Treatment of B cell malignancies using afucosylated pro-apoptotic anti-CD19 antibodies in combination with anti CD20 antibodies or chemotherapeutics

Abstract

The invention relates to novel combination therapies involving anti-CD19 antibodies for the treatment of cancer B cells expressing CD19. One preferred method is where the anti-CD19 proapoptotic MAb or a Fc optimized proapototic humanized MAb. In the methods of the present invention some anti-CD20 agents such as Rituxan®, or chemodrugs such as vincristine may be used in combitherapy. The methods of the present invention reduce the levels of B CD19 positive, more particularly in all diffuse large B cells lymphoma (DLBCL) subtypes and in Follicular lymphomas (FL).

Claims

1. Humanized monoclonal anti-CD19 antibody, having the following CDRs: TABLE-US-00020 Sequence SEQ SEQ SEQ (Common ID Sequence ID Sequence ID numbering NO: IMGT NO: Kabat NO: system) VH IDD001 CDR1 5 GYAFSSYW 11 SYWVN 16 SSYW CDR2 6 IYPGDGDT 12 QIYPGDGDT 6 IYPGDGDT NYNGKFKG CDR3 7 ARSITTVV 13 SITTVVGCA 13 SITTVVGC GCAMDY MDY AMDY VL IDD001 CDR1 41 QSINNW 42 KASQSINNW 41 QSINNW LA CDR2 43 GAS 44 GASTLET 43 GAS CDR3 10 QQSWNTP 10 QQSWNTPW 10 QQSWNT WT T PWT.

2. The humanized monoclonal anti-CD19 antibody of claim 1, having the VH sequence 1-121 of SEQ ID NO: 39 and the VL sequence 1-103 of SEQ ID NO: 37.

3. The humanized monoclonal anti-CD19 antibody of claim 1, having a Heavy Chain of SEQ ID NO: 39 and a Light Chain of SEQ ID NO: 37.

4. A method for treating Diffuse Large B Cell Lymphoma (DLBCL) in a patient in need thereof, comprising administering an efficient amount of a monoclonal antibody according to claim 1.

5. The method of claim 4, wherein said antibody has the VH sequence 1-121 of SEQ ID NO: 39 and the VL sequence 1-103 of SEQ ID NO: 37.

6. The method of claim 4, wherein said antibody has a Heavy Chain of SEQ ID NO: 39 and a Light Chain of SEQ ID NO: 37.

7. A method for treating Follicular Lymphoma (FL) in a patient in need thereof, comprising administering an efficient amount of a monoclonal antibody according to claim 1.

8. A method of claim 7, wherein said antibody has the VH sequence 1-121 of SEQ ID NO: 39 and the VL sequence 1-103 of SEQ ID NO: 37.

9. A method of claim 7, wherein said antibody has a Heavy Chain of SEQ ID NO: 39 and a Light Chain of SEQ ID NO: 37.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1D: The nucleotide and amino acids sequences of the murine MAbs R005-1 and R005-2 MAbs, (A): V.sub.H (B): VL. Amino acids are shown as one-letter codes.

(2) TABLE-US-00008 Amino acid Nucleic acid Amino acid Nucleic acid sequence VH sequence VH sequence VL sequence VL mR005-1 SEQ ID SEQ ID SEQ ID SEQ ID NO: 29 NO: 30 NO: 31 NO: 32 mR005-2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 33 NO: 34 NO: 35 NO: 36

(3) FIGS. 2A-2F: The amino acids and nucleic acid sequences of chimeric Fc24 (SEQ ID NO: 3 and 4) or Fc34 (SEQ ID NO: 1 and 2) variant MAb. The sequences of Fc39 is the same as sequence of Fc24, with an Ile at position 305. Amino acids are shown as one-letter codes.

