Heavy chain antibody that binds to PSMA

Abstract

Anti-PSMA heavy chain antibodies (e.g., UniAbs™), a method for preparing such antibodies, a composition including a pharmaceutical composition comprising such antibodies, and the use thereof for treating disorders characterized by the expression of PSMA are disclosed together.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims the benefit of priority from the filing date of U.S. Provisional Patent Application No. 62 / 830,130, filed on April 5, 2019, the full text of which is incorporated herein by reference.

[0003] Field of invention

[0004] The present invention relates to human heavy-chain antibodies (e.g., UniAbs™) that bind to PSMA. The present invention further relates to a method for producing such antibodies, a composition comprising a pharmaceutical composition containing such antibodies, and the use thereof for treating disorders characterized by the expression of PSMA. Background Technology

[0005] PSMA

[0006] PSMA, also known as prostate-specific membrane antigen and glutamate carboxypeptidase II (UniProt Q04609), is a type II transmembrane protein possessing N-acetylated-alpha-linked-acid dipeptidase, folate hydrolase, and dipeptidyl-peptidase activities. In humans, it is encoded by the FOLH1 gene and consists of a 19-amino acid cytoplasmic domain, a 24-amino acid transmembrane portion, and a 707-amino acid extracellular portion. The protein is enzymatically active as a non-covalent homomer. PSMA is expressed in prostate epithelial tissue and is upregulated in the neovascularization of prostate cancer and solid tumors. Furthermore, while it is expressed at low levels in healthy tissues such as the brain, kidneys, and salivary glands, it is overexpressed in malignant prostate tissue, making it an attractive target for the therapeutic treatment of prostate cancer. Additionally, considering its high expression in malignant neovascularization, it may be relevant for the therapy or imaging of solid tumors. Monoclonal antibodies, antibody-drug conjugates, and chimeric antigen receptor T-cells targeting PSMA have been described for the treatment of metastatic prostate cancer (Hernandez-Hoyos et al. 2016, PMID: 27406985; DiPippo et al. 2014, PMID: 25327986; Serganova et al. 2016, PMID: 28345023). In addition, radionuclide conjugates specific to PSMA are being investigated for the imaging and treatment of prostate cancer (e.g., Hofman et al., 2018, PMID: 29752180).

[0007] heavy chain antibody

[0008] In conventional IgG antibodies, the association of the heavy chain and light chain is partly attributed to hydrophobic interactions between the light chain constant region and the CH1 constant domain of the heavy chain. Additionally, there are additional residues in the heavy chain framework 2 (FR2) and framework 4 (FR4) regions attributed to these hydrophobic interactions between the heavy chain and the light chain.

[0009] However, serum from the Camelidae (the suborder Tyropoda, which includes camels, dromedary camels, and llamas) is known to contain the major type of antibody (heavy-chain monoclonal antibody or UniAbs™) composed solely of paired H-chains. Camelidae ( Camelidae )(Camellos Dromedarius( Camelus dromedarius ), Camelus Bactrianus Camelus bactrianus ), Rama Glama ( Lama glama ) , Rama Guanaco ( Lama guanaco ), Llama Alpaca Lama alpaca ) and Rama Vikugna ( Lama vicugna UniAbs™ possess a unique structure consisting of a single variable domain (VHH), a hinge region, and two constant domains (CH2 and CH3), which is highly homologous to the CH2 and CH3 domains of classical antibodies. These UniAbs™ lack the first domain of the constant region (CH1) present in the genome, but it is removed (spliced ​​out) during mRNA processing. The absence of the CH1 domain explains the absence of light chains in UniAbs™, as this domain serves as the anchorage site for the constant domain of the light chain. These UniAbs™ have naturally evolved to confer antigen-binding specificity and high affinity through three CDRs derived from conventional antibodies or fragments thereof (Muyldermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opinion Biol Ther Cartilaginous fish such as sharks have also evolved a unique type of immunoglobulin designated as IgNAR, which lacks light-chain polypeptides and is composed entirely of heavy chains. IgNAR molecules can be manipulated by molecular engineering to generate variable domains (vNARs) of single heavy-chain polypeptides (Nuttall et al. Eur. J. Biochem . 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

[0010] The ability of heavy-chain monoclonal antibodies, which lack light chains, to bind to antigens was established in the 1960s (Jaton et al. (1968) Biochemistry , 7, 4185-4195). Heavy chain immunoglobulins physically separated from light chains possessed 80% antigen-binding activity compared to tetrameric antibodies. Sitia et al. (1990) Cell , 60, 781-790 demonstrated that the removal of the CH1 domain from the rearranged mouse μ gene resulted in the production of heavy-chain-alone antibodies without light chains in mammalian cell cultures. The antibodies produced possessed VH binding specificity and effector function.

[0011] Heavy chain antibodies with high specificity and affinity can be generated against various antigens through immunization (van der Linden, RH, et al. Biochim. Biophys. Acta . 1431, 37-46 (1999)) The VHH portion can be readily cloned and expressed in yeast (Frenken, LGJ, et al. J. Biotechnol . 78, 11-21 (2000)). Their expression, solubility, and stability levels are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, MA et al.). FEBS Lett . 414, 521-526 (1997)).

[0012] Mice in which the λ (lambda) light chain (L) locus and / or the λ and κ (kappa) L chain loci are functionally silenced, and antibodies produced by such mice, are described in U.S. Patents No. 7,541,513 and No. 8,367,888. Recombinant production of heavy chain-alone antibodies in mice and rats is described, for example, WO2006008548; U.S. Application Publication No. 20100122358; Nguyen et al., 2003. Immunology ; 109(1), 93-101; Br ggemann 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 via embryonic microinjection of zinc-finger nuclease was reported in Geurts et al., 2009, Science , 325(5939):433. Transgenic rodents comprising soluble heavy-chain monoclonal antibodies and heterologous heavy-chain loci generating such antibodies are described in U.S. Patents No. 8,883,150 and 9,365,655. CAR-T structures comprising a monodomain antibody as a binding (targeting) domain are, for example, Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta It is recorded in , 1840:378-386.

[0013] An aspect of the present invention relates to heavy chain antibodies, including but not limited to UniAbs™, having binding affinity for PSMA. Further aspects of the present invention relate to a method for producing such antibodies, a composition comprising such antibodies, and the use thereof in the treatment of disorders characterized by the expression of PSMA.

[0014] In some embodiments, the antibody binding to PSMA comprises a first heavy chain variable region comprising (a) CDR1 having two or fewer substitutions in any sequence of the amino acid sequences of SEQ ID NO: 1 to 10; and / or (b) CDR2 having two or fewer substitutions in any sequence of the amino acid sequences of SEQ ID NO: 11 to 17; and / or (c) CDR3 having two or fewer substitutions in any sequence of the amino acid sequences of SEQ ID NO: 18 to 23. In some embodiments, the antibody further comprises a second heavy chain variable region comprising (a) CDR1 having two or fewer substitutions in any sequence of the amino acid sequences of SEQ ID NO: 1 to 10; and / or (b) CDR2 having two or fewer substitutions in any sequence of the amino acid sequences of SEQ ID NO: 11 to 17; and / or (c) CDR3 having two or fewer substitutions in any sequence of the amino acid sequences of SEQ ID NO: 18 to 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 the CH1 sequence.

[0015] In some embodiments, the first heavy chain variable region of the antibody comprises (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 to 10; and / or (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 11 to 17; and / or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18 to 23.

[0016] In some embodiments, the antibody further comprises a second heavy chain variable region comprising (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 to 10; and / or (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 11 to 17; and / or (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18 to 23.

[0017] In some embodiments, the antibody comprises (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 to 10; and (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 11 to 17; and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18 to 23. In some embodiments, the antibody comprises a second heavy chain variable region comprising (a) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 to 10; (b) a CDR2 sequence selected from the group consisting of SEQ ID NOs: 11 to 17; and (c) a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18 to 23.

[0018] In some embodiments, the antibody comprises (a) the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18; or (b) the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 95% sequence identity with respect to any one of the sequences of SEQ ID NO: 24 to 58. In some embodiments, the antibody comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 24 to 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.

[0019] In some embodiments, the antibody binding to PSMA is in a monovalent or divalent form and comprises a first heavy chain variable region comprising the following:

[0020] (a) CDR1 sequence of the following formula:

[0021] GGSISS X1X2Y X3 (Sequence No.: 67)

[0022] Here, X1 is S or N; X2 is S or N; X3 is Y or F; and

[0023] (b) CDR2 sequence of the following formula:

[0024] X4X5X6S G X7T (Sequence No.: 68)

[0025] Here, X4 is I or V; X5 is D or Y; X6 is Y or D; X7 is Y or S; and

[0026] (c) CDR3 sequence of the following formula:

[0027] ARHKAATADFDY (Sequence No.: 69).

[0028] In some embodiments, the antibody binding to PSMA is in a monovalent or divalent form and comprises a first heavy chain variable region comprising the following:

[0029] (a) CDR1 sequence of the following formula:

[0030] GF X1F X2X3Y G (Sequence No.: 70)

[0031] Here, X1 is S or I or T; X2 is S or T or R or I; X3 is R or S; and

[0032] (b) CDR2 sequence of the following formula:

[0033] I X4Y DGSN X5 (Sequence No.: 71)

[0034] Here, X4 is W or S; X5 is R or K; and

[0035] (c) CDR3 sequence of the following formula:

[0036] AREPR X6G YYY X7X8S GY X9S LDY (Sequence No.: 72)

[0037] Here, X6 is I or V; X7 is E or D; X8 is S or T; and X9 is Y or D.

[0038] In some embodiments, the antibody binding to PSMA comprises: a first heavy chain variable region comprising:

[0039] (a) CDR1 sequence of the following formula:

[0040] GGSISS X1X2Y X3 (Sequence No.: 67)

[0041] Here, X1 is S or N; X2 is S or N; X3 is Y or F; and

[0042] (b) CDR2 sequence of the following formula:

[0043] X4X5X6S G X7T (Sequence No.: 68)

[0044] Here, X4 is I or V; X5 is D or Y; X6 is Y or D; X7 is Y or S; and

[0045] (c) CDR3 sequence of the following formula:

[0046] ARHKAATADFDY (Sequence No.: 69),

[0047] and a second heavy chain variable region including the following:

[0048] (a) CDR1 sequence of the following formula:

[0049] GF X1F X2X3Y G (Sequence No.: 70)

[0050] Here, X1 is S or I or T; X2 is S or T or R or I; X3 is R or S; and

[0051] (b) CDR2 sequence of the following formula:

[0052] I X4Y DGSN X5 (Sequence No.: 71)

[0053] Here, X4 is W or S; X5 is R or K; and

[0054] (c) CDR3 sequence of the following formula:

[0055] AREPR X6G YYY X7X8S GY X9S LDY (Sequence No.: 72)

[0056] Here, X6 is I or V; X7 is E or D; X8 is S or T; and X9 is Y or D.

[0057] In some embodiments, the antibody comprises a first and a second heavy chain variable region, wherein the first heavy chain variable region is located closer to the N-terminus than the second heavy chain variable region. In some embodiments, the first heavy chain variable region is located closer to the C-terminus than the second heavy chain variable region.

[0058] In some embodiments, the antibody binding to PSMA comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences in a human VH framework, wherein the CDR sequence comprises a sequence having two or fewer substitutions in a CDR sequence selected from the group consisting of SEQ ID NOs 1-23.

[0059] In some embodiments, the antibody binding to PSMA comprises a heavy chain variable region containing 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.

[0060] In some embodiments, the antibody binding to PSMA comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18 in the human VH framework.

[0061] In some embodiments, the antibody binding to PSMA is in a monovalent or divalent configuration and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18 in the human VH framework.

[0062] In some embodiments, the antibody binding to PSMA comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20 in the human VH framework.

[0063] In some embodiments, the antibody binding to PSMA is in a monovalent or divalent configuration and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20 in the human VH framework.

[0064] In some embodiments, the antibody binding to PSMA comprises a first heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18 in a human VH framework; and a second heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20. In some embodiments, the antibody comprises a first heavy chain variable region located closer to the N-terminus than the second heavy chain variable region. In some embodiments, the first heavy chain variable region is located closer to the C-terminus than the second heavy chain variable region.

