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WO2024133890A1 - Anticorps ciblant cd45 - Google Patents

Anticorps ciblant cd45 Download PDF

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Publication number
WO2024133890A1
WO2024133890A1 PCT/EP2023/087612 EP2023087612W WO2024133890A1 WO 2024133890 A1 WO2024133890 A1 WO 2024133890A1 EP 2023087612 W EP2023087612 W EP 2023087612W WO 2024133890 A1 WO2024133890 A1 WO 2024133890A1
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Prior art keywords
seq
region
antibody
antibody fragment
heavy chain
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Ceased
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PCT/EP2023/087612
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Inventor
Stefanie Urlinger
Rosalba LEPORE
Romina MATTER-MARONE
Alessandro Sinopoli
Lukas JEKER
Anna HAYDN
Eva-Maria GROSSJOHANN
Andreja KNEZEVIC
Anna CAMUS
Anna DEVAUX
Frank Lehmann
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Cimeio Therapeutics Ag
Universitaet Basel
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Cimeio Therapeutics Ag
Universitaet Basel
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Priority to KR1020257017579A priority Critical patent/KR20250122457A/ko
Priority to EP23838073.7A priority patent/EP4637928A1/fr
Priority to CN202380088284.1A priority patent/CN120476145A/zh
Priority to IL321599A priority patent/IL321599A/en
Priority to AU2023410055A priority patent/AU2023410055A1/en
Publication of WO2024133890A1 publication Critical patent/WO2024133890A1/fr
Priority to MX2025007213A priority patent/MX2025007213A/es
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD45
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure relates to antibodies and antibody fragments that are specific for CD45.
  • the antibodies are improved humanized versions of a murine antibody.
  • the antibodies were also engineered to remove several detrimental motifs within the CDR region without losing any beneficial properties.
  • the antibodies are useful for the treatment of diseases associated with CD45.
  • STATEMENT REGARDING FUNDING The project leading to this application has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 818806).
  • CD45 also known as protein tyrosine phosphatase receptor type C (PTPRC) is an enzyme encoded by the PTPRC gene (Kaplan et al., PNAS 87:7000-7004 (1990)).
  • CD45 is a member of the protein tyrosine phosphatase (PTP) family, which includes signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation.
  • PTP protein tyrosine phosphatase
  • CD45 contains an extracellular domain, a single transmembrane segment, and two tandem intracytoplasmic catalytic domains, and thus belongs to the receptor type PTP family.
  • CD45 is a type I transmembrane protein that is present in various isoforms on differentiated hematopoietic cells (except e.g. erythrocytes) (Holmes, Immunology 7:145-55 (2006)). CD45 has been shown to be a regulator of T- and B-cell antigen receptor signaling. It functions through either direct interaction with components of the antigen receptor complexes via its extracellular domain, or by activating various Src family kinases (SFK), such as Lck, required for the antigen receptor signaling via its cytoplasmic domain. CD45 also suppresses JAK kinases, and thus functions as a negative regulator of cytokine receptor signaling.
  • Src family kinases such as Lck
  • CD45 is present on the surface of hematopoietic cells, including HSCs, leukocytes, and osteoclasts, which are of hematopoietic origin (Shivtiel et al., J Exp Med 205:2381 (2008)). Deletion mutations within CD45 in humans are associated with severe immunodeficiency. This is primarily due to the absence of CD45 on T cells, where it is typically abundant and required to modulate SFK activity during antigen responses. CD45- deficient (CD45-/-) mouse bone marrow contains normal numbers of hematopoietic cells, but the most primitive HSCs are reduced in number, and their mobilization in response to G-CSF is impaired.
  • CD45-/- HSCs are also deficient in G-CSF-stimulated mobilization and homing to the chemokine CXCL12/SDF-1, which negatively affects cell engraftment following transplantation. These deficiencies can be restored by supplementation with SFK inhibitors, indicating that this role is usually performed by CD45.
  • CD45-/- recipients also show deficient engraftment and subsequent mobilization of normal HSCs, indicating a role for CD45 in the stem cell niche, as well as in the HSC (Shivtiel et al, J Exp Med 205:2381 (2008)).
  • CD45 is expressed, for example, on HSCs and leukocytes, it presents a target for therapies including conditioning therapies, immune reset, and treatment of diseases.
  • anti-CD45 moieties are known in the art, some of which are currently in development.
  • BC8 is a mouse hybridoma antibody commercially available from IchorBio (#ICH1155).
  • the BC8 antibody is the basis for an anti-CD45 antibody-radioconjugate developed by Actinium Pharmaceuticals (WO2017155937, WO2019084258, WO2020159656).
  • Other anti-CD45 antibodies and antibody-based moieties are disclosed in WO2016016442, WO2019115791 and WO2020058495, WO2017009473, WO2019129178, WO2020018580, WO2020170254 and WO2020219959. These and other anti-CD45 moieties may be used in the context of the present disclosure.
  • the present disclosure relates to humanized antibodies or antibody fragments specific for human CD45, wherein said antibody or antibody fragment comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises at least one of the following mutations: a) the glycine in the LCDR1 region (SEQ ID NO: 7) is replaced by alanine, b) the second asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by lysine, c) the arginine in the LCDR2 region (SEQ ID NO: 8) is replaced by leucine, or d) the phenylalanine in the LCDR2 region (SEQ ID NO:
  • the present disclosure relates to a humanized antibodies or antibody fragments specific for human CD45, wherein said antibody or antibody fragment comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises at least one of the following mutations: a) the glycine in the LCDR1 region (SEQ ID NO: 7) is replaced by alanine, b) the second asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by lysine, or c) the arginine in the LCDR2 region (SEQ ID NO: 8) is replaced by leucine.
  • the humanized antibody or antibody fragment comprises at least one of the following mutations:
  • the present disclosure also relates to humanized antibodies or antibody fragments specific for human CD45, wherein said antibody or antibody fragment comprises a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 75, the LCDR2 region of SEQ ID NO: 96 and the LCDR3 region of SEQ ID NO: 9, b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9, c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a
  • the present disclosure also relates to aforementioned humanized antibodies or antibody fragments specific for human CD45, wherein said antibody or antibody fragment additionally comprises a mutation of the second glycine of the triple glycine motif of framework region 4 of the variable light chain to glutamine.
  • the present disclosure also relates to humanized antibodies or antibody fragments specific for human CD45, wherein said humanized antibodies or antibody fragments comprise a) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 98, b) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 99, c) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 101, d) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 102, e) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 103, f) a variable heavy chain of S
  • the present disclosure also relates to aforementioned humanized antibodies or antibody fragments specific for human CD45, wherein said humanized antibodies or antibody fragments comprise a mutation of a) the glycine in the LCDR1 region (SEQ ID NO: 7) is replaced by alanine, b) the second asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by lysine, and c) the arginine in the LCDR2 region (SEQ ID NO: 8) is replaced by leucine.
  • the present disclosure also relates to humanized antibodies or antibody fragments specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104.
  • the present disclosure also relates to aforementioned humanized antibodies or antibody fragments specific for human CD45, wherein said humanized antibody comprises a modification in the FC region, preferably wherein said Fc modification is a silencing mutation, more preferably wherein said mutation is a PA-LALA, a PG-LALA or an AEASS mutation.
  • the present disclosure also relates to aforementioned humanized antibodies or antibody fragments specific for human CD45, wherein said humanized antibody or antibody fragment is a monoclonal antibody or antibody fragment.
  • the present disclosure also relates to an antibody drug conjugate comprising aforementioned humanized antibodies or antibody fragments specific for human CD45and a cytotoxic drug.
  • said cytotoxic moiety is a pyrrolobenzodiazepine (PBD).
  • the present disclosure also relates to aforementioned humanized antibodies, antibody fragments and antibody drug conjugates comprising said humanized antibodies or antibody fragments for use in medicine.
  • the present disclosure also relates to aforementioned humanized antibodies, antibody fragments and antibody drug conjugates comprising said humanized antibodies or antibody fragments for use in the treatment of a hematological cancer, such as a leukemia, myeloma or lymphoma.
  • the present disclosure also relates to aforementioned humanized antibodies, antibody fragments and antibody drug conjugates comprising said humanized antibodies or antibody fragments for use in the treatment of an inflammatory or autoimmune disease such as multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn’s disease, Type 1 diabetes, rheumatoid arthritis or idiopathic arthritis
  • an inflammatory or autoimmune disease such as multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn’s disease, Type 1 diabetes, rheumatoid arthritis or idiopathic arthritis
  • nucleic acid compositions comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding aforementioned humanized antibodies and antibody fragments.
  • the present disclosure also relates to vectors comprising said nucleic acid compositions.
  • the present disclosure also relates to host cell comprising said vectors or nucleic acid compositions.
  • the present disclosure also relates to pharmaceutical compositions comprising aforementioned humanized antibodies, antibody fragments or antibody drug conjugates comprising said humanized antibodies or antibody fragments, and a pharmaceutically acceptable carrier or excipient.
  • Figure legends Figure 1 shows the alignment of five humanized variants to the original murine VH sequence of ⁇ tti.
  • VHO is the murine sequence and VH1-5 are the humanized variants. Key residues important for the VH/VL interface and canonical loop structure have been maintained as much as possible.
