Antibody that binds to CD19, CAR-T cell, pharmaceutical composition, its uses, kit, polynucleotide, vector, cell and method for producing an antibody or a CAR-T cell.

Description

1 / 81 Antibody that binds to CD19, CAR-T cell, pharmaceutical composition, its uses, kit, polynucleotide, vector, cell and METHOD FOR PRODUCING AN ANTIBODY OR A CAR-T CELL Divided from BR 11 2023 020450 0 CROSS REFERENCE TO RELATED ORDERS

[0001] This application claims priority benefit from the filing date of U.S. Provisional Application Serial Number 63 / 171,520, filed on April 6, 2021, the disclosure of which is incorporated herein in its entirety by reference. This application also claims priority benefit from the filing date of U.S. Provisional Application Serial Number 63 / 311,913, filed on February 18, 2022, the disclosure of which is incorporated herein in its entirety by reference. SEQUENCE LISTING

[0002] This request contains a Sequence Listing that was submitted electronically in ASCII format and is thus incorporated by reference in its entirety. The said ASCII copy, created on June 8, 2022, is named TNO-0040-WO_SL.txt and is 74,739 bytes in size. AREA OF THE INVENTION

[0003] The present invention relates to antibodies (e.g., UniAbs™) and CAR-T structures that bind to CD19. The invention further relates to methods of producing such antibodies and CAR-T structures, compositions, including pharmaceutical compositions comprising such antibodies and CAR-T structures, and their use in treating disorders characterized by CD19 expression. BACKGROUND OF THE INVENTION Differentiation cluster 19 (CD19)

[0004] CD19, also known as Surface Antigen of B lymphocytes B4 (UniProt P15391) is a cell surface receptor that is expressed on all human B cells, but not plasma cells. CD19 is a transmembrane protein that Petition 870260076930, dated 07 / 31 / 2026, page 15 / 122 CD19 2 / 81 recruits cytoplasmic signaling proteins to the membrane and acts within the CD19 / CD21 complex to lower the threshold for B-cell receptor signaling pathways. CD19 has a relatively large cytoplasmic tail of 240 amino acids. The extracellular Ig-like domains are divided by a non-Ig-like domain linked by a potential disulfide-linked carbohydrate addition sites. The cytoplasmic tail contains at least nine tyrosine residues near the C-terminus, some of which have been shown to be phosphorylated. Along with CD20 and CD22, the lineage-restricted expression of CD19 makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many CD19-specific monoclonal antibodies and antibody-drug conjugates have been described (Naddafi et al. 2015, PMC4644525).Additionally, T cells modified with anti-CD19 chimeric antigen receptors have been approved for the treatment of leukemia (Sadelain et al. 2017, PMID: 29245005). Heavy Chain Antibodies

[0005] In a conventional IgG antibody, the association of the heavy chain and the light chain is due in part to a hydrophobic interaction between the constant region of the light chain and the constant domain CH1 of the heavy chain. There are additional residues in the framework 2 (FR2) and framework 4 (FR4) regions of the heavy chain that also contribute to this hydrophobic interaction between the heavy and light chains.

[0006] It is known, however, that camelid serums (suborder Tylopoda (which includes camels, dromedaries, and llamas) contain a major type of antibody composed solely of paired H chains (heavy-chain-only antibodies or UniAbs™). Camelidae UniAbs™ (Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanaco, Lama alpaca, and Lama vicugna) have a unique structure consisting of a single variable domain (VHH), a hinge region, and two constant domains (CH2 and CH3). Petition 870260076930, dated 07 / 31 / 2026, page 16 / 122 3 / 81 that are highly homologous to the CH2 and CH3 domains of classical antibodies. These UniAbs™ lack the first constant region domain (CH1) that is present in the genome, but is removed during mRNA processing. The absence of the CH1 domain explains the absence of the light chain in UniAbs™, since this domain is the anchoring site for the constant domain of the light chain. Such UniAbs™ have naturally evolved to confer antigen-binding specificity and high affinity for three CDRs of conventional antibodies or fragments thereof (Muyldermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther 5:111-124). Cartilaginous fish, such as sharks, have also developed a distinct type of immunoglobulin, designated IgNAR, which lacks light polypeptide chains and is composed entirely of heavy chains.IgNAR molecules can be manipulated by molecular engineering to produce the variable domain of a single heavy chain polypeptide (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)).

[0007] The ability of heavy-chain-only antibodies lacking a light chain to bind to antigens was established in the 1960s (Jaton et al. (1968) Biochemistry, 7, 4185-4195). Immunoglobulin with a heavy chain physically separated from the light chain retained 80% of the antigen-binding activity compared to the tetrameric antibody. Sitia et al. (1990) Cell, 60, 781-790 demonstrated that removal of the CH1 domain from a rearranged mouse μ gene results in the production of a heavy-chain-only antibody, lacking a light chain, in mammalian cell culture. The antibodies produced retained VH binding specificity and effector functions.

[0008] Heavy chain antibodies with high specificity and Petition 870260076930, dated 07 / 31 / 2026, page 17 / 122 4 / 81 affinity fragments can be generated against a variety of antigens through 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)). Their expression levels, solubility, and stability are significantly higher than those of classic F(ab) or Fv fragments (Ghahroudi, MA et al. FEBS Lett. 414, 521-526 (1997)).

[0009] Mice in which the λ (lambda) (L) light chain locus and / or the L λ and κ (kappa) chain loci have been functionally silenced and antibodies produced by such mice are described in US Patents 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; US 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 production of knockout mice by means of microinjections into embryos of zinc finger nuclease is described in Geurts et al., 2009, Science, 325(5939):433. Soluble heavy chain-only antibodies and transgenic rodents comprising a heterologous heavy chain locus that produces such antibodies are described in US Patents 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

[0010] Aspects of the invention include antibodies that bind to CD19, comprising a variable heavy chain region comprising: (a) a CDR1 sequence having two or fewer substitutions at SEQ ID NO: 1; and / or (b) a CDR2 sequence having Petition 870260076930, dated 07 / 31 / 2026, p. 18 / 122 5 / 81 two or fewer substitutions in SEQ ID NO: 2; and / or (c) a CDR3 sequence having two or fewer substitutions in any of the SEQ ID NOs: 3-4.

[0011] In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in a human HV scaffold. In some embodiments, an antibody additionally comprises a heavy chain constant region sequence in the absence of a CH1 sequence.

[0012] In some embodiments, an antibody comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 and 38; and / or (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 2 and 39; and / or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 3-4. In some embodiments, an antibody comprises: (a) a CDR1 sequence comprising SEQ ID NO: 1 or SEQ ID NO: 38; and (b) a CDR2 sequence comprising SEQ ID NO: 2 or SEQ ID NO: 39; and (c) a CDR3 sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4.In some embodiments, an antibody comprises: (a) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 2, and a CDR3 sequence of SEQ ID NO: 3; or (b) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 2, and a CDR3 sequence of SEQ ID NO: 4; or (c) a CDR1 sequence of SEQ ID NO: 38, a CDR2 sequence of SEQ ID NO: 2, and a CDR3 sequence of SEQ ID NO: 3; or (d) a CDR1 sequence of SEQ ID NO: 38, a CDR2 sequence of SEQ ID NO: 2, and a CDR3 sequence of SEQ ID NO: 4; or (e) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 39, and a CDR3 sequence of SEQ ID NO: 3; or (f) a CDR1 sequence with SEQ ID NO: 1, a CDR2 sequence with SEQ ID NO: 39, and a CDR3 sequence with SEQ ID NO: 4.

[0013] In some embodiments, an antibody comprises a variable heavy chain region having at least 95% sequence identity with any of the SEQ ID sequences NOS: 5, 6, 40, and 41. In some embodiments, an antibody Petition 870260076930, dated 07 / 31 / 2026, page 19 / 122 6 / 81 comprises a variable region heavy chain sequence selected from the group consisting of SEQ ID NOs: 5, 6, 40, and 41. In some embodiments, an antibody comprises a variable region heavy chain sequence of SEQ ID NO: 5. In some embodiments, an antibody comprises a variable region heavy chain sequence of SEQ ID NO: 6. In some embodiments, an antibody comprises a variable region heavy chain sequence of SEQ ID NO: 40. In some embodiments, an antibody comprises a variable region heavy chain sequence of SEQ ID NO: 41.

[0014] Aspects of the invention include antibodies that bind to CD19, comprising a variable heavy chain region comprising CDR1, CDR2, and CDR3 sequences in a human HV scaffold, wherein the CDR sequences are sequences having two or fewer substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 38, and 39. In some embodiments, an antibody comprises a variable heavy chain region comprising CDR1, CDR2, and CDR3 sequences in a human HV scaffold, wherein the CDR sequences are selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 38, and 39.

[0015] Aspects of the invention include antibodies that bind to CD19, comprising a variable heavy chain region comprising: (a) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 2, and a CDR3 sequence of SEQ ID NO: 3, in a human HV scaffold; or (b) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 2, and a CDR3 sequence of SEQ ID NO: 4, in a human HV scaffold; or (c) a CDR1 sequence of SEQ ID NO: 38, a CDR2 sequence of SEQ ID NO: 2, and a CDR3 sequence of SEQ ID NO: 3, in a human HV scaffold; or (d) a CDR1 sequence with SEQ ID NO: 38, a CDR2 sequence with SEQ ID NO: 2, and a CDR3 sequence with SEQ ID NO: 4, in a human VH scaffold; or (e) a CDR1 sequence with SEQ ID NO: 1, a CDR2 sequence with SEQ ID NO: 39, and a Petition 870260076930, dated 07 / 31 / 2026, p. 20 / 122 7 / 81 CDR3 sequence with SEQ ID NO: 3, in a human HV scaffold; or (f) a CDR1 sequence with SEQ ID NO: 1, a CDR2 sequence with SEQ ID NO: 39, and a CDR3 sequence with SEQ ID NO: 4, in a human HV scaffold.

[0016] In some embodiments, an antibody is multispecific. In some embodiments, an antibody is bispecific. In some embodiments, an antibody binds to two different CD19 proteins. In some embodiments, an antibody binds to two different epitopes on the same CD19 protein. In some embodiments, an antibody binds to an effector cell. In some embodiments, an antibody binds to a T-cell antigen. In some embodiments, an antibody binds to CD3. In some embodiments, an antibody is in a CAR-T format.

[0017] Aspects of the invention include CAR-T cells comprising a CAR comprising an extracellular antigen-binding domain that binds to CD19, comprising a variable heavy chain region comprising: (a) a CDR1 sequence comprising SEQ ID NO: 1 or SEQ ID NO: 38; and (b) a CDR2 sequence comprising SEQ ID NO: 2 or SEQ ID NO: 39; and (c) a CDR3 sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4.

[0018] In some embodiments, the variable heavy chain region comprises (a) a CDR1 sequence with SEQ ID NO: 1, a CDR2 sequence with SEQ ID NO: 2, and a CDR3 sequence with SEQ ID NO: 3, in a human HV scaffold; or (b) a CDR1 sequence with SEQ ID NO: 1, a CDR2 sequence with SEQ ID NO: 2, and a CDR3 sequence with SEQ ID NO: 4, in a human HV scaffold; or (c) a CDR1 sequence with SEQ ID NO: 38, a CDR2 sequence with SEQ ID NO: 2, and a CDR3 sequence with SEQ ID NO: 3, in a human HV scaffold; or (d) a CDR1 sequence with SEQ ID NO: 38, a CDR2 sequence with SEQ ID NO: 2, and a CDR3 sequence with SEQ ID NO: 4, in a human VH scaffold; or (e) a CDR1 sequence with SEQ ID NO: 1, a CDR2 sequence with SEQ ID NO: 39, and a Petition 870260076930, dated 07 / 31 / 2026, p. 21 / 122 8 / 81 CDR3 sequence with SEQ ID NO: 3, in a human HV scaffold; or (f) a CDR1 sequence with SEQ ID NO: 1, a CDR2 sequence with SEQ ID NO: 39, and a CDR3 sequence with SEQ ID NO: 4, in a human HV scaffold.

[0019] In some embodiments, the extracellular antigen-binding domain that binds to CD19 comprises a variable heavy chain region having at least 95% sequence identity with any of the SEQ ID NOs: 5, 6, 40, and 41 sequences. In some embodiments, the extracellular antigen-binding domain that binds to CD19 comprises a variable heavy chain region sequence selected from the group consisting of SEQ ID NOs: 5-6. In some embodiments, the extracellular antigen-binding domain that binds to CD19 comprises a variable region heavy chain sequence with SEQ ID NO: 5, 6, 40, and 41. In some embodiments, the extracellular antigen-binding domain that binds to CD19 comprises a variable region heavy chain sequence with SEQ ID NO: 6. In some embodiments, the extracellular antigen-binding domain that binds to CD19 comprises a variable region heavy chain sequence with SEQ ID NO: 40.In some embodiments, the extracellular antigen-binding domain that binds to CD19 comprises a variable region sequence of heavy chain with SEQ ID NO: 41.

[0020] Aspects of the invention include pharmaceutical compositions comprising an antibody or a CAR-T cell as described herein.

[0021] Aspects of the invention include methods for treating a B-cell disorder characterized by CD19 expression, comprising administering to a subject with said disorder an antibody, a CAR-T cell, or a pharmaceutical composition as described herein.

[0022] In some forms, the disorder is diffuse large B-cell lymphoma (DLBCL). In some forms, the disorder Petition 870260076930, dated 07 / 31 / 2026, page 22 / 122 9 / 81 is acute lymphoblastic leukemia (ALL). In some forms, the disorder is non-Hodgkin lymphoma (NHL). In some forms, the disorder is systemic lupus erythematosus (SLE). In some forms, the disorder is rheumatoid arthritis (RA). In some forms, the disorder is multiple sclerosis (MS).

[0023] Aspects of the invention include polynucleotides encoding an antibody or a CAR of a CAR-T cell as described herein, vectors comprising such polynucleotides, and cells comprising such vectors.

