TREATMENT OF PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA

Abstract

The present invention relates to a method for the treatment, amelioration or elimination of pediatric acute lymphoblastic leukemia (ALL), the method comprising the administration of a pharmaceutical composition comprising a CD19×CD3 bispecific single chain antibody construct to a pediatric ALL patient in the need thereof.

Claims

1. A method for achieving minimal residual disease (MRD) negativity in a patient diagnosed with acute lymphoblastic leukemia (ALL), the method comprising administering to the patient a composition comprising a CD19×CD3 bispecific single chain antibody construct comprising (a) an anti-CD19 heavy chain complementarity determining region (CDR)1 amino acid sequence set forth in SEQ ID NO: 14, an anti-CD19 heavy chain CDR2 amino acid sequence set forth in SEQ ID NO: 15, an anti-CD19 heavy chain CDR3 amino acid sequence set forth in SEQ ID NO: 16, and an anti-CD19 light chain CDR1 amino acid sequence set forth in SEQ ID NO: 11 an anti-CD19 light chain CDR2 amino acid sequence set forth in SEQ ID NO: 12, and an anti-CD19 light chain CDR3 amino acid sequence set forth in SEQ ID NO: 13; and (b) an anti-CD3 heavy chain CDR1 amino acid sequence set forth in SEQ ID NO: 17, an anti-CD3 heavy chain CDR2 amino acid sequence set forth in SEQ ID NO: 18, an anti-CD3 heavy chain CDR3 amino acid sequence set forth in SEQ ID NO: 19, and an anti-CD3 light chain CDR1 amino acid sequence set forth in SEQ ID NO: 20, an anti-CD3 light chain CDR2 amino acid sequence set forth in SEQ ID NO: 21, and an anti-CD3 light chain CDR3 amino acid sequence set forth in SEQ ID NO: 22 in a daily constant dose of 10 μg to 100 μg per square meter patient body surface area as a continuous infusion for at least four weeks.

2. The method of claim 1, wherein the acute lymphoblastic leukemia (ALL) is B-lineage acute lymphoblastic leukemia.

3. The method of claim 1, wherein the acute lymphoblastic leukemia (ALL) is refractory to chemotherapy in patients non-eligible for allogeneic hematopoietic stem cell transplantation.

4-7. (canceled)

8. The method of claim 1, wherein MRD negativity is measured with quantitative detection of at least one cytogenetic abnormality or rearrangement selected from the group consisting of: t(12;21)[TEL-AML1]; t(1;19;)[E2A-PBX]; t(4;11)[AF4-MLL]; t(9;22)[BCR-ABL]; hyperdiploidy or trisomies of chromosomes 4, 10, and 17; hyperdiploidy or trisomy of chromosome 4; hyperdiploidy or trisomy of chromosome 10; hyperdiploidy or trisomy of chromosome 17; hypodiploidy; rearrangements of an immunoglobulin gene; and a T-cell receptor (TCR) rearrangement.

9. The method of claim 8, wherein the cytogenic abnormality or rearrangement is detected by at least one marker with a signal with a sensitivity of greater than or equal to one in ten thousand cells.

10. (canceled)

11. The method of claim 1, wherein the corresponding variable heavy chain regions (V.sub.H) and the corresponding variable light chain regions (V.sub.L) regions in the CD19×CD3 bispecific single chain antibody construct are arranged, from N-terminus to C-terminus, in the order, V.sub.L(CD19)−V.sub.H(CD19)−V.sub.H(CD3)−V.sub.L(CD3).

12. The method of claim 1, wherein the CD19×CD3 bispecific single chain antibody construct comprises an amino acid sequence comprising at least 90% identity to the amino acid sequence of SEQ ID NO. 1.

13. The method of claim 1, wherein the continuous infusion for at least four weeks is followed by a 2-week treatment-free interval.

14. The method of claim 13, wherein the treatment by continuous infusion is repeated at least three times after determination of a MRD negative status.

15. (canceled)

16. The method of claim 1, wherein the CD19×CD3 bispecific single chain antibody construct is administered in a daily dose of 15 μg to 30 μg per square meter patient body surface area.

