New treatment

Abstract

The present invention relates to a bispecific antibody or antigen-binding fragment thereof that binds to CD33 and CD7 for use in the treatment of CD33+CD7+ hematological malignancies, particularly acute myeloid leukemia (AML). In particular, the present invention relates to a bispecific antibody or antigen-binding fragment thereof that binds to CD33 and CD7, comprising a first binding region that binds to human CD33 comprising a sequence having at least 95% sequence identity to the sequences VH SEQ ID NO:81 and VL SEQ ID NO:85, and a VH sequence having at least 95% sequence identity to the following sequences VH SEQ ID NO:11, VH SEQ ID NO:21, VH SEQ ID NO:31, VH SEQ ID NO:51, VH SEQ ID NO:71, VL SEQ ID NO:15, VL SEQ ID NO:25, VL SEQ ID NO:35, VL SEQ ID NO:55, and VL SEQ ID NO:75. and a second binding region that binds to human CD7 comprising a VH sequence and a VL sequence having at least 95% sequence identity to the following sequences: VH SEQ ID NO:1, VH SEQ ID NO:51, VH SEQ ID NO:71, VL SEQ ID NO:5, VL SEQ ID NO:55, and VL SEQ ID NO:75.

Description

Technical Field

[0001] The present invention relates to bispecific antibodies that bind to CD33 and CD7 and antigen-binding fragments thereof for use in the treatment of hematological malignancies, particularly acute myeloid leukemia (AML).

Background Art

[0002] Acute myelogenous leukemia or acute myeloid leukemia (AML) is a heterogeneous hematological malignancy associated with clonal proliferation of myeloblasts in the bone marrow and peripheral blood. AML accounts for over 90% of adult acute leukemia and is an aggressive disease with a severe clinical course. Despite the progress of treatment regimens and current understanding of the success of hematopoietic stem cell transplantation (HSCT), the mortality rate of AML patients remains high.

[0003] It is well known in the prior art that both CD7 and CD33 are internalizing cell surface antigen receptors, with CD7 being a rapid and efficient internalizing cell surface antigen receptor (internalization is seen within 15 - 30 minutes), while CD33 is a slow and inefficient internalizing cell surface antigen receptor (internalization is seen after 60 minutes).

[0004] WO2019 / 102234 discloses the dual targeting of cell surface receptors CD7 and CD33 with cell inhibitors in the treatment of hematological malignancies.

[0005] Based on the current state of the art and previous experimental results regarding monovalent CD7 and CD33 binding antibodies, the CD33 Fab arm of a bispecific antibody is expected to be sufficient to render a bispecific antibody containing the CD33 Fab arm cytotoxic to CD33-expressing cells. Furthermore, exposure of CD7-expressing cells to a CD33+ / CD7+ bispecific antibody has been shown to cause substantial cytotoxicity.

[0006] The object of the present invention is to provide an improved treatment for hematological malignancies, particularly AML. It is desirable that such a treatment does not result in myelosuppression. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2019 / 102234 [Patent Document 2] US4,978,744 [Patent Document 3] US5,635,483 [Patent Document 4] US5,780,588 [Non-patent literature]

[0008] [Non-Patent Document 1] De Propris, MS et al. (2011) High CD33 expression levels in acute myeloid leukemia cells carrying the nucleophosmin (NPM1) mutation, haematological, 96 pp. 1548~1551 [Non-Patent Document 2] Ehninger, A. et al., (2014) Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia, Blood Cancer Journal, 4 pp. 1-10 [Non-Patent Document 3] Poeta, GD et al. (1995) CD7 Expression in Acute Myeloid Leukemia. Leuk. Lymphoma, 17, pp. 111-119 [Non-Patent Document 4] Rohrs, S. et al., (2010) CD7 in acute myeloid leukemia: correlation with loss of wild-type CEBPA, consequence of epigenetic regulation, Journal of Hematology & Oncology, 3 pp. 1-7 [Non-Patent Document 5] Rausei-Mills, V. et al. (2008) Aberrant Expression of CD7 in Myeloblasts Is Highly Associated With De Novo Acute Myeloid Leukemias With FLT3 / ITD Mutation, Am J Clin Pathol, 129 pp. 624-629 [Non-Patent Document 6] Shimamoto, T. et al. (1994) Clinical and Biological Characteristic of CD7+ Acute Myeloid Leukaemia, Cancer Genet Cytogenet 73 pp. 69-74 [Non-Patent Document 7] Reading, CL et al. (1993) Expression of unusual immunophenotype combinations in acute myelogenous leukemia, Blood 81 pp. 3083-3090 [Non-Patent Document 8] Ossenkoppele, GJ et al. (2011) Review of the relevance of aberrant antigen expression by flow cytometry in myeloid neoplasms British Journal of Haematology 153 pp. 421-436 [Non-Patent Document 9] Lo Coco, F. et al. (1989) CD7 positive acute myeloid leukaemia: a subtype associated with cell immaturity, British Journal of Haematology 73 pp. 480-485 [Non-Patent Document 10] Kita, K. et al. (1983) Clinical Importance of CD7 Expression in Acute Myelocytic Leukemia, Blood 81, pp. 2399-2405 [Non-Patent Document 11] Eto, T. et al. (1992) Biological characteristics of CD7 positive acute myelogenous leukaemia, British lournal olHaernatology 82 pp. 508-511 [Non-Patent Document 12] Chang, H. (2004) Prognostic relevance of immunophenotyping in 379 patients with acute myeloid leukemia, Leukemia Research 28 pp. 43-48 [Non-Patent Document 13] Kahl, C. et al. (2001) CD7+ and CD56+ Acute Myelogenous Leukemia is a Distinct Biologic and Clinical Disease Entity. Haematology and Blood Transfusion, 40 pp. 112-119 [Non-Patent Document 14] Tien, H. and Wang, C. (1998) CD7 Positive Hematopoietic Progenitors and Acute Myeloid Leukemia and other Minimally Differentiated Leukemia, Leukemia and Lymphoma, 3 pages 93~98 [Non-licensed Document 15] Barcena, A.ら, (1994) Tracing the Expression of CD7 and other Antigens during T- and Myeloid-cell Differentiation in the Human Fetal Liver and Thymus, Leukaemia and Lymphoma 17 pages 1~11 [Non-licensed Document 16] The Merck Manual, Sec. 11, Ch. 138 (17th edition, 1997): Estey, 2001, Cancer 92(5): 1059-1073 [Non-licensed Document 17] Riechmann, L.ら, Nature 332 (1988) pp. 323~327 [Non-licensed Document 18] Neuberger, MSら, Nature 314 (1985) pp. 268~270 [Non-licensed Document 19] Lefranc, M.-P., Current Protocols in Immunology (2000)-Appendix 1P A.1P.1-A.1P.37 [Non-licensed Document 20] http: / / imgt.cines.fr [Non-licensed Document 21] http: / / vbase.mrc-cpe.cam.ac.uk [Non-licensed Document 22] van Dijk, MA, van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) pp. 368~374 [Non-licensed Document 23] Jakobovits, A., Proc. Natl. Acad. Sci. USA 90 (1993) Pages 2551~2555 [Non-licensed Document 24] Jakobovits, A. Nature 362 (1993) pp. 255~258 [Non-licensed Document 25] Brueggemann, MD, Year Immunol. 7 (1993) pp. 33~40 [Non-licensed Document 26] Hoogenboom, HR and Winter, G., J. Mol. Biol. 227 (1992) pp. 381~388 [Non-licensed Document 27] Marks, J.D., J. Mol. Biol. 222 (1991) pp. 581~597 [Non-licensed Document 28] Cole, A., Monoclonal Antibodies and Cancer Therapy, Liss, AR (1985) 77 pages [Non-licensed Document 29] Boerner, P.ら, J. Immunol. 147 (1991) pp. 86~95 [Non-licensed Document 30] Huston, JS, Methods in Enzymol. 203 (1991) pp. 46~88 [Non-licensed Document 31] Khaw, BA, J. Nucl. Med. 23:1011~1019 (1982) [Non-licensed Document 32] Rousseaux, Methods Enzymology, 121:663~69 pages, Academic Press, 1986 [Non-licensed Document 33] Klein ら、2012, imAbs 4:6, pages 1~11 [Non-licensed Document 34] Jonsson, U., (1993) Ann. Biol. Clin. 51: pages 19~26 [Non-licensed Document 35] Jonsson, U., (1991) Biotechniques 11: pages 620~627 [Non-licensed Document 36] Johnsson, B. (1995) J. Mol. Recognit. 8: 125-131 [Non-licensed Document 37] Johnnson, B.ら, (1991) Anal. Biochem. 198:268~277 pages [Non-licensed Document 38] Weiner GJ. Monoclonal antibody mechanisms of action in cancer. Immunol Res. 2007, 39(l-3): pages 271~8 [Non-licensed Document 39] Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med. 2000 Apr; 6(4):443~6 [Non-licensed Document 40] Weng WK, Levy R. Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J Clin Oncol. November 1, 2003; 21(21):3940-7 (electronic version September 15, 2003) [Non-licensed Document 41] Presta LG Engineering of therapeutic antibodies to minimize immunogenicity and optimize function. Adv Drug Deliv Rev. August 7, 2006; 58(5-6): 640-56 (electronic version May 23, 2006) [Non-licensed Document 42] AL Smith, J. Med. Chem., 1996, 39,11, pages 2103~2117 [Non-licensed Document 43] D. Boger, Pure & Appl. Chem., 1994, 66, 4, pages 837~844 [Non-licensed Document 44] I. Bhatnagarら、Mar. Drugs 2010, 8, pages 2702~2720 [Non-licensed Document 45] TL Simmons, Mol. Cancer Ther. 2005, 4(2), pages 333~342 [Non-licensed Document 46] X. :Pivotら、European Oncology, 2008;4(2), pp. 42~45 [Non-licensed Document 47] Alley, Current Opinion in Chemical Biology 2010 14: Pages 1~9 [Non-licensed Document 48] Senter, Cancer J., 2008, 14(3): 154-169 [Non-licensed Document 49] Dubowchik and Walker, Pharm. Therapeutics 83:67~123, 1999 [Non-licensed Document 50] Johnson, Anticancer Res. 15:1387~93 pages, 1995 [Non-licensed Document 51] Lau, Bioorg-Med-Chem. 3: pp. 1299~1304, 1995 [Non-Patent Document 52] Lau et al., Bioorg-Med-Chem. 3: pp. 1305-1312, 1995. [Overview of the project] [Means for solving the problem]

[0009] According to a first aspect of the present invention, a bispecific antibody or antigen-binding fragment thereof that binds to CD33 and CD7, wherein the sequence is: VH Sequence ID 81 and VL Sequence ID 85, A first binding region that binds to human CD33, containing a sequence having at least 95% sequence identity with respect to, And the following sequence: VH Sequence ID 11, VH Sequence ID 21, VH Sequence ID 31, VH Sequence ID 51, VH Sequence ID 71, VL Sequence ID 15, VL Sequence ID 25 VL Sequence ID 35, VL Sequence ID 55 and VL Sequence ID 75 A second binding region that binds to human CD7, comprising a VH sequence and a VL sequence having at least 95% sequence identity with respect to, An antibody containing or an antigen-binding fragment thereof is provided.

[0010] The sequences may have at least 98%, at least 99%, or 100% sequence identity.

[0011] The second binding region that binds to human CD7 is the following sequence: a) VH Sequence ID 11 and VL Sequence ID 15, b) VH Sequence ID 21 and VL Sequence ID 25, c) VH Sequence ID 31 and VL Sequence ID 35, It may also include VH sequences and VL sequences having the following characteristics:

[0012] In a further embodiment, a bispecific antibody or antigen-binding fragment thereof that binds to CD33 and CD7, wherein the sequence: VH Sequence ID 97 and VL Sequence ID 101, A first binding region that binds to human CD33, containing a sequence having at least 95% sequence identity with respect to, And the following sequence: VH Sequence ID 1, VH Sequence ID 51 VH Sequence ID 71, VL Sequence ID 5, VL Sequence ID 55 and VL Sequence ID 75 A second binding region that binds to human CD7, comprising a VH sequence and a VL sequence having at least 95% sequence identity with respect to, An antibody containing or an antigen-binding fragment thereof is provided.

