Heavy chain antibody that binds to PSMA

Abstract

Anti-PSMA heavy chain antibodies (e.g., UniAbs™) are disclosed, along with methods for making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use to treat disorders characterized by expression of PSMA.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the filing date of U.S. Provisional Patent Application No. 62 / 830,130, filed April 5, 2019, the disclosure of which is incorporated herein by reference in its entirety. Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy was created on July 9, 2020, is named TNO-0016-WO_SL.txt, and is 121,310 bytes in size.

[0002] The present invention relates to human heavy chain antibodies (e.g., UniAbs™) that bind to PSMA. The invention further relates to methods for making such antibodies, compositions, such as pharmaceutical compositions, comprising such antibodies, 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 acid dipeptidase, folate hydrolase, and dipeptidyl peptidase activity. It is encoded by the human 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 in prostate epithelial tissue and is upregulated in the neovasculature of prostate cancer and solid tumors. While it is also expressed at low levels in healthy tissues such as the brain, kidney, and salivary gland, its overexpression in malignant prostate tissue makes it an attractive target for therapeutic treatment of prostate cancer. Its high expression in malignant neovasculature may also be useful for the treatment or diagnostic 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, radionuclide conjugates specific for PSMA are being investigated for 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. Additional residues in the heavy chain framework 2 (FR2) and framework 4 (FR4) regions also contribute to this hydrophobic interaction 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-only antibodies or UniAbs™). UniAbs™ from camelids (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 is spliced ​​out during mRNA processing. The absence of the CH1 domain explains the absence of light chains in UniAbs™, since this domain is the anchoring site for the constant domain of the light chain. Such UniAbs™ have naturally evolved with three CDRs derived from conventional antibodies or fragments thereof to confer their antigen-binding specificity and high affinity (Muyldermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther 5:111-124). Cartilaginous fish such as sharks have also evolved a unique type of immunoglobulin called IgNAR, which lacks light chain polypeptides and is composed entirely of heavy chains. IgNAR molecules can be engineered by molecular engineering to generate variable domains of a single heavy chain polypeptide (vNAR) (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

[0006] The ability of heavy-chain-only antibodies lacking light chains to bind 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 light-chain-depleted heavy-chain-only antibodies could be produced in mammalian cell culture by removing the CH1 domain from a rearranged mouse μ gene. The produced antibodies retained VH binding specificity and possessed effector functions.

[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 (L) chain 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-only antibodies in mice and rats has been reported, for example, in WO2006008548, U.S. Patent Application Publication No. 20100122358, Nguyen et al., 2003, Immunology; 109(1), 93-101, Bruggemann et al., Crit. Rev. Immunol.; 2006, 26(5):377-90, and Zou et al., 2007, J Exp Med; 204(13):3271-3283. The generation of knockout rats by embryonic microinjection of zinc finger nucleases is described in Geurts et al., 2009, Science, 325(5939):433. Soluble heavy chain-only antibodies and transgenic rodents with 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 that include single-domain antibodies as the binding (targeting) domain are described, for example, in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378-386. Summary of the Invention

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

[0010] In some embodiments, an antibody that binds to PSMA comprises a first heavy chain variable region comprising (a) a CDR1 with no more than two substitutions in any of the amino acid sequences of SEQ ID NOs: 1-10, and / or (b) a CDR2 with no more than two substitutions in any of the amino acid sequences of SEQ ID NOs: 11-17, and / or (c) a CDR3 with no more than two substitutions in any of the amino acid sequences of SEQ ID NOs: 18-23. In some embodiments, the antibody further comprises a second heavy chain variable region comprising (a) a CDR1 with no more than two substitutions in any of the amino acid sequences of SEQ ID NOs: 1-10, and / or (b) a CDR2 with no more than two substitutions in any of the amino acid sequences of SEQ ID NOs: 11-17, and / or (c) a CDR3 with no more than two substitutions in any of the amino acid sequences of SEQ ID NOs: 18-23. In some embodiments, the CDR1, CDR2, and CDR3 sequences are in a human framework. In some embodiments, the antibody further comprises a heavy chain constant region sequence and does not comprise a CH1 sequence.

[0011] In some embodiments, the first heavy chain variable region of the antibody comprises (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-10, and / or (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 11-17, and / or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18-23.

[0012] In some embodiments, the antibody further comprises a second heavy chain variable region comprising (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1-10, and / or (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 11-17, and / or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18-23.

[0013] In some embodiments, the antibody comprises (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 to 10, and (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 11 to 17, and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18 to 23. In some embodiments, the antibody comprises a second heavy chain variable region comprising (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 to 10, and (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 11 to 17, and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18 to 23.

[0014] In some embodiments, the antibody comprises (a) the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18, or (b) the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 24-58. In some embodiments, the antibody comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 24-58. In some embodiments, the antibody comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 25 and SEQ ID NO: 38.

[0015] In some embodiments, the antibody that binds to PSMA has (a) a CDR1 sequence of the following formula: GGSISSX1X2YX3 (SEQ ID NO: 67) wherein X1 is S or N, X2 is S or N, and X3 is Y or F, and (b) a CDR2 sequence of the formula: X4X5X6SGX7T (SEQ ID NO: 68) wherein X4 is I or V, X5 is D or Y, X6 is Y or D, and X7 is Y or S, and (c) a CDR3 sequence of the formula: A first heavy chain variable region comprising ARHKAATADFDY (SEQ ID NO: 69) in a monovalent or bivalent format.

[0016] In some embodiments, the antibody that binds to PSMA has (a) a CDR1 sequence of the following formula: GFX1FX2X3YG (SEQ ID NO: 70) wherein X1 is S or I or T, X2 is S or T or R or I, and X3 is R or S, and (b) a CDR2 sequence of the formula: IX4YDGSNX5 (SEQ ID NO: 71) wherein X4 is W or S and X5 is R or K, and (c) a CDR3 sequence of the formula: AREPRX6GYYYX7X8SGYX9SLDY (SEQ ID NO: 72) wherein X6 is I or V, X7 is E or D, X8 is S or T, and X9 is Y or D, in a monovalent or bivalent format.

[0017] In some embodiments, the antibody that binds to PSMA has (a) a CDR1 sequence of the following formula: GGSISSX1X2YX3 (SEQ ID NO: 67) wherein X1 is S or N, X2 is S or N, and X3 is Y or F, and (b) a CDR2 sequence of the formula: X4X5X6SGX7T (SEQ ID NO: 68) wherein X4 is I or V, X5 is D or Y, X6 is Y or D, and X7 is Y or S, and (c) a CDR3 sequence of the formula: A first heavy chain variable region comprising: ARHKAATADFDY (SEQ ID NO: 69), and (a) a CDR1 sequence of the following formula: GFX1FX2X3YG (SEQ ID NO: 70) wherein X1 is S or I or T, X2 is S or T or R or I, and X3 is R or S, and (b) a CDR2 sequence of the formula: IX4YDGSNX5 (SEQ ID NO: 71) wherein X4 is W or S and X5 is R or K, and (c) a CDR3 sequence of the formula: AREPRX6GYYYX7X8SGYX9SLDY (SEQ ID NO: 72) wherein X6 is I or V, X7 is E or D, X8 is S or T, and X9 is Y or D.

[0018] In some embodiments, the antibody comprises a first and a second heavy chain variable region, wherein the first heavy chain variable region is located more proximal to the N-terminus than the second heavy chain variable region, and in some embodiments, the first heavy chain variable region is located more proximal to the C-terminus than the second heavy chain variable region.

[0019] In some embodiments, an antibody that binds to PSMA comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences in a human VH framework, wherein the CDR sequences comprise sequences having no more than two substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs: 1-23.

[0020] In some embodiments, an antibody that binds to PSMA comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences in a human VH framework, wherein the CDR sequences are selected from the group consisting of SEQ ID NOs: 1-23.

[0021] In some embodiments, an antibody that binds to PSMA comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:2, the CDR2 sequence of SEQ ID NO:11, and the CDR3 sequence of SEQ ID NO:18 in a human VH framework.

[0022] In some embodiments, an antibody that binds to PSMA comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18, in a human VH framework, in a monovalent or bivalent configuration.

[0023] In some embodiments, an antibody that binds to PSMA comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20 in a human VH framework.

[0024] In some embodiments, an antibody that binds to PSMA comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20, in a human VH framework, in a monovalent or bivalent format.

[0025] In some embodiments, an antibody that binds to PSMA comprises a first heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18, and a second heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20, in a human VH framework. In some embodiments, the antibody comprises the first heavy chain variable region that is positioned more proximal to the N-terminus than the second heavy chain variable region. In some embodiments, the first heavy chain variable region is positioned more proximal to the C-terminus than the second heavy chain variable region.

[0026] In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific. In some embodiments, the antibody is bispecific. In some embodiments, the antibody has binding affinity for CD3 protein and PSMA protein. In some embodiments, the antibody has binding affinity for two different epitopes on the same PSMA protein. In some embodiments, the antibody has binding affinity for effector cells. In some embodiments, the antibody has binding affinity for a T cell antigen. In some embodiments, the antibody has binding affinity for CD3. In some embodiments, the antibody is in a CAR-T format.

[0027] Embodiments of the invention include bispecific antibodies comprising: (i) a heavy chain variable region with binding affinity to CD3, comprising, in a human VH framework, the CDR1 sequence of SEQ ID NO: 59, the CDR2 sequence of SEQ ID NO: 60, and the CDR3 sequence of SEQ ID NO: 61; (ii) a light chain variable region, in a human VL framework, the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64; and (iii) an antigen-binding domain of an anti-PSMA heavy chain antibody, comprising, in a human VH framework, the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18.

[0028] Embodiments of the invention include bispecific antibodies comprising (i) a heavy chain variable region with binding affinity to CD3, comprising the CDR1 sequence of SEQ ID NO: 59, the CDR2 sequence of SEQ ID NO: 60, and the CDR3 sequence of SEQ ID NO: 61, in a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64, in a human VL framework; and (iii) an antigen-binding domain of an anti-PSMA heavy chain antibody comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18, in a human VH framework, in a monovalent or bivalent configuration.

