PSMA antibodies and uses thereof

Abstract

Provided herein are prostate specific membrane antigen (PSMA) antibodies, e.g., human monoclonal antibodies against PSMA, nucleic acid molecules encoding the antibodies, expression vectors and host cells for expressing the antibodies, methods for making the same, and uses thereof, e.g., in the treatment of PSMA-related diseases, including cancer.

Description

[0001] Cross-reference

[0002] This application claims the benefit of priority patent applications PCT / CN2022 / 134047, filed on November 24, 2022, and PCT / CN2022 / 134163, filed on November 24, 2022, both of which are hereby incorporated by reference in their entirety.

[0003] Sequence Listing

[0004] The content of the electronic sequence listing (70351WO01 Seq List 16 Nov 2023.xml; size: 47 kilobytes; created on November 16, 2023) is hereby incorporated by reference in its entirety. Summary of the Invention

[0006] In one aspect, the present disclosure provides a isolated antibody or an antigen-binding portion thereof, which comprises 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 Description of the Drawings

[0007] Figure 1 Shows SDS-PAGE and SEC-HPLC analyses of the exemplary PSMA antibody W305042.

[0008] Figure 2 Binding of the antibodies (W305042, J591, and isotype control) to human PSMA was shown by ELISA.

[0009] Figure 3 Binding of the antibodies (W305042, J591, and isotype control) to cynomolgus monkey PSMA was shown by ELISA.

[0010] Figure 4 Binding of the antibodies (W305042, J591, and isotype control) to LNCaP cells was shown by FACS.

[0011] Figure 5 Shows binding of the antibodies (W305042, J591, and isotype control) to cynomolgus monkey PSMA-expressing CHO cells measured by FACS.

[0012] Figure 6 Shows the binding of antibodies (W305042 and isotype control) to murine PSMA measured by ELISA.

[0013] Figure 7 Shows the DSF spectrum of W305042.

[0014] Figure 8 Shows the HIC-HPLC spectrum of W305042.

[0015] Figure 9 Is a schematic representation of the bispecific CD3xPSMA antibody W308051, where T3 represents the anti-CD3 arm, in which the heavy chain variable domain of the anti-CD3 arm is fused to the modified TCR β constant domain of human IgG4 (represented by the grey rectangle) and the hinge-Fc region, and this human IgG4 has S228P mutation, Fc null mutations (F234A L235A) and knob mutations (S354C-T366W), and the VL of the anti-CD3 arm is fused to the modified TCR α constant domain (represented by another grey rectangle); and U5 represents the anti-PSMA arm, in which the heavy chain variable domain of the anti-PSMA arm is fused to the hinge-Fc region of human IgG4, and this human IgG4 has 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.

[0016] Figure 10A and 10B Shows the results of SDS-PAGE ( Figure 10A ) and SEC-HPLC ( Figure 10B ) analysis of W308051. In Figure 10A , the lanes from left to right show protein Marker, non-reduced antibody, reduced antibody and protein Marker respectively.

[0017] Figure 11 Shows the binding of antibodies (W308051, AMG160 and isotype hIgG4 control) to human PSMA measured by ELISA.

[0018] Figure 12 Shows the binding of antibodies (W308051, AMG340, AMG160 and isotype hIgG4 control) to human C4-2 (with high PSMA expression), LNCaP (with high PSMA expression), 22Rv1 (with low PSMA expression) and PC-3 cells (PSMA negative) measured by FACS.

[0019] Figure 13 Shows the binding of antibodies (W308051, AMG340, AMG160, and isotype hIgG4 control) to CD3-positive Jurkat cells and primary human T cells as measured by FACS.

[0020] Figure 14A and 14B Shows the binding of antibodies (W308051, AMG160, and isotype hIgG4 control) to cynomolgus monkey PSMA ( Figure 14A ) and mouse PSMA ( Figure 14B ) as measured by ELISA.

[0021] Figure 15 Shows the binding of antibodies (W308051, AMG340, AMG160, and isotype hIgG4 control) to cynomolgus monkey PSMA-positive cells as measured by FACS.

[0022] Figure 16 Shows the T cell cytotoxicity of C4-2 cells, LNCaP cells, and PC-3 cells co-cultured with CD3+ T cells and incubated with antibodies W308051, AMG340, AMG160, and isotype hIgG4 control.

[0023] Figure 17 Shows the cytokine release of C4-2 and PC-3 cells co-cultured with CD3+ T cells and incubated with antibodies W308051, AMG340, AMG160, and isotype hIgG4 control.

[0024] Figure 18 Shows the cytokine release of C4-2 cells co-cultured with PBMC and incubated with antibodies W308051, AMG340, AMG160, and isotype hIgG4 control.

[0025] Figure 19 Shows the thermal stability of antibody W308051 as measured by DSF.

[0026] Figure 20 Shows the results of antibody W308051 as measured by hydrophobic interaction chromatography HPLC (HIC-HPLC).

[0027] Figure 21 Shows the average serum concentration of antibody W308051 in a pharmacokinetic study.

[0028] Figure 22A and 22B Shows the in vivo efficacy of antibodies (W308051, AMG340, and AMG160) in the NPG-hPBMC model: (Figure 22A ) Tumor growth curve; and Figure 22B ) Body weight of tumor-bearing mice. DETAILED DESCRIPTION OF THE INVENTION

[0030] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. The singular forms "a", "an", and "the" may include plural referents unless the context clearly dictates otherwise. For example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes a mixture of cells, etc. In this application, the use of "or" means "and / or" unless stated otherwise. In addition, the use of the term "comprising", as well as other forms such as "comprises" and "comprised", is not limiting. In addition, the ranges provided in the specification and the appended claims include both endpoints and all endpoints between the endpoints.