(4) According to the literature, the amino acid numbering of Fc region is based to the Kabat data base, (CH1: aa n° 118 to 215; Hinge: aa n° 216 to 230; CH2: aa n° 231 to 340; CH3: aa n° 341 to 447). Variations between the various Fc used in the invention with respect to native Fc (Fc0):

(5) TABLE-US-00009 Name of the mutant Mutations with respect to native human IgG1 Fc (Fc0) Fc34 F243L/R292P/Y300L/V305L/K326A/P396L Fc24 F243L/R292P/Y300L/V305L/K326A/E333A/P396L Fc39 F243L/R292P/Y300L/V305I/K326A/E333A/P396L

(6) FIG. 3: In vivo inhibition of tumour growth of DLBCL subtype ABC with MAb anti-CD19/IDD001. 3A: xenograft model LY0257; 3B: xenograft model LY2264; 3C: xenograft model LY3604; 3D: xenograft model LY2264 with dose variations.

(7) FIG. 4: In vivo inhibition of tumour growth of DLBCL subtype GCB with MAb anti-CD19/IDD001. 4A: xenograft model LY2214; 4B: xenograft model LY2318; 4C: xenograft model LY2214 with dose variations.

(8) FIG. 5: In vivo inhibition of tumour growth of DLBCL subtype ABC/G CB with MAb anti-CD19/IDD001. 5A: xenograft model LY2345; 5B: xenograft model LY2266; 5C: xenograft model LY2345 with dose variations.

(9) FIG. 6: In vivo inhibition of tumour growth of DLBCL ABC subtype with MAb anti-CD19/IDD001 in combination with vincristine.

(10) FIG. 7: In vivo inhibition of tumour growth of DLBCL ABC subtype with MAb anti-CD19/IDD001 in combination with doxorubicine.

(11) FIG. 8: In vivo inhibition of tumour growth of FL with MAb anti-CD19/IDD001 in combination with MAb anti-CD20 (rituximab)

(12) FIG. 9: Co-targeting CD19 and CD20 increases the survival percentage with lower MAb concentration.

(13) FIGS. 10A-10B: Light and Heavy humanized sequences, antibody IDD001.

(14) Example 1: The anti-CD19 antibodies were produced as described in WO 2012/010562 or U.S. Pat. No. 9,120,856.

(15) The murine MAb screening regarding the ability to trigger the highest level of programmed cell death or apoptosis was performed. Two MAbs anti CD19 were selected with the nucleotide and amino acids sequences of the murine MAbs R005-1 and R005-2 MAbs described in FIG. 1.

(16) To improve the cancer cell depleting, Fc engineering was performed to enhance cytotoxicity potency dependent on ADCC and CDC. The amino acids and nucleic acid sequences of optimised Fc codified Fc24 (SEQ ID NO: 3 and 4) or Fc34 (SEQ ID NO: 1 and 2) variant MAb were described as one-letter codes in FIG. 2. According to the literature, the amino acid numbering of Fc region is based to the Kabat data base, (CH1: aa n° 118 to 215; Hinge: aa n° 216 to 230; CH2: aa n° 231 to 340; CH3: aa n° 341 to 447). Variations between the various Fc used in the invention with respect to native Fc (Fc0).

(17) The full sequence of the Fc optimised humanised MAb IDD001 is described in FIG. 10.

(18) Example 2: Different amino acid substitutions in the Fc region:

(19) In these experiments, several alternative monoclonal antibodies were generated based on the chR005-1 Fc24 of example 1. This antibody has variable regions binding to CD19 and an IgG1 Fc region comprising the mutations F243UR292P/Y300L/V305L/K326A/E333A/P396L. Variants of this antibody have thus been prepared wherein one substitution amino acid has been changed.

(20) These variants were assayed for CDC in the presence of human serum and ADCC in whole blood.

(21) The results obtained are presented in a Jan. 11, 2016 Declaration under Rule 132 in the File History of U.S. application Ser. No. 13/811,134, which Declaration is incorporated herein by reference. The results show that the following variant substitutions are functional on ADCC whole blood and CDC in human serum, with no significant impact being observed with respect to the original substitution of IDD001:

(22) F243: L could be subsituted by W, Y, R, Q, I, A

(23) R292: P could be subsituted by G or A

(24) V305: L (or I) could be subsituted by A

(25) P396: L could be subsituted by I or A

(26) Y300: L could be subsituted by K, F, I, A

(27) K326: A could be subsituted by V, E, D, M, S, N, W

(28) E333: A could be subsituted by V, G, D, K, S, N, R, Q.

