CN116472291A

CN116472291A - Multimerized CD45 binding molecules

Info

Publication number: CN116472291A
Application number: CN202180070222.9A
Authority: CN
Inventors: S·E·拉佩基; R·亚当斯; D·P·哈姆费雷斯; H·M·芬尼; R·F·比瑟尔
Original assignee: UCB Biopharma SRL
Current assignee: UCB Biopharma SRL
Priority date: 2020-10-15
Filing date: 2021-10-14
Publication date: 2023-07-21
Also published as: GB202100737D0

Abstract

The present invention provides one or more binding molecules capable of multimerizing CD45 to induce cell death of cells expressing CD45 without inducing significant cytokine release. For example, the invention provides antibodies against CD45, wherein the antibodies comprise at least two different paratopes each specific for a different epitope of CD45. The antibodies can be used to crosslink CD45 on the cell surface. The antibodies can be used in a variety of therapeutic methods, including depletion of cells, e.g., prior to cell transplantation.

Description

Multimerized CD45 binding molecules

Technical Field

The present invention relates to binding molecules, in particular antibodies, specific for CD45. The binding molecules may, for example, be used to kill target cells, particularly prior to transplantation of the cells.

Background

CD45 is the first prototype receptor-like protein tyrosine phosphatase that is expressed on nucleated hematopoietic cells and plays a central role in regulating cellular responses. CD45 is also known as PTPRC, T200, ly5, leukocyte Common Antigen (LCA) and B220.CD45 is the most abundant cell surface protein in T and B cells. It is critical for B and T cell development and activation. Studies on CD45 mutant cell lines, CD45 deficient mice and CD45 deficient humans initially demonstrated the necessary role of CD45 in T and B cell antigen receptor signaling and lymphocyte development. CD45 is now known to also modulate signals emanating from integrin and cytokine receptors. In contrast to its positive role in antigen receptor signaling, CD45 acts as a negative regulator in integrin-mediated signaling (e.g., in macrophages). CD45 may also play a role in regulating hematopoietic and interferon-dependent antiviral responses. CD45 may also play a role in cell survival.

CD45 comprises a highly variable glycosylated extracellular domain of about 400 to 550 amino acids, followed by a single transmembrane domain and a long intracellular domain of 705 amino acids comprising two tandem repeat phosphatase domains. Modulation of CD45 expression and expression of multiple alternatively spliced isoforms (which alternatively splice exons 4, 5 and 6 of the CD45 gene, and are designated A, B and C) largely modulated phosphatase activity and differential signaling. CD45 affects cellular responses by controlling the relative sensitivity threshold to external stimuli. This disturbance of function may lead to autoimmunity, immunodeficiency and malignancy.

All CD45 isoforms exhibit tyrosine phosphatase activity mediated by the cytoplasmic domain of the molecule comprising two tandem repeats of phosphatase domains D1 and D2, each tandem repeat comprising highly conserved HC (X) ₅ R motif. All tyrosine phosphatase activities of CD45 are thought to originate in the D1 domain, whereas the D2 domain may be involved in regulation. One of the major targets for CD45 tyrosine phosphatases is Src family kinase, reflecting the role of CD45 in cell signaling. Depending on the site of action of CD45 phosphatase activity, it may activate or down-regulate the activity of these Src family kinases.

Given the importance of CD45, there is a continuing need for agents that can target and modulate CD 45.

Disclosure of Invention

The invention provides, inter alia, binding molecules capable of multimerizing CD45 on target cells to induce cell death without inducing significant cytokine release. Without wishing to be bound by this theory, it is believed that the binding molecules of the invention are more capable of cross-linking CD45 molecules than known binding molecules and thus have an improved ability to induce cell death in target cells. The binding molecules of the invention may thus be used to kill target cells, particularly prior to transplanting the cells into a subject. In certain embodiments, binding molecules are provided. In other embodiments, a mixture of at least two different binding molecules is provided.

As discussed in detail herein, the binding molecules of the invention are provided in a variety of forms. In a particularly preferred embodiment, the binding molecule of the invention is an antibody. In another particularly preferred embodiment, the mixture of at least two different binding molecules of the invention is a mixture of at least two different antibodies. Antibody formats that may be used in various embodiments of the invention are discussed in detail herein. In a preferred embodiment, the antibody is an IgG antibody. In one embodiment, the IgG antibody is an IgG1, igG2, or IgG4 antibody. In a particularly preferred embodiment, the antibody is an IgG4 antibody.

Examples of preferred IgG forms include: igG with altered hinges (e.g., altered length and/or disulfide bonds); igG with altered glycans; igG with altered FcRn binding (e.g., with such altered binding to reduce serum half-life); igG with heavy chain modifications (e.g., knob-in-holes) that favor heterodimer formation over homodimer formation and/or charge modification; igG with a modification of the heavy chain that alters binding to the purifying agent (especially one heavy chain has a modification that alters binding to protein a, whereas the other heavy chain does not, in order to facilitate purification of the heterodimer but not of the homodimer); igG with altered effector function (e.g., altered FcGR binding and/or C1q binding); and/or IgG with reduced/no effector function. In a particularly preferred embodiment, such forms are used for IgG antibodies. In another particularly preferred embodiment, the antibody used in the present invention is an IgG4 antibody with a knob modification. In a further preferred embodiment, the antibody is an IgG4 antibody having a knob modification and a FALA modification. In a particularly preferred embodiment, such IgG forms will be used in cases where the antibody has two different specificities for CD 45.

The invention is not limited to antibodies in the form of IgG and any suitable binding molecule, particularly those described herein, may be used. For example, non-IgG antibodies may be used. Antibodies in the form of TrYbe and BYbe, particularly those described herein, may be used. Likewise, non-antibody binding molecules described herein may also be used.

Thus, the present invention provides:

an antibody comprising at least two different paratopes, each of said paratopes being specific for a different epitope of CD 45.

One or more nucleic acid molecules encoding an antibody of the invention.

One or more vectors encoding an antibody of the invention or comprising one or more nucleic acid molecules of the invention.

A pharmaceutical composition comprising: (a) An antibody of the invention, one or more nucleic acid molecules of the invention, or one or more vectors of the invention; and (b) a pharmaceutically acceptable carrier or diluent.

One or more binding molecules capable of multimerizing CD45 to induce cell death of CD45 expressing cells without inducing significant cytokine release.

One or more nucleic acid molecules encoding one or more binding molecules of the invention.

One or more vectors encoding one or more binding molecules of the invention or comprising one or more nucleic acid molecules of the invention.

A pharmaceutical composition comprising: (a) One or more binding molecules of the invention, one or more nucleic acid molecules of the invention, or one or more vectors of the invention; and (b) a pharmaceutically acceptable carrier or diluent.

The pharmaceutical composition of the invention for use in a method of treatment.

The pharmaceutical composition of the invention for use in a method of killing a disease-associated CD45 expressing cell in a subject.

A method of killing a disease-associated CD45 expressing cell in a subject, the method comprising administering to the subject a pharmaceutical composition of the invention.

Use of one or more binding molecules of the invention, one or more nucleic acid molecules of the invention or one or more vectors of the invention in the manufacture of a medicament for killing a cell expressing CD45 associated with a disease in a subject.

A method of screening for one or more binding molecules capable of multimerizing CD45 to induce cell death, the method comprising: (a) Contacting one or more binding molecules capable of binding CD45 with a target cell expressing CD 45; and (b) determining whether the target cell undergoes cell death.

An ex vivo method of depleting or killing target cells expressing CD45 in a population, tissue or organ of cells, said method comprising contacting said cells, tissue or organ with a binding molecule of the invention or an antibody of the invention.

A binding molecule of the invention or an antibody of the invention for use in a method of treating or preventing Graft Versus Host Disease (GVHD) in a subject, the method comprising: (a) Contacting a population, tissue or organ of cells ex vivo with a binding molecule of the invention or an antibody of the invention to kill target cells expressing CD 45; and (b) transplanting the treated cell population, tissue or organ into the subject.

A method of treating or preventing Graft Versus Host Disease (GVHD), comprising: (a) Contacting a population, tissue or organ of cells ex vivo with a binding molecule of the invention or an antibody of the invention to kill target cells expressing CD 45; and (b) transplanting the treated cell population, tissue or organ into a subject in need of such transplantation.

Use of a binding molecule of the invention or an antibody of the invention in the manufacture of a medicament for treating or preventing Graft Versus Host Disease (GVHD) in a method comprising: (a) Contacting a population, tissue or organ of cells ex vivo with a binding molecule of the invention or an antibody of the invention to kill target cells expressing CD 45; and (b) transplanting the treated cell population, tissue or organ into a subject in need of such transplantation.

Drawings

FIG. 1 is a bar graph showing the percentage of apoptotic cells in (A) lymphocyte count and (B) lymphocyte after incubation with a combination of Fab-X and Fab-Y against CD45 or an unrelated antigen. Apoptosis was measured by annexin V binding.

FIG. 2 is a graph showing the effect on CD4+ T cells by combined titration of Fab-X and Fab-Y with specificity for CD45 or an unrelated antigen. Values are percent reduction in T cell count relative to untreated cells.

FIG. 3 is a graph showing the effect on cell subsets in PBMC by (A) a combination of Fab-X and Fab-Y (6294-X/4133-Y) with specificity for CD45 or (B) a BYbe (Fab-scFv) with specificity for CD45 (4133-6294 BYbe) titration. Values are percent reduction in cell count relative to subpopulations of untreated cells.

FIG. 4 is a graph showing the titration of (A) lymphocytes and (B) CD4 in whole blood from donor HTA #051119-01 by a combination of Fab-X and Fab-Y (6294-X/4133-Y), anti-CD 45 BYbe (4133-6294 BYbe) or BYbe (NegCtrl BYbe) with irrelevant specificity specific for CD45 ⁺ Effect of cells on (C) lymphocytes and (D) CD 4-cells in whole blood from donor HTA # 051119-02. The values are percent reduction in cell count relative to untreated cells.

FIG. 5 is a bar graph showing the levels of anti-CD 45 BYbe (4133-6294 BYbe), BYbe (NegCtrl BYbe) with irrelevant specificity, campath or PBS induced in whole blood (A) CCL2, (B) GM-CSF, (C) IL-RA, (D) IL-6, (E) IL-8, (F) IL-10, (G) IL-11 or (H) M-CSF.

FIG. 6 is a bar graph showing the effect on T cell levels in whole blood by Fab-X and Fab-Y combinations (6294-X/4133-Y), anti-CD 45 BYbe (4133-6294 BYbe), BYbe (NegCtrl BYbe), campath or PBS with irrelevant specificity, with specificity to CD 45.

FIG. 7 is a graph showing the levels of (A) IFNγ, (B) IL-6 and (C) TNFα induced in whole blood by Fab-X and Fab-Y combinations (6294-X/4133-Y), anti-CD 45 BYbe (4133-6294 BYbe), BYbe (NegCtrl BYbe), campath or PBS with unrelated specificity, with specificity to CD 45.

FIG. 8 is usedThe images taken by the S3 system show (A) M1 macrophages and (B) M2 macrophages.

FIG. 9 is a bar graph showing the effect of anti-CD 45 BYbe (4133-6294 BYbe), BYbe (NegCtrl BYbe), camptothecin, staurosporine or PBS with unrelated specificity on the viability of (A) M1 macrophages and (B) M2 macrophages. The value is the original luminescence unit (RLU).

FIG. 10 is a graph showing the levels of anti-CD 45 BYbe (4133-6294 BYbe), BYbe (NegCtrl BYbe) with irrelevant specificity, camptothecins or PBS induced Caspase (Caspase) 3/7 in (A) M1 macrophages and (B) M2 macrophages.

FIG. 11 graphically shows mass spectral signals for mixtures of (A) CD45 ECD, (B) 4133-6294BYbe, and (C) CD45 ECD and 4133-6294 BYbe. The values are counts detected with respect to mass (kDa). Schematic of CD45 ECD was generated from PDB code 5 FMV. A schematic of 4133-6294BYbe is a model generated by ligating the internal crystal structures of Fab and scFv. A schematic of 4133-6294BYbe-CD45 ECD complex and higher order multimeric forms is a model. These models are for illustration purposes only and are not intended to indicate a specific location of an epitope.

FIG. 12 sequences of domains 1-4 of the V regions of antibodies 4133 and 6294, the humanized grafts of antibodies 4133 and 6294, the 4133-6294BYbe heavy and light chains and the extracellular domain of CD 45. Predicted N-linked glycosylation sites in CD45 ECD are underlined. Sequences of 4133 and 6294 chimeric light and heavy chain IgG4P FALA chains are also shown.

FIG. 13 is a graph showing the titration of anti-CD 45 6294-X/4133-Y ((A) and (C)) or anti-CD 454133-6294BYbe ((B) and (D)) against lymphocytes and CD34 in PBMC ⁺ Effects of cells. The values in (A) and (B) are shown as percent reduction in cell count relative to untreated cells, with actual cell counts shown in (C and D).

FIG. 14 is a graph showing the molar 1:1 mixture of CD45ECD and 4133-6294BYbe in minutesSedimentation velocity measured in analytical ultracentrifuge (solid black line). Superimposed on the graph are the CD45ECD (dotted line) and the settling velocity of 4133-6294BYbe (dashed line). The values are continuous distribution (frames/S) relative to the sedimentation coefficient (10 ^-13 Second). Schematic of CD45ECD is generated from PDB code 5 FMV. A schematic of 4133-6294BYbe is a model generated by ligating the internal crystal structures of Fab and scFv. A schematic of 4133-6294BYbe-CD45 ECD complex and higher order multimeric forms is a model. These models are for illustration purposes only and are not intended to indicate a specific location of an epitope.

FIG. 15 is a graph showing the effect of titrating anti-CD 45 4133-6294_IgG4P FALA KiH, anti-CD 454133-6294BYbe or anti-CD 454133_IgG4P FALA on lymphocytes in PBMC. Values are shown as percent reduction in cell count relative to untreated cells.

FIG. 16 is a graph showing the effect of titrating anti-CD 454133_IgG4P FALA, anti-CD 454133-6294BYbe, or a combination of anti-CD 454133 _Ig4P FALA and anti-CD 45 6294-X/6294-Y on lymphocytes in PBMC. Values are shown as percent reduction in cell count relative to untreated cells.

FIG. 17 is a graph showing the effect of titrating anti-CD 454133-6294 BYbe, anti-CD 454133-6294-645TrYbe or anti-CD 45 4133-6294IgG4 FALA KiH on lymphocytes in PBMC. Values are shown as percent reduction in cell count.

FIG. 18 is a graph showing the effect of titrating anti-CD 45 Bybe (4133-6294 Bybe) against cell lines (A) Jurkat, (B) CCRF-SB, (C) MC116, (D) Raji and Ramos, (E) SU-DHL-4, SU-DHL-5, SU-DHL-8, NU-DUL-1 and OCI-Ly3, (F) THP-1 and (G) Dakiki. Values are shown as percent reduction in cell count.

FIG. 19 is a bar graph showing the percent decrease in cell count for Negctrl Bybe (Bybe with irrelevant specificity showing only the highest concentration of 500nM for the dilution series), staurosporine, camptothecin, rituximab, campath, or anti-thymocyte globulin (ATG) versus cell line (A) Jurkat, (B) CCRF-SB, (C) MC116, (D) Raji, (E) Ramos, (F) SU-DHL-4, (G) SU-DHL-5, (H) SU-DHL-8, (I) NU-DUL-1, (J) OCI-Ly3, (K) THP-1, and (L) Dakiki. The highest percent cytopenia of 4133-6294BYbe is also marked.

Detailed Description

The invention provides, inter alia, binding molecules capable of multimerizing CD45 to induce cell death of target cells without significantly inducing cytokine release. In a particularly preferred embodiment, the binding molecule is an antibody. Further details of binding molecules and their uses are provided below.

CD45 molecules

The binding molecules of the invention are specific for CD 45. As explained above, CD45 is a member of the Protein Tyrosine Phosphatase (PTP) family. PTPs are known as signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. CD45 comprises an extracellular domain, a single transmembrane segment and two cytoplasmic catalytic domains in tandem and therefore belongs to the receptor type PTP. CD45 exists in a variety of isoforms: CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, CD45R (ABC). CD45 splice variant isoforms A, B and C are differentially expressed on many leukocyte subsets. Although different CD45 isoforms exist, they share a consensus sequence, meaning that all isoforms can be targeted by one binding molecule, in particular by one antibody.

The intracellular (COOH end) region of CD45 contains two PTP catalytic domains, while the extracellular region is highly variable due to alternative splicing of exons 4, 5 and 6 (designated A, B and C, respectively) and different levels of glycosylation. The CD45 isoforms detected are cell type, maturation and activation state specific. Typically, the long form of the protein (A, B or C) is expressed on naive or non-activated B cells, while the mature or truncated form (RO) of CD45 is expressed on activated or mature/memory B cells.

Human CD45 sequences are available in UniProt entry number P08575 and are provided herein in SEQ ID NO. 41 or amino acids 24-1304 of SEQ ID NO. 41 (lacking a signal peptide). The amino acid sequences of domains 1-4 of the extracellular domain of human CD45 are provided in SEQ ID NO. 113. The murine version of CD45 is provided in UniProt entry P06800. The present invention relates to all forms of CD45 from any species. In one embodiment, CD45 is mammalian CD45. In a particularly preferred embodiment, CD45 refers to the human form of the protein and its natural variants and isoforms. In a preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, are capable of binding to all CD45 isoforms expressed by a given species. For example, binding molecules (particularly antibodies) may bind all human CD45 isoforms. In one embodiment employing a mixture of binding molecules, particularly a mixture of antibodies, together they may be capable of binding all CD45 isoforms of a species, particularly all human CD45 isoforms. In an alternative embodiment, the binding molecules of the invention, in particular the antibodies of the invention, are specific for a particular CD45 isoform. In another embodiment, the binding molecule of the invention is capable of binding rodent CD45, e.g., it is capable of binding rodent and human CD45.

In a preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, are capable of binding to all CD45 isoforms expressed by a subject. In another preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, are capable of specifically binding to all CD45 isoforms expressed by a subject, without binding to other proteins. In another preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, recognize extracellular regions common to all CD45 isoforms expressed by a subject. In a preferred embodiment, the binding molecule of the invention, in particular the antibody of the invention, comprises at least two different specificities, each specifically binding to a different epitope within the extracellular domain of CD45, the sequence of the extracellular domain being provided as SEQ ID No. 113. In alternative embodiments, one or more binding molecules of the invention, in particular one or more antibodies of the invention, bind to the intracellular region of CD 45.

Binding molecules

The present invention provides binding molecules, in particular binding molecules specific for CD 45. In a particularly preferred embodiment, the binding molecules of the invention are antibodies. Alternatively, the binding molecules of the invention are not antibodies. Unless explicitly stated otherwise, what is stated herein with respect to antibodies also applies to the binding molecules of the invention and vice versa. In one embodiment, the binding molecules of the invention that are not antibodies may comprise a biocompatible framework structure for the binding domain of the molecule having a structure based on a protein scaffold or scaffold other than an immunoglobulin structure. Examples of alternative binding molecules of the invention include those based on fibronectin, ankyrin, lipocalin, neocarcinomycin, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain and tendriasat domain (see, e.g., nygren and Uhlen,1997,Current Opinion in Structural Biology,7,463-469). The term "binding molecule" as used herein also includes binding molecules based on biological scaffolds (including Adnectins, affibodies, darpins, phylomers, avimers, aptamers, anti, tetranectins, micro-bodies, affilins and Kunitz-type domains).

Small molecules capable of binding CD45 may also be used as binding molecules in the present invention. In one embodiment, small molecules that may be used include, for example, peptides, cyclized peptides, and macrocyclic compounds. For example, a peptide-mRNA library can be used to identify the desired peptide. In one embodiment, a library of these molecules is converted to cDNA-peptides, and then screened to identify peptides having the requisite ability to bind CD45, and then selected cDNA peptides having the desired properties are subjected to PCR to identify the sequence of the cDNA and peptides obtained therefrom. In one embodiment, extreme Diversity of Ra Pharma may be used ^TM The platform performs this screening. In another embodiment, libraries of peptides modified with scaffolds may be screened to assess their ability to bind CD45, such library screening being performed, for example, using the method of Bicycle Therapeutics.

The binding molecules of the invention will have at least one specificity for CD45. "specificity" of a binding molecule refers to the target to which the binding molecule binds, and generally also in the context of the present invention, to the position on the target to which the binding molecule binds. Thus, for example, both specificities of a binding molecule might be specific for CD45, but bind to different parts of CD45 itself, thus representing different specificities for CD45. Typically, one or more specific portions of the binding molecule will bind CD45, e.g., the binding site of the binding molecule will bind CD45. In the case of antibodies, the antigen-binding site will bind CD45 and confer specificity. In one embodiment, the portion of the antibody that binds CD45 is referred to as the paratope of the antibody that is specific for CD45. The binding portion of CD45 may, for example, be referred to as an antibody epitope.

In one embodiment, the binding molecule of the invention exhibits trans-binding, i.e., it binds more than one CD45 molecule simultaneously. This trans-binding typically results in crosslinking of CD45 and thus represents a particularly preferred embodiment of the present invention. In one embodiment, the binding molecules of the invention may exhibit cis-binding of CD45 such that their binding site binds only one CD45 molecule. In these embodiments, additional binding agents may be used to crosslink binding molecules that bind to different CD45 molecules.

The binding molecules of the invention, in particular the antibodies of the invention, may thus be multispecific, i.e. they may comprise at least two different specificities, each binding a different part of CD45, in particular a different epitope. Thus, in the context of the present invention, a multispecific or bispecific binding molecule need not necessarily bind to a different molecule: it encompasses binding molecules of the invention, in particular antibodies of the invention, comprising different binding sites that bind to the same target molecule and in particular different sites on CD 45. As discussed further below, the binding molecules of the invention may comprise other specificities for targets other than CD45, as well as for CD 45. In a further embodiment, the other specificity is for serum albumin.

In a preferred embodiment, the binding molecules of the invention may comprise two different specificities for CD 45. In a preferred embodiment, the two different specificities bind non-overlapping CD45 moieties. In one embodiment wherein the binding molecules are antibodies, it is possible that the specific binding is to a non-identical CD45 epitope. In one embodiment, the epitopes may overlap, but are not identical. In another embodiment, they may not overlap at all. In one embodiment, two different specificities may be defined as a specificity that does not compete with each other for binding to CD45 or that does not cross-block each other, or a specificity that does not compete significantly with each other or that cross-blocks each other. One preferred method of determining whether the specificity for CD45 is different is to perform a cross-blocking or competition assay, as discussed further below. The binding molecules preferably do not compete with each other or cross-block each other. They should generally be able to bind CD45 simultaneously, but on different epitopes.

The number of binding sites that a binding molecule (particularly an antibody) has can be referred to as its binding valency, each binding valency representing one binding site, in the case of an antibody, one antigen binding site of an antibody. Each binding valency may represent the same or different specificities; for example, bispecific IgG antibodies have two binding valencies and two different specificities. In one embodiment, the binding molecules of the invention, particularly the antibodies of the invention, may have at least two different specificities for CD 45. For example, it may have two, three, four, five, six, seven, eight, nine or ten different specificities for CD 45. In one embodiment, the binding molecules of the invention, in particular the antibodies of the invention, may comprise two or three different specificities for CD45, in particular at least two different antigen combining sites conferring different specificities for CD 45. In one embodiment, the binding molecules of the invention, in particular the antibodies of the invention, comprise three different specificities for CD 45. In a particularly preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, comprise two different specificities for CD 45. In one embodiment, the antibody may comprise at least two different paratopes, wherein each paratope is specific for a different epitope of CD 45. In one embodiment, the binding molecules of the invention, in particular the antibodies of the invention, have two binding valencies and have two different specificities for CD 45. In another embodiment, it has three binding valencies, and two of those binding valencies correspond to different specificities for CD 45. In one embodiment, the other binding valences are specific for serum albumin.

In another embodiment, the binding molecules of the invention (particularly antibodies) may have three binding valencies, each binding site of the molecule being specific for CD 45. In a preferred embodiment, all three binding sites have different specificities for CD 45. Thus, in a preferred embodiment, the binding molecules (particularly antibodies) of the invention may have three different specificities for CD 45. Such molecules may thus have, for example, three different paratopes to CD 45. Thus, in some embodiments of the invention, the binding molecules (particularly antibodies) are multivalent and preferably multispecific to CD 45. Binding molecules multispecific to CD45 are thus also provided. In particular, they are provided and they have multiple paratopes to CD 45. For example, in one embodiment, the binding molecules (particularly antibodies) may have three, four or more different specificities for CD45, and in particular such a plurality of paratopes. In a preferred embodiment, it has two, three or four different specificities for CD 45. In particular, it may have such many different paratopes to CD 45. In a particularly preferred embodiment, it has three different specificities for CD45, and preferably it has three different paratopes for CD 45. In another preferred embodiment, in addition to having such number of specificity/paratopes for CD45, the binding molecules (particularly antibodies) of the invention also have at least one other specificity for non-CD 45 antigen conferred by an independent binding site. For example, the binding molecule may also be capable of binding to albumin via binding sites that are separate from those binding sites that bind CD 45.

In a particularly preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, can bind CD45, resulting in CD45 multimerization. In one embodiment, the binding molecules of the invention, particularly antibodies of the invention, bind to the extracellular portion of CD45, resulting in CD45 multimerization. In alternative embodiments, the binding molecules of the invention, particularly antibodies of the invention, may bind to the intracellular portion of CD45. In a preferred embodiment, the binding molecules of the invention, particularly antibodies of the invention, can bind to the intracellular portion of CD45, resulting in CD45 multimerization.

The binding molecules of the invention may be used to multimerize CD45. In particular, they are useful for multimerizing CD45 on the surface of target cells. CD45 multimers are particularly high order structures composed of more than one CD45 molecule linked together by one or more binding molecules of the invention. For example, in one embodiment, a CD45 multimer may comprise at least two CD45 molecules. In a particularly preferred embodiment, the CD45 multimer comprises at least three CD45 molecules. In one embodiment, the CD45 multimer may comprise at least three, four, five, six, seven, or more CD45 molecules linked together by a binding molecule of the invention. Techniques such as mass spectrophotometry, as described herein, can be used to identify CD45 multimers that complex with binding molecules of the invention, thereby assessing the ability of one or more binding molecules of the invention to produce CD45 multimers. In one embodiment, one or more binding molecules of the invention are used to crosslink CD45. In a preferred embodiment, one or more binding molecules of the invention are used to crosslink CD45 molecules on the surface of target cells. In a further embodiment, they bind to the inner portion of CD45 and crosslink CD45 in this way, preferably creating a CD45 multimer.

Mixtures of binding molecules

In another embodiment, rather than providing a single binding molecule, a mixture of at least two different binding molecules may be provided. For example, in one embodiment, a mixture of at least two different binding molecules is provided, wherein a single binding molecule in the mixture has only one specificity for CD45, but the mixture of binding molecules has at least two different specificities for CD45 as a whole. Thus, the use of a mixture of binding molecules represents another approach to promote CD45 cross-linking. The invention also provides a mixture of binding molecules, particularly a mixture of antibodies, wherein individual binding molecules in the mixture have at least two different specificities for CD 45. In another embodiment, a mixture of binding molecules that are generally only one specific for CD45 may be used. Both the binding molecule and the plurality of binding molecules of the present invention may be provided as a mixture with other therapeutic agents.

Unless specifically stated, any reference herein to a binding molecule may be replaced with a mixture of binding molecules. For example, unless specifically stated otherwise, any reference herein to a single antibody may be replaced with a mixture of at least two different antibodies. And vice versa.

Screening for biomolecules

In addition to the binding molecules themselves, the present invention also provides methods for identifying the binding molecules of the present invention and determining the efficacy of these binding molecules. Various functional assays are disclosed herein and may be employed, for example.

For example, the invention provides a method of screening for one or more binding molecules capable of multimerizing CD45 to induce cell death, the method comprising: (a) Contacting one or more binding molecules capable of binding CD45 with a target cell expressing CD 45; and (b) determining whether the target cell undergoes cell death. In one embodiment, the method further comprises: (c) Determining whether the cytokine is released in the test sample, e.g., measuring the level of one or more of CCL2, GM-CSF, IL-1RA, IL-6, IL-8, IL-10, IL-11, and M-CSF. In one embodiment, one or more binding molecules have been identified as being capable of multimerizing CD45. In another embodiment, the method comprises first screening for the ability to multimerize CD45 of a binding molecule specific for CD45, for example by screening for the ability to multimerize CD45 in an aligned combination between two or more different binding molecules.

In one embodiment, the invention provides a method of identifying a biomolecule comprising at least two different specificities. For example, the method may comprise screening a library of pairwise permutations specific for CD45. In one embodiment, the pairwise permutations of multimerized CD45 are screened for their ability, for example by mass spectrophotometry. In another embodiment, they are screened for their ability to cause killing of target cells expressing CD45. In another embodiment, they are screened for their ability to kill these target cells while not triggering cytokine release. Various functional assay and screening formats are described herein, any of which may be used. In one embodiment, the Fab-X/Fab-Y format is used to screen for pairwise combinations. In one embodiment, the screening may also include comparison to equivalent molecules having only one such specificity when evaluating pairwise permutations.

In another embodiment, to identify a desired mixture of at least two binding molecules, various permutations of a mixture of different individual binding molecules specific for CD45 may be screened for a desired property. In one embodiment, the screening also compares the activity of the mixture to that of a single binding molecule. Accordingly, the present invention provides a method of identifying a mixture of binding molecules of the invention that are capable of multimerizing CD45 without inducing cytokines to significant levels, the method comprising screening a mixture comprising a set of individual binding molecules specific for CD45 in various permutations and identifying the mixture that yields the highest levels of desired properties. For example, the assay may identify a mixture that achieves the highest level of multimerization or alternatively a mixture that achieves the highest level of cell killing of target cells expressing CD 45. The method may involve identifying a mixture that achieves the highest level of cell killing without cytokine release.

Antibodies to

In a particularly preferred embodiment, the one or more binding molecules of the invention are one or more antibodies to CD 45. Thus, in any of the embodiments outlined herein, if one or more binding molecules are mentioned, it is preferred to use one or more antibodies. The term "antibody" includes the various antibody forms disclosed herein, including antibodies comprising the various heavy and/or light chain forms described herein. Thus, for example, the term "antibody" specifically includes the Fab-X/Fab-Y, BYbe, trYbe and in situ (on-site) multimerized IgG antibody forms described herein. The term "antibody" also includes antibody fragments, preferably those mentioned herein. As described herein, one particularly preferred antibody isotype is IgG4.

In a particularly preferred embodiment, the sequence of the antibody is such that it favors heterodimer formation rather than homodimer formation, such that the antibody comprises two different heavy chains and thus has two different specificities. Alternatively, it may have modifications that allow for purification of heterodimeric antibodies instead of homodimeric antibodies. These forms may be particularly useful in situations where an antibody is desired that is an antibody having two different specificities, and thus two different heavy chains, one for each specificity.

As described above, the specificity of a binding molecule may represent the target to which the binding molecule binds and the binding site of the binding molecule on the target. Thus, for an antibody, it refers to the target to which the antigen binding site of the antibody binds and the binding site on the target. In the context of antibodies, the antigen binding site of an antibody can be said to confer specificity to the antibody. Two antibodies can be said to have different CD45 specificities if they both bind to CD45 but at different positions. For example, the locations may overlap, but not be the same, or not overlap at all. An "paratope" of an antibody is a portion of an antibody antigen binding site that recognizes and binds an antigen. In particular, paratopes are part of antibodies that recognize and bind to epitopes of an antigen. In a preferred embodiment, when two different specificities or paratopes are mentioned, they will be different in the sense that they each bind to a different part of CD 45. In particular, they will each bind a different epitope of CD 45. In one embodiment, when referring to a different specificity or paratope for CD45, it may mean that a different and in particular a different CD45 epitope is bound. Thus, in a preferred embodiment, the bound CD45 epitopes are not identical. In one embodiment, it means that different specificities correspond to different paratopes to CD 45.

In one embodiment, the specificities, particularly paratopes, of the antibodies of the invention each bind to a different epitope of CD 45. Binding to a "different epitope" means that the two epitopes are not identical. In a preferred embodiment, the two different epitopes recognized do not overlap at all. For example, in a preferred embodiment, the recognized epitopes are separated by at least one amino acid in the linear amino acid sequence of CD 45. In another preferred embodiment, the two epitopes identified are at least 5, 10, 15, 20, 50, 100 or more amino acids apart in the linear sequence of CD 45. In another embodiment, two different epitopes may overlap by a small amount, e.g., five or fewer amino acids overlap in the linear sequence of CD 45. In another embodiment, an epitope may overlap four or fewer amino acids, for example three or fewer amino acids, preferably two or fewer amino acids. In another preferred embodiment, the epitope will overlap in the linear sequence of CD45 by only one amino acid or not at all. In one embodiment, where the epitopes are non-linear, e.g. they are conformational epitopes, there may be some overlap between the portions of CD45 that bind as epitopes, but the two portions of CD45 that bind are not identical. In one embodiment, conformational epitopes will not overlap at all.

In one embodiment, when it is desired to determine whether the two specificities of a binding molecule, particularly an antibody, are different, a binding molecule having only one putative specificity will be generated for each of the two specificities, preferably wherein the binding molecules have the same binding valency, but only differences in the specificities that are present. The ability of the two binding molecules to compete or cross-block in the binding assay will be determined. In particular, such an assay would determine whether two binding molecules are capable of binding CD45, but without significantly reducing each other's binding to CD45. Thus, for example, in a preferred embodiment, a cross-blocking or competition assay may compare the binding of each binding molecule to CD45 alone, or may compare the binding of two binding molecules to CD45 when mixed together with CD45. In one embodiment, the desired antibody does not reduce the binding of another antibody.

For example, in the context of antibodies, antibodies will be produced having the same binding valency, wherein the or each binding site of the antibody confers only one specificity. The ability of these antibodies to compete or cross-block with each other will be determined for each specificity. However, these antibodies should still all bind CD45. In a preferred embodiment, monovalent antibodies, such as scFv or Fab, will be produced for each specificity, and then the ability of the antibodies to cross-block or compete is measured for each specificity. In another embodiment, each specific double binding valency antibody will be generated and the ability of each binding valency to compete or cross block the other binding valency will be determined. In a preferred embodiment, the antibodies used for comparison are identical except for the regions that confer specificity, e.g. only the different variable regions, in particular only the paratopes. For example, two antibodies may differ only in the different variable regions of the paratope. In one embodiment, no cross-over blocking is seen when such a comparison is made. In another embodiment, no significant cross-blocking is seen. For example, the amount of cross-blocking by one antibody against another antibody may be less than 25%, preferably less than 20%, more preferably less than 10%. In another preferred embodiment, the degree of cross-blocking may be less than 5%. In another embodiment, the degree of cross-blocking will be less than 1%. In another preferred embodiment, a cross-blocking of 0% will be seen. These percentages refer to the extent to which the first antibody reduces the binding of the second antibody to CD45, for example in an ELISA.

In one embodiment, the binding domain of an antibody of the invention has an affinity for CD45 of about 100nM or more, such as about 50nM, 20nM, 10nM, 1nM, 500pM, 250pM, 200pM, 100pM or more. In one embodiment, the binding affinity is 50pM or greater. In one embodiment, at least one paratope of the antibody has such affinity for CD 45. In another embodiment, the antibody has two paratopes, each paratope having a different specificity for CD45, wherein all paratopes alone have such affinity for CD 45. In one embodiment, this is the overall affinity of the antibody for CD 45. In one embodiment, the affinity of a paratope for CD45 can be less than 1 μΜ, less than 750nM, less than 500nM, less than 250nM, less than 200nM, less than 150nM, less than 100nM, less than 75nM, less than 50nM, less than 10nM, less than 1nM, less than 0.1nM, less than 10pM, less than 1pM, or less than 0.1pM. In some embodiments, kd is from about 0.1pM to about 1 μΜ. In one embodiment, the antibody population of the invention has such affinity levels for CD 45. In one embodiment, the binding molecules of the invention will exhibit such affinity for CD 45. In another embodiment, when referring to specificity, it will display such a value. In a further embodiment, the binding molecule of the invention, or the specificity of the binding molecule, will exhibit such a value.

In one embodiment, when an antibody of the invention has more than one specificity, the antibody may be selected to have a particular specificity. For example, the different specificities may be selected such that the binding sites of each specificity have substantially similar affinities. For example, individual specific binding affinities may be selected to be within 100-fold, preferably within 50-fold, in particular within 10-fold of each other. In another embodiment, the different specificities of the antibodies of the invention may be selected such that they have different affinities. For example, in one embodiment they may differ from each other by at least a factor of 10. In another embodiment, they may differ from each other by at least a factor of 50. In further embodiments, the affinities may differ from each other by at least a factor of 100. In another embodiment, the affinities may differ by at least 1000-fold. Such a level of difference can be seen, for example, in the KD values.

In a preferred embodiment, the antibodies of the invention will have at least two specificities, in particular at least two different paratopes, each paratope binding a different epitope of CD45, and thus any suitable format allowing such antibodies may be employed. Preferably, while neither antibody will significantly block the binding of the other, they should still be able to bind CD45 simultaneously. In embodiments where the antibodies of the invention comprise at least two different paratopes specific for CD45, typically each paratope of a double paratope antibody is capable of specifically binding to CD45, wherein the two paratopes each specifically bind a different epitope of CD45. Thus, the presence of different specificities still allows simultaneous binding of the two.

In one embodiment, where there are two variable regions in each antigen binding site of an antigen binding site and/or antibody, the two variable regions may act synergistically to provide specificity for CD45, e.g. they are a pair of cognate pairs or are affinity matured to provide sufficient affinity such that the domain is specific for a particular antigen. Typically, they are pairs of heavy and light chain variable regions (VH/VL pairs). In one embodiment, two different antigen binding sites of an antibody of the invention will each comprise the same light chain, also referred to as a "common" light chain. For example, in one embodiment, the antibodies of the invention are in the form of IgG antibodies and comprise such a common light chain. In one embodiment, such a method may be combined with a knob-to-socket modification in the heavy chain that favors heterodimer formation.

Antibodies of the invention may include whole antibodies or fragments thereof having whole length heavy and light chains, e.g., fab, modified Fab, fab ', modified Fab ', F (ab ') ₂ Fv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibodies, bis-scFv, diabodies (diabodies), triabodies (triabodies), tetrabodies (tetrabodies) or epitope-binding fragments of any of the foregoing (see, e.g., holliger and Hudson,2005,Nature Biotech.23 (9): 1126-1136; adair and Lawson,2005,Drug Design Reviews-Online 2 (3), 209-217). In embodiments of the invention, when a binding molecule (particularly an antibody) has a certain number of binding sites but the type of fragment or antibody form referred to has fewer than that number of binding sites, it may still form part of the overall binding molecule. Methods for forming and making antibody fragments are well known in the art Known (see, e.g., verma et al, 1998,Journal of Immunological Methods,216,165-181). Other antibody fragments useful in the present invention include Fab and Fab' fragments described in International patent applications WO2005/003169, WO2005/003170 and WO 2005/003171. Multivalent antibodies may comprise a variety of specificities, e.g. bispecific or may be monospecific (see e.g. WO 92/22853, WO05/113605, WO2009/040562 and WO 2010/035012).

The antibodies of the invention may be in any of the forms discussed herein. In a particularly preferred embodiment, the antibodies of the invention are in the form of BYbe, trYbe or IgG antibodies. These antibody forms are particularly preferred in various embodiments of the invention in which the antibodies are used for therapy.

