Anti-PSMA antibodies and CAR-T constructs

Abstract

Anti-PSMA 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 their uses for treating disorders characterized by expression of PSMA.

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 / 153,884, filed February 25, 2021, 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 PSMA. 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 their use to treat disorders characterized by expression of PSMA. [Background technology]

[0003] PSMA PSMA, also known as prostate-specific membrane antigen and glutamate carboxypeptidase II (UniProt Q04609), is a type II transmembrane protein with N-acetylated-α-linked acidic dipeptidase, folate hydrolase, and dipeptidyl peptidase activities. In humans, it is encoded by the FOLH1 gene and consists of a 19-amino acid cytoplasmic domain, a 24-amino acid transmembrane segment, and a 707-amino acid extracellular portion. The protein is enzymatically active as a noncovalent homodimer. PSMA is expressed on prostate epithelial tissue and is upregulated in the neovasculature of prostate cancer and solid tumors. While it is expressed at low levels in healthy tissues such as the brain, kidney, and salivary gland, it is overexpressed in malignant prostate tissue, making it an attractive target for the therapeutic treatment of prostate cancer. Given its high expression in malignant neovasculature, it may also be relevant for the treatment or imaging of solid tumors. Monoclonal antibodies, antibody-drug conjugates, and chimeric antigen receptor T cells targeting PSMA have been described for the treatment of metastatic prostate cancer (Hernandez-Hoyos et al., 2016, PMID: 27406985; DiPippo et al., 2014, PMID: 25327986; Serganova et al., 2016, PMID: 28345023). In addition, radionuclide conjugates specific for PSMA are being investigated for the imaging and treatment of prostate cancer (e.g., Hofman et al., 2018, PMID: 29752180).

[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 hydrophobic interactions between the heavy and light chains.

[0005] However, it is known that the sera of camelids (the suborder Tylopoda, which includes camels, dromedaries, and llamas) contain a major type of antibody composed only of paired heavy chains (heavy chain antibodies or UniAbs™). 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), which are highly homologous to the CH2 and CH3 domains of classical antibodies. These UniAbs™ lack the first domain of the constant region (CH1), which is present in the genome but spliced ​​out during mRNA processing. The absence of the CH1 domain explains the absence of light chains in UniAbs™, as this domain is located at the anchor position of the light chain constant domain. These UniAbs™ have naturally evolved to confer antigen-binding specificity and high affinity through three CDRs derived from conventional antibodies or their fragments (Muyldermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther 5:111-124). Cartilaginous fish, such as sharks, have also evolved a unique type of immunoglobulin, termed IgNAR, which lacks light chain polypeptides and is composed entirely of heavy chains.IgNAR molecules can be engineered 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 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 their antigen-binding activity compared to tetrameric antibodies. Sitia et al. (1990) Cell, 60, 781-790 demonstrated that removal of the CH1 domain from a rearranged mouse μ gene resulted in the production of heavy-chain antibodies lacking light chains in mammalian cell culture. The antibodies produced retained VH binding specificity and effector function.

[0007] Heavy-chain antibodies with high specificity and affinity can be generated against various antigens 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 those of classical F(ab) or Fv fragments (Ghahroudi, MA, et al., FEBS Lett. 414, 521-526 (1997)).

[0008] Mice in which the λ (lambda) light chain (L) locus and / or the λ and κ (kappa) light chain loci are functionally silenced 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 Application Publication No. 20100122358, Nguyen et al., 2003, Immunology; 109(1), 93-101; Brueggemann 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 that produce such antibodies are described in U.S. Patent Nos. 8,883,150 and 9,365,655. CAR-T constructs containing single-domain antibodies as binding (targeting) domains are described, for example, in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378-386. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 7,541,513 [Patent Document 2] U.S. Patent No. 8,367,888 [Patent Document 3] International Publication No. 2006008548 Brochure [Patent Document 4] U.S. Patent Application Publication No. 20100122358 [Patent Document 5] U.S. Patent No. 8,883,150 [Patent Document 6] U.S. Patent No. 9,365,655

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[0011] It seems there is a formatting issue in the original text where the tag has an incorrect "?" in the text part. I've translated it as best as possible with the given text. Please check if this is what you need.Aspects of the invention include antibodies that bind to PSMA, comprising a heavy chain variable region comprising: (a) a CDR1 sequence comprising no more than two substitutions in any one of the amino acid sequences of SEQ ID NO: 1 or 4; and / or (b) a CDR2 sequence comprising no more than two substitutions in any one of the amino acid sequences of SEQ ID NO: 2 or 5; and / or (c) a CDR3 sequence comprising no more than two substitutions in any one of the amino acid sequences of SEQ ID NO: 3 or 6. In some embodiments, the CDR1, CDR2, and CDR3 sequences are in a human framework. In some embodiments, the antibody further comprises a heavy chain constant region sequence that is absent a CH1 sequence.

[0012] In some embodiments, the antibody comprises (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 and 4; and / or (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 2 and 5; and / or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 3 and 6. In some embodiments, the antibody comprises (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 and 4; and (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 2 and 5; and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 3 and 6.

[0013] 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: 4, a CDR2 sequence of SEQ ID NO: 5, and a CDR3 sequence of SEQ ID NO: 6.

[0014] In some embodiments, the antibody comprises a heavy chain variable region having at least 95% sequence identity to any of the sequences in SEQ ID NOs: 7-8. In some embodiments, the antibody comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 7-8. In some embodiments, the antibody comprises the heavy chain variable region sequence of SEQ ID NO: 7. In some embodiments, the antibody comprises the heavy chain variable region sequence of SEQ ID NO: 8.

[0015] Aspects of the invention include antibodies that bind to PSMA, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences in a human VH framework, wherein the CDR sequences have no more than two substitutions in the CDR sequences selected from the group consisting of SEQ ID NOs: 1 to 6. In some embodiments, the antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences in a human VH framework, wherein the CDR sequences are selected from the group consisting of SEQ ID NOs: 1 to 6.

[0016] Embodiments of the invention include antibodies that bind to PSMA, comprising: (a) a heavy chain variable region comprising, in a human VH framework, 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; or (b) a human VH framework, the CDR1 sequence of SEQ ID NO: 4, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence of SEQ ID NO: 6.

[0017] In some embodiments, the antibody is multispecific. In some embodiments, the antibody is bispecific. In some embodiments, the antibody binds to two different PSMA proteins. In some embodiments, the antibody binds to two different epitopes on the same PSMA 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 CAR-T cells comprised of a CAR comprising an extracellular antigen-binding domain that binds to PSMA, the CAR 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; or (b) the CDR1 sequence of SEQ ID NO: 4, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence of SEQ ID NO: 6.

[0019] In some embodiments, the extracellular antigen binding domain that binds PSMA comprises a heavy chain variable region having at least 95% sequence identity to any of the sequences in SEQ ID NOs: 7-8. In some embodiments, the extracellular antigen binding domain that binds PSMA comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 7-8. In some embodiments, the extracellular antigen binding domain that binds PSMA comprises the heavy chain variable region sequence of SEQ ID NO: 7. In some embodiments, the extracellular antigen binding domain that binds PSMA comprises the heavy chain variable region sequence of SEQ ID NO: 8.

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

[0021] Aspects of the invention include methods for treating a disorder characterized by expression of PSMA, comprising administering to a subject having said disorder an antibody as described herein, a CAR-T cell as described herein, or a pharmaceutical composition as described herein. In some embodiments, the disorder is prostate cancer.

