Anti-CD19 antibodies and CAR-T constructs

Abstract

Anti-CD19 antibodies (e.g., UniAbs™) and CAR-T constructs are disclosed, along with methods of making such antibodies and CAR-T constructs, compositions, including pharmaceutical compositions, comprising such antibodies and CAR-T constructs, and uses thereof for treating disorders characterized by expression of CD19.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to the filing date of U.S. Provisional Patent Application No. 63 / 171,520, filed April 6, 2021, the disclosure of which is incorporated herein by reference in its entirety. This application also claims the benefit of priority to the filing date of U.S. Provisional Patent Application No. 63 / 311,913, filed February 18, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to antibodies (e.g., UniAbs™) and CAR-T constructs that bind to CD19. The present invention further relates to methods of making such antibodies and CAR-T constructs, compositions, including pharmaceutical compositions, comprising such antibodies and CAR-T constructs, and uses thereof for treating disorders characterized by expression of CD19. [Background technology]

[0003] Cluster of differentiation 19 (CD19) CD19, also known as B-lymphocyte surface antigen B4 (UniProt P15391), is a cell surface receptor expressed on all human B cells but not on plasma cells. CD19 is a transmembrane protein that recruits cytoplasmic signaling proteins to the membrane, where it plays a role in lowering the threshold of the B cell receptor signaling pathway within the CD19 / CD21 complex. CD19 has a relatively large 240 amino acid cytoplasmic tail. This extracellular Ig-like domain is divided into a potentially disulfide-linked non-Ig-like domain and an N-linked glycosylation site. This cytoplasmic tail has at least nine tyrosine residues near the C-terminus, some of which have been shown to be phosphorylated. Together with CD20 and CD22, CD19 expression is restricted to the B cell lineage, making it an attractive target for therapeutic treatment of B cell malignancies. A number of monoclonal antibodies and antibody-drug conjugates specific for CD19 have been described (Naddafi et al. 2015, PMC4644525). In addition, anti-CD19 chimeric antigen receptor T cells have been approved for the treatment of leukemia (Sadelain et al. 2017, PMID:29245005).

[0004] Heavy Chain Antibodies In conventional IgG antibodies, the association of the heavy and light chains is due in part to hydrophobic interactions between the light chain constant region and the CH1 constant domain of the heavy chain. There are additional residues in the 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.

[0005] However, it is known that sera from camelids (the suborder Tylopoda, which includes camels, dromedaries and llamas) contain a major type of antibody composed only of paired H chains (heavy chain antibodies or UniAbs™). The UniAbs™ from Camelidae (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) that are highly homologous to the CH2 and CH3 domains of classical antibodies. These UniAbs™ lack the first domain (CH1) of the constant region, which is present in the genome but is spliced ​​out during mRNA processing. The absence of the CH1 domain explains the absence of light chains in the UniAbs™, since this domain is the anchor position for the constant domain of the light chain. Such UniAbs™ have naturally evolved to confer antigen-binding specificity and high affinity by three CDRs from a conventional antibody or a fragment thereof (Muyldermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther 5:111-124). Cartilaginous fishes, such as sharks, have also evolved a unique type of immunoglobulin, called IgNAR, which lacks light chain polypeptides and is entirely composed of heavy chains. IgNAR molecules can be engineered by molecular engineering to create variable domains of a single heavy chain polypeptide (vNAR) (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

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

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

[0008] Mice functionally silenced at the lambda (lambda) light chain (L) locus and / or at the lambda and kappa (kappa) light chain loci and antibodies produced by such mice are described in U.S. Patent Nos. 7,541,513 and 8,367,888. Recombinant production of heavy chain antibodies in mice and rats has been reported, for example, in WO 2006008548, U.S. Patent Publication No. 20100122358, Nguyen et al., 2003, Immunology; 109(1), 93-101; Bruggemann et al., Crit. Rev. Immunol.; 2006, 26(5):377-90; and Zou et al., 2007, J Exp Med; 204(13):3271-3283. The generation of knockout rats by embryonic microinjection of zinc finger nucleases is described in Geurts et al., 2009, Science, 325(5939):433. Soluble heavy chain antibodies and transgenic rodents containing heterologous heavy chain loci producing such antibodies are described in U.S. Patents 8,883,150 and 9,365,655. CAR-T structures containing single domain antibodies as binding (targeting) domains are described, for example, in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378-386. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 7,541,513 [Patent Document 2] U.S. Pat. No. 8,367,888 [Patent Document 3] International Publication No. 2006008548 Brochure [Patent Document 4] U.S. Patent Application Publication No. 2010 / 0122358 [Patent Document 5] U.S. Patent No. 8,883,150 [Patent Document 6] U.S. Patent No. 9,365,655 [Non-Patent Document]

[0010] [Non-Patent Document 1] Naddafi et al. 2015, PMC4644525 [Non-Patent Document 2] Sadelain et al. 2017, PMID: 29245005 [Non-Patent Document 3] Muyldermans, 2001; J Biotechnol 74: 277 - 302 [Non-Patent Document 4] Revets et al., 2005; Expert Opin Biol Ther 5: 111 - 124 [Non-Patent Document 5] Nuttall et al. Eur. J. Biochem. 270, 3543 - 3554 (2003) [Non-Patent Document 6] Nuttall et al. Function and Bioinformatics 55, 187 - 197 (2004) [Non-Patent Document 7] Dooley et al., Molecular Immunology 40, 25 - 33 (2003) [Non-Patent Document 8] Jaton et al. (1968) Biochemistry, 7, 4185 - 4195 [Non-Patent Document 9] Sitia et al. (1990) Cell, 60, 781 - 790 [Non-Patent Document 10] van der Linden, R.H., et al. Biochim. Biophys. Acta. 1431, 37 - 46 (1999) [Non-Patent Document 11] Frenken,LGJ,et al.J.Biotechnol.78,11-21(2000) [Non-Patent Document 12] Ghahroudi, MA et al. FEBS Lett. 414, 521-526 (1997) [Non-Patent Document 13] Nguyen et al.,2003,Immunology;109(1),93-101 [Non-Patent Document 14] Bruggemann et al.,Crit.Rev.Immunol.;2006,26(5):377-90 [Non-Patent Document 15] Zou et al.,2007,J Exp Med;204(13):3271-3283 [Non-Patent Document 16] Geurts et al.,2009,Science,325(5939):433 [Non-Patent Document 17] Iri-Sofla et al.,2011,Experimental Cell Research 317:2630-2641 [Non-Patent Document 18] Jamnani et al.,2014,Biochim Biophys Acta,1840:378-386 Summary of the Invention [Means for solving the problem]

[0011] Embodiments of the present invention include antibodies that bind to CD19, comprising a heavy chain variable region comprising: (a) a CDR1 sequence having no more than two substitutions in SEQ ID NO:1; and / or (b) a CDR2 sequence having no more than two substitutions in SEQ ID NO:2; and / or (c) a CDR3 sequence having no more than two substitutions in any one of SEQ ID NOs:3-4.

[0012] In some embodiments, the CDR1, CDR2 and CDR3 sequences are present in a human VH framework, hi some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence.

[0013] In some embodiments, the antibody comprises (a) a CDR1 sequence selected from the group consisting of SEQ ID NO:1 and 38; and / or (b) a CDR2 sequence selected from the group consisting of SEQ ID NO:2 and 39; and / or (c) a CDR3 sequence selected from the group consisting of SEQ ID NO:3-4. In some embodiments, the 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, the 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 of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 39, and a CDR3 sequence of SEQ ID NO: 4.

[0014] In some embodiments, the antibody comprises a heavy chain variable region having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 5, 6, 40, and 41. In some embodiments, the antibody comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 5, 6, 40, and 41. In some embodiments, the antibody comprises a heavy chain variable region sequence of SEQ ID NO: 5. In some embodiments, the antibody comprises a heavy chain variable region sequence of SEQ ID NO: 6. In some embodiments, the antibody comprises a heavy chain variable region sequence of SEQ ID NO: 40. In some embodiments, the antibody comprises a heavy chain variable region sequence of SEQ ID NO: 41.

[0015] Aspects of the invention include an antibody that binds to CD19, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, the CDR sequences being sequences with no more than two substitutions in the CDR sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 38 and 39. In some embodiments, the antibody comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences in a human VH framework, the CDR sequences being selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 38 and 39.

[0016] Embodiments of the invention include an antibody that binds CD19, comprising a heavy chain variable region comprising: (a) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 2 and the CDR3 sequence of SEQ ID NO: 3 in a human VH framework; or (b) 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 in a human VH framework; or (c) the CDR1 sequence of SEQ ID NO: 38, the CDR2 sequence of SEQ ID NO: 2 and the CDR3 sequence of SEQ ID NO: 3 in a human VH framework; or (d) 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 in a human VH framework; or (e) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 39 and the CDR3 sequence of SEQ ID NO: 3 in a human VH framework; or (f) 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 in a human VH framework.

[0017] In some embodiments, the antibody is multispecific. In some embodiments, the antibody is bispecific. In some embodiments, the antibody binds to two different CD19 proteins. In some embodiments, the antibody binds to two different epitopes on the same CD19 protein. In some embodiments, the antibody binds to an effector cell. In some embodiments, the antibody binds to a T cell antigen. In some embodiments, the antibody binds to CD3. In some embodiments, the antibody is in a CAR-T format.

[0018] Embodiments of the invention include a CAR-T cell comprising a CAR that comprises an extracellular antigen-binding domain that binds CD19, comprising a heavy chain variable 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.

[0019] In some embodiments, the heavy chain variable region comprises (a) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 2, and the CDR3 sequence of SEQ ID NO: 3 in a human VH framework; or (b) 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 in a human VH framework; or (c) the CDR1 sequence of SEQ ID NO: 38, the CDR2 sequence of SEQ ID NO: 2, and the CDR3 sequence of SEQ ID NO: 3 in a human VH framework; or (d) 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 in a human VH framework; or (e) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 39, and the CDR3 sequence of SEQ ID NO: 3 in a human VH framework; or (f) 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 in a human VH framework.

[0020] In some embodiments, the extracellular antigen binding domain that binds to CD19 comprises a heavy chain variable region having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 5, 6, 40, and 41. In some embodiments, the extracellular antigen binding domain that binds to CD19 comprises a heavy chain variable 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 heavy chain variable region sequence of SEQ ID NOs: 5, 6, 40, and 41. In some embodiments, the extracellular antigen binding domain that binds to CD19 comprises a heavy chain variable region sequence of SEQ ID NO: 6. In some embodiments, the extracellular antigen binding domain that binds to CD19 comprises a heavy chain variable region sequence of SEQ ID NO: 40. In some embodiments, the extracellular antigen binding domain that binds to CD19 comprises a heavy chain variable region sequence of SEQ ID NO: 41.