[0031] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to a polymer of amino acid residues, or an assembly of multiple polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been subsequently modified, such as, for example, hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as the naturally occurring amino acid. The α-carbon refers to the first carbon atom attached to a functional group (such as a carbonyl group). The β-carbon refers to the second carbon atom connected to the α-carbon, and the system continues to name the carbons in alphabetical order using Greek letters. Amino acid mimetics are compounds that have a structure different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. The term "protein" generally refers to a large polypeptide. The term "peptide" generally refers to a short polypeptide. A polypeptide sequence is typically described such that the left end of the polypeptide sequence is the amino terminus (N-terminus); the right end of the polypeptide sequence is the carboxyl terminus (C-terminus). As used herein, a "polypeptide complex" refers to a complex that includes one or more polypeptides associated with the performance of certain functions. In some instances, the polypeptides are immune-related.

[0032] The term "antibody" or "Ab" is used herein in its broadest sense and encompasses a variety of antibody structures, including polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies). A native intact antibody is typically a Y-shaped tetrameric protein that comprises two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be divided into kappa light chains and lambda light chains. The heavy chains can be divided into μ, δ, γ, α, and ε, which define the isotypes of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable region is joined to the constant region via a "J" region of about 12 or more amino acids, and the heavy chain further contains a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region / domain (V H ) and a heavy chain constant region / domain (C H ). The heavy chain constant region is composed of 3 domains (C H 1, C H 2, and C H 3). Each light chain is composed of a light chain variable region / domain (V L ) and a light chain constant region / domain (C L ). The V H and V L regions can be further divided into hypervariable regions (termed complementarity-determining regions (CDRs)), which are separated by relatively conserved regions (termed framework regions (FRs)). Each V H and V L is composed of 3 CDRs and 4 FRs in the following order: from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (V H and V L ) of each pair of heavy / light chains form the antigen-binding site, respectively. Antibodies can have different antibody isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0033] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody, which are used interchangeably in the context of the present application, refer to a polypeptide containing a fragment of a full-length antibody that retains the ability to specifically bind an antigen that the full-length antibody binds to, and / or the ability to compete with the full-length antibody for binding to the same antigen. Antigen-binding fragments of an antibody can be derived from, for example, intact antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding the variable domains and optionally the constant domains of the antibody. Such DNA is known and / or can be readily obtained from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and chemically manipulated, or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, generate cysteine residues, modify, add or delete amino acids, etc.

[0034] 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 that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment" as used herein. In some instances, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. The variable and constant domains can be directly linked to one another, or can be linked via a full or partial hinge or linker region. The hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule.

[0035] As used herein, the term "variable domain" with respect to an antibody refers to the variable region of an antibody or a fragment thereof that contains one or more CDRs. Although a variable domain can contain the full variable region (such as HCVR or LCVR), it can also contain less than the full variable region but still retain the ability to bind an antigen or form an antigen-binding site.

[0036] As used herein, the term "antigen-binding portion" refers to an antibody fragment formed by a part of an antibody that contains one or more CDRs, or any other antibody fragment that binds an antigen but does not contain the full native antibody structure. Examples of antigen-binding portions include, but are not limited to, variable domains, variable regions, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), multispecific antibodies (e.g., bispecific antibodies such as Het-mAb), camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding portion is capable of binding the same antigen to which the parent antibody binds. In some instances, an antigen-binding portion may contain one or more CDRs from a particular human antibody that are grafted onto a framework region from one or more different human antibodies.

[0037] The "Fab" of an antibody refers to the portion of the antibody consisting of a single light chain (variable and constant regions) that is bound via a disulfide bond to the variable region and the first constant region of a single heavy chain. In some instances, the constant regions of both the light and heavy chains are replaced with TCR constant regions.

[0038] "F(ab’)2" refers to a dimer of Fab’.

[0039] The "fragment difficult (Fd)" of an antibody refers to the amino-terminal half of the heavy chain fragment that can combine with a light chain to form a Fab.

[0040] The "Fc" of an antibody refers to the portion of the antibody consisting of the second (CH2) and third (CH3) constant regions of the first heavy chain that are bound via a disulfide bond to the second and third constant regions of the second heavy chain. The Fc portion of an antibody is responsible for a variety of effector functions, such as ADCC and CDC, but does not play a role in antigen binding.

[0041] With respect to an antibody, the "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 amino acid residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently.

[0042] As used herein, the "CH2 domain" refers to the portion of the heavy chain molecule that includes, using a conventional numbering scheme (amino acids 244 to 360, Kabat numbering system; and amino acids 231 - 340, EU numbering system), approximately amino acids 244, for example, to amino acids 360 of an IgG antibody.

[0043] The "CH3 domain" extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 amino acids. Certain immunoglobulin classes, such as IgM, further include a CH4 region.

[0044] The "Fv" of an antibody refers to the smallest fragment of an antibody that carries the complete antigen-binding site. The Fv fragment consists of the variable domain of a single light chain bound to the variable domain of a single heavy chain. Many Fv designs have been provided, including dsFv, in which the association between the two domains is enhanced by introduced disulfide bonds; and the two domains can be joined together as a single polypeptide using a peptide linker to form a scFv. Fvs constructs have also been generated that contain the variable domain of an immunoglobulin heavy or light chain bound to the variable and constant domains of the corresponding immunoglobulin heavy or light chain. Fvs are multimerized to form diabodies and triabodies.

[0045] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light-chain variable region and a heavy-chain variable region that are directly or via a peptide linker sequence connected to each other.

[0046] In some instances, a "scFv dimer" is a bivalent diabody or bivalent scFv (BsFv) containing V H -V L (linked by a peptide linker), where this V H -V L partially dimerizes with another V H -V L such that one part of the V H cooperates with other parts of the V L and forms two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes).

[0047] In some instances, a "scFv dimer" is a bispecific diabody containing V H1 -V L2 (linked by a peptide linker), where this V H1 -V L2 binds to V L1 -V H2 (also linked by a peptide linker) such that V H1 and V L1 cooperate, V H2 and V L2 cooperate, and each cooperating pair has different antigen specificities.

