EA045291B1 - ANTIBODIES CONTAINING ONLY HEAVY CHAINS THAT BIND TO CD22 - Google Patents

Description

Cross reference to related applications

This application claims priority to U.S. Provisional Patent Application No. 62/609,759, filed December 22, 2017, the disclosure of which is incorporated herein by reference in its entirety.

Field of technology

This invention relates to human antibodies containing only heavy chains (eg, UniAbs™) that bind to CD22. The present invention also relates to methods for producing such antibodies, compositions, including pharmaceutical compositions, containing such antibodies, and their use for the treatment of B cell disorders that are characterized by CD22 expression.

State of the art

CD22.

CD22, also known as SIGLEC-2 (UniProt P20273), is a cell surface receptor that is expressed on mature B cells. CD22 contains multiple Ig domains and is a member of the immunoglobulin superfamily. The extracellular domain of CD22 interacts with sialic acid moieties, including those present on the cell surface protein CD45. CD22 is thought to function as an inhibitory receptor for B cell receptor signaling. Along with CD20 and CD19, the restricted B cell expression of CD22 makes it an attractive target for the therapeutic treatment of B cell malignancies. Monoclonal antibodies specific for CD22 have been described in the literature (e.g., Jabbour, Elias, et al. Monoclonal antibodies in acute lymphoblastic leukemia. Blood 125.26 (2015): 4010–4016) and have been used therapeutically as standard monoclonal antibody preparations (e.g. epratuzumab), and also as antibody-drug conjugates (inotuzumab ozogamicin). In addition, anti-CD22 chimeric antigen receptor T cells have been used in clinical practice to treat leukemia (Fry, Terry J., et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19targeted CAR immunotherapy Nature medicine (2017)

Antibodies containing heavy chains.

In a conventional IgG antibody, the association of the heavy chain and the light chain is due in part to the hydrophobic interaction between the light chain constant region and the CH1 constant domain of the heavy chain. There are additional residues in the heavy chain regions of framework 2 (FR2) and framework 4 (FR4) that also contribute to this hydrophobic interaction between the heavy and light chains.

It is known, however, that serum from camelids (suborder Tylopoda, which includes camels, dromedaries and llamas) contains a major type of antibody consisting exclusively of paired H-chains (heavy chain-only antibodies or UniAbs™). UniAbs™ Camelidae (Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanaco, Lama alpaca and Lama vicugna) have a unique structure consisting of one variable domain (VHH), a hinge region and two constant domains (CH2 and CH3), which are highly homologous to the domains CH2 and CH3 of classical antibodies. These UniAbs™ do not have the first constant region domain (CH1), which is present in the genome but is spliced during mRNA processing. The absence of the CH1 domain explains the absence of a light chain in UniAbs™, since this domain is the anchoring site for the light chain constant domain. Such UniAbs™ have naturally evolved to impart antigen-binding specificity and high affinity to three CDRs from conventional antibodies or fragments thereof (Muyldermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther 5:111-124 ). Cartilaginous fish such as sharks have also developed a special type of immunoglobulin, designated IgNAR, which lacks light polypeptide chains and is composed entirely of heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce a single heavy chain polypeptide variable domain (vNAR) (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187 -197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

The ability of antibodies containing only heavy chains, lacking a light chain, to bind antigen was established in the 1960s (Jaton et al. (1968) Biochemistry, 7, 4185-4195). The immunoglobulin heavy chain, physically separated from the light chain, retained 80% of the antigen-binding activity relative to the tetrameric antibody. Sitia et al. (1990) Cell, 60, 781-790 demonstrated that deletion of the CH1 domain from a rearranged mouse μ gene results in a heavy chain-only antibody lacking a light chain in mammalian cell culture. The antibodies produced retained VH binding specificity and effector functions.

Heavy chain-only antibodies with high specificity and affinity can be generated against various antigens through immunization (van der Linden, R.H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH portion can be easily cloned and expressed in yeast (Frenken, L. G. J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility and stability are significantly higher than those of the classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. FEBS Lett. 414, 521-526 (1997)).

Mice in which the λ (lambda) light (L) chain locus and/or the λ and k (kappa) L chain loci were functional

- 1045291 are suppressed and the antibodies produced by such mice are described in US Pat. Nos. 7,541,513 and 8,367,888. Recombinant production of heavy chain-only antibodies in mice and rats has been described, for example, in WO 2006008548; publication of US patent application No. 20100122358; Nguyen et al., 2003, Immunology; 109(1), 93-101; Bruggemann et al., Crit. Rev. Immunol; 2006, 26(5):377-90; and Zou et al., 2007, J Exp Med; 204(13): 3271-3283. Generation of knockout rats by embryonic microinjection of zinc finger nuclease is described in Geurts et al., 2009, Science, 325(5939):433. Soluble heavy chain-only antibodies and transgenic rodents containing a heterologous heavy chain locus producing such antibodies are described in US Pat. Nos. 8,883,150 and 9,365,655. CAR-T structures containing single-domain antibodies as the binding (targeting) domain are described, e.g. , in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630–2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378–386.

The essence of the invention

Aspects of the invention relate to heavy chain-only antibodies, including, without limitation, UniAbs™, with binding affinity for CD22. Additional aspects of the invention relate to methods for producing such antibodies, compositions containing such antibodies, and their use for the treatment of B cell disorders that are characterized by CD22 expression.

In some embodiments, a heavy chain only antibody that binds to CD22 comprises a heavy chain variable region comprising: (a) a CDR1 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NOs: 1-10, and/or ( b) CDR2 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NO: 11-17, and/or (c) CDR3 having two or less substitutions in any of the amino acid sequences of SEQ ID NO: 18-23. In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in the human backbone. In some embodiments, the heavy chain-only antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence.

In some embodiments, the heavy chain-only antibody comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-10, and/or (b) a CDR2 sequence selected from the group consisting of SEQ ID NO: 11-17, and/or (c) a CDR3 sequence selected from the group consisting of SEQ ID NO: 18-23. In some embodiments, the heavy chain-only antibody comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-10, and (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 11-17, and (c) a CDR3 sequence selected from the group consisting of SEQ ID NO: 18-23.

In some embodiments, the heavy chain-only antibody comprises: (a) a CDR1 sequence SEQ ID NO: 1, a CDR2 sequence SEQ ID NO: 11, and a CDR3 sequence SEQ ID NO: 18, or (b) a CDR1 sequence SEQ ID NO: 1 , CDR2 sequence SEQ ID NO: 12 and CDR3 sequence SEQ ID NO: 19, or (c) CDR1 sequence SEQ ID NO: 1, CDR2 sequence SEQ ID NO: 12 and CDR3 sequence SEQ ID NO: 20. In some embodiments, the antibody , containing only heavy chains, contains a heavy chain variable region having at least 95% sequence identity with any of the sequences of SEQ ID NO: 24-84. In some embodiments, the heavy chain only antibody comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 24-84. In some embodiments, the heavy chain only antibody comprises the heavy chain variable region sequence of SEQ ID NO: 24.

In some embodiments, a heavy chain only antibody that binds to CD22 comprises a heavy chain variable region comprising (a) a CDR1 sequence of the formula:

G X1 SI X ₂ X3 X4 X5 X6 Y, where X1 represents D or G; X ₂ represents S, T, I or N; X ₃ represents S or D; X4 represents G, S or N; X ₅ represents D, G or S, and X ₆ represents Y or H; and (b) a CDR2 sequence of the formula:

X7 X8 Y X9 G X10 X11, where X ₇ represents I or V; X ₈ represents Y or H; X ₉ represents S or T; X ₁₀ represents A, V or S, and X _p represents T or A; and (c) a CDR3 sequence of the formula:

X12 R X13 DSS X14 WRS, where X ₁₂ represents T, A or K; X ₁₃ represents D or E, and X _m represents N or S.