[0021] Aspects of the invention include pharmaceutical compositions comprising the antibodies or CAR-T cells described herein.

[0022] An embodiment of the invention includes a method of treating a B cell disorder characterized by expression of CD19, comprising administering to a subject having said disorder an antibody, a CAR-T cell or a pharmaceutical composition described herein.

[0023] In some embodiments, the disorder is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the disorder is acute lymphoblastic leukemia (ALL). In some embodiments, the disorder is non-Hodgkin's lymphoma (NHL). In some embodiments, the disorder is systemic lupus erythematosus (SLE). In some embodiments, the disorder is rheumatoid arthritis (RA). In some embodiments, the disorder is multiple sclerosis (MS).

[0024] Aspects of the invention include polynucleotides encoding the antibodies or CAR of CAR-T cells described herein, vectors comprising such polynucleotides, and cells comprising such vectors.

[0025] Aspects of the invention include methods of producing an antibody described herein, the method comprising growing a cell described herein under conditions permissive for expression of the antibody, and isolating the antibody from the cell and / or the cell culture medium in which the cell is grown.

[0026] An embodiment of the invention includes a method of making the antibodies described herein, comprising immunizing a UniRat animal with CD19 and identifying a CD19-binding heavy chain sequence.

[0027] Aspects of the invention include methods of treatment comprising administering to an individual in need thereof an effective dose of an antibody, CAR-T cell or pharmaceutical composition described herein.

[0028] An embodiment of the invention includes the use of an antibody or CAR-T cell described herein in the preparation of a medicament for the treatment of a disease or disorder in an individual in need thereof.

[0029] Aspects of the invention include kits for treating a disease or disorder in an individual in need thereof, the kit comprising an antibody, CAR-T cell or pharmaceutical composition described herein and instructions for use. In some embodiments, the kit further comprises at least one additional reagent. In some embodiments, the at least one additional reagent comprises a chemotherapeutic agent.

[0030] These and additional aspects are further described in the remainder of the disclosure, including the Examples. [Brief description of the drawings]

[0031] [Figure 1] 1 is a table summarizing CD19 binding data for the indicated antibody constructs on CD19+ Raji cells and negative control cells. [Diagram 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). [Diagram 3] 1 is a table summarizing the cell binding EC50 values ​​of the indicated antibody constructs on CD19+ Raji and Daudi cells. [Figure 4] Panel A is a schematic of a CAR-T construct containing an anti-CD19 extracellular binding domain. Panels B and C are graphs showing antigen-specific binding and activation of CAR constructs containing an anti-CD19 extracellular binding domain tested in the indicated cell lines. [Diagram 5] Panels A and B are graphs showing comparative cytotoxicity results between T cells expressing CARs containing either the T29D VH binder or the S55D VH binder compared to the parental VH-034 binder. [Figure 6]1 is a table summarizing the improved in vitro T cell activation in T cells expressing CARs containing either the T29D VH binder or the S55D VH binder compared to the parental VH-034 binder. [Figure 7] 1 is a table showing that enriched CD19 VH binders in a CAR format have higher expression of the CAR in T cells compared to the parental VH-034 binder. [Figure 8] 1 is a table showing that T cells expressing a CAR comprising a T29D VH binder or an S55D VH binder exhibit improved T cell expansion in vivo. [Figure 9] 1 is a table showing that T cells expressing CARs comprising T29D or S55D CD19 VH binders exhibited less T cell depletion compared to the parental VH-034 CD19 binder. [Figure 10] Graph showing that T cells expressing CARs comprising T29D VH binders or S55D VH binders exhibited tumor control. [Figure 11] 11 is a graph showing unreduced individual mouse model data illustrating the same results as shown in FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are described in the literature, such as “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (MJ Gait, ed., 1984); “Animal Cell Culture” (RIFreshney, ed., 1987); Biology” (FMAusubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies:A Laboratory Manual:Portable Protocol No.I,Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0033] When a range of values ​​is provided, each intervening value between the upper and lower limit of that range (to one-tenth of the unit of the lower limit unless the context clearly indicates otherwise), and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any limit specifically excluded in that stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the invention.

[0034] 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)).

[0035] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those of ordinary skill in the art have not been described in order to avoid obscuring the present invention.

[0036] All references cited throughout this disclosure, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0037] I. Definition By "comprising," it is meant that the recited elements are required in the composition / method / kit, but other elements can be included to form a composition / method / kit / etc. within the scope of the claim.

[0038] "Consisting essentially of" means limiting the scope of the described composition or process to particular materials or steps that do not materially affect the basic and novel characteristics of the subject invention.

[0039] "Consisting of" means excluding from a composition, method, or kit any element, step, or ingredient not specified in the claim.

[0040] Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to residues in the variable domain (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 residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index in Kabat" refers to the numbering of residues in the human IgG1 EU antibody. Unless otherwise stated herein, reference to a residue number in the variable domain of an antibody refers to the residue numbering according to the Kabat numbering system. Unless otherwise stated herein, reference to a residue number in the constant domain of an antibody refers to the residue numbering according to the EU numbering system.

[0041] Antibodies, also called immunoglobulins, traditionally comprise at least one heavy chain and one light chain, and the sequences of the amino-terminal domains of the heavy and light chains are variable and are therefore commonly referred to as variable region domains or variable heavy (VH) or variable light (VL) domains. The two domains traditionally associate to form a specific binding region, although, as discussed herein, specific binding can also be obtained using the variable sequence of the heavy chain alone, and a variety of non-native structures of antibodies are known and used in the art.

[0042] A "functional" or "biologically active" antibody or antigen-binding molecule (including heavy chain antibodies and multispecific (e.g., bispecific) three-chain antibody-like molecules (TCAs) described herein) is one that is capable of exerting one or more of its native activities in structural, regulatory, biochemical or biophysical events. For example, a functional antibody or other binding molecule, such as a TCA, may have the ability to specifically bind to an antigen, which binding may then trigger or alter a cellular or molecular event, such as a signal transduction or enzymatic activity. A functional antibody or other binding molecule, such as a TCA, may block ligand activation of a receptor or may also act as an agonist or antagonist. The ability of an antibody or other binding molecule, such as a TCA, to exert one or more of its native activities depends on several factors, including the proper folding and assembly of the polypeptide chain.

[0043] The term "antibody" herein is used in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain antibodies, three-chain antibodies, TCAs, single chain Fvs (scFvs), nanobodies, etc., as well as antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species.

[0044] The term antibody may refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or any immunologically active portion of these polypeptide subunits, i.e., a polypeptide comprising an antigen binding site that immunospecifically binds to an antigen or a portion thereof of a target of interest, including, but not limited to, a cancer cell or a cell that produces an autoimmune antibody associated with an autoimmune disease. The immunoglobulins 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 modified subclasses with modified Fc portions that reduce or enhance effector cell activity. The light chain of the antibody may be a kappa light chain (Vkappa) or a lambda light chain (Vlambda). The immunoglobulin may be derived from any species. In one aspect, the immunoglobulin is primarily of human origin.

[0045] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for minor possible naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, in contrast to conventional (polyclonal) antibody preparations that 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 invention can be produced, for example, by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, and can also be produced by recombinant protein production methods (see, for example, U.S. Pat. No. 4,816,567).

[0046] The term "variable", when used in reference to antibodies, refers to the fact that the sequences of certain portions of antibody variable domains vary widely among antibodies and that these portions are used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FRs connected by three hypervariable regions that largely adopt a β-sheet structure and form loop connections and sometimes form part of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0047] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally include amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) of 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 amino acid residues from the "hypervariable loops" (e.g., residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, "CDR" refers to the complementarity determining regions of an antibody as defined in Lefranc, MP et al., IMGT, the 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.

[0048] Exemplary CDR names are provided herein, however, one of skill in the art will appreciate that several definitions of CDRs are commonly used, including the Kabat definition (see, "Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions." Mol Immunol. 2010;47:694-700), 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 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 al.“Identification of specificity-determining residues in antibodies.”Faseb J. 1995;9:133-139, each of which is expressly incorporated herein by reference.

[0049] The terms "heavy chain-only antibody" and "heavy chain antibody" are used interchangeably herein and refer broadly to an antibody or one or more portions of an antibody, such as one or more arms of an antibody lacking the light chain of a conventional antibody. The terms specifically include, but are not limited to, homodimeric antibodies comprising a VH antigen-binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain; functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NARs and functional fragments thereof comprising a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NAR); and soluble single domain antibodies (sUniDabs™). In one embodiment, a heavy chain antibody is composed of a variable region antigen-binding domain composed of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3 and framework 4. In another embodiment, the heavy chain antibody is composed of an antigen binding domain, at least a portion of the hinge region, and a CH2 and CH3 domain. In another embodiment, the heavy chain antibody is composed of an antigen binding domain, at least a portion of the hinge region, and a CH2 domain. In a further embodiment, the heavy chain antibody is composed of an antigen binding domain, at least a portion of the hinge region, and a CH3 domain. Also included herein are heavy chain antibodies in which the CH2 and / or CH3 domains are truncated. In a further embodiment, the heavy chain is composed of an antigen binding domain and at least one CH (CH1, CH2, CH3 or CH4) domain, but does not include the hinge region. Heavy chain antibodies may be in the form of a dimer, in which the two heavy chains are disulfide-bonded to each other or otherwise covalently or non-covalently bound to each other. Heavy chain antibodies may belong to the IgG subclass, but also antibodies belonging to other subclasses, such as IgM, IgA, IgD and IgE subclasses, are included herein. In particular embodiments, the heavy chain antibody is an IgG1, IgG2, IgG3 or IgG4 subtype, in particular an IgG1 or IgG4 subtype. In one embodiment, the heavy chain antibody is an IgG4 subtype and one or more of the CH domains are modified to alter the effector function of the antibody.In one embodiment, the heavy chain antibody is an IgG1 or IgG4 subtype, and one or more of the CH domains are modified to change the effector function of the antibody. Modifications of the CH domain to change the effector function are further described herein. Non-limiting examples of heavy chain antibodies are described, for example, in WO 2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0050] In some embodiments, the heavy chain antibodies herein are used as binding (targeting) domains of chimeric antigen receptors (CARs). This definition specifically includes human heavy chain antibodies produced by human immunoglobulin transgenic rats (UniRat™), called UniAbs™. The variable regions (VH) of UniAbs™, called UniDabs™, 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. Since homodimeric UniAbs™ lack light chains and thus VL domains, the antigen is recognized by one single domain, the variable domain of the heavy chain of the heavy chain antibody (VH or VHH).

