Multispecific heavy chain antibodies binding to CD22 and CD3

Abstract

To provide multispecific human heavy chain antibodies binding to CD22 and CD3, methods of making such antibodies, compositions including pharmaceutical compositions comprising such antibodies, and their use to treat disorders that are characterized by the expression of CD22.SOLUTION: The present invention provides a multispecific binding compound that binds to CD3, comprising a heavy chain variable region, having specific amino acid sequences, and a light chain variable region, as well as a multispecific binding compound comprising a first binding unit having binding affinity for CD22 and a second binding unit having binding affinity for CD3, wherein the first binding unit has specific amino acid sequences.SELECTED DRAWING: Figure 14A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the filing date of U.S. Provisional Patent Application No. 62 / 861,708, filed June 14, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to multispecific human heavy chain antibodies (e.g., UniAb™) that bind to CD22 and CD3. The invention further relates to methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use to treat disorders characterized by CD22 expression. [Background technology]

[0003] CD22 CD22, also known as SIGLEC-2 (UniProt P20273), is a cell surface receptor 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 CD45 cell surface protein. CD22 is thought to function as an inhibitory receptor for B cell receptor signaling. Along with CD20 and CD19, CD22 is an attractive target for therapeutic treatment of B cell malignancies due to its restricted B cell expression. 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 these have been used therapeutically as standard monoclonals (e.g., epratuzumab) as well as antibody-drug conjugates (inotuzumab ozogamicin). In addition, anti-CD22 chimeric antigen receptor T cells have been used clinically to treat leukemia (Fry, Terry J., et al. "CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy." Nature Medicine (2017)).

[0004] Heavy chain antibodies In conventional IgG antibodies, the association of the heavy and light chains is due in part to hydrophobic interactions between the light chain constant region and the CH1 constant domain of the heavy chain. There are additional residues in the heavy chain framework 2 (FR2) and framework 4 (FR4) regions that also contribute to the hydrophobic interactions between the heavy and light chains.

[0005] However, sera from Camelidae (a suborder of Camelidae that includes camels, dromedaries, and llamas) are known to contain a major type of antibody consisting only of paired heavy chains (heavy chain-only antibodies or UniAbs™). UniAbs™ from Camelidae (dromedaries, Bactrian camels, llamas, guanacos, alpacas, and vicuñas) have a unique structure consisting of a single variable domain (VHH), a hinge region, and two constant domains (CH2 and CH3), which are highly homologous to the CH2 and CH3 domains of classical antibodies. These UniAbs™ lack the first domain of the constant region (CH1), which is present in the genome but is spliced out during mRNA processing. The absence of the CH1 domain explains the absence of light chains in UniAbs™, as this domain is the anchoring site for the constant domain of the light chain. Such UniAbs™ have naturally evolved to confer antigen-binding specificity and high affinity by 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 evolved a unique type of immunoglobulin, called IgNAR, which lacks light polypeptide chains and is composed entirely of heavy chains. IgNAR molecules can be engineered by molecular engineering to generate variable domains of a single heavy chain polypeptide (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)).

[0006] The ability of heavy-chain-only antibodies lacking light chains to bind antigen was established in the 1960s (Jaton et al. (1968) Biochemistry, 7, 4185-4195). Heavy-chain immunoglobulins physically separated from light chains retained 80% of the antigen-binding activity for tetrameric antibodies. Sitia et al. (1990) Cell, 60, 781-790 showed that removal of the CH1 domain from a rearranged mouse μ gene resulted in the production of heavy-chain-only antibodies lacking light chains in mammalian cell culture. The antibodies produced retained VH binding specificity and effector function.

[0007] Heavy-chain antibodies with high specificity and affinity can be generated against various antigens through immunization (van der Linden, RH, et al., Biochim. Biophys. Acta. 1431, 37-46 (1999)), and VHH moieties can be easily cloned and expressed in yeast (Frenken, LGJ, et al., J. Biotechnol. 78, 11-21 (2000)). Their expression levels, solubility, and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, MA, et al., FEBS Lett. 414, 521-526 (1997)).

[0008] Mice in which the λ (lambda) light (L) chain locus and / or the λ or κ (kappa) light chain locus have been functionally silenced, as well as antibodies produced by such mice, are described in U.S. Patent Nos. 7,541,513 and 8,367,888. Recombinant production of heavy chain-only antibodies in mice and rats has been reported, for example, in WO2006008548, U.S. Patent Application Publication 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. The generation of knockout rats by embryonic microinjection of zinc finger nucleases is described in Geurts et al., 2009, Science, 325(5939):433. Soluble heavy chain-only antibodies and transgenic rodents containing heterologous heavy chain loci that produce such antibodies are described in U.S. Patent Nos. 8,883,150 and 9,365,655. CAR-T constructs containing single-domain antibodies as binding (targeting) domains are described, for example, in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378-386. Summary of the Invention

[0009] Aspects of the present invention relate to heavy chain antibodies, including but not limited to UniAbs™, that have binding affinity for CD22. Further aspects of the present invention relate to methods of making such antibodies, compositions comprising such antibodies, and their use in treating disorders characterized by expression of CD22.

[0010] Aspects of the invention include multispecific binding compounds that bind to CD3, the binding compounds comprising a heavy chain variable region comprising (a) a CDR1 sequence with no more than two substitutions from SEQ ID NO: 85, and / or (b) a CDR2 sequence with no more than two substitutions from SEQ ID NO: 86, and / or (c) a CDR3 sequence with no more than two substitutions from SEQ ID NO: 87, and a light chain variable region. In some embodiments, the heavy chain CDR1, CDR2, and CDR3 sequences are present in a human VH framework. In some embodiments, the heavy chain variable region comprises heavy chain CDR1, CDR2, and CDR3 sequences in a human VH framework, each CDR sequence comprising a sequence having at least 85% identity to any one of SEQ ID NOs: 85-87, and the binding compound also comprises a light chain variable region.

[0011] In some embodiments, the multispecific binding compound comprises a heavy chain variable region comprising (a) a CDR1 sequence with no more than two substitutions from SEQ ID NO: 85, and (b) a CDR2 sequence with no more than two substitutions from SEQ ID NO: 86, and (c) a CDR3 sequence with no more than two substitutions from SEQ ID NO: 87, and the binding compound also comprises a light chain variable region.

[0012] In some embodiments, the multispecific binding compound comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 85, the CDR2 sequence of SEQ ID NO: 86, and the CDR3 sequence of SEQ ID NO: 87, and the binding compound also comprises a light chain variable region.

[0013] In some embodiments, the light chain variable region comprises CDR1, CDR2, and CDR3 sequences in a human VL framework, where each CDR sequence comprises a sequence with no more than three amino acid substitutions relative to the CDR sequence or set of CDR sequences of SEQ ID NO: 92, or where these CDR sequences comprise a sequence with at least 85% identity to the CDR sequence or set of CDR sequences of SEQ ID NO: 92. In some embodiments, the light chain variable region comprises a CDR1 sequence of SEQ ID NO: 88, a CDR2 sequence of SEQ ID NO: 89, and a CDR3 sequence of SEQ ID NO: 90. In some embodiments, the heavy chain variable region comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 91. In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the light chain variable region comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 92. In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 92.

[0014] Aspects of the invention include multispecific binding compounds comprising a first binding unit having binding affinity for CD22 and a second binding unit having binding affinity for CD3, wherein the first binding unit comprises (a) a CDR1 having no more than two substitutions in any of the amino acid sequences of SEQ ID NOs: 1-10, and / or (b) a CDR2 having no more than two substitutions in any of the amino acid sequences of SEQ ID NOs: 11-17, and / or (c) a CDR3 having no more than two substitutions in any of the amino acid sequences of SEQ ID NOs: 18-23. In some embodiments, the CDR1, CDR2, and CDR3 sequences of the first binding unit are present in a human framework. In some embodiments, the first binding unit further comprises a heavy chain constant region sequence in the absence of a CH1 sequence.

[0015] In some embodiments, the first binding unit comprises a heavy chain variable region comprising (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 NOs: 11-17, and / or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18-23.

[0016] In some embodiments, the multispecific binding compound 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 NOs: 18-23.

[0017] In some embodiments, the multispecific binding compound comprises (a) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18, (b) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 12, and the CDR3 sequence of SEQ ID NO: 19, or (c) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 12, and the CDR3 sequence of SEQ ID NO: 20. In some embodiments, the multispecific binding compound comprises a heavy chain variable region having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 24-84. In some embodiments, the multispecific binding compound comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 24-84. In some embodiments, the multispecific binding compound comprises the heavy chain variable region sequence of SEQ ID NO: 24.

[0018] Embodiments of the invention include multispecific binding compounds comprising a first binding unit having binding affinity for CD22 and a second binding unit having binding affinity for CD3, wherein the first binding unit has (a) a CDR1 sequence of the formula G X1S I X2X3X4X5X6Y (SEQ ID NO: 104, wherein X1 is D or G, X2 is S, T, I or N, X3 is S or D, X4 is G, S or N, X5 is D, G or S, and X6 is Y or H), and (b) a CDR1 sequence of the formula X7X8Y X9G X 10 X 11 (SEQ ID NO: 105, wherein X7 is I or V, X8 is Y or H, X9 is S or T, and X 10 is A, V or S, and X 11 is T or A), and (c) a CDR2 sequence of formula X 12 RX 13 DSSX 14 WRS (SEQ ID NO: 106, wherein X 12is T, A or K, and X 13 is D or E, and X 14 is N or S).

[0019] Embodiments of the invention include multispecific binding compounds comprising a first binding unit that has binding affinity for CD22 and a second binding unit that has binding affinity for CD3, wherein the first binding unit comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences in a human VH framework, and the CDR sequences comprise sequences with no more than two substitutions in the CDR sequences selected from the group consisting of SEQ ID NOs: 1-23.

[0020] Embodiments of the invention include multispecific binding compounds comprising a first binding unit having binding affinity for CD22 and a second binding unit having binding affinity for CD3, wherein the first binding unit comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences in a human VH framework, and the CDR sequences are selected from the group consisting of SEQ ID NOs: 1-23.

[0021] Embodiments of the invention include multispecific binding compounds comprising a first binding unit that has binding affinity for CD22 and a second binding unit that has binding affinity for CD3, wherein the first binding unit comprises a heavy chain variable region that comprises, in a human VH framework, (a) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18, or (b) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 12, and the CDR3 sequence of SEQ ID NO: 19, or (c) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 12, and the CDR3 sequence of SEQ ID NO: 20.