[0029] Embodiments of the invention include bispecific antibodies comprising: (i) a heavy chain variable region with binding affinity to CD3, comprising, in a human VH framework, the CDR1 sequence of SEQ ID NO: 59, the CDR2 sequence of SEQ ID NO: 60, and the CDR3 sequence of SEQ ID NO: 61; (ii) a light chain variable region, in a human VL framework, the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64; and (iii) an antigen-binding domain of an anti-PSMA heavy chain antibody, comprising, in a human VH framework, the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20.

[0030] Embodiments of the invention include bispecific antibodies comprising (i) a heavy chain variable region with binding affinity to CD3, comprising the CDR1 sequence of SEQ ID NO: 59, the CDR2 sequence of SEQ ID NO: 60, and the CDR3 sequence of SEQ ID NO: 61, in a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64, in a human VL framework; and (iii) an antigen-binding domain of an anti-PSMA heavy chain antibody comprising the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20, in a human VH framework, in a monovalent or bivalent configuration.

[0031] Embodiments of the invention include multispecific antibodies comprising: (i) a heavy chain variable region having binding affinity to CD3, the heavy chain variable region comprising, in a human VH framework, the CDR1 sequence of SEQ ID NO: 59, the CDR2 sequence of SEQ ID NO: 60, and the CDR3 sequence of SEQ ID NO: 61; (ii) a light chain variable region comprising, in a human VL framework, the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64; and (iii) an antigen-binding domain of an anti-PSMA heavy chain antibody, the antigen-binding domain comprising, in a bivalent configuration, first and second antigen-binding regions, the first antigen-binding region comprising, in a human VH framework, the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18; and the second antigen-binding region comprising, in a human VH framework, the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20. In certain embodiments, the first antigen-binding region is located more proximal to the N-terminus than the second antigen-binding region, while in certain other embodiments, the first antigen-binding region is located more proximal to the C-terminus than the second antigen-binding region.

[0032] Aspects of the present invention include multispecific or bispecific antibodies in which the first and second antigen-binding regions of an anti-PSMA heavy chain antibody antigen-binding domain are connected by a polypeptide linker. In some embodiments, the peptide linker is a GS linker. In some embodiments, the GS linker consists of the sequence of SEQ ID NO: 73 or SEQ ID NO: 74. In some embodiments, the anti-PSMA heavy chain antibody antigen-binding domain is monoparatopic and induces less cytokine production compared to a biparatopic antigen-binding domain. In some embodiments, the anti-PSMA heavy chain antibody antigen-binding domain is monoparatopic and induces greater proliferation of CD8+ T cells compared to a biparatopic antigen-binding domain.

[0033] In some embodiments, the antibodies are biparatopic and have a higher affinity for PSMA compared to monoparatopic anti-PSMA antibodies, hi some embodiments, the antibodies are biparatopic and have a higher effector function compared to monoparatopic anti-PSMA antibodies.

[0034] Aspects of the invention relate to pharmaceutical compositions comprising the antibodies described herein.

[0035] Aspects of the invention relate to methods of treating a disorder characterized by PSMA expression, comprising administering to a subject having the disorder an antibody or pharmaceutical composition described herein. In certain other aspects, the invention relates to the use of an antibody described herein in the preparation of a medicament for treating a disorder characterized by PSMA expression. In yet other aspects, the invention relates to an antibody described herein for use in treating a disorder characterized by PSMA expression. In certain other aspects, the invention relates to a method of treatment, comprising administering to an individual in need thereof an effective dose of an antibody or pharmaceutical composition described herein. With regard to these aspects, and in some embodiments, the disorder is prostate cancer.

[0036] Aspects of the invention pertain to polynucleotides encoding the antibodies described herein, vectors comprising such polynucleotides, and cells comprising such vectors.

[0037] Aspects of the invention relate to methods of producing the antibodies described herein, comprising growing a cell described herein under conditions permissive for expression of the antibody, and isolating the antibody from the cell.

[0038] Aspects of the invention relate to methods for producing the antibodies described herein, including immunizing UniRat animals with PSMA protein and identifying PSMA-binding antibody sequences.

[0039] These and additional aspects are further described in the remainder of the disclosure, including the Examples. [Brief explanation of the drawings]

[0040] [Figure 1] Panels A-B provide a series of graphs showing serum titers as a function of dilution. [Figure 2] Panel A is a graph showing cell binding to human PSMA, and Panel B is a graph showing cell binding to cynomolgus monkey PSMA. [Figure 3] 1 is a graph showing binding competition between two antibody families according to embodiments of the present invention. [Figure 4] Panel A is a Scatchard plot showing the binding affinity to cell surface-expressed PSMA of a bispecific antibody having binding affinity to CD3 and PSMA, where the PSMA arm is monoparatopic and monovalent according to an embodiment of the invention. Panel B is a Scatchard plot showing the binding affinity to cell surface-expressed PSMA of a bispecific antibody having binding affinity to CD3 and PSMA, where the PSMA arm is biparatopic according to an embodiment of the invention. [Figure 5]Panels A-C show schematic diagrams of an anti-CD3 x monovalent, monospecific anti-PSMA antibody (Panel A), an anti-CD3 x bivalent, monospecific anti-PSMA antibody (Panel B), and an anti-CD3 x bivalent, biparatopic anti-PSMA antibody (Panel C), according to embodiments of the invention. [Figure 6] 1 is a graph showing T cell-mediated lysis of PSMA-positive cells using pre-activated T cells. [Figure 7] 1 is a graph showing T cell-mediated lysis of PSMA-positive cells using unstimulated T cells. [Figure 8] Figure 1 is a graph showing % specific lysis of PSMA-negative DU145 cells as a function of multispecific antibody concentration in the presence of pre-activated T cells. [Figure 9] 1 is a graph showing binding of PSMAxCD3 bispecific antibodies to PSMA-positive and -negative cells. [Figure 10] 1 is a graph showing T cell-mediated lysis of PSMA-positive cells. [Figure 11] Panel A is a graph showing T cell proliferation as a function of antibody concentration. Panel B is a graph showing T cell proliferation as a function of antibody concentration. Panel C is a graph showing the ratio of CD8 to CD4 of expanded T cells. Panel D is a graph showing the ratio of CD8 to CD4 of expanded T cells. [Figure 12] Panel A is a graph showing T cell-mediated lysis of PSMA-positive cells as a function of antibody concentration. Panel B is a graph showing cytokine (IFNγ) release as a function of antibody concentration. Panel C is a graph showing cytokine (IL-2) release as a function of antibody concentration. [Figure 13] 1 is a graph showing inhibition of 22Rv1 tumor growth in a tumor xenograft model. DETAILED DESCRIPTION OF THE INVENTION

[0041] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); Biology” (FMAusubel et al., eds., 1987, and periodic updates), “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994), “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988), “Phage Display: A Laboratory Manual” (Barbas et al., 2001), Harlow, Lane and Harlow, Using Antibodies:A Laboratory Manual:Portable Protocol No.I,Cold Spring Harbor Laboratory (1998), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0042] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed herein, subject to any specific excluded limits in the stated range. Where a stated range includes one or both of those upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the invention.

[0043] Unless otherwise specified, antibody residues herein are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0044] 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 are not described in order to avoid obscuring the present invention.

[0045] All references cited throughout this disclosure, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0046] I. Definition "Comprising" means that the recited element is necessary for the composition / method / kit, but other elements may be included to form the composition / method / kit, etc., within the scope of the claim.

[0047] "Consisting essentially of" means limiting the scope of the described composition or method to certain substances or steps that do not materially affect the basic and novel characteristic(s) of the invention.

[0048] "Consisting of" means that any element, step, or ingredient not specified in the claim is excluded from the composition, method, or kit.

[0049] 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 according to Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise specified herein, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise specified herein, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system.

[0050] Antibodies, also called immunoglobulins, traditionally comprise at least one heavy chain and one light chain, with the amino-terminal domains of the heavy and light chains being variable in sequence and therefore commonly referred to as variable region domains, or variable heavy (VH) domains or variable light (VL) domains. The two domains traditionally associate to form a specific binding region, although, as discussed herein, specific binding can also be obtained with variable sequence of the heavy chain alone, and various non-naturally occurring configurations of antibodies are known and used in the art.

[0051] A "functional" or "biologically active" antibody or antigen-binding molecule (including heavy-chain-only antibodies and multispecific (e.g., bispecific) three-chain antibody-like molecules (TCAs described herein)) is a molecule that can exert one or more of its native activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule, e.g., a TCA, can have the ability to specifically bind to an antigen, which binding can then trigger or alter a cellular or molecular event, such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, e.g., a TCA, can also block ligand activation of a receptor or act as an agonist or antagonist. The ability of an antibody or other binding molecule, e.g., a TCA, to exert one or more of its native activities depends on several factors, including proper folding and assembly of the polypeptide chain.

[0052] The term "antibody" as used herein is used in the broadest sense and includes, among others, monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain-only antibodies, three-chain antibodies, TCAs, single-chain Fvs (scFvs), nanobodies, and the like, as well as antibody fragments so long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0053] 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 of a target of interest or a portion thereof, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. 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 (including subclasses engineered with modified Fc portions that attenuate or enhance effector cell activity) of immunoglobulin molecule. The light chain of the subject antibody can be a kappa light chain (Vκ) or a lambda light chain (Vλ). The immunoglobulin can be derived from any species. In one aspect, the immunoglobulin is predominantly human in origin.

[0054] 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 minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies according to the present invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can also be produced, for example, via recombinant protein production methods (see, e.g., U.S. Pat. No. 4,816,567).

[0055] The term "variable" 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. It is concentrated in three segments called hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of variable domains are called framework regions (FRs). Native heavy-chain and light-chain variable domains each contain four FRs, which adopt a primarily β-sheet configuration and are connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are held in close proximity to the hypervariable regions of the other chain by the FRs and contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular cytotoxicity (ADCC).