[0051] As used herein, an "intact antibody chain" is an antibody chain that comprises a full-length variable region and a full-length constant region (Fc). An intact "conventional" antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and a heavy chain constant domain, CH1, hinge, CH2 and CH3, in secreted IgG. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effector functions," which refer to biological activities attributable to the Fc constant region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down-regulation of cell surface receptors. Constant region variants include those that alter effector profile, binding to Fc receptors, etc.

[0052] Depending on the amino acid sequence of the Fc (constant domain) of the heavy chain, antibodies and various antigen-binding proteins can be assigned to different "classes". There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains corresponding to the various classes of antibodies can be called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. Ig forms include hinge-modified or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US Patent Application Publication No. 2005 / 0048572; US Patent Application Publication No. 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 the amino acid sequences of their constant domains. Antibodies according to embodiments of the invention may comprise a kappa light chain sequence or a lambda light chain sequence.

[0053] A "functional Fc region" has the "effector functions" of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding; CDC; Fc receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require the Fc region to interact with receptors, e.g., FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors and low affinity FcRn receptors, 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, e.g., with respect to extending serum half-life, but does not activate high affinity Fc receptors or has reduced affinity for Fc receptors.

[0054] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include, for example, native sequence human IgG1 Fc region (non-A and A allotypes), native sequence human IgG2 Fc region, native sequence human IgG3 Fc region, and native sequence human IgG4 Fc region, and naturally occurring variants thereof.

[0055] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of the parent polypeptide, compared to the native sequence Fc region or the Fc region of the parent polypeptide. A variant Fc region herein preferably has at least about 80% homology with the native sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology therewith, and more preferably at least about 95% homology therewith.

[0056] The variant Fc sequence may contain three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitutions at positions 233-236 of human IgG1 or IgG2 residues and at positions 327, 330 and 331 of IgG4 residues significantly reduce ADCC and CDC (see, e.g., Armour KL.et al., 1999 Eur J Immunol.29(8):2613-24; and Shields RL.et al., 2001.J Biol Chem.276(9):6591-604). The human IgG4 Fc amino acid sequence (UniProtKB number P01861) is provided herein 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 disclosures of which are incorporated herein by reference in their entirety.

[0057] Other Fc variants are possible, including but not limited to those in which regions capable of forming disulfide bonds have been deleted, or specific amino acid residues have been removed at the N-terminus of the native Fc, or a methionine residue has been added. Thus, in some embodiments, one or more Fc portions of an antibody may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet another embodiment, the hinge region of the Fc may be completely removed. In yet another embodiment, an antibody may comprise an Fc variant.

[0058] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector functions by substituting (mutating), deleting or adding amino acid residues to effect complement binding or Fc receptor binding. For example, but not limited to, deletions can occur in complement binding sites, such as the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in WO 97 / 34631 and WO 96 / 32478. Furthermore, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0059] In some embodiments, the antibody comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation, which may optionally be referred to herein as an IgG4 CH3 knob sequence. In some embodiments, the antibody comprises a variant human IgG4 CH3 domain sequence comprising a T366S mutation, a L368A mutation and a Y407V mutation, which may optionally be referred to herein as an IgG4 CH3 hole sequence. The IgG4 CH3 mutations described herein may be utilized in any suitable manner to place a "knob" in the first heavy chain constant region of a first monomer in the antibody dimer and a "hole" in the second heavy chain constant region of a second monomer in the antibody dimer, thereby facilitating proper pairing (heterodimerization) of the desired pair of heavy chain polypeptide subunits in the antibody.

[0060] In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising a S228P, F234A, L235A, and T366W mutation (knob).In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising a S228P, F234A, L235A, T366S, L368A, and Y407V mutation (hole).

[0061] The term "Fc region-containing antibody" 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 purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Thus, antibodies with an Fc region according to the present invention can include antibodies with or without K447.

[0062] An embodiment of the present invention includes an antibody comprising a heavy chain-only variable region in a monovalent or bivalent structure. As used herein, the term "monovalent structure" when used in reference to a heavy chain-only variable region domain means that there is only one heavy chain-only variable region domain, with a single binding site. On the other hand, the term "bivalent structure" when used in reference to a heavy chain-only variable region domain means that there are two heavy chain-only variable region domains, each with a single binding site, connected by a linker sequence. Non-limiting examples of linker sequences are discussed further herein and include, but are not limited to, GS linker sequences of various lengths. When the heavy chain-only variable region is in a bivalent structure, each of the two heavy chain-only variable region domains can bind to the same antigen or different antigens (e.g., to different epitopes on the same protein; to two different proteins, etc.). However, unless otherwise specified, a heavy chain-only variable region designated as being a "bivalent structure" is understood to contain two identical heavy chain-only variable region domains linked by a linker sequence, each of which binds to the same target antigen.

[0063] Aspects of the invention include antibodies with multispecific structures, including but not limited to bispecific, trispecific, etc. A wide variety of methods and protein configurations are known and have been used to create bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc.

[0064] Various methods have been developed to generate multivalent artificial antibodies by recombinantly fusing the variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on a polypeptide are linked by a polypeptide linker. One non-limiting example of such a polypeptide linker is a GS linker having an amino acid sequence of four glycine residues followed by one serine residue, the sequence repeated n times, where n is an integer ranging from 1 to about 10, e.g., 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 38) (n=1) and GGGGSGGGGS (SEQ ID NO: 39) (n=2). Other suitable linkers may also be used, e.g., as described in Chen et al., Adv Drug Deliv Rev. 2013 October 15;65(10):1357-69, the disclosure of which is incorporated herein by reference in its entirety.

[0065] The term "three-chain antibody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule that comprises, consists essentially of, or consists of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy and one light chain of a monoclonal antibody or a functional antigen-binding fragment of such an antibody chain comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy chain antibody that comprises an Fc portion comprising a CH2, and / or CH3, and / or CH4 domain in the absence of a CH1 domain, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind to an epitope of a second antigen or a different epitope of a first antigen, such binding domains being derived from or having sequence identity with the variable regions of the antibody heavy or light chains. Part of such a variable region is the V H and / or V L Gene segments, D and J H Gene segment or J LThe variable region can be encoded by a rearranged V H DJ H , V L DJ H , V H J L Or V L J L It can be encoded by a gene segment.

[0066] The TCA binding compound uses the term "heavy chain only antibody" or "heavy chain-antibody" or "heavy chain polypeptide", as used herein, to mean a single chain antibody comprising the heavy chain constant regions CH2 and / or CH3 and / or CH4, but not the CH1 domain. In one embodiment, the heavy chain antibody is comprised of an antigen binding domain, at least a portion of a hinge region, and a CH2 and CH3 domain. In another embodiment, the heavy chain antibody is comprised of an antigen binding domain, at least a portion of a hinge region, and a CH2 domain. In a further embodiment, the heavy chain antibody is comprised of an antigen binding domain, at least a portion of a hinge region, and a CH3 domain. Also included herein are heavy chain antibodies in which the CH2 and / or CH3 domains are truncated. In a further embodiment, the heavy chain is comprised of an antigen binding domain and at least one CH (CH1, CH2, CH3 or CH4) domain, but not the hinge region. Heavy chain antibodies may be in the form of a dimer in which two heavy chains are disulfide-bonded or otherwise covalently or non-covalently attached to one another, and may optionally include an asymmetric interface (e.g., a knob-in-hole (KiH) interface) between one or more of the CH domains to promote correct pairing between the polypeptide chains. Heavy chain antibodies may belong to the IgG subclass, although antibodies belonging to other subclasses, such as IgM, IgA, IgD and IgE subclasses, are also included herein. In certain embodiments, the heavy chain antibodies are of the IgG1, IgG2, IgG3 or IgG4 subtype, particularly the IgG1 or IgG4 subtype. Non-limiting examples of TCA binding compounds are described, for example, in WO 2017 / 223111 and WO 2018 / 052503, the disclosures of which are incorporated herein by reference in their entirety.

[0067] Heavy chain antibodies constitute about one quarter of the IgG antibodies produced by camelids, such as camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains but lack light chains. As a result, the variable antigen-binding portion is called a VHH domain and represents the smallest naturally occurring intact antigen-binding site, only about 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with high specificity and affinity can be generated against various antigens by immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and VHH moieties can be easily cloned and expressed in yeast (Frenken, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Their expression, solubility and stability levels are significantly higher than classical F(ab) or Fv fragments (Ghahroudi, MA et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies, called VNAR. (Nuttall et al.Eur.J.Biochem.270,3543-3554(2003);Nuttall et al.Function and Bioinformatics 55,187-197(2004);Dooley et al.,Molecular Immunology 40,25-33(2003)).

[0068] The term "CD19," as used herein, refers to a 95 kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily that functions as a co-receptor for the B cell antigen receptor complex (BCR) on B lymphocytes. The term "CD19" includes the CD19 protein of any human and non-human animal species, and in particular includes human CD19 as well as non-human mammalian CD19.

[0069] The term "human CD19," as used herein, includes any variant, isoform, and species homologue of human CD19 (UniProt P15391), regardless of its 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.

[0070] The terms "anti-CD19 heavy chain-only antibody", "CD19 heavy chain antibody", "anti-CD19 heavy chain antibody" and "CD19 heavy chain antibody" are used interchangeably herein and refer to heavy chain antibodies, as defined herein above, that immunospecifically bind to CD19, including human CD19, as defined herein above. This definition includes, but is not limited to, human heavy chain antibodies produced by transgenic animals, such as transgenic rats or mice expressing human immunoglobulins, e.g., the UniRat™ antibody producing a human anti-CD19 UniAb™, as defined herein above.

[0071] "Percent (%) amino acid sequence identity" 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 the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2.

[0072] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that may interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified (1) to greater than 95% by weight, most preferably greater than 99% by weight, as determined by the Lowry method, (2) to the extent sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated antibodies include antibodies present in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0073] The antibodies of the present invention include multispecific antibodies. Multispecific antibodies have multiple binding specificities. The term "multispecific" specifically includes "bispecific" and "trispecific" as well as higher order independent specific binding affinities such as polyepitopic specificity and tetravalent antibodies and antibody fragments. The terms "multispecific antibody", "multi-specific heavy chain-only antibody", "multi-specific heavy chain antibody" and "multispecific UniAb™" are used herein in the broadest sense to include all antibodies with multiple binding specificities. Multispecific anti-heavy chain anti-CD19 antibodies of the present invention specifically include antibodies that immunospecifically bind to two or more non-overlapping epitopes on the CD19 protein, such as human CD19 (i.e. bivalent and biparatopic). Multispecific heavy chain anti-CD19 antibodies of the invention also include antibodies that specifically bind to an epitope on a CD19 protein, such as human CD19, and an epitope on a different protein, such as a CD3 protein, such as a human CD3 protein (i.e., bispecific and biparatopic). Multispecific heavy chain anti-CD19 antibodies of the invention also include antibodies that specifically bind to two or more non-overlapping or partially overlapping epitopes on a CD19 protein, such as a human CD19 protein, and an epitope on a different protein, such as a CD3 protein, such as a human CD3 protein (i.e., trivalent and biparatopic).