[0013] The sequences may have at least 98%, at least 99%, or 100% sequence identity.

[0014] The second binding region that binds to human CD33 is the following sequence: VH Sequence ID 97 and VL Sequence ID 101 It includes VH sequences and VL sequences having [a specific characteristic].

[0015] A further aspect of the present invention provides a bispecific antibody or antigen-binding fragment thereof that binds to CD33 and CD7, comprising a first binding region that binds to human CD33 and a second binding region that binds to human CD7, wherein the second binding region comprises one or more of the following: a VH CDR2 region containing the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 23, or SEQ ID NO: 53, and / or a VH CDR3 region containing the amino acid sequence of SEQ ID NO: 34, or SEQ ID NO: 74.

[0016] In an additional embodiment, a bispecific antibody or antigen-binding fragment thereof that binds to CD33 and CD7 is provided, comprising a first binding region that binds to human CD33 and a second binding region that binds to human CD7, wherein the first binding region comprises a VH CDR3 region containing the amino acid sequence of SEQ ID NO: 100.

[0017] In relation to a further embodiment, a bispecific antibody or antigen-binding fragment thereof that binds to CD33 and CD7, comprising a first binding region that binds to human CD33 and a second binding region that binds to human CD7, wherein the second binding region that binds to human CD7 is as follows a) VH CDR1 region containing the amino acid sequence of SEQ ID NO: 12, VH CDR2 region containing the amino acid sequence of SEQ ID NO: 13, VH CDR3 region containing the amino acid sequence of SEQ ID NO: 14, VL CDR1 region containing the amino acid sequence of SEQ ID NO: 16, VL CDR2 region containing the amino acid sequence of SEQ ID NO: 17, and VL CDR3 region containing the amino acid sequence of SEQ ID NO: 18. b) VH CDR1 region containing the amino acid sequence of SEQ ID NO: 22, VH CDR2 region containing the amino acid sequence of SEQ ID NO: 23, VH CDR3 region containing the amino acid sequence of SEQ ID NO: 24, VL CDR1 region containing the amino acid sequence of SEQ ID NO: 26, VL CDR2 region containing the amino acid sequence of SEQ ID NO: 27, and VL CDR3 region containing the amino acid sequence of SEQ ID NO: 28 c) VH CDR1 region containing the amino acid sequence of SEQ ID NO: 32, VH CDR2 region containing the amino acid sequence of SEQ ID NO: 33, VH CDR3 region containing the amino acid sequence of SEQ ID NO: 34, VL CDR1 region containing the amino acid sequence of SEQ ID NO: 36, VL CDR2 region containing the amino acid sequence of SEQ ID NO: 37, and VL CDR3 region containing the amino acid sequence of SEQ ID NO: 38. d) VH CDR1 region containing the amino acid sequence of SEQ ID NO: 52, VH CDR2 region containing the amino acid sequence of SEQ ID NO: 53, VH CDR3 region containing the amino acid sequence of SEQ ID NO: 54, VL CDR1 region containing the amino acid sequence of SEQ ID NO: 56, VL CDR2 region containing the amino acid sequence of SEQ ID NO: 57, and VL CDR3 region containing the amino acid sequence of SEQ ID NO: 58, and e) VH CDR1 region containing the amino acid sequence of SEQ ID NO: 72, VH CDR2 region containing the amino acid sequence of SEQ ID NO: 73, VH CDR3 region containing the amino acid sequence of SEQ ID NO: 74, VL CDR1 region containing the amino acid sequence of SEQ ID NO: 76, VL CDR2 region containing the amino acid sequence of SEQ ID NO: 77, and VL CDR3 region containing the amino acid sequence of SEQ ID NO: 78 An antibody or an antigen-binding fragment thereof is provided, which may contain one of the following.

[0018] The first binding region that binds to human CD33 preferably includes the wild-type VH CDR1, VH CDR2, and VH CDR3 amino acid sequences and VL CDR1, VL CDR2, and VL CDR3, or an associated sequence having two or more mutations across the CDR of a given VH or VL chain. More preferably, the first binding region that binds to human CD33 preferably includes the wild-type VH CDR1, VH CDR2, and VH CDR3 amino acid sequences and VL CDR1, VL CDR2, and VL CDR3, or an associated sequence having one or more mutations across the CDR of a given VH or VL chain.

[0019] According to a third aspect of the present invention, a bispecific antibody or antigen-binding fragment thereof that binds to CD33 and CD7, comprising a first binding region that binds to human CD33 containing wild-type VH and VL sequences, and the following a) Single or double mutations in the heavy chain, and / or b) Single mutation in the light chain A second binding region for human CD7, comprising VH and VL sequences derived from each heavy and light chain sequence having at least one or more of the following: An antibody or an antigen-binding fragment thereof is provided.

[0020] The CD7 VH and VL sequences derived from the respective heavy and light chain sequences are as follows: a) Single or double mutations in heavy chain residues 57, 104 and 108, and / or b) Single mutation at residue 27 of the light chain It may have at least one or more of these.

[0021] The mutation in the heavy chain would preferably be Gly or Lys at residue 57, or Ala at residue 104, or Ala at residue 108.

[0022] The mutation in the light chain would preferably be at residue 27, Ala.

[0023] In a preferred embodiment, the second binding region for human CD7 includes VH and VL sequences derived from the respective heavy and light chain sequences having a single mutation at Ala residue 104 of the heavy chain.

[0024] The antibodies or antigen-binding fragments thereof described herein with reference to all embodiments may be intended for use in the treatment of CD7+CD33+ malignancies, such as hematological malignancies.

[0025] The antibodies or antigen-binding fragments described herein with reference to all embodiments may be for a method of treating a CD7+CD33+ hematological malignancy in an individual requiring such treatment, the method comprising administering the antibody or antigen-binding fragment. The antibodies or antigen-binding fragments are preferably artificially produced.

[0026] In another related aspect of the present invention, antibodies or antigen-binding fragments thereof described herein are provided with reference to any aspect for use in the manufacture of pharmaceuticals for CD7+CD33+ hematological malignancies.

[0027] As used herein, “pharmaceutical” refers to a substance (i.e., a drug) used in a medical procedure. A pharmaceutical may, for example, be a T-cell preparation for use in adoptive cell transfer.

[0028] As used herein, CD7 is preferably human CD7, and CD33 is preferably human CD33. In certain embodiments, the bispecific antibody or its antigen-binding fragment specifically binds to CD7 and CD33 expressed on the cell surface. As used herein, the expression “cell surface expression” means one or more CD7 and / or CD33 proteins expressed on the cell surface in vitro or in vivo such that at least a portion of the CD7 and / or CD33 proteins are exposed to the extracellular side of the cell membrane and are accessible to the bispecific antibody or its antigen-binding fragment of the present invention.

[0029] The term "CD7+CD33+ hematological malignancies" refers to hematological malignancies characterized by the expression of both CD7 and CD33 on the surface of malignant cells (for example, hematological malignancies that overexpress CD33 and / or CD7 on the cell surface, and / or hematological malignancies that express CD33 and / or CD7 at levels where treatment with antibodies or antigen-binding fragments that specifically bind to CD7 and CD33 is considered acceptable).

[0030] Antibodies or their antigen-binding fragments may be able to induce internalization into CD33+ and / or CD7+ cells via CD33 and / or CD7 receptors.

[0031] CD7+CD33+ hematological malignancies include, but are not limited to, acute myeloid leukemia (AML), myelodysplastic syndromes, T-cell acute lymphoblastic leukemia, and blastic plasmacytoid dendritic cell neoplasms (BPDCN).

[0032] The antibody or its antigen-binding fragment bispecifically binds to CD33 and CD7, and CD33+ and CD7+ cells are AML cells.

[0033] Antibodies or their antigen-binding fragments may also mediate antibody-dependent cell-mediated cytotoxicity.

[0034] The antibody or its antigen-binding fragment may be bound to or formed with the immune effector cell. The immune effector cell may include T cells and / or NK cells. Preferably, the immune effector cell is a T cell. The immune effector cell may be a bispecific anti-CD33 anti-CD7 CAR-T cell. The T cells may include CD33+ T cells, CD7+ T cells, or a combination thereof.

[0035] The antibody or its antigen-binding fragment may include i) a cell-killing portion, ii) a CD7-binding portion, and iii) a CD33-binding portion.

[0036] In an alternative embodiment, the CD33 and / or CD7 binding portion includes an antigen-binding fragment of the antibody.

[0037] The cell-killing portion may be a cytotoxin, and those skilled in the art will understand that various cytotoxins will be compatible with the composition. The cytotoxin may be selected from i) peptide toxins, ii) chemotoxins, or iii) Bcl-2 or Bcl-axl inhibitors, iv) RNA polymerase inhibitors such as α-amanitin, v) spliceosome inhibitors, vi) microtubule-targeted payloads, or vii) DNA damage payloads. The antibody or its antigen-binding fragment may further include a linking portion that links the cell-killing portion to a CD7-binding portion and / or a CD33-binding portion. The antibody or its antigen-binding fragment may be in the form of an antibody-drug conjugate.

[0038] In embodiments of bispecific antibodies, such antibodies may be full-length antibodies.

[0039] Surprisingly, the inventors discovered that a bispecific antibody containing both a CD33-binding arm and a CD7-binding arm does not cause cytotoxicity in healthy human CD33-expressing myeloid cells. Therefore, this offers the potential for improved treatment of hematological malignancies, particularly AML, without myelosuppression.

[0040] CD7 is a pan-leucocytic receptor expressed on T cells, B lymphocytes, natural killer cells, and dendritic cell precursors (Hao, 2001; Sempowki, 1999), plays an adjunct role in T cell activation (Lazarovits, 1994; Stillwell, 2011), and is present on the surface of mature CD4<+> cells (Cotta, 2006; Lobac, 1985). CD7 has been widely studied as a delivery target for cytotoxic molecules for the treatment of leukemia and lymphoma (Peipp, 2002; Bremmer, 2006; Franker, 1997; Vallera, 1996; Waurzyniak, 1997).

[0041] CD33 is a 67kDa cell membrane protein that binds to sialic acid and is a member of the sialic acid-binding Ig-associated lectin (SIGLEC) family. CD33 is known to be expressed on myeloid cells, and its expression has also been reported on many malignant cells.

[0042] While CD33, a common bone marrow antigen, is expressed in the majority of AML cells (De Propris, MS et al., (2011) High CD33 expression levels in acute myeloid leukemia cells carrying the nucleophosmin (NPM1) mutation, haematological, 96 pp. 1548-1551; Ehninger, A. et al., (2014) Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia, Blood Cancer Journal, 4 pp. 1-10), CD7, a common marker for T cells and NK cells, is abnormally expressed in the chemotherapy-resistant subpopulation of AML cells (approximately 22%), resulting in a phenotype associated with a poor prognosis (Poeta, GD et al., (1995) CD7 Expression in Acute Myeloid Leukemia. Leuk. Lymphoma, 17, pp. 111-119; Rohrs, S. et al., (2010) CD7 in acute myeloid leukemia: correlation with loss of wild-type CEBPA, consequence of epigenetic regulation, Journal of Hematology & Oncology, 3 pp. 1-7; Rausei-Mills, V. et al., (2008) Aberrant Expression of CD7 in Myeloblasts Is Highly Associated With De Novo Acute Myeloid Leukemias With FLT3 / ITD Mutation, Am J Clin Pathol, 129 pp. 624-629; Shimamoto, T. et al. (1994) Clinical and Biological Characteristic of CD7+ Acute Myeloid Leukaemia, Cancer Genet Cytogenet 73 pp. 69-74; Reading, CLRaf, (1993) Expression of unusual immunophenotype combinations in acute myelogenous leukemia, Blood 81: 3083 - 3090; Ossenkoppele, G. J. et al., (2011) Review of the relevance of aberrant antigen expression by flow cytometry in myeloid neoplasms British Journal of Haematology 153: 421 - 436; Lo Coco, F. et al., (1989) CD7 positive acute myeloid leukaemia: a subtype associated with cell immaturity, British Journal of Haematology 73: 480 - 485; Kita, K. et al., (1983) Clinical Importance of CD7 Expression in Acute Myelocytic Leukemia, Blood 81, 2399 - 2405; Eto, T. et al., (1992) Biological characteristics of CD7 positive acute myelogenous leukaemia, British Journal of Haematology 82: 508 - 511; Chang, H. (2004) Prognostic relevance of immunophenotyping in 379 patients with acute myeloid leukemia, Leukemia Research 28: 43 - 48).