  • Figure 2 shows the alignment of five humanized variants to the original murine VL sequence of antibody ⁇ tti.
  • VLO is the murine sequence and VL1-6 are the humanized variants.
  • Figure 3 shows complete depletion of all human CD45+ cells and LT-HSCs 3 weeks after single dose of 0.3 mg/kg with Kiebitz-Teserine compared to PBS control group, whereas BC8-Teserine treatment resulted only in partial depletion.
  • Figure 4 shows that in both cell lines tested blocking with naked antibodies mimics shielding of the cells and is able to prevent cell viability decrease via the respective ADCs.
  • BC08-Tesirine shows lower potency than Kiebitz-Tesirine.
  • Panel A MV4-11; panel B: Molm-13.
  • Figure 5 shows that also in HSPC, blocking with naked Kiebitz antibody shields cells and is able to prevent cell viability decrease a respective ADC.
  • Jurkat CD45 knock-out cells have an IC50 for Kiebitz-Tesrine which is significantly higher than the IC50 in Jurkat wild-type cells (0.46 ⁇ g/ml vs.0.032 ⁇ g/ml).
  • CD45 refers to a protein also known as PTPRC, Leukocyte common antigen (L-CA) or T200.
  • Human CD45 has the following amino acid sequence (UniProt P08575-3, defined as canonical sequence) MTMYLWLKLLAFGFAFLDTEVFVTGQSPTPSPTGLTTAKMPSVPLSSDPLPTHTTAFSPASTFE RENDFSETTTSLSPDNTSTQVSPDSLDNASAFNTTGVSSVQTPHLPTHADSQTPSAGTDTQTFS GSAANAKLNPTPGSNAISDVPGERSTASTFPTDPVSPLTTTLSLAHHSSAALPARTSNTTITAN TSDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNE NVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTT ICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIK
  • antibody refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, which interacts with an antigen.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FR’s arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • antibody includes for example, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies and chimeric antibodies.
  • the antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., Igd , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.
  • antibody fragment refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing spatial distribution) an antigen.
  • binding fragments include, but are not limited to, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
  • F(ab)2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • a Fd fragment consisting of the VH and CH1 domains
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antibody fragment”.
  • Antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136).
  • Antibody fragments can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No.6,703,199, which describes fibronectin polypeptide monobodies).
  • Fn3 Fibronectin type III
  • Antibody fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1 -VH- CH1 ) which, together with complementary light chain polypeptides, form a pair of antigen- binding sites (Zapata et al., (1995) Protein Eng.8: 1057-1062; and U.S. Pat. No.5,641 ,870).
  • the structures and locations of immunoglobulin variable domains, e.g., CDRs may be defined using well known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g. Sequences of Proteins of Immunological Interest, U.S.
  • a “human antibody” or “human antibody fragment”, as used herein, is an antibody and antibody fragment having variable regions in which both the framework and CDR regions are from sequences of human origin.
  • Human antibodies can also be isolated from synthetic libraries or from transgenic mice (e.g. Xenomouse, OmniMouse, Harbour Mouse, ATX-Gx Mouse, Trianni Mouse) provided the respective system yield in antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin.
  • the antibody contains a constant region, the constant region also is derived from such sequences.
  • Human origin includes, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., (2000) J Mol Biol 296:57-86).
  • a “humanized antibody” or “humanized antibody fragment” is defined herein as an antibody molecule, which has constant antibody regions derived from sequences of human origin and the variable antibody regions or parts thereof or only the CDRs are derived from another species.
  • a humanized antibody can be CDR-grafted, wherein the CDRs of the variable domain are from a non-human origin, while one or more frameworks of the variable domain are of human origin and the constant domain (if any) is of human origin.
  • the term “chimeric antibody” or “chimeric antibody fragment” is defined herein as an antibody molecule, which has constant antibody regions derived from, or corresponding to, sequences found in one species and variable antibody regions derived from another species.
  • the constant antibody regions are derived from, or corresponding to, sequences found in humans
  • the variable antibody regions e.g. VH, VL, CDR or FR regions
  • isolated antibody refers to an antibody or antibody fragment that is substantially free of other antibodies or antibody fragments having different antigenic specificities. Moreover, an isolated antibody or antibody fragment may be substantially free of other cellular material and/or chemicals. Thus, in some aspects, antibodies provided are isolated antibodies, which have been separated from antibodies with a different specificity. An isolated antibody may be a monoclonal antibody. An isolated antibody may be a recombinant monoclonal antibody.
  • an isolated antibody that specifically binds to an epitope, isoform or variant of a target may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., species homologs).
  • the term “recombinant antibody” or “recombinant antibody fragment”, as used herein, includes all antibodies or antibody fragment that are prepared, expressed, created or segregated by means not existing in nature.
  • antibodies isolated from a host cell transformed to express the antibody antibodies selected and isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences or antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom.
  • recombinant antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • a recombinant antibody may be a monoclonal antibody.
  • an antibody “binds specifically to”, “specifically binds to”, is “specific to/for” or “specifically recognizes” an antigen, such as human CD45, if such antibody is able to discriminate between such antigen and one or more reference antigen(s), since binding specificity is not an absolute, but a relative property.
  • a standard ELISA assay or standard flow cytometry assay can be carried out.
  • the scoring may be carried out by standard color development (e.g. secondary antibody with horseradish peroxide and tetramethyl benzidine with hydrogen peroxide) or by binding of a secondary antibody labeled with PE or another dye or marker.
  • the reaction in certain wells is scored by the optical density (OD), for example, at 450 nm or by mean or median fluorescence intensity (MFI) in flow cytometry.
  • OD optical density
  • MFI median fluorescence intensity
  • binding specificity is performed by using not a single reference antigen, but a set of about three to five unrelated antigens, such as milk powder, BSA, transferrin or the like. For flow cytometry various antigen-negative cells can be used. An antibody that specifically binds to an antigen may however have cross-reactivity to the respective orthologous antigen from other species (e.g., species homologs). In certain embodiments such cross-reactivity to an orthologous antigen is even preferred.
  • affinity refers to the strength of interaction between the polypeptide and its target at a single site.
  • epitope includes any proteinaceous region which is specifically recognized by an antibody or antibody fragment thereof or otherwise interacts with a molecule.
  • epitopes are of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally may have specific three-dimensional structural characteristics, as well as specific charge characteristics. As will be appreciated by one of skill in the art, practically anything to which an antibody can specifically bind could be an epitope.
  • compositions or of the present disclosure may be used for therapeutic or prophylactic applications.
  • the present disclosure includes a pharmaceutical composition containing an antibody or antibody fragment as disclosed herein and a pharmaceutically acceptable carrier or excipient therefore.
  • the present disclosure provides a method for treating inflammatory diseases, autoimmune diseases, hematologic malignancies and potentially other diseases. Such method contains the steps of administering to a subject in need thereof an effective amount of the pharmaceutical composition that contains an antibody or antibody fragment as described herein.
  • the present disclosure provides therapeutic methods comprising the administration of a therapeutically effective amount of an antibody or antibody fragment as disclosed herein to a subject in need of such treatment.
  • a “therapeutically effective amount” or “effective amount”, as used herein, refers to the amount of an anti-CD45 antibody necessary to elicit the desired biological response.
  • the therapeutic effective amount is the amount of an anti-CD45 antibody necessary to treat and/or prevent a disease.
  • administerable form such as, for example, an intravenous, intramuscular, intradermal or subcutaneous route or mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestible solution, capsule or tablet.
  • the administration is by an injectable form.
  • treatment refers to clinical intervention in an attempt to alter the natural course of a disease in the subject being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies or antibody fragments according to the preset disclosure are used to delay development of a disease or to slow the progression of a disease.
  • Preventing or “prevention” refers to a reduction in risk of acquiring or developing a disease (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset).
  • Prevention also refers to methods which aim to prevent the onset of a disease or its symptoms or which delay the onset of a disease or its symptoms.
  • Subject or “species” or as used in this context refers to any mammal, including rodents, such as mouse or rat, and primates, such as cynomolgus monkey (Macaca fascicularis), Marmoset monkey (Callithrix jacchus), rhesus monkey (Macaca mulatta) or humans (Homo sapiens).
  • rodents such as mouse or rat
  • primates such as cynomolgus monkey (Macaca fascicularis), Marmoset monkey (Callithrix jacchus), rhesus monkey (Macaca mulatta) or humans (Homo sapiens).
  • the subject is a primate, most preferably a human.
  • effector function refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
  • Non-limiting examples of antibody effector functions include C1 q binding and complement dependent cytotoxicity (CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody- dependent cellular phagocytosis (ADCP); down regulation of cell surface receptors (e.g. B cell receptor); and direct cell activation or direct cell inhibition.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g. NK cells, neutrophils, and macrophages
  • NK cells express FcyRIII only, whereas monocytes/macrophages express FcyRI, FcyRII, and FcyRIII.
  • Complement-dependent cytotoxicity or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) of the present disclosure, which are bound to their cognate antigen.
  • ADCP antibody-dependent cellular phagocytosis
  • ADCP refers to a mechanism of elimination of antibody-coated target cells by internalization by phagocytic cells, such as macrophages or dendritic cells.
