Humanized anti-CD45 antibody

Abstract

A humanized anti-human CD45 antibody and a pharmaceutical composition comprising the antibody are provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 407,979, filed September 19, 2022, No. 63 / 405,237, filed September 9, 2022, and No. 63 / 323,257, filed March 24, 2022, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing. It has been submitted electronically in XML format and is incorporated herein by reference in its entirety. Created on Mar. 20, 2023, this XML copy is named ATNM-016PCT_SL_ST26.xml and is 853,669 bytes in size.

[0003] The present invention relates to the field of antibody-based therapeutics. [Background technology]

[0004] The CD45 antigen is a member of the protein tyrosine phosphatase (PTP) family and is a 180-240 kD transmembrane glycoprotein. It is also known as leukocyte common antigen (LCA), T200 or Ly-5. CD45 plays an important role in T-cell and B-cell receptor signaling. Different isoforms of CD45 exist due to alternative splicing of its exons. These isoforms are highly specific to the activation and maturation state of the cell, as well as the cell type. The various isoforms have the same transmembrane and cytoplasmic fragments, but different extracellular domains, and are differentially expressed on subpopulations of B- and T-cell lymphocytes. The main ligands for CD45 include galectin-1, CD1, CD2, CD3, CD4, TCR, CD22 and Thy-1. Monoclonal antibodies (mAbs) have been identified that restrict their specificity to one or other isoform of CD45, as well as mAbs that recognize epitopes common to all CD45 isoforms. A mAb designated CD45RA recognizes the product of exon A. A mAb designated CD45RB recognizes the product of exon B. A mAb designated CD45RO (exemplified by UCHL1) selectively binds to the 180 kD isoform (which does not have any of the variable exons A, B, or C), which is restricted to cortical thymocytes, activated T cells, and a subset of memory cells, and is absent from B cells. BC8, also known as apamistamab, is a murine IgG1 monoclonal antibody that recognizes all isoforms of human CD45. Hematopoietic cells, except mature erythrocytes and platelets, generally express CD45. High expression of CD45 is seen in many acute lymphoblastic and myeloid leukemias. Because CD45 is not found in non-hematopoietic tissues, its specific expression in leukemias makes it an excellent target for developing therapeutic approaches, including radioimmunotherapy. For example, CD45 is expressed on circulating leukocytes and malignant B cells at a density of approximately 200,000-300,000 sites per cell. What is needed and what is provided in various aspects of the invention are novel humanized antibodies against human CD45. Summary of the Invention

[0005] One aspect of the present invention is a method for producing a composition comprising the steps of: (i) the heavy chain of mCD45-HCparental, huCD45.HC1, huCD45.HC2, huCD45.HC3, huCD45.HC1, or huCD45.HC4, or any of said heavy chains without the signal sequence, or a heavy chain comprising the heavy chain variable region of any of said heavy chains; (ii) a light chain of mCD45-LCparental, huCD45.LC1, huCD45.LC2, huCD45.LC3, or huCD45.LC4, or any of said light chains without the signal sequence, or a light chain comprising the light chain variable region of any of said light chains; When the heavy chain is mCD45-HC parental, with or without a signal sequence, the light chain is not mCD45-LC parental, with or without a signal sequence. Anti-huCD45 antibodies, or human CD45 binding fragments of the antibodies, such as Fab or Fab2 fragments or corresponding scFv molecules, are provided. A related aspect of the invention is (i) an immunoglobulin heavy chain variable region comprising SEQ ID NO:2 (from mCD45-HC parental), 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); (ii) an immunoglobulin light chain variable region comprising SEQ ID NO:28 (from mCD45-LC parental), 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); When the heavy chain variable region comprises SEQ ID NO:2, the light chain variable region is not SEQ ID NO:28; Anti-huCD45 antibodies or human CD45 binding antibody fragments are provided. In a variation of the above aspect of the invention, the anti-huCD45 antibody or human CD45 binding antibody fragment comprises: (i) an immunoglobulin heavy chain sequence comprising SEQ ID NO:6 (from mCD45-HC parental), 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); (ii) an immunoglobulin light chain sequence comprising SEQ ID NO: 32 (from mCD45-LCparental), 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).

[0006] Another aspect of the present invention is a method for producing a (a) a set of: (i) SEQ ID NO: 53, SEQ ID NO: 56 and SEQ ID NO: 55, (ii) SEQ ID NO: 53, SEQ ID NO: 57, and SEQ ID NO: 55, (iii) SEQ ID NO: 53, SEQ ID NO: 58, and SEQ ID NO: 55, (iv) SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55, A set of immunoglobulin heavy chain complementarity determining regions (CDRs) each including a heavy chain CDR1, CDR2, and CDR3 amino acid sequence selected from (b) the following set: (i) SEQ ID NO: 62, SEQ ID NO: 60 and SEQ ID NO: 61, (ii) SEQ ID NO: 59, SEQ ID NO: 63, and SEQ ID NO: 61, (iii) SEQ ID NO: 59, SEQ ID NO: 64, and SEQ ID NO: 61, (iv) SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, and a set of immunoglobulin light chain complementarity determining regions (CDRs) each comprising a light chain CDR1, CDR2 and CDR3 amino acid sequence selected from when the set of heavy chain CDRs is (a)(iv), then the set of light chain CDRs is not (b)(iv); Anti-huCD45 antibodies or huCD45 binding antibody fragments are provided. A related aspect of the invention is (a) the following set: (i) SEQ ID NO: 53, SEQ ID NO: 56 and SEQ ID NO: 55, (ii) SEQ ID NO: 53, SEQ ID NO: 57, and SEQ ID NO: 55, (iii) SEQ ID NO: 53, SEQ ID NO: 58, and SEQ ID NO: 55, A set of immunoglobulin heavy chain complementarity determining regions (CDRs) each including a heavy chain CDR1, CDR2, and CDR3 amino acid sequence selected from (b) the following set: (i) SEQ ID NO: 62, SEQ ID NO: 60 and SEQ ID NO: 61, (ii) SEQ ID NO: 59, SEQ ID NO: 63, and SEQ ID NO: 61, (iii) SEQ ID NO: 59, SEQ ID NO: 64, and SEQ ID NO: 61, and a set of immunoglobulin light chain complementarity determining regions (CDRs) each comprising a light chain CDR1, CDR2 and CDR3 amino acid sequence selected from Anti-huCD45 antibodies or huCD45 binding antibody fragments are provided.

[0007] Additional features, advantages, and aspects of the present invention will be set forth or will become apparent from a review of the following detailed description, drawings, and claims. Moreover, it should be understood that both the foregoing summary of the invention and the following detailed description are exemplary and intended to further illustrate, but not limit, the scope of the invention as claimed.