[0022] Aspects of the invention include polynucleotides encoding antibodies as described herein or CARs of CAR-T cells as described herein. Aspects of the invention include vectors comprising polynucleotides as described herein. Aspects of the invention include cells comprising vectors as described herein.

[0023] An aspect of the invention is a method of producing an antibody as described herein, the method comprising growing a cell as 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.An aspect of the invention is a method of making an antibody as described herein, the method comprising immunizing a UniRat animal with PSMA and identifying a PSMA-binding heavy chain sequence.

[0024] Aspects of the invention include methods of treatment comprising administering to an individual in need thereof an effective dose of an antibody as described herein, a CAR-T cell as described herein, or a pharmaceutical composition as described herein.

[0025] Aspects of the invention include the use of an antibody as described herein, or a CAR-T cell as described herein, in the preparation of a medicament for treating a disease or disorder in an individual in need thereof.

[0026] Aspects of the invention include the use of an antibody as described herein, a CAR-T cell as described herein, or a pharmaceutical composition as described herein in the treatment of an individual in need thereof.

[0027] Aspects of the invention include kits for treating a disease or disorder in an individual in need thereof, comprising an antibody as described herein, a CAR-T cell as described herein, or a pharmaceutical composition as 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.

[0028] These and further aspects are further described in the remainder of the disclosure, including the Examples. [Brief explanation of the drawings]

[0029] [Figure 1] 1 is a table showing cell binding and ELISA data for the indicated antibody constructs. [Figure 2] Graph showing percent cell lysis as a function of antibody concentration for the indicated antibody constructs. [Figure 3] 1 is a table showing binding affinity data for the indicated antibody constructs. [Figure 4-1]Panel A is a schematic diagram of a CAR-T structure comprising an anti-PSMA extracellular binding domain. Panel B is a graph showing T cell activity of Jurkat cells transfected with an anti-PSMA CAR construct according to one embodiment of the present invention. Panel C is a graph showing T cell activity of Jurkat cells transfected with an anti-PSMA CAR construct according to one embodiment of the present invention. [Figure 4-2] Panel A is a schematic diagram of a CAR-T structure comprising an anti-PSMA extracellular binding domain. Panel B is a graph showing T cell activity of Jurkat cells transfected with an anti-PSMA CAR construct according to one embodiment of the present invention. Panel C is a graph showing T cell activity of Jurkat cells transfected with an anti-PSMA CAR construct according to one embodiment of the present invention. [Figure 5] Panel A is a graph showing tumor progression as a function of days after CAR-T infusion for the indicated antibody constructs. Panel B is a bar graph showing the area under the curve of the graph depicted in Panel A of Figure 5 for the indicated antibody constructs. [Figure 6] Panel A is a graph showing in vivo blood T cell counts as a function of days after CAR-T infusion for the indicated antibody constructs. Panel B is a bar graph showing the area under the curve of the graph depicted in Panel A of Figure 6 for the indicated antibody constructs. DETAILED DESCRIPTION OF THE INVENTION

[0030] 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, for example, “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).

[0031] Where a range of values ​​is given, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the scope of the disclosure. 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 specifically excluded limit in the 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 in the invention.

[0032] 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)).

[0033] 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 skilled 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 known to those skilled in the art have not been described to avoid obscuring the present invention.

[0034] All references cited throughout this disclosure, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0035] I. Definition "Comprising" means that the listed elements are required for the composition / method / kit, but that other elements may be included to form a composition / method / kit, etc. within the scope of the claim.

[0036] "Consisting essentially of" means that the scope of the described composition or process is limited to particular materials or steps that do not materially affect the basic and novel characteristics of the invention.

[0037] "Consisting of" means excluding from a composition, method, or kit any element, step, or ingredient not specified in the claim.

[0038] 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 to 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 specified herein, references to residue numbers in the variable domain of an antibody refer to the numbering of residues according to the Kabat numbering system. Unless otherwise specified herein, references to residue numbers in the constant domain of an antibody refer to the numbering of residues according to the EU numbering system.

[0039] Antibodies, also called immunoglobulins, traditionally comprise at least one heavy chain and one light chain, the amino-terminal domains of which are variable in sequence 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 achieved through variable sequence in the heavy chain alone, and various non-native antibody structures are known and used in the art.

[0040] 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, and this binding may then trigger or alter a cellular or molecular event, such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, such as a TCA, may also block ligand activation of a receptor or 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 proper folding and assembly of the polypeptide chain.

[0041] The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain antibodies, triple chain antibodies, TCAs, single chain Fvs (scFvs), nanobodies, etc., and also antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0042] The term antibody can refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptides, i.e., a polypeptide comprising an antigen-binding site that immunospecifically binds to an antigen or portion thereof of a desired target, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune disease. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule, including engineered subclasses with engineered Fc portions that confer reduced or enhanced effector cell activity. The light chain of the antibody can be a kappa light chain (Vkappa) or a lambda light chain (Vlambda). The immunoglobulin can be derived from any species. In one aspect, the immunoglobulin is predominantly an immunoglobulin of human origin.

[0043] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, 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 may be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be produced by recombinant protein production methods (see, e.g., U.S. Pat. No. 4,816,567).

[0044] 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 are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. This variability is concentrated in three segments called hypervariable regions in both the light- and heavy-chain variable domains. The more highly conserved portions of variable domains are called framework regions (FRs). Naturally occurring heavy- and light-chain variable domains each contain four FRs connected by three hypervariable regions that largely adopt a β-sheet structure and form loops connecting, and occasionally forming 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).

[0045] The term "hypervariable region," as used herein, refers to the amino acid residues of an antibody 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 those 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.

[0046] While exemplary CDR names are provided herein, those skilled in the art will appreciate that several definitions of CDRs are commonly used, including the Kabat definition, which is based on sequence variability and is the most commonly used (see, "Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions." Mol Immunol. 2010;47:694-700). 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 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.

[0047] The terms "heavy chain-only antibody" and "heavy chain antibody" are used interchangeably herein and broadly refer to an antibody or one or more portions of an antibody, e.g., one or more arms of an antibody lacking the light chain of a conventional antibody. The term specifically includes, but is not limited to, homodimeric antibodies that lack the CH1 domain and comprise a VH antigen-binding domain and CH2 and CH3 constant domains; 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-NARs); 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, a heavy chain antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In another embodiment, a heavy chain antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In a further embodiment, a heavy chain antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In a further embodiment, a 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 two heavy chains are disulfide-bonded or otherwise covalently or non-covalently linked to each other. 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 antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 or IgG4 subtype. In one embodiment, the heavy chain antibody is of the IgG4 subtype, and one or more of the CH domains have been modified to alter the effector function of the antibody.In one embodiment, the heavy chain antibody is an IgG1 or IgG4 subtype heavy chain antibody, and one or more of the CH domains have been modified to alter the effector function of the antibody. Modifications of CH domains to alter 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.

[0048] In some embodiments, the heavy chain antibodies herein are used as the binding (targeting) domain of a chimeric antigen receptor (CAR). This definition specifically includes human heavy chain antibodies produced by human immunoglobulin transgenic rats (UniRat™), referred to as UniAbs™. The variable regions (VH) of UniAbs™, referred to as UniDabs™, are versatile building blocks that can be linked to Fc regions or serum albumin for the development of novel therapeutics with improved potency, extended half-life, and multispecificity. Because homodimeric UniAbs™ lack a light chain, and thus a VL domain, antigens are recognized by one single domain: the variable domain of the heavy chain of the heavy chain antibody (VH or VHH).