[0024] Aspects of the invention include methods of producing an antibody as described herein, the methods comprising culturing a cell as described herein under conditions permissive for antibody expression, and isolating the antibody from the cell and / or from a cell culture medium where the cell is cultured.

[0025] Aspects of the invention include methods of manufacturing an antibody as described herein, the methods comprising immunizing a UniRat animal with CD19 and identifying CD19-binding heavy chain sequences.

[0026] Aspects of the invention include methods of treatment, comprising administering to a needy individual an effective dose of an antibody, a CAR-T cell, or a pharmaceutical composition as described herein.

[0027] Aspects of the invention include the use of an antibody or a CAR-T cell as described herein in the preparation of a medicament for the treatment of a disease or disorder in an individual in need.

[0028] Aspects of the invention include kits for the treatment of a disease or disorder in a needy individual, comprising an antibody, a CAR-T cell, or a pharmaceutical composition as described herein, and instructions for use. In some embodiments, a kit further comprises at least one additional reagent. In some embodiments, the at least one additional reagent comprises a chemotherapeutic drug. Petition 870260076930, dated 07 / 31 / 2026, page 23 / 122 10 / 81

[0029] These and other aspects will be further explained in the rest of the disclosure, including the Examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 is a table summarizing CD19 binding data, for the indicated antibody constructs, to CD19+ Raji cells and negative control cells.

[0031] FIG. 2, panels A and B, are graphs showing cell binding as a function of antibody concentration for the indicated antibody constructs, in the indicated cell types (Raji and Daudi).

[0032] FIG. 3 is a table summarizing EC50 values ​​of cell binding of the indicated antibody constructs to CD19+ Raji and Daudi cells.

[0033] FIG. 4, panel A, is a schematic diagram of a CAR-T structure comprising an anti-CD19 extracellular binding domain.

[0034] FIG. 4, panels B and C, are graphs showing antigen-specific binding and activation of CAR constructs comprising anti-CD19 extracellular binding domains, tested in the indicated cell lines.

[0035] FIG. 5, panels A and B, are graphs showing comparative cytotoxicity results between T cells expressing a CAR comprising a VH T28D ligand or a VH S54D ligand compared with a paternal VH-034 ligand.

[0036] FIG. 6 is a table summarizing enhanced in vitro T cell activation in T cells expressing a CAR comprising a VH T28D ligand or a VH S54D ligand compared with a paternal VH-034 ligand.

[0037] FIG. 7 is a table showing that VH-CD19 ligands enriched in a CAR format express higher levels of CAR in T cells compared to the paternal VH-034 ligand.

[0038] FIG. 8 is a table showing that T cells Petition 870260076930, dated 07 / 31 / 2026, page 24 / 122 11 / 81 expressing CARs comprising a VH T28D ligand or a VH S54D ligand exhibit enhanced T cell expansion in vivo.

[0039] FIG. 9 is a table showing that T cells expressing a CAR comprising the VH ligand CD19 T28D or S54D exhibited less T cell exhaustion compared with the paternal CD19 ligand VH-034.

[0040] FIG. 10 is a graph showing that T cells expressing a CAR comprising the VH ligand T28D or the VH ligand S54D exhibited tumor control.

[0041] FIG. 11 is a graph showing the uncondensed individual mouse model data illustrating the same result shown in FIG. 10. DETAILED DESCRIPTION OF PREFERRED OPTIONS

[0042] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the domain of the technique. Such techniques are fully explained in the literature, such as, for example, 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., eds., 1987, and periodic updates); PCR: The Polymerase Chain Reaction (Mullis et al., ed., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001); Harlow, Lane, and Harlow, “Using Antibodies: A Laboratory Manual: Portable Protocol No.I”, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory; (1988).

[0043] When a range of values ​​is given, if Petition 870260076930, dated 07 / 31 / 2026, p. 25 / 122 12 / 81 understands that each intervening value, up to the tenth of a unit of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of that range and any other stated or intervening value of that stated range is covered by the invention. The upper and lower limits of those smaller ranges may independently be included in the smaller ranges, and are also covered by the invention, subject to any limit specifically excluded in the indicated range. When the indicated range includes one or both limits, the ranges excluding one or both included limits are also included in the invention.

[0044] Unless otherwise indicated, antibody residues herein are numbered according to the Kabat numbering system (e.g., Kabat et al., “Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0045] In the following description, numerous specific details are presented to provide a more complete understanding of the present invention. However, it will be evident to a person skilled in the art that the present invention can be practiced without one or more of these specific details. In other cases, features and procedures well known to those skilled in the art have not been described to avoid obscuring the invention.

[0046] All references cited throughout this disclosure, including patent applications and publications, are incorporated herein by reference in their entirety. I. Definitions

[0047] By "understanding" it is understood that the cited elements are necessary in the composition / method / kit, but other elements may be included to form the composition / method / kit, etc. within the scope of the claim.

[0048] By consisting essentially in, it is understood a limitation of the scope of the composition or method described to Petition 870260076930, dated 07 / 31 / 2026, page 26 / 122 13 / 81 specified materials or steps that do not materially affect the basic and novel feature(s) of the present invention.

[0049] By consisting of is meant the exclusion from the composition, method or kit of any element, step or ingredient not specified in the claim.

[0050] Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-113 of the heavy chain) (e.g., Kabat et al., “Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)”). The EU numbering system or EU index is generally used when referring to a residue in a constant region of the immunoglobulin heavy chain (e.g., the EU index reported in Kabat et al., supra). The EU index as in Kabat refers to the numbering of residues of the human IgG1 EU antibody. Unless otherwise indicated herein, references to residue numbers in the variable domain of antibodies mean residue numbering by the Kabat numbering system.Unless otherwise indicated herein, references to residue numbers in the antibody constant domain mean residue numbering using the EU numbering system.

[0051] Antibodies, also referred to as immunoglobulins, conventionally comprise at least one heavy chain and one light chain, wherein the amino-terminal domain of the heavy and light chains has a variable sequence, therefore it is commonly referred to as a variable region domain or variable heavy (VH) or variable light (VL) domain. The two domains conventionally associate to form a specific binding region, although, as will be discussed here, specific binding can also be achieved with variable sequences of the heavy chain only, and a variety of unnatural antibody configurations are Petition 870260076930, dated 07 / 31 / 2026, page 27 / 122 14 / 81 known and used in the technique.

[0052] A functional or biologically active multispecific (e.g., bispecific) antibody or antigen-binding molecule (including heavy-chain-only antibodies and three-chain antibody-like molecules (TCAs, described herein)) is such that it is capable of exerting one or more of its natural activities on structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule, e.g., a TCA, may have the ability to bind specifically to an antigen, and the binding may in turn elicit or alter a cellular or molecular event, such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, e.g., a TCA, may also block the activation of receptor ligands or act as an agonist or antagonist.The ability of an antibody or other binding molecule, for example, a TCA, to exert one or more of its natural activities depends on several factors, including proper folding and assembly of the polypeptide chains.

[0053] The term antibody is used here in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain-only antibodies, three-chain antibodies, TCAs, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, provided they exhibit the desired biological activity (Miller et al (2003) Jour. de Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species.

[0054] The term antibody may refer to a complete heavy chain, a complete light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptides, that is, a polypeptide that Petition 870260076930, dated 07 / 31 / 2026, page 28 / 122 15 / 81 comprises an antigen-binding site that binds immunospecifically to an antigen of a target of interest or part thereof, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule, including subclasses manipulated with altered Fc portions that provide reduced or increased effector cellular activity. The light chains of the antibodies in question may be kappa (Vkappa) light chains or lambda (Vlambda) light chains. Immunoglobulins may be derived from any species. In one respect, immunoglobulin is largely of human origin.

[0055] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minimal quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies according to the present invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, and can also be prepared by recombinant protein production methods (see, for example, U Patent).S. No. 4,816,567), for example.

[0056] The term variable, as used in connection with Petition 870260076930, dated 07 / 31 / 2026, page 29 / 122 16 / 81 antibodies, refers to the fact that the sequences of certain portions of antibody variable domains differ extensively between antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, the variability is not uniformly distributed across antibody variable domains. It is concentrated in three segments called hypervariable regions, in the variable domains of both the light and heavy chains. The most highly conserved portions of the variable domains are called scaffold regions (SRs). The variable domains of native heavy and light chains each comprise four SRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form connecting loops and, in some cases, are part of the β-sheet structure.The hypervariable regions in each chain are kept close together by the FRs and, along with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions, such as the antibody's participation in antibody-dependent cellular cytotoxicity (ADCC).

[0057] The term hypervariable region, as used herein, refers to the amino acid residues of an antibody that are responsible for binding to antigens. The hypervariable region generally comprises amino acid residues from a 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 Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those Petition 870260076930, dated 07 / 31 / 2026, page 30 / 122 17 / 81 residues of the hypervariable loop residues” 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, CDR means a complementarity-determining region of an antibody as defined in Lefranc, MP et al., IMGT, International ImMunoGeneTics Database, Nucleic Acids Res., 27:209-212 (1999). Framework Region or FR residues are those variable domain residues other than the hypervariable region / CDR residues as defined herein.

[0058] Exemplary CDR designations are shown here; however, a person skilled in the art will understand that some definitions of CDRs are commonly used, including the Kabat definition (see Zhao et al. A germline knowledge-based computational approach for determining antibody complementarity-determining regions. Mol Immunol. 2010; 47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of structural loop regions (Chothia et al. Conformations of immunoglobulin hypervariable regions. Nature. 1989; 342:877-883). Alternative CDR definitions of interest include, without limitation, those published 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 Padlanet et al. Identification of specificity-determining residues in antibodies. Faseb J. 1995;9:133-139, each of which is specifically incorporated herein by way of reference. Petition 870260076930, dated 07 / 31 / 2026, page 31 / 122 18 / 81

[0059] The terms heavy chain-only antibody and heavy chain antibody are used interchangeably herein and refer, in the broadest sense, to antibodies, or one or more portions of an antibody, for example, one or more arms of an antibody, without the light chain of a conventional antibody. The terms specifically include, without limitation, homodimeric antibodies comprising the VH antigen-binding domain and the CH2 and CH3 constant domains, in the absence of the CH1 domain; functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NAR comprising a homodimer of a variable domain (V-NAR) and five C-type constant domains (CNAR) and functional fragments thereof; and soluble single-domain antibodies (sUniDabs™). In one embodiment, a heavy chain-only antibody is composed of a variable region antigen-binding domain composed of scaffold 1, CDR1, scaffold 2, CDR2, scaffold 3, CDR3, and scaffold 4.In another embodiment, a heavy chain-only antibody consists of an antigen-binding domain, at least part of a hinge region, and CH2 and CH3 domains. In another embodiment, a heavy chain-only antibody consists of an antigen-binding domain, at least part of a hinge region, and a CH2 domain. In a further embodiment, a heavy chain-only antibody consists of an antigen-binding domain, at least part of a hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains are truncated are also included here. In another embodiment, a heavy chain consists of an antigen-binding domain and at least one CH domain (CH1, CH2, CH3, or CH4), but no hinge region.A heavy chain-only antibody can be in the form of a dimer, in which two heavy chains are linked by disulfide bridges or otherwise, covalently or non-covalently, linked to each other. The heavy chain-only antibody may belong to the IgG subclass. Petition 870260076930, dated 07 / 31 / 2026, p. 32 / 122 19 / 81 but antibodies belonging to other subclasses, such as the IgM, IgA, IgD and IgE subclasses, are also included here. In one particular embodiment, a heavy chain antibody is of the IgG1, IgG2, IgG3 or IgG4 subtype, in particular of the IgG1 or IgG4 subtype. In one embodiment, a heavy chain antibody is of the IgG4 subtype, in which one or more of the CH domains are modified to alter an effector function of the antibody. In one embodiment, the heavy chain antibody is of the IgG1 or IgG4 subtype, in which one or more of the CH domains are modified to alter an effector function of the antibody. CH domain modifications that alter the effector function are further described herein. Non-limiting examples of heavy chain antibodies are described, for example, in WO2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0060] In some embodiments, heavy chain-only antibodies are used as the binding domain (selective approach) of a chimeric antigen receptor (CAR). The definition specifically includes human heavy chain-only antibodies produced by transgenic human immunoglobulin mice (UniRat™), termed UniAbs™. The variable (VH) regions of UniAbs™ are called UniDabs™ and are versatile building blocks that can be linked to Fc regions or serum albumin for the development of novel therapeutics with multispecificity, increased potency, and extended half-life. Because homodimeric UniAbs™ lack a light chain and therefore a VL domain, the antigen is recognized by a single domain, i.e., the variable domain of the heavy chain of a heavy chain antibody (VH or VHH).

[0061] An intact antibody chain, as used herein, is such that it comprises a complete variable region and a complete constant region (Fc). A conventional intact antibody comprises an intact light chain and an intact heavy chain, as well as a constant light chain domain (CL) and Petition 870260076930, dated 07 / 31 / 2026, page 33 / 122 20 / 81 constant heavy chain domains, CH1, hinge, CH2, and CH3 for secreted IgG. Other isotypes, such as IgM or IgA, may have different CH domains. Constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or their amino acid sequence variants. The intact antibody may have one or more effector functions, which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or an 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. Variants of the constant region include those that alter the effector profile, Fc receptor binding, and the like.

[0062] Depending on the amino acid sequence of the Fc (constant domain) of their heavy chains, antibodies and various antigen-binding proteins can be provided as different classes. There are five main classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains that correspond to the different antibody classes can be referenced as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different immunoglobulin classes are well known. Ig forms include hinge modifications or hingeless forms (Roux et al (1998) J. Immunol. 161:40834090; 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 κ (kappa) and λ (lambda), based on their... Petition 870260076930, dated 07 / 31 / 2026, p. 34 / 122 21 / 81 amino acid sequences of their constant domains. Antibodies according to embodiments of the invention may comprise kappa light chain sequences or lambda light chain sequences.