[0065] In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific. In some embodiments, the antibody is bispecific. In some embodiments, the antibody has binding affinity for CD3 protein and PSMA protein. In some embodiments, the antibody has binding affinity for two different epitopes on the same PSMA protein. In some embodiments, the antibody has binding affinity for effector cells. In some embodiments, the antibody has binding affinity for T-cell antigens. In some embodiments, the antibody has binding affinity for CD3. In some embodiments, the antibody is of the CAR-T type.

[0066] An aspect of the present invention comprises a bispecific antibody comprising: (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 a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64 in a human VL framework; and (iii) an antigen-binding domain of an anti-PSMA heavy chain antibody comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18 in a human VH framework.

[0067] An aspect of the present invention comprises a bispecific antibody comprising, in a monovalent or divalent configuration, (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 a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64 in a human VL framework; and (iii) an antigen-binding domain of an anti-PSMA heavy chain antibody comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18 in a human VH framework.

[0068] An aspect of the present invention comprises a bispecific antibody comprising (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 a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64 in a human VL framework; and (iii) an antigen-binding domain of an anti-PSMA heavy chain antibody comprising the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20 in a human VH framework.

[0069] An aspect of the present invention comprises a bispecific antibody comprising, in a monovalent or divalent configuration, (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 a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64 in a human VL framework; and (iii) an antigen-binding domain of an anti-PSMA heavy chain antibody comprising the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20 in a human VH framework.

[0070] An aspect of the present invention comprises a two-part configuration: (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 a human VH framework; (ii) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64 in a human VL framework; and (iii) a multispecific antibody comprising an antigen-binding domain of an anti-PSMA heavy chain antibody, wherein the antigen-binding domain comprises a first and a second antigen-binding region, wherein the first antigen-binding region comprises the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18 in a human VH framework; The second antigen-binding region comprises the CDR1 sequence of SEQ ID NO: 7, the CDR2 sequence of SEQ ID NO: 15, and the CDR3 sequence of SEQ ID NO: 20 in the human VH framework. In a specific embodiment, the first antigen-binding region is located closer to the N-terminus than the second antigen-binding region. In another specific embodiment, the first antigen-binding region is located closer to the C-terminus than the second antigen-binding region.

[0071] An aspect of the present invention comprises a multispecific or bispecific antibody, wherein the first and second antigen-binding regions of the antigen-binding domain of the anti-PSMA heavy chain 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 of SEQ ID NO: 73 or SEQ ID NO: 74. In some embodiments, the antigen-binding domain of the anti-PSMA heavy chain antibody is a single paratope and induces less cytokine production compared to a biparatope antigen-binding domain. In some embodiments, the antigen-binding domain of the anti-PSMA heavy chain antibody is a single paratope and expands CD8+ T-cells to a greater extent than a biparatope antigen-binding domain.

[0072] 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.

[0073] An aspect of the present invention relates to a pharmaceutical composition comprising the antibody described herein.

[0074] An aspect of the present invention relates to a method for treating a disorder, comprising the step of administering the antibody or pharmaceutical composition described herein to a subject having a disorder characterized by the expression of PSMA. In another specific aspect, the present invention relates to the use of the antibody described herein in the preparation of a drug for treating a disorder characterized by the expression of PSMA. In yet another aspect, the present invention relates to the antibody described herein for use in the treatment of a disorder characterized by the expression of PSMA. In yet another specific aspect, the present invention relates to a treatment method comprising the step of administering an effective amount of the antibody or pharmaceutical composition described herein to an individual in need thereof. In connection with these aspects, and in some embodiments, the disorder is prostate cancer.

[0075] An aspect of the present invention relates to a polynucleotide encoding an antibody described herein, a vector comprising such polynucleotide, and a cell comprising such vector.

[0076] An aspect of the present invention relates to a method for producing an antibody as described herein, comprising the steps of growing a cell as described herein under conditions allowing the expression of an antibody, and isolating the antibody from the cell.

[0077] An aspect of the present invention relates to a method for producing an antibody as described herein, comprising the steps of immunizing a UniRat animal with a PSMA protein and identifying a PSMA-binding antibody sequence.

[0078] These and additional aspects will be further described in the remainder of this invention, including the examples. Brief explanation of the drawing

[0079] Figure 1, panel AB provides a series of graphs showing serum titers as a function of dilution. Figure 2, Panel A is a graph showing cell binding to human PSMA. Figure 2, Panel B is a graph showing cell binding to synomolgus monkey PSMA. Figure 3 is a graph showing binding competition between two antibody families according to an embodiment of the present invention. FIG. 4, Panel A is a Scatchard plot showing the binding affinity of a bispecific antibody having binding affinity for CD3 and PSMA to PSMA expressed on the cell surface, where the PSMA arm is a single paratop and a monovalent according to an embodiment of the present invention. FIG. 4, Panel B is a scatched plot showing the binding affinity of a bispecific antibody having binding affinity for CD3 and PSMA to PSMA expressed on the cell surface, where the PSMA arm is a bispecific paratop according to an embodiment of the present invention. FIG. 5, Panel AC provides schematic diagrams of an anti-CD3 x 1-valent, monospecific anti-PSMA antibody (Panel A); an anti-CD3 x 2-valent, monospecific anti-PSMA antibody (Panel B); and an anti-CD3 x 2-valent, diparatopic anti-PSMA antibody (Panel C) according to an embodiment of the present invention. Figure 6 is a graph illustrating T-cell-mediated lysis of PSMA-positive cells using pre-activated T-cells. Figure 7 is a graph illustrating T-cell-mediated lysis of PSMA-positive cells using unstimulated T-cells. Figure 8 is a graph showing the specific lysis rate of PSMA-negative DU145 cells as a function of multispecific antibody concentration in the presence of pre-activated T-cells. Figure 9 is a graph showing the binding of PSMA x CD3 bispecific antibodies to PSMA-positive and negative cells. Figure 10 is a graph showing T-cell-mediated lysis of PSMA-positive cells. Figure 11, Panel A is a graph showing T-cell proliferation as a function of antibody concentration. Figure 11, Panel B is a graph showing T-cell proliferation as a function of antibody concentration. Figure 11, Panel C is a graph showing the ratio of CD8 to CD4 of proliferated T-cells. Figure 11, Panel D is a graph showing the ratio of CD8 to CD4 of proliferated T-cells. Figure 12, Panel A is a graph showing T-cell mediated lysis of PSMA-positive cells as a function of antibody concentration. Figure 12, Panel B is a graph showing cytokine (IFNγ) release as a function of antibody concentration. Figure 12, Panel C is a graph showing cytokine (IL-2) release as a function of antibody concentration. Figure 13 is a graph illustrating the inhibition of 22Rv1 tumor growth in a tumor xenograft model. Specific details for implementing the invention

[0080] Practice of the present invention will utilize the ordinary techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology within the art of the art, unless otherwise indicated. Such techniques include "Molecular Cloning: A Laboratory Manual", 2nd ed. (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (FM Ausubel et al., eds., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); It is sufficiently explained in literature such as "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).

[0081] Where a range of values ​​is provided, each intermediate value is understood to be included within the invention up to one-tenth of a lower limit unit, between the upper and lower limits of the range and any other specified value or intermediate value within the said range, unless otherwise clearly indicated by the context. The upper and lower limits of such smaller ranges may be independently included within the smaller ranges and are also included within the invention according to any specifically excluded limits from the stated ranges. Where the stated range includes one or both of the limits, the range excluding either or both of the included limits is also included in the invention.

[0082] Unless otherwise indicated, antibody residues herein are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0083] In the following description, numerous specific details are provided to provide a more complete understanding of the invention. However, it will be apparent to those skilled in the art that the invention may be practiced without one or more of these specific details. In other cases, well-known features and procedures that are familiar to those skilled in the art have not been described to avoid obscuring the invention.

[0084] All references cited throughout this Appendix, including patent applications and publications, are incorporated herein by reference in their entirety.

[0085] I. definition

[0086] "Including" means that while the cited element is necessary for the composition / method / kit, other elements may be included to form the composition / method / kit, etc. within the scope of the claims.

[0087] "Essentially composed of" means a limitation of the scope of the composition or method described for a specified material or step that does not substantially affect the basic and novel feature(s) of the subject invention.

[0088] "Consisting of" means exclusion from any element, step, or component composition, method, or kit not specified in the claims.

[0089] 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 when referring to residues within the variable domain (approximately residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The “EU numbering system” or “EU index” is generally used when referring to residues within the constant domain of the immunoglobulin heavy chain (e.g., the EU index reported in Kabat et al. above). The “EU index as in Kabat” refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise noted herein, references to residue numbers in the variable domain of an antibody mean residue numbering by the Kabat numbering system. Unless otherwise noted herein, references to residue numbering in the constant domain of an antibody mean residue numbering by the EU numbering system.

[0090] Antibodies, also referred to as immunoglobulins, typically comprise at least one heavy chain and one light chain, wherein the amino-terminal domains of the heavy and light chains are variable in sequence and are therefore generally referred to as variable region domains, or variable heavy chain (VH) or variable light chain (VL) domains. The two domains are typically associated to form a specific binding region, but as discussed herein, specific binding can also be obtained as a heavy chain-only variable sequence, and various non-natural compositions of antibodies are known and used in the art.

[0091] “Functional” or “biologically active” antibodies or antigen-binding molecules (including heavy-chain monoclonal antibodies and multispecific (e.g., bispecific) three-chain antibody-like molecules (TCAs, described herein)) are capable of exerting one or more natural activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule, e.g., a TCA, may have the ability to bind specifically to an antigen, and this binding may result in or alter cellular or molecular events such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, e.g., a TCA, may also block ligand activation of a receptor or act as an agonist or antagonist. The ability of an antibody or other binding molecule, e.g., a TCA, to exert one or more natural activities depends on several factors, including the proper folding and assembly of polypeptide chains.

[0092] In this document, the term “antibody” is used in a broad sense and specifically includes monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain monoclonal antibodies, tri-chain antibodies, TCAs, single-chain Fvs (scFvs), nanobodies, etc., and also includes antibody fragments insofar as they exhibit the desired biological activity (Miller et al. (2003) Journal of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimera, or derived from other species.

[0093] The term antibody may refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule; or a polypeptide comprising an immunologically active portion of any of these polypeptides, namely an antigen-binding site that binds immunospecifically to an antigen of a target of interest or a portion thereof, wherein such targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or engineered subclasses having a modified Fc portion that provides reduced or enhanced effector cell activity. The light chain of the target antibody may be a kappa light chain (V-kappa) or a lambda light chain (V-lambda). The immunoglobulin may be derived from any species. In one aspect, immunoglobulins are primarily of human origin.

[0094] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed to a single antigenic site. Furthermore, in contrast to a conventional (polyclonal) antibody population comprising different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. Monoclonal antibodies according to the present invention are Kohler et al. (1975) Nature It can be produced by the hybridoma method first described in 256:495, and also by, for example, through a recombinant protein production method (see, for example, U.S. Patent No. 4,816,567).

[0095] The term "variability," as used in relation to antibodies, refers to the fact that specific parts of an antibody's variable domain differ widely in sequence among antibodies and are utilized for the binding and specificity of each particular antibody to a specific antigen. However, variability is not evenly distributed throughout the antibody's variable domain. Three segments called hypervariability regions are concentrated in both the light and heavy chain variable domains. A more highly conserved part of the variable domain is called the framework region (FR). The variable domains of natural heavy and light chains each contain four FRs, which primarily adopt a β-sheet configuration and are connected by three hypervariability regions to form loop connections, and in some cases, form part of the β-sheet structure. The hypervariability regions of each chain are held together very closely by FRs and, together with hypervariability regions from other chains, contribute to the formation of the antibody's antigen-binding site (Kabat et al.). , Sequences of Proteins of Immunological Interest(See , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domain is not directly involved in binding the antibody to the antigen, but it exhibits various effector functions, such as the antibody's participation in antibody-dependent cytotoxicity (ADCC).