(29) Thus the person skilled in the art may appreciate that substitutions may be made in the Fc region while keeping substantially the effector functions of the Fc or the antibody comprising the Fc, and that these substitutions are encompassed by the present invention.

(30) Example 3: Rituxan® (rituximab) was purchased commercially by Roche.

(31) Example 4: Oncovin® (vincristine) was purchased commercially by Selleck (Catalog number: S1241)

(32) Example 5: Adriamycine® (doxorubicine) was purchased commercially by Selleck (Catalog number: S1208)

(33) Example 6: In vivo investigation on PDX models for DLBCL: Each mouse was inoculated subcutaneously at the right flank with one primary human tumor xenograft model (LY0257, LY2214, LY2264, LY2266, LY2318, LY2345 orLY3604) tumor fragment (2-3 mm in diameter) for tumour development. When average or individual tumor size reaches 100-250 mm3, mice was randomly (rolling enrollment will be involved if necessary) allocated into 3 groups. Each group contained 1 or 2 mice. The day of grouping and dosing initiation was denoted as day 0. The dosing volume was adjusted for body weight (Dosing volume=5 μL/g). After tumor inoculation, the animals were checked daily for morbidity and mortality. At the time of routine monitoring, the animals were checked for any effects of tumor growth and treatments on normal behavior such as mobility, food and water consumption, body weight gain/loss, eye/hair matting and any other abnormal effect. Death and observed clinical signs were recorded on the basis of the numbers of animals within each subset. Two weeks of dosing-free observation were applied after final treatment. The animals in vehicle group were sacrificed before study termination because of tumor volume (TV) over 3000 mm.sup.3. Tumor size was measured by caliper twice weekly in two dimensions. The tumor volume was expressed in mm3 using the formula: TV=0.5 a×b2 where a and b are the long and short diameters of the tumor, respectively. Body weight was measured twice weekly. When individual mouse has a body weight loss ≥15%, the mouse was given dosing holiday(s) until its body weight recovers to body weight loss. Under following conditions, the in-life experiment of individual animal or whole groups was terminated, by human euthanization, prior to death, or before reaching a comatose state.

(34) DLBCL in vivo proof of concept from patient derivates (PDX models): 7 HuPrime® lymphoma xenograft models (selected in CRO cell bank CD19+ for cDNA), LY0257, LY2214, LY2264, LY2266, LY2318, LY2345 and LY3604, were selected. The cancer subtype of those models is listed in the table below.

(35) TABLE-US-00010 TABLE 1 Study design of MAb impact regarding DLBCL subtype Model nomenclature Subtype LY0257 NHL (DLBCL, ABC, MYD88 L265P) LY2214 NHL (DLBCL, GCB) LY2264 NHL (DLBCL, ABC, MYD88 L265P) LY2266 NHL (B cell, ABC/GCB) LY2318 NHL (DLBCL, GCB) LY2345 NHL (DLBCL, ABC/GCB) LY3604 NHL (DLBCL, ABC)

(36) Study design for MAb impact regarding DLBCL subtypes Seven HuPrime® DLBCL lymphoma xenograft models as ABC (LY0257, LY2264, LY3604), GCB (LY2214, LY2318) or ABC/GCB (LY2345, LY2266) were tested.

(37) TABLE-US-00011 TABLE 2 Study design of MAb impact regarding DLBCL subtype Dose level Dose Dosing Group N Treatment (mg/kg) Route Frequency 1 1 No treatment NA NA BIW × 4 or irrelevant human IgG 2 2 IDD001 10 i.v. BIW × 4 MAb anti CD19 N: animal number per group

(38) Study design for MAb dose effect according the DLBCL subtypes Three HuPrime® DLBCL lymphoma xenograft models as ABC (LY2264), GCB (LY2214), or ABC/GCB (LY2345) were tested.