In some embodiments, a heavy chain-only antibody that binds to CD22 comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences in the human VH framework, wherein the CDR sequences are sequences having two or fewer substitutions in the CDR sequence, selected from the group consisting

- 2 045291 from SEQ ID NO: 1-23.

In some embodiments, the heavy chain only antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences in the human VH framework, wherein the CDR sequences are selected from the group consisting of SEQ ID NOs: 1-23.

In some embodiments, a heavy chain only antibody that binds to CD22 comprises a heavy chain variable region comprising: (a) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 11, and a CDR3 sequence of SEQ ID NO: 18, or (b) CDR1 sequence SEQ ID NO: 1, CDR2 sequence SEQ ID NO: 12 and CDR3 sequence SEQ ID NO: 19, or (c) CDR1 sequence SEQ ID NO: 1, CDR2 sequence SEQ ID NO: 12 and CDR3 sequence SEQ ID NO: 20 in human VH frame.

In some embodiments, the heavy chain-only antibody is multispecific. In some embodiments, the heavy chain-only antibody is bispecific. In some embodiments, the heavy chain-only antibody has binding affinity for two different CD22 proteins. In some embodiments, the heavy chain-only antibody has binding affinity to two different epitopes on the same CD22 protein. In some embodiments, the heavy chain-only antibody has binding affinity for the effector cell. In some embodiments, the heavy chain-only antibody has binding affinity for a T cell antigen. In some embodiments, the heavy chain-only antibody has binding affinity for CD3. In some embodiments, the heavy chain-only antibody is in CAR-T format.

Aspects of the invention relate to pharmaceutical compositions containing a heavy chain-only antibody as described herein.

Aspects of the invention relate to methods of treating a B cell disorder characterized by CD22 expression, comprising administering to a subject with the disorder an antibody or pharmaceutical composition as described herein. In certain other aspects, the invention relates to the use of an antibody described herein in the preparation of a medicament for the treatment of a B cell disorder characterized by CD22 expression. In still other aspects, the invention provides an antibody described herein for use in the treatment of a B cell disorder characterized by CD22 expression. In these aspects, in some embodiments, the disorder is diffuse large B cell lymphoma (DLBCL). In some embodiments, the disorder is non-Hodgkin's lymphoma (NHL). In some embodiments, the disorder is systemic lupus erythematosus (SLE). In some embodiments, the disorder is rheumatoid arthritis (RA). In some embodiments, the disorder is multiple sclerosis (MS).

Aspects of the invention relate to polynucleotides encoding an antibody described herein, vectors containing such polynucleotides, and cells containing such vectors.

Aspects of the invention relate to methods for producing an antibody described herein, including growing a cell described herein under conditions favorable for antibody expression and isolating the antibody from the cell.

Aspects of the invention relate to methods for producing antibodies described herein, including immunizing an animal with UniRat with CD22 and identifying CD22 heavy chain binding sequences.

These and other aspects will be further explained in the remainder of the disclosure, including examples.

Brief description of graphic materials

In fig. 1 shows the unique amino acid sequences of the heavy chain-only anti-CD22 antibody CDR.

In fig. 2 shows the amino acid sequences of the heavy chain-only variable domain of an anti-CD22 antibody.

In fig. 3 shows the amino acid sequences of CDR1, CDR2 and CDR3 of a heavy chain-only anti-CD22 antibody.

In fig. 4 shows the biological activity of an anti-CD22 antibody containing only heavy chains.

In fig. 5A is a graph depicting the percentage of specific lysis as a function of antibody concentration for Daudi cells.

In fig. 5B is a graph depicting the percentage of specific lysis as a function of antibody concentration for Raji cells.

In fig. 5C is a graph depicting the percentage of specific lysis as a function of antibody concentration for Ramos cells.

In fig. 5D is a schematic illustration of an anti-CD22xCD3 bispecific antibody in accordance with one embodiment of the invention.

- 3 045291

In fig. Figure 6 is a series of graphs showing serum titer as a function of dilution.

Detailed Description of Preferred Embodiments

In the practice of the present invention, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are known to specialists in the art, are used. Such methods are described in detail in the literature, for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, and periodic updates); PCR: The Polymerase Chain Reaction, (Mullis et al., ed., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

When a range of values is given, it is understood that this invention covers every intermediate value to the tenth decimal place of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of that range, as well as any other specified or intermediate value within such specified range. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in the present invention, subject to any specially excluded limit within the specified range. If the specified range includes one or both limits, ranges excluding one or both of these included limits are also included in this invention.

Unless otherwise noted, antibody residues herein are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) .

To provide a thorough understanding of the present invention, numerous specific details are set forth in the following detailed description. However, it will be obvious to one skilled in the art that the present invention can be practiced without one or more of the specified specific details. In other cases, well-known features and procedures well known to those skilled in the art have not been described to avoid making the invention difficult to understand.

All references contained herein, including patent applications and publications, are incorporated herein by reference in their entirety.

I. Definitions.

The term containing means that the listed elements are required for the composition/method/set, however, to form the composition/method/set, etc. Other elements may be included within the scope of the claims.

By the term consisting essentially of is meant the limitation of the described composition or method to specified materials or step-by-step actions that do not significantly affect the basic and novel characteristic(s) of the subject matter of the invention.

The term consisting of means the exclusion of any element, step or ingredient not specified in the claims from the composition, method or kit.

Antibody residues in this document are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is commonly used to designate a residue in the variable domain (approximately heavy chain residues 1-113) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ( 1991)). The EU numbering system or EU index is commonly used to designate a residue in the immunoglobulin heavy chain constant region (eg, EU index described in Kabat et al., supra). The EU index according to Kabat refers to the EU residue numbering of the human IgG1 antibody. Unless otherwise indicated, references herein to residue numbers in the antibody variable domain refer to residue numbering in accordance with the Kabat numbering system. Unless otherwise stated, references herein to residue numbers in the antibody constant domain refer to residue numbering in accordance with the EU numbering system.

The term monoclonal antibody as used herein refers to an antibody derived from a population of largely homogeneous antibodies, that is, the individual antibodies within the population are identical except for naturally occurring mutations that may be present in small quantities. Monoclonal antibodies are highly specific and directed against a single antigenic site. Additionally, unlike conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. Monoclonal antibodies in accordance with this invention can be obtained by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, can also be obtained, for example, by methods for producing recombinant protein (see, for example, US patent No. 4816567).

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The term variable, as used herein in connection with antibodies, refers to the fact that the sequences of certain portions of the variable domains of an antibody vary widely between antibodies and contribute to the binding and specificity of each particular antibody for its particular antigen. At the same time, variability is not evenly distributed across the variable domains of antibodies. It is concentrated in three segments of variable domains of both the light and heavy chains, called hypervariable regions. The more conserved portions of the variable domains are called framework regions (FR). The variable domains of the native light and heavy chains each contain four FRs, predominantly adopting a β-sheet configuration and connected by three hypervariable regions that form loops connecting β-type structures, and in some cases, being part of them. The hypervariable regions of each chain are held together in close proximity to the FR and, together with the hypervariable regions of the other chain, participate in the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Constant domains are not directly involved in the binding of antibodies to antigens, but exhibit various effector functions, for example, the participation of antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC).

The term hypervariable region as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region typically contains amino acid residues from the complementarity determining region or CDR (eg, residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)) and/or residues from hypervariable loop residues 26-32 (H1), 53-55 (H2) and 96101 (H3 ) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The framework region or FR residues, as defined herein, are hypervariable region residues other than variable domain residues.