[0096] As used herein, the term “hypervariable domain” refers to the amino acid residues of the antibody responsible for antigen-binding. The hypervariable domain is generally an amino acid residue from the “complementarity determining domain” or “CDR” (e.g., residues 31–35 (H1), 50–65 (H2), and 95–102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or such residues from "hypervariable loops" (residues 26-32(H1), 53-55(H2) and 96-101(H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. Includes 196:901-917 (1987)). In some embodiments, “CDR” refers to 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). The “framework region” or “FR” residue is a variable domain residue other than the hypervariable region / CDR residue as defined herein.

[0097] Although exemplary CDR designations are presented herein, those skilled in the art will understand that numerous definitions of CDR, including the Kabat definition, which is based on sequence variability and is the most commonly used, are generally in use ("Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions." Mol Immunol . 2010;47:694-700). The definition of Chothia is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature . 1989; 342:877-883). An alternative CDR definition of interest is 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 sizes: implications for the rational design of antibody repertoires." J Mol Recognit.... 2004;17:132-143; and Padlan et al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139. Each of these is specifically incorporated herein by reference.

[0098] The terms “heavy-chain-alone antibody” and “heavy-chain antibody” are used interchangeably herein and, in a broad sense, refer to an antibody, or one or more parts of an antibody, for example, one or more arms of an antibody lacking the light chain of a conventional antibody. The term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen-binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain; functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NARs and functional fragments thereof comprising homodimerics of one variable domain (V-NAR) and five C-like constant domains (C-NAR); and soluble single-domain antibodies (sUniDabs™). In one embodiment, the heavy-chain-alone antibody comprises a variable domain antigen-binding domain composed of Framework 1, CDR1, Framework 2, CDR2, Framework 3, CDR3, and Framework 4. In another embodiment, the heavy chain-alone antibody comprises an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In another embodiment, the heavy chain-alone antibody comprises an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In a further embodiment, the heavy chain-alone antibody comprises an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. A heavy chain-alone antibody in which the CH2 and / or CH3 domains are truncated is also included herein. In a further embodiment, the heavy chain comprises an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but without a hinge region. The heavy chain-alone antibody may be in the form of a dimer in which two heavy chains are disulfide-linked or otherwise attached to each other covalently or non-covalently. Heavy chain-only antibodies may belong to the IgG subclass, but antibodies belonging to other subclasses such as IgM, IgA, IgD, and IgE are also included herein.In certain embodiments, the heavy chain antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype. In one embodiment, the heavy chain-only antibody of the present invention is used as the binding (targeting) domain of a chimeric antigen receptor (CAR). The definition specifically includes human heavy chain-only antibodies produced by human immunoglobulin transplanted rats (UniRat™), called UniAbs™. The variable domain (VH) of UniAbs™ is called UniDabs™ and is a versatile building block that can be linked to the Fc domain or serum albumin for the development of novel therapeutics with multispecificity, increased efficacy, and extended half-life. Since the homomeric UniAbs™ lacks a light chain and therefore lacks a VL domain, the antigen is recognized by a single domain, namely the variable domain (VH or VHH) of the heavy chain of the heavy chain antibody.

[0099] As used herein, an “intact antibody chain” comprises a full-length variable region and a full-length constant region (Fc). An intact “conventional” antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and a heavy chain constant domain, CH1, hinge, CH2, and CH3 for secreted IgG. Other isoforms, such as IgM or IgA, may have different CH domains. The constant domain may be a natural sequence constant domain (e.g., human natural sequence constant domain) or an amino acid sequence variant thereof. An intact antibody may have one or more “effector functions” referring to biological activity attributable to the antibody’s Fc constant region (natural sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors. Invariant region variants include those that alter the effector profile, binding to Fc receptors, etc.

[0100] Depending on the amino acid sequence of the heavy chain Fc (constant domain), antibodies and various antigen-binding proteins can be provided as different classes. The heavy chain Fc region has five major classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains corresponding to the different classes of antibodies can be designated as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. Ig forms include hinge-modified or hinge-less 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). The light chain of an antibody from any vertebrate species may be assigned to one of two types called κ (kappa) and λ (lambda) based on the amino acid sequence of the constant domain. An antibody according to an embodiment of the present invention may comprise a kappa light chain sequence or a lambda light chain sequence.

[0101] "Functional Fc regions" possess "effector functions" of the natural-sequence Fc regions. Non-limiting examples of effector functions include C1q binding; CDC; Fc-receptor binding; ADCC; ADCP; and downregulation of cell-surface receptors (e.g., B-cell receptors). These effector functions generally require Fc regions to interact with receptors, e.g., FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors, and low-affinity FcRn receptors; and can be evaluated using various assays known in the art. "Dead" or "silenced" Fc are mutated to maintain activity, e.g., associated with serum half-life prolongation, but do not activate high-affinity Fc receptors, or have reduced affinity for Fc receptors.

[0102] "Natural-sequence Fc regions" contain amino acid sequences identical to the amino acid sequences of Fc regions found in nature. Natural-sequence human Fc regions include, for example, natural-sequence human IgG1 Fc regions (non-A and A homologous types); natural-sequence human IgG2 Fc regions; natural-sequence human IgG3 Fc regions; and natural-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0103] The “variant Fc region” comprises an amino acid sequence different from that of the natural-sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the natural-sequence Fc region or the Fc region of the parent polypeptide, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, compared to the natural-sequence Fc region or the Fc region of the parent polypeptide. In the invention, the variant Fc region will preferably have at least about 80% homology with the natural-sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology with it, and more preferably at least about 95% homology with it.

[0104] Variant Fc sequences may include three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement 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 with IgG4 residues at positions 327, 330, and 331 significantly reduces ADCC and CDC (see, e.g., Armour KL. et al., 1999 Eur J Immunol. 29(8):2613–24; and Shields RL. et al., 2001 J Biol Chem. 276(9):6591–604). The human IgG4 Fc amino acid sequence (UniProtKB No. P01861) is provided herein by SEQ ID NO: 76. Silenced IgG1 is described, for example, in Boesch, AW, et al., “Highly parallel characterization of IgG Fc binding interactions.” MAbs, 2014. 6(4): p. 915–27, the full text of which is incorporated herein by reference.

[0105] Other Fc variants are possible, including but not limited to cases where a region capable of forming a disulfide bond is deleted, where a specific amino acid residue is removed from the N-terminal end of the natural Fc, or where a methionine residue is added thereto. Accordingly, in some embodiments, one or more Fc portions of the antibody may include one or more mutations in the hinge region to remove the disulfide bond. Also, in another embodiment, the hinge region of the Fc may be completely removed. Also, in yet another embodiment, the antibody may include an Fc variant.

[0106] Additionally, Fc variants may be configured to eliminate or substantially reduce effector function by affecting complement binding or Fc receptor binding through the substitution (mutation), deletion, or addition of amino acid residues. For example, and without limitation, deletion may occur at complement-binding sites such as C1q-binding sites. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publications Nos. WO 97 / 34631 and WO 96 / 32478. Furthermore, the Fc domain may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0107] In some embodiments, the antibody comprises a variant human IgG4 CH3 domain sequence including the T366W mutation, which may optionally be referred herein as the IgG4 CH3 knob sequence. In some embodiments, the antibody comprises a variant human IgG4 CH3 domain sequence including the T366S mutation, the L368A mutation, and the Y407V mutation, which may optionally be referred herein as the IgG4 CH3 hole sequence. The IgG4 CH3 mutations described herein may be utilized in any suitable manner to facilitate the proper pairing (heterodimerization) of a desired pair of heavy chain polypeptide subunits in the antibody by placing a “knob” on the first heavy chain constant region of the first monomer in the antibody dimer and a “hole” on the second heavy chain constant region of the second monomer in the antibody dimer.

[0108] In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region including the S228P mutation, the F234A mutation, the L235A mutation, and the T366W mutation (NOB). In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region including the S228P mutation, the F234A mutation, the L235A mutation, the T366S mutation, the L368A mutation, and the Y407V mutation (HOL).

[0109] The term "Fc-region-containing antibody" refers to an antibody containing an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during the purification of the antibody or by recombination operations of the nucleic acid encoding the antibody. Accordingly, antibodies having an Fc region according to the present invention may include antibodies with or without K447.

[0110] An aspect of the present invention comprises an antibody comprising a heavy chain-only variable region in a monovalent or divalent configuration. As used herein, the term “monovalent configuration” as 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 “divalent configuration” as used in relation to a heavy chain-only variable region domain means that there are two heavy chain-only variable region domains (each having a single binding site) and are connected by a linker sequence (see FIG. 5, Panels B and C, right arm of the antibody). Non-limiting examples of linker sequences are further discussed herein and include, but are not limited to, GS linker sequences of various lengths. When a heavy chain-only variable region is in a divalent configuration, each of the two heavy chain-only variable region domains may have binding affinities for the same antigen or 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 indicated as being in a "divalent configuration" is understood to contain two identical heavy chain-only variable region domains connected by a linker sequence, wherein each of the two identical heavy chain-only variable region domains has binding affinities for the same target antigen.

[0111] An aspect of the present invention includes antibodies having a multispecific composition, including but not limited to bispecific, triplespecific, etc. A wide variety of methods and protein compositions are known and are used in bispecific monoclonal antibodies (BsMAB), triplespecific antibodies, etc.

[0112] Various methods for the production of polyvalent artificial antibodies have been developed by recombinantly fusing the variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on 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, wherein the sequence is repeated n times, where n is an integer in the range of 1 to about 10, e.g., 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkers include GGGGS (SEQ No.: 73) (n=1) and GGGGSGGGGS (SEQ No.: 74) (n=2). Other suitable linkers may also be used, such as those described, for example, in Chen et al., Adv Drug Deliv Rev. 2013 October 15; 65(10): 1357-69, the full text of which is incorporated herein by reference.

[0113] The terms “three-chain antibody-like molecule” or “TCA” are used herein to refer to an antibody-like molecule comprising, essentially consisting of, or consisting of three polypeptide subunits, two of which comprise one heavy chain and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such antibody chain, comprising an antigen-binding region and at least one CH domain. This heavy chain / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, essentially consisting of, or consisting of a heavy chain monoantibody comprising an Fc portion comprising CH2 and / or CH3 and / or CH4 domains in the absence of a CH1 domain, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind to an epitope of the second antigen or a different epitope of the first antigen, wherein such binding domains are derived from or have sequence identity with a variable region of the antibody heavy chain or light chain. Part of this variable region is V H and / or V L Gene segments, D and J H　 Gene segment, or J L It can be encoded by gene segments. The variable region is a rearranged V H DJ H , V L DJ H , V H J L , or V L J L It can be encoded by gene segments.

[0114] As used herein, the TCA conjugated compound uses the terms "heavy chain single antibody," "heavy chain antibody," or "heavy chain polypeptide," meaning a single-chain antibody comprising a heavy chain constant region CH2 and / or CH3 and / or CH4 but not a CH1 domain. In one embodiment, the heavy chain antibody consists of an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In another embodiment, the heavy chain antibody consists of an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In a further embodiment, the heavy chain antibody consists of an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. A heavy chain antibody in which the CH2 and / or CH3 domains are truncated is also included herein. In a further embodiment, the heavy chain consists of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain but lacks a hinge region. The heavy chain monoclonal antibody may be in a dimeric form, wherein the two heavy chains are otherwise covalently or non-covalently attached to each other and may include an asymmetric interface between one or more of the CH domains to facilitate appropriate pairing between the polypeptide chains. The heavy chain antibody may belong to the IgG subclass, but antibodies belonging to other subclasses such as the IgM, IgA, IgD, and IgE subclasses are also included herein. In certain embodiments, the heavy chain antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype or the IgG4 subtype. Non-limiting examples of TCA-conjugated compounds are described, for example, in WO2017 / 223111 and WO2018 / 052503, the full text of which is incorporated herein by reference.

[0115] Heavy chain antibodies constitute about one-quarter of the IgG antibodies produced by camelid animals, e.g., camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed of two heavy chains but lack a light chain. Consequently, the variable antigen-binding region is referred to as the VHH domain and represents the smallest naturally occurring intact antigen-binding site, with a length of approximately 120 amino acids (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with high specificity and affinity can be generated against various antigens through immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH portion can be easily cloned and expressed in yeast (Frenken, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Their expression, solubility, and stability levels are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, MA et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been suggested to possess a single VH-like domain called VNAR in their antibodies. (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)).

