PSMA antibodies and uses thereof

Abstract

The present application provides prostate-specific membrane antigen (PSMA) antibodies, such as human monoclonal antibodies against PSMA, nucleic acid molecules encoding the antibodies, expression vectors or host cells used to express the antibodies, methods for their preparation, and uses thereof, such as for the treatment of PSMA-related diseases, including cancer.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority patent application no. PCT / CN2022 / 134047, filed on November 24, 2022, and priority patent application no. PCT / CN2022 / 134163, filed on November 24, 2022, the disclosures of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Sequence Listing The contents of the electronic sequence listing (70351WO01 Seq List 16 Nov 2023.xml; size: 47 kilobytes; and creation date: November 16, 2023) are incorporated herein by reference in their entirety. Summary of the Invention

[0003] In one aspect, the present disclosure provides an isolated antibody, or antigen-binding portion thereof, comprising a prostate-specific membrane antigen (PSMA) binding portion capable of binding to PSMA, wherein the PSMA binding portion comprises: a heavy chain CDR1 comprising the sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the sequence of SEQ ID NO: 2; a heavy chain CDR3 comprising the sequence of SEQ ID NO: 3; a light chain CDR1 comprising the sequence of SEQ ID NO: 4; a light chain CDR2 comprising the sequence of SEQ ID NO: 5; and a light chain CDR3 comprising the sequence of SEQ ID NO: 6. [Brief explanation of the drawings]

[0004] [Figure 1-1] 1 shows SDS-PAGE and SEC-HPLC analysis of exemplary PSMA antibody W305042. [Figure 1-2] 1 shows SDS-PAGE and SEC-HPLC analysis of exemplary PSMA antibody W305042. [Figure 2]Binding of antibodies (W305042, J591, and isotope control) to human PSMA by ELISA. [Figure 3] Binding of antibodies (W305042, J591, and isotope control) to cynomolgus monkey PSMA by ELISA. [Figure 4] Binding of antibodies (W305042, J591, and isotope control) to LNCaP cells by FACS. [Figure 5] Binding of antibodies (W305042, J591, and isotope control) to cynomolgus monkey PSMA-expressing CHO cells as measured by FACS is shown. [Figure 6] Binding of antibodies (W305042, and isotope control) to mouse PSMA as measured by ELISA is shown. [Figure 7] The DSF profile of W305042 is shown below. [Figure 8] 1 shows the HIC-HPLC profile of W305042. [Figure 9] Schematic diagram of bispecific CD3 x PSMA antibody W308051, where T3 represents the anti-CD3 arm, in which the heavy chain variable domain of the anti-CD3 arm is fused to a modified TCR β constant domain (represented by a gray rectangle) and hinge-Fc region of human IgG4 containing the S228P mutation, Fc null mutations (F234A, L235A) and knob mutations (S354C-T366W), and the VL of the anti-CD3 arm is fused to a modified TCR α constant domain (represented by another gray rectangle); and U5 represents the anti-PSMA arm, in which the heavy chain variable domain of the anti-PSMA arm is fused to a hinge-Fc region of human IgG4 containing the S228P mutation, Fc null mutations (F234A L235A) and hole mutations (Y349C-T366S-L368A-Y407V), and the VL of the anti-PSMA arm is fused to the CL domain. [Figure 10A] The results of SDS-PAGE (FIG. 10A) and SEC-HPLC (FIG. 10B) analyses of W308051 are shown. In FIG. 10A, the lanes, from left to right, represent a protein marker, non-reduced antibody, reduced antibody, and protein marker, respectively. [Figure 10B] The results of SDS-PAGE (FIG. 10A) and SEC-HPLC (FIG. 10B) analyses of W308051 are shown. In FIG. 10A, the lanes, from left to right, represent a protein marker, non-reduced antibody, reduced antibody, and protein marker, respectively. [Figure 11] Binding of antibodies (W308051, AMG160, and isotopic hIgG4 control) to human PSMA as measured by ELISA is shown. [Figure 12-1] Binding of antibodies (W308051, AMG340, AMG160, and isotopic hIgG4 control) to human C4-2 (high PSMA expression), LNCaP (high PSMA expression), 22Rv1 (low PSMA expression), and PC-3 cells (PSMA negative) as measured by FACS is shown. [Figure 12-2] Binding of antibodies (W308051, AMG340, AMG160, and isotopic hIgG4 control) to human C4-2 (high PSMA expression), LNCaP (high PSMA expression), 22Rv1 (low PSMA expression), and PC-3 cells (PSMA negative) as measured by FACS is shown. [Figure 13] Binding of antibodies (W308051, AMG340, AMG160, and isotopic hIgG4 control) to CD3-positive Jurkat cells and primary human T cells as measured by FACS is shown. [Figure 14A] Binding of antibodies (W308051, AMG160, and isotopic hIgG4 control) to cynomolgus monkey PSMA (FIG. 14A) and mouse PSMA (FIG. 14B) as measured by ELISA is shown. [Figure 14B] Binding of antibodies (W308051, AMG160, and isotopic hIgG4 control) to cynomolgus monkey PSMA (FIG. 14A) and mouse PSMA (FIG. 14B) as measured by ELISA is shown. [Figure 15] Binding of antibodies (W308051, AMG340, AMG160, and isotopic hIgG4 control) to cynomolgus monkey PSMA-positive cells as measured by FACS is shown. [Figure 16-1]Figure 1 shows T cell cytotoxicity of C4-2, LNCaP, and PC-3 cells co-cultured with CD3+ T cells and incubated with antibodies W308051, AMG340, AMG160, and isotopic hIgG4 control. [Figure 16-2] Figure 1 shows T cell cytotoxicity of C4-2, LNCaP, and PC-3 cells co-cultured with CD3+ T cells and incubated with antibodies W308051, AMG340, AMG160, and isotopic hIgG4 control. [Figure 17] Cytokine release from C4-2 and PC-3 cells co-cultured with CD3+ T cells and incubated with antibodies W308051, AMG340, AMG160, and isotopic hIgG4 control is shown. [Figure 18-1] Cytokine release of C4-2 cells co-cultured with PBMCs and incubated with antibodies W308051, AMG340, AMG160, and isotopic hIgG4 control. [Figure 18-2] Cytokine release of C4-2 cells co-cultured with PBMCs and incubated with antibodies W308051, AMG340, AMG160, and isotopic hIgG4 control. [Figure 19] 1 shows the thermal stability of antibody W308051 as measured by DSF. [Figure 20] 1 shows the results of antibody W308051 measured by hydrophobic interaction chromatography HPLC (HIC-HPLC). [Figure 21] 1 shows the mean serum concentrations of antibody W308051 in a pharmacokinetic study. [Figure 22A] Shows the in vivo efficacy of antibodies (W308051, AMG340, and AMG160) in the NPG-hPBMC model: (Figure 22A) tumor growth curves; and (Figure 22B) body weights of tumor-bearing mice. [Figure 22B]Shows the in vivo efficacy of antibodies (W308051, AMG340, and AMG160) in the NPG-hPBMC model: (Figure 22A) tumor growth curves; and (Figure 22B) body weights of tumor-bearing mice. Specific Description of the Invention

[0005] Unless otherwise defined herein, scientific and technical terms used in the context of this disclosure have the meanings commonly understood by those of ordinary skill in the art. The singular forms "a," "an," and "the" can include plural referents unless the context clearly dictates otherwise. For example, a reference to "a protein" includes a plurality of proteins, a reference to "a cell" includes a mixture of cells, etc. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "comprising," as well as other forms such as "comprises" and "comprised," is non-limiting. Additionally, the ranges provided in this specification and the appended claims include both endpoints of the ranges and all points between the endpoints.

[0006] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues or a collection of multiple polymers of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to natural and unnatural amino acid polymers. The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds with the same basic chemical structure as natural amino acids, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino acid, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. The α-carbon refers to the primary carbon atom bonded to a functional group, such as a carbonyl. The β-carbon represents the secondary carbon atom bonded to the α-carbon, and this system continues alphabetically, naming the carbons using Greek letters. Amino acid mimetics refer to chemical compounds that have a structure different from the general structure of an amino acid, but function similarly to a natural amino acid. The term "protein" usually refers to a large polypeptide. The term "peptide" usually refers to a short polypeptide. Polypeptide sequences are usually described such that the left-hand side of the polypeptide sequence is the amino-terminus (N-terminus); and the right-hand side of the polypeptide sequence is the carboxyl-terminus (C-terminus). As used herein, a "polypeptide complex" refers to a complex comprising one or more polypeptides associated with a particular function. In some cases, the polypeptide is immune-related.

[0007] As used herein, the term "antibody" or "Ab" is used broadly to encompass a variety of antibody structures, including polyclonal, monospecific, and multispecific antibodies (e.g., bispecific antibodies). Natural, intact antibodies are generally Y-shaped tetrameric proteins comprising two heavy (H) and two light (L) polypeptide chains linked by covalent disulfide bonds and non-covalent interactions. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, and epsilon, which define antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable region is connected to the constant region via a "J" region of about 12 or more amino acids, and heavy chains further comprise a "D" region of about 3 or more amino acids. Each heavy chain comprises a heavy chain variable region / domain (V H ) and heavy chain constant region / domain (C H The heavy chain constant region consists of three domains (C H 1. C H 2 and C H Each light chain consists of a light chain variable region / domain (V L ) and light chain constant region / domain (C L ) V H and V L The region can be further divided into hypervariable regions (called complementarity determining regions (CDRs)), which are spaced apart by relatively conserved regions (called framework regions (FRs)). H and V L consists of 3 CDRs and 4 FRs in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L ) each form an antigen-binding site. The antibodies can be of different antibody isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0008] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody, which can be used interchangeably in the context of this application, refer to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to an antigen to which the full-length antibody specifically binds and / or competes with the full-length antibody for binding to the same antigen. Antigen-binding fragments of antibodies may be derived from whole antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and, optionally, constant domains of an antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA may be engineered by sequencing and chemically or using molecular biology techniques to place one or more variable and / or constant domains into the appropriate configuration, or by introducing codons, creating cysteine ​​residues, modifying, adding, or deleting amino acids, etc.

[0009] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides) of an antibody, or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other recombinant molecules such as shark variable IgNAR domains are also encompassed within the term "antigen-binding fragment" as used herein. In some cases, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. The variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region, which may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule.

[0010] As used herein, the term "variable domain" with respect to an antibody refers to an antibody variable region or fragment thereof comprising one or more CDRs. A variable domain may comprise an intact variable region (such as an HCVR or LCVR), but it may comprise less than an intact variable region and still retain the ability to bind to antigen or form an antigen-binding site.

[0011] As used herein, the term "antigen-binding portion" refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or other antibody fragments that bind to an antigen but do not contain an intact native antibody structure. Examples of antigen-binding portions include, but are not limited to, variable domains, variable regions, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), multispecific antibodies (e.g., bispecific antibodies such as Het-mAbs), camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding portion can bind to the same antigen as the parent antibody. In some cases, an antigen-binding portion can comprise one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies.

[0012] "Fab" in reference to an antibody refers to the portion of an antibody consisting of a single light chain (both variable and constant regions) associated by disulfide bonds with the variable region and first constant region of a single heavy chain. In some cases, the constant regions of both the light and heavy chains are replaced by TCR constant regions.

[0013] "F(ab')2" refers to a dimer of Fab'.

[0014] "Fragment difficult (Fd)" with respect to antibodies refers to the amino-terminal half of a heavy chain fragment that can combine with a light chain to form Fab.

[0015] "Fc" with respect to an antibody refers to the portion of the antibody consisting of the second (CH2) and third (CH3) constant regions of the first heavy chain connected by disulfide bonds to the second and third constant regions of the second heavy chain. The Fc portion of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not function in antigen binding.

[0016] With respect to antibodies, the "hinge region" includes the portion of the heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 amino acid residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently.

[0017] As used herein, a "CH2 domain" includes, for example, the portion of a heavy chain molecule extending from about amino acid 244 to amino acid 360 of an IgG antibody using conventional numbering schemes (amino acids 244-360, Kabat numbering system; and amino acids 231-340, EU numbering system).

[0018] The "CH3 domain" extends from the CH2 domain to the C-terminus of an IgG molecule and comprises approximately 108 amino acids. Certain immunoglobulin classes, such as IgM, further comprise a CH4 region.

[0019] "Fv," with respect to antibodies, refers to the smallest antibody fragment containing a complete antigen-binding site. Fv fragments consist of a single light-chain variable domain linked to a single heavy-chain variable domain. Several Fv designs are available, including dsFv, in which the association between the two domains is enhanced by an introduced disulfide bond; scFv can be formed using a peptide linker to link the two domains as a single polypeptide. Fv constructs containing immunoglobulin heavy or light chain variable domains linked to the corresponding immunoglobulin heavy or light chain variable and constant domains have also been produced. Fvs have also been multimerized to form diabodies and triabodies.

[0020] Single chain Fv antibody or "scFv" refers to a recombinant antibody consisting of a light chain variable region and a heavy chain variable region connected to each other either directly or by a peptide linker sequence.

[0021] In some cases, an "scFv dimer" may be a dimer of a portion of the V H Part V of other parts Land another V to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). H -V L V dimerized with the moiety H -V L (linked by a peptide linker).

[0022] In some cases, an "scFv dimer" is a V H1 and V L1 cooperated, V H2 and V L2 are linked, and each linked pair has a different antigen specificity. L1 -V H2 (linked by a peptide linker) and associated V H1 -V L2 (linked by a peptide linker).

[0023] "ScFab" refers to a fusion polypeptide comprising an Fd linked to a light chain by a polypeptide linker, resulting in the formation of a single-chain Fab fragment (scFab).

[0024] "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some cases, "(dsFv)2" or "(dsFv-dsFv')" refers to a three-peptide chain: two VV fragments linked by a peptide linker (e.g., a long flexible linker) and disulfide bridged, respectively. L 2V combined with part H In some cases, the dsFv-dsFv' is bispecific, in which each disulfide-paired heavy and light chain has a different antigen specificity.

[0025] "Additional IgG" refers to a fusion protein having a Fab arm fused to an IgG, forming a bispecific (Fab)2-Fc format. This can produce "IgG-Fab" or "Fab-IgG," with an Fab fused to the C-terminus or N-terminus of the IgG molecule, with or without a connector. In some cases, the addition IgG can be further modified to an IgG-Fab4 format.

[0026] As used herein, the term "anti-CD3 antibody" or "CD3 antibody" refers to an antibody, as defined herein, that is capable of binding to CD3, e.g., human CD3, e.g., to elicit a potential therapeutic effect.

[0027] The terms "CD3" and "CD3 protein" are used interchangeably herein. The CD3 protein is present in virtually all T cells. The CD3-TCR complex regulates T cell functions in both innate and adaptive immune responses, as well as cellular and humoral immune functions. These include the elimination of pathogenic microorganisms and the control of tumor growth through a wide range of cytotoxic effects. The CD3 T cell coreceptor is a protein complex consisting of four distinct chains: the CD3γ chain, the CD3δ chain, and two CD3ε chains. The four chains associate with a molecule known as the T cell receptor (TCR) and the ζ chain, which generates activation signals in T lymphocytes. The TCR, ζ chain, and CD3 molecule constitute the TCR complex; the TCR, as a subunit, recognizes and binds antigens, while the CD3 subunit transduces antigen stimulation into signal transduction pathways, ultimately regulating T cell activity. The term "CD3" can include human CD3 as well as its variants, isoforms, and species homologs. Thus, the antibodies or antigen-binding portions thereof as defined and disclosed herein may also bind to CD3 from species other than human, eg, cynomolgus monkey CD3.

[0028] As used herein, the term "human CD3" refers to CD3 of human origin, such as the complete amino acid sequence of human CD3.

[0029] As used herein, the term "cynomolgus CD3" refers to CD3 from cynomolgus monkeys, such as the complete amino acid sequence of rhesus CD3.

[0030] As used herein, the term "anti-PSMA antibody" refers to an antibody that specifically binds PSMA. An "anti-PSMA antibody" may include a monovalent antibody having a single specificity. Exemplary anti-PSMA antibodies are described elsewhere herein.

[0031] The term "prostate-specific membrane antigen (PSMA)" is a type II membrane glycoprotein of 750 amino acids that has folate hydrolase and NAALADase enzymatic activity.

[0032] As used herein, the term "bivalent" refers to an antibody or antigen-binding fragment having two antigen-binding sites; the term "monovalent" refers to an antibody or antigen-binding fragment having only one single antigen-binding site; the term "multivalent" refers to an antibody or antigen-binding fragment having multiple antigen-binding sites; and the term "monovalent" refers to an antibody or antigen-binding fragment having only one single antigen-binding site. In some instances, an antibody or antigen-binding portion thereof is bivalent.

[0033] As used herein, "bispecific" refers to an artificial antibody that can bind to or target two different epitopes, for example, having fragments derived from two different monoclonal antibodies. The binding of a bispecific antibody to two different epitopes can potentially produce a therapeutic effect. The two different epitopes may be present on the same antigen or on two different antigens. In some cases, the bispecific antibody is a Het-mAb.