[0074] The antibodies of the invention include monospecific antibodies having one binding specificity. Monospecific antibodies specifically include antibodies that contain a single binding specificity as well as antibodies that contain multiple binding units with the same binding specificity. The terms "monospecific antibody", "monospecific heavy chain-only antibody", "monospecific heavy chain antibody" and "monospecific UniAb™" are used herein in the broadest sense to encompass all antibodies that have one binding specificity. The monospecific heavy chain anti-CD19 antibodies of the invention specifically include antibodies that immunospecifically bind to one epitope on a CD19 protein, such as human CD19 protein (monovalent and monospecific). The monospecific heavy chain anti-CD19 antibodies of the invention also specifically include antibodies with multiple binding units that immunospecifically bind to an epitope on a CD19 protein, such as human CD19 (e.g., multivalent antibodies). For example, a monospecific antibody according to an embodiment of the invention may comprise a heavy chain variable region that comprises two antigen binding domains, each antigen binding domain binding to the same epitope on a CD19 protein (i.e., bivalent and monospecific).

[0075] An "epitope" is a 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 and conformational epitopes.

[0076] "Epitope mapping" is the process of identifying the binding site or epitope of an antibody on its target antigen. Antibody epitopes can be linear or conformational epitopes. Linear epitopes are formed by a contiguous sequence of amino acids in a protein. Conformational epitopes are formed from amino acids that are discontinuous in the protein sequence but come together when the protein folds into a three-dimensional structure.

[0077] "Polyepitopic specificity" refers to the ability to specifically bind two or more different epitopes on the same or different targets. As mentioned above, the present invention includes, in particular, anti-CD19 heavy chain antibodies with polyepitopic specificity, i.e., anti-CD19 heavy chain antibodies that bind one or more non-overlapping epitopes on the CD19 protein, such as human CD19; and anti-CD19 heavy chain antibodies that bind one or more epitopes on the CD19 protein and an epitope on a different protein, such as the CD3 protein. The term "non-overlapping epitopes" or "non-competing epitopes" of an antigen is defined herein to mean an epitope that is recognized by one member of a pair of antigen-specific antibodies but not by the other member. A pair of antibodies or antigen-binding regions that target the same antigen on a multispecific antibody that recognize non-overlapping epitopes do not compete for binding to the antigen and are capable of binding to the antigen simultaneously.

[0078] An antibody binds to "essentially the same epitope" as a reference antibody if the two antibodies recognize the same or sterically overlapping epitopes. The quickest and most widely used method for determining whether two epitopes bind to the same or sterically overlapping epitopes is a competitive assay, which can be configured in any number of different formats, using either labeled antigen or labeled antibody. Typically, antigen is immobilized on a 96-well plate, and the ability of unlabeled antibody to block the binding of labeled antibody is measured using a radioactive or enzyme label.

[0079] The term "valency" as used herein refers to the specific number of binding sites in an antibody molecule.

[0080] A "monovalent" antibody has one binding site. A monovalent antibody is therefore also monospecific.

[0081] 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 otherwise multivalent. A bivalent antibody according to embodiments of the invention may have two binding sites for the same epitope (i.e. bivalent, monoparatopic) or two different epitopes (i.e. bivalent, biparatopic).

[0082] A wide variety of methods and protein structures for preparing bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc. are known and in use.

[0083] The term "three-chain antibody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy and one light chain of a monoclonal antibody or a functional antigen-binding fragment of such an antibody chain comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises an Fc portion comprising a CH2 and / or CH3 and / or CH4 domain in the absence of a CH1 domain, and an antigen-binding domain that binds to an epitope of a second antigen or a different epitope of a first antigen, such binding domain comprising, consisting essentially of, or consisting of a heavy chain antibody derived from or having sequence identity with the variable region of an antibody heavy or light chain. Part of such a variable region is the V H and / or V L Gene segments, D and J H Gene segment or J L The variable region can be encoded by a rearranged V H DJ H , V L DJ H , V H J L Or V L JL The TCA protein may be encoded by a gene segment. The TCA protein utilizes a heavy chain antibody as defined herein above.

[0084] The term "chimeric antigen receptor" or "CAR" is used herein in the broadest sense to refer to an engineered receptor that grafts a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) onto a transmembrane domain and an intracellular signaling domain. Typically, this 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 engineered to generate an artificial T cell receptor for use in immunotherapy. In one embodiment, "CAR-T cells" refers to therapeutic T cells that express a transgene encoding one or more chimeric antigen receptors that are minimally composed of an extracellular domain, a transmembrane domain, and at least one cytoplasmic domain.

[0085] The term "human antibody" is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. The term "human antibody" specifically includes heavy chain antibodies having human heavy chain variable region sequences produced by transgenic animals, e.g., transgenic rats or mice, in particular UniAb™ produced by UniRat™ as defined above.

[0086] "Chimeric antibody" or "chimeric immunoglobulin" refers to an immunoglobulin molecule that contains amino acid sequences derived from at least two different Ig loci, e.g., a transgenic antibody that contains a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies with non-human or artificial Fc regions and human idiotypes. Such immunoglobulins can be isolated from animals of the invention that have been engineered to produce such chimeric antibodies.

[0087] 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 recognition and activation phases of an immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, are capable of inducing antibody-dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages that express FcR are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system or binding to cells that present antigens. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0088] A "human effector cell" is a leukocyte that expresses a receptor, such as a T cell receptor or FcR, and performs effector function. Preferably, the cell expresses at least FcγRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with NK cells being preferred. Effector cells may be isolated from their natural source, such as blood or PBMCs as described herein.

[0089] The term "immune cell" is used herein in the broadest sense and includes, but is not limited to, cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0090] Antibody "effector functions" refer to biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor, BCR), and the like.

[0091] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3, 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 U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0092] "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, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed.

[0093] "Binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X to its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high affinity antibodies generally bind antigens faster and tend to remain bound.

[0094] As used herein, "Kd" or "Kd value" refers to the dissociation constant determined by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetic mode. For example, an anti-mouse Fc sensor is loaded with mouse Fc fusion antigen and then immersed in an antibody-containing well to measure the concentration-dependent binding rate (kon). The antibody dissociation rate (koff) is measured at the final stage, where the sensor is immersed in a well containing only buffer. Kd is the ratio of koff / kon. (For more details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0095] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic, in that it partially or completely cures the disease and / or the deleterious effects caused by the disease. As used herein, "treatment" encompasses any treatment of disease in a mammal, including (a) preventing the disease from arising in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) arresting the disease, i.e., arresting its onset; or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of the disease or injury. Of particular interest is the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient. It is desirable that such treatment is performed before the complete loss of function in the affected tissue. The treatment of the subject may be administered during, and optionally after, the symptomatic stage of the disease.

[0096] By "therapeutically effective amount" is intended the amount of active agent required to provide a therapeutic benefit to a subject. For example, a "therapeutically effective amount" is an amount that induces, ameliorates or causes the amelioration of pathological symptoms, disease progression or physiological conditions associated with a disease, or improves resistance to a disorder.

[0097] The term "B cell neoplasm" or "mature B cell neoplasm" in the context of the present invention includes, but is not limited to, all lymphocytic leukemias and lymphomas, chronic lymphocytic leukemia, acute lymphoblastic leukemia, prolymphocytic leukemia, precursor B lymphoblastic leukemia, hairy cell leukemia, small lymphocytic lymphoma, B cell prolymphocytic lymphoma, B cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse large B cell lymphoma (DL-1 lymphoma), diffuse large B cell lymphoma (DL-2 lymphoma), ... BCL), multiple myeloma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell neoplasms such as plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, MALT lymphoma, nodal marginal zone B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, non-Hodgkin's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, primary effusion lymphoma, and AIDS-related non-Hodgkin's lymphoma.

[0098] The term "characterized by expression of CD19" refers broadly to any disease or disorder in which CD19 expression is associated with or implicated in one or more pathological processes that are characteristic of the disease or disorder, including, but not limited to, B-cell neoplasms.

[0099] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or treated. In one embodiment, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune disease, individuals with pathogen infection, and the like. Subjects can be humans, but also include other mammals, particularly mammals useful as experimental models for human disease, e.g., mice, rats, and the like.

[0100] The term "pharmaceutical formulation" refers to a formulation that is in such a form as to allow the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. A "pharmaceutical acceptable" excipient (vehicle, additive) is one that can be reasonably administered to a target mammal to provide an effective dose of the active ingredient employed.

[0101] A "sterile" formulation is sterile or free or essentially free of all living microorganisms and their spores. A "frozen" formulation is one at a temperature below 0°C.

[0102] A "stable" formulation is one in which the protein therein essentially retains its physical stability, and / or chemical stability, and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones. A. Adv. Drug Delivery Rev. 10: 29-90) (1993). Stability can be measured at a selected temperature for a selected period of time. Stability can be qualitatively and / or quantitatively assessed in a variety of different ways, including assessing aggregate formation (e.g., by measuring turbidity using size-exclusion chromatography and / or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, imaging capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; analysis of amino- or carboxy-terminal sequences; mass spectrometry; SDS-PAGE analysis to compare reduced and intact antibodies; peptide map (e.g., trypsin or LYS-C) analysis; evaluation of antibody biological activity or antigen-binding function, etc. Instability can include any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, differential glycosylation, etc.

[0103] II. Detailed Description Anti-CD19 antibody The present invention provides a family of closely related antibodies that bind to human CD19. The antibodies of this family comprise a set of CDR sequences as defined herein and shown in Table 1, and are exemplified by the provided heavy chain CDR1, CDR2 and CDR3 sequences shown in Table 2 and the heavy chain variable region (VH) sequences of SEQ ID NOs: 5 and 6 shown in Table 3. This antibody family offers a number of benefits that contribute to its usefulness as one or more clinical therapeutic agents. These antibodies include members with a range of binding affinities, allowing the selection of a specific sequence with a desired binding affinity.

[0104] [Table 1]

[0105] [Table 2]

[0106] [Table 3]

[0107] Suitable antibodies may be selected from those provided herein for development and use for therapeutic or other uses, including, without limitation, as bispecific antibodies or as part of a CAR-T construct, e.g., as depicted in Figure 4, panel B.

