Clever-1 inhibitor for promoting hematological recovery when using chemotherapy

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

[0001] CLEVER-1 INHIBITOR FOR PROMOTING HEMATOLOGICAL RECOVERY WHEN USING CHEMOTHERAPY

[0002] Field of the invention

[0003] The present invention relates to a promoting of hematological recovery in conjunction with chemotherapy. More detailed the present invention relates to a Clever-1 inhibitor for use to prevent and treat chemotherapy-induced suppression of the bone marrow, lymphopenia and / or neutropenia.

[0004] Background of the invention

[0005] All forms of cancer, both solid tumors and hematological malignancies, are widely treated with chemotherapy, but it can have side effects on rapidly dividing cells, including both cancer cells and normal cells. Lymphocytes, being rapidly dividing cells, are often affected by chemotherapy, leading to a reduction in their numbers. Chemotherapy-induced lymphopenia refers to a condition in which a patient's lymphocyte count in the blood decreases significantly as a result of undergoing chemotherapy treatment. Lymphocytes are a type of white blood cell that plays a crucial role in the immune system, defending the body against infections and diseases. Thus, the decline in lymphocyte count can weaken the immune system and increase the susceptibility of the patient to infections. The onset and duration of chemotherapy-induced lymphopenia can vary depending on the specific chemotherapy drugs used, the dosage, and the individual patient's response. Lymphocyte counts typically reach their lowest levels a few days to weeks after chemotherapy administration. In many cases, the lymphocyte count begins to recover after the completion of chemotherapy, but the recovery time can vary. Some patients may experience prolonged lymphopenia, and in some cases, it may persist even after the completion of chemotherapy.

[0006] In hematological malignancies the diseased hematopoietic cells including the hematopoietic stem cells (HSCs) are ablated using intensive chemotherapy and replaced by the healthy cells from a suitable donor. Bone marrow suppression is a serious side effect of chemotherapy. Bone marrow is the manufacturing center of blood cells, and the suppression of bone marrow activity causes a deficiency of blood cells. This condition can rapidly lead to life-threatening infection, as the body cannot produce leukocytes in response to invading bacteria and viruses, as well as leading to anemia due to a lack of red blood cells and spontaneous severe bleeding due to deficiency of platelets. Currently these states are treated with growth factors, such as G- CSF and GM-CSF, which enhance the production of required blood cells, but using these therapeutics agents often also feeds cancer cells, which the previously given chemotherapy aims to eliminate. Hence, anti-cancerous treatments to enhance the production of hematopoiesis are desperately needed in conjunction with chemotherapy.

[0007] Summary of the Invention

[0008] It is an object of the present invention to reduce or even eliminate the above- mentioned problems appearing in prior art.

[0009] It is an object of the present invention to provide a method for promoting hematological recovery when using chemotherapy, and agent(s) suitable for use in promoting hematological recovery in conjunction with chemotherapy.

[0010] In order to achieve among others the objects presented above, the invention is characterized by what is presented in the enclosed independent claim.

[0011] Some preferred embodiments of the invention will be described in the other claims.

[0012] The embodiments and advantages mentioned in this text relate, where applicable, both to the uses and methods as well as to the Clever-1 inhibitors according to the invention, even though it is not always specifically mentioned.

[0013] Surprisingly, it has now been found that Common Lymphatic Endothelial and Vascular Endothelial Receptor-1 (Clever-1) is an important regulator of hematopoiesis and its immunotherapeutic targeting can improve bone marrow recovery to repopulate blood with various immune cells after chemotherapy. It has been found that the inhibition of Clever-1 leads to the rapid recovery of hematopoietic stem cells in the bone marrow after chemotherapy. Consequently, according to the present invention a Clever-1 inhibitor is used in a prevention or a treatment of chemotherapy-induced suppression of the bone marrow and chemotherapy-induced disorders, such as lymphopenia and / or neutropenia. According to the present invention a Clever-1 inhibitor can be used in the treatment of cancer together with chemotherapy to prevent and / or treat chemotherapy-induced suppression of the bone marrow, lymphopenia and / or neutropenia.

[0014] The findings of the present invention may help to increase the count of the various immune cells, such as lymphocytes, monocytes and neutrophils in the blood during or after chemotherapy used for the treatment of cancer. In one aspect, the findings of the present invention provide a novel efficient method for treating cancer in combination with chemotherapy or for treating chemotherapy-induced disorders, such chemotherapy-induced lymphopenia and neutropenia, infections, anemia, thrompocytopenia etc. In one aspect, the present invention also provides a novel efficient method for treating hematological malignancies in combination with chemotherapy. The findings of the present invention may help to improve bone marrow recovery during or after the use of chemotherapy, enhance the effects of bone marrow transplantation and ameliorate the mobilization, harvesting and expansion of hematopoietic stem cells (HSCs). The findings of the present invention provide a method for treating a condition in which bone marrow does not function normally and the patient is in need of boosting his / her hematopoiesis.