[0116] The term “PSMA” as used herein refers to a type II transmembrane protein having N-acetylated-alpha-linked acidic peptidase, folate hydrolase, and dipeptidyl-peptidase activity. The term “PSMA” includes PSMA proteins of any human and non-human animal species, specifically including human PSMA as well as PSMA of non-human mammals.

[0117] The term “human PSMA” as used herein includes any variants, isoforms, and species homologs of human PSMA (UniProt Q04609), regardless of source or method of production. Accordingly, “human PSMA” includes human PSMA naturally expressed by cells and PSMA expressed on cells transfected with the human PSMA gene.

[0118] The terms "anti-PSMA heavy chain-only antibody," "PSMA heavy chain-only antibody," "anti-PSMA heavy chain antibody," and "PSMA heavy chain antibody" are used interchangeably herein to refer to heavy chain-only antibodies as defined above that bind immunospecifically to PSMA, including human PSMA, as defined above. The definition includes, but is not limited to, human heavy chain antibodies produced by transplanted animals, such as transplanted rats or transplanted mice expressing human immunoglobulin, including UniRats™ that produce human anti-PSMA UniAb™ antibodies, as defined above.

[0119] With respect to a reference polypeptide sequence, "percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in candidate sequences identical to amino acid residues in the reference polypeptide sequence, after aligning sequences and introducing gaps to achieve maximum percentage sequence identity where necessary, and any conservative substitutions are not considered as part of the sequence identity. Alignment to determine percentage amino acid sequence identity can be achieved using various methods within the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art may determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this invention, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2.

[0120] "Isolated" antibodies are identified, separated, and / or recovered from components of the natural environment. Contaminating components of the natural environment are substances that interfere with the diagnostic or therapeutic use of the antibodies and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody will be purified for homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining, to an extent sufficient to obtain (1) more than 95% by weight of the antibody as determined by the Lowry method, most preferably more than 99% by weight, (2) at least 15 residues of the N-terminal or internal amino acid sequence by the use of a spinning cup sequencer, or (3) Coomassie blue or, preferably, silver staining. The isolated antibody contains the antibody in situ within the recombinant cell, as at least one component of the antibody's natural environment will not be present. However, normally, the isolated antibody will be prepared by at least one purification step.

[0121] The antibodies of the present invention comprise multispecific antibodies. Multispecific antibodies have more than one binding specificity. The term “multispecific” specifically includes “bispecific” and “triplespecific,” as well as higher-order independent specific binding affinities such as higher-order polyepitope specificity, as well as quadrivalent antibodies and antibody fragments. The terms “multispecific antibody,” “multispecific heavy-chain monoantibody,” “multispecific heavy-chain antibody,” and “multispecific UniAb™” are used herein in a broad sense and encompass all antibodies having more than one binding specificity. The multispecific heavy-chain anti-PSMA antibodies of the present invention specifically comprise antibodies that immunospecifically bind to two or more non-overlapping epitopes (i.e., bivalent and biparatopes) on a PSMA protein such as human PSMA. The multispecific heavy chain anti-PSMA antibody of the present invention also comprises an antibody that specifically binds to epitopes on PSMA proteins such as human PSMA and epitopes on different proteins such as, for example, human CD3 proteins (i.e., divalent and double paratopes). The multispecific heavy chain anti-PSMA antibody of the present invention also comprises an antibody that specifically binds to two or more non-overlapping or partially overlapping epitopes on PSMA proteins such as human PSMA proteins, and epitopes on different proteins such as, for example, human CD3 proteins (i.e., trivalent and double paratopes).

[0122] The antibodies of the present invention comprise monospecific antibodies having a single binding specificity. Monospecific antibodies specifically comprise antibodies having a single binding specificity, as well as antibodies having more than one binding unit having the same binding specificity. The terms “monospecific antibody,” “monospecific heavy-chain-alone antibody,” “monospecific heavy-chain antibody,” and “monospecific UniAb™” are used herein in a broad sense and encompass all antibodies having a single binding specificity. The monospecific heavy-chain anti-PSMA antibodies of the present invention specifically comprise antibodies that immunospecifically bind to a single epitope (monovalent and monospecific) on a PSMA protein such as human PSMA. The monospecific heavy-chain anti-PSMA antibodies of the present invention also specifically comprise antibodies (e.g., polyvalent antibodies) having more than one binding unit that immunospecifically bind to an epitope on a PSMA protein such as human PSMA. For example, a monospecific antibody according to an embodiment of the present invention may include a heavy chain variable region comprising two antigen-binding domains, wherein each antigen-binding domain binds to the same epitope (i.e., divalent and monospecific) on the PSMA protein.

[0123] An "epitope" is a region on the surface of an antigen molecule to which a single antibody molecule binds. Generally, antigens have several or many different epitopes and react with many different antibodies. The term specifically includes linear epitopes and morphological epitopes.

[0124] "Epitope mapping" is the process of identifying antibody binding sites, or epitopes, on a target antigen. Antibody epitopes can be linear epitopes or morphological epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Morphological epitopes are discontinuous in the protein sequence but form amino acids that combine when the protein folds into a three-dimensional structure.

[0125] "Polyepitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). As mentioned above, the present invention specifically comprises an anti-PSMA heavy chain antibody having polyepitope specificity, namely, an anti-PSMA heavy chain antibody that binds to one or more non-overlapping epitopes on a PSMA protein such as human PSMA; and an anti-PSMA heavy chain antibody that binds to one or more epitopes on an anti-PSMA protein and epitopes on a different protein such as, for example, the CD3 protein. The terms "non-overlapping epitope(s)" or "non-competitive epitope(s)" of an antigen are defined herein to mean epitope(s) that are recognized by one member of a pair of antigen-specific antibodies but not by the other member. A pair of antibodies or antigen-binding regions on a multispecific antibody that recognizes a non-overlapping epitope and targets the same antigen can bind to the antigen simultaneously without competing for binding to the antigen.

[0126] When two antibodies recognize identical or stereoscopically overlapping epitopes, the antibody binds to the "essentially identical epitope" as a reference antibody. The most widely used rapid method to determine whether two epitopes bind to identical or stereoscopically overlapping epitopes is a competitive test, which can be configured in any number of different forms using labeled antigens or labeled antibodies. Typically, antigens are immobilized on 96-well plates, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzymatic labeling.

[0127] The term "valence" as used herein refers to a specified number of binding sites in an antibody molecule.

[0128] "Monovalent" antibodies have a single binding site. Therefore, monovalent antibodies are also monospecific.

[0129] “Multivalent” antibodies have two or more binding sites. Accordingly, the terms “divalent,” “trivalent,” and “tetravalent” refer to the presence of two binding sites, three binding sites, and four binding sites, respectively. Accordingly, the bispecific antibody according to the present invention may be at least divalent and may be trivalent, tetravalent, or otherwise multivalent. The divalent antibody according to an embodiment of the present invention may have two binding sites on the same epitope (i.e., divalent, single paratope) or two different epitopes (i.e., divalent, double paratope).

[0130] A wide variety of methods and protein compositions are known and used for the production of bispecific monoclonal antibodies (BsMABs), trispecific antibodies, and the like.

[0131] The terms “three-chain antibody-like molecule” or “TCA” are used herein to refer to an antibody-like molecule comprising, essentially consisting of, or consisting of three polypeptide subunits, two of which comprise one heavy chain and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such antibody chain, comprising an antigen-binding region and at least one CH domain. Such heavy chain / light chain pairs have binding specificity for a first antigen. A third polypeptide subunit comprises, essentially consisting of, or consisting of a heavy chain-only antibody comprising an Fc portion comprising CH2 and / or CH3 and / or CH4 domains in the absence of a CH1 domain, and an antigen-binding domain that binds to an epitope of the second antigen or a different epitope of the first antigen, wherein such binding domain is derived from or has sequence identity with a variable region of the antibody heavy chain or light chain. A portion of such variable region is V H and / or V L Gene segments, D and J H　Gene segment, or J L It can be encoded by gene segments. The variable region is a rearranged V H DJ H , V L DJ H , V H J L , or V L J L It can be encoded by a gene segment. The TCA protein uses a heavy chain-alone antibody as defined above.

[0132] The terms “chimeric antigen receptor” or “CAR” are used herein in a broad sense to refer to engineered receptors that transplant a desired binding specificity (e.g., the antigen-binding domain of a monoclonal antibody or other ligand) into a membrane-spanning and intracellular signaling domain. Typically, receptors are used to generate chimeric antigen receptors (CARs) by transplanting the specificity of a monoclonal antibody into T-cells. J Natl Cancer Inst, 2015; 108(7):dvj439; and Jackson et al., Nature Reviews Clinical Oncology , 2016; 13:370-383). CAR-T cells are T-cells that have been genetically engineered to produce artificial T-cell receptors for use in immunotherapy. In one embodiment, “CAR-T cell” refers to a therapeutic T-cell that expresses a transplant gene encoding one or more chimeric antigen receptors consisting of at least an extracellular domain, a transmembrane domain, and at least one cytoplasmic domain.

[0133] The term “human antibody” is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences, for example, mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo. The term “human antibody” specifically includes heavy-chain monoclonal antibodies having human heavy-chain variable region sequences produced by transplanted animals such as transplanted rats or mice, in particular UniAbs™ produced by UniRats™ as defined above.

[0134] "Chimeric antibody" or "chimeric immunoglobulin" means an immunoglobulin molecule comprising amino acid sequences from at least two different Ig loci, for example, a transplanted gene antibody comprising a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include a transplanted gene antibody having a non-human Fc-region or an artificial Fc-region, and a human individual-specific type. Such immunoglobulins may be isolated from the animals of the present invention engineered to produce such chimeric antibodies.

[0135] As used herein, the term “effector cell” refers to an immune cell involved in the effector phase of an immune response, in contrast to the recognition and activation phases of the immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, may induce antibody-dependent cytotoxicity (ADCC). For example, monocytes and macrophages expressing FcR are involved in the specific death of target cells and the presentation of antigens to other components of the immune system, or binding to antigen-presenting cells. In some embodiments, effector cells may phagocytose target antigens or target cells.

[0136] "Human effector cells" are leukocytes that express a receptor such as T-cell receptor or FcR and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T-cells, and neutrophils; NK cells are preferred. Effector cells may be isolated from their natural sources, for example, blood or PBMC as described herein.

[0137] The term “immune cell” is used herein in a broad sense and includes, but is not limited to, cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells including cytolytic T-cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells such as neutrophils, granulocytes, mast cells, and basophils.

[0138] Antibody "effector function" refers to biological activity attributed to the antibody's Fc region (natural sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B-cell receptors; BCR).

[0139] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which nonspecific cytotoxic cells expressing the Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently induce lysis of the target cells. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is Ravetch and Kinet, Annu. Rev. Immunol This is summarized in Table 3 on page 464 of 9:457-92 (1991). To evaluate the ADCC activity of the molecule of interest, in vitro ADCC assays such as those described in U.S. Patent No. 5,500,362 or No. 5,821,337 may be performed. Effector cells useful for such assays include peripheral blood monocytes (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo, for example, by Clynes et al. PNAS (USA) It can be evaluated in animal models such as that disclosed in 95:652-656 (1998).

[0140] "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 binding the first component (C1q) of the complement system to a molecule (e.g., an antibody) complexed with a cognate antigen. To evaluate complement activation, for example, Gazzano-Santoro et al., J. Immunol. Methods A CDC test as described in 202:163 (1996) can be performed.

[0141] "Binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., antibody) and its binding partner (e.g., antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity reflecting the 1:1 interaction between the members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art. Low-affinity antibodies generally tend to bind slowly to an antigen and dissociate easily, whereas high-affinity antibodies generally tend to bind rapidly to an antigen and remain bound.

[0142] As used herein, "Kd" or "Kd value" refers to the dissociation constant determined by biolayer interferometer using the Octet QK384 instrument (Fortebio Inc., Menlo Park, California) in kinetic mode. For example, the concentration-dependent association rate (kon) is measured by loading a mouse-Fc fused antigen onto an anti-mouse Fc sensor and then immersing it in an antibody-containing well. The antibody dissociation rate (koff) is measured at the final step by immersing the sensor in a well containing only buffer. Kd is the ratio of koff to kon. (For further details, see Concepcion, J, et al. Comb Chem High Throughput Screen See , 12(8), 791-800, 2009).