[0048] A "scFab" refers to a fusion polypeptide having an Fd linked to a light chain via a polypeptide linker, resulting in the formation of a single-chain Fab fragment (scFab).

[0049] “dsFv” refers to a disulfide-stabilized Fv fragment in which the connection between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some instances, “(dsFv)2” or “(dsFv-dsFv')” comprises three peptide chains: two V L portions that are connected by a peptide linker (e.g., a long flexible linker) and that are bound to two V H portions via disulfide bonds, respectively. In some instances, dsFv-dsFv’ is bispecific, wherein the heavy and light chains paired by each disulfide bond have different antigen specificities.

[0050] “Additional IgG” refers to a fusion protein having Fab arms that are fused to IgG to form a bispecific (Fab)2-Fc format. It can form “IgG-Fab” or “Fab-IgG”, wherein the Fab is fused to the C-terminus or N-terminus of the IgG molecule, with or without a linker. In some instances, the additional IgG can be further modified to an IgG-Fab4 format.

[0051] As used herein, the term “anti-CD3 antibody” or “CD3 antibody” refers to an antibody that, as defined herein, is capable of binding CD3, such as human CD3, e.g., for eliciting a potential therapeutic effect.

[0052] The terms “CD3” and “CD3 protein” are used interchangeably herein. The CD3 protein is present in almost all T cells. The CD3-TCR complex regulates T cell function in innate and adoptive immune responses, as well as cellular and humoral immune functions. These include elimination of pathogenic organisms and control of tumor growth through a wide range of cytotoxic effects. The CD3 T cell coreceptor is a protein complex composed of four different chains, the CD3γ chain, the CD3δ chain, and two CD3ε chains. These four chains associate with a molecule called the T cell receptor (TCR) and the ζ chain to generate an activation signal in T lymphocytes. The TCR, ζ chain, and CD3 molecules form the TCR complex, wherein the TCR, as a subunit, recognizes and binds antigen, and CD3, as a subunit, transfers and transmits the antigenic stimulus to the signaling pathway and ultimately regulates T cell activity. The term “CD3” can include human CD3, as well as its variants, isoforms, and species homologs. Thus, as defined and disclosed herein, an antibody or its antigen-binding portion can also bind CD3 from species other than human, such as cynomolgus monkey CD3.

[0053] As used herein, the term “human CD3” refers to CD3 of human origin, such as the complete amino acid sequence of human CD3.

[0054] As used herein, the term “cynomolgus monkey CD3” refers to CD3 derived from cynomolgus monkey, such as the complete amino acid sequence of cynomolgus monkey CD3.

[0055] As used herein, the term "anti-PSMA antibody" refers to an antibody that specifically binds to PSMA. An "anti-PSMA antibody" can include monovalent antibodies having a single specificity. Exemplary anti-PSMA antibodies are described elsewhere herein.

[0056] The term "prostate-specific membrane antigen (PSMA)" is a type II membrane glycoprotein composed of 750 amino acids with folate hydrolase and NAALADase enzymatic activities.

[0057] 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 a single antigen-binding site; and the term "multivalent" refers to an antibody or antigen-binding fragment having multiple antigen-binding sites. In some instances, the antibody or its antigen-binding portion is bivalent.

[0058] As used herein, a "bispecific" antibody refers to an engineered antibody capable of binding or targeting two different epitopes, e.g., having fragments derived from two different monoclonal antibodies. Binding of a bispecific antibody to two different epitopes can elicit a potential therapeutic effect. The two different epitopes can be present on the same antigen, or they can be present on two different antigens. In some instances, the bispecific antibody is a Het-mAb.

[0059] As used herein, a "Het-mAb" is an IgG-like molecule that can target two different epitopes on the same or different targets and has 4 different chains; 2 heavy chains and 2 light chains. These chains contain a set of mutations in the Fc portion of the molecule to drive heavy chain dimerization and a set of mutations on the Fab portion to drive proper heavy / light pairing, resulting in a bispecific mAb of the κ / κ or λ / κ subtype.

[0060] The term "bispecific antigen-binding molecule" means a protein, polypeptide, or molecular complex that includes 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 instances, a "bispecific antigen-binding molecule" is a "bispecific antibody". Each antigen-binding portion within a bispecific antibody contains at least one CDR that, alone or in combination with one or more additional CDRs and / or FRs, specifically binds a particular antigen. In some instances, the first antigen-binding site specifically binds a first antigen (e.g., PSMA or CD3), and the second antigen-binding site specifically binds a second different antigen (e.g., CD3 or PSMA).

[0061] As used interchangeably herein, the terms “anti-PSMA / anti-CD3 antibody”, “anti-PSMA / anti-CD3 bispecific antibody”, “antibody against PSMA and CD3”, “anti-PSMA×CD3 bispecific antibody”, “PSMA×CD3 antibody”, “anti-CD3 / anti-PSMA antibody”, “anti-CD3 / anti-PSMA bispecific antibody”, “antibody against CD3 and PSMA”, “anti-CD3×PSMA bispecific antibody”, “CD3×PSMA antibody” refer to bispecific antibodies that specifically bind to CD3 and PSMA, regardless of the order in which the targets are first mentioned.

[0062] As used herein, the term “monoclonal antibody” or “mAb” refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope.

[0063] As used herein, the term “human antibody” is intended to include antibodies having variable regions in which both the framework regions and the CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains constant regions, the constant regions are 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 introduced by in vitro random or site-directed mutagenesis or by in vivo somatic mutation). 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 have been grafted onto human framework sequences.

[0064] The term “humanized antibody” is intended to refer to an antibody 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.

[0065] 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 mouse antibody and the constant region sequences are derived from a human antibody.