[0056] As used herein, the term "hypervariable region" 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 residues from the "hypervariable loops" of the heavy chain variable domain, residues 26-32 (H1), 53-55 (H2), and 96-101 (H3); 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.

[0057] While exemplary CDR designations are provided herein, those skilled in the art will appreciate that several definitions of CDRs are commonly used, including the most commonly used Kabat definition (see "Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions." Mol Immunol. 2010;47:694-700), which is based on sequence variability. The Chothia definition is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989;342:877-883).Alternative CDR definitions of interest include, but are not limited to, those described in Honegger, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol. 2001;309:657-670; Ofran et al., "Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B-cell epitopes," J Immunol. 2008;181:6230-6235; Almagro, "Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires," J Mol Recognit. 2004;17:132-143; and Padlan et al., "Identification of specificity-determining residues in antibodies," Faseb J. 1995;9:133-139, each of which is specifically incorporated herein by reference.

[0058] The terms "heavy chain-only antibody" and "heavy chain antibody" are used interchangeably herein and refer, in the broadest sense, 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. These terms specifically include, but are not limited to, homodimeric antibodies comprising a VH antigen-binding domain and CH2 and CH3 constant domains but lacking a CH1 domain, functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NARs and functional fragments thereof comprising a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NARs), and soluble single-domain antibodies (sUniDabs™). In one embodiment, a heavy chain-only antibody comprises a variable region antigen-binding domain consisting of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In another embodiment, a heavy chain-only antibody comprises an antigen-binding domain, at least a portion of the hinge region, and the CH2 and CH3 domains. In another embodiment, the heavy chain-only antibody consists of an antigen-binding domain, at least a portion of the hinge region, and a CH2 domain. In a further embodiment, the heavy chain-only antibody consists of an antigen-binding domain, at least a portion of the hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In a further embodiment, the heavy chain consists 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-only antibodies may be in the form of a dimer in which two heavy chains are disulfide-linked or otherwise covalently or non-covalently bound to each other. Heavy chain-only 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 a specific embodiment, the heavy chain antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype. In one embodiment, the heavy chain-only antibodies herein are used as the binding (targeting) domain of a chimeric antigen receptor (CAR).This definition specifically includes human heavy chain-only antibodies produced by human immunoglobulin transgenic rats (UniRat™), referred to as UniAbs™. The variable regions (VH) of UniAbs™, also referred to as UniDabs™, are variable building blocks that can be conjugated to Fc regions or serum albumin to develop novel therapeutics with multispecificity, increased potency, and extended half-life. Homodimeric UniAbs™ lack light chains and therefore a VL domain; therefore, antigens are recognized by a single domain, i.e., the variable domain of the heavy chain of the heavy chain antibody (VH or VHH).

[0059] As used herein, an "intact antibody chain" refers to an antibody chain comprising 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) for secreted IgG and heavy chain constant domains, 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 (a native-sequence Fc region or an amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and down-regulation of cell surface receptors. Constant region variants include variants that alter effector properties, binding to Fc receptors, and the like.

[0060] Depending on the amino acid sequence of the Fc (constant domain) of the heavy chain, antibodies and various antigen-binding proteins can be provided as different classes. There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, some of which 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 different classes of immunoglobulins are well known. Forms of Ig include hinge-modified or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US2005 / 0048572; US2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types, called κ (kappa) and λ (lambda), based on the amino acid sequence of their constant domains. Antibodies according to embodiments of the invention can comprise a κ light chain sequence or a λ light chain sequence.

[0061] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding, CDC, Fc receptor binding, ADCC, ADCP, down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require the Fc region to interact with a receptor, e.g., FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and the low-affinity FcRn receptor, and can be assessed using various assays known in the art. A "dead" or "silenced" Fc is an Fc that has been mutated to retain activity, e.g., with respect to extended serum half-life, but does not activate high-affinity Fc receptors or has reduced affinity for Fc receptors.

[0062] 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.

[0063] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide. A variant Fc region herein preferably retains at least about 80% homology with a native-sequence Fc region and / or the Fc region of the parent polypeptide, and most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0064] The variant Fc sequence may contain three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitution of human IgG1 or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 with human IgG1 or IgG2 residues significantly reduces ADCC and CDC (see, e.g., Armour KL et al., 1999 Eur J Immunol. 29(8):2613-24, and Shields RL et al., 2001 J Biol Chem. 276(9):6591-604). The human IgG4 Fc amino acid sequence (UniProtKB No. P01861) is provided herein as SEQ ID NO: 76. Silenced IgG1 is described, for example, in Boesch, AW, et al., "Highly parallel characterization of IgG Fc binding interactions." MAbs, 2014.6(4):915-27, the disclosure of which is incorporated herein by reference in its entirety.

[0065] Other Fc variants are possible, including, but not limited to, variants in which regions capable of forming disulfide bonds are deleted, or in which specific amino acid residues are removed or a methionine residue is added at the N-terminus of a native Fc. Thus, in some embodiments, one or more Fc 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.

[0066] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector function by substituting (mutating), deleting, or adding amino acid residues to confer complement binding or Fc receptor binding. For example, but not limited to, deletions may be made in complement binding sites, such as the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478. Furthermore, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0067] 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 promoting proper pairing (heterodimerization) of the desired pair of heavy chain polypeptide subunits in the antibody.

[0068] In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P, F234A, L235A, and T366W mutation (knob). In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P, F234A, L235A, and T366S, L368A, and Y407V mutation (hole).

[0069] The term "antibody comprising an Fc region" refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Thus, antibodies having an Fc region according to the present invention may include antibodies that either contain or do not contain K447.

[0070] Embodiments of the present invention include antibodies comprising heavy chain-only variable regions in a monovalent or bivalent configuration. As used herein, the term "monovalent configuration" when used with respect to a heavy chain-only variable region domain means that only one heavy chain-only variable region domain is present, having a single binding site (see Figure 5, panel A, right arm of the antibody). In contrast, the term "bivalent configuration" when used with respect to a heavy chain-only variable region domain means that two heavy chain-only variable region domains are present (each having a single binding site) and are connected by a linker sequence (see Figure 5, panels B and C, right arm of the antibody). 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 configuration, each of the two heavy chain-only variable region domains has binding affinity for the same antigen or different antigens (e.g., for different epitopes on the same protein, for two different proteins, etc.). However, unless otherwise specified, a heavy chain-only variable region designated as being in a "bivalent configuration" is understood to comprise two identical heavy chain-only variable region domains connected by a linker sequence, where each of the two identical heavy chain-only variable region domains has binding affinity for the same target antigen.

[0071] Aspects of the present invention include antibodies with multispecific configurations, including but not limited to bispecific, trispecific, etc. A wide variety of methods and protein configurations for bispecific monoclonal antibodies (BsMAB), trispecific antibodies, etc. are known and used.

[0072] Various methods for producing multivalent artificial antibodies have been developed by recombinantly fusing the variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on a polypeptide are connected by a polypeptide linker. One non-limiting example of such a polypeptide linker is a GS linker, which has an amino acid sequence of four glycine residues followed by one serine residue, where this sequence is repeated n times in the GS linker, where n is an integer ranging from 1 to about 10, e.g., 2, 3, 4, 5, 6, 7, 8, or 9. (SEQ ID NO: 94) Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 73) (n=1) and GGGGSGGGGS (SEQ ID NO: 74 (n=2). Other suitable linkers can also be used, e.g., as described in Chen et al., Adv Drug Deliv Rev. 2013 October 15;65(10):1357-69, the disclosure of which is incorporated herein by reference in its entirety.

[0073] The term "tri-chain antibody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy chain and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain, comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy chain-only antibody comprising an Fc portion that does not include a CH1 domain but includes CH2 and / or CH3 and / or CH4 domains, 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, where such binding domains are derived from or have sequence identity to the variable regions of the heavy or light chains of the antibody. Portions of such variable regions are V H and / or V L Gene segments, D and JH gene segment, or J L The variable region can be encoded by a rearranged V H DJ H , V L DJ H , V H J L , or V L J L It can be encoded by a gene segment.

[0074] The TCA binding compounds utilize "heavy chain-only antibodies" or "heavy chain antibodies" or "heavy chain polypeptides," which, as used herein, refer to single-chain antibodies comprising heavy chain constant regions CH2 and / or CH3 and / or CH4, but not the CH1 domain. In one embodiment, a heavy chain antibody consists 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 consists 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 consists of an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domains have been truncated are also included herein. In a further embodiment, the heavy chain consists of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but not the hinge region. Heavy-chain-only antibodies may be in the form of a dimer in which two heavy chains are disulfide-bonded or covalently or non-covalently bound to each other, and may optionally contain an asymmetric interface between one or more 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 IgM, IgA, IgD, and IgE subclasses, are also encompassed herein. In certain embodiments, heavy-chain antibodies are of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 or IgG4 subtype. Non-limiting examples of TCA-binding compounds are described, for example, in WO2017 / 223111 and WO2018 / 052503, the disclosures of which are incorporated herein by reference in their entireties.

[0075] Heavy chain antibodies constitute 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 have no light chains. As a result, the variable antigen-binding portion is called a VHH domain, which represents the smallest naturally occurring intact antigen-binding site, only about 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy-chain antibodies with high specificity and affinity can be generated against a variety of 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, called VNARs. (Nuttall et al.Eur.J.Biochem.270,3543-3554(2003), Nuttall et al. Function and Bioinformatics 55,187-197(2004), Dooley et al.,Molecular Immunology 40,25-33(2003)).

[0076] As used herein, the term "PSMA" refers to a type II transmembrane protein with N-acetyl-α-linked acid depeptidase, folate hydrolase, and dipeptidyl peptidase activity. The term "PSMA" includes PSMA proteins of any human and non-human animal species, and specifically includes human PSMA and PSMA of non-human mammals.