[0049] As used herein, an "intact antibody chain" comprises a full-length variable region and a full-length constant region (Fc). An intact, "traditional" 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, which in the case of secreted IgG is CH1, hinge, CH2, and CH3. 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.

[0050] 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 some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains corresponding to the different classes of antibodies can be called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. Ig forms include hinge-modified or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; U.S. Patent Application Publication No. 2005 / 0048572; U.S. 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 sequence of their constant domain. Antibodies according to embodiments of the invention may comprise a kappa light chain sequence or a lambda light chain sequence.

[0051] A "functional Fc region" possesses 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 an 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 has been mutated to retain activity, e.g., for extended serum half-life, but does not activate high-affinity Fc receptors or has reduced affinity for Fc receptors.

[0052] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include, for example, native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0053] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region due to 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 one to about ten amino acid substitutions, preferably about one to about five 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 thereto, and more preferably at least about 95% homology thereto.

[0054] The variant Fc sequence may contain three amino acid substitutions in the CH2 region to reduce FcγRI binding at positions 234, 235, and 237 of the EU index (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at positions 330 and 331 of the EU index reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitutions at positions 233-236 in human IgG1 or IgG2 and at positions 327, 330, and 331 in IgG4 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 Fc amino acid sequence of human IgG4 (UniProtKB number P01861) is set forth 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):915-27, the disclosures of which are incorporated herein by reference in their entireties.

[0055] Other Fc variants are possible, including, but not limited to, those in which regions capable of forming disulfide bonds have been deleted, or in which specific amino acid residues have been deleted or a methionine residue has been added at the N-terminus of the native Fc. 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.

[0056] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector functions by substituting (mutating), deleting, or adding amino acid residues to confer complement binding or Fc receptor binding. For example, deletions can be made in complement binding sites, such as, but not limited to, the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in WO 97 / 34631 and WO 96 / 32478. Additionally, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0057] 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, an 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 can be utilized in any suitable manner to place a "knob" in the first heavy chain constant region of the first monomer of the antibody dimer and a "hole" in the second heavy chain constant region of the second monomer of the antibody dimer, thereby facilitating proper pairing (heterodimerization) of the desired pair of antibody heavy chain polypeptide subunits.

[0058] In some embodiments, the antibody comprises a heavy chain polypeptide subunit (knob) comprising a variant human IgG4 Fc region comprising a S228P mutation, a F234A mutation, a L235A mutation, and a T366W mutation. In some embodiments, the antibody comprises a heavy chain polypeptide subunit (hole) comprising a variant human IgG4 Fc region comprising a S228P mutation, a F234A mutation, a L235A mutation, a T366S mutation, a L368A mutation, and a Y407V mutation.

[0059] 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 antibody purification or by recombinant engineering of a nucleic acid encoding the antibody. Thus, antibodies with an Fc region according to the present invention can include antibodies with or without K447.

[0060] Embodiments of the present invention include antibodies comprising heavy chain-only variable regions in a monovalent or bivalent structure. As used herein, the term "monovalent structure" when used with respect to a heavy chain-only variable region domain means that only one heavy chain-only variable region domain is present, with a single binding site. In contrast, the term "bivalent structure" when used with respect to a heavy chain-only variable region domain means that two heavy chain-only variable region domains are present (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 in a "bivalent structure" is understood to contain two identical heavy chain-only variable region domains connected by a linker sequence, each of which binds to the same target antigen.

[0061] Aspects of the present invention include antibodies with multispecific structures, including but not limited to bispecific, trispecific, etc. A wide variety of methods and protein structures are known and have been used in bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc.

[0062] Various methods have been developed for generating 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, which has 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: 40) (n=1) and GGGGSGGGGS (SEQ ID NO: 41) (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.

[0063] 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 chain and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises an Fc portion composed of CH2, and / or CH3, and / or CH4 domains, absent a CH1 domain, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind to an epitope of a second antigen or a different epitope of the first antigen, such binding domains comprising, consisting essentially of, or consisting of a heavy chain antibody derived from or having sequence identity to the variable region of an antibody heavy or light chain. Portions of such variable regions include 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.

[0064] The TCA binding compound utilizes a "heavy chain only antibody" or "heavy chain antibody" or "heavy chain polypeptide," as used herein, refers to a single-chain antibody comprising heavy chain constant regions CH2 and / or CH3 and / or CH4, but not a CH1 domain. In one embodiment, a heavy chain antibody is comprised of an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In another embodiment, a heavy chain antibody is comprised of an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In a further embodiment, a heavy chain antibody is comprised of an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In 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 bound to each other, and may optionally include an asymmetric interface (e.g., a knobs-in-holes (KiH) interface) between one or more of the CH domains to promote proper pairing between the polypeptide chains. Heavy-chain antibodies may belong to the IgG subclass, although antibodies belonging to other subclasses, such as the IgM, IgA, IgD, and IgE subclasses, are also included herein. In certain embodiments, the heavy-chain antibody is 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 entireties.

[0065] Heavy chain antibodies account for approximately one-quarter of the IgG antibodies produced by camelids, such as camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains but lack light chains. As a result, the variable antigen-binding portion, called a VHH domain, represents the smallest naturally occurring intact antigen-binding site and is only approximately 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 those of classical F(ab) or Fv fragments (Ghahroudi, MA, et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies, termed VNAR. (Nuttall et al.Eur.J.Biochem.270,3543-3554(2003);Nuttall et al.Function and Bioinformatics 55,187-197(2004);Dooley et al.,Molecular Immunology 40,25-33(2003)).

[0066] As used herein, the term "PSMA" refers to a type II transmembrane protein with N-acetyl-α-linked acidic dipeptidase activity, folate hydrolase activity, and dipeptidyl peptidase activity. The term "PSMA" includes PSMA proteins from all human and non-human animal species, and specifically includes human PSMA as well as PSMA from non-human mammals.

[0067] As used herein, the term "human PSMA" includes any variant, isoform, and species homolog of human PSMA (UniProt Q04609), regardless of its source or method of preparation. Thus, "human PSMA" includes human PSMA naturally expressed by cells and PSMA expressed in cells transfected with the human PSMA gene.

[0068] The terms "anti-PSMA heavy chain-only antibody," "PSMA heavy chain-only antibody," "anti-PSMA heavy chain antibody," and "PSMA heavy chain antibody" are used interchangeably herein and refer to heavy chain antibodies as defined above that immunospecifically bind to PSMA, including human PSMA as defined above. This definition includes, but is not limited to, human heavy chain antibodies produced by transgenic animals, such as transgenic rats or transgenic mice expressing human immunoglobulins, including UniRats™ that produce human anti-PSMA UniAb™ as defined above.

[0069] "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 those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of one in 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 necessary 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.

[0070] 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 for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified (1) to greater than 95% by weight, and most preferably greater than 99% by weight, of the antibody as determined by the Lowry assay, (2) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0071] The antibodies of the present invention include multispecific antibodies. Multispecific antibodies have two or more binding specificities. The term "multispecific" specifically includes "bispecific" and "trispecific," as well as higher-order independent specific binding affinities, e.g., higher-order polyepitopic specificities, 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 in the broadest sense herein to encompass all antibodies with two or more binding specificities. The multispecific anti-heavy chain anti-PSMA antibodies of the present invention specifically include antibodies that immunospecifically bind to two or more non-overlapping epitopes on a PSMA protein, such as human PSMA (i.e., bivalent and biparatopic). Multispecific heavy chain anti-PSMA antibodies of the invention also specifically include antibodies that immunospecifically bind to an epitope on a PSMA protein, such as human PSMA, and to an epitope on a different protein, such as a CD3 protein, e.g., human CD3 (i.e., bispecific and biparatopic). Multispecific heavy chain anti-PSMA antibodies of the invention also specifically include antibodies that immunospecifically bind to two or more non-overlapping or partially overlapping epitopes on a PSMA protein, such as a human PSMA protein, and to epitopes on different proteins, such as a CD3 protein, e.g., human CD3 protein (i.e., trivalent and biparatopic).