Exemplary CDR designations are shown herein, however, one skilled in the art will appreciate that a number of CDR definitions are commonly used, including the Kabat definition (see Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions. Mol Immunol . 2010;47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of regions of a closed structure (Chothia et al. Conformations of immunoglobulin hypervariable regions. Nature. 1989; 342:877-883). Alternative CDR definitions of interest include, but are not limited to, those disclosed by Honegger, Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool. J Mol Biol. 2001;309:657-670; Ofran et al. Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes. J Immunol. 2008;181:6230-6235; Almagro Identification of differences in the specificitydetermining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires. J Mol Recognit. 2004;17:132-143; and Padlanet al. Identification of specificitydetermining residues in antibodies. Faseb J. 1995;9:133-139., the contents of each of which are incorporated herein in their entirety by reference.

The terms heavy chain only antibody and heavy chain antibody are used interchangeably herein and refer in the broadest sense to antibodies that lack the light chain of a conventional antibody. The terms specifically include, without limitation, homodimeric antibodies containing a VH antigen binding domain and CH2 and CH3 constant domains, in the absence of a CH1 domain; functional (antigen-binding) variants of such antibodies, soluble variants of VH, Ig-NAR containing a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NAR), and their functional fragments; and soluble single domain antibodies (sUniDabs™). In one embodiment, the heavy chain only antibody consists of a variable region antigen binding domain consisting of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In another embodiment, the heavy chain only antibody consists of from the antigen binding domain of at least part of the hinge region and the CH2 and CH3 domains. In another embodiment, the heavy chain-only antibody consists of an antigen binding domain of at least a portion of a hinge region and a CH2 domain. In a further embodiment, the heavy chain-only antibody consists of an antigen binding domain, at least a portion of a hinge region, and a CH3 domain. Antibodies containing only heavy chains in which the CH2 and/or CH3 domain is truncated are also included herein. In a further embodiment, the heavy chain consists of an antigen binding domain and at least one CH domain (CH1, CH2, CH3 or CH4), but without a hinge region. The heavy chain-only antibody may be in the form of a dimer in which the two heavy chains are disulfide-linked to each other, covalently linked, or non-covalently linked to each other. An antibody containing only heavy chains may belong to the IgG subclass, but also in this document

- 5 045291 includes antibodies belonging to other subclasses, such as the IgM, IgA, IgD and IgE subclass. In a specific embodiment, the heavy chain-only antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype. In one embodiment, heavy chain-only antibodies are used herein as the binding (targeting) domain of a chimeric antigen receptor (CAR). The definition specifically includes heavy chain-only human antibodies called UniAbs™ produced by human immunoglobulin transgenic rats (UniRat™). The variable regions (VH) of UniAbs™ are called UniDabs™, and are versatile building blocks that can be coupled to Fc regions or serum albumin to develop new therapeutics with multispecificity, increased potency and extended half-life. Since homodimeric UniAbs™ lack a light chain and therefore a VL domain, the antigen is recognized by a single domain, that is, the heavy chain variable domain of a heavy chain-only (VH) antibody.

The terms CD22 and cluster of differentiation-22 as used herein refer to the SIGLEC family of lectin molecules found on the surface of mature B cells and to a lesser extent on some immature B cells. The term CD22 includes the CD22 protein of humans and any non-human animal species, and particularly includes human CD22 as well as non-human mammalian CD22.

As used herein, the term human CD22 includes any variants, isoforms and species homologs of human CD22 (UniProt P20273), regardless of their source or method of preparation. Thus, human CD22 includes human CD22 naturally expressed by cells and CD22 expressed in cells transfected with the human CD22 gene.

The terms anti-CD22 heavy-chain-only antibody, anti-CD22 heavy-chain-only antibody, anti-CD22 heavy-chain-only antibody, and anti-CD22 heavy-chain-only antibody are used interchangeably herein to refer to heavy chain-only antibody. , as defined above, for immunospecific binding to CD22, including human CD22, as defined above. The definition includes, without limitation, human heavy chain-only antibodies produced by transgenic animals, such as transgenic rats or transgenic mice expressing human immunoglobulin, including UniRats™ producing UniAb™ anti-human CD22 antibodies as defined above.

Percentage (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after sequence alignment and the introduction of gaps where necessary to achieve the maximum percentage of sequence identity, and excluding any conservative substitutions of part of the sequence identity. Alignment to determine percent amino acid sequence identity can be performed in a variety of ways known to one skilled in the art, for example, using publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For the purposes of this document, however, % amino acid sequence identity values are obtained using the sequence comparison program ALIGN-2.

An isolated antibody is one that has been identified and separated and/or extracted from a component of its natural environment. Contaminants in the natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of the antibody, as determined by the Lowry method, and most preferably greater than 99% by weight, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spin-beaker sequencer, or (3) to homogeneity (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or, preferably, a silver-based dye. The isolated antibody includes the antibody in situ in the recombinant cells, since at least one component of the antibody's natural environment will be absent. Typically, however, the isolated antibody will be obtained through at least one purification step.

Antibodies of the present invention include multispecific antibodies. Multispecific antibodies have more than one binding specificity. The term multispecific specifically includes bispecific and trispecific, as well as higher order independent specific binding affinity, such as higher order polyepitope specificity, as well as tetravalent antibodies and antibody fragments. Multispecific antibodies include, but are not limited to:

- 6 045291 antibodies containing a combination of different binding entities, as well as antibodies containing more than the same binding entity. The terms multispecific antibody, multispecific heavy chain only antibody, multispecific heavy chain antibody, and multispecific UniAb™ are used herein in the broadest sense to cover all antibodies with more than one binding specificity. The multispecific heavy chain anti-CD22 antibodies of the present invention particularly include antibodies that immunospecifically bind to more than one non-overlapping epitope on a CD22 protein, such as human CD22.

An epitope is a region on the surface of an antigen molecule to which one antibody molecule binds. Typically an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes.

Epitope mapping is the process of identifying antibody binding sites or epitopes on their target antigens. Antibody epitopes can be linear or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed from amino acids that are discontinuous in the sequence of a protein, but which come together when the protein folds into its three-dimensional structure.

The term polyepitope specificity refers to the ability to specifically bind to two or more different epitopes on the same or different targets. As noted above, the present invention particularly includes heavy chain-only anti-CD22 antibodies with polyepitope specificity, that is, heavy chain-only anti-CD22 antibodies that bind to two or more non-overlapping epitopes on a CD22 protein, such as human CD22 . The term non-overlapping epitope(s) or non-competitive epitope(s) of an antigen is defined herein to mean epitope(s) that are recognized by one member of an antigen-specific antibody pair but not by the other member. Pairs of antibodies or antigen-binding regions targeting the same antigen on a multispecific antibody that recognize non-overlapping epitopes do not compete for binding to that antigen and are capable of binding that antigen simultaneously.

An antibody binds substantially the same epitope as a reference antibody when the two antibodies recognize identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competitive binding assays, which can be configured in a number of different formats using either a labeled antigen or a labeled antibody. Typically, the antigen is immobilized in a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzyme labels.

The term valence, as used herein, refers to the specified number of binding sites in an antibody molecule.

A polyvalent antibody has two or more binding sites. Thus, the terms divalent, trivalent and tetravalent refer to the presence of two binding sites, three binding sites and four binding sites, respectively. Thus, the bispecific antibody of the invention is at least bivalent and may be trivalent, tetravalent, or otherwise multivalent.

A wide variety of methods and protein configurations are known and are used to produce bispecific monoclonal antibodies (BsMAB), trispecific antibodies and the like.

The term bispecific three-chain antibody-like molecule or TCA is used herein to refer to antibody-like molecules comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which contain, consist essentially of, or consist of one heavy and one light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains containing an antigen-binding region and at least one CH domain. This heavy chain/light chain pair has binding specificity for the first antigen. The third polypeptide subunit contains, consists essentially of, or consists of a heavy chain-only antibody containing an Fc region containing CH2 and/or CH3 and/or CH4 domains, in the absence of a CH1 domain, and an antigen binding domain that binds an epitope of a second antigen or another epitope of the first antigen, wherein such binding domain is derived from or has sequence identity with the variable region of the heavy or light chain of the antibody. Portions of such a variable region may be encoded by VH and/or _VL gene segments, D and JH gene segments, or _JL gene segments. The variable region may be encoded by rearranged VHDJH, VLDJH, VHJ _L or V _L J _L gene segments. The TCA protein uses a heavy chain-only antibody as defined above.