Examples of possible antibody formats are known in the art, for example in review, "The coming of Age of Engineered Multivalent Antibodies, nunez-Prado et al, pages Drug Discovery Today Vol 20Number 5Mar 2015,588-594, d.holmes, nature Rev Drug Disc Nov 2011:10;798, chan and Carter, nature Reviews Immunology vol.10, may 2010,301, which review is incorporated herein by reference. In one embodiment, an antibody of the invention may comprise, consist essentially of, or consist of any of the following forms:

Tandem sdAb, tandem sdAb-sdAb (three sdabs);

·(scFv) ₂ (also known as tandem scFv), scFv-dsFv, dsscFv-dsFv (dsFv) ₂ ；

Diabodies, ds diabodies, dids diabodies;

sc diabody, dssc diabody, didsc diabody;

dart antibody, i.e. VL ₁ Linker VH ₂ Linker and VH ₁ Joint VL ₂ Wherein VH ₁ And VH ₂ Is linked by disulfide bonds at the C-terminus of (C);

·dsBiTE，didsBiTE；

di-diabody (see Nunez-Prado et al, particularly molecule 25 in FIG. 1 therein), dsdi-diabody, didsdi-diabody;

tri-antibodies, ds-tri-antibodies;

four antibodies, ds four antibodies, tetra four antibodies;

tandab (see Nunez-Prado et al, particularly molecule 22 in FIG. 1), dstandab, didsttandab, tristandab, tetradstandab;

antibodies in the form of ByBe or TrYbe;

·[sc(Fv) ₂ ] ₂ (see Nunez-Prado et al, particularly molecule 22 in FIG. 1), ds [ sc (Fv) ₂ ] ₂ ，dids[sc(Fv) ₂ ] ₂ ，trids[sc(Fv) ₂ ] ₂ ，tetrads[sc(Fv) ₂ ] ₂ ；

Five antibodies (see Nunez-Prado et al, particularly wherein molecule 27 in FIG. 1);

Fab-scFv (also known as a bi-targeting antibody (bipody)), fab 'scFv, fabdscfv (or BYbe), fab' dsscFv;

trisomy (trisomy), ds trisomy (also known as FabdidsscFv or TrYbe or Fab- (dsscFv) ₂ )，Fab’didsscFv；

·Fabdab，FabFv，Fab’dab，Fab’Fv；

Fab single linker Fv (also referred to herein as FabdsFv as disclosed in WO 2014/096390), fab 'single linker Fv (also referred to herein as Fab' dsFv);

FabscFv single linker Fv, fab' scFv single linker Fv;

FabdsscFv single linker Fv, fab' dsscFv single linker Fv;

·FvFabFv，FvFab’Fv，dsFvFabFv，dsFvFab’Fv，FvFabdsFv，FvFab’dsFv，dsFvFabdsFv，dsFvFab’dsFv；

·FabFvFv，Fab’FvFv，FabdsFvFv，Fab’dsFvFv，FabFvdsFv，Fab’FvdsFv，FabdsFvdsFv，Fab’dsFvdsFv；

DiFab, diFab 'includes chemically conjugated DiFab';

·(FabscFv) ₂ ，(Fab) ₂ scFvdsFv，(Fab) ₂ dsscFvdsFv，(FabdscFv) ₂ ；

·(Fab’scFv) ₂ ，(Fab’) ₂ scFvdsFv，(Fab’) ₂ dsscFvdsFv，(Fab’dscFv) ₂ ；

·V _H HC _K (see Nunez-Prado et al, particularly wherein molecule 6 in FIG. 1);

minibodies (minibodies), ds minibodies, dids minibodies;

minibodies (minibodies) (ZIPs) [ see Nunez-Prado et al, particularly wherein molecule 7 in FIG. 1 ], ds minibodies (ZIPs) and dids minibodies (ZIPs);

a tri bi minibody [ see Nunez-Prado et al, particularly wherein molecule No. 15 in fig. 1 ], a dstri bi minibody, a didstribi minibody, a trisribi minibody;

diabody-CH ₃ Ds diabody-CH ₃ Dids diabody-CH ₃ Sc diabody-CH ₃ Dssc diabody-CH ₃ Didssc diabody-CH ₃ ；

Tandem scFv-CH ₃ Tandem dsscFv-CH ₃ Tandem didscfv-CH ₃ Tandem trisscfv-CH ₃ Tandem tetradsscFv-CH ₃ ，

Scorpion molecule (Trubion), i.e.binding domain, linker-CH ₂ CH ₃ Binding domains as described in US8,409,577;

SMIP (Trubion), i.e. (scFv-CH) ₂ CH ₃ ) ₂ ；

·(dsFvCH ₂ CH ₃ ) ₂ Tandem scFv-Fc, tandem dsscFvscFv-Fc, tandem dsscFv-Fc,

·scFv-Fc-scFv，dsscFv-Fc-scFv，scFv-Fc-dsscFv；

diabody-Fc, ds diabody-Fc, triabody-Fc, ds triabody-Fc, tetrabody-Fc, ds tetrabody-Fc, dids tetrabody-Fc, triabody-Fc, tetrads tetrabody-Fc, ds tetrabody-Fc, dids tetrabody-Fc, triads tetrabody-Fc, tetrads tetrabody-Fc, sc diabody-Fc, dssc diabody, didsc diabody;

bifunctional or trifunctional antibodies, e.g.antibodies with different heavy chain variable regions and a common light chain, e.g.Merus bispecific antibody formsCommon light chain with fixed sequences and different heavy chains (including different CDRs) and engineered CH ₃ Domains drive dimerization between different heavy chains;

duobody (i.e., wherein one full length chain in an antibody has a different specificity than another full length chain in an antibody);

full length antibodies, wherein Fab arm exchange is used to form a bispecific format;

bifunctional or trifunctional antibodies, wherein the full length antibodies have a common heavy chain and different light chains, also known as kappa/lambda or kappa/lambda bodies, see for example WO2012/023053, which is incorporated herein by reference;

One, two, three or four Ig-scFvs from the C-terminus of a heavy or light chain, one, two, three or four scFvs from the N-terminus of a heavy or light chain, a single linker Ig-Fv, one, two, three or four Ig-dsscFvs (having one, two, three or four disulfide bonds) from the C-terminus of a heavy or light chain;

one, two, three or four Ig-dsscFv (with one, two, three or four disulfide bonds) from the N-terminus of the heavy or light chain;

ig single linker Fv (see PCT/EP 2015/064450);

·Ig-dab，dab-Ig，scFv-Ig，V-Ig，Ig-V；

scFabFvFc, scFabdsFvFc (scFavFv in the form of a single linker), (FabFvFc) ₂ ，(FabdsFvFc) ₂ ，scFab’FvFc，scFab’dsFvFc，(Fab’FvFc) ₂ ，(Fab’dsFvFc) ₂ The method comprises the steps of carrying out a first treatment on the surface of the And

DVDIg, which will be discussed in more detail below.

In one embodiment, the antibody formIncluding those known in the art and described herein, such as those in which the antibody molecule is in the form of, or includes, a member selected from the group consisting of, or consisting of: diabodies, bybe, sc diabodies, triabodies, tetrabodies, trYbe, tandem scFv, fabFv, fab' Fv, fabdsFv, fab-scFv, fab-dsscFv, fab- (dsscFv) ₂ DiFab, diFab', tandem scFv-Fc, scFv-Fc-scFv, sc diabody-Fc, sc diabody-CH ₃ Ig-scFv, scFv-Ig, V-Ig, ig-V, duobody and DVDIg, which are discussed in more detail below. Another preferred antibody format for use in the present invention is a bispecific antibody.

In a preferred embodiment, the antibody molecule of the invention does not comprise an Fc domain, i.e. does not comprise CH2 and CH3 domains. For example, the molecule may be selected from the group comprising: tandem scFv, scFv-dsFv, dsscFv-dsFv, diabodies, ds diabodies, sc diabodies (also known as (scFv) 2), dssc diabodies, triabodies, ds triabodies, tetrabodies, ds tetrabodies, triabodies, tetrads triabodies, trisomy, ds trisomy, fabdab, fabdFv, fab ' dab, fab ' Fv, fab single linker Fv (disclosed in WO 2014/096390), fab ' single linker Fv, fabdsFv, fab ' dsFv, fab-scFv (also known as a bi-targeting antibody), fab ' scFv, fabdsFv, fab ' dsscFv, fabddsscfv, fabdscfv single linker Fv, fabdscfvs single linker Fv, fab ' dsscFv single linker Fv, fvFabFv, fvFab ' Fv, dsFvFabFv, dsFvFab ' Fv, fvFabdsFv, fvFab ' dsFv, dsFvFabdsFv, dsFvFab ' dsFv, fabdfv, fab ' ffv, fabdsFv, fab ' dsFvFv, fabFvdsFv, fab ' FvdsFv, fabdsFvdsFv, fab ' dsFvdsFv, diFab ' including chemically conjugated diFab ', (fabdscfv) ₂ ，(Fab) ₂ scFvdsFv，(Fab) ₂ dsscFvdsFv，(FabdscFv) ₂ Minibodies, ds minibodies, dids minibodies, diabodies-CH ₃ Ds diabody-CH ₃ Dids diabody-CH ₃ Sc diabody-CH ₃ Dssc diabody-CH ₃ Didssc diabody-CH ₃ Tandem scFv-CH ₃ Tandem dsscFv-CH ₃ Tandem didscfv-CH 3, tandem trisscfv-CH ₃ And tandem tetradsscFv-CH ₃ . In one embodiment, the antibody of the invention is or comprises a diabody. In another embodiment, it is or includes a duobody.

Further explanation of antibody formats suitable for use in the present invention, whether as antibodies of the invention or as part of an overall antibody, is provided below:

as used herein, "single domain antibody" (also referred to herein as dab and sdAb) refers to an antibody fragment consisting of a single monomeric variable antibody domain. Examples of single domain antibodies include V _H Or V _L Or V _H H。

tandem-sdAb as used herein refers to two domain antibodies connected by a linker (e.g., a peptide linker), particularly when the domain antibodies are specific for different antigens.

tandem-sdAb as used herein refers to a three domain antibody connected in series by two linkers (e.g., peptide linkers), particularly when the domain antibodies are specific for different antigens.

dsFv as used herein refers to Fv's having an internally variable disulfide bond. dsFv may be a component of a larger molecule, for example, in which one variable domain may be linked to another antibody fragment/component by, for example, an amino acid linker.

As used herein (dsFv) ₂ Refers to having a group such as through a peptide linker or disulfide bond (e.g., two V _H C-terminal) to a domain in a second dsFv, in a form similar to that described below (scFv) ₂ But each pair of variable regions comprises an internal variable region disulfide bond.

A module as used herein refers to a building block or portion of an antibody of the invention, particularly when the module is an antibody fragment (such as scFv, fab or other fragment), particularly as described herein, which may be used as part of the whole antibody of the invention in some embodiments.

As used herein, single chain Fv or simply "scFv ", refers to an antibody fragment comprising V linked (e.g., by a peptide linker) to form a single polypeptide chain _H And V _L An antibody domain. In this form the constant regions of the heavy and light chains are omitted.

dsscFv as used herein refers to scFv having an internal variable region disulfide bond.

Tandem scFv (also referred to herein as discofv or (scFv) as used herein ₂ ) Refers to two scFvs linked by a single linker such that there is a single Fv linker.

Tandem dsscFv (also referred to herein as scfvdscfv or dsscfscfv) as used herein refers to two scFv joined by a single linker such that there is a single Fv-to-Fv linker and one of the scFv has an internal variable region disulfide bond.

Tandem didscfv (also referred to herein as didscfv) as used herein refers to two scFv joined by a single linker such that there is a single Fv-linker, and wherein each scFv comprises an internal variable region disulfide bond.

scFv-dsFv as used herein refers to an scFv that is linked, e.g., by a peptide linker, to an Fv domain comprising two variable domains linked by disulfide bonds to form a dsFv. In this form, the VH or VL of the scFv may be linked to the VH or VL of the dsFv.

As used herein, dsscFv-dsFv refers to a dsscFv that is linked, e.g., by a peptide linker, to an Fv domain comprising two variable domains linked by disulfide bonds to form a dsFv. In this form, the VH or VL of the dsscFv may be linked to the VH or VL of the dsFv.

Diabodies as used herein refer to two Fv pairs V having two Fv linkers _H /V _L And another V _H /V _L For V of the first Fv _H V with a second Fv _L Connected to V of the first Fv _L V with a second Fv _H Are connected.

As used herein, ds diabodies refer to diabodies comprising internal variable region disulfide bonds.

As used herein, a dids diabody refers to a diabody comprising two internal variable region disulfide bonds, i.e., one ds between each pair of variable regions.

Sc-diabodies as used herein refer to diabodies comprising an internal Fv linker such that the molecule comprises three linkers and forms two normal scFvs, e.g.VH ₁ Joint VL ₁ Linker VH ₂ Joint VL ₂ 。

Dssc-diabodies as used herein refer to sc-diabodies having internal variable region disulfide bonds.

As used herein, a didsc-diabody refers to a sc-diabody having one internal variable region disulfide bond between each pair of variable regions.

Dart as used herein refers to VL ₁ Linker VH ₂ Linker and VH ₁ Joint VL ₂ Wherein VH ₁ And VH ₂ Is linked by disulfide bonds to the C-terminus of Paul A.Moore et al Blood,2011;117 (17):4542-4551.

As used hereinRefers to a molecule comprising two pairs of variable domains in the following form: from pair 1 (e.g. VH ₁ ) Is linked to a domain from pair 2 (e.g., VH ₂ Or VL (VL) ₂ ) The second domain is linked to the domain from pair 1 (e.g., VL ₁ ) The latter is then linked to a domain from pair 2 (i.e.VL ₂ Or VH ₂ ) Is a domain of the remainder of the sequence.

Di-diabodies, see Nunez-Prado et al, particularly wherein molecule number 25 in FIG. 1.

Dsdi-diabodies as used herein are di-diabodies having internal variable region disulfide bonds.

Didsdi-diabodies as used herein are di-diabodies having internal variable region disulfide bonds between each pair of variable regions.

As used herein, a tri-antibody refers to a form similar to a diabody, comprising three Fv and three Fv-to-Fv linkers.

As used herein, ds autoantibodies refer to autoantibodies comprising an internal variable region disulfide bond between one of the variable domain pairs.

Dids trisomy as used herein refers to trisomy comprising two internal variable region disulfide bonds, i.e., one ds between each of the two variable domain pairs.

Trids trisomy as used herein refers to trisomy comprising three internal variable region disulfide bonds, i.e., one ds between each pair of variable regions.

A tetrabody as used herein refers to a diabody-like form comprising 4 Fv's and 4 Fv-to-Fv linkers.

As used herein, ds tetrabodies refers to tetrabodies comprising internal variable region disulfide bonds between one of the variable domain pairs.

As used herein, a Dids tetrabody refers to a tetrabody comprising two internal variable region disulfide bonds, i.e., one ds between each of the two variable domain pairs.

As used herein, a Trids tetrabody refers to a tetrabody comprising three internal variable region disulfide bonds, i.e., one ds between each of the three pairs of variable regions.

Tetrads tetrabodies, as used herein, refers to tetrabodies comprising four internal variable region disulfide bonds, i.e., one ds between each variable domain.

Trisomy (also known as Fab (scFv)) as used herein ₂ ) Refers to a Fab fragment in which a first scFv is attached to the C-terminus of the light chain and a second scFv is attached to the C-terminus of the heavy chain.

As used herein, ds trisomy refers to trisomy of dsscFv contained in one of two positions.

As used herein, a dids trisome or TrYbe refers to a trisome comprising two dsscFv.

As used herein, dsFab refers to Fab having internal variable region disulfide bonds.

As used herein, dsFab 'refers to Fab' having internal variable region disulfide bonds.

scFab is a single chain Fab fragment.

scFab 'is a single chain Fab' fragment.

dsscFab is dsFab as single chain.

dsscFab 'is dsFab' as single chain.

Fabdab, as used herein, refers to a Fab fragment in which a domain antibody is attached (optionally via a linker) to either the heavy or light chain.

Fab 'dab as used herein refers to a Fab' fragment to which a domain antibody is attached (optionally via a linker) to a heavy or light chain.

FabFv, as used herein, refers to Fab fragments with additional variable regions attached at the C-terminus of each of the following: CH1 of the heavy chain and CL of the light chain, see for example WO2009/040562. Said forms may be provided as their pegylated forms, see for example WO2011/061492.

Fab 'Fv as used herein is similar to FabFv, in that the Fab portion is replaced by Fab'. The form may be provided as its pegylated form.

As used herein, fabdsFv refers to FabdsFv in which an internal-Fv disulfide bond is stably attached to the C-terminal variable region, see, e.g., WO2010/035012. The form may be provided as its pegylated form.

Fab single linker Fv and Fab 'single linker as used herein refers to Fab or Fab' fragments linked to a variable domain, e.g. via a peptide linker, and the variable domain is linked to a second variable domain via an internal variable domain disulfide bond, thereby forming a dsFv, see e.g. WO2014/096390.

Fab-scFv (also referred to as a bi-targeted antibody) as used herein is a Fab molecule to which scFv is attached (optionally via a linker) at the C-terminus of a heavy or light chain.

Fab '-scFv as used herein is a Fab' molecule to which an scFv is attached (optionally via a linker) at the C-terminus of a heavy or light chain.

As used herein, fabdsscFv or BYbe is FabscFv having disulfide bonds between the variable regions of single chain Fv.

As used herein, a Fab 'dsscFv is a Fab' scFv having disulfide bonds between the variable regions of a single chain Fv.

FabscFv-dab as used herein refers to a Fab in which the C-terminus of one chain is attached to the scFv and the C-terminus of the other chain is attached to a domain antibody.

Fab 'scFv-dab as used herein refers to Fab' in which the C-terminus of one chain is attached to a scFv and the C-terminus of the other chain is attached to a domain antibody.

FabdsscFv-dab as used herein refers to a Fab in which one chain is attached to the dsscFv at the C-terminus and the other chain is attached to a domain antibody at the C-terminus.

Fab 'dsscFv-dab as used herein refers to a Fab' in which one chain is attached to the dsscFv at the C-terminus and the other chain is attached to a domain antibody at the C-terminus.

FabscFv single linker Fv as used herein refers to Fab single linker Fv in which the domains of Fv are linked to the heavy or light chain of Fab, scFv is linked to the other Fab chain, and the domains of Fv are linked by an internal variable region disulfide bond.

As used herein, fabdsscFv single linker Fv refers to FabscFv single linker Fv wherein the scFv comprises an internal variable region disulfide bond.

Fab 'scFv single linker Fv as used herein refers to Fab' single linker Fv wherein the domains of Fv are linked to the heavy or light chain of Fab, scFv is linked to the other Fab chain, and the domains of Fv are linked by an internal variable region disulfide bond.

Fab 'dsscFv single linker Fv as used herein refers to Fab' scFv single linker Fv wherein the scFv comprises internal variable region disulfide bonds.

FvFabFv, as used herein, refers to a Fab in which the domains of the first Fv are attached to the N-terminus of the heavy and light chains of the Fab and the domains of the second Fv are attached to the C-terminus of the heavy and light chains.

FvFab ' Fv as used herein refers to Fab ' wherein the domain of the first Fv is attached to the N-terminus of the heavy and light chains of Fab ' and the domain of the second Fv is attached to the C-terminus of the heavy and light chains.

As used herein, dsFvFabFv refers to a Fab in which the domains of the first Fv are attached to the N-terminus of the heavy and light chains of the Fab, wherein the first Fv comprises an internal variable region disulfide bond and the domain of the second Fv is attached to the C-terminus of the heavy and light chains.

FvFabdsFv as used herein refers to a Fab in which the domains of the first Fv are attached to the N-terminus of the heavy and light chains of the Fab and the domain of the second Fv is attached to the C-terminus of the heavy and light chains, and wherein the second Fv comprises an internal variable region disulfide bond.

As used herein, dsFvFab ' Fv refers to a Fab ' in which the domains of the first Fv are attached to the N-terminus of the heavy and light chains of the Fab ', wherein the first Fv comprises an internal variable region disulfide bond and the domain of the second Fv is attached to the C-terminus of the heavy and light chains.

FvFab ' dsFv as used herein refers to Fab ' in which the domain of the first Fv is attached to the N-terminus of the heavy and light chains of Fab ' and the domain of the second Fv is attached to the C-terminus of the heavy and light chains, and wherein the second Fv comprises an internal variable region disulfide bond.

As used herein, dsFvFabdsFv refers to a Fab in which the domains of the first Fv are attached to the N-terminus of the heavy and light chains of the Fab, wherein the first Fv comprises an internal variable region disulfide bond and the domain of the second Fv is attached to the C-terminus of the heavy and light chains, and wherein the second Fv also comprises an internal variable region disulfide bond.

As used herein, dsFvFab ' dsFv refers to a Fab ' in which the domains of the first Fv are attached to the N-terminus of the heavy and light chains of the Fab ', wherein the first Fv comprises an internal variable region disulfide bond and the domain of the second Fv is attached to the C-terminus of the heavy and light chains, and wherein the second Fv also comprises an internal variable region disulfide bond.

Fabfv Fv as used herein refers to Fab fragments having two pairs of Fv attached in tandem to the C-terminus of the heavy and light chains, see for example WO2011/086091.

Fab 'ffv as used herein refers to a Fab' fragment having two pairs of Fv attached in series to the C-terminus of the heavy and light chains, see for example WO2011/086091.

Fabdsfv as used herein refers to Fab fragments having two pairs of Fv's attached in series to the C-terminus of the heavy and light chains, see for example WO2011/086091, wherein the first Fv pair attached directly to the C-terminus comprises an internal variable region disulfide bond.

Fab ' dsfv Fv as used herein refers to a Fab ' fragment having two pairs of Fv's attached in series to the C-terminus of the heavy and light chains, see for example WO2011/086091, wherein the first Fv pair attached directly to the C-terminus comprises an internal variable region disulfide bond.

FabFvdsFv as used herein refers to Fab fragments having two pairs of Fv's attached in series to the C-terminus of the heavy and light chains, wherein the second Fv pair at the "C" -terminus of the molecule comprises an internal variable region disulfide bond.

Fab 'fvdfv as used herein refers to a Fab' fragment having two pairs of Fv attached in series to the C-terminus of the heavy and light chains, wherein the second Fv pair at the "C" -terminus of the molecule comprises an internal variable region disulfide bond.

FabdsFvdsFv, as used herein, refers to a Fab fragment having two pairs of Fv's attached in series to the C-terminus of the heavy and light chains, wherein the first and second Fv pairs comprise an internal variable region disulfide bond.

Fab 'dsfvdfv as used herein refers to a Fab' fragment having two pairs of Fv's attached in series to the C-terminus of the heavy and light chains, wherein the first and second Fv's comprise an internal variable region disulfide bond.

DiFab as used herein refers to two Fab molecules linked by the C-terminus of their heavy chains.

DiFab 'as used herein refers to two Fab' molecules that are linked by one or more disulfide bonds in their hinge region.

DiFab and DiFab' molecules include chemically conjugated versions thereof.

As used herein (FabscFv) ₂ Refers to a di fab molecule with two scFv attached, for example, to the C-terminus of a heavy or light chain (such as a heavy chain).

As used herein (Fab' scFv) ₂ Refers to a di fab' molecule with two scFv attached, e.g., to a heavy or light chain (such as a heavy chain)C-terminal.

As used herein (Fab) ₂ scFvdsFv refers to a di Fab with scFv and dsFv attached, e.g., one from the C-terminus of each heavy chain.

As used herein (Fab') ₂ scFvdsFv refers to a di Fab' to which scFv and dsFv are attached, e.g., one from the C-terminus of each heavy chain.

As used herein (Fab) ₂ dsscFvdsFv refers to a di Fab with dsscFv and dsFv attached, e.g., from the heavy chain C-terminus.

As used herein (Fab') ₂ dsscFvdsFv refers to dsFv and dsFv attached di Fab', e.g.from the heavy chain C-terminus.

Minibody as used herein refers to (VL/VH-CH ₃ ) ₂ 。

As used herein, ds minibodies refer to (VL/VH-CH ₃ ) ₂ One of the VL/VHs contains internal variable region disulphide bonds.

Dids minibodies as used herein refer to (dsFv-CH) ₃ ) ₂ 。

scFv-Fc as used herein refers to an scFv attached to the N-terminus of the CH2 domain of the constant region fragment- (CH 2CH 3), e.g., via a hinge, such that the molecule has 2 binding domains.

As used herein, dsscFv-Fc refers to a dsscFv attached to the N-terminus of the CH2 domain of the constant region fragment- (CH 2CH 3) 2 and a scFv attached to the N-terminus of the second CH2 domain, e.g., attached via a hinge, such that the molecule has 2 binding domains.

As used herein, a didscfv-Fc refers to a scFv attached to the N-terminus of the CH2 domain of the constant region fragment- (CH 2CH 3) 2, e.g., attached via a hinge, such that the molecule has 2 binding domains.

Tandem scFv-Fc as used herein refers to two tandem scFv, each of which is attached in tandem to a constant region fragment- (CH) ₂ CH ₃ ) CH of (2) ₂ The N-terminus of the domain is attached, for example via a hinge, such that the molecule has 4 binding domains.

Sc diabody-Fc as used herein is twosc diabodies, each of which is attached to a constant region segment-CH ₂ CH ₃ CH of (2) ₂ The N-terminus of the domain is attached, for example via a hinge.

ScFv-Fc-ScFv as used herein refers to 4 ScFv, each of which is attached to both the N-terminus and the C-terminus of the heavy and light chains of a-CH 2CH3 fragment.

Sc diabodies-CH as used herein ₃ Refers to two sc diabody molecules, each of which is linked to CH, e.g., via a hinge ₃ A domain.

The `kappa/lambda body` or `kappa/lambda body` is a form of normal IgG having two heavy chains and two light chains, wherein the two light chains are different from each other, one being a lambda light chain (VL-CL) and the other being a kappa light chain (VK-CK). The heavy chains are identical, even at the CDRs, as described in WO 2012/023053.

An IgG-scFv as used herein is a full length antibody, wherein the scFv is on the C-terminus of each heavy or each light chain.

As used herein, scFv-IgG is a full-length antibody in which the scFv is on the N-terminus of each heavy chain or each light chain.

V-IgG as used herein is a full length antibody in which the variable domain is on the N-terminus of each heavy or light chain.

IgG-V as used herein is a full length antibody in which the variable domain is at the C-terminus of each heavy or light chain.

DVD-Ig (also known as double V domain IgG) is a full length antibody with 4 additional variable domains, one on each heavy and each light chain at the N-terminus.

Duobody or 'Fab-arm exchange' as used herein is a bispecific IgG-format antibody in which matched and complementary engineered amino acid changes in the constant domains (typically CH 3) of two different monoclonal antibodies result in heterodimer formation after mixing. Heavy chain from the first antibody as a result of residue engineering: the light chain pair will preferentially hybridize to the heavy chain of the second antibody: the light chain pairs associate. See, for example, WO2008/119353, WO2011/131746, and WO2013/060867.

The antibodies of the invention may be antibody fragments, and thus reference herein to antibodies also includes antibody fragments. In one embodiment, an antibody of the invention may be any antibody fragment disclosed herein comprising at least two different paratopes to CD 45. In another embodiment, an antibody of the invention may comprise an antibody fragment discussed herein that comprises only a single antigen binding site for CD45, but may be part of a binding molecule of the invention, or as one of the antibody mixtures discussed herein. In another embodiment, monovalent antibody fragments may be used in the present invention, preferably in the antibody mixtures described herein. As used herein, "binding fragment" refers to a fragment capable of binding to a target peptide or antigen that has sufficient affinity to characterize the fragment as specific for the peptide or antigen.

The term "Fab fragment" as used herein refers to an antibody fragment comprising: light chain fragment, and V _H (variable heavy chain) domain and first constant domain of heavy chain (CH ₁ ) Wherein the light chain fragment comprises V _L (variable light chain) domain and light chain (C) _L ) Is a constant domain of (a). The term "Fv" refers to two variable domains, e.g., a synergistic variable domain, such as a cognate pair or affinity matured variable domain, i.e., V _H And V _L For each pair. In one embodiment, such fragments are used as antibody molecules of the invention. The synergistic variable domains as used herein are complementary to each other and/or all participate in antigen binding such that Fv (V _H /V _L A variable domain specific for the antigen of interest.

Antibodies of the invention may include any of the antibody forms discussed herein, including in particular the Fab-X/Fab-Y, byBe, trYbe and IgG forms discussed herein. Antibodies in the form of BYBe, trYbe and IgG are particularly useful therapeutically. Antibodies of the invention may include forms comprising heavy and/or light chain variable regions, optionally comprising linkers or other entities linking together different parts of the antibodies. These antibodies may also be referred to as molecules. In a particularly preferred embodiment, the antibodies of the invention are in the form of IgG. In another particularly preferred embodiment, the antibody of the invention is in the form of BYBe. In another particularly preferred embodiment, the antibody of the invention is in the form of a TrYbe.

The antibodies of the invention may also be of the IgA, igE, igD or IgM type.

The degree of specificity (or specificity) for a target molecule, in particular for CD45, as used herein may refer to that a partner or related portion thereof in an interaction can only recognize each other or have a significantly higher affinity than each other than a non-partner, e.g. an affinity that is at least 10-fold, at least 100-fold, at least 1000-fold, at least 10,000-fold, at least 100,000-fold or at least 1,000,000-fold compared to e.g. the background binding level or binding to another unrelated protein (e.g. egg white lysozyme). In one embodiment, this degree of specificity is for CD45. In another embodiment, this specificity is not only for CD45, but also for an epitope of CD45 that is specifically bound by an antigen binding site, and in particular for the paratope of an antibody, as compared to other epitopes of CD45.

As used herein, a "binding site" refers to a binding region, typically a polypeptide, capable of binding to a target antigen, e.g., having sufficient affinity to characterize the site as specific for the antigen. In a preferred embodiment, the binding site binds CD45. In one embodiment, the binding site comprises at least one variable domain or derivative thereof, e.g., a pair of variable domains or derivatives thereof, such as a pair of homologous variable domains or derivatives thereof. Typically this is the VH/VL pair.

Any suitable antigen binding site may be used in the antibodies of the invention. In one embodiment, the binding site, in particular the paratope, comprises at least one variable domain or derivative thereof, e.g. a pair of variable domains or derivatives thereof, e.g. a pair of homologous variable domains or derivatives thereof. Typically this is the VH/VL pair.

The variable region (also referred to herein as the variable domain) typically comprises 3 CDRs and a suitable framework. In one embodiment, the antigen binding site comprises two variable regions, a light chain variable region and a heavy chain variable region, which together determine the specificity of the antibody or binding fragment for binding interaction with CD45, particularly in terms of the location on CD45 at which the binding site binds. In one embodiment, the variable domains used in the antigen binding sites of the antibody molecules of the invention are cognate pairs. "cognate pair" as used herein refers to a pair of heavy and light chain variable domains (or derivatives thereof, such as humanized forms) that are isolated from a host as a preformed pair. This definition does not include variable domains isolated from libraries, where the original pairing of hosts is not preserved. Homologous pairs may be advantageous because they are typically affinity matured in a host, and thus may have a higher affinity for the antigen to which they are specific than the combination of variable domain pairs selected from a library, such as a phage library. In another embodiment, the heavy and light chains in the binding site of an antibody of the invention may not be cognate pairs. In one embodiment, for example where a common light chain is used, the light chain is not homologous to at least one heavy chain variable region, but is still capable of forming a functional antigen binding site.

Derivatives, modifications and humanizations

As used herein, "derivative" is intended to mean that one, two, three, four or five amino acids are replaced or deleted in a naturally occurring sequence, for example, in order to optimize a property, such as by eliminating an undesired property, but wherein a characterization feature is retained. Examples of modifications are removal of glycosylation sites, GPI anchors or solvent exposed lysines. These modifications can be made by substituting conservative amino acid substitutions for the relevant amino acid residues.

Other modifications in the CDRs may include, for example, substitution of one or more cysteines with, for example, serine residues. Asn may be a deaminated substrate and this tendency may be reduced by replacing Asn and/or adjacent amino acids with alternative amino acids, such as conservative substitutions. The amino acid Asp in the CDRs may be isomerised. The latter can be minimized by replacing Asp and/or adjacent amino acids with alternative amino acids, such as conservative substitutions.

In one embodiment, one or more variable regions (e.g., in an antigen binding site in an antibody molecule of the invention) are humanized. Humanized forms of variable regions are also derivatives thereof, and in the context of this specification, humanized may include replacement of a non-human framework with a human framework, and optionally back-mutation of one or more residues to a "donor residue". As used herein, a donor residue refers to a residue present in the original variable region isolated from the host, particularly a substitution of an amino acid at the corresponding position in the donor frame for a given amino acid in the human frame. In one embodiment, any of the non-human variable regions disclosed herein may also be present in humanized form in an antibody molecule of the invention. In one embodiment, the CDRs disclosed herein are present in a human variable region framework. In another embodiment, the framework donor residues may also be transferred with the CDRs. In another embodiment, the antibodies of the invention comprise a fully human variable region. In another embodiment, the antibodies of the invention are fully human.

Antibody constant region and Fc region function

In a preferred embodiment, the antibody of the invention does not comprise an Fc domain.

In one embodiment, the antibodies of the invention comprise an altered Fc domain as described below. In another preferred embodiment, the antibodies of the invention comprise an Fc domain, but the sequence of the Fc domain has been altered to remove one or more Fc effector functions. In another embodiment, the Fc region of an antibody of the invention has been modified to optimize a particular property of the antibody, such as any of those disclosed herein.

In one embodiment, an antibody of the invention comprises a "silenced" Fc region. For example, in one embodiment, the antibodies of the invention do not exhibit effector functions associated with a normal Fc region.

The Fc domain as used herein is generally referred to as- (CH) ₂ CH ₃ ) ₂ Unless the context clearly indicates otherwise.

In one embodiment, the present inventionThe light antibody does not contain-CH ₂ CH ₃ Fragments.

In one embodiment, the antibodies of the invention do not comprise CH ₂ A domain.

In one embodiment, the antibodies of the invention do not comprise CH ₃ A domain.

In one embodiment, the antibodies of the invention do not bind to Fc receptors.

In one embodiment, the antibodies of the invention do not bind complement. In a preferred embodiment, the antibodies of the invention do not bind to the first complement factor C1q or C1. In one embodiment, the antibody of the invention does not bind those factors because, for example, it lacks an Fc region. In another embodiment, the antibody of the invention does not bind to these factors because it has modifications in the constant region that prevent its ability to do so. In an alternative embodiment, the antibody of the invention does not bind fcγr, but binds complement. For example, in one embodiment, the antibodies of the invention do not bind fcγr, but bind C1q and/or C1.

In one embodiment, the antibody of the invention does not comprise an active Fc region, in the sense that the antibody does not trigger the release of one or more cytokines that the normal Fc region would trigger. For example, the Fc region of an antibody of the invention may not trigger release of a cytokine when bound to an Fc receptor, or may not significantly trigger release of a cytokine.

In one embodiment, the binding molecules of the invention may generally include modifications that alter the serum half-life of the binding molecule. Thus, in another embodiment, the antibodies of the invention have an Fc region modification that alters the half-life of the antibody. Such modifications may be present as well as modifications that alter Fc function. In a preferred embodiment, the antibodies of the invention have modifications that alter the serum half-life of the antibody. In a particularly preferred embodiment, the antibodies of the invention have modifications that reduce the serum half-life of the antibody compared to antibodies lacking such modifications. In another preferred embodiment, the antibodies of the invention include modifications that collectively silence the Fc region and reduce the serum half-life of the antibody as compared to an antibody lacking such modifications.

The antibody constant region domains of the antibody molecules of the invention, if present, may be selected to take into account the proposed function of the antibody molecule, in particular the effector function that may be required. In preferred embodiments, the antibody is one that lacks Fc or lacks one or more effector functions of the Fc region and preferably lacks all effector functions. In other embodiments of the invention, the effector function of the Fc region of the antibody may still be present. In one embodiment, an antibody of the invention may comprise a human constant region, such as IgA, igD, igE, igG or IgM domain. In particular, human IgG constant region domains may be used, particularly when the antibody molecule is intended for therapeutic uses requiring antibody effector functions, igG1 and IgG3 isotypes may be used. Alternatively, igG2 and IgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and antibody effector function is not required. Particularly preferred IgG isotypes are IgG2 and IgG4. In preferred embodiments, the constant region may be modified such that the antibody does not have effector function. Thus, it will be appreciated that sequence variants of these constant region domains may also be used. For example, an IgG4 molecule may be used in which serine at position 241 has been changed to proline, as described by Angal et al, 1993,Molecular Immunology,1993, 30:105-108. Thus, in this embodiment, wherein the antibody is an IgG4 antibody, the antibody may comprise the mutation S241P. In another embodiment, the antibodies of the invention may lack an Fc region.

In one embodiment, an antibody of the invention may have a silent Fc region. The terms "silent", "silenced" or "silenced" as used herein refer to an antibody having a modified Fc region as described herein that has reduced binding to fcgamma receptor (FcgR) relative to the binding of the same antibody comprising an unmodified Fc region to FcgR (e.g., reduced binding to FcgR by at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100%, e.g., as measured by BLI relative to the binding of the same antibody comprising an unmodified Fc region to FcgR). In some embodimentsIn this case, the Fc-silent antibodies had no detectable binding to FcgR. Binding of antibodies having modified Fc regions to FcgR can be determined using a variety of techniques known in the art, such as, but not limited to, equilibration methods (e.g., enzyme-linked immunosorbent assay (ELISA); kinExA, rathanaswami et al Analytical Biochemistry, vol.373:52-60, 2008; or Radioimmunoassay (RIA)), or by surface plasmon resonance assays or other kinetics-based assay mechanisms (e.g., BIACORE) ^TM Analysis or Octet ^TM Analysis (forteBIO)), and other methods such as indirect binding assays, competitive binding assays, fluorescence Resonance Energy Transfer (FRET), gel electrophoresis, and chromatographic (e.g., gel filtration) methods. In another embodiment, the antibodies of the invention can be modified to reduce or eliminate their binding to FcgR, but still allow for activation of complement. In another embodiment, the antibodies of the invention may have an Fc region modified such that it does not activate cytokine release, but still activates complement.

In one embodiment, the antibody heavy chain comprises CH ₁ Domain, while antibody light chain includes CL domain (kappa or lambda). In one embodiment, the antibody heavy chain comprises CH ₁ Domain, CH ₂ Structure and CH ₃ Domains, while antibody light chains include a CL domain (k or λ).

The four human IgG isotypes bind with different affinities to the activated fcγ receptor (fcγri, fcγriia, fcγriic, fcγriiia), the inhibited fcγriib receptor and the first component of complement (C1 q), resulting in very different effector functions (Bruhns p. Et al, 2009.Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses.Blood.113 (16): 3716-25), see also Jeffrey b.stavenhagen et al, cancer Research 2007sep 15;67 (18):8882-90. In one embodiment, the antibodies of the invention do not bind to Fc receptors. In another embodiment of the invention, the antibody binds to one or more types of Fc receptors.

Binding of IgG to fcγr or C1q depends on the hinge region and CH ₂ Amino acid residues in the domain. CH (CH) ₂ DomainIs critical for fcγr and C1q binding and has unique sequences in IgG2 and IgG 4. Substitution of residues 233-236 of IgG2 and residues 327, 330 and 331 of IgG4 into human IgG1 greatly reduced their ADCC and CDC activities (ArmourKL. Et al, 1999.Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities.Eur J Immunol.29 (8): 2613-24 and Shields RL. et al, 2001,High resolution mapping of the binding site on human IgG1 for Fc gamma RI,Fc gamma RII,Fc gamma RIII,and FcRn and design of IgG1 variants with improved binding to the Fc gamma R.J Biol Chem.276 (9): 6591-604). Furthermore, idusogie et al demonstrated that alanine substitutions at various locations, including K322, significantly reduced complement activation (Idusogie EE et al 2000.Mapping of the C1q binding site on rituxan,a chimeric antibody with a human IgG1 Fc.JImmunol.164 (8): 4178-84). Similarly, the CH of murine IgG2A ₂ Mutations in the domains were shown to reduce binding to Fcgamm and C1q (Steurer W. Et al 1995.Ex vivo coating of islet cell allografts with murine CTLA4/Fc promotes graft tolerance. J immunol.155 (3): 1165-74).

In one embodiment, the Fc region employed is mutated, particularly the mutations described herein. In one embodiment, the mutation is to remove binding and/or effector functions. In a preferred embodiment, the antibodies of the invention have been mutated so that they do not bind to Fc receptors. In another preferred embodiment, the antibodies of the invention do not comprise an Fc region and therefore do not exhibit Fc effector activity for this reason. In one embodiment, the Fc mutation is selected from the group comprising: mutations that remove or enhance binding of the Fc region to Fc receptors, mutations that increase or remove effector function, mutations that increase or decrease antibody half-life, and combinations thereof. In a preferred embodiment, the modification eliminates or reduces binding to Fc receptors. In another preferred embodiment, the modification eliminates or reduces Fc effector function. In another preferred embodiment, the modification reduces serum half-life. In another preferred embodiment, the constant region of the antibody includes one or more modifications that reduce or eliminate Fc receptor binding, and Fc effector function, as well as reduce serum half-life. In one embodiment, where the effect of a modification is mentioned, it can be demonstrated by comparison with an equivalent antibody lacking the modification.

In another embodiment of the invention, the antibodies may have heavy chain modifications to alter the ability to bind to protein a, particularly to eliminate binding to protein a. As described herein, such a method may be preferred to facilitate the purification of bispecific antibodies. However, in other embodiments, if the antibodies of the invention have an Fc region, they may be modified to alter binding to protein a. For example, both heavy chains may comprise the modification. Alternatively, both heavy chains may lack the modification. In a preferred embodiment, however, one heavy chain has this modification, while the other does not.

Some antibodies that selectively bind FcRn at pH 6.0 but not pH 7.4 exhibit longer half-lives in a variety of animal models. Located at CH ₂ And CH (CH) ₃ Several mutations at the interface between domains, such as T250Q/M428L (Hinton PR. et al 2004.Engineered human IgG antibodies with longer serum half-antibodies in matrices J Biol chem.279 (8): 6213-6) and M252Y/S254T/T256 E+H2433K/N434F (Vaccaro C et al 2005.Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels.Nat Biotechnol.23 (10): 1283-8), have been shown to increase binding affinity to FcRn and the half-life of IgG1 in vivo. Thus, modifications to alter serum half-life may be present in M252/S254/T256+H244/N434, and in particular M252Y/S254T/T256 E+H24K/N434F may be present. However, there is not always a direct link between increasing FcRn binding and half-life (Datta-Mannan a. Et al, 2007.Humanized IgG1 Variants with Differential Binding Properties to the Neonatal Fc Receptor:Relationship to Pharmacokinetics in Mice and Primates.Drug Metab.Dispos.35:86-94). In one embodiment, it is desirable to increase half-life. In another embodiment, it may actually be desirable to reduce the serum half-life of the antibody, so modifications that reduce the serum half-life may be present.

The IgG4 subclass shows reduced Fc receptor (fcγriiia) binding and antibodies of other IgG subclasses generally show strong binding. Reduced receptor binding in these other IgG subclasses can be achieved by altering, for example, replacement of one or more amino acids selected from the group consisting of Pro238, aps265, asp270, asn270 (loss of Fc glycosyl), pro329, leu234, leu235, gly236, gly237, ile253, ser254, lys288, thr307, gin 311, asn434, and His 435. In one embodiment, the molecule according to the invention has an Fc of the IgG subclass, e.g. IgG1, igG2 or IgG3, wherein the Fc is mutated in one, two or all of the following positions: s228, L234 and/or D265. In one embodiment, the mutation in the Fc region is independently selected from S228P, L234A, L235A, L235A, L235E and combinations thereof.

In one embodiment, the antibodies of the invention may include modifications that affect whether the antibody causes cytokine release. In particular, L234F and K274Q modifications have been demonstrated to reduce the ability of antibodies to elicit cytokine release. Thus, in one embodiment, an antibody of the invention may comprise modifications at L234 and/or K274 that alter cytokine release, in particular L234F and K274Q modifications. Furthermore, the L234 residue may have an effect on platelet activation, and thus the residue may additionally or alternatively be modified. For example, in one embodiment of the invention, an L234 modification, particularly an L234F modification, that alters platelet binding may be introduced. P331 has also been shown to play a role in C1q binding, and thus in one embodiment, P331 may not be modified in order to preserve complement activation. In another embodiment, modifications may be made to reduce or eliminate complement activation; for example, the heavy chain may comprise a P331S modification. In another embodiment, there is a P329 modification, particularly a P329A modification, that reduces or eliminates complement binding. In another embodiment, the antibody may comprise one or more modifications at positions P329, P331, K332 and/or D265. In a preferred embodiment, the antibody may comprise modifications of P329A, P331S, K332A and D265A to affect complement fixation, in particular to reduce C1q binding.

