Humanized anti-CD45 antibodies

Abstract

Humanized anti-human CD45 antibodies and pharmaceutical compositions comprising the antibodies are provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 407,979 filed on September 19, 2022, U.S. Provisional Application Serial No. 63 / 405,237 filed on September 9, 2022, and U.S. Provisional Application Serial No. 63 / 323,257 filed on March 24, 2022, each of which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on March 20, 2023, named ATNM-016PCT_SL_ST26.xml, and is 853,669 bytes in size. Technical Field

[0005] The present invention relates to the field of antibody-based therapeutic agents. Background Art

[0006] The CD45 antigen is a member of the protein tyrosine phosphatase (PTP) family and is a transmembrane glycoprotein of 180-240kD. It is also known as leukocyte common antigen (LCA), T200 or Ly-5. CD45 plays a key role in T cell and B cell receptor signal transduction. Due to the alternative splicing of its exons, CD45 has different isoforms. These isoforms have strong specificity for the activation and maturation state of cells and cell types. Various isoforms have the same transmembrane and cytoplasmic segments, but have different extracellular domains and are differentially expressed on B cell and T cell lymphocyte subsets. The main ligands described for CD45 include galectin-1, CD1, CD2, CD3, CD4, TCR, CD22 and Thy-1.

[0007] Monoclonal antibodies (mAbs) whose specificity is restricted to one or another isoform of CD45, as well as mAbs that recognize epitopes common to all CD45 isoforms, have been identified. The mAb designated CD45RA recognizes the product of exon-A. The mAb designated CD45RB recognizes the product of exon-B. The mAb designated CD45RO (exemplified by UCHL1) selectively binds to the 180 kD isoform (without any variable exons A, B, or C), which is restricted to a subset of cortical thymocytes, activated T cells, and memory cells, and is absent on B cells. BC8, also known as apamistamab, is a murine IgG1 monoclonal antibody that recognizes all isoforms of human CD45.

[0008] In addition to mature red blood cells and platelets, cells of hematopoietic origin usually express CD45. High expression of CD45 is found in most acute lymphoblastic and myeloid leukemias. Since CD45 is not found on tissues of non-hematopoietic origin, its specific expression in leukemia makes it a good target for the development of therapeutic drugs (including radioimmunotherapy drugs). For example, CD45 is expressed on circulating leukocytes and malignant B cells at a density of approximately 200,000 to 300,000 sites per cell.

[0009] What is desired and provided by various aspects of the present invention are novel humanized antibodies against human CD45. Summary of the invention

[0010] One aspect of the present invention provides an anti-huCD45 antibody comprising:

[0011] (i) a heavy chain which is mCD45-HC parent, huCD45.HC1, huCD45.HC2, huCD45.HC3, huCD45.HC1 or huCD45.HC4, or any of said heavy chains without a signal sequence, or a heavy chain comprising the heavy chain variable region of any of said heavy chains; and

[0012] (ii) a light chain which is mCD45-LC parent, huCD45.LC1, huCD45.LC2, huCD45.LC3 or huCD45.LC4, or any of said light chains without a signal sequence, or a light chain comprising the light chain variable region of any of said light chains,

[0013] Provided that when the heavy chain is the mCD45-HC parent with or without a signal sequence, the light chain is not the mCD45-LC parent with or without a signal sequence,

[0014] Or a human CD45 binding fragment of an antibody, such as a Fab fragment or a Fab2 fragment or a corresponding scFv molecule.

[0015] A related aspect of the present invention provides an anti-huCD45 antibody or human CD45 binding antibody fragment comprising:

[0016] (i) an immunoglobulin heavy chain variable region comprising SEQ ID NO: 2 (from mCD45-HC parent), SEQ ID NO: 7 (from huCD45.HC1), SEQ ID NO: 12 (from huCD45.HC2), SEQ ID NO: 17 (from huCD45.HC3), or SEQ ID NO: 22 (from huCD45.HC4); and

[0017] (ii) an immunoglobulin light chain variable region comprising SEQ ID NO: 28 (from mCD45-LC parent), SEQ ID NO: 33 (from huCD45.LC1), SEQ ID NO: 38 (from huCD45.LC2), SEQ ID NO: 43 (from huCD45.LC3), or SEQ ID NO: 48 (from huCD45.LC4),

[0018] Provided that when the heavy chain variable region comprises SEQ ID NO:2, the light chain variable region is not SEQ ID NO:28.

[0019] In a variant of the aforementioned aspect of the invention, the anti-huCD45 antibody or human CD45 binding antibody fragment comprises:

[0020] (i) an immunoglobulin heavy chain sequence comprising SEQ ID NO: 6 (from mCD45-HC parent), SEQ ID NO: 11 (from huCD45.HC1), SEQ ID NO: 16 (from huCD45.HC2), SEQ ID NO: 21 (from huCD45.HC3), or SEQ ID NO: 26 (from huCD45.HC4); and

[0021] (ii) an immunoglobulin light chain sequence comprising SEQ ID NO:32 (from mCD45-LC parent), SEQ ID NO:37 (from huCD45.LC1), SEQ ID NO:42 (from huCD45.LC2), SEQ ID NO:47 (from huCD45.LC3) or SEQ ID NO:52 (from huCD45.LC4).

[0022] Another aspect of the present invention provides an anti-huCD45 antibody or huCD45 binding antibody fragment, comprising:

[0023] (a) a set of immunoglobulin heavy chain complementarity determining regions (CDRs) comprising heavy chain CDR1, CDR2 and CDR3 amino acid sequences selected from the group consisting of:

[0024] (i) SEQ ID NO:53, SEQ ID NO:56 and SEQ ID NO:55, respectively,

[0025] (ii) SEQ ID NO:53, SEQ ID NO:57 and SEQ ID NO:55, respectively,

[0026] (iii) SEQ ID NO:53, SEQ ID NO:58 and SEQ ID NO:55, respectively, and

[0027] (iv) SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, respectively; and

[0028] (b) a set of immunoglobulin light chain complementarity determining regions (CDRs) comprising light chain CDR1, CDR2 and CDR3 amino acid sequences selected from the group consisting of:

[0029] (i) are SEQ ID NO:62, SEQ ID NO:60 and SEQ ID NO:61, respectively,

[0030] (ii) SEQ ID NO:59, SEQ ID NO:63 and SEQ ID NO:61, respectively,

[0031] (iii) SEQ ID NO:59, SEQ ID NO:64 and SEQ ID NO:61, respectively, and

[0032] (iv) SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61,

[0033] Provided that when the heavy chain CDR set is (a)(iv), the light chain CDR set is not (b)(iv).

[0034] A related aspect of the present invention provides an anti-huCD45 antibody or huCD45 binding antibody fragment comprising:

[0035] (a) a set of immunoglobulin heavy chain complementarity determining regions (CDRs) comprising heavy chain CDR1, CDR2 and CDR3 amino acid sequences selected from the group consisting of:

[0036] (i) SEQ ID NO:53, SEQ ID NO:56 and SEQ ID NO:55, respectively,

[0037] (ii) SEQ ID NO:53, SEQ ID NO:57 and SEQ ID NO:55, respectively, and

[0038] (iii) SEQ ID NO:53, SEQ ID NO:58 and SEQ ID NO:55, respectively; and

[0039] (b) a set of immunoglobulin light chain complementarity determining regions (CDRs) comprising light chain CDR1, CDR2 and CDR3 amino acid sequences selected from the group consisting of:

[0040] (i) are SEQ ID NO:62, SEQ ID NO:60 and SEQ ID NO:61, respectively,

[0041] (ii) SEQ ID NO:59, SEQ ID NO:63 and SEQ ID NO:61, respectively, and

[0042] (iii) SEQ ID NO:59, SEQ ID NO:64 and SEQ ID NO:61, respectively.

[0043] Additional features, advantages and aspects of the present invention may be set forth or become apparent in view of the following detailed description, drawings and claims. In addition, it should be understood that the foregoing summary of the present invention and the following detailed description are exemplary and intended to provide further explanation without limiting the scope of the claimed invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The amino acid sequences of various antibody heavy and light chains are as follows Figure 1-10 shown.

[0045] Figure 1 Shown is the amino acid sequence of the heavy chain of a mouse anti-huCD45 antibody ("mCD45-HC parent") that binds all isoforms of human CD45 (huCD45) and portions thereof and was used as the basis for the generation of humanized anti-huCD45 antibodies.