17. (canceled)

18. The method of claim 1, wherein the CD19×CD3 bispecific single chain antibody construct comprises a CD19 VH amino acid sequence as set forth in SEQ ID NO: 3 and/or a CD19 VL amino acid sequence as set forth in SEQ ID NO: 5.

19. The method of claim 1, wherein the CD19×CD3 bispecific single chain antibody construct comprises a CD3 VH amino acid sequence as set forth in SEQ ID NO: 7 and/or a CD3 VL amino acid sequence as set forth in SEQ ID NO: 9.

20. The method of claim 1, wherein the CD19×CD3 bispecific single chain antibody construct comprises a CD19 VH amino acid sequence as set forth in SEQ ID NO: 3, a CD19 VL amino acid sequence as set forth in SEQ ID NO: 5, and/or a CD3 VH amino acid sequence as set forth in SEQ ID NO: 7 and a CD3 VL amino acid sequence as set forth in SEQ ID NO: 9.

21. The method of claim 1, wherein the CD19×CD3 bispecific single chain antibody construct comprises the amino acid sequence set forth in SEQ ID NO: 1.

22. The method of claim 2, wherein the B-lineage ALL is pediatric B precursor ALL.

23. The method of claim 22, wherein the pediatric B-precursor ALL is pediatric pro-B ALL, pre-B ALL, or common ALL (cALL).

24. The method of claim 23, wherein the pediatric B precursor ALL is common ALL (cALL).

25. The method of claim 1, wherein the patient has no signs of graft versus host disease (GVHD).

26. The method of claim 1, wherein the patient does not suffer from adverse side effects resulting from the treatment.

27. The method of claim 1, wherein MRD negativity is measured as less than 1 leukemia cell per 10,000 bone marrow cells.

Description

[0143] The Figures show:

[0144] FIG. 1: CD19×CD3 bispecific single chain antibody (CD19×CD3 bscab) mode of action. CD19×CD3 bispecific single chain antibody (SEQ ID NO. 1) redirects CD3-positive cytotoxic T cells to eliminate human acute lymphoblastic leukemia cells carrying the CD19 antigen.

[0145] FIG. 2: Failure of conventional ALL therapy in a 7-year old patient with common ALL. One year after HSCT, the pediatric patient experienced a second bone marrow relapse and received subsequent chemotherapy including 1 cycle of Clofarabine/Cyclophosphamide/VP16, 2 cycles of Amsacrine, VP16, prednisone and 1 cycle of Melphalan/Cytarabine. Despite this aggressive chemotherapy, the patient had persistent morphologic disease throughout treatment, as demonstrated by the high MRD levels. Left y-axis indicates FACS-MRD %, right y-axis indicates PCR-MRD (from 10.sup.0 to <10.sup.−4), x-axis indicates days from first HSCT; see Example 4.

[0146] FIG. 3: Successful treatment of the pediatric patient by administration of CD19×CD3 bispecific single chain antibody (CD19×CD3 bscab; SEQ ID NO. 1), as demonstrated by MRD prior, during and after treatment with said antibody. After failure of conventional ALL therapy, the patient was treated with a continuous infusion of 15 μg/m.sup.2 CD19×CD3 bispecific single chain antibody (SEQ ID NO. 1) for 5 weeks. Upon antibody treatment, MRD negativity could be reached. In October 2008, i.e. 2 weeks after end of treatment with the CD19×CD3 bispecific single chain antibody (day 0 in FIGS. 3 to 5), he underwent a second HSCT from his haploidentical mother using a non-myeloablative preparative regimen consisting of Clofarabine, Thiotepa and melphalan (Lang) indicated by the conditioning bar. Left y-axis indicates FACS-MRD %, right y-axis indicates PCR-MRD (from 10.sup.0 to <10.sup.−4), x-axis indicates days from first HSCT, day “0” indicates the day of the second HSCT (following the antibody treatment); see Example 4.