The term chimeric antigen receptor or CAR is used herein in its broadest sense to refer to an engineered receptor that imparts a desired binding specificity (e.g., the antigen binding region of a monoclonal antibody or other

- 7 045291 ligand) to the membrane-spanning and intracellular signaling domains. Typically, the receptor is used to graft the specificity of a monoclonal antibody onto a T cell to create chimeric antigen receptors (CARs). (J Natl Cancer Inst, 2015; 108(7):dvj439; and Jackson et al., Nature Reviews Clinical Oncology, 2016; 13:370-383).

The term human antibody is used herein to include antibodies that have variable and constant regions derived from human germline immunoglobulin sequences. As used herein, human antibodies may include amino acid residues not encoded by germline human immunoglobulin sequences, such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. The term human antibody specifically includes heavy chain-only antibodies having human heavy chain variable region sequences produced by transgenic animals such as transgenic rats or mice, particularly UniAbs™ produced by UniRats™ as defined above.

By chimeric antibody or chimeric immunoglobulin is meant an immunoglobulin molecule containing amino acid sequences from at least two different Ig loci, for example, a transgenic antibody containing a portion encoded by the human Ig locus and a portion encoded by the rat Ig locus. Chimeric antibodies include transgenic antibodies with non-human Fc regions or artificial Fc regions, and human idiotypes. Such immunoglobulins can be isolated from animals of the invention that have been engineered to produce such chimeric antibodies.

As used herein, the term effector cell refers to an immune cell that participates in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, an effector cell, such as a natural killer cell, is capable of inducing antibody-dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages that express FcRs are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system or bind to cells that present antigens. In some embodiments, the effector cell may phagocytose the target antigen or target cell.

Human effector cells are white blood cells that express receptors, such as T cell receptors or FcRs, and exert effector functions. Preferably, such cells express at least FcyRIII and exert ADCC effector function. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with NK cells being preferred. Effector cells can be isolated from a native source, such as blood or PBMC, as described herein.

The term immune cell is used herein in its broadest sense, including, without limitation, cells of myeloid or lymphoid origin, such as lymphocytes (such as B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells such as neutrophils, granulocytes, mast cells and basophils.

The effector functions of an antibody refer to those biological activities that are attributable to the Fc region (native sequence Fc region or variant amino acid sequence Fc region) of the antibody. Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; suppression of cell surface receptors. (eg B cell receptor, BCR), etc.

Antibody-dependent cell-mediated cytotoxicity and ADCC correspond to a cell-mediated response in which nonspecific cytotoxic cells that express Fc receptors (FcR) (eg, natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on the target cell and thus thus lead to lysis of the target cell. The primary cells that mediate ADCC, natural killer cells, express only FcyRIII, whereas monocytes express FcyRI, FcyRII, and FcyRIII. Data on FcR expression on hematopoietic cells are summarized in Table. 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, as described in US Pat. No. 5,500,362 or 5,821,337. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or in addition, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model, for example, as described in Clynes et al. PNAS (USA) 95:652–656 (1998).

Complement dependent cytotoxicity or CDC refers to the ability of a molecule to lyse

- 8 045291 target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (eg, antibody) that forms a complex with the antigen being recognized. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

Binding affinity refers to the strength of the total number of noncovalent interactions between one binding site of a molecule (eg, antibody) and its binding partner (eg, antigen). Unless otherwise specified as used herein, binding affinity refers to self-binding affinity, which reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art. Low-affinity antibodies typically bind antigen slowly and tend to dissociate easily, whereas high-affinity antibodies typically bind antigen more quickly and tend to remain bound.

As used herein, the term Kd or Kd value refers to the dissociation constant determined by BioLayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, Calif.) in kinetics mode. For example, anti-mouse Fc sensors are loaded with a mouse Fc antigen fusion and then dipped into wells containing antibodies to measure concentration-dependent rates (kon). Antibody dissociation rates (koff) are measured in the final step, when the probes are immersed in wells containing only buffer. Kd is the koff/kon ratio. (For more information, see Conception, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

The terms treatment, treat, and the like are used herein to denote the achievement of a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms and/or may be therapeutic in terms of partially or completely curing a disease and/or an adverse event associated with that disease. As used herein, the term treatment covers any treatment of a disease in a mammal, and includes:

(a) preventing the onset of a disease in a subject who may be predisposed to the disease but in whom the disease has not yet been diagnosed;

(b) inhibition of the disease, i.e. cessation of its development; or (c) alleviation of the disease, i.e. regression of the disease.

The therapeutic agent may be administered before, during, or after the onset of the disease or injury. Treatment of ongoing disease in which the treatment stabilizes or reduces the patient's undesirable clinical symptoms is of particular interest. It is advisable to carry out such treatment until complete loss of function in the affected tissues. Therapy may be administered to a subject during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

The term therapeutically effective amount means the amount of active agent that is necessary to impart a therapeutic effect in a subject. For example, a therapeutically effective amount is an amount that causes, attenuates, or otherwise causes an improvement in pathological symptoms, disease progression, or physiological conditions associated with a disease, or that increases resistance to the disorder.

The terms B-cell neoplasms or mature B-cell neoplasms as used herein include small cell lymphocytic lymphoma, B-cell prolymphocytic lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma ( DLBCL), multiple myeloma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell neoplasms such as plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, MALT lymphoma, nodal marginal zone cell lymphoma, intravascular B- large cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, non-Hodgkin's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, primary effusion lymphoma and AIDS-associated non-Hodgkin's lymphoma.

The terms subject, individual, and patient are used interchangeably herein to refer to a mammal being evaluated for treatment and/or undergoing treatment. In one embodiment, the mammal is human. The terms subject, individual and patient include, without limitation, individuals with cancer, individuals with autoimmune diseases, pathogenic infections and the like. Subjects may be humans, but may also be other mammals, particularly those mammals that can be used as laboratory models of human disease, such as mice, rats, etc.

The term pharmaceutical composition refers to a composition that is in a form that allows the biological activity of the active ingredient to be effective and that does not

- 9 045291 contains additional components that are unacceptably toxic to the subject to whom the composition will be administered. Such compositions are sterile. Pharmaceutically acceptable excipients (carriers, adjuvants) are those that can reasonably be administered to a mammalian subject and provide an effective dose of the active ingredient used.

The sterile composition is aseptic or free from, or substantially free from, any living microorganisms and their spores. A frozen composition is a composition at a temperature below 0°C.

A stable composition is one in which the protein retains substantially all of its physical stability and/or chemical stability and/or biological activity during storage. Preferably, the composition retains substantially all of its physical or chemical stability as well as its biological activity. The storage period is generally selected based on the shelf life of the composition. A variety of techniques for measuring protein stability are known in the art and are discussed in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones. A. Adv. Drug Delivery Rev. 10: 29-90) (1993), for example. Stability can be measured at a selected temperature for a selected period of time. Stability can be assessed qualitatively and/or quantitatively in a variety of ways, including assessing the formation of aggregates (eg, using size exclusion chromatography, by measuring turbidity and/or visual inspection); by assessing charge heterogeneity using cation exchange chromatography, isoelectric image focusing (icIEF), or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map analysis (eg tryptic or LYS-C); assessing the biological activity or antigen-binding function of the antibody; and so on. Instabilities may include one or more of: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), excision/hydrolysis/fragmentation (e.g., hinge region fragmentation), formation succinimide, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.

II. Detailed description of the invention

Antibodies to CD22.