[0063] A functional Fc region has an effector function of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding; CDC; binding to Fc receptors; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptor), etc. Such effector functions generally require the Fc region to interact with a receptor, for example, FcyRI; FcyRIIA; FcyRIIBI; FcyRIIB2; FcyRIIIA; FcyRIIIB receptors and the low-affinity FcRn receptor; and can be assessed using various assays known in the art. A dead or silenced Fc is one that has been mutated to retain activity with respect to, for example, prolonging serum half-life, but that does not activate a high-affinity Fc receptor or that has reduced affinity for an Fc receptor.

[0064] A native sequence Fc region comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Human native sequence Fc regions include, for example, a native sequence human IgG1 Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as their naturally occurring variants.

[0065] A variant Fc region comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide. Petition 870260076930, dated 07 / 31 / 2026, page 35 / 122 22 / 81 for example, from about one to about ten amino acid substitutions and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably have at least about 80% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, and very preferably at least about 90% homology with the latter, more preferably at least about 95% homology with the latter.

[0066] The variant Fc sequences may include three amino acid substitutions in the CH2 region to reduce FcyRI binding at EU index positions 234, 235 and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions at the C1q binding site of complement at positions 330 and 331 of the EU index reduce complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J.Exp. Med. 173:1483 (1991)). Substitution in human IgG1 or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 greatly reduces ADCC and CDC (see, for example, 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 Fc amino acid sequence of human IgG4 (UniProtKB No. P01861) is given here as SEQ ID NO: 76. Silenced IgG1 is described, for example, in 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.

[0067] Other variants of Fc are possible, including, without limitation, one in which a region capable of forming a disulfide bond is deleted, or in which certain amino acid residues are eliminated at the N-terminal end of a native Fc, or a methionine residue is added to it.Thus, in some forms, one or more Fc portions of an antibody may comprise one or more mutations in the hinge region. Petition 870260076930, dated 07 / 31 / 2026, page 36 / 122 23 / 81 eliminate the formation of disulfide bonds. In yet another embodiment, the hinge region of an Fc can be completely removed. In yet another embodiment, an antibody can comprise an Fc variant.

[0068] Furthermore, an Fc variant can be constructed to substantially remove or reduce effector functions by substituting (mutating), deleting, or adding amino acid residues to effect complement binding or binding to Fc receptors. For example, and not as a limitation, a deletion can occur at a complement binding site, such as a C1q binding site. Techniques for preparing such immunoglobulin Fc fragment sequence derivatives are disclosed in International Patent Publications 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, and the like.

[0069] In some embodiments, an antibody comprises a variant sequence of the human IgG4 CH3 domain comprising a T366W mutation, which may optionally be referred to herein as the IgG4 CH3 knot sequence. In some embodiments, an antibody comprises a variant sequence of the human IgG4 CH3 domain comprising a T366S mutation, an L368A mutation, and a Y407V mutation, which may optionally be referred to herein as the IgG4 CH3 hole sequence. The IgG4 CH3 mutations described herein may be used in any suitable manner so as to place a knot in a first heavy chain constant region of a first monomer in an antibody dimer and a hole in a second heavy chain constant region of a second monomer in an antibody dimer, thereby facilitating proper pairing (heterodimerization) of the desired pair of heavy chain polypeptide subunits in the antibody.

[0070] In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a Petition 870260076930, dated 07 / 31 / 2026, page 37 / 122 24 / 81 variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, an L235A mutation, and a T366W (knot) mutation. In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, an L235A mutation, a T366S mutation, an L368A mutation, and a Y407V (hole) mutation.

[0071] The term antibody comprising an Fc region refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Consequently, an antibody having an Fc region according to this invention may comprise an antibody with or without K447.

[0072] Aspects of the invention include antibodies comprising a variable-only heavy chain region in a monovalent or bivalent configuration. As used herein, the term monovalent configuration as used in reference to a variable-only heavy chain region domain means that only one variable-only heavy chain region domain is present, having a single binding site. In contrast, the term bivalent configuration, as used in reference to a variable-only heavy chain region domain, means that two variable-only heavy chain region domains are present (each having a single binding site) and are connected by a linker sequence. Non-limiting examples of linker sequences are discussed further herein and include, without limitation, GS linker sequences of various lengths.When a variable-only heavy chain region is in a bivalent configuration, each of the two domains of the variable-only heavy chain region can bind to the same antigen or to different antigens (e.g., to...). Petition 870260076930, dated 07 / 31 / 2026, page 38 / 122 25 / 81 different epitopes on the same protein; for two different proteins, etc.). However, unless specifically indicated otherwise, a heavy chain-only variable region denoted as being in a bivalent configuration is understood as containing two identical heavy chain-only variable region domains connected by a linker sequence, wherein each of the two identical heavy chain-only variable region domains binds to the same target antigen.

[0073] Aspects of the invention include antibodies having multispecific configurations, which include, without limitation, bispecific, trispecific, etc. A wide variety of methods and protein configurations are known and used in bispecific monoclonal antibodies (BsMAB), trispecific antibodies, etc.

[0074] Several methods for the production of multivalent artificial antibodies have been developed by recombinant fusion of variable domains from two or more antibodies. In some embodiments, a first and a second antigen-binding domain in a polypeptide are connected by a polypeptide linker. A non-limiting example of such a polypeptide linker is a GS linker, having 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 (SEQ ID NO: 42). Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 43) (n=1) and GGGGSGGGGS (SEQ ID NO: 44) (n=2). Other suitable ligands may also be used and are described, for example, in Chen et al., Adv Drug Deliv Rev. 2013, October 15; 65(10): 1357-69, the disclosure of which is incorporated herein by reference in its entirety.

[0075] The term three-chain antibody-like molecule or TCA is used herein to refer to antibody-like molecules comprising, consisting essentially of, or Petition 870260076930, dated 07 / 31 / 2026, p. 39 / 122 26 / 81 consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of a heavy and a light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains comprising an antigen-binding region and at least one CH domain. 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 comprising CH2 and / or CH3 and / or CH4 domains, in the absence of a CH1 domain, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind an epitope of a second antigen or an epitope other than the first antigen, wherein such binding domain is derived from, or has sequence identity with, the variable region of an antibody heavy or light chain.Parts of this variable region can be encoded by VH and / or VL gene segments, D and JH gene segments, or JL gene segments. The variable region can be encoded by rearranged VHDJH, VLDJH, VhJl, or VlJl gene segments.

[0076] A TCA-binding compound makes use of a heavy chain-only antibody or heavy chain antibody or heavy chain polypeptide which, as used herein, means a single-chain antibody comprising constant CH2 and / or CH3 and / or CH4 heavy chain regions, but no CH1 domain. In one embodiment, the heavy chain antibody is composed of an antigen-binding domain, at least part of a hinge region, and CH2 and CH3 domains. In another embodiment, the heavy chain antibody is composed of an antigen-binding domain, at least part of a hinge region, and a CH2 domain. In yet another embodiment, the heavy chain antibody is composed of an antigen-binding domain, at least part of a hinge region, and a CH3 domain. The Petition 870260076930, dated 07 / 31 / 2026, page 40 / 122 27 / 81 Heavy chain antibodies in which the CH2 and / or CH3 domains are truncated are also included here. In another embodiment, the heavy chain is composed of an antigen-binding domain and at least one CH domain (CH1, CH2, CH3, or CH4), but no hinge region. The heavy chain-only antibody may be in the form of a dimer, in which two heavy chains are linked by disulfide bridges or otherwise covalently or non-covalently linked to each other and may optionally include an asymmetric interface (e.g., a knot-hole (KiH) interface) between one or more of the CH domains to facilitate proper pairing between the polypeptide chains. The heavy chain antibody may belong to the IgG subclass, but antibodies belonging to other subclasses, such as the IgM, IgA, IgD, and IgE subclasses, are also included here.In one particular embodiment, the heavy chain antibody is of the IgG1, IgG2, IgG3 or IgG4 subtype, in particular of the IgG1 subtype or the IgG4 subtype. Non-limiting examples of a TCA-binding compound are described in, for example, WO2017 / 223111 and WO2018 / 052503, the disclosures of which are incorporated herein by reference in their entirety.

[0077] Heavy chain antibodies constitute about a quarter of the IgG antibodies produced by camelids, for example, camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains, but are devoid of light chains. As a consequence, the variable antigen-binding portion is referred to as the VHH domain and represents the smallest naturally occurring intact antigen-binding site, with only about 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). High-specificity, high-affinity heavy chain antibodies can be generated against a variety of antigens through immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)) and the Petition 870260076930, dated 07 / 31 / 2026, p. 41 / 122 The 28 / 81 VHH portion can be easily cloned and expressed in yeast (Frenken, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Its expression levels, solubility, and stability are significantly higher than those of classic 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, termed VNAR (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

[0078] The term CD19 as used herein refers to a 95 kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily that acts as a coreceptor for the B-cell antigen receptor (BCR) complex on B lymphocytes. The term CD19 includes a CD19 protein from any human and non-human animal species, and specifically includes human CD19 as well as CD19 from non-human mammals.

[0079] The term human CD19 as used herein includes any variants, isoforms and species homologs of human CD19 (UniProt P15391), regardless of their source or mode of preparation. Thus, human CD19 includes human CD19 naturally expressed by cells and CD19 expressed in cells transfected with the human CD19 gene.

[0080] The terms anti-CD19 heavy chain-only antibody, CD19 heavy chain-only antibody, anti-CD19 heavy chain antibody, and CD19 heavy chain antibody are used herein interchangeably to refer to a heavy chain-only antibody as defined above that binds immunospecifically to CD19, including human CD19, as defined above. The definition includes, without limitation, human heavy chain antibodies produced by transgenic animals, such as transgenic rats or transgenic mice expressing human immunoglobulin, including UniRats™ producing antibodies. Petition 870260076930, dated 07 / 31 / 2026, page 42 / 122 29 / 81 UniAbTManti-CD19 in humans, as defined above.

[0081] Percent amino acid sequence identity (%) with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percentage sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved in several ways that are within the expertise of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Experts in the technique can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment along the entire length of the sequences being compared. For purposes here, however, % amino acid sequence identity values ​​are generated using the ALIGN-2 sequence comparison computer program.

[0082] An isolated antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are materials that will interfere with diagnostic or therapeutic uses for the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to more than 95% by weight of the antibody, as determined by the Lowry method and, most preferably, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 amino acid sequence residues from the N-terminus or internal by the use of a rotary cup sequencer or (3) to homogeneity by Petition 870260076930, dated 07 / 31 / 2026, page 43 / 122 30 / 81 SDS-PAGE under reduction or non-reduction conditions using Coomassie blue or, preferably, silver dye. The isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Usually, however, the isolated antibody will be prepared by at least one purification step.

[0083] The antibodies of the invention include multispecific antibodies. Multispecific antibodies have more than one binding specificity. The term multispecific specifically includes bispecific and trispecific, as well as independent higher-order specific binding affinities such as higher-order polyepitope specificity, as well as tetravalent antibodies and antibody fragments. The terms multispecific antibody, heavy chain-only multispecific antibody, heavy chain multispecific antibody, and multispecific UniAb™ are used herein in the broadest sense to encompass all antibodies with more than one binding specificity. The multispecific heavy chain anti-CD19 antibodies of the present invention specifically include antibodies that bind immunospecifically to two or more non-overlapping epitopes on a CD19 protein, such as human CD19 (i.e., bivalent and biparatopic).The multispecific anti-CD19 heavy chain antibodies of the present invention also specifically include antibodies that bind immunospecifically to an epitope on a CD19 protein, such as human CD19, and to an epitope on a different protein, such as, for example, a CD3 protein, such as human CD3 (i.e., bivalent and biparatopic). The multispecific anti-CD19 heavy chain antibodies of the present invention also specifically include antibodies that bind immunospecifically to two or more non-overlapping or partially overlapping epitopes on a CD19 protein, such as a protein. Petition 870260076930, dated 07 / 31 / 2026, p. 44 / 122 31 / 81 Human CD19, and an epitope on a different protein, such as, for example, a CD3 protein, like human CD3 protein (i.e., trivalent and biparatopic).

[0084] The antibodies of the invention include monospecific antibodies having a binding specificity. Monospecific antibodies specifically include antibodies comprising a single binding specificity, as well as antibodies comprising more than one binding unit having the same binding specificity. The terms monospecific antibody, monospecific heavy chain-only antibody, monospecific heavy chain antibody, and monospecific UniAb™ are used herein in the broadest sense and encompass all antibodies with a binding specificity. The monospecific anti-CD19 heavy chain antibodies of the present invention specifically include antibodies that bind immunospecifically to an epitope on a CD19 protein, such as a human CD19 protein (monovalent and monospecific).The monospecific heavy chain anti-CD19 antibodies of the present invention also specifically include antibodies having more than one binding unit (e.g., multivalent antibodies) that bind immunospecifically to an epitope on a CD19 protein, such as human CD19. For example, a monospecific antibody according to embodiments of the invention may include a variable heavy chain region comprising two antigen-binding domains, wherein each antigen-binding domain binds to the same epitope on a CD19 protein (i.e., bivalent and monospecific).

[0085] An epitope is the site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes. Petition 870260076930, dated 07 / 31 / 2026, page 45 / 122 32 / 81

[0086] Epitope mapping is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens. Antibody epitopes can be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed by amino acids that are discontinuous in the protein sequence but are joined together after the protein folds into its three-dimensional structure.

[0087] Polyepitope specificity refers to the ability to bind specifically to two or more different epitopes on the same target or on different target(s). As noted above, the present invention specifically includes anti-CD19 heavy chain antibodies with polyepitope specificities, that is, anti-CD19 heavy chain antibodies that bind to one or more non-overlapping epitopes on a CD19 protein, such as a human CD19; and anti-CD19 heavy chain antibodies that bind to one or more epitopes on a CD19 protein and to an epitope on a different protein, such as, for example, a CD3 protein. The term non-overlapping epitope(s) or non-competitive epitope(s) of an antigen is defined herein to mean epitope(s) that are recognized by one member of a pair of antigen-specific antibodies, but not by the other member.Pairs of antibodies or antigen-binding regions targeting the same antigen in a multispecific antibody, recognizing non-overlapping epitopes, do not compete for binding to that antigen and are able to bind to that antigen simultaneously.