[0066] As used herein, the term “recombinant antibody” refers to an antibody prepared, expressed, produced, or isolated by recombinant means, such as an antibody isolated from an animal transgenic for immunoglobulin genes of another species, an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant, combinatorial antibody library, or an antibody prepared, expressed, produced, or isolated by any other means involving splicing of immunoglobulin gene sequences to other DNA sequences.

[0067] As used herein, the term "spacer" refers to an artificial amino acid sequence having 1, 2, 3, 4, or 5 amino acid residues, or a length of 5 to 15, 20, 30, 50, or more amino acid residues, which are joined by peptide bonds and are used to link one or more polypeptides. The spacer may or may not have a secondary structure. For example, spacers useful in the present disclosure may be rich in glycine and proline residues. Examples include spacers having a single or repeated sequence consisting of threonine / serine and glycine, such as TGGGG, GGGGS, or SGGGG or tandem repeats thereof (e.g., 2, 3, 4, or more repeats).

[0068] The term "operably link" or "operably linked" refers to the juxtaposition of two or more biological sequences of interest, with or without intervening or linker sequences, such that they are in a relationship that permits them to function in their intended manner. When used with respect to polypeptides, it is intended to mean that the polypeptide sequences are linked in a manner that permits the resulting product to have the intended biological function. For example, an antibody variable region may be operably linked to a constant region to provide a stable product having antigen-binding activity. The term may also be used with respect to polynucleotides. By way of example, when a polynucleotide encoding a polypeptide is operably linked to regulatory sequences (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequences are linked in a manner that permits the expression of the polypeptide to be regulated from the polynucleotide.

[0069] As used herein, the term "epitope" refers to the portion on an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to as "antigenic determinant". An epitope or antigenic determinant typically consists of the chemically active surface groups of a molecule (e.g., amino acids, carbohydrates, or sugar side chains) and generally has a specific three-dimensional structure and specific charge characteristics. For example, an epitope typically contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which may be "linear" or "conformational". In a linear epitope, all of the interaction sites between a protein and an interacting molecule (e.g., an antibody) are linearly present along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites span amino acid residues that are separated from each other in the protein. For example, studies of competition or cross-competition 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). High-throughput methods for obtaining antibodies that bind to the same epitope are based on their cross-competition.

[0070] As used herein, the term "specific binding" or "specifically bind(s)" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen.

[0071] K D Used to refer to the ratio of the dissociation rate to the association rate (k off / k on ), which can be determined by surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). In some instances, the K D value can be appropriately determined by using flow cytometry.

[0072] When used with respect to amino acid sequences (e.g., peptides, polypeptides, or proteins), the term "fusion" or "fused" refers to the combination of two or more amino acid sequences into a single amino acid sequence that is not naturally occurring (e.g., by chemical bonding or recombinant means). Fusion amino acid sequences can be produced by genetic recombination of two coding polynucleotide sequences and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

[0073] The term "antigen specificity" refers to a specific antigen or its epitope that is selectively recognized by an antigen-binding molecule.

[0074] 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. "Percentage identity" means the percentage of identical residues between the amino acids or nucleotides in the molecules being compared and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) can be resolved by specific mathematical models or computer programs (i.e., "algorithms").

[0075] As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It refers not only to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells so as to ultimately generate immune effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response that can form antibodies or sensitized T lymphocytes in the immune system of an organism after stimulation with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors: the properties of the antigen, the reactivity of the host, and the mode of immunization.

[0076] As used herein, the term "substitution" with respect to an amino acid residue refers to the natural or induced replacement of one or more amino acids in a peptide, polypeptide, or protein with another amino acid. Substitutions in a polypeptide can result in a decrease, enhancement, or elimination of the polypeptide's function.

[0077] As used herein, the term "mutation" or "mutated" with respect to an amino acid residue refers to the replacement, insertion, or addition of an amino acid residue.

[0078] A natural "T cell receptor" or natural "TCR" is a heterodimeric T cell surface protein that binds to invariant CD3 chains to form a complex capable of mediating signal transduction. TCRs belong to the immunoglobulin superfamily and are similar to half-antibodies with a single heavy chain and a single light chain. Natural TCRs have an extracellular portion, a transmembrane portion, and an intracellular portion. The extracellular domain of TCRs has a membrane-proximal constant region and a membrane-distal variable region. In some instances, bispecific antibodies comprise soluble chimeric proteins having the variable domains of an antibody and the constant domains of a TCR, wherein the subunits of the TCR constant domains (such as the α and β domains) are linked by engineered disulfide bonds.

[0079] As used herein, the term "Ka" is intended to refer to the association rate of a particular antibody-antigen interaction, and as used herein, the term "Kd" is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using methods established in the art. As used herein, the term "K D " is intended to refer to the dissociation constant of a particular antibody-antigen interaction, which is obtained as the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). Exemplary methods for determining the Kd of an antibody are by using surface plasmon resonance, such as using a biosensor system such as a BIACORE system.

[0080] As used herein, the term "high affinity" of an IgG antibody refers to an antibody having a K -7 of 1 x 10 D M or lower for a target antigen, such as 5 x 10 -8 M or lower, 1 x 10 -8 M or lower, 5 x 10 -9 M or lower or 1 x 10 -9 M or lower.

[0081] As used herein, the term "EC 50 ", which is also referred to as the "half maximal effective concentration", refers to the concentration of a drug, antibody, or toxin that induces half of the response between the baseline and the maximum after a specified exposure time. In the context of the present application, EC 50 is expressed in units of "nM".

[0082] As used herein, the term "competitive binding" refers to the interaction of two antibodies in their binding to a binding target. If the binding of a first antibody to its cognate epitope is significantly lower in the presence of a second antibody than in its absence, the first antibody competes with the second antibody for binding. Optionally, the binding of the second antibody to its epitope may also be significantly reduced in the presence of the first antibody, which may occur but is not required. That is, the first antibody may inhibit the binding of the second antibody to its epitope, while the second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, when each antibody significantly inhibits the binding of the other antibody to its cognate epitope, regardless of whether the degree is the same, greater or smaller, these antibodies are said to "cross-compete" with each other for the binding of their respective epitopes.