[0072] The antibodies of the present invention include monospecific antibodies having one binding specificity. Monospecific antibodies specifically include antibodies comprising two or more binding units with the same binding specificity, as well as antibodies comprising a single binding specificity. The terms "monospecific antibody," "monospecific heavy chain-only antibody," "monospecific heavy chain antibody," and "monospecific UniAb™" are used in the broadest sense herein to encompass all antibodies with one binding specificity. The monospecific heavy chain anti-PSMA antibodies of the present invention specifically include antibodies that immunospecifically bind to one epitope on a PSMA protein, such as human PSMA protein (monovalent and monospecific). The monospecific heavy chain anti-PSMA antibodies of the present invention also specifically include antibodies with two or more binding units that immunospecifically bind to an epitope on a PSMA protein, such as human PSMA (e.g., multivalent antibodies). For example, a monospecific antibody according to embodiments of the present invention may comprise a heavy chain variable region comprising two antigen-binding domains, each of which binds to the same epitope on a PSMA protein (i.e., bivalent and monospecific).

[0073] 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.

[0074] "Epitope mapping" is the process of identifying the binding site, or epitope, of an antibody on a target antigen. Antibody epitopes can be linear or conformational. Linear epitopes are formed by a contiguous sequence of amino acids in a protein. Conformational epitopes are formed by amino acids that are not contiguous in the protein sequence but come together when the protein folds into a three-dimensional structure.

[0075] "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. As described above, the present invention specifically includes anti-PSMA heavy chain antibodies with polyepitopic specificity, i.e., anti-PSMA heavy chain antibodies that bind to one or more non-overlapping epitopes on a PSMA protein, such as human PSMA, and anti-PSMA heavy chain antibodies that bind to one or more epitopes on a PSMA protein and to 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. Pairs of antibodies or antigen-binding regions that target the same antigen on a multispecific antibody that recognize non-overlapping epitopes can simultaneously bind to the antigen without competing for binding to the antigen.

[0076] An antibody binds to "essentially the same epitope" as a reference antibody if the two antibodies recognize the same or sterically overlapping epitopes. The rapid 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, the antigen is immobilized on a 96-well plate and a radioactive or enzyme label is used to measure the ability of an unlabeled antibody to block the binding of the labeled antibody.

[0077] As used herein, the term "valency" refers to the specific number of binding sites on an antibody molecule.

[0078] A "monovalent" antibody has one binding site. A monovalent antibody is therefore also monospecific.

[0079] 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. Bispecific antibodies according to the invention are therefore at least bivalent and may be trivalent, tetravalent, or otherwise multivalent. Bivalent antibodies according to embodiments of the invention may have two binding sites for the same epitope (i.e., bivalent, monoparatopic), or two binding sites for two different epitopes (i.e., bivalent, biparatopic).

[0080] A wide variety of methods and protein structures are known and have been used to prepare bispecific monoclonal antibodies (BsMABs), trispecific antibodies, and the like.

[0081] 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 chain and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of an Fc portion composed of CH2, and / or CH3, and / or CH4 domains, absent the CH1 domain, and an antigen-binding domain that binds to an epitope of a second antigen or a different epitope of the first antigen, such binding domain being derived from or sharing sequence identity with the variable region of an antibody heavy or light chain. Portions of such variable regions include 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 Lor V L J L The TCA protein may be encoded by a gene segment. The TCA protein utilizes a heavy chain antibody as defined above.

[0082] The term "chimeric antigen receptor" or "CAR" is used herein in the broadest sense to refer to an engineered receptor in which a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) is grafted onto a transmembrane domain and an intracellular signaling domain. Typically, this receptor is used to transfer 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 genetically engineered to generate artificial T cell receptors for use in immunotherapy. In one embodiment, "CAR-T cells" refers to therapeutic T cells expressing a transgene encoding one or more chimeric antigen receptors minimally consisting of an extracellular domain, a transmembrane domain, and at least one cytoplasmic domain.

[0083] 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, particularly UniAbs™ produced by UniRats™ as defined above.

[0084] "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 modified to produce chimeric antibodies.

[0085] 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 the immune response. Some effector cells express specific Fc receptors to perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, can induce antibody-dependent cellular cytotoxicity (ADCC). For example, FcR-expressing monocytes and macrophages are involved in specifically killing target cells and presenting antigens to other components of the immune system or binding to cells that present antigens. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0086] "Human effector cells" are leukocytes that express a receptor, such as a T cell receptor or FcR, and perform effector function. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with NK cells being preferred. Effector cells can be isolated from their native source, e.g., blood or PBMCs, as described herein.

[0087] 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 (such as B cells and T cells, including cytotoxic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0088] Antibody "effector functions" refer to biological activities attributable to the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and down-regulation of cell surface receptors (e.g., B cell receptor, BCR).

[0089] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3, page 464, of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, 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).

[0090] "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), may be performed.

[0091] "Binding affinity" refers to the overall 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, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the 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 more quickly and tend to remain bound.

[0092] As used herein, "Kd" or "Kd value" refers to the dissociation constant measured by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetic mode. For example, an anti-mouse Fc sensor is loaded with a mouse Fc fusion antigen, then immersed in an antibody-containing well, and the concentration-dependent binding rate (k) is measured. In the final step, the sensor is immersed in a well containing only buffer, and the antibody dissociation rate (k) is measured. Kd is the ratio of k / k. (For further details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009.)

[0093] The terms "treatment," "treating," and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic, in that it completely or partially prevents a disease or its symptoms, and / or therapeutic, in that it partially or completely cures a disease and / or the deleterious effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the disease from occurring in a subject who may be predisposed to, but has not yet been diagnosed with, the disease; (b) arresting the disease, i.e., preventing its development; or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents can be administered before, during, or after the onset of disease or injury. Treatment of ongoing disease, in which treatment stabilizes or alleviates undesirable clinical symptoms in the patient, is of particular interest. Such treatment is desirably administered before complete loss of function in the affected tissue. Treatment of a subject may be administered during, and optionally after, the symptomatic stage of the disease.

[0094] By "therapeutically effective amount" is intended the amount of active agent required to confer a therapeutic effect on a subject. For example, a "therapeutically effective amount" is an amount that induces, ameliorates, or causes an improvement in pathological symptoms, disease progression, or physiological condition associated with a disease, or improves resistance to a disorder.

[0095] As used herein, the term "prostate cancer" refers to a malignant tumor of glandular origin in the prostate gland.

[0096] The term "characterized by expression of PSMA" broadly refers to any disease or disorder in which PSMA expression is associated with or contributes to one or more pathological processes characteristic of the disease or disorder, including, but not limited to, prostate cancer.

[0097] 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 diseases, individuals with pathogen infections, and the like. The subject can be a human, but also includes other mammals, particularly mammals useful as experimental models for human disease, e.g., mice, rats, and the like.

[0098] The term "pharmaceutical formulation" refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can reasonably be administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0099] 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.