[0077] As used herein, the term "human PSMA" includes any variant, isoform, and species homologue of human PSMA (UniProt Q04609), regardless of its source or mode of preparation. Thus, "human PSMA" includes human PSMA naturally expressed by cells and PSMA expressed on cells transfected with the human PSMA gene.

[0078] As defined above, 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 to refer to heavy chain-only antibodies 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™ antibodies, as defined above.

[0079] "Percentage of amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percentage of sequence identity (any conservative substitutions are not considered as part of sequence identity).Alignment for determining percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the entire length of the sequences being compared.However, for the purposes herein, the sequence comparison computer program ALIGN-2 is used to generate percentage amino acid sequence identity values.

[0080] An "isolated" antibody is one that has been identified, separated, and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses for the antibody, and these 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 method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue, or preferably silver stain. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0081] The antibodies of the present invention include multispecific antibodies. Multispecific antibodies have multiple binding specificities. The term "multispecific" specifically includes "bispecific" and "trispecific," as well as higher-order independent specific binding affinities such as higher-order polyepitopic specificities, as well as tetravalent antibodies and antibody fragments. The terms "multispecific antibody," "multispecific heavy chain-only antibody," "multispecific heavy chain antibody," and "multispecific UniAb™" are used in the broadest sense herein to encompass all antibodies with multiple binding specificities. Multispecific 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 present invention also specifically include antibodies that immunospecifically bind to an epitope on a PSMA protein, such as human PSMA, and an epitope on a different protein, such as a CD3 protein, e.g., human CD3 (i.e., bivalent 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 human PSMA, and epitopes on different proteins, such as, for example, the CD3 protein, e.g., the human CD3 protein (i.e., bivalent and biparatopic).

[0082] The antibodies of the present invention include monospecific antibodies having one binding specificity. Monospecific antibodies specifically include antibodies with a single binding specificity and antibodies containing multiple binding units with the same binding specificity. The terms "monospecific antibody," "monospecific heavy chain-only antibody," "monospecific heavy chain antibody," and "monospecific UniAb™" are used in the broadest sense herein to encompass all antibodies with a single 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 (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).

[0083] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Typically, an antigen has several or many different epitopes and will react with many different antibodies. The term specifically includes linear and conformational epitopes.

[0084] "Epitope mapping" is the process of identifying antibody binding sites, or epitopes, on a target antigen. Antibody epitopes can be linear or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed by amino acids that are discontinuous in the protein sequence but come together when the protein folds into its three-dimensional structure.

[0085] "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). As described above, the present invention specifically includes anti-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, as well as anti-PSMA heavy chain antibodies that bind to one or more epitopes on a PSMA protein and an epitope on a different protein, such as the CD3 protein. The term "non-overlapping epitopes" or "non-competing epitopes" of an antigen is defined herein to mean epitopes recognized by one member of an antigen-specific antibody pair but not by the other member. Antibody pairs that recognize non-overlapping epitopes, or antigen-binding regions that target the same antigen on a multispecific antibody, do not compete for binding to the antigen and can simultaneously bind to the antigen.

[0086] If two antibodies recognize the same or sterically overlapping epitopes, the antibodies bind to "essentially the same epitope" as a reference antibody. The most widely used rapid method for determining whether two epitopes bind to the same or sterically overlapping epitopes is a competitive assay, which can be configured in a variety of formats using either labeled antigen or labeled antibody. Typically, the antigen is immobilized on a 96-well plate, and the ability of an unlabeled antibody to block the binding of the labeled antibody is measured using a radioactive or enzyme label.

[0087] As used herein, the term "valency" refers to a specific number of binding sites within an antibody molecule.

[0088] A "monovalent" antibody has one binding site. A monovalent antibody is therefore also monospecific.

[0089] A "multivalent" antibody has two or more binding sites. Thus, the terms "bivalent," "trivalent," and "tetravalent" refer to the presence of two binding sites, three binding sites, and four binding sites, respectively. Thus, bispecific antibodies according to the invention are at least bivalent, and may be trivalent, tetravalent, or multivalent. Bivalent antibodies according to embodiments of the invention may have two binding sites for the same epitope (i.e., bivalent, monoparatopic), or for two different epitopes (i.e., bivalent, biparatopic).

[0090] A wide variety of methods and protein constructs for preparing bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc. are known and in use.

[0091] The term "tri-chain antibody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy chain and one light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains, comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy chain-only antibody comprising an Fc portion lacking a CH1 domain and including CH2 and / or CH3 and / or CH4 domains, and an antigen-binding domain that binds to an epitope of a second antigen or a different epitope of the first antigen, wherein such binding domain is derived from or has sequence identity to the variable region of the heavy or light chain of the antibody. Portions of such variable regions are 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 , VH J L , or V L J L The TCA proteins may be encoded by gene segments. The TCA proteins utilize heavy chain-only antibodies as defined above.

[0092] The term "chimeric antigen receptor" or "CAR" is used in the broadest sense herein to refer to an engineered receptor that grafts a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) onto a transmembrane domain and an intracellular signaling domain. Typically, receptors are used to graft the specificity of a monoclonal antibody onto a T cell to create a chimeric antigen receptor (CAR). (J Natl Cancer Inst, 2015;108(7):dvj439, and Jackson et al., Nature Reviews Clinical Oncology, 2016;13:370-383). CAR-T cells are T cells that have been genetically engineered to produce an artificial T cell receptor for use in immunotherapy. In one embodiment, "CAR-T cells" refers to therapeutic T cells that express a transgene encoding one or more chimeric antigen receptors minimally consisting of an extracellular domain, a transmembrane domain, and at least one cytosolic domain.

[0093] The term "human antibody" is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. The term "human antibody" specifically includes heavy chain-only antibodies having human heavy chain variable region sequences, as defined above, produced by transgenic animals such as transgenic rats or mice, particularly UniAbs™ generated by UniRats™.

[0094] "Chimeric antibody" or "chimeric immunoglobulin" refers to an immunoglobulin molecule that contains amino acid sequences 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 such chimeric antibodies.

[0095] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector stage of an immune response, as opposed to the recognition and activation stages of an immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, can induce antibody-dependent cellular cytotoxicity (ADCC). For example, FcR-expressing monocytes and macrophages are involved in the specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0096] "Human effector cells" are leukocytes that express receptors such as T cell receptors and FcRs and perform effector function. Preferably, these 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., from blood or PBMCs, as described herein.

[0097] The term "immune cell" is used herein in the broadest sense and includes, but is not limited to, cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0098] An "effector function" of an antibody refers to a biological activity attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), and the like.

[0099] "Antibody-dependent cellular cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0100] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0101] "Binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low-affinity antibodies usually bind antigens slowly and tend to dissociate easily, while high-affinity antibodies usually bind antigens more quickly and tend to remain bound to them.

[0102] 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 mouse Fc fusion antigen and immersed in a well containing antibody to measure the concentration-dependent association rate (k). In the final step, the sensor is immersed in a well containing only buffer solution to measure the dissociation rate (koff) of the antibody. Kd is the ratio of koff / koff. (For more details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009.)

[0103] The terms "treatment," "treating," and the like are used generally herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the onset of the disease in a subject who may be susceptible to, but has not yet been diagnosed with, the disease; (b) inhibiting the disease, i.e., preventing its development; or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. Treatment of ongoing diseases, where the 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 affected tissues. The therapeutic agent of interest may also be administered during, and in some cases after, the symptomatic period of the disease.

[0104] By "therapeutically effective amount" is intended the amount of active agent required to provide a therapeutic effect to a subject, e.g., 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.

[0105] As used herein, the term "prostate cancer" refers to a malignant tumor of glandular origin located in the prostate gland.

[0106] The term "characterized by PSMA expression" 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.Such disorders include, but are not limited to, prostate cancer.

[0107] 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. While the subject can be a human, other mammals are also included, particularly mammals useful as laboratory models of human disease, e.g., mice, rats, and the like.

[0108] The term "pharmaceutical formulation" refers to a formulation in which the biological activity of the active ingredient is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) is one that can be reasonably administered to a mammalian subject to provide an effective dose of the active ingredient used.

[0109] A "sterile" formulation is sterile or free or essentially free of all viable microorganisms and their spores. A "frozen" formulation is one at a temperature below 0°C.

[0110] A "stable" formulation is one in which the protein therein essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pub. (1991) and Jones. A. Adv. Drug Delivery Rev. 10:29-90) (1993). Stability can be measured at a selected temperature over a selected period of time. Stability can be qualitatively and / or quantitatively assessed in a variety of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection), cation exchange chromatography, by assessing charge heterogeneity using image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis, amino- or carboxy-terminal sequence analysis, mass spectrometry, SDS-PAGE analysis comparing reduced and intact antibody, peptide mapping (e.g., trypsin or LYS-C) analysis, assessment of antibody biological activity or antigen-binding function, etc. Instability can include any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, differential glycosylation, etc.

[0111] 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 comprise the set of CDR sequences defined herein and set forth in Table 1, and are exemplified by the provided heavy chain variable region (VH) sequences in SEQ ID NOS: 24-54 shown in Table 2. This family of antibodies offers many advantages that contribute to their usefulness as clinical therapeutic(s). The antibodies include members with a range of binding affinities, allowing selection of specific sequences with desired binding affinities.