[0008] The heavy and light chain amino acid sequences of various antibodies are shown in Figures 1-10. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the amino acid sequence of the heavy chain of a murine anti-huCD45 antibody ("mCD45-HC prental") that binds to all isoforms of human CD45 (huCD45) and some of these, and is used as the basis for generating humanized anti-huCD45 antibodies. [Diagram 2] FIG. 2 shows the amino acid sequence of the heavy chain of a humanized anti-huCD45 antibody ("huCD45.HC1") and a portion thereof. [Diagram 3] FIG. 3 shows the amino acid sequence of the heavy chain of a humanized anti-huCD45 antibody ("huCD45.HC2") and a portion thereof. [Figure 4] FIG. 4 shows the amino acid sequence of the heavy chain of a humanized anti-huCD45 antibody ("huCD45.HC3") and a portion thereof. [Diagram 5] FIG. 5 shows the amino acid sequence of the heavy chain of a humanized anti-huCD45 antibody ("huCD45.HC4") and a portion thereof. [Figure 6] FIG. 6 shows the amino acid sequence of the light chain of the murine anti-huCD45 antibody (“mCD45-LC parental”) and a portion thereof, used as the basis for producing a humanized anti-huCD45 antibody. [Figure 7] FIG. 7 shows the amino acid sequence of the light chain of a humanized anti-huCD45 antibody ("huCD45.LC1") and a portion thereof. [Figure 8] FIG. 8 shows the amino acid sequence of the light chain of a humanized anti-huCD45 antibody ("huCD45.LC2") and a portion thereof. [Figure 9] FIG. 9 shows the amino acid sequence of the light chain of a humanized anti-huCD45 antibody ("huCD45.LC3") and a portion thereof. [Figure 10] Figure 10 shows the amino acid sequence of the light chain of a humanized anti-huCD45 antibody ("huCD45.LC4") and a portion thereof. In the representation of the variable region sequences in the figure, the amino acid sequences of the complementarity determining regions (CDRs) are underlined. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] One aspect of the present invention is a method for producing a composition comprising the steps of: (i) the heavy chain of mCD45-HC parental, 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; (ii) a light chain of mCD45-LCparental, huCD45.LC1, huCD45.LC2, huCD45.LC3, or huCD45.LC4, or any of said light chains without the signal sequence, or a light chain comprising the light chain variable region of any of said light chains; When the heavy chain is mCD45-HC parental, with or without a signal sequence, the light chain is not mCD45-LC parental, with or without a signal sequence. anti-(human CD45) ["anti-huCD45"] antibody, or a human CD45-binding fragment of said antibody, such as a Fab fragment or a Fab2 fragment or a corresponding scFv molecule.

[0011] Parental murine monoclonal antibodies, containing parental heavy chains as shown in Figure 1 and parental light chains as shown in Figure 6, binding to all isoforms of human CD45, were used as the basis for designing humanized anti-huCD45 antibodies. Antibodies consisting of various combinations of humanized immunoglobulin heavy and 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 OctetHTX (Sartorius, Bohemia, NY, USA) to measure the KD of the antibodies. The antibodies were loaded onto an anti-human Fc capture (AHC) sensor. The loaded sensor was then bathed in a serial dilution of rhCD45His tag (starting at 900 nM, 1:3 dilution, 3 points; R&D Systems, Minneapolis, MN, USA). Reference sample wells (buffer) were used for data analysis. Rate constants were calculated using a monovalent (1:1) binding model. The results are shown in Table 1 below.

[0012] [Table 1] huCD45 HC3+LC3 retained similar binding affinity to the parental antibody, and all other humanized samples showed less than a 10-fold change in KD values ​​relative to the parental antibody.

[0013] Another aspect of the present invention is a method for producing a (i) an immunoglobulin heavy chain variable region comprising SEQ ID NO:2 (from mCD45-HC parental), 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); (ii) an immunoglobulin light chain variable region comprising SEQ ID NO:28 (from mCD45-LC parental), 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); When the heavy chain variable region comprises SEQ ID NO:2, the light chain variable region is not SEQ ID NO:28; Anti-huCD45 antibodies or human CD45 binding antibody fragments are provided. In a variation of the above aspect of the invention, the anti-huCD45 antibody or human CD45 binding antibody fragment comprises: (i) an immunoglobulin heavy chain sequence comprising SEQ ID NO:6 (from mCD45-HC parental), 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); (ii) an immunoglobulin light chain sequence comprising SEQ ID NO: 32 (from mCD45-LCparental), 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).

[0014] A further aspect of the present invention is (a) the following set: (i) SEQ ID NO: 53, SEQ ID NO: 56 and SEQ ID NO: 55, (ii) SEQ ID NO: 53, SEQ ID NO: 57, and SEQ ID NO: 55, (iii) SEQ ID NO: 53, SEQ ID NO: 58, and SEQ ID NO: 55, (iv) SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55, A set of immunoglobulin heavy chain complementarity determining regions (CDRs) each including a heavy chain CDR1, CDR2, and CDR3 amino acid sequence selected from (b) the following set: (i) SEQ ID NO: 62, SEQ ID NO: 60 and SEQ ID NO: 61, (ii) SEQ ID NO: 59, SEQ ID NO: 63, and SEQ ID NO: 61, (iii) SEQ ID NO: 59, SEQ ID NO: 64, and SEQ ID NO: 61, (iv) SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, and a set of immunoglobulin light chain complementarity determining regions (CDRs) each comprising a light chain CDR1, CDR2 and CDR3 amino acid sequence selected from when the heavy chain CDR set is (a)(iv), then the light chain CDR set is not (b)(iv); An anti-huCD45 antibody or huCD45 binding antibody fragment is provided, which should be understood to comprise a heavy chain variable region comprising the heavy chain CDRs, and a light chain variable region comprising the light chain CDRs. A related aspect of the invention is (a) a set of: (i) SEQ ID NO: 53, SEQ ID NO: 56 and SEQ ID NO: 55, (ii) SEQ ID NO: 53, SEQ ID NO: 57, and SEQ ID NO: 55, (iii) SEQ ID NO: 53, SEQ ID NO: 58, and SEQ ID NO: 55, A set of immunoglobulin heavy chain complementarity determining regions (CDRs) each comprising a heavy chain CDR1, CDR2, and CDR3 amino acid sequence selected from (b) the following set: (i) SEQ ID NO: 62, SEQ ID NO: 60 and SEQ ID NO: 61, (ii) SEQ ID NO: 59, SEQ ID NO: 63, and SEQ ID NO: 61, (iii) SEQ ID NO: 59, SEQ ID NO: 64, and SEQ ID NO: 61, A set of immunoglobulin light chain complementarity determining regions (CDRs) each comprising a light chain CDR1, CDR2, and CDR3 amino acid sequence selected from The present invention provides an anti-huCD45 antibody or a huCD45 binding antibody fragment comprising: Again, it should be understood that an antibody or antibody fragment comprises a heavy chain variable region comprising the heavy chain CDRs, and a light chain variable region comprising the light chain CDRs.