[0046] Figure 2 The amino acid sequence of the humanized anti-huCD45 antibody heavy chain ("hu CD45.HC1") and portions thereof are shown.

[0047] Figure 3 The amino acid sequence of the humanized anti-huCD45 antibody heavy chain ("hu CD45.HC2") and portions thereof are shown.

[0048] Figure 4 The amino acid sequence of the humanized anti-huCD45 antibody heavy chain ("hu CD45.HC3") and portions thereof are shown.

[0049] Figure 5 The amino acid sequence of the humanized anti-huCD45 antibody heavy chain ("hu CD45.HC4") and portions thereof are shown.

[0050] Figure 6 Shown is the amino acid sequence of the light chain of the mouse anti-huCD45 antibody ("mCD45-LC parent") and the portion thereof, which was used as the basis for generating the humanized anti-huCD45 antibody.

[0051] Figure 7 The amino acid sequence of the humanized anti-huCD45 antibody light chain ("hu CD45.LC1") and portions thereof are shown.

[0052] Figure 8 The amino acid sequence of the humanized anti-huCD45 antibody light chain ("hu CD45.LC2") and portions thereof are shown.

[0053] Fig. 9 The amino acid sequence of the humanized anti-huCD45 antibody light chain ("hu CD45.LC3") and portions thereof are shown.

[0054] Fig.10 The amino acid sequence of the humanized anti-huCD45 antibody light chain ("hu CD45.LC4") and portions thereof are shown.

[0055] In the presentation of the variable region sequences in the figures, the complementarity determining region (CDR) amino acid sequences are underlined. DETAILED DESCRIPTION

[0056] One aspect of the present invention provides an anti-(human CD45) ["anti-huCD45"] antibody comprising:

[0057] (i) a heavy chain which is mCD45-HC parent, huCD45.HC1, huCD45.HC2, huCD45.HC3, huCD45.HC1 or huCD45.HC4, or any of said heavy chains without a signal sequence, or a heavy chain comprising the heavy chain variable region of any of said heavy chains; and

[0058] (ii) a light chain which is mCD45-LC parent, huCD45.LC1, huCD45.LC2, huCD45.LC3 or huCD45.LC4, or any of said light chains without a signal sequence, or a light chain comprising the light chain variable region of any of said light chains,

[0059] Provided that when the heavy chain is the mCD45-HC parent with or without a signal sequence, the light chain is not the mCD45-LC parent with or without a signal sequence,

[0060] Or a human CD45 binding fragment of an antibody, such as a Fab fragment or a Fab2 fragment or a corresponding scFv molecule.

[0061] Parental mouse monoclonal antibodies include Figure 1 The parental heavy chain and Figure 6The parent light chain shown, which binds to all isoforms of human CD45, was used as the basis for designing humanized anti-huCD45 antibodies. Antibodies consisting of various combinations of humanized immunoglobulin heavy chains and immunoglobulin light chains were expressed in CHO cells and isolated for analysis and comparison with the parental antibodies. Binding experiments were performed at 25°C using Octet HTX (Sartorius, Bohemia, New York, USA) to determine the KD of the antibodies. The antibodies were loaded onto anti-human Fc capture (AHC) sensors. The loaded sensors were immersed in a series of dilutions of the rhCD45 His tag (900nM starting, 1:3 dilution, 3 points; R&D Systems, Minneapolis, MN, USA). Reference sample wells (buffer) were used for data analysis. Kinetic constants were calculated using a monovalent (1:1) binding model. The results are shown in Table 1 below.

[0062] Table 1

[0063]

[0064]

[0065] hu CD45 HC3+LC3 retained similar binding affinity to the parental antibody, while all other humanized samples showed less than 10-fold change in KD values ​​relative to the parental antibody.

[0066] Another aspect of the present invention provides an anti-huCD45 antibody or human CD45 binding antibody fragment, comprising:

[0067] (i) an immunoglobulin heavy chain variable region comprising SEQ ID NO: 2 (from mCD45-HC parent), SEQ ID NO: 7 (from huCD45.HC1), SEQ ID NO: 12 (from huCD45.HC2), SEQ ID NO: 17 (from huCD45.HC3), or SEQ ID NO: 22 (from huCD45.HC4); and

[0068] (ii) an immunoglobulin light chain variable region comprising SEQ ID NO: 28 (from mCD45-LC parent), SEQ ID NO: 33 (from huCD45.LC1), SEQ ID NO: 38 (from huCD45.LC2), SEQ ID NO: 43 (from huCD45.LC3), or SEQ ID NO: 48 (from huCD45.LC4),

[0069] Provided that when the heavy chain variable region comprises SEQ ID NO:2, the light chain variable region is not SEQ ID NO:28.

[0070] In a variant of the aforementioned aspect of the invention, the anti-huCD45 antibody or human CD45 binding antibody fragment comprises:

[0071] (i) an immunoglobulin heavy chain sequence comprising SEQ ID NO: 6 (from mCD45-HC parent), SEQ ID NO: 11 (from huCD45.HC1), SEQ ID NO: 16 (from huCD45.HC2), SEQ ID NO: 21 (from huCD45.HC3), or SEQ ID NO: 26 (from huCD45.HC4); and

[0072] (ii) an immunoglobulin light chain sequence comprising SEQ ID NO:32 (from mCD45-LC parent), SEQ ID NO:37 (from huCD45.LC1), SEQ ID NO:42 (from huCD45.LC2), SEQ ID NO:47 (from huCD45.LC3) or SEQ ID NO:52 (from huCD45.LC4).

[0073] Another aspect of the present invention provides an anti-huCD45 antibody or huCD45 binding antibody fragment, comprising:

[0074] (a) a set of immunoglobulin heavy chain complementarity determining regions (CDRs) comprising heavy chain CDR1, CDR2 and CDR3 amino acid sequences selected from the group consisting of:

[0075] (i) SEQ ID NO:53, SEQ ID NO:56 and SEQ ID NO:55, respectively,

[0076] (ii) SEQ ID NO:53, SEQ ID NO:57 and SEQ ID NO:55, respectively,

[0077] (iii) SEQ ID NO:53, SEQ ID NO:58 and SEQ ID NO:55, respectively, and

[0078] (iv) SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, respectively; and

[0079] (b) a set of immunoglobulin light chain complementarity determining regions (CDRs) comprising light chain CDR1, CDR2 and CDR3 amino acid sequences selected from the group consisting of:

[0080] (i) are SEQ ID NO:62, SEQ ID NO:60 and SEQ ID NO:61, respectively,

[0081] (ii) SEQ ID NO:59, SEQ ID NO:63 and SEQ ID NO:61, respectively,

[0082] (iii) SEQ ID NO:59, SEQ ID NO:64 and SEQ ID NO:61, respectively, and

[0083] (iv) SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61,

[0084] Provided that when the heavy chain CDR group is (a)(iv), the light chain CDR group is not (b)(iv). It should be understood that the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR and the light chain variable region comprises a light chain CDR.

[0085] A related aspect of the present invention provides an anti-huCD45 antibody or huCD45 binding antibody fragment comprising:

[0086] (a) a set of immunoglobulin heavy chain complementarity determining regions (CDRs) comprising heavy chain CDR1, CDR2 and CDR3 amino acid sequences selected from the group consisting of:

[0087] (i) SEQ ID NO:53, SEQ ID NO:56 and SEQ ID NO:55, respectively,

[0088] (ii) SEQ ID NO:53, SEQ ID NO:57 and SEQ ID NO:55, respectively, and

[0089] (iii) SEQ ID NO:53, SEQ ID NO:58 and SEQ ID NO:55, respectively; and

[0090] (b) a set of immunoglobulin light chain complementarity determining regions (CDRs) comprising light chain CDR1, CDR2 and CDR3 amino acid sequences selected from the group consisting of:

[0091] (i) are SEQ ID NO:62, SEQ ID NO:60 and SEQ ID NO:61, respectively,

[0092] (ii) SEQ ID NO:59, SEQ ID NO:63 and SEQ ID NO:61, respectively, and

[0093] (iii) SEQ ID NO:59, SEQ ID NO:64 and SEQ ID NO:61, respectively.

[0094] It should again be understood that the antibody or antibody fragment comprises a heavy chain variable region including the heavy chain CDRs and a light chain variable region including the light chain CDRs.