[0043] The CD7+ subtype of AML is associated with leukocytosis, poor response to chemotherapy, and poor overall survival and disease-free survival (Kahl, C. et al., (2001) CD7+ and CD56+ Acute Myelogenous Leukemia is a Distinct Biologic and Clinical Disease Entity. Haematology and Blood Transfusion, 40 pp. 112-119). Clinically, CD7 AML patients are more common in young men, have a higher incidence of central nervous system lesions, are often associated with less differentiated subtypes of AML, and further contribute to a poorer prognosis (Tien, H. and Wang, C. (1998) CD7 Positive Hematopoietic Progenitors and Acute Myeloid Leukemia and other Minimally Differentiated Leukemia, Leukemia and Lymphoma, 3 pp. 93-98). The immaturity of CD7+ AML cells is further supported by the high expression of CD34 in this population. A 1995 study by Poeta et al. showed that the complete response (CR) rate in CD7+ leukemia patients was significantly lower than in CD7- phenotypic leukemia patients (32% vs. 74%), indicating that this subtype is associated with a higher degree of relapse and / or refractory to standard treatment (Poeta, GD et al., (1995) CD7 Expression in Acute Myeloid Leukemia. Leuk. Lymphoma, 17, pp. 111-119).

[0044] CD7 is closely associated with, and is considered a defining characteristic of, the FLT3-ITD+ AML subgroup. This subtype is associated with worse clinical outcomes due to the de-distortion of the FLT3 tyrosine kinase receptor, which leads to the emission of signals that downregulate the translation of apoptotic proteins, resulting in resistance to chemotherapy-induced cell death in the AML cell population (Rausei-Mills, V. et al., (2008) Aberrant Expression of CD7 in Myeloblasts Is Highly Associated With De Novo Acute Myeloid Leukemias With FLT3 / ITD Mutation, Am J Clin Pathol, 129 pp. 624-629). The FLT3 AML subgroup, a subgroup of AML with a particularly poor prognosis, is a desirable target for novel drug developers, and many new therapeutic agents, including kinase inhibitors and monospecific ADCs, specifically target this population.

[0045] Several mechanisms have been reported to explain the abnormal expression of CD7 in AML. These include disease-specific irregular gene expression in leukocytes (lineage infidelity), malignancy of pluripotent progenitor cells capable of differentiating or proliferating into lymphoid or myeloid cells, and cessation of maturation of rare progenitor cells that transiently express markers of different cell lineages during the normal cell differentiation process (lineage promiscuity) (Tien, H. and Wang, C. (1998) CD7 Positive Hematopoietic Progenitors and Acute Myeloid Leukemia and other Minimally Differentiated Leukemia, Leukemia and Lymphoma, 3 pp. 93-98).

[0046] Transient CD7 expression has been reported in a subset of early progenitor cells capable of producing cells with both myeloid and lymphoid origins, but it disappears during the transformation into mature myeloid or lymphoid cells (Tien, H. and Wang, C. (1998) CD7 Positive Hematopoietic Progenitors and Acute Myeloid Leukemia and other Minimally Differentiated Leukemia, Leukemia and Lymphoma, 3 pp. 93-98). Similarly, in one study, pluripotent stem cells (CD33) low / CD7 + / - ), some myeloid progenitor cells (CD33 high / CD7 + / - ), some T cell progenitor cells (CD33 + / - / CD7 med These two antigens were found to be co-expressed at low levels in a specific subset of healthy hematopoietic cells, including ), but the co-expression was found to disappear during development (Barcena, A. et al., (1994) Tracing the Expression of CD7 and other Antigens during T- and Myeloid-cell Differentiation in the Human Fetal Liver and Thymus, Leukaemia and Lymphoma 17 pp. 1-11). Therefore, it is most likely that this co-expression pattern is the result of clonal proliferation of a specific subset of progenitor cells captured at a particular stage of development, thereby transiently bringing these two antigens together, and that this expression is amplified during malignancy.

[0047] As used herein, the terms “to treat,” “to treat,” and “treatment” include interventions undertaken for the purpose of preventing the onset of a disorder or symptom, or altering a pathological condition. Therefore, “treatment” refers to both therapeutic treatments and preventive or deterrent measures, the purpose of which is to prevent or delay (reduce) the disorder or symptom in question. Thus, the term “to treat” encompasses treating and / or preventing the onset of a disorder or symptom. As used herein, “treatment” refers to the prevention or treatment of a disease or disorder. Treatment may be preventive or therapeutic.

[0048] In this embodiment, the antibody or antigen-binding fragment thereof of the present invention is administered to a patient in remission of a hematological malignancy, thereby preventing or delaying the recurrence of the underlying hematological malignancy.

[0049] As used herein, “patient” typically refers to a human being who is being treated for or has been diagnosed with a hematological malignancy, preferably a CD7+CD33+ hematological malignancy. In some embodiments, an antibody or its antigen-binding fragment is administered to a patient in remission of a CD7+CD33+ hematological malignancy, thereby preventing or delaying recurrence of the hematological malignancy. In some embodiments, the patient lacks detectable cells of the hematological malignancy. As used herein, “lack of detectable cells” is determined by standard diagnostic or prognostic methods. Patients in remission of AML typically exhibit the disappearance of abnormal clinical features, the return of normal blood cell counts, and the disappearance of normal hematopoiesis and leukemia clones in the bone marrow, with less than 5% blasts, more than 1,000–1,500 neutrophils, and more than 100,000 platelets. For example, see The Merck Manual, Sec. 11, Ch. 138 (17th edition, 1997): Estey, 2001, Cancer 92(5): pp. 1059-1073.

[0050] In some embodiments, patients in remission from CD7+CD33+ hematological malignancies do not receive a bone marrow transplant. In other embodiments, patients in remission from CD7+CD33+ hematological malignancies receive a bone marrow transplant. The bone marrow transplant may be either an autologous bone marrow transplant or an allogeneic bone marrow transplant.

[0051] In the embodiment, treating CD7+CD33+ hematological malignancies (e.g., AML) to prevent or delay recurrence of CD7+CD33+ hematological malignancies (e.g., AML) includes inducing AML cancer cell death and / or inhibiting AML cancer cell proliferation.

[0052] An antibody or its antigen-binding fragment may be part of a composition (e.g., a therapeutic composition) comprising a compound (i.e., an antibody or its antigen-binding fragment) and one or more other components. The composition may be a therapeutic composition comprising an antibody or its antigen-binding fragment and pharmaceutically acceptable excipients, adjuvants, diluents and / or carriers. The therapeutic composition may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants and cytokines or other auxiliary immunostimulants, and optionally other therapeutic agents or compounds.

[0053] As used herein, “pharmaceutically acceptable” means a material that is biologically or otherwise desirable, i.e., a material that can be administered to an individual together with a selected compound without causing undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition in which it is contained.

[0054] Excipients are natural or synthetic substances formulated together with active ingredients (e.g., vaccines, cell cycle inhibitors, immunosuppressive mechanism modifiers, immune checkpoint inhibitors (where appropriate)) to increase the volume of the formulation or to impart therapeutic effects to the active ingredient in the final dosage form, such as by enhancing drug absorption or solubility. Excipients are also useful in the manufacturing process to assist in the handling of the active substance, such as by promoting powder fluidity and non-stickiness, as well as by assisting in vitro stability, such as by preventing denaturation during the expected storage period. Pharmaceutically acceptable excipients are well known in the art. Therefore, a suitable excipient can be easily identified by those skilled in the art. Examples of suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, and ethanol.

[0055] An adjuvant is a pharmacological and / or immunological agent that modifies the effects of other agents in a drug formulation. Pharmaceutically acceptable adjuvants are well known in the art. Therefore, a suitable adjuvant is readily identifiable to those skilled in the art.

[0056] A diluent is a chemical used to dilute a substance. Pharmaceutically acceptable diluents are well known in the art. Therefore, a suitable diluent is easily identifiable by those skilled in the art.

[0057] The carrier is non-toxic to the recipient at the dose and concentration used and is compatible with the other components of the formulation. The term "carrier" refers to a natural or synthetic organic or inorganic component to which the active ingredient is mixed for ease of application. Pharmaceutically acceptable carriers are well known in the art. Therefore, a suitable carrier is readily identifiable to those skilled in the art.

[0058] As used herein, the terms “effective dose” and “therapeutic dose” refer to an amount of active therapeutic agent sufficient to produce a desired therapeutic response without excessive side effects such as toxicity, irritation, or allergic reactions. The specific “effective dose” will obviously vary depending on factors such as the particular condition being treated, the patient’s physical condition, the type of animal being treated, the duration of treatment, the nature of any concurrent treatments, and the structure of any particular formulation and compound or derivative thereof employed. In this context, a dose is considered therapeutically effective if it results in one or more of the following: (a) inhibition of cancer cell proliferation (e.g., AML cells), and (b) death of cancer cells (e.g., AML cells).

[0059] The dosage of antibodies or their antigen-binding fragments and their therapeutic compositions administered to a patient may vary depending on the patient's age, physique, target disease, condition, and route of administration. The preferred dosage is typically calculated based on body weight or body surface area.

[0060] Methods of administering antibodies or their antigen-binding fragments and their therapeutic compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antibodies or their antigen-binding fragments and their therapeutic compositions may be administered by any convenient route, for example, by injection or bolus injection, or by absorption through the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa), or may be administered together with other biologically active agents. Administration can be systemic or topical.

[0061] Preferably, the dual-target therapies described herein will benefit the treatment of CD7+CD33+ hematological malignancies in subjects requiring them. For example, dual-target therapies may have additive or synergistic effects on the treatment of AML in subjects requiring them. A dual-target therapy is defined as producing an “additive effect,” a “synergistic effect,” or a “synergistic treatment” if its effect is therapeutically superior to what can be achieved by administering one or other components of the dual-target therapy at their conventional doses, as measured, for example, by the degree of response (e.g., apoptosis or cell viability), response rate, time to disease progression, or survival. For example, the effect of a dual-target therapy is additive if its effect is therapeutically superior to the effect achievable by an antibody or its antigen-binding fragment that specifically binds to CD33 or CD7 alone. For example, the effect of a combination treatment may be synergistic if the effect of the combination treatment outweighs the effect of the individual treatments combined. Furthermore, the combined effect is beneficial (e.g., additive or synergistic) if it is obtained in a control group that does not respond (or responds poorly) to a cell inhibitor that specifically binds to CD33 alone or a cell inhibitor that specifically binds to CD7 alone. Moreover, the effect of a combined treatment is defined as a benefit (e.g., additive or synergistic) if the therapeutic effect, measured by, for example, the degree of response, response rate, time to disease progression, or survival period, is equivalent to or greater than what could be achieved by administering either component of the combined treatment at the conventional dose, while administering the other component at a reduced dose.

[0062] As used herein, “target cell death” refers to, for example, inhibition of protein synthesis that reduces cell viability, or induction of apoptosis that results in the elimination or death of target cells. Assays for determining cell toxicity and apoptosis are well known in the art. Cytotoxicity assays assess the number of live and dead cells in a population after treatment with a pharmacological substance (e.g., LDH cytotoxicity assay, or live-dead cell assay). Apoptosis assays assess how cells are dying by measuring markers that are activated during cell death (e.g., PS exposure assay, caspase activation assay, DNA fragmentation assay, GSH / GSSG measurement, LDH cytotoxicity assay, live-dead cell assay, or non-caspase protease activation assay).