[0015] In either of the above two aspects of the invention, or in a variation of the other aspects of the invention in which CDR sequences are described, the antibody or antibody fragment or protein further comprises an additional N-terminal and / or C-terminal amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues adjacent to one or more CDRs (at either or both the N-terminus and C-terminus of the CDR), for any and all combinations of one or more of the CDRs described, as shown in any of the respective variable region amino acid sequences in Figures 1 to 10.

[0016] Another embodiment of the invention relates to the following light chain CDRs: 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); 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); CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO: 61), and an antibody light chain variable region comprising The combination of CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO: 59) and CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO: 60) is excluded. Proteins such as anti-CD45 antibodies or CD45 binding antibody fragments (such as Fab fragments, Fab2 fragments or scFv molecules) or immunoglobulin light chains are provided. The next amino acid residue after the sequence QHSRELPFT (SEQ ID NO: 61) of CDR-L3 in the protein may be, for example, FGQ.

[0017] A further aspect of the invention is the heavy chain CDRs: CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO:53) or GFDFSRYWMN (SEQ ID NO:85); 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); CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO:55); and an antibody heavy chain variable region comprising The combination of CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53) and CDR-H2 comprising the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO: 54) is excluded. Proteins such as anti-CD45 antibodies or CD45 binding antibody fragments (such as Fab fragments, Fab2 fragments or scFv molecules) or immunoglobulin heavy chains are provided.

[0018] Yet another aspect of the present invention is a method for producing a medicament for use in a method for the treatment of a pulmonary artery (a) a light chain CDR(s): 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); 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); CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO:61); an antibody light chain variable region comprising: (b) the heavy chain CDRs: CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO:53) or GFDFSRYWMN (SEQ ID NO:85); 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); CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO:55); and an antibody heavy chain variable region comprising The combination of CDR-LI 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. Proteins such as anti-CD45 antibodies or CD45 binding antibody fragments (such as Fab fragments, Fab2 fragments or scFv molecules) are provided. The next amino acid residue after the sequence QHSRELPFT (SEQ ID NO: 61) of CDR-L3 in the protein may be, for example, FGQ.

[0019] A further aspect of the invention is the following light chain CDRs: CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO:59) or KSSKSVSTSGYSYLH (SEQ ID NO:62); 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); CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO: 61), 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 an scFv molecule) or an immunoglobulin light chain, comprising an antibody light chain variable region comprising: The next amino acid residue after the sequence QHSRELPFT (SEQ ID NO: 61) of CDR-L3 in the protein may be, for example, FGQ. Another embodiment of the invention relates to the following heavy chain CDRs: CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO:53); CDR-H2 comprising the amino acid sequence EINPTSSTINFADSVKG (SEQ ID NO: 56), EINPTSSTINYADSVKG (SEQ ID NO: 57) or EINPTSSTINFVDSVKG (SEQ ID NO: 58); CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO:55); 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 an scFv molecule) or an immunoglobulin heavy chain, comprising an antibody heavy chain variable region comprising:

[0020] A further aspect of the present invention is (a) a light chain CDR(s): CDR-L1 comprising the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO:59) or KSSKSVSTSGYSYLH (SEQ ID NO:62); CDR-L2 comprising the amino acid sequences 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); CDR-L3 comprising the amino acid sequence QHSRELPFT (SEQ ID NO:61); an antibody light chain variable region comprising (b) the heavy chain CDRs: CDR-H1 comprising the amino acid sequence GFDFSRYWMS (SEQ ID NO:53); 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); CDR-H3 comprising the amino acid sequence GNYYRYGDAMDY (SEQ ID NO:55); and an antibody heavy chain variable region comprising 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. Proteins such as anti-CD45 antibodies or CD45 binding antibody fragments (such as Fab fragments, Fab2 fragments or scFv molecules) are provided. The next amino acid residue after the sequence QHSRELPFT (SEQ ID NO: 61) of CDR-L3 in the protein may be, for example, FGQ. CDR-H2 may, for example, comprise the amino acid sequence EINPTSSTINFADSVKG (SEQ ID NO: 56), EINPTSSTINYADSVKG (SEQ ID NO: 57) or EINPTSSTINFVDSVKG (SEQ ID NO: 58).

[0021] One aspect of the invention provides a protein, such as an immunoglobulin heavy chain variable region, or 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 that comprises a parent immunoglobulin heavy chain variable region disclosed herein in any combination with any non-parent immunoglobulin heavy chain variable region disclosed herein and that contains one or more of the amino acid substitutions disclosed, but otherwise comprises the same amino acid sequence as the parent immunoglobulin heavy chain variable region sequence. One aspect of the invention provides a protein, such as an immunoglobulin light chain variable region, or an anti-CD45 antibody or a CD45 binding antibody fragment, such as a Fab, Fab2 or scFv molecule, comprising a parent immunoglobulin light chain variable region disclosed herein in any combination with any non-parent immunoglobulin light chain variable region disclosed herein that contains one or more amino acid substitutions as disclosed, but otherwise comprises the same amino acid sequence as the parent immunoglobulin light chain variable region sequence.

[0022] One aspect of the present invention is a method for producing a composition comprising the steps of: (i) an immunoglobulin heavy chain variable region comprising a parent immunoglobulin heavy chain variable region disclosed herein in any combination with any non-parent immunoglobulin heavy chain variable region disclosed herein, the immunoglobulin heavy chain variable region comprising one or more of the disclosed amino acid substitutions, but otherwise comprising the same amino acid sequence as the parent immunoglobulin heavy chain variable region sequence; and (ii) a parent immunoglobulin light chain variable region disclosed herein in any combination with any non-parent immunoglobulin light chain variable region disclosed herein, comprising one or more of the amino acid substitutions disclosed, but otherwise comprising the same amino acid sequence as the parent immunoglobulin light chain variable region sequence; Proteins such as anti-CD45 antibodies or CD45 binding antibody fragments such as Fab, Fab2 or scFv molecules are provided.

[0023] One aspect of the present invention is a method for producing a composition comprising the steps of: (i) a parent immunoglobulin heavy chain variable region disclosed herein, or an immunoglobulin heavy chain variable region comprising one or more of the disclosed amino acid substitutions, but otherwise comprising the same amino acid sequence as the parent immunoglobulin heavy chain variable region sequence, in any combination with any of the non-parent immunoglobulin heavy chain variable regions disclosed herein for a parent immunoglobulin heavy chain variable region disclosed herein; and (ii) a parent immunoglobulin light chain variable region disclosed herein; or a parent immunoglobulin light chain variable region disclosed herein in any combination with any non-parent immunoglobulin light chain variable region disclosed herein, and an immunoglobulin light chain variable region that contains one or more of the amino acid substitutions disclosed, but otherwise contains the same amino acid sequence as the parent immunoglobulin light chain variable region sequence, does not include both a parent immunoglobulin heavy chain variable region disclosed herein and a parent immunoglobulin light chain variable region disclosed herein. Proteins such as anti-CD45 antibodies or CD45 binding antibody fragments such as Fab, Fab2 or scFv molecules are provided. In one variation of this embodiment, 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.