[0100] A "stable" formulation is one in which the protein therein essentially retains its physical stability, and / or chemical stability, and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pub. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90) (1993). Stability can be measured for a selected period of time at a selected temperature. Stability can be qualitatively and / or quantitatively assessed in a variety of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; analysis of amino- or carboxy-terminal sequences; mass spectrometry; SDS-PAGE analysis comparing reduced and intact antibodies; peptide map (e.g., trypsin or LYS-C) analysis; assessment of antibody biological activity or antigen-binding function, etc. Instability can involve any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, differential glycosylation, etc.

[0101] II. Detailed Description Anti-PSMA antibody The present invention provides a family of closely related antibodies that bind to human PSMA. The antibodies in this family are composed of 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 listed in Table 2, and the heavy chain variable region (VH) sequences of SEQ ID NOs: 5 and 6 listed in Table 3. This family of antibodies offers many benefits that contribute to their usefulness as clinical therapeutics. The antibodies include members with a range of binding affinities, allowing for the selection of specific sequences with desired binding affinities.

[0102] [Table 1]

[0103] [Table 2]

[0104] [Table 3]

[0105] Suitable antibodies may be selected from those provided herein for development and therapeutic or other uses (e.g., including but not limited to, use as part of a bispecific antibody or CAR-T structure as shown in FIG. 1).

[0106] Affinity measurements for candidate proteins can be performed using methods known in the art, such as Biacore measurements. Members of an antibody family are approximately 10 -6 ~about 10 -11 Affinity for PSMA with a Kd of around, for example, but not limited to, about 10 -6 ~about 10 -10 Around 10 -6 ~about 10 -9 Around 10 -6 ~about 10 -8 Around 10 -8 ~about 10 -11Around 10 -8 ~about 10 -10 Around 10 -8 ~about 10 -9 Around 10 -9 ~about 10 -11 Around 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 PSMA biological activity, including in vitro assays, preclinical models, and clinical trials, and evaluation of potential toxicity.

[0107] Members of the antibody family herein are not cross-reactive with the PSMA protein of Cynomolgus macaques, but can be modified, if desired, to confer cross-reactivity with the PSMA protein of Cynomolgus macaques, or PSMA of any other animal species.

[0108] The family of PSMA-specific antibodies herein includes a VH domain comprising CDR1, CDR2, and CDR3 sequences in a human VH framework. The CDR sequences may be located around amino acid residues 26-33, 51-58, and 97-116 for CDR1, CDR2, and CDR3, respectively, of the exemplary variable region sequences provided in SEQ ID NOS: 7-8. 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 the order of the sequences will generally remain the same.

[0109] In certain embodiments, the anti-PSMA antibody comprises the CDR1 sequence of any one of SEQ ID NOs: 1 or 4. In certain embodiments, the CDR1 sequence comprises SEQ ID NO: 1. In certain embodiments, the CDR1 sequence comprises SEQ ID NO: 4.

[0110] In certain embodiments, the anti-PSMA antibody comprises the CDR2 sequence of any one of SEQ ID NOs: 2 or 5. In certain embodiments, the CDR2 sequence comprises SEQ ID NO: 2. In certain embodiments, the CDR2 sequence comprises SEQ ID NO: 5.

[0111] In certain embodiments, the anti-PSMA antibody comprises the CDR3 sequence of any one of SEQ ID NOs: 3 or 6. In certain embodiments, the CDR3 sequence comprises SEQ ID NO: 3. In certain embodiments, the CDR3 sequence comprises SEQ ID NO: 6.

[0112] In a further embodiment, the anti-PSMA 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.

[0113] In a further embodiment, the anti-PSMA antibody comprises the CDR1 sequence of SEQ ID NO:4, the CDR2 sequence of SEQ ID NO:5, and the CDR3 sequence of SEQ ID NO:6.

[0114] In a further embodiment, the anti-PSMA antibody comprises any of the amino acid sequences of the heavy chain variable region of SEQ ID NOs: 7-8 (Table 3).

[0115] In still further embodiments, the anti-PSMA antibody comprises the heavy chain variable region sequence of SEQ ID NO:7.

[0116] In still further embodiments, the anti-PSMA antibody comprises the heavy chain variable region sequence of SEQ ID NO:8.

[0117] In some embodiments, the CDR sequences of the anti-PSMA antibodies of the invention contain one or two amino acid substitutions compared to the CDR1, CDR2 and / or CDR3 sequence of any one of SEQ ID NOs: 1-6 (Table 1; Table 2), or the set of CDR1, CDR2 and CDR3 sequences.

[0118] In some embodiments, the anti-PSMA antibody preferably comprises a heavy chain variable domain (VH) that binds PSMA, 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 the CDR3 sequence of any one of the antibodies whose CDR3 sequences are provided in Table 1 or 2.

[0119] In some embodiments, the anti-PSMA antibody preferably comprises a heavy chain variable domain (VH) that binds PSMA, wherein the full set of CDR1, 2, and 3 (combined) has eighty-five percent (85%) or greater sequence identity at the amino acid level to the CDR1, 2, and 3 (combined) of an antibody whose CDR sequences are provided in Table 1 or 2.

[0120] In some embodiments, the anti-PSMA antibody comprises a heavy chain variable region sequence having at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any of the heavy chain variable region sequences of SEQ ID NOs: 7-8 (shown in Table 3), and binds to PSMA.

[0121] In some embodiments, bispecific or multispecific antibodies are provided, which may have any of the structures discussed herein, including, but not limited to, bispecific three-chain antibody-like molecules (TCAs). In some embodiments, a multispecific antibody may comprise at least one heavy chain variable region with binding specificity for PSMA and at least one heavy chain variable region with binding specificity for a protein other than PSMA. In some embodiments, a multispecific antibody may comprise at least one heavy chain variable region that binds to PSMA and at least one heavy chain variable region that binds to a protein other than PSMA. In some embodiments, a multispecific antibody may comprise a heavy chain variable region comprising at least two antigen-binding domains, each of which binds to PSMA. In some embodiments, a multispecific antibody may comprise a heavy chain / light chain pair that binds to a first antigen (e.g., CD3) and a heavy chain derived from a heavy-chain antibody. In certain embodiments, the heavy chain derived from a heavy-chain antibody comprises an Fc portion composed of CH2 and / or CH3 and / or CH4 domains 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., a 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 PSMA.

[0122] 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: 9, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 11, in a human VH framework. In some embodiments, the CD3-binding VH domain comprises the CDR1 sequence of SEQ ID NO: 12, the CDR2 sequence of SEQ ID NO: 13, and the CDR3 sequence of SEQ ID NO: 14, in a human VH framework. In some embodiments, the fixed light chain comprises the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 16, and the CDR3 sequence of SEQ ID NO: 17, 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: 18. In some embodiments, the CD3-binding VH domain comprises the heavy chain variable region sequence of SEQ ID NO: 19. 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: 18 or 19. In some embodiments, the fixed light chain comprises a light chain variable region sequence of SEQ ID NO: 20. 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 heavy chain variable region sequence of SEQ ID NO: 20.

[0123] Multispecific antibodies comprising the CD3-binding VH domain and light chain variable domain described above 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 described herein with binding affinity for PSMA can be combined with the CD3-binding domain and fixed light chain domain described herein (see, e.g., Tables 4 and 5), and additional sequences such as those provided in Tables 6 and 7, to generate multispecific antibodies with binding affinity for one or more PSMA epitopes as well as CD3.