[0088] An antibody essentially binds to the same epitope as a reference antibody when the two antibodies recognize identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or overlapping epitopes Petition 870260076930, dated 07 / 31 / 2026, p. 46 / 122 33 / 81 Sterically, these are competitive assays, which can be set up in several different formats, using labeled antigen or labeled antibody. Typically, the antigen is immobilized in a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzymatic markers.

[0089] The term valent as used herein refers to a specified number of binding sites on an antibody molecule.

[0090] A monovalent antibody has one binding site. Thus, a monovalent antibody is also monospecific.

[0091] A multivalent antibody has two or more binding sites. Thus, the terms bivalent, trivalent, and tetravalent refer to the presence of two binding sites, three binding sites, and four binding sites, respectively. Thus, a bispecific antibody according to the invention is at least bivalent and may be trivalent, tetravalent, or multivalent. A bivalent antibody according to embodiments of the invention may have two binding sites for the same epitope (i.e., bivalent, monoparatopic) or for two different epitopes (i.e., bivalent, biparatopic).

[0092] A wide variety of methods and protein configurations are known and used for the preparation of bispecific monoclonal antibodies (BsMAB), trispecific antibodies and the like.

[0093] The term three-chain antibody-like molecule or TCA is used herein to refer to antibody-like molecules comprising, essentially consisting of, or consisting of three polypeptide subunits, two of which comprise, essentially consist of, or consist of a heavy chain and a light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains, comprising an antigen-binding region and Petition 870260076930, dated 07 / 31 / 2026, page 47 / 122 34 / 81 at least one CH domain. This heavy chain / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, essentially consists of, or consists of a heavy chain-only antibody comprising an Fc portion comprising CH2 and / or CH3 and / or CH4 domains, in the absence of a CH1 domain, and an antigen-binding domain that binds to an epitope of a second antigen or an epitope different from the first antigen, wherein such binding domain is derived from, or has sequence identity with, the variable region of an antibody heavy or light chain. Portions of this variable region may be encoded by segments of VH and / or VL genes, segments of D and JH genes, or segments of JL genes. The variable region may be encoded by rearranged VHDJH, VlDJh, VhJl, or VlJl gene segments. A TCA protein makes use of a heavy chain-only antibody as defined above.

[0094] The term chimeric antigen receptor or CAR is used here in the broadest sense to refer to a modified receptor that grafts a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) onto membrane-spanning and intracellular signaling domains. Typically, the receptor is used to graft the specificity of a monoclonal antibody onto a T cell to create 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). CAR-T cells are T cells that have been genetically modified to produce an artificial T cell receptor for use in immunotherapy.In one embodiment, CAR-T cell means a therapeutic T cell that expresses a transgene encoding one or more chimeric antigen receptors comprising at least an extracellular domain, a transmembrane domain, and at least one cytosolic domain.

[0095] The term human antibody is used here for Petition 870260076930, dated 07 / 31 / 2026, page 48 / 122 35 / 81 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 UniAbs™ produced by UniRats™, as defined above.

[0096] A chimeric antibody or chimeric immunoglobulin is understood to be an immunoglobulin molecule comprising amino acid sequences from at least two different Ig loci, for example, a transgenic antibody comprising a portion encoded by a human Ig locus and a portion encoded by a mouse Ig locus. Chimeric antibodies include transgenic antibodies with non-human Fc regions or artificial Fc regions and human idiotypes. Such immunoglobulins may be isolated from animals of the invention that have been manipulated to produce such chimeric antibodies.

[0097] As used herein, the term effector cell refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Some effector cells express specific Fc receptors and perform specific immunological functions. In some modes, an effector cell, such as a natural killer cell, is capable of inducing antibody-dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages, which express FcR, are involved in the specific killing of target cells and in the presentation of antigens to other components of the immune system. Petition 870260076930, dated 07 / 31 / 2026, page 49 / 122 36 / 81 immunological or in binding to cells that present antigens. In some modes, an effector cell may proceed to phagocytose a target antigen or target cell.

[0098] 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 FcyRIII and perform the ADCC effector function. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with NK cells being preferred. Effector cells can be isolated from a native source, for example, from blood or PBMCs as described herein.

[0099] The term immune cell is used here in the broadest sense, including, without limitation, cells of myeloid or lymphoid origin, for example, lymphocytes (such as B cells and T cells including cytotoxic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells such as neutrophils, granulocytes, mast cells and basophils.

[0100] Antibody effector functions refer to those biological activities attributable to the Fc region (a 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.

[0101] Antibody-dependent cell-mediated cytotoxicity and ADCC refer to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies. Petition 870260076930, dated 07 / 31 / 2026, p. 50 / 122 37 / 81 bound to a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express only FcyRIII, while monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and Natural Killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that reported in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0102] 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, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed.

[0103] Binding affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, binding affinity refers to intrinsic binding affinity reflecting a 1:1 interaction between the members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in Petition 870260076930, dated 07 / 31 / 2026, page 51 / 122 38 / 81 technique. Low-affinity antibodies generally bind to antigens slowly and tend to dissociate readily, while high-affinity antibodies generally bind to antigens more rapidly and tend to remain bound.

[0104] As used herein, Kd or Kd value refers to a dissociation constant determined by BioLayer Interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetic mode. For example, mouse anti-Fc sensors are loaded with mouse Fc fused antigen and then immersed in wells containing antibodies to measure concentration-dependent association rates (kon). Antibody dissociation rates (koff) are measured in the final step when the sensors are immersed in wells containing only buffer. Kd is the koff / kon ratio. (For more details see, Concepcion, J, et al., Comb Chem High Throughput Screen, 12 (8), 791-800, 2009).

[0105] The terms treatment, treat, and the like are used herein to generally mean the attainment of a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of total or partial prevention of a disease or symptom thereof and / or may be therapeutic in terms of partial or complete cure of a disease and / or adverse effect attributable to the disease. Treatment, as used herein, encompasses any treatment of a disease in a mammal and includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., halting 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. Treatment of ongoing disease, where treatment stabilizes or reduces the patient's undesirable clinical symptoms, is of particular interest.This treatment is ideally performed before complete loss of [the cell / capacity]. Petition 870260076930, dated 07 / 31 / 2026, page 52 / 122 39 / 81 function in the affected tissues. The therapy in question can be administered during the symptomatic stage of the disease and, in some cases, after the symptomatic stage of the disease.

[0106] A therapeutically effective amount means an amount of active agent that is needed to confer therapeutic benefit to a subject. For example, a therapeutically effective amount is an amount that induces, improves, or otherwise causes an improvement in pathological symptoms, disease progression, or physiological conditions associated with a disease, or that improves resistance to a disorder.

[0107] The terms “B-cell neoplasms” or “mature B-cell neoplasms” in the context of the present invention include, but are not limited to, all lymphoid leukemias and lymphomas, chronic lymphocytic leukemia, acute lymphoblastic leukemia, prolymphocytic leukemia, B-cell precursor lymphoblastic leukemia, hairy cell leukemia, small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt 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, marginal B-cell lymphoma nodal, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis,Non-Hodgkin lymphoma, Hodgkin lymphoma, hairy cell leukemia, primary effusion lymphoma, and AIDS-related non-Hodgkin lymphoma.

[0108] The term characterized by CD19 expression refers broadly to any disease or disorder in which CD19 expression is associated with or involved in one or more pathological processes that are characteristic of the disease or Petition 870260076930, dated 07 / 31 / 2026, page 53 / 122 40 / 81 disorder. Such disorders include, but are not limited to, B-cell neoplasms.

[0109] The terms subject, individual, and patient are used here interchangeably to refer to a mammal being evaluated for treatment and / or being treated. In one embodiment, the mammal is a human being. The terms subject, individual, and patient encompass, without limitation, individuals having cancer, individuals with autoimmune diseases, with infections by pathogens, and the like. Subjects may be humans, but also include other mammals, particularly those mammals useful as laboratory models for human diseases, e.g., mice, rats, etc.

[0110] The term pharmaceutical formulation refers to a preparation that is in a form such that it allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to an individual to whom the formulation will be administered. Such formulations are sterile. Pharmaceutically acceptable excipients (vehicles, additives) are those that can reasonably be administered to a mammalian individual to provide an effective dose of the active ingredient employed.

[0111] A sterilized formulation is aseptic or free or essentially free of all living microorganisms and their spores. A frozen formulation is one at a temperature below 0 °C.

[0112] A stable formulation is one in which the protein contained therein essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. Several analytical techniques for measuring the Petition 870260076930, dated 07 / 31 / 2026, page 54 / 122 41 / 81 Protein stability data are available in the art and are reviewed in 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), for example. Stability can be measured at a selected temperature for a selected period of time.Stability can be assessed qualitatively and / or quantitatively in several different ways, including evaluating aggregate formation (e.g., using size exclusion chromatography, measuring turbidity, and / or by visual inspection); assessing charge heterogeneity using cation exchange chromatography, capillary isoelectric focusing imaging (icIEF), or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map analysis (e.g., tryptic or LYS-C); assessment of the antibody's biological activity or antigen-binding function; etc.Instability can involve any one or more of: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc. II. Detailed Description Anti-CD19 antibodies

[0113] The present invention provides a family of closely related antibodies that bind to human CD19. The antibodies in this family comprise a set of CDR sequences as defined herein and shown in Table 1, and are exemplified by the provided CDR1, CDR2, and CDR3 heavy chain sequences presented in Table 2, and the variable region heavy chain (VH) sequences of SEQ ID NOs: 5 and 6 presented. Petition 870260076930, dated 07 / 31 / 2026, page 55 / 122 42 / 81 in Table 3. The antibody family provides some benefits that contribute to its usefulness as clinically therapeutic agent(s). Antibodies include members with a range of binding affinities, allowing the selection of a specific sequence with a desired binding affinity.

[0114] Table 1: Amino acid sequences of unique CDRs of anti-CD19 heavy chain antibodies. SEQ_aa_CDR1 SEQ_aa_CDR2 SEQ_aa_CDR3 GFTFSDYY (SEQ ID NO: 1) MSSSGSTI (SEQ ID NO: 2) ARGGIAATGT (SEQ ID NO: 3) ARGGIAAAGT (SEQ ID NO: 4) GFDFSDYY (SEQ ID NO: 38) MSSDGSTI (SEQ ID NO: 39)

[0115] Table 2: Amino acid sequences of CDR1, CDR2 and CDR3 heavy chain antibodies against CD19. Clone ID # SEQ_aa_CDR1 SEQ aa CDR2 SEQ_aa_CDR3 370083 GFTFSDYY NO: 1) (SEQ ID MSSSGSTI ID NO: 2) (SEQ ARGGIAATGT ID NO: 3) (SEQ 370034 GFTFSDYY NO: 1) (SEQ ID MSSSGSTI ID NO: 2) (SEQ ARGGIAAAGT ID NO: 4) (SEQ

[0116] Table 3: Amino acid sequences of the variable domain of anti-CD19 heavy chain antibodies. Clone ID # SEQ aa FR1 FR4 SEQ ID NO. 37008 3 QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG KGLEWVSY MSSSGSTIYYADSVKGRFTISRDNAKNSLYLQMTSLRAEDTA VYYCARGGI AATGTWGQGTLVTVSS 5 Petition 870260076930, dated 07 / 31 / 2026, page 56 / 122 43 / 81 Clone ID # SEQ aa FR1 FR4 SEQ ID NO. 37003 3 MSSSGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTA VYYCARGGI AAAGTWGQGTLVTVSS 40 S54D QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG KGLEWVSY MSSDGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTA VYYCARGGI AAAGTWGQGTLVTVSS 41

[0117] A suitable antibody may be selected from those provided herein for development and therapeutic or other use, including, without limitation, use as a bispecific antibody, or part of a CAR-T structure, as shown, for example, in FIG. 4, panel B.

[0118] The determination of affinity for a candidate protein can be performed using methods known in the art, such as Biacore measurements. Antibody family members may have affinity for CD19 with a Kd from about 10⁻⁶ to approximately about 10⁻¹¹, including, without limitation: from about 10⁻⁶ to approximately about 10¹⁰; from about 10⁻⁶ to approximately about 10⁻⁹; from about 10⁻⁶ to approximately about 10⁻⁸; from about 10⁻⁸ to approximately about 10⁻¹¹; from about 10⁻⁸ to approximately about 10⁻¹⁰; from about 10⁻⁸ to approximately about 10⁻⁹; from about 10⁻⁹ to Petition 870260076930, dated 07 / 31 / 2026, page 57 / 122 44 / 81 approximately about 10-11; from about 10-9 to approximately about 10-10; or any value within these ranges. Affinity selection can be confirmed with a biological assessment of modulation, for example, blockade, of a CD19 biological activity, including in vitro assays, preclinical models, and clinical trials, as well as assessment of potential toxicity.

[0119] Members of the antibody family here do not cross-react with Cynomolgus monkey CD19 protein, but can be manipulated to provide cross-reactivity with Cynomolgus monkey CD19 protein, or with CD19 from any other animal species, if desired.

[0120] The CD19-specific antibody family herein comprises a VH domain, comprising CDR1, CDR2, and CDR3 sequences in a human VH framework. The CDR sequences may be located, for example, in the region around amino acid residues 26-33; 51-58; and 97-116 for CDR1, CDR2, and CDR3, respectively, of the exemplary variable region sequences provided in SEQ ID Nos: 56. It will be understood by those usually skilled in the art that the CDR sequences may be in different positions if a different framework sequence is selected, although generally the sequence order remains the same.

[0121] In some embodiments, an anti-CD19 antibody comprises a CDR1 sequence of either SEQ ID NOs: 1 or 38. In a particular embodiment, an anti-CD19 antibody comprises a CDR1 sequence of SEQ ID NO: 1. In a particular embodiment, an anti-CD19 antibody comprises a CDR1 sequence of SEQ ID NO: 38.

