EA046268B1 - ANTIBODIES CONTAINING ONLY HEAVY CHAINS THAT BIND TO PSMA - Google Patents

Description

Cross reference to related applications

This application claims priority to 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.

List of sequences

This application contains a sequence listing that has been submitted electronically in ASCII format and is therefore incorporated herein by reference in its entirety. The specified ASCII copy, created on 2020-07-09, is named TNO-0016-WO_SL.txt and is 121310 bytes in size.

Field of technology

The present invention relates to human antibodies containing only heavy chains (for example, UniAb™) that bind to PSMA (PSMA -prostate specific membrane antigen). The present invention also relates to methods for producing such antibodies, to compositions, including pharmaceutical compositions, containing such antibodies, and their use for the treatment of disorders that are characterized by the expression of PSMA.

State of the art

PSMA.

PSMA, also known as prostate-specific membrane antigen and glutamate carboxypeptidase II (UniProt Q04609), is a type II transmembrane protein that has N-acetylated alpha-linked acid dipeptidase, folate hydrolase, and dipeptidyl peptidase activities. It is encoded by the FOLH1 gene in humans and consists of a cytoplasmic domain of 19 amino acids, a transmembrane portion of 24 amino acids, and an extracellular portion of 707 amino acids. The protein is enzymatically active as a non-covalent homodimer. PSMA is expressed in prostate epithelial tissue and its expression is upregulated in prostate malignancy and in new vasculature of solid tumors. It is also expressed at low levels in normal tissues such as the brain, kidney and salivary glands, but its overexpression in prostate malignancy makes it an attractive target for the therapeutic treatment of prostate malignancy. It may also be relevant for therapy or imaging of solid tumors, given its high expression in malignant neovascularization. Monoclonal antibodies, antibody-drug conjugates and chimeric antigen receptor T cells targeting PSMA have been described in the context of the treatment of metastatic prostate malignancy (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 explored for imaging and treatment of prostate malignancy (e.g., Hofman et al., 2018 PMID: 29752180).

Antibodies containing only heavy chains.

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

It is known, however, that serum from camelids (a subgroup of Tylopoda that includes camels, dromedaries and llamas) contains a major type of antibody consisting entirely of paired H-chains (heavy chain-only antibodies, or UniAb™). UniAb™ Camelidae (Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanaco, Lama alpaca and Lama vicugna) have a unique structure consisting of one variable domain (VHH), a hinge region and two constant domains (CH2 and CH3), which are highly homologous to the domains CH2 and CH3 of classical antibodies. These UniAbs™ do not have the first constant region domain (CH1), which is present in the genome but is removed during mRNA processing. The absence of the CH1 domain explains the absence of a light chain in UniAb™, since this domain is the attachment site for the light chain constant domain. Such UniAbs™ have naturally evolved to impart antigen-binding specificity and high affinity to three CDRs from conventional antibodies, or fragments thereof (Muyldermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther 5:111- 124). Cartilaginous fish such as sharks have also developed a special type of immunoglobulin, designated IgNAR, which lacks light polypeptide chains and is composed entirely of heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce a single heavy chain polypeptide variable domain (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)).

The ability of antibodies containing only heavy chains, lacking a light chain, to bind antigen was established in the 1960s (Jaton et al. (1968) Biochemistry, 7, 4185-4195). The immunoglobulin heavy chain, physically separated from the light chain, retained 80% of the antigen-binding activity relative to

- 1 046268 strongly tetrameric antibody. Sitia et al. (1990) Cell, 60, 781-790 demonstrated that deletion of the CH1 domain from a rearranged mouse μ gene results in a heavy chain-only antibody lacking a light chain in mammalian cell culture. The antibodies produced retained VH binding specificity and effector functions.

Heavy chain-only antibodies with high specificity and affinity can be generated against various antigens by immunization (van der Linden, R.H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH portion can be easily cloned and expressed in yeast (Frenken, L.G.J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility and stability are significantly higher than those of the classical F(ab) or Fv fragments (Ghahroudi, M.A. et al. FEBS Lett. 414, 521-526 (1997)).

Mice in which the λ (lambda) light (G) chain locus and/or λ and k (kappa) L chain loci have been functionally silenced, and the antibodies produced by such mice are described in US Patent Nos. 7,541,513 and 8,367,888. the production of heavy chain-only antibodies in mice and rats has been described, for example, in WO2006008548; publication of US patent application 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. Generation of knockout rats by embryonic microinjection of zinc finger nuclease is described in Geurts et al., 2009, Science, 325(5939):433. Soluble heavy chain-only antibodies and transgenic rodents containing a heterologous heavy chain locus producing such antibodies are described in US Pat. Nos. 8,883,150 and 9,365,655. CAR-T structures containing single-domain antibodies as the binding (targeting) domain are described, e.g. , in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630–2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378–386.

The essence of the invention

Aspects of the invention relate to heavy chain-only antibodies, including, but not limited to, UniAb™, with binding affinity for PSMA. Further aspects of the invention relate to methods for producing such antibodies, compositions containing such antibodies, and their use for the treatment of disorders that are characterized by the expression of PSMA.

In some embodiments, an antibody that binds to PSMA comprises a first heavy chain variable region comprising: (a) a CDR1 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NO: 1-10; and/or (b) a CDR2 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NO: 11-17; and/or (c) CDR3 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NO: 18-23. In some embodiments, the antibody further comprises a second heavy chain variable region comprising: (a) a CDR1 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NO: 1-10; and/or (b) a CDR2 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NO: 11-17; and/or (c) CDR3 having two or fewer substitutions in any of the amino acid sequences of SEQ ID NO: 18-23. In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in the human framework. In some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence.

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 NO: 1-10; and/or (b) a CDR2 sequence selected from the group consisting of SEQ ID NO: 11-17; and/or (c) a CDR3 sequence selected from the group consisting of SEQ ID NO: 18-23.

In some embodiments, the antibody further comprises a second heavy chain variable region that comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NO: 1-10; and/or (b) a CDR2 sequence selected from the group consisting of SEQ ID NO: 11-17; and/or (c) a CDR3 sequence selected from the group consisting of SEQ ID NO: 1823.

In some embodiments, the antibody comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NO: 1-10; and (b) a CDR2 sequence selected from the group consisting of SEQ ID NO: 11-17; and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18-23. In some embodiments, the antibody comprises a second heavy chain variable region that comprises: (a) a CDR1 sequence selected from the group consisting of SEQ ID NO: 1-10; and (b) a CDR2 sequence selected from the group consisting of SEQ ID NO:

11-17; and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18-23.

In some embodiments, the antibody comprises: (a) 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; or (b) a CDR1 sequence of SEQ ID NO: 7, a CDR2 sequence of SEQ ID NO: 15, and a CDR3 sequence of SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 95% identity afterbirth

- 2046268 activity with any of the sequences SEQ ID NO: 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.

In some embodiments, an antibody that binds to PSMA contains a first heavy chain variable region comprising: (a) a CDR1 sequence of the formula:

GGSISS X1 X ₂ Y X ₃ (SEQ ID NO: 67), where X is S or N; X ₂ represents S or N; and X ₃ represents Y or F; and (b) a CDR2 sequence of the formula:

X4 X5 X6 SG X7 T (SEQ ID NO: 68), where X4 represents I or V; X ₅ represents D or Y; X ₆ represents Y or D; and X ₇ is Y or S; and (c) is a CDR3 of the formula:

ARHKAATADFDY (SEQ ID NO: 69), in monovalent or divalent format.

In some embodiments, an antibody that binds to PSMA contains a first heavy chain variable region comprising: (a) a CDR1 sequence of the formula:

GF X1 F X ₂ X ₃ YG (SEQ ID NO: 70), where X ₁ represents S or I or T; X ₂ represents S or T or R or I; and X ₃ represents R or S; and (b) a CDR2 sequence of the formula:

I X4 YDGSN X ₅ (SEQ ID NO: 71), where X4 represents W or S; and X ₅ represents R or K; and (c) a CDR3 sequence of the formula:

AREPRX6GYYYX7X8SGYX9SLDY (SEQ ID NO: 72), where X6 represents I or V; X ₇ represents E or D; X8 represents S or T; and X9 is Y or D, in monovalent or divalent format.

In some embodiments, the PSMA-binding antibody comprises: a first heavy chain variable region comprising: (a) a CDR1 sequence of the formula:

GGSISS X1 X ₂ Y X ₃ (SEQ ID NO: 67), where X ₁ represents S or N; X ₂ represents S or N; and X ₃ represents Y or F; and (b) a CDR2 sequence of the formula:

X4 X5 X6 SG X7 T (SEQ ID NO: 68), where X4 represents I or V; X5 represents D or Y; X6 represents Y or D; and X ₇ is Y or S; and (c) is a CDR3 of the formula:

ARHKAATADFDY (SEQ ID NO: 69), and a second heavy chain variable region containing: (a) a CDR1 sequence of the formula:

GF X1 F X ₂ X ₃ YG (SEQ ID NO: 70), where X1 represents S or I or T; X2 represents S or T or R or I; and X3 is R or S; and (b) a CDR2 sequence of the formula:

I X4 YDGSN X ₅ (SEQ ID NO: 71), where X ₄ represents W or S; and X ₅ represents R or K; and (c) a CDR3 sequence of the formula:

AREPRX6GYYYX7X8SGYX9SLDY (SEQ ID NO: 72), where X6 represents I or V; X ₇ represents E or D; X8 represents S or T; and X9 is Y or D.

In some embodiments, the antibody comprises a first and a second heavy chain variable region, wherein the first heavy chain variable region is located proximal to the N-terminus of the second heavy chain variable region. In some embodiments, the first heavy chain variable region is located proximal to the C-terminus of the second heavy chain variable region.

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 a sequence having two or fewer substitutions in a CDR sequence selected from the group consisting of SEQ ID NO: 1-23.

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

In some embodiments, an antibody that binds to PSMA comprises: a heavy chain variable region comprising 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 in a human VH framework.

In some embodiments, an antibody that binds to PSMA comprises: a heavy chain variable region comprising 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 in a human VH framework in monovalent or divalent configuration.

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

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

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

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 the CD3 protein and the PSMA protein. In some embodiments, the antibody has binding affinity to two different epitopes on the same PSMA protein. In some embodiments, the antibody has binding affinity for an effector cell. 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 CAR-T format.

Aspects of the invention include a bispecific antibody comprising: (i) a heavy chain variable region having binding affinity for CD3, comprising a CDR1 sequence of SEQ ID NO: 59, a CDR2 sequence of SEQ ID NO: 60, and a CDR3 sequence of SEQ ID NO: 61 in human VH frame; (ii) a light chain variable region containing 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 the human VL framework; and (iii) an antigen binding domain of a heavy chain only antibody to PSMA comprising 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, in a human VH framework.

Aspects of the invention include a bispecific antibody comprising: (i) a heavy chain variable region having binding affinity for CD3, comprising a CDR1 sequence of SEQ ID NO: 59, a CDR2 sequence of SEQ ID NO: 60, and a CDR3 sequence of SEQ ID NO: 61 in human VH frame; (ii) a light chain variable region containing 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 the human VL framework; and (iii) an antigen binding domain of a heavy chain only antibody to PSMA 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 divalent configuration.

Aspects of the invention include a bispecific antibody comprising: (i) a heavy chain variable region having binding affinity for CD3, comprising a CDR1 sequence of SEQ ID NO: 59, a CDR2 sequence of SEQ ID NO: 60, and a CDR3 sequence of SEQ ID NO: 61 in human VH frame; (ii) a light chain variable region containing 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 the human VL framework; and (iii) an antigen binding domain of a heavy chain only antibody to PSMA 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.

Aspects of the invention include a bispecific antibody comprising: (i) a heavy chain variable region having binding affinity for CD3, comprising a CDR1 sequence of SEQ ID NO: 59, a CDR2 sequence of SEQ ID NO: 60, and a CDR3 sequence of SEQ ID NO: 61 in human VH frame; (ii) a light chain variable region containing 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 the human VL framework; and (iii) an antigen binding domain of a heavy chain only antibody to PSMA 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 divalent configuration.

Aspects of the invention include a multispecific antibody comprising: (i) a heavy chain variable region having binding affinity for CD3, comprising a CDR1 sequence of SEQ ID NO: 59, a CDR2 sequence of SEQ ID NO: 60, and a CDR3 sequence of SEQ ID NO:

- 4 046268 in human VH frame; (ii) a light chain variable region containing 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 the human VL framework; and (iii) an antigen binding domain of a heavy chain only antibody to PSMA, wherein the antigen binding domain comprises a first and a second antigen binding region in a bivalent configuration, wherein: the first antigen binding region comprises a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: NO: 11 and the CDR3 sequence of SEQ ID NO: 18 in the human VH framework; and the second antigen binding region comprises a CDR1 sequence of SEQ ID NO: 7, a CDR2 sequence of SEQ ID NO: 15, and a CDR3 sequence of SEQ ID NO: 20 in the human VH framework. In certain embodiments, the first antigen-binding region is located proximal to the N-terminus of the second antigen-binding region. In certain other embodiments, the first antigen-binding region is located closer to the C-terminus compared to the second antigen-binding region.

Aspects of the invention include polyspecific or bispecific antibodies in which the first and second antigen binding regions of the heavy chain-only antigen binding domain of the anti-PSMA antibody are connected by a polypeptide linker. In some embodiments, the polypeptide linker is a GS linker. In some embodiments, the GS linker consists of the sequence SEQ ID NO: 73 or SEQ ID NO: 74. In some embodiments, the antigen binding domain of the heavy chain only anti-PSMA antibody is monoparatope and induces less cytokine production compared to the biparatope antigen binding domain . In some embodiments, the antigen binding domain of a heavy chain only antibody in PSMA is monoparatope and increases the number of CD8+ T cells to a greater extent than the biparatope antigen binding domain.

In some embodiments, the antibody is biparatope and has increased affinity for PSMA compared to a monoparatope anti-PSMA antibody. In some embodiments, the antibody is biparatope and has increased effector function compared to a monoparatope anti-PSMA antibody.

Aspects of the invention relate to pharmaceutical compositions containing an antibody described herein.

Aspects of the invention relate to methods of treating a disorder characterized by the expression of PSMA, comprising administering to a subject with 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 the treatment of a disorder characterized by the expression of PSMA. In yet other aspects, the invention provides an antibody described herein for use in treating a disorder characterized by the expression of PSMA. In certain other aspects, the invention provides methods of treatment comprising administering to a person in need an effective dose of an antibody or pharmaceutical composition described herein. In these aspects, and in some embodiments, the disease is prostate cancer.

Aspects of the invention relate to polynucleotides encoding an antibody described herein, vectors containing such polynucleotides, and cells containing such vectors.

Aspects of the invention relate to methods for producing an antibody described herein, comprising culturing a cell described herein under conditions allowing expression of the antibody and isolating the antibody from the cell.