[0095] In the above two aspects of the invention or variants of other aspects of the invention where CDR sequences are described, the antibody or antibody fragment or protein further comprises, relative to one or more of the described CDRs, additional N-terminal and / or C-terminal amino acid sequences, in any and all combinations, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues directly adjacent to the one or more CDRs (on either or both sides of the N-terminus and C-terminus of the CDR), such as Figure 1-10 Any of the corresponding variable region amino acid sequences shown.

[0096] Another aspect of the present invention provides a protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab fragment, a Fab2 fragment or a scFv molecule) or an immunoglobulin light chain, comprising an antibody light chain variable region, wherein the antibody light chain variable region comprises the following light chain CDRs:

[0097] A CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO:59), KSSKSVSTSGYSYLH (SEQ ID NO:62), RASKSVSTSGYSYLA (SEQ ID NO:65), RASKSVSTSGYSYLS (SEQ ID NO:66), or RASKSVSTSGYSYLN (SEQ ID NO:67);

[0098] A CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO:60), LASNLA (SEQ ID NO:68), LASNLAT (SEQ ID NO:63), LASNLQ (SEQ ID NO:69), LASNLQS (SEQ ID NO:64), LASTRES (SEQ ID NO:70), LASTRAT (SEQ ID NO:71), LASNRAT (SEQ ID NO:72) or LASSQLS (SEQ ID NO:73);

[0099] A CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO: 61),

[0100] The proviso is that a combination of a CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO: 59) and a CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO: 60) is excluded.

[0101] The next amino acid residue after the CDR-L3 sequence QHSRELPFT (SEQ ID NO: 61) in the protein may be, for example, FGQ.

[0102] Another aspect of the present invention provides a protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab fragment, a Fab2 fragment or a scFv molecule) or an immunoglobulin heavy chain, comprising an antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises the following light chain CDRs:

[0103] a CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO:53) or GFDFSRYWMN (SEQ ID NO:85);

[0104] a CDR-H2 comprising the amino acid sequence EINPTSSSTINFTPSLKD (SEQ ID NO:54), EINPTSSTINFADSVKG (SEQ ID NO:56), EINPTSSTINYADSVKG (SEQ ID NO:57), EINPTSSTINFVDSVKG (SEQ ID NO:58), YINPTSSTIYYADSVKG (SEQ ID NO:74), AINPTSSTIYYADSVKG (SEQ ID NO:75), NINPTSSTIYYVDSVKG (SEQ ID NO:76), or SINPTSSTIYYADSVKG (SEQ ID NO:77);

[0105] A CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55),

[0106] Provided that a combination of a CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53) and a CDR-H2 comprising the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO: 54) is excluded.

[0107] Another aspect of the present invention provides a protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab fragment, a Fab2 fragment or a scFv molecule), comprising:

[0108] (a) an antibody light chain variable region comprising the following light chain CDRs:

[0109] A CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO:59), KSSKSVSTSGYSYLH (SEQ ID NO:62), RASKSVSTSGYSYLA (SEQ ID NO:65), RASKSVSTSGYSYLS (SEQ ID NO:66), or RASKSVSTSGYSYLN (SEQ ID NO:67);

[0110] a CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO:60), LASNLA (SEQ ID NO:68), LASNLAT (SEQ ID NO:63), LASNLQ (SEQ ID NO:69), LASNLQS (SEQ ID NO:64), LASTRES (SEQ ID NO:70), LASTRAT (SEQ ID NO:71), LASNRAT (SEQ ID NO:72), or LASSQLS (SEQ ID NO:73); and

[0111] A CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO:61); and

[0112] (b) an antibody heavy chain variable region comprising the following heavy chain CDRs:

[0113] a CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO:53) or GFDFSRYWMN (SEQ ID NO:85);

[0114] a CDR-H2 comprising the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO:54), EINPTSSTINFADSVKG (SEQ ID NO:56), EINPTSSTINYADSVKG (SEQ ID NO:57), EINPTSSTINFVDSVKG (SEQ ID NO:58), YINPTSSTIYYADSVKG (SEQ ID NO:74), AINPTSSTIYYADSVKG (SEQ ID NO:75), NINPTSSTIYYVDSVKG (SEQ ID NO:76), or SINPTSSTIYYADSVKG (SEQ ID NO:77); and

[0115] A CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55),

[0116] The premise is that the combination of CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO:59), CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO:60), CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO:61), CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO:53), CDR-H2 comprising the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO:54) and CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO:55) is excluded.

[0117] The next amino acid residue after the CDR-L3 sequence QHSRELPFT (SEQ ID NO: 61) in the protein may be, for example, FGQ.

[0118] Another aspect of the present invention provides a protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab fragment, a Fab2 fragment or a scFv molecule) or an immunoglobulin light chain, comprising an antibody light chain variable region, wherein the antibody light chain variable region comprises the following light chain CDRs:

[0119] A CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO: 59) or KSSKSVSTSGYSYLH (SEQ ID NO: 62);

[0120] A CDR-L2 comprising the amino acid sequence LASNLA (SEQ ID NO:68), LASNLAT (SEQ ID NO:63), LASNLQ (SEQ ID NO:69), or LASNLQS (SEQ ID NO:64); and

[0121] A CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO: 61).

[0122] The next amino acid residue after the CDR-L3 sequence QHSRELPFT (SEQ ID NO: 61) in the protein may be, for example, FGQ.

[0123] Another aspect of the present invention provides a protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab fragment, a Fab2 fragment or a scFv molecule) or an immunoglobulin heavy chain, comprising an antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises the following heavy chain CDRs:

[0124] a CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53);

[0125] A CDR-H2 comprising the amino acid sequence EINPTSSTINFADSVKG (SEQ ID NO:56), EINPTSSTINYADSVKG (SEQ ID NO:57), or EINPTSSTINFVDSVKG (SEQ ID NO:58); and

[0126] A CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55).

[0127] Another aspect of the present invention provides a protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab fragment, a Fab2 fragment or a scFv molecule), comprising:

[0128] (a) an antibody light chain variable region comprising the following light chain CDRs:

[0129] A CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO: 59) or KSSKSVSTSGYSYLH (SEQ ID NO: 62);

[0130] A CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO:60), LASNLA (SEQ ID NO:68), LASNLAT (SEQ ID NO:63), LASNLQ (SEQ ID NO:69), LASNLQS (SEQ ID NO:64); and

[0131] A CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO:61); and

[0132] (b) an antibody heavy chain variable region comprising the following heavy chain CDRs:

[0133] a CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53);

[0134] A CDR-H2 comprising the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO:54), EINPTSSTINFADSVKG (SEQ ID NO:56), EINPTSSTINYADSVKG (SEQ ID NO:57), or EINPTSSTINFVDSVKG (SEQ ID NO:58); and

[0135] A CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55),

[0136] The premise is that the combination of CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO:59), CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO:60), CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO:61), CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO:53), CDR-H2 comprising the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO:54) and CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO:55) is excluded.

[0137] The next amino acid residue after the CDR-L3 sequence QHSRELPFT (SEQ ID NO: 61) in the protein can be, for example, FGQ. For example, CDR-H2 can include the amino acid sequence EINPTSSTINFADSVKG (SEQ ID NO: 56), EINPTSSTINYADSVKG (SEQ ID NO: 57), or EINPTSSTINFVDSVKG (SEQ ID NO: 58).

[0138] One aspect of the present invention provides an immunoglobulin heavy chain variable region or protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab, Fab2 or scFv molecule), which includes an immunoglobulin heavy chain variable region sequence, which sequence includes one or more amino acid substitutions disclosed in any non-parent immunoglobulin heavy chain variable region disclosed herein relative to the parent immunoglobulin heavy chain variable region disclosed herein, but otherwise includes an amino acid sequence that is identical to the parent immunoglobulin heavy chain variable region sequence.

[0139] One aspect of the present invention provides an immunoglobulin light chain variable region or protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab, Fab2 or scFv molecule), which includes an immunoglobulin light chain variable region, which variable region includes one or more amino acid substitutions disclosed in any non-parent immunoglobulin light chain variable region disclosed herein relative to the parent immunoglobulin light chain variable region disclosed herein, but otherwise includes an amino acid sequence that is identical to the parent immunoglobulin light chain variable region sequence.