This invention relates to a family of closely related heavy chain-only antibodies that bind to human CD22. Antibodies of this family contain CDR sequences as defined herein and shown in FIG. 1 and are illustrated by the reported heavy chain variable region (VH) sequences of SEQ ID NO: 24-84 shown in FIG. 2. Antibody families provide a number of advantages that favor their use as clinical therapeutic agent(s). Antibodies include members with a range of binding affinities allowing the selection of a particular sequence with the desired binding affinity.

A suitable antibody may be selected from those presented herein for development and therapeutic or other use, including, without limitation, use as a bispecific antibody, such as that shown in FIG. 5B, or a trispecific antibody or part of a CAR-T structure.

Determination of affinity for a candidate protein can be performed using methods known in the art, such as Biacore measurements. Members of the antibody family may have an affinity for CD22 with a Kd of about 10 ^-6 to about 10 ^-11 , including but not limited to about 10 ⁶ to about 10 ¹⁰ ; from about 10 ⁶ to about 10 ⁹ ; from about 10 ^-6 to about 10 ^-8 ; from about 10 ^-8 to about 10'¹¹; from about 10 ^-8 to about 10 ^-10 ; from about 10 ^-8 to about 10 ^-9 ; from about 10 ^-9 to about 10 ^-11 ; from about 10 ^-9 to about 10 ^-10 ; or any value in these ranges. Affinity selection can be supported by biological assessment for modulation, such as blocking, CD22 biological activity, including in vitro assays, preclinical models and clinical trials, and assessment of potential toxicity.

Members of the antibody family described herein are not cross-reactive with Cynomolgus CD22 protein, but can be engineered to be cross-reactive with Cynomolgus CD22 protein or CD22 proteins of other species, if desired.

The family of CD22-specific antibodies herein includes the VH domain containing the CDR1, CDR2 and CDR3 sequences in the human VH framework. The CDR sequences may be located, for example, in the region around amino acid residues 26-35; 53-59; and 98-117, for CDR1, CDR2 and CDR3, respectively, of the provided exemplary variable region sequences set forth in SEQ ID NOs: 24-84. One skilled in the art will appreciate that the CDR sequences may be in other positions if a different framework sequence is selected, although generally the order of the sequences remains the same.

The CDR1, CDR2 and CDR3 sequences of the anti-CD22 antibodies of the present invention may be covered by the following structural formulas, where X denotes a variable amino acid that

- 10 045291 may be specific amino acids, as indicated below.

CDR1.

G X1 SI X ₂ X ₃ X ₄ X ₅ X ₆ Y, where X1 represents D or G;

X ₂ represents S, T, I or N;

X ₃ represents S or D;

X ₄ represents G, S, or N;

X ₅ represents D, G or S; A

X ₆ represents Y or H.

CDR2.

X7 X8 Y X9 G X10 X11, where X ₇ represents I or V;

X ₈ represents Y or H;

X ₉ represents S or T;

X ₁₀ represents A, V or S; A

X11 represents T or A.

CDR3.

X ₁₂ RX ₁₃ DSSX ₁₄ WRS, where X ₁₂ represents T, A or K;

X ₁₃ represents D or E; A

X14 represents N or S.

Exemplary CDR1, CDR2, and CDR3 sequences are shown in FIG. 1 and 3.

In some embodiments, the heavy chain-only anti-CD22 antibody of the present invention comprises a CDR1 sequence of any of SEQ ID NO: 1-10. In a specific embodiment, the CDR1 sequence is SEQ ID NO: 1.

In some embodiments, the heavy chain-only anti-CD22 antibody of the present invention comprises a CDR2 sequence of any of SEQ ID NO: 11-17. In a specific embodiment, the CDR2 sequence is SEQ ID NO: 11.

In some embodiments, the heavy chain-only anti-CD22 antibody of the present invention comprises a CDR3 sequence of any of SEQ ID NO: 18-23. In a specific embodiment, the CDR2 sequence is SEQ ID NO: 18.

In a further embodiment, the heavy chain-only anti-CD22 antibody of the present invention comprises the sequence CDR1 SEQ ID NO: 1; CDR2 sequence SEQ ID NO: 11 and CDR3 sequence SEQ ID NO: 18.

In further embodiments, the heavy chain-only anti-CD22 antibody of the present invention comprises any of the heavy chain variable region amino acid sequences of SEQ ID NO: 24 to 84 (FIG. 2).

In another further embodiment, the heavy chain only anti-CD22 antibody of the present invention comprises the heavy chain variable region of SEQ ID NO: 24.

In some embodiments, the CDR sequence in the heavy chain-only anti-CD22 antibody of the present invention contains one or two amino acid substitutions relative to the CDR1, CDR2 and/or CDR3 sequence or the set of CDR1, CDR2 and CDR3 sequences in any of SEQ ID NO: 1-23 (Fig. 1). In some embodiments, said amino acid substitution(s) are one or two amino acid positions 4-6 of CDR1 and/or one or two amino acid positions 2, 4-7 of CDR2, and/or one or two amino acid positions 5-12 of CDR3, relative to formulas proposed above. In some embodiments, heavy chain-only anti-CD22 antibodies will comprise a heavy chain variable region sequence with at least about 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any of the heavy chain variable region sequences of SEQ ID NO: 24-84 (shown in FIG. 2).

In some embodiments, bispecific or multispecific antibodies are provided, which may have any of the configurations discussed herein, including, without limitation, a bispecific triple-chain antibody-like molecule. In some embodiments, a bispecific antibody may comprise at least one heavy chain variable region having binding specificity for CD22 and at least one heavy chain variable region having binding specificity for a protein other than CD22. In some embodiments, a bispecific antibody may comprise a heavy chain/light chain pair that has binding specificity for a first antigen, and a heavy chain from a heavy chain-only antibody comprising an Fc portion containing CH2 and/or CH3 and/or CH4 domains, in the absence of a CH1 domain, and an antigen binding domain that binds an epitope of the second antigen or another epitope of the first antigen. In one specific embodiment, a bispecific

- 11 045291 a new antibody contains a heavy chain/light chain pair that has binding specificity for an antigen on an effector cell (for example, the CD3 protein on a T cell), and the heavy chain of a heavy chain-only antibody contains an antigen binding domain that has specificity binding for CD22.

In some embodiments, when the protein of the invention is a bispecific antibody, one arm of the antibody (one binding fragment) is specific for human CD22, while the other fragment may be specific for target cells, tumor-associated antigens, target antigens, for example, integrins, etc., pathogen antigens, checkpoint proteins and the like. Target cells specifically include cancer cells, including, without limitation, cells from a hematologic tumor, such as B cell tumors, as described above.

Various formats of bispecific antibodies are within the scope of the invention, including, without limitation, single chain polypeptides, double chain polypeptides, triple chain polypeptides, quadruple chain polypeptides, and polypeptides thereof. As used herein, bispecific antibodies specifically include T cell bispecific antibodies that bind to CD22, which is selectively expressed on mature B cells, and CD3 (anti-CD22xCD3 antibodies). Such antibodies induce potent T cell-mediated killing of CD22-expressing cells.

Preparation of anti-CD22 antibodies containing only heavy chains.

The heavy chain-only antibodies of this invention can be prepared using methods known in the art. In a preferred embodiment, the heavy chain only antibodies in this case are produced by transgenic animals, including transgenic mice and rats, preferably rats in which endogenous immunoglobulin genes are knocked out or disabled. In a preferred embodiment, the heavy chain-only antibodies of the present invention are produced in UniRat™. UniRat™ have silent endogenous immunoglobulin genes and utilize the human immunoglobulin heavy chain translocus to express a diverse, naturally optimized repertoire of fully human HCAbs. While endogenous immunoglobulin loci in rats can be knocked out or silenced using various technologies, in UniRat™ zinc finger (endo)nuclease (ZNF) technology was used to inactivate the endogenous rat heavy chain J locus, light chain K locus and Cλ locus light chain. ZNF constructs for microinjection into oocytes can produce IgH and IgL knockout (KO) lines. For more details see, for example, Geurts et al., 2009, Science 325:433. Characterization of Ig heavy chain knockout rats has been reported by Menoret et al., 2010, Eur. J. Immunol. 40:2932–2941. The advantages of ZNF technology are that non-homologous end joining to silence a gene or locus through deletions up to several kb in size. may also provide a target site for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). The human heavy chain antibodies produced in UniRat™ are called UniAbs™ and can bind epitopes that cannot be attacked by conventional antibodies. Their high specificity, affinity and small size make them ideal for mono- and polyspecific applications.