[0063] As used herein, “inhibits cell proliferation” (e.g., target cells) means a measurable reduction in the growth or proliferation of target cells upon contact with the antibody or antigen-binding fragment described herein, compared to the proliferation of the same cells that have not been in contact with the antibody or antigen-binding fragment described herein. For example, cell proliferation is inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Assays for determining cell viability or proliferation are well known in the art. Cell viability assays assess how healthy cells are by measuring markers of cell activity (e.g., ATP and ADP determination assays, cell cycle assays, cell proliferation assays, cell viability assays, LHD cytotoxicity assays, or live-dead cell assays). Cell proliferation assays assess the growth rate of a cell population or detect daughter cells in a growing population (e.g., cell cycle assays, cell proliferation assays, cell viability assays, or senescence assays).

[0064] As used herein, "CD33-expressing cells" and "CD33+ cells" refer to cells that have CD33 as a surface antigen. As used herein, "CD7-expressing cells" and "CD7+ cells" refer to cells that have CD7 as a surface antigen. As used herein, "CD33 and CD7-expressing cells" and "CD33+CD7+ cells" refer to cells that have both CD33 and CD7 as surface antigens.

[0065] As used herein, “target cells” refers to cells or cell types characterized by the expression or overexpression of the target molecules CD7 and CD33. Any type of cell expressing CD7 and CD33 may be considered a target cell for treatment with the antibody or its antigen-binding fragment of the present invention. In certain embodiments, the cells are tumor cells, such as tumor cells derived from hematological malignancies, like AML cells.

[0066] In certain embodiments, the antibodies or antigen-binding fragments described herein can induce the internalization of the antibody or antigen-binding fragment into CD33+ cells via the CD33 receptor, and / or the internalization of the antibody or antigen-binding fragment into CD7+ cells via the CD7 receptor. In certain embodiments, the antibody or antigen-binding fragment is an antibody or antigen-binding fragment that specifically binds to both CD33 and CD7, and the binding of both CD7 and CD33 on the cell surface can induce the internalization of the drug into CD7+CD33+ cells.

[0067] As used herein, "CD33 receptor-mediated internalization" refers to the internalization (i.e., invasion) of CD33+ cells via the binding of CD33 on the cell surface. In therapeutic applications, in vivo internalization is intended. As used herein, "CD7 receptor-mediated internalization" refers to the internalization (i.e., invasion) of CD7+ cells via the binding of CD7 on the cell surface. In therapeutic applications, in vivo internalization is intended.

[0068] For therapeutic purposes, a sufficient or appropriate number of internalized antibodies or their antigen-binding fragments may be necessary to kill CD33+CD7+ cells, particularly CD7+CD33+ hematological cancer cells such as AML cells. Depending on the ability of the antibody or its antigen-binding fragment, in some cases, the uptake of a single molecule into the cell may be sufficient to kill the target cells to which the drug binds.

[0069] In certain embodiments, the antibody or antigen-binding fragment of the present invention may be an ADC, a small molecule drug conjugate (SMDC), an immunotoxin, a peptide or non-peptide conjugate, an imaging agent, a therapeutic vaccine, or a nanoparticle.

[0070] As used herein, the term “antibody” refers to a molecule or active fragment of a molecule that binds to a known antigen, and in particular to immunoglobulin molecules, and molecules that include an immunologically active portion of an immunoglobulin molecule, i.e., a binding site that binds immunospecifically to an antigen (i.e., CD7 or CD33). The immunoglobulins described herein are any class (IgG, IgM, IgD, IgE, IgA and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass (isotype) of an immunoglobulin molecule (e.g., IgG in IgG1, IgG2, IgG3 and IgG4, or IgA in IgA1 and IgA2).

[0071] Within the scope of the present invention, the term "antibody" includes human antibodies and humanized antibodies, as well as their active fragments. Examples of active fragments of molecules that bind to known antigens include Fab, F(ab'), and F(ab'). 2 Examples include scFv and Fv fragments, and include products of the Fab immunoglobulin expression library and epitope-binding fragments of any of the antibodies and fragments mentioned above.

[0072] As used herein, the terms “humanized antibody” or “humanized version of an antibody” refer to an antibody in which the framework or “complementarity-determining region” (CDR) is modified to include an immunoglobulin CDR having different specificity compared to the CDR of the parent immunoglobulin. In some exemplary embodiments, the “humanized antibody” is prepared by transplanting VH and VL CDRs into the framework region of a human antibody. See, for example, Riechmann, L. et al., Nature 332 (1988) pp. 323–327; and Neuberger, MS et al., Nature 314 (1985) pp. 268–270. The heavy and light chain variable framework regions may be derived from the same or different human antibody sequences. The human antibody sequences may be sequences of naturally occurring human antibodies. Human heavy and light chain variable framework regions are listed, for example, in Lefranc, M.-P., Current Protocols in Immunology (2000)-Appendix 1P A.1P.1-A.1P.37, or can be accessed through IMGT, the international ImMunoGeneTics information system® (registered trademark) (http: / / imgt.cines.fr), or http: / / vbase.mrc-cpe.cam.ac.uk, etc. In some cases, the framework regions may be modified by further mutations. An exemplary CDR corresponds to a sequence that recognizes the antigen described above for a chimeric antibody. In some embodiments, such a humanized version is chimerized with a human constant region. As used herein, the term “humanized antibody” also includes such antibodies that have been modified in the constant region to produce the properties described herein, particularly with respect to C1q binding and / or FcR binding, for example, by “class switching,” i.e., changes or mutations in the Fc portion (e.g., IgG1 to IgG4 and / or IgG1 / IgG4 mutations).

[0073] As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) pp. 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) capable of producing the entire repertoire of human antibodies or selectively by immunotherapy in the absence of endogenous immunoglobulin production. When a human germline immunoglobulin gene array is introduced into such germline mutant mice, human antibodies are produced upon antigen loading (see, for example, Jakobovits, A. et al., Proc. Natl. Acad. Sci. USA 90 (1993) pp. 2551-2555; Jakobovits, A. et al., Nature 362 (1993) pp. 255-258; Brueggemann, MD et al., Year Immunol. 7 (1993) pp. 33-40). Human antibodies can also be produced using phage display libraries (Hoogenboom, HR and Winter, G., J. Mol. Biol. 227 (1992) pp. 381-388; Marks, JD et al., J. Mol. Biol. 222 (1991) pp. 581-597). The techniques of Cole, A. et al. and Boerner, P. et al. can also be used for the preparation of human monoclonal antibodies (Cole, A. et al., Monoclonal Antibodies and Cancer Therapy, Liss, AR (1985) p. 77; and Boerner, P. et al., J. Immunol. 147 (1991) pp. 86-95). As previously stated, as used herein, the term “human antibody” as used herein also includes such antibodies that have been modified in the constant region to produce the properties described herein, for example with respect to C1q binding and / or FcR binding, by “class switching,” i.e., a change or mutation in the Fc portion (e.g., IgG1 to IgG4 and / or IgG1 / IgG4 mutation).

[0074] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, and the term "antigen-binding fragment" refers to its variable domain, or at least its antigen-binding site, e.g., CDR. Examples of antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are described, for example, in Huston, JS, Methods in Enzymol. 203 (1991), pp. 46-88. Antibody fragments can be derived from the antibodies of the present invention by many known techniques. For example, a purified monoclonal antibody can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. A suitable fraction containing the Fab fragment can then be collected and concentrated by membrane filtration or the like. For a more detailed explanation of general techniques for isolating antibody active fragments, see, for example, the following publications: Khaw, BA et al., J. Nucl. Med. 23: pp. 1011-1019 (1982); Rousseaux et al., Methods Enzymology, 121: pp. 663-69, Academic Press, 1986.

[0075] As used herein, the term “bispecific antibody” refers to an antibody that binds to two (or more) different antigens. A bispecific antibody typically comprises at least two different variable domains, each of which can specifically bind to a different antigen. In certain embodiments, the bispecific antibody of the present invention is a human antibody. As used herein, the expression “bispecific antigen-binding molecule” means a protein, polypeptide, or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain within a bispecific antigen-binding molecule contains at least one CDR that binds specifically to a particular antigen, either alone or in combination with one or more additional CDRs. In the context of the present invention, the first antigen-binding domain specifically binds to a first antigen (e.g., CD7), and the second antigen-binding domain specifically binds to a second different antigen (e.g., CD33). In certain embodiments, the bispecific molecule can bind to human CD7 and human CD33 simultaneously.

[0076] In certain embodiments, bispecific antibodies may be referred to as "anti-CD7 × CD33" or "anti-CD7 / anti-CD33," etc.

[0077] Any bispecific antibody format or technique may be used to construct the bispecific antigen-binding molecule of the present invention. Specific exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-1g, Quadroma, knob-into-hole, common light chain (e.g., common light chain with knob-into-hole), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab(DAF)-IgG, Mab2 bispecific format (e.g., Klein et al., 2012, imAbs 4:6, pp. 1-11, and the literature cited therein for a review of the above formats), and Fab-based bispecific formats. In certain embodiments, the bispecific antibody is Fab-based anti-CD7×CD33 bispecific.

[0078] As used herein, the terms “specific” and “specifically” are used interchangeably to indicate that biomolecules other than CD7 or CD33 (or both CD7 and CD33 if the biomolecule is a bispecific molecule) do not significantly bind to the antibody. In some embodiments, the binding level to biomolecules other than CD7 or CD33 can be ignored (e.g., undeterminable) by ELISA or affinity determination.

[0079] "Negligible bond" means a bond that is at least approximately 85%, particularly at least approximately 90%, more particularly at least approximately 95%, even more particularly at least approximately 98%, especially at least approximately 99%, and up to 100% lower than the bond to CD7 or CD33.

[0080] The binding affinity of an antibody to a peptide or epitope may be determined using a standard binding assay such as surface plasmon resonance (BIAcore®, GE Healthcare, Uppsala, Sweden). As used herein, "surface plasmon resonance" refers to an optical phenomenon that enables real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia BiosensorAB, Uppsala, Sweden, and Piscataway, NJ). For further details, see Jonsson, U. et al., (1993) Ann. Biol. Clin. 51: pp. 19-26; Jonsson, U. et al., (1991) Biotechniques 11: pp. 620-627; Johnsson, B. et al., (1995) J. Mol. Recognit. 8: pp. 125-131; and Johnson, B. et al., (1991) Anal. Biochem. 198: pp. 268-277.

[0081] In one embodiment, the antibody or its antigen-binding fragment of the present invention can mediate antibody-dependent cell-mediated cytotoxicity. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism mediated by immune effector cells that may contribute to the antitumor activity of monoclonal antibodies (Weiner GJ. Monoclonal antibody mechanisms of action in cancer. Immunol Res. 2007, 39(l-3): pp. 271-278). The relevance of ADCC to antitumor effects has been demonstrated in preclinical models, such as mouse tumor models (e.g., Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med. April 2000; 6(4): pp. 443-446). Data from clinical trials support the association of ADCC with the clinical efficacy of antibody therapies (e.g., Weng WK, Levy R Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J Clin Oncol. November 1, 2003;21(21):3940-397, e-version September 15, 2003). The interaction between monoclonal antibodies and Fc receptors on immune cells contributes to ADCC. The Fc of antibodies can be modified to increase their affinity for Fc receptors (e.g., Presta LG Engineering of therapeutic antibodies to minimize immunogenicity and optimize function. Adv Drug Deliv Rev. August 7, 2006;58(5-6):640-56, e-version May 23, 2006). This increased affinity for Fc receptors may lead to increased ADCC activity and potentially enhanced antitumor effects in patients.

[0082] In alternative embodiments, the antigen-binding fragment of the present invention is an immune-responsive cell expressing a chimeric antigen T cell receptor protein (CAR), the chimeric T cell receptor protein specifically binds to CD7 and CD33. In one embodiment, the immune-responsive cell is bispecific, expressing a chimeric antigen T cell receptor protein (CAR), the chimeric T cell receptor protein specifically binds to CD7 and the chimeric antigen T cell receptor protein (CAR), and the chimeric T cell receptor protein specifically binds to CD33. The immune-responsive cell expressing CAR may be selected from the group consisting of T cells, hematopoietic stem cells, natural killer cells, natural killer T cells, B cells, and monocyte lineage cells. In a particular embodiment, the immune-responsive cell is a T cell.