  • the term “antibody-drug conjugate” or “ADC” refers to an antibody or an antibody fragment that is chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent.
  • cytotoxic drug as use herein is art recognized and includes, but is not limited to, daunorubicin, mitoxantrone, doxorubicin, cucurbitacin, chaetocin, chaetoglobosin, chlamydocin, calicheamicin, mertansine, nemorubicin, cryptophyscin, mensacarcin, ansamitocin, mitomycin C, geldanamycin, mechercharmycin, rebeccamycin, safracin, okilactomycin, oligomycin, actinomycin, sandramycin, hypothemycin, polyketomycin, hydroxyellipticine, thiocolchicine, methotrexate, triptolide, taltobul
  • pyrrolobenzodiazepine is tesirine.
  • the words “comprise”, “have” and “include” and their respective variations such as “comprises”, “comprising”, “has”, “having”, “includes” and “including” will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
  • the terms “engineered” or “modified” as used herein includes manipulation of nucleic acids or polypeptides by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques).
  • the antibodies or antibody fragments according to the present disclosure are engineered or modified to improve one or more properties, such as antigen binding, stability, half-life, effector function, immunogenicity, safety and the like.
  • “Variant” as used herein refers to a polypeptide that differs from a reference polypeptide by one or more modifications for example amino acid substitutions, insertions or deletions. Variant polypeptides typically retain most of the properties of the reference polypeptide, e.g. binding to the target antigen, but introduce a novel, additional feature or property, e.g. the variant polypeptide has a higher affinity to the target antigen compared to the reference polypeptide or the variant polypeptide is a humanized version of the reference polypeptide.
  • amino acid mutation as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made as long as the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor.
  • Amino acid sequence deletions and insertions include N-and/or C-terminal deletions and insertions of amino acid residues. Particular amino acid mutations are amino acid substitutions.
  • Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like.
  • EC50 refers to the concentration of an antibody or antibody fragment, which induces a response in an assay half-way between the baseline and maximum. It therefore represents the antibody or ligand concentration at which 50% of the maximal effect is observed.
  • Ka refers to the association rate of a particular antibody-antigen interaction
  • Kd refers to the dissociation rate of a particular antibody-antigen interaction.
  • Kd values for antibodies can be determined using methods well established in the art.
  • KD refers to the dissociation constant of a particular antibody- antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration.
  • a preferred method for determining the Kd of an antibody is by using surface plasmon resonance, preferably using a biosensor system, such as a Biacore system, or by using biolayer interferometry with the Octet BLI instrument.
  • inhibitors or “inhibit” or “reduction” or “reduce” or “neutralization” or “neutralize” refer to a decrease or cessation of any phenotypic characteristic (such as binding or a biological activity or function) or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. “Inhibition”, “reduction” or “neutralization” needs not to be complete as long as it is detectable using an appropriate assay. In some embodiments, by “reduce” or “inhibit” or “neutralize” is meant the ability to cause a decrease of 20% or greater. In another embodiment, by “reduce” or “inhibit” or “neutralize” is meant the ability to cause a decrease of 50% or greater.
  • reduce or “inhibit” or “neutralize” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
  • antagonistic antibody refers to an antibody or antibody fragment that interacts with an antigen and partially or fully inhibits or neutralizes a biological activity or function or any other phenotypic characteristic of a target antigen.
  • a “wild-type” protein is a version or variant of the protein as it is found in nature.
  • An amino acid sequence of a wildtype protein e.g., a Fc region of a human lgG1 antibody, is the amino acid sequence of the protein as it occurs in nature.
  • the “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain.
  • the Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain.
  • the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the C-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • numbering of amino acid residues in the Fc region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • Various Fc modifications are commonly used. For a review see for example Antibodies (2020) 9: 64.
  • Silencing functions include (numbering according EU index) the LALA (L234A/L235A), the PA-LALA (L234A/L235A/P329A) and the PG-LALA (L234A/L235A/P329G) mutations, as well as the AEASS mutations (L234A/L235E/G237A/A330S/P331S).
  • a preferred FC modification is PA-LALA.
  • the mutation may also be a mutation which leads to a reduced binding to FcRn, thereby decreasing the in vivo half- life of the antibody. Such mutations include I253A, H310A, H435A and H435Q.
  • the mutation may also a mutation which leads to an increased binding to FcRn, thereby increasing the in vivo half-life of the antibody.
  • Such mutations include T250Q/M428L, M252Y/S254T/T256E (YTE), H433K/N434F and M252Y/S254T/T256E/H433K/N434F.
  • the present disclosure relates to antibodies or antibody fragments that are specific for CD45.
  • the present disclosure relates to antibodies or antibody fragments that are specific for human CD45.
  • the present disclosure relates to humanized antibodies or antibody fragments that are specific for CD45.
  • the present disclosure relates to humanized antibodies or antibody fragments that are specific for human CD45. In certain embodiments, the present disclosure relates to humanized antibodies or antibody fragments that are specific for a polypeptide consisting of the amino acid sequence of SEQ ID No. 1. In certain embodiments, the present disclosure relates to humanized antibodies or antibody fragments that are specific for a polypeptide comprising the amino acid sequence of SEQ ID No. 1.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said antibody or antibody fragment comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said antibody or antibody fragment comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9, wherein the glycine in the LCDR1 region (SEQ ID NO: 7) of said humanized antibody or antibody fragment is replaced by alanine.
  • said antibody or antibody fragment comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said antibody or antibody fragment comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9, wherein the second asparagine in the LCDR1 region (SEQ ID NO: 7) of said humanized antibody or antibody fragment is replaced by lysine.
  • said antibody or antibody fragment comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCD
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said antibody or antibody fragment comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9, wherein the arginine in the LCDR2 region (SEQ ID NO: 8) of said humanized antibody or antibody fragment is replaced by leucine.
  • said antibody or antibody fragment comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said antibody or antibody fragment comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9, wherein the glycine in the LCDR1 region (SEQ ID NO: 7) of said humanized antibody or antibody fragment is replaced by alanine, wherein the second asparagine in the LCDR1 region (SEQ ID NO: 7) of said humanized antibody or antibody fragment is replaced by lysine, and wherein the arginine in the LCDR2 region (SEQ ID NO: 8) of said humanized antibody or antibody fragment is replaced by leucine.
  • said antibody or antibody fragment comprises a variable heavy chain comprising a HC
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said antibody or antibody fragment comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9, wherein said humanized antibody or antibody fragment comprises a mutation of the second glycine of the triple glycine motif of framework region 4 of the variable light chain to glutamine.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said antibody or antibody fragment comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9, wherein the glycine in the LCDR1 region (SEQ ID NO: 7) of said humanized antibody or antibody fragment is replaced by alanine, wherein the second asparagine in the LCDR1 region (SEQ ID NO: 7) of said humanized antibody or antibody fragment is replaced by lysine, wherein the arginine in the LCDR2 region (SEQ ID NO: 8) of said humanized antibody or antibody fragment is replaced by leucine, and wherein said humanized antibody or antibody fragment comprises a mutation of the
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 63, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 65, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 67, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 69, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 71, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 73, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 75, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 77, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 83, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 85, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 87, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 89, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 91, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 93 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 94 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 95 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 96 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 97 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 67, the LCDR2 region of SEQ ID NO: 94 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 61 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 67, the LCDR2 region of SEQ ID NO: 94 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 61 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 69, the LCDR2 region of SEQ ID NO: 94 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 75, the LCDR2 region of SEQ ID NO: 96 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 108, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 108, the LCDR2 region of SEQ ID NO: 96 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 110, the LCDR2 region of SEQ ID NO: 96 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 96 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 93 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 108, the LCDR2 region of SEQ ID NO: 93 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 34.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 34.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 23 and a variable light chain of SEQ ID NO: 34.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 38.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 39.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 40.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 41.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 42. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 43. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 44.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 45.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 46.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 47.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 48. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 49. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 50.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 51.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 52.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 53.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 54. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 55. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 56.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 57. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 58. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 60 and a variable light chain of SEQ ID NO: 58.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 60 and a variable light chain of SEQ ID NO: 59. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 98. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 99.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 100.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 101.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 102.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 103. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein said humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 105.
  • the present disclosure relates to a humanized antibody or antibody fragment, wherein said humanized antibody or antibody fragment comprises 6 six CDRs of one of the antibodies disclosed in Table 14. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein said humanized antibody or antibody fragment comprises 6 six CDRs as defined by Kabat of one of the antibodies disclosed in Table 14. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein said humanized antibody or antibody fragment comprises 6 six CDRs as defined by IMGT of one of the antibodies disclosed in Table 14.
  • the present disclosure relates to a humanized antibody or antibody fragment, wherein said humanized antibody or antibody fragment comprises a variable heavy chain and a variable light chain of one of the antibodies disclosed in Table 14.
  • the present disclosure relates to a humanized antibody or antibody fragment, wherein said humanized antibody or antibody fragment is a monoclonal antibody or antibody fragment.
  • the present disclosure relates to a humanized antibody or antibody fragment, wherein said humanized antibody or antibody fragment is a recombinant antibody or antibody fragment.