[0122] In some embodiments, an anti-CD19 antibody comprises a CDR2 sequence of either SEQ ID NO: 2 or 39. In one particular embodiment, an anti-CD19 antibody comprises a CDR2 sequence of SEQ ID NO: 2. In one embodiment Petition 870260076930, dated 07 / 31 / 2026, p. 58 / 122 45 / 81 in particular, an anti-CD19 antibody comprises a CDR2 sequence of SEQ ID NO: 39.

[0123] In some embodiments, an anti-CD19 antibody comprises a CDR3 sequence of any of the SEQ ID NOs: 34. In a particular embodiment, an anti-CD19 antibody comprises a CDR3 sequence of the SEQ ID NO: 3. In a particular embodiment, an anti-CD19 antibody comprises a CDR3 sequence of the SEQ ID NO: 4.

[0124] In a further embodiment, an anti-CD19 heavy chain-only antibody comprises the CDR1 sequence from SEQ ID NO: 1; the CDR2 sequence from SEQ ID NO: 2; and the CDR3 sequence from SEQ ID NO: 3.

[0125] In a further embodiment, an anti-CD19 antibody comprises the CDR1 sequence of SEQ ID NO: 1; the CDR2 sequence of SEQ ID NO: 2; and the CDR3 sequence of SEQ ID NO: 4.

[0126] In a further embodiment, an anti-CD19 antibody comprises the CDR1 sequence of SEQ ID NO: 38; the CDR2 sequence of SEQ ID NO: 2; and the CDR3 sequence of SEQ ID NO: 4.

[0127] In a further embodiment, an anti-CD19 antibody comprises the CDR1 sequence of SEQ ID NO: 1; the CDR2 sequence of SEQ ID NO: 39; and the CDR3 sequence of SEQ ID NO: 4.

[0128] In a further embodiment, an anti-CD19 antibody comprises any of the heavy chain variable region amino acid sequences of SEQ ID NOs: 5-6 and 40-41 (Table 3).

[0129] In yet another embodiment, an antiCD19 antibody comprises the variable region sequence of the heavy chain of SEQ ID NO: 5.

[0130] In yet another embodiment, an antiCD19 antibody comprises the variable region sequence of the heavy chain of SEQ ID NO: 6.

[0131] Yet another embodiment, an anti-CD19 antibody comprises variable region heavy chain sequences of Petition 870260076930, dated 07 / 31 / 2026, page 59 / 122 46 / 81 SEQ ID NO: 40.

[0132] In yet another embodiment, an antiCD19 antibody comprises the variable region sequence of the heavy chain of SEQ ID NO: 41.

[0133] In some embodiments, a CDR sequence in an anti-CD19 antibody of the invention comprises one or two amino acid substitutions relative to a CDR1, CDR2 and / or CDR3 sequence or set of CDR1, CDR2 and CDR3 sequences in any of the SEQ ID NOs: 1-4, 38 and 39 (Table 1; Table 2).

[0134] In some embodiments, an anti-CD19 antibody preferably comprises a variable heavy chain (VH) domain in which the CDR3 sequence has more than or equal to 80%, such as at least 85%, at least 90%, at least 95%, or at least 99% sequence identity at the amino acid level with a CDR3 sequence of any of the antibodies whose CDR3 sequences are provided in Table 1 or Table 2, and binds to CD19.

[0135] In some embodiments, an anti-CD19 antibody preferably comprises a variable heavy chain (VH) domain in which the complete set of CDRs 1, 2, and 3 (combined) has more than or equal to eighty-five percent (85%) amino acid sequence identity with the CDRs 1, 2, and 3 (combined) of the antibodies whose CDR sequences are provided in Table 1 or Table 2, and binds to CD19.

[0136] In some embodiments, an anti-CD19 antibody comprises a variable region heavy chain sequence with at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity with any of the variable region heavy chain sequences of SEQ ID NOs: 5-6 and 40-41 (shown in Table 3), and binds to CD19.

[0137] In some modalities, antibodies are provided. Petition 870260076930, dated 07 / 31 / 2026, p. 60 / 122 47 / 81 bispecific or multispecific antibodies, which may have any of the configurations discussed herein, including, without limitation, a molecule similar to a bispecific three-chain antibody (TCA). In some embodiments, a multispecific antibody may comprise at least one variable heavy chain region having binding specificity for CD19, and at least one variable heavy chain region having binding specificity for a protein other than CD19. In some embodiments, a multispecific antibody may comprise at least one variable heavy chain region that binds to CD19, and at least one variable heavy chain region that binds to a protein other than CD19. In some embodiments, a multispecific antibody may comprise a variable heavy chain region comprising at least two antigen-binding domains, wherein each of the antigen-binding domains binds to CD19.In some embodiments, a multispecific antibody may comprise a heavy chain / light chain pair that binds to a primary antigen (e.g., CD3), and a heavy chain of a single heavy chain antibody. In certain embodiments, the heavy chain of the single heavy chain antibody comprises an Fc portion comprising CH2 and / or CH3 and / or CH4 domains, in the absence of a CH1 domain. In a particular embodiment, a bispecific antibody comprises a heavy chain / light chain pair that binds to an antigen on an effector cell (e.g., a CD3 protein on a T cell), and a heavy chain of a single heavy chain antibody comprising an antigen-binding domain that binds to CD19.

[0138] In some embodiments, a multispecific antibody comprises a CD3-binding VH domain that is paired with a variable light chain 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 the SEQ Petition 870260076930, dated 07 / 31 / 2026, page 61 / 122 48 / 81 In some embodiments, the CD3-binding VH domain comprises a CDR1 sequence from SEQ ID NO: 10, a CDR2 sequence from SEQ ID NO: 11, and a CDR3 sequence from SEQ ID NO: 12, in a human VH scaffold. In some embodiments, the fixed light chain comprises a CDR1 sequence from SEQ ID NO: 13, a CDR2 sequence from SEQ ID NO: 14, and a CDR3 sequence from SEQ ID NO: 15, in a human VH scaffold. Together, the CD3-binding VH domain and the variable light chain domain have binding affinity for CD3. In some embodiments, a CD3-binding VH domain comprises a variable region heavy chain sequence of SEQ ID NO: 16. In some embodiments, a CD3-binding VH domain comprises a variable region heavy chain sequence of SEQ ID NO: 17.In some embodiments, a 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% percentage identity with the variable region heavy chain sequence of SEQ ID NO: 16 or 17. In some embodiments, a fixed light chain comprises a variable region light chain sequence of SEQ ID NO: 18. In some embodiments, a 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% percentage identity with the variable region heavy chain sequence of SEQ ID NO: 18.

[0139] Multispecific antibodies comprising the CD3-binding VH domain and variable light chain domain described above have advantageous properties, for example, as described in the published PCT application number WO2018 / 052503, the disclosure of which is incorporated herein in its entirety by reference. Any of the antibodies Petition 870260076930, dated 07 / 31 / 2026, page 62 / 122 49 / 81 multispecific and antigen-binding domains described herein, having binding affinity for CD19, can be combined with the CD3-binding domains and fixed light chain domains described herein (see, for example, Table 4 and Table 5), as well as additional sequences, such as those provided in Table 6 and Table 7, to generate multispecific antibodies having binding affinity for one or more epitopes of CD19 as well as CD3. Table 4. Amino acid sequences of CDR1, CDR2, CDR3 Chain Heavy and Light Anti-CD3. SEQ_aa_CDR1 SEQ_aa_CDR2 SEQ_aa_CDR3 Heavy Chain (F2B) GFTFDDYA (SEQ ID NO: 7) ISWNSGSI (SEQ ID NO: 8) AKDSRGYGDYRLGGAY (SEQ ID NO: 9) Heavy Chain (F2F) GFTFHNYA (SEQ ID NO: 10) ISWNSGSI (SEQ ID NO: 11) AKDSRGYGDYSLGGAY (SEQ ID NO: 12) Light Chain QSVSSN (SEQ ID NO: 13) GAS (SEQ ID NO: 14) QQYNNWPWT (SEQ ID NO: 15) Table 5. Amino acid sequences of variable regions of anti-CD3 heavy and light chains. VH (F2B) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEW VSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKDSRGYGDYRLGGAYWGQGTLVTVSS (SEQ ID NO: 16) VH (F2F) VL EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYG ASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQ GTKVEIK (SEQ ID NO: 18) Table 6: Fc region sequences of human IgG1 and IgG4. Petition 870260076930, dated 07 / 31 / 2026, page 63 / 122 50 / 81 IgG1 humana (UniProt No. P01857) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 19) IgG4 humana (UniProt No.P01861) ASTKGPSVFP LAPCRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 20) Human IgG1 with silencing mutations (Fc region) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVT CVVVDVSHED P EVKFNWYVD GVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 21). Petition 870260076930, de 31 / 07 / 2026, pág. 64 / 122 51 / 81 Human IgG4 with silencing mutations (Fc region) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 22) Table 7: Additional sequences. Sequence of constant region of anti-CD3 light chain (kappa light chain) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23) Sequence of anti-CD3 heavy chain (VH + Fc of IgG1 ts) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPG KGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMN SLRAEDTALYYCAKDSRGYGDYRLGGAYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVT CVVVDVSHED P EVKFNWYVD GVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24) Petition 870260076930, dated 07 / 31 / 2026, page 65 / 122 52 / 81 Sequence of EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPG heavy chain KGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMN anti-CD3 (with Fc SLRAEDTALYYCAKDSRGYGDYRLGGAYWGQGTLVTVSSAST of IgG1 KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA silenced) LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVT CVVVDVSHED P EVKFNWYVD GVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25) Sequence of EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPG constant region KGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMN heavy chain SLRAEDTALYYCAKDSRGYGDYRLGGAYWGQGTLVTVSSAST anti-CD3 (with Fc KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA of IgG4 ts) LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 26) Petition 870260076930, dated 07 / 31 / 2026, page 66 / 122 53 / 81 Sequence of constant region of anti-CD3 heavy chain (with Fc of IgG4 silenced) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPG KGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMN SLRAEDTALYYCAKDSRGYGDYRLGGAYWGQGTLVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 27) IgG4 silenced (hinge - CH2 - CH3; hole (S228P, F234A, L235A; T366S, L368A, Y407V)) ESKYGPPCPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK (SEQ ID NO: 28) IgG4 silenced (hinge - CH2 CH3; node (S228P, F234A, L235A;T366W)) ESKYGPPCPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK (SEQ ID NO: 29) Complete anti-CD3 light chain (VL + CL sheath) EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQ APRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFA VYYCQQYNNWPWTFGQGTKVEIKRTVAAP SVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 30); Petition 870260076930, dated 07 / 31 / 2026, page 67 / 122 54 / 81 Full anti-CD3 heavy chain (VH + Fc of silenced IgG4 + node (S228P, F234A, L235A;T366W)) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPG KGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMN SLRAEDTALYYCAKDSRGYGDYRLGGAYWGQGTLVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 31) Cadeia pesada monovalente para CD19 (clone ID 370083) + Fc de IgG4 silenciado, buraco (S228P, F234A, L235A, T366S, L368A, Y407V) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG KGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKNSLYLQMT SLRAEDTAVYYCARGGIAATGTWGQGTLVTVSSESKYGPPCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGK (SEQ ID NO: 32); Petition 870260076930, dated 07 / 31 / 2026, p. 68 / 122 55 / 81 Bivalent heavy chain for CD19 (clone ID 370083) + silenced IgG4 Fc, hole (S228P, F234A, L235A, T366S, L368A, Y407V) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG KGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKNSLYLQMT SLRAEDTAVYYCARGGIAATGTWGQGTLVTVSSGGGGSGGGG SQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP GKGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKNSLYLQM TSLRAEDTAVYYCARGGIAATGTWGQGTLVTVSSESKYGPPC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP PSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO: 33) Monovalent heavy chain for CD19 (clone ID 370034) + Fc of silenced IgG4, hole (S228P, F234A, L235A, T366S, L368A,Y407V) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG KGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKKSLYLQMN SLRAEDTAVYYCARGGIAAAGTWGQGTLVTVSSESKYGPPCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGK (SEQ ID NO: 34), Petition 870260076930, dated 07 / 31 / 2026, page 69 / 122 56 / 81 Bivalent weighted cell for CD19 (clone ID 370034) + silenced IgG4 protein, buraco (S228P, F234A, L235A, T366S, L368A, Y407V) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG KGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKKSLYLQMN SLRAEDTAVYYCARGGIAAAGTWGQGTLVTVSSGGGGSGGGG SQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP GKGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKKSLYLQM NSLRAEDTAVYYCARGGIAAAGTWGQGTLVTVSSESKYGPPC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP PSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO: 35) Bivalent weighted cell for CD19 (clone ID 370083 x clone ID 370034) + silenced IgG4 Fc, buraco (S228P, F234A, L235A, T366S, L368A,Y407V) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG KGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKNSLYLQMT SLRAEDTAVYYCARGGIAATGTWGQGTLVTVSSGGGGSGGGG SQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP GKGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKKSLYLQM NSLRAEDTAVYYCARGGIAAAGTWGQGTLVTVSSESKYGPPC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP PSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO: 36), Petição 870260076930, de 31 / 07 / 2026, pág. 70 / 122 57 / 81 Cadeia pesada bivalente para CD19 (clone ID 370034 x clone ID 370083) + Fc de IgG4 silenciado, buraco (S228P, F234A, L235A, T366S, L368A, Y407V) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG KGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKKSLYLQMN SLRAEDTAVYYCARGGIAAAGTWGQGTLVTVSSGGGGSGGGG SQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP GKGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKNSLYLQM TSLRAEDTAVYYCARGGIAATGTWGQGTLVTVSSESKYGPPC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP PSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO: 37)

[0140] In some embodiments, bispecific or multispecific antibodies are provided, which may have any of the configurations discussed herein, including, without limitation, a molecule similar to a bispecific three-chain antibody (TCA). In some embodiments, a bispecific antibody may comprise at least one variable heavy chain region that binds to CD19, and at least one variable heavy chain region that binds to a protein other than CD19. In some embodiments, a bispecific antibody may comprise a heavy chain / light chain pair that binds to a first antigen, and a heavy chain of a single heavy chain antibody comprising an Fc portion comprising CH2 and / or CH3 and / or CH4 domains, in the absence of a CH1 domain, and an antigen-binding domain that binds to an epitope of a second antigen or an epitope other than the first antigen.In one particular embodiment, a bispecific antibody comprises a heavy chain / light chain pair that binds to an antigen on an effector cell (e.g., a CD3 protein on a T cell), and a heavy chain of a heavy chain-only antibody comprising an antigen-binding domain that binds to CD19. Petition 870260076930, dated 07 / 31 / 2026, p. 71 / 122 58 / 81

[0141] In some embodiments, when an antibody of the invention is a bispecific antibody, one arm of the antibody (a binding fragment, or a binding unit) is specific for human CD19, while the other arm may be specific for target cells, tumor-associated antigens, targeting antigens, for example, integrins, etc., pathogenic antigens, checkpoint proteins, and the like. Target cells specifically include cancer cells, including, without limitation, cells associated with hematological malignancies characterized by CD19 expression, as well as pathogenic B cells associated with autoimmune disorders characterized by CD19 expression. In some embodiments, one arm of the antibody (a binding fragment, or a binding unit) is specific for human CD19, while the other arm is specific for CD3.