[0083] In some embodiments, the immune-responsive cells are self to the target. In other embodiments, the immune-responsive cells are not self to the target. In certain embodiments, the immune-responsive cells are T cells and are self to the target being treated.

[0084] In some embodiments, the antibody or its antigen-binding fragment includes a binding portion (i.e., a CD33 binding portion, a CD7 binding portion, or a CD7 and CD33 binding portion) and a cell-killing portion. In certain embodiments, the cell-binding portion is the antibody or its antigen-binding fragment. In certain embodiments, the cell-binding portion is the antibody or its antigen-binding fragment.

[0085] In some embodiments, the antibody or its antigen-binding fragment further comprises (or is incorporated into or associated with) a cytotoxic agent or cell proliferation inhibitor, i.e., a compound that kills or inhibits tumor cells. Such agents may confer cytotoxic and cell proliferation inhibitory effects through mechanisms including tubulin binding, DNA binding, proteasome and / or topoisomerase inhibition.

[0086] The cytotoxic agent or cell proliferation inhibitor may be, for example, a peptide toxin, a low-molecular-weight toxin, or a radioisotope.

[0087] In one embodiment, the cytotoxic agent or cell proliferation inhibitor may be a tubulin inhibitor or a DNA interacting agent. Tubulin inhibitors regulate tubulin polymerization. DNA interacting agents target cellular DNA.

[0088] In one embodiment, the cytotoxic agent or cell growth inhibitor is a tubulin inhibitor. In one embodiment, the tubulin inhibitor is selected from the group consisting of (a) auristatin and (b) maytansine derivatives. In one embodiment, the cytotoxic agent or cell growth inhibitor is auristatin. Auristatin includes synthetic derivatives of the naturally occurring compound drastatin-10. Auristatin is a family of antitumor / cell growth inhibitory pseudopeptides. Drastatin is structurally unique because it incorporates four rare amino acids (dolavaine, dolaisoleuine, dolaproine, and dolaphenine) identified in naturally occurring biosynthetic products. Furthermore, natural products of this class have numerous chiral centers as defined by total synthesis studies by Pett et al. (US4,978,744). Structure-activity relationships suggest that dry soroiine residues and draproine residues are necessary for antitumor activity (US5,635,483 and US5,780,588). In one embodiment, auristatin is selected from the group consisting of auristatin E (AE), monomethyl auristatin E (MMAE), auristatin F (MMAF), vcMMAE, vcMMAF, mcMMAE, and mcMMAF. In one embodiment, the cytotoxic agent or cell proliferation inhibitor is maytansine or a structural analog of maytansine. In one embodiment, the cytotoxic agent or cell proliferation inhibitor is maytansine. Maytansine contains structurally complex antimitotic polypeptides. Maytansine is a potent inhibitor of microtubule association that leads to apoptosis in tumor cells. In one embodiment, maytansine is selected from the group consisting of meltansine (DM1) and structural analogs of maytansine such as DM3 or DM4. Preferably, the drug is meltansine (DM1).

[0089] In one embodiment, the cytotoxic agent or cell growth inhibitor is a DNA interaction agent. In one embodiment, the DNA interaction agent is selected from the group consisting of (a) calicheamicin, (b) duocalmycin, and (c) pyrrolobenzodiazepine (PBD). In one embodiment, the cytotoxic agent or cell growth inhibitor is calicheamicin. Calicheamicin is a potent cytotoxic agent that causes double-strand DNA breaks and leads to cell death. Calicheamicin is a naturally occurring engine antibiotic (AL Smith et al., J. Med. Chem., 1996, 39, 11, pp. 2103-2117). Calicheamicin was discovered from the soil microorganism Micromonospora echinospora. In one embodiment, calicheamicin is calicheamicin γ1. In one embodiment, the drug is duocalmycin. Duocalmycin is a potent antitumor antibiotic that exerts its biological effect by sequence-selectively binding to the grooves of DNA double helix and alkylating the N3 group of adenine (D. Boger, Pure & Appl. Chem., 1994, 66, 4, pp. 837-844). In one embodiment, duocalmycin is selected from the group consisting of duocalmycin A, duocalmycin B1, duocalmycin B2, duocalmycin C1, duocalmycin C2, duocalmycin D, duocalmycin SA, cyclopropylbenzoindole (CBI) duocalmycin, centanamycin, rachermycin (CC-1065), adzeresin, bizeresin, and karzeresin. In one embodiment, the cytotoxic agent or cell proliferation inhibitor is pyrrolobenzodiazepine. Pyrrolobenzodiazepines (PBDs) are a class of naturally occurring antitumor antibiotics. Pyrrolobenzodiazepines belong to the genus Streptomyces. PBDs exert antitumor activity by specifically covalently binding to the small grooves of DNA in purine-guanine-purine units. PBDs are inserted into the N2 position of guanine via amine bonds, and their shape minimizes disruption of the DNA helix. The formation of DNA-PBD adducts is thought to inhibit nucleic acid synthesis and cause DNA helix cleavage-dependent single-strand and double-strand breaks.As a synthetic derivative, two PBD units are linked via a flexible polymethylene anchorage, causing the PBD dimer to crosslink opposing DNA strands and induce highly lethal lesions. In one embodiment, the cytotoxic or cell proliferation inhibitor is a synthetic derivative in which two pyrrolobenzodiazepine units are linked via a flexible polymethylene anchorage. In one embodiment, the pyrrolobenzodiazepine is selected from the group consisting of anthramycin (and its dimers), mazethramycin (and its dimers), tomaymycin (and its dimers), prothracarcin (and its dimers), chicamycin (and its dimers), neothramycin A (and its dimers), neothramycin B (and its dimers), DC-81 (and its dimers), sibiromycin (and its dimers), porothramycin A (and its dimers), porothramycin B (and its dimers), sibanomycin (and its dimers), abbeymycin (and its dimers), SG2000, and SG2285.

[0090] In one embodiment, the cytotoxic agent or cell growth inhibitor is a drug that targets interstrand crosslinks of DNA through alkylation. The drug that targets interstrand crosslinks of DNA through alkylation is selected from DNA-targeted mustard, guanine-specific alkylating agents, and adenine-specific alkylating agents. In one embodiment, the cytotoxic agent or cell growth inhibitor is a DNA-targeted mustard. For example, the DNA-targeted mustard may be selected from the group consisting of oligopyrrole, oligoimidazole, bis-(benzimidazole) carrier, polybenzamide carrier, and 9-anilinoacridin-4-carboxamide carrier.

[0091] In one embodiment, the cytotoxic agent or cell proliferation inhibitor is selected from the group consisting of netropsin, dystamycin, lexitropsin, talimustin, dibromotalimustin, PNU157977, and MEN10710.

[0092] In one embodiment, the cytotoxic agent or cell proliferation inhibitor is a bis-(benzimidazole) carrier. Preferably, the drug is Hoechst 33258.

[0093] Guanine-specific alkylating agents are highly regiospecific alkylating agents that react at specific nucleoside positions. In one embodiment, the cytotoxic agent or cell proliferation inhibitor is a guanine-specific alkylating agent selected from the group consisting of G-N2 alkylating agents, A-N3 alkylating agents, mitomycin, carmethizole analogs, and ectinacidin analogs. In one embodiment, mitomycin is selected from mitomycin A, mitomycin C, porphyromycin, and KW-2149. In one embodiment, the carmethizole analog is selected from bis-(hydroxymethyl)pyrrolizidine and NSC602668. In one embodiment, the ectinacidin analog is ectinacidin 743.

[0094] Adenine-specific alkylating agents are position-specific and sequence-specific small groove alkylating agents that react with the N3 position of adenine in polypyrimidine sequences.

[0095] Cyclopropanedrone and duocamycin are sometimes defined as adenine-specific alkylating agents. In one embodiment, the cytotoxic agent or cell proliferation inhibitor is a cyclopropanedrone analog. Preferably, the drug is selected from adzeresin and karzeresin.

[0096] In one embodiment, the cytotoxic agent or cell proliferation inhibitor is benz[e]indron. Preferably, the cytotoxic agent or cell proliferation inhibitor is selected from CBI-TMI and iso-CBI.

[0097] In one embodiment, the cytotoxic agent or cell proliferation inhibitor is bizeresin.

[0098] In one embodiment, the cytotoxic agent or cell proliferation inhibitor is a marine antitumor drug. Marine antitumor drugs are a developing field in the area of ​​antitumor drug development (I. Bhatnagar et al., Mar. Drugs 2010, 8, pp. 2702-2720, and TL Simmons et al., Mol. Cancer Ther. 2005, 4(2), pp. 333-342). Marine organisms such as sponges, sponge-microbial symbioses, gorgonian corals, actinomycetes, and soft corals are widely explored as candidates for anticancer drugs.

[0099] In one embodiment, the cytotoxic agent or cell proliferation inhibitor is selected from cytarabine, Ara-C, trabectedin (ET-743), and eribulin mesylate. In one embodiment, eribulin mesylate is (E7389), sobridotin (TZT 1027), squalamine lactate, semadolin prinabulin (NPI-2358), pritisin, erysidepsin, zalypsis, tacidotin, synthadotin, (ILX-651), discodermolide, HT1286, LAF389, kahalalide F, KRN7000, bryostatin 1, hemiasterin (E7974), marizomib, salinosporamide A (NPI-0052), LY355703, CRYPTO 52, Depsipeptide (NSC630176), Ectinacydin 743, Synsadotin, Kahalalide F, Squalamine, Dehydrodydemnin B, Didemnin B, Semadin, Sobridotin, E7389, NVP-LAQ824, Discodermorid, HTI-286, LAF-389, KRN-7000 (Agelasphin derivative), Curacin A, DMMC, Salinosporamide A, Laurimalid, Bitilevamide (V The following are selected: itilevuamide, diazonamide, eryuterobin, sarcodictiin, perolside A, salicylihalimide A and B, thiocoralin, ascididemin, variolin, lamelarin D, dictyodendrin, ES-285 (spithlosin), and halichondrin B.

[0100] The following cytotoxic or cell proliferation inhibitors are also included in the present invention: amatoxin (α-amanitin) bicyclic octapeptide, tubulysin, citricin, dolabellanin, and epothirons A, B, C, D, E, and F, produced by basidiomycetes of the genus Amanita, such as Green Deathcap mushroom. These constitute a group of epothirons-nontaxane tubulin polymerizers obtained by the natural fermentation of the myxobacterium Sorangium cellulosum. These sites possess potent cytotoxic activity and, in association with microtubule stabilization, lead to mitotic arrest at the G2 / M transition. Epothirons exhibit potent cytotoxicity in various cancer cell lines and often show higher activity than paclitaxel (X.: Pivot et al., European Oncology, 2008;4(2), pp. 42-45). In one embodiment, the drug is amatoxin. In one embodiment, the drug is tubricin. In one embodiment, the drug is citricin. In one embodiment, the drug is drabellanine. In one embodiment, the drug is epothilon.

[0101] The following cytotoxic agents or cell proliferation inhibitors are also included in the present invention. In one embodiment, the drug is doxorubicin, epirubicin, esorubicin, detrubicin, morpholino-doxorubicin, methotrexate, metopterin, bleomycin, dichloromethotrexate, 5-fluorouracil, cytosine-β-D-arabinofuranoside, taxol, angidin, melphalan, vinblastine, homopsin A, ribosome inactivating protein (RIP), daunorubicin, vinca alkaloid, idarubicin, melphalan, cisplatin, lysine Selected from saporin, anthracycline, indolino-benzodiazepine, 6-mercaptopurine, actinomycin, leulosin, leulosidine, carminomycin, aminopterin, tarisomycin, podophyllotoxin, etoposide, hairpin polyamide, etoposide phosphate, vinblastine, vincristine, vindesine, retinoic acid taxotere, N8-acetylspermidine, camptothecin, esperamicin, and engdiyne.