It may be desirable to reduce or enhance the effector function of the Fc region. In a preferred embodiment, it is desirable to reduce these effector functions. In another embodiment, it is desirable to optimize it. The use of antibodies that target cell surface molecules, particularly molecules on immune cells, generally requires elimination of effector functions. In other cases, particularly where the goal is to deplete cells, it is desirable to eliminate Fc effector function or reduce effector function to as low a level as possible. For example, in a particularly preferred embodiment, the antibodies of the invention are capable of inducing cell death (preferably apoptosis) in target cells expressing CD45, but do not exhibit Fc effector function. Thus, in a preferred embodiment, the antibodies of the invention lack an active Fc region. For example, an antibody may have no physical Fc region, or an antibody may comprise modifications that inactivate an Fc region. The latter may for example be referred to as Fc silencing. In one embodiment, fc silencing may mean that antibodies of the invention are less capable or do not cause release of one or more cytokines, whereas antibodies with unmodified Fc regions will normally trigger release of these cytokines. In a preferred embodiment, the antibodies of the invention are capable of stimulating cell death (preferably apoptosis), but do not exhibit Fc function. Further examples of Fc functions include stimulating degranulation of mast cells, which may also be reduced or absent in the antibodies of the invention. The degree of reduction in Fc function may be, for example, at least 65%, such as at least 75%. In one embodiment, the degree of reduction is at least 80%. In another embodiment, the degree of reduction is at least 90%. The degree of reduction may be, for example, at least 95%. In a preferred embodiment, the degree of reduction is at least 99%. In another embodiment, the decrease may be 100%, which means that the Fc function is completely eliminated in this case.

CH of human IgG1 ₂ The domains were subjected to a number of mutations and their effect on ADCC and CDC was tested in vitro (Idusogenie EE et al 2001.Engineered antibodies with increased activity to recruit complement.J Immunol.166 (4): 2571-5). Notably, alanine substitutions at position 333 are reported to increase ADCC and CDC. Thus, in one embodiment, there may be a location 333Modifications, particularly those that alter the ability to recruit supplements. Lazar et al describe triple mutants (S239D/I332E/a 330L) that have higher affinity for fcγriiia and lower affinity for fcγriib, resulting in enhanced ADCC (Lazar GA. et al, 2006). Thus, there may be modifications at S239/I332/A330, particularly those that alter the affinity for Fc receptors, especially S239D/I332E/A330L. The engineered antibody Fc variants have enhanced effector functions (PNAS 103 (11): 4005-4010). The same mutations were also used to generate antibodies with enhanced ADCC (Ryan MC. et al 2007.Antibody targeting of B-cell maturation antigen on malignant plasma cells. Mol. Cancer Ther., 6:3009-3018). Richards et al studied slightly different triple mutants (S239D/I332E/G236A) with improved FcgammaRIIIa affinity and FcgammaRIIIa/FcgammaRIIB ratios that mediated enhanced phagocytosis of target cells by macrophages (Richards JO et al (2008) Optimization of antibody binding to Fcgamma RIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther.7 (8): 2517-27). In one embodiment, the S239D/I332E/G236A modification may thus be present.

Due to the lack of effector functions of IgG4 antibodies, igG4 antibodies represent a suitable subclass of IgG for receptor blocking. IgG4 molecules can exchange half-molecules in a dynamic process called Fab arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous IgG 4. In a preferred embodiment, the antibody of the invention has a modification at S228, in particular S228P. The S228P mutation has been shown to prevent this recombination process, allowing the design of less unpredictable therapeutic IgG4 antibodies (Labrijn AF. Et al, 2009.Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG in vivo. Nat Biotechnol.27 (8): 767-71). This technique can be used to create bispecific antibody molecules. In a preferred embodiment, the modifications presented herein may be applied in the context of IgG 4.

WO 2008/145142 discloses examples of some modifications, in particular of IgG4 isotype antibodies that can be used in the invention. In one embodiment, the heavy chain of an antibody of the invention may comprise a human IgG4 constant region with substitution of an Arg residue at position 409, a Phe residue at position 405, and/or a Lys residue at position 370. For example, in a preferred embodiment, the heavy chain of the antibody comprises a modification at position 409, in particular selected from one of the Lys, ala, thr, met or Leu residues introduced at that position. In one embodiment, the modification is the introduction of a Lys, thr, met or Leu residue at position 409. In another embodiment, the modification is the introduction of a Lys, met, or Leu residue at position 409. In one embodiment, the hinge region of the antibody does not comprise Cys-Pro-Pro-Cys. In one embodiment, the antibody exhibits a reduced ability to induce Fab arm exchange in vivo. In one embodiment, the hinge region of the antibody comprises a CXPC or CPXC sequence, wherein X is any amino acid other than proline. In one embodiment, the antibodies of the invention may utilize the ability of a particular antibody type, antibody isotype, or antibody isotype to display a particular property. This natural diversity can be used to impart specific properties. For example, igG1 has R409 at position 409 of the heavy chain, while IgG4 has K409 at that position, which can naturally affect the ability of the antibody. An overview of various naturally occurring sequence variations is provided in Jefferis et al (2009) mAbs,1 (4): 332-338, which is incorporated by reference in its entirety, particularly with respect to the sequence variations discussed therein.

Those skilled in the art will also appreciate that antibodies may undergo various post-translational modifications. The type and extent of these modifications generally depend on the host cell line used to express the antibody and the culture conditions. These modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and aspartic acid deamidation. Frequent modifications are due to the action of carboxypeptidase enzymes leading to the loss of basic residues at the carboxy terminus, such as lysine or arginine (as described in Harris, RJ. Journal of Chromatography 705:705:129-134,1995). Thus, the C-terminal lysine of the antibody heavy chain may be deleted.

In one embodiment, the antibodies of the invention may be aglycosylated IgG, e.g., to reduce Fc function, particularly an almost Fc-empty phenotype. In one embodiment, the antibody of the invention has a modification at N297, in particular N297A. In one embodiment, the antibodies of the invention have modifications at F243 and/or F244, in particular meaning that the antibodies are aglycosylated IgG. In one embodiment, the antibodies of the invention may comprise F243A and/or F244A heavy chain modifications. In another embodiment, one or more of F241, F243, V262 and V264 may be modified, in particular to amino acids that affect glycosylation. In one embodiment, the antibodies of the invention may have modifications of F241A, F243A, V262E and V264E. These modifications are discussed in Yu et al (2013) 135 (26): 9723-9732, which is incorporated by reference in its entirety, particularly with respect to the modifications discussed therein. These modifications provide a means of modulating, for example, fc receptor binding. Modifications that affect antibody glycosylation may be present. Furthermore, the antibodies of the invention may be produced in cell types that affect glycosylation as a further means of glycosylation engineering. In one embodiment, fucosylation, sialylation, galactosylation and/or mannosylation of an antibody of the invention may be altered by sequence modification and/or via the cell type used to produce the antibody.

In one embodiment, the antibodies of the invention have modifications at positions 297 and/or 299. For example, in one embodiment, an antibody of the invention comprises an N297A modification in its heavy chain, preferably N297Q or a mutation of Ser or Thr at 299 to another residue. In one embodiment, it has both of these modifications.

In one embodiment, the antibodies of the invention may have modifications that facilitate the formation of the antibodies of the invention rather than unwanted species. For example, in one embodiment, the production of antibodies of the invention may involve two different antigenic sites, in particular two different paratopes, located on different units and associated together. Thus, it is desirable to form a heterodimer comprising two paratopes, rather than a homodimer comprising only one paratope. An example of a method that favors heterodimer formation is the use of heavy chain modifications that favor two different heavy chain associations (rather than two identical heavy chain associations). In one embodiment, one (or at least one) of the binding partners is incapable of forming a homodimer, e.g., the amino acid sequence of the binding partner is mutated to eliminate or minimize homodimer formation. Examples of such modifications include so-called "mortar and pestle" modifications. Possible socket modifications are described in Merchant et al (1998) Nature Biotechnology (7): 677-681 and Carter et al (2001) J Immunol Methods,248 (1-2): 7-15, the entire contents of both of which are incorporated by reference, particularly with respect to the socket modifications discussed therein. Charge modifications may alternatively or additionally be used to promote heterodimer formation relative to homodimers, for example these modifications may be present in the heavy chain. In another embodiment, charge modification is used to pair a particular light chain with a particular heavy chain.

In one embodiment, these modes of promoting heterodimer formation are used in combination with a common light chain mode. In another embodiment, it may not be the relative homodimer that promotes heterodimer formation, but rather modifications exist that mean that the heterodimer can be more easily separated from the homodimer, for example by chromatography. Also, in some embodiments, this approach may be used in combination with a common light chain approach. In another embodiment, the portions of the antibody bearing a particular paratope for CD45 can only be associated with those portions of the antibody that include different paratopes of the antibody.

In one embodiment, neither binding partner is capable of forming a homodimer, e.g., one of the binding partners is a peptide and the other binding partner is V specific for the peptide _HH . In one embodiment, the scFv employed in the molecules of the invention are incapable of forming homodimers.

The inability to form homodimers as used herein means that the tendency to form homodimers is low or 0. As used herein, "low" refers to 5% or less, such as 4%, 3%, 2%, 1%, 0.5% or less, of aggregates.

In another embodiment, the antibodies of the invention may have a modified hinge region and/or CH1 region. Alternatively, the isoform employed may be selected because it has a specific hinge region. IgG2CH1 and hinge regions impart specific properties, particularly with respect to disulfide bonds between heavy and light chains, as described in White et al (2015) Cancer Cell 27 (1): 138-148. For example, modifications that utilize a promotion of flexibility or a reduction of flexibility in the hinge region may also be employed in the antibodies of the invention. A way to change the flexibility of the hinge region is disclosed in Liu et al (2019) Nature Communications 10:4206. The entire contents of White et al (2015) and Liu et al (2019) are incorporated by reference, particularly with respect to the modifications in question. In one embodiment, the heavy chain of the antibody of the invention has IgG2CH1 and/or hinge regions, and in another embodiment, both heavy chains have these regions. In one embodiment, the antibody employed is an h2 antibody. In a particularly preferred embodiment, the antibody employed may be an IgG2 or IgG4 antibody with a hinge or CH1 modification, in particular an antibody with a modified hinge region, e.g. an antibody engineered to alter disulfide bond formation. In another embodiment, antibodies of the IgG2 or IgG4 isotype are employed, as the hinge regions of these isotypes exhibit less flexibility than the hinge regions of antibodies of the IgG3 isotype. In one embodiment, an IgG4 isotype antibody is employed, which is in a form that may be capable of causing CD32 to crosslink.

In another embodiment, the antibody exhibits the optimal ability to spatially induce cross-linking of CD45 molecules.

Bispecific antibodies

In a preferred embodiment, the binding molecules (particularly antibodies) of the invention are bispecific. Thus, in a preferred embodiment, bispecific antibodies are employed in the present invention, and in a particularly preferred embodiment, bispecific antibodies having two different specificities for CD45 are employed. There are a variety of bispecific antibody formats that can be used to facilitate the formation or purification of bispecific antibodies relative to monospecific antibodies when the different heavy and light chains for each specificity are expressed together, and these formats can be used in the present invention.

For example, shape or charge modifications may be present in the heavy chain for one or both specificities that favor heterodimer formation rather than homodimer formation. Examples of such modifications include mortar and pestle heavy chain modifications, which means that two different heavy chains for different specificities are more likely to interact, thereby favoring heterodimer formation. The strand-exchange engineering domain (SEEDbody) approach may also be used to promote heterodimer formation.

Heavy chain modifications may also be employed such that one heavy chain has a different affinity for binding agents than the other heavy chain. For example, two different heavy chains may have different affinities for protein a. In one embodiment, one heavy chain has modifications that eliminate protein a binding or isoforms that do not bind protein a, while the other heavy chain remains bound to protein a. Although this does not alter the proportion of heterodimers formed, it allows for the purification of heterodimeric antibodies from either homodimeric antibody based on protein a affinity. Antibodies of the invention may have modifications at positions 95 and 96 of one heavy chain that affect protein a binding. Examples of such modifications that may be employed include modifications with H95R in one heavy chain or modifications with H95R and Y96F, both modifications according to the IMGT exon numbering system. These modifications are H435R modifications, H435R and Y436F modifications according to the EU numbering system. In one embodiment, the antibodies of the invention may also have modifications at D16, L18, N44, K52, V57, and V82. In one embodiment, these modifications are present in the heavy chain, and one or more of the D16E, L18M, N44S, K N, V M and V82I modifications (according to IMGT numbering system). In one embodiment, such modifications are employed where the IgG is IgG1, igG2 or IgG 4. In a particularly preferred embodiment, where both heavy chains are of the IgG4 isotype, they are used for one of the two heavy chains. Such modifications affecting the binding of protein a are described in, for example, US 2010/0331527 A1, the entire contents of which are incorporated by reference, and in particular with respect to the modifications that it discloses in relation to protein a binding.

In further embodiments, the isotypes of the heavy chains used may be selected based on their ability to bind to protein a. For example, in humans, wild-type forms of IgG1, igG2 and IgG4 all bind to protein a, whereas wild-type human IgG3 cannot. In a particularly preferred embodiment, both heavy chains are IgG4, but one of the heavy chains has a modification that reduces or eliminates binding to protein a. This means that antibodies in heterodimeric form will be able to more easily separate from unwanted homodimeric forms based on affinity to protein a.

In one embodiment, the modification that promotes heterodimer formation may be combined with a modification that allows for heterodimer purification. In one embodiment, these modifications may be at positions F405 and K409. For example, a pair of modifications that can be introduced into two heavy chains to promote heterodimer formation are F405L and K409R. Those modifications may be used alone or in combination with heavy chain modifications that allow preferential purification of the heterodimer. In one embodiment, one heavy chain has modifications at positions 405, 409, 435, and 436, while the other heavy chain has modifications at position 409. In one embodiment, one heavy chain has the F405L modification and the other heavy chain has the K409R, H435R and Y436F modifications. In another embodiment, one heavy chain has the F405L, H435R and Y436F modifications and the other heavy chain has the K409R modifications. Examples of such means are described in Steinhardt et al (2020) pharmaceuticals, 12,3, the entire contents of which are incorporated by reference, particularly with respect to the bispecific antibody formats and heavy chain modifications described. In another embodiment, a light chain related approach, particularly in addition to the heavy chain approach described above, may be employed. For example, for one light chain portion of a desired pairing of a light chain and a heavy chain, the light chain and heavy chain portions may be interchanged with one another to promote the formation of the light chain heavy chain pairing, while the other specific heavy and light chain portions are not modified. In one embodiment, the Roche Cross-Mab approach is therefore used. In another embodiment, a common light chain may be employed, such that the same light chain is used for both specificities. Various bispecific antibody formats are reviewed in Spiess et al (2015) Molecular Immunology 67:95-106 and may be used in the present invention, including in particular those shown in FIG. 1 of that reference. Spiess et al (2015) are incorporated by reference, including in particular for the types of antibody formats shown in FIG. 1 of this reference.

Antibody production and screening

In one embodiment, the antibodies of the invention, or antibody/fragment components thereof, are treated to provide increased affinity for one or more target antigens, particularly for CD 45. Such variants can be obtained by a variety of affinity maturation assay protocols, including mutant CDRs (Yang et al, j. Mol. Biol.,254,392-403,1995), strand shuffling (Marks et al, bio/Technology,10,779-783,1992), use of e.coli mutants (Low et al, j. Mol. Biol.,250,359-368,1996), DNA shuffling (pattern et al, curr. Opin. Biotechnol.,8,724-733, 1997), phage display (Thompson et al, j. Mol. Biol.,256,77-88,1996) and sexual PCR (Crameri et al, nature,391,288-291,1998). Vaughan et al (supra) discuss these affinity maturation methods. The binding domains for use in the present invention may be generated by any suitable method known in the art, e.g., CDRs may be obtained from non-human antibodies (including commercially available antibodies) and grafted into a human framework, or alternatively chimeric antibodies may be made with non-human variable and human constant regions, and so forth.

Examples of CD45 antibodies are known in the art, and paratopes of these antibodies may be used in the antibodies of the invention, which have more than one specificity for CD45, or screened for their suitability using the methods described herein, and if desired subsequently modified, e.g., humanized using the methods described herein. Therapeutic anti-CD 45 antibodies have been described in the prior art, for example anti-CD 45 antibodies disclosed in US 2011/00746270. Examples of CD45 antibodies include rat monoclonal YTH54, YTH25.4, mouse monoclonal from Miltenyi clone 5B1 and clone 30F11, rat monoclonal YAML568, mouse monoclonal clone 2D1 from BD Bioscience under accession number 347460, mouse monoclonal antibody 5D3A3 from Novus under accession number NBP2-37293, mouse monoclonal HI30 under accession number NBP1-79127, mouse monoclonal 4A8A4C7A2 under accession number NBP1-47428, mouse monoclonal 2B11 under accession number NBP2-32934, rat monoclonal YTH24.5 under accession number NB100-63828, rabbit monoclonal Y321 under accession number NB110-55701, mouse monoclonal PD7/26/16 under accession number NB120-875, mouse monoclonal B8 from Santa Cruz under accession number sc-28369, the mouse monoclonal from clone F10-89-4 under accession number sc-52490, the rabbit monoclonal from clone H-230 under accession number sc-25590, the goat monoclonal from clone N-19 under accession number sc-1123, the mouse monoclonal from clone OX1 under accession number sc-53045, the rat monoclonal (T29/33) under accession number sc-18901, the rat monoclonal (YAML 501.4) under accession number sc65344, the rat monoclonal (YTH 80.103) under accession number sc-59071, the mouse monoclonal (351C 5) under accession number sc-53201, the mouse monoclonal (35-Z6) under accession number sc-1178, the mouse monoclonal (158-4D 3) under accession number sc-52386, the mouse monoclonal (UCH-L1) under accession number sc-1183 against CD45RO, the mouse monoclonal against CD45RO (2Q 1392) under the accession number sc-70712. CD45 antibodies are also disclosed in WO2005/026210, WO02/072832 and WO2003/048327, incorporated herein by reference. These commercially available antibodies may be a useful tool in the discovery of therapeutic antibodies. In a particularly preferred embodiment, the antibody of the invention is a human antibody or an antibody that has been humanized. Thus, in one embodiment, commercially available antibodies may be humanized. Although specific examples of preferred antibodies, and methods of identifying further antibodies, are set forth in this application.

The skilled artisan can use any suitable method known in the art to generate antibodies for use in the antibodies of the invention. Antigenic polypeptides for the production of antibodies, for example for immunization of a host or for panning (such as in phage display), may be prepared from genetically engineered host cells comprising an expression system by methods well known in the art or they may be recovered from natural biological sources. In this application, the term "polypeptide" includes peptides, polypeptides and proteins. Unless otherwise indicated, these terms are used interchangeably. In some cases, the antigen polypeptide may be part of a larger protein, such as a fusion protein fused to an affinity tag or the like. In one embodiment, the host may be immunized with cells transfected with CD45, e.g., expressing CD45 on the surface of the cells.

In the case where it is desired to immunize an animal, antibodies raised against the antigenic polypeptide may be obtained by injecting the polypeptide into the animal (preferably a non-human animal) using well known and conventional experimental protocols, see for example Handbook of Experimental Immunology, d.m. weir (editions), vol 4,Blackwell Scientific Publishers,Oxford,England,1986. Many warm-blooded animals, such as rabbits, mice, rats, sheep, cattle, camels or pigs, can be immunized. However, mice, rabbits, pigs and rats are generally most suitable. Monoclonal antibodies may be prepared by any method known in the art, such as the hybridoma technique (Kohler & Milstein,1975, nature, 256:495-497), the trioma technique (trioma technique), the human B cell hybridoma technique (Kozbor et al, 1983,Immunology Today,4:72) and the EBV-hybridoma technique (Cole et al, monoclonal Antibodies and Cancer Therapy, pages 77-96, alan R Lists, inc., 1985).

Antibodies can also be generated using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNA produced from single lymphocytes, such as by Babcook, J. Et al, 1996,Proc.Natl.Acad.Sci.USA 93 (15): 7843-7848l; WO92/02551; the methods described in WO2004/051268 and WO2004/106377 were chosen for the production of specific antibodies. Antibodies for use in the present invention may also be generated using a variety of phage display methods known in the art, and include those produced by Brinkman et al (in J.Immunol. Methods,1995, 182:41-50), ames et al (J.Immunol. Methods,1995, 184:177-186), kettlebough et al (Eur. J.Immunol.1994, 24:952-958), persic et al (Gene, 1997 187 9-18), burton et al (Advances in Immunology,1994, 57:191-280) and WO90/02809; WO91/10737; WO92/01047; WO92/18619; WO93/11236; WO95/15982; WO95/20401; and US 5,698,426;5,223,409;5,403,484;5,580,717;5,427,908;5,750,753;5,821,047;5,571,698;5,427,908;5,516,637;5,780,225;5,658,727;5,733,743;5,969,108, and those disclosed in WO 20011/30305. In a preferred embodiment, the antibodies of the invention have at least two different paratopes specific for CD45, possibly by first generating antibodies recognizing one paratope of CD45, and then using two of those antibodies, for example, to generate antibodies of the invention capable of binding to at least two different paratopes of CD 45. For example, a plurality of antibodies to CD45 can be generated using the methods described herein, and then screened for a desired property (such as binding affinity). The best candidate may then be used to generate antibodies of the invention.

In one example, the antibodies of the invention are fully human, particularly the one or more variable domains are fully human. Fully human molecules are those in which the variable and constant regions (when present) of both the heavy and light chains are of human origin, or are substantially identical to sequences of human origin, not necessarily from the same antibody. Examples of fully human antibodies may include, for example, antibodies produced by the phage display method described above, as well as antibodies produced by mice in which the mouse immunoglobulin variable region and optionally constant region genes have been replaced by their human counterpart genes, as generally described, for example, in EP0546073, US5,545,806, US5,569,825, US5,625,126, US5,633,425, US5,661,016, US5,770,429, EP 0438474 and EP 0463151. Single paratope antibodies may be produced first and then used to produce antibodies of the invention that include at least two different paratopes to CD 45.

In one example, the antigen binding site, particularly the variable region, of the antibodies of the invention is humanized. Humanization, including CDR-grafted antibodies, as used herein refers to molecules having one or more Complementarity Determining Regions (CDRs) from a non-human species and framework regions from a human immunoglobulin molecule (see, e.g., US5,585,089; wo 91/09967). It is to be understood that it may only be necessary to transfer specific determining residues of a CDR, not the whole CDR (see e.g. Kashmiri et al 2005, methods,36, 25-34). Nonetheless, in a preferred embodiment, one or more entire CDRs are grafted. The humanized antibody may optionally further comprise one or more framework residues derived from a non-human species from which the CDRs are derived. As used herein, the term "humanized antibody molecule" refers to an antibody molecule in which the heavy and/or light chain comprises one or more CDRs (including one or more modified CDRs, if desired) from a donor antibody (e.g., a murine monoclonal antibody) grafted into the heavy and/or light chain variable region framework of a recipient antibody (e.g., a human antibody). For review, see Vaughan et al, nature Biotechnology,16,535-539,1998. In one embodiment, not the entire CDR is transferred, but only one or more specificity determining residues from any one of the CDRs described herein are transferred to a human antibody framework (see, e.g., kashmiri et al 2005, methods,36, 25-34). In one embodiment, only the specificity determining residues from one or more of the CDRs described herein are transferred into a human antibody framework. In another embodiment, only the specificity determining residues from each CDR described herein are transferred into the human antibody framework.

When grafting CDRs or specificity determining residues, any suitable acceptor variable region framework sequence may be used, taking into account the class/type of donor antibody from which the CDRs are derived, including mouse, primate and human framework regions. Suitably, a humanized antibody according to the invention has variable domains comprising human acceptor framework regions and one or more CDRs provided herein. Examples of human frameworks that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al, supra). For example, KOL and nemm can be used for the heavy chain, REI can be used for the light chain, EU, LAY, and POM can be used for both the heavy and light chains. Alternatively, a human germline sequence may be used; these are available at the following web sites: http:// www2.Mrc-lmb. Cam. Ac. Uk/vbase/list2.Php.

In the humanized antibody molecules of the invention, the heavy and light chains of the receptor do not necessarily need to be derived from the same antibody, and may, if desired, comprise composite chains having framework regions derived from different chains. The framework regions need not have exactly the same sequence as the acceptor antibody framework regions. For example, for that acceptor chain class or type, unusual residues may be altered to more frequently occurring residues. Alternatively, selected residues in the acceptor framework region may be altered to correspond to residues found at the same position in the donor antibody (see Reichmann et al, 1998, nature,332, 323-324). This change should be kept to a minimum necessary to restore donor antibody affinity. An experimental protocol for selection of residues in a region of the acceptor framework that may need to be altered is given in WO 91/09967. Derivatives of the framework may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids replaced with alternative amino acids, for example with donor residues. The donor residues are residues from the donor antibody, i.e. the antibody from which the CDRs were originally derived, in particular residues from the corresponding positions of the donor sequence are employed. The donor residues may be replaced by suitable residues (acceptor residues) derived from the human acceptor framework.

Residues in the antibody variable domains are generally numbered according to the system designed by Kabat et al, which is described in Kabat et al, 1987,Sequences of Proteins of Immunological Interest,US Department of Health and Human Services,NIH,USA (hereinafter "Kabat et al (supra)"). This numbering system is used throughout this specification unless otherwise indicated. Kabat residue names do not always correspond directly to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to a shortening or insertion of the structural elements of the basic variable domain structure, whether framework or Complementarity Determining Regions (CDRs). For a given antibody, the correct Kabat residue number may be determined by aligning homologous residues in the antibody sequence to a "standard" Kabat numbering sequence. According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3). However, according to Chothia (Chothia, C. And Lesk, A.M.J.mol.biol.,196, 901-917 (1987)), the loop corresponding to CDR-H1 extends from residue 26 to residue 32. Thus, unless otherwise indicated, "CDR-H1" as used herein is intended to refer to residues 26 to 35 as described by the combination of the Kabat numbering system and the Chothia topology ring definition. According to the Kabat numbering system, the CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3).

In one embodiment, the invention extends to antibody sequences disclosed herein, in particular to humanized sequences disclosed herein.

In one example, the binding domain is humanized.

In one example, one or more CDRs provided herein can be modified to remove unwanted residues or sites, such as cysteine residues or aspartic acid (D) isomerization sites or asparagine (N) deamidation sites. In one example, an asparagine deamidation site may be removed from one or more CDRs by mutating the asparagine residue (N) and/or adjacent residues to any other suitable amino acid. In one example, an asparagine deamidation site such as NG or NS may be mutated, e.g., to NA or NT.

In one example, the aspartate isomerization site may be removed from one or more CDRs by mutating the aspartate residue (D) and/or adjacent residues to any other suitable amino acid. In one example, an aspartate isomerization site such as DG or DS may be mutated, e.g., to EG, DA or DT.

For example, one or more cysteine residues in any one CDR may be substituted with another amino acid, such as serine.

In one example, an N-glycosylation site such as NLS can be removed by mutating asparagine residue (N) to any other suitable amino acid (e.g., to SLS or QLS). In one example, an N-glycosylation site, such as NLS, can be removed by mutating serine residue (S) to any other residue than threonine (T).

The skilled artisan can test variants or humanized sequences of CDRs in any suitable assay, such as those described herein, to confirm whether activity is maintained.

Specific binding to an antigen may be tested using any suitable assay, including, for example, ELISA or surface plasmon resonance methods, such as BIAcore, where binding to an antigen (CD 45) may be measured. Such assays may use isolated native or recombinant CD45 or a suitable fusion protein/polypeptide. In one example, binding is measured by, for example, surface plasmon resonance (such as BIAcore) using recombinant CD45 (SEQ ID NO:41 or amino acids 23-1304 of SEQ ID NO: 41). Alternatively, the protein may be expressed on cells (such as HEK cells) and affinity measured using a flow cytometry-based affinity assay. In one embodiment, where it is desired to separately determine the nature of an antigen binding site (particularly the paratope), antibodies are generated using only that paratope. For example, antibodies of the invention are produced in the same form as antibodies having two different specificities, but only one specificity for CD45 is present. In one embodiment, antibodies from the antibodies of the invention having at least two paratopes may be raised against each paratope of CD45, e.g., to allow for determining the affinity of each paratope or to determine whether paratopes exhibit cross-blocking with each other. In one embodiment, the ability to bind to the extracellular region of CD45 is measured, for example using SEQ ID NO:113, a protein of 113. In one embodiment, monovalent antibodies, such as ScFv, are generated for comparison.

Antibodies comprising paratopes that cross-block binding of paratopes of an antibody molecule according to the invention may be similarly used to bind CD45 and thus similarly useful antibodies may be used, for example, in the antibodies of the invention. Thus, the use of such cross-blocking or competition assays can be a useful method for identifying and producing antibodies of the invention. In one embodiment, a single paratope from an antibody of the invention may be used to generate a bivalent antibody, wherein both antigen binding sites comprise the paratope, and then the bivalent antibody is used to perform a cross-blocking assay.

In another embodiment, antibodies capable of cross-blocking at least one paratope of one of the antibodies disclosed herein can be used to generate an antibody molecule of the invention. In one embodiment, an antibody of the invention may include one of the paratopes listed herein, or include paratopes that are capable of cross-blocking or competing therewith. Accordingly, the present invention also provides an antibody molecule comprising a binding domain specific for the antigen CD45, wherein the binding domain for CD45 cross-blocks the binding of at least one of the CD45 specific paratopes of any of the antibody molecules described above to CD45 and/or by any of those paratopes. Generally, although the different paratopes specific for CD45 and the different paratopes of CD45 in the antibodies of the invention do not cross-block or compete with each other for binding to CD45 or do not cross-block or compete significantly. For example, a cross-blocking of less than 30%, preferably less than 25%, more preferably less than 10% will be seen. In one embodiment, less than 5%, in particular less than 1% cross-blocking will be seen. In one embodiment, it is desirable to cross-block paratopes as a means of identifying more paratopes specific for CD45 to be used in the antibodies of the invention, e.g., to replace the existing paratopes of the cross-block. In another embodiment, the cross-blocked antibody binds to an epitope that is contiguous with and/or overlaps with an epitope to which the CD 45-specific paratope of the antibody described above binds. In another embodiment, the cross-blocked neutralizing antibody binds to an epitope that is contiguous with and/or overlaps with an epitope bound by the paratope of the antibody described above to CD45. The cross-blocking assay may also be used to check whether at least two different paratopes of the antibodies of the invention against CD45 bind to different epitopes of CD45 and thus do not cross-block each other's binding. In one embodiment, two antibodies may be produced, each comprising only one paratope for CD45, and the ability of each to cross-block the other is measured. While antibodies and/or specificities for CD45 should generally not cross-block or compete with other antibodies, they should be able to bind CD45 simultaneously.

The cross-blocking antibodies may be identified using any suitable method in the art, for example by using a competition ELISA or BIAcore assay, wherein binding of the cross-blocking antibodies to the antigen (CD 45) prevents binding of the antibodies of the invention, and vice versa. Such cross-blocking assays may use isolated native or recombinant CD45 or a suitable fusion protein/polypeptide. In one example, binding and cross-blocking are measured using recombinant CD45 (SEQ ID NO: 41), e.g., the cross-blocking of any of these antibodies is 80% or more, e.g., 85% or more, such as 90% or more, particularly 95% or more. In one embodiment, the cross-blocking assay may be performed using antibodies having only one of the CD 45-specific paratopes in the antibodies of the invention.

The degree of identity and similarity can be readily calculated (Computational Molecular Biology, lesk, a.m., edit, oxford University Press, new York,1988;Biocomputing.Informatics and Genome Projects,Smith,D.W, edit, academic Press, new York,1993;Computer Analysis of Sequence Data,Part 1,Griffin,A.M, and Griffin, h.g., edit, humana Press, new Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G, academic Press,1987,Sequence Analysis Primer,Gribskov,M, and deveerux, j., edit, MStockton Press, new York,1991, blast) ^TM Software is available from NCBI (Altschul, S.F. et al, 1990, J.mol. Biol.215:403-410; gish, W).&State, D.J.1993, nature Genet.3:266-272, madden, T.L. et al, 1996, meth. Enzymol.266:131-141; altschul, S.F. et al 1997,Nucleic Acids Res.25:3389-3402; zhang, J.&Madden,T.L.1997,Genome Res.7:649-656)。

The invention also extends to novel polypeptide sequences disclosed herein and sequences at least 80% similar or identical thereto, for example 85% or more, such as 90% or more, in particular 95%, 96%, 97%, 98% or 99% or more, similarity or identity. In one embodiment, the sequence may have at least 99% sequence identity to at least one specific sequence provided herein. "identity" as used herein means that at any particular position in an aligned sequence, the amino acid residues are identical between the sequences. "similarity" as used herein means that at any particular position in an aligned sequence, the amino acid residues are of a similar type between the sequences. For example, leucine may be substituted for isoleucine or valine. Other amino acids that may be commonly substituted for one another include, but are not limited to:

phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

Lysine, arginine and histidine (amino acids with basic side chains);

aspartic acid and glutamic acid (amino acids with acidic side chains);

asparagine and glutamine (amino acids with amide side chains); and

cysteine and methionine (amino acids with sulfur-containing side chains).

The degree of identity and similarity can be readily calculated (Computational Molecular Biology, lesk, a.m., edit, oxford University Press, new York,1988;Biocomputing.Informatics and Genome Projects,Smith,D.W, edit, academic Press, new York,1993;Computer Analysis of Sequence Data,Part 1,Griffin,A.M, and Griffin, h.g., edit, humana Press, new Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G, academic Press,1987,Sequence Analysis Primer,Gribskov,M, and Devereux, j., edit, MStockton Press, new York,1991, blast) ^TM Software is available from NCBI (Altschul, S.F. et al, 1990, J.mol. Biol.215:403-410; gish, W).&State, D.J.1993, nature Genet.3:266-272, madden, T.L. et al, 1996, meth. Enzymol.266:131-141; altschul, S.F. et al 1997,Nucleic Acids Res.25:3389-3402; zhang, J.&Madden,T.L.1997,Genome Res.7:649-656)。

It is to be understood that this aspect of the invention also extends to variants of these anti-CD 45 antibodies, including humanized versions and modified versions, including those in which amino acids in the CDRs are mutated to remove one or more isomerised, deamidated, glycosylation sites or cysteine residues, as described above. In one embodiment, one or both paratopes to CD45 may be from an antibody that is already known, but has not been used in the form of a double paratope antibody.

Preferred antibodies with FALA and knob modifications

In a particularly preferred embodiment of the invention, the antibodies employed comprise heavy chains with a FALA modification. In particular, FALA modifications alter Fc receptor binding. In a further preferred embodiment, the antibody comprises a modification in the hinge region of the antibody, in particular a modification at position 228, preferably 228P. In one embodiment, the antibody has a heavy chain comprising modifications at positions 228, 234 and 235. In a particularly preferred embodiment, the heavy chain of the antibody of the invention comprises S228P, F a and L235A FALA modifications. In a particularly preferred embodiment of the invention, the antibody provided is an IgG4 (P) isotype antibody and comprises such modifications.

In another particularly preferred embodiment, the antibodies of the invention will comprise a so-called "knob" modification. In one embodiment, one heavy chain of an antibody comprises a modification at T355 and the other heavy chain comprises modifications at T366, 368 and 407, particularly forming complementary shapes in two different heavy chains, meaning that they preferentially pair rather than two identical heavy chains. In particular, one specific heavy chain may have a T355W "knob" modification, while another specific heavy chain has a T366S, L368A, Y V "hole" modification. In a particularly preferred embodiment, the antibodies of the invention are IgG4 isotype antibodies and have such modifications.

In another particularly preferred embodiment of the invention, the FALA, hinge and "socket" modifications are combined. In a preferred embodiment, they are combined in the context of an IgG4 isotype antibody. In one embodiment, one heavy chain of an antibody has modifications at positions 228, 234, 235 and 355. In another embodiment, one heavy chain comprises modifications at positions 228, 234, 235, 366, 368 and 407. For example, in one embodiment, one heavy chain has a S228P, F234A, L235A, T355W modification (thus both FALA and "knob" modifications), and preferably the other heavy chain has a S228P, F234A, L235A, T366S, L368A, Y407V modification (thus both FALA and "knob" modifications).

In a particularly preferred embodiment, the antibody of the invention is a FALA IgG4 (P) antibody. In a further particularly preferred embodiment, it is a FALA knob IgG4 (P) antibody.

In another embodiment, the above forms may be combined with other forms/modifications discussed herein. For example, they may also include modifications discussed herein to remove protein a binding at positions 95 and 96. In further embodiments, they may comprise a common light chain, and may also comprise protein a binding modifications.

Further preferred antibody forms comprising BYbe and TrYbe

In one aspect, an antibody molecule is provided comprising or consisting of:

a) A polypeptide chain of formula (VII):

V _H -CH ₁ -W-(V ₁ ) _p ；

b) A polypeptide chain of formula (VIII):

V _L -C _L -Z-(V ₂ ) _q ；

wherein:

V _H represents a heavy chain constant domain;

CH ₁ a domain representing a heavy chain constant region, e.g., domain 1 thereof;

w represents a bond or a linker, e.g., an amino acid linker, unless p or q is zero, in which case they will also be zero;

z represents a bond or a linker, such as an amino acid linker;

V ₁ represent dab, scFv, dsscFv or dsFv;

V _L represents a variable domain, such as a light chain variable domain;

C _L represents a domain from a constant region, e.g., a light chain constant region domain, such as ck;

V ₂ represent dab, scFv, dsscFv or dsFv;

p is 0 or 1;

q is 0 or 1; and

q is 0 or 1 when p is 1, p is 0 or 1 when q is 1, i.e. p and q are not both 0,

wherein at least two antigen binding sites of the antibody are different paratopes to CD45, each recognizing a different epitope to CD 45.

In one embodiment, the binding domain specific for CD45 is selected from at least two of V1, V2 or VH/VL.

In one embodiment, q is 0 and p is 1.

In one embodiment, q is 1 and p is 1.

In one embodiment, V ₁ Is dab, V ₂ Are dabs that together form a single binding domain for a pair of co-operating variable regions, such as a cognate VH/VL pair, optionally linked by disulfide bonds.

In one embodiment, V _H And V _L Specific for CD 45.

In one embodiment, V ₁ Specific for CD 45.

In one embodiment, V ₂ Specific for CD 45.

In one embodiment, V ₁ And V ₂ Together (e.g., as binding domains) specific for CD45, while V _H And V _L Specific for CD 45.

In one embodiment, V ₁ Specific for CD 45.

In one embodiment, V ₂ Specific for CD 45.

In one embodiment, V ₁ And V ₂ Together (e.g., as a binding domain) specific for CD45, while V _H And V _L Specific for CD 45.

In one embodiment, V ₁ Specific for CD45, V ₂ Specific for CD45, V _H And V _L Specific for CD 45.

V in the above construct ₁ 、V ₂ 、V _H And V _L Binding domains may each be represented and any of the sequences provided herein added.

W and Z may represent any suitable linker, for example W and Z may independently be SGGGGSGGGGS (SEQ ID NO: 67) or SGGGGTGGGGS (SEQ ID NO: 114).

In one embodiment, when V ₁ And/or V ₂ In the case of dab, dsFv or dsscFv, V ₁ And/or V ₂ Variable domain V of (2) _H And V _L Disulfide bonds between are in position V _H 44 and V _L 100.

In a preferred embodiment of the invention, the antibody of the invention is in the form of a BYbe antibody. BYbe-form antibodies include Fab's linked to only one scFv or dsscFv, as for example WO 2013/068571 and Dave et al, (2016) Mabs,8 (7): 1319-1335. Thus, for example, in a preferred embodiment of the formulae given above, one of (V1) p and (V2) q will be ScFv or dsscFv, while the other will be absent such that the BYbe-form antibody comprises Fab and only one ScFv or dsscFv. For either of (V1) p and (V2) q to be zero, the corresponding W or Z will also be absent, and the other will be a bond or linker. Preferably, the BYbe form comprises Fab and dsscFv. In such a BYbe-form antibody, both antigen binding sites may preferably be specific for CD45, both antigen binding sites corresponding to two different paratopes of different epitopes of CD 45.

In a further particularly preferred embodiment of the invention, the antibody is in the form of a TrYbe. The TrYbe format comprises a Fab linked to two scFv or dsscFvs, each of which binds to the same or different targets (e.g., one scFv or dsscFv binds to a therapeutic target, one scFv or dsscFv increases half-life by binding to, e.g., albumin). Such antibody fragments are described in International patent application publication No. WO2015/197772, which is incorporated herein by reference in its entirety, particularly with respect to the structure and discussion of antibody fragments. With respect to the formulas given above, for the TrYbe antibody, p and q are both 1, and V1 and V2 are independently selected from ScFv and dsscFv. In a preferred embodiment, both V1 and V2 will be ScFv. In another embodiment, both V1 and V2 will be dsscFv. In another embodiment, one of V1 and V2 will be ScFv and the other is dsscFv. At least two antigen binding sites of the TrYbe will be specific for CD45 and the antibody will comprise two different paratopes, each paratope being specific for a different epitope of CD 45. In a particularly preferred embodiment, the third antigen binding site will be specific for albumin, in particular one of V1 and V2 will be specific for albumin. For example, VH/VL may be specific for CD45 (e.g., for a first epitope of CD 45), one of V1 and V2 may be specific for CD45 (e.g., for a second epitope of CD 45), and the other of V1 and V2 may be specific for albumin.

In a preferred embodiment, the antibodies of the invention comprise at least one paratope specific for albumin. In one embodiment, the antibody is a TrYbe-form antibody comprising two paratopes specific for different epitopes of CD45 and a third paratope specific for albumin. Examples of albumin binding antibody sequences that can be used to bind albumin include those disclosed in WO2017/191062, the entire contents of which are incorporated by reference, particularly when it relates to albumin binding sequences. Thus, the antibodies of the invention may comprise paratopes from one of the albumin specific antibodies in WO 2017191062.

In an alternative embodiment, the antibody as described above has only one specificity for CD45, rather than at least two different specificities. For example, one of the antigen binding sites of an antibody may be specific for CD 45. In another embodiment, both antigen binding sites are specific for CD45, but have the same specificity. In a further embodiment, all three antigen binding sites of the antibodies given above have the same specificity for CD 45. In another embodiment, two antigen binding sites have the same specificity for CD45 and the third site is specific for serum albumin. In a preferred embodiment, such antibodies are used in the antibody mixtures of the invention.

Disulfide bond

If one or more pairs of variable regions in an antibody of the invention comprise a disulfide bond between VH and VL, it may be in any suitable position, such as between the two residues listed below (Kabat numbering is used in the list below unless the context indicates otherwise). Where reference is made to Kabat numbering, the relevant reference is Kabat et al, 1987,Sequences of Proteins of Immunological Interest,US Department of Health and Human Services,NIH,USA.

In one embodiment, when V1 and/or V2 in the above formulas is dsFv or dsscFv, the disulfide bond between variable domains VH and VL of VI and/or V2 is located between two residues listed below (Kabat numbering is used in the following list unless the context indicates otherwise). Where reference is made to Kabat numbering, the relevant reference is Kabat et al, 1987,Sequences of Proteins of Immunological Interest,US Department of Health and Human Services,NIH,USA.

