 0 20 3 21 1 22 0 23 0 24 0 25 0 Mean 0.88 Standard Deviation 1.27

(49) With each of the 25 human sera assayed the L-asparaginase activity is closed to zero. The mean activity measured is 0.88 IU/L and the standard deviation is 1.27 IU/L. The maximum activity measured is 5 IU/L for serum 17 this basal activity signal is not likely to affect the measurement of L-asparaginase activity as it represents 1% of the activity measured for sera mixed with L-asparaginase at a final concentration of 500 IU/L.

Example 10

Inhibition of L-Asparaginase Activity by Anti-Asparaginase IgG Spiked Human Sera

(50) Two human sera, nave to L-asparaginase treatment, were pooled and spiked with anti-asparaginase IgG concentrations ranging between 1 and 100 g/mL (1, 2, 5, 10, 20, 40, 80, 100 g/mL). The anti-asparaginase IgG were obtained as described in examples 1 and 2.

(51) Then 500 IU/L of L-asparaginase was added. Samples were incubated 15 minutes at 37 C. and were centrifuged 10 minutes at 17500 g at 4 C. Residual L-asparaginase activity was measured in the supernatant. As a control, buffer 1PBS 4% BSA was used instead of the human sera pool. Results are presented in Table 10 and FIG. 3.

(52) TABLE-US-00010 TABLE 10 Added measured Added control Added anti-aspa L-asparaginase L-asparaginase inhibition IgG (g/mL) IgG (g/mL) (IU/L) activity (IU/L) (%) human 100 500 589 0.00 serum 40 500 537 0.00 pool 0 0 0 500 534 0.00 1 500 518 3.00 2 500 548 0.00 5 500 515 3.56 10 500 426 20.22 20 500 4 99.25 40 500 3 99.44 80 500 1 99.81 100 500 0 100.00 buffer 100 500 558 0.00 control 40 500 592 0.00 0 0 0 500 564 0.00 1 500 595 0.00 2 500 562 0.00 5 500 550 2.48 10 500 378 32.98 20 500 7 98.76 40 500 ND* 80 500 2 99.65 100 500 0 100.00 *Not Determined

(53) When L-asparaginase is mixed with increasing concentrations of anti-asparaginase IgG (specific IgG), in human serum or buffer control, total enzymatic activity inhibition occurs at an igG concentration of 20 g/mL and higher. Partial inhibition occurs when enzyme is mixed with 5 to 20 g/mL anti-asparaginase IgG. Below 5 g/mL anti-asparaginase IgG, L-asparaginase activity is not inhibited.

(54) No inhibition is observed when L-asparaginase is incubated with non-specific IgG (see added control IgG in Table 10).

(55) To refine the inhibition reaction of anti-asparaginase IgG concentration between 10 and 20 g/mL on L-asparaginase activity, an experiment was performed with IgG concentrations ranging from 8 to 22 g/mL. As usual, L-asparaginase is added to a final concentration of 500 IU/L. All the samples were incubated 15 minutes at 37 C. and were centrifuged 10 minutes at 17500 g at 4 C. Residual L-asparaginase activity is measured in the supernatant. The assay is performed with a human serum pool. Results are presented in Table 11 and FIG. 4.

(56) TABLE-US-00011 TABLE 11 Added Added measured anti-aspa L-asparaginase L-asparaginase inhibition IgG (g/mL) (IU/L) activity (IU/L) (%) human 2 serum 22 1 pool 500 580 8 500 510 12.07 10 500 410 29.31 12 500 98 83.10 14 500 65 88.79 16 500 5 99.14 18 500 1 99.83 20 500 0 100.00 22 500 2 99.66

(57) Total inhibition of L-asparaginase activity appears at an IgG concentration of 16 g/mL. Below 16 g/mL, L-asparaginase activity inhibition is partial.

(58) The opposite reaction was tested: a fixed anti-asparaginase IgG concentration (13.64 g/mL corresponding to 80% inhibition) was mixed with L-asparaginase concentrations ranging from 500 to 10000 IU/L. Samples were incubated 15 minutes at 37 C. and were centrifuged 10 minutes at 17500 g at 4 C. Residual L-asparaginase activity is measured in the supernatant. Results are shown in Table 12 and FIG. 5.

(59) TABLE-US-00012 TABLE 12 Added Added measured anti-aspa L-asparaginase L-asparaginase inhibition IgG (g/mL) (IU/L) activity (IU/L) (%) human 13.64 0 serum 13.64 500 36 93.00 pool 13.64 1000 716 32.00 13.64 5000 5010 9.00 13.64 10000 10780 3.00 500 548 1000 1052 5000 5480 10000 11080

(60) The more concentrated L-asparaginase is, the less the fixed IgG concentration (13.64 g/mL) inhibits its activity. A fixed quantity of anti-asparaginase IgG inhibits a fixed quantity of L-asparaginase. Therefore, L-asparaginase activity inhibition is dose-dependent.