  • the present disclosure relates to a humanized antibody or antibody fragment, wherein said humanized antibody or antibody fragment is of the IgG isotype.
  • the present disclosure relates to a humanized antibody or antibody fragment, wherein said humanized antibody or antibody fragment is of the lgG1 class. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein said humanized antibody or antibody fragment is of the human lgG1 class.
  • the antigen-binding regions of the antibodies disclosed herein are used as scFv’s. Such antibodies are particular useful for certain purposes, for example the use in CARs or on LNPs. Not all antigen-binding regions are equally suited for use as scFv’s, primarily because of low expression levels.
  • Antibodies that can be well expressed as scFv’s include the antibodies Jelly (VH2/ VL5.22), Lady Rosetta (VH2/ VL5.38), Levinata (VH2/ VL5.39), and Osira (VH2/ VL5.40). Therefore in certain embodiments, the present disclosure relates to a scFv comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising a LCDR1 region of SEQ ID NO: 67, a LCDR2 region of SEQ ID NO: 94 and a LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a scFv comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising a LCDR1 region of SEQ ID NO: 120, a LCDR2 region of SEQ ID NO: 94 and a LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a scFv comprising a variable heavy chain comprising a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 58.
  • the present disclosure relates to a scFv comprising a variable heavy chain comprising a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 121. In certain embodiments, the present disclosure relates to a scFv comprising a variable heavy chain comprising a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 122. In certain embodiments, the present disclosure relates to a scFv comprising a variable heavy chain comprising a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 123.
  • the isolated antibody or antibody fragment according to the present disclosure may or may not be fused to one or more other amino acid residues, polypeptides or moieties.
  • a fusion protein may be prepared in any suitable manner, including genetically or chemically approaches.
  • Said linked moieties may contain secretory or leader sequences, sequences that aid detection, expression, separation or purification, or sequences that confer to increased protein stability, for example, during recombinant production.
  • Non-limiting examples of potential moieties include beta-galactosidase, glutathione-S-transferase, luciferase, a T7 polymerase fragment, a secretion signal peptide, an antibody or antibody fragment, a toxin, a cytokine, a chemokine, a reporter enzyme, a moiety being capable of binding a metal ion like a poly-histidine tag, a tag suitable for detection and/or purification, a homo- or hetero-association domain, a moiety which increases solubility of a protein, or a moiety which comprises an enzymatic cleavage site.
  • the isolated antibody or antibody fragment according to the present disclosure may optionally contain one or more moieties for binding to other targets or target proteins of interest. It should be clear that such further moieties may or may not provide further functionality to the antibody and may or may not modify the properties of the
  • the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45 as disclosed herein, and (b) a cytotoxic drug.
  • said cytotoxic drug is selected from daunorubicin, mitoxantrone, doxorubicin, cucurbitacin, chaetocin, chaetoglobosin, chlamydocin, calicheamicin, mertansine, nemorubicin, cryptophyscin, mensacarcin, ansamitocin, mitomycin C, geldanamycin, mechercharmycin, rebeccamycin, safracin, okilactomycin, oligomycin, actinomycin, sandramycin, hypothemycin, polyketomycin, hydroxyellipticine, thiocolchicine, methotrexate, triptolide, taltobulin, lactacystin, dolastatin, auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), telomestatin, tubastatin A, combretastatin, may
  • said cytotoxic drug is a pyrrolobenzodiazepine. In other embodiments said pyrrolobenzodiazepine is tesirine.
  • antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45 as disclosed herein, and (b) a cytotoxic drug
  • the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug , wherein said humanized antibody or antibody fragment specific for human CD45 comprises i.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 63, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; ii.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 65, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; iii.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 67, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; iv.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 69, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; v.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 71, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; vi.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 73, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; vii.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 75, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; viii.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 77, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; ix.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; x.
  • avariable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; xi.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 83, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; xii.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 85, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; xiii.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 87, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; xiv.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 89, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; xv.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 91, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; xvi.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 93 and the LCDR3 region of SEQ ID NO: 9; xvii.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 94 and the LCDR3 region of SEQ ID NO: 9; xviii.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 95 and the LCDR3 region of SEQ ID NO: 9; xix.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 96 and the LCDR3 region of SEQ ID NO: 9; xx.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 97 and the LCDR3 region of SEQ ID NO: 9; xxi.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 67, the LCDR2 region of SEQ ID NO: 94 and the LCDR3 region of SEQ ID NO: 9; xxii.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 61 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 67, the LCDR2 region of SEQ ID NO: 94 and the LCDR3 region of SEQ ID NO: 9; xxiii.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 61 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 69, the LCDR2 region of SEQ ID NO: 94 and the LCDR3 region of SEQ ID NO: 9; xxiv.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 75, the LCDR2 region of SEQ ID NO: 96 and the LCDR3 region of SEQ ID NO: 9; xxv.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; xxvi.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 108, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; xxvii.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 108, the LCDR2 region of SEQ ID NO: 96 and the LCDR3 region of SEQ ID NO: 9; xxviii.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 110, the LCDR2 region of SEQ ID NO: 96 and the LCDR3 region of SEQ ID NO: 9; xxix.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 96 and the LCDR3 region of SEQ ID NO: 9; xxx.
  • variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 93 and the LCDR3 region of SEQ ID NO: 9; or xxxi.
  • said cytotoxic drug is a pyrrolobenzodiazepine.
  • said pyrrolobenzodiazepine is tesirine.
  • the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein said humanized antibody or antibody fragment specific for human CD45 comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 93 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein said humanized antibody or antibody fragment specific for human CD45 comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 93 and the LCDR3 region of SEQ ID NO: 9, and wherein said cytotoxic drug is a pyrrolobenzodiazepine.
  • the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein said humanized antibody or antibody fragment specific for human CD45 comprises a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 93 and the LCDR3 region of SEQ ID NO: 9, and wherein said cytotoxic drug is tesirine.
  • the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein said humanized antibody or antibody fragment specific for human CD45 comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104.
  • the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein said humanized antibody or antibody fragment specific for human CD45 comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104, and wherein said cytotoxic drug is a pyrrolobenzodiazepine.
  • the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein said humanized antibody or antibody fragment specific for human CD45 comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104, and wherein said cytotoxic drug is tesirine.
  • Nucleic acids In an embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD45 of the present disclosure.
  • the present disclosure relates to a nucleic acid composition
  • a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD45, wherein said antibody or antibody fragment comprises a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 63, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 65, the LCDR2 region of SEQ ID NO: 8 and
  • the present disclosure relates to a nucleic acid composition
  • a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD45, wherein said antibody or antibody fragment comprises a) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 34; b) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 34; c) a variable heavy chain of SEQ ID NO: 23 and a variable light chain of SEQ ID NO: 34; d) variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 38; e) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 39; f) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 40; g) a variable heavy chain of SEQ ID NO: 21 and
  • the present disclosure relates to a nucleic acid composition
  • a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD45, wherein said antibody or antibody fragment comprises 6 six CDRs as defined by Kabat of one of the antibodies disclosed in Table 14.
  • the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD45, wherein said antibody or antibody fragment comprises 6 six CDRs as defined by IMGT of one of the antibodies disclosed in Table 14.
  • the present disclosure relates to a nucleic acid composition
  • a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD45, wherein said antibody or antibody fragment comprises a variable heavy chain and a variable light chain of one of the antibodies disclosed in Table 13.
  • said nucleic acid composition and/or said nucleic acid sequence and/or said plurality of nucleic acid sequences are isolated.
  • the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding humanized antibody or antibody fragment specific for human CD45 according to the present disclosure.
  • the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the humanized antibody or antibody fragment specific for human CD45disclosed in Table 14.
  • the present disclosure provides a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a humanized antibody or antibody fragment specific for human CD45 according to the present disclosure.
  • the present disclosure refers to a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the humanized antibody or antibody fragment specific for human CD45 disclosed in Table 14.
  • the host cell according to the present disclosure is able to express the humanized antibody or antibody fragment specific for human CD45 encoded by the vector composition or the nucleic acid composition.
  • the host cell is an isolated host cell.
  • said host cell is a mammalian cell.
  • said mammalian cell is a human cell.
  • said mammalian cell is a CHO cell.
  • said cell is a HEK cell.
  • said cell is a PERC.6 cell.
  • said cell is a HKB11 cell.
  • nucleic acid sequence or the plurality of nucleic acid sequences encoding the heavy and/or light chain of an antibody or antibody fragment of the present disclosure can be cloned into different vectors or into the same vector.
  • the vectors can be introduced into the appropriate host cells such as prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast or mammalian) cells by methods well known in the art (see e.g., “Current Protocol in Molecular Biology”, Ausubel et al. (eds.), Greene Publishing Assoc and John Wiley Interscience, New York, 1989 and 1992).
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as “control” elements), so that the nucleic acid sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence.
  • such steps typically include transforming or transfecting a suitable host cell with a nucleic acid composition or vector composition or an infectious particle, which encodes the antibody, or antibody fragments. Further, such steps typically include culturing said host cells under conditions suitable for the proliferation (multiplication, growth) of said host cells and a culturing step under conditions suitable for the production (expression, synthesis) of the encoded antibody or antibody fragment. The culturing of host cells under conditions suitable for proliferation or expression is typically accomplished in the presence of media comprising components suitable for cell growth or induction of expression.