[0083] As used herein, the ability to "inhibit binding" refers to the ability of an antibody or its antigen-binding portion to inhibit the binding of two molecules (e.g., human CD3 / PSMA and human anti-CD3 / anti-PSMA antibodies) to any detectable level. In some instances, the binding of the two molecules can be inhibited by an antibody or its antigen-binding portion by at least 50%. In some instances, this inhibition can be greater than 60%, greater than 70%, greater than 80% or greater than 90%.

[0084] As used herein, the term "isolated" refers to a state obtained by artificial means from its natural state. If an "isolated" substance or component exists in nature, it may be because its natural environment has changed, or the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal body, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude a mixture of artificial or synthetic substances, nor other impure substances that do not affect the activity of the isolated substance.

[0085] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to the CD3 / PSMA protein is substantially free of antibodies that specifically bind to antigens of other proteins other than CD3 / PSMA). However, an isolated antibody that specifically binds to the human CD3 / PSMA protein may cross-react with other antigens (e.g., CD3 / PSMA proteins from other species). In addition, an isolated antibody can be substantially free of other cellular materials and / or chemicals.

[0086] As used herein, the term "vector" refers to a nucleic acid vehicle that can have polynucleotides inserted therein. When a vector permits the expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. A vector can have elements of genetic material carried thereon that are expressed in a host cell upon 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 virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). Vectors can contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors can contain origins of replication.

[0087] As used herein, the term "host cell" refers to a cell system 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 tissues or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term encompasses not only the particular subject cell but also the progeny of such a cell. Since certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not be identical to the parental cell, but are still included within the scope of the term "host cell".

[0088] As used herein, the term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Transfection protocols and techniques include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation.

[0089] As used herein, the term "SPR" or "surface plasmon resonance" refers to and includes an optical phenomenon that permits the analysis of real-time biospecific interactions by detecting alterations in protein concentration within a biosensor matrix, e.g., using a BIACORE system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

[0090] As used herein, the term "fluorescence-activated cell sorting" or "FACS" refers to a particular type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers (one cell at a time) based on the specific light scattering and fluorescence characteristics of each cell. Instruments used to perform FACS can include the FACS STAR PLUS, FACSCAN, and FACSORT instruments from Becton Dickinson (Foster City, Calif.), the EPICS C from Coulter Epics Division (Hialeah, Fla.), and the MOFLO from Cytomation (Colorado Springs, Colo.).

[0091] As used herein, the term "subject" or "individual" or "animal" or "patient" refers to a human or non-human animal in need of diagnosis, prognosis, amelioration, prevention, and / or treatment of a disease or condition, including mammals or primates. 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, and the like.

[0092] As used herein, the term "effector function" refers to the biological activities attributed to the binding of the Fc region of an antibody to its effectors such as the C1 complex and Fc receptors. Exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by the interaction of an antibody with C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the Fc region of an antibody to Fc receptors on effector cells; and phagocytosis.

[0093] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing the antigen and subsequently kill the target cells with cytotoxins. In some instances, antibody "arming" of the cytotoxic cells is required for this killing. The primary cell mediating ADCC, the NK cell, expresses only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay is performed. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. In some instances, the ADCC activity of a molecule of interest can be assessed in vivo.

[0094] 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 an antibody (appropriate subclass) bound to its cognate antigen. To assess complement activation, a CDC assay can be performed.

[0095] As used herein, the term "cancer" refers to any medical condition characterized by the growth of malignant cells or neoplasms, abnormal proliferation, invasion, or metastasis, and includes solid tumors and non-solid cancers (hematological malignancies) such as leukemia. As used herein, a "solid tumor" refers to a solid mass of neoplastic and / or malignant cells. Examples of cancers or tumors include hematological malignancies, oral cancers (e.g., lip, tongue, or pharyngeal cancers), digestive organs (e.g., esophagus, stomach, small intestine, colon, large intestine, or rectum), peritoneum, liver and biliary tract, pancreas, respiratory system such as larynx or lung (small cell and non-small cell), bone, connective tissue, skin (e.g., melanoma), breast, reproductive organs (fallopian tubes, uterus, cervix, testes, ovaries, or prostate), urinary tract (e.g., bladder or kidney), brain, and endocrine glands such as the thyroid. In some instances, the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, kidney cancer, bladder cancer, hepatocellular carcinoma, and colorectal cancer. In some instances, the cancer is selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and B-cell lymphoma.

[0096] As used herein, in the context of treating a condition, the terms "treatment", "treating", or "treated" generally refer to treatment and therapy, whether in humans or animals, in which some desired therapeutic effect is achieved, e.g., inhibition of the progression of the condition, and includes a decrease in the rate of progression, a halt in the rate of progression, regression of the condition, improvement of the condition, and cure of the condition. Also included is treatment as a preventive measure (i.e., prophylaxis, prevention). For cancer, "treatment" can refer to inhibition or slowing of tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. For a tumor, "treatment" includes removal of all or part of the tumor, inhibition or slowing of tumor growth and metastasis, prevention or delay of the development of the tumor, or some combination thereof.

[0097] As used herein, the term "effective amount" refers to the amount of an active compound or a material, composition, or dosage containing the active compound that, when administered according to a desired treatment regimen, is capable of effectively producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. For example, when used in connection with treating a CD3 / PSMA-related disease or condition, an "effective amount" is the amount or concentration of an antibody or its antigen-binding portion that effectively treats the disease or condition.

[0098] As used herein, with respect to a disease condition in a mammal, the terms "prevent", "prevention", or "preventing" refer to preventing or delaying the onset of the disease, or preventing the manifestation of its clinical or subclinical symptoms.

