Use of recombinant LAG-3 or the derivatives thereof for eliciting monocyte immune response

Abstract

The present invention relates to the use of a recombinant LAG-3 or derivatives thereof in order to boost a monocyte-mediated immune response, in particular to elicit an increase in the number of monocytes in blood. This finds use in the development of novel therapeutic agents for the treatment of an infectious disease or cancer.

Claims

1. A method of treating cancer in a human subject in need thereof, which comprises: administering to the subject an effective amount of: (i) a chemotherapy agent; and (ii) IMP321; wherein the IMP321 induces a systemic increase in the number of monocytes in blood of the subject, and elicits a systemic monocyte-mediated immune response; wherein the IMP321 is administered to the subject at a dose of more than 6 mg; and wherein the blood concentration of the IMP321 in the subject is greater than 1 ng/ml, for at least 24 hours after administration of the IMP321 to the subject.

2. The method of claim 1, wherein the chemotherapy agent and the IMP321 are administered to the subject simultaneously, separately, or sequentially.

3. The method of claim 1, wherein the IMP321 is administered 12 to 96 hours after administration of the chemotherapy agent.

4. The method of claim 1, wherein the subject is treated without administration of any additional antigen.

5. The method of claim 1, wherein the subject is administered an effective plurality of doses of the IMP321.

6. The method of claim 1, wherein the cancer is metastatic breast cancer or metastatic renal cell cancer.

7. The method of claim 1, wherein the chemotherapy agent is selected from the group consisting of taxanes, anthracyclines, and gemcitabine.

8. The method of claim 7, wherein the chemotherapy agent is a taxane.

9. The method of claim 8, wherein the taxane is paclitaxel.

10. The method of claim 1, wherein the IMP321 is administered to the subject at a dose of more than 6.25 mg.

11. The method of claim 1, wherein the IMP321 is administered to the subject at a dose of about 30 mg.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 represents fluorescence-activated cell sorting (FACS) analysis of monocytes (i.e. CD14.sup.+CD45.sup.+ cells) in PBMCs from from metastatic breast carcinoma patients.

(2) FIG. 2 represents fluorescence-activated cell sorting (FACS) analysis of monocytes (i.e. CD14.sup.+CD45.sup.+ cells) in fresh whole blood from metastatic breast carcinoma patients.

(3) FIG. 3 represents fluorescence-activated cell sorting (FACS) analysis of monocytes (i.e. CD14.sup.+CD45.sup.+ cells) in fresh whole blood from metastatic renal cell cancer patients.

(4) FIG. 4 represents fluorescence-activated cell sorting (FACS) analysis of monocytes (i.e. CD14.sup.+CD45.sup.+ cells) in fresh whole blood from metastatic breast carcinoma patients.

(5) FIG. 5 represents the pharmacokinetic profiles of IMP321 measured by ELISA in the plasma of metastatic renal cell cancer patients.

(6) FIG. 6 represents the flow cytometry analysis of PBMC cultured in different conditions with rituximab and/or IMP321.

EXAMPLES

Example 1: Monocytes Increase in Metastatic Breast Cancer (MBC) Patients Using Low IMP321 Dose

(7) Five MBC patients, receiving chemotherapy known to induce tumour cell apoptosis, each received one subcutaneous IMP321 dose of 0.25 mg 1-2 days after chemotherapy every other week, for 24 weeks, separated by 14-day administration-free intervals.

(8) Blood samples were collected in heparinated lithium tubes (Vacutainer; BD Biosciences) from each patient, 14 days after the last IMP321 injection (i.e. looking at lasting immunomodulatory effects of the product), at 3 months (Day 85) and 6 months (Day 170). PBMCs were isolated on Ficoll-Paque gradient (Pharmacia) using LeucoSep tubes (Greiner Bio-One), and used immediately.

(9) The increase in number of monocytes was analysed by fluorescence-activated cell sorting (FACS) in said fresh PBMC samples (because monocytes are sensitive to freezing), and compared with the monocyte counts carried out on fresh PBMC samples collected before IMP321 administration (Day 1).

(10) The results are represented in FIG. 1.

(11) The results showed a 2.5-fold (at 3 months, Day 85) and a 3.5-fold (at 6 months, Day 170) mean increase of monocyte counts at this low IMP321 dose clinical protocol.

(12) In order to confirm the above results, a more direct and probably more accurate approach was carried out, which was to quantify directly ex-vivo the number of monocytes in whole blood (i.e. without prior purification of PBMCs on Ficoll gradient) by first measuring the exact volume of blood to be analyzed with diluted fluorescent beads and then counting the number of CD14.sup.+ cells (i.e. monocytes) in the gated CD45.sup.+ (leukocytes) cells present in this whole blood volume.

(13) The results are represented in FIG. 2.

(14) The results showed a 4.4-fold mean increase at Day 170 (2.8-fold at Day 85) when IMP321 was given at low dose (0.25 mg) for a long period of time, 6 months, with 12 injections, showing strong and direct stimulation of the targeted MHC class II.sup.+ monocyte-like hematopoietic cells.