Example 11

Test of 57 Human Sera from 17 Patients Treated with L-Asparaginase

(61) To ensure the assay has the ability to quantify the neutralization of asparaginase in a patient: 57 human sera sampled from 17 Acute lymphoblastic leukaemia patients under treatment with L-asparaginase were tested according to the test protocol described in example 7. The samples were taken at different time of treatment course and a measurement of residual L-asparaginase enzyme activity was conducted to verify that this activity is negligible and will not interfere with the test procedure.

(62) The sera were then mixed with L-asparaginase at a final concentration of 500 IU/l and were incubated 15 minutes at room temperature. The L-asparaginase activity was then determined before and after a centrifugation step of 6 minutes at 7800 rpm. As a control, buffer 1PBS 4% BSA was mixed with L-asparaginase at a final concentration of 500 IU/L. Results are presented in the table 13 below and in FIG. 6.

(63) TABLE-US-00013 TABLE 13 Test of 57 human sera from 17 patients under treatment with L-asparaginase Activity after Activity after % of Initial addition of L- addition of L- % of inhibition inhibition activity asparaginase asparaginase and without after Patient Serum (IU/L) (IU/L) centrifugation (IU/L) centrifugation centrifugation Control 6 655 662 NA NA 1 1 2 551 515 15.88 22.21 2 2 440 342 32.82 48.34 3 1 559 518 14.66 21.75 4 2 658 655 0.46 1.06 2 5 7 NA 441 NA 33.38 6 13 767 727 17.1 9.82 3 7 1 706 701 7.79 5.89 8 2 724 716 10.53 8.16 9 11 692 731 5.65 10.42 10 2 680 666 3.82 0.6 11 1 711 733 8.55 10.73 12 1 754 711 15.11 7.4 13 2 726 730 10.84 10.27 4 14 2 664 744 1.37 12.39 15 15 804 667 22.75 0.76 Control 6 636 NA NA 5 16 1 690 516 8.49 18.87 17 2 649 678 2.04 6.6 18 2 684 575 7.55 9.59 19 ND 622 633 2.2 0.47 20 1 670 604 5.35 5.03 21 1 609 583 4.25 8.33 6 22 1 659 519 3.62 18.4 7 23 2 508 492 20.13 22.64 8 24 1 761 473 19.65 25.63 25 0 633 608 0.47 4.4 9 26 4 705 524 10.85 17.61 27 2 656 525 3.14 17.45 10 28 3 608 623 4.4 2.04 29 1 649 560 2.04 11.95 30 3 610 609 4.09 4.25 31 7 694 570 9.12 10.38 32 1 598 586 5.97 7.86 33 2 589 516 7.39 18.87 34 0 527 NA 17.14 NA Control 3 661 629 NA NA 11 35 2 713 587 7.87 6.68 36 1 499 667 24.51 6.04 37 3 596 666 9.83 5.88 38 2 701 579 6.05 7.95 39 2 850 408 28.59 35.14 12 40 2 798 446 20.73 29.09 41 2 599 596 9.38 5.25 42 5 814 448 23.15 28.78 43 3 752 265 13.77 57.87 13 44 1 705 570 6.66 9.38 14 45 1 685 659 3.63 4.77 15 46 1 573 388 13.31 38.31 47 2 492 408 25.57 35.14 48 1 605 678 8.47 7.79 49 2 571 355 13.62 43.56 50 2 725 275 9.68 56.28 51 2 464 437 29.8 30.52 52 3 497 475 24.81 24.48 53 0 540 445 18.31 29.25 16 54 3 550 524 16.79 16.69 55 2 568 637 14.07 1.27 56 1 595 379 9.98 39.75 17 57 1 709 585 7.26 7 ND: Not determined NA: Not Applicable

(64) All the samples have a residual asparaginase activity which is negligible, the highest residual activity is of 15 IU/L and should not interfere with the test procedure as it represents only 3% of the theoretical added L-asparaginase. The asparaginase activity measured for the control is higher than expected (655, 636 and 661 IU/L respectively for the 3 control compared to the 500 IU/L that were expected). The percentage of inhibition of asparaginase activity was calculated based on enzymatic activity measured for the control. The fact that numerous values of inhibition percentage are negative indicate a bias in the measurement procedure.

(65) The percentage of inhibition of asparaginase activity is higher after centrifugation suggesting that the centrifugation step has eliminated some immune complexes that were formed between asparaginase and anti-asparaginase antibodies.

(66) On the 17 patients assayed 14 experience an inhibition of the asparaginase activity by factors present in their serum (FIG. 6). Height patients have a percentage of inhibition above 20% but only three patients have a percentage of inhibition higher than 40% (FIG. 6).