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

These and other aspects will be further explained in the rest of the description, including examples.

Brief description of graphic materials

In fig. 1, panels A-B, are a series of graphs showing serum titer as a function of dilution.

In fig. 2, Panel A, is a graph showing the binding of cells to human PSMA.

In fig. 2, Panel B, is a graph showing the binding of cells to cynomolgus PSMA.

In fig. 3 is a graph showing binding competition between two families of antibodies in accordance with an embodiment of the invention.

In fig. 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, wherein the PSMA arm is monoparatopic and monovalent, according to an embodiment of the invention.

In fig. 4, Panel B, is a Scatchard plot showing the binding affinity of a bispecific antibody having the affinity to PSMA expressed on the cell surface

- 5 046268 binding to CD3 and PSMA, where the arm to PSMA is biparatopic according to an embodiment of the invention.

In fig. 5, panels A-C, show schematic images of: anti-CD3 x monovalent monospecific anti-PSMA antibody (panel A); anti-CD3 x divalent monospecific anti-PSMA antibody (panel B); and anti-CD3 x divalent biparatope anti-PSMA antibody (panel C) in accordance with embodiments of the invention.

In fig. 6 is a graph depicting T cell-mediated lysis of PSMA positive cells using pre-activated T cells.

In fig. 7 is a graph depicting T cell mediated lysis of PSMA positive cells using unstimulated T cells.

In fig. 8 is a graph depicting the percentage of specific lysis of PSMA-negative DU145 cells as a function of the concentration of polyspecific antibodies in the presence of pre-activated T cells.

In fig. 9 is a graph showing the binding of anti-PSMA x CD3 bispecific antibodies to PSMA positive and negative cells.

In fig. 10 is a graph showing T cell-mediated lysis of PSMA positive cells.

In fig. 11, Panel A, is a graph depicting T cell proliferation as a function of antibody concentration.

In fig. 11, Panel B, is a graph depicting T cell proliferation as a function of antibody concentration.

In fig. 11, Panel C, is a graph depicting the ratio of CD8 to CD4 proliferated T cells.

In fig. 11, Panel D, is a graph depicting the ratio of CD8 to CD4 proliferated T cells.

In fig. 12, Panel A, is a graph depicting T cell-mediated lysis of PSMA-positive cells as a function of antibody concentration.

In fig. 12, Panel B, is a graph depicting cytokine (IFN-γ) release as a function of antibody concentration.

In fig. 12, Panel C, is a graph depicting cytokine (IL-2) release as a function of antibody concentration.

In fig. 13 is a graph depicting tumor growth inhibition by 22Rvl in a tumor xenograft model.

Detailed Description of Preferred Embodiments

In the practice of this invention, unless otherwise indicated, standard methods of molecular biology (including recombinant methods), microbiology, cell biology, biochemistry and immunology, which are known to specialists in the art, are used. Such methods are fully described in the literature, for example, in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987, and periodic updates); PCR: The Polymerase Chain Reaction, (Mullis et al., ed., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

When a range of values is given, it is understood that this invention covers every intermediate value to the tenth decimal place of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of that range, as well as any other specified or intermediate value within such specified range. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in the present invention, subject to any specially excluded limit within the specified range. If the specified range includes one or both limits, ranges excluding one or both of these included limits are also included in this invention.

Unless otherwise noted, 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)) .

To provide a thorough understanding of the present invention, numerous specific details are set forth in the following detailed description. However, it will be obvious to one skilled in the art that the present invention can be practiced without one or more of the specified specific details. In other cases, well-known features and procedures well known to those skilled in the art have not been described to avoid making the invention difficult to understand.

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All references contained herein, including patent applications and publications, are incorporated herein by reference in their entirety.

I. Definitions.

By "comprising" it is meant that the listed elements are required for the composition/method/kit, however, other elements may be included within the scope of the claims to form the composition/method/kit and the like.

By the term consisting essentially of is meant the limitation of the described composition or method to specified materials or step-by-step actions that do not significantly affect the main and novel characteristic(s) of the subject matter of the invention.

The term consisting of means the exclusion of any element, step or ingredient not specified in the claims from the composition, method or kit.

Antibody residues in this document are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used to designate a residue in the variable domain (approximately heavy chain residues 1-113) (eg, 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 typically used to designate an amino acid residue in the immunoglobulin heavy chain constant region (eg, EU index presented in Kabat et al., earlier). The Kabat EU index refers to the numbering of amino acid residues of the human IgG1 antibody EU. Unless otherwise indicated, references to residue numbers in the antibody variable domain indicate residue numbering according to the Kabat numbering system. Unless otherwise noted, references to residue numbers in the antibody constant domain indicate residue numbering using the EU numbering system.

Antibodies, also called immunoglobulins, typically contain at least one heavy chain and one light chain, where the N-terminal domain of the heavy and light chains is variable in sequence and is therefore generally referred to as a variable region domain or a heavy chain variable domain. (VH) or light chain variable domain (VL). These two domains typically associate to form a specific binding region, although, as will be discussed herein, specific binding can also be achieved with heavy chain variable sequences only, and various non-natural antibody configurations are known and used in the art.

A functional or biologically active antibody or antigen-binding molecule (including heavy chain-only antibodies and multispecific (e.g., bispecific) triple-chain antibody-like molecules (TCA) described herein) is a molecule capable of exerting one or more of its natural activities in structural , regulatory, biochemical or biophysical events. For example, a functional antibody or other binding molecule, such as TCA, may have the ability to specifically bind an antigen, and the binding may in turn cause or alter a cellular or molecular event, such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, such as 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, such as TCA, to exhibit one or more of its natural activities depends on several factors , including correct folding and assembly of polypeptide chains.

The term antibody is used herein in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain only antibodies, triple chain antibodies, TCA, single chain Fv (scFv), nanoantibodies, etc., and also include antibody fragments if 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.

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, that is, a polypeptide that contains an antigen-binding site that immunospecifically binds an antigen of a target of interest or a portion thereof, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies, associated with an autoimmune disease. The immunoglobulin described herein can be of any type (eg, IgG, IgE, IgM, IgD and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of the immunoglobulin molecule, including engineered subclasses with altered Fc -parts that provide a decrease or increase in the activity of effector cells. The light chains of the antibodies in question may be kappa light chains (V-kappa) or lambda light chains (V-lambda). Immunoglobulins can be obtained from any species. In one aspect, the immunoglobulin is predominantly of human origin.

The term monoclonal antibody as used herein refers to an antibody derived from a population of largely homogeneous antibodies, that is, individual antibodies within a population

- 7 046268 are identical except for mutations occurring due to natural causes, which may be present in small quantities. Monoclonal antibodies are highly specific and directed against a single antigenic site. Additionally, unlike conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. Monoclonal antibodies in accordance with this invention can be obtained by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, can also be obtained, for example, by methods for producing a recombinant protein (see, for example, US patent No. 4816567).

The term variable, as used herein in connection with antibodies, refers to the fact that the sequences of certain portions of the variable domains of an antibody vary widely between antibodies and contribute to the binding and specificity of each particular antibody for its particular antigen. At the same time, variability is not evenly distributed across the variable domains of antibodies. It is concentrated in three segments of variable domains of both the light and heavy chains, called hypervariable regions. The more conserved portions of the variable domains are called framework regions (FR). The variable domains of the native light and heavy chains each contain four FRs, predominantly adopting a P-sheet configuration and connected by three hypervariable regions that form loops connecting P-type structures, and in some cases, being part of them. The hypervariable regions of each chain are linked to each other in close proximity by FR and, together with the hypervariable regions of the other chain, participate in 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)). Constant domains are not directly involved in the binding of antibodies to antigens, but exhibit various effector functions, for example, the participation of antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC).

The term hypervariable region as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region typically contains amino acid residues from the complementarity determining region or CDR (for example, residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or residues from hypervariable loop residues 26-32 (H1), 53-55 (H2) and 96 -101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, CDR means the complementarity determining region of an antibody, as defined in Lefranc, MP et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999). Framework region residues or FRs are variable domain residues other than hypervariable region/CDR residues as defined herein.

Exemplary CDR designations are shown herein, however, one skilled in the art will appreciate that a number of CDR definitions are commonly used, including the Kabat definition (see Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions. Mol Immunol. 2010 ; 47:694-700), which is based on sequence variability and is the most commonly used. The definition of Chothia is based on the location of the 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 disclosed by Honegger, Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool. J Mol Biol. 2001;309:657-670; Ofran et al. Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B-cell epitopes. J Immunol. 2008; 181:6230-6235; Almagro Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires. J Mol Recognit. 2004; 17:132-143; and Padlanet al. Identification of specificity-determining residues in antibodies. Faseb J. 1995; 9:133-139, the contents of each of which are incorporated herein in their entirety by reference.

The terms heavy chain-only antibody and heavy chain antibody are used interchangeably herein and refer in the broadest sense to antibodies or one or more portions of an antibody, such as one or more arms of an antibody, that lack the light chain of a conventional antibody. The terms include, but are not limited to, homodimeric antibodies containing a VH antigen binding domain and CH2 and CH3 constant domains, in the absence of a CH1 domain; functional (antigen-binding) variants of such antibodies, soluble variants of VH, Ig-NAR containing a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NAR), and their functional fragments; and soluble single domain antibodies (sUniDab™). In one embodiment, the heavy chain only antibody consists of a variable region antigen binding domain consisting of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In another embodiment, the heavy chain only antibody

- 8 046268 chain, consists of an antigen binding domain, at least part of the hinge region and the CH2 and CH3 domains. In yet another embodiment, the heavy chain-only antibody consists of an antigen binding domain of at least a portion of a 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 a hinge region, and a CH3 domain. Antibodies containing only heavy chains in which the CH2 and/or CH3 domain is truncated are also included herein. In another embodiment, the heavy chain consists of an antigen binding domain and at least one CH domain (CH1, CH2, CH3 or CH4), but without a hinge region. The heavy chain-only antibody may be in the form of a dimer in which the two heavy chains are disulfide-linked to each other, covalently linked, or non-covalently linked to each other. An antibody containing only heavy chains may belong to the IgG subclass, but antibodies belonging to other subclasses such as the IgM, IgA, IgD and IgE subclass are also included herein. In a specific embodiment, the heavy chain-only antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype. In one embodiment, heavy chain-only antibodies are used herein as the binding (targeting) domain of a chimeric antigen receptor (CAR). The definition specifically includes heavy chain-only human antibodies called UniAb™ produced by human immunoglobulin transgenic rats (UniRat™). The variable regions (VH) of UniAb™ are called UniDab™, and are versatile building blocks that can be coupled to Fc regions or serum albumin to develop new therapeutics with polyspecificity, increased potency and extended half-life. Because homodimeric UniAbs lack a light chain and therefore a VL domain, the antigen is recognized by a single domain, that is, the heavy chain variable domain of a heavy chain-only antibody (VH or VHH).

As used herein, the term intact antibody chain is a chain comprising a full-length variable region and a full-length constant region (Fc). The intact standard antibody contains an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, hinge, CH2 and CH3 for secreted IgG. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domains may be native sequence constant domains (eg, human native sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more effector functions that relate to those biological activities that are inherent in the constant Fc region (native sequence Fc region or variant amino acid sequence Fc region) of the antibody. Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and a decrease in the number of cell surface receptors. Constant region variants include those that alter the effector profile, Fc receptor binding, and the like.

Depending on the amino acid sequence of the Fc (constant domain) of their heavy chains, antibodies and various antigen-binding proteins can be classified into different classes. There are five main classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Fc constant domains, which correspond to different classes of antibodies, can be designated as a, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. Ig forms include hinged modifications or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US 2005/0048572; US 2004 /0229310). Antibody light chains from any vertebrate species can be classified into one of two types, called k (kappa) and λ (lambda), based on the amino acid sequences of their constant domains. Antibodies in accordance with embodiments of the invention may contain kappa light chain sequences or lambda light chain sequences.

The functional Fc region has the effector function of the native sequence Fc region. Non-limiting examples of effector functions include C1q binding; KZTs; Fc receptor binding; AZKTS; AZKF; reduction in the number of receptors on the cell surface (for example, B-cell receptor), etc. Such effector functions typically require the Fc region to interact with a receptor, such as FcyRI receptors; FcyRIIA; FcyRIIBI; FcyRIIB2; FcyRIIIA; FcyRIIIB, and the low affinity receptor FcRn; 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 for, for example, prolonging serum half-life, but that does not activate the high affinity Fc receptor or that has reduced affinity for the Fc receptor.

The native sequence Fc region contains the amino acid sequence

- 9 046268 identical to the amino acid sequence of the Fc region found in nature. Human native sequence Fc regions include, for example, the native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

The variant Fc region contains an amino acid sequence that differs from the native sequence Fc region sequence due to at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution relative to the native sequence Fc region or the Fc region of the parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the Fc region with the native sequence or in the Fc region of the original polypeptide. The variant Fc region herein will preferably have at least about 80% homology to the native sequence Fc region and/or the Fc region of the parent polypeptide, and most preferably at least about 90% homology thereto, more preferably at least about 95% homology with it.

Fc sequence variants may contain three amino acid substitutions in the CH2 region to reduce FcyRI binding at positions 234, 235, and 237 according to the EU index (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the C1q complement fixation site at EU index positions 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitution with human IgG1 or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 significantly reduces ADCC and CCZ (see, for example, Armor K.L. et al., 1999 Eur J Immunol. 29(8):2613 -24; and Shields R.L. et al., 2001. J Biol Chem. 276(9):6591-604). The amino acid sequence of human IgG4 Fc (UniProtKB No. P01861) is provided herein as SEQ ID NO: 76. Silenced IgG1 is described, for example, in Boesch, A.W., et al., Highly parallel characterization of IgG Fc binding interactions. MAbs, 2014. 6(4): p. 915-27, the description of which is incorporated herein by reference in its entirety.

Other Fc variants are possible, including, but not limited to, one in which the region capable of forming a disulfide bond is deleted, or in which certain amino acid residues at the N-terminus of the native Fc are deleted, or a methionine residue is added thereto. Thus, in some embodiments, one or more Fc portions of an antibody may contain one or more mutations in the hinge region to eliminate a disulfide bond. In yet another embodiment, the hinge region Fc may be removed completely. In yet another embodiment, the antibody may contain an Fc variant.

In addition, the Fc variant can be engineered to remove or substantially reduce effector functions by substituting, deleting, or adding amino acid residues to effect complement fixation or Fc receptor binding. For example, without limitation, the deletion may occur at a complement binding site such as the C1q binding site. Methods for obtaining such derivative immunoglobulin Fc sequences are described in international patent publications Nos. WO 97/34631 and WO 96/32478. In addition, the Fc domain can be modified by phosphorylation, sulfonation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

In some embodiments, the antibody comprises a variant human IgG4 CH3 domain sequence containing the T366W mutation, which may optionally be referred to herein as an IgG4 CH3 overhang 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 domain sequence. The IgG4 CH3 mutations described herein can be used in any suitable manner to place a knob in the first heavy chain constant region of a first monomer in an antibody dimer and a trough in the second heavy chain constant region of a second monomer in an antibody dimer, thereby promoting proper pairing (heterodimerization) of the desired pairs of heavy chain polypeptide subunits in an antibody.