[0034] As used herein, a "Het-mAb" is an IgG-like molecule that can target two different epitopes, either on the same or different targets, and has four different chains: two heavy chains and two light chains. These chains contain a set of mutations in the Fc portion of the molecule that promote heavy chain dimerization, and a set of mutations on the Fab portion that promote correct heavy / light chain pairing to form κ / κ or λ / κ subtype bispecific mAbs.

[0035] The term "bispecific antigen-binding molecule" refers to a protein, polypeptide, or molecular complex comprising at least a first antigen-binding portion (also referred to herein as a first antigen-binding site) and a second antigen-binding portion (also referred to herein as a second antigen-binding site). In some cases, a "bispecific antigen-binding molecule" is a "bispecific antibody." Each antigen-binding portion in a bispecific antibody comprises at least one CDR that specifically binds to a particular antigen, alone or in combination with one or more additional CDRs and / or FRs. In some cases, the first antigen-binding site specifically binds to a first antigen (e.g., PSMA or CD3), and the second antigen-binding site specifically binds to a second, different antigen (e.g., CD3 or PSMA).

[0036] As used interchangeably herein, the terms "anti-PSMA / anti-CD3 antibody," "anti-PSMA / anti-CD3 bispecific antibody," "antibody to PSMA and CD3," "anti-PSMA x CD3 bispecific antibody," "PSMA x CD3 antibody," "anti-CD3 / anti-PSMA antibody," "anti-CD3 / anti-PSMA bispecific antibody," "antibody to CD3 and PSMA," "anti-CD3 x PSMA bispecific antibody," and "CD3 x PSMA antibody" refer to bispecific antibodies that specifically bind to CD3 and PSMA, regardless of the order in which the targets are first mentioned.

[0037] As used herein, the terms "monoclonal antibody" or "mAb" refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0038] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0039] The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0040] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0041] As used herein, the term "recombinant antibody" refers to antibodies that have been prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from an animal transgenic for the immunoglobulin genes of another species, antibodies isolated from a recombinant combinatorial antibody library, or antibodies prepared, expressed, produced or isolated by other means that involve splicing immunoglobulin gene sequences into other DNA sequences.

[0042] As used herein, the term "spacer" refers to an artificial amino acid sequence having 1, 2, 3, 4, or 5 amino acid residues, or 5 to 15, 20, 30, 50, or more amino acid residues, linked by peptide bonds, used to link one or more polypeptides. Spacers may or may not have secondary structure. For example, spacers useful in the present disclosure may contain a high concentration of glycine and proline residues. Examples include spacers with single or repeating sequences consisting of threonine / serine and glycine, such as TGGGG, GGGGS, or SGGGG, or tandem repeats thereof (e.g., 2, 3, 4, or more repeats).

[0043] The term "operably linked" or "operably linked" refers to the juxtaposition of two or more biological sequences of interest, with or without spacers or linkers, in a relationship permitting them to function in their intended manner. When used in reference to a polypeptide, it is intended to mean that the polypeptide sequences are linked in a manner that allows the linked product to have the desired biological function. For example, an antibody variable region may be operably linked to a constant region to provide a stable product that has antigen-binding activity. The term may also be used in reference to polynucleotides. As an example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequences are linked in a manner that allows regulated expression of the polypeptide from the polynucleotide.

[0044] As used herein, the term "epitope" refers to a portion of an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also known as "antigenic determinant." Epitopes or antigenic determinants generally consist of chemically active surface groups of molecules, such as amino acids, carbohydrates, or sugar side chains, and generally have specific three-dimensional structures and specific charge characteristics. For example, epitopes generally comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique conformation, which may be "linear" or "conformational." In a linear epitope, all interaction sites between proteins and interacting molecules (e.g., antibodies) are linear along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites span amino acid residues that are separated from one another in the protein. For example, competitive or cross-competitive tests may be performed to obtain antibodies that compete or cross-compete with each other for binding to an antigen (e.g., an RSV fusion protein). A high-throughput method for obtaining antibodies that bind to the same epitope is based on these cross-competitions.

[0045] As used herein, the terms "specific binding" or "specifically binds" refer to a non-random binding reaction between two molecules, such as, for example, between an antibody and an antigen.

[0046] K D is the ratio of the dissociation rate to the association rate (k), which can be determined by surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (e.g., FACS). off / k on ) is sometimes used to represent K D The value can be suitably determined by using flow cytometry.

[0047] The term "fusion" or "fused" when used in reference to amino acid sequences (e.g., peptides, polypeptides, or proteins) refers to the joining of two or more amino acid sequences into a single, non-naturally occurring amino acid sequence, for example, by chemical conjugation or recombinant means. A fused amino acid sequence may be produced by genetic recombination of two coding polynucleotide sequences and expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

[0048] The term "antigen specificity" refers to the particular antigen or epitope thereof that is selectively recognized by an antigen-binding molecule.

[0049] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" refers to the percent of identical residues between the amino acids and nucleotides in the compared molecules, and is calculated based on the smallest size of the molecules being compared. For these calculations, gaps in the alignment, if any, can be accommodated by a specific mathematical model or computer program (i.e., an "algorithm").

[0050] As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in organisms.This term not only refers to the property of an antigen that stimulates specific immune cells to activate, proliferate and differentiate to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also refers to the specific immune response that can form antibodies or sensitized T lymphocytes in the immune system of an organism after stimulating the organism with the antigen.Immunogenicity is the most important property of an antigen.Whether an antigen can successfully induce the development of an immune response in a host depends on three factors: the characteristics of the antigen, the reactivity of the host, and the means of immunization.

[0051] As used herein, the term "substitution" with respect to amino acid residues refers to the natural or induced replacement of one or more amino acids with another amino acid in a peptide, polypeptide, or protein. The substitution in a polypeptide may result in a decrease, an enhancement, or an elimination of the function of the polypeptide.

[0052] As used herein, the term "mutation" or "mutated" in reference to an amino acid residue refers to the substitution, insertion or addition of an amino acid residue.

[0053] A native "T cell receptor" or native "TCR" is a heterodimeric T cell surface protein that associates with the invariant CD3 chain to form a complex capable of mediating signal transduction. TCRs belong to the immunoglobulin superfamily and resemble half antibodies, with a single heavy chain and a single light chain. Native TCRs have an extracellular portion, a transmembrane portion, and an intracellular portion. The extracellular domain of a TCR has a membrane-proximal constant region and a membrane-distal variable region. In some cases, bispecific antibodies comprise soluble chimeric proteins with antibody variable domains and TCR constant domains, where the subunits of the TCR constant domain (such as the α and β domains) are linked by engineered disulfide bonds.

[0054] As used herein, the term "Ka" is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "Kd" is intended to refer to the dissociation rate of a particular antibody-antigen interaction. Kd values ​​for antibodies can be determined using methods well established in the art. As used herein, the term "K D " is intended to represent the dissociation constant of a particular antibody-antigen interaction and is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is intended to be expressed as a molar concentration (M). An exemplary method of determining the Kd of an antibody is by using surface plasmon resonance, such as using a biosensor system such as a BIACORE system.

[0055] As used herein, the term "high affinity" for an IgG antibody refers to an antibody with an affinity of 1×10 to a target antigen. -7 M or less, e.g., 5 x 10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less, or 1 x 10 -9 K below M D The antibody has the formula:

[0056] As used herein, the term "EC 50 " represents the concentration of a drug, antibody, or toxin that elicits a response halfway between baseline and maximum after a specified exposure time. In the context of this application, EC 50 is expressed in units of "nM".

[0057] As used herein, the term "competing for binding" refers to the interaction of two antibodies in binding to their binding target. A first antibody competes with a second antibody for binding if the binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Alternatively, the option of the second antibody's binding to its epitope being detectably reduced in the presence of the first antibody can also be applied, but this is not necessarily the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope if the second antibody does not inhibit the binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of another antibody with its cognate epitope, whether to the same extent, a greater extent, or a lesser extent, the antibodies are said to "cross-compete" with each other for binding to their respective epitopes.

[0058] As used herein, the ability to "inhibit binding" refers to the ability of an antibody or antigen-binding portion thereof to inhibit the binding of two molecules (e.g., human CD3 / PSMA and a human anti-CD3 / anti-PSMA antibody) to a detectable level. In some cases, the binding of the two molecules can be inhibited by the antibody or antigen-binding portion thereof by at least 50%. In some cases, such an inhibitory effect can be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0059] As used herein, the term "isolated" refers to a state obtained from the natural state by artificial means. This is possible because when a particular "isolated" substance or component exists in nature, its natural environment is altered, or the substance is isolated from its natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide naturally occurs in a particular living animal, and a highly purified version of the same polynucleotide or polypeptide isolated from such a natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixtures of artificial or synthetic substances, nor does it exclude other impurities that do not affect the activity of the isolated substance.

[0060] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to CD3 / PSMA protein is substantially free of antibodies that specifically bind to antigenic proteins other than CD3 / PSMA). However, an isolated antibody that specifically binds to human CD3 / PSMA protein may have cross-reactivity to other antigens, such as CD3 / PSMA proteins from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0061] As used herein, the term "vector" refers to a nucleic acid vehicle capable of carrying a polynucleotide inserted therein. If the vector allows for the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can carry genetic material elements that are expressed in a host cell by transformation, transduction, or transfection into the host cell. Vectors can be plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and parvoviruses (such as SV40). A vector can comprise multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may comprise an origin of replication.

[0062] As used herein, the term "host cell" refers to a cell line that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, e.g., mammalian cultured cells derived from rodents (rat, mouse, guinea pig, or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0, or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissue. The term encompasses not only the particular subject cell but also the progeny of such cells. Certain modifications may occur in later generations, either due to mutation or environmental influences, and such progeny may not be identical to the parent cell, yet are still included within the scope of the term "host cell."

[0063] As used herein, the term "transfection" refers to a method of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, chemical and physical methods such as lipid transfection and electroporation.

[0064] As used herein, the term "SPR" or "surface plasmon resonance" refers to and includes the optical phenomenon that allows the analysis of real-time biospecific interactions by detection of changes in protein concentration within a biosensor matrix, for example, using the BIACORE system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ, USA).

[0065] As used herein, the term "fluorescence-activated cell sorting" or "FACS" refers to a specialized type of flow cytometry. It provides a method for sorting heterogeneous mixtures of biological cells into two or more containers, one at a time, based on the specific light scattering and fluorescence properties of each cell. Instruments that perform FACS include the FACS STAR PLUS, FACSCAN, and FACSORT instruments from Becton Dickinson (Foster City, California), the EPICS C from Coulter Epics Division (Hialeah, Florida), and the MOFLO from Cytomation (Colorado Springs, Colorado).

[0066] As used herein, the terms "subject" or "individual" or "animal" or "patient" refer to a human or non-human animal, including a mammal or primate, in need of diagnosis, prognosis, amelioration, prevention, and / or treatment of a disease or condition. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, etc.

[0067] As used herein, the term "effector function" refers to a biological activity resulting from the binding of the Fc region of an antibody to its effector, such as the C1 complex and an Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC), which is triggered by the interaction of an antibody on the C1 complex with C1q; antibody-dependent cell-mediated cytotoxicity (ADCC), which is triggered by the binding of the Fc region of an antibody to an Fc receptor on an effector cell; and phagocytosis.

[0068] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and then kill the target cells with cytotoxins. In some cases, antibody-armed cytotoxic cells are required for such killing. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay is used. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. In some cases, the ADCC activity of a molecule of interest may be assessed in vivo.

[0069] The term "complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) bound to their cognate antigen. To assess complement activation, CDC may be performed.

[0070] As used herein, the term "cancer" refers to a medical condition characterized by malignant cell growth or tumor, abnormal proliferation, invasion, or metastasis, and includes both solid tumors and non-solid cancers (malignant blood diseases) such as leukemia. As used herein, "solid tumor" refers to a solid mass of tumor cells and / or malignant cells. Examples of cancers or tumors include malignant blood diseases, oral cancer (e.g., lip, tongue, or pharynx), digestive tract (e.g., esophagus, stomach, small intestine, colon, large intestine, or rectum), peritoneum, dry and biliary tract, pancreas, respiratory system (small cell and non-small cell), such as pharynx, or lung, bone, connective tissue, skin (e.g., melanoma), breast, reproductive organs (fallopian tubes, uterus, cervix, testes, ovaries, or prostate), ureter (e.g., bladder or kidney), brain, and endocrine glands such as the thyroid gland. In some cases, the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, kidney cancer, bladder cancer, hepatocellular carcinoma, and colorectal cancer. In some cases, the cancer is selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and B-cell lymphoma.

[0071] As used herein in the context of treating a condition, the terms "treatment," "treating," or "treated" refer generally to treatments and therapies, whether in humans or animals, that achieve some desired therapeutic effect, e.g., inhibition of the progression of the condition, including slowing the rate of progression, stopping the rate of progression, alleviating the condition, ameliorating the condition, and curing the condition. Treatment as a preventative measure (i.e., prophylaxis, prevention) is also included. With respect to cancer, "treatment" can refer to attenuating or slowing tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. With respect to tumors, "treatment" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor onset, or some combination thereof.

[0072] As used herein, the term "effective amount" refers to an amount or dosage of an active compound, or a substance, or composition comprising an active compound, that, when administered in a desired treatment regimen, is effective to achieve some desired therapeutic effect, commensurate with a reasonable risk-benefit ratio. For example, when used in the context of treating a CD3 / PSMA-associated disease or condition, an "effective amount" refers to an amount or concentration of an antibody or antigen-binding portion thereof effective to treat the disease or condition.

[0073] The term "prevention" or "preventing" as used herein in reference to a particular disease state in a mammal refers to preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms thereof.

[0074] As used herein, the term "pharmaceutically acceptable" means that the vehicle, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients in the formulation and physiologically compatible with the recipient.

[0075] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmaceutically and / or physiologically compatible with the subject and active agent, including, but not limited to, pH adjusting agents, surfactants, adjuvants, and ionic enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween 80; ionic enhancers include, but are not limited to, sodium chloride.

[0076] As used herein, the term "adjuvant" refers to a nonspecific immunopotentiating agent that, when delivered to an organism together with or before an antigen, can enhance or change the type of immune response in the organism to the antigen. There are various adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, and the like.

[0077] Antibodies and antigen-binding portions thereof The antibodies disclosed herein are capable of binding to human PSMA and have the following properties: (a) 1×10 -8 K below M D binds to human PSMA having the formula: (b) CD4 + Inducing the production of cytokines (e.g., IL-2 or IFN-γ) in T cells; (c) Primary human CD4 + promoting T-cell proliferation; (d) Primary human CD4 + promoting the proliferation of T effector cells; (e) binds to human or rhesus PSMA, respectively; or (f) no cross-reactivity with human CD40, CD137, and CD271; It has one or more of the following.

[0078] Binding to PSMA can be evaluated using ELISA. Binding specificity can be determined by monitoring the binding of the antibody to cells expressing PSMA protein, for example, by flow cytometry. For example, the antibody can be tested by flow cytometry assay, in which the antibody reacts with a cell line expressing human PSMA, such as CHO cells transfected to express PSMA on the cell surface. Binding kinetics (e.g., K dBinding of antibodies containing 1×10 mAbs can be tested in a BIACORE binding assay. Still other suitable binding assays include, for example, ELISA assays using recombinant PSMA protein. For example, antibodies can be tested in a BIACORE binding assay using 1×10 mAbs. -8 M or less, 1×10 -9 M or less, 5×10 -10 M or less, 2×10 -10 M or less, 1×10 -10 M or less, 5×10 -11 M or less, 3×10 -11 M or less, or 2 x 10 -11 K below M D It can bind to human PSMA having the formula:

[0079] Variable regions and CDRs in antibody sequences can be identified according to general rules developed in the art (such as the Kabat numbering system, as described above) or by aligning the sequence to known variable region data bases. Exemplary databases of antibody sequences are described and accessible at the "Abysis" website maintained by the Department of Biochemistry & Molecular Biology, University College London, London, UK, and the VBASE2 website. Sequences can be analyzed using the Abysis database, which integrates sequence databases from Kabat, IMGT, and the Protein Data Bank (PDB), including structural data from the PDB. The Abysis database website also includes general rules developed to identify CDRs that can be used in accordance with the teachings herein. Unless otherwise specified, antibody CDR boundaries are defined or identified by the Kabat and IMGT conventions.

[0080] The percent identity between two amino acid sequences can be determined using the algorithm incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percentage identity between two amino acid sequences can be determined by the algorithm incorporated into the GAP program of the GCG software package using a BLOSSUM62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0081] The protein sequences of the present disclosure can further be used as a "query sequence" to search public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0). BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences that are homologous to the antibody molecules of the present disclosure. Gapped BLAST can be used to obtain gapped alignments for comparison purposes. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0082] In some cases, the amino acid sequences of the CDRs may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the respective sequences above. In some cases, the amino acid sequences of the variable regions may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the respective sequences above.