[0015] A typical method according to the invention for promoting hematological recovery after or in conjunction with chemotherapy comprises use of a Clever-1 inhibitor. A method according to the invention for promoting hematological recovery after or in conjunction with chemotherapy comprises administering a Clever-1 inhibitor into a patient. In an embodiment of the present invention, an administration of Clever-1 inhibitor is initiated prior to onset of chemotherapy, and / or Clever-1 inhibitor is administered during chemotherapy, and / or Clever-1 inhibitor is administered after chemotherapy for preventing and treating chemotherapy-induced suppression of the bone marrow and chemotherapy-induced disorders, such as lymphopenia and neutropenia. According to an embodiment of the present invention, a population of hematopoietic stem cells after or in conjunction with the use of chemotherapy can be expanded by administering a Clever-1 inhibitor into a patient. Hence, the present invention provides a method for producing an expanded population of hematopoietic stem cells with the use of chemotherapy. The present invention provides opportunities in the field of hematopoietic stem cell transplantation and use of chemotherapy more widely for the treatment of cancer without the subsequent use of growth factors, which may cancer growth.

[0016] Description of the drawings

[0017] The invention will be described in more detail with reference to appended Figure 1 and Figure 2.

[0018] Figure 1 shows that reconstitution of white blood cells (WBC) after 5- fluorouracil is improved in mice with genetic deletion of Clever-1 (KO). A) Left, photograph of bone marrow cells at study endpoint. Right, analysis of WBC with VETSCAN for different immune cell populations at day 6, 10 and 12 days after 5-fluorouracil treatment. B) Flow cytometry analysis of blood cells at study end point. Cells were first gated for CD45+ and thereafter for lineage markers: CD3+ T-cells, B220+ B cells, monocytes (CD11 b+Ly6C+), and granulocytes (CD11 b+Ly6G+).

[0019] Figure 2 shows that immunotherapeutic targeting of Clever-1 promotes bone marrow recovery after 5-fluorouracil treatment. C57BL / 6N mice were treated with either isotype control antibody or anti-Clever-1 antibody (mStab1.26) seven days every second day before 5-fluorouracil treatment (160 mg / kg) and continued until the study endpoint (day 14). Immune cell populations were analyzed from peripheral blood with flow cytometry by gating with CD45 and thereafter for lineage markers: CD3+ T-cells, B220+ B cells, monocytes (CD11b+Ly6C+), and granulocytes (CD11 b+Ly6G+).

[0020] Detailed description of the invention

[0021] Common Lymphatic Endothelial and Vascular Endothelial Receptor-1 (Clever-1) is scavenger receptor commonly found on macrophages, monocytes and lymphatic endothelium, and is known as an immunosuppressive receptor that can promote cancer growth and metastases. Clever-1 inhibition is considered a viable anti-cancer treatment because of its ability to re-program tumor associated macrophages from an immunosuppressive and pro-tumorigenic M2 state to an inflammatory and anti-tumorigenic M1 state. In the present invention, it has been found that Clever-1 is also an important regulator of hematopoiesis and its immunotherapeutic targeting can improve bone marrow recovery to repopulate blood with various immune cells after chemotherapy.

[0022] In the present invention, a Clever-1 inhibitor is used in the treatment and / or prevention of chemotherapy-induced suppression of the bone marrow and chemotherapy-induced disorders, such as lymphopenia and / or neutropenia, wherein Clever-1 inhibitor promotes bone marrow recovery and increases the count of immune cell populations in the patient’s blood. According to the present invention, a Clever-1 is inhibitor can be used in treatment of cancer, both solid tumors and hematological malignancies, together with chemotherapy to prevent and treatment of chemotherapy induced- suppression of the bone marrow, lymphopenia and / or neutropenia.

[0023] A Clever-1 inhibitor refers to agents including antibodies and fragments thereof, peptides or the like, which are capable of inhibiting Clever-1 expression or binding to Clever-1 for blocking the function of Clever-1 or blocking the interaction of Clever-1 and cells involved with disease etiology. The Clever-1 inhibitor may also be any other inhibitor, such as RNA therapy, small molecule inhibitor or macromolecule having an adequate affinity to bind to Clever-1 receptor or capability to reduce its expression and / or to inhibit the protein activity. The term “an antibody or fragment thereof’ is used in the broadest sense to cover any therapeutic agent whether be antibody, fragment or small molecule thereof which are capable to inhibit Clever-1 expression or bind Clever-1 molecule in an individual. Especially, it shall be understood to include chimeric, humanized or primatized antibodies, as well as antibody fragments and single chain antibodies (e.g. Fab, Fv), so long they exhibit the desired biological activities. Particular useful Clever-1 inhibitors are anti-Clever-1 antibodies and fragments thereof. Therefore, according to an embodiment of the present invention Clever-1 inhibitor is selected from the group consisting of an antibody or a fragment thereof, peptide(s), RNA, small molecule or macromolecule capable of binding to Clever-1 or capable of inhibiting Clever-1 expression, and any combination thereof.