In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a human IgG4 variant Fc region containing the S228P mutation, the F234A mutation, the L235A mutation, and the T366W (overhang) mutation. In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising the S228P mutation, the F234A mutation, the L235A mutation, the T366S mutation, the L368A mutation, and the Y407V (hollow) mutation.

The term antibody containing an Fc region refers to an antibody that contains an Fc region. The terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody purification or by constructing a recombinant nucleic acid encoding the antibody.

Accordingly, an antibody having an Fc region according to this invention may include

- 10 046268 antibody with or without K447.

Aspects of the invention include antibodies containing only the heavy chain variable region, in a monovalent or divalent configuration. As used herein, the term monovalent configuration when used in relation to a heavy chain only variable region domain means that there is only one heavy chain only variable region domain having a single binding site (see FIG. 5, panel A, right arm of the antibody). In contrast, the term bivalent configuration as used in relation 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 FIG. 5, panels B and C, right arms of antibodies). Non-limiting examples of linker sequences are further discussed herein and include, without limitation, GS linker sequences of various lengths. When the heavy chain only variable region has a bivalent configuration, each of the two heavy chain variable region domains may have binding affinity for the same antigen or for different antigens (e.g., different epitopes on the same protein; two different proteins etc.). However, unless specifically stated otherwise, a heavy chain only variable region designated as being in a divalent configuration is intended to comprise two identical heavy chain only variable region domains connected by a linker sequence, each of the two identical heavy chain only variable region domains , have binding affinity to the same target antigen.

Aspects of the invention include antibodies having multispecific configurations, which include, but are not limited to, bispecific, trispecific, etc. A wide variety of methods and protein configurations are known and used to produce bispecific monoclonal antibodies (bsmAbs), trispecific antibodies, and the like.

Various methods have been developed for producing multivalent artificial antibodies by recombinantly fusing the variable domains of two or more antibodies. In some embodiments, the first and second antigen binding domains on the polypeptide are connected by a polypeptide linker. One non-limiting example of such a polypeptide linker is a GS linker having an amino acid sequence of four glycine residues followed by one serine residue, and in which the sequence is repeated n times, where n is an integer from 1 to about 10, for example, 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, as described, for example, in Chen et al. Adv Drug Deliv Rev. 2013 October 15;65(10):1357-69, the disclosure of which is incorporated herein by reference in its entirety.

The term triple-chain antibody-like molecule or TCA is used herein to refer to antibody-like molecules comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which contain, consist essentially of, or consist of one heavy and one light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains containing an antigen-binding region and at least one CH domain. This heavy chain/light chain pair has binding specificity for the first antigen. The third polypeptide subunit includes, consists essentially of, or consists of a heavy chain-only antibody containing an Fc portion containing CH2 and/or CH3 and/or CH4 domains, in the absence of a CH1 domain, and one or more antigen binding domains (eg , two antigen-binding domains) that binds an epitope of a second antigen or another epitope of a first antigen, wherein such binding domain is derived from or has sequence identity with a heavy or light chain variable region of an antibody. Portions of such a variable region may be encoded by VH and/or VL gene segments, D and JH gene segments, or JL gene segments. The variable region may be encoded by rearranged gene segments V _H DJ _H , V _L DJ _H , V _H J _L or

VLJL.

The TCA binding compound uses a heavy chain only antibody or a heavy chain only antibody or a heavy chain only polypeptide, which as used herein means a single chain antibody containing the heavy chain constant regions CH2 and/ or CH3 and/or CH4, but without the CH1 domain. In one embodiment, the heavy chain-only antibody consists of an antigen binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In another embodiment, the heavy chain-only antibody consists of an antigen binding domain, at least a portion of a 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 a hinge region, and a CH3 domain.

Antibodies containing only heavy chains in which the CH2 and/or CH3 domain is truncated are also included herein. In another embodiment, the heavy chain consists of an antigen binding domain and at least one CH domain (CH1, CH2, CH3 or CH4), but without a hinge

- 11 046268 region. The heavy chain-only antibody may be in the form of a dimer in which two heavy chains are linked by a disulfide bond, the others otherwise covalently or non-covalently linked to each other, and may optionally include an asymmetric interaction surface between one or more CH domains to facilitate correct pairing between polypeptide chains. The heavy chain antibody may belong to the IgG subclass, but antibodies belonging to other subclasses such as the IgM, IgA, IgD and IgE subclass are also included. In a specific embodiment, the heavy chain-only antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype or the IgG4 subtype. Non-limiting examples of a TCA binding compound are described, for example, in WO2017/223111 and WO2018/052503, the disclosures of which are incorporated herein by reference in their entirety.

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

As used herein, the term PSMA refers to a type II transmembrane protein that has N-acetylated alpha-linked acid dipeptidase, folate hydrolase and dipeptidyl peptidase activities. The term PSMA includes the PSMA protein of humans and any non-human animal species, and particularly includes human PSMA as well as non-human mammalian PSMA.

As used herein, the term human PSMA includes any variants, isoforms and species homologues of human PSMA (UniProt Q04609), regardless of its source or method of preparation. Thus, human PSMA includes human PSMA naturally expressed by cells and PSMA expressed on cells transfected with the human PSMA gene.

The terms anti-PSMA heavy-chain-only antibody, anti-PSMA heavy-chain-only antibody, anti-PSMA heavy-chain-only antibody, and anti-PSMA heavy-chain-only antibody are used interchangeably herein to refer to an antibody that containing only heavy chains, as defined above, for immunospecific binding to PSMA, including human PSMA, as defined above. The definition includes, without limitation, human heavy chain-only antibodies produced by transgenic animals such as transgenic rats or transgenic mice expressing human immunoglobulin, including UniRat™ producing human anti-PSMA UniAb™ antibodies as defined above.

Percentage (%) identity of amino acid sequences relative 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 sequence alignment and the introduction of gaps, if necessary, to achieve maximum sequence identity, but without considering any conservative substitutions as part of sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways known to those skilled in the art, for example, using publicly available computer programs such as BLAST, BLAST2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this invention, % amino acid sequence identity values are generated using the ALIGN-2 sequence comparison computer program.

An isolated antibody is one that has been identified and separated and/or extracted from a component of its natural environment. Contaminants in the natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95 wt.% antibody, as determined by the Lowry method, and most preferably greater than 99 wt.%, (2) to a degree sufficient

- 12 046268 to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spin-beaker sequencer, or (3) to homogeneity by DSNPAAG electrophoresis under reducing or non-reducing conditions using Coomassie blue dye or, preferably, dye based on silver. The isolated antibody includes the antibody in situ in the recombinant cells, since at least one component of the antibody's natural environment will be absent. Typically, however, the isolated antibody will be obtained through at least one purification step.

Antibodies of the invention include multispecific antibodies. Polyspecific antibodies have more than one binding specificity. The term multispecific specifically includes bispecific and trispecific, as well as higher order independent specific binding affinity, such as higher order polyepitope specificity, as well as tetravalent antibodies and antibody fragments. The terms polyspecific antibody, polyspecific heavy chain antibody, polyspecific heavy chain only antibody, and polyspecific UniAb™ are used herein in the broadest sense to cover all antibodies with more than one binding specificity. Polyspecific heavy chain-only antibodies to PSMA of the present invention particularly include antibodies that immunospecifically bind to two or more non-overlapping epitopes on a PSMA protein, such as human PSMA (ie, bivalent and biparatopic). Polyspecific heavy chain-only antibodies to PSMA of the present invention also specifically include antibodies that immunospecifically bind to an epitope of a PSMA protein, such as human PSMA, and an epitope of another protein, such as, for example, a CD3 protein, such as human CD3 (i.e. e. divalent and biparatopic). Polyspecific heavy chain-only antibodies to PSMA of the present 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 protein, and an epitope on another protein, such as, for example , CD3 protein, such as human CD3 protein (ie, trivalent and biparatope).

Antibodies of the invention include monospecific antibodies having one binding specificity. Monospecific antibodies specifically include antibodies having a single binding specificity, as well as antibodies containing more than one binding unit having the same binding specificity. The terms monospecific antibody, monospecific heavy chain only antibody, monospecific heavy chain antibody, and monospecific UniAb™ are used herein in the broadest sense to cover all antibodies with a single binding specificity. The monospecific heavy chain only antibodies to PSMA of the present invention particularly include antibodies that immunospecifically bind to a single epitope on a PSMA protein, such as human PSMA (monovalent and monospecific). Monospecific heavy chain-only antibodies to PSMA of the present invention also specifically include antibodies having more than one binding unit (eg, multivalent antibodies) that immunospecifically bind to an epitope on a PSMA protein, such as human PSMA. For example, a monospecific antibody in accordance with embodiments of the invention may comprise a heavy chain variable region comprising two antigen binding domains, where each antigen binding domain binds to the same epitope on the PSMA protein (ie, bivalent and monospecific).

An epitope is a region on the surface of an antigen molecule to which one antibody molecule binds. Typically an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes.

Epitope mapping is the process of identifying antibody binding sites or epitopes on their target antigens. 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 from amino acids that are discontinuous in the sequence of a protein, but which come together when the protein folds into its three-dimensional structure.

The term polyepitope specificity refers to the ability to specifically bind to two or more different epitopes on the same or different targets. As noted above, the present invention particularly includes heavy chain only anti-PSMA antibodies with polyepitope specificity, that is, heavy chain only anti-PSMA antibodies that bind to one or more non-overlapping epitopes on a PSMA protein, such as PSMA person; and heavy chain-only antibodies to PSMA that bind to one or more epitopes on the PSMA protein and to an epitope on another protein, such as, for example, the CD3 protein. The term non-overlapping epitope(s) or non-competitive epitope(s) of an antigen is defined herein to mean epitope(s) that are recognized by one member of an antigen-specific antibody pair but not by the other member. Pairs of antibodies or antigen-binding regions targeting the same antigen

- 13 046268 on a polyspecific antibody that recognizes non-overlapping epitopes, does not compete for binding to this antigen and is capable of simultaneously binding this antigen.

An antibody binds substantially the same epitope as a reference antibody when the two antibodies recognize identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competitive binding assays, which can be configured in a number of different formats using either a labeled antigen or a labeled antibody. Typically, the antigen is immobilized in a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzyme labels.

The term valence, as used herein, refers to the specified number of binding sites in an antibody molecule.

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

A polyvalent antibody has two or more binding sites. Thus, the terms divalent, trivalent and tetravalent refer to the presence of two binding sites, three binding sites and four binding sites, respectively. Thus, the bispecific antibody of the invention is at least bivalent and may be trivalent, tetravalent, or otherwise multivalent. A divalent antibody in accordance with embodiments of the invention may have two binding sites to the same epitope (ie, divalent, monoparatope) or to two different epitopes (ie, divalent, biparatope).

A wide variety of methods and protein configurations are known and are used to produce bispecific monoclonal antibodies (bsmAbs), trispecific antibodies and the like.

The term bispecific three-chain antibody-like molecule or TCA is used herein to refer to antibody-like molecules comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which contain, 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 containing an antigen-binding region and at least one CH domain. This heavy chain/light chain pair has binding specificity for the first antigen. The third polypeptide subunit contains, consists essentially of or consists of a heavy chain-only antibody containing an Fc portion containing CH2 and/or CH3 and/or CH4 domains, in the absence of a CH1 domain, and an antigen binding domain that binds an epitope of a second antigen or another epitope of the first antigen, wherein such binding domain is derived from or has sequence identity with the heavy or light chain variable region of the antibody. Portions of such a variable region may be encoded by VH and/or VL gene segments, D and JH gene segments, or JL gene segments. The variable region may be encoded by rearranged VHDJH, VLDJH, VHJL or VLJL gene segments. The TCA protein uses a heavy chain-only antibody as defined above.

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

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 that are not encoded by human germline immunoglobulin sequences, for example, mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo. The term human antibody specifically includes heavy chain-only antibodies having human heavy chain variable region sequences produced by transgenic animals such as transgenic rats or mice, particularly UniAb™ produced by UniRat™ as defined above.

By chimeric antibody or chimeric immunoglobulin is meant an immunoglobulin molecule containing amino acid sequences from at least two different Ig loci, for example, a transgenic antibody containing a portion encoded by the human Ig locus and a portion

- 14 046268 encoded by the rat Ig locus. Chimeric antibodies include transgenic antibodies with non-human Fc regions or artificial Fc regions, and human idiotypes. Such immunoglobulins can be isolated from animals according to the invention that have been engineered to produce such chimeric antibodies.

As used herein, the term effector cell refers to an immune cell that participates in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, an effector cell, such as a natural killer cell, is capable of inducing antibody-dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages that express FcRs are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system or binding to cells that present antigens. In some embodiments, the effector cell may phagocytose the target antigen or target cell.

Human effector cells are white blood cells that express receptors, such as T-cell receptors or FcRs, and perform effector functions. Preferably, such cells express at least FcyRIII and exert ADCC effector function. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with NK cells being preferred. Effector cells can be isolated from a native source, such as blood or PBMC, as described herein.

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

The effector functions of an antibody refer to those biological activities that are attributable to the Fc region (native Fc region sequence or variant amino acid sequence of the Fc region) of the antibody. Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; decreased expression of cell surface receptors (eg B cell receptor, BCR), etc.

Antibody-dependent cell-mediated cytotoxicity and ADCC correspond to a cell-mediated response in which nonspecific cytotoxic cells that express Fc receptors (FcR) (eg, natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on the target cell and thus thus lead to lysis of the target cell. The primary cells that mediate ADCC, natural killer cells, express only FcyRIII, whereas monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression on hematopoietic cells is shown in Table 3 on p. 464 in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, as described in US Pat. No. 5,500,362 or 5,821,337. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or in addition, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model, for example, as described in Dynes et al., PNAS (USA) 95:652-656 (1998).

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 (eg, antibody) that forms a complex with the antigen being recognized. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

Binding affinity refers to the strength of the total number of noncovalent interactions between one binding site of a molecule (eg, antibody) and its binding partner (eg, antigen). Unless otherwise specified, the term binding affinity as used herein refers to the actual binding affinity, which reflects the 1:1 interaction between members of a binding pair (eg, antibody and 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 conventional methods known in the art. Low-affinity antibodies tend to bind antigen slowly and tend to dissociate easily, whereas high-affinity antibodies tend to bind antigen more quickly and tend to remain bound.