[0083] In some cases, the CDR of the isolated antibody or antigen-binding portion thereof contains conservative substitutions of no more than two amino acids, or no more than one amino acid. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the essential properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain, for example, a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar chemical properties, including size, shape, charge, ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with alkaline side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (threonine, valine, isoleucine, etc.), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, corresponding amino acid residues can be substituted with other amino acid residues from the same side chain family.

[0084] In some cases, the first antigen-binding portion and the second antigen-binding portion of the bispecific antibody may associate with each other by a knob-into-hole interaction.

[0085] In some cases, the first and / or second antigen-binding moiety is bivalent. The term "bivalent" refers to the presence of two binding sites in each antigen-binding molecule. This can provide stronger binding to an antigen or epitope than a monovalent counterpart. In some cases, in a bivalent antigen-binding moiety, the first valency of the binding site and the second valency of the binding site are structurally identical (i.e., have the same sequence).

[0086] In some cases, the antibodies and antigen-binding fragments thereof provided herein are bispecific. In some cases, the bispecific antibodies and antigen-binding portions thereof provided herein have a first specificity for PSMA and a second specificity for a second antigen different from PSMA, such that their blocking may have a synergistic (e.g., synergistic) effect relative to blocking one antigen alone.

[0087] In some cases, the second specificity is directed against a tumor-associated antigen or an epitope thereof. The term "tumor-associated antigen" refers to a target antigen that is expressed by tumor cells but may also be expressed by related cells (or healthy cells) before they transform into tumors. In some cases, tumor-associated antigens may be presented only by tumor cells and not by normal, i.e., non-tumor cells. In some cases, tumor-associated antigens may be expressed exclusively on tumor cells or may exhibit tumor-specific mutations compared to non-tumor cells. In some cases, tumor-associated antigens can be found in both tumor and non-tumor cells, but are more accessible to antibody binding in tumor cells compared to non-tumor cells due to their overexpression on tumor cells or the smaller, more compact structure of tumor tissue compared to non-tumor tissue. In some cases, tumor-associated antigens are present in the tumor vasculature.

[0088] Illustrative examples of tumor-associated antigens include LAG-3, CD10, CD19, CD20, CD22, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), epidermal growth factor receptor (EGFR), Her2neu, Her3, IGFR, IL3R, fibroblast activation protein (FAP), CDCP1, Delrin 1, tenascin, frizzled 1–10, and the vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-α (CD140a), PDGFR-β (CD140b), endoglin, CLEC14, Tem1–8, and Tie2. Further examples may include A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), carbonic anhydrase IX (MN / CA IX), de2-7 EGFR, EGFRvIII, EpCAM, Ep-CAM, folate binding protein, G250, Fms-like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (melanoma-associated cell surface chondroitin sulfate proteoglycan), Muc-1, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate-specific antigen (PSA), and TAG-72.

[0089] In some cases, the present disclosure includes bispecific antibodies or antigen-binding portions thereof comprising an antigen-binding site that specifically binds CD3 and an antigen-binding site that specifically binds PSMA. Such antibodies may be referred to herein as, for example, "anti-CD3 / anti-PSMA," or "anti-CD3 / PSMA," or "anti-CD3xPSMA," or "CD3xPSMA" bispecific antibodies, or other similar terms.

[0090] The bispecific antibodies of the present disclosure bind to human CD3 and human PSMA with high affinity. The binding of the antibodies of the present disclosure to CD3 or PSMA can be assessed using one or more techniques well established in the art, such as ELISA. The binding specificity of the antibodies of the present disclosure can be determined by monitoring the binding of the antibodies to cells expressing CD3 or PSMA protein, for example, by flow cytometry. For example, the antibodies can be tested by flow cytometry assays in which the antibodies react with cell lines expressing human CD3, such as CHO cells transfected to express CD3 on their cell surface. In some cases, the binding kinetics (e.g., K D The binding of antibodies containing the CD3 protein (e.g., IgG ...

[0091] In some cases, a bispecific antibody or antigen-binding portion thereof of the present disclosure comprises a CD3-binding portion and a PSMA-binding portion, wherein the CD3-binding portion comprises a chimeric Fab comprising a first heavy chain variable region of an anti-CD3 antibody operably linked to a first T cell receptor (TCR) constant region (C1), and a first light chain variable region of the anti-CD3 antibody operably linked to a second TCR constant region (C2), wherein C1 and C2 are capable of forming a dimer via a non-native interchain disulfide bond that is capable of stabilizing the dimer, and the PSMA-binding portion comprises a Fab comprising a second heavy chain variable region of the anti-PSMA antibody operably linked to a heavy chain CH1 constant region domain, and a second light chain variable region of the anti-PSMA antibody operably linked to a light chain constant region; (A) The PSMA binding moiety is a heavy chain CDR1 comprising or consisting of the sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising or consisting of the sequence of SEQ ID NO: 2; a heavy chain CDR3 comprising or consisting of the sequence of SEQ ID NO: 3; a light chain CDR1 comprising or consisting of the sequence of SEQ ID NO: 4; a light chain CDR2 comprising or consisting of the sequence of SEQ ID NO: 5; a light chain CDR3 comprising or consisting of the sequence of SEQ ID NO: 6; and (B) The CD3 binding moiety is a heavy chain CDR1 comprising or consisting of the sequence of SEQ ID NO: 7; a heavy chain CDR2 comprising or consisting of the sequence of SEQ ID NO: 8; a heavy chain CDR3 comprising or consisting of the sequence of SEQ ID NO: 9; a light chain CDR1 comprising or consisting of the sequence of SEQ ID NO: 10; a light chain CDR2 comprising or consisting of the sequence of SEQ ID NO: 11; a light chain CDR3 comprising or consisting of the sequence of SEQ ID NO: 12; and / or (A) the CD3 binding portion comprises a heavy chain variable region comprising SEQ ID NO: 13 and a light chain variable region comprising SEQ ID NO: 14; (B) The PSMA-binding portion comprises a heavy chain variable region comprising SEQ ID NO:15 and a light chain variable region comprising SEQ ID NO:16.

[0092] In some cases, a bispecific antibody or antigen-binding portion thereof may have the following properties: (a) It binds specifically and simultaneously to human CD3 and PSMA proteins with high affinity; (b) specifically binds to human CD3 and / or cynomolgus monkey CD3 protein; (c) specifically binds to human, mouse, and / or cynomolgus monkey PSMA protein; (d) its ability to induce potent T cell activation in the presence of PSMA-expressing tumor cells compared to anti-CD3 antibodies, anti-PSMA antibodies, their combinations, and other bispecific antibodies targeting CD3 and PSMA; (e) providing excellent thermal stability and being stable in human serum; and (f) providing superior anti-tumor efficacy compared to anti-CD3 antibodies, anti-PSMA antibodies, combinations thereof, and other bispecific antibodies targeting CD3 and PSMA; It has one or more of the following.

[0093] For example, when tested in tumor-bearing mouse models, bispecific antibodies of the present disclosure achieved desirable tumor growth inhibition (TGI) compared to known anti-CD3 and anti-PSMA antibodies.

[0094] Antigen-binding moiety that specifically binds to PSMA The antigen-binding portions provided herein specifically bind to PSMA and are also referred to in this disclosure as PSMA-binding portions.

[0095] The antigen-binding portion comprises a Fab comprising a heavy chain variable region of an anti-PSMA antibody operably linked to a heavy chain CH1 constant region domain, and a light chain variable region of an anti-PSMA antibody operably linked to a light chain constant region.

[0096] In some cases, the antigen-binding moiety: a heavy chain CDR1 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, or an amino acid sequence that differs from SEQ ID NO: 1 by an amino acid addition, deletion, or substitution of no more than two amino acids; a heavy chain CDR2 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO:2, or an amino acid sequence that differs from SEQ ID NO:2 by amino acid additions, deletions, or substitutions of no more than two amino acids; a heavy chain CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:3, or an amino acid sequence that differs from SEQ ID NO:3 by an amino acid addition, deletion, or substitution of one or less amino acids; a light chain CDR1 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 4, or an amino acid sequence that differs from SEQ ID NO: 4 by amino acid addition, deletion, or substitution of no more than two amino acids; a light chain CDR2 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 5, or an amino acid sequence that differs from SEQ ID NO: 5 by an amino acid addition, deletion, or substitution of one or less amino acids; a light chain CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 6, or an amino acid sequence that differs from SEQ ID NO: 6 by an amino acid addition, deletion, or substitution of one or less amino acids; The compound comprises:

[0097] In some cases, the antigen-binding moiety: a) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1; b) a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:2; c) a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3; d) a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4; e) a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5; f) a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6; The compound comprises:

[0098] In some cases, the antigen-binding moiety: a) a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1; b) a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2; c) a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; d) a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4; e) a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5; f) a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6; The compound comprises:

[0099] In some cases, the heavy chain variable region of the antigen-binding portion comprises: (i) the amino acid sequence of SEQ ID NO: 15, and (ii) an amino acid sequence that is at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, while maintaining binding specificity for PSMA (e.g., comprising the CDRs disclosed above); or (iii) an amino acid sequence having one or more amino acid additions, deletions, and / or substitutions (e.g., 1 to 18, 1 to 15, 1 to 10, or 1 to 5) compared to SEQ ID NO: 15, while maintaining binding specificity to PSMA (e.g., comprising the CDRs disclosed above); The compound comprises:

[0100] In some cases, the light chain variable region of the antigen-binding portion comprises: (i) the amino acid sequence of SEQ ID NO: 16, and (ii) an amino acid sequence that is at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, while maintaining binding specificity for PSMA (e.g., comprising the CDRs disclosed above); or (iii) an amino acid sequence having one or more amino acid additions, deletions, and / or substitutions (e.g., 1 to 16, 1 to 15, 1 to 10, or 1 to 5) compared to SEQ ID NO: 16, while maintaining binding specificity to PSMA (e.g., comprising the CDRs disclosed above); The compound comprises:

[0101] Optionally, the heavy chain variable region of the antigen-binding portion consists of the amino acid sequence of SEQ ID NO:15 and the light chain variable region of the antigen-binding portion consists of the amino acid sequence of SEQ ID NO:16.

[0102] In some cases, the heavy chain variable region of the PSMA binding moiety is operably linked to a human IgG Fc region, particularly a human IgG4 or IgG1 Fc region, e.g., a hinge Fc region such as a human IgG4 Fc region comprising a S228P mutation, an Fc null mutation (F234A L235A), and a hole mutation (Y349C-T366S-L368A-Y407V). In some cases, to construct a CD3xPSMA bispecific antibody, a DNA sequence encoding the VH region of an anti-CD3 antibody is fused to a modified TCR β constant domain and hinge Fc region of human IgG4 with S228P mutations, Fc null mutations (F234A L235A), and knob mutations (S354C-T366W); a DNA sequence encoding the VL region of an anti-CD3 antibody is fused to a modified TCR α constant domain; a DNA sequence encoding the VH region of an anti-PSMA antibody is fused to a hinge Fc region of human IgG4 with S228P mutations, Fc null mutations (F234A L235A), and hole mutations (Y349C-T366S-L368A-Y407V); and a DNA sequence encoding the VL region of an anti-PSMA antibody is fused to a CL domain.

[0103] In some cases, the antigen-binding moiety comprises two polypeptide chains: i) a second duplex comprising an amino acid sequence having at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19 and, at the same time, maintaining binding specificity for PSMA (e.g., including the CDRs or / and variable regions disclosed above); and ii) a second light chain comprising an amino acid sequence having at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20 and, at the same time, maintaining binding specificity to PSMA (e.g., including the CDRs or / and variable regions disclosed above); The compound comprises:

[0104] In some cases, the antigen-binding moiety comprises two polypeptide chains: i) a second duplex represented by SEQ ID NO: 19; and ii) a second light chain represented by SEQ ID NO: 20; The compound comprises:

[0105] In some cases, the antigen-binding moiety comprises two polypeptide chains: i) a second duplex represented by SEQ ID NO: 19; and ii) a second light chain represented by SEQ ID NO: 20; It consists of:

[0106] In some cases, the antigen-binding moiety comprises two polypeptide chains: i) a second duplex comprising an amino acid sequence having at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 31 and, at the same time, maintaining binding specificity for PSMA (e.g., including the CDRs or / and variable regions disclosed above); and ii) a second light chain comprising an amino acid sequence having at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 32 and, at the same time, maintaining binding specificity to PSMA (e.g., including the CDRs or / and variable regions disclosed above); The compound comprises:

[0107] In some cases, the antigen-binding moiety comprises two polypeptide chains: i) a second duplex represented by SEQ ID NO: 31; and ii) a second light chain represented by SEQ ID NO: 32; The compound comprises:

[0108] In some cases, the antigen-binding moiety comprises two polypeptide chains: i) a second duplex represented by SEQ ID NO: 31; and ii) a second light chain represented by SEQ ID NO: 32; It consists of:

[0109] Antigen-binding portion that specifically binds to CD3 The antigen-binding portion specifically binds to CD3 and is therefore also referred to as a CD3-binding portion in the present disclosure. The two terms can be used interchangeably.

[0110] The antigen-binding portion comprises a chimeric Fab comprising a heavy chain variable region of an anti-CD3 antibody operably linked to a first T cell receptor (TCR) constant region (C1), and a light chain variable region of an anti-CD3 antibody operably linked to a second TCR constant region (C2), wherein C1 and C2 are capable of forming a dimer through a non-native interchain disulfide bond that can stabilize the dimer.

[0111] In some cases, the antigen-binding moiety: a) a heavy chain CDR1 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 7, or an amino acid sequence that differs from SEQ ID NO: 7 by amino acid addition, deletion or substitution of no more than two amino acids; b) a heavy chain CDR2 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by amino acid addition, deletion or substitution of no more than two amino acids; c) a heavy chain CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 9 or an amino acid sequence that differs from SEQ ID NO: 9 by amino acid addition, deletion or substitution of no more than two amino acids; d) a light chain CDR1 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 10, or an amino acid sequence that differs from SEQ ID NO: 10 by amino acid additions, deletions or substitutions of no more than two amino acids; e) a light chain CDR2 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by an amino acid addition, deletion or substitution of not more than one amino acid; f) a light chain CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 12 or an amino acid sequence that differs from SEQ ID NO: 12 by an amino acid addition, deletion or substitution of not more than one amino acid; The compound comprises:

[0112] In some cases, the antigen-binding moiety: a) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7; b) a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8; c) a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9; d) a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10; e) a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11; f) a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; The compound comprises:

[0113] In some cases, the antigen-binding moiety: a) a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 7; b) a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 8; c) a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 9; d) a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10; e) a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11; f) a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 12; The compound comprises:

[0114] In some cases, the heavy chain variable region of the antigen-binding portion comprises: (i) the amino acid sequence of SEQ ID NO: 13, and (ii) an amino acid sequence that is at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, while maintaining binding specificity to CD3 (e.g., comprising the CDRs disclosed above); or (iii) An amino acid sequence having one or more amino acid additions, deletions, and / or substitutions (e.g., 1 to 18, 1 to 15, 1 to 10, or 1 to 5) compared to SEQ ID NO: 13, while maintaining CD3-binding specificity (e.g., comprising the CDRs disclosed above). The compound comprises:

[0115] In some cases, the light chain variable region of the antigen-binding portion comprises: (i) the amino acid sequence of SEQ ID NO: 14, and (ii) an amino acid sequence that is at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14, while maintaining binding specificity to CD3 (e.g., comprising the CDRs disclosed above); or (iii) an amino acid sequence having one or more amino acid additions, deletions, and / or substitutions (e.g., 1 to 17, 1 to 15, 1 to 10, or 1 to 5) compared to SEQ ID NO: 14, while maintaining CD3 binding specificity (e.g., comprising the CDRs disclosed above); The compound comprises:

[0116] Optionally, the heavy chain variable region of the antigen-binding portion comprises or consists of the amino acid sequence of SEQ ID NO:13 and the light chain variable region of the antigen-binding portion consists of or consists of the amino acid sequence of SEQ ID NO:14.

[0117] In some cases, the antigen-binding moiety comprises two polypeptide chains: i) a first heavy chain comprising an amino acid sequence having at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17 and, at the same time, maintaining binding specificity to CD3 (e.g., including the CDRs or / and variable regions disclosed above); and ii) a first light chain comprising an amino acid sequence having at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18 and at the same time maintaining binding specificity to CD3 (e.g., comprising the CDRs or / and variable regions disclosed above); The compound comprises:

[0118] In some cases, the antigen-binding moiety comprises two polypeptide chains: i) a first heavy chain represented by SEQ ID NO: 17; and ii) a first light chain represented by SEQ ID NO: 18; The compound comprises:

[0119] In some cases, the antigen-binding moiety comprises two polypeptide chains: i) a first heavy chain represented by SEQ ID NO: 17; and ii) a first light chain represented by SEQ ID NO: 18; It consists of:

[0120] In some embodiments, the heavy chain variable region of the antigen-binding portion is operably linked to a C1 and hinge Fc region of a human IgG Fc region, particularly a human IgG4 or IgG1 Fc region, e.g., a human IgG4 Fc region comprising an S228P mutation, an Fc null mutation (F234A L235A), a knob mutation (S354C-T366W), and / or a hole mutation (Y349C-T366S-L368A-Y407V). Optionally, the VH region is fused to a modified TCR β constant domain and a hinge Fc region of a human IgG4 with an S228P mutation, an Fc null mutation (F234A L235A), and a knob mutation (S354C-T366W). In some cases, the VH region is fused to the hinge Fc region of human IgG4 with the S228P mutation, the Fc null mutation (F234A L235A) and the hole mutation (Y349C-T366S-L368A-Y407V).