[0112] TIFF0007776987000001.tif131170

[0113] TIFF0007776987000002.tif251170TIFF0007776987000003.tif251170TIFF0007776987000004.tif92170

[0114] Suitable antibodies can be selected from the antibodies provided herein for development and therapeutic or other uses, including, but not limited to, bispecific or trispecific antibodies, or use as part of CAR-T structures, as shown, for example, in panels A-C of Figure 5. Panels A-C of Figure 5 provide illustrations of anti-CD3 x anti-PSMA multispecific antibodies, in which the anti-PSMA domain is monovalent and monospecific, bivalent and monospecific, or bivalent and bispecific (biparatopic). The anti-CD3 domain contains a CH1 domain and pairs with the light chain, while the anti-PSMA domain is derived from a heavy-chain-only antibody, does not contain a CH1 domain, and does not interact with the light chain. In some embodiments, the two heavy chains are paired, for example, using knob-into-hole technology. Turning to the antibodies shown in Figure 5, panel A illustrates an anti-CD3 x anti-PSMA bispecific antibody, in which the anti-PSMA binding arm is monovalent and monospecific, and the antigen-binding domain of the anti-PSMA arm is in a monovalent configuration, meaning that only one antigen-binding domain is present. Panel B shows an anti-CD3 x anti-PSMA bispecific antibody in which the anti-PSMA binding arms are bivalent and monospecific, and the antigen-binding domains of the anti-PSMA arms are in a bivalent configuration, meaning there are two identical antigen-binding domains arranged in tandem. Panel C shows an anti-CD3 x anti-PSMA bispecific antibody in which the anti-PSMA binding arms are bivalent and biparatopic, and the antigen-binding domains of the anti-PSMA arms are in a bivalent configuration.

[0115] Affinity measurements for candidate proteins can be performed using methods known in the art, such as Biacore measurements. Members of the antibody family can be, but are not limited to, approximately 10 -6 ~about 10 -10 , about 10 -6 ~about 10 -9 , about 10 -6 ~about 10 -8 , about 10 -8 ~about 10 -11 , about 10 -8 ~about 10 -10 , about 10 -8 ~about 10 -9, about 10 -9 ~about 10 -11 , about 10 -9 ~about 10 -10 , or any value within these ranges, inclusive, approximately 10 -6 ~about 10 -11 The selected affinity can be confirmed by biological evaluation to modulate, e.g., block, the biological activity of PSMA, including in vitro assays, preclinical models, and clinical trials, as well as evaluation of potential toxicity.

[0116] Members of the antibody family herein are not cross-reactive with the PSMA protein of cynomolgus macaques, but can be modified, if desired, to provide cross-reactivity with the PSMA protein of cynomolgus macaques or PSMA of any other animal species.

[0117] 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, by way of example, in regions around amino acid residues 26-33, 51-58, and 97-116 of CDR1, CDR2, and CDR3 of the exemplary variable region sequences provided, respectively, as set forth in SEQ ID NOS: 24-58. Those skilled in the art will appreciate that when different framework sequences are selected, the CDR sequences may be in different positions, although the order of the sequences generally remains the same.

[0118] The CDR1, CDR2, and CDR3 sequences of the anti-PSMA antibodies of the invention can be encompassed by the following structural formulas, where X represents a variable amino acid, which can be a specific amino acid as shown below: CDR1 GGSISSX1X2YX3 (SEQ ID NO: 67) wherein X1 is S or N; X2 is S or N, and X3 is Y or F, and CDR2 X4X5X6SGX7T (SEQ ID NO: 68) wherein X4 is I or V; X5 is D or Y; X6 is Y or D, and X7 is Y or S, and CDR3 ARHKAATADFDY (SEQ ID NO: 69)

[0119] The CDR1, CDR2, and CDR3 sequences of the anti-PSMA antibodies of the invention can be encompassed by the following structural formulas, where X represents a variable amino acid, which can be a specific amino acid as shown below: CDR1 GFX1FX2X3YG (SEQ ID NO: 70) wherein X1 is S, I, or T; X2 is S or T or R or I, and X3 is R or S, and CDR2 IX4YDGSNX5 (SEQ ID NO: 71) wherein X4 is W or S, and X5 is R or K, and CDR3 AREPRX6GYYYX7X8SGYX9SLDY (SEQ ID NO: 72) wherein X6 is I or V; X7 is E or D, X8 is S or T, and X9 is Y or D.

[0120] Representative CDR1, CDR2, and CDR3 sequences are shown in Tables 1 and 3. TIFF0007776987000005.tif251170TIFF0007776987000006.tif168170

[0121] In some embodiments, the anti-PSMA antibody comprises a CDR1 sequence of any one of SEQ ID NOs: 1 to 10. In particular embodiments, the CDR1 sequence is SEQ ID NO: 2 or 7.

[0122] In some embodiments, the anti-PSMA antibody comprises the CDR2 sequence of any one of SEQ ID NOs: 11 to 17. In particular embodiments, the CDR2 sequence is SEQ ID NO: 11 or 15.

[0123] In some embodiments, the anti-PSMA antibody comprises the CDR3 sequence of any one of SEQ ID NOs: 18 to 23. In particular embodiments, the CDR3 sequence is SEQ ID NO: 18 or 20.

[0124] In a further embodiment, the anti-PSMA heavy chain-only antibody comprises the CDR1 sequence of SEQ ID NO:2, the CDR2 sequence of SEQ ID NO:11, and the CDR3 sequence of SEQ ID NO:18.

[0125] In a further embodiment, the anti-PSMA antibody comprises the CDR1 sequence of SEQ ID NO:7, the CDR2 sequence of SEQ ID NO:15, and the CDR3 sequence of SEQ ID NO:20.

[0126] In further embodiments, the anti-PSMA antibody comprises any of the heavy chain variable region amino acid sequences of SEQ ID NOs: 24-58 (Table 2).

[0127] In yet another embodiment, the anti-PSMA antibody comprises the heavy chain variable region sequence of SEQ ID NO:25.

[0128] In yet another embodiment, the anti-PSMA antibody comprises the heavy chain variable region sequence of SEQ ID NO:38.

[0129] In some embodiments, the CDR sequences in the anti-PSMA antibodies of the invention contain one or two amino acid substitutions relative to the CDR1, CDR2 and / or CDR3 sequence of any one of SEQ ID NOs: 1-23 or the set of CDR1, CDR2 and CDR3 sequences (Table 1).

[0130] In some embodiments, the anti-PSMA antibody preferably comprises a heavy chain variable domain (VH) whose CDR3 sequence has 80% or more, e.g., at least 85%, at least 90%, at least 95%, or at least 99%, sequence identity at the amino acid level to the CDR3 sequence of any one of the antibodies (the CDR3 sequences of which are shown in Table 1) that bind to PSMA.

[0131] In some embodiments, the anti-PSMA antibody preferably comprises a heavy chain variable domain (VH) in which the full set (combination) of CDR1, 2, and 3 has eighty-five percent (85%) or greater sequence identity at the amino acid level to the CDR1, 2, and 3 (combination) of an antibody (the CDR sequences of which are shown in Table 1) that binds to PSMA.

[0132] In some embodiments, the anti-PSMA antibody preferably comprises a heavy chain variable domain (VH) in which the full set (combination) of CDR1, 2, and 3 has eighty-five percent (85%) or greater sequence identity at the amino acid level to the CDR1, 2, and 3 (combination) of an antibody (the CDR sequences of which are shown in Table 3) that binds to PSMA.

[0133] In some embodiments, the anti-PSMA antibody comprises a heavy chain variable region sequence that has 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: 24-58 (shown in Table 2) and binds to PSMA.

[0134] In some embodiments, bispecific or multispecific antibodies are provided, which may have any of the configurations described 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 a heavy chain variable region comprising at least two antigen-binding domains, each of which has binding specificity for PSMA. In some embodiments, a multispecific antibody may comprise a heavy chain / light chain pair with binding specificity for a first antigen (e.g., CD3) and a heavy chain derived from a heavy chain-only antibody. In certain embodiments, the heavy chain derived from a heavy chain-only antibody comprises an Fc portion that does not comprise a CH1 domain and comprises a CH2 and / or CH3 and / or CH4 domain. In one particular embodiment, the bispecific antibody comprises a heavy chain / light chain pair that has binding specificity for an antigen on an effector cell (e.g., a CD3 protein on a T cell), and a heavy chain derived from a heavy chain-only antibody that comprises an antigen-binding domain that has binding specificity for PSMA.

[0135] 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: 59, the CDR2 sequence of SEQ ID NO: 60, and the CDR3 sequence of SEQ ID NO: 61, in a human VH framework. In some embodiments, the fixed light chain comprises the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64, 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: 65. In some embodiments, the CD3-binding VH domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to the heavy chain variable region sequence of SEQ ID NO: 65. In some embodiments, the fixed light chain comprises the light chain variable region sequence of SEQ ID NO: 66. In some embodiments, the fixed light chain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to the heavy chain variable region sequence of SEQ ID NO: 66.

[0136] Multispecific antibodies comprising the above-described CD3-binding VH domains and light chain variable domains have advantageous properties, as described, for example, in published PCT application WO2018 / 052503, the disclosure of which is incorporated herein by reference in its entirety. Any of the multispecific antibodies and antigen-binding domains described herein that have binding affinity for PSMA can be combined with the CD3-binding domains and fixed light chain domains described herein (see, e.g., Tables 4 and 5), as well as additional sequences such as those shown in Tables 6 and 7, to generate multispecific antibodies with binding affinity for one or more PSMA epitopes and CD3. TIFF0007776987000007.tif38170TIFF0007776987000008.tif51170TIFF0007776987000009.tif216170TIFF000777698700 0010.tif215170TIFF0007776987000011.tif238170TIFF0007776987000012.tif241170TIFF0007776987000013.tif129170

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

[0138] In some embodiments in which the antibody of the present invention is a bispecific antibody, one arm of the antibody (one binding moiety or one binding unit) is specific for human PSMA, and the other arm can 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, but are not limited to, cancer cells, including cells from solid tumors, e.g., prostate tumors, as described below. In some embodiments, one arm of the antibody (one binding moiety or one binding unit) is specific for human PSMA, and the other arm is specific for CD3.

[0139] In some embodiments, the antibody comprises an anti-CD3 light chain polypeptide comprising the sequence of SEQ ID NO: 66 linked to the sequence of SEQ ID NO: 79, an anti-CD3 heavy chain polypeptide comprising the sequence of any one of SEQ ID NOs: 80, 81, 82, 83, 84, or 85, and an anti-PSMA heavy chain polypeptide comprising the sequence of any one of SEQ ID NOs: 24-58 in a monovalent or bivalent configuration linked to the sequence of any one of SEQ ID NOs: 75, 76, 77, 78, 84, or 85. These sequences can be combined in various ways to generate bispecific antibodies of desired IgG subclasses, e.g., IgG1, IgG4, silenced IgG1, or silenced IgG4. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 86, a second polypeptide comprising SEQ ID NO: 87, and a third polypeptide comprising SEQ ID NO: 88, 89, 90, 91, 92, or 93. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 86, a second polypeptide consisting of SEQ ID NO: 87, and a third polypeptide consisting of SEQ ID NO: 88, 89, 90, 91, 92 or 93.