[0124] [Table 4]

[0125] [Table 5]

[0126] [Table 6]

[0127] [Table 7]

[0128] [Table 8]

[0129] [Table 9]

[0130] [Table 10]

[0131] In some embodiments, bispecific or multispecific antibodies are provided that can have any of the structures discussed herein, including, but not limited to, bispecific three-chain antibody-like molecules (TCAs). In some embodiments, the bispecific antibody can comprise at least one heavy chain variable region that binds to PSMA and at least one heavy chain variable region that binds to a protein other than PSMA. In some embodiments, the bispecific antibody can comprise a heavy / light chain pair that binds to a first antigen, a heavy chain derived from a heavy-chain antibody that comprises an Fc portion composed of CH2 and / or CH3 and / or CH4 domains, with no CH1 domain present, 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, the bispecific antibody comprises a heavy / 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 PSMA.

[0132] In some embodiments where the antibody of the invention is a bispecific antibody, one arm of the antibody (one binding moiety or one binding unit) may be specific for human PSMA, while the other arm may be specific for a target cell, a tumor-associated antigen, a targeted antigen such as an integrin, a pathogen antigen, a checkpoint protein, etc. Target cells specifically include cancer cells, including, but not limited to, cells associated with hematological malignancies characterized by expression of PSMA, as well as pathogenic B cells associated with autoimmune diseases characterized by expression of PSMA. In some embodiments, one arm of the antibody (one binding moiety or one binding unit) is specific for human PSMA, while the other arm is specific for CD3.

[0133] In some embodiments, the antibody comprises an anti-CD3 light chain polypeptide comprising the sequence of SEQ ID NO: 32, an anti-CD3 heavy chain polypeptide comprising the sequence of SEQ ID NO: 18 or 19, and an anti-PSMA heavy chain polypeptide comprising the sequence of SEQ ID NO: 7 or 8, in a monovalent or bivalent configuration, linked to the sequence of any one of SEQ ID NOs: 30 or 31. These sequences can be combined in various ways to create bispecific antibodies of a desired IgG subclass, e.g., IgG1, IgG4, silenced IgG1, or silenced IgG4. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide consisting of SEQ ID NO: 32, a second polypeptide consisting of SEQ ID NO: 33, and a third polypeptide consisting of SEQ ID NO: 34, 35, 36, 37, 38, or 39.

[0134] 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 PSMA and CD3 (anti-PSMA x anti-CD3 antibodies). Such antibodies induce potent T cell-mediated killing of PSMA-expressing cells.

[0135] Preparation of anti-PSMA antibodies 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 produced in UniRat™. UniRat™ has silenced endogenous immunoglobulin genes and uses a human immunoglobulin heavy chain transgene to express a diverse, naturally optimized, fully human repertoire of HCAbs. While various techniques can be used to knock out or silence endogenous immunoglobulin loci in rats, 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. IgH and IgL knockout (KO) strains can be generated by microinjecting ZNF constructs into oocytes. For more details, see, for example, Geurts et al., 2009, Science 325:433. The characterization of Ig heavy chain knockout rats has been reported by Menoret et al., 2010, Eur. J. Immunol. 40:2932-2941. An advantage of ZNF technology is that it can also provide target sites for homologous integration by non-homologous end joining, which silences genes or loci by deletions of up to several kb (Cui et al., 2011, Nat Biotechnol 29:64-67). Human heavy chain antibodies produced in UniRat™ are called UniAbs™ and can bind to epitopes that cannot be targeted by conventional antibodies. Due to their high specificity, affinity, and small size, UniAbs™ are ideal for monospecific and multispecific applications.

[0136] In addition to UniAbs™, camelid heavy chain antibodies lacking VHH framework and mutations, as well as functional VH regions thereof, are specifically included herein. Such heavy chain antibodies can be produced, for example, in transgenic rats or mice containing a fully human heavy chain-only locus, as described, for example, in WO 2006 / 008548, although other transgenic mammals, such as rabbits, guinea pigs, and rats, can also be used, with rats and mice being preferred. Heavy chain antibodies (including their VHH or VH functional fragments) can also be produced by recombinant DNA technology, for example, by expressing encoding nucleic acids in suitable eukaryotic or prokaryotic hosts, including mammalian cells (e.g., CHO cells), E. coli, or yeast.

[0137] Heavy chain antibody domains combine the advantages of antibodies and small molecule drugs, can be monovalent or multivalent, have low toxicity, and are cost-effective to produce. These domains are easily administered, including orally or topically, due to their small size, and are characterized by high stability, including gastrointestinal stability, and their half-life can be tailored to the desired use or indication. In addition, VH and VHH domains of HCAbs can be produced cost-effectively.

[0138] In certain embodiments, heavy chain antibodies of the present invention, including UniAbs™, have a native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Kabat numbering system) substituted with another amino acid residue, thereby disrupting a surface-exposed hydrophobic patch containing or associated with the native amino acid residue at that position. Such a hydrophobic patch is normally buried at the interface with the antibody light chain constant region, but in HCAbs, it is at least partially surface-exposed, potentially contributing to undesirable aggregation and light chain binding of the HCAb. The substituted amino acid residue is preferably charged, preferably positively charged, such as lysine (Lys, K), arginine (Arg, R), or histidine (His, H), preferably arginine (R). In a preferred embodiment, heavy chain antibodies derived from transgenic animals contain a Trp-to-Arg mutation at position 101. The resulting HCAbs preferably have high antigen-binding affinity and solubility under physiological conditions in the absence of aggregation.

[0139] As part of the present invention, a human IgG anti-PSMA heavy chain antibody (UniAb™) with a unique sequence derived from UniRat™ animals was confirmed to bind to human PSMA in ELISA protein and cell binding assays. The identified heavy chain variable region (VH) sequences were positive for human PSMA protein binding and / or positive for binding to PSMA+ cells, and all negative for binding to cells that do not express PSMA.

[0140] Heavy chain antibodies that bind to non-overlapping epitopes on the PSMA protein, e.g., 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 a known antibody that binds to a target antigen and an antibody of interest can be utilized. Using this approach, a set of antibodies can be separated 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 bound by the reference antibody. Often, one antibody is immobilized and allowed to bind the antigen, and the ability of a second, labeled (e.g., biotinylated) antibody to bind to the captured antigen is tested in an ELISA assay. This assay can also be performed using surface plasmon resonance (SPR) platforms including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences) and MX96 SPR Imager (Ibis technologies BV), as well as on biolayer interference platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). For further details, see the Examples herein.

[0141] 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 measured 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.

[0142] Pharmaceutical Compositions, Methods of Use and Treatment Another aspect of the present invention is to provide pharmaceutical compositions comprising one or more antibodies of the present invention in admixture with a suitable pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier includes, but is not limited to, an adjuvant, a solid carrier, water, a buffer, or other carrier used in the art to carry therapeutic ingredients, or a combination thereof.

[0143] In one embodiment, the pharmaceutical composition comprises a heavy chain antibody (e.g., UniAb™) that binds to PSMA. In another embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb™) that has binding specificities for two or more non-overlapping epitopes on the PSMA 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 PSMA and has binding specificity for a binding target on an effector cell (e.g., a binding target on a T cell, such as the CD3 protein on a T cell). In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific and TCA) heavy chain antibody (e.g., UniAb™) that binds to PSMA and binds to a binding target on an effector cell (e.g., a binding target on a T cell, such as the CD3 protein on a T cell).

[0144] Pharmaceutical compositions of antibodies used according to the present invention are prepared by mixing proteins with the desired purity, for example, in the form of a lyophilized preparation or aqueous solution, with optional pharmaceutically acceptable carriers, excipients, or stabilizers for storage (see, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (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, Proteins such as gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0145] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., dosage form for a single administration). The formulation depends on the selected route of administration. The antibodies herein may be administered by intravenous injection or infusion, or may be administered subcutaneously. For injection administration, 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 reconstitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0146] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used for heavy chain antibodies, including the UniAbs™ of the present invention. Subcutaneous antibody formulations are described, for example, in U.S. Patent Application Publication Nos. 20160355591 and 20160166689.