[0108] Determination of affinity for a candidate protein may be performed using methods known in the art, such as Biacore measurements. - There are about 10 members -6 ~about 10 -10 Front and rear: Approx. 10 -6 ~10 -9 Front and rear: Approx. 10 -6 ~about 10 -8 Front and rear: Approx. 10 -8 ~about 10 -11 Front and rear: Approx. 10 -8 ~about 10 -10 Front and rear: Approx. 10 -8~about 10 -9 Front and rear: Approx. 10 -9 ~about 10 -11 Front and rear: Approx. 10 -9 ~about 10 -10 or any value within these ranges. Affinity selection can be confirmed by biological evaluation for modulation, e.g., blocking, of the biological activity of CD19, including in vitro assays, preclinical models, and clinical trials, as well as evaluation of potential toxicity.

[0109] The members of the antibody family herein do not exhibit cross-reactivity with the CD19 protein of cynomolgus monkeys, but can be engineered, if desired, to confer cross-reactivity with the CD19 protein of cynomolgus monkeys or the CD19 of any other animal species.

[0110] The family of CD19-specific antibodies herein comprises a VH domain comprising CDR1, CDR2 and CDR3 sequences in a human VH framework. The CDR sequences may be located in the regions around amino acid residues 26-33; 51-58; and 97-116 for CDR1, CDR2 and CDR3, respectively, of the representative variable region sequences provided, as shown, by way of example, in SEQ ID NOs: 5-6. It will be understood by those skilled in the art that if different framework sequences are selected, the CDR sequences may be in different positions, although generally the order of the sequences will remain the same.

[0111] In some embodiments, the anti-CD19 antibody comprises the CDR1 sequence of any one of SEQ ID NO: 1 or 38. In particular embodiments, the anti-CD19 antibody comprises the CDR1 sequence of SEQ ID NO: 1. In particular embodiments, the anti-CD19 antibody comprises the CDR1 sequence of SEQ ID NO: 38.

[0112] In some embodiments, the anti-CD19 antibody comprises the CDR2 sequence of any one of SEQ ID NO: 2 or 39. In particular embodiments, the anti-CD19 antibody comprises the CDR2 sequence of SEQ ID NO: 2. In particular embodiments, the anti-CD19 antibody comprises the CDR2 sequence of SEQ ID NO: 39.

[0113] In some embodiments, the anti-CD19 antibody comprises the CDR3 sequence of any one of SEQ ID NOs: 3 to 4. In particular embodiments, the anti-CD19 antibody comprises the CDR3 sequence of SEQ ID NO: 3. In particular embodiments, the anti-CD19 antibody comprises the CDR3 sequence of SEQ ID NO: 4.

[0114] In a further embodiment, the anti-CD19 heavy chain 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:3.

[0115] In a further embodiment, the 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.

[0116] In a further embodiment, the 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.

[0117] In a further embodiment, the 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.

[0118] In a further embodiment, the anti-CD19 antibody comprises any of the heavy chain variable region amino acid sequences of SEQ ID NOs: 5 to 6 and 40 to 41 (Table 3).

[0119] In yet a further embodiment, the anti-CD19 antibody comprises the heavy chain variable region sequence of SEQ ID NO:5.

[0120] In yet a further embodiment, the anti-CD19 antibody comprises the heavy chain variable region sequence of SEQ ID NO:6.

[0121] In yet a further embodiment, the anti-CD19 antibody comprises the heavy chain variable region sequence of SEQ ID NO:40.

[0122] In yet a further embodiment, the anti-CD19 antibody comprises the heavy chain variable region sequence of SEQ ID NO:41.

[0123] In some embodiments, the CDR sequences in the anti-CD19 antibodies of the invention comprise one or two amino acid substitutions compared to the CDR1, CDR2 and / or CDR3 sequences or a set of CDR1, CDR2 and CDR3 sequences in any one of SEQ ID NOs: 1-4, 38 and 39 (Table 1; Table 2).

[0124] In some embodiments, the anti-CD19 antibody preferably comprises a heavy chain variable domain (VH) whose CDR3 sequence has 80% or more, e.g. at least 85%, at least 90%, at least 95% or at least 99% sequence identity at the amino acid level to any one of the CDR3 sequences of an antibody having the CDR3 sequence provided in Table 1 or Table 2, and binds to CD19.

[0125] In some embodiments, the anti-CD19 antibody preferably comprises a heavy chain variable domain (VH) in which a full set of CDR1, 2 and 3 (combination) have eighty-five percent (85%) or greater sequence identity at the amino acid level to the CDR1, 2 and 3 (combination) of an antibody having the CDR sequences provided in Table 1 or Table 2, and binds to CD19.

[0126] In some embodiments, the anti-CD19 antibody comprises a heavy chain variable region sequence that is at least about 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to any of the heavy chain variable region sequences of SEQ ID NOs: 5-6 and 40-41 (shown in Table 3), and binds to CD19.

[0127] In some embodiments, bispecific or multispecific antibodies are provided, which may have any of the configurations discussed herein, including, without limitation, bispecific three-chain antibody-like molecules (TCAs). In some embodiments, the multispecific antibody may comprise at least one heavy chain variable region having binding specificity for CD19 and at least one heavy chain variable region having binding specificity for a protein other than CD19. In some embodiments, the multispecific antibody may comprise at least one heavy chain variable region that binds CD19 and at least one heavy chain variable region that binds a protein other than CD19. In some embodiments, the multispecific antibody may comprise a heavy chain variable region comprising at least two antigen binding domains, each of which binds to CD19. In some embodiments, the multispecific antibody may comprise a heavy chain / light chain pair that binds a first antigen (e.g., CD3) and a heavy chain from a heavy chain antibody. In certain embodiments, the heavy chain from the heavy chain antibody comprises an Fc portion comprising a CH2 and / or CH3 and / or CH4 domain in the absence of a CH1 domain. In one particular embodiment, the bispecific antibody comprises a heavy chain / light chain pair that binds to an antigen on an effector cell (e.g., the CD3 protein on a T cell) and a heavy chain from a heavy chain antibody that comprises an antigen-binding domain that binds to CD19.

[0128] In some embodiments, the multispecific antibody comprises a CD3-binding VH domain paired with a light chain variable domain. In certain embodiments, the light chain is a fixed light chain. In some embodiments, the CD3-binding VH domain comprises the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 8, and the CDR3 sequence of SEQ ID NO: 9 in a human VH framework. In some embodiments, the CD3-binding VH domain comprises the CDR1 sequence of SEQ ID NO: 10, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 12 in a human VH framework. In some embodiments, the fixed light chain comprises the CDR1 sequence of SEQ ID NO: 13, the CDR2 sequence of SEQ ID NO: 14, and the CDR3 sequence of SEQ ID NO: 15 in a human VL framework. Together, the CD3-binding VH domain and the light chain variable domain have binding affinity for CD3. In some embodiments, the CD3-binding VH domain comprises the heavy chain variable region sequence of SEQ ID NO: 16. In some embodiments, the CD3-binding VH domain comprises the heavy chain variable region sequence of SEQ ID NO: 17. In some embodiments, the CD3-binding VH domain comprises a sequence having a percent identity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% to the heavy chain variable region sequence of SEQ ID NO: 16 or 17. In some embodiments, the fixed light chain comprises a light chain variable region sequence of SEQ ID NO: 18. In some embodiments, the fixed light chain comprises a sequence having a percent identity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% to the light chain variable region sequence of SEQ ID NO: 18.

[0129] Multispecific antibodies comprising the above-described CD3-binding VH domain and light chain variable domain have advantageous properties, for example, as described in WO 2018 / 052503, the disclosure of which is incorporated herein by reference in its entirety. Any of the multispecific antibodies and antigen-binding domains with binding affinity for CD19 described herein can be combined with additional sequences, such as the CD3-binding domains and fixed light chain domains described herein (see, for example, Tables 4 and 5) and those provided in Tables 6 and 7, to create multispecific antibodies with binding affinity for one or more CD19 epitopes as well as CD3.

[0130] [Table 4]

[0131] [Table 5]

[0132] [Table 6]

[0133] [Table 7]

[0134] [Table 8]

[0135] [Table 9]

[0136] [Table 10]

[0137] In some embodiments, bispecific or multispecific antibodies are provided that may have any of the configurations discussed herein, including but not limited to bispecific three-chain antibody-like molecules (TCAs). In some embodiments, a bispecific antibody may comprise at least one heavy chain variable region that binds to CD19 and at least one heavy chain variable 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, a heavy chain derived from a heavy chain antibody that comprises an Fc portion that comprises a CH2 and / or CH3 and / or CH4 domain in the absence of a CH1 domain, and an antigen binding domain that binds to an epitope of a second antigen or a different epitope of the first antigen. In one particular embodiment, a bispecific antibody comprises a heavy chain / light chain pair that binds to an antigen on an effector cell (e.g., CD3 protein on a T cell), and a heavy chain derived from a heavy chain antibody that comprises an antigen binding domain that binds to CD19.

[0138] In some embodiments, when the antibody of the present invention is a bispecific antibody, one arm of the antibody (one binding moiety or one binding unit) may be specific for human CD19, while the other arm may be specific for a target cell, a tumor-associated antigen, a targeting antigen such as an integrin, a pathogen antigen, a checkpoint protein, etc. Target cells include, without limitation, cancer cells, including cells associated with hematological malignancies characterized by expression of CD19, as well as pathogenic B cells associated with autoimmune disorders characterized by expression of CD19. In some embodiments, one arm of the antibody (one binding moiety or one binding unit) is specific for human CD19, while the other arm is specific for CD3.

[0139] In some embodiments, the antibody comprises an anti-CD3 light chain polypeptide comprising the sequence of SEQ ID NO: 30, an anti-CD3 heavy chain polypeptide comprising the sequence of SEQ ID NO: 16 or 17, and an anti-CD19 heavy chain polypeptide comprising the sequence of SEQ ID NO: 5 or 6, in a monovalent or bivalent configuration, linked to any one of the sequences of SEQ ID NO: 28 or 29. These sequences can be combined in various ways to create bispecific antibodies of the desired IgG subclass, e.g., IgG1, IgG4, silent IgG1, silent IgG4. In a preferred embodiment, the 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.

[0140] Various formats of multispecific antibodies are within the scope of the present invention, including, but not limited to, single-chain polypeptides, two-chain polypeptides, three-chain polypeptides, four-chain polypeptides, and combinations thereof. Multispecific antibodies herein specifically include T cell multispecific (e.g., bispecific) antibodies that bind to CD19 and CD3 (anti-CD19 x anti-CD3 antibodies). Such antibodies induce potent T cell-mediated killing of CD19-expressing cells.