[0142] In some embodiments, an antibody comprises an anti-CD3 light chain polypeptide comprising the sequence SEQ ID NO: 30, an anti-CD3 heavy chain polypeptide comprising the sequence SEQ ID NO: 16 or 17, and an anti-CD19 heavy chain polypeptide comprising the sequence SEQ ID NO: 5 or 6, in a monovalent or bivalent configuration, linked to the sequence of any of the SEQ ID NOs: 28 or 29. These sequences can be combined in various ways to produce a bispecific antibody of a desired IgG subclass, for example, IgG1, IgG4, silenced IgG1, silenced IgG4. In a preferred embodiment, an antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 30, a second polypeptide comprising SEQ ID NO: 31, and a third polypeptide comprising SEQ ID NO: 32, 33, 34, 35, 36, or 37.

[0143] Various multispecific antibody formats are within the scope of the invention, including, without limitation, single-chain polypeptides, two-chain polypeptides, Petition 870260076930, dated 07 / 31 / 2026, page 72 / 122 59 / 81 three-chain polypeptides, four-chain polypeptides, and their multiples. Multispecific antibodies here specifically include multispecific (e.g., bispecific) T cell-mediated antibodies that bind to both CD19 and CD3 (anti-CD19 x anti-CD3 antibodies). Such antibodies induce potent T cell-mediated killing of CD19-expressing cells. Preparation of anti-CD19 antibodies

[0144] The antibodies of the present invention can be prepared by methods known in the art. In a preferred embodiment, the antibodies herein are produced by transgenic animals, including transgenic mice and rats, preferably rats, in which the endogenous immunoglobulin genes are eliminated or deactivated. In a preferred embodiment, the heavy chain antibodies herein are produced in UniRat™. UniRats™ have their endogenous immunoglobulin genes silenced and use a human immunoglobulin heavy chain translocus to express a diverse and naturally optimized repertoire of fully human HCAbs. Although endogenous immunoglobulin loci in rats can be deactivated or silenced using a variety of technologies, in UniRat™ zinc finger (ZNF) (endo)nuclease technology was used to inactivate the endogenous rat heavy chain J locus, light chain Ck locus, and light chain CA locus.ZNF constructs for microinjection into oocytes can produce knockout (KO) strains in IgH and IgL. For details, see, for example, Geurts et al., 2009, Science 325:433. Characterization of Ig heavy chain knockout mice was reported by Menoret et al., 2010, Eur. J. Immunol. 40:2932-2941. The advantages of ZNF technology are that non-homologous end joining to silence a gene or locus via deletions of up to several kb can also provide a target site for homologous integration (Cui et al., 2011, Nat Biotechnol 29: 64-67). Human heavy chain antibodies produced in UniRat™ are called UniAbsTMe. Petition 870260076930, dated 07 / 31 / 2026, p. 73 / 122 60 / 81 can bind to epitopes that cannot be targeted with conventional antibodies. Their high specificity, affinity, and small size make them ideal for mono- and polyspecific applications.

[0145] In addition to UniAbs™, specifically included here are heavy chain-only antibodies without the camelid VHH framework and mutations and their functional VH regions. These heavy chain-only antibodies can, for example, be produced in transgenic rats or mice comprising fully human heavy chain-only gene loci, as described, for example, in WO2006 / 008548, but other transgenic mammals such as rabbit, guinea pig, rat, can also be used, with rats and mice being preferred. Heavy chain-only antibodies, including their functional VHH or VH fragments, can also be produced by recombinant DNA technology, by expression of the coding nucleic acid in a suitable eukaryotic or prokaryotic host, including, for example, mammalian cells (e.g., CHO cells), E. coli, or yeast.

[0146] Heavy chain-only antibody domains combine the advantages of antibodies and small molecule drugs: they can be mono- or multivalent; they have low toxicity; and they have reduced manufacturing costs. Due to their small size, these domains are easy to administer, including oral or topical administration, are characterized by high stability, including gastrointestinal stability; and their half-life can be adjusted for the desired use or indication. In addition, VH and VHH domains of HCAbs can be manufactured economically.

[0147] In a particular embodiment, the heavy chain antibodies of the present invention, including UniAbs™, have the native amino acid residue in the first position of the FR4 region (amino acid position 101 according to the Kabat numbering system), Petition 870260076930, dated 07 / 31 / 2026, page 74 / 122 61 / 81 is replaced by another amino acid residue, which is capable of interrupting a hydrophobic extension exposed to the surface comprising or associated with the native amino acid residue at that position. Such hydrophobic extensions are normally buried at the interface with the constant region of the antibody light chain, but are exposed to the surface in HCAbs and are intended, at least partially, for the aggregation and association of unwanted HCAb light chains. The substituted amino acid residue is preferentially 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, heavy chain-only antibodies derived from transgenic animals contain a Trp mutation to Arg at position 101. The resulting HCAbs preferentially have high antigen-binding affinity and solubility under physiological conditions in the absence of aggregation.

[0148] As part of the present invention, human IgG anti-CD19 heavy chain antibodies with unique animal UniRat™(UniAb™) sequences that bind to human CD19 in ELISA protein and cell binding assays have been identified. The identified variable region heavy chain (VH) sequences are positive for binding to human CD19 protein and / or for binding to CD19+ cells, and are all negative for binding to cells that do not express CD19.

[0149] Heavy chain antibodies that bind to non-overlapping epitopes on a CD19 protein, for example, UniAbs™, can be identified by competitive binding assays, such as enzyme-linked immunosorbent assays (ELISA assays) or flow cytometry competitive binding assays. For example, it is possible to use competition between known antibodies that bind to the target antigen and the antibody of interest. Using this approach, a set of antibodies can be divided into those that compete with the reference antibody and those that do not. Petition 870260076930, dated 07 / 31 / 2026, p. 75 / 122 62 / 81 compete. Non-competing antibodies are identified as binding to a distinct epitope that does not overlap with the epitope bound by the reference antibody. Frequently, one antibody is immobilized, the antigen is ligated, and a second labeled (e.g., biotinylated) antibody is tested in an ELISA assay for its ability to bind to the captured antigen. This can also be accomplished using surface plasmon resonance (SPR) platforms, including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and the MX96 SPR imager (Ibis technologies BV), as well as biolayer interferometry platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). For more details, see the examples here.

[0150] Typically, an antibody competes with a reference antibody if it causes approximately a 15-100% reduction in the binding of the reference antibody to the target antigen, as determined by standard techniques such as the competition binding assays described above. In various embodiments, the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more. Pharmaceutical Compositions, Uses and Treatment Methods

[0151] Another aspect of the present invention is to provide pharmaceutical compositions comprising one or more antibodies of the present invention in a mixture with a suitable pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers used herein are exemplified, but not limited to, by adjuvants, solid carriers, water, buffers, or other carriers used in the art to contain Petition 870260076930, dated 07 / 31 / 2026, page 76 / 122 63 / 81 therapeutic components or combinations thereof.

[0152] In one embodiment, a pharmaceutical composition comprises a heavy chain antibody (e.g., UniAb™) that binds to CD19. In another embodiment, a pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb™) with binding specificity for two or more non-overlapping epitopes on a CD19 protein. In a preferred embodiment, a pharmaceutical composition comprises a multispecific (including bispecific and TCA) heavy chain antibody (e.g., UniAb™) with binding specificity for CD19 and with binding specificity for a binding target on an effector cell (e.g., a binding target on a T cell, such as, for example, a CD3 protein on a T cell).In a preferred embodiment, a pharmaceutical composition comprises a multispecific heavy chain antibody (e.g., UniAb™) (including bispecific and TCA) that binds to CD19 and binds to a binding target on an effector cell (e.g., a binding target on a T cell, such as, for example, a CD3 protein on a T cell).

[0153] The pharmaceutical compositions of the antibodies used according to the present invention are prepared for storage by mixing proteins having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (see, for example, “Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)”), such as in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are not toxic to receptors at the dosages and concentrations employed and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, chloride of Petition 870260076930, dated 07 / 31 / 2026, page 77 / 122 64 / 81 benzalkonium, benzethonium chloride, phenol, butyl or benzyl alcohol; alkylparabens such as methyl- or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (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 non-ionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0154] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practices (GMP) conditions. The pharmaceutical compositions may be provided in unit dose form (i.e., the dosage for a single administration). The formulation depends on the chosen route of administration. The antibodies described herein may be administered by intravenous or subcutaneous injection or infusion. For administration by injection, the antibodies described herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers to reduce discomfort at the injection site. The solution may contain carriers, excipients, or stabilizers as discussed above. Alternatively, the antibodies may be in lyophilized form for formulation with a suitable vehicle, e.g., pyrogen-free sterile water, prior to use.

[0155] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used for heavy chain antibodies, including UniAbs™. Petition 870260076930, dated 07 / 31 / 2026, p. 78 / 122 65 / 81 of this invention. Subcutaneous antibody formulations are described, for example, in US20160355591 and US20160166689. Methods of Use

[0156] The anti-CD19 antibodies and pharmaceutical compositions described herein may be used for the treatment of diseases and conditions characterized by CD19 expression, including, without limitation, the conditions and diseases further described herein.

[0157] CD19 is a cell surface receptor that is expressed on all human B cells but not found on plasma cells. It has a relatively large cytoplasmic tail, 240 amino acids long. The extracellular Ig-like domains are divided by a potential disulfide-linked non-Ig-like domain and N-linked carbohydrate addition sites. The cytoplasmic tail contains at least nine tyrosine residues near the C-terminus, some of which have been shown to be phosphorylated. Along with CD20 and CD22, the lineage-restricted expression of CD19 makes it an attractive target for the therapeutic treatment of B-cell malignancies.Due to its observed expression in some hematological malignancies, CD19 is a promising target for antibody-based therapies.

[0158] In one aspect, anti-CD19 antibodies (e.g., UniAbsTM) and pharmaceutical compositions herein can be used to treat disorders characterized by CD19 expression, including, without limitation, the diseases and disorders described further herein.

[0159] The anti-CD19 heavy chain-only antibodies (UniAbs) of the present invention can be used to develop therapeutic agents for the treatment of hematological malignancies characterized by CD19 expression, including, without limitation, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin lymphoma, chronic lymphocytic leukemia of B cells (CLL), and acute lymphoblastic leukemia of B cells (ALL).

[0160] Diffuse large B-cell lymphoma (DLBCL or DLBL) is Petition 870260076930, dated 07 / 31 / 2026, page 79 / 122 Lymphoma of the lymphoma (LDBCL) is the most common form of non-Hodgkin lymphoma among adults (Blood 1997 89 (11): 3909-18), with an estimated annual incidence of 7 to 8 cases per 100,000 people per year in the US and UK. It is characterized as an aggressive cancer that can arise in virtually any part of the body. The causes of DLBCL are not well understood, and it can arise from normal B cells as well as malignant transformation of other types of lymphoma or leukemia cells. Treatment approaches generally involve chemotherapy and radiation, and have resulted in an overall five-year survival rate of approximately 58% for adults. Although some monoclonal antibodies have shown promise in treating DLBCL, consistent clinical efficacy has not yet been conclusively demonstrated. Therefore, new therapies, including immunotherapies, are much needed for DLBCL.

[0161] In another aspect, heavy chain antibodies to CD19 (e.g., UniAbsTM) and pharmaceutical compositions herein may be used to treat autoimmune disorders characterized by pathogenic B cells expressing CD19, including, without limitation, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS).

[0162] In one embodiment, the antibodies here may be in the form of anti-CD19-CAR heavy chain-only antibody structures, that is, anti-CD19 T cell heavy chain-only antibody structures transduced with CAR.

[0163] The effective doses of the compositions of the present invention for the treatment of diseases vary depending on many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human being, but non-human mammals can also be treated, for example, companion animals such as dogs, cats, horses, etc. Petition 870260076930, dated 07 / 31 / 2026, page 80 / 122 67 / 81 laboratory mammals, such as rabbits, mice, rats, etc., and the like. Treatment dosages may be titrated to optimize safety and efficacy.

[0164] Dosage levels can be easily determined by the clinician with ordinary expertise and can be modified as needed, for example, as needed to modify a subject's response to therapy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending on the host being treated and the particular mode of administration. Unit dosage forms generally contain between about 1 mg and about 500 mg of an active ingredient.