[0147] How to use The anti-PSMA antibodies and pharmaceutical compositions described herein can be used to treat diseases and conditions characterized by expression of PSMA, including, but not limited to, the conditions and diseases further described herein.

[0148] PSMA is a type II transmembrane protein expressed on prostate epithelial tissue and upregulated in the neovasculature of prostate cancer and solid tumors. It is expressed at low levels in healthy tissues such as the brain, kidney, and salivary gland, but is overexpressed in malignant prostate tissue, making it an attractive target for therapeutic treatment of prostate cancer. Given its high expression in malignant neovasculature, it may also be relevant for the treatment or imaging of solid tumors. Monoclonal antibodies, antibody-drug conjugates, and chimeric antigen receptor T cells targeting PSMA have been described for the treatment of metastatic prostate cancer (Hernandez-Hoyos et al., 2016, PMID: 27406985; DiPippo et al., 2014, PMID: 25327986; Serganova et al., 2016, PMID: 28345023). Additionally, PSMA-specific radionuclide conjugates are being investigated for the imaging and treatment of prostate cancer (e.g., Hofman et al., 2018 PMID;29752180).

[0149] In one aspect, the anti-PSMA antibodies (e.g., UniAbs™) and pharmaceutical compositions herein can be used to treat disorders characterized by expression of PSMA, including, but not limited to, prostate cancer and solid tumors.

[0150] In one embodiment, the antibodies herein may be in the form of a heavy chain only anti-PSMA antibody-CAR construct, i.e., a heavy chain only anti-PSMA antibody-CAR transduced T cell construct.

[0151] The effective dose of the composition of the present invention for treating a 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, the administration of other drugs, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, such as companion animals such as dogs, cats, and horses, and laboratory mammals such as rabbits, mice, and rats, can also be treated. Treatment dosages can be titrated to optimize safety and efficacy.

[0152] Dosage levels can be easily determined by ordinary skilled clinicians and can be changed as needed, for example, to modify the subject's response to treatment. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the host treated and the specific mode of administration. Unit dosage forms generally contain about 1 mg to about 500 mg of active ingredient.

[0153] In some embodiments, therapeutic dosages of agents can range from about 0.0001 to 100 mg / kg, more typically 0.01 to 5 mg / kg, of host body weight. For example, dosages can be 1 mg / kg or 10 mg / kg body weight, or within the range of 1 to 10 mg / kg. Exemplary treatment regimens involve administration once every two weeks, once a month, or once every three to six months. Therapeutic agents of the present invention are typically administered multiple times. The interval between single doses can be weekly, monthly, or yearly. Intervals can also be irregular, as indicated by measuring the patient's blood levels of the therapeutic agent. Alternatively, therapeutic agents of the present invention can be administered as sustained-release formulations, requiring less frequent administration. The dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0154] Typically, the compositions are prepared as injectables, either as solutions or suspensions; 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 formulations can also be emulsified or encapsulated in liposomes or microparticles (e.g., polylactide, polyglycolide, or copolymers) to enhance adjuvant effect, as discussed above. Langer, Science 249:1527, 1990, and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of the present invention can be administered in the form of depot injections or implants, which can be formulated in a manner that allows sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally sterile, substantially isotonic, and formulated in full compliance with all U.S. Food and Drug Administration Good Manufacturing Practice (GMP) regulations.

[0155] Toxicity of the antibodies and antibody constructs described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is non-toxic for use in humans. The dosage of the antibodies described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. Dosage can vary within this range depending on the dosage form 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.

[0156] Compositions for administration generally contain an antibody or other ablative agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the active agent in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0157] Also within the scope of the present invention is a kit comprising the active agent of the present invention and its formulation, and instructions for use. The kit may further contain at least one additional agent, such as a chemotherapeutic agent. The kit typically includes a label indicating the intended use of the contents of the kit. As used herein, the term "label" includes any written or recorded material provided on or with the kit, or separately attached to the kit.

[0158] 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 thereto without departing from the spirit or scope of the invention. [Example]

[0159] Example 1: Analysis of binding to PSMA Binding to PSMA-positive cells was assessed by flow cytometry (Guava easyCyte 8HT, EMD Millipore) using the LNCaP cell line (ATCC:CRL-1740). 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 stained with goat F(ab')2 anti-human IgG conjugated to R-phycoerythrin (PE) (Southern Biotech, catalog no. 2042-09) to detect cell-bound antibodies. After 20 minutes of incubation at 4°C, cells were washed twice with flow cytometry buffer and the mean fluorescence intensity (MFI) was measured by flow cytometry. The MFI of cells stained with secondary antibody alone was used to measure background signal, and the binding of each antibody was converted to fold over background. The table in Figure 1 summarizes the binding data for LNCaP cells.

[0160] The two indicated antibodies were tested for binding in a standard ELISA assay against human PSMA. Human PSMA protein was used at a concentration of 2 μg / mL and captured with 50 ng / mL of UniAbs. Antibody binding was detected with a goat anti-human IgG HRP-conjugated antibody (ThermoFisher 31413). All antibodies were diluted in 1x PBS containing 0.05% Tween-20 and 1% dry milk powder. The table in Figure 1 summarizes the results of the ELISA binding analysis.

[0161] Example 2: Bispecific antibody-mediated oncolysis of 22Rv1 human tumor cells by redirecting human T cells Incubation of the PSMA-positive tumor cell line 22Rv1 (ATCC:CRL-2505) with increasing amounts of bispecific antibodies in the presence of activated human T cells resulted in specific tumor cell lysis. Briefly, PBMCs were thawed, washed, and activated for 3 days on plates coated with 1 μg / mL OKT3 and 1 ng / mL IL-2. Pan T cells were then isolated from the activated PBMCs (Miltenyi Biotec, Cat. No. 130-096-535), and 50,000 pan T cells were added together with the bispecific antibodies to 5,000 target cells loaded with 7.5 μM calcein-AM (ThermoFisher, Cat. No. C1430). After 2 hours of incubation, calcein release was measured to measure cytotoxicity. The results are shown in Figure 2. The bispecific antibodies were composed of an anti-CD3 binding arm paired with the indicated anti-PSMA VH binding domain (clone ID: 325795 or 325972). A negative control antibody containing a VH binding domain that does not bind PSMA did not show specific lysis. PSMA-negative cells did not show specific lysis (data not shown).

[0162] Example 3: Analysis of binding kinetics using Biolayer Interferometry (BLI) The antibody binding kinetics of clone ID numbers 325972 and 325795 were analyzed using an Octet QK-384 (ForteBio) instrument. Briefly, an anti-Penta HIS capture (HIS1K) sensor was used to immobilize the antigen, recombinant human PSMA (R&D Systems catalog number 4234-ZN), for 120 seconds. After a baseline reading, the sensor was immersed in a solution containing the test antibody (clone ID number 325972 or 325795), and the association and dissociation rates were measured. Data analysis was performed using Octet Data Analysis HT v11.0 (ForteBio), and the data are summarized in the table shown in Figure 3.