[0024] One embodiment of the invention relates to a nucleotide sequence comprising the amino acid sequence: EVKLLESGGGLVQPGGSLKLSCAAS GFDFSRYWMX 1WVRQAPGKGLEWX2X3 X 4 INPTSSTIX 5 X 6 X 7 X 8 SLKX 9X 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) and an immunoglobulin heavy chain variable region sequence comprising 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, X 19 is S or M, The amino acid sequence is the sequence EVKLLESGGGLVQPGGSLKLSCAAS GFDFSRYWMS WVRQAPGKGLEWIG EINPTSTINFTPSLKD KVFISRDNAKNTLYLQMSKVRSEDTALYYCAR GNYYRYGDAMDY WGQGTSVTVSS (SEQ ID NO: 2) Does not include Proteins such as immunoglobulin heavy chain variable regions, or anti-CD45 antibodies or CD45 binding antibody fragments such as Fab, Fab2 or scFv molecules are provided. The heavy chain variable region amino acid sequence of this embodiment may be referred to herein as a "modified heavy chain variable region sequence" or "MHCVR sequence."

[0025] One embodiment of the invention relates to a nucleotide sequence comprising the 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 XL13 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 QHSRELPFT FGX L31 GTKLEIK (SEQ ID NO:79) and an immunoglobulin light chain variable region sequence comprising: X L1 is D or E, X L2 is A or V or Q, X L3 is L or M, X L4 is A, D or S, X L5 is S or T, X L6 is A or S, X L7 is V, L or A, X L8 is L, P or V, X L9 is Q, E, or D, X L10 is A or V, X L11 is I or L, X L12 is S, 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 is N, T or S, X L18 is L or R, X L19 is E, A or Q, X L20 is S or T, X L21 is V or I, X L22 is A, 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 is E, A or P, X L29 is A, V, or F, X L30 is T or V, X L31 is S or Q, X L32 is R or K, The amino acid sequence is the sequence DIALTQSPASLAVSLGQRATISC RASKSVSTSGYSYLH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHSRELPFT FGSGTKLEIK (SEQ ID NO:28) Does not include Proteins such as immunoglobulin light chain variable regions, or anti-CD45 antibodies or CD45 binding antibody fragments such as Fab, Fab2 or scFv molecules are provided. The light chain variable region amino acid sequence of this embodiment may be referred to herein as a "modified light chain variable region sequence" or "MLCVR sequence."

[0026] One aspect of the present invention is a method for producing a composition comprising the steps of: (i) a heavy chain variable region comprising an MHCVR sequence or a parent heavy chain variable region amino acid sequence disclosed herein; (ii) a light chain variable region comprising an MLCVR sequence or a parent light chain variable region amino acid sequence disclosed herein, does not 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, Proteins such as anti-CD45 antibodies or CD45 binding antibody fragments such as Fab, Fab2 or scFv molecules are provided. One aspect of the present invention is a method for producing a composition comprising the steps of: (i) a heavy chain variable region comprising an MHCVR sequence; (ii) a light chain variable region comprising an MLCVR sequence; 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:

[0027] One aspect of the invention provides a protein, such as a protein comprising a CD45 binding scFv molecule or at least one scFv fragment, that comprises a contiguous (in amino to carboxy terminal order) amino acid sequence comprising amino acids 1-120 (amino acids through and including the sequence VTVS or corresponding amino acid positions) 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, but not both amino acids 1-120 of a parent heavy chain variable region sequence disclosed herein, and not a parent light chain variable region sequence disclosed herein. In one variation, the protein does not comprise either amino acids 1-120 of a parent heavy chain variable region sequence disclosed herein, or a parent light chain variable region sequence disclosed herein. The linker amino acid sequence may be, for example, the amino acid sequence: KISGGGGSGGGGSGGGGSGGGGSGGGGSS (SEQ ID NO:80); SPNSASHSGSAPQTSSAPGSQ (SEQ ID NO:81); (G3S) 4, i.e., GGGSGGGSGGGSGGGS (SEQ ID NO: 82), for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (G3S) n or (G4S)5, i.e., GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 84), for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (G4S) n , that is, (GGGGS [sequence number 83]) n , may include any of the following: The heavy chain variable region and light chain variable region disclosed herein each contain three complementarity determining regions (CDRs). The CDRs are surrounded by immunoglobulin framework regions (FRs) as follows: FR1 is the amino acid sequence preceding CDR1 (N-terminus), 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 following CDR3 to the end of the variable region sequence (C-terminus).

[0028] 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 having covalently or non-covalently bound chains, scFv molecules, and rIgG fragments, also known as half IgG ("rIgG" refers to reduced IgG (approximately 75,000 daltons)). Proteins such as antibodies and antibody fragments can be conjugated, directly or indirectly, by, for example, a chemically linked chelator, to a radionuclide to direct cytotoxic radiation to CD45 expressing cells in a mammalian subject, such as a human patient, or to non-cytotoxically image CD45 expression in a mammalian subject, such as a human patient. For example, antibodies can be conjugated according to the methods disclosed in U.S. Pat. No. 10,420,851. 131 The antibody can be directly labeled with I or, according to the procedures described in U.S. Pat. No. 9,603,954, the antibody can be chemically coupled to a chelating agent such as p-SCN-DOTA to label the radionuclide. 225 It can be labeled with Ac. Radionuclides are e.g.131 I、 125 I、 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 can be selected from Th.

[0029] Chelators in various embodiments of the present invention include, for example, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) or a derivative thereof; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) or a derivative thereof; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) or a derivative thereof; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA ) or its derivatives;1,4,7-triazacyclononane, 1-glutaric acid-4,7-diacetic acid (NODAGA) or its derivatives;1,4,7,10-tetraazacyclododecane, 1-glutaric 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-diazacyclooctadec-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-(bis-carboxymethylamino)-ethyl]-7- Carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid (NETA) or its derivatives; Diamsar or its derivatives; 1,4,7-triazacyclononane-1,4,7-tris[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-(carboxymethyl) N,N''-[[6-(carboxy)pyridin-2-yl]methyl]diethylenetriamine-N,N',N''-triacetic acid (H5decapa) or its derivatives;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] Pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) or a derivative thereof; desferrioxamine B (DFO) or a derivative thereof; N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]methyl-1,2-diaminoethane (H6phospa) or a derivative thereof; 1,4,7,10,13,16-hexaazacyclohexadecane-N,N',N'',N'',N'''',N''''-hexaacetic acid (HEHA) or a derivative thereof; 1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N'''',N''''-pentaacetic acid (PEPA) or a derivative thereof; or 3,4,3-LI (1,2-HOPO) or a derivative thereof.