  • the methods for the production of the antibodies or antibody fragments of the present disclosure further comprise the step of isolating and purifying the produced antibody or antibody fragment from the host cells or medium.
  • the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
  • the antibody or antibody fragment of the present disclosure can then be purified by a number of techniques as known to the person skilled in the art. In an embodiment, the present disclosure refers to a method of producing a humanized antibody or antibody fragment specific for human CD45 of any of the antibodies disclosed in Table 14.
  • a method of producing an isolated antibody or antibody fragment according to the present disclosure comprises culturing a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment according to the present disclosure, under conditions suitable for expression of the antibody or antibody fragment, and isolating the antibody or antibody fragment from the host cell or host cell culture medium.
  • An antibody or antibody fragment isolated as described herein may be purified techniques know in the art, such as high performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • HPLC high performance liquid chromatography
  • ion exchange chromatography gel electrophoresis
  • affinity chromatography size exclusion chromatography
  • the conditions used to purify a particular antibody or antibody fragment will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art.
  • affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the antibody or antibody fragment binds.
  • a matrix with protein A or protein G may be used for affinity chromatography purification of antibody or antibody fragment according to the present disclosure.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45. In an embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45 disclosed in Table 14. In an embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for a polypeptide encoded by the amino acid sequence of SEQ ID NO: 1. In an embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment cross-competes with antibody ⁇ tti for binding to CD45.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment cross-competes with an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9 for binding to CD45.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment binds to the same epitope on CD45 as antibody ⁇ tti.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment binds to the same epitope on CD45 as an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment retains the binding specificity of antibody ⁇ tti.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment retains the binding specificity of an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment binds to CD45 with about the same affinity as antibody ⁇ tti.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment binds to CD45 with about the same affinity as an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment binds to CD45 with a higher affinity than antibody ⁇ tti.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment binds to CD45 with a higher affinity than an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment binds to CD45 with an at least two-fold higher affinity than an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9, and wherein the humanized antibody or antibody fragment comprises at least one of the following mutations: a) the first asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by arginine, or b) the arginine in the LCDR2 region (SEQ ID NO: 8) is replaced by leucine.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment has a lower number of critical deamidation sequence motifs than antibody ⁇ tti.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment has a lower number of critical deamidation sequence motifs than an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment has a lower number of critical sequence motifs for T cell epitopes than antibody ⁇ tti.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment has a lower number of critical sequence motifs for T cell epitopes than an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment is functionally equivalent to antibody ⁇ tti.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment is functionally equivalent to an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment inhibits the biological function of CD45 to at least the same degree as antibody ⁇ tti.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment inhibits the biological function of CD45 to at least to the same degree as an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable heavy chain comprising, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9.
  • the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein said antibody or antibody fragment is coupled to a toxin and thereby inhibits proliferation of CD45-positive cells.
  • Effector function The Fc region of an immunoglobulin generally confers to the favorable pharmacokinetic properties of antibodies, such as prolonged half-life in serum and to the ability to induce effector function via binding to Fc receptors expressed on cells.
  • binding to Fc receptors might also results in an undesirable activation of certain cell surface receptors leading to unwanted cytokine release and severe side effects upon systemic administration.
  • the wild-type Fc region of an antibody such as of an wild-type IgG Fc region to one or more or all of Fc receptors and/or binding to a complement component, such as C1q in order to reduce or abolish the ability of the antibody to induce effector function.
  • a complement component such as C1q
  • Effector function can include, but is not limited to, one or more of the following: complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen- presenting cells, binding to NK cells, binding to macrophages, binding to monocytes, binding to polymorphonuclear cells, direct signaling inducing apoptosis, crosslinking of target-bound antibodies, dendritic cell maturation, or T cell priming.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • cytokine secretion immune complex-mediated antigen uptake by antigen- presenting cells
  • NK cells binding to macrophages
  • monocytes binding to monocytes
  • polymorphonuclear cells binding to polymorphonuclear cells
  • a reduced or abolished binding of an Fc region to an Fc receptor and/or to C1q is typically achieved by mutating a wild-type Fc region, such as of an lgG1 Fc region, more particular a human lgG1 Fc region, resulting in a variant or engineered Fc region of said wild-type Fc region, e.g., a variant human lgG1 Fc region.
  • a wild-type Fc region such as of an lgG1 Fc region, more particular a human lgG1 Fc region
  • substitutions that result in reduced binding can be useful.
  • non-conservative amino acid substitutions i.e., replacing one amino acid with another amino acid having different structural and/or chemical properties, are preferred.
  • the isolated antibody or antibody fragment specific for human CD45 according to the present disclosure comprises a variant Fc region having a reduced or abolished binding to an Fc receptor and/or to C1q when compared to the wild-type Fc region.
  • the isolated antibody or antibody fragment according to the present disclosure comprises a variant Fc region that reduces or abolishes the ability of the antibody to induce effector function.
  • the isolated antibody or antibody fragment according to the present disclosure does not substantially induce effector function.
  • the effector function is one or more selected from the group consisting of CDC, ADCC and ADCP.
  • the effector function is ADCC.
  • the effector function is CDC.
  • the effector function is ADCP.
  • the isolated antibody or antibody fragment according to the present disclosure does not substantially induce ADCC and/or CDC and/or ADCP.
  • the isolated antibody or antibody fragment according to the present disclosure does not induce ADCC or ADCP in vitro.
  • the variant Fc region of the isolated antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions that reduce or abolish the binding of the variant Fc region to one or more Fc receptors and/or to C1 q when compared to the wild-type Fc region.
  • the variant Fc region of the isolated antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions that reduce or abolish the ability of the antibody to induce effector function when compared to the wild-type Fc region.
  • the one or more amino acid substitutions may reduce the binding affinity of the variant Fc region for one or more Fc receptors and/or to C1q by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold or even at least 50-fold when compared to the wild-type Fc region.
  • the one or more amino acid substitutions may reduce the ability of the isolated antibody or antibody fragment according to the present disclosure to induce effector function by at least 2-fold, at least 5-fold, at least 10- fold, at least 20-fold or even at least 50-fold when compared to the wild-type Fc region.
  • the variant Fc region of the isolated antibody or antibody fragment according to the present disclosure does not substantially bind to one or more Fc receptors and/or C1q.
  • the variant Fc region of the antibody according to the present disclosure does substantially abolish the ability of said antibody to induce effector function.
  • the antibody or antibody fragment according to the present disclosure does not substantially induce effector function.
  • said effect function is ADCC and/or ADCP and/or CDC.
  • the antibody or antibody fragment according to the present disclosure does not substantially induce effector function meaning that the level of induced effector function is not significantly above the background as measured in the absence of said antibody.
  • the Fc receptor is a human Fc receptor.
  • the Fc receptor is an Fcy receptor.
  • the Fc receptor is a human FcyRIIIa, FcyRI, FcyRIla and/or FcyRIlb.
  • the isolated antibody or antibody fragment according to the present disclosure comprises a variant human lgG1 Fc region, which comprises one or more amino acid substitutions compared to the wild-type human lgG1 Fc region.
  • one or more amino acid substitutions reduce or abolish the binding of the variant Fc region to an Fc receptor and/or to C1q and/or reduces the ability of said antibody to induce effector function when compared to the wild-type Fc region.
  • Fc modifications are commonly used. For a review see for example Antibodies (2020) 9: 64.
  • Silencing functions include (numbering according EU index) the LALA (L234A/L235A), the PA-LALA (L234A/L235A/P329A) and the PG-LALA (L234A/L235A/P329G) mutations, as well as the AEASS mutations (L234A/L235E/G237A/A330S/P331S).
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies have a silenced Fc region.
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies or antibody fragments are in the AEASS format.
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a Fc region in which at least the following modifications are present: L234A, L235E, G237A, A330S, P331S (numbering according EU index).
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a Fc region in which the following modifications are present: L234A, L235E, G237A, A330S, P331S (numbering according EU index).
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies or antibody fragments are in the PA-LALA format.
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a Fc region in which at least the following modifications are present: L234A, L235A, P329A (numbering according EU index).
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a Fc region in which the following modifications are present: L234A, L235A, P329A (numbering according EU index).
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies or antibody fragments are in the PG-LALA format.
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a Fc region in which at least the following modifications are present: L234A, L235A, P329G (numbering according EU index).
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a Fc region in which the following modifications are present: L234A, L235A, P329G (numbering according EU index).
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies or antibody fragments have mutations that leads to a reduced binding to FcRn, thereby decreasing the in vivo half-life of the antibody.
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a Fc region with a I253A mutation (numbering according EU index). In certain embodiments, the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a Fc region with a H310A mutation (numbering according EU index). In certain embodiments, the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a Fc region with a H435A mutation (numbering according EU index).
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a Fc region with a H435Q mutation (numbering according EU index).
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies or antibody fragments have mutations that leads to a increased binding to FcRn, thereby increasing the in vivo half-life of the antibody.