[0143] Terms such as "treatment" and "treating" are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects attributable to the disease. As used herein, "treatment" encompasses any treatment of disease in mammals and includes (a) preventing the onset of disease in subjects who may be susceptible but have not yet been diagnosed with the disease; (b) suppressing the disease, i.e., inhibiting its development; or (c) alleviating the disease, i.e., causing the disease to induce regression. Therapeutic agents may be administered before, during, or after the onset of disease or injury. Treatment of an ongoing disease is of particular interest if the treatment stabilizes or reduces the patient's undesirable clinical symptoms. Such treatment is preferably performed before the complete loss of function in the affected tissues. Targeted therapy may be administered during the symptomatic phase of the disease, and in some cases, after the symptomatic phase.

[0144] "Therapeutically effective dose" is intended as the amount of activator necessary to impart a therapeutic benefit to the subject. For example, a "therapeutically effective dose" is an amount that induces, enhances, or otherwise causes improvement in pathological symptoms associated with a disease, disease progression, or physiological status, or improves tolerance to a disorder.

[0145] The term "prostate cancer" as used herein refers to a malignant tumor of congenital origin within the prostate.

[0146] The term "characterized by the expression of PSMA" broadly refers to any disease or disorder associated with or accompanying one or more pathological processes in which the expression of PSMA is characteristic of the disease or disorder. Such disorders include, but are not limited to, prostate cancer.

[0147] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to mammals being evaluated and / or treated for treatment. In an embodiment, the mammal is a human. The terms “subject,” “individual,” and “patient” include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals infected with pathogens, etc. The subject may include humans, but also other mammals, particularly mammals useful for laboratory models of human diseases, e.g., mice, rats, etc.

[0148] The term “pharmaceutical formulation” refers to a form that allows the biological activity of an active ingredient to be effective and does not contain any additional ingredients that are unacceptably toxic to the subject to which the formulation is to be administered. Such formulations are sterile. “Pharmaceutically acceptable” excipients (vehicles, additives) are those that can be reasonably administered to the subject mammal to provide an effective amount of the active ingredient used.

[0149] "Sterile" formulations are sterile or free of all living microorganisms and their spores or are essentially non-existent. "Frozen" formulations are at a temperature below 0°C.

[0150] A "stable" formulation is one in which the protein contained therein essentially maintains physical stability and / or chemical stability and / or biological activity during storage. Preferably, the formulation essentially maintains physical and chemical stability, as well as biological activity, during storage. The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones. A. Adv. Drug Delivery Rev. 10: 29-90) (1993). Stability can be measured at a selected temperature for a selected period. Stability is evaluated by assessing aggregation formation (e.g., using size exclusion chromatography, turbidity measurement, and / or visual inspection); evaluating charge heterogeneity using cation exchange chromatography, image capillary isoelectric point electrophoresis (icIEF), or capillary zone electrophoresis; It may be evaluated qualitatively and / or quantitatively in various different ways, including amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis for comparison of reduced and intact antibodies; peptide map (e.g., trypsin or LYS-C) analysis; evaluation of the biological activity or antigen-binding function of the antibody, etc. Instability may involve any one or more of the following: agglutination, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.

[0151] II. details

[0152] Anti-PSMA antibody

[0153] The present invention provides a closely related family of antibodies that bind to human PSMA. Antibodies of this family provide a set of CDR sequences as defined herein and presented in Table 1, and are exemplified by the provided heavy chain variable region (VH) sequences of SEQ ID NOs: 24 to 54 presented in Table 2. The antibody family provides many advantages that contribute to clinical utility as therapeutic(s). The antibodies include members with various binding affinities, allowing for the selection of specific sequences with desired binding affinities.

[0154] Table 1: Intrinsic CDR amino acid sequence of anti-PSMA heavy chain antibody.

[0155]

[0156] Table 2. Anti-PSMA heavy chain antibody variable domain amino acid sequence.

[0157]

[0158]

[0159]

[0160] Suitable antibodies may be selected from those provided herein for development, treatment, or other uses, including but not limited to bispecific antibodies or trispecific antibodies as presented in Panel AC of FIG. 5, for example, or for use as part of a CAR-T structure. Panel AC of FIG. 5 provides an example of an anti-CD3 x anti-PSMA multispecific antibody, wherein the anti-PSMA domain is monovalent and monospecific, divalent and monospecific, or divalent and bispecific (double paratop). The anti-CD3 domain contains a CH1 domain and is paired with a light chain, whereas the anti-PSMA domain is derived from a heavy chain-alone antibody and does not contain a CH1 domain or does not interact with a light chain. In some embodiments, the two heavy chains are paired, for example, using a knop-into-hole technique. Returning to the antibodies illustrated in Fig. 5, Panel A illustrates an anti-CD3 x anti-PSMA bispecific antibody, wherein the anti-PSMA binding arm is monovalent and monospecific, and the antigen-binding domain of the anti-PSMA arm is monovalent, meaning that only one antigen-binding domain is present. Panel B illustrates an anti-CD3 x anti-PSMA bispecific antibody, wherein the anti-PSMA binding arm is divalent and monospecific, and the antigen-binding domain of the anti-PSMA arm is divalent, meaning that two identical antigen-binding domains are arranged in a tandem. Panel C illustrates an anti-CD3 x anti-PSMA bispecific antibody, wherein the anti-PSMA binding arm is divalent and biparatope, and the antigen-binding domain of the anti-PSMA arm is divalent.

[0161] The determination of affinity for candidate proteins can be performed using methods known in the art, such as Biacore measurements. Members of the antibody family are approximately 10 -6 to approximately 10 -10 ; Approx. 10 -6 to approximately 10 -9; Approx. 10 -6 to approximately 10 -8 ; Approx. 10 -8 to approximately 10 -11 ; Approx. 10 -8 to approximately 10 -10 ; Approx. 10 -8 to approximately 10 -9 ; Approx. 10 -9 to approximately 10 -11 ; Approx. 10 -9 to approximately 10 -10 ; or approximately 10 including, but not limited to, any value within these ranges -6 to approximately 10 -11 It has an affinity for PSMA through its Kd. Affinity selection can be confirmed through in vitro assays, preclinical models, and clinical trials, as well as biological evaluations to modulate, for example, block the biological activity of PSMA, including the assessment of potential toxicity.

[0162] At this institution, a member of the antibody family is synomolgus ( Cynomolgus It does not cross-react with the PSMA protein of macaques, but can be engineered to provide cross-reactivity with the PSMA protein of synomolgus macaques, or, if preferred, with the PSMA of any other animal species.

[0163] In this document, the family of PSMA-specific antibodies comprises a VH domain containing the sequences CDR1, CDR2, and CDR3 in a human VH framework. The CDR sequences may be located in the regions near amino acid residues 26-33; 51-58; and 97-116, respectively, for CDR1, CDR2, and CDR3 of the provided exemplary variable region sequences presented in SEQ ID NOs. 24 to 58. The CDR sequences may be located at different positions when different framework sequences are selected, but it will be understood by those skilled in the art that the order of the sequences generally remains the same.

[0164] The CDR1, CDR2, and CDR3 sequences of the anti-PSMA antibody of the present invention may be included by the following structural formula, wherein X represents a variable amino acid that may be a specific amino acid as shown below.

[0165] CDR1

[0166] GGSISS X1X2Y X3 (Sequence No.: 67)

[0167] Here, X1 is S or N and;

[0168] X2 is S or N and;

[0169] X3 is Y or F;

[0170] CDR2

[0171] X4X5X6S G X7T (Sequence No.: 68)

[0172] Here, X4 is I or V and;

[0173] X5 is D or Y;

[0174] X6 is Y or D;

[0175] X7 is Y or S;

[0176] CDR3

[0177] ARHKAATADFDY (Sequence No.: 69)

[0178] The CDR1, CDR2, and CDR3 sequences of the anti-PSMA antibody of the present invention may be included by the following structural formula, wherein X represents a variable amino acid that may be a specific amino acid as shown below.

[0179] CDR1

[0180] GF X1F X2X3Y G (Sequence No.: 70)

[0181] Here, X1 is S or I or T and;

[0182] X2 is S or T or R or I;

[0183] X3 is R or S;

[0184] CDR2

[0185] I X4Y DGSN X5 (Sequence No.: 71)

[0186] Here, X4 is W or S and;

[0187] X5 is R or K;

[0188] CDR3

[0189] AREPR X6G YYY X7X8S GY X9S LDY (Sequence No.: 72)

[0190] Here, X6 is I or V and;

[0191] X7 is E or D;

[0192] X8 is S or T;

[0193] X8 is Y or D.

[0194] Representative CDR1, CDR2, and CDR3 sequences are presented in Tables 1 and 3.

[0195] Table 3: Anti-PSMA heavy chain antibody CDR1, CDR2, and CDR3 amino acid sequences.

[0196]

[0197]

[0198] In some embodiments, the anti-PSMA antibody comprises any one of the CDR1 sequences from SEQ ID NOs: 1-10. In certain embodiments, the CDR1 sequence is SEQ ID NOs: 2 or 7.

[0199] In some embodiments, the anti-PSMA antibody comprises any one of the CDR2 sequences from SEQ ID NOs: 11-17. In certain embodiments, the CDR2 sequence is SEQ ID NO: 11 or 15.

[0200] In some embodiments, the anti-PSMA antibody comprises any one of the CDR3 sequences SEQ ID NOs: 18-23. In certain embodiments, the CDR3 sequence is SEQ ID NO: 18 or 20.

[0201] In an additional embodiment, the anti-PSMA heavy chain-alone antibody comprises the CDR1 sequence of SEQ ID NO: 2; the CDR2 sequence of SEQ ID NO: 11; and the CDR3 sequence of SEQ ID NO: 18.

[0202] In an additional embodiment, the anti-PSMA antibody comprises the CDR1 sequence of SEQ ID NO: 7; the CDR2 sequence of SEQ ID NO: 15; and the CDR3 sequence of SEQ ID NO: 20.

[0203] In an additional embodiment, the anti-PSMA antibody comprises any of the heavy chain variable region amino acid sequences of SEQ ID NOs: 24 to 58 (Table 2).

[0204] In addition, in a further embodiment, the anti-PSMA antibody comprises the heavy chain variable region sequence of SEQ ID NO: 25.

[0205] In addition, in a further embodiment, the anti-PSMA antibody comprises the heavy chain variable region sequence of SEQ ID NO: 38.

[0206] In some embodiments, the CDR sequence in the anti-PSMA antibody of the present invention comprises one or two amino acid substitutions for the CDR1, CDR2 and / or CDR3 sequence or the set of CDR1, CDR2 and CDR3 sequences in any one of SEQ ID NOs: 1 to 23 (Table 1).

[0207] In some embodiments, the anti-PSMA antibody preferably comprises a heavy chain variable domain (VH) having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or at least 99% sequence identity at the amino acid level with respect to any one of the CDR3 sequences provided in Table 1, and binds to PSMA.

[0208] In some embodiments, the anti-PSMA antibody preferably comprises a heavy chain variable domain (VH) having at least 85% sequence identity at the amino acid level with respect to the CDR 1, 2, and 3 (combined) of the antibody having the CDR sequence provided in Table 1, and binds to PSMA.

[0209] In some embodiments, the anti-PSMA antibody preferably comprises a heavy chain variable domain (VH) having at least 85% sequence identity at the amino acid level with respect to the CDR 1, 2, and 3 (combined) of the antibody having the CDR sequence provided in Table 3, and binds to PSMA.

[0210] In some embodiments, the anti-PSMA antibody comprises a heavy chain variable region sequence having at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity with respect to any of the heavy chain variable regions of SEQ ID NOs: 24 to 58 (presented in Table 2), and binds to PSMA.