As used herein, the term Kd or Kd value refers to the dissociation constant determined by BioLayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, Calif.) in kinetics mode. For example, sensors with anti-mouse Fc antibody are loaded

- 15 046268 with a mouse Fc fusion antigen and then dipped into wells containing antibodies to measure concentration-dependent rates (kon). Antibody dissociation rates (koff) are measured at the last stage, when the sensors are immersed in wells containing only buffer. Kd is the koff/kon ratio (for details, see Conception, J., et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

The terms treatment, treat, and the like are used herein to denote the achievement of a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms and/or may be therapeutic in terms of partially or completely curing a disease and/or an adverse event associated with that disease. As used herein, the term treatment encompasses any treatment of a disease in a mammal and includes: (a) preventing the onset of the disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) suppression of the disease, that is, stopping its development; or (c) alleviation of the disease, i.e. causing regression of the disease. The therapeutic agent may be administered before, during, or after the onset of the disease or injury. Treatment of ongoing disease, in which the treatment stabilizes or reduces the patient's adverse clinical symptoms, is of particular interest. It is advisable to carry out such treatment until complete loss of function in the affected tissues. Therapy may be administered to a subject during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

The term therapeutically effective amount means the amount of active agent that is necessary to impart a therapeutic effect in a subject. For example, a therapeutically effective amount is an amount that causes, attenuates, or otherwise causes an improvement in pathological symptoms, disease progression, or physiological conditions associated with a disease, or that increases resistance to a disorder.

As used herein, the term prostate malignancy refers to a malignant tumor of glandular origin in the prostate gland.

The term characterized by PSMA expression broadly refers to any disease or disorder in which the expression of PSMA is associated with or involved in one or more pathological processes that are characteristic of the disease or disorder. Such disorders include, but are not limited to, prostate cancer.

The terms subject, individual, and patient are used interchangeably herein to refer to a mammal being evaluated for treatment and/or undergoing treatment. In one embodiment, the mammal is human. The terms subject, individual and patient include, without limitation, individuals with cancer, individuals with autoimmune diseases, pathogenic infections and the like. Subjects may be humans, but may also be other mammals, particularly those mammals that can be used as laboratory models of human disease, such as mice, rats, etc.

The term pharmaceutical formulation refers to a formulation that is in such a form as to provide the biological activity of the active ingredient to be effective and that does not contain any additional components that are unacceptably toxic to the subject to whom the formulation will be administered. Such compositions are sterile. Pharmaceutically acceptable excipients (carriers, adjuvants) are those that can reasonably be administered to a mammalian subject and provide an effective dose of the active ingredient used.

The sterile composition is aseptic or free from, or substantially free from, any living microorganisms and their spores. A frozen composition is a composition at a temperature below 0°C.

A stable composition is one in which the protein retains substantially all of its physical stability and/or chemical stability and/or biological activity during storage. Preferably, the composition retains substantially all of its physical or chemical stability as well as its biological activity. The storage period is generally selected based on the shelf life of the formulation. A variety of techniques for measuring protein stability are known in the art and are discussed, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones. A. Adv. Drug Delivery Rev. 10: 2990) (1993). Stability can be measured at a selected temperature for a selected period of time. Stability can be assessed qualitatively and/or quantitatively in a variety of ways, including assessing the formation of aggregates (eg, using size exclusion chromatography, by measuring turbidity and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, capillary isoelectric focusing with in-capillary detection (icIEF), or capillary zone electrophoresis; N-terminal or C-terminal sequence analysis; mass spectrometric analysis; analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis to compare reduced and injected

- 16 046268 tact antibody; peptide map analysis (eg tryptic or LYS-C); assessing the biological activity or antigen-binding function of the antibody; and so on. Instability may include one or more of the following: aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomerization), truncation/hydrolysis/fragmentation (e.g. hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.

II. Detailed description.

Antibodies to PSMA.

The present invention relates to a family of closely related antibodies that bind to human PSMA. Antibodies of this family contain a set of CDR sequences as defined herein and shown in table. 1, and are illustrated by the proposed heavy chain variable region (VH) sequences of SEQ ID NO: 24-54 shown in Table 2. The family of antibodies provides a number of advantages that favor their use as clinical therapeutic agent(s). . Antibodies include members with a range of binding affinities allowing the selection of a particular sequence with the desired binding affinity.

Table 1

Unique amino acid sequences of the CDR of a heavy chain-only antibody to PSMA

SEQ aa CDR1 SEQ aa CDR2 SEQ aa CDR3 GGSISSSSYY (SEQ ID NO: 1) IDYSGYT (SEQ ID NO: 11) ARHKAATADFDY (SEQ ID NO: 18) GGSISSSNYF (SEQ ID NO: 2) VDYSGYT (SEQ ID NO: 12) AREPRIGYYYESSGYDSLDY (SEQ ID NO: 19) GGSISSNSYY (SEQ ID NO: 3) IYDSGST (SEQ ID NO: 13) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) GFSFRSYG (SEQ ID NO: 4) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYDSSGYDSLDY (SEQ ID NO: 21) GFSFSSYG (SEQ ID NO: 5) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYDSSGYYSLDY (SEQ ID NO: 22) GFIFRSYG (SEQ ID NO: 6) ISYDGSNK (SEQ ID NO: 16) AREPRVGYYYETSGYYSLDY (SEQ ID NO: 23) GFSFSRYG (SEQ ID NO: 7) ISYDGSNR (SEQ ID NO: 17) GFSFTSYG (SEQ ID NO: 8) GFTFISYG (SEQ ID NO: 9) GFTFSSYG (SEQ ID NO: 10)

table 2

Amino acid sequences of the variable domain of an antibody containing only heavy chains to PSMA

Clone ID no. SEQ aa_FRl FR4 SEQ ID NO. 325920 QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIG SIDYSGYTYYNPSLQSRVTISVDTSKNQFSLKLSSVTAADTAVYNCARHK AATADFD YRGQGTLVTVS S 24 346181 QLQLQESGPGLVKPSETLSLTCTVSGGSISSSNYFWGWIRQSPGKGLEWIG SIDYSGYTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYNCARHK AATADFD YRGQGTLVTVS S 25 346165 QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIG SVDYSGYTYYNPSLQSRVTISVDTSKNQFSLKLSSVTAADTAVYNCARHK AATADFD YRGQGTLVTVS S 26 346172 QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIG SIDYSGYTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYNCARHK AATADFD YRGQGTLVTVS S 27 326109 QLQLQESGPGLVKPSETLSLTCTVSGGSISSNSYYWGWIRQSPGKGLEWLG SIYDSGSTHYNPSLKSRVIISGDTSKNQFSLKLSSVTAADTAVYYCARHKA ATADFD YRGQGTLVTVS S 28 325867 QVQLVESGGGVVQPGRSLRLSCAASGFSFRSYGMHWVRQAPGKGLEWV AVIWYDGSNKYYADSVKGRFTISRDYSKNTLYLQMNSLRAEDTAVYYCA REPRIGYYYES SGYDSLD YRGQGTLVTVS S 29 325742 QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEWV thirty

- 17 046268

AVIWYDGSNKYYADSVKGRFTISRDYSKNTLYLQMNSLRAEDTAVYYCA REPRIGYYYES SGYDSLDYRGQGTLVTVS S 325748 QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEGVA VIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EPRIGYYYES SGYYSLDYRGQGTLVTVS S 31 325940 QVQLVESGGGVVQPGRSLRLSCAASGFIFRSYGMHWVRQAPGKGPEWVA VIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EPRIGYYYES SGYYSLDYRGQGTLVTVS S 32 325836 QVQLVESGGGVVQPGRSLRLSCAASGFSFRSYGMHWVRQAPGKGLEWV AVIWYDGSNKYYADSVKGRFTISRDYSKNTLYLQMNSLRAEDTAVYYCA REPRIGYYYDSSGYDSLDYRGQGTLVTVSS 33 326027 QVQLVESGGGVVQPGRSLRLSCAASGFSFRSYGMHWVRQAPGKGLEWV AVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA REPRIGYYYDSSGYDSLDYRGQGTLVTVSS 34 326087 QVQLVESGGGVVQPGRSLRLSCAASGFIFRSYGMHWVRQAPGKGPEWVA VIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EPRIGYYYDSSGYDSLDYRGQGTLVTVSS 35 326084 QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEGVA VIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EPRIGYYYDS SGYYSLDYRGQGTLVTVS S 36 326028 QVQLVESGGGVVQPGRSLRLSCAASGFSFRSYGMHWVRQAPGKGLEWV AVISYDGSNKYYADSVKGRFTISRDYSKNTLYLQMNSLRAEDTAVYYCA REPRIGYYYDSSGYDSLDYRGQGTLVTVSS 37 345497 QVQLVESGGGVVQPGRSLRLSCAASGFSFSRYGMHWVRQAPGKGLEGV AVIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA REPRIGYYYES SGYYSLDYRGQGTLVTVS S 38 326029 QVQLVESGGGVVQPGRSLRLSCAASGFSFSRYGMHWVRQAPGKGLEWV AVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA REPRVGYYYETSGYYSLDYRGQGTLVTVS S 39 345461 QVQLVESGGGVVQPGRSLRLSCAASGFSFTSYGMHWVRQAPGKGLEGVA VIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EPRIGYYYES SGYYSLDYRGQGTLVTVS S 40 345493 QVQLVESGGGVVQPGRSLRLSCAASGFSFSRYGMHWVRQAPGKGLEWV AVIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA REPRIGYYYES SGYYSLDYRGQGTLVTVS S 41 345436 QVQLVESGGGVVQPGRSLRLSCAASGFSFSRYGMHWVRQAPGKGLEGV AVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA REPRIGYYYES SGYYSLDYRGQGTLVTVS S 42 345443 QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEGVA VIWYDGSNRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAR EPRIGYYYES SGYYSLDYRGQGTLVTVS S 43 345490 QVQLVESGGGLVKPGGSLRLSCAASGFSFSSYGMHWVRQAPGKGLEGVA VIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EPRIGYYYES SGYYSLDYRGQGTLVTVS S 44 345482 QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEGVA VIWYDGSNRYYADSVKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCAR EPRIGYYYES SGYYSLDYRGQGTLVTVS S 45 345485 QVQLVESGGGVVQPGRSLRLSCAASGFSFRSYGMHWVRQAPGKGLEWV AVIWYDGSNKYYADSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYC AREPRIGYYYES SGYYSLDYRGQGTLVTVS S 46 345463 QVQLVESGGGVVQPGRSLRLSCAASGFIFRSYGMHWVRQAPGKGPEWVA VIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EPRIGYYYES SGYYSLDYRGQGTLVTVS S 47 325932 QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEGVA 48

- 18 046268

VIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EPRIGYYYES SGYYSLDYRGQGTLVTVS S 345505 QVQLVESGGGVVQPGRSLRLSCAASGFFISYGMHWVRQAPGKGLEGVA VIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EPRIGYYYES SGYYSLDYRGQGTLVTVS S 49 345508 QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGPEWV AVIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA REPRIGYYYES SGYYSLDYRGQGTLVTVS S 50 345480 QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEWV AVIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA REPRIGYYYES SGYYSLDYRGQGTLVTVS S 51 326116 QVQLVESGGGVVQPGRSLRLSCAASGFSFSRYGMHWVRQAPGKGLEGV AVISYDGSNKYYADSVKGRFTISRDYSKNTLYLQMNSLRAEDTAVYYCA REPRIGYYYDSSGYDSLDYRGQGTLVTVSS 52 345509 QVQLVESGGGLVQPGGSLRLSCAASGFSFSSYGMHWVRQAPGKGLEGVA VIWYDGSNKYYADSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCA REPRIGYYYES SGYYSLDYRGQGTLVTVS S 53 345444 S 54 345421 QVQLVESVGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEGVA VIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EPRIGYYYES SGYYSLDYRGQGTLVTVS S 55 345447 QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEGVA VISYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARE PRIGYYYES SGYYSLDYRGQGTLVTVS S 56 345510 QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEWV AVIWYDGSNKYYADSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYC AREPRIGYYYES SGYYSLDYRGQGTLVTVS S 57 345438 QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGPEWV AVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA REPRIGYYYES SGYYSLDYRGQGTLVTVS S 58

A suitable antibody may be selected from those provided herein for development and therapeutic or other use, including, without limitation, use as a bispecific, for example, as shown in FIG. 5, panels A-C, or a trispecific antibody or part of a CAR-T structure. In fig. Panels 5 A-C show illustrations of polyspecific antibodies to CD3 x PSMA, where the anti-PSMA domain is monovalent and monospecific, bivalent and monospecific, or bivalent and bispecific (biparatope). The anti-CD3 domain contains a CH1 domain and is paired with the light chain, while the anti-PSMA domains are derived from heavy chain-only antibodies and do not contain a CH1 domain and do not interact with the light chain. In some embodiments, the two heavy chains are separated using, for example, a ridge-to-valve technology. Referring to the antibodies depicted in FIG. 5, Panel A shows an anti-CD3 x 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 a CD3 x PSMA bispecific antibody in which the anti-PSMA binding arm is bivalent and monospecific, and the anti-PSMA arm antigen-binding domain is in a bivalent configuration, meaning that there are two identical antigen-binding domains arranged in tandem. Panel C depicts an anti-CD3 x PSMA bispecific antibody in which the anti-PSMA binding arm is bivalent and biparatopic and the antigen-binding domains of the anti-PSMA arm are in a divalent configuration.

Determination of affinity for a candidate protein can be performed using methods known in the art, such as Biacore measurements. Members of the antibody family may have an affinity for PSMA with a Kd of from about 10 ^-6 to about 10' ¹¹ , including, but not limited to: from about 10 ^-6 to about 10 - ¹⁰ ; from about 10 ^-6 to about 10 ^-9 ; from about 10 ^-6 to about 10 ^-8 ; from about 10 ^-8 to about 10'¹¹; from about 10 ^-8 to about ^10-10 ; from about 10 ^-8 to about 10 ^-9 ; from about 10 ^-9 to about 10 ^-11 ; from about 10 ^-9 to about ^10-10 ; or any value in these ranges. Affinity selection can be supported by biological evaluation for modulation, e.g. blocking, of PSMA biological activity, including in vitro assays, preclinical models and clinical studies, as well as assessment of potential toxicity.

Members of the antibody family described herein are not cross-reactive with cynomolgus PSMA protein, but can be designed to be cross-reactive with cynomolgus PSMA protein or PSMA proteins of other animal species, if desired.

The family of PSMA-specific antibodies herein includes the VH domain containing the CDR1, CDR2 and CDR3 sequences in the human VH framework. CDR sequences can be

- 19 046268 are located, for example, in the region around amino acid residues 26-33; 51-58; and 97-116 for CDR1, CDR2 and CDR3, respectively, of the provided exemplary variable region sequences set forth in SEQ ID NOs: 24-58. One skilled in the art will appreciate that the CDR sequences may be in other positions if a different framework sequence is selected, although generally the order of the sequences remains the same.