In one embodiment, the disulfide bond is at a position selected from the group consisting of:

·V _H 37+V _L 95C, see, e.g., protein Science 6,781-788Zhu et al (1997);

·V _H 44+V _L 100, see, e.g., biochemistry 33 5451-5459Reiter et al (1994); or Journal of Biological Chemistry Vol.269No.28 pages 18327-18331, reiter et al (1994); or Protein Engineering, vol.10No.12, pages 1453-1459, rajagopal et al (1997);

·V _H 44+V _L 105, see, e.g., J biochem.118,825-831Luo et al (1995);

·V _H 45+V _L 87, see, e.g., protein Science 6,781-788Zhu et al 1997);

·V _H 55+V _L 101, see, e.g., FEBS Letters 377 135-139Young et al (1995);

·V _H 100+V _L 50, see, e.g., biochemistry 29 1362-1367Glockshuber et al (1990);

·V _H 100b+V _L 49；

·V _H 98+V _L 46, see, e.g., protein Science 6,781-788Zhu et al (1997);

·V _H 101+V _L 46；

·V _H 105+V _L 43, see for example; proc.Natl.Acad.Sci.USA Vol.90, pages 7538-7542, brinkmann et al (1993); or protein 19,35-47Jung et al (1994), and

·V _H 106+V _L 57, see, e.g., FEBS Letters 377 135-139Young et al (1995)

And a position corresponding to the variable region pair located in the molecule.

In one embodiment, at V _H 44 and V _L Disulfide bonds are formed between the 100 positions.

The amino acid pairs listed above are in positions that favor substitution by cysteines so that disulfide bonds can be formed. Cysteines may be engineered into these desired positions by known techniques. Thus, in one embodiment, an engineered cysteine according to the present disclosure refers to a position at a given amino acid position where a naturally occurring residue has been replaced with a cysteine residue.

The introduction of the engineered cysteine may be performed using any method known in the art. These methods include, but are not limited to, PCR extension overlap mutagenesis, site-directed mutagenesis or cassette mutagenesis (see generally Sambrook et al, molecular Cloning, A Laboratory Manual, cold Spring Harbour Laboratory Press, cold Spring Harbour, NY,1989; ausbel et al, current Protocols in Molecular Biology, greene Publishing) &Wiley-Interscience, N.Y., 1993). Kits for site-directed mutagenesis are commercially available, e.gSite-directed mutagenesis kit (Stratagen, la Jolla, calif.). Cassette mutagenesis may be based on Wells et al, 1985, gene,34: 315-323. Alternatively, mutants may be prepared by total gene synthesis by annealing, ligation and PCR amplification and cloning of overlapping oligonucleotides.

WO 2015/197772 details preferred positions of disulfide bonds associated with BYbe and TrYbe forms of antibodies. The entire content of WO 2015/197772 is incorporated by reference, in particular with regard to the position of the disulfide bonds.

As described herein, the ability to alter residues in the hinge region of an antibody is one potential method of affecting binding to CD45 and may be used in the present invention.

Joint

The teachings herein regarding linkers in one instance are equally applicable to linkers in different instances where linkers are used, such as in any binding molecule of the invention, particularly antibodies, particularly those involving linking entities, each having a different antigen binding site thereon. In one embodiment, linkers may be employed to join together components of binding molecules, particularly components of antibodies of the invention. For example, in one embodiment, a linker may be used to attach a component of the binding molecule, particularly to attach an antibody to a portion of a heterodimeric tether, e.g., a Fab or ScFv may be attached to one of the two units of the heterodimeric tether via the linker.

In one embodiment, the linker employed in the molecules of the invention is an amino acid linker of 50 residues or less in length, e.g., selected from the sequences shown in sequences 149 to 214.

TABLE 1 hinge linker sequences

SEQ ID NO:	Sequence(s)
		42	DKTHTCAA
43	DKTHTCPPCPA
		44	DKTHTCPPCPATCPPCPA
45	DKTHTCPPCPATCPPCPATCPPCPA
		46	DKTHTCPPCPAGKPTLYNSLVMSDTAGTCY
47	DKTHTCPPCPAGKPTHVNVSVVMAEVDGTCY
		48	DKTHTCCVECPPCPA
49	DKTHTCPRCPEPKSCDTPPPCPRCPA
		50	DKTHTCPSCPA

TABLE 2 Flexible linker sequences

/>

(S) optionally in sequences 160 to 164.

Examples of rigid linkers include peptide sequence GAPAPAAPAPA (SEQ ID NO: 92), PPPP (SEQ ID NO: 93), and PPP.

Other joints are shown in table 3:

TABLE 3 other hinge linker sequences

SEQ ID NO:	Sequence(s)
		94	DLCLRDWGCLW
95	DICLPRWGCLW
		96	MEDICLPRWGCLWGD
97	QRLMEDICLPRWGCLWEDDE
		98	QGLIGDICLPRWGCLWGRSV
99	QGLIGDICLPRWGCLWGRSVK
		100	EDICLPRWGCLWEDD
101	RLMEDICLPRWGCLWEDD
		102	MEDICLPRWGCLWEDD
103	MEDICLPRWGCLWED
		104	RLMEDICLARWGCLWEDD
105	EVRSFCTRWPAEKSCKPLRG
		106	RAPESFVCYWETICFERSEQ
107	EMCYFPGICWM

Tether-form binding molecules and antibodies

In one embodiment, the binding molecules of the invention, particularly antibodies, may comprise two moieties that are held together by a heterodimerization system chain. For example, an antibody of the invention may comprise two portions, each portion comprising a different antibody fragment having a different paratope for CD45, and further comprising a tether region that allows it to form an integral antibody molecule with the other half of the antibody. In one embodiment, the antibodies of the invention are in the form of Fab-X/Fab-Y antibodies (also known as Fab-Kd-Fab forms). The Fab-X/Fab-Y antibody format is particularly useful for screening as it allows for rapid screening of permutation combinations against different paratopes of CD 45.

Thus, in one embodiment, the antibody molecule of the invention is an antibody comprising at least two different paratopes specific for different epitopes of CD45, having the formula a-X: Y-B, wherein:

A-X is a first fusion protein;

Y-B is a second fusion protein;

y is a heterodimer-tether;

is the binding interaction between X and Y;

a is a first protein component of an antibody selected from Fab or Fab' fragments;

b is a second protein component of an antibody selected from Fab or Fab';

x is a first binding partner independently selected from a binding pair of an antigen or antibody or binding fragment thereof; and

y is a second binding partner independently selected from a binding pair of an antigen or antibody or binding fragment thereof;

with the proviso that when X is an antigen, Y is an antibody or binding fragment thereof specific for the antigen represented by X, and when Y is an antigen, X is an antibody or binding fragment thereof specific for the antigen represented by Y.

Illustrative examples of CD45 antibodies and sequences

Any suitable paratope specific for CD45 may be used in the present invention. Illustrative examples of such antibodies are set forth herein.

In one embodiment, an antibody of the invention may comprise at least one of the following CDRs:

For example, in one embodiment, the antibody may comprise a sequence derived from SEQ ID NO:1-14, at least one, two, three, four, five or six CDRs. In one embodiment, it may comprise a sequence derived from SEQ ID NO:1-14, and at least one CDR1, CDR2, and/or CDR3 sequence of those listed in seq id no. In another embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a sequence from SEQ ID NO: CDRH1, CDRH2 and/or CDRH3 sequences of those listed in 1-8. In another embodiment, an antibody of the invention may comprise a light chain variable region comprising a sequence from SEQ ID NO: CDRL1, CDRL2 and/or CDRL3 sequences of those listed in 9-14. In one embodiment, the antibody may comprise a sequence derived from SEQ ID NO:1-14, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 are selected from the group consisting of SEQ ID NOs: 1-14, wherein the specific CDR may be the original CDR specific to 4133, or the CDR of SEQ ID NO: 1-14. In one embodiment, an antibody may comprise: comprising the paratopes of the CDRs in SEQ ID No. 1, 3, 5, 9, 13 and 14. In one embodiment, the antibodies of the invention comprise at least one paratope for CD45 comprising such CDR sequences. In one embodiment, the invention provides the CD45 antibodies described herein in any suitable antibody format. Thus, in one embodiment, the invention provides an anti-CD 45 antibody or fragment thereof comprising one or more binding domains described herein, which comprises CDRs, which can be any of the antibody formats listed herein.

In one embodiment, the antibodies of the invention may comprise any of the variable regions of SEQ ID NOs 17-22. In one embodiment, one paratope of an antibody comprises a sequence selected from the group consisting of SEQ ID nos: 17 or 18. In another embodiment, one paratope of the antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 19-22. In one embodiment, the heavy chain variable regions listed herein are used in combination with the light chain variable regions listed herein. In a preferred embodiment, the antibody of the invention comprises a paratope specific for CD45 comprising a sequence selected from the group consisting of SEQ ID nos: 17 or 18 and a light chain variable region selected from SEQ ID NO: 19-22.

In one embodiment, the above is used in an antibody or antibody mixture of the invention, the antibody of the invention having at least two specificities for CD 45. In one embodiment, the antibody employed does not comprise one of the sequences described above.

for example, in one embodiment, the antibody may comprise a sequence derived from SEQ ID NO:23-31, at least one, two, three, four, five or six CDRs. In one embodiment, it may comprise a sequence derived from SEQ ID NO:23-31, at least one CDR1, CDR2, and/or CDR3 sequence of those listed. In another embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a sequence from SEQ ID NO:23-28, CDRH1, CDRH2 and/or CDRH3 sequences of those listed. In another embodiment, an antibody of the invention may comprise a light chain variable region comprising a sequence from SEQ ID NO:29-31, CDRL1, CDRL2 and/or CDRL3 sequences of those listed in seq id no. In one embodiment, the antibody may comprise a sequence derived from SEQ ID NO:23 to 31, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 are selected from the group consisting of SEQ ID NOs: 23-31, wherein the specific CDR may be the original CDR specific to 6294 or the CDR sequence of SEQ ID NO: 23-31. In one embodiment, the antibodies of the invention comprise paratopes for CD45 comprising the CDR sequences. In one embodiment, the paratope of the antibodies of the invention comprises the CDR sequences of SEQ ID Nos. 23-25 and 29-31. In one embodiment, the invention provides the CD45 antibodies described herein in any suitable antibody format. Thus, in one embodiment, the invention provides an anti-CD 45 antibody or fragment thereof comprising one or more binding domains described herein, which comprises CDRs, which can be any of the antibody formats listed herein.

In one embodiment, an antibody of the invention may comprise SEQ ID NO: 32-37. In one embodiment, one paratope of an antibody comprises a sequence selected from the group consisting of SEQ ID nos: 36 and 37. In another embodiment, one paratope of the antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 34 or 35. In one embodiment, the heavy chain variable regions listed herein are used in combination with the light chain variable regions listed herein. In a preferred embodiment, the antibody of the invention comprises a paratope specific for CD45 comprising a sequence selected from the group consisting of SEQ ID nos: 36 and 37 and a light chain variable region selected from SEQ ID NOs: 34 and 35.

In one embodiment of the invention, one paratope of an antibody of the invention comprises the amino acid sequence set forth above in SEQ ID NO:1-22 or a sequence derived from SEQ ID NO:1-22, and the other paratope of the antibody comprises the sequence set forth above in SEQ ID NO:23-37 or a sequence derived from SEQ ID NO: 23-37. For example, in one embodiment, one paratope of an antibody of the invention specific for CD45 comprises one, two, three, four, five or six CDRs from SEQ ID nos 1-14 as described above, while the other paratope of the antibody comprises one, two, three, four, five or six CDRs from SEQ ID nos 23-31 as described above. In another embodiment, one paratope of an antibody of the invention comprises the variable region sequences of SEQ ID nos 17-21 or variable region sequences derived from SEQ ID nos 17-21 as described above, while the other paratope specific for CD45 comprises the sequence of SEQ ID nos: 34-37 or a variable region sequence derived from SEQ ID No: 34-37.

In a particularly preferred embodiment, at least one paratope of an antibody of the invention specific for CD45 comprises one, two, three, four, five or six CDRs from the 4133CD45 specificities discussed herein. In another particularly preferred embodiment, at least one paratope of the antibody comprises a sequence selected from the group consisting of SEQ ID nos: 17 and 18 for a 4133 specific light chain variable region. In another particularly preferred embodiment, at least one paratope of the antibody specific for CD45 comprises a heavy chain variable region for 4133 specificity. Which is selected from the group consisting of SEQ ID No:19-21. In another particularly preferred embodiment, at least one paratope of the antibody specific for CD45 comprises such light and heavy chain variable region sequences. In one embodiment, rather than one of the specific CDR or variable region sequences listed herein, the antibodies of the invention comprise a CDR or variable region that is at least 95% identical or similar (such as 96, 97, 98, 99% identical or similar) to the specific sequences identified herein. For example, it may have one or more amino acid sequence changes, particularly conservative sequence changes. In one embodiment, the antibodies of the invention may comprise one of the framework modifications listed in the examples (particularly example 13).

In one embodiment, the heavy chain variable region human framework employed in the paratope of the antibodies of the invention is selected from the group comprising IGHV3-21, IGHV4-4 and variants of any of them, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more amino acids are replaced by amino acids other than cysteine, e.g. by residues at corresponding positions in the donor antibody, e.g. from a specific donor VH sequence listed herein. In another embodiment, the framework is selected from the group comprising IGHV3-48, IGHV1-19 or variants of any of them, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more amino acids are replaced with amino acids other than cysteine, e.g. with residues at corresponding positions in the donor antibody, e.g. from a specific donor VH sequence listed herein. In one embodiment, the human framework further comprises a suitable J region sequence, such as a JH4 or JH 1J region.

In one embodiment, the human VH framework employed in the antibody molecules of the present disclosure has amino acids substituted at least one position (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 positions) selected from the group consisting of 24, 37, 44, 48, 49, 67, 69, 71, 73, 76 and 78, for example wherein the original amino acid in the human framework is replaced with another amino acid other than cysteine, particularly by a residue at the corresponding position in the framework of the donor antibody.

In one embodiment, when the VH framework is IGHV type 3, then the substitution may be made at least five positions (typically 5 or 6 positions) selected from the group consisting of: 48. 49, 69, 71, 73, 76 and 78, such as 48, 71, 73, 76 and 78 (especially for IGHV 3-7), or 48, 69, 71, 73, 76 and 78 (especially for IGHV 3-7), or 48, 49, 71, 73, 76 and 78 (especially for IGHV 3-21). In one embodiment, when the VH framework is IGHV type 4, then substitution may be made at one or more (1, 2, 3, 4, 5, 6 or 7) positions, such as at 5 positions selected from 24, 37, 48, 49, 67, 69, 71, 73, 76 and 78, for example at all positions 24, 71, 73, 76 and 78, and optionally further 48 and 67 (which are particularly applicable to IGHV 4-4) or all positions 24, 37, 49, 67, 69, 71, 73, 76 and 78 (which are particularly applicable to IGHV 4-31).

In one embodiment, an antibody of the invention may comprise a light chain variable region in which amino acids 2, 3 and/or 70 from the original framework from which the CDRs originate are also transferred. For example, in the case of 4133 light chain variable region, glutamine (Q2), valine (V3) and/or glutamine (Q70) from the original antibody can also be transferred to the humanized light chain variable region employed, especially in the case where the acceptor framework is IGKV 1D-13. In some embodiments, CDRL1 may be mutated to remove potential N-glycosylation sites. In one embodiment, in the case of the 4133 heavy chain variable region, residues at positions 48, 49, 71, 73, 76 and/or 78 may be transferred into a new framework along with one or more CDRs. For example, isoleucine (I48), glycine (G49), lysine (K71), serine (S73), threonine (T76) and/or valine (V78) may also be transferred separately, especially in the case of IGHV3-21 as the acceptor framework. In some cases, CDRH1 and CDRH2 may be mutated to remove cysteine residues (CDRH 1 variant and CDRH2 variant, respectively). CDRH3 may also be mutated to modify potential aspartic acid isomerisation sites (CDRH 3 variants 1-3). In another embodiment, wherein CDRs from the 4133 heavy chain variable region are used, one or more of the following framework residues (donor residues) from the 4133VH gene may be retained at positions 24, 71, 73, 76 and 78, particularly when the acceptor framework is an IGHV4-4 sequence. For example, alanine (a 24), lysine (K71), serine (S73), threonine (T76) and valine (V78) may also be transferred together with the CDRs, respectively. In some cases, CDRH1 and CDRH2 may be mutated to remove cysteine residues (CDRH 1 variant and CDRH2 variant, respectively). CDRH3 may also be mutated to modify potential aspartic acid isomerisation sites (CDRH 3 variants 1-3). Example 13 of the present application also lists framework residues that may be retained if 6294 specificity is used as a CDR source, and in some embodiments employed those residues may be retained when one or more CDRs are from 6294 specificity.

In a preferred embodiment, the human framework further comprises a suitable human J region, such as a JH1 or JH4J region. In a preferred embodiment, a JH 1J region is employed.

Unless the context indicates otherwise, kabat numbering is used herein.

In one embodiment, the light chain variable region human framework employed in the humanized antibody molecules of the present disclosure is selected from the group comprising IGKV1-5, IGKV1-12, IGKV1D-13 and variants of any of them, wherein 1, 2, 3, 4 or 5 amino acids (such as 2 amino acids) are replaced by an amino acid other than cysteine, e.g., by a donor residue at the corresponding position in the original donor antibody, e.g., from SEQ ID NO: 60. 69, 78 or 88. Typically, the human framework also comprises a suitable human J region sequence, such as the JK 4J region.

In one embodiment, one or more changes in the light chain and/or heavy chain frameworks are shown in the sequences listed herein.

In one embodiment, the human VL framework employed in the antibody molecules of the present disclosure has an amino acid substituted at least one position selected from the group consisting of 2, 3 and 70, e.g., wherein the original amino acid in the human framework is substituted with another amino acid other than cysteine, particularly with a residue at the corresponding position in the donor antibody framework. In one embodiment, when using an IGKV1D-13 human framework, one, two, or three substitutions may be made at positions independently selected from 2, 3, and 70. In one embodiment, position 2 of the post-substitution VL framework is glutamine. In one embodiment, position 3 of the VL framework after substitution is valine. In one embodiment, position 70 of the post-substitution position VL framework is glutamine.

It is to be understood that one or more of the substitutions described herein may be combined to produce a humanized VL region for use in an antibody molecule of the invention.

In an independent aspect, there is provided a humanized VL variable domain comprising an amino acid sequence independently selected from SEQ ID NO:17 and 18, and a humanized sequence that is at least 95% identical or similar to any of the sequences (such as 96, 97, 98, or 99% identical or similar to any of the sequences). In an alternative embodiment, the humanized VL variable domain comprises a sequence independently selected from SEQ ID NOs: 36. 37, and a humanized sequence that is at least 95% identical or similar to any of the sequences (such as 96, 97, 98, or 99% identical or similar to any of the sequences).

In an independent aspect, there is provided a humanized VH variable domain comprising a sequence independently selected from the group consisting of SEQ ID NOs: 19-22 and a humanized sequence that is at least 95% identical or similar to any of the sequences (such as 96, 97, 98, or 99% identical or similar), and a humanized VL variable domain is provided comprising a sequence independently selected from SEQ ID NOs 17, 18, and a humanized sequence that is at least 95% identical or similar to any of the sequences (such as 96, 97, 98, or 99% identical or similar to any of the sequences). In an alternative aspect, there is provided a humanized VH variable domain comprising a sequence independently selected from SEQ ID NO: 34. 35, and a humanized sequence that is at least 95% identical or similar to any of the sequences (such as 96, 97, 98, or 99% identical or similar), and a humanized VL variable domain comprising a sequence independently selected from SEQ ID NOs: 36, 37, and a humanized sequence that is at least 95% identical or similar to any of the sequences (such as 96, 97, 98, or 99% identical or similar to any of the sequences).

In an independent aspect, there is provided a humanized VH variable domain comprising a sequence independently selected from the group consisting of SEQ ID NOs: 19-22, and a humanized VL variable domain comprising a sequence independently selected from SEQ ID NOs: 17 and 18. In an alternative aspect, there is provided a humanized VH variable domain comprising a sequence independently selected from SEQ ID NO:34 and 35, and provides a sequence independently selected from SEQ ID NOs: 36 and 37, or wherein the heavy chain CDR3 is selected from the group consisting of SEQ ID NOs: 26. 27 and 28. For example, in a preferred embodiment, an antibody is provided comprising the HCDR1 sequence of SEQ ID NO. 23, SEQ ID NO:24, and an HCDR2 sequence selected from SEQ ID No: 26. 27 and 28, and a variant HCDR3 sequence of one of claims 27 and 28. In a preferred embodiment, an antibody is provided comprising SEQ ID NO:29, LCDR1 sequence of SEQ ID NO:30 and the LCDR2 sequence of SEQ ID NO: 31. In another embodiment, the antibody comprises heavy and light chain CDR sequences such that the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:23, HCDR1, SEQ ID NO:24 and an HCDR2 sequence selected from SEQ ID No: 26. 27 and 28, and a variant HCDR3 sequence comprising one of SEQ ID NOs: 29 LCDR1, SEQ ID NO:30 and LCDR2 of SEQ ID NO:31, and a light chain of LCDR 3. The invention also encompasses antibodies comprising a set of such six CDRs, generally not limited to biparatopic antibodies.

Also provided are antibodies or binding fragments comprising paratopes that bind the same epitope as the paratopes of the antibodies or binding fragments explicitly disclosed herein.

In a preferred embodiment, the antibody of the invention comprises a polypeptide having the sequence of SEQ ID NO:115 or a sequence at least 95% identical or similar to said sequence (such as 96, 97, 98 or 99% identical or similar). In another embodiment, an antibody of the invention comprises a polypeptide having the amino acid sequence of SEQ ID NO:118 or a sequence at least 95% identical or similar to said sequence (such as 96, 97, 98 or 99% identical or similar). In another preferred embodiment, the antibodies of the invention, in particular antibodies having two different specificities, will comprise two such light chains.

In another preferred embodiment, the antibody of the invention comprises a polypeptide having the sequence of SEQ ID NO:116 or a sequence at least 95% identical or similar to said sequence (such as 96, 97, 98 or 99% identical or similar to any of said sequences), preferably while retaining the S228P, F234A, L a modification. In another embodiment, an antibody of the invention comprises a polypeptide having the amino acid sequence of SEQ ID NO:117 or a sequence at least 95% identical or similar to any of the sequences (such as 96, 97, 98 or 99% identical or similar to any of the sequences), preferably while retaining the S228P, F234A, L235A and T355W modifications.

In another preferred embodiment, the antibody of the invention comprises a polypeptide having the sequence of SEQ ID NO:119 or a sequence at least 95% identical or similar to said sequence (such as 96, 97, 98 or 99% identical or similar), preferably while retaining the S228P, F234A, L235A, T366S, L368A and Y407V modifications.

In one embodiment, the antibody of the invention comprises the amino acid sequence set forth above as SEQ ID No:115-119 or a sequence having at least 95% identity or similarity (such as at least 96, 97, 98 or 99% identity or similarity) to said sequence, while retaining said modification to FALA and/or mortar. In one embodiment, the antibodies of the invention have light chains of SEQ ID NOS 115 and 118 and heavy chains of SEQ ID NOS 117 and 119. In another embodiment, there is at least 95% identity or similarity to any or all of the sequences (such as 96, 97, 98, or 99% identical or similar to any of the sequences) while retaining the FALA and the knob modifications.

The invention also provides antibodies comprising CDRs, variable regions, light and/or heavy chain sequences of the 6294 antibody, or variants of the 6294 sequence, which antibodies do not necessarily have a double paratope for CD 45. For example, the invention provides antibodies, wherein one specificity of the antibody comprises the sequence of 6294 antibody or a variant thereof. In another embodiment, a monospecific antibody comprising the sequence of 6294 antibody is provided. For example, such an antibody may comprise six CDRs from SEQ ID NO. 23-31, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 are selected from the corresponding CDR sequences of SEQ ID NO. 23-31, wherein the specific CDR may be the original CDR specific for 6294 or one of the variant sequences listed in SEQ ID NO. 23-31. In an alternative aspect, an antibody is provided comprising a humanized VH variable domain comprising a sequence independently selected from SEQ ID NOs 34 and 35 and a sequence independently selected from SEQ ID NOs: 36 and 37. The invention also provides 6294 antibodies and humanized versions and other variants thereof. Various aspects listed herein in connection with antibodies, such as vectors, nucleic acids, pharmaceutical compositions, and the like, may also be used for such antibodies. Illustrative examples of albumin antibodies and sequences

Antibodies specific for albumin for use in the present invention may have the following CDR sequences:

such antibodies may comprise the VL sequence of SEQ ID NO. 126 and the VH sequence of SEQ ID NO. 127. Such antibodies may alternatively comprise disulfide-linked VL and VH sequences of SEQ ID NO:128 and SEQ ID NO:129, respectively. In the case of a TrYbe comprising two CD45 paratopes and one albumin paratope, the heavy chain may comprise the amino acid sequence of SEQ ID NO:130, the light chain may comprise the sequence of SEQ ID NO: 131.

Effector molecules

The binding molecules of the invention (particularly antibodies) may be conjugated to effector molecules. Thus, if desired, the binding molecules (particularly antibodies) used in the present invention may be conjugated to one or more effector molecules. It is to be understood that an effector molecule may comprise a single effector molecule or two or more such molecules that are linked to form a single moiety that may be linked to a binding molecule (particularly an antibody) of the invention. In case it is desired to obtain a binding molecule according to the invention (in particular an antibody) linked to an effector molecule, this can be prepared by standard chemical or recombinant DNA procedures, wherein the binding molecule (in particular an antibody) is linked to the effector molecule directly or via a coupling agent. Techniques for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al, controlled Drug Delivery, second edition, robinson et al, eds., 1987, pages 623-53; thorpe et al, 1982, immunol. Rev.,62:119-58 and Dubowchik et al, 1999,Pharmacology and Therapeutics,83,67-123). For example, specific chemical procedures include those described in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and WO 03/031581. Alternatively, where the effector molecule is a protein or polypeptide, ligation may be achieved using recombinant DNA procedures, for example as described in WO 86/01533 and EP 0392745. In one embodiment, the binding molecules (particularly antibodies) of the invention may comprise effector molecules. The term effector molecule as used herein includes, for example, antineoplastic agents, drugs, toxins, bioactive proteins, e.g., enzymes, antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof, e.g., DNA, RNA and fragments thereof, radionuclides, in particular radioiodides, radioisotopes, chelated metals, nanoparticles, and reporter groups, such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy.

Examples of effector molecules may include cytotoxins or cytotoxic agents, including any agents that are detrimental to (e.g., kill) cells. Examples include combretastatin (combretastatin), dolastatin (dolastatin), epothilones (epothilones), staurosporines, maytansinoids, spongostains, rhizomycin, halichondins (halichondrins), cercosporins (rotidins), haractelins (hemiasterlins), taxol, cytochalasin B, ponin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax, mitoxantrone, mithramycin (mithramycin), actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs thereof, or homologs thereof. Effector molecules also include, but are not limited to: antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., dichloromethyldiethylamine, chlorambucil thiobutyrate, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cisplatin (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin (AMC), carbo Li Ji, or multiple carcinomycin) and antimitotics (e.g., vincristine and vinblastine).

Other effector molecules may include chelated radionuclides, such as ¹¹¹ In and ⁹⁰ Y、Lu ¹⁷⁷ bismuth and bismuth ²¹³ Californium ²⁵² Iridium (Iridium) ¹⁹² And tungsten (W) ¹⁸⁸ Rhenium ¹⁸⁸ The method comprises the steps of carrying out a first treatment on the surface of the Or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxanes, and suramin. Other effector molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides, and peptides of interest include, but are not limited to: immunoglobulins, toxins such as abrin (brin), ricin a, pseudomonas exotoxin or diphtheria toxin, proteins such as insulin, tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, thrombosisA forming or anti-angiogenic agent, for example angiostatin or endostatin, or a biological response modifier, such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve Growth Factor (NGF) or other growth factors and immunoglobulins.

Other effector molecules may include detectable substances that may be used, for example, in diagnostics. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radionuclides, positron emitting metals (for positron emission tomography) and non-radioactive paramagnetic metal ions. See generally U.S. Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostic agents. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent substances include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; suitable luminescent substances include luminol; suitable bioluminescent materials include luciferase, luciferin and aequorin; suitable radionuclides include ¹²⁵ I、 ¹³¹ I、 ¹¹¹ In and ⁹⁹ Tc。

in another embodiment, the effector molecule may increase or decrease the in vivo half-life of the binding molecule (particularly an antibody), and/or decrease immunogenicity and/or enhance delivery across the epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin binding proteins or albumin binding compounds, such as those described in WO 05/117984. When the effector molecule is a polymer, it may generally be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, such as a homo-or hetero-polysaccharide. Specific optional substituents that may be present on the synthetic polymers described above include one or more hydroxy, methyl or methoxy groups. Specific examples of synthetic polymers include optionally substituted linear or branched poly (ethylene glycol), poly (propylene glycol) poly (vinyl alcohol) or derivatives thereof, particularly optionally substituted poly (ethylene glycol) such as methoxy poly (ethylene glycol) or derivatives thereof.

The binding molecules (particularly antibodies) of the invention may be conjugated to molecules that modulate or alter serum half-life. The binding molecules of the invention (particularly antibodies) may bind to albumin, for example in order to modulate serum half-life. In one embodiment, the binding molecules (particularly antibodies) of the invention will also include paratopes specific for albumin. In another embodiment, the binding molecules (particularly antibodies) of the invention may include a peptide linker that is an albumin binding peptide. Examples of albumin binding peptides are included in WO2015/197772 and WO2007/106120, the entire contents of which are incorporated by reference.

In another embodiment, the binding molecules (particularly antibodies) of the invention are not conjugated to effector molecules. In one embodiment, the binding molecules (particularly antibodies) of the invention are not conjugated to toxins. In another embodiment, the binding molecules of the invention (particularly antibodies) are not conjugated to a radioisotope. In another embodiment, the binding molecules (particularly antibodies) of the invention are not conjugated to a useful imaging agent.

In a preferred embodiment, what leads to cell death (preferably apoptosis) is the ability of the binding molecules of the invention, particularly antibodies, to bind CD45, rather than conjugated effector molecules. In a preferred embodiment, it is the ability of CD45 to crosslink that results in cell death (preferably apoptosis).

Cell death and killing

In a particularly preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, are capable of inducing cell death in target cells. Cell death types that can be induced to kill target cells include endogenous apoptosis, exogenous apoptosis, mitochondrial Permeability Transition (MPT) -driven necrosis, programmed necrosis (necroptosis), iron death, cell coke death, PARP-1 dependent programmed cell death (parthanatos), endoplasmic cell death (entotic cell death), NET-mediated cell death (NETotic cell death), lysosomal dependent cell death, autophagy-dependent cell death, immunogenic cell death, cell senescence, and mitotic catastrophe. In a particularly preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, are used to induce apoptosis. In one embodiment, the binding molecules (particularly antibodies) of the invention are used to kill target cells.

In one embodiment, the target cell is a cell expressing CD45, in particular a cell expressing CD45 on the cell surface. In a preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, can induce cell death (preferably apoptosis) in at least T cells. In another preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, may induce cell death (preferably apoptosis) in at least B cells. In another preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, may be capable of inducing cell death (preferably apoptosis) in B cells and T cells. In a preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, are capable of inducing cell death (preferably apoptosis) in hematopoietic stem cells. In one embodiment, the binding molecules of the invention, in particular the antibodies of the invention, do not induce cell death in all immune cells, e.g., in granulocytes, macrophages and monocytes. In one embodiment, the binding molecules of the invention, in particular the antibodies of the invention, induce cell death in all immune cells except granulocytes, macrophages and monocytes. In one embodiment, the effect of inducing cell death in hematopoietic stem cells is effective to enable replacement of all hematopoietic cells. In one embodiment, the binding molecules of the invention are used to kill the target cells described above by inducing cell death.

In another particularly preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, induce cell death (preferably apoptosis) but do not result in significant cytokine release. In another preferred embodiment, the binding molecules of the invention, in particular the antibodies of the invention, induce cell death (preferably apoptosis), but do not exhibit Fc effector function, for example because the antibodies lack an Fc region or have an Fc region with silent modifications.

Cytokines and methods of use

In a particularly preferred embodiment, the binding molecules of the invention (particularly antibodies) do not result in significant cytokine release. In a particularly preferred embodiment, the binding molecules (particularly antibodies) of the invention are capable of inducing cell death in target cells, but do not result in significant cytokine release. The reduced or absence of cytokine release may mean that the subject does not suffer from unnecessary cytokine-driven inflammation. For example, the treatment of the invention may kill target cells in a subject without triggering inflammation, particularly without the so-called "cytokine storm" associated with certain treatments.

In one embodiment, the binding molecules of the invention, and in particular the antibodies of the invention, do not significantly induce the release of one or more of interferon-gamma, IL-6, TNF-alpha, IL-1β, MCP1 and IL-8. In a preferred embodiment, the binding molecules of the invention (particularly antibodies) do not result in significant release of any of these cytokines. In another embodiment, the binding molecules (particularly antibodies) of the invention do not significantly induce the release of one or more of CCL2, IL-1RA, IL-6 and IL-8. In another preferred embodiment, it does not significantly induce the release of any of these cytokines. In one embodiment, such levels would be the case for one or more of interferon-gamma, IL-6, TNF-alpha, IL-1β, MCP1, and IL-8. In another embodiment, such levels would be the case for one or more of CCL2, IL-1RA, IL-6, and IL-8. In another embodiment, such levels will be seen for at least one of CCL2, IL-1RA, IL-6, IL-8, IL-10, and IL-11. In another embodiment, such levels would be the case for at least one of CCL2, IL-1RA, IL-6, IL-8.

Cytokine release may be measured using any suitable assay. For example, the ability of a binding molecule of the invention (particularly an antibody) to cause cytokine release can be determined by culturing cells with the binding molecule in vitro and measuring cytokine release. In one embodiment, whole blood is incubated with the antibodies, and then cytokine levels, such as the levels of any of those cytokines mentioned above, are measured. In another embodiment, leukocytes isolated from a whole blood sample may be incubated with the binding molecules of the invention and the level of one or more cytokines measured. Alternatively, one or more cytokine levels may be measured in a sample from a subject to which a binding molecule of the invention is administered, in particular one or more cytokine levels may be measured in a serum sample from the subject.

In one embodiment, not "significantly inducing" cytokine release means that the binding molecules of the invention induce cytokine release no more than five, four, three or two times that seen in a negative control, e.g., as compared to a negative control treated in vitro with PBS alone. In some embodiments, the level of cytokine release will be compared to a positive control, e.g., in vitro treatment with campath. In one embodiment, the binding molecules of the invention will trigger no more than 50%, 40%, 30%, 20%, 10% or less cytokine release than seen with Campath treatment. In one embodiment, the level of cytokine release seen with the binding molecules of the invention will be less than one tenth of the level seen with Campath. In one embodiment, the level of cytokine release after incubation with Campath will be at least twice, three times, four times, five times, ten times or more as seen after incubation with a binding molecule of the invention. In one embodiment, the level seen with Campath will be the level compared to the binding molecule of the invention after 24 hours of incubation of whole blood. In another embodiment, the comparator used to define the non-significant induction is another binding molecule. For example, where the binding molecules of the invention comprise modifications designed to reduce cytokine release, then the comparator will be an equivalent binding molecule, but without such modifications. In another embodiment, where the binding molecule is an antibody, it has either an Fc region modification aimed at reducing cytokine release, or no Fc region, and the comparison performed is compared to an equivalent antibody lacking such modification or having an Fc region.

In another embodiment, the comparison for not significantly releasing the cytokine will be performed in vivo. For example, when a binding molecule of the invention is administered to a subject, it will exhibit any of the levels of cytokine release described above, as compared to the comparison discussed above. In another embodiment, the lack of significant induction of cytokine release may be compared to prior to administration of the antibodies of the invention in terms of the level of one or more cytokines. For example, it can be said that the level of cytokines does not rise more than ten times, five times or less after administration of the binding molecules of the invention. The measurement may be performed, for example, immediately prior to or concurrently with administration of the antibody, and for example, one day, one week, or two weeks or more after administration. In one embodiment, the measurement is made one day to one week after administration. In another embodiment, the binding molecules of the invention do not significantly induce cytokine release, as the subject being treated does not experience adverse effects associated with unwanted cytokine release, e.g., the subject does not experience fever, hypotension, or an irregular or rapid heartbeat.

Functional assay

In one embodiment, a functional assay may be employed to determine whether one or more binding molecules of the invention have particular properties, such as any of those mentioned herein, for example. Thus, functional assays can be used to evaluate binding molecules, particularly antibodies, of the invention. As used herein, a "functional assay" is an assay that can be used to determine one or more desired properties or activities of one or more binding molecules of the invention. Suitable functional assays may be binding assays, cell death (preferably apoptosis) assays, antibody Dependent Cellular Cytotoxicity (ADCC) assays, complement Dependent Cytotoxicity (CDC) assays, cell growth or proliferation inhibition (cytostatic effect) assays, cell killing (cytotoxic effect) assays, cell signaling assays, cytokine production assays, antibody production and isotype switching assays. In one embodiment, the assay can measure the extent of cell depletion, e.g., for a particular cell type, by employing an antibody of the invention. In a preferred embodiment, the assay may measure the ability of an antibody of the invention to induce cell death (preferably apoptosis) in a target cell expressing CD 45. In a further preferred embodiment, the functional assay may measure the ability of a binding molecule of the invention (particularly an antibody) to induce cytokine release. In a preferred embodiment, a functional assay can be used to determine whether binding molecules (particularly antibodies) of the invention can be used to kill cells without significantly inducing cytokines.

The functional assay may be repeated as many times as necessary to improve the reliability of the results. Various statistical tests known to those skilled in the art can be used to identify statistically significant results, and thus binding molecules (particularly antibodies) that have biological functions. In one embodiment, a plurality of binding molecules are tested in parallel or substantially simultaneously. As used herein, "simultaneously" refers to the case where the sample/molecule/complex is analyzed in the same assay, e.g., in the same "run". In one embodiment, simultaneous refers to a concomitant analysis in which the signal output is analyzed by the instrument substantially simultaneously. The signal may need to be deconvoluted to interpret the results obtained. Advantageously, testing multiple biparatopic protein complexes allows for more efficient screening of large numbers of antibodies and identification of new and interesting relationships. Clearly, the different variable regions of CD45 of interest directed against the target antigen may allow for subtle differences in biological function to be obtained.

In one embodiment, where the binding molecules of the invention comprise more than one specificity of CD45, a functional assay may be used to compare the properties of the binding molecule to, for example, a binding molecule having the same binding valency but only one specificity of the binding molecule of the invention. In one embodiment, such an assay can be used to demonstrate that the binding molecules of the invention having at least two different specificities for CD45 are superior to the comparator binding molecules. Thus, in a preferred embodiment, the efficacy of a binding molecule of the invention (in particular an antibody according to the invention) comprising at least two different specificities for CD45 can be compared to a single "comparator" binding molecule (in particular a "comparator" antibody) comprising only one specificity for CD45 from a binding molecule of the invention. For example, in the case of assays to study the crosslinking or crosslinking effect of CD45, binding molecules (particularly antibodies) having the same binding valency but only one specificity may be used as a comparison. In one embodiment, where the binding molecule is an antibody of the invention, it can be compared to an antibody comprising one of the same paratopes from the antibody of the invention at all antigen binding sites of the antibody. In one embodiment, the antibodies of the invention can be compared to antibodies having the same binding valency and form as the antibodies of the invention, but in the latter there is one of the same paratopes from the antibodies of the invention at all antigen binding sites. In one embodiment, a bivalent antibody comprising two different paratopes specific for different epitopes of CD45 may be compared to each of two possible bivalent antibodies comprising only one of those paratopes. In one embodiment, this comparison is performed with one comparator antibody for each different specificity of the antibodies of the invention specific for CD45, in particular each different paratope. In one embodiment, the antibodies of the invention will exhibit better results than one such comparator antibody. In another embodiment, each specificity of the antibody, particularly the paratope, specific for CD45 will show better results than all the comparator antibodies.

In another embodiment, where the binding molecules of the invention comprise at least two different specificities for CD45, the monospecific binding molecules, particularly monospecific antibodies, are first evaluated, and then the selected candidates are used to generate antibodies of the invention having at least two different specificities for CD 45. In one embodiment, a plurality of binding molecules (particularly antibodies) are tested and subjected to one or more functional assays using the multiplex method described above.

The mixture of at least two binding molecules of the invention, in particular antibodies of the invention, can be compared with the individual binding molecules comprising the mixture of the invention using a functional assay. In a preferred embodiment, the mixture gives results superior to any single binding molecule, in particular a single antibody.

The term "biological function" as used herein refers to the natural activity of the biological entity being tested or the activity of interest, e.g. the natural activity of a cell, protein or the like. Ideally, an in vitro functional assay can be used to test for the presence of this function, including assays that utilize living mammalian cells. As used herein, natural functions include abnormal functions, such as functions associated with cancer.

In one embodiment, the binding molecules (particularly antibodies) of the invention are capable of cross-linking CD45 to a greater extent than the comparator binding molecules (particularly as compared to the comparator antibodies, such as those discussed above). For example, when the two are mixed (such as in equal amounts), the binding molecules of the invention (particularly antibodies) can be studied to form antibodies: ability of CD45 multimers of CD45 ECD. The multimer may be a binding molecule having at least two binding molecules: structure of CD45 ECD cell. One suitable technique is mass spectrophotometry, in which a binding molecule, particularly an antibody, is mixed with an equal concentration of CD45 ECD (such as CD45 ECD of SEQ ID No: 113) and a mass spectrophotometry is performed on a test sample. The control with antibody and CD45 ECD alone can be measured. Binding molecules (particularly antibodies) of the invention can produce more multimers than a comparison binding molecule (particularly a comparison antibody). The binding molecules of the invention (particularly antibodies) can produce greater amounts of binding molecules having two, three, four or more than one compared to the comparison: multimers of CD45 ECD units. This can be performed for all possible comparisons for each specificity (in particular paratope) specific for CD45. Another suitable technique for such comparison is Analytical Ultracentrifugation (AUC). Likewise, the comparison performed may also be between a mixture of binding molecules and each single type of binding molecule in the mixture itself.