[0022] In some embodiments, the multispecific binding compound is bispecific. In some embodiments, the multispecific binding compound is in the CAR-T format.

[0023] Embodiments of the invention include multispecific binding compounds comprising: (i) a heavy chain variable region with binding affinity to CD3, comprising the CDR1 sequence of SEQ ID NO: 85, the CDR2 sequence of SEQ ID NO: 86, and the CDR3 sequence of SEQ ID NO: 87 in a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 88, the CDR2 sequence of SEQ ID NO: 89, and the CDR3 sequence of SEQ ID NO: 90 in a human VL framework; and (iii) an antigen-binding domain of an anti-CD22 heavy chain antibody, comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18 in a human VH framework.

[0024] Embodiments of the invention include multispecific binding compounds comprising: (i) a heavy chain variable region with binding affinity to CD3, comprising the CDR1 sequence of SEQ ID NO: 85, the CDR2 sequence of SEQ ID NO: 86, and the CDR3 sequence of SEQ ID NO: 87 in a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 88, the CDR2 sequence of SEQ ID NO: 89, and the CDR3 sequence of SEQ ID NO: 90 in a human VL framework; and (iii) an antigen-binding domain of an anti-CD22 heavy chain antibody, comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 12, and the CDR3 sequence of SEQ ID NO: 19 in a human VH framework.

[0025] Embodiments of the invention include multispecific binding compounds comprising: (i) a heavy chain variable region with binding affinity to CD3, comprising the CDR1 sequence of SEQ ID NO: 85, the CDR2 sequence of SEQ ID NO: 86, and the CDR3 sequence of SEQ ID NO: 87 in a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 88, the CDR2 sequence of SEQ ID NO: 89, and the CDR3 sequence of SEQ ID NO: 90 in a human VL framework; and (iii) an antigen-binding domain of an anti-CD22 heavy chain antibody, comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 12, and the CDR3 sequence of SEQ ID NO: 20 in a human VH framework.

[0026] In some embodiments, the multispecific binding compound comprises a human IgG1 Fc region. In some embodiments, the human IgG1 Fc region is a silenced human IgG1 Fc region. In some embodiments, the multispecific binding compound comprises a human IgG4 Fc region. In some embodiments, the human IgG4 Fc region is a silenced human IgG4 Fc region.

[0027] Aspects of the present invention include pharmaceutical compositions comprising the multispecific binding compounds described herein.

[0028] Embodiments of the invention include methods for the treatment of a B cell disorder characterized by expression of CD22, comprising administering to a subject having the disorder a multispecific binding compound or pharmaceutical composition described herein.

[0029] An embodiment of the invention includes the use of a multispecific binding compound in the preparation of a medicament for the treatment of a B cell disorder characterized by expression of CD22.

[0030] 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).

[0031] Aspects of the invention include polynucleotides encoding the multispecific binding compounds described herein. Aspects of the invention include vectors comprising the polynucleotides described herein. Aspects of the invention include cells comprising the vectors described herein.

[0032] Aspects of the invention include methods of producing the multispecific binding compounds described herein, comprising growing a cell described herein under conditions permissive for expression of the binding compound, and isolating the binding compound from the cell.

[0033] An embodiment of the present invention includes a method of making the multispecific binding compounds described herein, comprising immunizing a UniRat animal with CD22 and identifying a CD22-binding heavy chain sequence.

[0034] Aspects of the invention include methods of treatment comprising administering to an individual an effective amount of a multispecific binding compound described herein or a pharmaceutical composition described herein.

[0035] These and additional aspects are further described in the remainder of the disclosure, including the examples. [Brief explanation of the drawings]

[0036] [Figure 1A] 1 is a graph showing T cell-mediated cytotoxicity of CD22 positive cells (Daudi) using resting human pan T cells. [Figure 1B] FIG. 1 is a graph showing dose response curves of cytokine release by resting human pan T cells incubated with CD22 positive cells (Daudi) and treated with anti-CD22×CD3_F2F multispecific binding compounds and positive controls. [Figure 2A] 1 is a graph showing T cell-mediated cytotoxicity of CD22 positive cells (SUDHL10) using resting human pan T cells. [Figure 2B] FIG. 1 is a graph showing dose response curves of cytokine release by resting human pan T cells incubated with CD22 positive cells (SUDHL10) and treated with anti-CD22×CD3_F2F multispecific binding compound and positive control. [Figure 3A] 1 shows a series of graphs depicting T cell-mediated cytotoxicity of CD22 positive cells (RI-1) using resting human pan T cells. [Figure 3B] 1 shows a series of graphs depicting dose response curves of cytokine release by resting human pan T cells incubated with CD22 positive cells (RI-1) and treated with anti-CD22xCD3_F2F multispecific binding compounds and positive controls. [Figure 4]1 shows a series of graphs depicting T cell-mediated cytotoxicity of CD22 positive cells using activated human pan T cells. [Figure 5] 1 shows a series of graphs showing cell binding of bispecific antibodies to CD22 and CD3. [Figure 6] FIG. 1 shows a treatment regimen for determining the in vivo efficacy of anti-CD22×CD3_F2F multispecific binding compounds in Daudi xenografts. [Figure 7] 1 is a graph showing mean tumor volume as a function of days after tumor implantation in murine Daudi xenografts. [Figure 8] 1 is a graph showing body weight as a function of days after tumor implantation in murine Daudi xenografts. [Figure 9] 1 is a graph showing percent body weight change as a function of days after tumor implantation in murine Daudi xenografts. [Figure 10] 1 is a graph showing mean tumor volume as a function of days after tumor implantation in murine Daudi xenografts. [Figure 11] 1 shows a series of graphs depicting individual tumor measurements as a function of days after tumor implantation in murine Daudi xenografts. [Figure 12] 1 is a graph showing body weight as a function of days after tumor implantation in murine Daudi xenografts. [Figure 13] 1 is a graph showing percent body weight change as a function of days after tumor implantation in murine Daudi xenografts. [Figure 14A] FIG. 1 is a schematic diagram of a bispecific binding compound having one binding unit that specifically binds to CD3 and one binding unit that specifically binds to CD22. [Figure 14B] FIG. 1 is a diagram of various CAR-T constructs that can incorporate one or more binding domains according to embodiments of the present invention. [Figure 15A] FIG. 1 is a schematic diagram of a bispecific binding molecule having one binding unit that specifically binds to CD3 and one binding unit that specifically binds to CD22 (monovalent and monospecific for CD22). [Figure 15B] FIG. 1 is a schematic diagram of a bispecific binding molecule having one binding unit that specifically binds to CD3 and two binding units that specifically bind to CD22 (bivalent and monospecific for CD22). [Figure 15C] FIG. 1 is a schematic diagram of a bispecific binding molecule having one binding unit that specifically binds to CD3 and two binding units that specifically bind to CD22 (bivalent and biparatopic to CD22). [Figure 16] 1 is a table showing data regarding various biological activities of anti-CD22 antibodies according to embodiments of the present invention. [Figure 17] 1 is a series of graphs showing serum titers as a function of dilution. DETAILED DESCRIPTION OF THE INVENTION

[0037] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); Biology” (FMAusubel 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).

[0038] Where a range of values is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of those smaller ranges may independently be included in the smaller ranges and are also encompassed herein, subject to any specific excluded limits in the stated range. When a stated range includes one or both of those upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the invention.

[0039] Unless otherwise specified, 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)).

[0040] In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures that are readily known to those skilled in the art are not described in order to avoid obscuring the present invention.

[0041] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

[0042] I. Definition "Comprising" means that the listed elements are required for the composition / method / kit, but other elements may be included to form a composition / method / kit, etc. within the scope of the claim.

[0043] "Consisting essentially of" means limiting the scope of the described composition or process to certain materials or steps that do not materially affect the basic and novel characteristic(s) of the invention.

[0044] "Consisting of" means the exclusion of any element, step, or ingredient of a composition, method, or kit not specified in the claim.

[0045] Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to residues in the variable domain (residues approximately 1-113 of the heavy chain) (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 generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise specified herein, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise specified herein, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system.

[0046] Antibodies, also called immunoglobulins, traditionally comprise at least one heavy chain and one light chain, with the amino-terminal domains of the heavy and light chains being variable in sequence and therefore commonly referred to as variable region domains, or variable heavy (VH) or variable light (VH) domains. The two domains traditionally associate to form a specific binding region, although, as discussed herein, specific binding can also be obtained with variable sequence in the heavy chain alone, and a variety of non-native antibody structures are known and used in the art.

[0047] A "functional" or "biologically active" antibody or antigen-binding molecule (including heavy-chain-only antibodies and multispecific (e.g., bispecific) three-chain antibody-like molecules (TCAs) described herein) is one that can exert one or more of its native activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule, e.g., a TCA, can have the ability to specifically bind to an antigen, and the binding can then trigger or modify a cellular or molecular event, such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, e.g., a TCA, can also block ligand activation of a receptor or function as an agonist or antagonist. The ability of an antibody or other binding molecule, e.g., a TCA, to exert one or more of its native activities depends on several factors, including proper folding and assembly of the polypeptide chain.

[0048] The term "antibody" as used herein is used in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, three-chain antibodies, single-chain Fvs (scFvs), nanobodies, etc., as well as antibody fragments so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0049] The term antibody may refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptides, i.e., a polypeptide comprising an antigen-binding site that immunospecifically binds to an antigen of a desired target or portion thereof, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune disease. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule, including engineered subclasses with modified Fc portions that provide reduced or enhanced effector cell activity. The immunoglobulin may be derived from any species. In one aspect, the immunoglobulin is substantially human.

[0050] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies according to the present invention can be produced, for example, by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can also be produced, for example, via recombinant protein production methods (see, e.g., U.S. Pat. No. 4,816,567).

[0051] The term "variable" when used in reference to antibodies refers to the fact that the sequences of certain portions of antibody variable domains vary extensively among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy- and light-chain variable domains each contain four FRs, primarily adopting a β-sheet configuration, connected by three hypervariable regions, which form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions within each chain are held together in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to 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)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular cytotoxicity (ADCC).