Example 2: Monocytes Increase in Metastatic Renal Clear-Cell Carcinoma (MRCC) Patients Using High IMP321 Dose

(15) Three MRCC patients each received one subcutaneous IMP321 dose of 6.25 mg every other week, for 12 weeks, separated by 14-day administration-free intervals.

(16) Blood samples were collected as described above from each patient, days after the last IMP321 injection (i.e. looking at lasting immunomodulatory effects of the product), at 2 months (Day 57) and 3 months (Day 85), and used immediately.

(17) The expansion of CD14.sup.+CD45.sup.+ cells was analysed by FACS in fresh blood samples (because monocytes are sensitive to freezing), and compared with the monocyte counts carried out on fresh blood samples collected before IMP321 administration (Day 1).

(18) The results are represented in FIG. 3.

(19) The results showed a 2-fold (at 3 months, Day 85) mean increase of monocyte counts with this high IMP321 dose clinical protocol where patients received only 6 injections.

Example 3: Monocytes Increase in Metastatic Breast Carcinoma Patients Receiving Paclitaxel and IMP321 Doses

(20) Patients receiving as a first line chemotherapy for metastatic breast carcinoma 6 cycles of paclitaxel (80 mg/m.sup.2 given i.v.) on days 1, 8, and 15 of a 28 day cycle, received 1-30 mg s.c. (sub-cutaneous) IMP321 on days 2 and 16 of each 28-day cycle. Alternatively, IMP321 was administered at days 3 or 17.

(21) Accordingly, each patient received a standard 6-month course of weekly paclitaxel with 12 s.c. injections of IMP321, each injection being given one to two days after paclitaxel administration on an every-two-week schedule.

(22) The increase in absolute monocyte counts per microliter of fresh blood was analysed by fluorescence-activated cell sorting (FACS), 14 days after the last injection, at 3 months (Day 85) and 6 months (Day 170) compared to day 1.

(23) The results obtained in patients injected with a low dose IMP321 (1.25 mg) are represented in FIG. 4.

(24) These data showed that doses of 1.25 mg in most if not all patients (FIG. 4) induce an expansion of the monocyte subset pool in the blood.

(25) It is predicted that the optimal dose regimen for IMP321 will be between 6 and 30 mg/injection.

(26) These doses have been shown to be safe and give an acceptable systemic exposure based on the results of pharmacokinetics data obtained in metastatic renal cell cancer patients (FIG. 5). A blood concentration of IMP321 superior to 1 ng/ml for at least 24 hours after s.c. injection could be obtained in patients injected by IMP321 doses of more than 6 mg (FIG. 5).

Example 4: Treatment of Advanced Pancreas Cancer Patients Receiving Gemcitabine and IMP321 Doses

(27) Patients, receiving as a first line chemotherapy for advanced pancreas cancer (or patients not eligible for surgical removal of the tumor) 6 cycles of standard gemcitabine (1 gm/m.sup.2 given i.v. over 30 min) on days 1, 8, and 15 of a 28 day cycle, receive in addition 6 to 30 mg s.c. IMP321 on days 2 and 16 of each 28-day cycle. Alternatively, IMP321 is administered at days 3 or 17.

(28) Accordingly, each patient receives a standard 6-month course of gemcitabine with 12 s.c. injections of IMP321, each injection being given one to two days after gemcitabine administration on an every-two-week schedule.

(29) The number of monocytes is analysed by fluorescence-activated cell sorting (FACS) as in example 1.

Example 5: Induction of Increased Rituximab-Mediated ADCC by Low Doses IMP321

(30) PBMCs are first incubated for 40 hours with IL-2 (100 U/ml), with or without IMP321 (at the concentrations 0 μg/m, 0.03 μg/ml or 0.1 μg/ml). PBMCs are then incubated with increasing concentrations of rituximab (0, 0.5 and 5 μg/ml) in the presence of target cells (i.e. human CD20.sup.+ Raji B cells).

(31) Raji cells were first labeled with CFSE (carboxy-fluorescein succinimidyl ester), incubated in medium with rituximab at 0, 0.5 or 5 μg/ml and cocultured with effector cells at an effector-target ratio of 25:1 for 6 hours at 37° C.

(32) The cells were then incubated with 7-AAD (7-amino-actinomycin-D) for 15 min on ice and analyzed by flow cytometry to determine the percentage of dead CFSE.sup.+7-AAD.sup.+ Raji target cells (i.e. % of cytotoxicity).

(33) The results are presented in FIG. 6.

(34) Increasing the concentration of rituximab increased the percentage of cytotoxicity, clearly showing a dose-dependent ADCC activity.

(35) When 0.03 or 0.1 μg/ml IMP321 is added, the percentage of cytotoxicity greatly increased. For instance, a 30% cytotoxicity is observed with 0.5 μg/ml rituximab in the presence of 0.1 μg/ml IMP321 which is superior to the 25% cytotoxicity value obtained with 5 μg/ml rituximab in the absence of IMP321.

(36) Thus, adding 0.1 μg/ml IMP321 potentializes 10-15 fold the activity of rituximab because a superior cytotoxicity is obtained with 10 time less antibody when a low dose IMP321 (0.1 μg/ml) is added.

(37) These data show the synergistic effect between rituximab and IMP321.

REFERENCE LIST

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