(39) TABLE-US-00012 TABLE 3 Study design of MAb dosing efficacy study for ABC/LY2264 and GCB/LY2214 in Nod SCID mice Dose level Dose Dosing Group N Treatment (mg/kg) Route Frequency 1 3 No treatment — — BIW × 4 or irrelevant human IgG 2 3 IDD001 10   i.v. BIW × 4 3 3 MAb anti  1   i.v. BIW × 4 4 3 CD19  0.1 i.v. BIW × 4

(40) TABLE-US-00013 TABLE 4 Study design of of MAb dosing efficacy study for ABC/GCB LY2345 in NPG mice Dose level Dose Dosing Group N Treatment (mg/kg) Route Frequency 1 3 No treatment — — BIW × 4 or irrelevant human IgG 2 3 IDD001 10 i.v. BIW × 4 3 3 MAb anti 25 i.v. BIW × 4 4 3 CD19 50 i.v. BIW × 4 Note: N: animal number per group

(41) Study design for Combitherapy for DLBCL ABC subtype The HuPrime® DLBCL lymphoma xenograft model ABC (LY0257) was tested.

(42) TABLE-US-00014 TABLE 5 Study design of combitherapy for LY0257 in BALB/c nude mice Dose level Dose Dosing Group N Treatment (mg/kg) Route Frequency 1 3 BIW × 4 — — BIW × 4 2 3 IDD001 10   i. v. BIW × 4 MAb anti CD19 3 3 Vincristine  0.2 i.p. QD × 7 4 3 Doxorubicin  5   i.v. Q3D × 4 5 3 IDD001 10   i.v. BIW.4 MAb anti CD19 Vincristine  0.2 i.p. QD × 7 6 3 IDD001 10   i.v. BIW × 4 MAb anti CD19 Doxorubicin  5   i.v. Q3D × 4

(43) In vivo inhibition of tumor growth of DLBCL subtypes with MAb anti-CD19

(44) Results show in FIG. 3 revealed that DLBCL-ABC lymphoma was reduced partially as exemplified from three independent human tumors in PDX grafted mice produced. 3A: xenograft model LY0257; 3B: xenograft model LY2264; 3C: xenograft model LY3604. Following the dose-dependent manner, IDD001 mediated cytotoxicity of LY2264 PDX model was very sensitive at the highest MAb dose of 10 mg/kg. 3D: xenograft model LY2264 with dose variations.

(45) Results show in FIG. 4 revealed that IDD001 mediated cytotoxicity against two DLBCL-GCB lymphoma at varying degrees of cell killing. The LY2214 PDX model was very sensitive, whereas the LY2318 PDX model was moderately sensitive. 4A: xenograft model LY2214; 4B: xenograft model LY2318. Following the dose-dependent manner, IDD001 mediated cytotoxicity of LY2214 PDX model was very sensitive at the highest MAb dose of 10 mg/kg. 4C: xenograft model LY2214 with dose variations.

(46) Results show in FIG. 5 revealed that DLBCL-ABC/GCB lymphoma was reduced partially 5A: xenograft model LY2345; 5B: xenograft model LY2266. Even at highest MAb concentration (50 mg/ml), no complete tumor regression was observed. 5C: xenograft model LY2345 with dose variations.

(47) In vivo inhibition of tumor growth of Diffuse Large B cell lymphoma (DLBCL) subtypes with combitherapy based on MAb anti-CD19 and chemodrugs

(48) In order to enhance tumor regression, the cytotoxic effects of a combination of IDD001 and Vincristine were presented in FIG. 6. The combitherapy with IDD001 and vincristine of LY0257 DLBCL-ABC model produced synergistic cell killing compared with either single agent.

(49) By contrast results show in FIG. 7 revealed that no similar stronger growth tumor control was observed following the combitherapy with IDD001 and doxorubicine of LY0257 DLBCL-ABC model.