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a T250Q/M428L mutation in the Fc region (numbering according EU index).
  • the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a M252Y/S254T/T256E (YTE) mutation in the Fc region (numbering according EU index). In certain embodiments, the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a H433K/N434F mutation in the Fc region (numbering according EU index). In certain embodiments, the present disclosure relates to humanized antibodies that are specific for CD45, wherein said humanized antibodies comprise a M252Y/S254T/T256E/H433K/N434F mutation in the Fc region (numbering according EU index).
  • the present disclosure relates to a humanized antibody, wherein said humanized antibody comprises a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 63, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6 and a variable light chain comprising, the LCDR1 region of SEQ ID NO: 65, the LCDR2 region of SEQ ID NO: 8 and the LCDR3 region of SEQ ID NO: 9; c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2
  • the Fc region of said humanized antibodies comprises the modifications L234A, L235A and P329A (numbering according EU index).
  • Therapeutic methods can be used for the treatment of various conditions.
  • such antibodies can be used in ablation of endogenous hematopoietic stem and progenitor cells (HSPCs) in a subject in need thereof.
  • Ablation of endogenous HSPCs is an initial step in stem cell replacement therapy.
  • Stem cell replacement therapy generally involves reducing or eliminating endogenous HSPCs, which are defective in some respect, and replacing them with replacement HSPCs.
  • the replacement HSPCs can be autologous, allogenic or xenogenic.
  • Endogenous HSPCs may be defective as a result of hereditary mutation impairing function or expression (e.g., sickle cell anemia or thalassemia), as a result of a hematologic cancer, or as a result of damage from chemotherapy used in treating a cancer.
  • Endogenous HSPCs may also be replaced in conjunction with an organ transplant because the endogenous HSPCs would result in immune attack of the transplant.
  • Antibodies against CD45 can also be used in treatment of cancers expressing CD45.
  • Such cancers include hematological cancer, such as a leukemia, myeloma or lymphoma.
  • Antibodies against CD45 can also be used in treatment of an inflammatory or autoimmune disease such as multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn’s disease, Type 1 diabetes, rheumatoid arthritis or idiopathic arthritis.
  • an inflammatory or autoimmune disease such as multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn’s disease, Type 1 diabetes, rheumatoid arthritis or idiopathic arthritis.
  • the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in medicine.
  • the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of cancer.
  • the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of a hematological cancer.
  • the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of leukemia, myeloma or lymphoma. In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of an inflammatory or autoimmune disease. In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn’s disease, Type 1 diabetes, rheumatoid arthritis or idiopathic arthritis.
  • the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of a disease associated with the undesired presence of CD45. In certain embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of a disease associated with the undesired presence of CD45-positive cells. In an embodiment, the present disclosure provides a method for the treatment of a disease. In an embodiment, the present disclosure provides a method for the treatment of a disease comprising administering to a patient an antibody or antibody fragment of the present disclosure. In an embodiment, the present disclosure provides a method for the treatment of a disease comprising administering to a subject in need there of an antibody or antibody fragment of the present disclosure.
  • the present disclosure provides a method for the prevention of a disease. In an embodiment, the present disclosure provides a method for the prevention of a disease comprising administering to a subject an antibody or antibody fragment of the present disclosure. In an embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for the treatment of a disease. In an embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use in the treatment of a disease. In an embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use in the treatment of a disease in a subject in need thereof. In an embodiment, the present disclosure provides the use of an isolated antibody or antibody fragment according to the present disclosure for the manufacture of a medicament.
  • the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use as a medicament. In an embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use in medicine. In an embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use as a medicament for the treatment of a subject in need thereof. In an embodiment, the present disclosure provides an isolated antibody or antibody fragment specific for human CD45 according to the present disclosure for use in a method of treating a subject having a disease comprising administering to the subject a therapeutically effective amount of an antibody or antibody fragment according to the present disclosure. In an embodiment, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent.
  • the subject in need of treatment is typically a mammal, more specifically a human.
  • an isolated antibody or antibody fragment according to the present disclosure would be formulated, dosed, and administered in a way consistent with good medical practice.
  • Pharmaceutical compositions In an embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated antibody or antibody fragment according to the present disclosure and a pharmaceutically acceptable carrier or excipient.
  • Antibodies are administered in an effective regime meaning a dosage, route of administration and frequency of administration that achieves the intended purpose, such as reduction of endogenous HSPCs, tissue resident or circulating immune cells or of cancer cells expressing CD45. In some instances, efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient.
  • the pharmaceutical compositions may further comprise at least one other pharmaceutically active compound.
  • the pharmaceutical composition according to the present disclosure can be used in the diagnosis, prevention and/or treatment of diseases associated with the undesired presence of CD45, in particular human CD45.
  • the pharmaceutical composition according to the present disclosure can be used in the diagnosis, prevention and/or treatment of diseases associated with the undesired presence of CD45-positive cells, in particular CD45-positive human cells.
  • the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure that is suitable for prophylactic, therapeutic and/or diagnostic use in a mammal, more particular in a human.
  • an antibody or antibody fragment according to the present disclosure may be formulated as a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure and at least one pharmaceutically acceptable carrier or excipient, and optionally one or more further pharmaceutically active compounds.
  • a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure may be administered parenterally, such as intravenously, or intramuscularly, or subcutaneously.
  • an antibody of the invention may be administered via a non-parenteral route, such as per-orally or topically.
  • a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure is administered intravenously or subcutaneously.
  • an antibody or antibody fragment according to the present disclosure may be used in combination with one or more pharmaceutically active compounds that are or can be used for the prevention and/or treatment of the diseases in which a target antigen of interest is involved, as a result of which a synergistic effect may or may not be obtained. Examples of such compounds, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician.
  • the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use in the prevention and/or treatment of a disease associated with the undesired presence of CD45, in particular human CD45.
  • the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use in the prevention and/or treatment of a disease associated with the undesired presence of CD45- positive cells, in particular CD45-positive human cells.
  • the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for the use as a medicament.
  • the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use in the prevention and/or treatment of an autoimmune disease and/or inflammatory disease and/or cancer.
  • the present disclosure provides a method for the treatment of an autoimmune disease and/or inflammatory disease and/or cancer in a subject in need thereof using a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure.
  • a method of producing an antibody or antibody fragment according to the present disclosure in a form suitable for administration in vivo comprising (a) obtaining an antibody or antibody fragment by a method according to the present disclosure, and (b) formulating said antibody or antibody fragment with at least one pharmaceutically acceptable carrier or excipient, whereby a preparation of antibody or antibody fragment is formulated for administration in vivo.
  • compositions according to the present disclosure comprise a therapeutically effective amount of one or more antibodies or antibody fragments according to the present disclosure dissolved in a pharmaceutically acceptable carrier or excipient.
  • Diagnostic use In an embodiment, the present disclosure provides the use of an isolated antibody or antibody fragment specific for human CD45 according to the present disclosure for the diagnosis of a disease. In an embodiment, the present disclosure provides the use of an antibody or antibody fragment according to the present disclosure for the detection of CD45, in particular human CD45. In an embodiment, the present disclosure provides a method for detecting CD145 in a subject or a sample, comprising the step of contacting said subject or sample with an isolated antibody or antibody fragment specific for human CD45 of the present disclosure.
  • the present disclosure provides a method for diagnosing a disease in a subject, comprising the step of contacting said subject or sample with an isolated antibody or antibody fragment according to the present disclosure.
  • the antibodies may also be used to determine CD45 expression levels in cells from patients.
  • the CD45 expressions levels may serve as therapeutic biomarkers, for example for patient stratification.
  • Antibody ⁇ tti is a mouse anti-CD45 antibody.
  • Example 2 Humanization of murine antibody ⁇ tti
  • the humanization process relied on a combination of CDR-grafting technologies coupled with the latest research on antibody structure and up-date database of mature human lgG sequences.
  • a number of human framework sequences were identified that were used as “acceptor” frameworks for the target CDR sequences. All acceptor sequences come from mature human IgG.
  • the humanized sequences are expected not to be immunogenic and retain the canonical structure of the CDR loops.
  • Example 2.1 Design of humanized variants of the variable heavy chain
  • the closest human germline gene V-region that was identified in Homo sapiens was IGHV3-23: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO.15).
  • Databases of human lgG sequences were searched for comparison to the murine VH domain using BLAST search algorithms, and candidate human variable domains were selected from the top 200 BLAST results. These were reduced to four candidates based on a combination of framework homology, maintaining key framework residues and canonical loop structure.
  • the four acceptor frameworks are: >QDF60926 EVQLVESGGGLVQPGGSLRLSCAASGFTLSNYDMNWVRQAPGKRLEWVSYISSSGSTTYYVDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGHNNWFLYFDSWGQGTLVTVSS (SEQ ID NO.16) >ABF83355 QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARDGSWYFDLWGRGTLVTVSS (SEQ ID NO.17) >QAV55037 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMSWVRQAPGKGLEWVSYISSSANTIYYADSVKGR FTISRDNAKKSLFLQMNSLRAEDTAVYYCARVVLGYWYFDLWGRGTLVTVSS (
  • the four acceptor frameworks are: >QEP26792 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSG SGAGTDFTLKISRVEAEDVGVYYCMQATHFPPYTFGQGTKLEIK (SEQ ID NO.26) >BAH04700 EIVLTQSPLSLPVTLGQPASISCRPSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPWTFGQGTKVEIK (SEQ ID NO.27) >ABA26062 DIVMTQTPLSLPVTLGQPASISCRSSQSLLYSDGNTYLNWFHQRPGQSPRRLIYRVSNRDSGIPDRFSG SGSGTDFTLKISRVEAE
  • FIG. 2 An alignment of the humanized variants to the original murine ⁇ tti sequence is shown in Figure 2.