The CDR1, CDR2, and CDR3 sequences of the anti-PSMA antibodies of the present invention may be covered by the following structural formulas, wherein X represents a variable amino acid, which may be a variable amino acid as defined below.

CDR1

GGSISSX ₁ X ₂ Y X ₃ (SEQ ID NO: 67) where X ₁ represents S or N;

X ₂ represents S or N; And

X ₃ represents Y or F; And

CDR2

X ₄ X ₅ X, SGX ₇ T (SEQ ID NO: 68) where X ₄ represents I or V;

X ₅ represents D or Y;

X ₆ represents Y or D; And

X ₇ represents Y or S; And

CDR3

ARHKAATADFDY (SEQ ID NO: 69).

The CDR1, CDR2, and CDR3 sequences of the anti-PSMA antibodies of the present invention may be covered by the following structural formulas, wherein X represents a variable amino acid, which may be a variable amino acid as defined below.

CDR1

GFX ₁ F X ₂ X ₃ YG (SEQ ID NO: 70) where X ₁ represents S or I or T;

X ₂ represents S or T or R or I; And

X ₃ represents R or S; And

CDR2

I X ₄ YDGSN X ₅ (SEQ ID NO: 71) where X ₄ represents W or S; And

X ₅ represents R or K; And

CDR3

AREPRX6GYYYX7X8SGYX9SLDY (SEQ ID NO: 72) where X ₆ represents I or V;

X ₇ represents E or D;

X ₈ represents S or T; And

X9 represents Y or D.

Representative sequences of CDR1, CDR2 and CDR3 are shown in Table. 1 and 3.

- 20 046268

Table 3

Amino acid sequences of CDR1, CDR2 and CDR3 of a heavy chain-only antibody to PSMA

Clone ID no. SEQaaCDRl SEQ_aa_CDR2 SEQ_aa_CDR3 325920 GGSISSSSYY (SEQ ID NO: 1) IDYSGYT (SEQ ID NO: 11) ARHKAATADFDY (SEQ ID NO: 18) 346181 GGSISSSNYF (SEQ ID NO: 2) IDYSGYT (SEQ ID NO: 11) ARHKAATADFDY (SEQ ID NO: 18) 346165 GGSISSSSYY (SEQ ID NO: 1) VDYSGYT (SEQ ID NO: 12) ARHKAATADFDY (SEQ ID NO: 18) 346172 GGSISSSSYY (SEQ ID NO: 1) IDYSGYT (SEQ ID NO: 11) ARHKAATADFDY (SEQ ID NO: 18) 326109 GGSISSNSYY (SEQ ID NO: 3) IYDSGST (SEQ ID NO: 13) ARHKAATADFDY (SEQ ID NO: 18) 325867 GFSFRSYG (SEQ ID NO: 4) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYESSGYDSLDY (SEQ ID NO: 19) 325742 GFSFSSYG (SEQ ID NO: 5) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYESSGYDSLDY (SEQ ID NO: 19) 325748 GFSFSSYG (SEQ ID NO: 5) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 325940 GFIFRSYG (SEQ ID NO: 6) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 325836 GFSFRSYG (SEQ ID NO: 4) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYDSSGYDSLDY (SEQ ID NO: 21) 326027 GFSFRSYG (SEQ ID NO: 4) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYDSSGYDSLDY (SEQ ID NO: 21) 326087 GFIFRSYG (SEQ ID NO: 6) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYDSSGYDSLDY (SEQ ID NO: 21) 326084 GFSFSSYG (SEQ ID NO: 5) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYDSSGYYSLDY (SEQ ID NO: 22) 326028 GFSFRSYG (SEQ ID NO: 4) ISYDGSNK (SEQ ID NO: 16) AREPRIGYYYDSSGYDSLDY (SEQ ID NO: 21) 345497 GFSFSRYG (SEQ ID NO: 7) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 326029 GFSFSRYG (SEQ ID NO: 7) ISYDGSNK (SEQ ID NO: 16) AREPRVGYYYETSGYYSLDY (SEQ ID NO: 23) 345461 GFSFTSYG (SEQ ID NO: 8) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345493 GFSFSRYG (SEQ ID NO: 7) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345436 GFSFSRYG (SEQ ID NO: 7) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345443 GFSFSSYG (SEQ ID NO: 5) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345490 GFSFSSYG (SEQ ID NO: 5) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345482 GFSFSSYG (SEQ ID NO: 5) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345485 GFSFRSYG (SEQ ID NO: 4) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345463 GFIFRSYG (SEQ ID NO: 6) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 325932 GFSFSSYG (SEQ ID NO: 5) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20)

- 21 046268

345505 GFTFISYG (SEQ ID NO: 9) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345508 GFSFSSYG (SEQ ID NO: 5) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345480 GFSFSSYG (SEQ ID NO: 5) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 326116 GFSFSRYG (SEQ ID NO: 7) ISYDGSNK (SEQ ID NO: 16) AREPRIGYYYDSSGYDSLDY (SEQ ID NO: 21) 345509 GFSFSSYG (SEQ ID NO: 5) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345444 GFTFSSYG (SEQ ID NO: 10) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345421 GFSFSSYG (SEQ ID NO: 5) IWYDGSNR (SEQ ID NO: 15) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345447 GFSFSSYG (SEQ ID NO: 5) ISYDGSNR (SEQ ID NO: 17) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345510 GFSFSSYG (SEQ ID NO: 5) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20) 345438 GFSFSSYG (SEQ ID NO: 5) IWYDGSNK (SEQ ID NO: 14) AREPRIGYYYES SGYYSLDY (SEQ ID NO: 20)

In some embodiments, the anti-PSMA antibody comprises a CDR1 sequence of any of SEQ ID NO: 1-10. In a specific embodiment, the CDR1 sequence is SEQ ID NO: 2 or 7.

In some embodiments, the anti-PSMA antibody comprises a CDR2 sequence with any of SEQ ID NOs: 11-17. In a specific embodiment, the CDR2 sequence is SEQ ID NO: 11 or 15.

In some embodiments, the anti-PSMA antibody comprises a CDR3 sequence with any of SEQ ID NOs: 18-23. In a specific embodiment, the CDR3 sequence is SEQ ID NO: 18 or 20.

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

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

In a further embodiment, the anti-PSMA antibody comprises any of the heavy chain variable region amino acid sequences of SEQ ID NO: 24-58 (Table 2).

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

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

In some embodiments, the CDR sequence in the anti-PSMA antibody of the invention contains one or two amino acid substitutions relative to the CDR1, CDR2 and/or CDR3 sequence or the set of CDR1, CDR2 and CDR3 sequences in any of SEQ ID NO: 1-23 (Table 1) .

In some embodiments, the anti-PSMA antibody preferably comprises a heavy chain variable domain (VH) in which the CDR3 sequence is greater than or equal to 80%, such as at least 85%, at least 90%, at least 95%, or at least 99% sequence identity at the amino acid level with the CDR3 sequence of any of the antibodies whose CDR3 sequences are presented in table. 1, and binds to PSMA.

In some embodiments, the anti-PSMA antibody preferably comprises a heavy chain variable domain (VH) in which the full set of CDRs 1, 2, and 3 (combined) has greater than or equal to eighty-five percent (85%) sequence identity at the amino acid level with CDR 1. 2 and 3 (combined) antibodies, whose CDR sequences are presented in Table 1, and bind to PSMA.

In some embodiments, the anti-PSMA antibody preferably comprises a heavy chain variable domain (VH) in which the full set of CDRs 1, 2, and 3 (combined) has greater than or equal to eighty-five percent (85%) sequence identity at the amino acid level with CDR 1. 2 and 3 (combined) antibodies, whose CDR sequences are presented in Table 3, and bind to PSMA.

In some embodiments, the anti-PSMA antibody comprises a heavy chain variable region sequence with at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity with any of the heavy chain variable region sequences of SEQ ID NO: 24-58 (shown in Table 2) and binds to PSMA.

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In some embodiments, bispecific or polyspecific antibodies are provided, which may have any of the configurations discussed herein, including, without limitation, a bispecific triple chain antibody (TCA) molecule. In some embodiments, a multispecific antibody may comprise at least one heavy chain variable region having a binding specificity for PSMA and at least one heavy chain variable region having a binding specificity for a protein other than PSMA. In some embodiments, the polyspecific antibody may comprise a heavy chain variable region comprising at least two antigen binding domains, wherein each of the antigen binding domains has a binding specificity for PSMA. In some embodiments, a polyspecific antibody may comprise a heavy chain/light chain pair that has binding specificity for a first antigen (eg, CD3) and a heavy chain from a heavy chain-only antibody. In some embodiments, the heavy chain of a heavy chain-only antibody comprises an Fc portion comprising CH2 and/or CH3 and/or CH4 domains, in the absence of a CH1 domain. In one specific embodiment, a bispecific antibody comprises a heavy chain/light chain pair that has binding specificity for an antigen on an effector cell (e.g., CD3 protein on a T cell), and a heavy chain from a heavy chain-only antibody comprising an antigen binding domain having binding specificity for PSMA.

In some embodiments, the multispecific antibody comprises a CD3 binding VH domain that is paired with a light chain variable domain. In certain embodiments, the light chain is a fixed light chain. In some embodiments, the CD3 binding domain of the VH comprises a CDR1 sequence of SEQ ID NO: 59, a CDR2 sequence of SEQ ID NO: 60, and a CDR3 sequence of SEQ ID NO: 61 in the human VH backbone. In some embodiments, the fixed light chain comprises a CDR1 sequence of SEQ ID NO: 62, a CDR2 sequence of SEQ ID NO: 63, and a CDR3 sequence of SEQ ID NO: 64 in the human VL backbone. Together, the CD3 binding VH domain and the light chain variable domain have binding affinity for CD3. In some embodiments, the VH CD3 binding domain comprises a heavy chain variable region sequence of SEQ ID NO: 65. In some embodiments, the VH CD3 binding domain comprises a sequence having at least about 80%, at least about 85%, of 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 with the heavy chain variable region sequence of SEQ ID NO: 66.

Multispecific antibodies containing the CD3 binding VH domain and light chain variable domain described above have useful properties, for example, as described in PCT Application Publication No. WO2018/052503, the disclosure of which is incorporated herein by reference in its entirety. Any of the polyspecific antibodies and antigen binding domains described herein having binding affinity for PSMA can be combined with the CD3 binding domains and fixed light chain domains described herein (see, for example, Table 4 and Table 5 ), as well as additional sequences such as those presented in Table. 6 and table. 7, to generate multispecific antibodies having binding affinity for one or more PSMA epitopes as well as CD3.

Table 4

Amino acid sequences of CDR1, CDR2, CDR3 of heavy and light chains to CD3

SEQ aa CDR1 SEQ aa CDR2 SEQ aa CDR3 Heavy chain GFTFDDYA (SEQ ID NO: 59) ISWNSGSI (SEQ ID NO: 60) AKDSRGYGDYRLGGAY (SEQ ID NO: 61) Light chain QSVSSN (SEQ ID NO: 62) G.A.S. (SEQ ID NO: 63) QQYNNWPWT (SEQ ID NO: 64)

Table 5

Amino acid sequences of CD3 heavy and light chain variable regions

VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEW VSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKDSRGYGDYRLGGAYWGQGTLVTVSS (SEQ ID NO: 65) VL EIVMTQSPATLSVSPGERATLLSCRASQSVSSNLAWYQQKPGQAPRLLIYG ASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQ GTKVEIK (SEQ ID NO: 66)

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Table 6

Sequences of the Fc regions of human IgG1 and IgG4

Human IgGl (UniProt No. P01857) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 75) Human IgG4 (UniProt No. P01861) ASTKGPSVFP LAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 76) Silent human IgGl mutation (Fc region) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQS SGLYSLS S WTVPS S SLGTQIYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVV LTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 77) Silenced human IgG4 mutation (Fc region) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQS SGLYSLS S WTVPS S SLGTKIYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV LTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQWTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 78)

Table 7

Additional Sequences

Const sequence lung area chains to CD3 (kappa light chain) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKWACEVTHQGL SSPVTKSFNRGEC (SEQ ID NO: 79) Anti-CD3 heavy chain sequence (VH + Fc IgGl WT) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLE WVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAL YYCAKDSRGYGDYRLGGAYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQWTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 80)

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CD3 heavy chain sequence (c Silenced Fc IgGl) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLE WVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAL YYCAKDSRGYGDYRLGGAYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81) Const sequence heavy chain region to CD3 (with Fc IgG4 WT) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLE WVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAL YYCAKDSRGYGDYRLGGAYWGQGTLVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSWTVPSSSLGTK I AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 82) Const sequence heavy chain regions to CD3 (with Fc silencing IgG4) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLE WVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAL YYCAKDSRGYGDYRLGGAYWGQGTLVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSWTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 83) Silenced IgG4 (hinge - CH2 CH3; depression (S228P, F234A, L235A; T366S, L368A, Y407V)) ESI<YGPPCPPCPAPEAAGGPSVFLFPPI<PI<DTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLVS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 84) Silenced IgG4 (hinge - CH2 CH3; protrusion (S228P, F234A, L235A; T366W)) ESI<YGPPCPPCPAPEAAGGPSVFLFPPI<PI<DTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 85) Full length anti-CD3 light chain (VL + kappa CL) EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLI YGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPW TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACE VTHQGLSSPVTKSFNRGEC (SEQ ID NO: 86)

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Full length heavy chain CD3(VH+silenced Fc IgG4 + protrusion (S228P, F234A, L235A; T366W)) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLE WVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAL YYCAKDSRGYGDYRLGGAYWGQGTLVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSWTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYP SDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 87) Monovalent heavy chain to PSMA (clone ID 346181) + Silenced Fc IgG4, depression (S228P, F234A, L235A, T366S, L368A, Y407V QLQLQESGPGLVKPSETLSLTCTVSGGSISSSNYFWGWIRQSPGKGLE WIGSIDYSGYTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYN CARHKAATADFDYRGQGTLVTVSSESKYGPPCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK (SEQ ID NO: 88) Divalent heavy chain to PSMA (clone ID 346181) + Silenced Fc IgG4, depression (S228P, F234A, L235A, T366S, L368A, Y407V QLQLQESGPGLVKPSETLSLTCTVSGGSISSSNYFWGWIRQSPGKGLE WIGSIDYSGYTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYN CARHKAATADFDYRGQGTLVTVSSGGGGSGGGGSQLQLQESGPGLV KPSETLSLTCTVSGGSISSSNYFWGWIRQSPGKGLEWIGSIDYSGYTYY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYNCARHKAATADFD YRGQGTLVTVSSESI<YGPPCPPCPAPEAAGGPSVFLFPPI<PI<DTLMISR TPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQV YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK (SEQ ID NO: 89) Monovalent heavy chain to PSMA (clone ID 345497) + Silenced Fc IgG4, depression (S228P, F234A, L235A, T366S, L368A, Y407V QVQLVESGGGVVQPGRSLRLSCAASGFSFSRYGMHWVRQAPGKGLE GVAVIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYC AREPRIGYYYES SGYYSLDYRGQGTLVTVS SESKYGPPCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWW DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 90) Divalent heavy chain to PSMA (clone ID 345497) + Silenced Fc IgG4, depression (S228P, F234A, L235A, T366S, L368A, Y407V QVQLVESGGGVVQPGRSLRLSCAASGFSFSRYGMHWVRQAPGKGLE GVAVIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAREPRIGYYYESSGYYSLDYRGQGTLVTVSSGGGGSGGGGSQV QLVESGGGVVQPGRSLRLSCAASGFSFSRYGMHWVRQAPGKGLEGV AVIWYDG GLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 91)