[0165] In some embodiments, the therapeutic dosage of the agent may vary from about 0.0001 to 100 mg / kg and, more commonly, from 0.01 to 5 mg / kg of the host's body weight. For example, dosages may be 1 mg / kg of body weight or 10 mg / kg of body weight or within the range of 1-10 mg / kg. An exemplary treatment regimen involves administration once every two weeks or once a month or once every 3 to 6 months. The therapeutic entities of the present invention are generally administered on multiple occasions. The intervals between single dosages may be weekly, monthly, or yearly. The intervals may also be irregular as indicated by measuring the blood levels of the therapeutic entity in the patient. Alternatively, the therapeutic entities of the present invention may 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.

[0166] Typically, compositions are prepared as injectables, as solutions or liquid suspensions; solid forms suitable for solution or suspension in liquid vehicles prior to injection may also be prepared. Compositions Petition 870260076930, dated 07 / 31 / 2026, page 81 / 122 The pharmaceutical compositions presented herein are suitable for intravenous or subcutaneous administration, directly or after reconstitution of solid compositions (e.g., lyophilized). The preparation may also be emulsified or encapsulated in liposomes or microparticles, such as polylactic, polyglycolide, or copolymer 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 this invention may be administered in the form of a depot injection or implant preparation that may be formulated to allow sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated in a sterile, substantially isotonic form and in full compliance with all Good Manufacturing Practices (GMP) regulations of the US Food and Drug Administration.

[0167] 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 (the lethal dose for 50% of the population) or the LD100 (the lethal dose for 100% of the population). The dose-to-therapeutic ratio is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for human use. The dosage of the antibodies described herein preferably falls within a range of circulating concentrations that includes the effective dose with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration used.The exact formulation, route of administration, and dosage can be chosen by the individual physician taking into account the patient's condition.

[0168] The compositions for administration will include Petition 870260076930, dated 07 / 31 / 2026, page 82 / 122 69 / 81 generally an antibody or other ablative agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, for example, buffered saline solution and the like. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by well-known conventional sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.The concentration of the active agent in these formulations can vary widely and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, according to the particular mode of administration selected and the patient's needs (e.g., “Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, “The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0169] Also included within the scope of the invention are kits comprising the active agents and their formulations, of the invention and instructions for use. The kit may also contain at least one additional reagent, for example, a chemotherapeutic drug, etc. Kits typically include a label indicating the intended use of the kit's contents. The term label, as used herein, includes any writing or engraved material provided in or with a kit, or otherwise accompanying a kit.

[0170] The invention having now been fully described, it will be evident to the person usually skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. III. Examples Petition 870260076930, dated 07 / 31 / 2026, p. 83 / 122 70 / 81 Example 1: Binding to Raji CD19+ cells

[0171] Binding to CD19-positive Raji cells was assessed by flow cytometry. In summary, 50,000 target cells were stained with a series of dilutions of purified UniAbs™ for 30 minutes at 4°C. After incubation, cells were washed twice with flow cytometry buffer (1X PBS, 1% BSA, 0.1% NaN3) and stained with R-phycoerythrin (PE)-conjugated goat anti-human F(ab')2 IgG (Southern Biotech, cat. #204209) to detect cell-bound antibodies. After a 20-minute incubation at 4°C, cells were washed twice with flow cytometry buffer and the mean fluorescence intensity (MFI) was measured by flow cytometry. The MFI of cells stained with secondary antibody only was used to determine the background signal, and the binding of each antibody was converted to times relative to the background.

[0172] The results are shown in FIG. 1, which summarizes the target binding activity of the indicated anti-CD19 antibodies. Column 1 indicates the HCAb clone ID. Column 2 indicates binding to Raji cells measured as folds relative to the MFI background signal. Column 3 indicates binding to CHO cells that do not express the CD19 protein (negative control) measured as folds relative to the MFI background signal. Example 2: Binding to Raji and Daudi CD19+ cells

[0173] Cell binding dose curves were constructed in two cell lines: Raji (A) and Daudi (B), as described in Example 1. Antibodies were tested at a starting concentration of 200 nM followed by serial 3-fold dilutions for an 8-point dose curve. The mean fluorescence intensity of PE was plotted as times relative to the background (cells incubated only with secondary detection antibody).

[0174] The results are presented in FIG. 2, panel A (Raji cells) and panel B (Daudi cells). Petition 870260076930, dated 07 / 31 / 2026, page 84 / 122 71 / 81 Example 3: EC50 values ​​of cell binding in CD19+ cell lines

[0175] To determine EC50 values ​​of cell binding, dose-cell binding curves were constructed in the CD19 Raji and Daudi expressing cell lines, as described in Example 2. Antibodies were tested at a starting dose of 200 nM, followed by 3-fold serial dilutions to an 8-point dose curve. The transformed data were plotted as an xy plot using nonlinear regression curve fitting (available in GraphPad Prism 8.4.3) to obtain the EC50s (nM).

[0176] The results are presented in FIG. 3. Example 4: CAR-T-mediated T cell activation by human tumor cell lines

[0177] CAR-T cell activity was measured by cotransfecting Jurkat T lymphocytes with an anti-CD19 CAR and a 6x NFAT TK nanoluciferase reporter. Transfected Jurkats were cocultured for 24 hours with Raji, Ramos, and SU-DHL-10 CD19+ or CD19-negative K562 cells. Luciferase activity was measured using the Promega Nano-Glo Luciferase Assay System (catalog # N1110) and data were normalized to the coculture containing the CAR-transfected and CD19-negative K562 Jurkat cell lines. Statistical significance was determined using an unpaired two-tailed t-test.

[0178] The results are presented in FIG. 4, panels B and C.

[0179] FIG. 4, panel A, is a schematic illustration of a CAR-T structure comprising an anti-CD19 extracellular binding domain comprising an antibody sequence according to aspects of the invention. Panel B illustrates the activity of Jurkats T cells transfected with an anti-CD19 CAR 370034 with Raji (**p=0.0055), Ramos (**p=0.0058), and SU Petition 870260076930, dated 07 / 31 / 2026, page 85 / 122 72 / 81 DHL-10 (***p=0.00025). Panel C illustrates the activity of Jurkats T cells transfected with an anti-CD19 CAR 370083 with Raji (****p=0.000009), Ramos (**p=0.004), and SU-DHL-10 (*p=0.016). Example 5: Generation of CD19 VH Ligands Exhibiting Enhanced Biological Properties via Site-Saturated Mutagenesis

[0180] In this example, site-saturated mutagenesis was used to generate a collection of unique CD19 VH ligand sequences in which a wild-type amino acid coding sequence was systematically replaced by one of the coding sequences of the other 19 amino acids to generate a collection of mutants that can be screened to identify CD19 VH ligands with improved in vitro and / or in vivo properties compared to the parent VH ligand.

[0181] The VH CD19 ligand VH-034 comprises three complementarity-determining regions (CDRs): CDR1 (8 amino acids; SEQ ID NO. 1), CDR2 (8 amino acids; SEQ ID NO. 2), and CDR3 (10 amino acids; SEQ ID NO. 4). Site-saturated mutagenesis was employed to systematically replace each of the 26 combined CDR amino acids with one of the other 19 remaining non-wild-type amino acid residues to generate a library of 513 unique VH CD19 ligand sequences comprising a single CDR amino acid substitution spanning all three CDR sequences.

[0182] In summary, the nucleotide sequence encoding VH034 served as a DNA template for saturated mutagenesis at sites (e.g., Chronopoulou and Labrou (2011) Curr Protocol Protein Sci Chapter 26 doi: 10.1002 / 0471140864.ps2606s63). Using PCR and degenerate synthetic oligonucleotides as primers, one codon was substituted for all 20 amino acid codons in the same reaction, which was then systematically applied to each of the 26 codons of CDR. The resulting assembly of 513 nucleic acid members, each encoding a single amino acid substitution, and the parental wild-type sequence were Petition 870260076930, dated 07 / 31 / 2026, page 86 / 122 73 / 81 clones were formed into a nanoplasmid vector, and the frequency of each of the 513 unique VH ligand sequences in the plasmid assembly was determined by next-generation sequencing (NGS).

[0183] The nanoplasmic vector nPBv2-EF1a-iC9-CD19.VH034-DHFR comprises the following, in a 5' to 3' orientation: right-end ITR - 5'UTR - insulator - EF1a promoter - inducible iCaspase9 gene - T2A - CD8a leader peptide - VH034 ligand site - CD19BBZ CAR cassette - T2A - selectable DHFR marker - SV40 polyA - insulator - 3'-UTR and a left-end ITR. Each unique ligand sequence was cloned into the structure in the CD19BBZ CAR cassette to generate a complete CAR (CD19 VH ligand - CD28 hinge - 41-BB transmembrane domain and CD3zeta domain) under the control of the EF1a promoter. Example 6: Generation of a T Cell Assembly Expressing CAR Comprising Mutant CD19 VH Ligands

[0184] In this example, a T cell cluster was generated from individual T cells expressing CARs comprising one of the 513 VH CD19 ligand sequences.

[0185] A T cell assembly comprising the library of 513 CD19 VH ligand nanoplasmids generated in Example 5 was prepared by electroporating T cells with an mRNA encoding SuperpiggyBac transposase and an aliquot of the DNA nanoplasmid assembly at a low DNA copy number to ensure that the electroporated T cells comprised only a single copy of one of the 513 nanoplasmids. The electroporated T cell population was incubated in 250 nM methotrexate to select transposed T cells comprising an integrated genomic copy of the CAR-expressing transposon. The frequency of each of the 513 unique ligand sequences in the T cell assembly was determined by NGS. Example 7: Enrichment of T cells expressing a CAR Petition 870260076930, dated 07 / 31 / 2026, page 87 / 122 74 / 81 Understanding a CD19 Ligand Exhibiting Enhanced CD19 Binding by Repeated Stimulation

[0186] This example illustrates that selected T cell clones can be enriched within a T cell population by repeated exposure to a particular antigen and selection of T clones that preferentially expand in numbers due to antigenic stimulation.

[0187] In an experiment, in Grex-6M well plates, approximately 20 x 10e6 T cells / well of T cells from the T cell assembly generated in Example 6 were added to each well in duplicate sets. To one set were added 2 x 10e6 RAJI (CD19+) cells and to the other set were added 2 x 10e6 control RAJI cells having an inactive CD19 gene (RAJI KO) to stimulate activation of CD19-CAR-expressing T cells. T cells were re-stimulated with 2 x 10e6 RAJI or RAJI KO cells, respectively, on Days 3, 6, and 9. After 12 days, the unsorted expansion of the RAJI-stimulated T cell assembly was approximately 20-fold, whereas little to no expansion was observed using RAJI KO cells.

[0188] CD19+ cells were analyzed by NGS to determine the frequency of each of the 513 mutant ligand sequences within the expanded CD19+ T cell population, and this frequency of each member was analyzed in comparison with the original plasmid assemblies and T cell populations.

[0189] In a second experiment, the T cell pool generated in Example 6 was treated with a rabbit anti-human IgG antibody to capture CAR-positive cells, and the CAR cells were screened and analyzed by FACS analysis. The screened CAR-positive T cell pool was then subjected to restimulation with RAJI CD19+ or RAJI-KO cells and, after 15 days (6 RAJI cell stimulations), a Petition 870260076930, dated 07 / 31 / 2026, page 88 / 122 75 / 81 T cell expansion approximately 40 times. CAR+ T cells were analyzed by NGS to determine the frequency of each of the 513 mutant ligand sequences within the expanded CAR+ T cell population, and this frequency of each member was analyzed in comparison with the original plasmid assemblies and T cell populations.

[0190] By comparing the relative frequency of each VH ligand within the original plasmid population, the T cell assembly, and under each of the two enrichment conditions, the relative change in the frequency of each VH ligand sequence can be determined. Most of the 513 VH ligand sequences were depleted from the RAJI-treated T cell assemblies, suggesting that these mutations were detrimental to CD19 binding, whereas the frequency of a proportion of the VH ligand sequences remained relatively unchanged by CD19 stimulation, suggesting that the introduced mutation may have had little effect on CD19 binding. Certain T cell clones were enriched by CD19 stimulation with both enrichment assays, producing overlapping lists of T cell clones appearing enriched in both assays.

[0191] The twelve main HV ligands observed in both enrichment assays were further analyzed. Two particular T cell clones were highly enriched in the T cell population after repeated stimulation using RAJI cells. The first T cell clone comprised a CAR expressing a CD19 HV ligand comprising a T28D mutation in CDR1 (SEQ ID NO. 38), and the second T cell clone comprised a CAR expressing a CD19 HV ligand comprising an S54D mutation in CDR2 (SEQ ID NO. 39).

[0192] The two VH CD19-enriched ligands T28D and S54D were further characterized in in vitro and in vivo assays. Petition 870260076930, dated 07 / 31 / 2026, page 89 / 122 76 / 81 Example 8: T Cells Expressing a CAR Comprising VH T28D or S54D Ligands Exhibit Enhanced In Vitro Cytotoxicity

[0193] This example illustrates that T cells expressing a CAR comprising a VH T28D ligand or a VH S54D ligand exhibit higher levels of in vitro cytotoxicity against CD19-expressing cells compared to the paternal VH034 ligand. FIG. 5, panels A and B, are graphs showing comparative in vitro cytotoxicity results for T cells expressing a CAR comprising a VH T28D ligand or a VH S54D ligand compared to a paternal VH-034 ligand. More specifically, the results show in vitro cytotoxicity upon stimulation with CD19 using Nalm6 cells (See FIG. 5, panel A) or RAJI cells (See FIG. 5, panel B) compared to the paternal VH-034 ligand.