[0052] The term "hypervariable region," as used herein, refers to the amino acid residues of an antibody responsible for antigen binding. Hypervariable regions generally comprise amino acid residues from the "complementarity-determining regions" or "CDRs" (e.g., 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 the "hypervariable loops" (e.g., residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0053] While exemplary CDR designations are provided herein, those skilled in the art will understand that several definitions of CDRs 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). The Kabat definition is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989;342:877-883).Alternative CDR definitions of interest include, but are not limited to, 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 specificity-determining 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 Padlan et al., "Identification of specificity-determining residues in antibodies," Faseb. J. 1995; 9: 133-139. each of which is specifically incorporated herein by reference.

[0054] The terms "heavy chain-only antibody" and "heavy chain antibody" are used interchangeably herein and refer, in their broadest sense, to an antibody lacking the light chain of a conventional antibody. These terms specifically include, but are not limited to, homodimeric antibodies comprising a VH antigen-binding domain in the absence of a CH1 domain, and CH2 and CH3 constant domains, functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NARs comprising a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NARs), and functional fragments thereof, and soluble single-domain antibodies (sUniDab™). In one embodiment, a heavy chain-only antibody is composed of a variable region antigen-binding domain consisting of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In another embodiment, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of the hinge region, and CH2 and CH3 domains. In another embodiment, the heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of the hinge region, and a CH2 domain. In a further embodiment, the heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of the hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains have been truncated are also included herein. In a further embodiment, the heavy chain is composed of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but not the hinge region. In a further embodiment, the heavy chain is composed of an antigen-binding domain, at least one CH (CH1, CH2, CH3, or CH4) domain, and at least a portion of the hinge region. Heavy chain-only antibodies may be in the form of a dimer in which two heavy chains are disulfide-bonded or otherwise covalently or non-covalently bound to each other. Heavy chain-only antibodies may belong to the IgG subclass, but antibodies belonging to other subclasses, such as the IgM, IgA, IgD, and IgE subclasses, are also included herein. In particular embodiments, the heavy chain antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype.In one embodiment, the heavy chain antibody is of the IgG4 subtype, and one or more of the CH domains are modified to alter the effector function of the antibody. In one embodiment, the heavy chain antibody is of the IgG1 subtype, and one or more of the CH domains are modified to alter the effector function of the antibody. Modifications of the CH domain to alter effector function are further described herein. Non-limiting examples of heavy chain antibodies are described, for example, in WO2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0055] In one embodiment, the heavy chain-only antibodies herein are used as the binding (targeting) domain of a chimeric antigen receptor (CAR). This definition specifically includes human heavy chain-only antibodies produced by human immunoglobulin transgenic rats (UniRat™), referred to as UniAb™. The variable region (VH) of UniAb™, referred to as UniDab™, is a versatile building block that can be linked to an Fc region or serum albumin to develop new therapeutics with multispecificity, increased potency, and extended half-life. Because homodimeric UniAb™ lacks a light chain and therefore a VL domain, antigens are recognized by one single domain: the variable domain of the heavy chain of the heavy chain antibody (antigen binding domain, VH).

[0056] As used herein, an "intact antibody chain" includes a full-length variable region and a full-length constant region (Fc). An intact, "traditional" antibody includes an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, hinge, CH2, and CH3, for secreted IgG. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domains may be native-sequence constant domains (e.g., human native-sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effector functions," which refer to biological activities attributable to the Fc constant region (native-sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and down-regulation of cell surface receptors. Constant region variants include those that alter the effector profile, Fc receptor binding, etc.

[0057] Antibodies and various antigen-binding proteins can be provided as different classes depending on the amino acid sequence of the Fc (constant domain) of their heavy chains. There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains corresponding to the different classes of antibodies can be referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modified or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090, Lund et al (2000) Eur. J. Biochem. 267:7246-7256, US2005 / 0048572, US2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0058] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding, CDC, Fc receptor binding, ADCC, ADCP, down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require the Fc region to interact with a receptor, e.g., FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and the low-affinity FcRn receptor, and can be assessed using a variety of assays well known in the art. A "dead" or "silenced" Fc is one that has been mutated to retain activity, e.g., with respect to extended serum half-life, but does not activate high-affinity Fc receptors or has reduced affinity for Fc receptors.

[0059] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include, for example, native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0060] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or Fc region of the parent polypeptide. A variant Fc region herein preferably has at least about 80% homology with the native-sequence Fc region and / or Fc region of the parent polypeptide, most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0061] The variant Fc sequence may have three amino acid substitutions in the CH2 region at positions 234, 235, and 237 in the EU index to reduce FcγRI binding (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at positions 330 and 331 in the EU index reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitutions with human IgG1 or IgG2 residues at positions 233-236, and IgG4 residues at positions 327, 330, and 331, significantly reduce ADCC and CDC (see, e.g., Armour KL et al., 1999 Eur J Immunol. 29(8):2613-24, and Shields RL et al., 2001 J Biol Chem. 276(9):6591-604). The human IgG1 amino acid sequence (UniProtKB No. P01857) is provided herein as SEQ ID NO: 93. The human IgG4 amino acid sequence (UniProtKB No. P01861) is provided herein as SEQ ID NO: 94. Silenced IgG1 is described, for example, in Boesch, AW, et al., "Highly parallel characterization of IgG Fc binding interactions." MAb, 2014.6(4):pp.915-27, the disclosure of which is incorporated herein by reference in its entirety.

[0062] Other Fc variants are possible, including, but not limited to, variants in which regions capable of forming disulfide bonds are deleted or in which specific amino acid residues are removed or a methionine residue is added at the N-terminus of a native Fc. Thus, in some embodiments, one or more Fc moieties of a binding compound may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet another embodiment, the hinge region of the Fc may be completely removed. In yet another embodiment, a binding compound may comprise an Fc variant.

[0063] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector function by substituting (mutating), deleting, or adding amino acid residues to confer complement binding or Fc receptor binding. For example, but not limited to, deletions can be made in complement binding sites, such as the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publications WO 97 / 34631 and WO 96 / 32478. Additionally, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0064] The term "Fc region-containing antibody" refers to an antibody that contains an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Thus, antibodies with an Fc region of the present invention can include antibodies with or without K447.

[0065] Aspects of the present invention include binding compounds with multispecific structures, including but not limited to bispecific, trispecific, etc. A wide variety of methods and protein structures are known and used for bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc.

[0066] Various methods have been developed for producing multivalent artificial antibodies by recombinantly fusing the variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on a polypeptide are connected by a polypeptide linker. One non-limiting example of such a polypeptide linker is a GS linker, which has an amino acid sequence of four glycine residues followed by one serine residue, repeated n times, where n is an integer ranging from 1 to about 10, such as 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 102) (n=1) and GGGGSGGGGS (SEQ ID NO: 103) (n=2). Other suitable linkers can also be used, e.g., as described in Chen et al., Adv Drug Deliv Rev. 2013 October 15;65(10):1357-69, the disclosure of which is incorporated herein by reference in its entirety.

[0067] The term "tri-chain antibody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy chain and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain, comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy chain-only antibody comprising an Fc portion comprising CH2 and / or CH3 and / or CH4 domains in the absence of a CH1 domain, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind to an epitope of a second antigen or a different epitope of the first antigen, wherein the binding domains are derived from or share sequence identity with the variable regions of the antibody heavy or light chains. Portions of such variable regions are V H and / or V L Gene segments, D and J Hgene segment, or J L The variable region can be encoded by a rearranged V H DJ H , V L DJ H , V H J L、 or V L J L The TCA proteins may be encoded by gene segments. The TCA proteins utilize heavy chain-only antibodies as defined above.

[0068] The TCA binding compounds utilize "heavy chain-only antibodies" or "heavy chain antibodies" or "heavy chain polypeptides," which, as used herein, refer to single-chain antibodies comprising heavy chain constant regions CH2 and / or CH3 and / or CH4, but lacking a CH1 domain. In one embodiment, a heavy chain antibody is comprised of an antigen-binding domain, at least a portion of the hinge region, and CH2 and CH3 domains. In another embodiment, a heavy chain antibody is comprised of an antigen-binding domain, at least a portion of the hinge region, and a CH2 domain. In a further embodiment, a heavy chain antibody is comprised of an antigen-binding domain, at least a portion of the hinge region, and a CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domains have been truncated are also included herein. In a further embodiment, the heavy chain is comprised of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but not the hinge region. Heavy-chain-only antibodies may be in the form of a dimer in which two heavy chains are disulfide-bonded or otherwise covalently or non-covalently bound to each other, and may optionally contain an asymmetric interface between two or more CH domains to facilitate proper pairing between the polypeptide chains. Heavy-chain antibodies may belong to the IgG subclass, although antibodies belonging to other subclasses, such as the IgM, IgA, IgD, and IgE subclasses, are also included herein. In certain embodiments, heavy-chain antibodies are of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 or IgG4 subtype. Non-limiting examples of TCA-binding compounds are described, for example, in WO2017 / 223111 and WO2018 / 052503, the disclosures of which are incorporated herein by reference in their entireties.

[0069] Heavy chain antibodies account for approximately one-quarter of the IgG antibodies produced by camelids, such as camels and llamas (Hamers-Casterman C., et al. Nature 363, 446-448 (1993)). These antibodies are formed by two heavy chains but lack light chains. As a result, the variable antigen-binding site is called a VHH domain, which represents the smallest naturally occurring intact antigen-binding site and is only about 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy-chain antibodies with high specificity and affinity can be generated against various antigens through immunization (van der Linden, RH, et al., Biochim. Biophys. Acta. 1431, 37-46 (1999)), and VHH moieties can be easily cloned and expressed in yeast (Frenken, LGJ, et al., J. Biotechnol. 78, 11-21 (2000)). Their expression levels, solubility, and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, MA, et al., FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies, called 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)).

[0070] The terms "CD22" and "cluster of differentiation 22," as used herein, refer to a molecule belonging to the SIGLEC family of lectins 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 any human and non-human animal species, and specifically includes human CD22 and CD22 of non-human mammals.

[0071] The term "human CD22," as used herein, includes any variant, isoform, and species homologue of human CD22 (UniProt P20273), regardless of its source or mode of preparation. Thus, "human CD22" includes human CD22 naturally expressed by cells and CD22 expressed on cells transfected with the human CD22 gene.