[0099] As used herein, the term "pharmaceutically acceptable" means that the carrier, diluent, excipient, and / or its salts are chemically and / or physically compatible with the other ingredients in the formulation and are physiologically compatible with the recipient.

[0100] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0101] As used herein, the term "adjuvant" refers to a non-specific immune enhancer that, when delivered to an organism together with an antigen or pre-delivered to an organism, can enhance the immune response against the antigen or alter the type of immune response in the organism. There are many types of adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, and the like.

[0102] Antibodies and antigen-binding portions thereof

[0103] The antibodies disclosed herein can bind to human PSMA and have one or more of the following characteristics:

[0104] (a) Bind to human PSMA with a K -8 of 1 x 10 D M or less;

[0105] (b) Induce the production of cytokines (e.g., IL-2 or IFN-γ) in CD4 + T cells;

[0106] (c) Enhance the proliferation of primary human CD4 + T cells;

[0107] (d) Enhance the proliferation of primary human CD4 + T effector cells in the presence of Treg cells;

[0108] (e) Bind to human or rhesus monkey PSMA, respectively; or

[0109] (f) does not cross-react with human CD40, CD137, and CD271.

[0110] Binding to PSMA can be evaluated using ELISA. Binding specificity can be determined by monitoring the binding of the antibody to cells expressing the PSMA protein, for example, by flow cytometry. For example, the antibody can be tested in a flow cytometry assay where the antibody reacts with a cell line expressing human PSMA, such as CHO cells that have been transfected to express PSMA on their cell surface. The binding of the antibody, including binding kinetics (e.g., K d value), can be tested in a BIACORE binding assay. Other suitable binding assays include ELISA assays, for example, using recombinant PSMA protein. By way of example, the antibody can bind human PSMA at 1x10 -8 M or less, 1x10 -9 M or less, 5x10 -10 M or less, 2x10 -10 M or less, 1x10 -10 M or less, 5x10 -11 M or less, 3x10 -11 M or less, or 2x10 -11 M or less K D binding.

[0111] The variable regions and CDRs in the antibody sequence can be identified according to general rules developed in the art (as described above, e.g., the Kabat numbering system) or by aligning the sequence with a database of known variable regions. Exemplary databases of antibody sequences are described on the "Abysis" website (maintained by the Department of Biochemistry and Molecular Biology, University College London, London, UK) and the VBASE2 website, and can be accessed through these websites. The Abysis database can be used to analyze sequences, which integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB) with structural data from the PDB. The Abysis database website further includes general rules developed for identifying CDRs that can be used according to the teachings herein. Unless otherwise indicated, the CDR boundaries of the antibody are defined or identified by Kabat and IMGT conventions.

[0112] The percent identity between two amino acid sequences can be determined using an algorithm that has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined by an algorithm that has been incorporated into the GAP program in the GCG software package, using either the BLOSSUM 62 matrix or the 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.

[0113] The protein sequences of the present disclosure can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such a search can be performed using the XBLAST program (version 2.0). A BLAST protein search can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the antibody molecules of the present disclosure. To obtain a gapped alignment for comparison purposes, Gapped BLAST can be utilized. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0114] In some instances, the amino acid sequences of the CDRs can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding sequences described above. In some instances, the amino acid sequences of the variable regions can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding sequences described above.

[0115] In some instances, the CDRs of the isolated antibody or its antigen-binding portion contain no more than 2 amino acid or no more than 1 amino acid conservative substitutions. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the basic properties of the protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can 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, e.g., a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having a similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid and glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (such as threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue can be replaced with another amino acid residue from the same side chain family.

[0116] In some instances, the first antigen-binding portion and the second antigen-binding portion of the bispecific antibody can bind to each other via a stud-and-hole interaction.

[0117] In some instances, the first and / or second antigen-binding portion is bivalent. The term "bivalent" indicates the presence of two binding sites, respectively, in the antigen-binding molecule. This can provide stronger binding to an antigen or epitope than the monovalent counterpart. In some instances, in the bivalent antigen-binding portion, the first valence of the binding site and the second valence of the binding site are structurally identical (i.e., having the same sequence).

[0118] In some instances, the antibodies and their antigen-binding fragments provided herein are bispecific. In some instances, the bispecific antibodies and their antigen-binding portions provided herein have a first specificity for PSMA and a second specificity for a second antigen different from PSMA, and their blockade can produce a synergistic (e.g., synergistic effect) compared to blocking only one antigen alone.

[0119] In some instances, the second specificity is directed against a tumor-associated antigen or an epitope thereof. The term "tumor-associated antigen" refers to a target antigen expressed by tumor cells, however, which may be expressed by homologous cells (or healthy cells) prior to transformation into a tumor. In some instances, the tumor-associated antigen may be presented only by tumor cells and not by normal cells (i.e., non-tumor cells). In some instances, the tumor-associated antigen may be expressed only on tumor cells or may exhibit tumor-specific mutations as compared to non-tumor cells. In some instances, the tumor-associated antigen may be found in both tumor cells and non-tumor cells, but is overexpressed on tumor cells as compared to non-tumor cells or is available for antibody binding in tumor cells due to the less compact structure of tumor tissue as compared to non-tumor tissue. In some instances, the tumor-associated antigen is located on the vasculature of the tumor.

[0120] Illustrative examples of tumor-associated antigens are 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, Derlin1, tenascin, frizzled protein 1-10, vascular antigen 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.

[0121] In some instances, the present disclosure includes a bispecific antibody or an antigen-binding portion thereof that includes 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.