[0194] In a 96-well plate, approximately 20,000 T cells / well expressing a CAR comprising the paternal VH-034 ligand, a VH T28D ligand, or a VH S54D ligand were added to each well in duplicate sets. One set was supplemented with 10,000 RAJI-GFP cells and the other set with 10,000 Nalm6-GFP cells to stimulate activation of the CD19-CAR-expressing cells. T cells were re-stimulated with 30,000 RAJI-GFP or Nalm6-GFP, respectively, at 48 hr, 116 hr, and 172 hr. In vitro cytotoxicity was measured in real time over a period of 252 (for Nalm6-GFP) or 188 (for RAJI-GFP) hours by Incucyte. Example 9: T Cells Expressing a CAR Comprising VH T28D or S54D Ligands Exhibit Enhanced In Vitro T Cell Activation

[0195] This example illustrates that T cells expressing a CAR comprising CD19-enriched VH034 ligands exhibit higher levels of T cell activation in vitro compared with the paternal VH034 ligand. Petition 870260076930, dated 07 / 31 / 2026, pages 90 / 122 77 / 81

[0196] FIG. 6 is a table summarizing the enhanced in vitro T cell activation in T cells expressing a CAR comprising a VH T28D ligand or a VH S54D ligand compared with a paternal VH-034 ligand. Approximately 0.2 x 10e6 T cells expressing a CAR comprising a paternal VH034 ligand, a VH T28D ligand, or a VH S54D ligand were activated by the addition of 0.1 x 10e6 Nalm6 or RAJI cells, and the cultures were incubated in RPMI 1640 medium supplemented with 10% FBS at 37 °C for 16 hours. The T cells were then analyzed by FACS analysis to quantify the expression of CD25, which is a marker of T cell activation. The geometric mean fluorescent intensity (gMFI) of CD25 from activated T cells for each VH ligand is shown in the table illustrated in FIG. 6.

[0197] As shown in the table illustrated in FIG. 6, the VH CD19 ligands T28D and S54D exhibited improved T cell activation compared to the paternal CD19 VH-034 ligand, demonstrating that these substitutions enhance T cell activation by CD19-expressing cells. Example 10: T Cells Expressing CARs Comprising a T28D VH Ligand or S54D VH Ligand Express Higher Levels of CAR In Vivo

[0198] This example illustrates that VH-CD19 ligands enriched in a CAR format express higher levels of CAR in T cells compared to the paternal VH-034 ligand.

[0199] FIG. 7 is a table showing that VH CD19 ligands enriched in a CAR format express higher levels of CAR in T cells compared to the paternal VH-034 ligand. Approximately 10 x 10e6 T cells expressing a CAR comprising the paternal VH-034 ligand, a VH T28D ligand, or a VH S54D ligand were administered to MICE (n=5). After 14 days, blood samples were collected from treated animals and the blood was subjected to ACK buffer lysis to Petition 870260076930, dated 07 / 31 / 2026, pages 91 / 122 78 / 81 remove red blood cells. Circulating T cells were then analyzed by FACS analysis with CD19+ cell clustering, and the number of cells expressing CAR was determined for each sample. The results are shown in the table illustrated in FIG. 7.

[0200] As shown in the table illustrated in FIG. 7, the average number of circulating CAR+ CD19 T cells was higher for the VH034 ligands T28D and S54D compared to the paternal VH034 ligand by approximately 35% and 65%, respectively. Example 11: T Cells Expressing CARs Comprising a VH T28D Ligand or a VH S54D Ligand Demonstrate Enhanced T Cell Expansion In Vivo

[0201] This example illustrates that T cells expressing a CAR comprising a VH T28D ligand or a VH S54D ligand exhibit enhanced T cell expansion in vivo compared to T cells expressing a CAR comprising the paternal VH-034.

[0202] FIG. 8 is a table showing that T cells expressing CARs comprising a VH T28D ligand or a VH S54D ligand exhibit enhanced T cell expansion in vivo. NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl / Szj) (n=5 / group) were administered with 10 x 10e6 T cells expressing a CAR comprising the wild-type VH-034 ligand, a VH T28D ligand, or a VH S54D ligand. Blood samples were collected from treated mice on Days 3, 7, and 14, and the number of circulating T cells per microliter of blood was determined. The results are shown in the table illustrated in FIG. 8.

[0203] As shown in the table illustrated in FIG. 8, the kinetics and magnitude of T cell expansion differ between T cells expressing the paternal VH-034 ligand or either of the two mutant VH ligands. For example, T cells expressing a CAR comprising the VH ligand S54D exhibit different kinetics from T cells expressing a CAR comprising the VH ligand Petition 870260076930, dated 07 / 31 / 2026, pages 92 / 122 79 / 81 T28D, since the peak expansion of S54D T cells is 3 days, whereas the peak expansion for T28D T cells is 14 days. Furthermore, each of the two mutant VH ligands demonstrated improved kinetics compared to T cells with paternal VH-034. Example 12: T cells expressing a CAR comprising a VH T28D ligand or VH S54D ligand demonstrate decreased T cell exhaustion in vivo.

[0204] This example illustrates that T cells expressing a CAR comprising one of the CD19-enriched VH-034 ligands exhibit decreased T cell exhaustion in vivo compared to the paternal VH-034 ligand.

[0205] Approximately 10 x 10e6 T cells expressing paternal VH-034, VH T28D ligand, or VH S54D ligand were administered to adult NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl / Szj) mice (n=5 / group). After 21 days, a blood sample of approximately 100 pL was collected from treated animals, and the red blood cells from each sample were removed by ACK lysis buffer. The degree of T cell exhaustion in each sample was determined by FACS analysis, grouping according to cells expressing PD-1 or TIGIT-1, which are known markers of T cell exhaustion. FIG. Table 9 shows that T cells expressing a CAR comprising the VH-034 CD19 ligand T28D or S54D exhibited less T cell exhaustion compared with the paternal VH-034 CD19 ligand.

[0206] As shown in the table in FIG. 9, T cells expressing a CAR comprising the VH ligand CD19 T28D or S54D exhibited less T cell exhaustion on Day 21, evidenced by a decrease in the number of T cells expressing PD-1 or TIGIT, demonstrating that a higher percentage of these cells remain in a non-exhausted state compared to the paternal CD19 ligand VH-034. Example 13: T Cells Expressing CARs Comprising a T28D VH Ligand or S54D VH Ligand Exhibit In Vivo Efficacy Petition 870260076930, dated 07 / 31 / 2026, page 93 / 122 80 / 81 Improved

[0207] This example illustrates that T cells expressing a CAR comprising a VH T28D ligand or a VH S54D ligand exhibit improved in vivo efficacy compared to T cells expressing a CAR comprising the paternal VH-034 ligand.

[0208] On Day 0, adult female NGS (NOD.CgPrkdcscidll2rgtm1Wjl / Szj) mice aged six to seven weeks (n=5 / group) were intravenously administered 0.5 x 10e6 RAJI cells genetically engineered to express luciferase. On Day 5, mice were administered 10 x 10e6 T cells expressing a CAR comprising paternal VH-034 ligand, a VH T28D ligand, or a VH S54D ligand. Mice were monitored for a period of 55 days.

[0209] Whole-body bioluminescent imaging (BLI) was performed on anesthetized mice at weekly intervals throughout the study to monitor tumor progression, and whole blood was collected from treated animals on Days 8, 12, 18, 25, 32, 39, and 55. On Day 55, mice were euthanized and their blood, spleen, and femoral bone marrow were subsequently collected for further analysis.

[0210] FIG. 10 is a graph with condensed data showing that T cells expressing a CAR comprising the VH ligand T28D or the VH ligand S54D exhibited tumor control. FIG. 11 is a graph showing uncondensed individual mouse model data illustrating the same result shown in FIG. 10. As shown, T cells expressing the CAR comprising the paternal VH-034 exhibited modest effects on tumor growth control throughout the study. In contrast, 5 / 5 mice administered with T cells expressing the CAR comprising the VH ligand T28D exhibited complete tumor control and regression by Day 55. Similarly, 3 / 5 administered with T cells expressing the CAR comprising the VH ligand Petition 870260076930, dated 07 / 31 / 2026, pp. 94 / 122 81 / 81 S54D exhibited complete tumor control and regression at least until Day 30. These results indicate that enriched VH CD19 ligands comprising a T28D and S54D mutation contribute to the improved in vivo efficacy observed in a RAJI model.

[0211] Notwithstanding that preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, alterations, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. The following claims are intended to define the scope of the invention and that methods and structures within the scope of these claims and their equivalents are covered by them. Petition 870260076930, dated 07 / 31 / 2026, pp. 95 / 122

Claims

1 / 4 CLAIMS 1. An antibody that binds to CD19, characterized in that it comprises a variable heavy chain region comprising: (a) a CDR1 sequence of SEQ ID NO: 38, a CDR2 sequence of SEQ ID NO: 2 and a CDR3 sequence of SEQ ID NO: 3; or (b) a CDR1 sequence of SEQ ID NO: 38, a CDR2 sequence of SEQ ID NO: 2 and a CDR3 sequence of SEQ ID NO: 4; or (c) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 39 and a CDR3 sequence of SEQ ID NO: 3; or (d) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 39 and a CDR3 sequence of SEQ ID NO:

4.

2. Antibody, according to claim 1, characterized in that the CDR1, CDR2 and CDR3 sequences are present in a human HV scaffold.

3. Antibody, according to claim 1, characterized in that it comprises a variable heavy chain region having at least 95% sequence identity with either of the SEQ ID sequences NOS: 40 and 41.

4. Antibody that binds to CD19, characterized in that it comprises a variable region sequence of heavy chain with SEQ ID NO:

40.

5. Antibody that binds to CD19, characterized in that it comprises a variable region sequence of heavy chain with SEQ ID NO:

41.

6. CAR-T cell, characterized in that it comprises a CAR comprising an extracellular antigen-binding domain that binds to CD19, comprising a variable heavy chain region comprising: (a) a CDR1 sequence of SEQ ID NO: 38, a CDR2 sequence of SEQ ID NO: 2 and a CDR3 sequence of SEQ ID NO: 3; or (b) a CDR1 sequence of SEQ ID NO: 38, a CDR2 sequence of SEQ ID NO: 2 and a CDR3 sequence of SEQ ID NO: 4; or Petition 870260076930, dated 07 / 31 / 2026, page 96 / 122 2 / 4 (c) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 39 and a CDR3 sequence of SEQ ID NO: 3; or (d) a CDR1 sequence with SEQ ID NO: 1, a CDR2 sequence with SEQ ID NO: 39 and a CDR3 sequence with SEQ ID NO:

4.

7. CAR-T cell, according to claim 6, characterized in that the variable heavy chain region comprises a CDR1 sequence with SEQ ID NO: 38, a CDR2 sequence with SEQ ID NO: 2, and a CDR3 sequence with SEQ ID NO:

3.

8. CAR-T cell, according to claim 6, characterized in that the variable heavy chain region comprises a CDR1 sequence with SEQ ID NO: 38, a CDR2 sequence with SEQ ID NO: 2 and a CDR3 sequence with SEQ ID NO:

4.

9. CAR-T cell, according to claim 6, characterized in that the variable heavy chain region comprises a CDR1 sequence with SEQ ID NO: 1, a CDR2 sequence with SEQ ID NO: 39 and a CDR3 sequence with SEQ ID NO:

3.

10. CAR-T cell, according to claim 6, characterized in that the variable heavy chain region comprises a CDR1 sequence with SEQ ID NO: 1, a CDR2 sequence with SEQ ID NO: 39 and a CDR3 sequence with SEQ ID NO:

4.

11. CAR-T cell, according to any one of claims 6 to 10, characterized in that the CDR1, CDR2 and CDR3 sequences are present in a human VH scaffold.

12. CAR-T cell, according to claim 6, characterized in that the extracellular antigen-binding domain that binds to CD19 comprises a variable heavy chain region having at least 95% sequence identity with either of the SEQ ID sequences NOS: 40 and 41.

13. CAR-T cell, characterized in that it comprises an extracellular antigen-binding domain that binds to CD19, comprising a variable region sequence of heavy chain SEQ ID NO:

40. Petition 870260076930, dated 07 / 31 / 2026, page 97 / 122 3 / 4 14. CAR-T cell, characterized by comprising an extracellular antigen-binding domain that binds to CD19, comprising a variable region sequence of heavy chain with SEQ ID NO:

41.

15. Pharmaceutical composition, characterized in that it comprises an antibody, as defined in any one of claims 1 to 5, or a CAR-T cell, as defined in any one of claims 6 to 14.

16. Use of an antibody, as defined in any one of claims 1 to 5, or a CAR-T cell, as defined in any one of claims 6 to 14, or a pharmaceutical composition, as defined in claim 15, characterized in that it is for the manufacture of a medicament for treating a B-cell disorder in a subject.

17. Use, according to claim 16, characterized in that the disorder is diffuse large B-cell lymphoma (DLBCL).

18. Use, according to claim 16, characterized in that the disorder is acute lymphoblastic leukemia (ALL).

19. Use, according to claim 16, characterized in that the disorder is non-Hodgkin lymphoma (NHL).

20. Use, according to claim 16, characterized in that the disorder is systemic lupus erythematosus (SLE).

21. Use, according to claim 16, characterized in that the disorder is rheumatoid arthritis (RA).

22. Use, according to claim 16, characterized in that the disorder is multiple sclerosis (MS).

23. Kit for treating a disease or disorder in a needy individual, characterized in that it comprises the CAR-T cell, as defined in claim 6.

24. Kit according to claim 23, characterized in that it further comprises at least one additional reagent. Petition 870260076930, dated 07 / 31 / 2026, p. 98 / 122 4 / 4 25. Kit according to claim 24, characterized in that at least one additional reagent comprises a chemotherapeutic drug.

26. Polynucleotide, characterized in that it encodes an antibody as defined in any one of claims 1 to 5, or a CAR of a CAR-T cell, as defined in any one of claims 6 to 14.

27. Vector, characterized in that it comprises the polynucleotide as defined in claim 26.

28. Cell, characterized in that it comprises the vector as defined in claim 27.

29. Method for producing an antibody, as defined in any one of claims 1 to 5, or a CAR-T cell, as defined in any one of claims 6 to 14, characterized in that it comprises cultivating a cell, as defined in claim 28, under conditions permissive for the expression of the antibody or CAR, and isolating the antibody from the cell and / or from a cell culture medium in which the cell is cultivated, or isolating the CAR-T cell. Petition 870260076930, dated 07 / 31 / 2026, pp. 99 / 122

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