In another embodiment, the comparison may be made in terms of the ability of the binding molecules (particularly antibodies) of the invention to induce cell death. In particular, in a preferred embodiment, the ability of binding molecules (particularly antibodies) to induce apoptosis can be studied. For example, a binding molecule (particularly an antibody) of the invention can induce more target cells expressing CD45 to undergo apoptosis than a comparator (e.g., a comparator antibody). When measured using T cells, it can induce a greater amount of apoptosis. For example, T cells isolated from PBMCs may be used. Any binding molecule of the invention, particularly antibodies, can induce higher levels of apoptosis in cd4+ T cells. It can induce higher levels of apoptosis in cd8+ T cells. It can induce higher levels of apoptosis in cd4+ memory T cells. It can induce higher levels of apoptosis in cd4+ naive T cells. In another embodiment, total cell count in whole blood may be measured after incubation with binding molecules of the invention (particularly antibodies) and compared to the results seen in the comparison. In one embodiment, total cell counts may be measured and the binding molecules of the invention (particularly antibodies) compared to control binding molecules (particularly antibodies). In a particularly preferred embodiment of the invention, annexin V can be used to measure apoptosis. Thus, for example, a binding molecule of the invention (particularly an antibody) will result in a greater proportion of annexin V stained cells than a comparison.

In one embodiment, in vivo assays, such as animal models, including mouse tumor models, autoimmune disease models, rodent or primate models of viral or bacterial infection, and the like, can be used to test binding molecules of the invention. In another embodiment, the degree of depletion of a particular cell type can be measured, for example, in vivo. In one embodiment, the binding molecules of the invention (particularly antibodies) will result in greater levels of depletion in animal models of disease and in preferred embodiments in animal models of cancer than in the comparison.

In one embodiment, the binding molecules (in particular antibody molecules) according to the invention have novel or synergistic functions. The term "synergistic function" as used herein refers to the absence of or higher biological activity observed when one or more of the comparison products are used than when one or more of the comparison products are used. Thus, "synergistic" includes novel biological functions. In one embodiment, the binding molecule of the invention (particularly an antibody) comprising at least two specificities for CD45 is synergistic in that it is more effective than binding molecules (particularly antibodies) comprising either specificity for CD45 alone (such as the comparisons discussed above). In a preferred embodiment, this synergy is shown with respect to CD45 crosslinking. In one embodiment, the mixture of binding molecules exhibits a synergistic effect compared to any of the individual binding molecules that make up the mixture itself.

In one embodiment, a novel biological function as used herein refers to a function that is not apparent or present or previously unidentified before two or more synergistic entities [ protein a and protein B ] are bound together. As used herein, "higher" refers to an increase in activity, including an increase from zero, i.e., some activity in one or more binding molecules, wherein the comparator molecule lacks that activity in a related functional assay, which activity is also referred to herein as a new activity or new biological function. "higher" as used herein also includes an increase in the relative activity of greater than the addition function of an antibody, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300% or more, compared to a single paratope in a relative function assay.

In one embodiment, the novel synergistic function is a higher inhibitory activity.

In a particularly preferred embodiment of the invention, the synergy is associated with cell depletion of a target cell type expressing CD 45. In one embodiment, the synergy is associated with cell killing.

Suitable binding domains for use in the present invention may also be identified by testing one or more binding domain pairs in a functional assay. For example, a binding molecule (e.g., an antibody) comprising at least one binding site specific for antigen CD45 may be tested in one or more functional assays.

In one embodiment, the ability of a binding molecule to kill a cancer cell line expressing CD45 may be determined. Examples of cancer cell lines that can be used in such cell killing assays include leukemia and lymphoma cell lines. In one embodiment, any of the following cell lines, which represent various leukemia and lymphoma cell lines, classified by ATCC (www.atcc.org /), can be used to study the ability to induce cancer cell killing: jurkat-acute T cell leukemia; acute lymphoblastic leukemia of CCRF-SB-B cells; MC116-B cell undifferentiated lymphoma; raji, ramos-burkitt lymphoma (rare forms of B-cell non-hodgkin lymphoma); SU-DHL-4, SU-DHL-5, SU-DHL-8, NU-DUL-1, OCI-Ly 3-diffuse large B cell lymphoma; THP-1-acute monocytic leukemia; and Dakiki-B cell nasopharyngeal carcinoma. The methods employed in the examples of the present application to assess the ability of binding molecules to cause killing of these cell lines can be used to investigate the ability of a given binding molecule to kill cells. In one embodiment, the variant binding molecules of the invention have the same or greater ability to kill cancer cells in such assays as one of the specific binding molecules set forth herein. In one embodiment, in such an assay, they will have at least 50%, 75%, 80%, 90%, 100% or more of the activity of one of the specific binding molecules given herein to kill one of the cancer cell lines mentioned above. In one embodiment, the binding molecules of the invention will kill at least 25%,40%,50%,60% or 75% of the cancer cells in such an assay. In another embodiment, the binding molecules of the invention will kill 100% of cancer cells in such an assay.

Pathological conditions, medical uses and cell depletion

The present invention provides the use of a binding molecule, particularly an antibody, of the invention in a method of treating the human or animal body. The binding molecules of the invention, particularly antibodies, can be used in any situation where targeting CD45 may have therapeutic benefit, particularly where killing such cells may have therapeutic benefit. The binding molecules of the invention, particularly antibodies, may also be used for diagnosis or detection of CD 45. The invention also provides pharmaceutical compositions of the invention for such use. The invention also provides one or more nucleic acid molecules and one or more vectors of the invention for such use.

Thus, the binding molecules of the invention are useful in therapy. In one embodiment, rather than administering one or more binding molecules of the invention, one or more nucleic acid molecules or one or more vectors of the invention may be administered to cause expression of the one or more binding molecules within the target cell. In another embodiment, the pharmaceutical composition of the invention is a preferred therapeutic agent for administration. Although binding molecules (particularly antibodies) are listed below as preferred therapeutic agents, the pharmaceutical compositions, one or more nucleic acid molecules, and one or more vectors of the invention may also be used in any of the embodiments listed. Nevertheless, in a preferred embodiment, one or more binding molecules or pharmaceutical compositions comprising them are preferred therapeutic agents. In a particularly preferred embodiment, the one or more antibodies or pharmaceutical compositions comprising them are therapeutic agents.

In a particularly preferred embodiment, the invention may be used to deplete target cells expressing CD 45. In a particularly preferred embodiment, the invention is used to deplete disease-causing CD45 expressing cell types. In particular, the invention is useful for depleting target cells expressing CD45 on the cell surface. In a particularly preferred embodiment, the binding molecules employed or encoded by the nucleic acid molecules or vectors are binding molecules having at least two different specificities for CD 45. While not being bound by any particular theory, it is believed that by having at least two different specificities for CD45, particularly at least two different paratopes for different epitopes of CD45, the binding molecules (particularly antibodies) of the invention are better able to crosslink CD45 on the target cell surface, which in turn may more effectively lead to cell death (preferably apoptosis). As mentioned above, this effect can also be achieved by using a mixture of binding molecules.

In a preferred embodiment employing one or more antibodies of the invention, induction of cell death (preferably apoptosis) in a target cell by one or more antibodies of the invention may mean that the one or more antibodies of the invention do not necessarily exhibit one or more Fc region effector functions normally exhibited by antibodies. In a particularly preferred embodiment, one or more antibodies of the invention are thus capable of inducing cell death (preferably apoptosis) in a target cell, but without an active Fc region. In particularly preferred embodiments, the one or more antibodies induce cell death, but do not induce significant cytokine release.

In a particularly preferred embodiment, cells are depleted by the present invention and subsequently the cells or tissue are transferred to a subject. In a further particularly preferred embodiment, the transferred cells or tissues replace those cells or tissues that have been depleted using the present invention. Thus, the treatments discussed herein include, without targeting the actual mechanism of the disease, replacement of all or part of the cell type involved in the disease or killing (particularly replacement) of that cell type may simply be of therapeutic benefit. In one embodiment, the invention thus provides a method of depleting cells comprising using the invention. In another embodiment, the methods of the invention may include both depleting cells and subsequently transferring cells or tissue. Cell depletion can be used in a variety of therapeutic settings to effectively kill target cells.

In a preferred embodiment, the binding molecules (particularly antibodies) of the invention are used to kill immune cells. As used herein, the term "immune cell" is intended to include, but is not limited to, cells of hematopoietic origin and that play a role in an immune response. In one embodiment, the invention is used to deplete T cells in a subject. In one embodiment, the invention is used to deplete B cells in a subject. In another embodiment, the present invention is used to deplete both. In another embodiment, the invention is used to deplete T cells, but not to deplete macrophages. In another embodiment, the invention is used to deplete B cells, but not macrophages. In another embodiment, the invention is used to deplete B cells and T cells, but does not result in the depletion of macrophages. In one embodiment, the invention is used to deplete Hematopoietic Stem Cells (HSCs). In another embodiment, the invention is used to deplete hematopoietic stem cells. In a preferred embodiment, HSCs in the subject are depleted by the present invention prior to transferring HSCs to repopulate the immune system of the subject. In another embodiment, the invention depletes specific cell types, but does not deplete hematopoietic stem cells. In another embodiment, the invention is used to kill the cell types described above. Thus, in any of the embodiments mentioned herein for cell depletion, the invention can be used to kill the cells.

In one embodiment, the subject treated by the invention is a subject suffering from an autoimmune disease, a hematological disease, a metabolic disorder, a cancer, or an immunodeficiency (such as severe combined immunodeficiency disease or SCID). The ability to treat a condition by first depleting and then replacing cells means that the binding molecules (particularly antibodies) of the invention are particularly useful in the treatment of cancer. In a particularly preferred embodiment, the condition to be treated is therefore cancer. In one embodiment, the invention is thus used to deplete cancer cells, such as cancer cells derived from cells of the immune system. In a preferred embodiment, the invention provides a method of treating cancer comprising administering the invention to deplete CD45 expressing cancer cells. The method may further comprise transplanting the cells to a subject. In one embodiment, the transferred cells replace the depleted cells. In one embodiment, the transferred cells are hematopoietic stem cells.

In a particularly preferred embodiment, the condition to be treated is a blood cancer. In a preferred embodiment, the cancer is a bone marrow-related cancer, in particular a cancer involving cells of the hematopoietic system.

In a preferred embodiment, the cancer may be leukemia. In one embodiment, the leukemia may be pediatric leukemia. In another embodiment, the leukemia may be adult leukemia. The leukemia may be acute leukemia. The leukemia may be chronic leukemia. Non-limiting examples of leukemias that can be treated by the use of the present invention include lymphoblastic leukemia (such as acute lymphoblastic leukemia or chronic lymphoblastic leukemia) and myelogenous leukemia (such as acute myelogenous leukemia or chronic myelogenous leukemia). The leukemia may be a B-cell leukemia, such as B-cell acute lymphoblastic leukemia, or B-cell prolymphocytic leukemia. In one embodiment, the invention is useful for treating adult acute lymphoblastic leukemia, pediatric acute lymphoblastic leukemia, refractory pediatric acute lymphoblastic leukemia, prolymphocytic leukemia, chronic lymphoblastic leukemia, or acute myelogenous leukemia.

In one embodiment of the invention, the blood cancer to be treated may be lymphoma. Non-limiting examples of lymphomas that can be treated include: b-cell lymphoma, recurrent or refractory B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, recurrent or refractory diffuse large cell lymphoma, anaplastic large cell lymphoma, primary mediastinal B-cell lymphoma, recurrent mediastinal, refractory mediastinal large B-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, recurrent or refractory non-hodgkin's lymphoma, refractory invasive non-hodgkin's lymphoma, B-cell non-hodgkin's lymphoma and refractory non-hodgkin's lymphoma.

In one embodiment, the blood cell cancer to be treated is myeloma. Non-limiting examples of treatable myelomas include relapsed plasma cell myelomas, refractory plasma cell myelomas, multiple myelomas, relapsed or refractory multiple myelomas and multiple myelomas of bone.

In one embodiment, the cancer may be one selected from the group consisting of acute T-cell leukemia, B-cell acute lymphoblastic leukemia, acute monocytic leukemia, and B-cell nasopharyngeal carcinoma. In another embodiment, the cancer may be one selected from the group consisting of acute T-cell leukemia, B-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma, acute monocytic leukemia, and B-cell nasopharyngeal carcinoma. In another embodiment, the cancer may be one selected from the group consisting of acute T-cell leukemia, B-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma, acute monocytic leukemia, B-cell nasopharyngeal carcinoma, B-cell undifferentiated lymphoma, and burkitt's lymphoma. In another embodiment, the cancer may be one selected from the group consisting of acute T-cell leukemia, B-cell acute lymphoblastic leukemia, acute monocytic leukemia, B-cell nasopharyngeal carcinoma, B-cell undifferentiated lymphoma, and burkitt lymphoma. In a preferred embodiment, in the case of treatment of such cancers, the binding molecules of the invention employed are BYbe antibodies.

In another embodiment, the subject to be treated has an autoimmune disease. In a particularly preferred embodiment, the autoimmune disease is multiple sclerosis. In another particularly preferred embodiment, the disease is scleroderma. Further examples of autoimmune diseases include scleroderma, ulcerative colitis, crohn's disease, type 1 diabetes, or another autoimmune pathological condition described herein. In some embodiments, the subject has or is otherwise affected by a metabolic storage disorder. The subject may suffer from or otherwise be affected by a metabolic disorder selected from the group consisting of: glycogen storage disease, mucopolysaccharidosis, gaucher disease, hurers disease, sphingolipid storage disease, metachromatic leukodystrophy, and any other disease or condition that may benefit from the treatment and therapies disclosed herein, including, but not limited to, severe combined immunodeficiency disease, wiscot-Aldrich syndrome, hyperimmune globulin M (IgM) syndrome, chediak-Higashi disease, hereditary lymphohistiocytosis, bone sclerosis, osteogenesis imperfecta, storage disease, severe thalassemia, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and those described in "Bone Marrow Transplantation for Non-Malignant Disease," ASH Education Book,1:319-338 (2000), as it relates to pathological conditions that can be treated by administration of hematopoietic stem cell transplantation therapies.

In one embodiment, the disorder to be treated is a condition known to involve abnormal expression of CD 45. In a particularly preferred embodiment, the treatment depletes cell types expressing CD45, CD45 playing a role in the disorder in the subject. Examples of such diseases include Alzheimer's disease, multiple sclerosis and lupus. Other conditions known to involve altered CD45 expression include immunodeficiency diseases such as Severe Combined Immunodeficiency Disease (SCID).

In one embodiment, the present invention is used to deplete cells prior to cell transplantation, and thus in some embodiments, the methods of the present invention may include a step of depleting with a therapeutic agent, particularly a binding molecule of the present invention, particularly an antibody, followed by a step of transferring the cells to a subject, e.g., to aid in the replacement of the depleted cells. In one embodiment, the metastasis may be allogeneic cells. In another embodiment, the transfer may be autologous cells. In one embodiment, the transferred cell may be a cell expressing a Chimeric Antigen Receptor (CAR). In some embodiments, the subject is in need of chimeric antigen receptor T Cell (CART) treatment. For example, such treatment may form part of the methods of the invention.

In another preferred embodiment, the invention provides a method of promoting implantation of a population of cells in a subject, wherein the method further comprises depleting the cells using a binding molecule (particularly an antibody) of the invention prior to implantation of the population of cells. Accordingly, the present invention provides a method of facilitating implantation of a metastatic cell, the method comprising depleting cells expressing CD45 in a subject by administration of a binding molecule (particularly an antibody) of the invention, and then transferring the cell of interest. In one embodiment, the invention provides a method of promoting the implantation of stem cells, particularly hematopoietic stem cells. In one embodiment, the hematopoietic stem cells are administered to a subject deficient or deficient in one or more cell types of the hematopoietic lineage to reconstitute or partially reconstitute the deficient or deficient cell population in vivo. In one embodiment, the invention is used to treat stem cell deficiency, for example, the invention is used to deplete target cells and replace them with transplanted cells, wherein the transplanted cells address the problem of stem cell deficiency. In one embodiment, the reintroduced cells have been genetically engineered. In one embodiment, after the invention is used to kill target cells (e.g., unmodified cells of the type that are still present in the subject), the cells from the subject have been removed and genetically modified and then returned to the subject. In a preferred embodiment, the genetically modified transfer cell is a hematopoietic stem cell.

In a preferred embodiment, the depleted cells and the transferred cells are or comprise the same cell type. In a preferred embodiment, the cells that are depleted are hematopoietic cells, in particular hematopoietic stem cells. In one embodiment, the invention is used to deplete cells prior to bone marrow transplantation. In another embodiment, the invention is used to deplete cells instead of irradiation. In another embodiment, the invention is employed in addition to irradiation to deplete cells.

In another embodiment, the invention provides a method of helping to reduce the chance of rejection of transplanted cells, the method comprising administering a therapeutic agent of the invention to deplete cells prior to transferring the cells. In another embodiment, the invention can be used to promote the acceptance of transplanted immune cells by a subject by depleting target cells expressing CD45 prior to immune cell transfer. The target cell may be any of those discussed herein. In one embodiment, the cells transplanted or transferred into the subject are stem cells.

Any of the methods discussed herein for eliminating CD45 expressing cells may be used for cell depletion or killing. However, in a particularly preferred embodiment of the invention, the invention may be used to cause cell death (preferably apoptosis) of cells expressing CD45 and thus deplete such cells. In particular, the present invention may lead to cross-linking of CD45 and thus to cell death (preferably apoptosis), preferably with the improved ability of the invention to cause cross-linking of CD45 also leading to more cell death (preferably apoptosis).

In one embodiment, as part of cell transfer, the subject may administer bone marrow as a means of transferring cells. In another embodiment, the subject may administer cord blood, or cells isolated from cord blood, as a means of transferring cells. In another embodiment, the transplanted cells may be from differentiated stem cells, such as in the case where the stem cells have been differentiated in vitro and then transplanted.

In one embodiment where the invention is used to deplete or kill cells, additional cell depleting or killing agents may also be used. In a preferred embodiment, the binding molecules (particularly antibodies) of the invention are the only cell depleting agents administered to a subject. In one embodiment, the level of depletion of target cells is sufficient to be effective, e.g., about at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of target cells are depleted. For example, in one embodiment, at least 50% of the target cells are depleted. In another embodiment, at least 75% of the target cells are depleted. In another embodiment, at least about 90% of the target cells are depleted. In another embodiment, at least about 95% of the target cells are depleted.

As mentioned above, in particularly preferred embodiments, the invention may be used to cause cell killing, particularly apoptosis, of cells expressing CD 45. CD 45-induced cell death (preferably apoptosis) may be identified, for example, by one or more of the following: cell contraction, membrane loosening, phosphatidylserine (PS) externalization resides in the outer leaflet of the plasma membrane, reduction of mitochondrial transmembrane potential, and production of reactive oxygen species. In one embodiment, the measurement or identification of these can be used to identify cell death (preferably apoptosis) of cells expressing CD 45. In one embodiment, stimulation of cells undergoing cell death (preferably apoptosis) by the present invention may exhibit one or more, preferably all, of Phosphatidylserine (PS) exposure (allowing annexin V staining), membrane foaming, retention of membrane integrity, nuclear aggregation, and RNA/protein synthesis that are not required for cell death (preferably apoptosis) to occur. Since the integrity of the cells is generally preserved and PS is present on the outer surface of the cells, staining (e.g. with annexin V) can be used to identify cell death, in particular apoptosis of the cells. Thus, in a preferred embodiment, annexin V staining is used to identify apoptotic cells. However, any suitable method may be used to assess cell viability and thus cell killing.

In another embodiment, the invention may be used for Graft Versus Host Disease (GVHD). In one embodiment, GVHD is acute. In another embodiment, the GVHD is chronic. For example, the invention may be used to avoid the development of GVHD or to alleviate GVHD, resulting in a reduction in its severity. For example, the invention may be used to deplete and/or kill target cells in cells, tissues or organs to be transplanted prior to transplantation into a subject. In one embodiment, the invention thus provides an ex vivo method of treating a population of cells, tissue or organ with a binding molecule (particularly an antibody) to deplete the cells.

In another embodiment, the invention provides a method of treatment comprising first performing such ex vivo treatment, and then performing transplantation. In another embodiment, the invention is used to deplete or kill cells in a subject prior to transplantation, such that fewer host cells are able to attack the transplanted material as a way to reduce the chance of GVHD. Accordingly, the present invention also provides a method of treating or preventing GVHD comprising administering a binding molecule (particularly an antibody) of the invention to deplete cells in a cell population, tissue or organ prior to transplanting the cell population, tissue or organ. The method may also include the transplanting itself. The depleted cells and transplanted cells, tissues or organs may be any of those mentioned herein. In a preferred embodiment, the transplanted cells are hematopoietic stem cells. In a preferred embodiment, the cells that are depleted are T cells. In another preferred embodiment, the ability of the invention to treat or prevent GVHD is used in heart, lung, kidney or liver transplants.

In another embodiment, the invention provides a method of depleting and/or killing cells in a cell population, tissue or organ prior to transplantation of the cell population, tissue or organ, rather than a method of treating a recipient. Accordingly, the present invention also provides a method of removing target cells from a cell population, tissue or organ prior to transplantation, comprising treating the cell population, tissue or organ prior to transplantation and then performing transplantation.

In one embodiment, the invention may be used to deplete immune cells in an organ or tissue, particularly if conventional therapies are not readily available or would result in exacerbation of inflammation due to some of their inherent mechanisms. In one embodiment, the invention is used to deplete cells in a closed organ, such as cells in the brain, spinal cord, eye or testes. In one embodiment, the invention can be used to deplete CD45 in immune-immune organs ⁺ And (3) cells. The binding molecules of the invention deplete CD45 without the use of Fc mediated functions ⁺ The ability of the cells may help to avoid unwanted side effects and damage. In one embodiment, the invention can be used to immunosilent deplete cells without the need for antibody effector mechanisms. This may have the advantage of minimizing or at least reducing unnecessary damage, for example, because the enclosed organ may contain fragile and often non-dividing tissue cells that may be destroyed by infiltrating leukocytes. When the invention is applied directly to an organ, such as the brain, spinal cord, eye or testis, it can result in the elimination of CD45 positive cells without causing further damage or inflammation or further reduction of damage to the tissue. In one embodiment, the target cells in the occluded organ are selected from lymphocytes, B cells and T cells. In one embodiment, the target cells in the occluded organ are or include cd4+ T cells. In another embodiment, the target cell is or comprises a cd8+ T cell.

In a further preferred embodiment, the condition to be treated by the present invention may be selected from one of the following:

viral encephalitis;

glaucoma, particularly glaucoma characterized by T-cell infiltration of the retina;

parkinson's disease;

·ALS；

side tumor syndrome of central nervous system involvement;

neuromyelitis optica;

autoimmune encephalitis;

autoimmune uveitis; and

chronic/autoimmune orchitis or other testicular disease leading to infertility.

In a further preferred embodiment, the condition in which the invention is applied is a condition characterized by infiltration of cd8+ T cells.

Pharmaceutical composition

In one aspect, a pharmaceutical composition comprises: (a) One or more binding molecules, one or more nucleic acid molecules, or one or more vectors of the invention; and (b) a pharmaceutically acceptable carrier or diluent. In a preferred embodiment, the pharmaceutical composition comprises one or more binding molecules of the invention. In one aspect, a pharmaceutical composition comprising one or more antibodies of the invention is provided. In a particularly preferred embodiment, it comprises one or more antibodies of the invention. Various components may be included in the composition, including pharmaceutically acceptable carriers, excipients, and/or diluents. The composition may optionally comprise other molecules capable of altering the properties of the molecules of the invention, for example, reducing, stabilizing, delaying, modulating and/or activating the function of said molecules. The composition may be in solid or liquid form and may be in the form of a powder, tablet, solution or aerosol, among others.

The invention also provides a pharmaceutical or diagnostic composition comprising a molecule of the invention in combination with one or more pharmaceutically acceptable excipients, diluents or carriers. Thus, there is provided the use of binding molecules (particularly antibodies) of the invention for the treatment of pathological conditions or disorders and for the manufacture of a medicament for the treatment of pathological conditions or disorders. In one embodiment, the therapeutic agent of the invention is administered to a subject who is also administered a second therapeutic agent, both of which may be administered, for example, simultaneously, sequentially or separately. In one embodiment, both are administered in the same pharmaceutical composition. In another embodiment, both are administered in separate pharmaceutical compositions. In one embodiment, the invention provides a binding molecule (particularly an antibody) of the invention for use in a method wherein the subject is also being treated with a second therapeutic agent. In another embodiment, the invention provides a second therapeutic agent for use in a method of treating a subject with a binding molecule (particularly an antibody) of the invention. One or more nucleic acid molecules of the invention and one or more vectors may also be administered in such a combination.

The compositions of the present invention are typically supplied as sterile pharmaceutical compositions. The pharmaceutical composition of the present invention may additionally comprise a pharmaceutically acceptable adjuvant. In another embodiment, such an adjuvant is not present in the compositions of the present invention. The invention also provides a method of preparing a pharmaceutical or diagnostic composition comprising adding and mixing a binding molecule of the invention (particularly an antibody) with one or more pharmaceutically acceptable excipients, diluents or carriers.

The term "pharmaceutically acceptable excipient" as used herein refers to a pharmaceutically acceptable formulation vehicle, solution or additive to enhance the desired properties of the compositions of the present invention. Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. The solution or suspension may be encapsulated in liposomes or biodegradable microspheres. The formulations are typically provided in a substantially sterile form using a sterile manufacturing process.

This may include production and sterilization by filtration of a buffered solvent solution for the formulation, sterile suspension of the antibody in the sterile buffered solvent solution, and dispensing of the formulation into sterile containers by methods familiar to those of ordinary skill in the art.

The pharmaceutically acceptable carrier should not itself induce the production of antibodies harmful to the individual receiving the composition, and should not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.

Pharmaceutically acceptable salts may be used, for example inorganic acid salts such as hydrochloride, hydrobromide, phosphate and sulfate, or organic acid salts such as acetate, propionate, malonate and benzoate. The pharmaceutically acceptable carrier in the therapeutic composition may additionally contain liquids such as water, saline, glycerol and ethanol. Such vehicles enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by a patient.

A full discussion of pharmaceutically acceptable vehicles can be found in Remington' sPharmaceutical Sciences (Mack Publishing Company, N.J.1991).

The term "therapeutically effective amount" as used herein refers to the amount of therapeutic agent required to treat, ameliorate or prevent a targeted disease or condition or to exhibit a detectable therapeutic or prophylactic effect. For any binding molecule (particularly antibodies), a therapeutically effective amount can be estimated initially in cell culture assays or animal models, typically in rodents, rabbits, dogs, pigs or primates. Animal models can also be used to determine the appropriate concentration ranges and route of administration. This information can then be used to determine useful dosages and routes of administration for humans.

The precise therapeutically effective amount of a human subject will depend on the severity of the disease state, the general health of the subject, the age, weight and sex of the subject, the diet, the time and frequency of administration, the combination of drugs, the sensitivity and tolerance/response to treatment. This amount can be determined by routine experimentation and is within the discretion of the clinician. Typically, the therapeutically effective amount is from 0.01mg/kg to 50mg/kg, for example from 0.1mg/kg to 20mg/kg per day. Alternatively, the dose may be 1 to 500mg per day, such as 10 to 100, 200, 300 or 400mg per day. The pharmaceutical compositions may conveniently be presented in unit dosage form containing a predetermined amount of the active agent of the present invention. In one embodiment, the amount of a given dose is at least sufficient to result in a particular function.

The compositions may be administered to the patient alone, or may be administered in combination (e.g., simultaneously, sequentially, or separately) with other agents, drugs, or hormones. The dosage to which the invention is applied depends on the nature of the condition to be treated, the degree of inflammation present and whether the binding molecule (particularly the antibody) is used prophylactically or for treating the existing condition.

The frequency of doses may depend on the half-life of the binding molecule (particularly the antibody) and the duration of its action. If the half-life is short (e.g., 2 to 10 hours), it may be desirable to administer one or more doses per day. Alternatively, if its half-life is long (e.g., 2 to 15 days), it may only be required to be administered once a day, once a week, or even once every 1 or 2 months. In some embodiments, it may be desirable for the therapeutic agents of the present invention to be cleared from the system rapidly after reaching their desired effect, so that the binding molecules (particularly antibodies) of the present invention may be deliberately selected and thus have a short half-life. For example, in embodiments of the invention, which aim to deplete target cells and transfer cells to a subject, if the binding molecules (particularly antibodies) of the invention employed are also targeted to the transferred cells, it may be desirable that the binding molecules (particularly antibodies) employed have a short half-life to avoid that they also target the transferred cells. This may mean that, for example, the interval between depletion of cells and transfer of new cells may be smaller.

In the present invention, the pH of the final formulation is not similar to the value of the isoelectric point of the binding molecule (particularly antibody) of the present invention, since if the pH of the formulation is 7, a pI of 8 to 9 or higher may be suitable. While not wishing to be bound by theory, it is believed that this ultimately may provide a final formulation, such as a binding molecule, particularly an antibody, that remains in solution with improved stability.

The binding molecules, particularly antibodies, and pharmaceutical compositions of the invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal (see, e.g., WO 98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal, or rectal routes. Needleless syringes (hypospray) may also be used to administer the pharmaceutical compositions of the present invention. The direct delivery of the composition will typically be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or to the interstitial space of the tissue. The composition may also be applied to specific tissues of interest. The administration therapy may be a single dose regimen or a multiple dose regimen. When the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and it may contain formulatory agents such as suspending, preservative, stabilizing and/or dispersing agents. Alternatively, the binding molecules (particularly antibodies) may be in dry form for reconstitution with a suitable sterile liquid prior to use. If the composition is to be administered by a route that uses the gastrointestinal tract, the composition needs to contain an agent that protects the antibody from degradation, but once it is absorbed from the gastrointestinal tract, the bispecific protein complex is released.

The nebulizable formulation according to the invention can be provided, for example, as single dose units (e.g. sealed plastic containers or vials) packaged in foil envelopes. Each vial contains a unit dose in a volume (e.g., 2ml solvent/solution buffer).

The invention also provides a method of preparing a pharmaceutical or diagnostic composition comprising adding and mixing together a binding molecule of the invention, particularly an antibody, and one or more pharmaceutically acceptable excipients, diluents or carriers.

The binding molecule (particularly an antibody), nucleic acid molecule or vector may be the only active ingredient in a pharmaceutical or diagnostic composition or may be accompanied by other active ingredients, including antibody or non-antibody ingredients, such as steroids or other pharmaceutical molecules.

The pharmaceutical composition suitably comprises a therapeutically effective amount of a binding molecule of the invention, in particular an antibody. The term "therapeutically effective amount" as used herein refers to the amount of therapeutic agent required to treat, ameliorate or prevent a targeted disease or condition or to exhibit a detectable therapeutic or prophylactic effect. A "therapeutically effective amount" may be an amount required to result in a desired level of cellular depletion. For any binding molecule, particularly antibodies, a therapeutically effective amount can be estimated initially in cell culture assays or animal models, typically in rodents, rabbits, dogs, pigs or primates. Animal models can also be used to determine the appropriate concentration ranges and route of administration. This information can then be used to determine useful dosages and routes of human use.

The pharmaceutical compositions may conveniently be presented in unit dosage form, each dosage containing a predetermined amount of the active agent of the present invention. The pharmaceutical compositions of the invention may be provided in a container that provides a means of administration to a subject. The pharmaceutical composition of the present invention may be provided in a prefilled syringe. The present invention thus provides such a loaded syringe. An automatic injector containing the pharmaceutical composition of the present invention is also provided.

The compositions may be administered to the patient alone or in combination (e.g., simultaneously, sequentially or separately) with other agents, drugs or hormones.

As used herein, an agent refers to an entity that has a physiological effect upon administration. As used herein, a drug refers to a chemical entity that has an appropriate physiological effect at the time of therapeutic dose.

The dosage of one or more molecules of the invention to be administered depends on the nature of the condition to be treated, the degree of inflammation present, and whether the invention is to be used prophylactically or to treat an existing condition. The frequency of dosage will depend on the half-life of the binding molecule (particularly the antibody) and the duration of its action. If the binding molecule, particularly the antibody, has a short half-life (e.g. 2 to 10 hours), it may be desirable to administer one or more doses per day. Alternatively, if the binding molecule (particularly the antibody) has a long half-life (e.g. 2 to 15 days) and/or long lasting Pharmacodynamic (PD) profile, it may be required to administer a dose only once a day, once a week or even every 1 or 2 months. In one embodiment, the dose is delivered once every two weeks, i.e., twice a month.

In one embodiment, a single dose is administered. In one embodiment, the method of the invention comprises administering until the target cell population is depleted, and then stopping all administration. The method may comprise allowing the subject to discontinue administration prior to administering the cell transplant to the subject, such that the antibody has time to clear from the subject's system. In one embodiment, the doses are administered at intervals to allow for a reduction in the anti-drug (in this case anti-antibody) response prior to administration of further doses.

Half-life as used herein is intended to refer to the duration of a molecule in the circulation (e.g., in serum/plasma). Pharmacodynamics as used herein refers to the general character and in particular the duration of the biological action of the molecules according to the invention.

Pharmaceutically acceptable salts may be used, for example inorganic acid salts such as hydrochloride, hydrobromide, phosphate and sulfate, or organic acid salts such as acetate, propionate, malonate and benzoate. The pharmaceutically acceptable carrier in the therapeutic composition may additionally contain liquids such as water, saline, glycerol and ethanol. Furthermore, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such vehicles enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by a patient.

Suitable forms of administration include forms suitable for parenteral administration, for example by injection or infusion, for example by bolus injection or continuous infusion. When the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulatory agents such as suspending, preservative, stabilizing and/or dispersing agents. Alternatively, the antibody molecules may be in dry form for reconstitution with a suitable sterile liquid prior to use.

Once formulated, the compositions of the invention may be administered directly to a subject. The subject to be treated may be an animal. However, in one or more embodiments, the composition is suitable for administration to a human subject.

In the formulation according to the invention, the pH of the final formulation is suitably not similar to the value of the isoelectric point of the antibody or fragment, for example if the pI of the protein is in the range of 8-9 or higher, a pH of 7 of the formulation may be suitable. While not wishing to be bound by theory, it is believed that this ultimately may provide a final formulation, such as a binding molecule, particularly an antibody, that remains in solution with improved stability. In one example, a pharmaceutical formulation having a pH in the range of 4.0 to 7.0 comprises: 1-200mg/mL of a binding molecule according to the invention, in particular an antibody, 1-100mM buffer, 0.001-1% surfactant, a) 10-500mM stabilizer, b) 10-500mM stabilizer and 5-500mM tonicity agent, or c) 5-500mM tonicity agent.

The pharmaceutical compositions of the present invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal (see, for example, WO 98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes. Needleless syringes may also be used to administer the pharmaceutical compositions of the present invention. In general, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms may also be prepared that are dissolved or suspended in a liquid vehicle prior to injection.

The direct delivery of the composition will typically be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or to the interstitial space of the tissue. The composition may also be applied to a lesion. The administration therapy may be a single dose regimen or a multiple dose regimen. It will be appreciated that the active ingredient in the composition will be an antibody molecule. Thus, it can be easily degraded in the gastrointestinal tract. Thus, if the composition is to be administered by a route that uses the gastrointestinal tract, the composition may contain an agent that protects the antibody from degradation, but once it is absorbed from the gastrointestinal tract, the antibody is released. In one embodiment, the compositions of the invention may be injected into a closed organ, such as any of those mentioned herein.

In one embodiment, the formulation is provided as a formulation for topical administration, including inhalation. Suitable inhalable formulations include inhalable powders, metered dose aerosols containing propellant gases or inhalable solutions without propellant gases. The inhalable powders containing an active substance according to the invention may consist of the above-mentioned active substance alone or in a mixture with physiologically acceptable excipients. These inhalable powders may include monosaccharides (e.g. glucose or arabinose), disaccharides (e.g. lactose, sucrose, maltose), oligo-and polysaccharides (e.g. dextran), polyols (e.g. sorbitol, mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or mixtures of these substances with each other. The mono-or disaccharides are suitably used, lactose or glucose are used, in particular but not exclusively in the form of their hydrates.

Particles for deposition in the lungs need to be less than 10 microns in size, such as 1-9 microns, e.g. 1-5 microns. The particle size of the active ingredient (such as an antibody or fragment) is of paramount importance.

Propellant gases useful for preparing inhalable aerosols are known in the art. Suitable propellant gases are selected from chlorinated and/or fluorinated derivatives of hydrocarbons such as n-propane, n-butane or isobutane and halogenated hydrocarbons such as methane, ethane, propane, butane, cyclopropane or cyclobutane. The propellant gases mentioned above may be used alone or in mixtures.

Particularly suitable propellant gases are haloalkane derivatives selected from TG 11, TG 12, TG134a and TG 227. Of the above-mentioned halogenated hydrocarbons, the halogenated hydrocarbons, TG134a (1, 2-tetrafluoroethane) TG227 (1, 2, 3) heptafluoropropane) and mixtures thereof are particularly suitable.

The propellant gas containing inhalable aerosols may also contain other components such as co-solvents, stabilizers, surface active agents (surfactants), antioxidants, lubricants and means for adjusting the pH. All of these ingredients are known in the art.

The propellant gas-containing inhalable aerosols according to the invention may contain up to 5% by weight of active substance. The aerosols according to the invention contain, for example, from 0.002 to 5% by weight, from 0.01 to 3% by weight, from 0.015 to 2% by weight, from 0.1 to 2% by weight, from 0.5 to 2% by weight or from 0.5 to 1% by weight of active ingredient.

Alternatively, topical administration to the lungs may also be by administration of a liquid solution or suspension formulation, for example using a device such as a nebulizer, for example a nebulizer connected to a compressor (e.g., a Pari LC-Jet Plus (R) nebulizer connected to a Pari Master (R) compressor, manufactured by Pari Respiratory Equipment, inc., richmond, va.).

The binding molecules of the invention (particularly antibodies) may be delivered dispersed in a solvent, for example in the form of a solution or suspension. It may be suspended in a suitable physiological solution, for example, physiological saline or other pharmacologically acceptable solvents or buffers. Suspensions may employ, for example, lyophilized binding molecules, particularly lyophilized antibodies.

Therapeutic suspension or solution formulations may also contain one or more excipients. Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. The solution or suspension may be encapsulated in liposomes or biodegradable microspheres. The formulations are typically provided in a substantially sterile form using a sterile manufacturing process. This may include production and sterilization by filtration of the buffer solvent/solution for the formulation, sterile suspension of the antibody in the sterile buffer solvent solution, and dispensing of the formulation into sterile containers by methods familiar to those of ordinary skill in the art.

The present invention may be suitable for delivery by nebulization.

The invention also provides a syringe containing a composition comprising a binding molecule of the invention, in particular an antibody. In one embodiment, a prefilled syringe is provided containing a unit dose of a binding molecule of the invention, particularly an antibody. In another embodiment, an auto-injector loaded with a binding molecule (particularly an antibody) of the invention is provided. In a further embodiment, an IV bag containing a binding molecule (particularly an antibody) of the invention is provided. Binding molecules (particularly antibodies) of the invention in lyophilized form in vials or in lyophilized form in similar containers are also provided.

It is also contemplated that binding molecules (particularly antibodies) of the invention may be administered using gene therapy. To achieve this, when the binding molecule is an antibody, DNA sequences encoding the heavy and light chains of the antibody molecule are introduced into the patient under the control of appropriate DNA components, such that the antibody chains are expressed from the DNA sequences and assembled in situ.

In one embodiment, the binding molecules (particularly antibodies) of the invention may be used to functionally alter the activity of one or more antigens of interest, particularly to modulate CD45. For example, the invention may directly or indirectly neutralize, antagonize or agonize the activity of the one or more antigens.

The invention also extends to a kit comprising a binding molecule, in particular an antibody, of the invention. In one embodiment, a kit comprising any binding molecule of the invention (in particular an antibody), optionally with instructions for administration, is provided.

In yet another embodiment, the kit further comprises one or more reagents for performing one or more functional assays.

In one embodiment, the molecules of the invention, including the antibodies of the invention, are provided for use as laboratory reagents.

Further aspects

In a further aspect, there is provided a nucleotide sequence, e.g. a DNA sequence, encoding an antibody molecule of the invention as described herein. In one embodiment, a nucleotide sequence, e.g., a DNA sequence, encoding a binding molecule (particularly an antibody) of the invention as described herein is provided. In one embodiment, the nucleotide sequences are co-present on more than one polynucleotide, but together they are collectively capable of encoding a binding molecule, particularly an antibody, of the invention.

The invention also extends herein to a vector comprising a nucleotide sequence as described above. The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. An example of a vector is a "plasmid" which is a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell, and subsequently are replicated along with the host genome. In the present specification, the terms "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. General methods of constructing vectors, transfection methods and culture methods are well known to those skilled in the art. In this regard, reference is made to "Current Protocols in Molecular Biology",1999, f.m. ausubel (edit), wiley Interscience, new York and the Maniatis Manual produced by Cold Spring Harbor publishing.

The term carrier herein also includes, for example, particles comprising a carrier, such as LNP (lipid nanoparticle) particles and in particular LNP-mRNA particles. Also included are viral particles for transferring the vectors of the invention.

The term "selectable marker" as used herein refers to a protein whose expression allows one to recognize cells that have been transformed or transfected with a vector containing a marker gene. A wide variety of selectable markers are known in the art. For example, in general, selectable marker genes confer resistance to drugs (such as G418, hygromycin or methotrexate) on host cells into which the vector has been introduced. The selectable marker may also be a visually identifiable marker, such as, for example, a fluorescent marker. Examples of fluorescent markers include rhodamine, FITC, TRITC, alexa Fluors and various conjugates thereof.

In one embodiment, the invention provides a vector encoding a binding molecule (particularly an antibody) of the invention. In another embodiment, the invention provides vectors that collectively encode the binding molecules (particularly antibodies) of the invention.

Also provided is a host cell comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding an antibody of the invention. Any suitable host cell/vector system may be used to express the DNA sequences encoding the antibody molecules of the invention. Bacteria such as E.coli and other microbial systems may be used or eukaryotic, such as mammalian, host cell expression systems may also be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells. Host cells comprising the nucleic acid molecules or vectors of the invention are also provided.

The invention also provides a method of producing a molecule according to the invention or a component thereof, the method comprising culturing a host cell containing a vector of the invention under conditions suitable to cause expression of a protein from DNA encoding the molecule of the invention, and isolating the molecule.