[0024] In an embodiment according to the present invention, a Clever-1 inhibitor is a humanized monoclonal anti-CLEVER-1 antibody. In an embodiment of the present invention, the anti-Clever-1 antibody is a humanized monoclonal immunoglobulin G4K antibody bexmarilimab (International Nonproprietary Name (INN)) as disclosed in WHO Drug Information, Vol. 33, No.4, 2019, or bexmarilimab variant or the antibody in a bexmarilimab biosimilar.

[0025] A bexmarilimab biosimilar means a biological product which is approved by a regulatory agency in any country for marketing as a bexmarilimab biosimilar. In an embodiment, a bexmarilimab biosimilar comprises a bexmarilimab variant as the drug substance. In an embodiment, a bexmarilimab biosimilar has substantially the same amino acid sequence of heavy and light chains as bexmarilimab. As used herein, a "bexmarilimab variant" means an antibody which comprises sequences of heavy chain and light chain that are identical to those in bexmarilimab, except for having one or more conservative amino acid substitutions at positions that are located outside of the light chain CDRs and / or one or more conservative amino acid substitutions that are located outside of the heavy chain CDRs, e.g. the variant positions are located in the framework regions or the constant region. In other words, bexmarilimab and a bexmarilimab variant comprise identical CDR sequences, but differ from each other due to having a conservative amino acid substitution at other positions in their full-length light and heavy chain sequences. A bexmarilimab variant is substantially the same as bexmarilimab with respect to binding affinity to Clever-1 .

[0026] According to an embodiment of the present invention, a cell line producing the therapeutic anti-Clever-1 antibody bexmarilimab (FP-1305) has been deposited on 27 May 2020 under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure with the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany, and has the accession number DSM ACC3361. The present invention is not to be limited in scope by the culture deposited, since the deposited embodiment is intended as a single illustration of one aspect of the invention and any culture that is functionally equivalent is within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustration that it represents.

[0027] According to an embodiment of the present invention, a Clever-1 inhibitor(s) can be used in the treatment of bone marrow suppression by improving an expansion of hematopoietic stem cells (HSCs) and thereby boosting hematopoiesis and increasing the count of of immune cell populations in the patient’s blood. Bone marrow suppression, also referred to as myelosuppression is a condition in which bone marrow activity to produce blood cells is decreased, resulting in fewer red blood cells, white blood cells and platelets. This is often a result of the use of chemotherapy for the treatment of a wide range of cancers. The present invention is used to prevent or treat chemotherapy-induced bone marrow suppression. Also, in bone marrow failure an insufficient amount of red blood cells, white blood cells or platelets are produced. Bone marrow failure or bone marrow suppression can be in association with multiple diseases or conditions, such as leukemia, multiple myeloma, aplastic anemia, mentioned as an example. In an embodiment of the present invention, bone marrow failure or bone marrow suppression can be treated by administering a Clever-1 inhibitor or a compound comprising a Clever-1 inhibitor to a patient receiving chemotherapy for the treatment of cancer, and / or with stem cells transplant.

[0028] In an embodiment of the present invention, a Clever-1 inhibitor is used to prevent or treat chemotherapy-induced lymphopenia, which refers to a condition in which a patient's lymphocyte count in the blood decreases significantly as a result of undergoing chemotherapy treatment. Correspondingly, a Clever-1 inhibitor can be used to prevent or treat chemotherapy-induced neutropenia, which refers to a condition in which a patient's neutrophils count in the blood decreases significantly as a result of undergoing chemotherapy treatment. In an embodiment of the present invention, chemotherapy-induced lymphopenia and neutropenia can be treated by administering a Clever-1 inhibitor or a compound comprising a Clever-1 inhibitor to a patient receiving chemotherapy for the treatment of cancer.

[0029] According to an embodiment of the invention, a method for treating a chemotherapy-induced suppression of the bone marrow, chemotherapy- induced lymphopenia and / or chemotherapy-induced neutropenia comprises administering to an individual of therapeutically effective amounts of a Clever-1 inhibitor or a pharmaceutical composition comprising a Clever-1 inhibitor. The term “treatment” or “treating” shall be understood to include complete curing of a disease or disorder, as well as amelioration or alleviation of said disease or disorder. The term “therapeutically effective amount” is meant to include any amount of a Clever-1 inhibitor according to the present invention that is sufficient to inhibit Clever-1 activity and produce expanded population of hematopoietic stem cells and increases the count of of immune cell populations in the patient’s blood. Therapeutically effective amount may comprise single or multiple doses of Clever-1 inhibitor. The dose(s) chosen should be sufficient on inhibition of Clever-1 activity and to promote an expansion of HSCs in an individual.