[0102] In one embodiment, the cell-killing portion is a peptide toxin, such as auristatin, like MMAE. In one embodiment, an antibody or its antigen-binding fragment comprises a binding portion and a cell-killing portion, the binding portion being an anti-CD7 anti-CD33 bispecific antibody or its binding portion, and the cell-killing portion being a peptide toxin, such as auristatin, like MMAE.

[0103] In certain embodiments, the antibody or its antigen-binding fragment includes a binding site conjugated to the cell-killing portion. Such conjugates can be prepared by in vitro methods known to those skilled in the art. Techniques for conjugating cytotoxic agents or cell proliferation inhibitors to proteins, particularly antibodies, are well known. (See, for example, Alley et al., Current Opinion in Chemical Biology 2010 14: pp. 1-9; Senter, Cancer J., 2008, 14(3): pp. 154-169).

[0104] In certain embodiments, linking groups are used to conjugate the binding and cytotoxic portions.

[0105] The linker is cleavable under intracellular conditions, and cleavage of the linker releases a cell-killing portion from the binding site in the intracellular environment. Cleavable linkers can be peptidyl linkers that are cleaved by intracellular peptidases or protease enzymes, such as lysosomal proteases or endosomal proteases. Examples of cleavage agents include cathepsin B and D, plasmin, etc. (see, for example, Dubowchik and Walker, Pharm. Therapeutics 83: pp. 67-123, 1999). The most typical is a peptidyl linker that can be cleaved by enzymes present in NTB-A expressing cells. For example, a peptidyl linker that can be cleaved by cathepsin B, a thiol-dependent protease highly expressed in cancer tissue, can be used (e.g., a linker containing Phe-Leu or Val-Cit peptides).

[0106] Cleavable linkers are pH-sensitive, meaning they are susceptible to hydrolysis at a specific pH value. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-unstable linkers that are hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) can be used.

[0107] Other linkers are cleavable under reducing conditions (e.g., disulfide linkers). Cleavable linkers may also be malonic acid linkers (Johnson et al., Anticancer Res. 15: pp. 1387-1393, 1995), maleimide benzoyl linkers (Lau et al., Bioorg-Med-Chem. 3: pp. 1299-1304, 1995), or 3'-N-amide analogs (Lau et al., Bioorg-Med-Chem. 3: pp. 1305-1312, 1995).

[0108] In some embodiments, the linker may be a protease-cleavable linker, such as valine-citrulline, which may be cleaved by cathepsin B in lysosomes.

[0109] The linker can also be a non-cleavable linker, such as a maleimide-alkylene or maleimide-aryl linker, which binds directly to the therapeutic agent and is released by proteolysis of the binding site.

[0110] Throughout this specification and in the claims, the words “includes” and “contains” and their variations mean “includes, but not limited to,” and are not intended (and are not intended) to exclude other parts, additives, ingredients, integers, or steps. Throughout this specification and in the claims, unless otherwise specified, singular forms include plural forms. In particular, where the indefinite article is used, this specification should be understood as plural and singular unless otherwise specified.

[0111] Features, integers, properties, compounds, chemical parts, or groups described in relation to a particular aspect, embodiment, or example of the present invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless otherwise incompatible. All features disclosed herein (including the appended claims, abstract, and drawings), and / or all steps of any method or process so herein disclosed may be combined in any combination, except for combinations in which at least some of such features and / or steps are mutually exclusive. The present invention is not limited to the details of the embodiments described above. The present invention extends to any novel one or any novel combination of any features disclosed herein (including the appended claims, abstract, and drawings), or any novel one or any novel combination of any step of any method or process so herein disclosed.

[0112] Embodiments of the present invention are described below only illustratively with reference to the following figures. [Brief explanation of the drawing]

[0113] [Figure 1] This bar graph shows the binding of reduced-affinity bi-Fab at 10 nM to CD33+ / CD7+ cell lines, normal PBMC monocytes, and T cells isolated from healthy human donors. 50,000 cells from HNT-34 (CD33+ / CD7+), Kasumi-3 (CD33+ / CD7+), Jurkat (CD33- / CD7+), SHI-1 (CD33+ / CD7-), healthy PBMC monocytes (CD33+ / CD7-), and healthy T cells (CD33- / CD7+) were tested for CD33+ / CD7+ bi-Fab antibody. The cells were resuspended in 100 μl of 10 nM bi-Fab on ice for 1 hour. After incubation, the cells were pelleted and incubated for another hour on ice with 50 μl of mouse anti-human IgG Fab secondary antibody diluted to 1 / 17 in ice-cold PBS / 0.1% BSA and PE. The cells were washed, resuspended in PBS, and analyzed using FACS Calibur. The data were plotted in Excel. [Figure 2] This bar graph shows the binding of reduced-affinity bi-Fab at 10 nM to CD33+ / CD7+ cell lines, normal PBMC monocytes, and T cells isolated from healthy human donors. 50,000 cells from HNT-34 (CD33+ / CD7+), Kasumi-3 (CD33+ / CD7+), Jurkat (CD33- / CD7+), SHI-1 (CD33+ / CD7-), healthy PBMC monocytes (CD33+ / CD7-), and healthy T cells (CD33- / CD7+) were tested for CD33+ / CD7+ bi-Fab antibodies. The cells were resuspended in 100 μl of 10 nM bi-Fab on ice for 1 hour. After incubation, the cells were pelleted and incubated for another hour on ice with 50 μl of mouse anti-human IgG Fab secondary antibody diluted to 1 / 17 in ice-cold PBS / 0.1% BSA and PE. The cells were washed, resuspended in PBS, and analyzed using FACS Calibur. The data were plotted in Excel. [Figure 3]This graph illustrates a cytotoxicity assay performed using a 10-point dose-response method with BVX130-MMAF directly conjugated to 2 × 10⁴ Kasumi-3 (CD33+ / CD7+) cells per well. Plates were incubated at 37°C and 5% CO₂ for 96 hours. After incubation, 5 μl of Alamar Blue reagent was added per well, and the plates were further incubated at 37°C and 5% CO₂ for 4 and 6 hours before readings were taken. Data for each reading were plotted in GraphPad PRISM, and IC50 values ​​were recorded. IC50 was calculated using the 4-hour reading data for the Kasumi-3 cell line. The IC50 was 0.1235 nM. [Figure 4] This graph illustrates a cytotoxicity assay performed using a 10-point dose-response system with BVX130-MMAF directly conjugated to 2 × 10⁴ HNT-34 (CD33+ / CD7+) cells per well. Plates were incubated at 37°C and 5% CO₂ for 96 hours. After incubation, 5 μl of Alamar Blue reagent was added per well, and the plates were further incubated at 37°C and 5% CO₂ for 4 and 6 hours before readings were taken. Data for each reading were plotted in GraphPad PRISM, and IC50 values ​​were recorded. IC50 was calculated using the 4-hour reading data for the HNT-34 cell line. The IC50 was 0.1204 nM. [Figure 5] This graph illustrates a cytotoxicity assay performed using a 10-point dose-response system with BVX100-MMAF directly conjugated to 2 × 10⁴ Kasumi-3 (CD33+ / CD7+) cells per well. Plates were incubated at 37°C and 5% CO₂ for 96 hours. After incubation, 5 μl of Alamar Blue reagent was added per well, and the plates were further incubated at 37°C and 5% CO₂ for 4 and 6 hours before readings were taken. Data for each reading were plotted in GraphPad PRISM, and IC50 values ​​were recorded. IC50 was calculated using the 4-hour reading data for the Kasumi-3 cell line. The IC50 was 0.0651 nM. [Figure 6] This graph illustrates a cytotoxicity assay performed using a 10-point dose-response method with BVX100-MMAF directly conjugated to 2 × 10⁴ HNT-34 (CD33+ / CD7+) cells per well. Plates were incubated at 37°C and 5% CO₂ for 96 hours. After incubation, 5 μl of Alamar Blue reagent was added per well, and the plates were further incubated at 37°C and 5% CO₂ for 4 and 6 hours before readings were taken. Data for each reading were plotted in GraphPad PRISM, and IC50 values ​​were recorded. IC50 was calculated using the 4-hour reading data for the HNT-34 cell line. The IC50 was 0.2437 nM. [Figure 7] This graph shows the dose-response curves of a cytotoxicity assay performed using nine doses of BVX130-MMAE or BVX100-MMAE directly conjugated to 2 × 10⁴ Jurkat cells (CD33- / CD7+) per well, up to a maximum final concentration of 30 nM. The plates were incubated at 37°C and 5% CO₂ for 96 hours. After incubation, 10 μl of CellTiter 96 AQueous One Solution was pipettered into each well, and the plates were incubated for a further 3 hours at 37°C and 5% CO₂. Absorbance was read at 492 nm and 690 nm. OD492 nm was subtracted from OD690 nm, and the data were plotted using GraphPad PRISM software. Error bars represent the standard deviation of four consecutive repeats. [Figure 8]This graph shows the dose-response curves of a cytotoxicity assay performed using a 10-point dose-response system with BVX130-MMAF or BVX100-MMAF directly conjugated to 2 × 10⁴ Jurkat cells (CD33- / CD7+) per well, up to a peak final concentration of 30 nM. Plates were incubated at 37°C and 5% CO₂ for 96 hours. After incubation, 5 μl of Alamar Blue reagent was added per well, and the plates were further incubated at 37°C and 5% CO₂ for 4 and 6 hours before readings were taken. Data for each reading were plotted in GraphPad PRISM, and IC50 values ​​were recorded. IC50 was calculated using the 4-hour reading data for the Jurkat cell line. Error bars represent the standard deviation of four consecutive repeats. [Figure 9] This graph shows the dose-response curves of a cytotoxicity assay performed using a 10-point dose-response system with BVX130-MMAF or BVX100-MMAF directly conjugated to 2 × 10⁴ LOUCY cells (CD33- / CD7+) per well, up to a peak final concentration of 30 nM. Plates were incubated at 37°C and 5% CO₂ for 96 hours. After incubation, 5 μl of Alamar Blue reagent was added per well, and the plates were further incubated at 37°C and 5% CO₂ for 4 and 6 hours before reading. Data for each reading were plotted in GraphPad PRISM, and the IC50 value was recorded. IC50 was calculated using the 4-hour reading data for the LOUCY cell line. Error bars represent the standard deviation of four consecutive repeats. [Modes for carrying out the invention]

[0114] (Example 1) Evaluation of the binding of different CD33+ / CD7+ bi-Fabs in cell lines and primary cells. The binding of different CD33+ / CD7+ conjugate bispecific antibodies was evaluated in various cell lines and primary cells derived from healthy human donors. Each conjugate bispecific antibody showed a different pair of binding arms targeting either CD33 or CD7 (Table 1). The binding arms of each bispecific antibody exhibited different affinities to CD33 or CD7.

[0115] [Table 1]

[0116] [Table 2]

[0117] reagent Mouse anti-human IgG Fab secondary antibody, PE ThermoFisher, #MA110377 Jurkat cells DSMZ282 Kasumi-3 cells DSMZ714 HNT-34 cells DSMZ600 SHI-1 cells DSMZ645 Healthy PBMC donor 1, Cambridge Bioscience Inc. ID PR18E125592 PBS / A (Catalog number 50086470) VWR BSA (Bovine Serum Albumin Fraction V 100ml 7.5% Catalog No. 15260037) ThermoFisher BVX1001 BVX1101 BVX1201 BVX1301 BVX1401 BVX1501 BVX1601 BVX1011 BVX1611 BVX1021 BVX1521 BVX1621 BVX1631

[0118] method Each bi-Fab agent was prepared in 1 ml of PBS / 0.1% BSA to a 10 nM concentration. Cells were collected and counted to a sufficient number to test each bi-Fab in its respective dilution. 50,000 cells were then used per test, and samples labeled with secondary antibody only were added. 100,000 cells were used per test for PBMC samples. Cells were pelleted at 1000 rpm at 4°C for 5 minutes and resuspended in ice-cold PBS. 100 μl aliquots of each cell sample were pipetted into each well of a V-bottom 96-well plate on ice. Cells were pelleted at 1000 rpm at 4°C for 5 minutes, and the supernatant was aspirated. Cells were resuspended in 100 μl of 10 nM bi-Fab. The plate was incubated on ice for 1 hour. 75 μl of ice-cold PBS was added to each well, and cells were pelleted at 1000 rpm at 4°C for 5 minutes. The supernatant was aspirated, and the cells were resuspended in 50 μl of mouse anti-human IgG Fab secondary antibody diluted to 1 / 17th in ice-cold PBS / 0.1% BSA, and PE. The plate was incubated on ice for 1 hour. 100 μl of ice-cold PBS was added to each well, and the cells were pelleted at 1000 rpm at 4°C for 5 minutes. The cells were resuspended in 300 μl of ice-cold PBS, transferred to a FACS tube, and fluorescence analysis was performed using FACS Calibur, detecting PE staining with FL2.