[0122] The bispecific antibodies of the present disclosure bind human CD3 and human PSMA with high affinity. The binding of the antibodies of the present disclosure to CD3 or PSMA can be evaluated using one or more well-established techniques in the art (e.g., ELISA). The binding specificity of the antibodies of the present disclosure can also be determined by monitoring the binding of the antibody to cells expressing the CD3 protein or the PSMA protein, e.g., flow cytometry. For example, the antibody can be tested by a flow cytometry assay in which the antibody reacts with a cell line expressing human CD3, such as CHO cells that have been transfected to express CD3 on their cell surface. In some instances, the binding of the antibody, including binding kinetics (e.g., K d values) can be tested in a BIACORE binding assay. Other suitable binding assays include ELISA or FACS assays, e.g., using recombinant CD3 protein.

[0123] In some instances, the bispecific antibody or an antigen-binding portion thereof of the present disclosure includes a CD3-binding portion and a PSMA-binding portion, wherein the CD3-binding portion includes a chimeric Fab that includes 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 an anti-CD3 antibody operably linked to a second TCR constant region (C2), and wherein C1 and C2 are capable of forming a dimer via a non-native inter-chain disulfide bond that can stabilize the dimer, and the PSMA-binding portion includes a Fab that includes a second heavy-chain variable region of an anti-PSMA antibody operably linked to a heavy-chain CH1 constant region domain, and a second light-chain variable region of an anti-PSMA antibody operably linked to a light-chain constant region, and wherein:

[0124] (A) The PSMA-binding portion includes:

[0125] a heavy-chain CDR1 that includes SEQ ID NO: 1 or consists of the same,

[0126] a heavy-chain CDR2 that includes SEQ ID NO: 2 or consists of the same,

[0127] a heavy-chain CDR3 that includes SEQ ID NO: 3 or consists of the same,

[0128] a light chain CDR1 comprising or consisting of SEQ ID NO: 4,

[0129] a light chain CDR2 comprising or consisting of SEQ ID NO: 5, and

[0130] a light chain CDR3 comprising or consisting of SEQ ID NO: 6,

[0131] and

[0132] (B) the CD3 binding portion comprises:

[0133] a heavy chain CDR1 comprising or consisting of SEQ ID NO: 7,

[0134] a heavy chain CDR2 comprising or consisting of SEQ ID NO: 8,

[0135] a heavy chain CDR3 comprising or consisting of SEQ ID NO: 9,

[0136] a light chain CDR1 comprising or consisting of SEQ ID NO: 10,

[0137] a light chain CDR2 comprising or consisting of SEQ ID NO: 11, and

[0138] a light chain CDR3 comprising or consisting of SEQ ID NO: 12;

[0139] and / or

[0140] (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, and

[0141] (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.

[0142] In some instances, the bispecific antibody or its antigen-binding portion has one or more of the following properties:

[0143] (a) specifically binds to human CD3 and PSMA proteins simultaneously with high affinity;

[0144] (b) specifically binds to human CD3 and / or cyno CD3 proteins;

[0145] (c) specifically binds to human, mouse and / or cyno PSMA proteins;

[0146] (d) Compared with anti-CD3 antibodies, anti-PSMA antibodies, combinations thereof, and other bispecific antibodies targeting CD3 and PSMA, it can induce effective T cell activation in the presence of PSMA-expressing tumor cells;

[0147] (e) Provide good thermal stability and are stable in human serum; and

[0148] (f) Compared with anti-CD3 antibodies, anti-PSMA antibodies, combinations thereof, and other bispecific antibodies targeting CD3 and PSMA, provide superior anti-tumor effects.

[0149] For example, as studied in a tumor-bearing mouse model, compared with known anti-CD3 and anti-PSMA antibodies, the bispecific antibodies of the present disclosure achieved desired tumor growth inhibition (TGI).

[0150] Antigen-binding portion that specifically binds to PSMA

[0151] The antigen-binding portion provided herein specifically binds to PSMA and is also referred to as a PSMA-binding portion in the present disclosure.

[0152] The antigen-binding portion comprises a Fab, which comprises 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.

[0153] In some instances, the antigen-binding portion comprises:

[0154] Heavy chain CDR1, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 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 2 amino acids,

[0155] Heavy chain CDR2, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 100% sequence identity with SEQ ID NO: 2, or an amino acid sequence that differs from SEQ ID NO: 2 by an amino acid addition, deletion or substitution of no more than 2 amino acids,

[0156] Heavy chain CDR3, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 100% sequence identity with 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 no more than 1 amino acid,

[0157] A light chain CDR1, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 100% sequence identity with SEQ ID NO: 4, or an amino acid sequence that is different from SEQ ID NO: 4 by amino acid addition, deletion or substitution of no more than 2 amino acids,

[0158] A light chain CDR2, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 100% sequence identity with SEQ ID NO: 5, or an amino acid sequence that is different from SEQ ID NO: 5 by amino acid addition, deletion or substitution of no more than 1 amino acid, and

[0159] A light chain CDR3, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 100% sequence identity with SEQ ID NO: 6, or an amino acid sequence that is different from SEQ ID NO: 6 by amino acid addition, deletion or substitution of no more than 1 amino acid.

[0160] In some instances, the antigen-binding portion comprises:

[0161] a) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1,

[0162] b) A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2,

[0163] c) A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3,

[0164] d) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4,

[0165] e) A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and

[0166] f) A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6.

[0167] In some instances, the antigen-binding portion comprises:

[0168] a) A heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1,

[0169] b) A heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2,

[0170] c) a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3,

[0171] d) a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4,

[0172] e) a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and

[0173] f) consisting of the amino acid sequence represented by SEQ ID NO: 6.

[0174] In some instances, the heavy chain variable region of the antigen-binding portion comprises:

[0175] (i) the amino acid sequence of SEQ ID NO: 15, and

[0176] (ii) an amino acid sequence that is at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15 and that simultaneously retains the binding specificity for PSMA (e.g., contains the CDRs disclosed above); or

[0177] (iii) an amino acid sequence having one or more amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 15 (e.g., 1 to 18, 1 to 15, 1 to 10 or 1 to 5 amino acids) and that simultaneously retains the binding specificity for PSMA (e.g., contains the CDRs disclosed above).