[0147] FIG. 4: Temporal development of blast counts before and after treatment with the CD19×CD3 bispecific single chain antibody (CD19×CD3 bscab; SEQ ID NO. 1). A bone marrow analysis at day 10 of CD19×CD3 bispecific single chain antibody treatment revealed a complete elimination of the bone marrow blasts (MRD<10.sup.−4) by the antibody. Another BM analysis at the end of CD19×CD3 bispecific single chain antibody treatment at day 35 confirmed complete absence of leukemic blasts from the bone marrow (MRD<10.sup.−4). Left y-axis indicates blast count by flow cytometry in %, right y-axis indicates blast count by microscopy in %, x-axis indicates days from HSCT, with negative days indicating days from first HSCT, day “0” indicating the day of the second HSCT (following the antibody treatment) and positive days indicating days after the second HSCT.

[0148] FIG. 5: Decrease of MRD levels upon treatment with CD19×CD3 bispecific single chain antibody (CD19×CD3 bscab; SEQ ID NO. 1). Upon antibody treatment, MRD negativity could be reached. As of June 2009, the patient remains in MRD-negative complete molecular remission. Left y-axis indicates FACS-MRD %, right y-axis indicates PCR-MRD (from 10.sup.0 to <10.sup.−4), x-axis indicates days from HSCT, with negative days indicating days from first HSCT and “0” indicating day of second HSCT and positive days indicating days from second HSCT.

[0149] The invention is further illustrated by the following examples:

EXAMPLES

1. CD19×CD3 Bispecific Single Chain Antibody

[0150] The generation, expression and cytotoxic activity of the CD19×CD3 bispecific single chain antibody has been described in WO 99/54440. The corresponding amino and nucleic acid sequences of the CD19×CD3 bispecific single chain antibody are shown in SEQ ID NOs. 1 and 2, respectively. The VH and VL regions of the CD3 binding domain of the CD19×CD3 bispecific single chain antibody are shown in SEQ ID NOs. 7 to 10, respectively, whereas the VH and VL regions of the CD19 binding domain of the CD19×CD3 bispecific single chain antibody are shown in SEQ ID NOs 3 to 6, respectively.

2. Phenotypic Analysis of Lymphocytes and Chimerism Analysis

[0151] For the phenotypic analysis of lymphocytes and chimerism analysis, the patient's blood samples were collected prior, during and after treatment with CD19×CD3 bispecific single chain antibody using EDTA-containing collection tubes. Absolute number of lymphocytes in the blood samples were determined through differential blood analysis on a Advia. Lymphocytes were stained using fluorescence-labeled antibodies directed against CD3, CD4, CD8, CD19 and CD56 (all obtained from Becton-Dickinson, Heidelberg, Germany). The analysis of labelled cells and data collection were performed with a FACSCalibur (Becton-Dickinson).

3. Detection of MRD

[0152] For the detection of MRD, either a polymerase chain reaction (PCR)-based assay (Bader et al., loc. cit.) or FACS analysis was used. Briefly, DNA was isolated by DNeasy Blood&Tissue Kit (Qiagen GmbH, Hilden, Germany). Immunoglobulin and T-cell receptor gene rearrangement as PCR-based targets have recently been shown to be stable markers for monitoring MRD in acute lymphoblastic leukemia after stem cell transplantation (Kreyenberg, Leukemia, 2009).