  • the homology of the humanized variants to original murine VL sequence is shown in the following table.
  • Table 3 Identical amino acids Consensus amino acids VL1 87.6% 93.8% VL2 89.4% 96.5% VL3 88.5% 96.5% VL4 90.3% 97.3% VL5 92.0% 97.3%
  • Example 2.3 Expression and purification of the humanized variants DNA coding for the amino acid sequence of each antibody was synthesized and cloned into the mammalian transient expression plasmid pETE V2 (Fusion Antibodies, Northern Ireland)).
  • Antibodies were expressed using a CHO based transient expression system and the resulting antibody containing cell culture supernatants were clarified by centrifugation and filtration. Antibodies were purified using a state-of-the-art chromatography equipment from cell culture supernatants via affinity chromatography. Purified antibodies were buffer exchanged into phosphate buffered saline solution. The purity of these antibodies was determined to be >95%, as judged by reducing and denaturing Sodium Dodecyl Sulfate Polyacrylamide gels. Antibody concentration was determined by measuring absorbance at 280 nm. The five humanized variable heavy chains were tested in combination with five of the humanized variable light chains, leading to a total of twenty-five (25) combinations.
  • Example 2.4 Affinity determination of the humanized variants lgG antibodies were immobilized on biosensors using suitable capture surfaces and binding of soluble antigen to immobilized antibodies was monitored by BLI (Octet). The resulting sensorgrams were analyzed using the supplied software (Fortebio). As antigen human recombinant CD45 from R&D Systems was used (Catalog No.1430-CD-050; Lot# NUZ142001).
  • lgG-captured sensors were dipped into wells containing only buffer and blank sensors were also dipped into wells containing the antigen.
  • This referencing provided a means of compensating for both the natural dissociation of the capture lgG and non-specific binding of the antigen to the sensor surface. Steps were performed at 25°C at a constant shaking of 1000 rpm. New sensors were used for each sample. Dissociation rate constants (KD) were calculated using the ForteBio Data Analysis software. All consumables used were those recommended by ForteBio. All samples were diluted in freshly prepared running buffer. Antibody variants were immobilized onto the surface of a series of biosensors using the capture methods described.
  • Antigen was passed over the surface to generate a binding response. Binding data for the IgG:antigen interactions were collected at 25°C on the biosensors. A dilution series of the antigen was used in the association step, in order to fit results globally and get the best values for ka, kd, and KD. The response data for the binding of antigen to the surface immobilized lgG were fitted to a 1:1 binding model. Kinetic parameters are summarized in the table below. HC0 LC0 refers to murine ⁇ tti. Table 5: NF: does not fit to 1:1 binding model Five of the antibodies showed a dissociation constant KD that is within the range of that of the original antibody ⁇ tti (HC0 LC0).
  • conformational stability is indicative of a protein’s propensity for non-native aggregation.
  • Thermal stability is accepted as being a viable method to monitor a protein’s conformational stability, through the determination of its melting point (Tm).
  • Tm melting point
  • DSF Differential scanning fluorimetry
  • DSC differential scanning calorimetry
  • DSF uses a fluorescent dye, which is quenched in aqueous environments, but fluoresces under hydrophobic conditions.
  • Fab melting transition is generally well defined with a peak up to 2-3 times greater than that of CH2 or CH3.
  • solutions of 5 ⁇ l of Sypro Orange (diluted 1/200 in water; Sigma) and 45 ul of 0.3 mg/ml of the antibody to be tested were added to a tube (Bio-Rad; TLS0831).
  • the tubes were sealed with optical flat cap (Bio-Rad; TCS0803) and heated in an i-Cycler iQ5 real-time PCR detection system (Bio-Rad) from 20 to 95 °C in increments of 0.5°C. Fluorescence changes in the wells of the plate were monitored simultaneously with a charge-coupled (CCD) camera.
  • CCD charge-coupled
  • the wavelengths for excitation and emission were 485 and 575 nm, respectively.
  • the temperature midpoint for the protein unfolding transition, Tm was calculated using the Bio-Rad iQ5 software.
  • SEC size exclusion chromatography
  • samples were diluted to a final concentration of 0.1 mg/ml using phosphate buffered saline (PBS).
  • Highly purified samples of antibodies were loaded independently onto a Superdex 200 increase 10/300 GL gel-filtration column.50 ⁇ l samples were injected and the column flow rate was maintained at 0.75 ml/min. Separations and equilibration steps were performed in phosphate buffered saline at 19°C.
  • Protein peaks were monitored using absorbance at 214 nm and spectra were analyzed using the Unicorn evaluation software package (Cytiva). Melting curves were successfully generated for all samples. All antibodies tested display single-phase transition melting profiles. VH2/VL5 exhibited the highest TM1 melting temperature, indicating that this IgG is the most conformation stable of the molecules tested. Tm values are summarized in the table below. Also SEC analysis has been performed successfully for all samples. The peak quality for SEC analysis was good for all samples. All antibodies eluted as one major peak. From the column calibration, the major peak corresponds to monomeric lgG.
  • Example 3 Removal of critical sites that are prone to PTM, immunogenicity, increased flexibility or hydrophobicity
  • Example 3.1 Design of the engineered, humanized variants In the next engineering step, antibodies were screened for sites that are prone to Fv glycosylation, deamidation, isomerization, fragmentation, other kinds of post-translational modification (PTM) sequence hot spots, increased flexibility or hydrophobicity. Several such sites were identified in the CDRs of the antibodies. See the following table.
  • Table 7 Motif Type Occurrence CDR NG deamidation RSSQSIVHSNGNTYLE (SEQ ID No.35) LCDR1 N T deamidation RSSQSIVHSNGNTYLE (SEQ ID No.35) LCDR1 RF T-cell epitope LLIYKVSNRFSGVPD (SEQ ID No.36) LCDR2 GGG flexible site FGGGTKLEIK (SEQ ID No.37) LC framework WWY Hydrophobic HCDR3 site To remove these detrimental amino acid motifs, variants of antibody VH2/VL5 were generated. The approach for the respective changes were rationalized by analyses as described in the following.
  • Sequence and structure-based descriptors were computed in silico to identify suitable variants that would preserve the structural integrity and bioactivity of the antibody while enhancing developability, binding affinity, solubility.
  • physicochemical descriptors were assessed against a dataset of therapeutic antibodies retrieved from the TABS database (https://tabs.craic.com/) and a second dataset consisting of paired antibody repertoires from the Observed Antibody Space database (http://opig.stats.ox.ac.uk/webapps/oas/).
  • HMMs Hidden Markov Models
  • Three-dimensional models were built based on methods described in Nucleic Acid Res (2017) 45: W17-W23 and Bioinformatics (2014) 30: 2733-40, using a template-based approach for the frameworks, canonical structure modelling for the CDRs and a random-forest machine learning modelling approach for the heavy chain loop H3.
  • a template based approach based on global similarity and identity at residue position L44 as described in FEBS J. (2011) 278: 2858-66 was used.
  • the antibody solubility profile was computed as a linear combination of different physicochemical properties such as hydrophobicity, electrostatic charge at neutral pH, ⁇ -helix and ⁇ -strand propensity of each amino acid (Nat Struct Biol (1996) 3: 842-8, J Mol Biol (1994) 238: 693-708).
  • we employed a sliding window approach where the per-residue solubility descriptor is averaged over a window of seven consecutive amino acids and corrected for the occurrence of polar/non-polar aggregation prone patterns J Mol Biol (2000) 296: 961-8; Methods Mol Biol (2022) 2313: 57-113.
  • Binding affinities were determined using an Octet system Red96e or R8 at 25 °C and 1000 rpm using 1x kinetic buffer (Sartorius, PN: 18-1105).
  • the antibodies were captured on antihuman capturing biosensors (Sartorius, PN_ 18-5060) at 0.5-1 ug/mL for 300-600 s.
  • the antigen were titrated at different concentrations ranging from 200 nM to 0.5 nM and association/dissociation were monitored for 300-600 s and 1000-2000 s. Reference subtraction was performed against buffer only wells. AHC tips were regenerated using 10 mM Gly-HCl pH 1.7.
  • Example 4 Removal of multiple sites that are prone to PTM or fragmentation
  • Example 4.1 Design of the further engineered, humanized variants Encouraged by the successful removal of detrimental sequences motifs from the CDRs of antibody ⁇ tti, the inventors next investigated if it would even be possible to remove multiple such motifs without losing affinity or functionality of the binders. The following variants were generated.