The method of producing an antibody comprising a heterodimeric tether may further comprise mixing two portions of the antibody and allowing association of the binding partners of the heterodimeric tether. The method may also include purification, e.g., removal of any material other than the desired heterodimer.

The binding molecules, particularly antibodies, of the invention are useful in diagnostic/detection kits. In one embodiment, the antibodies of the invention are immobilized on a solid surface. The solid surface may be, for example, a chip or an ELISA plate.

For example, the binding molecules (particularly antibodies) of the invention may be conjugated to fluorescent markers that facilitate detection of bound antibody-antigen complexes. They are useful for immunofluorescence microscopy. Alternatively, binding molecules, in particular antibodies, may also be used for western blotting or ELISA.

In one embodiment, a method of purifying a binding molecule (particularly an antibody) or component thereof of the invention is provided. In one embodiment, a method of purifying a binding molecule (particularly an antibody) or a component thereof according to the invention is provided, comprising the steps of: anion exchange chromatography is performed in non-binding mode such that impurities remain on the column and antibodies remain in the unbound fraction. This step may be performed, for example, at a pH of about 6-8. The method may further comprise an initial capture step using cation exchange chromatography, for example at a pH of about 4-5. The method may also include additional chromatographic step(s) to ensure proper resolution of product and process related impurities from the product stream. The purification process may also include one or more ultrafiltration steps, such as concentration and diafiltration steps.

The term "purified form" as used above is intended to mean at least 90% pure, e.g. 91, 92, 93, 94, 95, 96, 97, 98, 99% w/w or more pure.

In the context of this specification, "comprising" should be interpreted as "including". Aspects of the invention that include certain elements are also intended to extend to alternative embodiments "consisting of" or "consisting essentially of" the relevant elements.

The positively cited embodiments may be employed herein as the basis for disclaimers.

Where the singular is referred to herein, the plural is contemplated unless otherwise indicated or clearly contradicted by context. In particular, the singular forms "a," "an," "the," and the like include plural referents unless the context clearly dictates otherwise.

All references cited herein are specifically incorporated by reference.

The headings herein are used to assist in organizing the specification and are not intended to establish the meaning of the technical terms herein.

The sequences of the present invention are provided below.

In the context of this specification, "comprising" is to be interpreted as "including". Aspects of the present disclosure including certain elements are also intended to extend to alternative embodiments "consisting of" or "consisting essentially of" the relevant elements.

All references cited herein are specifically incorporated by reference.

Reference to the literature

1.Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries.Hanes J,Jermutus L,Weber-Bornhauser S,Bosshard HR,Plückthun A.(1998)Proc.Natl.Acad.Sci.U.S.A.95,14130-14135

2.Directed in Vitro Evolution and Crystallographic Analysis of aPeptide-binding Single Chain Antibody Fragment(scFv)withLow Picomolar Affinity.Zhand C,Spinelli S,Luginbuhl B,Amstutz P,Cambillau C,Pluckthun A.(2004)J.Biol.Chem.279,18870-18877

3.Antigen recognition by conformational selection.Berger C,Weber-Bornhauser S,Eggenberger Y,Hanes J,Pluckthun A,Bosshard H.R.(1999)F.E.B.S.Letters 450,149-153

Examples

The term Fab-X/-Fab-Y (or Fab-KD-Fab) as used in the examples describes a protein complex form having the formula A-X: Y-B, wherein:

A-X is a first fusion protein;

Y-B is a second fusion protein;

y is a heterodimeric tether;

a comprises a Fab fragment specific for an antigen (such as CD 45);

b comprises a Fab fragment specific for an antigen (such as CD 45);

x is a first binding partner of a binding pair, such as an scFv (e.g., an scFv that binds a GCN4 peptide);

y is a second binding partner of the binding pair, such as a peptide (e.g., GCN peptide); and

is the interaction between X and Y (such as a binding interaction).

-is a bond or a linker.

Many of the antibody molecules used in the examples are in this form. Others are in the form of BYbe. The BYbe antibodies used in the examples are of the following format:

c) Polypeptide chain: v (V) _H CH1-ScFv；

d) Polypeptide chain: v (V) _L C _L ；

Wherein:

V _H CH ₁ and V _L C _L Pairing each other to form Fab;

CH1 and ScFv are linked by a bond or linker "-".

Example 1-Production of Fab-X (Fab-52SR4scFv) and Fab-Y (Fab-GCN 4 peptide) for functional assays

Method

Cloning strategy

Antibody variable region DNA is produced by PCR or gene synthesis and contains flanking restriction enzyme sites. These sites are HindIII and XhoI for the variable heavy chain and HindIII and BsiWI for the variable light chain. This makes the heavy chain variable region suitable for ligation to two heavy chain vectors (pnfh linked to Fab-Y and pnfh linked to Fab-X ds [ disulfide stabilized ]), as they have complementary restriction sites. This links the variable region upstream (or 5') to the murine constant region and either peptide Y (GCN 4) or scFv X (52 SR 4), resulting in the entire reading frame. The light chain was cloned into a standard internal murine constant kappa vector (pMmCK or pMmCK S171C) which again used the same complementary restriction sites. If the variable regions were isolated from rabbits, the pMmCK S171C vector was used. Cloning events were confirmed by sequencing using primers flanking the entire open reading frame.

Cultivation of CHOS

Suspension CHOS cells were pre-adapted to CDCHO medium (Invitrogen) supplemented with 2mM (100X) glutamax. Shaking at 140rpm on a shaker incubator (Kuner AG, birsfelden, switzerland) and incubation at 37℃supplemented with 8% CO ₂ Cells were maintained in the logarithmic growth phase.

Electroporation transfection

Cell number and viability were determined using CEDEX cell counter (Innovatis AG. Bielefield, germany) prior to transfection, and the required amount of cells (2X 10 ⁸ Individual cells/ml) was transferred to a centrifuge cone and spun at 1400rpm for 10 minutes. The pelleted cells were resuspended in sterile Earls balanced salt solution and spun at 1400rpm for an additional 10 minutes. The supernatant was aspirated and the pellet resuspended to the desired cell density.

Vector DNA was used at a final concentration of 400. Mu.g for 2X10 ⁸ Mu.l of the cell/ml mixture was pipetted into a cuvette (Biorad) and electroporated using an internal electroporation system.

The transfected cells were transferred directly into a 3L Erlenmeyer flask containing ProCHOS medium enriched with 2mM glutamx and antibiotic anti-mitotic (100X) solution (1:500) and the cells were incubated at 37℃in 5% CO ₂ And a Kuhner shaker incubator with shaking at 140 rpm. Feed supplement 2g/L ASF (AJINOMOTO) was added 24 hours after transfection and the temperature was reduced to 32℃for further 13 days. On day 4, 3mM sodium butyrate (sodium n-butyrate, sigma B-5887) was added to the culture. On day 14, the cultures were transferred to tubes and after centrifugation at 4000rpm for 30 minutes the supernatant was separated from the cells. The remaining supernatant was further passed through a 0.22 μm m SARTO BRAN PMillipore followed by filtration through a 0.22 μm Gamma gold filter. The final expression level was determined by protein G-HPLC.

Large scale of(1.0L) purification

Fab-X and Fab-Y were purified by affinity capture using the AKTA Xpress system and a HisTrap Excel pre-loaded nickel column (GE Healthcare). The culture supernatant was sterile filtered at 0.22 μm and the pH was adjusted to neutral with a weak acid or base, if necessary, prior to loading onto the column. Any weakly bound host cell protein/non-specific His conjugate was displaced from the nickel resin using a secondary wash step comprising 15-25mM imidazole. Elution was performed with 10mM sodium phosphate, pH7.4+1M NaCl+250mM imidazole, and 2ml fractions were collected. One column volume was added to the eluate, and the system was suspended for 10 minutes to tighten the elution peak and thus reduce the total elution volume. The purest fractions were pooled and buffer exchanged into PBS (Sigma), pH7.4 and 0.22 μm filtered. The final pool was determined by A280Scan, SE-HPLC (G3000 method), SDS-PAGE (reduced and non-reduced) and using the Endosafe nexgen-PTS system (Charles River) (for endotoxin).

Example 2-Fab and BYbe production

Method

To generate Fab fragments of anti-CD 45 antibodies 4133 and 6294, genes encoding their respective light and heavy chain V regions were designed and constructed by automated synthesis methods (ATUM). The V region gene of rabbit antibody 4133 was cloned into an expression vector containing DNA encoding the rabbit ck 1 region and heavy chain ych 1 region, respectively. The V region gene of mouse antibody 6294 was cloned into an expression vector containing DNA encoding the mouse ck region and heavy chain γ1ch1 region, respectively.

Likewise, the full length of the heavy chain (Fab HC-G4S linker-scFv) of 4133-6294 and NegCtrl BYbe was designed and constructed by automated synthesis (ATUM). Both heavy chains were cloned into an internal mammalian expression vector. 4133-6294BYbe heavy chain paired with the 4133 light chain described above. The light chain V region gene of NegCtrl BYbe was designed and constructed by automated synthesis (ATUM) and then cloned into an expression vector containing DNA encoding the mouse ck region. NegCtrl BYbe has antigen-independent specificity in both the Fab and scFv positions.

According to the manufacturer's instructions, the Gibco ExpiFectamine CHO transfection kit (cat No. a29133, thermoFisher Scientific) the relevant heavy and light chain constructs were paired and transfected into CHO-SXE cells. Cells were incubated at 37℃with 5% CO ₂ And an incubator with shaking at 140rpm for 7 days. After incubation, the cultures were transferred to tubes and the supernatant was separated from the cells after centrifugation at 4000rpm for 30 minutes. The remaining supernatant was filtered through 0.22 μm SARTO BRAN P Millipore, then through 0.22 μm Gamma gold filter.

According to the manufacturer's instructions, all proteins from the supernatant were purified in series on an AKTA Pure purification system (GE Healthcare Life Sciences) using two 5ml HiTrap protein G HP columns (cat. No. GE29-0405-01, sigmaAldrich). Fractions of eluted protein were pooled and concentrated to <5ml using an Amicon Ultra-15 centrifugal filtration device (UFC 9010, sigmaAldrich) with an Ultra-10 membrane of 10 kDa.

To obtain a Pure fraction, the protein was then passed through a HiLoad Superdex 200pg 16/60HPLC size exclusion column on an AKTA Pure purification system. Protein concentration was determined using Thermo Scientific NanoDrop 2000 (cat# ND-2000). In addition, fractions were analyzed on 4-20% Tris-glycine gels and tested for endotoxin using the Endosafe nexgen-MCS system (Charles River).

Example 3-Production of CD45 extracellular Domain

Method

The coding gene for domains 1-4 of the CD45 extracellular domain (UniProtKB-P08575, residue positions 225-573) was designed and constructed by automated synthesis (ATUM). To aid purification, a TEV cleavage site and a 10-His tag were incorporated at the C-terminus of the expressed protein. This gene was cloned into an internal mammalian expression vector and then transfected into HEK293 cells using a Gibco ExpiFectamine 293 transfection kit (cat No. a14525, thermoFisher Scientific) according to the manufacturer's instructions. Cells were incubated at 37℃with 5% CO ₂ And an incubator with shaking at 140rpm for 7 days. After incubation, the cultures were transferred to tubes and the supernatant was separated from the cells after centrifugation at 4000rpm for 30 minutes. The remaining supernatant was filtered through 0.22. Mu. m SARTO BRAN P Millipore, then through 0.22 μm Gamma gold And (5) filtering.

His tagged proteins from the supernatant were purified in series on an AKTA Pure purification system (GE Healthcare Life Sciences) using two 1ml HisTrap Excel columns (cat. No. GE17-3712-05, sigmaAldrich) according to the manufacturer's instructions. Fractions of eluted protein were pooled and concentrated to <5ml using an Amicon Ultra-15 centrifugal filtration device (UFC 900308, sigmaAldrich) with an Ultra-3 membrane of 3 kDa. To obtain a Pure fraction, the protein was then passed through a HiLoad Superdex 75pg 16/60HPLC size exclusion column on an AKTA Pure purification system. Protein concentration was determined using Thermo Scientific NanoDrop 2000 (cat# ND-2000). In addition, fractions were analyzed on 4-20% Tris-glycine gels.

Example 4-Determination of anti-CD 45 Fab-KD-Fab induced apoptosis method by annexin V binding

Human PBMCs derived from the platelet-monocompartment of blood leukocytes (NHSBT Oxford) were stored as frozen aliquots. Before performing the assay, 2 vials of frozen cells per donor cone (each vial containing 5x10 in 1ml ⁷ Individual cells) were thawed in a 37 ℃ water bath, then 50ml of complete medium (RPMI 1640+2mM GlutaMAX+1%Pen/Strep, all supplied by Invitrogen, +10% Fetal Bovine Serum (FBS), sigma Aldrich) was added. The cells were centrifuged (500 g,5 min, room temperature) and resuspended in 30ml of complete medium for washing and re-centrifugation. Cells were resuspended in 20ml complete medium, counted and counted on ChemoMetec NucleoCounter NC-3000 to determine concentration and viability, then diluted to 1.25x10 ⁶ Individual cells/ml. Then 80. Mu.l of each well 10 ⁵ Individual cells were added to each well of a Corning Costar 96 well U-bottom microplate (cat. No. 07-200-95) treated for cell culture and incubated at 37℃with 5% CO ₂ The incubator was left to stand for 2 hours. PBMCs from three donor UCB-packages 652, 658 and 686 were used for this assay.

The Fab-KD-Fab reagent can be combined by premixing the two separate halves (labeled X and Y) to form a non-covalently linked Fab-Fab combination. Combining Fab-X and Fab-Y with NegCtrl-X/4133-Y, 6294-X/NegCtrl-Y, 6294-X/4133-Y and 6294-X/6294-A/A in Greiner 96 well non-binding microwell platesY was added to the complete medium to give a Fab-KD-Fab concentration of 500 nM. Microwell plates were incubated at 37℃with 5% CO ₂ Incubate for 1 hour. NegCtrl-X and NegCtrl-Y are negative controls that are specific for unrelated antigens.

Mu.l of each Fab-KD-Fab preparation was then added to the cells (final Fab-KD-Fab concentration 100 nM) and incubated at 37℃with 5% CO ₂ Incubate for 24 hours. After incubation, the plates were centrifuged at 500g for 5 min at room temperature and the medium was aspirated using a BioTek ELx405 microplate washer (20 μ l U bottom aspiration setup) leaving the cells in 20ul of residual medium.

The Multicyt apoptosis kit (intelllicyt cat No. 90054) was used according to the manufacturer's instructions. Staining mixtures were prepared at 2X working concentration in complete medium. Mu.l of antibody staining mixture was added to the cells and the plates were incubated at 37℃with 5% CO ₂ Incubate for 1 hour. Live cells were analyzed using Intellicyt iQue Screener PLUS. Viable cell count was extracted using Graphpad Prism version 8.1 (Graphpad) to generate an index and graphical representation.

Results

Data from a representative donor (UCB Cone-686) are shown in FIGS. 1 (A) and 1 (B). (A) A significant decrease in lymphocyte count was observed in 6294-X/4133-Y treated cells compared to cells treated or untreated with NegCtrl-X/4133-Y, 6294-X/NegCtrl-Y, 6294-X/6294-Y. (B) Of the cells surviving in 6294-X/4133-Y wells, 38% showed annexin V binding. This indicates that the cells are undergoing apoptosis. Furthermore, annexin V binding levels were 3-fold higher than other treated and untreated wells Gao Yao.

Example 5-Purification of apoptosis of T cells

Method

Human PBMCs derived from the platelet-monocompartment of blood leukocytes (NHSBT Oxford) were stored as frozen aliquots. Before performing the assay, 2 vials of frozen cells per donor cone (each vial containing 5x10 in 1ml ⁷ Individual cells) were thawed in a 37 ℃ water bath, then 50ml RPMI medium (RPMI 1640+2mm glutamine+1% penicillin/streptomycin, by Invitro) was addedgen, cat# H3667-20ML,Sigma Aldrich) 5% heat-inactivated human AB serum. The cells were centrifuged (500 g,5 min, room temperature) and resuspended in 30ml of complete medium for washing and re-centrifugation. Cells were resuspended in 20ml RPMI medium, counted and counted on ChemoMetec NucleoCounter NC-3000 to determine concentration and viability, then diluted to 1.25x10 ⁶ Individual cells/ml. Then 80. Mu.l of each well 10 ⁵ Individual cells were added to each well of a Corning Costar 96 well U-bottom microplate (cat. No. 07-200-95) treated for cell culture and incubated at 37℃with 5% CO ₂ The incubator was left to stand for 2 hours.

T cells were purified using a cd4+ T cell isolation kit according to the manufacturer's instructions (cat No. 130-096-533,Miltenyi Biotec). Briefly, PBMC were washed in cold MACS buffer (PBS pH 7.2,0.5% bovine serum albumin and 2mM EDTA,Sigma Aldrich) and incubated at 10 ⁷ Individual cells were resuspended in 40 μl MACS buffer. Mu.l of CD4+ T cell biotin-antibody mixture (per 10) ⁷ Individual cells), and then incubated at 4℃for 5 minutes). Then a further 30. Mu.l MACS buffer (per 10 ⁷ Individual cells), followed by 20 μl of cd4+ T cell microbead mixture (per 10 a) ⁷ Individual cells). The cells were mixed and then incubated at 4℃for 10 minutes. To separate cd4+ T cells from other cells, they were placed on a magnetic selection column (LS column) and washed three times with 3ml MACS buffer. Purified cd4+ T cells were collected from the column eluate. Cells were then washed in RPMI medium (as described above) and counted to assess recovery and viability (measured as 97% cell viability). Then 100 μl of each well 10 ⁵ Individual cells were added to each well of Corning Costar 96 well U-bottom microplates (cat No. 07-200-95) treated for cell culture.

In Greiner 96 well unbound microplates, the Fab-X and Fab-Y combinations 6294-X/4133-Y, 4133-Y/6294-Y, 6294-X/6294-Y and NegCtrl-X/4133-Y were added to complete medium to give a Fab-KD-Fab concentration of 200 nM. Microwell plates were incubated at 37℃with 5% CO ₂ Incubate for 1 hour. After incubation, the Fab-KD-Fab reagent is then serially diluted 7 times in RPMI medium at 1:5 to form an 8-point dose curve. It should be noted thatWhen two Y reagents are added together (because a combination of 4133-Y and 6294-Y is used herein), they form a mixture rather than a linked molecule.

Mu.l of each Fab-KD-Fab or BYbe dilution (final well concentration 100-0.00128 nM) was then added to the plates of CD4+ cells and incubated at 37℃with 5% CO ₂ Incubate for 24 hours. After incubation, the plates were centrifuged at 500g for 5 min at room temperature. Buffer was then aspirated (using a BioTek ELx405 microplate washer, 15 μ l U bottom aspiration setup), sealed plate and centrifuged again at 1800rpm for 30 seconds. Ice-cold FACS buffer (PBS+1% BSA+0.1% NaN) ₃ +2mM EDTA) plates were topped up and centrifuged again. The buffer was removed, the plate was centrifuged again and 20 μl 1 was added to each well: 1000 near infrared dye (Invitrogen). After 20 minutes, cells were washed in 200 μl FACS buffer, added and resuspended in 15 μl FACS buffer, and then analyzed using Intellicyt iQue Screener PLUS. Viable cell count was extracted using Graphpad Prism version 8.1 (Graphpad) to generate an index and graphical representation. Asymmetric (five parameter) curve fitting was applied to derive the maximum% cytopenia and EC50 values.

Results

The percentage decrease in the number of purified cd4+ T cells is shown in figure 2. Combination 6294-X/4133-Y showed the highest level of reduction, 97%, and was the most effective with an EC50 value of 0.32nM. All other combinations did not reach 50% of the maximum cytopenia level and were not effective enough to produce EC50 readings.

EXAMPLE 6-Method for inducing apoptosis of PBMC by anti-CD 45 antibodies in the form of Fab-X/Fab-Y and Bybe

Human PBMCs derived from the platelet-monocompartment of blood leukocytes (NHSBT Oxford) were stored as frozen aliquots. Before performing the assay, 2 vials of frozen cells (each vial containing 5x10 in 1ml ⁷ Individual cells) were thawed in a 37 ℃ water bath, then 50ml of complete medium (RPMI 1640+2mM GlutaMAX+1%Pen/Strep, all supplied by Invitrogen, +10% Fetal Bovine Serum (FBS), sigma Aldrich) was added. Cells were centrifuged (500 g,5 min, room temperature) and resuspended in 30ml complete mediumTo be washed and centrifuged again. Cells were resuspended in 10ml of complete medium and then counted using ChemoMetec NucleoCounter NC-3000. Then 100 μl of each well 10 ⁵ Individual cells were added to each well of Corning Costar 96 well U-bottom microplates (cat No. 07-200-95) treated for cell culture. The plates were incubated at 37℃with 5% CO ₂ The mixture was allowed to stand in the incubator for 2 hours. PBMC from two donor UCB-Cones 801 and 802 were used for this assay.

Fab-X and Fab-Y combinations 6294-X/4133-Y were added to complete medium in Greiner 96 well non-binding microwell plates to give a Fab-KD-Fab concentration of 1500nM. Likewise, 1500nM stock solution of 4133-6294BYbe was prepared in complete medium. Microwell plates were incubated at 37℃with 5% CO ₂ Incubate for 1 hour. After incubation, the Fab-KD-Fab and BYbe reagents were then serially diluted 9 times in complete medium at 1:5 to generate a 10-point dose curve.

Mu.l of each Fab-KD-Fab or BYbe dilution (final well concentration 250-0.000128 nM) was added to the cells and incubated at 37℃with 5% CO ₂ Incubate for 24 hours. After incubation, the plates were centrifuged at 500g for 5 min at room temperature, the buffer was aspirated with a BioTek ELx405 microplate washer, and the cells were resuspended in FACS buffer (pbs+1% Bovine Serum Albumin (BSA) +0.1% nan) ₃ +2mM EDTA,Sigma Aldrich) and then centrifuged again and the buffer aspirated to leave the cells in 20 μl of residual medium. Mu.l of the cell-specific marker antibody mixture solution was added to the wells and incubated at 4℃for 1 hour. The antibody mixtures are detailed in table 4 below.

Living cells were analyzed using Intellicyt iQue Screener PLUS. Viable cell count was extracted using Graphpad Prism version 8.1 (Graphpad) to generate an index and graphical representation. Asymmetric (five parameter) curve fitting was applied to derive the maximum% cytopenia and EC50 values.

Table 4.

Results

The percentage reduction in the number of PBMC cell subsets resulting from (A) 6294-X/4133-Y and (B) 4133-6294BYbe for the representative donor (UCB Cone-802) is shown in FIG. 3 and Table 5 and Table 6 below. 6294-X/4133-Y and 4133-6294Bybe showed almost the greatest decrease in T cells (> 95%) and B cells (87%), with a high efficiency EC50 of 0.19-0.52nM in T cells and 0.65-1.50 in B cells.

Table 5. Highest and lowest level of cell subpopulation reduction, and EC50 (nM) values for 6294-X/4133-Y.

Table 6. Highest and lowest level of cell subpopulation reduction, and EC50 (nM) values for 4133-6294 Bybe.

EXAMPLE 7-Apoptosis of lymphocytes in whole blood

Method

Human whole blood (lithium heparin tubes) was collected from two donors (HTA #051119-01 and # 051119-02) in the uk UCB Pharma Slough according to an approved ethical sample collection protocol.

Fab-X and Fab-Y combinations 6294-X/4133-Y and NegCtrl-X/4133-Y were added to PBS in Greiner 96 well non-binding microwell plates to give a Fab-KD-Fab concentration of 2750nM. Likewise, 2750nM stock solution of 4133-6294BYbe was prepared in PBS. Microwell plates were incubated at 37℃with 5% CO ₂ Incubate for 1 hour. After incubation, the Fab-KD-Fab and BYbe reagents were then serially diluted 9 times in PBS at 1:5 to generate a 10-point dose curve.

Mu.l of each Fab-KD-Fab or BYbe dilution was added to Nunc ^TM 96-well polypropylene DeepWell ^TM Plates (thermo fisher). Then 50 μl of blood was added to each well, the plates were gently mixed and sealed with a flat seal allowing gas exchange. These cells were then incubated at 37℃with 5% CO ₂ Incubate for 5 hours. Short time operation testThe need to add anticoagulants is avoided.

After incubation, 950. Mu. l BD Phosflow BD Lyse/Fix (product number BD558049, fisher scientific) was added to each well and the plate was incubated at 37℃with 5% CO ₂ Incubate for 10 minutes. The plates were then centrifuged at 500g for 8 minutes at 4 ℃. The buffer was aspirated and 1ml of FACS buffer (PBS+1% Bovine Serum Albumin (BSA) +0.1% NaN) was added using an integral Viaflo 96 channel pipette ₃ +2mM EDTA,Sigma Aldrich) to wash the cells. The plates were centrifuged at 500g for 8 min at 4 ℃. As before, the buffer was aspirated and 1ml FACS buffer was added to wash the cells. Followed by centrifugation at 250g for 10 minutes at 4 ℃. The buffer was again aspirated and 1ml FACS buffer was added to wash the cells. The plates were centrifuged again at 500g for 8 minutes at 4 ℃. The buffer was aspirated, leaving the cells in the minimum residual volume for cell-specific antibody staining. Mu.l of the cell-specific antibody mixture (as shown in Table 7 below) was added to the wells and the plates were incubated at 4℃for 1 hour.

After incubation with the antibody mixture, the cells were washed twice as described above and the buffer was aspirated leaving the cells in 20 μl of residual buffer. Mu.l of FACS buffer was then added to the sample to dilute the cells. Living cells were analyzed using Intellicyt iQue Screener PLUS. Viable cell count was extracted using Graphpad Prism version 8.1 (Graphpad) to generate an index and graphical representation. Asymmetric (five parameter) curve fitting was applied to derive the maximum% cytopenia and EC50 values.

TABLE 7

Results

6294-X/4133-Y and 4133-6294BYbe resulted in (A) total lymphocytes and (B) CD4 in whole blood ⁺ Cells (donor # 051119-01), total lymphocytes (C) and CD4 (D) ⁺ The percent reduction of cells (donor # 051119-02) is shown in figure 4 and table 8 below. The data for both donors are approximately similar. Negative control NegCtrl-X/4133-Y shows total lymphocytes or CD4 of either donor ⁺ CellsThe number is not reduced. In donor #051119-02, the peak of cytopenia at the highest concentration of NegCtrl-X/4133-Y is a single data point and is not considered to reflect true activity. In contrast, 6294-X/4133-Y and 4133-6294BYbe each showed a maximum reduction of 34-44% of total lymphocytes and 48-54% of CD4+ cells within only 5 hours. The potency of these agents and their potential for in vivo activity are demonstrated by EC50 values for total lymphocytes between 0.37-5.99nM and for cd4+ cells between 0.05-0.33 nM.

TABLE 8 reduction of Total lymphocyte and CD4+ T cell levels

Example 8-Cytokine release in whole blood at 24 hours as measured by Luminex bead assay

Method

Human whole blood (lithium heparin tube) was collected from two donors (HTA #300120-1 and # 300120-2) in the uk UCB Pharma Slough according to an approved ethical sample collection protocol.

Stock solutions of 5000nM 4133-6294BYbe and negative control BYbe (NegCtrl BYbe, with unrelated antigen specificity at both Fab and scFv positions) were prepared in Greiner 96 well non-binding microwell plates in PBS. The BYbe reagent was then serially diluted three times at 1:5 in PBS to form a 4-point dose curve. Transfer 12.5 μl of BYbe dilution to Corning Costar 96 well U-bottom microplates (cat. No. 07-200-95) treated for cell culture and add 237.5 μl of whole blood to each well. The final pore concentrations of BYbes were 250nM, 50nM, 10nM and 2nM. Campath (clinical grade, diluted from 30mg/ml stock to 1mg/ml in PBS, lot F1002H 29) was used as positive control, at a final concentration of 10 μg/ml. The flat plate is sealed by an air-permeable adhesive sealing strip, and the plate cover is replaced. The plates were then humidified at 37℃and 5% CO ₂ Incubate at undisturbed sites for 24 hours.

Cytokine release was then assessed using an R & D Systems Luminex 13-plex human cytokine assay (the cytokine customization was selected as follows: IL-1RA, IL-4, IL-5, IL-6, IL-10, IL-11, IL-13, CCL2, IL-8, CXCL1, CX3CL1, GM-CSF, and M-CSF). After 24 hours incubation, the plates were centrifuged at 1000g for 10 minutes and 50 μl of plasma was transferred to a separate plate containing 50 μl of assay dilution buffer (RD 6-52 from Luminex kit). Samples were resuspended well using a multichannel pipette and 50 μl was transferred to Luminex assay plates. Luminex assay standards were diluted 7 times at 1:2 to construct a standard curve and added to the plates. Mu.l of the microparticle mixture was then added to each well and the plates were incubated for 2 hours at Room Temperature (RT) and mixed at 800 rpm. Plates were washed 3 times by adding 150 μl wash buffer to each well, and then magnetic beads were magnetically coupled to a BioTek ELx405 microplate washer prior to aspiration of supernatant. Mu.l of biotin antibody mixture was added to each well and the plate was incubated with shaking at room temperature for 1 hour. The plates were then washed as before and finally 50. Mu.l of streptavidin-PE was added to each well. The plates were incubated for 30 minutes at room temperature with shaking, then the final wash step was performed and 50 μl wash buffer was added to each well. Luminex assay plates were run using an iQUEplus flow cytometer (Sartorius). A standard curve was generated (using the assay control provided) and extrapolated cytokine values were generated using Forecyt software (Sartorius). The data is then transmitted to Graphpad Prism version 8.1 (Graphpad) to generate visual data.

Results

After 24 hours incubation with the test reagents, the levels of each cytokine detected in whole blood were similar between the two donors. Figure 5 shows the data for donor #300120-1 as representative of two donors. The levels of individual cytokines are shown below as (A) CCL2, (B) GM-CSF, (C) IL-1RA, (D) IL-6, (E) IL-8, (F) IL-10, (G) IL-11, and (H) M-CSF. Cytokines IL-4, IL-5, IL-13, CXCL1 and CX3CL1 were not detected in any of the wells (data not shown). Campath induces cytokines (a) CCL2, (C) IL-1RA and (E) IL-8 to levels exceeding the standard curve and is therefore plotted as the maximum signal in this assay. Campath also induced significantly higher IL-6 (D) levels than PBS treated wells. Significantly, 4133-6294BYbe was observed to induce little or no inflammatory cytokines at levels that matched those in PBS and NegCtrl BYbe treated wells.

Example 9-Cytokine release in 24 hours whole blood as measured by MSD assay in combination with T cell count

Method

Human whole blood (lithium heparin tube) was collected from one supply (HTA # 031219-06) of uk UCB Pharma Slough according to approved ethical sample collection protocols.

Fab-KD-Fab concentration of 2000nM was obtained by adding the Fab-X and Fab-Y combination 6294-X/4133-Y to PBS in Greiner 96 well non-binding microwell plates. Likewise, stock solutions of 4133-6294BYbe and NegCtrl BYbe were prepared at 2000nM in PBS. Microwell plates were incubated at 37℃with 5% CO ₂ Incubate for 1 hour. After incubation, the Fab-KD-Fab and BYbe reagents were then serially diluted 7 times in PBS at 1:5 to generate an 8-point dose curve.

12.5 μl of Fab-KD-Fab or BYbe dilutions were transferred to Corning Costar 96 wells as cell culture treated U-bottom microplates (cat. No. 07-200-95) and 237.5 μl of whole blood was added to each well. The final well concentration of Fab-KD-Fab or BYbe was 100-0.00128nM. Campath (clinical grade, diluted from 30mg/ml stock to 1mg/ml in PBS, lot F1002H 29) was used as positive control, at a final concentration of 10 μg/ml. The flat plate is sealed by an air-permeable adhesive sealing strip, and the plate cover is replaced. The plates were then humidified at 37℃and 5% CO ₂ Incubate at undisturbed sites for 24 hours.

After 24 hours incubation, the plates were centrifuged at 1000g for 10 minutes and 50 μl of plasma was transferred to a separate plate and stored at-80 ℃ until cytokine release assay. To determine the level of cell depletion, the remaining cells were resuspended in PBS and 50 μl from untreated (PBS), campath, and Fab-KD-Fab or BYbe 100nM wells were transferred into 96-deep well plates. 950 mu l BD Phosflow BD Lyse/Fix (product number BD558049, fisher scientific) was added to each well and the plate was incubated at 37℃with 5% CO ₂ Incubate for 10 minutes. The plates were then centrifuged at 500g for 8 minutes at 4 ℃. The buffer was aspirated and 1ml of FACS buffer (PBS+1% bovine serum albumin) was added using an integral Viaflo 96 channel pipetteProtein (BSA) +0.1% NaN ₃ +2mM EDTA,Sigma Aldrich) to wash the cells. The plates were centrifuged at 500g for 8 min at 4 ℃. As before, the buffer was aspirated and 1ml FACS buffer was added to wash the cells. Followed by centrifugation at 250g for 10 minutes at 4 ℃. The buffer was again aspirated and 1ml FACS buffer was added to wash the cells. The plates were centrifuged again at 500g for 8 minutes at 4 ℃. The buffer was aspirated, leaving the cells in the minimum residual volume for cell-specific antibody staining. Mu.l of the cell-specific antibody mixture (as shown in Table 9 below) was added to the wells and the plates were incubated at 4℃for 1 hour.

After incubation with the antibody mixture, the cells were washed twice as described above and the buffer was aspirated leaving the cells in 20 μl of residual buffer. Mu.l of FACS buffer was then added to the sample to dilute the cells. Living cells were analyzed using Intellicyt iQue Screener PLUS. Viable cell count was extracted using Graphpad Prism version 8.1 (Graphpad) to generate an index and graphical representation. An asymmetric (five parameter) curve fit is applied.

TABLE 9

Cytokine measurements were performed using V-PLEX human pro-inflammatory group I (4-PLEX) (IFN-. Gamma., IL-1β, IL-6, TNF-. Alpha., accession number K15052D, meso Scale Discovery) according to the manufacturer's instructions. Briefly, plasma samples were thawed at room temperature and diluted 1:4 with diluent 2. Mu.l of sample or standard curve calibrator was added to the pro-inflammatory group I plates and incubated for 2 hours at room temperature on a plate shaker. Plates were washed with PBS (supplemented with 0.05% Tween-20) using a BioTek ELx405 microplate washer and 30. Mu.l of detection antibody was added to each well. Plates were incubated for an additional 2 hours at room temperature on a plate shaker. Plates were washed as before and 150 μl of read buffer (at dH ₂ Diluted 1:2 in O). Plates were then analyzed on a SECTOR Imager 6000 (Meso Scale Discovery).

Results

T cell counts in whole blood after 24 hours incubation with the test reagents are shown in figure 6. Campath showed a reduction in T cell number by about 8-fold compared to PBS and NegCtrl BYbe treated wells. 4133-6294BYbe and 6294-X/4133-Y also showed a significant 5-fold and 3.6-fold reduction in T cell numbers, respectively. The levels of inflammatory cytokines detected are shown in FIG. 7 (A) IFN- γ, (B) IL-6, and (C) TNF- α. IL-1β levels were lower than the detection levels of all reagents except Campath, which recorded significant levels (data not shown). Notably, it was observed that 4133-6294BYbe and 6294-X/4133-Y induced little or no inflammatory cytokines at levels that matched those in PBS and NegCtrl BYbe treated wells.

Example 10-Resistance of macrophages to apoptosis by anti-CD 45 BYbe

Isolation of monocytes from PBMC

Human PBMCs derived from the platelet-monocompartment of blood leukocytes (NHSBT Oxford) were stored as frozen aliquots. Before performing the assay, 2 vials of frozen cells (each vial containing 5x10 in 1ml ⁷ Individual cells) were thawed in a 37 ℃ water bath, then 50ml of complete medium (RPMI 1640+2mM GlutaMAX+1%Pen/Strep, all supplied by Invitrogen, +10% Fetal Bovine Serum (FBS), sigma Aldrich) was added. The cells were centrifuged (500 g,5 min, room temperature) and resuspended in 30ml of complete medium for washing and re-centrifugation. Cells were resuspended in 20ml MACS buffer (PBS, pH 7.2,0.5% Bovine Serum Albumin (BSA), and 2mM EDTA,Sigma Aldrich) and counted on ChemoMetec NucleoCounter NC-3000 to determine concentration and viability. PBMC from one donor (UCB-Cones 802) was used for this assay.

Monocyte isolation was performed using a Pan monocyte isolation kit (Miltenyi Biotec, cat# 130-096-537) and LS column (Miltenyi Biotec, cat# 130-042-401) according to manufacturer's instructions. 100 μl of cells were removed and stored on ice to check the purity of the isolated monocytes. Isolated cells were stained with BV421 mouse anti-human CD14 (BD Biosciences, cat No. 563743) to check the purity of monocytes using FACS.

The cells were centrifuged at 500g for 5 minutes at room temperature,the buffer was aspirated with a BioTek ELx405 microplate washer and the cells resuspended in FACS buffer (PBS+1% Bovine Serum Albumin (BSA) +0.1% NaN) ₃ +2mM EDTA,Sigma Aldrich) and then centrifuged again and the buffer aspirated to leave the cells in 20 μl of residual medium. Mu.l of biotin-antibody mixture was added to the wells and the plates were incubated for 1 hour at 4 ℃. After incubation, the plates were centrifuged as before, the cells were washed once in FACS buffer, centrifuged again as before, and the excess buffer was aspirated to leave the cells in 50 μl of residual buffer.

Living cells were analyzed using Intellicyt iQue Screener PLUS. Viable cell count was extracted using Graphpad Prism version 8.1 (Graphpad) to generate an index and graphical representation.

Deriving macrophages from monocytes

M-CSF (Sigma Aldrich, cat. SRP 3110) and GM-CSF (R)&D Systems, cat# 215-GM/CF) was prepared at 100. Mu.g/ml. At 2.5X10 in M1 medium (complete medium+50 ng/ml GM-CSF) or M2 medium (complete medium+50 ng/ml M-CSF) ⁵ Cells were prepared at a concentration of individual cells/ml. 200 μl of cells were inoculated into two Corning 96-well black polystyrene microwell plates (Sigma Aldrich) and incubated at 37deg.C, 5% CO ₂ And (5) incubating.

At 37 ℃,5% CO ₂ After 3 days of incubation, the plates were centrifuged (500 g,5 min, RT), the buffer aspirated, the cells washed once with PBS and then resuspended in 200 μ l M medium or M2 medium. Incubation was carried out for an additional 4 days (at 37 ℃,5% co ₂ ) After centrifugation plates, the buffer was aspirated, cells were washed once with PBS and then resuspended in 150. Mu. l M1 medium (supplemented with 50ng/ml IFNγ, sigma Aldrich, cat# SRP 3058) or M2 medium (supplemented with 20ng/ml IL-4, gibco cat# PHC 0044). Cells were incubated in the presence of 5% CO ₂ Incubate overnight at 37 ℃.

Macrophage treatment with BYbe

On day 8 after isolation, 4133-6294-BYbe or NegCtrl BYbe was added to M1 medium or M2 medium at 4000nM in Greiner 96 well unbound microplates. The BYbe protein was then serially diluted three times at 1:5 to yield 4 working concentrations. Mu.l of each BYbe dilution was added to 150ul of cells in 96 well black polystyrene microwell wells. The final concentration in the wells was 250nM, 50nM, 10nM and 2nM. Camptothecins (cat No. C9911-100MG,Sigma Aldrich) and staurosporine (cat No. S6942-200UL,Sigma Aldrich) were added as positive controls for apoptosis. Both were diluted in M1 or M2 medium and added to the wells to give a final concentration of 5 μm.

One microplate was incubated with 5% CO ₂ Is incubated in an incubator at 37℃for a further 24 hours and then used for the incubationCell viability was assessed. />The luminous cell viability assay (Promega, cat No. G9681) was performed according to the manufacturer's instructions. Will 150 μl->Add to the wells and mix gently on a shaker for 2 minutes. Plates were then incubated at room temperature for 10 minutes, followed by transfer of 100 μl of solution in each well to Corning ^TM 96-well solid white polystyrene plates (thermo fisher). Plates were then read on a BMG Labtech PHERAstar FSX microplate reader using the CellTiter-Glo program.

10 μl of diluted was added to another microplate on day 8Caspase-3/7 green apoptosis assay reagent (cat No. 4440) and +.>Cytotox Red reagent (cat. No. 4632) (final concentrations 5 μm and 2.5 μm, respectively). Then put it into +.>S3 livingIn the cell analysis system, a 10-fold objective lens was used and imaging was performed once per hour for 6 days. Caspase dye was measured with a green laser (350 ms) and cytotox dye was measured with a red laser (650 ms). Green dye Signal use->The analysis was performed by Zoom 2016B. The red cytox dye signal was poor and therefore the channel was not analyzed.

Results

Monocyte-derived macrophages M1 and M2 macrophages look phenotypically different (fig. 8). (A) M1 macrophages are round, while (B) M2 macrophages are more elongated. M2 macrophages also showed higher confluency than M1 macrophages, as expected from M-CSF treatment. Cell viability was assessed at 24 hours to reflect the assay period of PBMCs (fig. 3A and 3B). Both camptothecins and staurosporines reduced the viability of M1 (fig. 9 (a)) and M2 (fig. 9 (B)) macrophages compared to untreated cells. The effects of staurosporine are marked as few or no living cells are detected. In contrast, 4133-6294BYbe treated cells showed similar viability to NegCtrl BYbe and untreated wells.

During the course of 6 days, significant levels of caspase-3/7 could be detected in camptothecin-treated macrophages only (fig. 10 (a) and 10 (B)). The signal in staurosporine treated macrophages is very low and is therefore excluded in both figures. The level of caspase-3/7 in M1 and M2 macrophages treated with 4133-6294 Bybe was consistent with Negctrl Bybe treated and untreated macrophages. This suggests that macrophages are largely resistant to 4133-6294 BYbe-induced apoptosis. At about 16 hours, M2 macrophages showed small peaks in caspase-3/7 levels in all treatments. This is thought to be due to stress caused by high cell density.

EXAMPLE 11-Mass photometry

Method

Data were collected on a refynonemp mass photometer (refyn Ltd, oxford, UK) using AcquireMP (refyn Ltd, v2.2.1) software and images were processed and analyzed using discover mp (v2.3.dev 12) software.