[0140] Various forms 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 multiples 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 cell death of cells expressing PSMA.

[0141] 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, including transgenic mice and rats, preferably rats, in which endogenous immunoglobulin genes have been knocked out or disabled. In a preferred embodiment, the heavy chain antibodies herein are produced in UniRat™, which has silenced endogenous immunoglobulin genes and uses a human immunoglobulin heavy chain transgene to express a diverse, naturally optimized repertoire of fully human HCAbs. While endogenous immunoglobulin loci in rats can be knocked out or silenced using various techniques, in UniRat™, zinc finger (endo)nuclease (ZNF) technology was used to inactivate the endogenous rat heavy chain J locus, light chain Cκ locus, and light chain Cλ locus. ZNF constructs for microinjection into oocytes can generate IgH and IgL knockout (KO) strains. For details, see, e.g., Geurts et al., 2009, Science 325:433. Characterization of Ig heavy chain knockout rats has been reported by Menoret et al., 2010, Eur. J. Immunol. 40:2932-2941. An advantage of ZNF technology is that non-homologous end joining to silence genes or loci via deletions of up to several kb can also provide target sites for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). Human heavy chain antibodies produced in UniRat™, called UniAbs™, can bind to epitopes that cannot be targeted by conventional antibodies. Their high specificity, affinity, and small size make them ideal for mono- and multispecific applications.

[0142] In addition to UniAbs™, specifically included herein are camelid VHH frameworks and mutations, as well as heavy chain-only antibodies lacking their functional VH regions. Such heavy chain-only antibodies can be produced in transgenic rats or mice containing a fully human heavy chain-only locus, as described, for example, in WO 2006 / 008548, although other transgenic mammals, such as rabbits, guinea pigs, and rats, can also be used, with rats and mice being preferred. Heavy chain-only antibodies containing 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.

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

[0144] In certain embodiments, heavy chain antibodies of the present invention, including UniAbs™, have a substitution of the native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Kabat numbering system) 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's light chain constant region, but in HCAbs, it is surface-exposed and is used, at least in part, to prevent undesired aggregation of HCAbs and association with the light chain. The substituted amino acid residue is preferably charged, more preferably positively charged, such as lysine (Lys, K), arginine (Arg, R), or histidine (His, H), preferably arginine (R). In a preferred embodiment, heavy chain-only antibodies derived from transgenic animals contain a Trp to Arg mutation at position 101. The resulting HCAbs preferably have high antigen-binding affinity and solubility under physiological conditions without aggregation.

[0145] As part of the present invention, human IgG anti-PSMA heavy chain antibodies (UniAbs™) with unique sequences derived from UniRat™ animals were identified that bind to human PSMA in ELISA protein and cell binding assays. The identified heavy chain variable region (VH) sequences are all positive for human PSMA protein binding and / or binding to PSMA+ cells, and negative for binding to cells that do not express PSMA. See, e.g., Table 8.

[0146] 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 used. Using this approach, a set of antibodies can be classified into those that compete with a reference antibody and those that do not. Non-competing antibodies are identified as those that bind to a distinct epitope that does not overlap with the epitope bound by the reference antibody. Often, one antibody is immobilized and allowed to bind to the antigen, and a second, labeled (e.g., biotinylated) antibody is tested for its ability to bind to the captured antigen in an ELISA assay. This can also be performed using surface plasmon resonance (SPR) platforms such as the ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and MX96 SPR Imager (Ibis Technologies BV), as well as biolayer interferometry platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). See the Examples herein for details.

[0147] Generally, an antibody "competes" with a reference antibody if it causes about a 15-100% reduction in binding of the reference antibody to the 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.

[0148] Pharmaceutical Compositions, Uses and Methods of Treatment It is another aspect of the present invention 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 is exemplified by, but not limited to, an adjuvant, a solid carrier, water, a buffer, or other carrier used in the art to carry therapeutic ingredients, or a combination thereof.

[0149] 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 binding specificity for a binding target on an effector cell (e.g., a binding target on a T cell, e.g., the CD3 protein on a T cell).

[0150] Pharmaceutical compositions of antibodies used according to the present invention are prepared for storage, for example, in the form of a lyophilized formulation or aqueous solution, by mixing the protein having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (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 octadecyldimethylbenzyl ammonium 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, These include 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).

[0151] Pharmaceutical compositions for oral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., single-administered doses). Formulation depends on the selected route of administration. The antibodies herein can be administered by intravenous injection or infusion, or subcutaneously. For administration by injection, the antibodies herein can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain carriers, excipients, or stabilizers, as described above. Alternatively, the antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0152] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used for heavy chain antibodies, including the UniAbs™, of the present invention. Subcutaneous antibody formulations are described, for example, in U.S. Patent No. 20160355591 and U.S. Patent No. 20160166689.

[0153] 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 disorders further described herein.

[0154] PSMA is a type II transmembrane protein expressed in prostate epithelial tissue and upregulated in the neovasculature of prostate cancer and solid tumors. It is also expressed at low levels in healthy tissues such as the brain, kidney, and salivary gland. However, its overexpression in malignant prostate tissue makes it an attractive target for prostate cancer therapeutics. Its high expression in malignant neovasculature may also make it suitable 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 prostate cancer imaging and treatment (e.g., Hofman et al., 2018 PMID:29752180).

[0155] 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.

[0156] The effective dose of the compositions of the present invention for treating a disease will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, such as companion animals such as dogs, cats, and horses, and laboratory mammals such as rabbits, mice, and rats, can also be treated. Treatment doses can be titrated to optimize safety and efficacy.

[0157] Dosage levels can be easily determined by those skilled in the art, but can be varied as needed, for example, to accommodate changes in the subject's response to treatment. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the host being treated and the specific mode of administration. Generally, a unit dosage form contains about 1 mg to about 500 mg of active ingredient.

[0158] In some embodiments, the therapeutic dose of the agent can range from about 0.0001 to 100 mg / kg of host body weight, more typically 0.01 to 5 mg / kg. For example, the dose 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 determined by measuring the blood level of the therapeutic agent in the patient. Alternatively, therapeutic agents of the present invention can be administered as sustained-release formulations, in which case less frequent administration is required. The dose and frequency will vary depending on the half-life of the polypeptide in the patient.

[0159] Generally, the compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolving or suspending 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, such as polylactides, polyglycolides, or copolymers, to enhance adjuvant effects, as described above. Langer, Science 249:1527, 1990, and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of the present invention can be administered in the form of depot injections or implants, which can be formulated in a manner that allows sustained or pulsed release of the active ingredient. The pharmaceutical compositions are generally sterile, substantially isotonic, and formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0160] The toxicity of the antibodies and antibody constructs described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used in formulating a non-toxic dosage range for use in humans. The dosage of the antibodies described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition.

[0161] Compositions for administration will generally contain the antibody or other abrasive dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions (pH adjusting and buffering agents, toxicity adjusting agents, etc., e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.). The concentration of the active agent in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, body weight, etc., 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)).

[0162] Also within the scope of the present invention are kits containing the active agents of the present invention and their formulations and instructions for use. The kits may further include at least one additional reagent, such as a chemotherapeutic agent. The kits typically include a label indicating the intended use of the contents of the kit. As used herein, the term "label" includes any writing or recorded material supplied on or with the kit, or otherwise accompanying the kit.

[0163] Now that the present invention is fully described, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. [Example]

[0164] Materials and Methods Example 1: UniRat™ Immunization with Recombinant Human PSMA Twelve UniRat™ animals were immunized with recombinant human PSMA protein fused to a his-tag (R&D Systems catalog number: 4234-ZN). Animals were immunized twice a week for eight weeks. After a 35-day immunization period, serum was collected from the rats and serum titers were measured.

[0165] Serum titer measurement results Summary information on serum titers is shown in Figure 1, Panels A-B. In the graphs shown in Figure 1, Panels A-B, each line represents an individual animal. The graph legend indicates the individual animal's ID number. A 12-point dilution series of sera was tested for binding activity by ELISA against the huPSMA+His-tagged protein and His-tagged off-target proteins. A range of serum reactivity levels was observed against the human PSMA protein within this group of animals. No serum reactivity was observed against His-tagged off-target proteins.

[0166] Example 2: Flow cytometry analysis of binding to PSMA-positive and -negative cells by anti-PSMA UniAbs™ Binding to PSMA-positive cells was assessed by flow cytometry (Guava easyCyte 8HT, EMD Millipore) using LNCaP (ATCC:CRL-1740), 22Rv1 (ATCC CRL-2505), and PC3 (ATCC CRL-1435) cell lines stably transfected to express human PSMA or DU-145 (ATCC HTB-81) cell lines. Briefly, 50,000 target cells were stained with serial dilutions of purified UniAbs™ for 30 minutes at 4°C. After incubation, cells were washed twice with flow cytometry buffer (1x PBS, 1% BSA, 0.1% NaN3) and stained with R-phycoerythrin (PE)-conjugated goat F(ab')2 anti-human IgG (Southern Biotech, catalog no. 2042-09) to detect cell-bound antibodies. After 20 min of incubation at 4°C, cells were washed twice with flow cytometry buffer, and mean fluorescence intensity (MFI) was measured by flow cytometry. The MFI of cells stained with secondary antibody alone was used to measure background signal, and binding of each antibody was converted to fold background. Binding to cynomolgus PSMA-positive cells was measured using the same protocol with the following modification: target cells were derived from Freestyle 293-F cells (ThermoFisher R79007) transiently transfected to express the extracellular domain of cynomolgus PSMA. In some experiments, EC50 values ​​were calculated using GraphPad Prism 7.