(50) Example 7: In vivo investigation on CDX models for FL

(51) Human follicular cell line RL was subcutaneously injected in SCID mice, with a concentration of 5.10.sup.6 cells per injection (200 μL). Mice were randomized when the tumors reached a mean volume of about 100 mm.sup.3 for the 9 groups (total 45 mice). All the mice were observed in order to detect any toxic effects of the product. The endpoint was defined by animal ethics as a tumor diameter of >18 mm, significant weight loss or alteration of animal well-being. In order to assess the effectiveness of the compounds on tumorigenesis, tumor volume was measured two times a week. The sizes of the primary tumors were measured using calipers and the tumor volume (TV) was extrapolated to a sphere using the formula TV=4/3 λ×r.sup.3, by calculating the mean radius from the two measurements. The median and standard deviation were also calculated for each group. Median is preferred to mean in order to exclude the extreme values. MAb treatment was administered by intraperitoneal injection twice a week during three weeks at 30, 10 or 1 mg/kg doses. The product was prepared in accordance with the sponsor's guidelines, i.e. diluted in PBS. Mice were sacrificed when the tumors reached a maximum volume of 1600 mm.sup.3. The endpoints were defined by clinical trial ethics as a tumor diameter of >18 mm or weight loss of >10% of body weight, or when the tumors are dangerous for mice (necrosis). Statistical analysis was performed with GraphPad Prism software. GraphPad Prism combined scientific graphing, comprehensive curve fitting, understandable statistics, and data organization. The t-test (two-tailed test) was performed on the tumor volume values (mm.sup.3) measured on the day of sacrifice.

(52) Results shown in FIG. 8 that the combination treatment resulted in additive cytotoxicity of tumour growth of FL with MAb anti-CD19/IDD001 in combination with MAb anti-CD20 (rituximab).

(53) Co-targeting CD19 and CD20 increased the survival percentage. Whereas 33% of mice treated with rituximab at 1 mg/kg are in complete remission, 50% mice treated with rituximab and IDD001 are in complete remission at day 45. Detailed values of survival at day 55 are presented in the following table:

(54) TABLE-US-00015 Tumor Complete Identification progression Remission Day  55 55 control 100  0 rituximab-1 mg/kg  67 33 IDD001-1 mg/kg 100  0 rituximab-1 mg/kg  50 50 +IDD001-1 mg/kg

(55) The invention will now be described by the following numbered paragraphs: 1- A pharmaceutical composition comprising an anti-CD19 antibody which comprises (i) the CDRs of one of the antibodies R005-1, R005-2 or IDD001 whose VH and VL amino acid sequences are depicted on the following table,

(56) TABLE-US-00016 Amino acid sequence VH Amino acid sequence VL R005-1 SEQ ID NO: 29 SEQ ID NO: 31 R005-2 SEQ ID NO: 33 SEQ ID NO: 35 IDD001 SEQ ID NO: 39 SEQ ID NO: 37

(57) (2i) a variant human IgG1 Fc region, wherein this variant region comprises an amino acid substitution at each of the amino acid positions 243, 292, 300, 305, 396, or 243, 292, 300, 305, 326, 396 or 243, 292, 300, 305, 326, 333, 396 of the human IgG Fc region, wherein the numbering of the amino acid residues in the Fc region is the one of the Kabat, for use in treating Diffuse Large B Cell Lymphoma (DLBCL) expressing CD19 or Follicular Lymphoma (FL) expressing CD19 in a patient in need thereof.