Measurements were performed using a clean glass coverslip (high precision coverslip, no.1.5, 24×50 mm, marienfeld) fitted with a silicon spacer (culturwe well) cut into 2×2 Kong Qiepian pieces ^TM The shims may be reused, grace biolabs). Protein stock was directly diluted in Dulbecco phosphate buffered saline (DPBS, thermoFisher). Typical working concentrations of protein complexes are 1-100nM, depending on the dissociation characteristics of the protein complex.

The instrument lens was cleaned with isopropyl alcohol (IPA), dried, and a drop of Olympus Low Auto fluorescent immersion oil (NC 0297589, thermo fisher) was dropped onto the lens, and then the microscope coverslip and sample were placed on the light stage. To find the focus, 15 μl of fresh DPBS was pipetted into the silicone well, the focus position was identified and fixed in place based on the total internal reflection of the entire measurement using an autofocus system. For each acquisition, 5 μl of diluted protein was introduced into the wells, mixed well (before autofocus stabilization) and a movie was recorded with a duration of 90 seconds. One measurement per sample, with new wells and buffer used for each measurement. The mixture of CD45 ECD and 4133-6294BYbe was not pre-incubated, so that complexing occurred when 2 proteins were added to the wells.

Results

FIG. 11 shows the mass spectrophotometric signals of (A) CD45 ECD, (B) 4133-6294BYbe or (C) a mixture of CD45 ECD and 4133-6294 BYbe. A single peak of CD45 ECD was observed, indicating a homogeneous formulation (a). The predicted mass of CD45 ECD was 41.3kDa, but the peak represented a mass of 62 kDa. This difference may be attributable to glycosylation because there are ten predicted N-linked glycosylation sites (see FIG. 12). A single peak corresponding to a mass of 76kDa was observed for 4133-6294Bybe (B). This is believed to be consistent with the predicted mass of 73.5 kDa.

A plurality of peaks were observed for mixture (C) of CD45 ECD and 4133-6294 BYbe. The peak at 75kDa may correspond to unbound BYbe. Additional peaks were observed at 136, 274, 415 and 555 kDa. Based on the observed masses of 62 and 76kDa, respectively, the mass of the CD45 ECD and 4133-6294BYbe complex was predicted to be 138kDa. Thus, the peak at 136kDa may correspond to the CD45 ECD-4133-6294 BYbe complex. Furthermore, the peaks at 274, 415 and 555kDa may be attributed to multimeric forms of the CD45-BYbe complex containing 2 copies, 3 copies and 4 copies, respectively (Table 10).

TABLE 10 theoretical weight and observed weight of CD45-BYbe composite

Species of type	Theoretical mass (kDa)	Quality of observation (kDa)
			1x(CD45+BYbe)	138	136
2x(CD45+BYbe)	276	274
			3x(CD45+BYbe)	414	415
4x(CD45+BYbe)	552	555

Example 12-Affinity of 4133 and 6294 Fab measured using surface plasmon resonance

Method

Surface Plasmon Resonance (SPR) experiments were performed on a Biacore 3000 system at 25℃using a CM5 sensor chip (GE Healthcare Bio-Sciences AB, uppsala, sweden) and HBS-EP running buffer (10mM HEPES,150mM NaCl,EDTA 2mM and 0.005% (v/v) P20, pH 7.4). Respectively using polyclonal goat F (ab) ₂ Fragment anti-rabbit F (ab) ₂ (Jackson Labs product code # 111-006-047) and polyclonal goat F (ab) ₂ Fragment anti-mouse F (ab) ₂ (Jackson Labs product code # 115-006-072) 4133 rabbit Fab and 6294 mouse Fab were captured. Covalent immobilization of the capture antibody is achieved by standard amine coupling chemistry to levels of 1000-3000 Response Units (RU).

CD 45D 1-D4 was titrated from 50nM to 0.05nM on captured purified antibody. Each assay cycle consisted of: antibody Fab fragments were first captured using 1 minute injection at a flow rate of 10. Mu.l/min, followed by an association period consisting of 3 minutes injection of CD 45D 1-D4 at a flow rate of 30. Mu.l/min. The subsequent dissociation phase was monitored for at least 3 minutes. After each cycle, the capture surface was regenerated by feeding 40mM HCl at a flow rate of 10. Mu.l/min for 1 minute, followed by 5mM NaOH for 30 seconds. Reference subtraction was performed using a blank flow cell and included buffer blank sample injection to subtract instrument noise and instrument zero drift. Kinetic parameters were determined using BIA evaluation software (version 4.1.1).

Results

The affinities of 4133 and 6294Fab were demonstrated to be 61nM and 85pM, respectively. Association constant (K) _a ) Dissociation constant (K) _d ) And affinity constant (K) _D ) As shown in table 11 below.

Tables 11.4133 and 6294 affinity for Fab

	K _a (1/Ms)	K _d (1/s)	K _D (M)
				4133Fab	5.7E+05	3.5E-02	6.1E-08
6294Fab	2.6E+06	2.2E-04	8.5E-11

EXAMPLE 13-Humanization

Method

Humanized versions of rabbit antibody 4133 and mouse antibody 6294 were designed by grafting CDRs from a donor antibody V region onto a human germline antibody V region framework. To increase the likelihood of restoring antibody activity, some framework residues from the donor V region are also retained in the humanized sequence. These residues were selected using the experimental protocol outlined by Adair et al (1991) (humanized antibody WO 91/09967). CDRs grafted from the donor to the acceptor sequence were defined by Kabat (Kabat et al, 1987), except CDRH1, using the comprehensive Chothia/Kabat definition (see Adair et al, 1991 humanized antibody WO 91/09967). In addition, the VH gene of a rabbit antibody is typically shorter than the selected human VH receptor gene. When aligned with human receptor sequences, framework 1 of the VH region of a rabbit antibody typically lacks the N-terminal residues that remain in the humanized antibody. Framework 3 of the rabbit antibody VH region is also typically devoid of one or two residues (75, or 75 and 76) in the loop between β -sheet chains D and E: in humanized antibodies, the gaps are filled by corresponding residues from the selected human acceptor sequence.

Humanized sequences and CDR variants are listed in fig. 12, described below.

CD45 antibody 4133

Human V region IGKV1D-13 plus JK 4J region (IMGT, http:// www.imgt.org /) was selected as the receptor for the light chain CDR of antibody 4133. In addition to the CDRs, one or more of the following framework residues (donor residues) from the 4133VK gene may be retained at positions 2, 3 and 70 (Kabat numbering), respectively: glutamine (Q2), valine (V3) and glutamine (Q70). In some cases, CDRL1 may be mutated to remove a potential N-glycosylation site (CDRL 1 variant 1-2).

Human V region IGHV3-21 plus JH 1J region (IMGT, http:// www.imgt.org /) was selected as the receptor for the heavy chain CDR of antibody 4133. In addition to the CDRs, one or more of the following framework residues (donor residues) from the 4133VH gene may be retained at positions 48, 49, 71, 73, 76 and 78 (Kabat numbering), respectively: isoleucine (I48), glycine (G49), lysine (K71), serine (S73), threonine (T76) and valine (V78). In some cases, CDRH1 and CDRH2 may be mutated to remove cysteine residues (CDRH 1 variant and CDRH2 variant, respectively). CDRH3 may also be mutated to modify potential aspartic acid isomerisation sites (CDRH 3 variants 1-3).

Human V region IGHV4-4 plus JH 1J region (IMGT, http:// www.imgt.org /) was selected as the candidate receptor for the heavy chain CDR of antibody 4133. In addition to the CDRs, one or more of the following framework residues (donor residues) from the 4133VH gene may be retained at positions 24, 71, 73, 76 and 78 (Kabat numbering), respectively: alanine (a 24), lysine (K71), serine (S73), threonine (T76) and valine (V78). The glutamine residue at position 1 of the human framework is replaced with glutamic acid (E1) to provide for expression and purification of the homogeneous product. In some cases, CDRH1 and CDRH2 may be mutated to remove cysteine residues (CDRH 1 variant and CDRH2 variant, respectively). CDRH3 may also be mutated to modify potential aspartic acid isomerisation sites (CDRH 3 variants 1-3).

CD45 antibody 6294

Human V region IGKV1D-33 plus JK 4J region (IMGT, http:// www.imgt.org /) was selected as the receptor for the light chain CDR of antibody 6294. In addition to the CDRs, one or more of the following framework residues (donor residues) from the 6294VK gene may be retained at positions 49, 63, 67, 85 and 87 (Kabat numbering), respectively: phenylalanine (F49), threonine (T63), tyrosine (Y67), valine (V85) and phenylalanine (F87).

The human V region IGKV4-1 plus JK 4J region (IMGT, http:// www.imgt.org /) was selected as the receptor for the light chain CDR of antibody 6294. In addition to the CDRs, one or more of the following framework residues from the 6294VK gene (donor residues) may be retained at positions 49, 63, 67 and 87 (Kabat numbering), respectively: phenylalanine (F49), threonine (T63) and phenylalanine (F87).

Human V region IGHV1-69 plus JH 4J region (IMGT, http:// www.imgt.org /) was selected as the candidate receptor for the heavy chain CDR of antibody 6294. In addition to the CDRs, one or more of the following framework residues (donor residues) from the 6294VH gene may be retained at positions 1, 48 and 73 (Kabat numbering), respectively: glutamic acid (E1), isoleucine (I48) and lysine (K73). In some cases, CDRH3 may be mutated to modify potential aspartic acid isomerisation sites (CDRH 3 variants 1-3).

Human V region IGHV3-48 plus JH 4J region (IMGT, http:// www.imgt.org /) was selected as the receptor for the heavy chain CDR of antibody 6294. In addition to the CDRs, one or more of the following framework residues (donor residues) from the 6294VH gene may be retained at positions 48, 49, 71, 73 and 76 (Kabat numbering), respectively: isoleucine (I48), glycine (G49), alanine (A71), lysine (K73) and serine (S76). In some cases, CDRH3 may be mutated to modify potential aspartic acid isomerisation sites (CDRH 3 variants 1-3).

Example 14-Apoptosis of peripheral blood hematopoietic stem cells induced by anti-CD 45 antibodies

Method

Human whole blood (K2 EDTA tubes) was received from an 18 year old donor (#pr 20T386505, from Cambridge Bioscience, UK). PBMCs were isolated from whole blood using pre-filled LeucoSep tubes (Greiner). Whole blood was layered onto a LeucoSep filter and the tube centrifuged (800 g,15 min, slow acceleration and deceleration at room temperature). Buffy coats were extracted and cells were washed twice in sterile PBS. PBMC were resuspended in 50ml complete medium (RPMI 1640+2mM GlutaMAX+1% Pen/Strep, all supplied beforehand by Invitrogen, +10% Fetal Bovine Serum (FBS), sigma Aldrich. Cells were counted using ChemoMetec NucleoCounter NC-3000. Then 1X10 per well in 80. Mu.l ⁶ Individual cells were added to each well of Corning Costar 96 well U-bottom microplates (cat No. 07-200-95) treated for cell culture.

Fab-X and Fab-Y combinations 6294-X/4133-Y were added to complete medium in Greiner 96 well non-binding microwell plates to give a Fab-KD-Fab concentration of 1000nM. Likewise, 1000nM stock solutions of 4133-6294BYbe and NegCtrl BYbe were prepared in complete medium. Microwell plates were incubated at 37℃with 5% CO ₂ Incubate for 1 hour. After incubation, the Fab-KD-Fab and BYbe reagents were then serially diluted 7 times in a semilog dilution series in complete medium to generate an 8-point dose curve.

Mu.l of each Fab-KD-Fab or BYbe dilution (final well concentration 200-0.00632 nM) was added to the cells and incubated at 37℃with 5% CO ₂ Incubate for 24 hours. After incubation, plates were centrifuged at 500g for 5 min at room temperature, buffer was aspirated with a BioTek ELx405 microplate washer, and cells were resuspended in FACS buffer (pbs+1% Bovine Serum Albumin (BSA) +0.1% nan) ₃ +2mM EDTA,Sigma Aldrich) and then centrifuged again and the buffer aspirated to leave the cells in 20 μl of residual medium. Mu.l of the cell-specific marker antibody mixture solution was added to the wells and incubated at 4℃for 30 minutes. The antibody mixtures are detailed in table 12 below. The washing and aspiration steps were repeated leaving the cells in a residual volume of 20. Mu.l. Then use LIVE/DEAD ^TM Near infrared DEAD cell stain (LIVE/DEAD) can be immobilized ^TM Fixable Near-IR Dead Cell Stain, invitrogen) stained cells in a 1:1000 dilution and incubated at 4℃for 10 minutes. The washing and aspiration steps were repeated leaving the cells in a residual volume of 20. Mu.l. Cells were fixed by adding 100. Mu.l BD Cytofix fixation buffer (BD Bioscience) for 15 min at 4 ℃. The washing and aspiration steps were repeated and the final volume of FACS collection was adjusted to 200 μl per well.

Cells were analyzed using a Bio-Rad ZE5 cell analyzer. Viable cell count was extracted using Graphpad Prism version 8.1 (Graphpad) to generate an index and graphical representation. Hematopoietic stem cells are defined as lymphocyte lineage negative, CD45 positive and CD34 positive populations.

Table 12

Results

The effect of 6294-X/4133-Y and 4133-6294BYbe on CD34+ stem cells in PBMC is shown in FIG. 13. Both agents showed significant reductions in stem cells, 54% and 53% ((a) and (B), respectively). Consistent with previous experiments in PBMC, 99% and 98% ((C) and (D)) reduction in total lymphocytes, respectively, was observed. It must be noted that the initial number of cd34+ stem cells is about 250, compared to about 300,000 total lymphocytes. This is unexpected because circulating stem cells are known to be present at very low levels.

EXAMPLE 15-Sedimentation velocity

Method

CD45 ECD and 4133-6294BYbe were set at 1:1 and incubated at room temperature for 1 hour. The molar ratio was determined using the predicted mass of 4133-6294Bybe 73.5kDa and the mass of CD45 ECD of 62kDa as determined by mass spectrophotometry.

CD45 ECD-4133-6294Bybe mixtures, CD45 ECD only or 4133-6294Bybe only were loaded into a cell with a 2 channel charcoal-epon center piece (optical path length 12mm, glass quartz glass window). The corresponding buffers were loaded into the reference channel of each cell (the instrument functions similarly to a dual beam spectrometer). These loaded pools were then placed in AN-60Ti analytical rotor, loaded into a Beckman-coulter Optima analytical ultracentrifuge and raised to 20 ℃. The rotor was then set to 3,000rpm and the sample scanned at 280nm to confirm proper cell loading and to properly adjust the laser by laser delay settings. Then, the final running speed of the rotor was brought to 50,000rpm. Scans were recorded every 20 seconds for 8 hours. The radial scan range is 5.75 to 7.25cm.

The data were analyzed using the c(s) method developed by Peter gluck, n.i.h, and implemented in his analysis program SEDFIT version 14.6 e. In this approach, many raw data scans are fitted directly (in this case 36,000 data points per sample) to derive a distribution of sedimentation coefficients, while the effect of diffusion on the data is modeled to improve resolution. The principle of operation of this method is to assign a diffusion coefficient to each sedimentation coefficient value based on the assumption that all species have the same overall hydrodynamic shape (shape is defined by the coefficient of friction relative to the sphere, f/f 0). Varying the f/f0 values found the best overall fit of the data for each sample. A maximum entropy regularization probability of 0.95 is used and time invariant noise is removed. Analysis was performed using a standard solvent model.

Results

CD45ECD monomer, 4133-6294BYbe monomer, and CD45ECD and 4133-6294BYbe moles 1 measured in analytical ultracentrifuge: the sedimentation velocity of the 1 mixture is shown in fig. 14. The sedimentation coefficient value of CD45ECD was 3.547, giving a mass of 58kDa. This is greater than the predicted mass 41.3kDa for CD45ECD, but is consistent with the mass observed by mass spectrophotometry (62 kDa) in example 11. The sedimentation coefficient value of 4133-6294BYbe was 4.395, giving a mass of 72kDa. This is consistent with a predicted mass of 73.5 kDa.

Multiple peaks were observed for the mixture of CD45ECD and 4133-6294BYbe, indicating the presence of CD45ECD-BYbe multimeric complexes. For the purpose of complexing, the crystal structures of CD45ECD (PDB code 5 FMV) and 4133-6294BYbe (modeled from the single internal crystal structures of Fab and scFv) were modeled and complexed together in coarse grain fashion. The hydrodynamic parameters extracted from these structures revealed that the S values calculated from these structures correspond to the data we observed, with acceptable errors (+/-0.5S). Concluding that: we can be confident about the stoichiometry of the complexes (table 13).

TABLE 13

The area under the trace curve was calculated (Table 14) to show that the mixture consisted essentially of 37.7% 2xCD45-BYbe and 35.7% 3 xCD 45-BYbe.

TABLE 14

Composite material	Area percent under curve
		CD45	9.21
CD45 1:1BYbe	11.99
		CD45 2:2BYbe	37.7
CD45 3:3BYbe	35.7
		CD45 4:4BYbe	3.46
Higher orders greater than 4:4	0.81,0.54,0.59

EXAMPLE 16-Production of IgG4P FALA and IgG4P FALA KiH

Method

To generate the anti-CD 45 antibodies 4133igg4p FALA and 4133-6294igg4p FALA knob, genes encoding the light and heavy chain V-regions of antibodies 4133 and 6294, respectively, were designed and constructed by automated synthesis methods (ATUM). The light chain V-region genes of antibodies 4133 and 6294 were cloned into an expression vector containing DNA encoding the human C.kappa.region. The heavy chain V region gene of antibody 4133 was cloned into an expression vector containing DNA encoding the IgG4P FALA (human IgG4 sequence plus S228P, F234A, L a) or IgG4P FALA pestle (human IgG4 sequence plus S228P, F234A, L235A, T355W) constant region. The heavy chain V region gene of antibody 6294 was cloned into an expression vector containing DNA encoding the IgG4P FALA mortar (human IgG4 sequence plus S228P, F234A, L235A, T366S, L368A, Y407V) constant region.

The 4133 light chain construct was paired with 4133igg4p FALA and 4133igg4p FALA pestle heavy chain constructs. 6294 light chain construct was paired with 6294igg4p FALA mortar construct. The DNA was transfected into CHO-SXE cells using a Gibco ExpiFectamine CHO transfection kit (cat No. a29133, thermoFisher Scientific) according to the manufacturer's instructions. Cells were incubated at 32℃with 5% CO ₂ Is cultured in an incubator at 140rpm for 11 days. After incubation, the cultures were transferred to tubes and the supernatant was separated from the cells after centrifugation at 4000rpm for 2 hours. The remaining supernatant was filtered through 0.22 μm SARTO BRAN P Millipore, then through 0.22 μm Gamma gold filter.

Antibodies 4133IgG4P FALA, 4133IgG4P FALA pestle and 6294IgG4P FALA mortar were purified from the supernatant using a 5ml MabSelect Sure column (GE Healthcare) on an AKTA Pure purification system (GE Healthcare Life Sciences) according to the manufacturer's instructions. Fractions eluting protein were pooled and concentrated to <5ml using an Amicon Ultra-15 centrifugal filtration device with an Ultra-50 membrane 50kDa (SigmaAldrich). To obtain a Pure fraction, the protein was then passed through a HiLoad Superdex 200pg 26/60HPLC size exclusion column on an AKTA Pure purification system. Protein concentration was determined using Thermo Scientific NanoDrop 2000 (cat# ND-2000).

To generate 4133-6294igg4p FALA pestle, purified 4133igg4p FALA pestle and 6294igg4p FALA mortar proteins were mixed at 1:1 molar ratio, cysteamine (SigmaAldrich) was added to a final concentration of 5mM, and the mixture was incubated overnight at room temperature. The mixture was then passed through a HiLoad Superdex 200pg 26/60HPLC size exclusion column on an AKTA Pure purification system. Protein concentration was determined using Thermo Scientific NanoDrop 2000 (cat# ND-2000). In addition, fractions were analyzed by SDS-PAGE on 4-20% Tris-glycine gels using ACQUITY BEH200 column on Waters ACQUITY UPLC SEC system. Endotoxin was tested using the Endosafe nexgen-MCS system (Charles River).

EXAMPLE 17-anti-CD 45 antibody-induced PBMC in the forms Bybe, igG4P FALA and IgG4P FALA KiH Apoptosis of (a)

Method

Human PBMCs derived from the platelet-monocompartment of blood leukocytes (NHSBT Oxford) were stored as frozen aliquots. Before performing the assay, 2 vials of frozen cells (each vial containing 5x10 in 1ml ⁷ Individual cells) were thawed in a 37 ℃ water bath, then 50ml of complete medium (RPMI 1640+2mM GlutaMAX+1% Pen/Strep, all previously supplied by Invitrogen, +10% Fetal Bovine Serum (FBS), sigma Aldrich) was added. The cells were centrifuged (500 g,5 min, room temperature) and resuspended in 30ml of complete medium for washing and re-centrifugation. Cells were resuspended in 10ml of complete medium and then counted using ChemoMetec NucleoCounter NC-3000. Then 80. Mu.l of each well 10 ⁵ Individual cells were added to each well of Corning Costar 96 well U-bottom microplates (cat No. 07-200-95) treated for cell culture. The plates were incubated at 37℃with 5% CO ₂ The mixture was allowed to stand in the incubator for 2 hours. PBMC from two donor UCB-Cones 811 and 831 were used for this assay.

Stock solutions of 4133-6294 Bybe, 4133-6294 IgG4P FALA KiH and 4133IgG4P FALA were prepared in complete medium at 2500 nM. In Greiner 96 well unbound microplates, reagents were serially diluted 7 times in complete medium at 1:5 to generate an 8-point dose curve.

Mu.l of each dilution (final well concentration 500-0.0064 nM) was added to the cells and incubated at 37℃with 5% CO ₂ Incubate for 24 hours. After incubation, the plates were centrifuged at 500g for 5 min at room temperature and the plates were plated with BioTek ELx405 microwell platesThe washer aspirates the buffer and resuspended cells in FACS buffer (PBS+1% Bovine Serum Albumin (BSA) +0.1% NaN) ₃ +2mM EDTA,Sigma Aldrich) and then centrifuged again and the buffer aspirated to leave the cells in 20 μl of residual medium. 20 μl LIVE/DEAD ^TM Near infrared dead cell stain (Invitrogen, 1:1000 dilution) can be fixed into the wells and incubated for 1 hour at 4 ℃.

Living cells were analyzed using Intellicyt iQue Screener PLUS. Viable cell count was extracted using Graphpad Prism version 8.1 (Graphpad) to generate an index and graphical representation. Asymmetric (five parameter) curve fitting was applied to obtain EC50 values.

Results

The percent lymphocyte decrease of 4133-6294 BYbe,4133-6294 IgG4PFALA KiH and 4133 IgG4P FALA of representative donors (UCB Cone-811) is shown in FIG. 15. Both 4133-6294 BYbe and 4133-6294 IgG4P FALA KiH showed high potency EC50 of 0.10nM and 0.17nM, respectively. In contrast, 4133 IgG4P FALA had an EC50 of 44nM.

EXAMPLE 18- Apoptosis of PBMCs induced by anti-CD 45 antibody combinations

Method

Human PBMCs derived from the platelet-monocompartment of blood leukocytes (NHSBT Oxford) were stored as frozen aliquots. Before performing the assay, 2 vials of frozen cells (each vial containing 5x10 in 1ml ⁷ Individual cells) were thawed in a 37 ℃ water bath, then 50ml of complete medium (RPMI 1640+2mM GlutaMAX+1%Pen/Strep, all supplied by Invitrogen, +10% Fetal Bovine Serum (FBS), sigma Aldrich) was added. The cells were centrifuged (500 g,5 min, room temperature) and resuspended in 30ml of complete medium for washing and re-centrifugation. Cells were resuspended in 10ml of complete medium and then counted using ChemoMetec NucleoCounter NC-3000. Then 80. Mu.l of each well 10 ⁵ Individual cells were added to each well of Corning Costar 96 well U-bottom microplates (cat No. 07-200-95) treated for cell culture. The plates were incubated at 37℃with 5% CO ₂ The mixture was allowed to stand in the incubator for 2 hours. PBMC from two donor UCB-Cones 811 and 831 were used for thisAnd (5) measuring.

In Greiner 96 well unbound microplates, the Fab-X and Fab-Y combinations 6294-X/6294-Y were added to complete medium to give a Fab-KD-Fab concentration of 1250nM. A1250 nM stock of 4133 IgG4P FALA was prepared in complete medium and mixed with 6294-X/6294-Y in an equimolar mixture to give a final total antibody concentration of 2500nM. Stock solutions of 2500nM 4133-6294 BYbe and 4133 IgG4P FALA were also prepared in complete medium. In Greiner 96 well unbound microplates, reagents were serially diluted 7 times in complete medium at 1:5 to generate an 8-point dose curve.

Mu.l of each dilution (final well concentration 500-0.0064 nM) was added to the cells and incubated at 37℃with 5% CO ₂ Incubate for 24 hours. After incubation, the plates were centrifuged at 500g for 5 min at room temperature, the buffer was aspirated with a BioTek ELx405 microplate washer, and the cells were resuspended in FACS buffer (pbs+1% Bovine Serum Albumin (BSA) +0.1% nan) ₃ +2mM EDTA,Sigma Aldrich) and then centrifuged again and the buffer aspirated to leave the cells in 20 μl of residual medium. 20 μl LIVE/DEAD ^TM Near infrared dead cell stain (Invitrogen, 1:1000 dilution) can be fixed into the wells and incubated for 10 minutes at 4 ℃.

Results

The percent lymphocyte decrease for representative donors (UCB Cone-811) by 4133-6294 Bybe, 4133 IgG4P FALA and 4133 IgG4P FALA combinations is shown in FIG. 16. 4133-6294 BY shows a high potency EC50 of 0.10 nM. In contrast, 4133 IgG4 FALA had an EC50 of 44nM.4133 The potency of the IgG4P FALA and 6294-X/6294-Y combinations was similar to that of 4133 IgG4P FALA used alone at 46 nM.

EXAMPLE 19-Production of TrYbe

Method

To generate 4133-6294-645TrYbe, the full length of the heavy chain (4133 Fab HC-G4S linker-6294 scFv) and the full length of the light chain (4133 Fab LC-G4S linker-645 scFv) were designed and constructed by automated synthesis (ATUM). Both strands were cloned into an internal mammalian expression vector. 645 bind to human and mouse serum albumin (WO 2011/036460, WO 2010/035012, WO 2013/068571). It confers a longer serum half-life to TrYbe.

The heavy and light chain constructs were paired and transfected into CHO-SXE cells using a Gibco ExpiFectamine CHO transfection kit (cat No. a29133, thermoFisher Scientific) according to the manufacturer's instructions. Cells were incubated at 37℃with 5% CO ₂ Is cultured in an incubator at 140rpm for 7 days. After incubation, the cultures were transferred to tubes and the supernatant was separated from the cells after centrifugation at 4000rpm for 30 minutes. The remaining supernatant was filtered through 0.22 μm SARTO BRAN PMillipore, then through 0.22 μm Gamma gold filter.

4133-6294-645TrYbe protein was purified by a native protein G capture step followed by a preparative size exclusion purification step using the AKTA Pure purification system (GE Healthcare Life Sciences). The clarified supernatant was loaded onto a 50ml Gammabind Plus agarose column (Resin cytova, internally packed column) providing a 25 minute contact time and washed with 2.5x column volume of PBS, ph 7.4. Wash fractions with Ultraviolet (UV) readings >25mAU were collected by fractionation. The bound material was eluted by a step elution with 0.1M glycine pH2.7, fractionated and neutralized with 2M Tris/HCl pH 8.5. Both the wash material and the elution material were quantified by absorbance at 280 nm.

Size exclusion chromatography (SE-UPLC) was used to determine the purity status of the washed samples and eluted products. Protein (. About.3. Mu.g) was loaded onto BEH200,1.7 μm, 4.6mm by 300mm column (Waters ACQUITY) and developed with an isocratic gradient of 0.2M phosphate pH7 at 0.35 mL/min. Continuous detection was performed by absorbance at 280nm and a multi-channel Fluorescence (FLR) detector (Waters).

Combining the washed fraction and the eluted fraction containing TrYbe monomers and allowingConcentrated with an Amicon Ultra-15 concentrator (membrane with a molecular weight cut-off of 30 kDa) and centrifuged at 4000g in a swing-out rotor. Concentrated samples were loaded onto HiLoad 16/600Superdex 200pg column (Cytiva) equilibrated in PBS, pH7.4 and expanded at 1ml/min with an isocratic gradient of PBS, pH 7.4. At BEH200,Fractions were collected and analyzed by size exclusion chromatography on a 1.7 μm, 4.6mm ID x 300mm column (acquisition) and developed at 0.35mL/min with an isocratic gradient of 0.2M phosphate pH7, detected by absorbance at 280nm and a multichannel Fluorescence (FLR) detector (Waters). Selected monomer fractions were pooled, sterile filtered at 0.22 μm and the final sample concentration was determined by a280 scan on a Varian Cary 50 uv spectrometer (Agilent Technologies). Endotoxin levels below 1.0EU/mg, e.g.by Charles River +. >The portable test system was evaluated using a limulus amoebocyte lysate (Limulus Amebocyte Lysate, LAL) test kit.

By the method of the preparation of the composition in BEH200,The monomer status of the final TrYbe was determined by size exclusion chromatography on a 1.7 μm, 4.6mm ID by 300mm column (acquisition) and developed with an isocratic gradient of 0.2M phosphate pH 7 at 0.35mL/min and detected by absorbance at 280nm and a multichannel Fluorescence (FLR) detector (Waters). Discovery of the final TrYbe antibodies>99% is monomer.

For analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), samples were prepared by adding 4X Novex NuPAGE LDS sample buffer (Life Technologies) and 10X NuPAGE sample reducing agent (Life Technologies) or 100mM N-ethylmaleimide (Sigma-Aldrich) to 3 μg of purified protein and heating to 98℃for 3 minutes. Samples were loaded onto a 15-well Novex 4-20% Tris-glycine 1.0mm SDS-polyacrylamide gel (Life Technologies) and separated in Tris-glycine SDS running buffer (Life Technologies) for 40 minutes at a constant voltage of 225V. Novex Mark12 broad range protein standard (Life Technologies) was used as standard. The gel was stained with coomassie fast stain (Generon) and destained in distilled water.

EXAMPLE 20-anti-CD 45 antibody-induced apoptosis of PBMC in the form of TrYbe, BYbe and IgG4P FALA KiH Apoptosis of

Method

Human PBMCs derived from the platelet-monocompartment of blood leukocytes (NHSBT Oxford) were stored as frozen aliquots. Before performing the assay, 2 vials of frozen cells (each vial containing 5x10 in 1ml ⁷ Individual cells) were thawed in a 37 ℃ water bath, then 50ml of complete medium (RPMI 1640+2mM GlutaMAX+1%Pen/Strep, all supplied by Invitrogen, +10% Fetal Bovine Serum (FBS), sigma Aldrich) was added. The cells were centrifuged (500 g,5 min, room temperature) and resuspended in 30ml of complete medium for washing and re-centrifugation. Cells were resuspended in 10ml of complete medium and then counted using ChemoMetec NucleoCounter NC-3000. Then 100 μl of each well 10 ⁵ Individual cells were added to each well of Corning Costar 96 well U-bottom microplates (cat No. 07-200-95) treated for cell culture. The plates were incubated at 37℃with 5% CO ₂ The mixture was allowed to stand in the incubator for 2 hours. PBMC from two donor UCB-Cones 802 and 812 were used for this assay.

2500nM stock solutions of 4133-6294-645 TrYbe, 4133-6294 BYbe and 4133-6294 IgG4P FALA KiH were prepared in complete medium. In Greiner 96 well unbound microwell plates, reagents were then serially diluted 11 times in complete medium at 1:3.5 dilution series to generate a 12-point dose curve. Mu.l of each reagent dilution (final well concentration 500-0.000518 nM) was added to the cells and incubated at 37℃with 5% CO ₂ Incubate for 24 hours. After incubation, plates were centrifuged at 500g for 5 min at room temperature, buffer was aspirated with a BioTek ELx405 microplate washer, and cells were resuspended in FACS buffer (pbs+1% Bovine Serum Albumin (BSA) +0.1% nan) ₃ +2mM EDTA,Sigma Aldrich) and then centrifuged again and the buffer aspirated to remove the cellsLeave in 20. Mu.l of residual medium. 20 μl LIVE/DEAD ^TM Near infrared dead cell stain (Invitrogen, 1:1000 dilution) can be fixed into the wells and incubated for 10 minutes at 4 ℃. Cells were analyzed using a Bio-Rad ZE5 cell analyzer. Cell counts were extracted using Graphpad Prism version 8.1 (Graphpad) to generate indicators and graphical representations. Asymmetric (five parameter) curve fitting was applied to obtain EC50 values.

Results

The percent lymphocyte depletion of 4133-6294-645 TrYbe, 4133-6294 BYbe and 4133-6294IgG4P FALA KiH of representative donors (UCB Cone-802) is shown in FIG. 17. The potency of 4133-6294 TrYbe, 4133-6294 BYbe and 4133-6294IgG4P FALA KiH were similar with EC50 values of 0.35nM, 0.15nM and 0.09nM, respectively.

EXAMPLE 21-Apoptosis of anti-CD 45 4133-6294 Bybe induced cell lines

Method

The following cell lines, classified by ATCC (www.atcc.org /), representing various leukemias and lymphomas were used: jurkat-acute T cell leukemia; acute lymphoblastic leukemia of CCRF-SB-B cells; MC116-B cell undifferentiated lymphoma; raji, ramos-burkitt lymphoma (rare forms of B-cell non-hodgkin lymphoma); SU-DHL-4, SU-DHL-5, SU-DHL-8, NU-DUL-1, OCI-Ly 3-diffuse large B cell lymphoma; THP-1-acute monocytic leukemia; and Dakiki-B cell nasopharyngeal carcinoma.

Prior to performing the assay, 1 vial of each of the above cell lines was thawed in a 37℃water bath, and then 20ml of complete medium (RPMI 1640+2mM GlutaMAX+1%Pen/Strep, all supplied previously by Invitrogen, +10% Fetal Bovine Serum (FBS), sigma Aldrich) was added. The cells were centrifuged (500 g,5 min, room temperature) and resuspended in 20ml of complete medium for washing and re-centrifugation. Cells were resuspended in 10ml of complete medium and then counted using ChemoMetec NucleoCounter NC-3000. 6X10 of each cell line ⁶ Individual cells were centrifuged (500 g,5 min, room temperature) and resuspended in 4.8ml complete medium. Then 1X10 in 80. Mu.l ⁵ Addition of individual cells to Corning Costar96 wells are in each well of a cell culture treated U-bottom microplate (cat# 07-200-95). The plates were incubated at 37℃with 5% CO ₂ The mixture was allowed to stand in the incubator for 2 hours.

Stock solutions of 4133-6294BYbe and NegCtrl BYbe were prepared in 2500nM in complete medium. In Greiner 96 well unbound microwell plates, the two reagents were serially diluted 7 times in complete medium at 1:5 to form an 8-point dose curve. Mu.l of each dilution (final well concentration 500-0.0064 nM) was added to the cells. Each concentration was generated in triplicate. Camptothecins (cat No. C9911-100MG,Sigma Aldrich) and staurosporine (cat No. S6942-200UL,Sigma Aldrich) were added as positive controls for apoptosis. Both were diluted in complete medium and added to the wells to give a final concentration of 5 μm. Other positive controls included anti-thymocyte globulin (ATG, approved by the FDA for conditioning regimens), rituximab (anti-CD 20, approved by the FDA for non-hodgkin's lymphoma and chronic lymphocytic leukemia) and Campath (anti-CD 52, approved by the FDA for B-cell chronic lymphocytic leukemia). Anti-thymocyte globulin, rituximab and Campath were added to wells to give final concentrations of 200 μg/ml, 500nM and 200 μg/ml, respectively. Each concentration of the control was generated in triplicate, except for only 2 replicates of Jurkat. The plates were incubated at 37℃with 5% CO ₂ Incubate for 21 hours.

After incubation, the plates were centrifuged at 500g at room temperature for 5 min, the buffer was aspirated with a BioTek ELx405 microplate washer, and the cells were resuspended in FACS buffer (PBS+1% Bovine Serum Albumin (BSA) +0.1% NaN) ₃ +2mM EDTA,Sigma Aldrich) and then centrifuged again and the buffer aspirated to leave the cells in 20 μl of residual medium. 20. Mu.l LIVE/DEAD ^TM Near infrared dead cell stain (Invitrogen, 1:1000 dilution) can be fixed into the wells and incubated for 10 minutes at 4 ℃. Cells were analyzed using a Bio-Rad ZE5 cell analyzer. Cell counts were extracted using Graphpad Prism version 8.1 (Graphpad) to generate indicators and graphical representations. Asymmetric (five parameter) curve fitting was applied, and constraint type HillSlope was set to "constant equal to 1" to obtain the best fit and derive the maximum reduction of cells and EC50 values.

Results

The maximum decrease in 4133-6294 Bybe-induced Jurkat (99.27%), CCRF-SB (83.40%), OCI-Ly3 (39.84%), THP-1 (60.72%) and Dakiki (76.37%) cells was significantly greater than rituximab (6.52%, 59.87%, 19.05%, 23.12% and 58.55%, respectively) and Campath (5.64%, 24.86%, 6.72%, 15.40% and 8.83%, respectively) (Table 15, FIGS. 18 and 19). The maximum reduction in 4133-6294BYbe induced MC116 (99.45%), raji (74.02%) and Ramos (96.11%) cells was significantly greater than Campath (70.24%, 36.00% and 39.40%, respectively) similar to rituximab induced maximum reductions (98.13%, 87.89% and 91.05%, respectively). The maximum reduction of SU-DHL-8 induced by 4133-6294BYbe (16.77%) was similar to 15.25% of rituximab and 7.97% of Campath, respectively. Although 4133-6294BYbe induced significant reductions in SU-DHL-4 (91.88%) and SU-DHL-5 (91.01%), negCtrl BYbe also induced reductions of 32.09% and 25.63%, respectively. The maximum reduction of NU-DHL-1 induced by 4133-6294Bybe (44.87%) was less than 87.04% of rituximab and 67.49% of Campath.

Tables 15.4133-6294 percent (%) cell reduction and highest and lowest levels of EC50 (nM) values for BYbe and average percent cell reduction for NegCtrl BYbe, rituximab and Campath. Diseases associated with each cell line are also shown: t cell acute lymphoblastic leukemia (T-ALL), B cell non-hodgkin lymphoma (NHL), burkitt Lymphoma (BL), diffuse Large B Cell Lymphoma (DLBCL) and acute monocytic leukemia (AMoL).

* Although the average percent cell reduction at the lowest concentration was 33.85%, the shape of the curve was not sigmoid, which resulted in a calculated minimum of-65.46%.