4. Case Reports

4.1 Case Report Patient 1

[0153] This 7-year old patient was diagnosed with high risk CD10+ common ALL (CD19−, CD34-positive; CD45 reduced; TCR rearrangement; CNS negative) in 2004. After treatment, he experienced a bone marrow relapse in June 2006 and was treated according to the ALL-REZ BFM study in the S3 arm. After two chemotherapy cycles, the patient had persistent disease and achieved a complete remission after 3 courses of clofarabine. In 2007, he received an allogeneic HSCT from a 9 out of ten HLA-allele matched unrelated donor after conditioning with total body irradiation and Etoposide. One year after HSCT, he experienced another bone marrow relapse and received subsequent chemotherapy including 1 cycle of Clofarabine/Cyclophosphamide/VP16 (Nobuko), 2 cycles of Amsacrine, VP16, prednisone (Hamburg) and 1 cycle of Melphalan/Cytarabine; see FIG. 2. Despite this aggressive chemotherapy, the patient had persistent morphologic disease throughout treatment, as evident from the high MRD levels in FIG. 2. He was then treated under compassionate use with a continuous infusion of 15 μg/m.sup.2 of the CD19×CD3 bispecific single chain antibody (blinatumomab; SEQ ID NO. 1) for 5 weeks; see FIG. 3. Serial analysis of the lymphocyte populations prior (day 0) and during treatment with the mentioned antibody showed an impressive expansion of CD8+T-lymphocytes, whereas no change in CD4+ T-cells and CD56+NK cells was noted. Upon antibody treatment, MRD negativity could be achieved. Concomitant chimerism analysis showed 100% donor chimerism. As shown in FIG. 4, bone marrow analysis at the 10.sup.th day of CD19×CD3 bispecific single chain antibody treatment revealed a complete elimination of the bone marrow blasts (MRD<10.sup.−4). Another BM analysis at the end of CD19×CD3 bispecific single chain antibody treatment showed again complete absence of leukemic blasts from the bone marrow (MRD<10.sup.−4). The serial analysis of MRD prior, during and after treatment with said antibody is depicted in FIG. 5. Before antibody treatment, the pediatric patient had a very poor prognosis and was not eligible to HSCT due to his bad clinical condition. Upon antibody treatment, MRD negativity could be achieved. Besides from transient discrete signs of ataxia, no major side effects were observed and no signs of GvHD were seen despite the impressive expansion of donor-derived CD8+T-lymphocytes. In October 2008, 2 weeks after end of treatment with CD19×CD3 bispecific single chain antibody (i.e. day 0 in FIGS. 3 to 5), he underwent a second HSCT from his haploidentical mother using a nonmyeloablative preparative regimen consisting of Clofarabine, Thiotepa and melphalan (Lang). As of November 2009, the patient has remained in MRD-negative complete remission.

4.2 Case Report Patient 2

[0154] This fifteen-year old patient was diagnosed with Philadelphia chromosome- and CD19-positive B-precursor ALL in April 2001. After chemotherapy, he received a HSCT from a HLA-identical sibling in October 2001 after conditioning with TBI of 12 Gy and etoposide. In 2002, a bone marrow relapsed was diagnosed and another remission was achieved with matinib and chemotherapy. He then received a second HSCT from an HLA-identical unrelated donor in October 2004. In March 2008, a second relapse was diagnosed and he was treated with low-dose chemotherapy and Dasatinib due to resistance to Imatinib. After additional chemotherapy with Clofarabin and Cytosin/Arabinosid, he achieved a molecular remission and received a third allogeneic HSCT from his 3 out of 6 HLA-allele-mismatched haploidentical father with post-transplant treatment with Dasatinib. Due to gastrointestinal bleeding and dilatative cardiomyopathy, Dasatinib was discontinued 5 months after transplantation. In April 2009, a combined central nervous system (CNS) relapse with 7×10.sup.9/L blasts in the CNS and 3% blasts in the bone marrow was diagnosed. The patient was then treated with Nilotinib, intrathecal chemotherapy and fractionated irradiation of the CNS with 18 Gy. Three months after this treatment, the patients' bone marrow remained MRD-positive at a level of 1.1×10.sup.3 while the CNS was free of blasts. Chimerism analysis of peripheral blood revealed a complete donor-derived hematopoiesis from his haploidentical father.

[0155] The patient was then treated under compassionate use with single-agent blinatumomab at 15 μg/m.sup.2/day for 4 weeks by continuous infusion without any side effects. A bone marrow aspiration at the end of the treatment showed complete remission with undetectable MRD in the bone marrow below <1×10.sup.−4. As patient 1, patient 2 did not show any signs of GvHD during or after treatment with blinatumomab. Two weeks after end of the treatment with blinatumomab, the patient experienced a transient haemolytic reaction triggered by an infection. Patient 2 is currently without relapse four weeks after treatment. He is well and attending school.