[0167] Table 8 summarizes the target binding activity of several anti-PSMA heavy chain antibodies (HCAbs) described herein. Column 1 shows the clone ID of the HCAb. Column 2 shows the binding to LNCaP cells measured as fold over background MFI signal. TIFF0007776987000014.tif214170

[0168] As shown in Figure 2, panels A and B, the differences in binding to cynomolgus monkey PSMA support the differences in the human PSMA epitopes recognized by HCAbs 346181 and 345497.

[0169] Example 3: Binding of recombinant proteins by Biolayer Interferometry (BLI) Biolayer interferometry was used to evaluate binding competition between the two antibody families of which clone ID345497 and clone ID346181 are members. Antigen-antibody epitope binning analysis was performed on an Octet QK-384 (ForteBio). Briefly, the anti-pentaHIS capture (HIS1K) sensor ("Penta HIS" disclosed as SEQ ID NO: 95) Antigen-recombinant human PSMA (R&D Systems catalog number: 4234-ZN) was immobilized for 120 seconds using a fluorophore. After a baseline reading, the sensor was immersed in a solution containing antibody 1 (325867) for 300 seconds, and another baseline was established for 60 seconds. The sensor was then immersed in a well containing either antibody 1 (325867) as a positive blocking control or antibody 2 (325920). Association and dissociation rates were measured for 300 and 600 seconds, respectively. Data analysis was performed using Octet Data Analysis HT v11.0 (ForteBio). As shown in Figure 3, 325920 bound to the PSMA protein pre-bound with antibody 325867, indicating that these two antibodies recognize non-overlapping epitopes on PSMA. The binding signal shift is reported in nanometers.

[0170] Example 4: Composition of biparatopic and bivalent anti-PSMA antibodies As shown in Table 9, clone ID350123 consists of the clone ID346181 sequence linked to the clone ID345497 sequence by the bridging sequence GGGGSGGGGS (SEQ ID NO: 74). Clone ID350122 consists of two repeats of clone ID346181 linked by the same linker sequence. Clone ID350123 is biparatopic because it consists of two anti-PSMA domains that recognize different epitopes on PSMA. Clone ID350122 is bivalent but not biparatopic because it consists of tandem identical anti-PSMA domains. Schematic diagrams of various anti-PSMA x anti-CD3 antibodies are shown in Figure 5, panels A-C. TIFF0007776987000015.tif43170

[0171] Example 5: Measurement of affinity to cell surface-expressed human PSMA PSMA cell surface affinity was measured by Scatchard analysis using the human prostate cancer cell line 22Rv1. First, the PSMA×CD3 polyspecific antibody was labeled with Alexa Fluor 488 using the Alexa Fluor 488 5-SDP Ester kit (ThermoFisher A30052). Binding to 22Rv1 was then assessed by flow cytometry (Guava easyCyte 8HT, EMD Millipore). Briefly, 100,000 target cells were stained with a dilution series of Alexa Fluor 488-labeled polyspecific antibody for 1 hour at 4°C. After incubation, the cells were washed twice with flow cytometry buffer, and then the mean fluorescence intensity was measured by flow cytometry.

[0172] To establish a standard curve for calculating equivalent molecules of soluble fluorophore (MESF), Bangs Lab Quantum Alex Fluor 488 MESF bead populations 1–4 were combined in a single tube and run on a Guava easyCyte 8HT. Blank beads were analyzed in a separate tube. The MFI of each bead population was measured for the FITC channel. A linear regression of Log10(MFI) against Log10(MESF) was plotted using GraphPad Prism 7.

[0173] The MFI of each experimental sample was interpolated with the standard curve to determine the MESF for each sample. The average number of bound antibodies per cell (ABC) was then calculated by dividing the average MESF by the degree of antibody labeling (DOL). The total concentration of bound antibody was determined by multiplying the number of ABCs by the cell concentration. The free antibody concentration was calculated by subtracting the bound antibody concentration from the staining concentration (starting dose). The free antibody concentration was plotted against the bound antibody concentration in GraphPad Prism 7. The resulting plot was fitted to a nonlinear regression single-site-specific binding function to determine the affinity, as shown in Figure 4, panels A and B.

[0174] Example 6: Multispecific antibody-mediated cell killing of PSMA-positive prostate tumor cells by T cell redirection Assays using resting T cells Target cells were seeded at 15,000 cells per well in 96-well plates and grown overnight at 37°C. After incubation, a large amount of multispecific antibody was added along with resting human T cells at a 10:1 effector-to-target cell ratio and incubated for an additional 48 or 72 hours at 37°C (48 hours for assays with LNCaP, MDA-PCa-2b, and PC3-PSMA cells, and 72 hours for assays with 22Rv1 cells). Cell death was measured using either the cell proliferation reagent WST-1 (Sigma catalog number: 11644807001) or flow cytometry. In some experiments, after incubation but prior to analysis of target cell viability, a small sample of each supernatant was collected and saved for analysis of cytokine production. When cell viability was analyzed with the WST-1 reagent, the reagent stock was added to each well at a 1:10 dilution and incubated for 90 minutes at 37°C. The absorbance was then measured at 450 nm (reference 690 nm) and the specific lysis rate (%) was calculated.

[0175] When target cell viability was analyzed by flow cytometry, target cells were then labeled before the assay was initiated with the membrane dye DiR (ThermoFisher D12731). After incubation with T cells and antibodies, the supernatant was either saved for cytokine analysis or discarded. The wells were then washed once to collect dead tumor cells and T cells and transfer them to the flow cytometry plate. Remaining adherent tumor cells were trypsinized and then added to the corresponding wells of the flow cytometry plate. Dead cells were stained using Annexin V reagent, and flow cytometry was performed (BD FACSCelesta) to quantify the percentage of dead tumor cells in each sample gated by DiR staining. Wells containing untreated target cells were used to normalize to spontaneous cell death. In some experiments, a negative control antibody was used, consisting of the same CD3-targeting arm as the PSMAxCD3 multispecific molecule, but with the tumor-targeting arm replaced with a VH specific for the HIV protein gp120.

[0176] Figure 7 shows T cell-mediated lysis of PSMA-positive cells using unstimulated T cells. Unstimulated human T cells were incubated with PSMA-expressing cells (LNCaP) and various concentrations of multispecific antibodies. The biparatopic anti-PSMA x CD3 antibody (350123 x CD3) was superior to the monoparatopic anti-PSMA x CD3 antibody (346181 x CD3).

[0177] Assays using pre-activated T cells Human pan-T cells were preactivated with plate-bound OKT3 and IL-2 for 3 days, followed by an additional day of incubation in fresh IL-2. Target cells were trypsinized, loaded with Calcein-AM (ThermoFisher C3100MP), mixed with activated T cells at a 20:1 E:T ratio, and added to wells of a 96-well plate. A dilution series of different polyspecific antibodies was added, followed by incubation at 37°C for 4 hours. The supernatant was then transferred to a black 96-well plate, and absorbance was measured at 480 nm / 520 nm ex / em to quantify calcein release. Target cells incubated in the absence of T cells were used to normalize spontaneous calcein release from intact tumor cells. Addition of 2% Triton-X to control wells containing target cells allowed the calculation of the calcein signal corresponding to maximum cell lysis. This value was used to report each experimental well as a percentage of maximum cell lysis. Data analysis was performed using GraphPad Prism 7.

[0178] Figure 6 shows T cell-mediated lysis of PSMA-positive cells using preactivated T cells. Preactivated human T cells were incubated with human PSMA-expressing cells (LNCaP) and various concentrations of multispecific antibodies. Tumor cell death was measured by calcein release and normalized to the spontaneous release of tumor cells in the absence of T cells. The biparatopic anti-PSMA x CD3 antibody (350123 x CD3) outperformed both monoparatopic PSMA x CD3 antibodies.

[0179] Figure 8 shows that multispecific antibodies do not lyse PSMA-negative cells. Preactivated human T cells were incubated with PSMA-negative prostate cancer cells (DU145) and various concentrations of multispecific antibodies. None of the antibodies tested induced lysis of these cells.

[0180] Figure 9 shows the binding of PSMAxCD3 multispecific antibodies to PSMA-positive and -negative cells. The multispecific anti-PSMAxCD3 antibody shows binding to PSMA-positive prostate tumor cells (22Rv1) but not to PSMA-negative prostate tumor cells (DU145). The biparatopic molecule (350123) showed the strongest on-target cell binding.

[0181] Figure 10 shows T cell-mediated lysis of PSMA-positive cells. The data in Figure 10 demonstrate that binding to PSMA via two different epitopes increases cell killing compared to a bivalent but monospecific version of the antibody.

[0182] Example 7: Monoparatopic PSMAxCD3 bispecific antibodies do not induce cytokine production compared to biparatopic PSMAxCD3 polyspecific antibodies Cytokine production was analyzed in tumor cytotoxicity assays using resting T cells. The design of these assays has been described in detail elsewhere. Supernatants were collected at the completion of the assay (after 72 hours of incubation for assays using 22Rv1 cells and 48 hours for all other cell lines). ELISA kits were used to detect IL-2 (Biolegend 431804) and IFNγ (Biolegend 430104) according to the manufacturer's protocol. Test supernatants were diluted prior to ELISA analysis so that cytokine levels fell within the linear portion of the standard curve provided with each kit. In some cases, cytokines could not be detected in the test wells, and values ​​were reported as below the lower limit of quantitation of the assay.

[0183] Panels A-C of Figure 12 show T cell-mediated lysis of PSMA-positive cells and a comparison with cytokine production. Multispecific PSMAxCD3 antibodies induce T cell-mediated lysis of the PSMA-positive prostate cancer cell line LNCaP. The biparatopic molecule (350123) more potently stimulated tumor cell killing compared with the monoparatopic molecule (346181), but also induced higher levels of the cytokines interferon-γ (IFNγ) and interleukin-2 (IL-2), as shown in Panels B and C of Figure 12.