[0030] Proteins such as antibodies and antibody fragments can be conjugated, for example, to one or more cytotoxic drugs to target and deplete CD45-expressing cells in a mammalian subject, such as a human patient. Thus, one aspect of the invention provides an antibody-drug conjugate (ADC) that includes an antibody or antigen-binding antibody fragment of the invention as a component. The ADC can be conjugated to a cytotoxin that contains a benzodiazepine moiety, such as a pyrrolobenzodiazepine (PBD) or an indolinobenzodiazepine (IGN), as disclosed, for example, in US Patent Publication No. 20220175951.

[0031] The use of the word "comprising" and the various forms of the word "comprising," as well as the word "including" and the various forms of the word "including," in the specification and claims, does not limit the inclusion of elements other than those referred to. Additionally, throughout this disclosure, various aspects or elements thereof are described by "including" or "comprising," but corresponding aspects or elements thereof are described by "consisting essentially of" or "consisting of" and are similarly disclosed. For example, although some aspects of the invention are described in terms of a protein "including" or "comprising" one or more particular amino acid sequences, corresponding aspects which instead describe a protein "consisting essentially of" or "consisting of" one or more particular amino acid sequences are also within the scope of the aspects and disclosed by this disclosure.

[0032] The composition comprising the radiolabeled antibody may also include one or more pharma- ceutically acceptable carriers or pharma-ceutically 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 polymeric injections, and may include excipients such as solubility-altering agents (e.g., ethanol, propylene glycol, and sucrose) and polymers (e.g., polycaprylactones and PLGA). Exemplary formulations are substantially as described in U.S. Pat. No. 10,420,851 or WO 2017 / 155937, which are incorporated herein by reference. For example, according to some embodiments, the formulation may include 0.5% to 5.0% (weight / volume) of an excipient selected from the group consisting of ascorbic acid, polyvinylpyrrolidone (PVP), human serum albumin (HSA), water-soluble salts of HSA, and mixtures thereof. Some formulations can include 0.5-5% ascorbic acid in 50 mM PBS buffer, pH 7; 0.5-4% polyvinylpyrrolidone (PVP); and a monoclonal antibody.

[0033] The various humanized anti-huCD45 antibodies disclosed herein can be used, e.g., 133 I, 177 Lu, 211 At, 227 Th or 225It can be labeled with a radionuclide such as Ac, 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) for treating hematological cancers, or in preparation for the administration of genetically engineered cell therapies such as CAR-T therapy for treating cancer, or for use as a direct treatment for hematological cancers, such as myeloid or lymphoid hematological cancers, such as leukemia, for example acute myeloid leukemia (AML), or lymphomas, such as non-Hodgkin's lymphoma (NHL), or for use as an immune resetting agent in the treatment of autoimmune diseases, such as multiple sclerosis. The hematological cancers or diseases to be treated include, for example, leukemia (e.g., acute myeloid leukemia (AML), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed lineage leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, or large granular lymphocytic leukemia), myelodysplastic syndromes (MDS), myeloproliferative disorders (e.g., polycythemia vera, essential thrombocythemia, primary myeloblastoma, myelodysplastic syndromes (MDS), myeloproliferative disorders (e.g., polycythemia vera, essential thrombocythemia, primary myeloblastoma, myelodysplastic syndromes), myeloproliferative disorders (e.g., polycythemia vera, essential thrombocythemia, primary myeloblastoma, myelodysplastic syndromes), myelodysplastic syndromes (MDS ... myelofibrosis or chronic myelogenous leukemia), multiple myeloma, MGUS and similar diseases, lymphoma (e.g., Hodgkin's lymphoma (HL), non-Hodgkin's 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 embodiment, a radiolabeled or drug-conjugated anti-huCD45 antibody is administered as maintenance therapy to a subject who has previously undergone BMT or HSCT in the treatment of a hematological cancer, such as AML or another described herein, e.g., to treat or prevent relapse or recurrence of the cancer. In each case described herein, the subject can be a mammal, such as a human patient. EXAMPLES

[0034] Example 1: Generation of radiolabeled chelator-conjugated anti-huCD45 antibodies Conjugation to chelators: Reconstitute a vial of lyophilized p-SCN-Bn-DOTA in metal-free water to a concentration of 10 mg / ml. To an actinium reaction vial, add 0.02 ml of ascorbic acid solution (150 mg / ml) and 0.05 ml of reconstituted p-SCN-Bn-DOTA and adjust the pH to 5-5.5 with 2 M tetramethylammonium acetate (TMAA). Then heat the mixture at 55 ± 4 °C for 30 min. 225 To measure the labeling efficiency of Ac-p-SCN-Bn-DOTA, take an aliquot of the reaction mixture and inject it onto a 1 ml column of Sephadex C25, a cation exchange resin. Elute the product with 0.9% saline in 2-4 ml fractions. 225 The fraction with Ac activity was 225 The fraction retained on the column was Ac-p-SCN-Bn-DOTA, and the unreacted fraction was 225 Ac. Typically, the labeling efficiency is greater than 95%. To the reaction mixture, add 0.22 ml of previously prepared anti-CD45 mAb in DTPA (1 mg) and 0.02 ml ascorbic acid. DTPA is added to bind any trace metals that would compete with the labeling of the antibody. Ascorbic acid is added as a radioprotectant. The pH is adjusted to pH 8.5-9 with carbonate buffer. The mixture is heated at 37 ± 3 °C for 30 min. The final product can be purified by size exclusion chromatography using 10DG resin and eluted with 2 ml of 1% HSA.

[0035] 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), and the conjugation reaction is aimed at achieving a DOTA-antibody ratio of 3:1 to 5:1. 177 Lu, 90 Y or 225 Chelation using a radionuclide such as AC can be performed, and the efficiency and purity of the resulting radiolabeled antibody, such as an anti-CD45 antibody, can be determined by HPLC and iTLC. 225 An exemplary labeling reaction for Ac is as follows: A reaction containing 15 μl of 0.15 M NH4OAc buffer, pH=6.5 and 2 μL (10 μg) of DOTA-anti-CD45 (5 mg / ml) was mixed in an Eppendorf reaction tube, followed by 4 μL of 0.05 M HCl. 225 Add Ac (10 μCi). Mix the contents of the tube with a pipette tip and incubate the reaction mixture at 37 °C for 90 min with shaking at 100 rpm. At the end of the incubation, add 3 μL of a 1 mM DTPA solution to the reaction mixture and incubate at room temperature for 20 min to remove unreacted DTPA. 225 Binding to Ac 225 Instant thin layer chromatography using a 10 cm silica gel strip and a 10 mM EDTA / saline mobile phase was used to obtain the free 225 Ac( 225 Ac-DTPA) 225 Ac-labeled anti-CD45 ( 225 Ac-DOTA-anti-CD45) 225 The radiochemical purity of Ac-DOTA-anti-CD45 can be measured. In this system, the radiolabeled antibody remains at the destination and 225 Ac-DTPA has a migrating solvent front. Cut the silica gel piece in half and measure the chromatogram in a gamma counter with a multichannel analyzer. 225 The number can be counted using channels 72 to 110 for Ac, and the daughter isotopes can be excluded.