[0163] Example 4: CAR-T-mediated T cell activation by human tumor cells CAR-T cell activity was measured by transfecting Jurkat T lymphocyte cells with an anti-PSMA CAR and a 6x NFAT TK nanoluciferase reporter. Transfected Jurkat cells were co-cultured for 24 hours with PSMA+ PC3 cells stably transfected to express human PSMA, LNCaP, and 22Rv1, or with PSMA-negative DU145 cells. Luciferase activity was measured using Promega's Nano-Glo Luciferase Assay System (Cat. No. N1110), and data were normalized to co-cultures containing the CAR-transfected Jurkat and PSMA-negative DU145 cell lines. Statistical significance was determined using an unpaired, two-tailed t-test. The results are shown in Figure 4, panels B and C.

[0164] Panel A of Figure 4 is a schematic diagram of a CAR-T construct comprising an anti-PSMA extracellular binding domain, including an antibody sequence according to an embodiment of the invention. Panel B shows T cell activity of Jurkat transfected with an NFAT luciferase reporter of T cell signaling and the anti-PSMA 325972 CAR, co-cultured with prostate tumor cell lines PC3-PSMA (**p=0.0016), LNCaP (**p=0.0011), and 22Rv1 (****p=0.000077). Panel C shows T cell activity of Jurkat transfected with an NFAT luciferase reporter of T cell signaling and the anti-PSMA 325795 CAR, co-cultured with prostate tumor cell lines PC3-PSMA (***p=0.0004), LNCaP (****p=0.00004), and 22Rv1 (***p=0.00014). These results indicate that T cell activation was specific to PSMA target binding, as coculture with the PSMA-negative DU145 cell line or incubation of transfected Jurkats alone did not result in a discernible luciferase reporter signal.

[0165] Example 5: In vivo evaluation of tumor suppression by CAR-T-mediated T cell activation Preclinical evaluation of tumor growth was performed using a mouse xenograft model for antibody constructs corresponding to clone ID numbers 325795 and 325972. CBG99 and luciferase-expressing LNCaP cell line (LNCaP-CBG99-Luc) were transfected with NOD.Cg-Prkdc. scid Il2rg tm1Wjl The in vivo antitumor efficacy of VHH chimeric antigen receptor (VCAR) was evaluated by subcutaneous injection into / Szj (NSG) mice. For the in vivo study, all CAR-T cells were generated using PiggyBac (PB) delivery of the VCAR plasmid. Mice were injected with LNCaP in the axilla (n=25, given the poor "uptake" rate" of LNCaP) and treated when tumors were established (100-300 mm as measured by vernier caliper). 3 , 17 days after injection). Mice were treated intravenously with several doses of P-PSMA-101, including an ultra-low dose (1x10^6), a "stress" dose (5x10^6), and a standard dose (10x10^6).

[0166] Panel A of Figure 5 is a graph showing tumor progression as a function of days after CAR-T injection for the indicated antibody constructs in preclinical evaluation. Tumor volume assessment was completed by caliper measurement. CAR-T cells containing the indicated antibody constructs (clone ID: 325795 or 325972) demonstrated robust tumor control compared to untreated mice. For example, immediately after treatment on day 17, clone IDs 325795 and 325972 demonstrate a rapid decrease in tumor volume. In contrast, tumor volume in untreated mice continued to increase after day 17, peaking around day 49.

[0167] Figure 5, Panel B, is a bar graph showing the area under the curve (AUC) of the graph depicted in Figure 5, Panel A, for the indicated antibody constructs. Untreated mice showed a significantly higher AUC compared to clone ID numbers 325795 and 325972.

[0168] Example 6: T cell expansion for in vivo CAR-T evaluation T cell expansion was assessed using the same preclinical evaluation described in Example 5. Panel A of Figure 6 is a graph showing in vivo blood T cell counts as a function of days after CAR-T infusion for the indicated antibody constructs. Mice treated with CAR-T cells containing the indicated antibody constructs (clone ID: 325795 or 325972) demonstrate robust expansion of P-PSMA8-101 CD8+ T cells. At days 7 and 14, prior to treatment, mice exhibited low T cell counts. After 17 days of treatment with CAR-T cells containing the indicated antibody constructs (clone ID: 325795 or 325972), a sharp increase in T cell counts was observed at day 21 compared to untreated mice, with no change. T cell counts decreased at days 28 and 42 for both treatments. At day 63, an increase in T cell counts could be observed with both antibodies. In untreated mice, T cell counts remained consistently low and did not change over the course of the study.

[0169] Figure 6, Panel B, is a bar graph showing the area under the curve (total T cell counts) of the graph depicted in Figure 6, Panel A, for the indicated antibody constructs. The total T cell counts for both antibody constructs are significant compared to the total T cell counts of untreated mice.

[0170] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the embodiments of the invention described herein may be used in practicing 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

1. 1. An antibody that binds to PSMA, (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: 4, a CDR2 sequence of SEQ ID NO: 5, and a CDR3 sequence of SEQ ID NO: 6 An antibody comprising a heavy chain variable region comprising:

2. A CAR-T cell comprising a CAR that comprises an extracellular antigen-binding domain that binds to PSMA, (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: 4, a CDR2 sequence of SEQ ID NO: 5, and a CDR3 sequence of SEQ ID NO: 6 A CAR-T cell comprising a heavy chain variable region comprising:

3. The CAR-T cell described in claim 2, wherein the CDR1, CDR2 and CDR3 sequences are present in a human framework.

4. The CAR-T cell described in claim 2, wherein the heavy chain variable region comprises a CDR1 sequence of sequence number 1, a CDR2 sequence of sequence number 2, and a CDR3 sequence of sequence number 3.

5. A CAR-T cell as described in claim 2, wherein the heavy chain variable region comprises a CDR1 sequence of sequence number 4, a CDR2 sequence of sequence number 5, and a CDR3 sequence of sequence number 6.

6. The CAR-T cell of claim 2, wherein the extracellular antigen-binding domain that binds to PSMA comprises a heavy chain variable region having at least 95% sequence identity to SEQ ID NO: 7 or 8.

7. The CAR-T cell of claim 2, wherein the extracellular antigen-binding domain that binds to PSMA comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 7-8.

8. The CAR-T cell of claim 7, wherein the extracellular antigen-binding domain that binds to PSMA comprises the heavy chain variable region sequence of SEQ ID NO:

7.

9. The CAR-T cell of claim 7, wherein the extracellular antigen-binding domain that binds to PSMA comprises the heavy chain variable region sequence of SEQ ID NO:

8.

10. A pharmaceutical composition comprising the antibody of claim 1 or the CAR-T cell of any one of claims 2 to 9.

11. A drug for use in a method for treating a disorder characterized by expression of PSMA, the drug comprising an antibody of claim 1, a CAR-T cell of any one of claims 2 to 9, or a pharmaceutical composition of claim 10, the method comprising administering the drug to a subject having the disorder.

12. The method of claim 11, wherein the disorder is cancer.

13. The method of claim 12, wherein the cancer is prostate cancer.

14. A polynucleotide encoding the antibody of claim 1 or the CAR of the CAR-T cell of any one of claims 2 to 9.

15. A vector comprising the polynucleotide of claim 14.

16. A cell comprising the vector of claim 15.

17. A method for producing an antibody according to claim 1 or a CAR-T cell according to any one of claims 2 to 9, comprising growing a cell according to claim 16 under conditions permissive for expression of said antibody or said CAR-T cell, and isolating said antibody from said cell and / or the cell culture medium in which said cell is grown, or isolating said CAR-T cell.

18. A kit for treating a disease or disorder in an individual in need thereof, comprising the antibody of claim 1 or the CAR-T cell of any one of claims 2 to 9, and instructions for use.

19. 20. The kit of claim 18, further comprising at least one additional reagent.

20. 20. The kit of claim 19, wherein the at least one additional reagent comprises a chemotherapeutic agent.

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