[0072] The terms "anti-CD22 heavy chain-only antibody," "CD22 heavy chain-only antibody," "anti-CD22 heavy chain antibody," and "CD22 heavy chain antibody" are used interchangeably herein and refer to a heavy chain-only antibody, as defined above, that immunospecifically binds to CD22, as defined above, including human CD22. This definition includes human heavy chain antibodies produced by transgenic animals, such as transgenic rats or transgenic mice expressing human immunoglobulins, including, but not limited to, UniRat™, which produces the human anti-CD22 UniAb™ antibody, as defined above.

[0073] "Amino acid sequence identity percentage (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity.Alignment for determining amino acid sequence identity percentage can be achieved by various methods within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.However, for the purposes of this specification, amino acid sequence identity percentage values are generated using the sequence comparison computer program ALIGN-2.

[0074] An "isolated" antibody is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified to the following extent: (1) greater than 95% by weight of the antibody, and most preferably greater than 99% by weight, as determined by the Lowry method; (2) sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions with Coomassie blue, or preferably silver stain. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0075] The antibodies of the present invention include multispecific antibodies. Multispecific antibodies have two or more binding specificities. The term "multispecific" specifically includes "bispecific," "trispecific," higher-order independent specific binding affinities, such as higher-order polyepitopic specificities, and tetravalent antibodies and antibody fragments. The terms "multispecific antibody," "multispecific heavy chain-only antibody," "multispecific heavy chain antibody," "multispecific UniAb™," and "multispecific binding compound" are used in the broadest sense herein to encompass all antibodies with two or more binding specificities. Multispecific heavy chain anti-CD22 antibodies of the present invention specifically include antibodies that immunospecifically bind to one single epitope on the CD22 protein, such as human CD22, and to an epitope on a different protein, e.g., the CD3 protein (i.e., bivalent and monoparatopic). Multispecific heavy chain anti-CD22 antibodies of the present invention specifically include antibodies that immunospecifically bind to two or more non-overlapping epitopes on the CD22 protein, such as human CD22 (i.e., bivalent and biparatopic). Multispecific heavy chain anti-CD22 antibodies of the invention also specifically include antibodies that immunospecifically bind to an epitope on a CD22 protein, such as human CD22, and to an epitope on a different protein, such as a CD3 protein, e.g., human CD3 (i.e., bivalent and biparatopic). Multispecific heavy chain anti-CD22 antibodies of the invention also specifically include antibodies that immunospecifically bind to two or more non-overlapping or partially overlapping epitopes on a CD22 protein, such as human CD22 protein, and to epitopes on a different protein, such as a CD3 protein, e.g., human CD3 protein (i.e., trivalent and biparatopic).

[0076] Antibodies of the present invention include monospecific antibodies having one binding specificity. Monospecific antibodies specifically include antibodies containing a single binding specificity and antibodies containing two or more binding units with the same binding specificity. The terms "monospecific antibody," "monospecific heavy chain-only antibody," "monospecific heavy chain antibody," and "monospecific UniAb™" are used in the broadest sense herein to encompass all antibodies with one binding specificity. Monospecific heavy chain anti-CD22 antibodies of the present invention specifically include antibodies that immunospecifically bind to one epitope on the CD22 protein, such as human CD22 (monovalent and monospecific). Monospecific heavy chain anti-CD22 antibodies of the present invention also specifically include antibodies with two or more binding units that immunospecifically bind to an epitope on the CD22 protein, such as human CD22 (e.g., multivalent antibodies). For example, a monospecific antibody according to embodiments of the present invention may comprise a heavy chain variable region comprising two antigen-binding domains, each of which binds to the same epitope on the CD22 protein (i.e., bivalent and monospecific).

[0077] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Typically, an antigen has several or many different epitopes and will react with many different antibodies. The term specifically includes linear and conformational epitopes.

[0078] "Epitope mapping" is the process of identifying the binding site, or epitope, of an antibody on its target antigen. Antibody epitopes can be linear or conformational. 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 protein sequence but combine when the protein folds into its three-dimensional structure.

[0079] "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). As described above, the present invention specifically includes anti-CD22 heavy chain antibodies with polyepitopic specificity, i.e., anti-CD22 heavy chain antibodies that bind to one or more non-overlapping epitopes on the CD22 protein, such as human CD22, and anti-CD22 heavy chain antibodies that bind to one or more epitopes on the CD22 protein and epitopes on other proteins, such as the CD3 protein. The terms "non-overlapping epitope(s)" or "non-competing epitope(s)" of an antigen are defined herein to mean epitope(s) that are recognized by one member of a pair of antigen-specific antibodies but not by the other member. A pair of antigen-binding regions or antibodies targeting the same antigen on a multispecific antibody that recognize non-overlapping epitopes do not compete for binding to that antigen and can simultaneously bind to that antigen.

[0080] If two antibodies recognize the same or sterically overlapping epitopes, the antibodies bind to "essentially the same epitope" as the reference antibody. The most widely used rapid method for determining whether two epitopes bind to the same or sterically overlapping epitopes is the competitive assay, which can be configured in any format using labeled antigen or labeled antibody. Typically, the antigen is immobilized on a 96-well plate, and the ability of unlabeled antibody to block the binding of the labeled antibody is measured using radioactive or enzyme labels.

[0081] The term "valency" as used herein refers to a specific number of binding sites in an antibody molecule.

[0082] A "monovalent" antibody has one binding site. A monovalent antibody is therefore also monospecific.

[0083] A "multivalent" antibody has two or more binding sites. Thus, the terms "bivalent," "trivalent," and "tetravalent" refer to the presence of two binding sites, three binding sites, and four binding sites, respectively. Thus, bispecific antibodies according to the invention are at least bivalent and may be trivalent, tetravalent, or otherwise multivalent. Bivalent antibodies according to embodiments of the invention may have two binding sites for the same epitope (i.e., bivalent and monoparatopic) or two binding sites for two different epitopes (i.e., bivalent and biparatopic).

[0084] A wide variety of methods and protein structures are known and used to prepare bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc.

[0085] The term "chimeric antigen receptor" or "CAR" is used in the broadest sense herein to refer to an artificial receptor (e.g., the antigen-binding region of a monoclonal antibody or other ligand) that fuses a desired binding specificity to a transmembrane domain and an intracellular signaling domain. Typically, receptors are used to fuse the specificity of a monoclonal antibody onto T cells 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).

[0086] The term "human antibody" is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., 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 as defined above, specifically UniAb™ produced by UniRat™.

[0087] "Chimeric antibody" or "chimeric immunoglobulin" refers to an immunoglobulin molecule that contains amino acid sequences from at least two different Ig loci, such as a transgenic antibody that contains a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies with non-human 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.

[0088] As used herein, the term "effector cell" refers to an immune cell that is involved 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, effector cells, such as natural killer cells, can induce antibody-dependent cellular cytotoxicity (ADCC). For example, FcR-expressing monocytes and macrophages are involved in the specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0089] A "human effector cell" is a leukocyte that expresses a receptor, such as a T cell receptor or FcR, and performs effector function. Preferably, the cell expresses at least FcγRIII and performs 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 their native source, e.g., from blood or PBMCs, as described herein.

[0090] The term "immune cell" is used herein in the broadest sense and includes, but is not limited to, cells of myeloid or lymphoid origin, such as lymphocytes (e.g., 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.

[0091] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (a native sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), and the like.

[0092] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0093] " Complement-dependent cytotoxicity " or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) with a molecule (e.g., antibody) complexed with a cognate antigen. To assess complement activation, the CDC assay described in, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996) can be carried out.

[0094] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens faster and tend to remain bound.

[0095] As used herein, " Kd " or " Kd value " refers to the dissociation constant determined by BioLayer interferometry in kinetic mode using Octet QK384 instrument (ForteBio Inc., Menlo Park, CA). For example, the anti-mouse Fc sensor is loaded with mouse-Fc fusion antigen, and then immersed in the well containing antibody to measure the concentration-dependent association rate (k). The antibody dissociation rate (koff) is measured in the final step, and the sensor is immersed in the well containing only buffer. Kd is the ratio of koff / koff (for further details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0096] The terms "treatment," "treating," and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the onset of the disease in a subject who is susceptible to the disease but has not yet been diagnosed with it; (b) inhibiting the disease, i.e., blocking its development; or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents can be administered before, during, or after the onset of a disease or injury. Treatment of ongoing disease is particularly interesting if the treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is desirably administered before complete loss of function in the affected tissue. The therapy may be administered during, or in some cases after, the symptomatic stage of the disease.

[0097] "Therapeutically effective amount" refers to the amount of active agent required to provide a therapeutic benefit to a subject. For example, a "therapeutically effective amount" is an amount that reduces or otherwise causes improvement in pathological symptoms, disease progression, or physiological conditions associated with a disease, or induces improved resistance to the disorder.

[0098] The term "B cell neoplasm" or "mature B cell neoplasm" in the context of the present invention includes small 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, lymphoblastic lymphoma, splenic marginal zone lymphoma, plasma cell neoplasms such as plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, Malt's lymphoma, nodal marginal B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granuloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, primary effusion lymphoma, and AIDS-related non-Hodgkin's tumors.

[0099] The term "characterized by CD22 expression" refers broadly to any disease or disorder in which CD22 expression is associated with or contributes to one or more pathological processes that are characteristic of that disease or disorder, including, but not limited to, B-cell neoplasms.

[0100] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for and / or treated for therapy. In one embodiment, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, and the like. Subjects can be humans, but can also include other mammals, particularly those mammals useful as laboratory models of human disease, e.g., mice, rats, and the like.

[0101] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can reasonably be administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0102] A "sterile" formulation is sterile or free or essentially free of all living microorganisms and their spores. A "frozen" formulation is a formulation at a temperature below 0°C.

[0103] A "stable" formulation is one in which the protein therein essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pub. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90) (1993). 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 different ways, including assessment of aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection), assessment of charge heterogeneity using cation exchange chromatography, imaging capillary isoelectric focusing (icIEF), or capillary zone electrophoresis, amino- or carboxy-terminal sequence analysis, mass spectrometry, SDS-PAGE analysis to compare reduced and intact antibodies, peptide map (e.g., tryptic or LYS-C) analysis, assessment of antibody biological activity or antigen-binding function, etc. Instability can involve any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, differential glycosylation, etc.