[0121] TCR constant region The human TCR β chain constant region has two different variants known as TRBC1 and TRBC2 (IMGT nomenclature). In the present disclosure, the sequence of the wild-type TCR β domain has the NCBI accession number of A0A5B9. The modified TCR β constant domain of the present disclosure is: [ka] is.

[0122] In some cases, the first T cell receptor (TCR) constant region (C1) comprises a modified TCR β constant region comprising the amino acid sequence of SEQ ID NO: 29, and in some cases, C1 comprises or consists of a modified TCR β constant region represented by SEQ ID NO: 29.

[0123] The human TCR alpha chain constant region is known as TRAC and has the NCBI accession number P01848. The modified TCR alpha constant domain of the present disclosure is: [ka] is.

[0124] In some cases, the second T cell receptor (TCR) constant region (C2) comprises a modified TCR alpha constant region comprising the amino acid sequence of SEQ ID NO: 30, and in some cases, C2 comprises or consists of a modified TCR alpha constant region represented by SEQ ID NO: 30.

[0125] In the present disclosure, the first and second TCR constant regions of the polypeptide complexes provided herein can form a dimer comprising at least one non-native interchain bond between the TCR constant regions that can stabilize the dimer.

[0126] As used herein, the term "dimer" refers to an associated structure formed by two molecules, such as polypeptides or proteins, through covalent or non-covalent interactions. A homodimer or homodimerization is formed by two identical molecules, while a heterodimer or heterodimerization is formed by two different molecules. A dimer formed by a first and second TCR constant region is a heterodimer.

[0127] The interchain bond is formed between one amino acid residue on the TCR constant region and another amino acid residue on the other TCR constant region. In some cases, the non-natural interchain bond can be any bond or interaction that can associate the two TCR constant regions into a dimer. Examples of suitable non-natural interchain bonds include disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridges, or hydrophilic-hydrophobic interactions, knobs-into-holes, or combinations thereof.

[0128] "Disulfide bond" refers to a covalent bond having the structure RSSR. The amino acid cysteine, for example, contains a thiol group that can form a disulfide bond with a second thiol group from another cysteine ​​residue. Disulfide bonds can form between the thiol groups of two cysteine ​​residues on each of two polypeptide chains, thereby forming interchain crosslinks or bonds.

[0129] As used herein, a "non-natural" interchain bond refers to an interchain bond that is not found in the natural association of natural counterpart TCR constant regions. For example, a non-natural interchain bond can be formed between a mutated amino acid residue and a natural amino acid residue, each present on a respective TCR constant region; or between two mutated amino acid residues, each present on a TCR constant region. In some cases, at least one non-natural interchain bond is formed between a first mutated residue contained in a first TCR constant region and a second mutated residue contained in a second TCR constant region of the polypeptide complex.

[0130] As used herein, the term "contact interface" refers to a specific region on a polypeptide where the polypeptides interact / associate with each other. The contact interface comprises one or more amino acid residues that can interact with corresponding amino acid residues that contact or associate when an interaction occurs. The amino acid residues in the contact interface may or may not be in a continuous sequence. For example, if the interface is three-dimensional, the amino acid residues in the interface may be separated at different positions on the linear sequence.

[0131] Generation of antibody-producing hybridomas To generate hybridomas producing the antibodies disclosed herein, for example, human monoclonal antibodies, splenocytes and / or lymph node cells from immunized mice can be isolated and fused with a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies.

[0132] Generation of antibody-producing transfectomas The antibody of the present disclosure can also be produced in a host cell transfectoma, for example, by using a combination of recombinant DNA technology and gene transfection methods. In some cases, DNA encoding partial or full-length light and heavy chains obtained by standard molecular and biochemical techniques is inserted into one or more expression vectors so that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector so that the transcriptional and translational control sequences in the vector perform their intended function of regulating the transcription and translation of the antibody gene.

[0133] The antibody light chain gene and antibody heavy chain gene can be inserted into the same or separate expression vectors. In some cases, the variable regions are used to generate full-length antibody genes of any antibody isotype by inserting them into an expression vector already encoding the heavy and light chain constant regions of the desired isotype, such that the heavy chain variable domain is operably linked to a CH segment in the vector and the heavy chain variable domain is operably linked to a CL segment in the vector. The recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0134] For expression of the light and heavy chains, expression vectors encoding the light and heavy chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. The antibodies of the present invention can be expressed in either prokaryotic or eukaryotic host cells, for example, mammalian host cells, that are capable of assembling and secreting properly folded and immunoreactive antibodies.

[0135] Mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary (CHO cells), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody within the host cell or secretion of the antibody into the culture medium in which the host cell grows. The antibody can be recovered from the culture medium using standard protein purification methods.

[0136] Generation of bispecific antibodies The bispecific antibodies and antigen-binding fragments provided herein can be made using any suitable method, for example, two immunoglobulin heavy chain-light chain pairs can be co-expressed in a host cell to produce the bispecific antibody by recombinant means, which can then be purified by affinity chromatography.

[0137] A recombinant approach may be used in which sequences encoding antibody heavy chain variable domains for the two specificities are each fused to an immunoglobulin constant domain sequence and then inserted into an expression vector that is co-transfected with an expression vector for the light chain sequence into a suitable host cell for recombinant expression of the bispecific antibody. Similarly, scFv dimers can also be recombinantly constructed and expressed from host cells.

[0138] Alternatively, the leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. The linked antibodies were reduced at the hinge region to four half antibodies (i.e., monomers) and then re-oxidized to form heterodimers.

[0139] Two antigen-binding sites may be conjugated or cross-linked to generate bispecific antibodies or antigen-binding fragments. For example, one antibody can be conjugated to biotin while the other antibody is conjugated to avidin; the strong association of biotin and avidin will complex the two antibodies to generate a bispecific antibody.

[0140] Bispecific antigen-binding fragments may be generated from bispecific antibodies, for example, by proteolytic cleavage or chemical conjugation. For example, antigen-binding fragments of antibodies (e.g., Fab fragments) may be generated from bispecific antibodies, for example, by proteolytic cleavage or chemical conjugation. 5 ) is prepared and converted into a Fab'-thiol derivative, and then another converted Fab with a different antigen specificity is prepared. 5 It may be reacted with a derivatizer to generate a bispecific antigen-binding fragment.

[0141] Nucleic acid molecules encoding antibodies of the present disclosure In some aspects, the present disclosure is directed to isolated nucleic acid molecules, wherein the isolated nucleic acid molecule comprises a nucleic acid sequence encoding a bispecific antibody or antigen-binding portion disclosed herein, e.g., the nucleic acid sequence comprises any combination of the heavy or light chain sequences of SEQ ID NOs: 35-38, such as SEQ ID NOs: 35 and 36, or SEQ ID NOs: 37 and 38. For example, the nucleic acid sequence can encode the heavy and / or light chain of a bispecific antibody, e.g., the nucleic acid sequence comprises all of the sequences of SEQ ID NOs: 35-38.

[0142] The isolated nucleic acid molecule encoding the heavy chain variable region of the CD3 binding moiety is: (A) a nucleic acid sequence encoding the heavy chain variable region set forth in SEQ ID NO: 13; (B) the nucleic acid sequence set forth in SEQ ID NO: 21; and (C) a nucleic acid sequence that hybridizes under high stringency with the complement of the nucleic acid sequence of (A) or (B); The nucleic acid sequence may comprise a nucleic acid sequence selected from:

[0143] The isolated nucleic acid molecule encoding the light chain variable region of the CD3 binding moiety is: (A) a nucleic acid sequence encoding a light chain variable region set forth in SEQ ID NO: 14; (B) the nucleic acid sequence set forth in SEQ ID NO: 22; and (C) a nucleic acid sequence that hybridizes under high stringency with the complement of the nucleic acid sequence of (A) or (B); The nucleic acid sequence may comprise a nucleic acid sequence selected from:

[0144] The isolated nucleic acid molecule encoding the heavy chain variable region of the PSMA binding moiety is: (A) a nucleic acid sequence encoding a heavy chain variable region set forth in SEQ ID NO: 15; (B) the nucleic acid sequence set forth in SEQ ID NO: 23; and (C) a nucleic acid sequence that hybridizes under high stringency with the complement of the nucleic acid sequence of (A) or (B); The nucleic acid sequence may comprise a nucleic acid sequence selected from:

[0145] The isolated nucleic acid molecule encoding the light chain variable region of the PSMA binding moiety is: (A) a nucleic acid sequence encoding a light chain variable region set forth in SEQ ID NO: 16; (B) the nucleic acid sequence set forth in SEQ ID NO: 24; and (C) a nucleic acid sequence that hybridizes under high stringency with the complement of the nucleic acid sequence of (A) or (B); The nucleic acid sequence may comprise a nucleic acid sequence selected from:

[0146] In some cases, the disclosure provides an isolated nucleotide sequence encoding a heavy chain of a CD3 binding moiety, wherein the isolated nucleotide sequence encoding the heavy chain of the CD3 binding moiety is: (A) a nucleic acid sequence encoding the heavy chain set forth in SEQ ID NO: 17; (B) a nucleic acid sequence set forth in SEQ ID NO: 25 or 35; or (C) a nucleic acid sequence that hybridizes under high stringency with the complement of the nucleic acid sequence of (A) or (B); or

[0147] In some cases, the disclosure provides an isolated nucleotide sequence encoding a light chain of a CD3 binding moiety, wherein the isolated nucleotide sequence encoding the light chain of the CD3 binding moiety is: (A) a nucleic acid sequence encoding a light chain set forth in SEQ ID NO: 18; (B) a nucleic acid sequence set forth in SEQ ID NO: 26 or 36; or (C) a nucleic acid sequence that hybridizes under high stringency with the complement of the nucleic acid sequence of (A) or (B); or

[0148] In some cases, the disclosure provides an isolated nucleotide sequence encoding a heavy chain of a PSMA-binding moiety, wherein the isolated nucleotide sequence encoding the heavy chain of the PSMA-binding moiety is: (A) a nucleic acid sequence encoding the heavy chain set forth in SEQ ID NO: 19; (B) a nucleic acid sequence set forth in SEQ ID NO: 27 or 37; or (C) a nucleic acid sequence that hybridizes under high stringency with the complement of the nucleic acid sequence of (A) or (B); or

[0149] In some cases, the disclosure provides an isolated nucleotide sequence encoding a light chain of a PSMA-binding moiety, wherein the isolated nucleotide sequence encoding the light chain of the PSMA-binding moiety is: (A) a nucleic acid sequence encoding a light chain set forth in SEQ ID NO: 20; (B) a nucleic acid sequence set forth in SEQ ID NO: 28 or 38; or (C) a nucleic acid sequence that hybridizes under high stringency with the complement of the nucleic acid sequence of (A) or (B); or

[0150] In some cases, the disclosure provides an isolated nucleotide sequence encoding a heavy chain of a PSMA-binding moiety, wherein the isolated nucleotide sequence encoding the heavy chain of the PSMA-binding moiety is: (A) the nucleic acid sequence of SEQ ID NO: 33; (B) a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 31; or (C) a nucleic acid sequence that hybridizes under high stringency with the complement of the nucleic acid sequence of (A) or (B); or

[0151] In some cases, the disclosure provides an isolated nucleotide sequence encoding a light chain of a PSMA-binding moiety, wherein the isolated nucleotide sequence encoding the light chain of the PSMA-binding moiety is: (A) the nucleic acid sequence of SEQ ID NO: 34; (B) a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 32; or (C) a nucleic acid sequence that hybridizes under high stringency with the complement of the nucleic acid sequence of (A) or (B); or

[0152] In some aspects, the disclosure is directed to a vector comprising the nucleic acid sequences disclosed herein. Optionally, the expression vector further comprises a nucleotide sequence encoding a bispecific antibody constant region.

[0153] The vectors relevant to the present disclosure may be any suitable vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences comprising an appropriate set of expression control elements). Examples of such vectors include SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In some cases, the CD3 or PSMA antibody-encoding nucleic acid is contained in naked DNA or RNA, including, for example, linear expression elements, compact nucleic acid vectors, plasmid vectors such as pBR322, pUC 19 / 18, or pUC 118 / 119, "midge" minimal size nucleic acid vectors, or precipitated nucleic acid vector constructs such as the CaP04 precipitated construct.

[0154] In some cases, the vector is suitable for expressing anti-CD3 antibodies and / or anti-PSMA antibodies in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors, pET vectors (Novagen, Madison, Wisconsin), and the like. The vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha-factor, alcohol oxidase, and PGH.

[0155] The vector may also, or alternatively, be a vector suitable for expression in mammalian cells, for example a vector comprising glutamine synthetase as a selectable marker.

[0156] The nucleic acid and / or vector may comprise a nucleic acid sequence encoding a secretion / localization sequence capable of targeting a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or cell culture medium. Such sequences may include a secretory leader or signal peptide.

[0157] The vectors can comprise or be associated with any suitable promoters, enhancers, and other expression-facilitating elements. Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter / enhancer and RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), efficient poly(A) termination sequences, origins of replication for plasmid production in E. coli, antibiotic resistance genes as selectable markers, and / or convenient cloning sites (e.g., polylinkers). The nucleic acid may also comprise an inducible promoter as opposed to a constitutive promoter, such as CMV IE.

[0158] In a still further aspect, the present disclosure relates to a host cell comprising a vector as defined herein above.

[0159] Thus, the present disclosure also relates to recombinant eukaryotic or prokaryotic host cells, such as transfectomas, that produce the bispecific antibodies of the present disclosure.

[0160] CD3-specific antibodies may be expressed in recombinant eukaryotic or prokaryotic host cells, such as transfectomas, that produce the antibodies of the present disclosure as defined herein or the bispecific antibodies of the present disclosure as defined herein. PSMA-specific antibodies may similarly be expressed in recombinant eukaryotic or prokaryotic host cells, such as transfectomas, that produce the antibodies of the present disclosure as defined herein or the bispecific antibodies of the present disclosure as defined herein.

[0161] Examples of host cells include yeast, bacteria, plants, and mammalian cells, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NSO cells, or lymphocytic cells. For example, in some cases, the host cell can comprise the first and second nucleic acid constructs stably integrated into the cellular genome. In some cases, the present disclosure provides cells comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element comprising the first and second nucleic acid constructs defined above.

[0162] In a still further aspect, the present disclosure relates to a transgenic non-human animal or plant comprising nucleic acids encoding one or two sets of human heavy chains and human light chains, said animal or plant producing a bispecific antibody of the present disclosure.

[0163] In a further aspect, the present disclosure relates to a hybridoma that produces an antibody for use in a bispecific antibody of the present disclosure as defined herein. In a still further aspect, the present disclosure relates to a transgenic non-human animal or plant that comprises nucleic acids encoding one or two sets of human heavy and light chains, said animal or plant producing an antibody for use in a bispecific antibody or a bispecific antibody of the present disclosure.

[0164] In one aspect, the present disclosure provides: (i) a nucleic acid sequence encoding a heavy chain variable region of a first antigen-binding moiety and / or a heavy chain variable region of a second antigen-binding moiety according to any one of the examples or embodiments disclosed herein, optionally further encoding a CH1 domain or a CL domain; (ii) a nucleic acid sequence encoding the light chain variable region of the first antigen-binding moiety and / or the heavy chain variable region of the light chain variable region of the second antigen-binding moiety according to any one of the examples or embodiments disclosed herein; (iii) a nucleic acid sequence encoding a modified TCR β constant domain or a modified TCR α constant domain; (iv) a nucleic acid sequence encoding an Fc region; (v) a nucleic acid sequence encoding a linker; or (vi) a combination of at least two of the above; The present invention relates to an expression vector comprising:

[0165] In one aspect, the present disclosure relates to nucleic acid constructs that encode one or more of the amino acid sequences set forth in the Sequence Listing.

[0166] In one aspect, the present disclosure relates to a method for producing a bispecific antibody according to any one of the examples or embodiments disclosed herein, the method comprising culturing a host cell disclosed herein comprising an expression vector or expression vectors disclosed herein that express a bispecific antibody disclosed herein, and purifying the antibody from the culture medium. In one aspect, the disclosure relates to a host cell comprising an expression vector as defined above. Optionally, the host cell is a recombinant eukaryotic host cell, a recombinant prokaryotic host cell, or a recombinant microbial host cell.