[0140] One aspect of the present invention provides a protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab, Fab2 or scFv molecule), comprising:

[0141] (i) an immunoglobulin heavy chain variable region comprising one or more amino acid substitutions disclosed in any non-parent immunoglobulin heavy chain variable region disclosed herein, relative to a parent immunoglobulin heavy chain variable region disclosed herein, in any combination, but otherwise comprising an amino acid sequence that is identical to the parent immunoglobulin heavy chain variable region sequence; and

[0142] (ii) an immunoglobulin light chain variable region comprising, in any combination, one or more amino acid substitutions disclosed in any non-parent immunoglobulin light chain variable region disclosed herein relative to a parent immunoglobulin light chain variable region disclosed herein, but otherwise comprising an amino acid sequence that is identical to the parent immunoglobulin light chain variable region sequence.

[0143] One aspect of the present invention provides a protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab, Fab2 or scFv molecule), comprising:

[0144] (i) a parent immunoglobulin heavy chain variable region disclosed herein, or

[0145] An immunoglobulin heavy chain variable region comprising one or more amino acid substitutions disclosed in any non-parent immunoglobulin heavy chain variable region disclosed herein, relative to a parent immunoglobulin heavy chain variable region disclosed herein, in any combination, but otherwise comprising an amino acid sequence that is identical to the parent immunoglobulin heavy chain variable region sequence; and

[0146] (ii) a parent immunoglobulin light chain variable region disclosed herein, or

[0147] An immunoglobulin light chain variable region comprising one or more amino acid substitutions disclosed in any non-parent immunoglobulin light chain variable region disclosed herein, relative to a parent immunoglobulin light chain variable region disclosed herein, in any combination, but otherwise comprising an amino acid sequence that is identical to the parent immunoglobulin heavy chain variable region sequence.

[0148] The proviso is that the protein does not simultaneously include both a parent immunoglobulin heavy chain variable region disclosed herein and a parent immunoglobulin light chain variable region disclosed herein.

[0149] In a variation of this aspect, the protein does not include a parent immunoglobulin heavy chain variable region disclosed herein, and does not include a parent immunoglobulin light chain variable region disclosed herein.

[0150] One aspect of the present invention provides an immunoglobulin heavy chain variable region or protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab, Fab2 or scFv molecule), comprising an immunoglobulin heavy chain variable region sequence, wherein the immunoglobulin heavy chain variable region sequence comprises the following amino acid sequence: EVKLLESGGGLVQPGGSLKLSCAAS GFDFSRYWMX1 WVRQAPGKGLEWX2X3 X4INPTSSTIX5X6X7X8SLKX9 X 10 X 11 X 12 ISRDNX 13 KNX 14 LYLQMSX 15 X 16 RX 17 EDTAX 18 YYCAR GNYYRYGDAMDY WGQGTX 19 VTVSS (SEQ ID NO: 78),

[0151] in

[0152] X1 is S or N,

[0153] X2 is I or V,

[0154] X3 is G, S or A,

[0155] X4 is E, Y, A, N or S,

[0156] X5 is N or Y,

[0157] X6 is F or Y,

[0158] X7 is T, A or V,

[0159] X8 is P or D,

[0160] X9 is D or G,

[0161] X 10 is K or R,

[0162] X 11 is V or F,

[0163] X 12 is F or T,

[0164] X 13 is A or S,

[0165] X 14 is T or S,

[0166] X 15 is K or S,

[0167] X 16 is V or L,

[0168] X 17 Is S or A

[0169] X 18 is L or V, and

[0170] X 19 It is S or M,

[0171] Provided that the amino acid sequence does not include the sequence EVKLLESGGGLVQPGGSLKLSCAAS GFDFSRYWMS WVRQAPGKGLEWIG EINPTSSTINFTPSLKD KVFISRDNAKNTLYLQMSKVRSEDTALYYCAR GNYYRYGDAMDY WGQGTSVTVSS (SEQ ID NO: 2).

[0172] The heavy chain variable region amino acid sequence in this regard may be referred to herein as a "modified heavy chain variable region sequence" or a "MHCVR sequence."

[0173] One aspect of the present invention provides an immunoglobulin light chain variable region or protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab, Fab2 or scFv molecule), comprising an immunoglobulin light chain variable region sequence, wherein the immunoglobulin light chain variable region sequence comprises the following amino acid sequence: DIX L1 X L2 QTCE L3 X L4 LX L5 X L6 SX L7 GX L8 RX L9 TX L10 X L1 1C X L32 X L13 SKSVSTSGYSY L X L14 WQX L15 PX L16 LLY LASX L17 X L18 X L19 X L20 GX L21 PX L22 RFSGSGSGTDFTLX L23 IX L24 X L25 X L26 X L27 X L28 EDX L29 AX L30 YYC QHSRELPFT FGX L31GTKLEIK (SEQ ID NO: 79),

[0174] in

[0175] X L1 is D or E,

[0176] X L2 It's A or V or Q,

[0177] X L3 It is L or M.

[0178] X L4 It is A or D or S,

[0179] X L5 is S or T,

[0180] X L6 is A or S,

[0181] X L7 It is V or L or A,

[0182] X L8 It is L or P or V,

[0183] X L9 It's Q or E or D,

[0184] X L10 is A or V,

[0185] X L11 is I or L,

[0186] X L12 It is S or N or T,

[0187] X L13 is A or S,

[0188] X L14 is H, A, S or N,

[0189] X L15 is P or A,

[0190] X L16 is K or R,

[0191] X L17 It is N or T or S,

[0192] X L18 is L or R,

[0193] X L19 It's E or A or Q,

[0194] X L20 is S or T,

[0195] XL21 is V or I,

[0196] X L22 It is A or D or S,

[0197] X L23 is N or T,

[0198] X L24 is H or S,

[0199] X L25 is P or S,

[0200] X L26 is V or L,

[0201] X L27 is E or Q,

[0202] X L28 It is E or A or P,

[0203] X L29 It's A or V or F,

[0204] X L30 is T or V,

[0205] X L31 is S or Q, and

[0206] X L32 Is R or K

[0207] Provided that the amino acid sequence does not include the sequence

[0208] DIALTQSPASLAVSLGQRATISC RASKSVSTSGYSYLH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHSRELPFT FGSGTKLEIK (SEQ ID NO: 28).

[0209] The light chain variable region amino acid sequence in this regard may be referred to herein as a "modified light chain variable region sequence" or a "MLCVR sequence."

[0210] One aspect of the present invention provides a protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab, Fab2 or scFv molecule), comprising:

[0211] (i) a heavy chain variable region comprising an MHCVR sequence disclosed herein or a parent heavy chain variable region amino acid sequence; and

[0212] (ii) a light chain variable region comprising the MLCVR sequence disclosed herein or a parent light chain variable region amino acid sequence,

[0213] The proviso is that the protein does not simultaneously include both the parent heavy chain variable region amino acid sequence disclosed herein and the parent light chain variable region amino acid sequence disclosed herein.

[0214] One aspect of the present invention provides a protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab, Fab2 or scFv molecule), comprising:

[0215] (i) a heavy chain variable region comprising an MHCVR sequence; and

[0216] (ii) a light chain variable region comprising the MLCVR sequence.

[0217] One aspect of the invention provides a protein, such as a CD45 binding scFv molecule or a protein comprising at least one scFv segment, comprising a contiguous (amino to carboxyl terminal order) amino acid sequence comprising amino acids 1-120 (up to and including the amino acid of the sequence VTVS or the corresponding amino acid position) or amino acids 1-121 of a heavy chain variable region disclosed herein, followed by a linker amino acid sequence, followed by an amino acid sequence of a light chain variable region disclosed herein, with the proviso that the protein does not include both amino acids 1-120 of a parent heavy chain variable region sequence disclosed herein and a parent light chain variable region sequence disclosed herein. In one variant, the protein does not include any of amino acids 1-120 of a parent heavy chain variable region sequence disclosed herein and a parent light chain variable region sequence disclosed herein.

[0218] The linker amino acid sequence may, for example, include any of the following amino acid sequences:

[0219] KISGGGGSGGGGSGGGGSGGGGSGGGGSS (SEQ ID NO:80);

[0220] SPNSASHSGSAPQTSSAPGSQ(SEQ ID NO:81);

[0221] (G3S) n , wherein, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, such as (G3S)4, i.e., GGGSGGGSGGGSGGGS (SEQ ID NO: 82); or

[0222] (G4S) n , that is (GGGGS[SEQ ID NO:83]) n, wherein, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, such as (G4S)5, i.e., GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 84).