[0119] Monocyte populations in PBMC samples were identified using side-scatter / forward-scatter dot plots. The data were plotted in Excel.

[0120] result The binding of each bispecific antibody (1 nM and 10 nM) to each cell line was evaluated simultaneously with its binding to monocytes and T cells isolated from healthy human donors (Figures 1 and 2). As is evident from this data, the specific CD7 and CD33 constructs used within each bispecific construct affect the binding to bi-(CD33+ / CD7+) versus mono(CD33+ / CD7- or CD33- / CD7+) antigen-positive cell lines and primary cells isolated from healthy human donors.

[0121] [Table 3-1]

[0122] [Table 3-2]

[0123] [Table 3-3]

[0124] [Table 3-4]

[0125] Figures 1 and 2 show that the decrease in binding affinity of the CD7-binding arm of bispecific antibodies, including the wild-type CD33-binding arm, does not affect the binding affinity to CD7+ / CD33+ cell lines. Surprisingly, the binding of the bispecific construct to CD33- / CD7+ cells was affected by the affinity of both the CD7 and CD33 arms used within the construct. Table 2 (Table 3) above shows the results for the bispecific antibodies tested, and this data is summarized in Table 3 (Table 4), which includes the multiplier of decrease in binding affinity of each binding arm for each bispecific antibody.

[0126] [Table 4]

[0127] (Example 2) Evaluation of cytotoxicity in CD7+CD33+ immortalized cell lines The cytotoxic (cytotoxic) efficiency of CD33+ / CD7+ bispecific antibody-drug conjugates was evaluated in multiple CD33+ / CD7+ cell lines.

[0128] Test reagents • BVX100-MMAF - bi-Fab process using CD33 monobinder × CD7 monobinder conjugated to mcMMAF (cytotoxic payload) • BVX130-MMAF - A bi-Fab of CD33 monobinder × CD7 monobinder conjugated to a partially optimized mcMMAF (cytotoxic payload) containing a CD7 monobinder (i.e., the CD7 protein sequence is different compared to BVX100-MMAF). • CD33 x CD33-MMAF - bi-Fab of CD33 monobinder x CD33 monobinder conjugated to mcMMAF (cytotoxic payload) • Gemtuzumab-MMAF - Gemtuzumab is a commercially available CD33 monospecific IgG antibody component of Pfizer's ADC Mylotarg™, conjugated to mcMMAF (cytotoxic payload). • All ADC conjugates used the same payload, conjugation technology, and drug-to-antibody ratio.

[0129] reagent · Kasumi-3 cells DSMZ714 · HNT-34 cells DSMZ600 • BVX130-MMAF (P01-32, 10.5μM) - In-house - Head of Chemical Department • BVX130-MMAE (193-27-3, 49μM) - In-house - Head of Chemical Department • BVX100-MMAF (193-15, 13μM) - In-house - Head of Chemistry Department • BVX100-MMAE (193-28, 34 μM) - In-house - Head of Chemical Department • BSA (Bovine Serum Albumin Fraction V 100ml 7.5% Catalog No. 15260037) ThermoFisher • PBS / A (Catalog number 50086470) VWR Corning 384-well transparent flat-bottom polystyrene TC-treated microplate SLS • alamarBlue (trademark) Cell viability reagent (DAL1025) ThermoFisher • RPMI-1640 medium 21875034 Gibco, Life Technologies • GlutaMAX (trademark) nutritional supplement 35050061 Gibco, Life Technologies • Fetal bovine serum, thermoinactivated 10500064 Gibco, Life Technologies • Penicillin-streptomycin (10,000 U / mL) 15140122 Gibco, Life Technologies

[0130] method Kasumi-3 and HNT-34 cell lines were collected and counted, and the amount of medium required to seed 2000 cells per well across 100 wells in a 384-well plate was calculated using 40 μl of medium per well. Ten dose-response assays for BVX130-MMAE, BVX130-MMAF, BVX100-MMAE, and BVX100-MMAF were prepared in assay medium (RPMI, 10% FBS, 1% Glutamax, 1% Pen / Strep) at 5 times the final concentration, with a maximum final concentration of 30 nM. 10 μl of each dose was pipettered into triple-well sections of a 384-well plate, and a separate plate was prepared for each cell line tested. 500,000 cells from each cell line were pipettered into 5 ml of assay medium and pelletized. Each cell pellet was resuspended in 10 ml of assay medium and pipetted into 40 μl of marked wells. 50 μl of assay medium was pipetted into blank control wells, and 50 μl of PBS was pipetted into all reserve wells. The plates were incubated at 37°C, 5% CO2 for 4 or 7 days. After 4 days of incubation, 5 μl of Alamar Blue reagent was added per well, and the plates were further incubated at 37°C, 5% CO2 for 4 and 6 hours, after which the plates were read. The data for each reading was plotted in GraphPad PRISM, and the IC50 values ​​were recorded. The 4-hour reading data for cell lines Kasumi-3 and HNT-34 was used in the IC50 summary table.

[0131] result Examples of dose-response curves for each of these assays are shown in Figures 3 to 6. Briefly, each CD33+ / CD7+ bispecific antibody-drug conjugate exhibited robust cytotoxicity in different CD33+ / CD7+ cell lines. Table 4 (Table 5) below summarizes the mean cytotoxicity IC50 of each bispecific antibody in each cell type across multiple experiments.

[0132] [Table 5]

[0133] Figure 3 shows the dose-response curve of cytotoxicity occurring when Kasumi-3 cells are exposed to increasing concentrations of BVX130-MMAF. Kasumi-3 cells are CD33+ / CD7+, and BVX130-MMAF is a low-affinity CD33+ / CD7+ bispecific antibody conjugated with a cytotoxic payload. An example of the IC50 of BVX130-MMAF in Kasumi-3 cells is 0.1235 nM, and the average IC50 over 13 experiments is 0.16 nM. The average IC50 over 12 experiments with the same bispecific antibody conjugate in HNT-34 cells (CD33+ / CD7+) is 0.37 nM. Figure 4 shows an example of the dose-response curve occurring when HNT-34 cells are exposed to increasing concentrations of BVX130-MMAF.

[0134] BVX130-MMAF contains a partially optimized CD7 sequence compared to BVX100-MMAF. Figures 5 and 6 show examples of dose-response curves using BVX100-MMAF as a bispecific antibody-drug conjugate. Similar to BVX130-MMAF, the cell types tested were Kasumi-3 and HNT-34, both of which are CD33+ / CD7+. The mean IC50 of BVX100-MMAF in Kasumi-3 cells was 0.11 nM over 6 experiments. The mean IC50 of BVX100-MMAF in HNT-34 cells was 0.41 nM over 4 experiments.

[0135] As shown in Table 4 (Table 5), MMAF conjugate bispecific antibodies generally exhibit higher IC50 values ​​in each cell type. For example, the average IC50 of BVX130-MMAF in Kasumi-3 cells is 0.16 nM, while the IC50 of BVX130-MMAE in the same cell type is 0.45 nM. The same applies when considering BVX100-MMAF versus BVX100-MMAE in the same cell line.

[0136] overview The results above demonstrate that each of the bispecific CD33+ / CD7+ antibodies conjugated with a cytotoxic payload effectively induces cell death in two CD33+ / CD7+ cell lines. The next step was to determine whether exposure to these bispecific antibody-drug conjugates causes off-target cytotoxicity in cells expressing only CD33 or CD7 as a single target, representing a single-antigen-positive healthy cell population in the blood. This was evaluated by determining the selectivity ratio in cell death using a panel of bispecific antibodies conjugated with cytotoxic payloads in CD33+ / CD7+ cell lines, CD33+ / CD7- cell lines, and CD33- / CD7+ cell lines.

[0137] (Example 3) Evaluation of CD33+ / CD7+ selectivity for CD33+ / CD7- and CD33- / CD7+ cells. To investigate whether specific bispecific antibodies conjugated to cytotoxic payloads preferentially target CD33+ / CD7+ cells over CD33+ / CD7- cells and CD33- / CD7+ cells, a panel of biantigen-positive and monoantigen-positive immortalized cell lines were exposed to various conjugated CD7+ / CD33+ bispecific antibodies, and cytotoxicity was measured.

[0138] Cytotoxic activity of Bi-Fab-MMAE conjugates in dual and mono-positive CD33 and CD7 cell lines reagent · Kasumi-3 cells DSMZ714 · HNT-34 cells DSMZ600 · SHI-1 cells DSMZ645 · MV4.11 cells DSMZ102 · Jurkat cells DSMZ282 Internal Department - Head of Chemicals · BVX100-MMAE · BVX110-MMAE · BVX120-MMAE · BVX130-MMAE · BVX140-MMAE · BVX150-MMAE · BVX160-MMAE • BSA (Bovine Serum Albumin Fraction V 100ml 7.5% Catalog No. 15260037) ThermoFisher • PBS / A (Catalog number 50086470) VWR Corning 384-well transparent flat-bottom polystyrene TC-treated microplate SLS • alamarBlue (trademark) Cell viability reagent (DAL1025) ThermoFisher • RPMI-1640 medium 21875034 Gibco, Life Technologies • GlutaMAX (trademark) nutritional supplement 35050061 Gibco, Life Technologies • Fetal bovine serum, thermoinactivated 10500064 Gibco, Life Technologies • Penicillin-streptomycin (10,000 U / mL) 15140122 Gibco, Life Technologies

[0139] method The cells were harvested, counted, and divided into 2 × 10⁶ cells per 90 μl of growth medium. 4 The cells were resuspended. Eight dose-response tests of bi-Fab-MMAE were prepared in growth medium at 5 times the final assay concentration, with a peak final concentration of 30 nM. 10 μl of bi-Fab-MMAE dose settings were pipettered into a 384-well plate, and each concentration was tested in two wells. 40 μl of cells were pipettered into the wells, and the plate was incubated at 37°C, 5% CO2 for 96 hours. After incubation, 5 μl of MTS reagent was pipettered into each well, and the plate was incubated for a further 3 hours at 37°C, 5% CO2. Absorbance was read at 492 nm and 690 nm. OD492 nm was subtracted from OD690 nm, and the data was plotted using GraphPad PRISM software.

[0140] [Table 6]

[0141] result Table 5 (Table 6) shows the determined cytotoxicity of each conjugate bispecific antibody tested across the entire cell line panel, along with the selectivity ratio of each construct against bipositive CD33+ / CD7+ cell lines compared to CD33+ / CD7- or CD33- / CD7+ cell lines. Based on these results, BVX130 was selected for further analysis due to its potent cytotoxicity observed in CD33+ / CD7+ cell lines and the increased selectivity ratio observed against both CD33+ / CD7- and CD33- / CD7+ cell lines.