[0167] Pharmaceutical Composition In some aspects, the present disclosure is directed to pharmaceutical compositions comprising at least one antibody or antigen-binding portion thereof disclosed herein and a pharmaceutically acceptable carrier.

[0168] Ingredients of the composition The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present disclosure can also be administered in combination with, for example, another immunostimulant, anticancer drug, antiviral drug, or vaccine, such that the anti-CD3 / anti-PSMA bispecific antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers can include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonicity agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, ingredients, or many other various combinations.

[0169] Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene, and / or propylgalactone. As disclosed herein, in a solvent containing an antibody or antigen-binding fragment of the present disclosure, the composition may contain one or more antioxidants, such as methionine, to oxidize the reduced antibody or antigen-binding portion thereof. Redox may prevent or reduce binding affinity, thereby enhancing antibody stability and shelf life. In some cases, the present disclosure provides one or more antibodies or antigen-binding portions thereof and one or more antioxidants, such as methionine. The present disclosure further provides various methods in which antibodies or antigen-binding portions thereof can be mixed with one or more antioxidants, such as methionine, to prevent the antibodies or antigen-binding portions thereof from oxidizing, thereby extending their shelf life and / or increasing their activity.

[0170] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; emulsifying agents such as polysorbate 80 (Tween 80); sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antibacterial agents used as carriers may be added to pharmaceutical compositions in multi-dose containers, including phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, p-hydroxybenzoic acid methyl and propyl esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, dissolution promoters, or sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0171] Administration, Formulation and Dosage The pharmaceutical compositions of the present disclosure may be administered to a subject in need thereof by various routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or other routes by implantation or inhalation. The subject compositions may be formulated into solid, semi-solid, liquid, or gaseous formulations, including but not limited to tablets, capsules, powders, granules, ointments, liquids, suppositories, enemas, injections, inhalants, and aerosols. The appropriate formulation and administration route may be selected depending on the intended application and treatment regimen.

[0172] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets including coated tablets, elixirs, suspensions, syrups or inhalants and sustained release forms thereof.

[0173] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may further comprise other pharmaceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickeners, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, the specific administration regimen, including dosage, timing, and repetition, will depend on the particular individual and their medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

[0174] The frequency of administration may be determined and adjusted over the course of treatment based on a reduction in the number of proliferative or tumorigenic cells, a maintenance of such a reduction in neoplastic cells, a reduction in the proliferation of neoplastic cells, or a delay in the onset of metastasis. In some cases, the administered dosage may be adjusted or reduced to manage potential side effects and / or toxicity. Alternatively, sustained continuous release formulations of the subject therapeutic compositions may be appropriate.

[0175] In some cases, antibodies or antigen-binding portions thereof of the present disclosure may be administered within a suitable range, including about 5 μg / kg to about 100 mg / kg of body weight per dose; about 50 μg / kg to about 5 mg / kg of body weight per dose; and about 100 μg / kg to about 10 mg / kg of body weight per dose. Other ranges include about 100 μg / kg to about 20 mg / kg of body weight per dose and about 0.5 mg / kg to about 20 mg / kg of body weight per dose. In some cases, the dosage is at least about 100 μg / kg, at least about 250 μg / kg, at least about 750 μg / kg, at least about 3 mg / kg, at least about 5 mg / kg, or at least about 10 mg / kg of body weight per dose.

[0176] In some cases, a course of treatment involving an antibody or antigen-binding portion thereof of the present disclosure will comprise multiple doses of the selected drug product over a period of several weeks or months. More specifically, an antibody or antigen-binding portion thereof of the present disclosure may be administered once daily, every two days, every four days, once weekly, once ten days, once every two weeks, once every three weeks, once monthly, once every six weeks, once every two months, once every ten weeks, or once every three months. In this regard, it will be understood that dosages may vary or intervals may be adjusted based on patient response and clinical practice.

[0177] Dosages and regimens may be empirically determined for a disclosed therapeutic composition in an individual given one or more doses. For example, an individual may be given increasing doses of a therapeutic composition prepared as described herein. In some cases, the dosage may be gradually increased or decreased or decreased based on empirically determined or observed side effects or toxicity, respectively. To evaluate the effectiveness of a selected composition, markers of a particular disease, disorder, or condition can be tracked as previously described. With respect to cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; improvement as assessed by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor markers (e.g., PSA for prostate cancer) or identified tumorigenic antigens by the methods described herein, reduction in pain or anesthesia; improved speech, vision, breathing, or other physical impairments associated with the tumor; increased appetite; or improved quality of life or prolonged survival as measured by recognized tests. It will be apparent to one skilled in the art that dosages will vary depending on the individual, the type of neoplastic condition, the stage of the neoplastic condition, whether the neoplastic condition has begun to metastasize to other locations in the individual, and previous and concurrent treatments being used.

[0178] Formulations suitable for parenteral administration (e.g., intravenous injection) comprise an antibody or antigen-binding portion thereof disclosed herein at a concentration of about 10 μg / ml to about 100 mg / ml. In some cases, the concentration of the antibody or antigen-binding portion thereof comprises 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, or 1 mg / ml. In some cases, the ADC concentration comprises 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml or 100 mg / ml.

[0179] Medical Uses / Methods In some aspects, the present disclosure provides methods of treating a disorder in a subject, the method comprising administering a therapeutically effective amount of an antibody or antigen-binding portion thereof disclosed herein to a subject (e.g., a mammal, e.g., a human) in need of treatment. For example, the disorder is cancer.

[0180] Various cancers that involve PSMA, whether malignant or benign, whether primary or secondary, can be treated or prevented using the method provided by the present disclosure.Cancer can be solid cancer.Examples of such cancer include lung cancer, such as bronchogenic carcinoma (for example, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondroitin hamartoma (non-cancerous), and sarcoma (cancerous); renal cancer; breast cancer; gastric cancer; colorectal cancer; glioblastoma; prostate cancer; pancreatic cancer; or ovarian cancer.In some cases, cancer is prostate cancer, particularly metastatic castration-resistant prostate cancer (mCRPC).

[0181] Combination with chemotherapy The antibody or antigen-binding portion thereof may be used in combination with an anti-cancer agent, a cytotoxic agent, or a chemotherapeutic agent.

[0182] The term "anti-cancer agent" or "anti-proliferative agent" refers to an agent that can be used to treat cell proliferative disorders, such as cancer, including, but not limited to, cytotoxic agents, cytostatic agents, angiogenesis inhibitors, tumor debulking agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormonal therapy, radiotherapy, and anti-metastatic and immunotherapeutic agents. As discussed above, it will be understood that in some cases, such anti-cancer agents may comprise a conjugate and may be associated with the disclosed site-specific antibodies prior to administration. More specifically, in some cases, a selected anti-cancer agent is linked to the unpaired cysteine ​​of an engineered antibody to provide the engineered conjugate described herein. Accordingly, such engineered conjugates are expressly intended to be within the scope of the present disclosure. In some cases, the disclosed anti-cancer agents are given in combination with a site-specific conjugate comprising a different therapeutic agent as described above.

[0183] As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells, reduces or inhibits the function of cells, and / or causes destruction of cells. In some cases, the substance is a naturally occurring molecule of biological origin. Examples of cytotoxic agents include, but are not limited to, small molecule or enzymatically active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas aeruginosa toxin and exotoxin, Staphylococcal enterotoxin A), fungi (e.g., α-sarcin, restrictocin), plants (e.g., abrin, ricin, modeccin, viscamine, pokeweed antiviral protein, saporin, gelonin, peach toxin, trichosatin, barley toxin, tallow fruit protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcin, crotin, chlorambu officinalis inhibitor, gelonin, mitegelin, restrictocin, phenomycin, neomycin, and trichothecenes), or animals (e.g., cytotoxic RNases such as extracellular pancreatic RNase; DNase I, including fragments and / or variants thereof).

[0184] For purposes of this disclosure, a "chemotherapeutic agent" comprises a chemical compound (e.g., a cytotoxic or cytostatic agent) that nonspecifically reduces or inhibits the growth, proliferation, and / or viability of cancer cells. Such chemical agents often target intracellular processes necessary for cell growth or differentiation and are therefore particularly effective against rapidly growing and differentiating cancer cells in general. For example, vincristine depolymerizes microtubules, thus inhibiting cells from entering mitosis. Overall, chemotherapeutic agents can include chemical agents that inhibit or are designed to inhibit cancer cells or cells likely to become cancerous or produce tumorigenic progeny. Such agents are often administered in combination, for example, in regimens such as CHOP or FOLFIRI, and are often most effective.

[0185] Examples of anti-cancer agents (either site-specifically conjugated or unconjugated) that may be used in combination with the site-specific constructs of the present disclosure include abiraterone, apalutamide, bicalutamide, alkylating agents, alkyl sulfonates, aziridines, ethylenimines and methylamelamines, acetogenins, camptothecin, bryostatin, kallistatin, CC-1065, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictin, spongiostatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, dextromethorphan, phenoxyethanol ... Torubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, erlotin Nibs, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogues, purine analogues, androgens, antiadrenal agents, folic acid supplements such as floric acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziconazole, elfornithine, elliptinium acetate, epothilone, etoglucide, gallium nitrate;Hydroxyurea, lentinan, lonidynin, maytansinoids, mitoguazone, mitoxantrone, mopidamol, nitraelin, pentostatin, fenameth, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex (JHS Natural Products, Eugene, Oregon, USA), razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, chlorambucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine, platinum; ethan These include, but are not limited to, toposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (camptosar, CPT-11), the topoisomerase inhibitor RFS2000; difluoromethylornithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; inhibitors of PKC-α, Raf, H-Ras, EGFR and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. Additionally included in this definition are antihormonal agents that serve to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, and antiandrogens; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, proleukin rIL-2; lurtotecan topoisomerase 1 inhibitors; abarelix rmRH;Vinorelbine and esperamicins, and pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0186] Combined with radiation therapy The present disclosure also provides for the use of antibodies or antigen-binding portions thereof in combination with radiation therapy (i.e., any mechanism for locally inducing DNA damage in tumor cells, such as gamma radiation, X-rays, UV radiation, microwaves, electron radiation, and the like). Combination therapy using directed delivery of radioisotopes to tumor cells is also contemplated, and the conjugates of the present disclosure may be used in conjunction with targeted anti-cancer drugs or other targeting means. Typically, radiation therapy is administered in pulses over a period of about 1 to about 2 weeks. Radiation therapy may be administered to subjects with head and neck cancer for about 6 to 7 weeks. Depending on the need, radiation therapy may be administered as a single dose or multiple, sequential doses.

[0187] Medicine Packs and Kits Pharmaceutical packs and kits are also provided, comprising one or more containers and containing one or more doses of an antibody or antigen-binding portion thereof. In some cases, the unit dose is provided containing a predetermined amount of a composition comprising, for example, an antibody or antigen-binding portion thereof, with or without one or more additional agents. In some cases, such unit doses are provided in a single-use prefilled syringe for injection. In some cases, the composition contained in the unit dose may comprise a buffer, such as saline, sucrose, or the like; phosphate, or the like; and / or may be formulated within a stable and effective pH range. Alternatively, in some cases, the conjugate composition may be provided as a lyophilized powder that can be reconstituted upon addition of an appropriate liquid, e.g., sterile water or saline. In some cases, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. A label on or associated with the container indicates that the enclosed conjugate composition is used to treat a selected neoplastic disease state.

[0188] The present disclosure also provides kits for obtaining single or multiple doses of a site-specific conjugate and, optionally, one or more anti-cancer agents. The kits comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers may be formed from a variety of materials, such as glass or plastic, and hold a pharmaceutically effective amount of a conjugate of the present disclosure, conjugated or unconjugated. Optionally, the container comprises a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). Such kits will generally comprise, in suitable containers, a pharmaceutically acceptable formulation of the engineered conjugate and, optionally, one or more anti-cancer agents in the same or different containers. The kits may also comprise other pharmaceutically acceptable formulations for either diagnosis or combination therapy. For example, in addition to an antibody or antigen-binding portion thereof of the present disclosure, such kits may comprise any one or more of a range of anti-cancer agents, such as chemotherapeutic or radiotherapeutic agents; angiogenesis inhibitors; anti-metastatic agents; targeted anti-cancer agents; cytotoxic agents; and / or other anti-cancer agents.

[0189] More specifically, the kit may have a single container containing the disclosed antibody or antigen-binding portion thereof, with or without additional components, or may have separate containers for each desired agent. When combination therapeutic agents are provided for conjugation, the single agents may be premixed, either in molar equivalent amounts or with one component in excess of the other. Alternatively, the conjugate and any optional anticancer agent of the kit may be kept separately in different containers prior to administration to a patient. The kit may also comprise second / third container means for containing a sterile pharmaceutically acceptable buffer or other diluent, such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution, and dextrose solution.

[0190] When the components of the kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution, for example, a sterile aqueous solution or a physiological saline solution.However, the components of the kit can also be provided as a dry powder.When a reagent or component is provided as a dry powder, the powder can be reconstituted by adding a suitable solvent.It is also conceivable that the solvent can be provided in a separate container.

[0191] As briefly indicated above, the kits may include a means for administering the antibody or antigen-binding portion thereof and any optional components to a patient, e.g., one or more needles, intravenous, bag, or syringe, or an eye dropper, pipette, or other such device by which the formulation may be injected or introduced into an animal or applied to a diseased area of ​​the body. Kits of the present disclosure will typically also include vials, or a means for providing such, and other components in a sealed container for commercial sale, such as, for example, a blow-molded plastic container into which the syringes or desired vials and other devices may be placed and retained.

[0192] Exemplary Antibodies One exemplary antibody is an anti-CD3 / anti-PSMA bispecific antibody designated W308051-T3U5.E17-61.uIgG4V322, or W308051. CDR numbering is defined using IMGT+Kabat and includes all residues defined by both IMGT and Kabat.

[0193] [Table 1]

[0194] [Table 2]

[0195] [Table 3]

[0196] [Table 4]

[0197] [Table 5] JPEG2025540716000008.jpg213170

[0198] [Table 6] JPEG2025540716000010.jpg196169

[0199] Another exemplary antibody is a fully human anti-PSMA monoclonal antibody designated W305042-1.135.2-uIgG1L, or WBP305042. CDR numbering is defined using IMGT+Kabat and includes all residues defined by both IMGT and Kabat.

[0200] [Table 7]

[0201] [Table 8]

[0202] [Table 9]

[0203] [Table 10]

[0204] Exemplary Embodiments Embodiment 1: An isolated antibody or antigen-binding portion thereof comprising a prostate-specific membrane antigen (PSMA) binding portion capable of binding to PSMA, wherein the PSMA binding portion comprises: a heavy chain CDR1 comprising the sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the sequence of SEQ ID NO: 2; a heavy chain CDR3 comprising the sequence of SEQ ID NO: 3; a light chain CDR1 comprising the sequence of SEQ ID NO: 4; a light chain CDR2 comprising the sequence of SEQ ID NO: 5; and a light chain CDR3 comprising the sequence of SEQ ID NO: 6.

[0205] Embodiment 2: The isolated antibody or antigen-binding portion thereof of embodiment 1, wherein the PSMA-binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 or the amino acid sequence encoded by SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16 or the amino acid sequence encoded by SEQ ID NO: 24.

[0206] Embodiment 3: The isolated antibody or antigen-binding portion thereof of embodiment 1 or 2, which is a bispecific antibody or antigen-binding portion thereof and comprises a CD3 binding portion capable of binding to CD3.

[0207] Embodiment 4: The isolated antibody or antigen-binding portion thereof of embodiment 3, wherein the CD3 binding portion comprises: a heavy chain CDR1 comprising the sequence of SEQ ID NO:7; a heavy chain CDR2 comprising the sequence of SEQ ID NO:8; a heavy chain CDR3 comprising the sequence of SEQ ID NO:9; a light chain CDR1 comprising the sequence of SEQ ID NO:10; a light chain CDR2 comprising the sequence of SEQ ID NO:11; and a light chain CDR3 comprising the sequence of SEQ ID NO:12.

[0208] Embodiment 5: The isolated antibody or antigen-binding portion thereof of embodiment 3 or 4, wherein the CD3-binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 or the amino acid sequence encoded by SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 or the amino acid sequence encoded by SEQ ID NO: 22.

[0209] Embodiment 6: The isolated antibody or antigen-binding portion thereof of embodiment 5, wherein the CD3 binding portion comprises a heavy chain TCR beta constant region comprising the sequence of SEQ ID NO: 29, and a light chain TCR beta constant region comprising the sequence of SEQ ID NO: 30.

[0210] Embodiment 7: The isolated antibody or antigen-binding portion thereof of embodiment 6, wherein the CD3 binding portion comprises a heavy chain comprising the sequence of SEQ ID NO: 17 and a light chain comprising the sequence of SEQ ID NO: 18.

[0211] Embodiment 8: The isolated antibody or antigen-binding portion thereof of any one of embodiments 1 to 7, wherein the PSMA-binding portion comprises a heavy chain comprising the sequence of SEQ ID NO: 19 and a light chain comprising the sequence of SEQ ID NO: 20.