[0223] The heavy chain variable region and light chain variable region disclosed herein each include three (3) complementarity determining regions (CDRs). The CDRs are surrounded by immunoglobulin framework regions (FRs) in the following manner: FR1 is the amino acid sequence before (N-terminal to) CDR1, FR2 is the amino acid sequence between CDR1 and CDR2, FR3 is the amino acid sequence between CDR2 and CDR3, and FR4 is the amino acid sequence after (C-terminal to) CDR3 to the end of the variable region sequence.

[0224] As used herein, the term "antibody fragment" includes, but is not limited to, proteolytic fragments of antibodies, such as Fab or Fab2 (F(ab′)2) fragments, recombinant antibody fragments with covalently or non-covalently associated chains, scFv molecules, and rIgG fragments ("rIgG" refers to reduced IgG (~75,000 Daltons)) (also known as half-IgG).

[0225] The proteins (e.g., antibodies and antibody fragments) can be linked directly or indirectly to a radionuclide, for example, via a chemically conjugated chelator, for example, to target cytotoxic radiation to cells expressing CD45 in a mammalian subject (e.g., a human patient), or to perform non-cytotoxic imaging of CD45 expression in a mammalian subject (e.g., a human patient). For example, according to the methods disclosed in U.S. Pat. No. 10,420,851, antibodies can be used directly to 131 Alternatively, the antibody can be chemically conjugated to a chelator (such as p-SCN-DOTA) and labeled with a radionuclide according to the procedure described in U.S. Pat. No. 9,603,954. 225 Ac label.

[0226] For example, the radionuclide may be selected from 131 I. 125I , 123 I. 32 P. 213 Po, 134 Ce, 43 Sc, 44 Sc, 47 Sc, 55 Co. 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga,68 Ga, 82 Rb, 86 Y, 87 Y, 90 Y, 89 Zr, 97 Ru, 105 Rh, 109 Pd, 111 In, 117m Sn, 149 Pm, 149 Tb, 161 Tb, 153 Sm, 177 Lu, 186 Re, 188 Re, 199 Au, 201 Tl, 137 Cs, 223 Ra, 203 Pb, 212 Pb, 211 At, 212 Bi, 213 Bi, 225 Ac and 227 Th.

[0227] The chelating agent groups of various aspects of the present invention may include, for example: 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) or its derivatives; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) or its derivatives; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) or its derivatives; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DO3A) or its derivatives. OTA) or its derivatives; 1,4,7-triazacyclononane, 1-pentanedioic acid-4,7-diacetic acid (NODAGA) or its derivatives; 1,4,7,10-tetraazacyclodecane, 1-pentanedioic acid-4,7,10-triacetic acid (DOTAGA) or its derivatives; 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) or its derivatives; 1,4,8,11-tetraazabicyclo[6.6.2] hexadecane-4,11-diacetic acid (CB-TE2A) or its derivatives; diethylenetriaminepentaacetic acid (DTPA), its diesters or its derivatives; 2-cyclohexyldiethylenetriaminepentaacetic acid (CHX-A″-DTPA) or its derivatives; deforoxamine (DFO) or its derivatives; 1,2-[[6-carboxypyridin-2-yl]methylamino]ethane (H2dedpa) or its derivatives; DADA or its derivatives; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra( methylenephosphonic acid) (DOTP) or its derivatives; 4-amino-6-[[16-[(6-carboxypyridin-2-yl)methyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl]methyl]pyridine-2-carboxylic acid (MACROPA-NH2) or its derivatives; MACROPA or its derivatives; 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (TCMC) or its derivatives; {4-[2-(biscarboxymethylamino)-ethyl]-7-carboxymethyl -[1,4,7]triazacyclononane-1-yl}-acetic acid (NETA) or its derivatives; Diamsar or its derivatives; 1,4,7-triazacyclononane-1,4,7-tri[methyl(2-carboxyethyl)phosphinic acid (TRAP, PRP9, TRAP-Pr) or its derivatives; N,N′-bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N′-diacetic acid (H4octapa) or its derivatives; N,N′-[1-benzyl-1,2,3-triazol-4-yl]methyl-N,N′-[6-(carboxy) N,N'-bis(2-hydroxy-5-sulfobenzyl)ethylenediamine-N,N'-diacetic acid (SHBED) or its derivatives; N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) or its derivatives; 3,6,9,15-tetraazabicyclo[9.3.1] Pentadecane-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) or its derivatives; desferrioxamine B (DFO) or its derivatives; N,N′-(methylenephosphonate)-N,N′-[6-(methoxycarbonyl)pyridin-2-yl]methyl-1,2-diaminoethane (H6phospa) or its derivatives; 1,4,7,10,13,16-hexaazacyclohexadecane-N,N′,N″,N″′,N″″,N″″′-hexaacetic acid (HEHA) or its derivatives; 1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N″′,N″″-pentaacetic acid (PEPA) or its derivatives; or 3,4,3-LI (1,2-HOPO) or its derivatives. .

[0228] The proteins (such as antibodies and antibody fragments) can, for example, be linked to one or more cytotoxic drugs to target and deplete cells expressing CD45 in mammalian subjects such as human patients. Therefore, one aspect of the invention provides an antibody-drug-conjugate (ADC) comprising an antibody or antigen-binding antibody fragment according to the invention as a component. For example, the ADC can be a conjugate conjugated to a cytotoxin containing a benzodiazepine moiety, such as a pyrrolobenzodiazepine (PBD) or indolebenzodiazepine (IGN) moiety, as disclosed in U.S. Publication No. 20220175951.

[0229] The word "comprising" and various forms of the word "comprising" and the word "include" and various forms of the word "including" used in the specification and claims do not limit the elements included beyond what is mentioned. In addition, although various aspects or elements thereof are described as "comprising" or "including" throughout the disclosure, corresponding aspects or elements thereof described as "consisting essentially of" or "consisting of" are similarly disclosed. For example, although certain aspects of the present invention have been described in terms of proteins "comprising" or "including" one or more specific amino acid sequences, corresponding aspects that alternatively recite that the protein "consists essentially of" or "consists of" the one or more specific amino acid sequences are also within the scope of the aspects and disclosed by the present disclosure.

[0230] In addition, the composition including the radiolabeled antibody may include one or more pharmaceutically acceptable carriers or pharmaceutically acceptable excipients. Such carriers are well known to those skilled in the art. For example, injectable drug delivery systems include solutions, suspensions, gels, microspheres, and polymer injections, and may include excipients such as solubility modifiers (e.g., ethanol, propylene glycol, and sucrose) and polymers (e.g., polycaprolactone and PLGA). An exemplary formulation may be substantially as described in U.S. Patent No. 10,420,851 or International Publication No. WO 2017 / 155937, which are incorporated herein by reference. For example, according to certain aspects, the formulation may include 0.5% to 5.0% (w / v) of an excipient selected from the group consisting of ascorbic acid, polyvinylpyrrolidone (PVP), human serum albumin (HSA), a water-soluble salt of HSA, and a mixture thereof. Certain formulations may include 0.5-5% ascorbic acid; 0.5-4% polyvinylpyrrolidone (PVP); and the monoclonal antibody in 50 mM PBS buffer (pH 7).

[0231] The humanized and various anti-huCD45 antibodies disclosed herein can be treated, for example, with radionuclides such as 131 I. 177 Lu, 211 At 227 Th or 225Ac-labeled, or conjugated to a cytotoxic drug, for use as a conditioning agent in preparation for bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), e.g., for the treatment of blood cancers, or in preparation for administration of genetically engineered cell therapies, e.g., CAR-T therapy for cancer treatment, or as a direct treatment of blood cancers, e.g., myeloid or lymphoid blood cancers, such as leukemias, e.g., acute myeloid leukemia (AML), or lymphomas, e.g., non-Hodgkin lymphoma (NHL), or as an immune resetting agent for the treatment of autoimmune diseases such as multiple sclerosis. The hematological cancer or disorder treated can be, for example, a leukemia (e.g., acute myeloid leukemia (AML), acute promyelocytic leukemia, acute lymphocytic leukemia (ALL), acute mixed cell leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, or large granular lymphocytic leukemia), myelodysplastic syndrome (MDS), a myeloproliferative disorder (e.g., polycythemia vera, essential thrombocythemia, primary myelofibrosis, or chronic myeloid leukemia), multiple myeloma, MGUS, and similar disorders, a lymphoma (e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, or T-cell lymphoma), or another B-cell malignancy. In one aspect, the radiolabeled or drug-conjugated anti-huCD45 antibody is administered as maintenance therapy to a subject who has previously received BMT or HSCT in the treatment of a hematological cancer (such as AML or other types described herein), e.g., to treat or prevent relapse or recurrence of the cancer. In each of the cases described herein, the subject can be a mammal, such as a human patient.