[0211] In some embodiments, a bispecific or multispecific antibody is provided that may have any of the configurations described herein, including but not limited to a bispecific three-chain antibody-like molecule (TCA). In some embodiments, the multispecific antibody may include at least one heavy chain variable region having binding specificity for PSMA, and at least one heavy chain variable region having binding specificity for proteins other than PSMA. In some embodiments, the multispecific antibody may include a heavy chain variable region having at least two antigen-binding domains, each of which has binding specificity for PSMA. In some embodiments, the multispecific antibody may include a heavy chain / light chain pair having binding specificity for a first antigen (e.g., CD3), and a heavy chain from a heavy chain-alone antibody. In certain embodiments, the heavy chain from a heavy chain-alone antibody comprises an Fc portion having CH2 and / or CH3 and / or CH4 domains in the absence of a CH1 domain. In one specific embodiment, the bispecific antibody comprises a heavy chain / light chain pair having binding specificity to an antigen on an effector cell (e.g., CD3 protein on a T-cell), and a heavy chain from a middle chain-alone antibody having an antigen-binding domain having binding specificity to PSMA.

[0212] In some embodiments, the multispecific antibody comprises a CD3-binding VH domain paired with a light chain variable domain. In certain embodiments, the light chain is a fixed light chain. In some embodiments, the CD3-binding VH domain comprises the CDR1 sequence of SEQ ID NO: 59, the CDR2 sequence of SEQ ID NO: 60, and the CDR3 sequence of SEQ ID NO: 61 in a human VH framework. In some embodiments, the fixed light chain comprises the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64 in a human VL framework. Together, the CD3-binding VH domain and the light chain variable domain have binding affinity for CD3. In some embodiments, the CD3-binding VH domain comprises the heavy chain variable region sequence of SEQ ID NO: 65. In some embodiments, the CD3-binding VH domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity with respect 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% percent identity with respect to the heavy chain variable region sequence of SEQ ID NO: 66.

[0213] The multispecific antibody comprising the CD3-binding VH domain and light chain variable domain described above has advantageous properties as described, for example, in published PCT application publication number WO2018 / 052503, the full text of which is incorporated herein by reference. Any of the multispecific antibodies and antigen-binding domains described herein having binding affinity for PSMA may be combined with additional sequences such as those provided in Tables 6 and 7, as well as the CD3-binding domain and immobilized light chain domain described herein (e.g., see Tables 4 and 5), to produce one or more PSMA epitopes, as well as a multispecific antibody having binding affinity for CD3.

[0214] Table 4. Anti-CD3 heavy chain and light chain CDR1, CDR2, CDR3 amino acid sequences.

[0215]

[0216] Table 5. Anti-CD3 heavy chain and light chain variable region amino acid sequences.

[0217]

[0218] Table 6: Human IgG1 and IgG4 Fc region sequences.

[0219]

[0220] Table 7: Additional sequences

[0221]

[0222]

[0223]

[0224]

[0225] In some embodiments, a bispecific or multispecific antibody is provided that may have any of the configurations discussed herein, including but not limited to a bispecific three-chain antibody-like molecule (TCA). In some embodiments, the bispecific antibody may include at least one heavy chain variable region having binding specificity for PSMA, and at least one heavy chain variable region having binding specificity for proteins other than PSMA. In some embodiments, the bispecific antibody may include a heavy chain / light chain pair having binding specificity for a first antigen, a heavy chain from a heavy chain-alone antibody comprising an Fc portion including a CH2 and / or CH3 and / or CH4 domain in the absence of a CH1 domain, and an antigen binding domain that binds to an epitope of the second antigen or a different epitope of the first antigen. In one specific embodiment, the bispecific antibody comprises a heavy chain / light chain pair having binding specificity to an antigen on an effector cell (e.g., CD3 protein on a T-cell), and a heavy chain from a heavy chain-alone antibody having an antigen-binding domain having binding specificity to PSMA.

[0226] In some embodiments, where the antibody of the present invention is a bispecific antibody, one arm of the antibody (one binding moiety or one binding unit) may be specific to human PSMA, while the other arm may be specific to target cells, tumor-associated antigens, targeting antigens, e.g., integrins, etc., pathogen antigens, checkpoint proteins, etc. Target cells include, specifically, but not limited to, cancer cells from solid tumors, e.g., prostate tumors, as discussed below. In some embodiments, one arm of the antibody (one binding moiety or one binding unit) is specific to human PSMA, while the other arm is specific to CD3.

[0227] 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 any one of the sequences of SEQ ID NO: 80, 81, 82, 83, 84, or 85, and an anti-PSMA heavy chain polypeptide comprising any one of the sequences of SEQ ID NO: 24-58 linked to any one of the sequences of SEQ ID NO: 75, 76, 77, 78, 84, or 85 in a monovalent or divalent configuration. These sequences can be combined in various ways to produce bispecific antibodies against a desired IgG subclass, e.g., IgG1, IgG4, silencing IgG1, or silencing IgG4. 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 comprising a first polypeptide composed of SEQ ID NO: 86, a second polypeptide composed of SEQ ID NO: 87, and a third polypeptide composed of SEQ ID NO: 88, 89, 90, 91, 92, or 93.

[0228] Various forms of multispecific antibodies, including but not limited to single-strand polypeptides, two-strand polypeptides, three-strand polypeptides, four-strand polypeptides, and many of these, are within the scope of the present invention. The multispecific antibodies of the present invention specifically include T-cell multispecific (e.g., bispecific) antibodies (anti-PSMA x anti-CD3 antibodies) that bind to PSMA and CD3. Such antibodies induce potent T-cell-mediated death of cells expressing PSMA.

[0229] Preparation of anti-PSMA antibodies

[0230] The antibodies of the present invention may be produced by methods known in the art. In a preferred embodiment, the antibodies of the present invention are produced by transplanted animals, including transplanted mice and rats, preferably rats, wherein the endogenous immunoglobulin genes are knocked out or incapacitated. In a preferred embodiment, the heavy chain antibodies of the present invention are produced in UniRat™. UniRat™ possesses silenced endogenous immunoglobulin genes and expresses a diverse and naturally optimized repertoire of fully human HCAb using human immunoglobulin heavy chain translocus. While endogenous immunoglobulin loci in rats can be knocked out or silenced using various techniques, in UniRat™, the endogenous rat heavy chain J-locus, light chain Cκ locus, and light chain Cλ locus were inactivated using zinc-finger (endo)nuclease (ZNF) technology. ZNF constructs for microinjection into oocytes can produce IgH and IgL knockout (KO) cell lines. For details, refer, for example, to Geurts et al., 2009, Science 325:433. Characterization of Ig heavy chain knockout rats was reported by Menoret et al., 2010, Eur. J. Immunol. 40:2932-2941. An advantage of ZNF technology is that non-homologous terminal binding, which silences genes or loci through deletions of up to several kb, can also provide target sites for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). Human heavy chain antibodies produced in UniRat™ are called UniAbs™ and can bind to epitopes that cannot be attacked by conventional antibodies. Their high specificity, affinity, and small size make them ideal for single-specific and multi-specific applications.

[0231] UniAbs™ In addition, specifically, heavy-chain monoclonal antibodies lacking the camelid VHH framework and mutations, and their functional VH regions are included herein. For example, such heavy-chain monoclonal antibodies may be produced in transplanted rats or mice containing a complete human heavy-chain monoclonal gene locus as described, for example, in WO2006 / 008548, but other transplanted mammals such as rabbits, guinea pigs, and rats may also be used, with rats and mice preferred. Heavy-chain monoclonal antibodies containing VHH or VH functional fragments may also be produced by recombinant DNA technology, for example, by the expression of coding nucleic acids in suitable eukaryotic or prokaryotic hosts including mammalian cells (e.g., CHO cells), E. coli, or yeast.

[0232] The domains of heavy-chain monoclonal antibodies combine the benefits of antibodies and small molecule drugs; they can be monovalent or multivalent; have low toxicity; and can be manufactured cost-effectively. Due to their small size, these domains are easy to administer, including oral or topical administration, and feature high stability, including gastrointestinal stability; and their half-lives can be tailored to desired uses or indications. Additionally, the VH and VHH domains of HCAbs can be manufactured in a cost-effective manner.

[0233] In a specific embodiment, the heavy chain antibody of the present invention, including UniAbs™, has a natural amino acid substituted by another amino acid residue at a first position (amino acid position 101 according to the Kabat numbering system) of the FR4 region, which may disrupt a surface-exposed hydrophobic patch containing or associated with the natural amino acid at that position. This hydrophobic patch is normally buried at the boundary with the antibody light chain constant region but becomes surface-exposed in HCAb and is intended at least partially for unwanted aggregation and light chain association of HCAb. The substituted amino acid residue is preferably charged, more preferably positively charged, such as lysine (Lys, K), arginine (Arg, R), or histidine (His, H), preferably arginine (R). In a preferred embodiment, the heavy chain-alone antibody derived from a transplanted animal contains a mutation from Trp to Arg at position 101. The generated HCAb preferably has high antigen-binding affinity and solubility under physiological conditions in the absence of aggregation.

[0234] As part of the present invention, a human IgG anti-PSMA heavy chain antibody (UniAb™) having a unique sequence from UniRat™ animals that binds to human PSMA in ELISA protein and cell-binding assays was identified. The identified heavy chain variable region (VH) sequence is positive for binding to human PSMA protein and / or to PSMA+ cells, and negative for binding to cells that do not express PSMA. See, for example, Table 8.

[0235] Heavy chain antibodies that bind to non-overlapping epitopes on the PSMA protein, such as UniAbs™, can be identified by competitive binding assays, such as enzyme-linked immunoassays (ELISA assays) or flow cytometry competitive binding assays. For example, competition between a known antibody binding to a target antigen and an antibody of interest can be used. By using this approach, a set of antibodies can be divided into those that compete with the reference antibody and those that do not. Non-competitive antibodies are identified as binding to distinct epitopes that do not overlap with the epitope bound by the reference antibody. Often, one antibody is immobilized and bound to the antigen, and a second labeled (e.g., biotinylated) antibody is tested in an ELISA assay for its ability to bind to the capture antigen. This can also be performed using surface plasmon resonance (SPR) platforms including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and the MX96 SPR imager (Ibis technologies BV), as well as on biolayer interferometer platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). For more details, refer to the embodiments of the present invention.

[0236] Typically, the antibody "competes" with the reference antibody when it causes a reduction of about 15-100% in the binding of the reference antibody to the target antigen, as determined by standard techniques such as the competitive binding assay described above. In various embodiments, the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more.

[0237] Pharmaceutical composition, uses and treatment methods

[0238] Another aspect of the present invention is to provide a pharmaceutical composition comprising one or more antibodies of the present invention mixed with a suitable pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers as used herein exemplify, but are not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof.

[0239] In one embodiment, the pharmaceutical composition comprises a heavy chain antibody (e.g., UniAb™) that binds to PSMA. In another embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb™) having binding specificity to two or more non-overlapping epitopes on the PSMA protein. In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific and TCA) heavy chain antibody (e.g., UniAb™) having binding specificity to PSMA and binding specificity to a binding target on an effector cell (e.g., a binding target on a T-cell such as the CD3 protein on a T-cell).

[0240] The pharmaceutical composition of the antibody used according to the present invention is prepared for storage by mixing a protein of desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer, such as in a lyophilized formulation or an aqueous solution (see, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Suitable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; and preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). Low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulin; 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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0241] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage forms (i.e., dosages for a single administration). The formulation depends on the selected route of administration. The antibodies of the present invention may be administered by intravenous injection or infusion, or subcutaneously. For injection administration, the antibodies of the present invention may be formulated into an aqueous solution, preferably a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain a carrier, excipient, or stabilizer as discussed above. Alternatively, the antibodies may be in a lyophilized form to be composed of a suitable vehicle, e.g., sterile non-pyrogenic water, before use.

[0242] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations may be used for the heavy-chain antibodies of the present invention, including UniAbs™. Subcutaneous antibody formulations are described, for example, in US20160355591 and US20160166689.

[0243] How to use

[0244] The anti-PSMA antibodies and pharmaceutical compositions described herein may be used to treat diseases and conditions characterized by the expression of PSMA, including but not limited to the conditions and diseases further described herein.