[0141] Preparation of anti-CD19 antibody 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, such as transgenic mice and rats, preferably rats, in which endogenous immunoglobulin genes have been knocked out or disabled. In a preferred embodiment, the heavy chain antibodies herein are made in UniRat™. The endogenous immunoglobulin genes of UniRat™ are silenced and a human immunoglobulin heavy chain transgene locus is used to express a diverse, naturally optimized repertoire of fully human HCAbs. While rat endogenous immunoglobulin loci can be knocked out or silenced using a variety of techniques, in UniRat™, zinc finger (endo)nuclease (ZNF) technology was used to inactivate the endogenous rat heavy chain J locus, light chain Cκ locus and light chain Cλ locus. ZNF constructs for microinjection into oocytes can produce IgH and IgL knockout (KO) strains. For details, see, for example, Geurts et al., 2009, Science 325:433. The properties of Ig heavy chain knockout rats have been reported by Menoret et al., 2010, Eur. J. Immunol. 40:2932-2941. The advantage of ZNF technology is that the non-homologous ends that are linked to silence genes or loci by deletions of up to several kb can also provide target sites for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). The human heavy chain antibodies produced in UniRat™ are called UniAbs™ and can bind epitopes that cannot be attacked by conventional antibodies. Due to their high specificity, affinity and small size, they are ideal for monospecific and multispecific applications.

[0142] In addition to UniAbs™, heavy chain antibodies lacking camelid VHH frameworks and mutations, as well as functional VH regions thereof, are specifically included herein. Such heavy chain antibodies can be produced in transgenic rats or mice containing a fully human heavy chain single locus, for example as described in WO 2006 / 008548, although other transgenic mammals such as rabbits, guinea pigs, rats, etc. may also be used, with rats and mice being preferred. Heavy chain antibodies (including their VHH or VH functional fragments) can also be produced by recombinant DNA techniques, for example by expression of the encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including mammalian cells (e.g. CHO cells), E. coli, or yeast.

[0143] Heavy chain antibody domains combine the advantages of antibodies and small molecule drugs, can be monovalent or polyvalent, have low toxicity and are cost-effective to produce. Due to their small size, these domains are easy to administer, including orally or topically, are characterized by high stability, including gastrointestinal stability, and their half-life can be tailored to the desired use or indication. Furthermore, the VH and VHH domains of HCAbs can be produced in a cost-effective manner.

[0144] In a particular embodiment, the heavy chain antibodies of the invention, including UniAb™, have a native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Kabat numbering system) replaced by another amino acid residue, which can disrupt the surface-exposed hydrophobic patch that contains the native amino acid residue or is associated with the natural amino acid residue at that position. Such a hydrophobic patch is usually buried at the interface with the antibody light chain constant region, but is surface-exposed in the HCAb, at least in part due to undesired aggregation and light chain binding of the HCAb. The replaced amino acid residue is preferably charged, more preferably positively charged, for example, lysine (Lys, K), arginine (Arg, R) or histidine (His, H), preferably arginine (R). In a preferred embodiment, the heavy chain antibody from the transgenic animal contains a Trp to Arg mutation at position 101. The resulting HCAb preferably has high antigen binding affinity and solubility under physiological conditions without aggregation.

[0145] As part of the present invention, human IgG anti-CD19 heavy chain antibodies (UniAbs™) with unique sequences derived from UniRat™ animals were identified that bind to human CD19 in ELISA protein and cell binding assays. The identified heavy chain variable region (VH) sequences are positive for human CD19 protein binding and / or for binding to CD19+ cells, and are all negative for binding to cells that do not express CD19.

[0146] Heavy chain antibodies that bind to non-overlapping epitopes on CD19 protein, such as UniAbs™, can be identified by competitive binding assays, such as enzyme-linked immunosorbent assays (ELISA assays) or flow cytometry competitive binding assays. For example, competition between known antibodies that bind to target antigens and antibodies of interest can be used. Using this approach, a set of antibodies can be divided into those that compete with the reference antibody and those that do not. Non-competing antibodies are identified as binding to distinct epitopes that do not overlap with the epitope that the reference antibody binds. Often, one antibody is immobilized and allowed to bind antigen, 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 be done using surface plasmon resonance (SPR) platforms including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences) and MX96 SPR Imager (Ibis technologies BV), and also on biolayer interference platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). For further details, see the Examples herein.

[0147] Typically, an antibody "competes" with a reference antibody if it causes about a 15-100% reduction in binding of the reference antibody to a target antigen as determined by standard techniques, such as the competitive binding assays described above. In various embodiments, the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more.

[0148] Pharmaceutical Compositions, Methods of Use and Treatment Another aspect of the invention is to provide pharmaceutical compositions comprising one or more antibodies of the invention in admixture with a suitable pharma- ceutically acceptable carrier, as used herein, includes but is not limited to adjuvants, solid carriers, water, buffers, or other carriers used in the art to carry therapeutic ingredients, or combinations thereof.

[0149] In one embodiment, the pharmaceutical composition comprises a heavy chain antibody (e.g., UniAb™) that binds to CD19. In another embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb™) that has binding specificity for two or more non-overlapping epitopes on the CD19 protein. In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific and TCA) heavy chain antibody (e.g., UniAb™) that has binding specificity for CD19 and has binding specificity for a binding target on an effector cell (e.g., a binding target on a T cell, such as, for example, the CD3 protein on a T cell). In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific and TCA) heavy chain antibody (e.g., UniAb™) that binds to CD19 and has binding specificity for a binding target on an effector cell (e.g., a binding target on a T cell, such as, for example, the CD3 protein on a T cell).

[0150] Pharmaceutical compositions of antibodies used according to the present disclosure are prepared by mixing the protein having the desired purity, such as in the form of a lyophilized formulation or an aqueous solution for storage, with optional pharma- ceutically acceptable carriers, excipients, or stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; 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).

[0151] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., dosage form for single administration). Formulations depend on the route of administration selected. The antibodies herein may be administered by intravenous injection or infusion, or subcutaneously. For administration by injection, the antibodies herein may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, 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 constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0152] 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™, of the present invention. Subcutaneous antibody formulations are described, for example, in U.S. Patent Application Publication No. 20160355591 and U.S. Patent Application Publication No. 20160166689.

[0153] How to use The anti-CD19 antibodies and pharmaceutical compositions described herein can be used to treat diseases and conditions characterized by expression of CD19, including, without limitation, the conditions and diseases further described herein.

[0154] CD19 is a cell surface receptor expressed on all human B cells but not on plasma cells. It has a relatively large 240 amino acid cytoplasmic tail. The extracellular Ig-like domain is divided into a potentially disulfide-linked non-Ig-like domain and an N-linked glycosylation site. The cytoplasmic tail contains at least nine tyrosine residues near the C-terminus, some of which have been shown to be phosphorylated. Together with CD20 and CD22, CD19 expression is restricted to the B cell lineage, making it an attractive target for the therapeutic treatment of B cell malignancies. CD19 is a promising target for antibody-based therapeutics because its expression is observed in a number of hematological malignancies.

[0155] In one aspect, the anti-CD19 antibodies (e.g., UniAbs™) and pharmaceutical compositions herein can be used to treat disorders characterized by expression of CD19, including, without limitation, the diseases and disorders further described herein.

[0156] The anti-CD19 heavy chain antibodies (UniAbs) of the present invention can be used for the development of therapeutics for the treatment of hematological malignancies characterized by expression of CD19, including, without limitation, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia (CLL), and B-cell acute lymphoblastic leukemia (ALL).

[0157] Diffuse large B-cell lymphoma (DLBCL or DLBL) is the most common form of non-Hodgkin's lymphoma in adults (Blood 1997 89(11):3909-18), with an estimated annual incidence in the United States and the United Kingdom of 7-8 cases per 100,000 people each year. It is characterized as an aggressive cancer that can occur virtually anywhere in the body. The causes of DLBCL are poorly understood, and it can arise from malignant transformation of normal B cells as well as other types of lymphoma or leukemia cells. Treatment procedures generally involve chemotherapy and radiation, with an average 5-year overall survival rate of approximately 58% for adults. Some monoclonal antibodies have shown promise in the treatment of DLBCL, but consistent clinical efficacy has yet to be conclusively demonstrated. Thus, there is a great need for novel therapies, including immunotherapy, for DLBCL.

[0158] In another aspect, the CD19 heavy chain antibodies (e.g., UniAbs™) and pharmaceutical compositions herein can be used to treat autoimmune disorders characterized by pathogenic B cells that express CD19, including, without limitation, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS).

[0159] In one embodiment, the antibody herein may be in the form of a heavy chain only anti-CD19 antibody-CAR construct, i.e., a heavy chain only anti-CD19 antibody-CAR transduced T cell construct.

[0160] The effective dose of the composition of the present invention for the treatment of disease varies depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, administration of other drugs and whether the treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals, such as companion animals such as dogs, cats, horses, and laboratory mammals such as rabbits, mice, and rats, can also be treated. Treatment dosages can be titrated to optimize safety and efficacy.

[0161] Dosage levels can be easily determined by a clinician with ordinary skill, and can be changed as necessary, for example, to modify the subject's response to treatment. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the host treated and the specific mode of administration. A unit dosage form generally contains about 1 mg to about 500 mg of active ingredient.

[0162] In some embodiments, the therapeutic dosage of the agent may range from about 0.0001 to 100 mg / kg, more typically 0.01 to 5 mg / kg of host body weight. For example, dosages may be 1 mg / kg body weight or 10 mg / kg body weight or within the range of 1 to 10 mg / kg. Exemplary treatment regimes involve administration every two weeks or once a month or every 3 to 6 months. The therapeutic agent of the invention is usually administered multiple times. Intervals between single doses may be weekly, monthly or yearly. Intervals may also be irregular as indicated by measuring blood levels of the therapeutic agent in the patient. Alternatively, the therapeutic agent of the 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.

[0163] Typically, the compositions are prepared as injections, either as solutions or suspensions, and solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared. The pharmaceutical compositions herein are suitable for intravenous or subcutaneous administration, either directly or after reconstitution of a solid (e.g., lyophilized) composition. The preparations can also be emulsified or encapsulated in liposomes or microparticles, such as polylactides, polyglycolides or copolymers, to enhance adjuvant effects, as described above. Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of the present invention can be administered in the form of depot injection or implant formulations, which can be formulated in such a way as to allow sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally sterile, substantially isotonic, and formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0164] Toxicity of the antibodies and antibody constructs described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a non-toxic dosage range for use in humans. The dosage of the antibodies described herein is preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0165] The compositions for administration generally comprise an antibody or other ablative agent dissolved in a pharma- ceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline, etc. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of active agent in these formulations can vary widely and is selected primarily based on fluid volumes, viscosities, body weight, etc., according to the particular mode of administration selected and the needs of the patient (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0166] A kit comprising the active agent of the present invention and its formulation and instructions for use is also within the scope of the present invention.The kit may further comprise at least one additional agent, such as a chemotherapeutic agent.The kit typically comprises a label indicating the intended use of the contents of the kit.The term "label" as used herein includes any written or recorded material that is provided on or with the kit, or that otherwise accompanies the kit.