4.3 Summary

[0156] The drugs so far available for treatment of ALL are of low specificity and affect a variety of other cells resulting in severe side effects such as immunosuppression, bone marrow aplasia, mucositis, neuropathy, cardiotoxicity, and alopecia. Better targeted approaches with fewer side effects are therefore urgently needed. Furthermore, in some patients, ALL clones develop complete resistance to conventional chemotherapy, in particular those clones arising post HSCT, such that drugs with a distinct mode of action are desirable. The efficacy of antibody-dependent cytotoxicity via the Fc receptors of NK cells and granulocytes in combination with chemotherapy could be demonstrated in pediatric ALL using monoclonal chimeric antibody rituximab. Currently, blinatumomab is the only antibody in clinical trials allowing the engagement of cytotoxic T cells for targeting CD19 in B-cell non-Hodgkin lymphomas and lymphatic leukemias. T cells are considered to have a higher cytotoxic potential than those immune cells engaged by conventional monoclonal antibodies.

[0157] The allogeneic GvL effect is supposed to be one of the main reasons for the anti-leukemic efficacy of HSCT. Unfortunately, the occurrence of GvL is often associated with GvHD, which is still a major cause of morbidity and mortality after allogeneic HSCT. Therefore, the induction of GvL in the absence of GvHD is a topic of intensive research. One approach for the induction of a GvL effect is donor lymphocyte infusion (DLI). While DLI is very effective in the treatment of CML, it is less effective in the post-transplant treatment of relapsed ALL and often leads to the occurrence of chronic GvHD with significant impairment of quality of life for the affected children.

[0158] One possible approach to induce GvL without GvHD is the in-vivo activation of donor-derived T lymphocytes after HSCT using low doses of the T-cell engaging antibody blinatumomab, which can direct T-lymphocytes against the patients' CD19-positive ALL blasts. This antibody has shown impressive anti-lymphoma and anti-leukemic activity in the autologous situation, but has never been tested in relapsed ALL after HSCT or in children. In the autologous situation, molecular remissions have been induced in 13 out of 16 evaluable adult patients with MRD-positive ALL. Both pediatric patients described above showed an impressive anti-leukemic response after initiation of treatment. In both cases, MRD dropped below the level of detection without any signs of GvHD, despite an extensive expansion of donor-derived T-cells. This could be due to the fact that the action of blinatumomab is independent from regular peptide antigen presentation and may not involve engagement of the naïve T cell repertoire.

[0159] Dose levels of blinatumomab as low as 15 μg/m.sup.2 per day were sufficient to induce an expansion of donor-derived T-lymphocytes and to eliminate the ALL blasts below the detection level of MRD. This highlights that T-cell engagement is very different from the mode of action of conventional monoclonal antibodies, which require much higher doses and cannot engage cytotoxic T-cells due to their lack of Fc receptors.

[0160] Blinatumomab was well tolerated in both patients causing only fatigue, mild ataxia and tremor of CTCAE grade 1-2. In patient 2, no side effects were seen at all despite his intensive pre-treatments. The continuous i.v. infusion over several weeks was well accepted by both pediatric patients.

[0161] From this first clinical experience in pediatric ALL, the inventors conclude that blinatumomab is well tolerated and can rapidly induce complete haematological and MRD-negative remissions in children with therapy-refractory disease after multiple relapses of CD19-positive B-precursor ALL post-allogeneic HSCT. It is noteworthy that none of the patients showed any signs of GvHD despite an expansion of donor-derived T-lymphocytes, even after mismatched haploidentical transplantation.

[0162] These first results indicate that treatment of pediatric acute lymphoblastic leukemia (ALL) patients with the CD19×CD3 bispecific single chain antibody is able to completely eliminate acute lymphoblastic leukemia cells from the body of the pediatric patients. Importantly, this treatment resulted not only in complete hematologic remission but in complete molecular remission in that minimal residual disease (MRD) positive acute lymphoblastic leukemia (ALL) has been converted into an MRD negative status. The treatment was well tolerated. In light of this, the administration of the CD19×CD3 bispecific single chain antibody described herein provides an improved treatment option for pediatric acute lymphoblastic leukemia (ALL), in particular to ALL refractory to chemotherapy and/or allogeneic HSCT and/or relapsed ALL.

[0163] The results may be briefly summarized as follows: [0164] Two pediatric patients have been treated on a compassionate use basis. [0165] Ongoing MRD negativity in said patients. [0166] No major adverse events (AEs) observed. [0167] All AEs transient, no treatment interruption necessary.