[0245] PSMA is a type II transmembrane protein expressed in prostate epithelial tissue and upregulated in the neovascularization of prostate cancer and solid tumors. It is also expressed at low levels in healthy tissues such as the brain, kidneys, and salivary glands, but is overexpressed in malignant prostate tissue, making it an attractive target for the therapeutic treatment of prostate cancer. Furthermore, considering its high expression in malignant neovascularization, it may be relevant for the therapy or imaging of solid tumors. Monoclonal antibodies, antibody-drug conjugates, and chimeric antigen receptor T-cells targeting PSMA have been described for the treatment of metastatic prostate cancer (Hernandez-Hoyos et al., 2016, PMID: 27406985; DiPippo et al., 2014, PMID: 25327986; Serganova et al., 2016, PMID: 28345023). In addition, radionuclide conjugates specific to PSMA are being investigated for the imaging and treatment of prostate cancer (e.g., Hofman et al., 2018 PMID: 29752180).

[0246] In one aspect, the anti-PSMA antibody (e.g., UniAbs™) and pharmaceutical composition of the present invention may be used to treat disorders characterized by the expression of PSMA, including but not limited to prostate cancer and solid tumors.

[0247] The effective dose of the composition of the present invention for the treatment of a disease depends on many different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Generally, the patient is a human, but non-human mammals, such as companion animals like dogs, cats, and horses, and laboratory mammals like rabbits, mice, and rats, may also be treated. The therapeutic dose can be titrated to optimize safety and efficacy.

[0248] The dosage level can be easily determined by a skilled clinician and, if necessary, can be modified, for example, as needed to modify the subject's response to treatment. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form depends on the treated host and the specific mode of administration. The dosage unit forms generally contain about 1 mg to about 500 mg of the active ingredient.

[0249] In some embodiments, the therapeutic dose formulation may be in the range of about 0.0001 to 100 mg / kg of host body weight, more generally 0.01 to 5 mg / kg. For example, the dose may be 1 mg / kg body weight or 10 mg / kg body weight, or within the range of 1-10 mg / kg. An exemplary therapeutic regimen involves administration once every two weeks, once every month, or once every three to six months. The therapeutic subjects of the present invention are generally administered in multiple instances. The interval between single doses may be weekly, monthly, or annually. The interval may also be irregular, as indicated by measuring the blood levels of the therapeutic subjects in the patient. Alternatively, the therapeutic subjects of the present invention may be administered as sustained-release formulations, in which case less frequent administration is required. The dose and frequency depend on the half-life of the polypeptide in the patient.

[0250] Typically, the composition is prepared as an injectable substance as a liquid solution or suspension; additionally, a solid form suitable for a solution or suspension of a liquid vehicle may be prepared prior to injection. The pharmaceutical compositions of the present invention are suitable for intravenous or subcutaneous administration immediately after or after the reconstitution of a solid (e.g., lyophilized) composition. The formulations may also be emulsified or encapsulated in liposomes or microparticles, such as polylactide, polyglycolide, or copolymers, for an enhanced adjuvant effect as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The formulations of the present invention may be administered in the form of depot injections or implantable preparations that can be formulated in a manner that allows for sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally sterile, substantially isotonic, and formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0251] The toxicity of the antibodies and antibody structures described herein may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (lethal dose for 50% of the population) or LD100 (lethal dose for 100% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index. Data obtained from these cell culture assays and animal studies may be used to formulate a dosage range that is not toxic for use in humans. The dosage of the antibodies described herein is preferably within a circulating concentration range containing an effective amount with little to no toxicity. The dosage may vary within this range depending on the formulation used and the route of administration utilized. The precise formulation, route of administration, and dosage may be selected by the individual physician in consideration of the patient's condition.

[0252] The composition for administration will typically comprise an antibody or other lysant dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous carriers, e.g., buffered saline, etc., may be used. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized by commonly known sterilization techniques. The compositions may, if necessary, contain pharmaceutically acceptable auxiliary substances to access physiological conditions, such as pH adjusters and buffers, toxicity modifiers, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. In these formulations, the concentration of the active agent may vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., depending on the specific mode of administration selected and the patient's needs (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0253] Additionally, within the scope of the present invention there is a kit comprising the active agent of the present invention, a formulation thereof, and instructions for use. The kit may additionally contain at least one additional reagent, e.g., a chemotherapy drug. The kit typically includes a label indicating the intended use of the contents of the kit. The term “label” as used herein includes any written or recorded material supplied with or accompanying the kit, or otherwise accompanying the kit.

[0254] The present invention has now been sufficiently described, and it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit or scope of the invention.

[0255] Examples

[0256] Materials and Methods

[0257] Example 1: UniRat™ Immunization Using Recombinant Human PSMA

[0258] Twelve UniRat™ animals were immunized with recombinant human PSMA protein fused to his tag (R&D Systems catalog number: 4234-ZN). The animals were immunized twice a week for 8 weeks. After 35 days of immunization, serum was collected from the rats to determine serum titers.

[0259] Serum titer results

[0260] Summary information on serum titers is presented in Panel AB of Fig. 1. In the graphs shown in Panel AB of Fig. 1, each line represents an individual animal. The legend of the graph indicates the ID number of each individual animal. Binding activity for a 12-point dilution series of serum was tested by ELISA for the huPSMA+His tag protein and the His tag extratarget protein. Among the animals in this group, a range of serum reactivity levels for the human PSMA protein was observed. No serum reactivity was observed for the His tag extratarget protein.

[0261] Example 2: Flow cytometry analysis of binding of anti-PSMA UniAbs™ to PSMA-positive and negative cells

[0262] Binding to PSMA-positive cells was evaluated by flow cytometry (Guava easyCyte 8HT, EMD Millipore) using LNCaP cell lines (ATCC: CRL-1740), 22Rv1 cell lines (ATCC CRL-2505), PC3 cell lines stably transfected to express human PSMA (ATCC CRL-1435), or DU-145 cell lines (ATCC HTB-81). Briefly, 50,000 target cells were stained with a series of dilutions of purified UniAbs™ at 4°C for 30 minutes. After incubation, cells were washed twice with flow cytometry buffer (1X PBS, 1% BSA, 0.1% NaN3) and stained with goat F(ab')2 anti-human IgG conjugated to R-phycoerythrin (PE) (Southern Biotech, cat. #2042-09) to detect cell-bound antibodies. After incubation at 4°C for 20 minutes, cells were washed twice with flow cytometry buffer, and mean fluorescence intensity (MFI) was measured by flow cytometry. The MFI 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 multiple of the background. Binding to synomolgus PSMA-positive cells was determined using the same protocol according to the following modification: Target cells were derived from Freestyle 293-F cells (ThermoFisher R79007) transiently transfected to express the extracellular domain of synomolgus PSMA. In some experiments, EC50 values ​​were calculated using GraphPad Prism 7.

[0263] Table 8 summarizes the target binding activities of several anti-PSMA heavy chain antibodies (HCAbs) described herein. Column 1 indicates the clonal IDs of the HCAbs. Column 2 indicates binding to LNCaP cells measured as a multiple relative to the background MFI signal.

[0264] Table 8: Binding to PSMA-expressing cell lines

[0265]

[0266] As shown in Figure 2, Panels A and B, the difference in binding to synomolgus PSMA supports the difference in human PSMA epitopes recognized by HCAb 346181 and 345497.

[0267] Example 3: Recombinant protein binding by biolayer interferometer (BLI)

[0268] Using a biolayer interferometer, binding competition between two antibody families, whose members are clone ID 345497 and clone ID 346181, was evaluated. Antigen-antibody epitope binning analysis was performed on an Octet QK-384 (ForteBio). Briefly, antigen-recombinant human PSMA (R&D Systems catalog number: 4234-ZN) was immobilized for 120 seconds using an anti-Penta HIS Capture (HIS1K) sensor. After the baseline reading, the sensor was immersed in a solution containing antibody 1 (325867) for 300 seconds, and another baseline was established for 60 seconds. The sensor was then immersed in wells containing either antibody 1 or antibody 2 (325920) as a positive control for blocking. Association and dissociation rates were measured for 300 and 600 seconds, respectively. Data analysis was performed using Octet Data Analysis HT v11.0 (ForteBio). As shown in Figure 3, 325920 binds to the PSMA protein pre-conjugated with antibody 325867, indicating that these two antibodies recognize non-overlapping epitopes on PSMA. The movement of the binding signal is recorded in nanometers.

[0269] Example 4: Composition of diparatope and divalent anti-PSMA antibodies

[0270] As shown in Table 9, clone ID 350123 consists of the sequence of clone ID 346181 linked to the sequence of clone ID 345497, which has the bridging sequence GGGGSGGGGS (Sequence No. 74). Clone ID 350122 consists of two repeats of clone ID 346181 linked by the same linker sequence. Clone ID 350123 is a double paratope because it consists of two anti-PSMA domains that recognize different epitopes on PSMA. Clone ID 350122 is divalent but not a double paratope because the same anti-PSMA domain is composed of a tandem. Schematic diagrams of various anti-PSMA x anti-CD3 antibodies are shown in panel AC of Fig. 5.

[0271] Table 9: Description of amino acid sequences of diparatope and divalent anti-PSMA antibodies

[0272]

[0273] Example 5: Determination of affinity for human PSMA expressed on cell surface

[0274] PSMA cell surface affinity was determined by scatched analysis using the human prostate carcinoma cell line 22Rv1. First, the PSMAxCD3 multispecific antibody was labeled with Alexa Fluor 488 using the Alexa Fluor 488 5-SDP Ester Kit (ThermoFisher A30052). Then, binding to 22Rv1s was evaluated by flow cytometry (Guava easyCyte 8HT, EMD Millipore). Briefly, 100,000 target cells were stained with a series of dilutions of the Alexa Fluor 488-labeled multispecific antibody at 4°C for 1 hour. After incubation, the cells were washed twice with flow cytometry buffer, and the mean fluorescence intensity was measured by flow cytometry.

[0275] To establish a standard curve for the molecular calculation of equivalent soluble fluorophores (MESF), Bangs Lab Quantum Alex Fluor 488 MESF bead populations 1 through 4 were combined into a single tube and run on a Guava easyCyte 8HT. Blank beads were analyzed in a separate tube. The MFI of each bead population was measured against the FITC-channel. The linear regression of Log10(MFI) against Log10(MESF) was plotted using GraphPad Prism 7.

[0276] The MFI of each experimental sample was interpolated using a calibration curve, and the MESF was determined for each sample. Subsequently, the average MESF was divided by the degree of antibody labeling (DOL) to calculate the average number of bound antibodies (ABC) per cell. The total concentration of bound antibodies was determined by multiplying the number of ABCs by the cell concentration. The free antibody concentration was calculated by subtracting the bound antibody concentration from the staining concentration (starting dose). The free antibody concentration was plotted against the bound antibody concentration in GraphPad Prism 7. The affinity was determined by fitting the generated plot to a non-linear regression, single-site specific binding function, as presented in Figure 4, Panels A and B.

[0277] Example 6: Multispecific antibody-mediated death of PSMA-positive prostate tumor cells via T-cell pathway modification

[0278] Test using resting T-cells

[0279] Target cells were seeded into 96-well plates at a ratio of 15,000 cells per well and grown overnight at 37°C. After incubation, increasing amounts of multispecific antibodies were added along with resting human T-cells at an effector-to-target cell ratio of 10:1, and the cells were incubated at 37°C for an additional 48 or 72 hours (48 hours for assays using LNCaP, MDA-PCa-2b, and PC3-PSMA cells, and 72 hours for assays using 22Rv1 cells). Cell death was measured using the cell proliferation reagent WST-1 (Sigma Catalog No.: 11644807001) or flow cytometry. In some experiments, small samples of the supernatant were collected after incubation but prior to the target cell viability assay and stored for cytokine production analysis. When analyzing cell viability with the WST-1 reagent, the reagent stock was added to each well at a 1:10 dilution and incubated at 37°C for 90 minutes. Then, the absorbance was measured at 450 nm (reference 690 nm), and the percentage-specific dissolution was calculated.

[0280] When target cell viability was analyzed by flow cytometry, target cells were labeled with the membrane dye DiR (ThermoFisher D12731) before initiating the assay. After incubation with T-cells and antibodies, the supernatant was either saved for cytokine analysis or discarded. The wells were then washed once to collect dead tumor cells and T-cells, which were transferred to a flow cytometry plate. The remaining attached tumor cells were trypsinized and added to the corresponding wells of the flow cytometry plate. Dead cells were stained using Annexin-V reagent, and flow cytometry was performed (BD FACSCelesta) to quantify the percentage of dead tumor cells in each sample gated by DiR staining. This was normalized to spontaneous cell death using wells containing untreated target cells. In some experiments, a negative control antibody was used, consisting of the same CD3-targeting arm as in the PSMAxCD3 multispecific molecule, but with the tumor-targeting arm replaced by VH, which is specific to the HIV protein gp120.