[0104] II. Detailed Description Anti-CD22 antibody Embodiments of the present invention include multispecific binding compounds comprising an anti-CD22 binding domain. Provided herein is a family of closely related heavy chain-only antibody binding domains that bind to human CD22. Antibodies in this family comprise the set of CDR sequences defined herein and set forth in Table 1, and are exemplified by the heavy chain variable region (VH) sequences provided in SEQ ID NOS: 24-84 in Table 2. The antibodies described herein offer numerous advantages that contribute to their usefulness as clinical therapeutic(s). The antibodies include members with a range of binding affinities, allowing for the selection of specific sequences with desired binding affinities. [Table 1] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0105] Suitable antibodies can be selected from those provided herein for development and therapeutic or other uses, including, but not limited to, bispecific antibodies, such as those shown in FIG. 14A, or trispecific antibodies, or for use as part of a CAR-T structure (e.g., as shown in FIG. 14B). FIG. 14A is a diagram of a non-limiting example of an anti-CD3 x anti-CD22 multispecific antibody, in which the anti-CD22 domain is monovalent and monospecific. In some embodiments, the anti-CD3 domain contains a CH1 domain and pairs with a light chain, while the anti-CD22 domain(s) are derived from a heavy-chain-only antibody, do not contain a CH1 domain, and do not interact with the light chain. In some embodiments, the two heavy chains are paired using, for example, knobs-into-holes technology.

[0106] Referring to the antibodies shown in Figure 15, Figure 15A shows an anti-CD3 x anti-CD22 bispecific antibody, in which the anti-CD22 binding arm is monovalent and monospecific, and the antigen-binding domain of the anti-CD22 arm is in a single configuration, meaning that only one antigen-binding domain is present. Figure 15B shows an anti-CD3 x anti-CD22 bispecific antibody, in which the anti-CD22 binding arm is bivalent and monospecific, and the antigen-binding domain of the anti-CD22 arm is in a tandem configuration, meaning that two identical antigen-binding domains are present in tandem. Figure 15C shows an anti-CD3 x anti-CD22 bispecific antibody, in which the anti-CD22 binding arm is bivalent and biparatopic, and the antigen-binding domain of the anti-CD22 arm is in a tandem configuration.

[0107] Determining affinity for a candidate protein can be performed using methods known in the art, such as Biacore measurements. Members of an antibody family are approximately 10 -6 ~about 10 -11 and, without limitation, may have affinity for CD22 with a Kd of about 10 -6 ~about 10 -10 , about 10 -6 ~about 10 -9 , about 10 -6 ~about 10 -8 , about 10-8 ~about 10 -11 , about 10 -8 ~about 10 -10 , about 10 -8 ~about 10 -9 , about 10 -9 ~about 10 -11 , about 10 -9 ~about 10 -10 , or any value within these ranges. Affinity selection may be confirmed by biological evaluation to modulate, e.g., block, CD22 biological activity, including in vitro assays, preclinical models, and clinical trials, as well as evaluation of potential toxicity.

[0108] The members of the antibody family herein are not cross-reactive with cynomolgus monkey CD22 protein, but can be engineered to provide cross-reactivity with cynomolgus monkey CD22 protein, or CD22 of any other animal species, if desired.

[0109] The family of CD22-specific antibodies herein comprises a VH domain comprising CDR1, CDR2, and CDR3 sequences within a human VH framework. The CDR sequences may be located within the regions surrounding amino acid residues 26-35, 53-59, and 98-117 for CDR1, CDR2, or CDR3, respectively, of the exemplary variable region sequences provided in SEQ ID NOS: 24-84. While the order of the sequences generally remains the same, one skilled in the art will understand that the CDR sequences may be in different positions if different framework sequences are selected.

[0110] The CDR1, CDR2, and CDR3 sequences of the anti-CD22 antibodies of the invention may be encompassed by the following structural formulas, where X represents a variable amino acid, which may be a specific amino acid as shown below. CDR1 G X1S I X2X3X4X5X6Y (SEQ ID NO: 104) wherein X1 is D or G; X2 is S, T, I, or N; X3 is S or D, X4 is G, S, or N, X5 is D, G, or S, X6 is Y or H. CDR2 X7X8Y X9G X 10 X 11 (SEQ ID NO: 105) wherein X7 is I or V; X8 is Y or H, X9 is S or T, X 10 is A, V, or S, X 11 is T or A. CDR3 X 12 RX 13 DSSX 14 WRS (SEQ ID NO: 106) In the formula, X 12 is T, A, or K, X 13 is D or E, X 14 is N or S.

[0111] Representative CDR1, CDR2, and CDR3 sequences are shown in Tables 1 and 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0112] In some embodiments, an anti-CD22 heavy chain-only antibody of the invention comprises the CDR1 sequence of any one of SEQ ID NOs: 1 to 10. In a particular embodiment, the CDR1 sequence is SEQ ID NO: 1.

[0113] In some embodiments, an anti-CD22 heavy chain-only antibody of the invention comprises the CDR2 sequence of any one of SEQ ID NOs: 11 to 17. In a specific embodiment, the CDR2 sequence is SEQ ID NO: 11.

[0114] In some embodiments, an anti-CD22 heavy chain-only antibody of the invention comprises the CDR3 sequence of any one of SEQ ID NOs: 18 to 23. In certain embodiments, the CDR2 sequence is SEQ ID NO: 18.

[0115] In a further embodiment, an anti-CD22 heavy chain-only antibody of the invention comprises the CDR1 sequence of SEQ ID NO:1, the CDR2 sequence of SEQ ID NO:11, and the CDR3 sequence of SEQ ID NO:18.

[0116] In further embodiments, the anti-CD22 heavy chain-only antibody of the invention comprises any of the heavy chain variable region amino acid sequences of SEQ ID NOs: 24-84 (Table 2).

[0117] In still further embodiments, the anti-CD22 heavy chain-only antibody of the invention comprises the heavy chain variable region sequence of SEQ ID NO:24.

[0118] In some embodiments, the CDR sequences of the anti-CD22 heavy chain-only antibodies of the invention comprise one or two amino acid substitutions relative to the CDR1, CDR2, and / or CDR3 sequence, or set of CDR1, CDR2, and CDR3 sequences, of any one of SEQ ID NOS: 1-23 (Figure 1). In some embodiments, the amino acid substitution(s) are at one or two of amino acid positions 4-6 of CDR1, and / or one or two of amino acid positions 2, 4-7 of CDR2, and / or one or two of amino acid positions 5 or 12 of CDR3, relative to the formula provided above. In some embodiments, the heavy chain-only anti-CD22 antibodies herein may comprise a heavy chain variable region sequence having at least about 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of the heavy chain variable region sequences of SEQ ID NOS: 24-84 (shown in Table 2).

[0119] In some embodiments, bispecific or multispecific antibodies are provided, which may have any of the configurations discussed herein, including, but not limited to, bispecific three-chain antibody-like molecules. In some embodiments, a multispecific antibody may comprise at least one heavy chain variable region that has binding specificity for CD22. In some embodiments, a multispecific antibody may comprise a heavy chain variable region that comprises at least two antigen-binding domains, each of which has binding specificity for CD22. In some embodiments, a multispecific antibody may comprise a heavy chain / light chain pair that has binding specificity for a first antigen (e.g., CD3) and a heavy chain from a heavy chain-only antibody. In certain embodiments, the heavy chain from the heavy chain-only antibody comprises an Fc portion that comprises a CH2 and / or CH3 and / or CH4 domain in the absence of a CH1 domain. In one particular embodiment, a bispecific antibody comprises a heavy chain / light chain pair that has binding specificity for an antigen on an effector cell (e.g., CD3 protein on a T cell) and a heavy chain from a heavy chain-only antibody that comprises an antigen-binding domain that has binding specificity for CD22.

[0120] In some embodiments, the multispecific antibody comprises a CD3-binding VH domain paired with a light chain variable domain. In certain embodiments, the light chain is a fixed light chain. In some embodiments, the CD3-binding VH domain comprises the CDR1 sequence of SEQ ID NO: 85, the CDR2 sequence of SEQ ID NO: 86, and the CDR3 sequence of SEQ ID NO: 87, in a human VH framework. In some embodiments, the fixed light chain comprises the CDR1 sequence of SEQ ID NO: 88, the CDR2 sequence of SEQ ID NO: 89, and the CDR3 sequence of SEQ ID NO: 90, in a human VL framework. The CD3-binding VH domain and the light chain variable domain, when combined, have binding affinity for CD3. In some embodiments, the CD3-binding VH domain comprises the heavy chain variable region sequence of SEQ ID NO: 91. In some embodiments, the CD3-binding VH domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to the heavy chain variable region sequence of SEQ ID NO: 91. In some embodiments, the fixed light chain comprises the light chain variable region sequence of SEQ ID NO: 92. In some embodiments, the fixed light chain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to the heavy chain variable region sequence of SEQ ID NO: 92.

[0121] Multispecific antibodies comprising the above-described CD3-binding VH domain and light chain variable domain have advantageous properties, as described, for example, in published PCT application WO2018 / 052503, the disclosure of which is incorporated herein by reference in its entirety. Any of the multispecific antibodies and antigen-binding domains described herein that have binding affinity for CD22 can be combined with a CD3-binding domain and a fixed light chain domain described herein to generate multispecific antibodies that have binding affinity for one or more CD22 epitopes and for CD3. [Table 4] [Table 5] [Table 6-1] [Table 6-2] [Table 7-1] [Table 7-2]

[0122] In some embodiments, bispecific or multispecific antibodies are provided, which may have any of the configurations discussed herein, including, but not limited to, bispecific three-chain antibody-like molecules. In some embodiments, a bispecific antibody may comprise at least one heavy chain variable region with binding specificity for CD22 and at least one heavy chain variable region with binding specificity for a protein other than CD22. In some embodiments, a bispecific antibody may comprise a heavy / light chain pair with binding specificity for a first antigen, a heavy chain from a heavy chain-only antibody comprising an Fc portion comprising a CH2 and / or CH3 and / or CH4 domain in the absence of a CH1 domain, and an antigen-binding domain that binds to an epitope of a second antigen or a different epitope of the first antigen. In one particular embodiment, a bispecific antibody comprises a heavy / light chain pair with binding specificity for an antigen on an effector cell (e.g., CD3 protein on a T cell) and a heavy chain from a heavy chain-only antibody comprising an antigen-binding domain with binding specificity for CD22.

[0123] In some embodiments where the binding compound of the invention is a bispecific antibody, one arm of the antibody (one binding moiety, or one binding unit) is specific for human CD22, and the other arm can be specific for a target cell, a tumor-associated antigen, a target antigen such as an integrin, a pathogen antigen, a checkpoint protein, etc. Target cells specifically include cancer cells, including, but not limited to, cells from hematological tumors, e.g., B-cell tumors, as described below. In some embodiments, one arm of the antibody (one binding moiety, or one binding unit) is specific for human CD22, and the other arm is specific for CD3.

[0124] In some embodiments, the binding compound comprises an anti-CD3 light chain polypeptide comprising the sequence of SEQ ID NO: 92 linked to the sequence of SEQ ID NO: 97, an anti-CD3 heavy chain polypeptide comprising the sequence of any one of SEQ ID NOs: 98, 99, 100, or 101, and an anti-CD22 heavy chain polypeptide comprising the sequence of any one of SEQ ID NOs: 24-84 linked to the sequence of any one of SEQ ID NOs: 93, 94, 95, or 96. These sequences can be combined in various ways to produce bispecific antibodies of a desired IgG subclass, e.g., IgG1, IgG4, silenced IgG1, silenced IgG4.

[0125] Various formats of bispecific antibodies are within the scope of the present invention, including, but not limited to, single-chain polypeptides, two-chain polypeptides, three-chain polypeptides, four-chain polypeptides, and multiples thereof. Multispecific antibodies herein specifically include T cell multispecific (e.g., bispecific) antibodies that bind to CD22 (anti-CD22 x anti-CD3 antibodies) and CD3, which are selectively expressed on mature B cells. Such antibodies induce potent T cell-mediated killing of cells expressing CD22.

[0126] Antibody preparation The multispecific binding compounds of the present invention can be prepared by methods well known in the art. In a preferred embodiment, the heavy chain antibodies of the present invention are produced by transgenic animals, including transgenic mice and rats, preferably rats, in which endogenous immunoglobulin genes have been knocked out or disabled. In a preferred embodiment, the heavy chain antibodies of the present invention are produced in UniRat™. UniRat™ has silenced endogenous immunoglobulin genes and uses a human immunoglobulin heavy chain translocus to express a diverse and naturally optimized repertoire of fully human HCAbs. While endogenous immunoglobulin loci in rats can be knocked out or silenced using various techniques, in UniRat™, zinc finger (endo)nuclease (ZNF) technology was used to inactivate the endogenous rat heavy chain J locus, light chain Cκ locus, and light chain Cλ locus. ZNF constructs for microinjection into oocytes can produce IgH and IgL knockout (KO) strains. For 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. An advantage of ZNF technology is that the non-homologous ends joined to silence genes or loci through deletions of up to several kb can also provide target sites for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). Human heavy chain antibodies produced in UniRat™ are called UniAbs™ and can bind to epitopes that cannot be attacked by conventional antibodies. Their high specificity, affinity, and small size make UniAbs™ ideal for mono- and polyspecific applications.

[0127] In addition to UniAbs™, the present specification specifically includes heavy chain-only antibodies lacking camelid VHH frameworks and mutations, as well as functional VH regions thereof. Such heavy chain-only antibodies can be produced in transgenic rats or mice containing a fully human heavy chain-only locus, as described, for example, in WO 2006 / 008548, although other transgenic mammals, such as rabbits, guinea pigs, and rats, can also be used, with rats and mice being preferred. Heavy chain-only antibodies, including their VHH or VH functional fragments, can also be produced by recombinant DNA technology, for example, by expression of encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including mammalian cells (e.g., CHO cells), E. coli, or yeast.

[0128] Heavy-chain-only antibody domains combine the advantages of antibodies and small molecule drugs: they can be monovalent or polyvalent, have low toxicity, and are cost-effective to manufacture. Their small size allows for easy administration, including oral or topical administration, and they are characterized by high stability, including gastrointestinal stability, and their half-lives can be tailored to the desired use or indication. Additionally, VH and VHH domains of HCAbs can be produced cost-effectively.

[0129] In certain embodiments, heavy chain antibodies of the present invention, including UniAb™, have a native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Kabat numbering system) that is replaced with another amino acid residue capable of disrupting a surface-exposed hydrophobic patch comprising or associated with the native amino acid residue at that position. Such a hydrophobic patch, normally buried at the interface with the antibody light chain constant region, is surface-exposed in HCAbs and is responsible, at least in part, for undesired aggregation and light chain association of HCAbs. The substituted amino acid residue is preferably charged, more preferably 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 derived from transgenic animals contain a Trp to Arg mutation at position 101. The resulting HCAbs preferably have high antigen-binding affinity and solubility under physiological conditions in the absence of aggregation.

[0130] As part of the present invention, human anti-CD22 heavy chain antibodies (UniAbs™) with unique sequences from UniRat™ animals were identified that bind to human CD22 in ELISA protein and cell binding assays. The identified heavy chain variable region (VH) sequences (see, e.g., Table 2) are positive for human CD22 protein binding and / or binding to CD22+ cells, and are all negative for binding to cells that do not express CD22.

[0131] Heavy chain antibodies that bind to non-overlapping epitopes on the CD22 protein, such as UniAb™, can be identified by competitive binding assays, such as enzyme-linked immunosorbent assays (ELISA assays) or flow cytometry competitive binding assays. For example, competition between a known antibody that binds to a target antigen and an antibody of interest can be used. Using this approach, a set of antibodies can be divided into those that compete with the reference antibody and those that do not. Non-competing antibodies are identified as those that bind to distinct epitopes that do not overlap with the epitope bound by the reference antibody. Often, one antibody is immobilized, the antigen is bound, and a second labeled (e.g., biotinylated) antibody is tested for its ability to bind to the captured antigen in an ELISA assay. This can also be accomplished using surface plasmon resonance (SPR) platforms including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and MX96 SPR Imager (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 herein.

[0132] Typically, an antibody "competes" with a reference antibody if it causes about a 15-100% reduction in binding of the reference antibody to a target antigen, as determined by standard techniques, 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 about 80%, at least about 85%, at least about 90%, at least about 95% or more.

[0133] Pharmaceutical Compositions, Uses and Methods of Treatment Another aspect of the present invention is to provide pharmaceutical compositions comprising one or more multispecific binding compounds of the present invention in admixture with a suitable pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier is exemplified by, but not limited to, an adjuvant, a solid carrier, water, a buffer, or other carrier used in the art to carry therapeutic ingredients, or a combination thereof.

[0134] In one embodiment, the pharmaceutical composition comprises a heavy chain antibody (e.g., UniAb™) that binds to CD22. In another embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb™) that has binding specificities for two or more non-overlapping epitopes on the CD22 protein. In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb™) that has binding specificity for CD22 and binding specificity for a binding target on an effector cell (e.g., a binding target on a T cell, such as the CD3 protein on a T cell).

[0135] Pharmaceutical compositions of antibodies used according to the present invention are prepared for storage by mixing the protein having the desired purity, such as in the form of a lyophilized formulation or an aqueous solution, with any pharmaceutically acceptable carrier, excipient, or stabilizer (see, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). 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 (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; The composition may comprise a protein such as albumin, gelatin, or immunoglobulin, a hydrophilic polymer such as polyvinylpyrrolidone, an amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine, a monosaccharide, disaccharide, and other carbohydrate including glucose, mannose, or dextrin, a chelating agent such as EDTA, a sugar such as sucrose, mannitol, trehalose, or sorbitol, a salt-forming counterion such as sodium, a metal complex (e.g., a Zn-protein complex), and / or a non-ionic surfactant such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0136] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a dose for a single administration). The formulation depends on the selected route of administration. The antibodies herein can be administered by intravenous injection or infusion, or subcutaneously. For injection administration, the antibodies herein can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution can contain carriers, excipients, or stabilizers as described above. Alternatively, the antibodies can be in lyophilized form for constitution with a suitable vehicle, such as sterile, pyrogen-free water, before use.

[0137] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used for the heavy chain antibodies of the invention, including UniAb™. Subcutaneous antibody formulations are described, for example, in U.S. Patent No. 20160355591 and U.S. Patent No. 20160166689.

[0138] How to use The heavy chain-only anti-CD22 antibodies, multispecific antibodies, and pharmaceutical compositions described herein can be used to treat diseases and conditions characterized by expression of CD22, including, but not limited to, the conditions and diseases further described herein.

[0139] CD22 is a 135 kDa type I transmembrane protein that is expressed at low levels on premature and immature B cells, maximally on mature B cells, and ultimately downregulated on plasma cells (e.g., Walker et al., Immunology, 2008 Mar;123(3)314-25). CD22 is strongly expressed on follicular (primary and secondary B cell zones), mantle, and marginal zone B cells and has been reported to be present in 60% to 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 several hematological malignancies, CD22 is a promising target for antibody-based therapies.

[0140] In one aspect, the CD22 heavy chain antibodies (e.g., UniAb™) and pharmaceutical compositions herein can be used to treat hematological malignancies characterized by expression of CD22, 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).

[0141] Diffuse large B-cell lymphoma (DLBCL or DLBL) 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 in the United States and the United Kingdom. It is characterized as an aggressive cancer that can arise in virtually any part of the body. The cause of DLBCL is poorly understood; it can arise from malignant transformation of normal B cells as well as other types of lymphoma or leukemia cells. Treatment approaches generally involve chemotherapy and radiation, with an overall average 5-year survival rate of approximately 58% for adults. Several monoclonal antibodies have shown promise for treating DLBCL, but consistent clinical efficacy has not yet been conclusively demonstrated. Therefore, new therapies, including immunotherapy, for DLBCL are greatly needed.

[0142] In another aspect, the CD22 heavy chain antibodies (e.g., UniAb™) and pharmaceutical compositions herein 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).

[0143] The effective amount of the compositions of the invention for treating a disease will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, such as companion animals such as dogs, cats, and horses, and laboratory animals such as rabbits, mice, and rats, can also be treated. Treatment dosages can be titrated to optimize safety and efficacy.

[0144] Dosage levels can be easily determined by those skilled in the art and can be modified as needed, for example, to modify the subject's response to treatment. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the host treated and the specific mode of administration. Generally, a unit dosage form contains between about 1 mg and about 500 mg of active ingredient.

[0145] In some embodiments, the therapeutic dosage of the agent can range from about 0.0001 to 100 mg / kg, and more usually 0.01 to 5 mg / kg, of host body weight. For example, dosages can be 1 mg / kg or 10 mg / kg body weight, or within the range of 1 to 10 mg / kg. Exemplary treatment regimens involve administration once every two weeks, once a month, or once every three to six months. The therapeutic entities of the present invention are typically administered on multiple occasions. The intervals between single administrations can be weekly, monthly, or yearly. The intervals can also be irregular, as determined by measuring the patient's blood levels of the therapeutic entity. Alternatively, the therapeutic entities of the present invention can be administered as sustained-release formulations, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0146] Typically, the compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquid vehicles prior to injection can also be prepared. The pharmaceutical compositions herein are suitable for intravenous or subcutaneous administration, either directly or after reconstitution of a solid (e.g., lyophilized) composition. The preparations can also be emulsified or encapsulated in liposomes or microparticles such as polylactides, polyglycolides, or copolymers for enhanced adjuvant effect, as described above. Langer, Science 249:1527, 1990, and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of the present invention can be administered in the form of depot injections or implant preparations, which can be formulated in a manner that allows sustained or pulsed release of the active ingredient. The pharmaceutical compositions are generally sterile, substantially isotonic, and formulated in full compliance with all U.S. Food and Drug Administration Good Manufacturing Practice (GMP) regulations.

[0147] The toxicity of the antibodies and antibody constructs described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate a non-toxic dosage range for use in humans. The dosage of the antibodies described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. Dosage can vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition.

[0148] Compositions for administration will generally contain an antibody or other agent (e.g., another abrasive) dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. These solutions are sterile and generally free of undesirable material. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions of the present invention can contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions (pH adjusting agents, buffers, and toxicity adjusting agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate). The concentration of the active agent in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, weight, and the like, in accordance with the particular mode of administration selected and the patient's needs (see, e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, *The Pharmacological Basis of Therapeutics* (Hardman et al., eds., 1996)).

[0149] Also within the scope of the present invention are kits comprising the active agents of the present invention and their formulations, as well as instructions for use. The kits may further contain at least one additional reagent, such as a chemotherapeutic agent. The kits typically include a label indicating the intended use of the contents of the kit. As used herein, the term "label" includes any writing or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0150] Now that the present invention is fully described, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. [Example]

[0151] 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 interference. Anti-human IgG Fc capture (AHC) biosensors (ForteBio, part number: 18-5064) were hydrated in assay buffer (1x PBS, 0.1% BSA, 0.02% Tween-20, pH 7.2) and pretreated in 100 mM glycine pH 1.5. A baseline was established in assay buffer for 120 seconds. The AHC biosensors were then immobilized with UniAb™ at a concentration of 5 μg / mL for 120 seconds. Another baseline (120 seconds) was established in assay buffer. Next, they were immersed in a seven-point 1:2 dilution series 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 the loaded biosensor and served as a reference well. Association was observed for 600 seconds, followed by dissociation for 900 seconds. Data analysis was performed using Octet Data Analysis v9.0 (ForteBio). Binding kinetics were analyzed using a standard 1:1 binding model.

[0152] 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 UniAb™ for 30 minutes at 4°C. After incubation, cells were washed twice with flow cytometry buffer (1x PBS, 1% BSA, 0.1% NaN3) and stained with goat F(ab')2 anti-human IgG conjugated to R-phycoerythrin (PE) (Southern Biotech, catalog no. 2042-09) to detect cell-bound antibodies. After 20 minutes of 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 cynomolgus monkey CD22-positive cells was determined using the same protocol with the following modifications: target cells were from CHO cells stably transfected to express the extracellular domain of cynomolgus monkey CD22, and each antibody was tested at a single concentration (approximately 1.7 μg / mL), so EC50 values were not calculated.

[0153] Example 1: Genetically engineered rats expressing heavy chain-only antibodies The "human-rat" IgH locus was constructed and assembled in several parts, including the human J H Following downstream modification and ligation of rat C region genes, human V H This was followed by the upstream addition of the 6-D fragment region. H Two BACs [BAC6 and BAC3] carrying separate clusters of genes were cloned to contain human V H 6. All Ds, all Js H , and modified rat Cγ2a / 1 / 2b (ΔC H A BAC called Georg encoding the assembled and modified region containing 1) was co-injected.

[0154] Transgenic rats were generated that contained an artificial heavy chain immunoglobulin locus in an unrearranged configuration. H 1), IgG1(ΔC H 1), IgG2b (ΔC H 1) The gene is C H The transgenic rats lacked a segment of the IgE gene. The constant region genes IgE and IgA, as well as the 3' enhancer, were included in the Georg BAC. RT-PCR and serum analysis (ELISA) of the transgenic rats demonstrated productive rearrangement of the transgenic immunoglobulin loci and expression of heavy-chain-only antibodies of various isotypes in the serum. The transgenic rats were crossed with rats carrying the mutant endogenous heavy and light chain loci previously described in U.S. Patent Publication No. 2009 / 0098134A1. Analysis of such animals demonstrated inactivation of rat immunoglobulin heavy and light chain expression and high-level expression of heavy-chain antibodies with variable regions encoded by human V, D, and J genes. Immunization of the transgenic rats resulted in the generation of high-titer serum responses of antigen-specific heavy-chain antibodies. These transgenic rats expressing heavy-chain antibodies with human V, D, and J regions were designated UniRat™.

[0155] Example 2: Immunization Immunization with recombinant extracellular domain of CD22 Twelve UniRat animals (6 HC27 and 6 HC28) were immunized with recombinant human CD22 protein. Animals were immunized according to standard protocols using Titermax / Alhydrogel adjuvant. Recombinant CD22 extracellular domain was purchased from R&D Systems, diluted in sterile saline, and combined with adjuvant. The immunogen was combined with Titermax and Alhydrogel adjuvant. A prime immunization with immunogen in Titermax was administered in the left and right legs. Subsequent boosts were administered in the presence of Alhydrogel, and a boost with immunogen in PBS 3 days prior to harvest. Serum was collected from the rats at the time of the final bleed to determine serum titers.

[0156] Serum titer results Serum titer summary information is shown in Figure 17. In the graph shown in Figure 17, each row represents an individual animal. The graph legend indicates the identification number of each individual animal. The binding activity of an eight-point dilution series of sera was tested by ELISA against huCD22+Fc protein, huCD22+His-tag protein, rhesus CD22+His-tag protein, and His-tagged off-target protein. A wide range of serum reactivity levels against both human and rhesus CD22 proteins was observed within this group of animals. Serum responses against the His protein tag were also observed.

[0157] Example 3: Binding to CD22-expressing cell lines Figure 16 summarizes the target binding activity of anti-CD22 heavy chain only antibodies described herein. Column 1 shows the clone identification number of the anti-CD22 heavy chain only antibody. Column 2 shows the binding affinity (KD) to the protein measured in molar concentration. Column 3 shows the dissociation constant (K-off rate) for binding to the protein measured in seconds. Column 4 shows binding to Daudi cells measured as fold over background MFI signal. Column 5 shows binding to CHO cells stably expressing cynomolgus monkey CD22 measured as fold over background MFI signal. Column 6 shows binding to CHO cells not expressing CD22 protein measured as fold over background MFI signal.

[0158] Example 4: T cell-mediated cytotoxicity of CD22-positive cells using resting human pan-T cells Unstimulated human T cells were incubated with CD22-positive cells (Daudi) and different concentrations of bispecific antibodies. After 48 hours, the cells were subjected to flow cytometry to measure cytotoxicity. Supernatants from the cell cultures were used to measure the release of the cytokine IL-2. POS CTRL antibodies refer to antibodies containing the same anti-CD22 arm but with a stronger affinity anti-CD3 arm. The results are shown in Figure 1A and Figure 1B.

[0159] Unstimulated human T cells were incubated with CD22-positive cells (SUDHL10) and different concentrations of bispecific antibodies. After 72 hours, the cells were subjected to flow cytometry to measure cytotoxicity. Supernatants from the cell cultures were used to measure the release of the cytokine IL-2. POS CTRL antibodies refer to antibodies containing the same anti-CD22 arm but with a stronger affinity anti-CD3 arm. The results are shown in Figures 2A and 2B.

[0160] Unstimulated human T cells were incubated with a CD22-positive DL-BCL cell line (RI-1) and different concentrations of bispecific antibodies at various effector:target (E:T) cell ratios of 10:1, 5:1, or 1:1. After 72 hours, the cells were subjected to flow cytometry to measure cytotoxicity. Supernatants from the cell cultures were used to measure the release of the cytokine IL-2. POS CTRL antibodies refer to antibodies containing the same anti-CD22 arm but with a stronger affinity anti-CD3 arm. The data show that the % cytotoxicity depends on the E:T ratio. The results are shown in Figures 3A and 3B.

[0161] Example 5: T cell-mediated cytotoxicity of CD22-positive cells using activated human pan-T cells Activated human T cells were incubated with CD22-positive cells (Daudi and RI-1) or CD22-negative cell lines (K562) and different concentrations of bispecific antibodies. Cell lysis was measured using a calcein-based fluorescent readout. The bispecific CD22×CD3_F2F binding compound specifically induced lysis of CD22+ cells but not CD22-K562 cells. POS CTRL antibody refers to an antibody containing the same anti-CD22 arm but a stronger affinity anti-CD3 arm. NEG CTRL refers to an antibody with a nonspecific tumor arm and the same anti-CD3 arm as anti-CD3_F2F. The results are shown in Figure 4.

[0162] Example 6: Cell binding of bispecific antibodies against CD22 and CD3 CD22-positive cells, Daudi, Raji, and Ramos, and CD22-negative cells, K562, were incubated with the bispecific antibody. Cell binding was measured by flow cytometry using an anti-human IgG secondary antibody reagent. The data show that the bispecific antibody binds to CD22+ cells but not to CD22- cells. POS CTRL antibody refers to an antibody containing the same anti-CD22 arm but a stronger affinity anti-CD3 arm. NEG CTRL refers to an antibody with a nonspecific tumor arm and the same anti-CD3 arm as anti-CD3_F2F. The results are shown in Figure 5.

[0163] Example 7: In vivo efficacy study using CD22-1 x CD3_F2F in Daudi xenografts To test the in vivo efficacy of CD22-1xCD3_F2F, various doses of CD22-1xCD3_F2F were administered to female NSG mice implanted with Daudi cells (5e6 cells / mouse), as shown in Figure 6. The treatment schedule is shown in Table 7 below. The efficacy of treatment was assessed using mean tumor volume, body weight, percent body weight change, and individual tumor volume. [Table 8]

[0164] Data from CD22-1xCD3_F2F are shown in Figures 7-13 in comparison with the negative control and rituximab, demonstrating the efficacy of CD22-1xCD3_F2F.

[0165] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended to cover methods and structures within the scope of these claims and their equivalents.

Claims

1. 1. A multispecific binding compound that binds to CD3, comprising: A heavy chain variable region comprising: (a) a CDR1 sequence having no more than two substitutions in SEQ ID NO: 85, and / or (b) a CDR2 sequence having no more than two substitutions in SEQ ID NO: 86, and / or (c) the heavy chain variable region comprising a CDR3 sequence with no more than two substitutions in SEQ ID NO: 87; a light chain variable region.

2. 2. The multispecific binding compound of claim 1, wherein the heavy chain CDR1, CDR2, and CDR3 sequences are in a human VH framework.

3. a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences in a human VH framework, wherein each CDR sequence comprises a sequence having at least 85% identity to any one of SEQ ID NOs: 85-87; and a light chain variable region.

4. A heavy chain variable region comprising: (a) a CDR1 sequence having no more than two substitutions in SEQ ID NO: 85; and (b) a CDR2 sequence having no more than two substitutions in SEQ ID NO: 86; and (c) the heavy chain variable region comprising a CDR3 sequence with no more than two substitutions in SEQ ID NO: 87; and a light chain variable region.

5. a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 85, the CDR2 sequence of SEQ ID NO: 86, and the CDR3 sequence of SEQ ID NO: 87; and a light chain variable region.

6. 6. The multispecific binding compound of any one of claims 1 to 5, wherein the light chain variable region comprises CDR1, CDR2, and CDR3 sequences in a human VL framework, and each CDR sequence comprises a sequence with no more than three amino acid substitutions relative to the CDR sequence or set of CDR sequences of SEQ ID NO: 92, or wherein the CDR sequences comprise sequences with at least 85% identity to the CDR sequence or set of CDR sequences of SEQ ID NO:

92.

7. 7. The multispecific binding compound of any one of claims 1 to 6, wherein the light chain variable region comprises the CDR1 sequence of SEQ ID NO: 88, the CDR2 sequence of SEQ ID NO: 89, and the CDR3 sequence of SEQ ID NO:

90.

8. The multispecific binding compound of any one of claims 1 to 7, wherein the heavy chain variable region comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

91.

9. The multispecific binding compound of any one of claims 1 to 7, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

91.

10. The multispecific binding compound of any one of claims 1 to 9, wherein the light chain variable region comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

92.

11. The multispecific binding compound of any one of claims 1 to 9, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

92.

12. 1. A multispecific binding compound comprising a first binding unit having binding affinity for CD22 and a second binding unit having binding affinity for CD3, wherein the first binding unit comprises: (a) a CDR1 having no more than two substitutions in any of the amino acid sequences of SEQ ID NOs: 1-10; and / or (b) a CDR2 having no more than two substitutions in any of the amino acid sequences of SEQ ID NOs: 11-17; and / or (c) the multispecific binding compound, comprising a CDR3 having no more than two substitutions in any of the amino acid sequences of SEQ ID NOs: 18-23.

13. 13. The multispecific binding compound of claim 12, wherein the CDR1, CDR2, and CDR3 sequences of the first binding unit are in a human framework.

14. 14. The multispecific binding compound of claim 12 or 13, wherein the first binding unit further comprises a heavy chain constant region sequence in the absence of a CH1 sequence.

15. the first linking unit is (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 NOs: 11-17, and / or (c) a heavy chain variable region comprising a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18-23.

16. (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) the multispecific binding compound of claim 15, comprising a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18-23.

17. (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; (b) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 12, and a CDR3 sequence of SEQ ID NO: 19, or (c) the multispecific binding compound of any one of claims 12 to 16, comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 12, and the CDR3 sequence of SEQ ID NO:

20.

18. 13. The multispecific binding compound of claim 12, comprising a heavy chain variable region having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 24-84.

19. 19. The multispecific binding compound of claim 18, comprising a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 24-84.

20. 20. The multispecific binding compound of claim 19, comprising the heavy chain variable region sequence of SEQ ID NO:

24.

21. 1. A multispecific binding compound comprising a first binding unit having binding affinity for CD22 and a second binding unit having binding affinity for CD3, wherein the first binding unit comprises: a heavy chain variable region, the heavy chain variable region comprising: (a) a CDR1 sequence of the formula: GX １ 6X ２ X ３ X ４ X ５ X ６ Y (SEQ ID NO: 104) (In the formula, X １ is D or G, X ２ is S, T, I, or N; X ３ is S or D, X ４ is G, S, or N, X ５ is D, G, or S; X ６ is Y or H), and (b) a CDR2 sequence of the formula: X ７ X ８ Y X ９ GX １０ X １１ (SEQ ID NO: 105) (In the formula, X ７ is I or V, X ８ is Y or H, X ９ is S or T, X １０ is A, V, or S; X １１ is T or A), and (c) a CDR3 sequence of the following formula: X １２ R X １３ D S S X １４ W R S (SEQ ID NO: 106) (In the formula, X １２ is T, A, or K; X １３ is D or E, X １４ is N or S).

22. 1. A multispecific binding compound comprising a first binding unit having binding affinity for CD22 and a second binding unit having binding affinity for CD3, wherein the first binding unit comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences in a human VH framework, and the CDR sequences comprise sequences with no more than two substitutions in the CDR sequences selected from the group consisting of SEQ ID NOs: 1-23.

23. 1. A multispecific binding compound comprising a first binding unit having binding affinity for CD22 and a second binding unit having binding affinity for CD3, wherein the first binding unit comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences in a human VH framework, and the CDR sequences are selected from the group consisting of SEQ ID NOs: 1-23.

24. 1. A multispecific binding compound comprising a first binding unit having binding affinity for CD22 and a second binding unit having binding affinity for CD3, wherein the first binding unit comprises: a heavy chain variable region, the heavy chain variable region comprising, in a human VH framework: (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) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 12, and a CDR3 sequence of SEQ ID NO: 19, or (c) the multispecific binding compound, comprising a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 12, and a CDR3 sequence of SEQ ID NO:

20.

25. 25. The multispecific binding compound of claim 24, which is bispecific.

26. 10. The multispecific binding compound of any one of the preceding claims, which is in a CAR-T format.

27. 1. A multispecific binding compound comprising: (i) a heavy chain variable region having binding affinity for CD3, comprising the CDR1 sequence of SEQ ID NO: 85, the CDR2 sequence of SEQ ID NO: 86, and the CDR3 sequence of SEQ ID NO: 87 in a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 88, the CDR2 sequence of SEQ ID NO: 89, and the CDR3 sequence of SEQ ID NO: 90 in a human VL framework; (iii) an antigen-binding domain of an anti-CD22 heavy chain antibody comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18 in a human VH framework.

28. 1. A multispecific binding compound comprising: (i) a heavy chain variable region having binding affinity for CD3, comprising the CDR1 sequence of SEQ ID NO: 85, the CDR2 sequence of SEQ ID NO: 86, and the CDR3 sequence of SEQ ID NO: 87 in a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 88, the CDR2 sequence of SEQ ID NO: 89, and the CDR3 sequence of SEQ ID NO: 90 in a human VL framework; (iii) an antigen-binding domain of an anti-CD22 heavy chain antibody comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 12, and the CDR3 sequence of SEQ ID NO: 19 in a human VH framework.

29. 1. A multispecific binding compound comprising: (i) a heavy chain variable region having binding affinity for CD3, comprising the CDR1 sequence of SEQ ID NO: 85, the CDR2 sequence of SEQ ID NO: 86, and the CDR3 sequence of SEQ ID NO: 87 in a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 88, the CDR2 sequence of SEQ ID NO: 89, and the CDR3 sequence of SEQ ID NO: 90 in a human VL framework; (iii) an antigen-binding domain of an anti-CD22 heavy chain antibody comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 12, and the CDR3 sequence of SEQ ID NO: 20 in a human VH framework.

30. The multispecific binding compound of any one of claims 1 to 29, comprising a human IgG1 Fc region.

31. 31. The multispecific binding compound of claim 30, wherein the human IgGl Fc region is a silenced human IgGl Fc region.

32. The multispecific binding compound of any one of claims 1 to 29, comprising a human IgG4 Fc region.

33. 33. The multispecific binding compound of claim 32, wherein the human IgG4 Fc region is a silenced human IgG4 Fc region.

34. A pharmaceutical composition comprising the multispecific binding compound of any one of claims 1 to 33.

35. 35. A method for the treatment of a B-cell disorder characterized by expression of CD22, comprising administering to a subject having said disorder a multispecific binding compound of any one of claims 1 to 33, or a pharmaceutical composition of claim 34.

36. 34. Use of a multispecific binding compound according to any one of claims 1 to 33 in the preparation of a medicament for the treatment of a B cell disorder characterized by expression of CD22.

37. 34. The multispecific binding compound of any one of claims 1 to 33 for use in the treatment of a B cell disorder characterized by expression of CD22.

38. 38. The method, use or multispecific binding compound of any one of claims 35 to 37, wherein the disorder is diffuse large B-cell lymphoma (DLBCL).

39. 38. The method, use or multispecific binding compound of any one of claims 35 to 37, wherein the disorder is non-Hodgkin's lymphoma (NHL).

40. 38. The method, use or multispecific binding compound of any one of claims 35 to 37, wherein the disorder is systemic lupus erythematosus (SLE).

41. 38. The method, use or multispecific binding compound of any one of claims 35 to 37, wherein the disorder is rheumatoid arthritis (RA).

42. 38. The method, use or multispecific binding compound of any one of claims 35 to 37, wherein said disorder is multiple sclerosis (MS).

43. A polynucleotide encoding the multispecific binding compound of any one of claims 1 to 33.

44. A vector comprising the polynucleotide of claim 43.

45. A cell comprising the vector of claim 44.

46. 46. A method of producing a multispecific binding compound of any one of claims 1 to 33, comprising growing a cell of claim 45 under conditions permissive for expression of said binding compound, and isolating said binding compound from said cell.

47. 34. A method of making a multispecific binding compound of any one of claims 1 to 33, comprising immunizing a UniRat animal with CD22 and identifying a CD22-binding heavy chain sequence.

48. 35. A method of treatment comprising administering to an individual an effective dose of a multispecific binding compound of any one of claims 1 to 33, or a pharmaceutical composition of claim 34.

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