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Divalent heavy chain to PSMA (clone ID 346181 x clone ID 345497) + Silenced Fc IgG4, depression (S228P, F234A, L235A, T366S, L368A, Y407V Y YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREPRIGYY YESSGYYSLDYRGQGTLVTVSSESKYGPPCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KA KGQPREPQVYTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK (SEQ ID NO: 92) Divalent heavy chain to PSMA (clone ID 345497 x clone ID 346181) + Silenced Fc IgG4, depression (S228P, F234A, L235A, T366S, L368A, Y407V QVQLVESGGGVVQPGRSLRLSCAASGFSFSRYGMHWVRQAPGKGLE GVAVIWYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAREPRIGYYYESSGYYSLDYRGQGTLVTVSSGGGGSGGGGSQL QLQESGPGLVKPSETLSLTCTVSGGSISSSNYFWGWIRQSPGKGLEWIG SIDYSGY KGQ PREPQVYTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK (SEQ ID NO: 93)

In some embodiments, bispecific or polyspecific antibodies are provided, which may have any of the configurations discussed herein, including, without limitation, a bispecific triple chain antibody (TCA) molecule. In some embodiments, a bispecific antibody may comprise at least one heavy chain variable region having binding specificity for PSMA and at least one heavy chain variable region having binding specificity for a protein other than PSMA. In some embodiments, a bispecific antibody may comprise a heavy chain/light chain pair that has binding specificity for a first antigen, and a heavy chain from a heavy chain-only antibody comprising an Fc portion comprising CH2 and/or CH3 domains CH4, in the absence of a CH1 domain, and an antigen binding domain that binds an epitope of a second antigen or another epitope of a first antigen. In one specific embodiment, the bispecific antibody comprises a heavy chain/light chain pair that has binding specificity for an antigen on an effector cell (e.g., CD3 protein on a T cell), and a heavy chain from a heavy chain-only antibody containing an antigen binding domain , having binding specificity for PSMA.

In some embodiments, when the antibody of the invention is a bispecific antibody, one arm of the antibody (one binding fragment or one binding unit) is specific for human PSMA, while the other arm may be specific for target cells associated with tumor antigens, targeting antigens such as integrins, etc., pathogen antigens, checkpoint proteins, etc. Target cells specifically include cancer cells, including, but not limited to, solid tumor cells, such as prostate tumors, as discussed below. In some embodiments, one arm of the antibody (one binding fragment or one binding unit) is specific for human PSMA, while the other arm is specific for CD3.

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 of SEQ ID NO: 80, 81, 82, 83 , 84 or 85, and an anti-PSMA heavy chain polypeptide containing the sequence of any of SEQ ID NOs: 24-58, in a monovalent or divalent configuration, linked to the sequence of any of SEQ ID NOs: 75, 76, 77, 78, 84 or 85 These sequences can be combined in various ways to produce a bispecific antibody of the desired IgG subclass, eg IgG1, IgG4, IgG1-silenced, IgG4-silenced. In one 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 one 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.

The invention includes various formats of multispecific antibodies, including, without limitation, single chain polypeptides, double chain polypeptides, triple chain polypeptides, quadruple chain polypeptides and polypeptides thereof. Polyspecific antibodies herein specifically include polyspecific (eg, bispecific) T cell antibodies that bind to PSMA and CD3 (anti-PSMA x CD3). Such antibodies cause potent T cell-mediated killing of cells expressing PSMA.

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Obtaining antibodies to PSMA.

Antibodies of the present invention can be obtained by methods known in the art. In a preferred embodiment of the present invention, the antibodies are produced by transgenic animals, including transgenic mice and rats, preferably rats in which the endogenous immunoglobulin genes are knocked out or disabled. In a preferred embodiment, the heavy chain only antibodies of the present invention are produced in UniRat™. UniRat™ silences endogenous immunoglobulin genes and utilizes the human immunoglobulin heavy chain translocus to express a diverse, naturally optimized repertoire of fully human HCAbs. While endogenous immunoglobulin loci in rats can be knocked out or silenced using a variety of technologies, UniRat™ used (endo) technology to inactivate the endogenous rat heavy chain J locus, light chain K locus, and light chain Ολ locus. Zinc finger nucleases (ZNFs). ZNF constructs for microinjection into oocytes can produce IgH and IgL knockout (KO) lines. For more details see, for example, Geurts et al., 2009, Science 325:433. Characterization of Ig heavy chain knockout rats has been reported by Menoret et al., 2010, Eur. J. Immunol. 40:2932–2941. The advantages of ZNF technology are that non-homologous end joining to silence a gene or locus through deletions up to several kb. may also provide a target site for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). Human antibodies containing only the heavy chains produced in UniRat™ are called UniAb™ and can bind epitopes that cannot be attacked by conventional antibodies. Their high specificity, affinity and small size make them ideal for mono- and polyspecific applications.

In addition to UniAb™, specifically included herein are antibodies containing only heavy chains free of the camel backbone and VHH mutations and their functional VH regions. Such heavy chain-only antibodies can, for example, be produced in transgenic rats or mice that contain gene loci consisting entirely of human heavy chains, as described, for example, in WO2006/008548, but also in other transgenic mammals, such as rabbit, guinea pig, rats can also be used, with rats and mice being preferred. Antibodies containing only heavy chains, including functional VHH or VH fragments thereof, can also be produced using recombinant DNA technology by expressing the encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including, for example, mammalian cells (eg, CHO cells), E coli or yeast.

Heavy chain-only antibody domains combine the benefits of antibodies and small molecule drugs: can be monovalent or multivalent; have low toxicity; and are cost effective to produce. Due to their small size, these domains are easy to administer, including oral or topical administration, and are characterized by high stability, including stability in the gastrointestinal tract; and their half-life can be tailored to the desired use or indication. In addition, the VH and VHH domains of HCAbs can be produced in a cost-effective manner.

In a specific embodiment, the heavy chain only antibodies of the present invention, including UniAb™, have a native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Kabat numbering system) replaced by another amino acid residue that is capable of disrupting the surface exposed hydrophobic a region containing or linked to a native amino acid residue at that position. Such hydrophobic regions are typically buried at the surface interacting with the antibody light chain constant region but become surface accessible in the HCAb and, at least in part, serve to promote undesired aggregation and association of the HCAb light chain. The substituted amino acid residue is preferably charged and more preferably positively charged, for example lysine (Lys, K), arginine (Arg, R) or histidine (His, H), preferably arginine (R). In a preferred embodiment, heavy chain only antibodies obtained from transgenic animals contain a Trp to Arg mutation at position 101. The resulting HCAbs preferably have high antigen binding affinity and solubility under physiological conditions in the absence of aggregation.

The present invention has identified human heavy chain-only IgG antibodies to PSMA with unique sequences from animal UniRat™ (UniAb™) that bind to human PSMA in ELISA-based protein and cell binding assays. The variable heavy chain (VH) sequences identified are positive for human PSMA protein binding and/or binding to PSMA+ cells, and all are negative for binding to cells that do not express PSMA; see, for example, table. 8.

Heavy chain-only antibodies that bind to non-overlapping epitopes on the PSMA protein, such as UniAb™, can be identified using competitive assays

- 28 046268 binding, such as enzyme-linked immunosorbent assays (ELISAs) or flow cytometry-based competitive binding assays. For example, competition between known antibodies that bind to a target antigen and the antibody of interest can be used. Using this approach, it is possible to separate a set of antibodies into those that compete with the reference antibody and those that do not. Non-competing antibodies are identified as binding to a distinct epitope that does not overlap with the epitope bound by the reference antibody. Often, one antibody is immobilized, the antigen is bound, and a second, labeled (eg, biotinylated) antibody is tested in an ELISA assay for its ability to bind the captured antigen. This can also be done using surface plasmon resonance (SPR) platforms including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences) and the SPR MX96 imaging system (Ibis technologies BV), as well as other biolayer interferometry platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). For more information, see the examples in this document.

Typically, an antibody competes with a reference antibody if it causes approximately 15-100% reduction in binding of the reference antibody to the target antigen, as determined by standard methods, 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 at least about 80%, at least about 85%, at least about 90%, at least about 95% or higher.

Pharmaceutical compositions, uses and methods of treatment

Another aspect of the present invention is to provide pharmaceutical compositions containing one or more antibodies of the present invention in admixture with a suitable pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers used herein include, for example, but are not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art for storing therapeutic components or combinations thereof.

In one embodiment, the pharmaceutical composition contains a heavy chain-only antibody (eg, UniAb™) that binds to HCMA. In another embodiment, the pharmaceutical composition contains a multispecific (including bispecific) heavy chain-only antibody (eg, UniAb™) with binding specificity for two or more non-overlapping epitopes on the HCMA protein. In a preferred embodiment, the pharmaceutical composition contains a multispecific (including bispecific and TCA) heavy chain-only antibody (eg, UniAb™) with binding specificity for HCMA and with binding specificity for a binding target on an effector cell (eg, a binding target on T- cell, such as the CD3 protein on a T cell).

Pharmaceutical compositions of antibodies used in accordance with the present invention are prepared for storage by mixing proteins having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (see, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), for example, in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. The pharmaceutical compositions may be provided in unit dosage form (eg, unit dosage). The composition depends on the chosen route of administration. The antibodies described herein can be administered by intravenous injection or infusion, or subcutaneously. For injection administration, the antibodies described herein can be formulated in aqueous solutions, preferably in physiologically compatible buffers

- 29 046268 to reduce discomfort at the injection site. The solution may contain carriers, auxiliaries or stabilizers as described above. Alternatively, the antibodies can be lyophilized for mixing with a suitable vehicle, such as sterile pyrogen-free water, before use.

Antibody formulations are described, for example, in US Pat. No. 9,034,324. Similar formulations may be used for heavy chain-only antibodies, including UniAb™, of the present invention. Antibody formulations for subcutaneous administration are described, for example, in patents US20160355591 and US20160166689.

Methods of application.

The anti-PSMA antibodies and pharmaceutical compositions described herein can be used to treat diseases and conditions characterized by expression of PSMA, including, without limitation, the conditions and diseases described later herein.

PSMA is a type II transmembrane protein that is expressed in prostate epithelial tissue and its expression is upregulated in prostate malignancy and in new vasculature of solid tumors. It is also expressed at low levels in normal tissues such as the brain, kidney and salivary glands, but its overexpression in prostate malignancy makes it an attractive target for the therapeutic treatment of prostate malignancy. It may also be relevant for therapy or imaging of solid tumors, given its high expression in malignant neovascularization. Monoclonal antibodies, antibody-drug conjugates, and chimeric antigen receptor T cells targeting PSMA have been described for the treatment of metastatic prostate malignancy (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 explored for imaging and treatment of prostate malignancy (e.g., Hofman et al., 2018 PMID: 29752180).

In one aspect, anti-PSMA antibodies (eg, UniAb™) and pharmaceutical compositions provided herein can be used to treat disorders characterized by PSMA expression, including, but not limited to, prostate cancer and solid tumors.

Effective dosages of the compositions of this invention for treating a disease vary depending on many different factors, including routes of administration, destination, physiological condition of the patient, whether the patient is human or animal, whether other drugs are administered, and whether the treatment is prophylactic or therapeutic. Typically the patient is a human, but non-human mammals may also be treated, such as domestic animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc. , etc. Drug dosages can be adjusted to optimize safety and effectiveness.

Dosage levels can be readily determined by a person of ordinary skill in the art and may be adjusted as necessary, for example, to change the subject's response to therapy. The amount of active ingredient that can be combined with carrier materials to form a unit dosage form varies depending on the host being treated and the particular route of administration. Unit dosage forms typically contain from about 1 mg to about 500 mg of active ingredient.

In some embodiments, the therapeutic dosage of the agent may be from about 0.0001 to 100 mg/kg, and more typically from 0.01 to 5 mg/kg, based on the body weight of the host. For example, the dosage may be 1 mg/kg body weight or 10 mg/kg body weight, or in the range of 1-10 mg/kg. An exemplary treatment regimen includes administration once every two weeks or once a month or once every 3-6 months. The therapeutic agents of the present invention are typically administered multiple times. The intervals between single doses may be weekly, monthly or annual. The intervals may also be irregular, as indicated by measuring the level of the therapeutic substance in the patient's blood. Alternatively, the therapeutic agents of the present invention may be administered as a sustained release composition, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.

Typically, the compositions are prepared as injectable preparations, either as liquid solutions or suspensions; Solid forms suitable for dissolution or suspension in liquid vehicles prior to injection may also be prepared. The pharmaceutical compositions described herein are suitable for intravenous or subcutaneous administration, directly or after reconstitution from a solid (ie, lyophilized) composition. The drug may also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide or copoly

- 30 046268 measures to enhance the adjuvant effect, as described above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation, which can be formulated to provide prolonged or pulsatile release of the active substance. Pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and fully compliant with all US Food and Drug Administration (GMP) regulations.

The toxicity of the antibodies and antibody structures described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (dose lethal to 50% of a population) or LD100 (dose lethal to 100% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic indicator. Data obtained from these cell culture assays and animal studies can be used to develop dosage ranges that are not toxic for use in humans. The dosage of the antibodies described herein is preferably within a range of circulating concentrations that include an effective dose with little or no toxicity. The dosage may vary within this range depending on the dose used and the route of administration used. The exact composition, route of administration and dosage can be selected individually by the doctor, taking into account the patient’s condition.

Compositions for administration typically contain an antibody or other ablative agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous vehicle. Various aqueous vehicles can be used, for example, buffered saline and the like. These solutions are sterile and generally free of unwanted substances. These compositions can be sterilized by conventional, well known sterilization methods. The compositions may contain pharmaceutically acceptable excipients necessary to approximate physiological conditions, such as pH adjusting agents and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like . The concentration of active agent in these formulations can vary widely and will be selected based primarily on fluid volumes, viscosity, body weight, and the like, in accordance with the particular route of administration chosen and the needs of the patient (eg, Remington's Pharmaceutical Science (15th ed., 1980) ) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al, eds., 1996)).