In addition to UniAbs™, specifically included herein are antibodies containing only heavy chains free of camel backbone and VHH mutations and their functional VH regions. Such heavy chain only antibodies can, for example, be produced in transgenic rats or mice that contain fully human heavy chain only gene loci, as described, for example, in WO 2006/008548, but other transgenic mammals can also be used. such as rabbit, guinea pig, rat, rats and mice are preferred. Antibodies containing only heavy chains, including functional VHH or VH fragments thereof, can also be produced using recombinant DNA technology by expressing the encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including, for example, mammalian cells (eg, CHO cells), intestinal stick or yeast.

Antibody domains containing only heavy chains combine the advantages of antibodies and small-molecule drugs: they can be mono- or multivalent; have low toxicity; and are economical to produce. Due to their small size, these domains are easy to administer, including oral or topical administration, and are characterized by high stability, including gastrointestinal stability; and their half-life can be tailored to the desired use or indication. In addition, the VH and VHH domains of HCAbs can be produced in a cost-effective manner.

In a specific embodiment, the heavy chain-only antibodies of the present invention, including UniAbs™, have a native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Kabat numbering system) replaced by another amino acid residue that is capable of disrupting a surface-exposed hydrophobic patch , containing or linked to a native amino acid residue at this position. Such hydrophobic

- 12 045291 patches are usually hidden at the interface with the constant region of the antibody light chain, but become exposed to the surface in the HCAb and, at least in part, to unwanted aggregation and association of the HCAb light chain. The substituted amino acid residue is preferably charged and more preferably is positively charged, such as lysine (Lys, K), arginine (Arg, R) or histidine (His, H), preferably arginine (R). In a preferred embodiment, heavy chain-only antibodies obtained from transgenic animals contain a Trp-Arg mutation at position 101. The resulting HCAbs preferably have high antigen-binding affinity and solubility under physiological conditions in the absence of aggregation.

The present invention has identified human heavy chain-only anti-CD22 IgG antibodies with unique sequences from animal UniRat™ (UniAb™) that bind human CD22 in a protein-cell binding ELISA (recombinant extracellular domain of CD22). The identified heavy chain variable region (VH) sequences (see FIG. 2) are positive for binding to human CD22 protein and/or for binding to CD22+ cells, and all are negative for binding to cells that do not express CD22.

The antibodies described herein bind CD22-positive Burkitt lymphoma in the Daudi cell line (ATCC® CCL-213™), and some cross-react with cynomolgus CD22 protein. In addition, they can be designed to be cross-reactive with the CD22 protein of any animal species if desired.

Heavy chain-only anti-CD22 antibodies, such as UniAbs™, may have an affinity for CD22 with a Kd of about 10 ^-6 to about 10 ^-11 , including but not limited to about 10 ^-6 to about 10 ^-10 ; from about 10 ^-6 to about 10 ^-9 ; from about 10 ^-6 to about 10 ^-8 ; from about 10 ^-8 to about 10 ^-11 ; from about 10 ^-8 to about 10 ^-10 ; from about 10 ^-8 to about 10 ^-9 ; from about 10 ^-9 to about 10 ^-11 ; from about 10 ^-9 to about 10 ^-10 ; or any value in these ranges. Affinity selection can be supported by biological assessment for modulation, such as blocking, CD22 biological activity, including in vitro assays, preclinical models and clinical trials, and assessment of potential toxicity.

Heavy chain-only antibodies that bind to non-overlapping epitopes on the CD22 protein, such as UniAbs™, can be identified using competitive binding assays such as enzyme-linked immunosorbent assays (ELISAs) or competitive binding assays using flow cytometry, e.g. known antibodies that bind to the target antigen and the antibody of interest. Using this approach, it is possible to separate a set of antibodies into those that compete with the reference antibody and those that do not. Noncompetitive antibodies are identified as binding to a distinct epitope that does not overlap with the epitope bound to the reference antibody. Often one antibody is immobilized, the antigen is bound, and a second labeled (eg, biotinylated) antibody is tested in an ELISA for its ability to bind the captured antigen. It can also be performed using surface plasmon resonance (SPR) platforms, including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and MX96 SPR tomograph (Ibis technologies BV), as well as biolayer interferometry platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). For further details, see the examples in this document.

Typically, an antibody competes with a reference antibody if it causes a reduction in binding of the reference antibody to the target antigen by about 15-100%, as determined by standard methods such as the competitive binding assays described above. In various embodiments, the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least at least about 80%, at least about 85%, at least about 90%, at least about 95% or higher.

Pharmaceutical compositions, uses and methods of treatment.

Another aspect of the present invention is to provide pharmaceutical compositions containing one or more antibodies of the present invention in admixture with a suitable pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers used herein include, for example, but are not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art for storing therapeutic components or combinations thereof.

In one embodiment, the pharmaceutical composition contains a heavy chain-only antibody (eg, UniAb™) that binds to CD22. In another embodiment, the pharmaceutical composition contains a multispecific (including bispecific) heavy chain-only antibody (eg, UniAb™) with binding specificity for two or more non-overlapping epitopes on the CD22 protein. In a preferred embodiment, the pharmaceutical composition contains a multispecific (including bispecific) antibody, containing

- 13 045291 only heavy chains, (eg UniAb™) with binding specificity to CD22 and with binding specificity to a binding target on the effector cell (eg a binding target on a T cell, such as, for example, the CD3 protein on a T cell ).

The pharmaceutical composition of antibodies used in accordance with the present invention is prepared for storage by mixing proteins having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (see, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. ( 1980)), for example, in the form of lyophilized compositions or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. The pharmaceutical compositions may be provided in unit dosage form (eg, single administration dosage form). The appropriate dosage form depends on the route of administration chosen. The antibodies described herein can be administered by intravenous injection or infusion, or subcutaneously. For injection, the antibodies described herein can be formulated in aqueous solutions, preferably in physiologically compatible buffers to reduce discomfort at the injection site. The solution may contain carriers, auxiliaries or stabilizers as described above. Alternatively, the antibodies can be lyophilized for mixing with a suitable vehicle, such as sterile pyrogen-free water, before use.

Antibody formulations are disclosed, for example, in US Pat. No. 9,034,324. Similar formulations may be used for heavy chain-only antibodies, including UniAbs™, of the present invention. Antibody compositions for subcutaneous administration are described, for example, in US patents 20160355591 and US 20160166689.

Methods of application.

The heavy chain-only anti-CD22 antibodies, multispecific antibodies and pharmaceutical compositions described herein can be used to treat diseases and conditions characterized by CD22 expression, including, without limitation, the conditions and diseases described later herein. Aspects of the present invention also relate to the use of an antibody described herein in the preparation of a medicament for the treatment of a B-cell disorder characterized by CD22 expression. Aspects of the present invention also relate to the antibody described herein for use in the treatment of a B cell disorder characterized by CD22 expression.

CD22 is a 135-kDa type I transmembrane protein that is expressed at low levels in pre- and immature B cells, maximally in mature B cells, and ultimately downregulated in plasma cells. (E.g. Walker et al., Immunology, 2008 Mar; 123(3) 314-25). CD22 is highly expressed in follicular (primary and secondary zone B cells), mantle and marginal zone B cells and is reported to be present in 60–80% of samples from patients with B cell malignancies (Alderson et al., Clin Cancer Res 2009;15(3) February 11, 2009). Because of its observed expression in a number of hematologic malignancies, CD22 is a promising target for antibody-based therapy.