[0232] Example 1: Generation of Radiolabeled Chelator-Conjugated Anti-huCD45 Antibodies

[0233] Conjugation with chelating agents: A vial of lyophilized p-SCN-Bn-DOTA was reconstituted with metal-free water to a concentration of 10 mg / ml. 0.02 ml of ascorbic acid solution (150 mg / ml) and 0.05 ml of reconstituted p-SCN-Bn-DOTA were added to the actinium reaction bottle, and the pH was adjusted to between 5 and 5.5 with 2M tetramethylammonium acetate (TMAA). The mixture was then heated at 55 ± 4 °C for 30 minutes.

[0234] To determine 225 To determine the labeling efficiency of Ac-p-SCN-Bn-DOTA, an aliquot of the reaction mixture was taken and applied to a 1 ml Sephadex C25 cation exchange resin column. The product was eluted in 2-4 ml fractions using 0.9% saline solution.225 The Ac active fraction is 225 Ac-p-SCN-Bn-DOTA, and the fraction retained on the column is the unchelated, unreacted 225 Ac. Typically, the labeling efficiency is greater than 95%.

[0235] To the reaction mixture was added 0.22 ml of previously prepared anti-CD45 mAb in DTPA (1 mg) and 0.02 ml of ascorbic acid. DTPA was added to bind any trace metals that might compete with the antibody labeling. Ascorbic acid was added as a radioprotectant. The pH was adjusted to 8.5-9 with carbonate buffer. The mixture was heated at 37±3°C for 30 minutes.

[0236] The final product can be purified by size exclusion chromatography using 10DG resin and eluted with 2 ml 1% HSA.

[0237] Radiolabeling: The antibody can be conjugated to a linker, such as any of the linkers described in the above patent applications. Exemplary linkers include at least dodecanetetraacetic acid (DOTA), where the goal of the conjugation reaction is to achieve a DOTA-antibody ratio of 3:1 to 5:1. Conjugation with a radionuclide such as 177 Lu, 90 Y or 225 Ac chelation, and the efficiency and purity of the resulting radiolabeled antibody (such as anti-CD45 antibody) can be determined by HPLC and iTLC.

[0238] 225 An exemplary labeling reaction of Ac is as follows: a reaction including 15 μl of 0.15 M NH4OAc buffer (pH = 6.5) and 2 μL (10 μg) DOTA-anti-CD45 (5 mg / ml) can be mixed in an Eppendorf reaction tube, and then 4 μL of 0.05 M HCl in 225 Ac (10 μCi). The contents of the tube can be mixed with a pipette tip and the reaction mixture can be incubated at 37°C with shaking at 100 rpm for 90 min. At the end of the incubation period, 3 μL of 1 mM DTPA solution can be added to the reaction mixture and incubated at room temperature for 20 min to remove unreacted 225 Ac binds to 225 Ac-DTPA complex. 225 Ac-labeled anti-CD45 ( 225 Ac-DOTA-anti-CD45) and free 225 Ac( 225Ac-DTPA) separation can be determined using instant thin layer chromatography with a 10 cm silica strip and a 10 mM EDTA / saline mobile phase. 225 Radiochemical purity of Ac-DOTA-anti-CD45. In this system, the radiolabeled antibody remains at the point of application, and 225 Ac-DTPA moves with the solvent front. The strip can be cut in half and used in a gamma counter equipped with a multichannel analyzer using channels 72-110. 225 Ac is counted to exclude its progeny.

[0239] purification: Exemplary radiolabeled targeting agents (such as 225 Ac-DOTA-antibody) can be purified on a PD10 column pre-blocked with 1% HSA; or on a Vivaspin centrifugal concentrator with a MW cutoff of 50 kDa, washing 2×1.5 mL, centrifuging for 3 min each time. 225 HPLC analysis of Ac-DOTA-antibody can be performed using a Waters HPLC system equipped with a flow-through Waters UV and Bioscan radiation detector, using a TSK3000SW XL column, eluting with PBS (pH=7.4), and a flow rate of 1 ml / min.

[0240] Example 2: Radioiodination of anti-huCD45 antibody and purification in the presence of ascorbic acid

[0241] The anti-CD45 antibody can be radioiodinated according to the following method.

[0242] In the presence of chloramine-T (23 μg), 20 to 30 mCi of 131 I-Na (30 mCi) label 1 mg of anti-CD45 antibody. Add aqueous sodium thiosulfate (69 micrograms) to quench the reaction and dilute with cold NaI (1 mg). Next, add concentrated ascorbic acid solution prepared in 50 mM PBS (pH 7) to achieve an ascorbic acid concentration of 2.5% (w / v) in the quenched reaction mixture. For example, labeling reactions up to 3,000 mCi per batch can be successfully performed using this method.

[0243] Radiolabeled antibodies (e.g., radiolabeled immunoglobulins) can be purified by gel filtration on a sterile, prepackaged commercially available Sephadex G25 column (GE HiPrep 26 / 10 column, column bed volume 53 mL), using a PBS (50 mM, pH 7) mobile phase supplemented with 2.5% (w / v) ascorbic acid to stabilize the radiolabeled product. A reaction volume of up to 1,000 mCi can be purified on a single column. The product can be collected from the column in 5 to 35 mL elution volumes.

[0244] Radioiodination batches with <200 mCi can be purified in a similar manner on a smaller desalting column (GE PD10 column, 8.6 mL bed volume).

[0245] Example 3: Radioiodination of anti-CD45 immunoglobulin and purification in the presence of ascorbic acid and HSA

[0246] Radioiodination and purification can be performed essentially as described in Example 2, except that 2% or 4% (w / v) HSA is added to the quenched reaction and elution buffer along with 2.5% (w / v) ascorbic acid during purification.

[0247] Although various specific embodiments have been shown and described herein, it should be understood that various changes may be made without departing from the spirit and scope of the invention. In addition, features described in conjunction with one aspect of the invention may be used in combination with other aspects of the invention, even if not explicitly combined in an example therein.

Claims

1. An anti-huCD45 antibody or huCD45 binding antibody fragment comprising: (i) an immunoglobulin heavy chain variable region comprising SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 22 or SEQ ID NO: 2; and (ii) an immunoglobulin light chain variable region comprising SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:33, SEQ ID NO:38 or SEQ ID NO:28, Provided that when the heavy chain variable region comprises SEQ ID NO:2, the light chain variable region is not SEQ ID NO:

28.

2. The anti-huCD45 antibody or huCD45 binding antibody fragment according to claim 1, comprising: (i) an immunoglobulin heavy chain sequence comprising SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:11, SEQ ID NO:16 or SEQ ID NO:6; and (ii) an immunoglobulin light chain sequence comprising SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:37, SEQ ID NO:42 or SEQ ID NO:

32.

3. An anti-huCD45 antibody or huCD45 binding antibody fragment comprising: (a) a set of immunoglobulin heavy chain complementarity determining regions (CDRs) comprising heavy chain CDR1, CDR2 and CDR3 amino acid sequences selected from the group consisting of: (i) SEQ ID NO:53, SEQ ID NO:58 and SEQ ID NO:55, respectively, (ii) SEQ ID NO:53, SEQ ID NO:56 and SEQ ID NO:55, respectively, and (iii) SEQ ID NO:53, SEQ ID NO:57 and SEQ ID NO:55, respectively; and (b) a set of immunoglobulin light chain complementarity determining regions (CDRs) comprising light chain CDR1, CDR2 and CDR3 amino acid sequences selected from the group consisting of: (i) SEQ ID NO:59, SEQ ID NO:63 and SEQ ID NO:61, respectively, and (ii) SEQ ID NO:59, SEQ ID NO:64 and SEQ ID NO:61, respectively, and (iii) are SEQ ID NO:62, SEQ ID NO:60 and SEQ ID NO:61, respectively.