[0184] Table 10 shows T cell-mediated lysis and cytokine production against four PSMA-positive prostate tumor cell lines. PSMAxCD3 multispecific antibodies were tested in in vitro tumor cell cytotoxicity assays using unstimulated T cells and a range of antibody doses against a panel of four PSMA-positive tumor cell lines. After 72 hours (22Rv1) or 48 hours (MDA-PCa-2b, LNCAP, PC3-PSMA), the percentage of tumor cell death was calculated and reported as an EC50. The highest achieved killing rate was also reported. Supernatants from these test wells were collected and analyzed by ELISA for the cytokines interferon-γ (IFNγ) or interleukin-2 (IL-2). The monoparatopic molecule (3461881) induced similar levels of tumor cytotoxicity against all four cell lines tested compared with the biparatopic molecule, but had a higher EC50 for cytokine production and, in most cases, a lower level of stimulation of maximal cytokine production.

[0185] TIFF0007776987000016.tif94170

[0186] Example 8: PSMAxCD3 multispecific antibodies induce T cell proliferation PSMA-positive tumor cells were seeded at 25,000 cells per well in a 96-well plate and grown overnight at 37°C. Human pan T cells (Miltenyi 130-096-535) isolated from resting PBMCs were labeled with the lineage-tracking dye CFSE according to the manufacturer's instructions (ThermoFisher C34554). 100,000 labeled pan T cells were then added to wells containing tumor cells, followed by a series of diluted antibodies and incubation at 37°C and 8% CO2. After 5 days of incubation, the cells were gently mixed and transferred to a flow cytometry plate. The cells were pelleted, the supernatant removed, and then stained with anti-CD8 conjugated to APC (Biolegend 301049) and anti-CD4 conjugated to PE (Biolegend 317410) for 20 minutes on ice. The cells were then washed and resuspended in flow cytometry buffer (BD FACSCelesta) for analysis. Cells were gated by forward and side scatter and CD4 or CD8 expression. The percentage of proliferated T cells, as indicated by positive CD4 or CD8 staining and low or negative CFSE signal, was calculated for the entire T cell population and for CD4 and CD8 subsets. Flow cytometry data were analyzed using FlowJo and plotted in GraphPad Prism 7.

[0187] Panels A–D of Figure 11 show that a PSMA×CD3 polyspecific antibody stimulates T cell proliferation in the presence of PSMA-positive tumor cells, and a monoparatopic PSMA bispecific antibody preferentially activates CD3 T cells. The polyspecific antibody was incubated with PSMA-expressing tumor cells and T cells labeled with the lineage-tracing dye CFSE. After 5 days of incubation, T cell proliferation and the composition of expanded T cells (CD8+ vs. CD4+) were analyzed by flow cytometry. Panels A and B show total T cell proliferation, while panels C and D show the ratio of CD8+ to CD4+ T cells in expanded wells. The horizontal dashed line indicates the CD8:CD4 ratio of unstimulated T cells, which is approximately 1:2 (actual value = 0.64). The monoparatopic PSMAxCD3 bispecific antibody (346181) preferentially activates CD8 T cells (CD8:CD4 ratio after expansion is approximately 2:1), whereas the biparatopic PSMAxCD3 polyspecific antibody (350123) less preferentially activates CD8+ T cells (CD8:CD4 ratio is approximately 1:1).

[0188] Example 9: Multispecific antibodies inhibit prostate tumor growth in xenograft models Five- to six-week-old male immunodeficient CIEA-NOG mice (Taconic) were implanted subcutaneously with 10 million 22Rv1 cells in the right lower flank, and 10 million human PBMCs were added via tail vein injection one day after tumor implantation. The animals were treated with 100 μg of multispecific antibody or vehicle on days 1, 5, 9, and 13, starting via tail vein injection one day after tumor implantation. Tumor volume was quantified using calipers and recorded for 25 days.

[0189] Figure 13 shows the results of a 22Rv1 tumor xenograft model. The biparatopic PSMAxCD3 molecule (350123) demonstrated inhibition of 22Rv1 tumor growth in the tumor xenograft model. Three mice were tested for each treatment group, and the change in tumor volume for each animal was plotted in cubic millimeters. Animals were administered PBMCs on day 1 after tumor implantation and treated with antibody on days 1, 5, 9, and 13. Two of the three animals treated with the multispecific antibody demonstrated delayed tumor progression.

[0190] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will recognize numerous variations, changes, and substitutions that do not depart from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A multispecific antibody that binds to the CD3 protein and the PSMA protein: (a)(i) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 11, and a CDR3 sequence of SEQ ID NO: 18; or (ii) a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 12, and a CDR3 sequence of SEQ ID NO: 18; or (iii) a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 11, and a CDR3 sequence of SEQ ID NO: 18; The heavy chain variable region of an anti-PSMA heavy chain antibody comprising and (b) a CD3-binding light chain variable region and a CD3-binding heavy chain variable region that pairs with the CD3-binding light chain variable region, the CD3-binding light chain variable region comprises the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64; and the CD3-binding heavy chain variable region comprises the CDR1 sequence of SEQ ID NO:59, the CDR2 sequence of SEQ ID NO:60, and the CDR3 sequence of SEQ ID NO:61; and the CD3-binding light chain variable region comprises a sequence having at least 95% sequence identity to the light chain variable region of SEQ ID NO: 66; and a CD3-binding light chain variable region and a CD3-binding heavy chain variable region, wherein the CD3-binding heavy chain variable region comprises a sequence having at least 95% sequence identity with the heavy chain variable region sequence of SEQ ID NO: 65; A multispecific antibody comprising:

2. The multispecific antibody of claim 1 , which is bispecific.

3. the CD3-binding heavy chain variable region comprises, in a human VH framework, the CDR1 sequence of SEQ ID NO: 59, the CDR2 sequence of SEQ ID NO: 60, and the CDR3 sequence of SEQ ID NO: 61; and 3. The multispecific antibody of claim 1 or 2, wherein the CD3-binding light chain variable region comprises the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64, in a human VL framework.

4. the CD3-binding heavy chain variable region comprises the heavy chain variable region sequence of SEQ ID NO: 65; and 4. The multispecific antibody of claim 1 , wherein the CD3-binding light chain variable region comprises the light chain variable region sequence of SEQ ID NO:

66.

5. The multispecific antibody of any one of claims 1 to 4, wherein the heavy chain variable region of the anti-PSMA heavy chain antibody comprises the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO:

18.

6. 6. The multispecific antibody of any one of claims 1 to 5, wherein the heavy chain variable region of the anti-PSMA heavy chain antibody comprises the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18 in a monovalent configuration.

7. 7. The multispecific antibody of any one of claims 1 to 6, wherein the heavy chain variable region of the anti-PSMA heavy chain antibody comprises a heavy chain variable region sequence having at least 95% sequence identity to the heavy chain variable region sequence of SEQ ID NO:

25.

8. The multispecific antibody of any one of claims 1 to 7, wherein the heavy chain variable region of the anti-PSMA heavy chain antibody comprises the heavy chain variable region sequence of SEQ ID NO:

25.

9. 9. The multispecific antibody of claim 1, further comprising a heavy chain constant region sequence and not comprising a CH1 sequence.

10. the CD3-binding heavy chain variable region comprises the heavy chain variable region sequence of SEQ ID NO: 65; and the CD3-binding light chain variable region comprises the light chain variable region sequence of SEQ ID NO: 66; and 3. The multispecific antibody of claim 1 or 2, wherein the heavy chain variable region of the anti-PSMA heavy chain antibody comprises the heavy chain variable region sequence of SEQ ID NO:

25.

11. a CD3-binding heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 59, the CDR2 sequence of SEQ ID NO: 60, and the CDR3 sequence of SEQ ID NO: 61, in a human VH framework; a CD3-binding light chain variable region comprising, in a human VL framework, the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64; and a heavy chain variable region of an anti-PSMA heavy chain antibody comprising, in a human VH framework, the CDR1 sequence of SEQ ID NO:2, the CDR2 sequence of SEQ ID NO:11, and the CDR3 sequence of SEQ ID NO:18; The multispecific antibody of claim 10, comprising:

12. 12. The multispecific antibody of any one of claims 1 to 11, comprising a silenced Fc, wherein said silenced Fc has been mutated to retain activity with respect to extended serum half-life, but does not activate high affinity Fc receptors or has reduced affinity for Fc receptors.

13. The multispecific antibody according to any one of claims 1 to 12, which is a three-chain antibody-like molecule.

14. a first polypeptide comprising the sequence of SEQ ID NO: 86; A second polypeptide comprising the sequence of SEQ ID NO: 87; and 14. The multispecific antibody of claim 1, 2 or 13, comprising a third polypeptide comprising the sequence of SEQ ID NO: 88 or 89.

15. 15. The multispecific antibody of claim 14, wherein the third polypeptide comprises the sequence of SEQ ID NO:

88.

16. a first polypeptide consisting of the sequence of SEQ ID NO: 86; A second polypeptide consisting of the sequence of SEQ ID NO: 87; and 16. The multispecific antibody of any one of claims 1, 2 or 13 to 15, comprising a third polypeptide consisting of the sequence of SEQ ID NO:

88.

17. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1 to 16 and a pharmaceutically acceptable excipient.

18. A pharmaceutical composition for use in the treatment of a disorder characterized by expression of PSMA, the pharmaceutical composition comprising a multispecific antibody according to any one of claims 1 to 16.

19. 19. The pharmaceutical composition for use according to claim 18, wherein the disorder is prostate cancer.

20. 19. The pharmaceutical composition for use according to claim 18, wherein the disorder is a solid tumor.

21. A polynucleotide encoding the multispecific antibody of any one of claims 1 to 16.

22. A vector comprising the polynucleotide of claim 21.

23. A cell comprising the vector of claim 22.

24. 27. A method for producing a multispecific antibody according to any one of claims 1 to 16, comprising growing the cells of claim 23 under conditions permissive for expression of the multispecific antibody, and isolating said multispecific antibody from said cells.

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