In one aspect, heavy chain-only anti-CD22 antibodies (e.g., UniAbs™) and pharmaceutical compositions of this invention can be used to treat hematologic malignancies characterized by CD22 expression, including, but not limited to, diffuse large B-cell lymphoma (DLBCL). , non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia (CLL) and B-cell acute lymphoblastic leukemia (ALL).

Diffuse large B-cell lymphoma (DLBCL or DLBCL) is the most common form of non-Hodgkin's lymphoma in adults (Blood 1997 89(11):3909–18), with an estimated annual incidence of 7 to 8 cases per 100,000 people per year in the US and UK. She's a character

- 14 045291 is called an aggressive cancer that can occur in almost any part of the body. The causes of DLBCL are not entirely understood, and it can be caused by normal B cells as well as malignant transformation of other types of lymphoma or leukemia cells. Treatment approaches typically include chemotherapy and radiation, and the resulting overall five-year survival rate averages about 58% for adults. Although some monoclonal antibodies have shown promise in the treatment of DLBCL, consistent clinical efficacy has not yet been conclusively demonstrated. Therefore, there is a great need for new treatments, including immunotherapy, for the treatment of DLBCL.

In another aspect, heavy chain-only anti-CD22 antibodies (e.g., UniAbs™) and pharmaceutical compositions of this invention can be used to treat autoimmune disorders characterized by pathogenic B cells that express CD22, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and multiple sclerosis (MS).

Effective dosages of the compositions of this invention for treating a disease vary depending on many different factors, including routes of administration, destination, physiological condition of the patient, whether the patient is human or animal, whether other drugs are administered, and whether the treatment is prophylactic or therapeutic. Typically the patient is a human, but non-human mammals may also be treated, such as domestic animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc. , etc. Drug dosages can be adjusted to optimize safety and effectiveness.

Dosage levels can be readily determined by a person of ordinary skill in the art and can be adjusted as necessary, for example, to change the subject's response to therapy. The amount of active ingredient that can be combined with carrier materials to form a unit dosage form varies depending on the host being treated and the particular route of administration. Unit dosage forms typically contain from about 1 to about 500 mg of active ingredient.

In some embodiments, the therapeutic dosage of the agent may be from about 0.0001 to 100 mg/kg, and more typically from 0.01 to 5 mg/kg, based on the body weight of the host. For example, the dosage may be 1 mg kg body weight or 10 mg/kg body weight or in the range of 1-10 mg/kg. An exemplary treatment regimen includes administration once every two weeks or once a month or once every 3 to 6 months. The therapeutic agents of the present invention are typically administered multiple times. The intervals between single doses may be weekly, monthly or yearly. The intervals may also be irregular, as indicated by measuring the level of the therapeutic substance in the patient's blood. Alternatively, the therapeutic agents of the present invention may be administered as a sustained release composition, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.

Typically, the compositions are prepared as injectable preparations, either as liquid solutions or suspensions; Solid forms suitable for dissolution or suspension in liquid vehicles prior to injection may also be prepared. The pharmaceutical compositions described herein are suitable for intravenous or subcutaneous administration, directly or after reconstitution from a solid (ie, lyophilized) composition. The drug may also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide or copolymer to enhance the adjuvant effect as described above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The substances of this invention can be administered in the form of a depot injection or implant preparation, which can be formulated to provide prolonged or pulsatile release of the active substance. Pharmaceutical compositions are typically formulated to be sterile, substantially isotonic, and fully compliant with all US Food and Drug Administration (GMP) regulations.

The toxicity of the antibodies and antibody structures described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD ₅₀ (dose lethal to 50% of the population) or LD ₁₀₀ (dose lethal to 100% of the population ).

The dose ratio between toxic and therapeutic effects is the therapeutic indicator. Data obtained from these cell culture assays and animal studies can be used to develop dosage ranges that are not toxic for use in humans. The dosage of the antibodies described herein is preferably within a range of circulating concentrations that include an effective dose with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. The exact composition, route of administration and dosage can be selected

- 15 045291 us individually by a doctor, taking into account the patient’s condition.

Compositions for administration typically contain an antibody or other ablative agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous vehicle. Various aqueous vehicles can be used, such as buffered saline and the like. These solutions are sterile and generally free of unwanted substances. These compositions can be sterilized by conventional, well known sterilization methods. The compositions may contain pharmaceutically acceptable excipients necessary to approximate physiological conditions, such as pH adjusting agents and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like . The concentration of active agent in these formulations can vary widely and will be selected based primarily on fluid volumes, viscosity, body weight, and the like, in accordance with the particular route of administration chosen and the needs of the patient (eg, Remington's Pharmaceutical Science (15th ed., 1980) ) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

Also included within the scope of the invention are kits containing active agents and their compositions, inventions and instructions for use. The kit may further contain at least one additional component, for example, a chemotherapy drug, etc. Kits typically include a label indicating the intended use of the contents of the kit. The term label as used herein includes any written or recorded materials supplied in or with the kit or otherwise accompanying the kit.

Now that the invention has been fully described, it will be apparent to one skilled in the art that various changes and modifications may be made without departing from the spirit or scope of the invention.

Examples

Materials and methods.

CD22 protein binding.

Kinetic binding experiments to determine antigen-antibody affinity were performed on an Octet QK-384 system (ForteBio) using dual-layer interferometry. Anti-Human IgG Fc Capture (AHC) Biosensors (Forte Bio, Lot No: 18-5064) were hydrated in assay buffer (1x PBS, 0.1% BSA, 0.02% Tween-20, pH 7.2) and pre-treated in 100 mM glycine pH 1.5. The baseline was set in the assay buffer at 120 s. The ANS biosensors were then immobilized with UniAbs™ at a concentration of 5 μg/mL for 120 s. A different baseline (120 s) was set in the assay buffer. They were then immersed in a 7-point series of 1:2 dilutions of human CD22 protein in assay buffer starting at 250 nM. The last well of the analyte column contained only assay buffer to test for nonspecific binding between the buffer and loaded biosensors and was used as a control well. Association was observed for 600 s, followed by dissociation for 900 s. Data analysis was performed using Octet Data Analysis v9.0 (ForteBio). Binding kinetics were analyzed using a 1:1 binding model.

CD22 cell binding.

Binding to CD22-positive cells was assessed by flow cytometry (Guava easyCyte 8HT, EMD Millipore) using the Daudi cell line (ATCC). Briefly, 100,000 target cells were stained with a dilution series of purified UniAbs™ for 30 min at 4°C. After incubation, cells were washed twice with flow cytometry buffer (1X PBS, 1% BSA, 0.1% NaN ₃ ) and stained with goat F(ab') ₂ anti-human IgG conjugated to R-phycoerythrin (PE) (Southern Biotech, Cat. No. 2042-09) for the detection of cell-bound antibodies. After 20 min incubation at 4°C, cells were washed twice with flow cytometry buffer and then mean fluorescence intensity (MFI) was measured by flow cytometry. EC50 values were calculated using GraphPad Prism 7. Binding to CD22-positive cynomolgus cells was determined using the same protocol with the following modifications: target cells were CHO cells stably transfected to express the extracellular domain of cynomolgus CD22, and each antibody was tested at one concentration (~1.7 µg/ml) therefore EC50 values were not calculated.

Example 1: Genetically engineered rats expressing antibodies containing only heavy chains.

The human and rat IgH locus were designed and assembled from several parts. This involved modifying and combining genes from the rat C region downstream of the human _JH and then adding the human _VH 6 -D segment region upstream. Two BACs with distinct human VH gene clusters [BAC6 and BAC3] were then co-injected with a BAC called Georg, encoding an assembled and modified region including human VH6, all D, all JH, and modified rat Cγ2a/1/2b (ACH1).