4. A pharmaceutical composition comprising the antibody or antibody fragment according to any one of the preceding claims and at least one pharmaceutically acceptable excipient.

5. A radiopharmaceutical composition comprising: The antibody or antibody fragment according to any one of claims 1 to 3; a radionuclide attached to the antibody or antibody fragment, and At least one pharmaceutically acceptable excipient.

6. The radiopharmaceutical composition of claim 5, wherein the radionuclide is an alpha particle emitter or a beta particle emitter.

7. The radiopharmaceutical composition of claim 5, wherein the radionuclide comprises 131 I.

8. The radiopharmaceutical composition of claim 5, wherein the radionuclide comprises 225 Ac, 177 Lu or 90 Y.

9. A composition comprising the antibody or antibody fragment according to any one of claims 1 to 3 chemically conjugated to a chelating agent.

10. The composition of claim 9, wherein the chelating agent comprises DOTA or a DOTA derivative.

11. The composition according to claim 9 or 10, further comprising a radionuclide chelated by the chelator.

12. A protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab fragment, a Fab2 fragment or a scFv molecule) or an immunoglobulin light chain, comprising an antibody light chain variable region, wherein the antibody light chain variable region comprises the following light chain CDRs: a CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO:59), KSSKSVSTSGYSYLH (SEQ ID NO:62), RASKSVSTSGYSYLA (SEQ ID NO:65), RASKSVSTSGYSYLS (SEQ ID NO:66), or RASKSVSTSGYSYLN (SEQ ID NO:67); a CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO:60), LASNLA (SEQ ID NO:68), LASNLAT (SEQ ID NO:63), LASNLQ (SEQ ID NO:69), LASNLQS (SEQ ID NO:64), LASTRES (SEQ ID NO:70), LASTRAT (SEQ ID NO:71), LASNRAT (SEQ ID NO:72) or LASSQLS (SEQ ID NO:73); a CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO: 61), The proviso is that a combination of a CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO: 59) and a CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO: 60) is excluded.

13. The protein of claim 12, wherein the next amino acid residue after the CDR-L3 sequence QHSRELPFT (SEQ ID NO: 61) in the protein is FGQ.

14. A protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab fragment, a Fab2 fragment or a scFv molecule) or an immunoglobulin heavy chain, comprising an antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises the following light chain CDRs: a CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO:53) or GFDFSRYWMN (SEQ ID NO:85); Contains the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO: 54), EINPTSSTINFADSVKG (SEQ ID NO: 56), EINPTSSTINYADSVKG (SEQ ID NO: 57), EINPTSSTINFVDSVKG (SEQ ID NO: 58), YINPTSSTIYYADSVKG (SEQ ID NO:74), AINPTSSTIYYADSVKG (SEQ ID NO:75), NINPTSSTIYYVDSVKG (SEQ ID NO:76) or SINPTSSTIYYADSVKG (SEQ ID NO:77) CDR-H2; a CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55), The proviso is that a combination of a CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53) and a CDR-H2 comprising the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO: 54) is excluded.

15. A protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab fragment, a Fab2 fragment or a scFv molecule), comprising: (a) an antibody light chain variable region comprising the following light chain CDRs: a CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO:59), KSSKSVSTSGYSYLH (SEQ ID NO:62), RASKSVSTSGYSYLA (SEQ ID NO:65), RASKSVSTSGYSYLS (SEQ ID NO:66), or RASKSVSTSGYSYLN (SEQ ID NO:67); a CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO:60), LASNLA (SEQ ID NO:68), LASNLAT (SEQ ID NO:63), LASNLQ (SEQ ID NO:69), LASNLQS (SEQ ID NO:64), LASTRES (SEQ ID NO:70), LASTRAT (SEQ ID NO:71), LASNRAT (SEQ ID NO:72) or LASSQLS (SEQ ID NO:73); and a CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO:61); and (b) an antibody heavy chain variable region comprising the following heavy chain CDRs: a CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO:53) or GFDFSRYWMN (SEQ ID NO:85); a CDR-H2 comprising the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO:54), EINPTSSTINFADSVKG (SEQ ID NO:56), EINPTSSTINYADSVKG (SEQ ID NO:57), EINPTSSTINFVDSVKG (SEQ ID NO:58), YINPTSSTIYYADSVKG (SEQ ID NO:74), AINPTSSTIYYADSVKG (SEQ ID NO:75), NINPTSSTIYYVDSVKG (SEQ ID NO:76), or SINPTSSTIYYADSVKG (SEQ ID NO:77); and a CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55), The premise is that the combination of CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO:59), CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO:60), CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO:61), CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO:53), CDR-H2 comprising the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO:54) and CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO:55) is excluded.

16. The protein of claim 15, wherein the next amino acid residue after the CDR-L3 sequence QHSRELPFT (SEQ ID NO: 61) in the protein is FGQ.

17. A protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab fragment, a Fab2 fragment or a scFv molecule) or an immunoglobulin light chain, comprising an antibody light chain variable region, wherein the antibody light chain variable region comprises the following light chain CDRs: a CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO: 59) or KSSKSVSTSGYSYLH (SEQ ID NO: 62); a CDR-L2 comprising the amino acid sequence LASNLA (SEQ ID NO:68), LASNLAT (SEQ ID NO:63), LASNLQ (SEQ ID NO:69) or LASNLQS (SEQ ID NO:64); A CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO: 61).

18. The protein of claim 17, wherein the next amino acid residue after the CDR-L3 sequence QHSRELPFT (SEQ ID NO: 61) in the protein is FGQ.

19. A protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab fragment, a Fab2 fragment or a scFv molecule) or an immunoglobulin heavy chain, comprising an antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises the following heavy chain CDRs: a CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53); a CDR-H2 comprising the amino acid sequence EINPTSSTINFADSVKG (SEQ ID NO:56), EINPTSSTINYADSVKG (SEQ ID NO:57), or EINPTSSTINFVDSVKG (SEQ ID NO:58); and A CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55).

20. A protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment (such as a Fab fragment, a Fab2 fragment or a scFv molecule), comprising: (a) an antibody light chain variable region comprising the following light chain CDRs: a CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO: 59) or KSSKSVSTSGYSYLH (SEQ ID NO: 62); a CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO:60), LASNLA (SEQ ID NO:68), LASNLAT (SEQ ID NO:63), LASNLQ (SEQ ID NO:69), LASNLQS (SEQ ID NO:64); and a CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO:61); and (b) an antibody heavy chain variable region comprising the following heavy chain CDRs: a CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53); a CDR-H2 comprising the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO:54), EINPTSSTINFADSVKG (SEQ ID NO:56), EINPTSSTINYADSVKG (SEQ ID NO:57), or EINPTSSTINFVDSVKG (SEQ ID NO:58); and a CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55), The premise is that the combination of CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO:59), CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO:60), CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO:61), CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO:53), CDR-H2 comprising the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO:54) and CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO:55) is excluded.

21. The protein of claim 20, wherein the next amino acid residue after the CDR-L3 sequence QHSRELPFT (SEQ ID NO: 61) in the protein is FGQ.

22. The protein of claim 20 or 21, wherein the CDR-H2 comprises the amino acid sequence EINPTSSTINFADSVKG (SEQ ID NO: 56), EINPTSSTINYADSVKG (SEQ ID NO: 57), or EINPTSSTINFVDSVKG (SEQ ID NO: 58).

23. An immunoglobulin heavy chain variable region or protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment, such as a Fab, Fab2 or scFv molecule, comprising an immunoglobulin heavy chain variable region sequence, the immunoglobulin heavy chain variable region sequence comprising one or more amino acid substitutions disclosed in any non-parent immunoglobulin heavy chain variable region disclosed herein, relative to the parent immunoglobulin heavy chain variable region disclosed herein, in any combination, but otherwise comprising the same amino acid sequence as the parent immunoglobulin heavy chain variable region sequence.

24. An immunoglobulin light chain variable region or protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment, such as a Fab, Fab2 or scFv molecule, comprising an immunoglobulin light chain variable region, said immunoglobulin light chain variable region comprising, in any combination, one or more amino acid substitutions disclosed in any non-parent immunoglobulin light chain variable region disclosed herein relative to the parent immunoglobulin light chain variable region disclosed herein, but otherwise comprising an amino acid sequence identical to the parent immunoglobulin light chain variable region sequence.