[0212] Embodiment 9: The isolated antibody or antigen-binding portion thereof of embodiment 1 or 2, wherein the PSMA-binding portion comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 or encoded by SEQ ID NO: 33, and a light chain comprising the amino acid sequence of SEQ ID NO: 32 or encoded by SEQ ID NO: 34.

[0213] Embodiment 10: The isolated antibody or antigen-binding portion thereof of any one of embodiments 1 to 9, wherein the isolated antibody or antigen-binding portion thereof is a monoclonal antibody, a chimeric antibody, or a humanized antibody.

[0214] Embodiment 11: The isolated antibody or antigen-binding portion thereof of embodiment 10, wherein the isolated antibody or antigen-binding portion thereof is a monoclonal antibody.

[0215] Embodiment 12: The isolated antibody or antigen-binding portion thereof of embodiment 11, wherein the isolated antibody or antigen-binding portion thereof is a monoclonal antibody.

[0216] Embodiment 13: The isolated antibody or antigen-binding portion thereof of any one of embodiments 1 to 12, wherein said isolated antibody or antigen-binding portion thereof is fused to a constant region of an IgG, optionally a human IgG, optionally a human IgG1 or a human IgG4.

[0217] Embodiment 14: A pharmaceutical composition comprising the isolated antibody or antigen-binding portion thereof of any one of embodiments 1 to 13 and a pharmaceutically acceptable carrier.

[0218] Embodiment 15: A conjugate comprising the isolated antibody or antigen-binding portion thereof of any one of embodiments 1 to 13 and one or more moieties conjugated to said isolated antibody or antigen-binding portion thereof.

[0219] Embodiment 16: An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the isolated antibody or antigen-binding portion thereof of any one of Embodiments 1 to 13, wherein optionally the nucleic acid sequence comprises any combination of the sequences of SEQ ID NOs: 35 to 38.

[0220] Embodiment 17: A vector comprising the nucleic acid molecule of embodiment 16.

[0221] Embodiment 18: A host cell comprising the isolated nucleic acid molecule of embodiment 16 or the vector of embodiment 17.

[0222] Embodiment 19: A method for preparing the isolated antibody or antigen-binding portion thereof of any one of Embodiments 1 to 14, the method comprising: a) expressing the antibody or antigen-binding portion thereof in a host cell of Embodiment 18; and b) isolating the antibody or antigen-binding portion thereof from the host cell.

[0223] Embodiment 20: A method of inhibiting tumor cell growth or metastasis in a subject (e.g., a human subject), the method comprising administering to the subject an effective amount of the isolated antibody or antigen-binding portion thereof of any one of embodiments 1 to 13, or the pharmaceutical composition of embodiment 14.

[0224] Embodiment 21: A method of modulating an immune response in a subject (e.g., a human subject), the method comprising administering to the subject an effective amount of the isolated antibody or antigen-binding portion thereof of any one of embodiments 1 to 13, or the pharmaceutical composition of embodiment 14.

[0225] Embodiment 22: A method for treating or preventing a proliferative disease, an autoimmune disease, an inflammatory disease, or an infectious disease in a subject (e.g., a human subject), the method comprising administering to the subject an effective amount of the isolated antibody or antigen-binding portion thereof of any one of embodiments 1 to 13 or the pharmaceutical composition of embodiment 14.

[0226] Embodiment 23: The method of embodiment 22, wherein the method treats a proliferative disease that is cancer, optionally prostate cancer, lung cancer, bronchogenic carcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, adenocarcinoma, alveolar cell carcinoma, bronchial adenoma, chondroid hamartoma (non-cancerous), sarcoma, renal cancer, breast cancer, gastric cancer, colorectal cancer, glioblastoma, pancreatic cancer, ovarian cancer, or metastatic castration-resistant prostate cancer.

[0227] Embodiment 24: The method of embodiment 23, wherein the cancer is prostate cancer.

[0228] Embodiment 25: The method of any one of embodiments 20 to 24, wherein the isolated antibody or antigen-binding portion thereof of any one of embodiments 1 to 13 or the pharmaceutical composition of embodiment 14 is administered in combination with a chemotherapeutic agent, radiation, and / or another cancer immunotherapy.

[0229] Embodiment 26: A therapeutic or diagnostic kit for a proliferative, immune, inflammatory, or infectious disease, comprising a container comprising at least one isolated antibody or antigen-binding portion thereof of any one of embodiments 1 to 13. [Example]

[0230] The invention having thus generally been described will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention, and are not intended to represent that the experiments described below are all or the only experiments performed.

[0231] The commercially available materials used in Examples 1-4 are listed in the table immediately below. [Table 11]

[0232] Example 1. Preparation of materials 1.1 Establishment of stable cell lines The cynomolgus PSMA-expressing cell line WBP3xx042-FlpinCHO.cPro1.B7 (cynomolgus PSMA CHO cells) was generated using Flp-in cells (Thermo, R75807) transfected with a plasmid encoding full-length cynomolgus PSMA (XM_005579322.1, NCBI) according to the user's instructions.

[0233] 1.2 Benchmark antibody production The DNA sequence encoding the J591 antibody (W3XX042-BMK1) was synthesized at Genewiz (Suzhou, China) according to sequences 21 and 22 from WO 02 / 098897(A2) and then subcloned into a modified pcDNA3.3 expression vector (Thermo).

[0234] Plasmids encoding the heavy and light chains were co-transfected into Expi293 cells. The cells were cultured for 5 days, and the supernatant was collected for protein purification using a Protein A column (GE Healthcare, 175438). The resulting antibodies were analyzed by SDS-PAGE and SEC-HPLC, and then stored at -80°C.

[0235] Example 2. Antibody hybridoma production 2.1 Immunization and cell fusion Three OMT rats, 6-8 weeks old, were alternately immunized with human PSMA ECD protein and cynomolgus monkey PSMA ECD protein. Serum antibody titers against the antigen were monitored by ELISA and FACS. For ELISA, 96-well plates (Nunc) were coated with 100 μL of 2 μg / mL human PSMA antigen overnight at 4°C and then blocked with blocking buffer (1x PBS / 2% BSA) for 1 hour at ambient temperature. Rat serum was serially diluted 3-fold starting from a 1:100 dilution in blocking buffer and incubated for 1 hour at ambient temperature. Wells without serum samples served as negative controls. The plates were then washed and incubated with the secondary antibody, goat anti-rat IgG-Fc-HRP (Bethyl) for 1 hour. After washing, TMB substrate was added, and the interaction was stopped with 2 M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Devices). For FACS, 96-well plates were pre-coated with 3 × 10 LNCaP cells per well and cultured for 2 days in an incubator set at 37°C and 5% CO2. The plates were blocked with blocking buffer (1 × PBS / 5% milk) for 1 hour at ambient temperature. Then, 50 μL of hybridoma supernatant was added to the plate and incubated for 1 hour at ambient temperature. After washing the plate three times with PBS, the plate was incubated with the secondary antibody, goat anti-rat Fc-Alexa647 (1:500), for 1 hour at ambient temperature. The mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer and analyzed using FLOWJO.

[0236] Lymph nodes and spleens from immunized animals were homogenized and filtered to remove blood clots and cell debris. B cells and Sp2 / 0 myeloma cells were treated separately with pronase solution, and the reaction was stopped with 100% FBS. Cells were washed and counted. B cells were fused 1:1 with Sp2 / 0 myeloma cells in electrofusion solution according to standard electrofusion procedures. Fused cells were resuspended in DMEM medium supplemented with 20% FBS and 1X HAT and then transferred to a 96-well plate. Fused cells were cultured for 10-14 days in an incubator set at 37°C and 5% CO2.

[0237] 2.2 High-throughput screening of hybridoma supernatants and IgG conversion High-throughput screening methods using hybridoma culture supernatants include a primary screen by ELISA binding to human PSMA and a confirmatory screen by FACS / ELISA binding to human and cynomolgus PSMA.

[0238] Total RNA was isolated from hybridoma cells using the RNeasy Plus Mini Kit (Qiagen). First-strand cDNA was reverse transcribed using oligo-dT. The VH and VL genes of the above antibodies were amplified from the cDNA using a 3'-constant region degenerate primer and a 5'-degenerate primer set. The 5'-degenerate primer was designed based on the upstream signal sequence coding region of the Ig variable sequence. The PCR product was then ligated into the pMD18-T vector, and 10 μL of the ligation product was transformed into Top10 competent cells. The transformed cells were plated onto 2xYT plates with carbocinin and incubated at 37°C overnight. A total of 12 positive colonies were randomly picked for DNA sequencing.

[0239] After sequence analysis and functional screening, candidates were selected for fully human antibody production. The DNA sequences of the variable domains of the candidates were synthesized and cloned into a modified pcDNA3.4 vector containing human IgG1 Fc. After sequence confirmation, expression vectors containing the whole IgG of fully human antibodies were used for transient transfection for antibody production.

[0240] The purified IgG antibodies were further screened by ELIAS and FACS binding with human and cynomolgus monkey PSMA. Antibody W305042 was one of the antibodies.

[0241] Example 3. Fully human antibody molecule construction and purification The heavy and light chain expression plasmids for antibody W305042 were co-transfected into Expi293 cells using the Expi293 Expression System Kit (ThermoFisher-A14635). Five days after transfection, the supernatant was collected and used for protein purification using a Protein A column. The antibody concentration was measured using a NanoDrop. The protein purity was assessed by SDS-PAGE and SEC-HPLC (Figure 1). After purification, the yield of W305042 was 223.16 mg / L, and the purity by SEC-HPLC was 99.19%.

[0242] Example 4. Antibody Characterization 4.1 Binding Ability Test by ELISA Binding of the W305042 antibody to human PSMA was determined by ELISA. Plates were precoated with 1 μg / mL of THE™ His-tag antibody overnight in a refrigerator set at 4°C. After blocking with 200 μL of 1× PBS / 2% BSA for 1 hour, the plates were washed three times with 1× PBST. 0.5 μg / mL of human PSMA ECD protein diluted in 1× PBS / 2% BSA was then added to the plate at a volume of 50 μL / well and incubated at ambient temperature for 1 hour. After washing the plate three times with 1× PBST, various concentrations of the W305042 antibody, J591 (positive control), and a human IgG isotype antibody (negative control) were added to the plate at a volume of 50 μL / well (4-fold serial dilutions from 100 nM to 0.095 pM in 1× PBS / 2% BSA). The plates were then incubated at ambient temperature for 2 hours. After washing the plate three times with 1x PBST, 50 μL / well of HRP-conjugated goat anti-human IgG antibody (diluted 1:5000 in 1x PBS / 2% BSA) was added to the plate and incubated for 1 hour at ambient temperature. After washing the plate six times with 1x PBST, color was developed by adding 50 μL / well of TMB substrate for 4-8 minutes, and the reaction was then stopped by adding 50 μL / well of 2M HCl. Absorbance was read at 450 nm using a microplate reader. Binding EC50 values ​​were calculated using GraphPad Prism by plotting OD450 values ​​(y-axis) against antibody concentration (x-axis) and analyzing the results as a nonlinear regression (curve fit)—log(agonist) vs. response—variable slope (4 parameters).

[0243] The results of antibody binding to human PSMA protein are shown in Figure 2 and Table 1. W305042 could effectively bind to human PSMA ECD with an EC50 of 0.017 nM, which was slightly more potent than the reference antibody J591. A human IgG isotype antibody used as a negative control showed no obvious binding to human PSMA protein. The results suggested the excellent binding ability of W305042 to human PSMA.

[0244] [Table 12]

[0245] 4.2 Binding analysis by flow cytometry Fluorescence-activated cell sorting (FACS) was used to detect binding of the W305042 antibody to human PSMA. Briefly, 1 x 105 cells per well of LNCaP cells were incubated with various concentrations of W305042 antibody (100 nM to 0.095 pM, serially diluted 4-fold in 1x PBS / 1% BSA) in a volume of 100 μL per well for 1 hour in a refrigerator set at 4°C. J591 was used as a positive control, and a human IgG isotype antibody was used as a negative control. After washing the cells twice with 1x PBS / 1% BSA, Alexa Fluor 647-labeled goat anti-human antibody (diluted 1:500 in 1x PBS / 1% BSA) was added to the cells and incubated in the dark for 0.5 hour in a refrigerator set at 4°C. After washing the cells twice with 1x PBS / 1% BSA, the mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer and analyzed using FLOWJO. Binding EC50 was calculated with GraphPad Prism by plotting antibody concentration (x-axis) against MFI (y-axis) and analyzing as a nonlinear regression (curve fit) - log (agonist) vs. response - variable slope (4 parameters).

[0246] The antibody binding results for human PSMA-expressing LNCaP cells are shown in Figure 3 and Table 2. W305042 effectively bound to LNCaP cells with an EC50 of 0.25 nM, comparable to the reference antibody J591. A human IgG isotype antibody used as a negative control showed no significant binding to LNCaP cells. The results indicated the excellent binding ability of W305042 to LNCaP cells.

[0247] [Table 13]

[0248] 4.3 Orthologous (cross-species) binding test 4.3.1 Binding to cynomolgus monkey PSMA by ELISA Binding of the W305042 antibody to cynomolgus monkey PSMA was determined by ELISA. Plates were precoated with 1 μg / mL of THE™ His-tag antibody overnight in a refrigerator set at 4°C. After blocking with 200 μL of 1× PBS / 2% BSA for 1 hour, the plates were washed three times with 1× PBST. Then, 0.5 μg / mL of cynomolgus monkey PSMA ECD protein diluted in 1× PBS / 2% BSA was added to the plate at a volume of 50 μL / well and incubated at ambient temperature for 1 hour. After washing the plate three times with 1× PBST, various concentrations of the W305042 antibody, J591 (positive control), and a human IgG isotype antibody (negative control) were added to the plate at a volume of 50 μL / well (4-fold serial dilutions from 10 nM to 0.0095 pM in 1× PBS / 2% BSA). The plates were incubated at ambient temperature for 2 hours. After washing the plate three times with 1x PBST, 50 μL / well of HRP-labeled goat anti-human IgG antibody (diluted 1:5000 in 1x PBS / 2% BSA) was added to the plate and incubated for 1 hour at ambient temperature. After washing the plate six times with 1x PBST, color was developed by adding 50 μL / well of TMB substrate for 4-8 minutes, and the reaction was then stopped by adding 50 μL / well of 2M HCl. Absorbance was read at 450 nm using a microplate reader. EC50 was determined as described above.

[0249] The antibody binding results for cynomolgus monkey PSMA protein are shown in Figure 4 and Table 3. W305042 effectively bound to cynomolgus monkey PSMA ECD protein with an EC50 of 0.006 nM, comparable to the reference antibody J591. A human IgG isotype antibody used as a negative control showed no significant binding to cynomolgus monkey PSMA protein. The results suggested the excellent binding ability of W305042 to cynomolgus monkey PSMA.

[0250] [Table 14]

[0251] 4.3.2 Binding to cynomolgus monkey PSMA by FACS Fluorescence-activated cell sorting (FACS) was used to detect binding of the W305042 antibody to cynomolgus monkey PSMA. This method allows for quantitative analysis and identification of specific molecules expressed on the surface of live cells. Unlabeled cells were used as a control to set a threshold before detection, and the percentage change in each group above the fluorescence intensity threshold was analyzed. Engineered cells expressing cynomolgus monkey PSMA (W3xx042.FIpinCHO.cPro1.B7) were maintained in F-12 medium containing 10% FBS and 600 μg / mL hygromycin. Briefly, 1 x 105 cells per well of W3xx042.FIpinCHO.cPro1.B7 were incubated with various concentrations of W305042 antibody (100 nM to 6.1 pM, serially diluted 4-fold in 1x PBS / 1% BSA) in a volume of 100 μL per well for 1 h in a refrigerator set at 4 °C. J591 was used as a positive control, and a human IgG isotype antibody was used as a negative control. After washing the cells twice with 1x PBS / 1% BSA, Alexa fluor 647-labeled goat anti-human antibody (diluted 1:500 in 1x PBS / 1% BSA) was added to the cells and incubated in the dark for 0.5 hours in a refrigerator set at 4°C. After washing the cells twice with 1x PBS / 1% BSA, the mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer and analyzed using FLOWJO. The MFI and EC50 were determined as described above.

[0252] The antibody binding results with engineered CHO cells expressing cynomolgus monkey PSMA are shown in Figure 5 and Table 4. W305042 was able to effectively bind to cynomolgus monkey PSMA+ CHO cells with an EC50 of 2.25 nM, comparable to the reference antibody J591. A human IgG isotype antibody used as a negative control showed no significant binding to cynomolgus monkey PSMA+ CHO cells. The results suggested the excellent binding ability of W305042 to cynomolgus monkey PSMA.