[0281] Figure 7 shows T-cell-mediated lysis of PSMA-positive cells using unstimulated T-cells. Unstimulated human T-cells were cultured with PSMA-expressing cells (LNCaP) and different concentrations of multispecific antibodies. The dual paratop anti-PSMAxCD3 antibody (350123xCD3) outperformed the single paratop PSMAxCD3 antibody (346181xCD3).

[0282] assay using pre-activated T-cells

[0283] Human pan T-cells were pre-activated with plate-conjugated OKT3 and IL-2 for 3 days, followed by an additional 1 day of culture in fresh IL-2. Target cells were trypsinized, loaded with Calcein-AM (ThermoFisher C3100MP), mixed with activated T-cells at an E:T ratio of 20:1, and added to the wells of a 96-well plate. After adding a series of dilutions of different multispecific antibodies, the cells were incubated at 37°C for 4 hours. The supernatant was then transferred to a black 96-well plate, and calcein release was quantified by measuring absorbance at 480 nm / 520 nm ex / em. This was normalized to the spontaneous calcein release of intact tumor cells using target cells cultured without T-cells. Adding 2% Triton-X to control wells containing target cells allowed for the calculation of the calcein signal corresponding to maximum cell lysis. Using this value, each experimental well was recorded as a percentage of maximum cell lysis. Data analysis was performed using GraphPad Prism 7.

[0284] Figure 6 shows T-cell-mediated lysis of PSMA-positive cells using pre-activated T-cells. Pre-activated human T-cells were cultured with human PSMA-expressing cells (LNCaP) and different concentrations of multispecific antibodies. Tumor cell death was measured by calcein release and normalized to the spontaneous release of tumor cells in the absence of T-cells. The dual paratop anti-PSMAxCD3 antibody (350123xCD3) outperformed two single paratop PSMAxCD3 antibodies.

[0285] Figure 8 shows that multispecific antibodies do not lyse PSMA-negative cells. Pre-activated human T-cells were cultured with PSMA-negative prostate cancer cells (DU145) and different concentrations of multispecific antibodies. Lysis of these cells did not occur with any of the antibodies tested.

[0286] Figure 9 shows the binding of the PSMAxCD3 multispecific antibody to PSMA-positive and negative cells. The multispecific anti-PSMA x anti-CD3 antibody shows binding to PSMA-positive prostate tumor cells (22Rv1) but not to PSMA-negative prostate tumor cells (DU145). The dual paratop molecule (350123) showed the strongest target cell binding.

[0287] Figure 10 illustrates T-cell-mediated lysis of PSMA-positive cells. The data in Figure 10 demonstrate that binding to PSMA through two different epitopes results in increased cell death compared to only two single-specific versions of the antibody.

[0288] Example 7: A single paratopic PSMAxCD3 bispecific antibody induces less cytokine production than a bispecific paratopic PSMAxCD3 multispecific antibody.

[0289] Cytokine production was analyzed in a tumor cytotoxicity assay using resting T-cells. The design of this assay is described in detail elsewhere. Supernatants were collected upon completion of the assay (after 72 hours of culture for assays using 22Rv1 cells, and after 48 hours for all other cell lines). ELISA kits were used according to the manufacturer's protocol for the detection of IL-2 (Biolegend 431804) and IFNγ (Biolegend 430104). The experimental supernatants were diluted prior to analysis in the ELISA so that cytokine levels fell within the linear portion of the standard curve supplied with each kit. In some cases, cytokines may not be detected in the experimental wells, and values ​​were reported below the lower limit of quantification for the assay.

[0290] Fig. 12, Panel AC shows a comparison of T-cell mediated lysis and cytokine production in PSMA-positive cells. Multispecific PSMAxCD3 antibodies induce T-cell mediated lysis in the PSMA-positive prostate cancer cell line LNCaP. The dual paratop molecule (350123) stimulated more potent tumor cell death compared to the single paratop molecule (346181), but also induced higher levels of cytokine interferon gamma (IFNγ) and interleukin 2 (IL-2), as exemplified by Fig. 12, Panels B and C.

[0291] Table 10 shows T-cell-mediated lysis and cytokine production for four PSMA-positive prostate tumor cell lines. PSMAxCD3 multispecific antibodies were tested in in vitro tumor cell cytotoxicity assays using dose series of antibodies against unstimulated T-cells and 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 the EC50 as well as the highest percentage death achieved were recorded. Supernatants were collected from these experimental wells and analyzed by ELISA for the cytokines interferon-gamma (IFNγ) or interleukin-2 (IL-2). A single paratop molecule (3461881) induced roughly equivalent levels of tumor cytotoxicity in all four cell lines tested compared to a double paratop molecule, but had a higher EC50 for cytokine production and stimulated lower levels of maximum cytokine production in most cases.

[0292] Table 10: T-cell-mediated lysis and cytokine production for 4 PSMA-positive prostate tumor cell lines.

[0293]

[0294] Example 8: PSMAxCD3 multispecific antibody induces T-cell proliferation

[0295] PSMA-positive tumor cells were seeded into 96-well plates at 25,000 cells per well and grown overnight at 37°C. Human pan T-cells isolated from resting PBMC (Miltenyi 130-096-535) were labeled with the lineage tracer dye CFSE according to the manufacturer's instructions (ThermoFisher C34554). Then, 100,000 labeled pan T-cells were added to the wells containing the tumor cells, a series of antibody dilutions were added, and the cells were incubated at 37°C, 8% CO2. After 5 days of incubation, the cells were gently mixed and transferred to flow cytometry plates. The cells were pelleted, and after removing the supernatant, the anti-CD8 (Biolegend) conjugated to APC Cells were stained with anti-CD4 (Biolegend 317410) conjugated to PE (301049) and on ice for 20 minutes. Then, cells were washed and resuspended in flow cytometry buffer (BD FACSCelesta). Cells were gated for forward and lateral scattering and for CD4 or CD8 expression. The percentage of proliferated T-cells was calculated for the total T-cell population, as well as for the CD4 and CD8 subsets, as indicated by CD4 or CD8 positive staining and low or negative CFSE signals. Flow cytometry data were analyzed using FlowJo and plotted on GraphPad Prism 7.

[0296] Figure 11, Panel AD, indicates that the PSMAxCD3 multispecific antibody stimulates T-cell proliferation in the presence of PSMA-positive tumor cells, and that the single paratop PSMA bispecific antibody preferentially activates CD3 T-cells. The multispecific antibodies were cultured with PSMA-expressing tumor cells and T-cells labeled with the lineage tracer dye CFSE. After 5 days of culture, T-cell proliferation and the composition of proliferated T-cells (CD8+ to CD4+) were analyzed by flow cytometry. Panels A and B represent total T-cell proliferation, while Panels C and D represent the ratio of CD8+ to CD4+ T-cells in the proliferated wells. The dashed horizontal line represents the CD8:CD4 ratio of unstimulated T-cells, which is approximately 1:2 (actual value = 0.64). A single paratop PSMAxCD3 bispecific antibody (346181) preferentially activates CD8 T-cells (CD8:CD4 ratio after expansion of approximately 2:1), whereas a double paratop PSMAxCD3 multispecific antibody (350123) less preferentially activates CD8+ T-cells (CD8:CD4 ratio of approximately 1:1).

[0297] Example 9: Multispecific antibodies induce inhibition of prostate tumor growth in a xenograft model

[0298] After subcutaneously transplanting 10 million 22Rv1 cells into the right lower flank of 5-6 week old male immunodeficient CIEA-NOG mice (Taconic), an additional 10 million human PBMCs were transplanted via tail vein injection the day after tumor transplantation. Animals were treated with 100 μg of multispecific antibody or vehicle via tail vein injection starting 1 day after tumor transplantation on days 1, 5, 9, and 13. Tumor volume was quantified using a caliper and recorded for 25 days.

[0299] Figure 13 shows the results of a 22Rv1 tumor xenograft model. The dual paratop PSMAxCD3 molecule (350123) demonstrated inhibition of 22Rv1 tumor growth in the tumor xenograft model. Three mice were tested for each treatment group, and the change in tumor volume for each animal was plotted in cubic millimeters. The animals received PBMC on day 1 after tumor transplantation and were treated with antibodies on days 1, 5, 9, and 13. Two of the three animals treated with the multispecific antibody showed a delay in tumor progression.

[0300] Although preferred embodiments of the present invention have been presented and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Numerous modifications, variations, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used to practice the invention. The following claims define the scope of the invention and are intended to encompass methods and structures within the scope of such claims and equivalents.

Claims

Claim 1 A bispecific tri-chain antibody-like molecule comprising: (a) a heavy chain variable (VH) region of an anti-PSMA heavy chain antibody comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18 in a monovalent configuration; and (b) a CD3-binding VH region paired with a light chain variable (VL) region, wherein (i) the CD3-binding VH region comprises the CDR1 sequence of SEQ ID NO: 59, the CDR2 sequence of SEQ ID NO: 60, and the CDR3 sequence of SEQ ID NO: 61; and (ii) the VL region comprises the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64, and the CD3-binding VH region paired with the light chain variable (VL) region. Claim 2 A bispecific tri-chain antibody-like molecule according to claim 1, wherein the VH region of the anti-PSMA heavy-chain antibody comprises the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 18 in a monovalent configuration in a human VH framework, the CD3-binding VH region comprises the CDR1 sequence of SEQ ID NO: 59, the CDR2 sequence of SEQ ID NO: 60, and the CDR3 sequence of SEQ ID NO: 61 in a human VH framework, and the VL region comprises the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 63, and the CDR3 sequence of SEQ ID NO: 64 in a human VL framework. Claim 3 A bispecific tri-chain antibody-like molecule according to claim 1, wherein the CD3-binding VH region comprises a sequence having at least 95% sequence identity with respect to the heavy chain variable region sequence of SEQ ID NO: 65, and the VL region comprises a sequence having at least 95% sequence identity with respect to the light chain variable region of SEQ ID NO:

66. Claim 4 A bispecific tri-chain antibody-like molecule according to claim 1, wherein the VH region of the anti-PSMA heavy chain antibody comprises a heavy chain variable region sequence having at least 95% sequence identity with respect to SEQ ID NO:

25. Claim 5 A bispecific tri-chain antibody-like molecule according to claim 1, wherein the VH region of the anti-PSMA heavy chain antibody comprises the heavy chain variable region sequence of SEQ ID NO:

25. Claim 6 A bispecific tri-chain antibody-like molecule according to claim 1, wherein the CD3-binding VH region comprises the heavy chain variable region sequence of SEQ ID NO: 65 and the VL region comprises the light chain variable region sequence of SEQ ID NO:

66. Claim 7 A bispecific tri-chain antibody-like molecule according to claim 1, wherein the CD3-binding VH region comprises the heavy chain variable region sequence of SEQ ID NO: 65, the VL region comprises the light chain variable region sequence of SEQ ID NO: 66, and the VH region of the anti-PSMA heavy chain antibody comprises the heavy chain variable region sequence of SEQ ID NO:

25. Claim 8 A bispecific three-chain antibody-like molecule that binds to CD3 and PSMA, 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. Claim 9 A pharmaceutical composition comprising a bispecific three-chain antibody-like molecule of any one of claims 1 to 8 for use in the treatment of prostate cancer. Claim 10 A polynucleotide encoding a bispecific three-chain antibody-like molecule of any one of claims 1 to 8. Claim 11 A vector containing the polynucleotide of claim 10. Claim 12 Isolated host cells containing the vector of claim 11. Claim 13 A method for producing a bispecific three-chain antibody-like molecule according to any one of claims 1 to 8, comprising the steps of: growing an isolated host cell comprising a vector containing a polynucleotide encoding the bispecific three-chain antibody-like molecule under conditions allowing the expression of the bispecific three-chain antibody-like molecule; and isolating the bispecific three-chain antibody-like molecule from the isolated host cell. Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete Claim 58 delete Claim 59 delete Claim 60 delete

Images