[0030] According to an embodiment of the present invention, a Clever- 1 inhibitor is administered into a patient in combination with a chemotherapy. In an embodiment of the present invention, an administration of Clever-1 inhibitor into a patient is initiated prior to onset of chemotherapy and / or a Clever-1 inhibitor is administered during the chemotherapy and / or a Clever-1 inhibitor is administered after the chemotherapy. Onset and duration of a Clever-1 inhibitor treatment with the chemotherapy can vary depending on the chemotherapy drugs used, the dosage, and the individual patient's response. Chemotherapy can comprise any suitable and commonly used anti-cancer drug(s). According to an exemplary embodiment of the present invention, chemotherapy comprises use of 5-fluorouracil as a chemotherapy drug.

[0031] EXPERIMENTAL

[0032] Example 1

[0033] Clever-1 knockout (KO) mice with a mixed C57BL / 6N and 129SvJ background were generated as previously described in the publication of Karikoski et. al. (2014) and used with their wild type (WT) counterparts (both sexes in a randomized fashion) at an age between 6 and 12 weeks.

[0034] Clever-1 KO mice and their WT counterparts were administered intraperitoneal injections (i.p.) 160 mg / kg of 5-fluorouracil (Sigma). Blood samples were collected at day 6, 10 and 12 days after 5-fluorouracil treatment from the saphenous vein into BD Microtainer tubes containing EDTA. These samples were subsequently analyzed using the VETSCAN Hematology Analyzer (Abaxis) and through flow cytometry. For flow cytometry, cells were stained with the following antibodies: CD45-PE (BD 553081 ), CD11 b-APC-Cy7 (BD 557657), Ly6C-BV605 (BD 563011), Ly6G- BV510 (Biolegend 127633), CD8-PE-Cy7 (BD 552877), CD4-AlexaFluor 647 (BD 557681 ), CD3-Percp.Cy5.5 (BD 560527), and B220-Alexa Fluor 420 (BD 562922). Stained samples were analyzed using an LSR Fortessa flow cytometer (BD) and data were processed with FlowJo software (Treestar). The results are presented in Figure 1. Clever-1 knock-out (KO) mice have better blood cell recovery compared to wild type after treatment with 5- fluorouracil, a common chemotherapy regime.

[0035] Example 2

[0036] C57BL / 6 mice, purchased from Charles River Laboratories, were administered i.p. of 500 pg of mStab-1.26 antibody (Invivo Biotech) or an equivalent mouse lgG1 isotype control (BioXcell). This regimen was initiated seven days prior to the administration of 5-fluorouracil and was repeated every other day until the endpoint of the study. The results are presented in Figure 2. Improved count of immune cell populations has been observed, and consequently anti-Clever-1 antibody (mStab 1.26) treated mice have better hematological stem cell recovery compared to isotype control treated mice after treatment with 5-fluorouracil, a common chemotherapy regime.

[0037] Cited reference

[0038] Karikoski M, Marttila-lchihara F, Elima K, Rantakari P, Hollmen M, Kelkka T, et al. Clever-1 / stabilin-1 controls cancer growth and metastasis. Clin Cancer Res (2014) 20(24):6452-64. doi: 10.1158 / 1078-0432.CCR-14-1236

Claims

Claims1. Clever-1 inhibitor for use in the treatment of chemotherapy-induced suppression of the bone marrow, lymphopenia and / or neutropenia.

2. Clever-1 inhibitor for use in the treatment of chemotherapy-induced suppression of the bone marrow, lymphopenia and / or neutropenia according to claim 1 , characterized in that Clever-1 inhibitor is selected from the group consisting of an antibody or a fragment thereof, peptide(s), RNA, small molecule or macromolecule capable of binding to Clever-1 or capable of inhibiting Clever-1 expression, and any combination thereof.

3. Clever-1 inhibitor for use in the treatment of chemotherapy-induced suppression of the bone marrow, lymphopenia and / or neutropenia according to claim 1 or 2, characterized in that Clever-1 inhibitor is administered into a patient in combination with a chemotherapy.

4. Clever-1 inhibitor for use in the treatment of chemotherapy-induced suppression of the bone marrow, lymphopenia and / or neutropenia according to any one of the preceding claims 1-3, characterized in that an administration of Clever-1 inhibitor into a patient is initiated prior to a chemotherapy.

5. Clever-1 inhibitor for use in the treatment of chemotherapy-induced suppression of the bone marrow, lymphopenia and / or neutropenia according to any one of the preceding claims 1-4, characterized in that a Clever-1 inhibitor is administered during the chemotherapy and / or after the chemotherapy.