[0253] [Table 15]

[0254] 4.3.3 Binding to mouse PSMA by ELISA Briefly, 2 μg / mL mouse PSMA protein (His tag) was coated onto the wells of an ELISA plate in coating buffer for 16 hours in a refrigerator set at 4°C. After washing the plate once with 1x PBST, 200 μL / well of 2% BSA was added to the wells and incubated at ambient temperature for 1 hour. After washing three times with 1x PBST, various concentrations of antibodies (W305042-1.135.2-uIgG1L and isotype control, diluted 6-fold from 100 nM to 0.357 pM) diluted in 2% BSA in a volume of 100 μL / well were added to the wells and incubated at ambient temperature for 1 hour. After washing three times with 1x PBST, 100 μL of the secondary antibody, goat anti-human IgG-Fc-HRP (1:5000 dilution) against W305042-1.135.2-uIgG1L and the isotype control, was added to the wells and incubated for 1 hour at ambient temperature. After washing six times with 1x PBST, 100 μL of TMB substrate solution was added to the wells and incubated for 5 minutes in the dark. The reaction was then stopped by adding 100 μL / well of 2M HCl to the wells. Relative light units (RLU) were measured at OD450 and OD540 using a SPECTRAMAX M5E. EC50 was determined as described above.

[0255] The results of antibody binding to mouse PSMA protein are shown in Figure 6 and Table 5. W305042 was able to effectively bind to mouse PSMA ECD protein with an EC50 of 0.026 nM. A human IgG isotype antibody used as a negative control showed no significant binding to mouse PSMA protein. The results suggested the excellent binding ability of W305042 to mouse PSMA.

[0256] [Table 16]

[0257] 4.4 Binding affinity testing by surface plasmon resonance (SPR) The binding affinities of W305042 and J591 to human and cynomolgus monkey PSMA were detected by SPR assay using a BIACORE 8K. Each antibody was captured on an anti-human IgG Fc antibody-immobilized CM5 sensor chip (Cytiva). Human and cynomolgus monkey PSMA at different concentrations was injected over the sensor chip at a flow rate of 30 μL / min for an association phase of 120–180 s, followed by a dissociation phase of 600–3600 s. After each binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5).

[0258] The sensorgrams of the blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data were fitted with a 1:1 binding model. The molecular weights of 81.4 and 82.7 kDa were used to calculate the molar concentrations of human and cynomolgus PSMA.

[0259] The on-rate constants (ka), off-rate constants (kd), and affinity constants (KD) of the antibodies are listed in Table 6. The binding affinity of W305042 to human PSMA was higher than that of J591, and the binding affinity of W305042 to cynomolgus PSMA was similar to that of J591.

[0260] [Table 17]

[0261] 4.5 Development feasibility test Thermal stability with DSF The melting temperature (Tm) of each antibody was tested using a QUANTSTUDIO 7 Flex real-time PCR system (Applied Biosystems). 19 μL of antibody solution was mixed with 1 μL of 80× SYPRO Orange protein detection gel stain and transferred to a 96-well plate. The plate was sealed with optical adhesive film and centrifuged at 3,000 rpm for 5 minutes to remove any air bubbles. The plate was heated from 26°C to 95°C at a rate of 0.9°C / min, and the resulting fluorescence data was collected. The negative derivative of the fluorescence change with temperature was calculated, and the maximum value was defined as the melting point Tm. If a protein has multiple unfolding transitions, the first two Tms were reported and designated Tm1 and Tm2. Data collection and Tm calculation were performed automatically using the QUANTSTUDIO real-time PCR software (v1.3). W305042 exhibited excellent thermal stability, with a Tm1 of 69.5°C and a Tm2 of 71.1°C (Figure 7).

[0262] Determination of the diffusion interaction parameter (kD) by DLS kD measurements were performed using a DYNAPRO plate reader III (Wyatt Technology). During the sample preparation process, the appearance of the samples was recorded during thawing, filtration, and concentration. 7.5 μL of sample solution was then added to a 1536-well microplate. Data collection was performed using DYNAMICS operation software (v7.8.1.3). Data were collected five times for each protein sample, with each collection time being 5 seconds. For each measurement, the diffusion coefficient was determined and plotted against the protein concentration. kD values ​​were automatically calculated by the software.

[0263] W305042 exhibited high kD values ​​and a monodisperse size distribution, indicating that W305042 has excellent solubility properties. See Table 7.

[0264] [Table 18]

[0265] Hydrophobic interaction chromatography HPLC (HIC-HPLC) The hydrophobicity of the antibody was detected by HPLC 1260 Infinity II system (Agilent Technologies™) using a TSK Gel Butyl-NPR column. The sample was diluted in PBS buffer, and 20 μL of the diluted sample was injected onto the column and separated for 61 minutes at a flow rate of 0.5 mL / min. Peak retention was detected using UV light at wavelengths of 280 nm and 230 nm. Retention times were analyzed by HIC-HPLC analysis, and the total peak area from 20 to 40 minutes was integrated. The operating and analytical software was OPEN LAB CDS Workstation (v2.6.0.691). The retention time of W305042 by HIC-HPLC was 24.57 minutes, indicating its low hydrophobicity (Figure 8).

[0266] Example 5. Materials, cell lines and preparation of benchmark (BMK) antibodies Information regarding commercially available materials used in the following examples is provided in the table immediately below. [Table 19] JPEG2025540716000024.jpg208170

[0267] Generation of stable cell lines The cynomolgus PSMA-expressing cell line WBP3xx042-FlpinCHO.cPro1.B7 (cynomolgus PSMA CHO cells) was generated using Flp-in cells (Thermo, R75807) transfected with a plasmid encoding full-length cynomolgus PSMA (XM_005579322.1, NCBI) according to the user's instructions.

[0268] Benchmark (BMK) antibody production The DNA sequence encoding the CD3xPSMA reference antibody, AMG340 / TNB-585 (BMK1), was synthesized according to SEQ ID NOs: 49, 56, and 61 from WO 2021 / 222578(A1) and then subcloned into a modified pcDNA3.3 expression vector (Thermo).

[0269] The DNA sequence encoding the CD3xPSMA reference antibody, AMG160 (BMK2), was synthesized according to SEQ ID NO: 382 from US Patent Application Publication No. 2017 / 0218079(A1) and then subcloned into a modified pcDNA3.3 expression vector (Thermo).

[0270] Recombinant plasmids expressing the reference antibodies were transfected into Expi293 cells. The cells were cultured for 5 days, and the supernatant was collected for protein purification using a Protein A column (GE Healthcare, 175438) and / or an SEC column (Cytiva, 28990944). The resulting antibodies were analyzed by SDS-PAGE and HPLC-SEC, and then stored at -80°C.

[0271] Example 6. Preparation of bispecific antibodies Anti-CD3 monoclonal antibodies were discovered in mice immunized by hybridoma technology (WO 2019 / 057099(A1)). Anti-PSMA monoclonal antibodies were discovered in transgenic rats immunized by hybridoma technology.

[0272] Construction of bispecific antibodies was performed using standard molecular biology protocols. As illustrated in Figure 9, for construction of the CD3 x PSMA bispecific antibody W308051-T3U5.E17-61.uIgG4V322 (W308051), a DNA sequence encoding the VH region of an anti-CD3 antibody was fused to a modified TCR β constant domain and hinge-Fc region of human IgG4 with a S228P mutation, an Fc null mutation (F234A L235A), and a knob mutation (S354C-T366W); a DNA sequence encoding the VL region of an anti-CD3 antibody was fused to a modified TCR α constant domain; and a DNA sequence encoding the VH region of an anti-PSMA antibody was fused to a modified TCR α constant domain with a S228P mutation, an Fc null mutation (F234A L235A), and a knob mutation (S354C-T366W). The DNA sequence encoding the VL region of an anti-PSMA antibody was fused to the CL domain of a human IgG4 hinge-Fc region carrying the mutations Y349C-T366S-L368A-Y407V (Y349C-T366S-L368A-Y407V). The coding region was then cloned into a modified pcDNA3.3 expression vector.

[0273] Plasmids encoding bispecific antibodies were transfected into Expi293 cells at a 1000 ml scale. Cells were cultured for 5 days, and the supernatant was collected for protein purification using a Protein A column (Cytiva, 17549802) and / or a CEX column (Cytiva, 17118001). Antibody concentrations were detected by Nano Drop at 280 nm. Antibody purity was analyzed by SDS-PAGE and SEC-HPLC. Endotoxin levels were determined by PTS / MCS cartridge. Antibodies were stored at -80°C.

[0274] As shown in Figures 10A and 10B, the yield of W308051 was 79.22 mg / L, and the purity by SEC-HPLC was 98.18%.

[0275] Example 7. In vitro characterization 7.1 Target Binding Measured by ELISA / FACS Binding to human PSMA measured by ELISA Briefly, 1 μg / mL human PSMA protein (His tag) was coated onto the wells of an ELISA plate in coating buffer for 16 hours at 4°C. After washing the plate once with 1x PBST, the plate was blocked with 200 μL / well of 2% BSA for 1 hour. After washing the plate three times with 1x PBST, various concentrations of antibody (5-fold serial dilutions from 200 nM to 0.004096 pM) diluted in 2% BSA were added to the wells, and the plate was incubated at ambient temperature for 1 hour. After washing the plate three times with 1x PBST, goat anti-human IgG-Fc-HRP (1:5000 dilution) was added to the wells and incubated for 1 hour. After washing six times with 1x PBST, 100 μL of TMB substrate solution was added to the wells and incubated for 5 minutes in the dark. The reaction was then stopped by adding 100 μL / well of 2M HCl to the wells. Relative light units (RLU) were measured using a SPECTRAMAX M5E. 450 and O.D. 540 Binding EC 50 ,OD 450~540 The antibody concentration (x-axis) was plotted against the agonist concentration (y-axis) and analyzed as a nonlinear regression (curve fit) - log(agonist) vs. response - with variable slope (4 parameters) calculated using GraphPad Prism7 software.

[0276] The results are shown in Figure 11 and Table 8, and W308051 had an EC 50 It bound to human PSMA protein with greater potency than the reference antibody AMG160.

[0277] [Table 20]

[0278] Binding to human PSMA and CD3 measured by FACS Four human prostate cancer cells, human C4-2 (with high PSMA expression), LNCaP (with high PSMA expression), and 22Rv1 (with low PSMA expression), PC-3 (PSMA negative), and Jurkat2B8 (CD3 positive) cells, and primary T cells (5 × 10) isolated from fresh PBMCs were used. 4 Cells were incubated with various concentrations of antibodies (5-fold serial dilutions from 200 nM to 2.56 pM for W308051, AMG160, and isotype control, and 5-fold serial dilutions from 500 nM to 6.40 pM for AMG340) at 4°C for 1 hour. After washing twice with 1x PBS / 1% BSA / 0.1 mM EDTA, Alexa fluor 647-labeled goat anti-human IgG Fc (1:500 dilution) was added, and the plate was incubated for 0.5 hours at 4°C in the dark. After washing twice with 1x PBS / 1% BSA / 0.1 mM EDTA, cells were resuspended in 1x PBS / 1% BSA / 0.1 mM EDTA, and mean fluorescence intensity (MFI) was measured by flow cytometry and analyzed by FLOWJO. Bound EC 50 ,OD 450~540 The antibody concentration (x-axis) was plotted against the agonist concentration (y-axis) and analyzed as a nonlinear regression (curve fit) - log(agonist) vs. response - with variable slope (4 parameters) calculated using GraphPad Prism7 software.

[0279] The results are shown in Figures 12-13 and Table 9. As shown in Figure 12 and Table 9, W308051 had EC values ​​of 1.48 nM, 0.58 nM, and 0.29 nM, respectively. 50 13 , W308051 bound to human PSMA-positive C4-2 (high PSMA expression), LNCaP (high PSMA expression), and 22Rv1 (low PSMA expression) human tumor cells, demonstrating that W308051 was more potent than AMG160 and AMG340. At the same time, W308051 did not bind to the PSMA-negative PC-3 cell line. As shown in Figure 13 , W308051 bound to CD3-positive Jurkat cells and primary human T cells with lower binding affinity than AMG160.

[0280] [Table 21]

[0281] 3.2 Cross-species binding measured by ELISA / FACS Binding to cynomolgus monkey and mouse PSMA measured by ELISA Briefly, 1 μg / mL cynomolgus monkey PSMA protein (His-tagged) and 2 μg / mL mouse PSMA protein (His-tagged) were coated onto the wells of an ELISA plate in coating buffer for 16 hours at 4°C. After washing the plate once with 1x PBST, the plate was blocked with 200 μL / well of 2% BSA for 1 hour. After washing three times with 1x PBST, various concentrations of antibodies (W308051, AMG160, and isotype control for cynomolgus monkey PSMA, serially diluted from 200 nM to 0.004 pM; W308051, AMG160, and isotype control for mouse PSMA, serially diluted from 100 nM to 0.357 pM; AMG340 for mouse PSMA, serially diluted from 500 nM to 1.786 pM) diluted in 2% BSA were added and incubated at ambient temperature for 1 hour. After washing three times with 1x PBST, goat anti-human IgG-Fc-HRP (1:5000 dilution) was added and incubated for 1 hour. After washing six times with 1x PBST, 100 μL of TMB substrate solution was added to each well and incubated for 5 minutes in the dark. The reaction was then stopped by adding 100 μL / well of 2M HCl to each well. Relative light units (RLU) were measured using a SPECTRAMAX M5E as an OD meter. 450 and O.D. 540 Measured at EC 50 was determined as above.

[0282] As shown in Tables 10-11 and Figures 14A and 14B, W308051 cross-reacted with cynomolgus monkey and mouse PSMA. W308051 had an EC of 0.045 nM. 50W308051 bound to cynomolgus monkey PSMA ECD with an EC of 1.34 nM, which was more potent than AMG160. 50 AMG160 and AMG340 bound to mouse ECD PSMA protein, but AMG160 and AMG340 did not bind to mouse PSMA protein.

[0283] [Table 22]

[0284] [Table 23]

[0285] Binding of cynomolgus monkey PSMA-positive cells measured by FACS WBP3xx042-FlpinCHO.cPro1.B7(5×10 4 Cells) were incubated with various concentrations of antibodies for 1 hour at 4°C. After washing twice with 1x PBS / 1% BSA / 0.1mM EDTA, secondary antibody, Alexa fluor 647-labeled goat anti-human IgG Fc, was added and the plates were incubated for half an hour at 4°C in the dark. After washing twice with 1x PBS / 1% BSA / 0.1mM EDTA, cells were resuspended in 1x PBS / 1% BSA / 0.1mM EDTA. MFI and EC 50 was determined as above.

[0286] As shown in Figure 15 and Table 12, W308051 had an EC 50 It bound to cynomolgus monkey PSMA-positive cells at 1000 kJ / mL, which was more potent than AMG160 and AMG340.

[0287] [Table 24]

[0288] 7.3 Affinity measured by SPR The binding affinity of antibodies W308051, AMG340, and AMG160 to human PSMA was detected by SPR assay using a BIACORE 8K. Biotinylated human PSMA was captured on a streptavidin-immobilized CM5 sensor chip (Cytiva). Antibodies at different concentrations were injected over the sensor chip in a single-cycle injection mode at a flow rate of 30 μL / min for a 180-second association phase, followed by a 600-3600-second dissociation phase. The chip was then regenerated with 10 mM glycine (pH 1.5) for a final binding cycle.

[0289] The sensorgrams of the blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data were fitted with a 1:1 binding model. The molecular weights of 147, 111, and 106 kDa were used to calculate the molar concentrations of W308051, AMG340, and AMG160, respectively.

[0290] The binding affinities of antibodies W308051, AMG340, and AMG160 to human CD3δ and CD3ε were detected by SPR assay using a BIACORE 8K. Each antibody was captured on an anti-human IgG Fc antibody-immobilized CM5 sensor chip (Cytiva). Human CD3δ and CD3ε heterodimers at different concentrations were injected over the sensor chip at a flow rate of 30 μL / min for a 120-second association phase, followed by a 240-second dissociation phase. After each binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5).

[0291] The sensorgrams of the blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data were fitted with a 1:1 binding model. The molar concentrations of human CD3δ and CD3ε heterodimers were calculated using a molecular weight of 31 kDa.

[0292] The complete kinetic affinity of W308051 for human PSMA and CD3 is shown in Table 13. The affinity of W308051 for human PSMA and CD3 was 1.58×10 -11 M and 6.02 × 10-8 It was M.

[0293] [Table 25]

[0294] 7.4 T-cell cytotoxicity and cytokine release The efficacy of bispecific antibodies in mediating tumor cell lysis by human T cells was evaluated using a CTG-based cytotoxicity assay. Briefly, C4-2, LNCaP, and PC-3 cells were used as target cells, and human T cells were isolated from fresh human PBMCs and used as effector cells. 150 μL / well of antibody was added to a 96-well plate. Effector cells (1 × 10 5 cells / 50 μL / well) and target cells (1 × 10 4 Cells / 50 μL / well) were added to the corresponding wells (E / T ratio = 10:1). After 72 h incubation, the plates were washed once with DPBS, and CTG solution (75 μL / well) was added and incubated at ambient temperature for 10 min. Relative light unit (RLU) signals were measured by an invasion reader. The % cytotoxicity was calculated as (1 - (RLU) サンプル -RLU エフェクター細胞のみ ) / (RLU エフェクター細胞+標的細胞 -RLU エフェクター細胞のみ )) × 100%. Binding IC 50 was calculated with GraphPad Prism7 software by plotting antibody concentration (x-axis) against cytotoxicity (y-axis) and analyzing as a nonlinear regression (curve fit) - log(inhibitor) vs. response - variable slope (4 parameters).