[0036] purification: 225 Exemplary radiolabeled targeted agents such as Ac-DOTA-antibodies can be purified either on a PD10 column preblocked with 1% HSA or by Vivaspin centrifugal concentration with 50 kDa MW cutoff, 2x1.5 mL washes, 3 min per spin. After purification, a Waters HPLC system equipped with a Waters flow-through UV detector and a Bioscan Radiation detector is used with a TSK3000SW XL column eluted with PBS at pH=7.4 and a flow rate of 1 ml / min.225 HPLC analysis of the Ac-DOTA-antibody can be performed.

[0037] Example 2: Radioiodination of anti-huCD45 antibodies and purification in the presence of ascorbic acid The anti-CD45 antibody can be radioiodinated according to the following method. In PBS buffer (pH 7.2), 20-30 mCi in the presence of chloramine T (23 micrograms) 131 Label 1 mg of anti-CD45 antibody with I-Na (30 mCi). The reaction is stopped by the addition of aqueous sodium thiosulfate (69 micrograms) and diluted with cold NaI (1 mg). Immediately after, add concentrated ascorbic acid solution made up in 50 mM PBS (pH 7) to achieve a 2.5% (w / v) ascorbic acid concentration in the stopped reaction mixture. For example, labeling reactions of up to 3,000 mCi per batch can be successfully performed using this method. Radiolabeled antibodies, such as radiolabeled immunoglobulins, can be purified by gel filtration on a sterile, prepackaged, commercially available Sephadex G25 column (GE HiPrep26 / 10 column, 53 mL bed volume) using a mobile phase of PBS (50 mM, pH 7) supplemented with 2.5% (weight / volume) ascorbic acid to stabilize the radiolabeled product. Reaction volumes up to 1,000 mCi can be purified on a single column. The product can be collected from the column in elution volumes of 5-35 mL. Batches of radioiodination reactions of <200 mCi can be purified similarly on a smaller desalting column (GE PD10 column, 8.6 mL bed volume).

[0038] Example 3: Radioiodination of anti-CD45 immunoglobulins and purification in the presence of ascorbic acid and HSA Radioiodination and purification can be performed essentially as described in Example 2, except that 2% or 4% (wt / vol) HSA is also added to the stopped reaction and elution buffers along with 2.5% (wt / vol) ascorbic acid during the purification process.

[0039] While various specific embodiments have been illustrated and described herein, it should be understood that various modifications can be made without departing from the spirit and scope of the invention. Moreover, features described in connection with one aspect of the invention can be used in conjunction with other aspects of the invention, even if not explicitly illustrated in that combination.

Claims

1. (i) an immunoglobulin heavy chain variable region including SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 22, or SEQ ID NO: 2, (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, When the heavy chain variable region includes sequence number 2, the light chain variable region is not sequence number 28. Anti-huCD45 antibody or huCD45-conjugated antibody fragment.

2. (i) an immunoglobulin heavy chain sequence including SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 16, or SEQ ID NO: 6; (ii) an immunoglobulin light chain sequence containing SEQ ID NO: 47, SEQ ID NO: 52, SEQ ID NO: 37, SEQ ID NO: 42, or SEQ ID NO: 32, The anti-huCD45 antibody or huCD45-conjugated antibody fragment according to claim 1, comprising:

3. (a) The following set: (i) Sequence ID 53, Sequence ID 58, and Sequence ID 55, respectively (ii) Sequence ID 53, Sequence ID 56 and Sequence ID 55, respectively, (iii) Sequence ID 53, Sequence ID 57 and Sequence ID 55, respectively A set of immunoglobulin heavy chain complementarity-determining regions (CDRs) containing selected heavy chain CDR1, CDR2, and CDR3 amino acid sequences, (b) The following set: (i) Sequence ID 59, Sequence ID 63, and Sequence ID 61, respectively (ii) Sequence ID 59, Sequence ID 64 and Sequence ID 61, respectively, (iii) Sequence ID 62, Sequence ID 60 and Sequence ID 61, respectively A set of immunoglobulin light chain complementarity-determining regions (CDRs) containing selected light chain CDR1, CDR2, and CDR3 amino acid sequences, An anti-huCD45 antibody or huCD45-conjugated antibody fragment containing a huCD45 antibody.

4. A pharmaceutical composition comprising an antibody or antibody fragment according to any one of claims 1 to 3, and at least one pharmaceutically acceptable excipient.

5. The antibody or antibody fragment according to any one of claims 1 to 3, The radionuclide bound to the antibody or antibody fragment, and At least one pharmaceutically acceptable excipient, A radiopharmaceutical composition containing [the specified substance].

6. The radiopharmaceutical composition according to claim 5, wherein the radioactive nuclide is an alpha particle emitter or a beta particle emitter.

7. The aforementioned radioactive nuclide is 131 The radiopharmaceutical composition according to claim 5, comprising I.

8. The aforementioned radioactive nuclide is 225 Ac, 177 Lu or 90 The radiopharmaceutical composition according to claim 5, comprising Y.

9. A composition comprising an antibody or antibody fragment according to any one of claims 1 to 3, which is chemically bonded to a chelating agent.

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

11. The composition according to claim 9, further comprising a radionuclide chelated with the chelating agent.

12. The following light chain CDRs: CDR-L1 containing the amino acid sequences 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); CDR-L2 containing the amino acid sequences 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); CDR-L3 containing the amino acid sequence QHSRELPFT (SEQ ID NO: 61), Includes an antibody light chain variable region, The combination of CDR-L1 containing the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO: 59) and CDR-L2 containing the amino acid sequence LASNLES (SEQ ID NO: 60) is excluded. Anti-CD45 antibody or CD45-conjugated antibody fragment (Fab fragment, Fab 2 Proteins such as fragments or scFv molecules, or immunoglobulin light chains.

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

14. The following heavy chain CDRs: CDR-H1 containing the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53) or GFDFSRYWMN (SEQ ID NO: 85); CDR-H2 containing the amino acid sequences EINPTSSTINFPSLGD (SEQ ID NO: 54), EINPTSSTINFADSVKG (SEQ ID NO: 56), EINPTSSTINYADSVKG (SEQ ID NO: 57), EINPTSSTINFVDSVKG (SEQ ID NO: 58), YINPTSSTIYYYADSVKG (SEQ ID NO: 74), AINPTSSTIYYYADSVKG (SEQ ID NO: 75), NINPTSSTIYYYVDSVKG (SEQ ID NO: 76), or SINPTSSTIYYYADSVKG (SEQ ID NO: 77); CDR-H3 containing the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55), Includes an antibody heavy chain variable region, The combination of CDR-H1 containing the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53) and CDR-H2 containing the amino acid sequence EINPTSSTINFTPSLKD (SEQ ID NO: 54) is excluded. Anti-CD45 antibody or CD45-conjugated antibody fragment (Fab fragment, Fab 2 Proteins such as fragments or scFv molecules, or immunoglobulin heavy chains.

15. (a) The following light chain CDRs: CDR-L1 containing the amino acid sequences 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); CDR-L2 containing the amino acid sequences 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 CDR-L3 containing the amino acid sequence QHSRELPFT (SEQ ID NO: 61); The antibody light chain variable region includes, (b) The following heavy chain CDRs: CDR-H1 containing the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53) or GFDFSRYWMN (SEQ ID NO: 85); CDR-H2 containing the amino acid sequences EINPTSSTINFPSLGD (SEQ ID NO: 54), EINPTSSTINFADSVKG (SEQ ID NO: 56), EINPTSSTINYADSVKG (SEQ ID NO: 57), EINPTSSTINFVDSVKG (SEQ ID NO: 58), YINPTSSTIYYYADSVKG (SEQ ID NO: 74), AINPTSSTIYYYADSVKG (SEQ ID NO: 75), NINPTSSTIYYYVDSVKG (SEQ ID NO: 76), or SINPTSSTIYYYADSVKG (SEQ ID NO: 77); and CDR-H3 containing the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55), The antibody heavy chain variable region includes, The following combinations are excluded: CDR-L1 containing the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO: 59), CDR-L2 containing the amino acid sequence LASNLES (SEQ ID NO: 60), CDR-L3 containing the amino acid sequence QHSRELPFT (SEQ ID NO: 61), CDR-H1 containing the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53), CDR-H2 containing the amino acid sequence EINPTSSTINFTPSLGD (SEQ ID NO: 54), and CDR-H3 containing the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55). Anti-CD45 antibody or CD45-conjugated antibody fragment (Fab fragment, Fab 2 Proteins such as fragments or scFv molecules.

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

17. The following light chain CDRs: CDR-L1 containing the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO: 59) or KSSKSVSTSGYSYLH (SEQ ID NO: 62); CDR-L2 containing the amino acid sequences LASNLA (SEQ ID NO: 68), LASNLAT (SEQ ID NO: 63), LASNLQ (SEQ ID NO: 69), or LASNLQS (SEQ ID NO: 64); CDR-L3 containing the amino acid sequence QHSRELPFT (SEQ ID NO: 61), Includes an antibody light chain variable region, Anti-CD45 antibody or CD45-conjugated antibody fragment (Fab fragment, Fab 2 Proteins such as fragments or scFv molecules, or immunoglobulin light chains.

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

19. The following heavy chain CDRs: CDR-H1 containing the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53); CDR-H2 containing the amino acid sequence EINPTSSTINFADSVKG (SEQ ID NO: 56), EINPTSSTINYADSVKG (SEQ ID NO: 57), or EINPTSSTINFVDSVKG (SEQ ID NO: 58); CDR-H3 containing the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55), Includes an antibody heavy chain variable region, An anti-CD45 antibody or a CD45-binding antibody fragment (such as a Fab fragment, a Fab 2 fragment or a scFv molecule, etc.) or a protein such as an immunoglobulin heavy chain.

20. (a) The following light chain CDRs: CDR-L1 containing the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO: 59) or KSSKSVSTSGYSYLH (SEQ ID NO: 62); CDR-L2 containing the amino acid sequences 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 CDR-L3 containing the amino acid sequence QHSRELPFT (SEQ ID NO: 61), The antibody light chain variable region includes; (b) The following heavy chain CDRs: CDR-H1 containing the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53); CDR-H2 containing the amino acid sequences EINPTSSTINFTPSLKD (SEQ ID NO: 54), EINPTSSTINFADSVKG (SEQ ID NO: 56), EINPTSSTINYADSVKG (SEQ ID NO: 57), or EINPTSSTINFVDSVKG (SEQ ID NO: 58); and CDR-H3 containing the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55), The antibody heavy chain variable region includes, Includes, The following combinations are excluded: CDR-L1 containing the amino acid sequence RASKSVSTSGYSYLH (SEQ ID NO: 59), CDR-L2 containing the amino acid sequence LASNLES (SEQ ID NO: 60), CDR-L3 containing the amino acid sequence QHSRELPFT (SEQ ID NO: 61), CDR-H1 containing the amino acid sequence GFDFSRYWMS (SEQ ID NO: 53), CDR-H2 containing the amino acid sequence EINPTSSTINFTPSLGD (SEQ ID NO: 54), and CDR-H3 containing the amino acid sequence GNYYRYGDAMDY (SEQ ID NO: 55). Anti-CD45 antibody or CD45-conjugated antibody fragment (Fab fragment, Fab 2 Proteins such as fragments or scFv molecules.

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

22. The protein according to claim 20, 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 an anti-CD45 antibody or Fab, Fab 2 Alternatively, proteins such as CD45-binding antibody fragments, including scFv molecules.

24. An immunoglobulin light chain variable region comprising an immunoglobulin light chain variable region having the same amino acid sequence as the aforementioned promiscuous immunoglobulin light chain variable region, but including one or more amino acid substitutions disclosed in any combination in any of the non-promiscuous immunoglobulin light chain variable regions disclosed herein, or an anti-CD45 antibody or Fab, Fab 2 Alternatively, proteins such as CD45-binding antibody fragments, including scFv molecules.

25. (i) an immunoglobulin heavy chain variable region having the same amino acid sequence as the aforementioned immunoglobulin heavy chain variable region, but comprising one or more amino acid substitutions disclosed in any combination in any of the aforementioned non-implantiary immunoglobulin heavy chain variable regions; (ii) an immunoglobulin light chain variable region comprising, with respect to the promiscuous immunoglobulin light chain variable region disclosed herein, one or more amino acid substitutions disclosed in any combination in any of the non-promiscuous immunoglobulin light chain variable regions disclosed herein, but otherwise having the same amino acid sequence as the promiscuous immunoglobulin light chain variable region sequence, Anti-CD45 antibody or Fab, Fab 2 Alternatively, proteins such as CD45-binding antibody fragments, including scFv molecules.

26. (i) the variable regions of the progenitor immunoglobulin heavy chain disclosed herein, or A proximate immunoglobulin heavy chain variable region disclosed herein, comprising one or more amino acid substitutions disclosed in any combination in any of the non-proximate immunoglobulin heavy chain variable regions disclosed herein, but otherwise comprising the same amino acid sequence as the proximate immunoglobulin heavy chain variable region sequence; (ii) The variable regions of the progenitor immunoglobulin light chain disclosed herein, or The present invention provides for the present immunoglobulin light chain variable region, wherein the present invention provides for the present immunoglobulin light chain variable region, the present invention provides for one or more amino acid substitutions disclosed in any combination in any of the present non-implant immunoglobulin light chain variable regions, but otherwise provides for the same amino acid sequence as the present immunoglobulin light chain variable region sequence, This document does not include both the aforementioned pro-imoglobulin heavy chain variable region and the aforementioned pro-imoglobulin light chain variable region disclosed herein. Anti-CD45 antibody or Fab, Fab 2 Alternatively, proteins such as CD45-binding antibody fragments, including scFv molecules.

27. Amino acid sequence: EVKLLESGGG LVQPGGS LKLSCAA SGFDFSR YWMX 1 WVRQAPG KGLEWX 2 X 3 X 4 INPTSS TIX 5 X 6 X 7 X 8 SLKX 9 X 10 X 11 X 12 ISRD NX 13 KNX 14 LYLQM SX 15 X 16 RX 17 EDTA X 18 YYCAR GNYYR YGDAMD YWGQG TX 19 VTVS S (SEQ ID NO: 78) It includes an immunoglobulin heavy chain variable region sequence, X 1 is S or N, X 2 is I or V, X 3 is G, S, or A, X 4 is E, Y, A, N, or S, X 5 is N or Y, X 6 is F or Y, X 7 is T, A, or V, X 8 is P or D, X 9 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 is S or M, The aforementioned amino acid sequence is a sequence EVKLLESGGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPTTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTALYYYCARGNYYYRYGDAMDYWGQGTSVTVSS (Sequence ID 2) is not included. Immunoglobulin heavy chain variable region, or anti-CD45 antibody or Fab, Fab 2 Alternatively, proteins such as CD45-binding antibody fragments, including scFv molecules.

28. Amino acid sequence: .︩ L1 ︸ L2 4.

3. L3 ︸ L4 ︹ L5 ︸ L6 3, L7 ︹ L8 ︹ L9 ︹ L10 ︸ L11 ︃ L32 ︸ L13 The L14 صلى الله عليه وسلم? L15 ︃ L16 _______ L17 ︸ L18 ︸ L19 ︸ L20 ︹ L21 ︃ L22 __________________________________________ L23 ︸ L24 ︸ L25 ︸ L26 ︸ L27 ︸ L28 ︤ L29 1? L30 [||||________________________________________________ L31 ))EE()))) It includes an immunoglobulin light chain variable region sequence, X L1 is D or E, X L2 is A, V, or Q, X L3 is L or M, X L4 is A, D, or S, X L5 is S or T, X L6 is A or S, X L7 is V, L, or A, X L8 is L, P, or V, X L9 is Q, E, or D, X L10 is A or V, X L11 is I or L, X L12 is S, 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 is N, T, or S, X L18 is L or R, X L19 is E, A, or Q, X L20 is S or T, X L21 is V or I, X L22 is A, 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 is E, A, or P, X L29 is A, V, or F, X L30 is T or V, X L31 is S or Q, and X L32 is R or K, The aforementioned amino acid sequence is a sequence DIALTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPPARFSGSGSGTDFTLNIHPVEEEEDAAATYYCQHSRELPFTFGSGTKLEIK (Sequence ID 28) is not included. Immunoglobulin light chain variable region, or anti-CD45 antibody or Fab, Fab 2 Alternatively, proteins such as CD45-binding antibody fragments, including scFv molecules.

29. (i) A heavy chain variable region comprising the MHCVR sequence or parent heavy chain variable region amino acid sequence disclosed herein, and (ii) A light chain variable region comprising the MLCVR sequence or parent light chain variable region amino acid sequence disclosed herein, This document does not include both the parent heavy chain variable region amino acid sequence and the parent light chain variable region amino acid sequence disclosed herein. Anti-CD45 antibody or Fab, Fab 2 Alternatively, proteins such as CD45-binding antibody fragments, including scFv molecules.

30. (i) the heavy chain variable region including the MHCVR sequence, and (ii) Light chain variable region including the MLCVR sequence, The protein according to claim 29, comprising:

31. A protein, such as a protein comprising a CD45-binding scFv molecule or at least one scFv fragment, comprising amino acids 1-120 (including amino acids up to sequence VTVS or the corresponding amino acid position) or amino acid 1-121 of the heavy chain variable region disclosed herein, followed by a linker amino acid sequence, followed by a sequence of amino acids of the light chain variable region disclosed herein, and not comprising either amino acids 1-120 of the parent heavy chain variable region sequence disclosed herein or the parent light chain variable region sequence disclosed herein.

32. The protein according to claim 31, wherein the protein comprises amino acids 1-120 of the heavy chain variable region disclosed herein (amino acids up to (including) the sequence VTVS or the corresponding amino acid position), and does not comprise amino acids 1-121 of the heavy chain variable region disclosed herein.

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

34. The aforementioned linker amino acid sequence is KISSGGGGGGGGGGGGGGGGGGGGGGGGGGGGSS (No. 80); SPNSASHGSAPQTSSAPGSQ (Sequence ID 81); n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (G3S) n ; or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (G4S) n The protein according to claim 31, comprising

35. A pharmaceutical composition comprising the protein described in any one of claims 12 to 34.

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

37. The pharmaceutical composition according to claim 35, wherein at least a portion of the protein in the composition is radiolabeled or conjugated to a cytotoxic agent.

38. The pharmaceutical composition according to claim 35 for treating hematological cancer or solid tumor cancer in mammals.

39. The composition according to claim 4, for use as a conditioning agent in the preparation of bone marrow transplantation (BMT), hematopoietic stem cell transplantation (HSCT), or recombinant cell therapy such as CAR-T cell therapy in mammals.

40. The composition according to claim 35, for use as a conditioning agent in the preparation of bone marrow transplantation (BMT), hematopoietic stem cell transplantation (HSCT), or recombinant cell therapy such as CAR-T cell therapy in mammals.

41. The pharmaceutical composition according to claim 38, wherein the target mammal is a human patient.

42. The pharmaceutical composition according to claim 39, wherein the target mammal is a human patient.

43. The pharmaceutical composition according to claim 40, wherein the target mammal is a human patient.