Transgenic rats carrying artificial heavy chain immunoglobulin loci in a disordered configuration were obtained. IgG2a(AC _H 1), IgG1(AC _H 1), IgG2b(AC _H 1) genes were absent in

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CH1 segment. The IgE, IgA, and 3' enhancer constant region genes were included in the Georg BAC. RT-PCR and serum analysis (ELISA) of transgenic rats revealed productive rearrangement of transgenic immunoglobulin loci and expression of only heavy chain-only antibodies of various isotypes in the serum. Transgenic rats were crossed with rats with mutated endogenous heavy chain and light chain loci previously described in US Patent Publication 2009/0098134 A1. Analysis of these animals demonstrated inactivation of rat immunoglobulin heavy and light chain expression and high levels of expression of heavy chain only antibodies with variable regions encoded by the human V, D and J genes. Immunization of transgenic rats resulted in serum responses with high titers of antigen-specific antibodies containing only heavy chains. These transgenic rats expressing heavy chain-only antibodies with the human VDJ region were named UniRats™.

Example 2: Immunization.

Immunization with recombinant extracellular domain of CD22.

Twelve UniRat animals (6 HC27, 6 HC28) were immunized with recombinant human CD22 protein. Animals were immunized according to a standard protocol using Titermax/Alhydrogel adjuvant. Recombinant CD22 extracellular domain was purchased from R&D Systems and diluted in sterile saline and combined with adjuvant. The immunogen was combined with Titermax and Alhydrogel adjuvants. The first immunization (priming) with the immunogen in Titermax was carried out on the left and right legs. Subsequent booster immunizations were carried out in the presence of Alhydrogel and three days before the selection of boosts they were carried out with immunogen in the PBS. Serum was collected from rats at the last blood draw to determine serum titers.

Serum titer results.

A summary of serum titer information is presented in FIG. 6. In the graphs shown in FIG. 6, each line represents a different animal. The graph legends show the identification number of each individual animal. Binding activity for a series of 8-point dilution sera was tested by ELISA against huCD22+ Fc protein, huCD22+His tag, rhesus monkey CD22+His tag protein, and target protein without His tag. Among this group of animals, a range of levels of serum reactivity to both human and rhesus monkey CD22 was observed. The serum response to the His tag of the protein was also observed.

Example 3 Binding to CD22-expressing cell lines.

In fig. 4 shows the target binding activity of anti-CD22 antibodies containing only the heavy chains described herein. Column 1 shows the identification number (ID) of the heavy chain-only anti-CD22 antibody clone. Column 2 shows the protein binding affinity (KD), measured in molarity. Column 3 shows the protein binding dissociation constant (K-off rate), measured in sec. Column 4 shows binding to Daudi cells measured as fold over background MFI signal. Column 5 shows binding to CHO cells stably expressing cynomolgus CD22, measured as fold over background MFI signal. Column 6 shows binding to CHO cells that do not express CD22, measured as fold over background MFI signal.

Example 4: Bispecific Antibody-Mediated Killing of Human Tumor Cells by Redirecting Activated T Cells.

Three different CD22-positive Burkitt's lymphoma tumor cell lines (Daudi, Raji, and Ramos) were dye-labeled and incubated with increasing amounts of bispecific antibody in the presence of preactivated human T cells. The bispecific antibody consisted of an anti-CD3 binding arm coupled to an anti-CD22 VH binding domain, as schematically depicted in FIG. 5D. The negative control antibody contained a VH binding domain that did not bind CD22. CD22-negative K562 cells did not exhibit specific lysis (data not shown). Data from three bispecific antibodies containing three different heavy chain-only anti-CD22 binding domains paired with the same anti-CD3 binding domain are presented in FIG. 5A were compared to a negative control and demonstrated antibody-mediated killing of CD22-positive Daudi tumor cells through redirection of activated T cells. Data for two bispecific antibodies containing the same heavy chain-only anti-CD22 binding domain paired with two different anti-CD3 binding domains are presented in FIG. 5B was compared to a negative control and demonstrated antibody-mediated killing of CD22-positive Raji tumor cells through redirection of activated T cells. Data for two bispecific antibodies containing the same heavy chain-only anti-CD22 binding domain paired with two different anti-CD3 binding domains are presented in FIG. 5C were compared to a negative control and demonstrated antibody-mediated killing of CD22-positive Ramos tumor cells through redirection of activated T cells.

Although preferred embodiments have been shown and described herein

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Claims (18)

implementation of the present invention, it will be apparent to those skilled in the art that these embodiments are provided for purposes of illustration only. Many variations, changes and substitutions will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. It should be understood that various alternative embodiments of the present invention described herein may be used in the practice of the present invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents are thereby covered. CLAIM 1. A heavy chain only antibody that binds to CD22 containing a heavy chain variable region containing: (a) CDR1 sequence SEQ ID NO: 1, CDR2 sequence SEQ ID NO: 11 and CDR3 sequence SEQ ID NO: 18; or (b) CDR1 sequence SEQ ID NO: 1, CDR2 sequence SEQ ID NO: 12 and CDR3 sequence SEQ ID NO: 19; or (c) the CDR1 sequence SEQ ID NO: 1, the CDR2 sequence SEQ ID NO: 12, and the CDR3 sequence SEQ ID NO: 20. 2. An antibody containing only heavy chains according to claim 1, wherein the sequences CDR1, CDR2 and CDR3 are in the human VH framework. 3. The heavy chain only antibody of claim 1, wherein the heavy chain variable region comprises a sequence having at least 95% sequence identity to SEQ ID NO: 24. 4. The heavy chain only antibody according to claim 3, wherein the heavy chain variable region comprises SEQ ID NO: 24. 5. The heavy chain only antibody of claim 1, wherein the heavy chain variable region comprises a sequence having at least 95% sequence identity to SEQ ID NO: 25. 6. The heavy chain only antibody according to claim 5, wherein the heavy chain variable region comprises SEQ ID NO: 25. 7. The heavy chain only antibody of claim 1, wherein the heavy chain variable region comprises a sequence having at least 95% sequence identity to SEQ ID NO: 32. 8. The heavy chain only antibody of claim 7, wherein the heavy chain variable region comprises SEQ ID NO: 32. 9. The heavy chain-only antibody of claim 1, further comprising a heavy chain constant region sequence. 10. The heavy chain-only antibody according to claim 9, wherein the heavy chain constant region sequence lacks the CH1 sequence. 11. The heavy chain-only antibody according to claim 9, wherein the heavy chain constant region sequence comprises a CH2 domain and a CH3 domain. 12. The heavy chain-only antibody of claim 1, further comprising a human IgG4 Fc region. 13. The heavy chain-only antibody of claim 12, wherein the human IgG4 Fc region is a variant of the human IgG4 Fc region. 14. The heavy chain-only antibody of claim 12, wherein the human IgG4 Fc region is a silenced human IgG4 Fc region. 15. A multispecific antibody comprising a first binding fragment that has a binding specificity for CD22 and a second binding fragment, where the first binding fragment comprises a heavy chain variable region containing: (a) CDR1 sequence SEQ ID NO: 1, CDR2 sequence SEQ ID NO: 11 and CDR3 sequence SEQ ID NO: 18; or (b) CDR1 sequence SEQ ID NO: 1, CDR2 sequence SEQ ID NO: 12 and CDR3 sequence SEQ ID NO: 19; or (c) the CDR1 sequence SEQ ID NO: 1, the CDR2 sequence SEQ ID NO: 12, and the CDR3 sequence SEQ ID NO: 20. 16. The multispecific antibody according to claim 15, which is a bispecific antibody. 17. The multispecific antibody of claim 15, wherein the second binding moiety has binding affinity for the effector cell. 18. The multispecific antibody of claim 15, wherein the second binding moiety has binding affinity for a T cell antigen. -