[0295] The released cytokine levels in the supernatant were determined by an ELISA-based quantitative assay. Briefly, after 20 hours of incubation, 125 μL of supernatant was collected and stored at 4°C. Human IFN-γ release was measured by ELISA, and a standard curve was generated using recombinant human IFN-γ. Plates were pre-coated overnight at 4°C with 50 μL / well of a capture antibody specific for human IFN-γ (1:250). After blocking with 2% BSA for 1 hour, 50 μL of standard or sample was added to each well and incubated for 2 hours at ambient temperature. After washing three times with 1× PBST, 50 μL of biotin-conjugated human IFN-γ detection antibody (1:250) and peroxidase-conjugated streptavidin (1:250) were added to each well and incubated for 1 hour at ambient temperature. After washing six times with 1x PBST, color development was achieved by adding 50 μL of TMB substrate and then stopping with 50 μL of 2M HCl. Relative light units (RLU) were measured using a SPECTRAMAX M5E. 450 and O.D. 540 The concentration of human IFN-γ in the supernatant was quantified using a standard curve.

[0296] As shown in Figures 16-17 and Tables 14-15, the results showed that W308051 induced potent cytotoxicity and minimal IFN-γ release in co-cultures of PSMA+ tumor cells and CD3+ T cells, but no cytotoxicity or cytokine release in co-cultures of PSMA-negative PC-3 cells and CD3+ T cells.

[0297] [Table 26]

[0298] [Table 27]

[0299] 7.5 Cytokine release from C4-2 cells co-cultured with PBMCs Briefly, 100 μL / well of antibodies (10-fold serial dilutions from 20 nM to 0.2 pM for W308051, AMG160, and isotype control, and 10-fold serial dilutions from 500 nM to 5.0 pM for AMG340) were added to a 96-well plate. Effector cells (fresh human PBMCs, 2 × 10 5 cells / 50 μL / well) and C4-2 target cells (2 × 10 4 Cells / 50 μL / well) were added to the corresponding wells (E:T ratio = 10:1). After 20 hours of incubation, the supernatant was collected and stored at -80°C. The released human IFN-γ, IL-2, TNF-α, and IL-6 in the supernatant were measured by ELISA. Relative light units (RLU) were calculated by OD analysis using a SPECTRAMAX M5E. 450 and O.D. 540 The concentrations of human cytokines in the supernatants were quantified using standard curves. 50 was calculated with GraphPad Prism7 software by plotting antibody concentration (x-axis) against percentage (y-axis) and analyzing as a nonlinear regression (curve fit) - log(agonist) vs. response - variable slope (4 parameters).

[0300] As shown in Figure 18 and Table 16, W308051 induced a low release of a panel of cytokines in PBMC and C4-2 cell co-culture assays.

[0301] [Table 28]

[0302] 7.6 Thermal stability measured by DSF T of each antibody mThe melting point (T) was examined using a QUANTSTUDIO 7 Flex real-time PCR system (Applied Biosystems). 19 μL of antibody solution was mixed with 1 μL of 80×SYPRO Orange protein detection gel staining reagent and transferred to a 96-well plate. The plate was sealed with optical adhesive film and centrifuged at 3,000 rpm for 5 minutes to remove any air bubbles. The plate was heated from 26°C to 95°C at a rate of 0.9°C / min, and the resulting fluorescence data was collected. The negative derivative of the fluorescence change at different temperatures was calculated, and the maximum value was determined as the melting point (T). m If a protein has multiple unfolding transitions, the first two Tm are reported, and T m 1 and T m 2. Data collection and T m Calculations were performed automatically by QUANTSTUDIO real-time PCR software (v1.3). As shown in Figure 19, W308051 had a T of 62.9°C. m 1 and T of 69.3°C m 2 showed excellent thermal stability.

[0303] 7.7 Hydrophobic Interaction Chromatography HPLC (HIC-HPLC) The hydrophobicity of the antibody was detected using an HPLC 1260 Infinity II system (Agilent Technologies™) with a TSK Gel Butyl-NPR column. The sample was diluted in PBS buffer, and 20 μL of the diluted sample was injected onto the column and separated for 61 minutes at a flow rate of 0.5 ml / min. Peak retention was detected using UV light at wavelengths of 280 nm and 230 nm. Retention times were analyzed by HIC-HPLC analysis, and the total peak area from 20 to 40 minutes was integrated. The operation and analysis software was OpenLab CDS Workstation (v2.6.0.691). As shown in Figure 20, the retention time of W308051 by HIC-HPLC was 25.15 minutes, indicating that W308051 has low hydrophobicity.

[0304] 3.8 Determination of the diffusion interaction parameter (kD) by DLS kD measurements were performed using a DYNAPRO plate reader III (Wyatt Technology). During the sample preparation process, the appearance of the samples was recorded during thawing, filtration, and concentration. 7.5 μL of sample solution was then added to a 1536-well microplate. Data collection was performed using DYNAMICS operation software (v7.8.1.3). Data were collected five times for each protein sample, with each collection time being 5 seconds. For each measurement, the diffusion coefficient was determined and plotted against the protein concentration. kD values ​​were automatically calculated by the software.

[0305] As shown in Table 17, W308051 exhibited a high kD value and a monodisperse size distribution, indicating that W308051 has excellent dissolution properties.

[0306] [Table 29]

[0307] Example 8. In vivo characterization 8.1 Rat PK studies A preliminary pharmacokinetic study of WBP308051 was conducted in female CD(SD)IGS rats by a single intravenous bolus administration. Briefly, 8-10 week-old female CD(SD)IGS rats (Beijing Charles River) were used in this study. Five animals per group were administered W308051 at 10 mg / kg by a single intravenous bolus. PK serum samples were collected, and serum concentrations of WBP308051 were determined using three bioanalytical ELISA methods. In Method 1 (Fc+Fc), 96-well ELISA plates were coated with goat anti-human IgG overnight at 4°C, followed by serially diluted plasma samples. Biotin-labeled goat anti-human IgG Fc was used as the detection antibody. In Method 2 (PSMA+CD3), 96-well ELISA plates were coated with human PSMA ECD protein overnight at 4°C, followed by serially diluted plasma samples. Biotin-labeled human CD3ε was used as the detection protein. In method 3 (CD3 + PSMA), a 96-well ELISA plate was coated with recombinant human CD3ε overnight at 4°C, and then serially diluted plasma samples were added. Human PSMA ECD protein was used as the detection protein. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer (SPECTRAMAX M5E). The serum concentration of the WBP308051 lead antibody in rats was analyzed for non-septum pharmacokinetics using Phoenix WINNONLIN software (version 8.1, Pharsight, Mountain View, CA, USA). The linear / log trapezoidal rule was applied to obtain PK parameters.

[0308] As shown in Figure 21 and Table 18, for intravenous PK studies at 10 mg / kg using three ELISA configurations (i.e., coated with Fc, PSMA, or CD3, and detected with Fc, CD3, or PSMA, respectively), WBP308051 exhibited mean serum clearances of 6.05, 8.07, and 7.51 mL / day / kg, respectively; mean half-lives of 177, 142, and 132 hours, respectively; volumes of distribution (Vss) of 59.1, 65.8, and 58.8 mL / kg, respectively; and AUCs of 30,332, 24,087, and 26,745 h·μg / mL, respectively. Similar results from the three different ELISA configurations suggested excellent in vivo stability of WBP308051.

[0309] [Table 30]

[0310] 8.2 In vivo efficacy in the NPG-hPBMC model The in vivo efficacy of WBP308051 was tested in a LNCaP xenograft model in male NPG mice with human PBMC reconstitution. 6 LNCaP tumor cells were implanted (subcutaneous injection) into the right flank of NPG mice. Each mouse received 2 × 10 6 They also received PBMC cells. The tumor volume was approximately 105 mm 3 When tumor-bearing mice reached 100 mg / kg, they were randomly divided into nine groups. Each of the nine groups received six intraperitoneal injections twice weekly: vehicle (PBS); 0.30 mg / kg AMG340; 1.51 mg / kg AMG340; 0.057 mg / kg AMG160; 0.29 mg / kg AMG160; 1.44 mg / kg AMG160; 0.08 mg / kg W308051; 0.4 mg / kg W308051; and 2 mg / kg W308051. Mouse weight and tumor growth were measured twice weekly. Tumor volume was calculated using the formula (1 / 2) × (length × width). 2All procedures regarding the handling, care, and treatment of animals in the study were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi Biologics' LARC, which follows the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

[0311] The results of the tumor growth curve are shown in Figure 22A. The mean tumor volume in the PBS group was 1527.3 ± 216.73 mm on day 21 after treatment. 3 Treatment with AMG160 at 0.057, 0.29, and 1.44 mg / kg showed a stronger antitumor effect compared with treatment with PBS, with tumor sizes of 86.63 ± 10.42, 74.85 ± 10.52, and 50.36 ± 4.54 mm 3 Treatment with AMG340 at 0.30 and 1.51 mg / kg demonstrated antitumor efficacy compared to PBS treatment, with mean tumor volumes of 1061.97 ± 296.65 and 441.45 ± 297.15 (TGI = 32.75, 78.53%). Treatment with W308051 at 0.4 and 2 mg / kg demonstrated potent antitumor efficacy, with mean tumor volumes of 193.46 ± 264.26 and 69.09 ± 10.48 mm. 3 The mean tumor burden was 2022.29 ± 328.64 mm (TGI = 93.86, 102.63%). However, treatment with W308051 at 0.08 did not show any antitumor effect, with a mean tumor burden of 2022.29 ± 328.64 mm (TGI = 93.86, 102.63%). 3 The mean tumor burden was (TGI=-34.84%).

[0312] Mouse weights are shown in Figure 22B and slight weight loss was observed during the study, which may be due to GVHD (graft versus host disease) effects in the human PBMC reconstitution model.

[0313] In summary, W308051 inhibited LNCaP cell proliferation in vivo in a dose-dependent manner. At high dose levels (0.40 and 2.00 mg / kg), W308051 induced tumor growth inhibition comparable to that of equimolar AMG160 and more potent than that of equimolar AMG340.

[0314] Those skilled in the art will further appreciate that the present disclosure may be embodied in other specific forms without departing from the spirit or central characteristics thereof. In that the foregoing description of the present disclosure discloses only exemplary embodiments thereof, it should be understood that other variations are intended to be within the scope of the present disclosure. Accordingly, the present disclosure is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims, which indicate the scope and content of the present disclosure.

Claims

1. 1. An isolated antibody or antigen-binding portion thereof, comprising a PSMA-binding portion capable of binding to prostate-specific membrane antigen (PSMA), wherein said PSMA-binding portion is: a heavy chain CDR1 comprising the sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the sequence of SEQ ID NO:2; a heavy chain CDR3 comprising the sequence of SEQ ID NO: 3; a light chain CDR1 comprising the sequence of SEQ ID NO: 4; a light chain CDR2 comprising the sequence of SEQ ID NO:5; a light chain CDR3 comprising the sequence of SEQ ID NO: 6; 1. An isolated antibody or antigen-binding portion thereof comprising:

2. The isolated antibody or antigen-binding portion thereof of claim 1, wherein the PSMA-binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 or encoded by SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16 or encoded by SEQ ID NO:

24.

3. The isolated antibody or antigen-binding portion thereof according to claim 1 or 2, wherein the isolated antibody or antigen-binding portion thereof is a bispecific antibody or antigen-binding portion thereof and comprises a CD3 binding portion capable of binding to CD3.

4. the CD3 binding moiety a heavy chain CDR1 comprising the sequence of SEQ ID NO: 7; a heavy chain CDR2 comprising the sequence of SEQ ID NO: 8; a heavy chain CDR3 comprising the sequence of SEQ ID NO: 9; a light chain CDR1 comprising the sequence of SEQ ID NO: 10; a light chain CDR2 comprising the sequence of SEQ ID NO: 11; a light chain CDR3 comprising the sequence of SEQ ID NO: 12; 4. The isolated antibody or antigen-binding portion thereof of claim 3, comprising:

5. The CD3 binding portion of an isolated antibody or antigen-binding portion thereof according to claim 3 or 4, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 or encoded by SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 or encoded by SEQ ID NO:

22.

6. The isolated antibody or antigen-binding portion thereof of claim 5, wherein the CD3 binding portion comprises a heavy chain TCRβ constant region comprising the sequence of SEQ ID NO: 29 and a light chain TCRβ constant region comprising the sequence of SEQ ID NO:

30.

7. 7. The isolated antibody or antigen-binding portion thereof of claim 6, wherein the CD3 binding portion comprises a heavy chain comprising the sequence of SEQ ID NO: 17 and a light chain comprising the sequence of SEQ ID NO:

18.

8. The isolated antibody or antigen-binding portion thereof of any one of claims 1 to 7, wherein the PSMA-binding portion comprises a heavy chain comprising the sequence of SEQ ID NO: 19 and a light chain comprising the sequence of SEQ ID NO:

20.

9. The isolated antibody or antigen-binding portion thereof of claim 1 or 2, wherein the PSMA-binding portion comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 or encoded by SEQ ID NO: 33, and a light chain comprising the amino acid sequence of SEQ ID NO: 32 or encoded by SEQ ID NO:

34.

10. The isolated antibody or antigen-binding portion thereof of any one of claims 1 to 9, wherein the isolated antibody or antigen-binding portion thereof is a monoclonal antibody, a chimeric antibody, or a humanized antibody.

11. The isolated antibody or antigen-binding portion thereof of claim 10, wherein the isolated antibody or antigen-binding portion thereof is a monoclonal antibody.

12. 12. The isolated antibody or antigen-binding portion thereof of claim 11, wherein the isolated antibody or antigen-binding portion thereof is a human monoclonal antibody.

13. 13. The isolated antibody or antigen-binding portion thereof of any one of claims 1 to 12, wherein the isolated antibody or antigen-binding portion thereof is fused to a constant region of IgG, optionally human IgG, optionally human IgG1 or human IgG4.

14. A pharmaceutical composition comprising the isolated antibody or antigen-binding portion thereof of any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. A complex comprising the isolated antibody or antigen-binding portion thereof of any one of claims 1 to 13 and one or more moieties conjugated to said isolated antibody or antigen-binding portion thereof.

16. 14. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the isolated antibody or antigen-binding portion thereof of any one of claims 1 to 13, optionally wherein the nucleic acid sequence comprises any combination of the sequences of SEQ ID NOs: 35 to 38.

17. A vector comprising the nucleic acid molecule of claim 16.

18. 18. A host cell comprising the isolated nucleic acid molecule of claim 16 or the vector of claim 17.

19. 14. A method for preparing an isolated antibody or antigen-binding portion thereof according to any one of claims 1 to 13, said method comprising: a) expressing the antibody or antigen-binding portion thereof in the host cell of claim 18; and b) isolating the antibody or antigen-binding portion thereof from the host cell; The method comprising:

20. 15. A method for inhibiting tumor cell growth or metastasis in a subject, the method comprising administering to the subject an effective amount of the isolated antibody or antigen-binding portion thereof of any one of claims 1 to 13 or the pharmaceutical composition of claim 14.

21. 15. A method of modulating an immune response in a subject, the method comprising administering to the subject an effective amount of the isolated antibody or antigen-binding portion thereof of any one of claims 1 to 13 or the pharmaceutical composition of claim 14.

22. 15. A method for treating or preventing a proliferative disease, autoimmune disease, inflammatory disease, or infectious disease in a subject, said method comprising administering to the subject an effective amount of the isolated antibody or antigen-binding portion thereof of any one of claims 1 to 13 or the pharmaceutical composition of claim 14.

23. 23. The method of claim 22, wherein the method treats a proliferative disorder that is cancer, optionally prostate cancer, lung cancer, bronchogenic carcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, adenocarcinoma, alveolar cell carcinoma, bronchial adenoma, chondroid hamartoma (non-cancerous), sarcoma, renal cancer, breast cancer, gastric cancer, colorectal cancer, glioblastoma, pancreatic cancer, ovarian cancer, or metastatic castration-resistant prostate cancer.

24. 24. The method of claim 23, wherein the cancer is prostate cancer.

25. 25. The method of any one of claims 20 to 24, wherein the isolated antibody or antigen-binding portion thereof of any one of claims 1 to 13 or the pharmaceutical composition of claim 14 is administered in combination with a chemotherapeutic agent, radiation, and / or another cancer immunotherapy.

26. 14. A therapeutic or diagnostic kit for a proliferative, immune, or infectious disease, comprising a container comprising at least one isolated antibody or antigen-binding portion thereof of any one of claims 1 to 13.

Images