  • Table 12 LCDR1 LCDR2 LCDR3 VL Kabat IMGT Kabat IMGT Kabat IMGT V L5.24 98 75 76 96 14 9 9 VL5.25 99 106 107 8 14 9 9 V L5.26 100 108 109 8 14 9 9 V L5.27 101 108 109 96 14 9 9 V L5.28 102 110 111 96 14 9 9 V L5.29 103 106 107 96 14 9 9 V L5.30 104 106 107 93 14 9 9 V L5.31 105 108 109 93 14 9 9 SEQ ID No.106: RSSQSIVHSNAKTYLE SEQ ID No.107: RSSQSIVHSRGQTYLE SEQ ID No.109: QSIVHSRGQTY SEQ ID No.110: RSSQSIVHSRGKTYLE SEQ ID No.111: QSIVHSRGKTY Example 4.2: Testing of the further engineered, humanized variants The engineered variant were subjected
  • Example 5 Summary of the sequences of the antibodies disclosed in the present patent The following table summarizes the key antibodies produced and tested in the present disclosure. Indicated are the VH and the VL sequences of the antibodies. The respective VH sequences, the VL sequences, and the CDR sequences can be found in the preceding Examples.
  • MPA Membrane Proteome Array
  • the optimal concentration of the antibody to be tested for screening was determined on cells expressing positive (membrane-tethered Protein A) and negative (mock-transfected) binding controls, followed by detection by flow cytometry using a fluorescently-labeled secondary antibody (Jackson ImmunoResearch, Cat#109-606-008).
  • the optimal screening concentration of antibody Kiebitz was determined to be 20 ⁇ g/ml. Kiebitz was added to the MPA at 20 ⁇ g/mL, and binding across the protein library was measured on an Intellicyt iQue using a fluorescently labeled secondary antibody (see above).
  • Each array plate contains both positive (Fc-binding) and negative (empty vector) controls to ensure plate- by-plate reproducibility.
  • Interactions of antibody Kiebitz above background was observed for five targets. Binding to these five targets (NOX5, CPOX, FRFL2, PHKG1 and LYSMD4) was evaluated in more details in a second flow cytometry experiment using serial dilutions of Kiebitz antibody (0.3, 1.3, 5, and 20 ⁇ g/mL). Binding to the five antigens identified in the large scale MPA assay could not be confirmed. Binding was less than 2-fold above background levels on the two highest concentrations tested The Kiebitz antibody is highly specific for CD45.
  • Example 7 Depletion of CD45+ cells and LT-HSCs in humanized mice NSG mice were humanized by transplantation of human cord blood derived CD34+ HSPCs by The Jackson Laboratory.18 weeks after HSPC injection humanized mice were i.v. injected with Kiebitz-Teserine or BC8-Teserine conjugates at a single dose of 0.3mg/kg. Control animals were treated with phosphate-buffered saline (PBS) solution. 21 days after treatment the mice were euthanized and blood, spleen and bone marrow were analysed by FACS. The VH, the VL and the CDR sequences of antibody BC8 according to Kabat are shown in the following table.
  • PBS phosphate-buffered saline
  • LT-HSCs were gated as live/hCD45+/ CD34+/CD38- /CD90+/CD45RA-. Results show a complete depletion of all human CD45+ cells and LT-HSCs 3 weeks after a single dose of 0.3 mg/kg with the ADC Kiebitz-Teserine compared to PBS control group, whereas BC8-Teserine treatment resulted only in partial depletion.
  • Example 8 Blocking with a naked antibody mimics shielding of cells and is able to prevent a decrease of cell viability of cell lines by subsequent treatment with an ADC
  • various human cell lines were with a Teserine conjugate of selected naked anti-CD45 antibodies.
  • MV4-11 cells were seeded at a concentration of 75000 cells/mL in 135ul of medium (IMDM supplemented with GlutaMAX + 10% heat inactivated FBS) in a 96-well plate.
  • Kiebitz or Ref043 Some wells were pre-treated with 50ug/mL naked antibody (Kiebitz or Ref043) or PBS as a control for 1h, before adding various concentrations of Kiebitz-Tesrine or BC08-Tesirine (starting concentration for naked antibody pre-treated wells was 10 ug/ml and, starting concentrations for PBS pre- treated wells was 1 ug/ml for both cell lines) for 72h.
  • Cell viability measurement was performed by adding 50ul CTG2.0 (CellTiter-Glo® 2.0 Cell Viability Assay #G9241, Promega) per well, incubating plates 10 minutes at room temperature on a shaker and reading luminescence (EnVision, Perkin Elmer).
  • Molm-13 cells were analyzed according to the same protocol as described. Cells were seeded at a concentration of 75000 cells/mL in 135ul of medium (RPMI1640 supplemented with GlutaMAX + 20% heat inactivated FBS) in a 96-well plate. Starting concentration for naked antibody pre-treatment was 3 ug/ml. Results are shown in Figure 4. It can be seen that in both cell lines tested blocking with naked antibodies mimics shielding of the cells and is able to prevent cell viability decrease via the respective ADCs. BC08-Tesirine shows lower potency than Kiebitz-Tesirine. Example 9: Blocking with a naked antibody can also be observed with HSPCs The experiment of Example 8 was repeated with HSPCs.
  • CD34-positive HSPC’s were isolated from peripheral blood (CliniMACS Prodigy, Miltenyi Biotech). Cells were seeded at a concentration of 75000 cells/ml in 135ul of either low or high cytokine medium in a 96-well plate.
  • Low cytokine medium consists of StemPro media (Gibco), StemPro nutrients (Gibco), 50 ng/ml LDL (STEMcell Technologies), 1% Penicillin/Streptomycin (Thermo Fisher), 1% glutamine (Thermo Fisher), 20 ng/ml Flt3 (Miltenyi Biotec), 50 ng/ml TPO (Milteny Biotec ) and 100 ng/ml SCF (Milteny Biotec).
  • High cytokine medium consists of the same ingredients plus 50 ng/ml IL6 (Milteny Biotec), 10 ng/ml IL3 (Milteny Biotec), 10 ng/ml IL2 (Milteny Biotec), 20 ng/ml IL7 (Milteny Biotec), 50 ng/ml IL11 (Milteny Biotec), 3 ng/ml EPO (STEMcell Technologies) and 20 ng/ml GM- CSF (Milteny Biotec).
  • Kiebitz-Tesrine starting concentration for naked antibody pre-treated wells was 10 ug/ml and, starting concentrations for PBS pre-treated wells was 1 ug/ml for both cell lines
  • Cell viability measurement was performed by adding 50ul CTG2.0 (CellTiter-Glo® 2.0 Cell Viability Assay #G9241, Promega) per well, incubating plates 10 minutes at room temperature on a shaker and reading luminescence (EnVision, Perkin Elmer). Results are shown in Figure 5.
  • Jurkat or Jurkat cells engineered for CD45 KO were seeded at a concentration of 75,000 cells/mL in 135ul of medium (RPMI1640 (ATCC modification) + 10% heat inactivated FBS) in a 96-well plate. Various concentrations of Kiebitz-Tesrine were added. Starting concentration was 100 ug/ml. Cells were incubated for 72h. Cell viability measurement was performed by adding 50ul CTG2.0 (CellTiter- Glo® 2.0 Cell Viability Assay #G9241, Promega) per well, incubating plates 10 minutes at room temperature on a shaker and reading luminescence (EnVision, Perkin Elmer). Results are shown in Figure 6.
  • CTG2.0 CellTiter- Glo® 2.0 Cell Viability Assay #G9241, Promega
  • Jurkat CD45 knock-out cells have an IC50 for Kiebitz-Tesrine which is significantly higher than the IC50 in Jurkat wild-type cells (0.46 ⁇ g/ml vs.0.032 ⁇ g/ml).
  • Example 11 Antibodies bind to CD45 endogenously expressed on cells KASUMI-1 cells (ATCC No. CRL-2724) were stained with different concentrations of antibody ⁇ tti or the humanized derivatives Amandine or Ballerina, using an IgG Goat anti-Human AF488 secondary antibody. Cells stained with a human IgG1 isotype control were used to determine background staining. Results are shown in Figure 7. All three antibodies tested bind equally well to CD45 endogenously expressed on Jurkat cells.
  • Such antibodies are particular useful for certain purposes, for example the use in CARs or on LNPs. Certain additional antibodies were generated that all could be well expressed as scFv-Fc’s. These antibodies are shown in Table 16. Table 16: Lady Rosetta SEQ ID Comment Sequence No.

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Abstract

La présente invention concerne des anticorps et des fragments d'anticorps qui sont spécifiques de CD45. Les anticorps sont des versions humanisées améliorées d'un anticorps murin. En plus d'une campagne d'humanisation sophistiquée, les anticorps ont également été modifiés pour éliminer plusieurs motifs nuisibles à l'intérieur de la région CDR sans perte de quelconques propriétés bénéfiques. Les anticorps sont utiles pour le traitement de maladies associées à CD45.
PCT/EP2023/087612 2022-12-23 2023-12-22 Anticorps ciblant cd45 Ceased WO2024133890A1 (fr)

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CN202380088284.1A CN120476145A (zh) 2022-12-23 2023-12-22 靶向cd45的抗体
IL321599A IL321599A (en) 2022-12-23 2023-12-22 Antibodies targeting cd45
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