[0167] Now that the present invention is fully described, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit or scope of the invention. EXAMPLES

[0168] III. Examples Example 1: Binding to CD19+ Raji cells Binding to CD19-positive Raji cells was assessed by flow cytometry. Briefly, 50,000 target cells were stained with a dilution series 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 antibody bound to cells was detected by staining with goat F(ab')2 anti-human IgG conjugated to R-phycoerythrin (PE) (Southern Biotech, Cat. No. 2042-09). After 20 minutes of incubation at 4°C, cells were washed twice with flow cytometry buffer and mean fluorescence intensity (MFI) was measured by flow cytometry. The MFI of cells stained with secondary antibody alone was used to determine background signal, and binding of each antibody was converted to fold over background.

[0169] The results are provided in Figure 1, which summarizes the target binding activity of the indicated anti-CD19 antibodies. Column 1 shows the clone ID of the HCAb. Column 2 shows the binding to Raji cells measured as fold over background MFI signal. Column 3 shows the binding to CHO cells (negative control), which do not express CD19 protein, measured as fold over background MFI signal.

[0170] Example 2: Binding to CD19+ Raji and Daudi cells Cell binding dose curves were performed as described in Example 1 for two cell lines: Raji (A) and Daudi (B). Antibodies were tested at a starting concentration of 200 nM followed by 3-fold serial dilutions in an 8-point dose curve. PE mean fluorescence intensity was plotted as fold over background (cells incubated with secondary detection antibody only).

[0171] The results are presented in Figure 2, Panel A (Raji cells) and Panel B (Daudi cells).

[0172] Example 3: Cell binding EC50 values ​​against CD19+ cell lines To determine cell binding EC50 values, cell binding dose curves were performed on the CD19 expressing cell lines Raji and Daudi as described in Example 2. Antibodies were tested at a starting dose of 200 nM followed by 3-fold serial dilutions in an 8-point dose curve. EC50 (nM) was determined by plotting the transformed data as an xy graph using nonlinear regression curve fitting (available in GraphPad Prism 8.4.3).

[0173] The results are presented in FIG.

[0174] Example 4: CAR-T-mediated T cell activation by human tumor cell lines CAR-T cell activity was measured by co-transfecting Jurkat T lymphocyte cells with anti-CD19 CAR and 6xNFAT TK nanoluciferase reporter. Transfected Jurkats were co-cultured with CD19+ Raji, Ramos and SU-DHL-10 or CD19-negative K562 cells for 24 hours. Luciferase activity was measured using the Promega Nano-Glo Luciferase Assay System (Cat. No. N1110) and data were normalized to co-cultures containing CAR-transfected Jurkat and CD19-negative K562 cell lines. Statistical significance was determined using unpaired two-tailed t-tests.

[0175] The results are presented in FIG.

[0176] FIG. 4, panel A, is a schematic diagram of a CAR-T construct comprising an anti-CD19 extracellular binding domain comprising an antibody sequence according to an embodiment of the invention. Panel B shows Raji( ** p = 0.0055), Ramos ( ** p = 0.0058) and SU-DHL-10 ( *** Panel C depicts T cell activity in Jurkat transfected with anti-CD19 370083 CAR, Raji ( **** p=0.000009), Ramos(** p = 0.004) and SU-DHL-10 ( * p=0.016).

[0177] Example 5: Creation of CD19 VH binders exhibiting enhanced biological properties by site-saturation mutagenesis In this example, site-saturation mutagenesis was used to generate a pool of unique CD19 VH binding sequences in which the wild-type amino acid coding sequence was systematically replaced with one of 19 other amino acid coding sequences, generating a pool of mutants that can be screened to identify CD19 VH binders with improved in vitro and / or in vivo properties compared to the parent VH binder.

[0178] CD19 VH binder VH-034 contains 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 saturation mutagenesis was used to systematically replace each of the 26 combinations of CDR amino acids with one of the other 19 remaining non-wild type amino acid residues to generate a library of 513 unique CD19 VH binder sequences containing single CDR amino acid substitutions ranging across all three CDR sequences.

[0179] Briefly, the nucleotide sequence encoding VH-034 served as a DNA template for site-saturation mutagenesis (e.g., Chronopoulou and Labrou (2011) Curr Protocol Protein Sci Chapter 26 doi:10.1002 / 0471140864.ps2606s63). By using PCR and degenerate synthetic oligonucleotides as primers, one codon was replaced with all 20 amino acid codons in the same reaction, which was then systematically applied to each of the 26 CDR codons. The resulting nucleic acid pool of 513 members, each encoding a single amino acid substitution, and the parental wild-type sequence were cloned into the nanoplasmid vector, and the frequency of each of the 513 unique VH binder sequences in this plasmid pool was determined by next-generation sequencing (NGS).

[0180] Nanoplasmid vector nPBv2-EF1a-iC9-CD19.VH034-DHFR. contains from 5' to 3' direction: right end ITR-5'UTR-insulator-EF1a promoter-inducible iCaspase9 gene-T2A-CD8a leader peptide-site for VH034 binder-CD19BBZ CAR cassette-T2A-DHFR selectable marker-SV40 polyA-insulator-3'-UTR and left end ITR. Each unique binder sequence was cloned in frame into the CD19BBZ CAR cassette to generate a full-length CAR (CD19 VH binder-CD28 hinge-41-BB transmembrane domain and CD3 zeta domain) under the control of the EF1a promoter.

[0181] Example 6: Generation of CAR-expressing T cell pools containing mutant CD19 VH binders In this example, a T cell pool was generated that contained individual T cells expressing a CAR containing one of 513 CD19 VH binder sequences.

[0182] A T cell pool containing the library of 513 CD19 VH binder nanoplasmids generated in Example 5 was prepared by electroporating T cells with an aliquot of mRNA encoding the SuperpiggyBac transposase and a low DNA copy number DNA nanoplasmid pool, ensuring that the electroporated T cells contained a single copy of one of the 513 nanoplasmids. The electroporated T cell population was incubated in 250 nM methotrexate to select for transferred T cells containing an integrated genomic copy of the CAR-expressing transposon. The frequency of each of the 513 unique binder sequences in the T cell pool was determined by NGS.

[0183] Example 7: Enrichment of T cells expressing CARs containing CD19 VH binders that exhibit improved binding by repeated CD19 stimulation This example demonstrates that selection of T-clones that have preferentially increased in number due to repeated exposure to a particular antigen and antigenic stimulation can enrich for T cell clones of choice within a T cell population.

[0184] In one experiment, approximately 20x10e6 T cells / well of T cells from the T cell pool generated in Example 6 were added to each well in duplicate sets in Grex-6 M-well plates. To one set, 2x10e6 RAJI cells (CD19+) were added, and to the other set, 2x10e6 control RAJI cells (RAJI KO) with an inactive CD19 gene were added to stimulate activation of CD19-CAR expressing T cells. On days 3, 6, and 9, T cells were re-challenged with 2x10e6 RAJI cells or RAJI KO, respectively. After 12 days, there was an approximately 20-fold unsorted expansion of the T cell pool stimulated by RAJI cells, while little to no expansion was observed with the use of RAJI KO cells.

[0185] CD19+ cells were analyzed by NGS to determine the frequency of each of the 513 mutant binder sequences within the expanded CD19+ T cell population, and this frequency of each member was analyzed relative to the original plasmid and T cell pool populations.

[0186] In the second experiment, the T cell pool generated in Example 6 was treated with rabbit anti-human IgG antibody to capture CAR-positive cells, and the CAR cells were selected and analyzed by FACS analysis. The selected CAR-positive T cell pool was then subjected to restimulation with RAJI CD19+ cells or RAJI-KO, and about 40-fold expansion of T cells was observed after 15 days (after 6 stimulations with Raji cells). The CAR+ T cells were analyzed by NGS to determine the frequency of each of the 513 mutant binder sequences in the expanded CAR+ T cell population, and this frequency of each member was analyzed in comparison with the original plasmid and T cell pool population.

[0187] By comparing the relative frequency of each VH binder within the original plasmid population, within the T cell pool, and in each of the two enrichment conditions, the relative change in frequency of each VH binder sequence can be determined. The majority of the 513 VH binder sequences were depleted from the RAJI-treated T cell pool, suggesting that their mutations were deleterious in terms of CD19 binding, whereas the population frequency of VH binder sequences remained relatively unchanged upon CD19 stimulation, suggesting that the introduced mutations may have had little effect on CD19 binding. Certain T cell clones are enriched by CD19 stimulation, and these two enrichment assays produce overlapping lists of T cell clones that appear to be enriched in both assays.

[0188] The top 12 VH binders observed in both enrichment assays were further analyzed. Two particular T cell clones were highly enriched in the T cell population after repeated stimulation with RAJI cells. The first T cell clone contained a CAR expressing a CD19 VH binder containing a T29D mutation in CDR1 (SEQ ID NO: 38), and the second T cell clone contained a CAR expressing a CD19 VH binder containing a S55D mutation in CDR2 (SEQ ID NO: 39).

[0189] These two enriched CD19 VH binders, T29D and S55D, were further characterized in in vitro and in vivo assays.

[0190] Example 8: T cells expressing CARs containing T29D or S55D VH binders exhibit enhanced in vitro cytotoxicity In this example, we show that T cells expressing CARs containing T29D VH conjugates or S55D VH conjugates exhibit enhanced in vitro cytotoxicity against CD19-expressing cells when compared to the parental VH034 conjugates. Figure 5, panels A and B, are graphs showing comparative in vitro cytotoxicity results between T cells expressing CARs containing either T29D VH conjugates or S55D VH conjugates compared to the parental VH-034 conjugates. More specifically, these results show in vitro cytotoxicity upon CD19 stimulation using Nalm6 (see Figure 5, panel A) or RAJI (see Figure 5, panel B) cells compared to the parental VH-034 conjugates.

[0191] Approximately 20,000 T cells / well of T cells expressing CARs containing parental VH-034 conjugates, T29D VH conjugates or S55D VH conjugates were added to each well in duplicate sets in a 96-well plate. One set was added with 10,000 RAJI-GFP cells, and the other set was added with 10,000 Nalm6-GFP cells to stimulate activation of CD19-CAR expressing cells. At 48, 116 and 172 hours, T cells were re-challenged with 30,000 RAJI-GFP or Nalm6-GFP, respectively. In vitro cytotoxicity was measured in real time by Incucyte over a period of 252 hours (for Nalm6-GFP) or 188 hours (for RAJI-GFP).