(58) 2—A combination of (1) an anti-CD19 antibody comprising (i) the CDRs of one of the antibodies R005-1, R005-2 or IDD001 whose VH and VL amino acid sequences are depicted on the following table,

(59) TABLE-US-00017 Amino acid sequence VH Amino acid sequence VL R005-1 SEQ ID NO: 29 SEQ ID NO: 31 R005-2 SEQ ID NO: 33 SEQ ID NO: 35 IDD001 SEQ ID NO: 39 SEQ ID NO: 37

(60) (2i) a variant human IgG1 Fc region, wherein this variant region comprises an amino acid substitution at each of the amino acid positions 243, 292, 300, 305, 396, or 243, 292, 300, 305, 326, 396 or 243, 292, 300, 305, 326, 333, 396 of the human IgG Fc region, wherein the numbering of the amino acid residues in the Fc region is the one of the Kabat, and (2) a chemotherapeutic drug of the vinca alcloid group, for use in treating Diffuse Large B Cell Lymphoma (DLBCL) expressing CD19 in a patient in need thereof.

(61) 3—The combination for the use of paragraph 2, wherein the chemotherapeutic drug is vincristine.

(62) 4—The combination for the use of paragraph 2 or 3, which is for use to treat DLBCL subtype GCB, ABC or ABC/GCB.

(63) 5—The combination for the use of any one of paragraphs 2 to 4, which is for use in combination with vincristine to treat DLBCL subtype ABC or GCB or ABC/GCB, wherein the combined use of the antibody and the chemotherapeutic drug is simultaneous, separate or sequential.

(64) 6—The combination for the use of any one of paragraphs 2 to 5, wherein the anti-CD19 antibody in combination with vincristine leads to higher rate of regression compared with either single agent.

(65) 7—A combination of (1) an anti-CD19 antibody comprising (i) the CDRs of one of the antibodies R005-1, R005-2 or IDD001 whose VH and VL amino acid sequences are depicted on the following table,

(66) TABLE-US-00018 Amino acid sequence VH Amino acid sequence VL R005-1 SEQ ID NO: 29 SEQ ID NO: 31 R005-2 SEQ ID NO: 33 SEQ ID NO: 35 IDD001 SEQ ID NO: 39 SEQ ID NO: 37

(67) (2i) a variant human IgG1 Fc region, wherein this variant region comprises an amino acid substitution at each of the amino acid positions 243, 292, 300, 305, 396, or 243, 292, 300, 305, 326, 396 or 243, 292, 300, 305, 326, 333, 396 of the human IgG Fc region, wherein the numbering of the amino acid residues in the Fc region is the one of the Kabat, and (2) an anti-CD20 antibody for use in treating Follicular Lymphoma (FL) expressing CD19 and CD20 in a patient in need thereof.

(68) 8—The combination for the use of paragraph 8, wherein the anti-CD20 antibody is rituxan.

(69) 9—The combination for the use of paragraph 7 or, wherein the combination of both antibodies leads to a higher rate of regression or remission compared with either single agent.

(70) 10—The combination for the use of any one of paragraphs 2 to 9, wherein the anti-CD19 antibody comprises VH and VL amino acid sequences are depicted on the following table,

(71) TABLE-US-00019 Amino acid sequence VH Amino acid sequence VL R005-1 SEQ ID NO: 29 SEQ ID NO: 31 R005-2 SEQ ID NO: 33 SEQ ID NO: 35 IDD001 SEQ ID NO: 39 SEQ ID NO: 37

(72) 11—The composition or combination for the use of any one of paragraphs 1 to 10, wherein the antibody comprises an Fc region in which Phe243 is substituted by Leu, Arg292 is substituted by Pro, Tyr300 is substituted by Leu, Va1305 is substituted by Leu or Ile, Lys326 is substituted by Ala and Pro396 is substituted by Leu.

(73) 12—The composition or combination for the use of any one of paragraphs 1 to 10, wherein the antibody comprises an Fc region in which Phe243 is substituted by Leu, Arg292 is substituted by Pro, Tyr300 is substituted by Leu, Va1305 is substituted by Leu or Ile, Lys326 is substituted by Ala and Pro396 is substituted by Leu, and Glu333 is substituted by Ala.

(74) 13—Humanized monoclonal anti-CD19 antibody, having the VH sequence SEQ ID NO: 39 and the VL sequence SEQ ID NO: 37.