[0142] Cytotoxic activity of BVX100 and BVX130MMAF conjugates in CD7 monopositive cell lines reagent Jurkat cells DSMZ282 LOUCY cells DSMZ394 BVX130-MMAF (P01-32, 10.5μM) - In-house - Head of Chemical Department BVX100-MMAF (193-15, 13μM) - In-house - Head of Chemistry Department BSA (Bovine Serum Albumin Fraction V 100ml 7.5% Catalog No. 15260037) ThermoFisher PBS / A (Catalog number 50086470) VWR Corning 384-well transparent flat-bottom polystyrene TC-treated microplate SLS alamarBlue® Cell Viability Test (DAL1025) ThermoFisher RPMI-1640 Medium 21875034 Gibco, Life Technologies GlutaMAX (trademark) nutritional supplement 35050061 Gibco, Life Technologies Fetal bovine serum, thermoinactivated, 10500064 Gibco, Life Technologies Penicillin-Streptomycin (10,000 U / mL) 15140122 Gibco, Life Technologies

[0143] method Jurkat and LOUCY cell lines were collected and counted, and the amount of medium required to seed 2000 cells per well across 100 wells in a 384-well plate was calculated. Ten dose-response assays for BVX130-MMAF and BVX100-MMAF were prepared in assay medium (RPMI, 10% FBS, 1% Glutamax, 1% Pen / Strep) at five times the final concentration, with a maximum final concentration of 30 nM. 10 μl of each dose was pipettered into triple-well sections of a 384-well plate, creating a separate plate for each cell line tested. 500,000 cells from each cell line were pipettered into 5 ml of assay medium to form a cell pellet. Each cell pellet was resuspended in 10 ml of assay medium, and 40 μl was pipettered into the indicated well. 50 μl of assay medium was pipetteed into a blank control well, and 50 μl of PBS was pipetteed into all spare wells. The plate was incubated at 37°C and 5% CO2 for 4 days. After 4 days of incubation, 5 μl of Alamar Blue reagent was added per well, and the plate was incubated for 4 and 6 hours at 37°C and 5% CO2 before reading the plates. The data for each reading was plotted in GraphPad PRISM, and the IC50 values ​​were recorded.

[0144] result Affinity-modulated CD33+ / CD7+ bispecific antibody BVX130, conjugated with an MMAF or MMAE cytotoxic payload, exhibits reduced cytotoxicity in CD7 single antigen-positive cells compared to BVX100, as shown in Figures 7 to 9.

[0145] As shown, CD33+ / CD7+ bispecific antibodies conjugated with cytotoxic payloads induce robust cytotoxicity in immortalized CD33+ / CD7+ cell lines (Figures 3 to 6). In CD33+ / CD7- cell lines (Table 5 (Table 6)) and CD33- / CD7+ expressing cell lines (Figures 7 to 9), reduced cytotoxicity was observed in constructs with a low-affinity CD7 binding arm and a high-affinity CD33 binding arm. This was also true when two different cytotoxic payloads were conjugated to the bispecific antibody. This suggests that CD33+ / CD7+ bispecific antibodies conjugated with cytotoxic payloads can induce robust cytotoxicity in CD33+ / CD7+ cells while reducing cytotoxicity in CD33 and CD7 single-antigen positive cells such as monocytes (CD33+ / CD7-) and T cells (CD33- / CD7+).

[0146] The constructs of BVX102, BVX152, BVX162, BVX110, BVX120, and BVX130 all showed promise as therapeutic agents, with BVX130 in particular exhibiting similar activity to BVX110 and BVX120.

[0147] The embodiments described above are not intended to limit the scope of protection granted by the claims, but rather to illustrate examples of how the present invention can be implemented. Sequence List

[0148] CD7-PP-1-WT CD7 coupling arm arrangement: VH:

[0149] [ka]

[0150] VH CDR1:

[0151] [ka]

[0152] VH CDR2:

[0153]

change

[0154] VH CDR3:

[0155]

change

[0156] VL:

[0157]

change

[0158] VL CDR1:

[0159]

change

[0160] VL CDR2:

[0161]

change

[0162] VL CDR3:

[0163]

change

[0164] Relock:

[0165]

change

[0166] Light chain:

[0167]

Chem.

[0168] CD7-PP-2 CD7 binding arm sequence: VH:

[0169]

Chem.

[0170] VH CDR1:

[0171]

Chem.

[0172] VH CDR2:

[0173]

Chem.

[0174] VH CDR3: <x

[0175]

Chem.

[0176] [[ID=(62)]] VL:

[0177]

Chem.

[0178] VL CDR1:

[0179]

Chem.

化

Chem.

[0175] might be a misprint in the original, but it's preserved as is per the instructions. And for the tag <000093{}2>, it's not clear what the "{}" is supposed to represent, but the tag is left as is in the translation.

[0180] VL CDR2:

[0181]

Chem.

[0182] VL CDR3:

[0183]

Chem.

[0184] Heavy chain:

[0185]

Chem.

[0186] Light chain:

[0187]

Chem.

[0188] CD7-PP-3 CD7 binding arm sequence: VH:

[0189]

Chem.

[0190] VH CDR1:

[0191]

Chem.

[0192] VH CDR2:

[0193]

Chem.

[0194] VH CDR3:

[0195] [ka]

[0196] VL:

[0197] [ka]

[0198] VL CDR1:

[0199] [ka]

[0200] VL CDR2:

[0201] [ka]

[0202] VL CDR3:

[0203] [ka]

[0204] Heavy chain:

[0205] [ka]

[0206] Light chain:

[0207] [ka]

[0208] CD7-PP-6 binding sequence: VH:

[0209]

change

[0210] VH CDR1:

[0211]

change

[0212] VH CDR2:

[0213]

change

[0214] VH CDR3:

[0215]

change

[0216] VL:

[0217]

change

[0218] VL CDR1:

[0219]

change

[0220] VL CDR2:

[0221]

change

[0222] VL CDR3:

[0223] [ka]

[0224] Heavy chain:

[0225] [ka]

[0226] Light chain:

[0227] [ka]

[0228] CD7-PP-7 CD7 coupling arm arrangement: VH:

[0229] [ka]

[0230] VH CDR1:

[0231] [ka]

[0232] VH CDR2:

[0233] [ka]

[0234] VH CDR3:

[0235] [ka]

[0236] VL:

[0237] [ka]

[0238] VL CDR1:

[0239] [ka]

[0240] VL CDR2:

[0241] [ka]

[0242] VL CDR3:

[0243] [ka]

[0244] Heavy chain:

[0245] [ka]

[0246] Light chain:

[0247] [ka]

[0248] CD7-PP-8 CD7 coupling arm arrangement: VH:

[0249] [ka]

[0250] VH CDR1:

[0251]

change

[0252] VH CDR2:

[0253]

change

[0254] VH CDR3:

[0255]

change

[0256] VL:

[0257]

change

[0258] VL CDR1:

[0259]

change

[0260] VL CDR2:

[0261]

change

[0262] VL CDR3:

[0263]

change

[0264] Heavy chain:

[0265] [ka]

[0266] Light chain:

[0267] [ka]

[0268] CD7-PP-12 CD7 coupling arm arrangement: VH:

[0269] [ka]

[0270] VH CDR1:

[0271] [ka]

[0272] VH CDR2:

[0273] [ka]

[0274] VH CDR3:

[0275] [ka]

[0276] VL:

[0277] [ka]

[0278] VL CDR1:

[0279] [ka]

[0280] VL CDR2:

[0281] [ka]

[0282] VL CDR3:

[0283] [ka]

[0284] Heavy chain:

[0285] [ka]

[0286] Light chain:

[0287] [ka]

[0288] CD7-PP-13 CD7 coupling arm arrangement: VH:

[0289] [ka]

[0290] VH CDR 1:

[0291] [ka]

[0292] VH CDR 2:

[0293]

change

[0294] VH CDR 3:

[0295]

change

[0296] VL:

[0297]

change

[0298] VL CDR1:

[0299]

change

[0300] VL CDR2:

[0301]

change

[0302] VL CDR3:

[0303]

change

[0304] Relock:

[0305]

change

[0306] Light lock:

[0307]

change

[0308] CD33-PP-1-WT binding peptide sequence: VH:

[0309]

change

[0310] VH CDR 1:

[0311]

change

[0312] VH CDR 2:

[0313]

change

[0314] VH CDR 3:

[0315]

change

[0316] VL:

[0317]

change

[0318] VL CDR 1:

[0319]

change

[0320] VL CDR 2:

[0321]

change

[0322] VL CDR 3:

[0323]

change

[0324] CD33-PP-4 binding sequence: VH:

[0325]

change

[0326] VH CDR 1:

[0327]

change

[0328] VH CDR 2:

[0329]

change

[0330] VH CDR 3:

[0331]

change

[0332] VL:

[0333]

change

[0334] VL CDR 1:

[0335]

change

[0336] VL CDR 2:

[0337]

change

[0338] VL CDR 3:

[0339]

change

[0340] CD33-PP-7 binding sequence: VH:

[0341]

change

[0342] VH CDR 1:

[0343]

change

[0344] VH CDR 2:

[0345]

change

[0346] VH CDR 3:

[0347]

change

[0348] VL:

[0349] [ka]

[0350] VL CDR 1:

[0351] [ka]

[0352] VL CDR 2:

[0353] [ka]

[0354] VL CDR 3:

[0355] [ka]

[0356] Selective signal sequences upstream of the VH and VL domains

[0357] [ka]

Claims

1. A bispecific antibody that binds to CD33 and CD7, or an antigen-binding fragment thereof, VH sequence containing sequence of sequence number 81 and VL sequence containing sequence number 85 A first binding region that binds to human CD33, and The sequence of sequence number 31 includes VH CDR1, VH CDR2, VH CDR3 in the VH region, and VL CDR1, VL CDR2, VL CDR3 in the VL region of sequence number 35, and the sequence is defined by the IMGT numbering system, and comprises a second binding region that binds to human CD7. A bispecific antibody or its antigen-binding fragment, including the above.

2. The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein the second binding region that binds to human CD7 comprises a VH sequence containing the sequence of SEQ ID NO: 31 or a sequence having 95% sequence identity thereto, and a VL sequence containing the sequence of SEQ ID NO: 35 or a sequence having 95% sequence identity thereto.

3. A pharmaceutical composition for use in the treatment of CD7+CD33+ hematological malignancies, comprising the bispecific antibody or antigen-binding fragment thereof as described in claim 1 or 2.

4. The pharmaceutical composition according to claim 3, wherein the bispecific antibody or its antigen-binding fragment can induce internalization to CD33+ and / or CD7+ cells via CD33 and / or CD7 receptors.

5. The pharmaceutical composition according to claim 3 or 4, wherein the CD33+ and CD7+ cells are AML cells.

6. The pharmaceutical composition according to claim 4 or 5, wherein the bispecific antibody or its antigen-binding fragment can mediate antibody-dependent cell injury.

7. The pharmaceutical composition according to claim 4 or 5, wherein the bispecific antibody or its antigen-binding fragment is expressed by immune effector cells, and optionally the immune effector cells include T cells and / or NK cells.

8. The pharmaceutical composition according to claim 7, wherein the immune effector cells are bispecific anti-CD33 anti-CD7 CAR-T cells.

9. The pharmaceutical composition according to claim 8, wherein the T cells include CD33+ T cells, CD7+ T cells, or a combination thereof.

10. The pharmaceutical composition according to claim 3 or 4, wherein the bispecific antibody or antigen-binding fragment thereof comprises i) a cell-killing portion, ii) a CD7-binding portion, and iii) a CD33-binding portion.

11. The pharmaceutical composition according to claim 10, wherein the CD33 binding portion contains an antigen-binding fragment of an antibody, and / or the CD7 binding portion contains an antigen-binding fragment of an antibody.

12. The pharmaceutical composition according to claim 11, wherein the cell-killing portion is a cytotoxin, and the cytotoxin is optionally selected from i) a peptide toxin or ii) a chemical toxin.

13. The pharmaceutical composition according to claim 11 or 12, wherein the bispecific antibody or antigen-binding fragment thereof further comprises a linking portion that links the cell-killing portion to a CD7-binding portion and / or a CD33-binding portion.

14. The pharmaceutical composition according to any one of claims 11 to 13, wherein the bispecific antibody or its antigen-binding fragment is in the form of an antibody-drug conjugate.

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