/>

Sequence listing

<110> UCB biopharmaceutical Limited liability company (UCB BIOPHARMA SRL)

<120> multimerized CD45 binding molecules

<130> N420031WO

<150> GB 2016386.1

<151> 2020-10-15

<150> GB 22100737.2

<151> 2021-01-20

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<170> PatentIn version 3.5

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Gly Phe Ser Phe Ser Gly Asn Tyr Tyr Met Cys

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Gly Phe Ser Phe Ser Gly Asn Tyr Tyr Met Ser

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Cys Leu Tyr Thr Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala

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Lys Gly

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Ser Leu Tyr Thr Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala

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Lys Gly

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Asp Leu Gly Tyr Glu Ile Asp Gly Tyr Gly Gly Leu

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Asp Leu Gly Tyr Glu Ile Asp Ser Tyr Gly Gly Leu

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Asp Leu Gly Tyr Glu Ile Asp Ala Tyr Gly Gly Leu

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Asp Leu Gly Tyr Glu Ile Asp Thr Tyr Gly Gly Leu

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Gln Ala Ser Gln Ser Val Tyr Asn Asn Asn Asn Leu Ser

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Gln Ala Ser Gln Ser Val Tyr Asn Asn Asn Ser Leu Ser

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Gln Ala Ser Gln Ser Val Tyr Asn Asn Asn Gln Leu Ser

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Gln Ala Ser Gln Ser Val Tyr Asn Asn Asn Asn Leu Ala

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Asp Ala Ser Lys Leu Ala Ser

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Leu Gly Gly Tyr Tyr Ser Ser Gly Trp Tyr Phe Ala

1 5 10

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<223> Rabbit antibody 4133 VL region

<400> 15

Ala Gln Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly

1 5 10 15

Gly Thr Val Ser Ile Ser Cys Gln Ala Ser Gln Ser Val Tyr Asn Asn

20 25 30

Asn Asn Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu

35 40 45

Leu Ile Tyr Asp Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val

65 70 75 80

Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Tyr Ser

85 90 95

Ser Gly Trp Tyr Phe Ala Phe Gly Gly Gly Thr Lys Val Val Val Lys

100 105 110

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<212> PRT

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<223> Rabbit antibody 4133 VH region

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Gln Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Glu Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Gly Asn

20 25 30

Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Cys Leu Tyr Thr Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser

50 55 60

Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val

65 70 75 80

Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe

85 90 95

Cys Ala Arg Asp Leu Gly Tyr Glu Ile Asp Gly Tyr Gly Gly Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 17

<211> 112

<212> PRT

<213> artificial sequence

<220>

<223> 4133 g L7V-region-IGKV 1D-13 framework

<400> 17

Ala Gln Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Val Tyr Asn Asn

20 25 30

Asn Asn Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Asp Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu

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Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Tyr Ser

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Ser Gly Trp Tyr Phe Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

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<212> PRT

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<223> 4133 g L1V-region-IGKV 1D-13 framework

<400> 18

Ala Gln Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Val Tyr Asn Asn

20 25 30

Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Asp Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Tyr Ser

85 90 95

Ser Gly Trp Tyr Phe Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 19

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> 4133 gH1V-region-IGHV 3-21 framework

<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Gly Asn

20 25 30

Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Cys Leu Tyr Thr Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser

50 55 60

Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser Ala Lys Thr Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Leu Gly Tyr Glu Ile Asp Gly Tyr Gly Gly Leu

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

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<212> PRT

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<223> 4133 gH4V-region-IGHV 3-21 framework

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Gly Asn

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Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

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Ile Gly Ser Leu Tyr Thr Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser

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Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser Ala Lys Thr Ser

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Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

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Tyr Cys Ala Arg Asp Leu Gly Tyr Glu Ile Asp Gly Tyr Gly Gly Leu

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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

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<212> PRT

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Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ala Ser Gly Phe Ser Phe Ser Gly Asn

20 25 30

Tyr Tyr Met Cys Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Cys Leu Tyr Thr Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser

50 55 60

Trp Ala Lys Gly Arg Val Thr Ile Ser Lys Asp Ser Ser Lys Thr Gln

65 70 75 80

Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Leu Gly Tyr Glu Ile Asp Gly Tyr Gly Gly Leu

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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

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Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ala Ser Gly Phe Ser Phe Ser Gly Asn

20 25 30

Tyr Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

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Ile Gly Ser Leu Tyr Thr Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser

50 55 60

Trp Ala Lys Gly Arg Val Thr Ile Ser Lys Asp Ser Ser Lys Thr Gln

65 70 75 80

Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Leu Gly Tyr Glu Ile Asp Gly Tyr Gly Gly Leu

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 23

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<212> PRT

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Gly Tyr Thr Phe Thr Ser Tyr Thr Met His

1 5 10

<210> 24

<211> 17

<212> PRT

<213> artificial sequence

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<223> CDRH2

<400> 24

Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Glu Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

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<212> PRT

<213> artificial sequence

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Val Gly Asp Gly Phe Tyr Pro Ser Trp Leu Ala Tyr

1 5 10

<210> 26

<211> 12

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<400> 26

Val Gly Asp Ser Phe Tyr Pro Ser Trp Leu Ala Tyr

1 5 10

<210> 27

<211> 12

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<223> CDRH3 variant 2

<400> 27

Val Gly Asp Ala Phe Tyr Pro Ser Trp Leu Ala Tyr

1 5 10

<210> 28

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> CDRH3 variant 3

<400> 28

Val Gly Asp Thr Phe Tyr Pro Ser Trp Leu Ala Tyr

1 5 10

<210> 29

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> CDRL1

<400> 29

Lys Ala Ser Gln Ser Val Arg Asn Asp Val Ala

1 5 10

<210> 30

<211> 7

<212> PRT

<213> artificial sequence

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Tyr Ala Ser Lys Arg Tyr Thr

1 5

<210> 31

<211> 9

<212> PRT

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<220>

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<400> 31

Gln Gln Asp Tyr Ser Ser Pro Thr Thr

1 5

<210> 32

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> mouse antibody 6294 VL region

<400> 32

Asp Ile Gln Met Thr Gln Ser Pro Lys Phe Leu Leu Val Ser Ala Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Arg Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Phe Tyr Ala Ser Lys Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala

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Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Thr

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 33

<211> 121

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Glu Val Gln Leu Val Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Glu Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Asp Gly Phe Tyr Pro Ser Trp Leu Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 34

<211> 121

<212> PRT

<213> artificial sequence

<220>

<223> 6294 g H2V-region-IGHV 3-48 framework

<400> 34

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Glu Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Ala Asp Lys Ala Lys Ser Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Asp Gly Phe Tyr Pro Ser Trp Leu Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 35

<211> 121

<212> PRT

<213> artificial sequence

<220>

<223> 6294 gH4V-region-IGHV 1-69 framework

<400> 35

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Glu Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Asp Gly Phe Tyr Pro Ser Trp Leu Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 36

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> 6294 g L2V-region-IGKV 1D-33 framework

<400> 36

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Arg Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Phe Tyr Ala Ser Lys Arg Tyr Thr Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Thr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 37

<211> 107

<212> PRT

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<220>

<223> 6294 g L3V-region-IGKV 4-1 framework

<400> 37

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Arg Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Phe Tyr Ala Ser Lys Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Thr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 38

<211> 483

<212> PRT

<213> artificial sequence

<220>

<223> 4133-6294 BYbe heavy chain

<400> 38

Gln Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Glu Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Gly Asn

20 25 30

Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Cys Leu Tyr Thr Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser

50 55 60

Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val

65 70 75 80

Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe

85 90 95

Cys Ala Arg Asp Leu Gly Tyr Glu Ile Asp Gly Tyr Gly Gly Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr

130 135 140

Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr

145 150 155 160

Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro

165 170 175

Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser

180 185 190

Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala

195 200 205

Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

225 230 235 240

Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser

245 250 255

Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Thr Met His Trp Val

260 265 270

Lys Gln Arg Pro Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro

275 280 285

Ser Ser Gly Tyr Thr Glu Tyr Asn Gln Lys Phe Lys Asp Lys Thr Thr

290 295 300

Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Leu Gln Leu Ser Ser

305 310 315 320

Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Val Gly Asp

325 330 335

Gly Phe Tyr Pro Ser Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val

340 345 350

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

355 360 365

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro

370 375 380

Lys Phe Leu Leu Val Ser Ala Gly Asp Arg Val Thr Ile Thr Cys Lys

385 390 395 400

Ala Ser Gln Ser Val Arg Asn Asp Val Ala Trp Tyr Gln Gln Lys Pro

405 410 415

Gly Gln Ser Pro Lys Leu Leu Ile Phe Tyr Ala Ser Lys Arg Tyr Thr

420 425 430

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Tyr Gly Thr Asp Phe Thr

435 440 445

Phe Thr Ile Ser Thr Val Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys

450 455 460

Gln Gln Asp Tyr Ser Ser Pro Thr Thr Phe Gly Cys Gly Thr Lys Leu

465 470 475 480

Glu Ile Lys

<210> 39

<211> 216

<212> PRT

<213> artificial sequence

<220>

<223> 4133-6294 BYbe light chain

<400> 39

Ala Gln Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly

1 5 10 15

Gly Thr Val Ser Ile Ser Cys Gln Ala Ser Gln Ser Val Tyr Asn Asn

20 25 30

Asn Asn Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu

35 40 45

Leu Ile Tyr Asp Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val

65 70 75 80

Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Tyr Ser

85 90 95

Ser Gly Trp Tyr Phe Ala Phe Gly Gly Gly Thr Lys Val Val Val Lys

100 105 110

Arg Thr Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp

115 120 125

Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr

130 135 140

Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr

145 150 155 160

Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr

165 170 175

Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser

180 185 190

His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val

195 200 205

Gln Ser Phe Asn Arg Gly Asp Cys

210 215

<210> 40

<211> 369

<212> PRT

<213> artificial sequence

<220>

<223> CD45 Domains 1-4 with TEV cleavage site and 10-histidine tag

<400> 40

Lys Pro Thr Cys Asp Glu Lys Tyr Ala Asn Ile Thr Val Asp Tyr Leu

1 5 10 15

Tyr Asn Lys Glu Thr Lys Leu Phe Thr Ala Lys Leu Asn Val Asn Glu

20 25 30

Asn Val Glu Cys Gly Asn Asn Thr Cys Thr Asn Asn Glu Val His Asn

35 40 45

Leu Thr Glu Cys Lys Asn Ala Ser Val Ser Ile Ser His Asn Ser Cys

50 55 60

Thr Ala Pro Asp Lys Thr Leu Ile Leu Asp Val Pro Pro Gly Val Glu

65 70 75 80

Lys Phe Gln Leu His Asp Cys Thr Gln Val Glu Lys Ala Asp Thr Thr

85 90 95

Ile Cys Leu Lys Trp Lys Asn Ile Glu Thr Phe Thr Cys Asp Thr Gln

100 105 110

Asn Ile Thr Tyr Arg Phe Gln Cys Gly Asn Met Ile Phe Asp Asn Lys

115 120 125

Glu Ile Lys Leu Glu Asn Leu Glu Pro Glu His Glu Tyr Lys Cys Asp

130 135 140

Ser Glu Ile Leu Tyr Asn Asn His Lys Phe Thr Asn Ala Ser Lys Ile

145 150 155 160

Ile Lys Thr Asp Phe Gly Ser Pro Gly Glu Pro Gln Ile Ile Phe Cys

165 170 175

Arg Ser Glu Ala Ala His Gln Gly Val Ile Thr Trp Asn Pro Pro Gln

180 185 190

Arg Ser Phe His Asn Phe Thr Leu Cys Tyr Ile Lys Glu Thr Glu Lys

195 200 205

Asp Cys Leu Asn Leu Asp Lys Asn Leu Ile Lys Tyr Asp Leu Gln Asn

210 215 220

Leu Lys Pro Tyr Thr Lys Tyr Val Leu Ser Leu His Ala Tyr Ile Ile

225 230 235 240

Ala Lys Val Gln Arg Asn Gly Ser Ala Ala Met Cys His Phe Thr Thr

245 250 255

Lys Ser Ala Pro Pro Ser Gln Val Trp Asn Met Thr Val Ser Met Thr

260 265 270

Ser Asp Asn Ser Met His Val Lys Cys Arg Pro Pro Arg Asp Arg Asn

275 280 285

Gly Pro His Glu Arg Tyr His Leu Glu Val Glu Ala Gly Asn Thr Leu

290 295 300

Val Arg Asn Glu Ser His Lys Asn Cys Asp Phe Arg Val Lys Asp Leu

305 310 315 320

Gln Tyr Ser Thr Asp Tyr Thr Phe Lys Ala Tyr Phe His Asn Gly Asp

325 330 335

Tyr Pro Gly Glu Pro Phe Ile Leu His His Ser Thr Ser Gly Thr Lys

340 345 350

Glu Asn Leu Tyr Phe Gln Gly His His His His His His His His His

355 360 365

His

<210> 41

<211> 1304

<212> PRT

<213> artificial sequence

<220>

<223> human CD45

<400> 41

Met Tyr Leu Trp Leu Lys Leu Leu Ala Phe Gly Phe Ala Phe Leu Asp

1 5 10 15

Thr Glu Val Phe Val Thr Gly Gln Ser Pro Thr Pro Ser Pro Thr Gly

20 25 30

Leu Thr Thr Ala Lys Met Pro Ser Val Pro Leu Ser Ser Asp Pro Leu

35 40 45

Pro Thr His Thr Thr Ala Phe Ser Pro Ala Ser Thr Phe Glu Arg Glu

50 55 60

Asn Asp Phe Ser Glu Thr Thr Thr Ser Leu Ser Pro Asp Asn Thr Ser

65 70 75 80

Thr Gln Val Ser Pro Asp Ser Leu Asp Asn Ala Ser Ala Phe Asn Thr

85 90 95

Thr Gly Val Ser Ser Val Gln Thr Pro His Leu Pro Thr His Ala Asp

100 105 110

Ser Gln Thr Pro Ser Ala Gly Thr Asp Thr Gln Thr Phe Ser Gly Ser

115 120 125

Ala Ala Asn Ala Lys Leu Asn Pro Thr Pro Gly Ser Asn Ala Ile Ser

130 135 140

Asp Val Pro Gly Glu Arg Ser Thr Ala Ser Thr Phe Pro Thr Asp Pro

145 150 155 160

Val Ser Pro Leu Thr Thr Thr Leu Ser Leu Ala His His Ser Ser Ala

165 170 175

Ala Leu Pro Ala Arg Thr Ser Asn Thr Thr Ile Thr Ala Asn Thr Ser

180 185 190

Asp Ala Tyr Leu Asn Ala Ser Glu Thr Thr Thr Leu Ser Pro Ser Gly

195 200 205

Ser Ala Val Ile Ser Thr Thr Thr Ile Ala Thr Thr Pro Ser Lys Pro

210 215 220

Thr Cys Asp Glu Lys Tyr Ala Asn Ile Thr Val Asp Tyr Leu Tyr Asn

225 230 235 240

Lys Glu Thr Lys Leu Phe Thr Ala Lys Leu Asn Val Asn Glu Asn Val

245 250 255

Glu Cys Gly Asn Asn Thr Cys Thr Asn Asn Glu Val His Asn Leu Thr

260 265 270

Glu Cys Lys Asn Ala Ser Val Ser Ile Ser His Asn Ser Cys Thr Ala

275 280 285

Pro Asp Lys Thr Leu Ile Leu Asp Val Pro Pro Gly Val Glu Lys Phe

290 295 300

Gln Leu His Asp Cys Thr Gln Val Glu Lys Ala Asp Thr Thr Ile Cys

305 310 315 320

Leu Lys Trp Lys Asn Ile Glu Thr Phe Thr Cys Asp Thr Gln Asn Ile

325 330 335

Thr Tyr Arg Phe Gln Cys Gly Asn Met Ile Phe Asp Asn Lys Glu Ile

340 345 350

Lys Leu Glu Asn Leu Glu Pro Glu His Glu Tyr Lys Cys Asp Ser Glu

355 360 365

Ile Leu Tyr Asn Asn His Lys Phe Thr Asn Ala Ser Lys Ile Ile Lys

370 375 380

Thr Asp Phe Gly Ser Pro Gly Glu Pro Gln Ile Ile Phe Cys Arg Ser

385 390 395 400

Glu Ala Ala His Gln Gly Val Ile Thr Trp Asn Pro Pro Gln Arg Ser

405 410 415

Phe His Asn Phe Thr Leu Cys Tyr Ile Lys Glu Thr Glu Lys Asp Cys

420 425 430

Leu Asn Leu Asp Lys Asn Leu Ile Lys Tyr Asp Leu Gln Asn Leu Lys

435 440 445

Pro Tyr Thr Lys Tyr Val Leu Ser Leu His Ala Tyr Ile Ile Ala Lys

450 455 460

Val Gln Arg Asn Gly Ser Ala Ala Met Cys His Phe Thr Thr Lys Ser

465 470 475 480

Ala Pro Pro Ser Gln Val Trp Asn Met Thr Val Ser Met Thr Ser Asp

485 490 495

Asn Ser Met His Val Lys Cys Arg Pro Pro Arg Asp Arg Asn Gly Pro

500 505 510

His Glu Arg Tyr His Leu Glu Val Glu Ala Gly Asn Thr Leu Val Arg

515 520 525

Asn Glu Ser His Lys Asn Cys Asp Phe Arg Val Lys Asp Leu Gln Tyr

530 535 540

Ser Thr Asp Tyr Thr Phe Lys Ala Tyr Phe His Asn Gly Asp Tyr Pro

545 550 555 560

Gly Glu Pro Phe Ile Leu His His Ser Thr Ser Tyr Asn Ser Lys Ala

565 570 575

Leu Ile Ala Phe Leu Ala Phe Leu Ile Ile Val Thr Ser Ile Ala Leu

580 585 590

Leu Val Val Leu Tyr Lys Ile Tyr Asp Leu His Lys Lys Arg Ser Cys

595 600 605

Asn Leu Asp Glu Gln Gln Glu Leu Val Glu Arg Asp Asp Glu Lys Gln

610 615 620

Leu Met Asn Val Glu Pro Ile His Ala Asp Ile Leu Leu Glu Thr Tyr

625 630 635 640

Lys Arg Lys Ile Ala Asp Glu Gly Arg Leu Phe Leu Ala Glu Phe Gln

645 650 655

Ser Ile Pro Arg Val Phe Ser Lys Phe Pro Ile Lys Glu Ala Arg Lys

660 665 670

Pro Phe Asn Gln Asn Lys Asn Arg Tyr Val Asp Ile Leu Pro Tyr Asp

675 680 685

Tyr Asn Arg Val Glu Leu Ser Glu Ile Asn Gly Asp Ala Gly Ser Asn

690 695 700

Tyr Ile Asn Ala Ser Tyr Ile Asp Gly Phe Lys Glu Pro Arg Lys Tyr

705 710 715 720

Ile Ala Ala Gln Gly Pro Arg Asp Glu Thr Val Asp Asp Phe Trp Arg

725 730 735

Met Ile Trp Glu Gln Lys Ala Thr Val Ile Val Met Val Thr Arg Cys

740 745 750

Glu Glu Gly Asn Arg Asn Lys Cys Ala Glu Tyr Trp Pro Ser Met Glu

755 760 765

Glu Gly Thr Arg Ala Phe Gly Asp Val Val Val Lys Ile Asn Gln His

770 775 780

Lys Arg Cys Pro Asp Tyr Ile Ile Gln Lys Leu Asn Ile Val Asn Lys

785 790 795 800

Lys Glu Lys Ala Thr Gly Arg Glu Val Thr His Ile Gln Phe Thr Ser

805 810 815

Trp Pro Asp His Gly Val Pro Glu Asp Pro His Leu Leu Leu Lys Leu

820 825 830

Arg Arg Arg Val Asn Ala Phe Ser Asn Phe Phe Ser Gly Pro Ile Val

835 840 845

Val His Cys Ser Ala Gly Val Gly Arg Thr Gly Thr Tyr Ile Gly Ile

850 855 860

Asp Ala Met Leu Glu Gly Leu Glu Ala Glu Asn Lys Val Asp Val Tyr

865 870 875 880

Gly Tyr Val Val Lys Leu Arg Arg Gln Arg Cys Leu Met Val Gln Val

885 890 895

Glu Ala Gln Tyr Ile Leu Ile His Gln Ala Leu Val Glu Tyr Asn Gln

900 905 910

Phe Gly Glu Thr Glu Val Asn Leu Ser Glu Leu His Pro Tyr Leu His

915 920 925

Asn Met Lys Lys Arg Asp Pro Pro Ser Glu Pro Ser Pro Leu Glu Ala

930 935 940

Glu Phe Gln Arg Leu Pro Ser Tyr Arg Ser Trp Arg Thr Gln His Ile

945 950 955 960

Gly Asn Gln Glu Glu Asn Lys Ser Lys Asn Arg Asn Ser Asn Val Ile

965 970 975

Pro Tyr Asp Tyr Asn Arg Val Pro Leu Lys His Glu Leu Glu Met Ser

980 985 990

Lys Glu Ser Glu His Asp Ser Asp Glu Ser Ser Asp Asp Asp Ser Asp

995 1000 1005

Ser Glu Glu Pro Ser Lys Tyr Ile Asn Ala Ser Phe Ile Met Ser

1010 1015 1020

Tyr Trp Lys Pro Glu Val Met Ile Ala Ala Gln Gly Pro Leu Lys

1025 1030 1035

Glu Thr Ile Gly Asp Phe Trp Gln Met Ile Phe Gln Arg Lys Val

1040 1045 1050

Lys Val Ile Val Met Leu Thr Glu Leu Lys His Gly Asp Gln Glu

1055 1060 1065

Ile Cys Ala Gln Tyr Trp Gly Glu Gly Lys Gln Thr Tyr Gly Asp

1070 1075 1080

Ile Glu Val Asp Leu Lys Asp Thr Asp Lys Ser Ser Thr Tyr Thr

1085 1090 1095

Leu Arg Val Phe Glu Leu Arg His Ser Lys Arg Lys Asp Ser Arg

1100 1105 1110

Thr Val Tyr Gln Tyr Gln Tyr Thr Asn Trp Ser Val Glu Gln Leu

1115 1120 1125

Pro Ala Glu Pro Lys Glu Leu Ile Ser Met Ile Gln Val Val Lys

1130 1135 1140

Gln Lys Leu Pro Gln Lys Asn Ser Ser Glu Gly Asn Lys His His

1145 1150 1155

Lys Ser Thr Pro Leu Leu Ile His Cys Arg Asp Gly Ser Gln Gln

1160 1165 1170

Thr Gly Ile Phe Cys Ala Leu Leu Asn Leu Leu Glu Ser Ala Glu

1175 1180 1185

Thr Glu Glu Val Val Asp Ile Phe Gln Val Val Lys Ala Leu Arg

1190 1195 1200

Lys Ala Arg Pro Gly Met Val Ser Thr Phe Glu Gln Tyr Gln Phe

1205 1210 1215

Leu Tyr Asp Val Ile Ala Ser Thr Tyr Pro Ala Gln Asn Gly Gln

1220 1225 1230

Val Lys Lys Asn Asn His Gln Glu Asp Lys Ile Glu Phe Asp Asn

1235 1240 1245

Glu Val Asp Lys Val Lys Gln Asp Ala Asn Cys Val Asn Pro Leu

1250 1255 1260

Gly Ala Pro Glu Lys Leu Pro Glu Ala Lys Glu Gln Ala Glu Gly

1265 1270 1275

Ser Glu Pro Thr Ser Gly Thr Glu Gly Pro Glu His Ser Val Asn

1280 1285 1290

Gly Pro Ala Ser Pro Ala Leu Asn Gln Gly Ser

1295 1300

<210> 42

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 42

Asp Lys Thr His Thr Cys Ala Ala

1 5

<210> 43

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 43

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10

<210> 44

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 44

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Thr Cys Pro Pro Cys

1 5 10 15

Pro Ala

<210> 45

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 45

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Thr Cys Pro Pro Cys

1 5 10 15

Pro Ala Thr Cys Pro Pro Cys Pro Ala

20 25

<210> 46

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 46

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Gly Lys Pro Thr Leu

1 5 10 15

Tyr Asn Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr

20 25 30

<210> 47

<211> 31

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 47

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Gly Lys Pro Thr His

1 5 10 15

Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys Tyr

20 25 30

<210> 48

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 48

Asp Lys Thr His Thr Cys Cys Val Glu Cys Pro Pro Cys Pro Ala

1 5 10 15

<210> 49

<211> 26

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 49

Asp Lys Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp

1 5 10 15

Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala

20 25

<210> 50

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 50

Asp Lys Thr His Thr Cys Pro Ser Cys Pro Ala

1 5 10

<210> 51

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 51

Ser Gly Gly Gly Gly Ser Glu

1 5

<210> 52

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 52

Asp Lys Thr His Thr Ser

1 5

<210> 53

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 53

Ser Gly Gly Gly Gly Ser

1 5

<210> 54

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 54

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 55

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 55

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 56

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 56

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Gly Ser

20

<210> 57

<211> 26

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 57

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 58

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 58

Ala Ala Ala Gly Ser Gly Gly Ala Ser Ala Ser

1 5 10

<210> 59

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<400> 59

Ala Ala Ala Gly Ser Gly Xaa Gly Gly Gly Ser Gly Ala Ser Ala Ser

1 5 10 15

<210> 60

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa can be any naturally occurring amino acid

<400> 60

Ala Ala Ala Gly Ser Gly Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser

1 5 10 15

Gly Ala Ser Ala Ser

20

<210> 61

<211> 26

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (17)..(17)

<223> Xaa can be any naturally occurring amino acid

<400> 61

Ala Ala Ala Gly Ser Gly Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser

1 5 10 15

Xaa Gly Gly Gly Ser Gly Ala Ser Ala Ser

20 25

<210> 62

<211> 31

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (17)..(17)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (22)..(22)

<223> Xaa can be any naturally occurring amino acid

<400> 62

Ala Ala Ala Gly Ser Gly Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser

1 5 10 15

Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser Gly Ala Ser Ala Ser

20 25 30

<210> 63

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<400> 63

Ala Ala Ala Gly Ser Gly Xaa Ser Gly Ala Ser Ala Ser

1 5 10

<210> 64

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 64

Pro Gly Gly Asn Arg Gly Thr Thr Thr Thr Arg Arg Pro Ala Thr Thr

1 5 10 15

Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser His Tyr

20 25

<210> 65

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 65

Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr

1 5 10

<210> 66

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 66

Ala Thr Thr Thr Gly Ser

1 5

<210> 67

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 67

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 68

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 68

Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Ser Pro Pro Ser Lys Glu

1 5 10 15

Ser His Lys Ser Pro

20

<210> 69

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 69

Gly Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

1 5 10 15

<210> 70

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 70

Gly Gly Gly Gly Ile Ala Pro Ser Met Val Gly Gly Gly Gly Ser

1 5 10 15

<210> 71

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 71

Gly Gly Gly Gly Lys Val Glu Gly Ala Gly Gly Gly Gly Gly Ser

1 5 10 15

<210> 72

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 72

Gly Gly Gly Gly Ser Met Lys Ser His Asp Gly Gly Gly Gly Ser

1 5 10 15

<210> 73

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 73

Gly Gly Gly Gly Asn Leu Ile Thr Ile Val Gly Gly Gly Gly Ser

1 5 10 15

<210> 74

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 74

Gly Gly Gly Gly Val Val Pro Ser Leu Pro Gly Gly Gly Gly Ser

1 5 10 15

<210> 75

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 75

Gly Gly Glu Lys Ser Ile Pro Gly Gly Gly Gly Ser

1 5 10

<210> 76

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 76

Arg Pro Leu Ser Tyr Arg Pro Pro Phe Pro Phe Gly Phe Pro Ser Val

1 5 10 15

Arg Pro

<210> 77

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 77

Tyr Pro Arg Ser Ile Tyr Ile Arg Arg Arg His Pro Ser Pro Ser Leu

1 5 10 15

Thr Thr

<210> 78

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 78

Thr Pro Ser His Leu Ser His Ile Leu Pro Ser Phe Gly Leu Pro Thr

1 5 10 15

Phe Asn

<210> 79

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 79

Arg Pro Val Ser Pro Phe Thr Phe Pro Arg Leu Ser Asn Ser Trp Leu

1 5 10 15

Pro Ala

<210> 80

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 80

Ser Pro Ala Ala His Phe Pro Arg Ser Ile Pro Arg Pro Gly Pro Ile

1 5 10 15

Arg Thr

<210> 81

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 81

Ala Pro Gly Pro Ser Ala Pro Ser His Arg Ser Leu Pro Ser Arg Ala

1 5 10 15

Phe Gly

<210> 82

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 82

Pro Arg Asn Ser Ile His Phe Leu His Pro Leu Leu Val Ala Pro Leu

1 5 10 15

Gly Ala

<210> 83

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 83

Met Pro Ser Leu Ser Gly Val Leu Gln Val Arg Tyr Leu Ser Pro Pro

1 5 10 15

Asp Leu

<210> 84

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 84

Ser Pro Gln Tyr Pro Ser Pro Leu Thr Leu Thr Leu Pro Pro His Pro

1 5 10 15

Ser Leu

<210> 85

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 85

Asn Pro Ser Leu Asn Pro Pro Ser Tyr Leu His Arg Ala Pro Ser Arg

1 5 10 15

Ile Ser

<210> 86

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 86

Leu Pro Trp Arg Thr Ser Leu Leu Pro Ser Leu Pro Leu Arg Arg Arg

1 5 10 15

Pro

<210> 87

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 87

Pro Pro Leu Phe Ala Lys Gly Pro Val Gly Leu Leu Ser Arg Ser Phe

1 5 10 15

Pro Pro

<210> 88

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 88

Val Pro Pro Ala Pro Val Val Ser Leu Arg Ser Ala His Ala Arg Pro

1 5 10 15

Pro Tyr

<210> 89

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 89

Leu Arg Pro Thr Pro Pro Arg Val Arg Ser Tyr Thr Cys Cys Pro Thr

1 5 10 15

Pro

<210> 90

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 90

Pro Asn Val Ala His Val Leu Pro Leu Leu Thr Val Pro Trp Asp Asn

1 5 10 15

Leu Arg

<210> 91

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Flexible linker sequence

<400> 91

Cys Asn Pro Leu Leu Pro Leu Cys Ala Arg Ser Pro Ala Val Arg Thr

1 5 10 15

Phe Pro

<210> 92

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> rigid joint

<400> 92

Gly Ala Pro Ala Pro Ala Ala Pro Ala Pro Ala

1 5 10

<210> 93

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> rigid joint

<400> 93

Pro Pro Pro Pro

1

<210> 94

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 94

Asp Leu Cys Leu Arg Asp Trp Gly Cys Leu Trp

1 5 10

<210> 95

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 95

Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10

<210> 96

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 96

Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly Asp

1 5 10 15

<210> 97

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 97

Gln Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10 15

Glu Asp Asp Glu

20

<210> 98

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 98

Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10 15

Gly Arg Ser Val

20

<210> 99

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 99

Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10 15

Gly Arg Ser Val Lys

20

<210> 100

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 100

Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp

1 5 10 15

<210> 101

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 101

Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu

1 5 10 15

Asp Asp

<210> 102

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 102

Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp

1 5 10 15

<210> 103

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 103

Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp

1 5 10 15

<210> 104

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 104

Arg Leu Met Glu Asp Ile Cys Leu Ala Arg Trp Gly Cys Leu Trp Glu

1 5 10 15

Asp Asp

<210> 105

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 105

Glu Val Arg Ser Phe Cys Thr Arg Trp Pro Ala Glu Lys Ser Cys Lys

1 5 10 15

Pro Leu Arg Gly

20

<210> 106

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 106

Arg Ala Pro Glu Ser Phe Val Cys Tyr Trp Glu Thr Ile Cys Phe Glu

1 5 10 15

Arg Ser Glu Gln

20

<210> 107

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> hinge linker sequence

<400> 107

Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Met

1 5 10

<210> 108

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 108

Ala Ser Gly Gly Gly Gly

1 5

<210> 109

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 109

Ala Ser Gly Gly Gly Gly Ser Gly

1 5

<210> 110

<211> 132

<212> DNA

<213> artificial sequence

<220>

<223> GCN4 sequence

<400> 110

gctagcggag gcggaagaat gaaacaactt gaacccaagg ttgaagaatt gcttccgaaa 60

aattatcact tggaaaatga ggttgccaga ttaaagaaat tagttggcga acgccatcac 120

catcaccatc ac 132

<210> 111

<211> 44

<212> PRT

<213> artificial sequence

<220>

<223> GCN4 sequence

<400> 111

Ala Ser Gly Gly Gly Arg Met Lys Gln Leu Glu Pro Lys Val Glu Glu

1 5 10 15

Leu Leu Pro Lys Asn Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys

20 25 30

Lys Leu Val Gly Glu Arg His His His His His His

35 40

<210> 112

<211> 792

<212> DNA

<213> artificial sequence

<220>

<223> 52SR4 sequence

<400> 112

gatgcggtgg tgacccagga aagcgcgctg accagcagcc cgggcgaaac cgtgaccctg 60

acctgccgca gcagcaccgg cgcggtgacc accagcaact atgcgagctg ggtgcaggaa 120

aaaccggatc atctgtttac cggcctgatt ggcggcacca acaaccgcgc gccgggcgtg 180

ccggcgcgct ttagcggcag cctgattggc gataaagcgg cgctgaccat taccggcgcg 240

cagaccgaag atgaagcgat ttatttttgc gtgctgtggt atagcgacca ttgggtgttt 300

ggctgcggca ccaaactgac cgtgctgggt ggaggcggtg gctcaggcgg aggtggctca 360

ggcggtggcg ggtctggcgg cggcggcagc gatgtgcagc tgcagcagag cggcccgggc 420

ctggtggcgc cgagccagag cctgagcatt acctgcaccg tgagcggctt tctcctgacc 480

gattatggcg tgaactgggt gcgccagagc ccgggcaaat gcctggaatg gctgggcgtg 540

atttggggcg atggcattac cgattataac agcgcgctga aaagccgcct gagcgtgacc 600

aaagataaca gcaaaagcca ggtgtttctg aaaatgaaca gcctgcagag cggcgatagc 660

gcgcgctatt attgcgtgac cggcctgttt gattattggg gccagggcac caccctgacc 720

gtgagcagcg cggccgccca tcaccatcac catcacgaac agaaactgat tagcgaagaa 780

gatctgtaat ag 792

<210> 113

<211> 262

<212> PRT

<213> artificial sequence

<220>

<223> 52SR4 sequence

<400> 113

Asp Ala Val Val Thr Gln Glu Ser Ala Leu Thr Ser Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Ser Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly

35 40 45

Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Val Leu Trp Tyr Ser Asp

85 90 95

His Trp Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu Gly Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Asp Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Ala Pro

130 135 140

Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Leu Leu Thr

145 150 155 160

Asp Tyr Gly Val Asn Trp Val Arg Gln Ser Pro Gly Lys Cys Leu Glu

165 170 175

Trp Leu Gly Val Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala

180 185 190

Leu Lys Ser Arg Leu Ser Val Thr Lys Asp Asn Ser Lys Ser Gln Val

195 200 205

Phe Leu Lys Met Asn Ser Leu Gln Ser Gly Asp Ser Ala Arg Tyr Tyr

210 215 220

Cys Val Thr Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

225 230 235 240

Val Ser Ser Ala Ala Ala His His His His His His Glu Gln Lys Leu

245 250 255

Ile Ser Glu Glu Asp Leu

260

<210> 114

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 114

Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser

1 5 10

<210> 115

<211> 219

<212> PRT

<213> artificial sequence

<220>

<223> 4133 light chain

<400> 115

Ala Gln Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly

1 5 10 15

Gly Thr Val Ser Ile Ser Cys Gln Ala Ser Gln Ser Val Tyr Asn Asn

20 25 30

Asn Asn Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu

35 40 45

Leu Ile Tyr Asp Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val

65 70 75 80

Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Tyr Ser

85 90 95

Ser Gly Trp Tyr Phe Ala Phe Gly Gly Gly Thr Lys Val Val Val Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 116

<211> 449

<212> PRT

<213> artificial sequence

<220>

<223> 4133 IgG4P FALA

<400> 116

Gln Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Glu Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Gly Asn

20 25 30

Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Cys Leu Tyr Thr Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser

50 55 60

Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val

65 70 75 80

Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe

85 90 95

Cys Ala Arg Asp Leu Gly Tyr Glu Ile Asp Gly Tyr Gly Gly Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

Lys

<210> 117

<211> 451

<212> PRT

<213> artificial sequence

<220>

<223> 4133 IgG4P FALA pestle

<400> 117

Gln Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Glu Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Gly Asn

20 25 30

Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Cys Leu Tyr Thr Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser

50 55 60

Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val

65 70 75 80

Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe

85 90 95

Cys Ala Arg Asp Leu Gly Tyr Glu Ile Asp Gly Tyr Gly Gly Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ser Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Thr Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Leu Gly Lys

450

<210> 118

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> 6294 light chain

<400> 118

Asp Ile Gln Met Thr Gln Ser Pro Lys Phe Leu Leu Val Ser Ala Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Arg Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Phe Tyr Ala Ser Lys Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Thr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 119

<211> 448

<212> PRT

<213> artificial sequence

<220>

<223> 6294 IgG4P FALA mortar

<400> 119

Glu Val Gln Leu Val Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Glu Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Asp Gly Phe Tyr Pro Ser Trp Leu Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

210 215 220

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys

355 360 365

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

Claims

1. An antibody comprising at least two different paratopes, each of said paratopes being specific for a different epitope of CD45.

2. The antibody of claim 1, wherein the antibody is a bi-paratope antibody, wherein each of two different paratopes of the antibody is specific for a different epitope of CD45.

3. The antibody of claim 1 or 2, wherein the CD45 is human CD45.

4. The antibody of any one of the preceding claims, wherein the antibody is capable of inducing cell death of cells expressing CD45, preferably wherein the antibody does not induce release of cytokines.

5. The antibody of any one of the preceding claims, wherein the antibody lacks an Fc region or comprises an Fc region that has been silenced to remove one or more Fc effector functions and/or modified to alter serum pharmacokinetics.

6. The antibody of any one of the preceding claims, which is selected from the group consisting of: BYbe antibodies, trYbe antibodies, diabodies, duobodies, igG (e.g., igG with modifications to the homodimers that promote heterodimer formation and/or purify heterodimers, such as knob-to-socket modifications, charge-to-charge modifications, and/or modifications that alter the ability of one heavy chain binding protein a or IgG4 (P) antibodies with FALA and knob-to-socket modifications).

7. The antibody of any one of the preceding claims, wherein the antibody is a humanized antibody or a fully human antibody.

8. One or more nucleic acid molecules encoding an antibody as defined in any one of the preceding claims.

9. One or more vectors encoding an antibody as defined in any one of claims 1-7 or comprising one or more nucleic acid molecules of claim 8.

10. A pharmaceutical composition comprising:

(a) The antibody of any one of claims 1-7, the one or more nucleic acid molecules of claim 8, or the one or more vectors of claim 9; and

(b) A pharmaceutically acceptable carrier or diluent.

11. The pharmaceutical composition according to claim 10 for use in a method of treatment.

12. The pharmaceutical composition of claim 11 for use in a method of killing a disease-associated CD45 expressing cell in a subject.

13. Pharmaceutical composition according to claim 11 or 12 for use in a method of treatment of a blood cancer, such as leukemia, lymphoma or multiple myeloma.

14. Pharmaceutical composition according to claim 11 or 12 for use in a method of treatment of an autoimmune disease, such as multiple sclerosis or scleroderma.

15. The pharmaceutical composition for use in the method of any one of claims 11-14, wherein the method further comprises transferring cells into the subject after cell depletion.

16. A method of depleting cells expressing CD45 causing a disease in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 10.

17. The method of claim 16, for the treatment of a blood cancer, such as leukemia, lymphoma or multiple myeloma.

18. The method according to claim 16, for the treatment of autoimmune diseases, such as multiple sclerosis or scleroderma.

19. The method of any one of claims 16, 17 and 18, wherein the method further comprises transferring cells into the subject after cell depletion.

20. Use of the antibody of any one of claims 1-7, the nucleic acid molecule of claim 8, or the one or more vectors of claim 9 in the manufacture of a medicament for killing a disease-associated CD45 expressing cell in a subject.

21. The use according to claim 20, wherein the medicament is for the treatment of a blood cancer, such as leukemia, lymphoma or multiple myeloma.

22. The use according to claim 20, wherein the medicament is for the treatment of autoimmune diseases, such as multiple sclerosis or scleroderma.

23. The use of any one of claims 20-22, wherein the medicament is for use further comprising a method of transferring cells into the subject after killing cells.

24. One or more binding molecules capable of multimerizing CD45 to induce cell death of CD45 expressing cells without inducing significant cytokine release.

25. The one or more binding molecules of claim 24, wherein the one or more binding molecules are antibodies that specifically bind CD45 or a mixture of at least two different antibodies that specifically bind CD 45.

26. The one or more binding molecules of claim 25, wherein the antibody or antibodies in the mixture have an Fc region that is modified:

(a) An Fc region that is optimized for effector;

(b) Increased heterodimer formation relative to homodimers (such as with a knob-to-hole modification);

(c) The presence of charged residues that promote heterodimer formation relative to homodimers;

(d) Has altered serum pharmacokinetics; and/or

(e) With altered protein a binding.

27. The one or more binding molecules of claim 25 or 26, wherein the antibody or the antibodies in the mixture of antibodies have a silenced Fc region.

28. The one or more binding molecules of claim 25, wherein the antibody or the mixture of antibodies lacks an Fc region.

29. The one or more binding molecules of claim 25, wherein the antibody or antibodies in the mixture are selected from the group consisting of: BYbe antibodies, trYbe antibodies, diabodies, duobody, igG, or knob-modified IgG, particularly wherein one or more of the antibodies is in the form of an IgG4 (P) FALA knob.

30. The one or more binding molecules of any one of claims 24-29, wherein:

(a) The binding molecule is an antibody comprising at least two antigen binding sites having different specificities for CD 45; or (b)

(b) The plurality of binding molecules is a mixture of antibodies, wherein the plurality of antibodies in the mixture collectively comprise at least two different antigen binding sites having different specificities for CD 45.

31. The one or more binding molecules of claim 30, which is a mixture of antibodies, wherein each antibody has a single specificity for CD45, but the mixture comprises at least two different antibodies having different specificities for CD 45.

32. The one or more binding molecules of any one of claims 24-31, wherein one or more antibodies are chimeric, humanized, or fully human antibodies.

33. The one or more binding molecules of any one of claims 24-32, wherein the antibody or antibodies in the mixture comprise an antigen binding site specific for serum albumin.

34. One or more nucleic acid molecules encoding one or more binding molecules as defined in any one of claims 24-33.

35. One or more vectors encoding one or more binding molecules as defined in any one of claims 24-33 or comprising one or more nucleic acid molecules according to claim 34, e.g. wherein the vector is LNP-mRNA.

36. A pharmaceutical composition comprising:

(a) One or more binding molecules of any one of claims 24-33, one or more nucleic acid molecules of claim 34, or one or more vectors of claim 35; and

(b) A pharmaceutically acceptable carrier or diluent.

37. The pharmaceutical composition of claim 36 for use in a method of treatment.

38. The pharmaceutical composition of claim 37 for use in a method of killing a disease-associated CD45 expressing cell in a subject.

39. The pharmaceutical composition according to claim 37 or 38 for use in a method of treatment of a blood cancer, such as leukemia, lymphoma or multiple myeloma.

40. Pharmaceutical composition according to claim 37 or 38 for use in a method of treatment of an autoimmune disease, such as multiple sclerosis or scleroderma.

41. The pharmaceutical composition of any one of claims 37 or 38, wherein the method further comprises transferring cells into the subject after killing cells.

42. A method of killing a disease-associated CD45 expressing cell in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 36.

43. The method of claim 42 for treating a blood cancer, such as leukemia, lymphoma or multiple myeloma.

44. The method of claim 42 for treating an autoimmune disease, such as multiple sclerosis or scleroderma.

45. The method of any one of claims 42-44, wherein the method further comprises transferring cells into the subject after killing cells.

46. Use of one or more binding molecules of any one of claims 24-33, one or more nucleic acid molecules of claim 34, or one or more vectors of claim 35 in the manufacture of a medicament for killing a cell expressing CD45 associated with a disease in a subject.

47. The use according to claim 46 wherein the medicament is for the treatment of a blood cancer, such as leukemia, lymphoma or multiple myeloma.

48. The use according to claim 46, wherein the medicament is for the treatment of an autoimmune disease, such as multiple sclerosis or scleroderma.

49. The use of any one of claims 46-48, wherein the medicament is for use further comprising a method of transferring cells into the subject after killing cells.

50. A method of screening for one or more binding molecules capable of multimerizing CD45 to induce cell death, the method comprising:

(a) Contacting one or more binding molecules capable of binding CD45 with a target cell expressing CD45; and

(b) Determining whether the target cell undergoes cell death.

51. The method of claim 50, wherein the method further comprises:

(c) Determining whether there is cytokine release in the test sample, e.g., wherein the level of one or more of CCL2, GM-CSF, IL-1RA, IL-6, IL-8, IL-10, IL-11, and M-CSF is measured.

52. The method of claim 50 or 51, wherein:

(i) The one or more binding molecules have been identified as capable of multimerizing CD45; or (b)

(ii) The method comprises first screening for the ability to multimerize CD45 of a binding molecule specific for CD45, for example by screening for the ability to multimerize CD45 of an permutation of two or more different binding molecules.

53. An ex vivo method of depleting or killing a target cell expressing CD45 in a population, tissue or organ, the method comprising contacting the cell, tissue or organ with the antibody of any one of claims 1-7 or the binding molecule of any one of claims 24-33.

54. The antibody of any one of claims 1-7 or the binding molecule of any one of claims 24-33 for use in a method of treating or preventing Graft Versus Host Disease (GVHD) in a subject, the method comprising:

(a) Contacting a population, tissue or organ of cells ex vivo with the antibody of any one of claims 1-7 or the binding molecule of any one of claims 24-33 to kill target cells expressing CD 45; and

(b) Transplanting the treated cell population, tissue or organ into the subject.

55. A method of treating or preventing Graft Versus Host Disease (GVHD), comprising:

(b) Transplanting the treated cell population, tissue or organ into a subject in need of such transplantation.

56. Use of the antibody of any one of claims 1-7 or the binding molecule of any one of claims 24-33 in the manufacture of a medicament for treating or preventing Graft Versus Host Disease (GVHD) in a method comprising: