Multispecific heavy chain antibodies that bind to CD22 and CD3

Abstract

Disclosed are multispecific human heavy chain antibodies (e.g., UniAbs) that bind to CD22 and CD3. TM ), together with methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use for treating disorders characterized by CD22 expression.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to the filing date of U.S. Provisional Patent Application Serial No. 62 / 861,708, filed on June 14, 2019, the disclosure of which is incorporated herein by reference in its entirety. Field of the Invention

[0003] The present invention relates to multispecific human heavy chain antibodies (e.g., UniAbs) that bind to CD22 and CD3. TM The present invention further relates to methods of preparing such antibodies, compositions (including pharmaceutical compositions) comprising such antibodies, and their use for treating disorders characterized by CD22 expression. Background of the Invention

[0005] CD22

[0006] 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. Together with CD20 and CD19, the restricted B cell expression of CD22 makes it an attractive target for the treatment of B cell malignancies. Monoclonal antibodies specific for CD22 have been described in the literature (e.g., Jabbour, Elias, et al. "Monoclonal antibodies in acute lymphoblastic leukemia." Blood 125.26 (2015): 4010-4016) and have been used therapeutically as standard monoclonal drugs (e.g., epratuzumab) and 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 toCD19-targeted CAR immunotherapy." Nature medicine (2017)).

[0007] Heavy chain antibody

[0008] 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 heavy chain CH1 constant region. Additional residues are present in the heavy chain framework 2 (FR2) and framework 4 (FR4) regions that also contribute to this hydrophobic interaction between the heavy and light chains.

[0009] However, it is known that sera of camelids (the suborder Tylopoda, which includes camels, dromedaries, and llamas) contain the main type of antibodies composed only of paired H-chains (heavy chain-only antibodies or UniAbs). TM UniAbs of Camelidae (Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanaco, Lama alpaca and Lama vicugna) TM They have a unique structure consisting of a single variable domain (VHH), a hinge region, and two constant domains (CH2 and CH3) that are highly homologous to the CH2 and CH3 domains of classical antibodies. TM The first domain of the constant region (CH1) is missing, which is present in the genome but is spliced ​​during mRNA processing. The absence of the CH1 domain explains the UniAbs TM The light chain is not present because this domain is the anchoring site for the constant domain of the light chain. TM Natural evolution is 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 (named IgNAR) that lacks polypeptide light chains and is composed entirely of heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce variable domains (vNARs) of single heavy chain polypeptides (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)).

[0010] 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 relative to tetrameric antibodies. Sitia et al. (1990) Cell, 60, 781-790 demonstrated 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 resulting antibodies retained VH binding specificity and effector function.

[0011] Heavy chain antibodies with high specificity and affinity for a variety of antigens can be produced by immunization (van der Linden, RH et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH portion can be easily cloned and expressed in yeast (Frenken, LGJ et al. J. Biotechnol. 78, 11-21 (2000)). Its expression level, 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)).

[0012] Mice in which the lambda light (L) chain locus and / or the lambda and kappa L chain loci have been functionally silenced and antibodies produced by such mice are described in U.S. Pat. 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. Application Publication No. 20100122358; Nguyen et al., 2003, Immunology; 109(1), 93-101; Brüggemann 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 via 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 comprising heterologous heavy chain loci that produce such antibodies are described in U.S. Pat. Nos. 8,883,150 and 9,365,655. CAR-T structures comprising 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

[0013] Various aspects of the present invention relate to heavy chain antibodies having binding affinity for CD22, including but not limited to UniAbs TM Other aspects of the invention relate to methods of making such antibodies, compositions comprising such antibodies, and their use for treating disorders characterized by CD22 expression.

[0014] Aspects of the invention include multispecific binding compounds that bind to CD3, comprising: a heavy chain variable region comprising: (a) a CDR1 sequence having two or fewer substitutions in SEQ ID NO: 85; and / or (b) a CDR2 sequence having two or fewer substitutions in SEQ ID NO: 86; and / or (c) a CDR3 sequence having two or fewer substitutions in 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 a heavy chain CDR1, CDR2, and CDR3 sequence 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 the binding compound further comprises a light chain variable region.

[0015] In some embodiments, a multispecific binding compound comprises: a heavy chain variable region comprising: (a) a CDR1 sequence having two or fewer substitutions in SEQ ID NO:85; and (b) a CDR2 sequence having two or fewer substitutions in SEQ ID NO:86; and (c) a CDR3 sequence having two or fewer substitutions in SEQ ID NO:87; and the binding compound also comprises a light chain variable region.

[0016] In some embodiments, the multispecific binding compound comprises: a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO:85, a CDR2 sequence of SEQ ID NO:86, and a CDR3 sequence of SEQ ID NO:87; and the binding compound further comprises a light chain variable region.

[0017] In some embodiments, the light chain variable region comprises CDR1, CDR2 and CDR3 sequences in a human VL framework, wherein each CDR sequence comprises a sequence having 3 or fewer amino acid substitutions relative to a CDR sequence or a set of CDR sequences in SEQ ID NO: 92; or wherein the CDR sequence comprises a sequence having at least 85% identity to a CDR sequence or a set of CDR sequences in 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 having at least 95% identity to SEQ ID NO: 91. In one embodiment, the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the light chain variable region comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 92. In some embodiments, the light chain variable region comprises an amino acid sequence of SEQ ID NO: 92.

[0018] 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 two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 1 to 10; and / or (b) a CDR2 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 11 to 17; and / or (c) a CDR3 having two or fewer substitutions in any one of the amino acid sequences of SEQ ID NOs: 18 to 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 without a CH1 sequence.

[0019] 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 to 10; and / or (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 11 to 17; and / or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18 to 23.

[0020] In some embodiments, the multispecific binding compound comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 to 10; and (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 11 to 17; and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18 to 23.

[0021] In some embodiments, the multispecific binding compound comprises: (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) 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. In some embodiments, the multispecific binding compound comprises a heavy chain variable region that has at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 24 to 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 to 84. In some embodiments, the multispecific binding compound comprises a heavy chain variable region sequence of SEQ ID NO: 24.

[0022] 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 heavy chain variable region comprising: (a) a CDR1 sequence of the formula: G X1 SI X2 X3 X4 X5 X6 Y (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 CDR2 sequence of the formula: X7 X8 YX9 GX 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; and X 11 is T or A; and (c) a CDR3 sequence of the formula: 12 RX 13 DSSX 14 WRS (SEQ ID NO: 106), wherein X 12 is T, A or K; X 13 is D or E; and X14 It is N or S.

[0023] Various 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 heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, wherein the CDR sequences comprise a sequence having two or fewer substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 1-23.

[0024] Various 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 heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, wherein the CDR sequences are selected from the group consisting of SEQ ID NOs: 1-23.

[0025] Various 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 heavy chain variable region comprising: (a) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 11, and a CDR3 sequence of SEQ ID NO: 18 in a human VH framework; 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) 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.

[0026] In some embodiments, the multispecific binding compound is bispecific. In some embodiments, the multispecific binding compound is in the form of a CAR-T.

[0027] Various aspects of the invention include multispecific binding compounds comprising: (i) a heavy chain variable region having binding affinity for CD3, comprising a CDR1 sequence of SEQ ID NO:85, a CDR2 sequence of SEQ ID NO:86, and a CDR3 sequence of SEQ ID NO:87 in a human VH framework; (ii) a light chain variable region comprising 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 a human VL framework; and (iii) an antigen binding domain of an anti-CD22 heavy chain antibody, comprising 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 in a human VH framework.

[0028] Various aspects of the invention include multispecific binding compounds comprising: (i) a heavy chain variable region having binding affinity for CD3, comprising a CDR1 sequence of SEQ ID NO:85, a CDR2 sequence of SEQ ID NO:86, and a CDR3 sequence of SEQ ID NO:87 in a human VH framework; (ii) a light chain variable region comprising 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 a human VL framework; and (iii) an antigen binding domain of an anti-CD22 heavy chain antibody, 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:19 in a human VH framework.

[0029] Various aspects of the invention include multispecific binding compounds comprising: (i) a heavy chain variable region having binding affinity for CD3, comprising a CDR1 sequence of SEQ ID NO:85, a CDR2 sequence of SEQ ID NO:86, and a CDR3 sequence of SEQ ID NO:87 in a human VH framework; (ii) a light chain variable region comprising 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 a human VL framework; and (iii) an antigen binding domain of an anti-CD22 heavy chain antibody, 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 in a human VH framework.

[0030] In some embodiments, the multispecific binding compound comprises a human IgG1 Fc region. In some embodiments, the human IgG1 Fc region is a silent 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 silent human IgG4 Fc region.

[0031] Aspects of the invention include pharmaceutical compositions comprising a multispecific binding compound as described herein.

[0032] Aspects of the invention include methods for treating a B cell disorder characterized by CD22 expression, comprising administering to a subject suffering from the disorder a multispecific binding compound or pharmaceutical composition as described herein.

[0033] Aspects of the invention include the use of a multispecific binding compound in the preparation of a medicament for treating a B cell disorder characterized by CD22 expression.

[0034] In some embodiments, the disease is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the disease is non-Hodgkin lymphoma (NHL). In some embodiments, the disease is systemic lupus erythematosus (SLE). In some embodiments, the disease is rheumatoid arthritis (RA). In some embodiments, the disease is multiple sclerosis (MS).

[0035] Aspects of the invention include polynucleotides encoding multispecific binding compounds as described herein. Aspects of the invention include vectors comprising polynucleotides as described herein. Aspects of the invention include cells comprising vectors as described herein.

[0036] Aspects of the invention include methods of producing a multispecific binding compound as described herein, comprising growing a cell as described herein under conditions that permit expression of the binding compound, and isolating the binding compound from the cell.

[0037] Aspects of the invention include methods of making a multispecific binding compound as described herein, comprising immunizing a UniRat animal with CD22 and identifying a CD22 binding heavy chain sequence.

[0038] Aspects of the invention include methods of treatment comprising administering to a subject an effective dose of a multispecific binding compound as described herein or a pharmaceutical composition as described herein.

[0039] These and other aspects will be further explained in the remainder of this disclosure including the examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1A is a graph depicting T cell-mediated cytotoxicity of CD22-positive cells (Daudi) using resting human pan T cells.

[0041] Figure 1B Graph depicting the dose response curve of cytokine release from resting human pan T cells incubated with CD22 positive cells (Daudi) and treated with anti-CD22xCD3_F2F multispecific binding compound and positive control.

[0042] Figure 2A is a graph depicting T cell-mediated cytotoxicity of CD22-positive cells (SUDHL10) using resting human pan T cells.

[0043] Figure 2B is a graph depicting the dose response curve of cytokine release from resting human pan T cells incubated with CD22 positive cells (SUDHL10) and treated with anti-CD22xCD3_F2F multispecific binding compound and positive control.

[0044] Figure 3A Shown is a series of graphs depicting T cell-mediated cytotoxicity of CD22-positive cells (RI-1) using resting human pan T cells.

[0045] Figure 3B is a series of graphs depicting dose response curves for cytokine release from resting human pan T cells incubated with CD22 positive cells (RI-1) and treated with anti-CD22xCD3_F2F multispecific binding compound and positive control.

[0046] Figure 4 Shown is a series of graphs depicting T cell-mediated cytotoxicity of CD22-positive cells using activated human pan T cells.

[0047] Figure 5 Shown is a series of graphs depicting cellular binding of bispecific antibodies against CD22 and CD3.

[0048] Figure 6 A treatment plan to determine the in vivo efficacy of anti-CD22xCD3_F2F multispecific binding compounds in Daudi xenografts is shown.

[0049] Figure 7 is a graph depicting mean tumor volume in mouse Daudi xenografts as a function of days after tumor implantation.

[0050] Figure 8is a graph depicting body weight as a function of days post tumor implantation in mouse Daudi xenografts.

[0051] Fig. 9 is a graph depicting percent change in body weight in mouse Daudi xenografts as a function of days post tumor implantation.

[0052] Fig.10 is a graph depicting mean tumor volume in mouse Daudi xenografts as a function of days after tumor implantation.

[0053] Fig.11 Shown is a series of graphs depicting individual tumor measurements in mouse Daudi xenografts as a function of days post tumor implantation.

[0054] Fig.12 is a graph depicting body weight as a function of days post tumor implantation in mouse Daudi xenografts.

[0055] Fig.13 is a graph depicting percent change in body weight in mouse Daudi xenografts as a function of days post tumor implantation.

[0056] Fig.14A 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.

[0057] Fig. 14B is a schematic representation of various CAR-T constructs that may incorporate one or more binding domains according to embodiments of the present invention.

[0058] Fig.15A 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, monospecific for CD22).

[0059] Fig. 15B 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, monospecific for CD22).

[0060] Fig. 15C 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, biparatopic for CD22).

[0061] Fig.16 is a table showing data of various biological activities of anti-CD22 antibodies according to embodiments of the present invention.

[0062] Fig.17 is a series of graphs showing serum titer as a function of dilution. DETAILED DESCRIPTION

[0063] 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 fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (FM Ausubel et al., ed., 1987, and regularly updated); "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).

[0064] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range (unless the context clearly indicates otherwise, the intervening value is to one-tenth of the unit of the lower limit) and any other specified or intervening values ​​in that specified range are encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, and any limit in the specified range may be specifically excluded. When the specified range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0065] Unless otherwise indicated, antibody residues herein are numbered according to the Kabat numbering system (eg, Kabat et al., Sequences of Immunological Interest. 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0066] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention may be practiced without one or more of these specific details. In other cases, well-known features and procedures well known to those skilled in the art are not described in order to avoid obscuring the present invention.

[0067] All references cited throughout this disclosure, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0068] I. definition

[0069] "Comprising" means that the recited elements are required in the composition / method / kit, but other elements may be included to form a composition / method / kit etc. within the scope of the claim.

[0070] "Consisting essentially of" is meant to limit the scope of the described compositions or methods to the specified materials or steps that do not materially affect the basic and novel characteristics of the invention.

[0071] "Consisting of" means excluding from the composition, method or kit any elements, steps or ingredients not specified in the claims.

[0072] The antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. When referring to the residues in the variable domain (approximately the residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to the residues in the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" (e.g., Kabat et al., EU index reported in the same) is generally used. "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise specified herein, the reference to the residue numbering in the antibody variable domain means the residue numbering by the Kabat numbering system. Unless otherwise specified herein, the reference to the residue numbering in the antibody constant domain means the residue numbering by the EU numbering system.

[0073] Antibodies, also known as immunoglobulins, conventionally comprise at least one heavy chain and one light chain, wherein the amino terminal domains of the heavy and light chains are variable in sequence and are therefore often referred to as variable region domains or variable heavy (VH) domains or variable light (VH) domains. The two domains conventionally associate to form a specific binding region, although as will be discussed herein, specific binding can also be obtained with only heavy chain variable sequences, and a variety of non-natural antibody configurations are known and used in the art.

[0074] A "functional" or "biologically active" antibody or antigen binding molecule (including heavy chain only antibodies and multispecific (e.g., bispecific) triabody-like molecules (TCAs, described herein) is one that is able to exert one or more of its natural activities in a structural, regulatory, biochemical, or biophysical event. For example, a functional antibody or other binding molecule (e.g., TCA) can have the ability to specifically bind to an antigen, and the binding can in turn cause or alter a cellular or molecular event such as signal transduction or enzymatic activity. A functional antibody or other binding molecule (e.g., TCA) can also block ligand activation of a receptor or act as an agonist or antagonist. The ability of an antibody or other binding molecule (e.g., TCA) to exert one or more of its natural activities depends on several factors, including the correct folding and assembly of the polypeptide chains.

[0075] The term "antibody" herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain antibodies only, three-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, as long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854-4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species.

[0076] The term antibody can refer to full-length heavy chains, full-length light chains, complete immunoglobulin molecules; or the immunologically active portion of any of these polypeptides, i.e., a polypeptide or portion thereof comprising an antigen binding site of an antigen that immunospecifically binds to a target of interest, such targets include but are not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulin disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecules, including engineered subclasses of Fc parts with changes that provide reduced or enhanced effector cell activity. The immunoglobulin can be derived from any species. On the one hand, the immunoglobulin is mainly of human origin.

[0077] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical, except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies have a high degree of specificity (for a single antigenic site). In addition, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is for a single determinant on the antigen. Monoclonal antibodies according to the present invention can be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, and can also be prepared, for example, via a recombinant protein production method (see, for example, U.S. Patent number 4,816,567).

[0078] As used with respect to antibodies, the term "variable" refers to that some parts of the variable domains of antibodies are widely different in the sequence of antibodies, and are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed in the entire variable domain of antibodies. In the light chain and heavy chain variable domains, it is concentrated in three segments called hypervariable regions. The more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FRs, which mainly adopt a β-folded configuration, connected by three hypervariable regions, which form a loop connecting the β-folded structure and form a part of the β-folded structure in some cases. The hypervariable region in each chain is kept together very closely by FR, and together with the hypervariable region from another chain, it helps to form the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition 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 participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0079] The term "hypervariable region" when used herein refers to the amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from the "complementarity determining region" or "CDR" (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 Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from the "hypervariable loop" 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 defined herein.

[0080] Exemplary CDR names are shown herein, but those skilled in the art will appreciate that a variety of CDR definitions are commonly used, including the Kabat definition (see "Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions." Mol Immunol. 2010; 47: 694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the position of the structural loop region (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989; 342: 877-883).Alternative CDR definitions of interest include, but are not limited to, those disclosed by: Honegger, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool." J Mol Biol. 2001; 309: 657–670; Ofran et al. "Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B-cell epitopes." J Immunol. 2008; 181: 6230–6235; Almagro "Identification of differences in the 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.

[0081] The terms "heavy chain antibody only" and "heavy chain antibody" are used interchangeably herein and refer in the broadest sense to antibodies lacking the light chains of conventional antibodies. The term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen binding domain and CH2 and CH3 constant domains but without a CH1 domain; functional (antigen binding) variants of such antibodies, soluble VH variants, homodimeric Ig-NARs comprising one variable domain (V-NAR) and five C-like constant domains (C-NAR) and functional fragments thereof; and soluble single domain antibodies (sUniDabs TM). In one embodiment, only the heavy chain antibody consists of the variable region antigen binding domain, which consists of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3 and framework 4. In another embodiment, only the heavy chain antibody consists of the antigen binding domain, at least a portion of the hinge region, and the CH2 and CH3 domains. In another embodiment, only the heavy chain antibody consists of the antigen binding domain, at least a portion of the hinge region, and the CH2 domain. In another embodiment, only the heavy chain antibody consists of the antigen binding domain, at least a portion of the hinge region, and the CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In another embodiment, the heavy chain consists of the antigen binding domain and at least one CH (CH1, CH2, CH3 or CH4) domain, but does not contain a hinge region. In another embodiment, the heavy chain consists of the antigen binding domain, at least one CH (CH1, CH2, CH3 or CH4) domain, and at least a portion of the hinge region. Only heavy chain antibodies can be in the form of dimers, wherein two heavy chains are connected with disulfide bonds, or otherwise covalently or non-covalently attached to each other. Only heavy chain antibodies can belong to IgG subclasses, but antibodies belonging to other subclasses such as IgM, IgA, IgD and IgE subclasses are also included herein. In a specific embodiment, the heavy chain antibody has an IgG1, IgG2, IgG3 or IgG4 subtype, particularly an IgG1 subtype. In one embodiment, the heavy chain antibody has an IgG4 subtype, wherein one or more of the CH domains are modified to change the effector functions of the antibody. In one embodiment, the heavy chain antibody has an IgG1 subtype, wherein one or more of the CH domains are modified to change the effector functions of the antibody. The modification of the CH domains that change the effector functions is further described herein. Non-limiting examples of heavy chain antibodies are described in, for example, WO2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0082] In one embodiment, only heavy chain antibodies herein are used as the binding (targeting) domain of a chimeric antigen receptor (CAR). The definition specifically includes antibodies derived from human immunoglobulin transgenic rats (UniRat TM ) produces only heavy chain antibodies, called UniAbs TM .UniAbs TM The variable region (VH) is called UniDabs TM , and is a universal building block that can be attached to the Fc region or serum albumin to develop novel therapeutic agents with multispecificity, increased potency, and extended half-life. TMLacking a light chain and therefore a VL domain, the antigen is recognized by one single domain, the variable domain (antigen binding domain) of the heavy chain of a heavy chain antibody (VH).

[0083] As used herein, a "complete antibody chain" is a chain comprising a full-length variable region and a full-length constant region (Fc). For secretory IgG, a complete "conventional" antibody comprises a complete light chain and a complete heavy chain, as well as a light chain constant domain (CL) and a heavy chain constant domain CH1, hinge, CH2 and CH3. Other isotypes (such as IgM or IgA) may have different CH domains. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. A complete antibody may have one or more "effector functions," which refer to those 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 downregulation of cell surface receptors. Constant region variants include those that alter effector profiles, binding to Fc receptors, and the like.

[0084] Depending on the amino acid sequence of the Fc (constant domain) of its heavy chain, antibodies and a variety of antigen-binding proteins can be provided in different classes. There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The Fc constant domains corresponding to different antibody classes 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; US 2005 / 0048572; US 2004 / 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 sequence of their constant domains.

[0085] A "functional Fc region" has the "effector functions" of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding; CDC; Fc-receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require that the Fc region interacts with receptors, such as FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors and low affinity FcRn receptors; and can be assessed using a variety of assays known in the art. A "dead" or "silent" Fc is one that has been mutated to retain activity with respect to, for example, extended serum half-life, but does not activate high affinity Fc receptors, or has a reduced affinity for Fc receptors.

[0086] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence 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.

[0087] A "variant Fc region" comprises an amino acid sequence that is distinguished from the amino acid sequence of a native sequence Fc region by at least one amino acid modification (preferably one or more amino acid substitutions). Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parent polypeptide, for example, there are about one to about ten amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide, and preferably about one to about five amino acid substitutions. The variant Fc region herein will preferably have at least about 80% homology with the native sequence Fc region and / or with the Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0088] Variant Fc sequences may include three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitutions in human IgG1 or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 greatly 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. Silent IgG1 is described in, e.g., Boesch, AW et al., "Highly parallel characterization of IgG Fc binding interactions." MAbs, 2014. 6(4): p. 915-27, the disclosure of which is incorporated herein by reference in its entirety.

[0089] Other Fc variants are also possible, including but not limited to variants in which regions capable of forming disulfide bonds are deleted, or in which certain amino acid residues are eliminated at the N-terminus of the native Fc, or a methionine residue is added thereto. Thus, in some embodiments, one or more Fc portions 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 include an Fc variant.

[0090] In addition, Fc variants can be constructed by substitution (mutation), deletion or addition of amino acid residues to achieve complement binding or Fc receptor binding to remove or substantially reduce effector function. For example, but not limited to, deletions can occur in complement binding sites, such as C1q binding sites. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478. In addition, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0091] The term "Fc region-containing antibody" refers to an antibody comprising 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 purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Therefore, the antibody with an Fc region according to the present invention may include an antibody with or without K447.

[0092] Various aspects of the invention include binding compounds with multispecific configurations, including but not limited to bispecific, trispecific, etc. Various methods and protein configurations are known and used for bispecific monoclonal antibodies (BsMAB), trispecific antibodies, etc.

[0093] A variety of methods for making multivalent artificial antibodies have been developed by recombinantly fusing the variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on the polypeptide are connected by a polypeptide linker. A non-limiting example of such a polypeptide linker is a GS linker, which has an amino acid sequence of four glycine residues followed by a serine residue, and wherein the sequence is repeated n times, wherein 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 may also be used, and the linkers are described in, for example, 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.

[0094] The term "three-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 a heavy chain and a 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), consisting essentially of a heavy chain and a light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain, or consisting of a heavy chain and a light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain. This heavy chain / 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, wherein the Fc portion comprises a CH2 and / or CH3 and / or CH4 domains, but does not comprise 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 a first antigen, wherein such binding domains are derived from or have sequence identity with a variable region of an antibody heavy chain or light chain. Such a portion of a variable region may consist of a V H and / or V L Gene segments, D and J H Gene segment or J L The variable region may be encoded by a rearranged V H DJ H 、V L DJ H 、V H J L or V L J L The gene segments encode the TCA proteins using heavy chain only antibodies as defined above.

[0095] TCA-binding compounds use "heavy chain antibodies only" or "heavy chain antibodies" or "heavy chain polypeptides", as used herein, which means a single chain antibody comprising a heavy chain constant region CH2 and / or CH3 and / or CH4 but not a CH1 domain. In one embodiment, the heavy chain antibody consists of an antigen binding domain, at least a portion of a hinge region, and a CH2 and CH3 domain. In another embodiment, the heavy chain antibody consists of an antigen binding domain, at least a portion of a hinge region, and a CH2 domain. In another embodiment, the heavy chain antibody consists of an antigen binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In another embodiment, the heavy chain consists of an antigen binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but does not contain a hinge region. The heavy chain antibody can be in the form of a dimer, wherein the two heavy chains are connected by disulfide bonds, the others are covalently or non-covalently attached to each other in other ways, and can optionally include an asymmetric interface between one or more CH domains to promote appropriate pairing between polypeptide chains. The heavy chain antibody may belong to the IgG subclass, but antibodies belonging to other subclasses such as IgM, IgA, IgD and IgE subclasses are also included herein. In a specific embodiment, the heavy chain antibody has an IgG1, IgG2, IgG3 or IgG4 subtype, particularly an IgG1 subtype or an IgG4 subtype. Non-limiting examples of TCA-binding compounds are described in, for example, WO2017 / 223111 and WO2018 / 052503, the disclosures of which are incorporated herein by reference in their entirety.

[0096] Heavy chain antibodies constitute about one quarter of the IgG antibodies produced by camelids (e.g., camels and llamas) (Hamers-Casterman C. et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains, but do not contain light chains. Therefore, the variable antigen-binding portion is called a VHH domain and it represents the smallest naturally occurring complete antigen-binding site, which is only about 120 amino acids long (Desmyter, A. et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with high specificity and affinity for a variety of antigens can be produced by immunization (vander Linden, RH et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)) and the VHH portion can be easily cloned and expressed in yeast (Frenken, LGJ et al. J. Biotechnol. 78, 11-21 (2000)). Its expression level, 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 VNARs). (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)).

[0097] As used herein, the terms "CD22" and "cluster of differentiation-22" refer to molecules belonging to the SIGLEC family of lectins, present on the surface of mature B cells, and to a lesser extent on some immature B cells. The term "CD22" includes CD22 proteins of any human and non-human animal species, and specifically includes human CD22 as well as CD22 of non-human mammals.

[0098] As used herein, the term "human CD22" includes any variants, isoforms and species homologs of human CD22 (UniProt P20273), regardless of their origin or preparation method. Therefore, "human CD22" includes human CD22 naturally expressed by cells and CD22 expressed on cells transfected with the human CD22 gene.

[0099] 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 to refer to a heavy chain only antibody as defined above that immunospecifically binds to CD22 (including human CD22 as defined above). The definition includes, but is not limited to, human heavy chain antibodies produced by transgenic animals expressing human immunoglobulins (such as transgenic rats or transgenic mice), including UniRats as defined above. TM , which produces human anti-CD22 UniAb TM Antibody.

[0100] " Amino acid sequence identity percentage (%) " about reference polypeptide sequence is defined as after comparing sequence and introducing gap to realize maximum percentage sequence identity when necessary, and when any conservative substitution is not considered as part of sequence identity, the percentage of amino acid residue in candidate sequence identical with the amino acid residue in described reference polypeptide sequence.Comparison for measuring amino acid sequence identity percentage can be realized in a variety of ways within the technical scope of this area, 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 comparing sequence, including any algorithm required for realizing maximum comparison on the total length of compared sequence.However, for purposes herein, sequence comparison computer program ALIGN-2 is used to generate % amino acid sequence identity values.

[0101] "Separated" antibodies are antibodies that have been identified and separated and / or recovered from the components of their natural environment. The contaminating components of their natural environment are materials that will interfere with the diagnosis or therapeutic use of the antibody, and may include enzymes, hormones, and other proteins or non-protein solutes. In a preferred embodiment, the antibody will be purified (1) to more than 95% by weight of the antibody, as measured by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) to homogeneity found by SDS-PAGE using Coomassie blue or preferably silver staining under reducing or non-reducing conditions. The separated antibody includes an in situ antibody in a recombinant cell, because at least one component of the natural environment of the antibody will not exist. However, typically, the separated antibody will be prepared by at least one purification step.

[0102] The antibodies of the present invention include multispecific antibodies. Multispecific antibodies have more than one binding specificity. The term "multispecific" specifically includes "bispecific" and "trispecific", as well as higher order independent specific binding affinities, such as higher order multi-epitope specificity, and tetravalent antibodies and antibody fragments. The terms "multispecific antibody", "multispecific heavy chain only antibody", "multispecific heavy chain antibody", "multispecific UniAb" and "multispecific antibody" are also used herein. TM " and "multispecific binding compound" are used herein in the broadest sense and encompass all antibodies with more than one binding specificity. The multispecific heavy chain anti-CD22 antibodies of the invention also specifically include antibodies that immunospecifically bind to a single epitope on a CD22 protein (such as human CD22) and an epitope on a different protein (such as, for example, the CD3 protein) (i.e., bivalent and monoparatopic). The multispecific heavy chain anti-CD22 antibodies of the invention specifically include antibodies that immunospecifically bind to two or more non-overlapping epitopes on a CD22 protein (such as human CD22) (i.e., bivalent and bicomplementary). The multispecific heavy chain anti-CD22 antibodies of the present invention also specifically include antibodies that immunospecifically bind to an epitope on a CD22 protein (such as human CD22) and an epitope on a different protein (such as, for example, a CD3 protein, such as human CD3) (i.e., bivalent and biparatopic). The multispecific heavy chain anti-CD22 antibodies of the present invention also specifically include antibodies that immunospecifically bind to two or more non-overlapping or partially overlapping epitopes on a CD22 protein (such as a human CD22 protein) and an epitope on a different protein (such as, for example, a CD3 protein, such as human CD3) (i.e., trivalent and biparatopic).

[0103] The antibodies of the present invention include monospecific antibodies with one binding specificity. Monospecific antibodies specifically include antibodies comprising a single binding specificity, as well as antibodies comprising more than one binding unit with the same binding specificity. The terms "monospecific antibody", "monospecific heavy chain only antibody", "monospecific heavy chain antibody" and "monospecific UniAb" are used interchangeably. TM " is used herein in the broadest sense and encompasses all antibodies with one binding specificity. The monospecific heavy chain anti-CD22 antibodies of the present invention specifically include antibodies (monovalent and monospecific) that immunospecifically bind to one epitope on a CD22 protein (such as human CD22). The monospecific heavy chain anti-CD22 antibodies of the present invention also specifically include antibodies (e.g., multivalent antibodies) having more than one binding unit that immunospecifically binds to an epitope on a CD22 protein (such as human CD22). For example, a monospecific antibody according to an embodiment of the present invention may include a heavy chain variable region comprising two antigen binding domains, wherein each antigen binding domain binds to the same epitope on a CD22 protein (i.e., bivalent and monospecific).

[0104] An "epitope" is a site on the surface of an antigen molecule that binds to a single antibody molecule. Generally speaking, an antigen has several or more different epitopes and reacts with a variety of different antibodies. The term specifically includes linear epitopes and conformational epitopes.

[0105] "Epitope mapping" is the process of identifying the binding site, or epitope, of an antibody on its target antigen. Antibody epitopes can be linear epitopes or conformational epitopes. Linear epitopes are formed by contiguous sequences of amino acids in a protein. Conformational epitopes are formed by amino acids that are not contiguous in the protein sequence, but come together when the protein folds into its three-dimensional structure.

[0106] "Multi-epitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. As described above, the present invention specifically includes anti-CD22 heavy chain antibodies with multi-epitope specificity, i.e., anti-CD22 heavy chain antibodies that bind to one or more non-overlapping epitopes on CD22 proteins (such as human CD22); and anti-CD22 heavy chain antibodies that bind to one or more epitopes on CD22 proteins and epitopes on different proteins (such as, for example, CD3 proteins). The term "non-overlapping epitopes" or "non-competitive epitopes" of an antigen is defined herein to mean an epitope recognized by one member of a pair of antigen-specific antibodies but not the other member. Antibody pairs or antigen-binding regions on multispecific antibodies that recognize non-overlapping epitopes that target the same antigen will not compete for binding to that antigen and are able to bind to that antigen simultaneously.

[0107] An antibody binds "substantially the same epitope" as a reference antibody when the two antibodies recognize the same or spatially overlapping epitopes. The most widely used and rapid method for determining whether two epitopes bind to the same or spatially overlapping epitopes is a competition assay, which can be configured in a variety of different formats, using either labeled antigens or labeled antibodies. Typically, the antigen is immobilized on a 96-well plate, and a radioactive or enzymatic label is used to measure the ability of the unlabeled antibody to block the binding of the labeled antibody.

[0108] As used herein, the term "valent" refers to a specified number of binding sites in an antibody molecule.

[0109] A "monovalent" antibody has one binding site. Therefore, a monovalent antibody is also monospecific.

[0110] "Multivalent" antibodies have two or more binding sites. Thus, the terms "bivalent," "trivalent," and "tetravalent" refer to the presence of two, three, 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, monoparatope) or for two different epitopes (i.e., bivalent, biparatope).

[0111] A variety of methods and protein configurations are known and used to prepare bispecific monoclonal antibodies (BsMABs), trispecific antibodies, and the like.

[0112] The term "chimeric antigen receptor" or "CAR" is used in the broadest sense herein and refers to an engineered receptor that transplants the desired binding specificity (e.g., the antigen binding region of a monoclonal antibody or other ligand) to a transmembrane and intracellular signaling domain. Typically, the receptor is used to transplant the specificity of a monoclonal antibody to a T cell to create a chimeric antigen receptor (CAR). (J Natl Cancer Inst, 2015; 108 (7): dvj439; and Jackson et al., Nature Reviews Clinical Oncology, 2016; 13: 370–383).

[0113] 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, for example 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), in particular UniRats as defined above. TM Generated UniAbs TM .

[0114] "Chimeric antibody" or "chimeric immunoglobulin" means an immunoglobulin molecule comprising amino acid sequences from at least two different Ig loci, such as a transgenic antibody comprising a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies having a non-human Fc-region or an artificial Fc-region and a human idiotype. Such immunoglobulins can be isolated from animals of the present invention that have been engineered to produce such chimeric antibodies.

[0115] As used herein, the term "effector cell" refers to an immune cell that participates in the effector phase of an immune response, such as relative 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) are capable of inducing antibody-dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages expressing FcR participate in the specific killing of target cells and present antigens to other components of the immune system, or in combination with cells presenting antigens. In some embodiments, effector cells can engulf target antigens or target cells.

[0116] "Human effector cells" are leukocytes that express receptors (such as T cell receptors or FcRs) and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; NK cells are preferred. Effector cells can be isolated from their natural sources, such as blood or PBMCs as described herein.

[0117] The term "immune cell" is used herein in the broadest sense and includes, but is not limited to, cells of myeloid or lymphoid origin, for example, lymphocytes (such as B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0118] Antibody "effector functions" refer to those biological activities attributable to the Fc 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 (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

[0119] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcRs) recognize bound antibodies on target cells and subsequently cause the lysis of target cells. Primary cells (NK cells) used to mediate ADCC express only FcγRIII, while 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). In order to assess the ADCC activity of target molecules, an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337, can be performed. Effector cells that can be used for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0120] "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) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), can be performed.

[0121] "Binding affinity" refers to the strength of all 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 represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind antigen slowly and tend to dissociate rapidly, while high-affinity antibodies generally bind antigen faster and tend to remain bound.

[0122] As used herein, "Kd" or "Kd value" refers to the dissociation constant determined by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetic mode. For example, an anti-mouse Fc sensor is loaded with a mouse-Fc fusion antigen and then immersed in a well containing an antibody to measure the concentration-dependent association rate (kassociation). The antibody dissociation rate (kdissociation) is measured in a final step in which the sensor is immersed in a well containing only a buffer. Kd is the ratio of kdissociation / kassociation. (For more details, see Concepcion, J et al., Comb Chem HighThroughput Screen, 12(8), 791-800, 2009).

[0123] 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 preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or an adverse effect attributable to the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; or (c) alleviating the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during, or after the onset of the disease or injury. The treatment of an ongoing disease in which the treatment stabilizes or reduces the patient's undesirable clinical symptoms is of particular concern. It is desirable to perform such treatment before the function of the affected tissue is completely lost. The therapy of the present invention may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

[0124] "Therapeutically effective amount" means the amount of an active agent necessary to confer a therapeutic benefit to a subject. For example, a "therapeutically effective amount" is an amount that induces, ameliorates, or otherwise causes an improvement in pathological symptoms, disease progression, or physiological conditions associated with a disease, or improves resistance to a condition.

[0125] 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, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell neoplasms such as plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, MALT lymphoma, nodal marginal 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 lymphoma.

[0126] The term "characterized by CD22 expression" refers broadly to any disease or condition in which CD22 expression is associated with or implicated in one or more pathological processes characteristic of the disease or condition. Such conditions include, but are not limited to, B cell neoplasms.

[0127] The terms "subject", "individual" and "patient" are used interchangeably herein and refer to a mammal that is being evaluated for treatment and / or is undergoing treatment. In one embodiment, the mammal is a human. The terms "subject", "individual" and "patient" encompass, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infection, and the like. The subject can be a human, but also includes other mammals, particularly those mammals that can be used as laboratory models of human disease, such as mice, rats, and the like.

[0128] The term "pharmaceutical preparation" refers to a preparation in a form that allows the biological activity of the active ingredient to be effective, and which does not contain other components that are unacceptably toxic to the subject to which the preparation is to be administered. Such preparations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can be reasonably administered to a subject mammal to provide an effective dose of the active ingredient used.

[0129] A "sterile" preparation is one that is sterile or free or substantially free of all viable microorganisms and their spores. A "frozen" preparation is one that is at a temperature below 0°C.

[0130] A "stable" formulation is one in which the protein substantially maintains its physical stability and / or chemical stability and / or biological activity during storage. Preferably, the formulation substantially maintains its physical and chemical stability, as well as its biological activity during storage. The storage period is generally selected based on the expected shelf life of the formulation. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed in, for example, Peptide and Protein Drug Delivery, 247-301. Vincent Lee, ed., Marcel Dekker, Inc., New York, NY, Pubs. (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 assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); assessing charge heterogeneity by using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; amino-terminal or carboxyl-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis to compare reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessing the biological activity or antigen binding function of the antibody; etc. Instability may involve any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge fragmentation), succinimide formation, unpaired cysteine, N-terminal extension, C-terminal processing, glycosylation differences, etc.

[0131] II. Details

[0132] Anti-CD22 Antibody

[0133] Aspects of the invention include multispecific binding compounds comprising anti-CD22 binding domains. A family of closely related heavy chain-only antibody binding domains that bind to human CD22 is provided herein. The antibodies of this family comprise a collection of CDR sequences as defined herein and shown in Table 1, and are exemplified by the provided heavy chain variable region (VH) sequences of SEQ ID NOs: 24 to 84 set forth in Table 2. The antibodies described herein provide a number of benefits that facilitate use as clinical therapeutic agents. The antibodies include members with a range of binding affinities, thereby allowing selection of a specific sequence with a desired binding affinity.

[0134] Table 1: Unique CDR amino acid sequences of anti-CD22 heavy chain antibodies.

[0135]

[0136] Table 2. Anti-CD22 heavy chain antibody variable domain amino acid sequences.

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143] Suitable antibodies can be selected from those provided herein for development and therapeutic or other uses, including but not limited to as bispecific antibodies (e.g., Fig.14A ) or a portion of a trispecific antibody or CAR-T structure (e.g., as Fig. 14B shown). 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 is paired with a light chain, while the anti-CD22 domain is derived from a heavy chain-only antibody and does not contain a CH1 domain or does not interact with a light chain. In some embodiments, the two heavy chains are sheared off using, for example, knobs-into-holes technology.

[0144] Turning to the antibodies depicted in FIG. 15 , Fig.15A Depicted are anti-CD3 x anti-CD22 bispecific antibodies 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. Fig. 15B Depicted are anti-CD3 x anti-CD22 bispecific antibodies in which the anti-CD22 binding arm is bivalent and monospecific and the antigen binding domains of the anti-CD22 arm are in a tandem configuration, meaning there are two identical antigen binding domains placed in tandem. Fig. 15C Depicted are anti-CD3 x anti-CD22 bispecific antibodies wherein the anti-CD22 binding arm is bivalent and biparatopic, and the antigen binding domains of the anti-CD22 arm are in a tandem configuration.

[0145] Determination of affinity for a candidate protein can be performed using methods known in the art, such as Biacore measurements. Members of the antibody family may have an affinity for CD22 with a Kd of about 10 -6 Up to about 10 -11 , including but not limited to: about 10 -6 Up to about 10 -10 ; about 10 -6 Up to about 10 -9 ; about 10 -6 Up to about 10 -8 ; about 10 -8 Up to about 10 -11 ; about 10 -8 Up to about 10 -10 ; about 10 -8 Up to about 10 -9 ; about 10 -9 Up to about 10 -11 ; about 10 -9 Up to about 10 -10 ; or any value within these ranges. Affinity selection can be confirmed by biological assessments for modulating (e.g., blocking) CD22 bioactivity, including in vitro assays, preclinical models, and clinical trials, as well as assessments of potential toxicity.

[0146] Members of the antibody families herein may not cross-react with cynomolgus CD22 protein, but may be engineered, if necessary, to provide cross-reactivity with cynomolgus CD22 protein or with CD22 of any other animal species.

[0147] The family of CD22-specific antibodies herein comprises a VH domain comprising CDR1, CDR2 and CDR3 sequences in a human VH framework. For example, for CDR1, CDR2 and CDR3, respectively, the CDR sequences may be located in the region around amino acid residues 26-35; 53-59; and 98-117 of the provided exemplary variable region sequences set forth in SEQ ID NOs: 24 to 84. One of ordinary skill in the art will appreciate that if a different framework sequence is selected, the CDR sequences may be located in a different position, although in general, the order of the sequences will remain the same.

[0148] The CDR1, CDR2 and CDR3 sequences of the anti-CD22 antibodies of the invention may be encompassed by the following formula, wherein X indicates a variable amino acid, which may be a specific amino acid as indicated below.

[0149] CDR1

[0150] G X1 SI X2 X3 X4 X5 X6 Y(SEQ ID NO:104)

[0151] Where X1 is D or G;

[0152] X2 is S, T, I or N;

[0153] X3 is S or D;

[0154] X4 is G, S or N;

[0155] X5 is D, G or S; and

[0156] X6 is Y or H.

[0157] CDR2

[0158] X7 X8 Y X9 GX 10 X 11 (SEQ ID NO: 105)

[0159] Where X7 is I or V;

[0160] X8 is Y or H;

[0161] X9 is S or T;

[0162] X 10 is A, V, or S; and

[0163] X 11 It is T or A.

[0164] CDR3

[0165] X 12 RX 13 DSSX 14 WRS (SEQ ID NO: 106)

[0166] Where X 12 is T, A or K;

[0167] X 13 is D or E; and

[0168] X 14 It is N or S.

[0169] Representative CDR1, CDR2 and CDR3 sequences are shown in Tables 1 and 3.

[0170] Table 3: Anti-CD22 heavy chain antibody CDR1, CDR2 and CDR3 amino acid sequences.

[0171]

[0172]

[0173]

[0174]

[0175] In some embodiments, the anti-CD22 heavy chain only antibodies of the invention comprise a CDR1 sequence of any one of SEQ ID NOs: 1- 10. In a specific embodiment, the CDR1 sequence is SEQ ID NO: 1.

[0176] In some embodiments, the anti-CD22 heavy chain-only antibodies of the invention comprise a CDR2 sequence of any one of SEQ ID NOs: 11- 17. In a specific embodiment, the CDR2 sequence is SEQ ID NO:11.

[0177] In some embodiments, the anti-CD22 heavy chain only antibodies of the invention comprise a CDR3 sequence of any one of SEQ ID NOs: 18-23. In a specific embodiment, the CDR2 sequence is SEQ ID NO:18.

[0178] In another embodiment, an anti-CD22 heavy chain only antibody of the invention comprises 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.

[0179] In another embodiment, an anti-CD22 heavy chain only antibody of the invention comprises any one of the heavy chain variable region amino acid sequences of SEQ ID NOs: 24 to 84 (Table 2).

[0180] In yet another embodiment, the anti-CD22 heavy chain only antibody of the invention comprises the heavy chain variable region sequence of SEQ ID NO:24.

[0181] In some embodiments, the CDR sequences in 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 sequences in any one of SEQ ID NOs: 1 to 23 ( FIG. 1 ), or a set of CDR1, CDR2 and CDR3 sequences. In some embodiments, the amino acid substitutions are 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 and 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 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 to 84 (shown in Table 2).

[0182] 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, the multispecific antibody may include at least one heavy chain variable region with binding specificity to CD22. In some embodiments, the multispecific antibody may include a heavy chain variable region including at least two antigen binding domains, each of which has binding specificity to CD22. In some embodiments, the multispecific antibody may include a heavy chain / light chain pair with binding specificity to a first antigen (e.g., CD3), and a heavy chain from a heavy chain antibody alone. In certain embodiments, the heavy chain from the heavy chain antibody alone includes an Fc portion, and the Fc portion includes a CH2 and / or CH3 and / or CH4 domain, but does not contain a CH1 domain. In a specific embodiment, the bispecific antibody includes a heavy chain / light chain pair with binding specificity to an antigen on an effector cell (e.g., a CD3 protein on a T cell), and a heavy chain from a heavy chain antibody alone, the heavy chain including an antigen binding domain with binding specificity to CD22.

[0183] 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 a CDR1 sequence of SEQ ID NO: 85, a CDR2 sequence of SEQ ID NO: 86, and a CDR3 sequence of SEQ ID NO: 87 in a human VH framework. In some embodiments, the fixed light chain 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 a human VL framework. Together, the CD3 binding VH domain and the light chain variable domain have binding affinity for CD3. In some embodiments, the CD3 binding VH domain comprises a 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% percent identity to the heavy chain variable region sequence of SEQ ID NO: 91. In some embodiments, the fixed light chain comprises a 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% percent identity to the heavy chain variable region sequence of SEQ ID NO: 92.

[0184] Multispecific antibodies comprising the above-described CD3 binding VH domain and light chain variable domain have advantageous properties, for example, as described in published PCT application publication number 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 having binding affinity for CD22 can be combined with the CD3 binding domains and fixed light chain domains described herein to generate multispecific antibodies having binding affinity for one or more CD22 epitopes as well as CD3.

[0185] Table 4. Anti-CD3 heavy and light chain CDR1, CDR2, CDR3 amino acid sequences.

[0186]

[0187] Table 5. Anti-CD3 heavy and light chain variable region amino acid sequences.

[0188]

[0189] Table 6: Human IgG1 and IgG4 Fc region sequences.

[0190]

[0191]

[0192] Table 7: Additional sequences.

[0193]

[0194]

[0195]

[0196] 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, the bispecific antibody may include at least one heavy chain variable region with binding specificity to CD22 and at least one heavy chain variable region with binding specificity to proteins other than CD22. In some embodiments, the bispecific antibody may include a heavy chain / light chain pair with binding specificity to a first antigen, and a heavy chain from a heavy chain antibody alone, the heavy chain comprising an Fc portion, the Fc portion comprising a CH2 and / or CH3 and / or CH4 domain, but without a CH1 domain, and an antigen binding domain that binds to an epitope of a second antigen or a different epitope of a first antigen. In a specific embodiment, the bispecific antibody comprises a heavy chain / light chain pair with binding specificity to an antigen on an effector cell (e.g., a CD3 protein on a T cell), and a heavy chain from a heavy chain antibody alone, the heavy chain comprising an antigen binding domain with binding specificity to CD22.

[0197] In some embodiments, when the binding compound of the present invention is a bispecific antibody, one arm of the antibody (a binding moiety or a binding unit) is specific to human CD22, while the other arm can be specific to target cells, tumor-associated cell antigens, targeted antigens (e.g., integrins, etc.), pathogen antigens, checkpoint proteins, etc. Target cells specifically include cancer cells, including but not limited to cells from blood tumors (e.g., B cell tumors) as discussed below. In some embodiments, one arm of the antibody (a binding moiety or a binding unit) is specific to human CD22, while the other arm is specific to CD3.

[0198] 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 NO: 98, 99, 100, or 101, and an anti-CD22 heavy chain polypeptide comprising the sequence of any one of SEQ ID NO: 24-84 linked to the sequence of any one of SEQ ID NO: 93, 94, 95, or 96. These sequences can be combined in a variety of ways to generate bispecific antibodies of the desired IgG subclass, e.g., IgG1, IgG4, silent IgG1, silent IgG4.

[0199] Various forms 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. The multispecific antibodies herein specifically include T cell multispecific (e.g., bispecific) antibodies (anti-CD22 x anti-CD3 antibodies) that bind to CD22 (which is selectively expressed on mature B cells) and CD3. Such antibodies induce effective T cell-mediated killing of cells expressing CD22.

[0200] Antibody preparation

[0201] The multispecific binding compounds of the present invention can be prepared by methods known in the art. In a preferred embodiment, the heavy chain antibodies herein are produced by transgenic animals, including transgenic mice and rats, preferably rats, in which endogenous immunoglobulin genes are knocked out or disabled. In a preferred embodiment, in UniRat TM The heavy chain antibodies described herein were generated in UniRat TM UniRat has silenced endogenous immunoglobulin genes and uses human immunoglobulin heavy chain translocation sites to express a diverse, naturally optimized full human HCAb repertoire. While a variety of techniques can be used to knock out or silence endogenous immunoglobulin loci in rats, the UniRat TMIn the present invention, zinc finger (endo) nuclease (ZNF) technology is used to inactivate endogenous rat heavy chain J-locus, light chain Cκ locus and light chain Cλ locus. ZNF constructs used for microinjection into oocytes can produce IgH and IgL knockout (KO) lines. 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. The advantage of ZNF technology is that non-homologous end joining to silence genes or loci via deletions of up to several kb can also provide target sites for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). UniRat TM Human heavy chain antibodies produced in TM They can bind to epitopes that conventional antibodies cannot. Their high specificity, affinity, and small size make them ideal for monospecific and multispecific applications.

[0202] In addition to UniAbs TM In addition, only heavy chain antibodies lacking camelid VHH frameworks and mutations, as well as functional VH regions thereof, are specifically included herein. Such heavy chain antibodies can be produced, for example, in transgenic rats or mice comprising a fully human heavy chain locus as described, for example, in WO2006 / 008548, but other transgenic mammals such as rabbits, guinea pigs, rats, preferably rats and mice, can also be used. Only heavy chain antibodies, including VHH or VH functional fragments thereof, can also be produced by recombinant DNA technology by expressing encoding nucleic acids in suitable eukaryotic or prokaryotic hosts, including, for example, mammalian cells (e.g., CHO cells), Escherichia coli or yeast.

[0203] The domains of heavy chain antibodies only combine the advantages of antibodies and small molecule drugs: they can be monovalent or multivalent; have low toxicity; and are inexpensive to manufacture. Due to their small size, these domains are easily administered, including oral or topical administration, are characterized by high stability, including gastrointestinal stability; and their half-life can be tailored to the desired use or indication. In addition, the VH and VHH domains of HCAbs can be manufactured in a cost-effective manner.

[0204] In a specific embodiment, the heavy chain antibodies of the invention (including UniAbs TM) The native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Kabat numbering system) is replaced by another amino acid residue, and the other amino acid residue is capable of destroying the surface-exposed hydrophobic patch containing the native amino acid residue at the position or associated with the native amino acid residue. Such hydrophobic patches are usually embedded in the interface with the constant region of the light chain of the antibody, but become surface-exposed in HCAb and are at least partially used for undesirable aggregation and light chain association of HCAb. The substituted amino acid residue is preferably charged, and more preferably positively charged, such as lysine (Lys, K), arginine (Arg, R) or histidine (His, H), preferably arginine (R). In a preferred embodiment, the heavy chain antibody derived from a transgenic animal contains a mutation from Trp to Arg at position 101. The resulting HCAb preferably has high antigen binding affinity and solubility under physiological conditions without aggregation.

[0205] As part of the present invention, proteins with the ability to bind to human CD22 were identified in ELISA protein and cell binding assays. TM UniAb TM The identified heavy chain variable region (VH) sequences (see, e.g., Table 2) were positive for binding to human CD22 protein and / or binding to CD22+ cells, and were all negative for binding to cells that do not express CD22.

[0206] Heavy chain antibodies (e.g., UniAbs) that bind to non-overlapping epitopes on the CD22 protein can be identified by competitive binding assays, such as enzyme-linked immunosorbent assays (ELISA assays) or flow cytometry competitive binding assays. TM). For example, competition between a known antibody and a target antibody that binds to a target antigen can be used. By using this method, the collection of antibodies can be divided into those that compete with a reference antibody and those that do not compete with a reference antibody. Non-competitive antibodies are identified as binding to different epitopes, and the epitope does not overlap with the epitope bound to the reference antibody. Typically, an antibody is fixed, an antigen is bound, and the ability of a second labeled (e.g., biotinylated) antibody to bind to the captured antigen is tested in an ELISA assay. This can also be performed using a surface plasmon resonance (SPR) platform, including ProteOn XPR36 (BioRad, Inc), Biacore2000 and Biacore T200 (GE Healthcare Life Sciences) and MX96 SPR imager (Ibis technologies B.V.), and bio-layer interferometry platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). For further details, see Examples herein.

[0207] Typically, an antibody "competes" with a reference antibody if the competition results in a decrease in binding of the reference antibody to the target antigen by about 15%-100%, as determined by standard techniques, such as by the competition binding assay 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.

[0208] Pharmaceutical compositions, uses and methods of treatment

[0209] Another aspect of the present invention provides a pharmaceutical composition comprising a mixture of one or more multispecific binding compounds of the present invention and a suitable pharmaceutically acceptable carrier. As used herein, pharmaceutically acceptable carriers are, for example, but not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to support therapeutic components, or combinations thereof.

[0210] In one embodiment, the pharmaceutical composition comprises a heavy chain antibody (e.g., UniAb) that binds to CD22. TM In another embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (eg, UniAb) having binding specificity for two or more non-overlapping epitopes on the CD22 protein. TM In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (eg UniAbTM ), which has binding specificity for CD22 and has 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).

[0211] Pharmaceutical compositions of antibodies used in accordance with the invention are prepared by mixing a protein having a desired degree of purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) for storage, such as in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations used, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0212] The pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic, and is manufactured under good manufacturing practice (GMP) conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., for a single dose). The formulation depends on the selected route of administration. The antibody herein can be administered by intravenous injection or infusion or subcutaneous administration. For injection administration, the antibody herein can be formulated in an aqueous solution, preferably in a physiologically compatible buffer to reduce discomfort at the injection site. The solution may contain a carrier, excipient, or stabilizer as discussed above. Alternatively, the antibody may be in a lyophilized form to be composed of a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[0213] Antibody formulations are disclosed, for example, in U.S. Pat. No. 9,034,324. Similar formulations can be used for the heavy chain antibodies of the invention, including UniAbs TMSubcutaneous antibody formulations are described, for example, in US20160355591 and US20160166689.

[0214] How to use

[0215] The anti-CD22 heavy chain only antibodies, multispecific antibodies, and pharmaceutical compositions described herein can be used to treat diseases and conditions characterized by CD22 expression, including but not limited to the conditions and diseases further described herein.

[0216] CD22 is a 135 kDa type I transmembrane protein that is expressed at low levels on premature and immature B cells, expressed most on mature B cells, and ultimately downregulated on plasma cells. (e.g., Walker et al., Immunology, March 2008; 123 (3) 314-25). CD22 is strongly expressed in follicular (primary and secondary B cell zones), mantle zone, and marginal zone B cells, and is reported to be present in samples from 60% to 80% of patients with B cell malignancies (Alderson et al. Clin. Cancer Res 2009; 15 (3) February 11, 2009). Since its expression is observed in many hematological malignancies, CD22 is a promising target for antibody-based therapy.

[0217] In one aspect, the CD22 heavy chain antibodies (e.g. UniAbs TM ) and pharmaceutical compositions can be used to treat hematological malignancies characterized by CD22 expression, including but not limited to diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia (CLL) and B-cell acute lymphoblastic leukemia (ALL).

[0218] Diffuse large B-cell lymphoma (DLBCL or DLBL) is the most common form of non-Hodgkin 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 by an aggressive cancer that can arise in almost any part of the body. The cause of DLBCL is unknown, and it may arise from the malignant transformation of normal B cells as well as other types of lymphoma or leukemia cells. Treatment typically involves chemotherapy and radiation therapy and achieves an overall five-year survival rate averaging approximately 58% for adults. Although some monoclonal antibodies have shown promise in treating DLBCL, consistent clinical efficacy has not been conclusively demonstrated. Therefore, new therapies for DLBCL, including immunotherapy, are greatly needed.

[0219] In another aspect, the CD22 heavy chain antibodies (eg, UniAbs TM) and pharmaceutical compositions can be used to treat autoimmune disorders characterized by pathogenic B cells expressing CD22, including but not limited to systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS).

[0220] The effective dose of the composition of the present invention for treating a disease varies depending on a variety of different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other drugs administered, and whether the treatment is preventive or therapeutic. Typically, the patient is a human, but non-human mammals, such as companion animals (such as dogs, cats, horses, etc.), laboratory mammals (such as rabbits, mice, rats, etc.), etc., can also be treated. The therapeutic dose can be measured by titration to optimize safety and efficacy.

[0221] Dosage levels can be readily determined by an ordinary skilled clinician and can be modified as needed, for example, as needed to modify the subject's response to therapy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the host being treated and the particular mode of administration. Dosage unit forms generally contain between about 1 mg and about 500 mg of active ingredient.

[0222] In some embodiments, the therapeutic dose of the agent can be between about 0.0001 to 100 mg / kg and more generally 0.01 to 5 mg / kg of host body weight. For example, the dosage can be 1 mg / kg body weight or 10 mg / kg body weight or in the range of 1-10 mg / kg. Exemplary treatment regimens require administration once every two weeks or once a month or once every 3 to 6 months. The therapeutic entity of the present invention is generally administered in a variety of situations. The time interval between individual doses can be weekly, monthly or annual. The time interval can also be irregular, as indicated by measuring the blood level of the therapeutic entity in the patient. Alternatively, the therapeutic entity of the present invention can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0223] Typically, compositions are prepared in the form of injectables (liquid solutions or suspensions); solid forms that can be dissolved or suspended in liquid vehicles prior to injection can also be prepared. The pharmaceutical compositions herein can be used for intravenous or subcutaneous administration directly or after reconstitution of solid (e.g., lyophilized) compositions. The products can also be emulsified or encapsulated in liposomes or microparticles (such as polylactide, polyglycolide, or copolymers) to enhance the adjuvant effect, as discussed 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 reservoir injections or implant products, which can be formulated in such a way as to allow for sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and fully comply with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0224] The toxicity of the antibodies and antibody structures described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (a dose lethal to 50% of the population) or the LD100 (a dose lethal to 100% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index. The data obtained from these cell culture analyses and animal studies can be used to formulate a non-toxic dose range for humans. The dosage of the antibodies described herein is preferably within a circulating concentration range that includes an effective dose with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration used. The exact formulation, route of administration and dosage can be selected by an individual physician based on the patient's condition.

[0225] The composition for administration will generally comprise an antibody or other agent (e.g., ablative agent) dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, such as buffered saline, etc. These solutions are sterile and generally do not contain undesirable substances. These compositions may be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances such as pH regulators and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc., as needed for simulating physiological conditions. The concentration of the active agent in these preparations may vary widely, and will be selected based on the selected specific mode of administration and the needs of the patient (e.g., Remington's Pharmaceutical Science (15th edition, 1980) and Goodman and Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., ed., 1996)), mainly based on fluid volume, viscosity, body weight, etc.

[0226] Kits comprising the active agents of the invention and formulations thereof and instructions for use are also within the scope of the invention. The kits may also contain at least one additional agent, such as a chemotherapeutic drug, etc. The kits typically include a label indicating the intended use of the contents of the kit. As used herein, the term "label" includes any written or recorded material on or supplied with the kit or otherwise accompanying the kit.

[0227] Now that the present invention has been 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 present invention.

[0228] Example

[0229] Materials and methods

[0230] CD22 protein binding

[0231] Kinetic binding experiments were performed on an Octet QK-384 system (ForteBio) using biolayer interferometry to determine antigen-antibody affinity. Anti-human IgG Fc capture (AHC) biosensors (Forte Bio, 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. UniAbs were then added at a concentration of 5 μg / mL. TMThe AHC biosensors were fixed for 120 seconds. Another baseline (120 seconds) was established in assay buffer. Next, they were immersed in a 7-point, 1:2 dilution series of human CD22 protein in assay buffer, starting at 250nM. The last well of the analyte column contained only assay buffer to test for nonspecific binding between the buffer and the loaded biosensor and was used as a reference well. Association was observed for 600 seconds and dissociation was then observed 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.

[0232] CD22 cell binding

[0233] Binding to CD22-positive cells was assessed by flow cytometry (Guava easyCyte 8HT, EMD Millipore) using the Daudi cell line (ATCC). Briefly, purified UniAbs were incubated at 4°C for 1 hr. TM 100,000 target cells were stained with a dilution series of 100,000 for 30 minutes. 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 (SouthernBiotech, catalog #2042-09) conjugated to R-phycoerythrin (PE) to detect cell-bound antibodies. After incubation at 4°C for 20 minutes, cells were washed twice with flow cytometry buffer and then measured by flow cytometry for mean fluorescence intensity (MFI). EC50 values ​​were calculated using GraphPad Prism 7. The same protocol was used to determine the binding of cynomolgus CD22-positive cells, but with the following modifications: target cells were from CHO cells stably transfected to express the extracellular domain of cynomolgus CD22, and each antibody was tested at a single concentration (about 1.7 μg / mL), so EC50 values ​​were not calculated.

[0234] Example 1: Genetically engineered rats expressing heavy chain-only antibodies

[0235] The 'human-rat' IgH locus was constructed and assembled into several parts. This involved modifying and ligating the human J H The rat C region gene was added downstream, followed by the human V H 6-D-segment area. There will be different people V H Two BACs of the gene cluster [BAC6 and BAC3] were co-injected with a BAC called Georg, which encodes a gene containing human V H 6. All Ds, All Js H and modified rat Cγ2a / 1 / 2b (ΔC H 1) The assembly and modification regions.