25. A protein, such as a CD45 antibody or a CD45 binding antibody fragment, such as a Fab, Fab2 or scFv molecule, comprising: (i) an immunoglobulin heavy chain variable region comprising one or more amino acid substitutions disclosed in any non-parent immunoglobulin heavy chain variable region disclosed herein, relative to a parent immunoglobulin heavy chain variable region disclosed herein, in any combination, but otherwise comprising an amino acid sequence identical to the sequence of the parent immunoglobulin heavy chain variable region; and (ii) an immunoglobulin light chain variable region comprising one or more amino acid substitutions disclosed in any non-parent immunoglobulin light chain variable region disclosed herein, relative to a parent immunoglobulin light chain variable region disclosed herein, in any combination, but otherwise comprising an amino acid sequence identical to the sequence of the parent immunoglobulin light chain variable region.

26. A protein, such as a CD45 antibody or a CD45 binding antibody fragment, such as a Fab, Fab2 or scFv molecule, comprising: (i) a parent immunoglobulin heavy chain variable region disclosed herein, or An immunoglobulin heavy chain variable region comprising one or more amino acid substitutions disclosed in any non-parent immunoglobulin heavy chain variable region disclosed herein, relative to a parent immunoglobulin heavy chain variable region disclosed herein, in any combination, but otherwise comprising an amino acid sequence identical to the parent immunoglobulin heavy chain variable region sequence; and (ii) a parent immunoglobulin light chain variable region disclosed herein, or An immunoglobulin light chain variable region comprising one or more amino acid substitutions disclosed in any non-parent immunoglobulin light chain variable region disclosed herein, relative to a parent immunoglobulin light chain variable region disclosed herein, in any combination, but otherwise comprising an amino acid sequence identical to the parent immunoglobulin heavy chain variable region sequence, The proviso is that the protein does not simultaneously comprise both a parent immunoglobulin heavy chain variable region disclosed herein and a parent immunoglobulin light chain variable region disclosed herein.

27. An immunoglobulin heavy chain variable region or protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment, such as a Fab, Fab2 or scFv molecule, comprising an immunoglobulin heavy chain variable region sequence, wherein the immunoglobulin heavy chain variable region sequence comprises the following amino acid sequence: EVKLLESGGGLVQPGGSLKLSCAAS GFDFSRYWMX1 WVRQAPGKGLEWX2X3 X4INPTSSTIX5X6X7X8SLKX 9 X 10 X 11 X 12 ISRDNX 13 KNX 14 LYLQMSX 15 X 16 RX 17 EDTAX 18 YYCAR GNYYRYGDAMDY WGQGTX 19 VTVSS(SEQ IDNO:78) in X1 is S or N, X2 is I or V, X3 is G, S or A, X4 is E, Y, A, N or S, X5 is N or Y, X6 is F or Y, X7 is T, A or V, X8 is P or D, X9 is D or G, X 10 is K or R, X 11 is V or F, X 12 is F or T, X 13 is A or S, X 14 is T or S, X 15 is K or S, X 16 is V or L, X 17 Is S or A X 18 is L or V, and X 19 It is S or M, Provided that the amino acid sequence does not contain the sequence EVKLLESGGGLVQPGGSLKLSCAAS GFDFSRYWMS WVRQAPGKGLEWIG EINPTSSTINFTPSLKD KVFISRDNAKNTLYLQMSKVRSEDTALYYCAR GNYYRYGDAMDY WGQGTSVTVSS (SEQ ID NO: 2).

28. An immunoglobulin light chain variable region or protein, such as an anti-CD45 antibody or a CD45 binding antibody fragment, such as a Fab, Fab2 or scFv molecule, comprising an immunoglobulin light chain variable region sequence, wherein the immunoglobulin light chain variable region sequence comprises the following amino acid sequence: DIX L1 X L2 TQSPX L3 X L4 LX L5 X L6 SX L7 GX L8 RX L9 TX L10 X L11 C X L32 X L13 SKSVSTSGYSYL X L14 WYQQKPGQX L15 PX L16 LLIY LASX L17 X L18 X L19 X L20 GX L21 PX L22 RFSGSGSGTDFTLX L23 IX L24 X L25 X L26 X L27 X L28 EDX L29 AX L30 YYC Q HSRELPFT FGX L31 GTKLEIK(SEQ ID NO:79) in X L1 is D or E, X L2 It's A or V or Q, X L3 It is L or M. X L4 It is A or D or S, X L5 is S or T, X L6 is A or S, X L7 It is V or L or A, X L8 It is L or P or V, X L9 It's Q or E or D, X L10 is A or V, X L11 is I or L, X L12 It is S or N or T, X L13 is A or S, X L14 is H, A, S or N, X L15 is P or A, X L16 is K or R, X L17 It is N or T or S, X L18 is L or R, X L19 It's E or A or Q, X L20 is S or T, X L21 is V or I, X L22 It is A or D or S, X L23 is N or T, X L24 is H or S, X L25 is P or S, X L26 is V or L, X L27 is E or Q, X L28 It is E or A or P, X L29 It's A or V or F, X L30 is T or V, X L31 is S or Q, and X L32 Is R or K Provided that the amino acid sequence does not contain the sequence DIALTQSPASLAVSLGQRATISC RASKSVSTSGYSYLH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHSRELPFT FGSGTKLEIK(SEQ ID NO:28)。 29. A protein, such as a CD45 antibody or a CD45 binding antibody fragment, such as a Fab, Fab2 or scFv molecule, comprising: (i) a heavy chain variable region comprising the MHCVR sequence disclosed herein or a parent heavy chain variable region amino acid sequence; and (ii) a light chain variable region comprising the MLCVR sequence disclosed herein or a parent light chain variable region amino acid sequence, The proviso is that the protein does not simultaneously comprise both a parent heavy chain variable region amino acid sequence disclosed herein and a parent light chain variable region amino acid sequence disclosed herein.

30. The protein of claim 29, comprising: (i) a heavy chain variable region comprising an MHCVR sequence; and (ii) a light chain variable region comprising the MLCVR sequence.

31. A protein, such as a CD45 binding scFv molecule or a protein comprising at least one scFv segment, comprising a contiguous (amino to carboxyl terminal order) amino acid sequence comprising amino acids 1-120 (up to and including the amino acids of the sequence VTVS or the corresponding amino acid position) or amino acids 1-121 of a heavy chain variable region disclosed herein, followed by a linker amino acid sequence, followed by an amino acid sequence of a light chain variable region disclosed herein, with the proviso that the protein does not simultaneously comprise both amino acids 1-120 of a parent heavy chain variable region sequence disclosed herein and a parent light chain variable region sequence disclosed herein.

32. The protein of claim 31, wherein the protein comprises amino acids 1-120 of the heavy chain variable region disclosed herein (up to and including the amino acids of the sequence VTVS or the corresponding amino acid positions), and excludes amino acids 1-121 of the heavy chain variable region disclosed herein.

33. The protein of claim 31 or 32, wherein the protein does not include amino acids 1-120 of a parent heavy chain variable region sequence disclosed herein and any of a parent light chain variable region sequence disclosed herein.

34. The protein of any one of claims 31-33, wherein the linker amino acid sequence comprises: KISGGGGSGGGGSGGGGSGGGGSGGGGSS (SEQ ID NO:80); SPNSASHSGSAPQTSSAPGSQ(SEQ ID NO:81); (G3S) n , wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; or (G4S) n , where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

35. A pharmaceutical composition comprising the protein according to any one of claims 12-34.

36. The pharmaceutical composition of claim 35, further comprising at least one pharmaceutically acceptable excipient.

37. The pharmaceutical composition of claim 35 or 36, wherein at least a portion of the proteins in the composition are radiolabeled or conjugated to a cytotoxic drug.

38. The pharmaceutical composition according to any one of claims 35-37, for use in treating a hematological cancer or a solid tumor cancer in a mammalian subject.

39. A composition according to any one of claims 4-8 and 35-38, for use as a conditioning agent in preparation for bone marrow transplantation (BMT), hematopoietic stem cell transplantation (HSCT) in a mammalian subject, or in the administration of genetically modified cell therapy such as CAR-T cell therapy.

40. The pharmaceutical composition of claim 38 or 39, wherein the mammalian subject is a human patient.

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