Also included within the scope of the invention are kits containing active agents and their compositions, inventions and instructions for use. The kit may further contain at least one additional component, for example, a chemotherapy drug, etc. Kits typically include a label indicating the intended use of the contents of the kit. As used herein, the term label includes any written or recorded materials supplied in or with the kit or otherwise accompanying the kit.

Now that the invention has been fully described, it will be apparent to one skilled in the art that various changes and modifications may be made without departing from the spirit or scope of the invention.

Examples

Materials and methods.

Example 1: Immunization of UniRat™ with recombinant human PSMA.

Twelve UniRat™ animals were immunized with recombinant human PSMA protein fused to a His tag (R&D Systems, Cat. No: 4234-ZN). Animals were immunized twice a week for eight weeks. After a 35-day course of immunization, serum was collected from rats to determine serum titers.

Results for serum titers.

A summary of serum titers is shown in Fig. 1, panels A-B. In the graphs shown in Fig. 1, panels A-B, each line represents a different animal. The legends of the graphs show the identification number of each individual animal. Binding activity for a 12-point serum dilution series was tested by ELISA against the His-tagged human PSMA protein and the His-tagged nontarget protein. A range of levels of serum reactivity with human PSMA protein were observed among this group of animals. No serum response to the non-target His-tagged protein was observed.

Example 2: Flow cytometry analysis of anti-PSMA UniAb™ binding to PSMA positive and negative cells.

Binding to PSMA-positive cells was assessed by flow cytometry (Guava easyCyte 8HT, EMD Millipore) using the LNCaP cell line (ATCC: CRL-1740), 22Rv1 cell line (ATCC CRL-2505), PC3 cell line (ATCC CRL-1435 ), stably transfected to express human PSMA, or the DU-145 cell line (ATCC NTV-81). Briefly, 50,000 cells

- 31 046268 targets were stained with a series of dilutions of purified UniAb™ for 30 minutes at 4°C. After incubation, cells were washed twice with flow cytometry buffer (1X PBS, 1% BSA, 0.1% NaN ₃ ) and stained with goat F(ab')2 anti-human IgG antibody conjugated to R-phycoerythrin (PE) (Southern Biotech, Cat. No. 2042-09) for the detection of cell-bound antibodies. After 20 min incubation at 4°C, cells were washed twice with flow cytometry buffer and mean fluorescence intensity (MFI) was measured by flow cytometry. The SIF of cells stained with the secondary antibody alone was used to determine the background signal, and the binding of each antibody was converted to a fold value above the background signal. Binding to cynomolgus PSMA-positive cells was determined using the same protocol with the following modifications: 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.

In table 8 shows the target binding activity of several heavy chain only anti-PSMA antibodies (HCAbs) described herein. Column 1 shows the HCAb clone ID. Column 2 shows binding to LNCaP cells, measured as fold above background CIF signal.

Table 8

Binding to a cell line expressing PSMA

Column 1: CLONE ID Column 2: LNCaP 325920 282 346181 264 346165 243 346172 216 326109 25 325867 210 325742 200 325748 193 325940 169 325836 163 326027 138 326087 129 326084 125 326028 117 345497 112 326029 109 345461 102 345493 101 345436 87 345443 84 345490 80 345482 80 345485 71 345463 68 325932 64 345505 59 345508 55 345480 47 326116 38 345509 37 345444 23 345421 22 345447 14 345510 13 345438 13

Differences in cynomolgus PSMA binding as shown in FIG. 2, panels A and B, confirms the difference in the human PSMA epitope recognized by HCAbs 346181 and 345497.

Example 3. Binding of recombinant proteins using biolayer interferometry (BLI).

Using biolayer interferometry, binding competition between two families of antibodies, whose members are clone ID 345497 and clone ID 346181, was assessed. Epitope-specific antigen-antibody sorting analysis was performed on an Octet QK-384 (ForteBio). Briefly, pentagistidine sequence (HIS1K) sensors (Penta-HIS, described as SEQ ID NO: 95) were used to immobilize recombinant human PSMA antigen (R&D Systems, cat. no: 4234-ZN) for 120 seconds. After determining the baseline level, the sensors were immersed in solutions containing antibody 1 (325867) for 300 s, and another baseline level was established within

- 32 046268 seconds. The sensors were then dipped into wells containing either Antibody 1 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 FIG. 3, 325920 binds PSMA protein prebound to antibody 325867, indicating that the two antibodies recognize non-overlapping epitopes on PSMA. The binding signal shift is expressed in nanometers.

Example 4. Composition of biparatopic and divalent antibodies to PSMA.

As shown in table. 9, clone ID 350123 consists of the sequence of clone ID 346181 linked to the sequence of clone ID 345497 via the bridging sequence GGGGSGGGGS (SEQ ID NO: 74). Clone ID 350122 consists of two repeats of clone ID 346181 connected by the same linker sequence. Clone ID 350123 is biparatopic because it consists of two PSMA domains that recognize different epitopes on PSMA. Clone ID 350122 is bivalent but not biparatope because it consists of the same PSMA domain in tandem. Schematic illustrations of various anti-PSMA x CD3 antibodies are shown in FIG. 5, panels A-C.

Table 9

Description of the amino acid sequence of biparatopic and divalent antibodies to PSMA

Clone ID Sequence 1 Linker sequence Sequence 2 350123 346181 GGGGSGGGGS (SEQ ID NO: 74) 345497 350122 346181 GGGGSGGGGS (SEQ ID NO: 74) 346181

Example 5: Determination of affinity for human PSMA expressed on the cell surface.

Affinity for cell surface PSMA was determined by Scatchard assay using the human prostate carcinoma cell line 22Rv1. First, anti-PSMA x CD3 polyspecific antibodies were labeled with Alexa Fluor 488 using the Alexa Fluor 488 5-SDP Ester kit (ThermoFisher A30052). Binding to 22Rvl 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 antibodies for 1 h at 4°C. After incubation, cells were washed twice with flow cytometry buffer, and mean fluorescence intensity was measured by flow cytometry.

To obtain a standard curve for calculating equivalent soluble fluorochrome (MESF) molecules, Bangs Lab Quantum Alex Fluor 488 MESF bead populations 1 to 4 were combined in one tube and run on a Guava easyCyte 8HT. Empty beads were analyzed in a separate tube. The SIF of each set of beads was measured in the FITC channel. Linear regression of Log10 (LIF) versus Log10 (MESF) was constructed using GraphPad Prism 7.

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

Example 6: Multispecific Antibody-Mediated Killing of PSMA-Positive Prostate Tumor Cells via T-Cell Redirection.

Assays using resting T cells.

Target cells were seeded at 15,000 cells per well in a 96-well plate and cultured overnight at 37°C. After incubation, increasing amounts of polyspecific antibody were added along with resting human T cells at a ratio of effector cells to target cells of 10:1 and incubated for an additional 48 or 72 hours at 37°C (48 hours for assays with LNCaP, MDA-PCa cells -2b and PC3-PSMA and 72 hours for assays with 22Rv1 cells). Cell death was measured using cell proliferation reagent WST-1 (Sigma cat. no: 11644807001) or by flow cytometry. In some experiments, a small sample of each supernatant was collected after incubation but before target cell viability assays and stored for analysis of cytokine production. When cell viability was assayed using WST-1 reagent, the stock reagent was added to each well at a 1:10 dilution and incubated for 90 min at 37°C. The absorbance was then measured at 450 nm (reference wavelength 690 nm) and the percentage of specific lysis was calculated.

If target cell viability was analyzed by flow cytometry, the target cells were labeled with DiR membrane dye (ThermoFisher D12731) before analysis. After incubation with T cells and antibodies, supernatants were either saved for cytokine analysis or

- 33 046268 disposed of. The wells were then washed once to collect dead tumor cells and T cells, which were transferred to a flow cytometry plate. The remaining adherent tumor cells were trypsinized and then added to the appropriate wells of the flow cytometry plate. Annexin-V reagent was used to stain dead cells, and flow cytometry (BD FACSCelesta) was performed to quantify the percentage of dead tumor cells in each sample gated by DiR staining. Wells containing untreated target cells were used to normalize spontaneous cell death. Some experiments used a negative control antibody consisting of the same CD3-targeting arm as in the PSMA x CD3 polyspecific molecules, but replacing the tumor-targeting arm with a VH specific for the HIV gp120 protein.

In fig. 7 shows T cell-mediated lysis of PSMA-positive cells using unstimulated T cells. Unstimulated human T cells were incubated with cells expressing PSMA (LNCaP) and various concentrations of polyspecific antibodies. The biparatope anti-PSMA x CD3 antibody (350123xCD3) was superior to the monoparatope anti-PSMA x CD3 antibody (346181xCD3).

Assays using preactivated T cells.

Human pan-T cells were preactivated with plate-bound OKT3 and IL-2 for three days, followed by an additional day of incubation with fresh IL-2. Target cells were trypsinized, loaded with calcein-AM (ThermoFisher C3100MP), mixed with activated T cells to an E:T ratio of 20:1, and added to the wells of a 96-well plate. A series of dilutions of various polyspecific antibodies were added, followed by incubation for 4 hours at 37°C. The supernatants were then transferred to black 96-well plates and absorbance was measured at 480 nm/520 nm excitation/emission to quantify calcein release. Target cells incubated without T cells were used to normalize the spontaneous release of calcein by intact tumor cells. The addition of 2% Triton-X to control wells containing target cells allowed calculation of the calcein signal corresponding to maximum cell lysis. Using this value, each experimental well was reported as the percentage of maximum cell lysis. Data analysis was performed using GraphPad Prism 7.

In fig. 6 shows T cell-mediated lysis of PSMA-positive cells using pre-activated T cells. Pre-activated human T cells were incubated with human PSMA-expressing cells (LNCaP) and various concentrations of polyspecific antibodies. Tumor cell death was measured by calcein release and normalized by spontaneous tumor cell release in the absence of T cells. The biparatope anti-PSMA x CD3 antibody (350123xCD3) outperformed both monoparatope anti-PSMA x CD3 antibodies.

In fig. Figure 8 shows that polyspecific antibodies do not lyse PSMA-negative cells. Pre-activated human T cells were incubated with PSMA-negative prostate cancer cells (DU145) and various concentrations of polyspecific antibodies. None of the antibodies tested resulted in lysis of these cells.

In fig. Figure 9 shows the binding of polyspecific antibodies to PSMA x CD3 to PSMA positive and negative cells. Anti-PSMA x CD3 polyspecific antibodies show binding to PSMA-positive prostate tumor cells (22Rv1) but do not bind to PSMA-negative prostate tumor cells (DU145). The biparatope molecule (350123) showed the strongest binding to the target cell.

In fig. 10 shows T cell-mediated lysis of PSMA-positive cells. Data in Fig. 10 demonstrates that binding to PSMA via two different epitopes results in increased cell death compared to the bivalent but monospecific version of the antibody.

Example 7. Monoparatopic bispecific antibody to PSMA x CD3 induces less cytokine production than biparatopic polyspecific antibody to PSMA x CD3

Cytokine production was analyzed in tumor cytotoxicity assays with resting T cells. The design of these assays is described in detail elsewhere in this document. Supernatants were collected upon completion of assays (after 72 h of incubation for assays using 22Rv1 cells, 48 h for all other cell lines). ELISA kits were used to detect IL-2 (Biolegend 431804) and IFN-γ (Biolegend 430104) according to the manufacturer's protocol. Experimental 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 experimental wells and values were reported as less than or equal to the lower limit of quantitation for the assay.

In fig. 12, panels A-C, shows T cell-mediated lysis of PSMA-positive cells and comparison with cytokine production. Anti-PSMA x CD3 polyspecific antibodies induce T-cell-mediated lysis of the PSMA-positive prostate cancer cell line LNCaP. The biparatope molecule (350123) stimulated greater tumor cell killing compared to the monoparatope molecule (346181), but also caused

- 34 046268 work of higher levels of cytokines, gamma interferon (IFN-γ) and interleukin 2 (IL-2), as shown in Fig. 12, panels B and panels C.

In table 10 shows T cell-mediated lysis and cytokine production against four PSMA-positive prostate tumor cell lines. Anti-PSMA x CD3 polyspecific antibodies were tested in in vitro tumor cell cytotoxicity assays using unstimulated T cells and a series 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 recorded using EC50, and if the highest percentage of killing was achieved. Supernatants from these experimental wells were collected and analyzed by ELISA for cytokines, interferon gamma (IFN-γ), or interleukin-2 (IL-2). The monoparatope molecule (3461881) induced approximately equivalent levels of antitumor cytotoxicity in all four cell lines tested compared to the biparatope molecule, but had higher EC50s for cytokine production and in most cases stimulated lower levels of maximal cytokine production.

Table 10

T cell lysis and cytokine production against four PSMA-positive prostate tumor cell lines

Cell line Antibody Cell binding, EC50 (nM) Maximum cytotoxicity (% lysis) V negation, EC50 (nM) Max. IFN-γ (pg/ml) IFN- 7, EC50 (nM) Max. IL-2 (pg/ml) IL2, EC50 (nM) 22Rvl 346181Х CD3 58 45 52.8 21 150 173.8 <NPKO N/A 350123х CD3 3 53 0.42 73 031 380.2 <NPKO N/A MDA- RSa-2b 346181Х CD3 28 26 23.6 14 309 116.5 524 38.5 350123х CD3 2 29 0.41 12,026 0.90 1111 1.11 LNCAP 346181Х CD3 17 79 14.6 32 237 63.0 183 575 350123х CD3 2 75 0.84 60 397 3.29 1057 3.73 RSZPSMA 346181Х CD3 thirty 42 3.7 7 340 10.1 1569 4.1 350123х CD3 6 51 0.40 10 136 1.01 3480 1.1

Example 8. Polyspecific antibodies to PSMA x CD3 induce proliferation of T cells.

PSMA-positive tumor cells were seeded at 25,000 cells per well in a 96-well plate and cultured overnight at 37°C. Human pan T cells isolated from resting PBMCs (Miltenyi 130-096-535) were labeled with CFSE lineage tracing dye 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 antibody dilutions, and incubated at 37°C, 8% CO2. After 5 days of incubation, cells were gently mixed and transferred to a flow cytometry plate. Cells were pelleted and the supernatant was removed, followed by staining with APC-conjugated anti-CD8 (Biolegend 301049) and PE-conjugated anti-CD4 (Biolegend 317410) for 20 min on ice. Cells were then washed and resuspended in flow cytometry assay buffer (BD FACSCelesta). Cells were gated for forward and side scatter and CD4 or CD8 expression. The percentage of T cells that proliferated, as indicated by positive CD4 or CD8 staining and low or negative CFSE signal, was calculated for the entire T cell population and for the CD4 and CD8 subsets. Flow cytometry data were analyzed using FlowJo and plotted in GraphPad Prism 7.