[0192] Example 9: T cells expressing CARs containing T29D or S55D VH binders exhibit improved in vitro T cell activation This example shows that T cells expressing a CAR containing a CD19 VH conjugate enriched by CD19 stimulation exhibit enhanced in vitro T cell activation compared to the parental VH034 conjugate.

[0193] Figure 6 is a table summarizing the improved in vitro T cell activation in T cells expressing CARs containing either T29D VH conjugates or S55D VH conjugates compared to the parental VH-034 conjugates. Approximately 0.2x10e6 T cells expressing CARs containing the parental VH-034 conjugates, T29D VH conjugates or S55D VH conjugates were activated by adding 0.1x10e6 Nalm6 or RAJI cells, and these cultures were incubated for 16 hours at 37°C in RPMI 1640 medium supplemented with 10% FBS. T cells were then analyzed by FACS analysis to quantify CD25 expression, a marker of T cell activation. In the table depicted in Figure 6, the geometric mean fluorescence intensity (gMFI) of CD25 of activated T cells is shown for each VH conjugate.

[0194] As can be seen from the table depicted in FIG. 6, the T29D and S55D CD19 VH binders exhibited improved T cell activation compared to the parental CD19 VH-034 binder, demonstrating that these substitutions improve T cell activation by CD19-expressing cells.

[0195] Example 10: T cells expressing a CAR containing a T29D VH binder or an S55D VH binder express the CAR more highly in vivo This example shows that enriched CD19 VH conjugates in a CAR format result in higher expression of the CAR in T cells compared to the parental VH-034 conjugates.

[0196] Figure 7 is a table showing that enriched CD19 VH conjugates in CAR format express CARs at higher levels in T cells compared to parental VH-034 conjugates. Approximately 10x10e6 T cells expressing CARs containing parental VH-034 conjugates, T29D VH conjugates or S55D VH conjugates were administered to mice (n=5). After 14 days, blood samples were taken from treated animals and blood was lysed with ACK buffer to remove red blood cells. Circulating T cells were then analyzed by FACS analysis, gating on CD19+ cells, to determine the number of CAR-expressing cells per sample. The results are shown in the table depicted in Figure 7.

[0197] As can be seen from the table depicted in Figure 7, the mean CD19 CAR+ circulating T cell numbers were approximately 35% and 65% higher for the T29D and S55D VH conjugates, respectively, when compared to the parental VH-034 conjugate.

[0198] Example 11: T cells expressing CARs containing T29D VH binders or S55D VH binders demonstrate improved in vivo T cell expansion This example shows that T cells expressing a CAR comprising the T29D VH binder or the S55D VH binder exhibit improved in vivo T cell expansion compared to T cells expressing a CAR comprising the parental VH-034.

[0199] FIG. 8 is a table showing that T cells expressing CARs containing T29D VH binders or S55D VH binders exhibit improved T cell expansion in vivo. scid Il2rg tm1Wjl / Szj (n=5 / group) were administered 10x10e6 T cells expressing CARs containing wild type VH-034 conjugates, T29D VH conjugates or S55D VH conjugates. Blood samples were taken from treated mice on days 3, 7 and 14 to determine the number of circulating T cells per microliter of blood. The results are shown in the table depicted in Figure 8.

[0200] As can be seen from the table depicted in Figure 8, the kinetics and magnitude of T cell expansion differ between T cells expressing the parental VH-034 binder or either of the two mutant VH binders. For example, T cells expressing a CAR comprising the S55D VH binder exhibit different kinetics compared to T cells expressing a CAR comprising the T29D VH binder, in that the S55D T cells expand at a peak of 3 days, whereas the T29D T cells expand at a peak of 14 days. Moreover, these two mutant VH binders each demonstrated improved kinetics when compared to the parental VH-034 T cells.

[0201] Example 12: T cells expressing CARs containing T29D VH binders or S55D VH binders demonstrate reduced in vivo T cell depletion This example shows that T cells expressing a CAR containing one of the CD19 VH binders enriched for CD19 binding exhibit reduced in vivo T cell depletion compared to the parental VH-034 binder.

[0202] Approximately 10x10e6 T cells expressing parental VH-034, T29D VH binders or S55D VH binders were cultured in adult NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl100μl of blood samples were taken from the treated animals and red blood cells were removed from each sample with ACK lysis buffer. The degree of T cell depletion in each sample was determined by FACS analysis by gating on cells expressing PD-1 or TIGIT-1, known markers of T cell depletion. Figure 9 is a table showing that T cells expressing CARs containing T29D or S55D CD19 VH conjugates exhibited less T cell depletion compared to the parental VH-034 CD19 conjugate.

[0203] As can be seen from the table in Figure 9, T cells expressing CARs comprising T29D or S55D CD19 VH binders exhibited less T cell depletion at day 21 as evidenced by the reduced numbers of T cells expressing PD-1 or TIGIT, demonstrating that a higher percentage of these cells remained non-depleted compared to the parental VH-034 CD19 binders.

[0204] Example 13: T cells expressing CARs containing T29D VH binders or S55D VH binders exhibit improved in vivo efficacy This example shows that T cells expressing a CAR comprising the T29D VH binder or the S55D VH binder exhibit improved in vivo efficacy compared to T cells expressing a CAR comprising the parental VH-034 binder.

[0205] Day 0, 6-7 week-old adult female NGS (NOD.Cg-Prkdc scid ll2rg tm1Wjl / Szi) mice (n=5 / group) were administered 0.5x10e6 RAJI cells engineered to express luciferase intravenously. On day 5, mice were administered 10x10e6 T cells expressing CARs containing the parental VH-034 conjugate, the T29D VH conjugate, or the S55D VH conjugate. Mice were monitored over a period of 55 days.

[0206] Tumor progression was monitored by whole body bioluminescence imaging ("BLI") on anesthetized mice at weekly intervals throughout the course of the study, 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, spleens, and femoral bone marrow were subsequently collected for further analysis.

[0207] Figure 10 is a graph with reduced data showing that T cells expressing CARs containing T29D VH conjugates or S55D VH conjugates exhibited tumor control. Figure 11 is a graph showing unreduced individual mouse model data illustrating the same results shown in Figure 10. As shown, T cells expressing CARs containing parental VH-034 had a modest effect on tumor growth control over the course of the study. In contrast, five out of five mice administered T cells expressing CARs containing T29D VH conjugates exhibited complete tumor control and regression by day 55. Similarly, three out of five mice administered T cells expressing CARs containing S55D VH conjugates exhibited complete tumor control and regression by at least day 30. These results indicate that enriched CD19 VH conjugates containing T29D and S55D mutations contribute to the improved in vivo efficacy observed in the RAJI model.

[0208] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will now recognize many different modifications and substitutions without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in the practice of the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

(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) 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, 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 An antibody that binds to CD19, comprising a heavy chain variable region comprising:

2. The antibody described in claim 1, wherein the CDR1, CDR2, and CDR3 sequences are present in a human VH framework.

3. The antibody described in claim 1, wherein the extracellular antigen-binding domain that binds to CD19 comprises a heavy chain variable region having at least 95% sequence identity to either of SEQ ID NOs: 40 and 41.

4. An antibody comprising an extracellular antigen-binding domain that binds to CD19, the antibody comprising a heavy chain variable region sequence of SEQ ID NO:

40.

5. An antibody comprising an extracellular antigen-binding domain that binds to CD19, the antibody comprising a heavy chain variable region sequence of SEQ ID NO:

41.

6. A CAR-T cell comprising a CAR comprising an extracellular antigen-binding domain that binds to CD19, (a) the CDR1 sequence of SEQ ID NO: 38, the CDR2 sequence of SEQ ID NO: 2, and the CDR3 sequence of SEQ ID NO: 3, or (b) 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, 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 A CAR-T cell comprising a heavy chain variable region comprising:

7. The CAR-T cell described in claim 6, wherein the heavy chain variable region comprises 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.

8. The CAR-T cell described in claim 6, wherein the heavy chain variable region comprises a CDR1 sequence of sequence number 38, a CDR2 sequence of sequence number 2, and a CDR3 sequence of sequence number 4.

9. The CAR-T cell described in claim 6, wherein the heavy chain variable region comprises 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.

10. The CAR-T cell described in claim 6, wherein the heavy chain variable region comprises 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.

11. The CAR-T cell described in claim 6, wherein the CDR1, CDR2, and CDR3 sequences are present in a human VH framework.

12. The CAR-T cell described in claim 7, wherein the CDR1, CDR2, and CDR3 sequences are present in a human VH framework.

13. The CAR-T cell described in claim 8, wherein the CDR1, CDR2, and CDR3 sequences are present in a human VH framework.

14. The CAR-T cell of claim 9, wherein the CDR1, CDR2, and CDR3 sequences are present in a human VH framework.

15. The CAR-T cell of claim 10, wherein the CDR1, CDR2, and CDR3 sequences are present in a human VH framework.

16. The CAR-T cell described in claim 6, wherein the extracellular antigen-binding domain that binds to CD19 comprises a heavy chain variable region having at least 95% sequence identity to either of SEQ ID NOs: 40 and 41.

17. A CAR-T cell comprising an extracellular antigen-binding domain that binds to CD19, the CAR-T cell comprising a heavy chain variable region sequence of SEQ ID NO:

40.

18. A CAR-T cell comprising an extracellular antigen-binding domain that binds to CD19, said CAR-T cell comprising a heavy chain variable region sequence of SEQ ID NO:

41.

19. A pharmaceutical composition comprising an antibody of any of claims 1 and 3-5, or a CAR-T cell of any of claims 6-18.

20. A drug for use in a method for treating a B cell disorder characterized by expression of CD19, said drug comprising an antibody of any of claims 1 and 3-5, or a CAR-T cell of any of claims 6-18, said method comprising administering said antibody or said CAR-T cell to a subject having the disorder.

21. The drug described in claim 20, wherein the disorder is diffuse large B-cell lymphoma (DLBCL).

22. The method of claim 20, wherein the disorder is acute lymphoblastic leukemia (ALL).

23. The drug described in claim 20, wherein the disorder is non-Hodgkin's lymphoma (NHL).

24. The drug described in claim 20, wherein the disorder is systemic lupus erythematosus (SLE).

25. The drug described in claim 20, wherein the disorder is rheumatoid arthritis (RA).

26. The drug described in claim 20, wherein the disorder is multiple sclerosis (MS).

27. A kit for treating a disease or disorder in an individual in need thereof, comprising the CAR-T cells of claim 6.

28. The kit of claim 27, further comprising at least one additional reagent.

29. The kit of claim 28, wherein at least one additional reagent comprises a chemotherapeutic agent.

30. A polynucleotide encoding the CAR of an antibody of any of claims 1 and 3-5, or a CAR of a CAR-T cell of any of claims 6-18.

31. A vector comprising the polynucleotide of claim 30.

32. A cell containing the vector of claim 31.

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