[0236] Generation of transgenic rats carrying an artificial heavy chain immunoglobulin locus in an unrearranged configuration. H 1). IgG1(C H 1). IgG2b(C H 1) Genetic deficiency C H 1 segment. The constant region genes IgE, IgA and 3' enhancer are contained in the Georg BAC. RT-PCR and serum analysis (ELISA) of transgenic rats revealed efficient 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 in which the endogenous heavy chain and light chain loci were mutated as previously described in U.S. Patent Publication 2009 / 0098134A1. Analysis of such animals showed that rat immunoglobulin heavy and light chain expression was inactivated, and heavy chain antibodies with variable regions encoded by human V, D and J genes were expressed at high levels. Immunization of transgenic rats resulted in high titer serum responses producing antigen-specific heavy chain antibodies. These transgenic rats expressing heavy chain antibodies with human VDJ regions are called UniRats. TM .

[0237] Example 2: Immunization

[0238] Immunization was performed with the recombinant extracellular domain of CD22.

[0239] Twelve UniRat animals (6HC27, 6HC28) were immunized with recombinant human CD22 protein. Animals were immunized according to standard protocols using Titermax / Alhydrogel adjuvant. The recombinant extracellular domain of CD22 was purchased from R&D Systems and diluted with sterile saline and mixed with adjuvant. The immunogen was mixed with Titermax and Alhydrogel adjuvant. The first immunization (start) was performed in the left and right legs using the immunogen in Titermax. Subsequent booster immunizations were performed in the presence of Alhydrogel and were boosted with the immunogen in PBS three days before harvesting. Serum was collected from rats at the last blood draw to determine serum titer.

[0240] Serum titer results

[0241] Serum titer overview information is available at Fig.17 In Fig.17In the depicted figures, each line represents an individual animal. The legend of the figure shows the ID number of each individual animal. The binding activity of the sera was tested by ELISA against huCD22+Fc protein, huCD22+His tag, rhesus CD22+His tag protein and His tag off-target protein in an 8-point dilution series. In this group of animals, a range of serum reactivity levels against human and rhesus CD22 proteins were observed. Serum reactivity against the His protein tag was also observed.

[0242] Example 3: Binding to cell lines expressing CD22

[0243] Fig.16 The target binding activity of the anti-CD22 heavy chain only antibodies described herein is summarized. Column 1 represents the clone ID number of the anti-CD22 heavy chain only antibody. Column 2 represents the binding affinity (KD) for the protein measured in molar concentration. Column 3 represents the dissociation constant (K-dissociation rate) for binding to the protein measured in seconds. Column 4 represents binding to Daudi cells measured as multiples of the background MFI signal. Column 5 represents binding to CHO cells stably expressing cyno CD22 measured as multiples of the background MFI signal. Column 6 represents binding to CHO cells that do not express CD22 protein measured as multiples of the background MFI signal.

[0244] Example 4: T cell-mediated cytotoxicity of CD22-positive cells using resting human pan T cells

[0245] 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. The supernatant from the cell culture was used to measure the release of the cytokine IL-2. The POS CTRL antibody refers to an antibody containing the same anti-CD22 arm, but with a stronger affinity anti-CD3 arm. The results are in Figure 1A and Figure 1B Available in.

[0246] Unstimulated human T cells were incubated with CD22 positive cells (SUDHL10) and different concentrations of the bispecific antibody. After 72 hours, the cells were subjected to flow cytometry to measure cytotoxicity. The supernatant from the cell culture was used to measure the release of the cytokine IL-2. The POS CTRL antibody refers to an antibody that contains the same anti-CD22 arm, but with a stronger affinity anti-CD3 arm. The results are in Figure 2A and Figure 2B Available in.

[0247] Unstimulated human T cells were incubated with a CD22-positive DL-BCL cell line (RI-1) and different concentrations of the bispecific antibodies with an effector-target (E:T) cell ratio 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 that contain 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 in Figure 3A and Figure 3B Available in.

[0248] Example 5: T cell-mediated cytotoxicity of CD22-positive cells using activated human pan T cells

[0249] 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 fluorescence reader. The bispecific CD22xCD3_F2F binding compound specifically caused lysis of CD22+ cells but not CD22- K562 cells. POSCTRL antibodies refer to antibodies that contain the same anti-CD22 arm but with a stronger affinity anti-CD3 arm. NEG CTRL refers to antibodies with a non-specific tumor arm and the same anti-CD3 arm as anti-CD3_F2F. The results are shown in Figure 4 Available in.

[0250] Example 6: Cellular binding of bispecific antibodies against CD22 and CD3

[0251] CD22 positive cells Daudi, Raji, 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 showed that the bispecific antibody bound to CD22+ cells but not to CD22- cells. POS CTRL antibodies refer to antibodies that contain the same anti-CD22 arm but with a stronger affinity anti-CD3 arm. NEG CTRL refers to antibodies that have a non-specific tumor arm and the same anti-CD3 arm as anti-CD3_F2F. The results are in Figure 5 Available in.

[0252] Example 7: In vivo efficacy study with CD22-1 x CD3_F2F in Daudi xenografts

[0253] To test the in vivo efficacy of CD22-1 x CD3_F2F, different doses of CD22-1 x CD3_F2F were administered to female NSG mice implanted with Daudi cells (5e6 cells / mouse), as Figure 6 The treatment schedule is shown in Table 7 below. Mean tumor volume, body weight, percent change in body weight, and individual tumor volume were used to evaluate treatment efficacy.

[0254] Table 7: Sample Treatment Schedule

[0255]

[0256] The data of CD22-1 x CD3_F2F is Figure 7-13 Shown in Figure 2 is a comparison of negative control and rituximab and demonstrates the efficacy of CD22-1 x CD3_F2F.

[0257] Although 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. Without departing from the present invention, those skilled in the art will now carry out multiple modifications, changes and substitutions. It should be understood that a variety of alternatives to the embodiments of the invention described herein may be used to implement the present invention. This means that the following claims limit the scope of the present invention and the methods and structures within the scope of these claims and their equivalents are therefore encompassed. This means that the following claims limit the scope of the present invention and are thus encompassed within the scope of these claims and their equivalents Method and structure.

Claims

1. A bispecific antibody 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 having binding affinity for CD22 is a heavy chain binding unit only and comprises a heavy chain variable region, the heavy chain variable region 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: 19; And wherein the second binding unit comprises: (a) a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 85, a CDR2 sequence of SEQ ID NO: 86, and a CDR3 sequence of SEQ ID NO: 87; and (b) a light chain variable region, comprising 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.

2. The bispecific antibody of claim 1, wherein the first binding unit having binding affinity for CD22 comprises a heavy chain variable region sequence that is at least 95% identical to SEQ ID NO:

25.

3. The bispecific antibody of claim 2, wherein the first binding unit having binding affinity for CD22 comprises a heavy chain variable region sequence comprising SEQ ID NO:

25.

4. The bispecific antibody of claim 1, wherein the second binding unit having binding affinity for CD3 comprises a heavy chain variable region sequence that is at least 95% identical to SEQ ID NO:

91. 5 . The bispecific antibody of claim 4 , wherein the second binding unit having binding affinity for CD3 comprises a heavy chain variable region sequence comprising SEQ ID NO:

91.

6. The bispecific antibody of claim 1, wherein the second binding unit having binding affinity for CD3 comprises a heavy chain variable region sequence that is at least 95% identical to SEQ ID NO:

92.

7. The bispecific antibody of claim 6, wherein the second binding unit having binding affinity for CD3 comprises a heavy chain variable region sequence comprising SEQ ID NO:

92.

8. The bispecific antibody of claim 1, wherein the first binding unit having binding affinity for CD22 further comprises a heavy chain constant region sequence without a CH1 sequence.

9. The bispecific antibody of claim 1, comprising a human IgG4 Fc region.

10. The bispecific antibody of claim 9, wherein the human IgG4 Fc region is a silent human IgG4 Fc region. The bispecific antibody of claim 9 , wherein the human IgG4 Fc region comprises a hinge region mutation.

12. The bispecific antibody of claim 9, wherein the human IgG4 Fc region comprises a plurality of knob-to-hole mutations.

13. The bispecific antibody of claim 9, wherein the human IgG4 Fc region comprises a C-terminal lysine. The bispecific antibody of claim 9 , wherein the human IgG4 Fc region does not contain a C-terminal lysine. The bispecific antibody of claim 1 , comprising a human IgG1 Fc region. The bispecific antibody of claim 15 , wherein the human IgG1 Fc region is a silent human IgG1 Fc region. The bispecific antibody of claim 15 , wherein the human IgG1 Fc region comprises a hinge region mutation.

18. The bispecific antibody of claim 15, wherein the human IgG1 Fc region comprises a plurality of knob-to-hole mutations.

19. The bispecific antibody of claim 15, wherein the human IgG1 Fc region comprises a C-terminal lysine.

20. The bispecific antibody of claim 15, wherein the human IgG1 Fc region does not contain a C-terminal lysine.

21. A polynucleotide encoding the bispecific antibody of claim 1.

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