In fig. 11, panels A-D, show that polyspecific anti-PSMA x CD3 antibodies stimulated T cell proliferation in the presence of PSMA-positive tumor cells, and that monoparatopic bispecific anti-PSMA antibodies preferentially activated CD3 T cells. Polyspecific antibodies were incubated with PSMA-expressing tumor cells and CFSE-labeled T cells to trace lineage. After 5 days of incubation, T cell proliferation and the composition of proliferated T cells (CD8+ and 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 proliferating wells. The dashed horizontal line represents the CD8:CD4 ratio of unstimulated T cells and is approximately 1:2 (actual value = 0.64). The monoparatope bispecific anti-PSMA x CD3 antibody (346181) preferentially activates CD8 T cells (CD8:CD4 ratio after expansion is about 2:1), whereas the biparatope polyspecific anti-PSMA x CD3 antibody (350123) activates CD8+ T cells to a lesser extent ( the CD8:CD4 ratio is about 1:1).

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Claims (34)

Example 9. A polyspecific antibody causes inhibition of prostate tumor growth in a xenograft model. Male immunocompromised CIEA-NOG mice, 5-6 weeks old (Taconic), were implanted with 10 million 22Rv1 cells subcutaneously in their lower right flank, followed by the addition of 10 million human PBMCs via tail vein injection one day after tumor implantation. Animals were treated with 100 μg of polyspecific antibody or vehicle by tail vein injection starting one day after tumor implantation on days 1, 5, 9, and 13. Tumor volume was determined using a caliper and recorded for 25 days. In fig. Figure 13 shows the results of the 22Rv1 tumor xenograft model. The biparatope anti-PSMA x CD3 molecule (350123) demonstrated inhibition of 22Rv1 tumor growth in a tumor xenograft model. Three mice were tested for each treatment group, and the change in tumor volume for each animal was plotted in millimeters. Animals received PBMC on day 1 after tumor implantation and received antibody treatment on days 1, 5, 9, and 13. Two out of three animals receiving polyspecific antibodies showed a slowdown in tumor progression. Although preferred embodiments of the present invention have been shown and described herein, those skilled in the art will appreciate that these embodiments are provided for purposes of illustration only. Many variations, changes and substitutions will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. It should be understood that various alternative embodiments of the present invention described herein may be used in the practice of the present 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 are thereby covered. CLAIM 1. A polyspecific three-chain antibody-like molecule that binds to the PSMA protein and the CD3 protein, containing: (a) a first polypeptide subunit comprising a heavy chain variable region (VH) CD3 binding domain; (b) a second polypeptide subunit comprising a light chain variable domain, wherein the CD3 binding VH domain of the first polypeptide subunit is paired with said light chain variable domain of the second polypeptide subunit; and (c) a third polypeptide subunit comprising a heavy chain variable region of an anti-PSMA antibody containing only heavy chains, wherein the heavy chain variable region of the third polypeptide subunit comprises: i) CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 11 and CDR3 of SEQ ID NO: 18; or ii) CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 12 and CDR3 of SEQ ID NO: 18; or iii) CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 11 and CDR3 of SEQ ID NO: 18; or iv) CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 13 and CDR3 of SEQ ID NO: 18; or v) CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 14 and CDR3 of SEQ ID NO: 19; or vi) CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 14 and CDR3 of SEQ ID NO: 20; or vi i) CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 14 and CDR3 of SEQ ID NO: 21; or vii i) CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 16 and CDR3 of SEQ ID NO: 21; or ix) CDR1 of SEQ ID NO: 5, CDR2 of SEQ ID NO: 14 and CDR3 of SEQ ID NO: 19; or x) CDR1 of SEQ ID NO: 5, CDR2 of SEQ ID NO: 14 and CDR3 of SEQ ID NO: 20; or xi) CDR1 of SEQ ID NO: 5, CDR2 of SEQ ID NO: 15 and CDR3 of SEQ ID NO: 20; or xii) CDR1 of SEQ ID NO: 5, CDR2 of SEQ ID NO: 15 and CDR3 of SEQ ID NO: 22; or xiii) CDR1 of SEQ ID NO: 5, CDR2 of SEQ ID NO: 17 and CDR3 of SEQ ID NO: 20; or - 36 046268 xiv) CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 14 and CDR3 of SEQ ID NO: 20; or xv) CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 14 and CDR3 of SEQ ID NO: 21; or xvi) CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 14 and CDR3 of SEQ ID NO: 20; or xvi i) CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 15 and CDR3 of SEQ ID NO: 20; or xvii i) CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 16 and CDR3 of SEQ ID NO: 21; or xix) CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 16 and CDR3 of SEQ ID NO: 23; or xx) CDR1 of SEQ ID NO: 8, CDR2 of SEQ ID NO: 15 and CDR3 of SEQ ID NO: 20; or xxi) CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 15 and CDR3 of SEQ ID NO: 20; or xxii) CDR1 of SEQ ID NO: 10, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 20. 2. The polyspecific three-chain antibody-like molecule according to claim 1, wherein the third polypeptide subunit heavy chain variable region CDR1, CDR2 and CDR3 sequences are present in the human framework. 3. Polyspecific three-chain antibody-like molecule according to claim 1 or 2, where the variable region of the heavy chain of the third polypeptide subunit contains: (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) a CDR1 sequence of SEQ ID NO: 7, a CDR2 sequence of SEQ ID NO: 15, and a CDR3 sequence of SEQ ID NO: 20. 4. The polyspecific three-chain antibody-like molecule according to claim 1 or 2, wherein the heavy chain variable region sequence of the third polypeptide subunit has at least 95% sequence identity with any of the sequences of SEQ ID NO: 24-58. 5. The polyspecific three-chain antibody-like molecule according to claim 1 or 2, wherein the heavy chain variable region sequence of the third polypeptide subunit is selected from the group consisting of SEQ ID NO: 24-58. 6. The polyspecific three-chain antibody-like molecule of claim 5, wherein the heavy chain variable region sequence of the third polypeptide subunit is SEQ ID NO: 25 or SEQ ID NO: 38. 7. The polyspecific three-chain antibody-like molecule of claim 1 or 2, wherein the heavy chain variable region of the third polypeptide subunit comprises the sequence CDR1 SEQ ID NO: 2, the sequence CDR2 SEQ ID NO: 11 and the sequence CDR3 SEQ ID NO: 18 in the human VH framework . 8. The polyspecific three-chain antibody-like molecule of claim 1 or 2, wherein the heavy chain variable region of the third polypeptide subunit comprises the sequence CDR1 SEQ ID NO: 2, the sequence CDR2 SEQ ID NO: 11 and the sequence CDR3 SEQ ID NO: 18 in the human VH framework in monovalent or divalent configuration. 9. The polyspecific three-chain antibody-like molecule according to claim 1 or 2, wherein the heavy chain variable region of the third polypeptide subunit comprises the CDR1 sequence SEQ ID NO: 7, the CDR2 sequence SEQ ID NO: 15 and the CDR3 sequence SEQ ID NO: 20 in the human VH framework . 10. The polyspecific three-chain antibody-like molecule according to claim 1 or 2, wherein the heavy chain variable region of the third polypeptide subunit contains the sequence CDR1 SEQ ID NO: 7, the sequence CDR2 SEQ ID NO: 15 and the sequence CDR3 SEQ ID NO: 20 in the human VH framework in monovalent or divalent configuration. 11. Polyspecific three-chain antibody-like molecule that binds PSMA protein and CD3 protein, containing: (a) a first polypeptide subunit comprising a heavy chain variable region (VH) CD3 binding domain; (b) a second polypeptide subunit comprising a light chain variable domain, wherein the CD3 binding VH domain of the first polypeptide subunit is paired with said light chain variable domain of the second polypeptide subunit; And - 37 046268 (c) a third polypeptide subunit containing the first heavy chain variable region of an anti-PSMA antibody containing only heavy chains, and the second variable region of the heavy chain of an anti-PSMA antibody containing only heavy chains, where: i) the first heavy chain variable region contains the sequence CDR1 SEQ ID NO: 2, the sequence CDR2 SEQ ID NO: 11 and the sequence CDR3 SEQ ID NO: 18 in the human VH framework; a ii) the second heavy chain variable region contains the sequence CDR1 SEQ ID NO: 7, the sequence CDR2 SEQ ID NO: 15 and the sequence CDR3 SEQ ID NO: 20 in the human VH framework. 12. The polyspecific three-chain antibody-like molecule of claim 11, wherein the first heavy chain variable region of the third polypeptide subunit is located proximal to the N-terminus of the second heavy chain variable region of the third polypeptide subunit. 13. The polyspecific three-chain antibody-like molecule of claim 11, wherein the first heavy chain variable region of the third polypeptide subunit is located proximal to the C-terminus of the second heavy chain variable region of the third polypeptide subunit. 14. Polyspecific three-chain antibody-like molecule according to any one of claims 1 to 13, characterized in that: The CD3 binding domain VH of the first polypeptide subunit contains a sequence that is at least 95% identical to the heavy chain variable region sequence of SEQ ID NO: 65; and the light chain variable domain of the second polypeptide subunit comprises a sequence having at least 95% sequence identity with the light chain variable region of SEQ ID NO: 66. 15. Polyspecific three-chain antibody-like molecule according to any one of claims 1 to 13, characterized in that The CD3 binding domain VH of the first polypeptide subunit contains the heavy chain variable region sequence SEQ ID NO: 65; and the light chain variable domain of the second polypeptide subunit comprises the light chain variable domain of SEQ ID NO: 66. 16. A polyspecific three-chain antibody-like molecule that binds to the PSMA protein and the CD3 protein, containing a first polypeptide subunit containing the sequence SEQ ID NO: 87; a second polypeptide subunit containing the sequence SEQ ID NO: 86; and a third polypeptide subunit comprising the sequence SEQ ID NO: 88, 89, 90, 91, 92 or 93. 17. The polyspecific three-chain antibody-like molecule according to claim 16, wherein the third polypeptide subunit contains the sequence SEQ ID NO: 88. 18. Bispecific antibody that binds to the PSMA protein and the CD3 protein, containing: (i) a heavy chain variable region having binding affinity for 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 the human VH framework; (ii) a light chain variable region containing the sequence CDR1 SEQ ID NO: 62, the sequence CDR2 SEQ ID NO: 63 and the sequence CDR3 SEQ ID NO: 64 in the human VL framework; and (iii) a heavy chain-only anti-PSMA antibody antigen binding domain 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 the human VH framework. 19. Bispecific antibody that binds to the PSMA protein and the CD3 protein, containing: (i) a heavy chain variable region having binding affinity for 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 the human VH framework; (ii) a light chain variable region containing the sequence CDR1 SEQ ID NO: 62, the sequence CDR2 SEQ ID NO: 63 and the sequence CDR3 SEQ ID NO: 64 in the human VL framework; and (iii) a heavy chain-only anti-PSMA antibody antigen binding domain 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 divalent configuration. 20. Bispecific antibody that binds to the PSMA protein and the CD3 protein, containing: (i) a heavy chain variable region having binding affinity for 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 the human VH framework; - 38 046268 (ii) a light chain variable region containing the sequence CDR1 SEQ ID NO: 62, the sequence CDR2 SEQ ID NO: 63 and the sequence CDR3 SEQ ID NO: 64 in the human VL framework; and (iii) a heavy chain-only anti-PSMA antibody antigen binding domain 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 the human VH framework. 21. Bispecific antibody that binds to the PSMA protein and the CD3 protein, containing: (i) a heavy chain variable region having binding affinity for 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 the human VH framework; (ii) a light chain variable region containing the sequence CDR1 SEQ ID NO: 62, the sequence CDR2 SEQ ID NO: 63 and the sequence CDR3 SEQ ID NO: 64 in the human VL framework; and (iii) a heavy chain-only anti-PSMA antibody antigen binding domain 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 divalent configuration. 22. Polyspecific antibody that binds to the PSMA protein and the CD3 protein, containing: (i) a heavy chain variable region having binding affinity for 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 the human VH framework; (ii) a light chain variable region containing the sequence CDR1 SEQ ID NO: 62, the sequence CDR2 SEQ ID NO: 63 and the sequence CDR3 SEQ ID NO: 64 in the human VL framework; and (iii) a heavy chain-only anti-PSMA antibody antigen binding domain, wherein the antigen binding domain comprises first and second antigen binding regions in a bivalent configuration, wherein the first antigen binding region comprises the sequence CDR1 SEQ ID NO: 2, the sequence CDR2 SEQ ID NO: 11 and CDR3 sequence SEQ ID NO: 18 in the human VH framework; a the second antigen binding region contains the CDR1 sequence SEQ ID NO: 7, the CDR2 sequence SEQ ID NO: 15 and the CDR3 sequence SEQ ID NO: 20 in the human VH framework. 23. The multispecific antibody of claim 22, wherein the first antigen-binding region is located N-terminal to the second antigen-binding region. 24. The multispecific antibody of claim 22, wherein the first antigen-binding region is located proximal to the C-terminus of the second antigen-binding region. 25. The polyspecific antibody according to any one of claims 22 to 24, wherein the first and second antigen binding regions of the anti-PSMA heavy chain only antigen binding domain are connected by a polypeptide linker. 26. The polyspecific antibody of claim 25, wherein the polypeptide linker is a GS linker. 27. The multispecific antibody of claim 26, wherein the GS linker consists of the sequence SEQ ID NO: 73 or SEQ ID NO: 74. 28. A pharmaceutical composition containing a polyspecific three-chain antibody-like molecule according to any one of claims 1 to 17, or a polyspecific antibody according to any of claims 22 to 27, or a bispecific antibody according to any of claims 18 to 21. 29. A method of treating a disorder characterized by the expression of PSMA, comprising administering to a subject with said disorder a polyspecific three-chain antibody-like molecule according to any one of claims 1 to 17, or a polyspecific antibody according to any one of claims 22 to 27, or a bispecific antibody according to any one of claims 18 -21, or the pharmaceutical composition according to claim 28. 30. The method of claim 29, wherein the disorder is prostate cancer. 31. Use of a polyspecific three-chain antibody-like molecule according to any one of claims 1 to 17, or a polyspecific antibody according to any one of claims 22 to 27, or a bispecific antibody according to any one of claims 18 to 21 in the manufacture of a medicament for the treatment of a disorder characterized by the expression of PSMA . 32. Use according to claim 31, wherein the disorder is prostate cancer. 33. A polyspecific triple-chain antibody-like molecule according to any one of claims 1 to 17, or a polyspecific antibody according to any one of claims 22 to 27, or a bispecific antibody according to any one of claims 1821 for treating a disorder characterized by the expression of PSMA. 34. The polyspecific three-chain antibody-like molecule, or polyspecific antibody, or bispecific antibody according to claim 33, wherein the disorder is prostate cancer. -