[0178] In some instances, the light chain variable region of the antigen-binding portion comprises:

[0179] (i) the amino acid sequence of SEQ ID NO: 16, and

[0180] (ii) an amino acid sequence that is at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16 and that simultaneously retains the binding specificity for PSMA (e.g., contains the CDRs disclosed above); or

[0181] (iii) an amino acid sequence having one or more amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 16 (e.g., 1 to 16, 1 to 15, 1 to 10 or 1 to 5 amino acids) and that simultaneously retains the binding specificity for PSMA (e.g., contains the CDRs disclosed above).

[0182] In some instances, 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.

[0183] In some instances, the heavy chain variable region of the PSMA-binding portion is operably linked to a hinge-Fc region, such as a human IgG Fc region, particularly a human IgG4 or IgG1 Fc region, e.g., a human IgG4 Fc region containing the S228P mutation, the Fc null mutation (F234A L235A), and the psi mutation (Y349C-T366S-L368A-Y407V). In some instances, to construct a CD3xPSMA bispecific antibody, the DNA sequence encoding the VH region of an anti-CD3 antibody is fused to a modified TCRβ constant domain and hinge-Fc region of human IgG4 having the S228P mutation, the Fc null mutation (F234A L235A), and the psi mutation ((S354C-T366W)); the DNA sequence encoding the VL region of an anti-CD3 antibody is fused to a modified TCRα constant domain; the DNA sequence encoding the VH region of an anti-PSMA antibody is fused to the hinge-Fc region of human IgG4 having the S228P mutation, the Fc null mutation (F234A L235A), and the psi mutation (Y349C-T366S-L368A-Y407V); and the DNA sequence encoding the VL region of an anti-PSMA antibody is fused to a CL domain.

[0184] In some instances, the antigen-binding portion comprises two polypeptide chains:

[0185] I) A second heavy chain that comprises 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 even 100% sequence identity to SEQ ID NO: 19 and that simultaneously retains the binding specificity to PSMA (e.g., contains the CDRs and / or variable regions disclosed above); and

[0186] ii) A second light chain that comprises 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 even 100% sequence identity to SEQ ID NO: 20 and that simultaneously retains the binding specificity to PSMA (e.g., contains the CDRs and / or variable regions disclosed above).

[0187] In some instances, the antigen-binding portion comprises two polypeptide chains:

[0188] i) A second heavy chain represented by SEQ ID NO: 19; and

[0189] ii) A second light chain represented by SEQ ID NO: 20.

[0190] In some instances, the antigen-binding portion consists of two polypeptide chains:

[0191] i) A second heavy chain represented by SEQ ID NO: 19; and

[0192] ii) A second light chain represented by SEQ ID NO: 20.

[0193] In some instances, the antigen-binding portion comprises two polypeptide chains:

[0194] I) A second heavy chain comprising an amino acid sequence having at least 85%, such as, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to SEQ ID NO: 31, and simultaneously maintaining the binding specificity to PSMA (e.g., containing the CDRs and / or variable regions disclosed above); and

[0195] ii) A second light chain comprising an amino acid sequence having at least 85%, such as, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to SEQ ID NO: 32, and simultaneously maintaining the binding specificity to PSMA (e.g., containing the CDRs and / or variable regions disclosed above).

[0196] In some instances, the antigen-binding portion comprises two polypeptide chains:

[0197] i) A second heavy chain represented by SEQ ID NO: 31; and

[0198] ii) A second light chain represented by SEQ ID NO: 32.

[0199] In some instances, the antigen-binding portion consists of two polypeptide chains:

[0200] i) A second heavy chain represented by SEQ ID NO: 31; and

[0201] ii) A second light chain represented by SEQ ID NO: 32.

[0202] An antigen-binding portion that specifically binds to CD3

[0203] The antigen-binding portion specifically binds to CD3 and is thus also referred to as a CD3-binding portion in the present disclosure. These two terms can be used interchangeably.

[0204] The antigen-binding portion comprises a chimeric Fab that comprises 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), and wherein C1 and C2 are capable of forming a dimer via a non-native interchain disulfide bond capable of stabilizing the dimer.

[0205] In some instances, the antigen-binding portion comprises:

[0206] a) a heavy chain CDR1 that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 100% sequence identity with SEQ ID NO: 7, or an amino acid sequence that differs from SEQ ID NO: 7 by an amino acid addition, deletion or substitution of no more than 2 amino acids,

[0207] b) a heavy chain CDR2 that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 100% sequence identity with SEQ ID NO: 8, or an amino acid sequence that differs from SEQ ID NO: 8 by an amino acid addition, deletion or substitution of no more than 2 amino acids,

[0208] c) a heavy chain CDR3 that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 100% sequence identity with SEQ ID NO: 9, or an amino acid sequence that differs from SEQ ID NO: 9 by an amino acid addition, deletion or substitution of no more than 2 amino acids,

[0209] d) a light chain CDR1 that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 100% sequence identity with SEQ ID NO: 10, or an amino acid sequence that differs from SEQ ID NO: 10 by an amino acid addition, deletion or substitution of no more than 2 amino acids,

[0210] e) a light chain CDR2 that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 100% sequence identity with 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 no more than 1 amino acid, and

[0211] 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 even 100% sequence identity with 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 no more than 1 amino acid.

[0212] In some instances, the antigen-binding portion comprises:

[0213] a) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7,

[0214] b) A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8,

[0215] c) A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9,

[0216] d) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10,

[0217] e) A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and

[0218] f) A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12.

[0219] In some instances, the antigen-binding portion comprises:

[0220] a) A heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 7,

[0221] b) A heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 8,

[0222] c) A heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 9,

[0223] d) A light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10,

[0224] e) A light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and

[0225] f) A light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 12.

[0226] In some instances, the heavy chain variable region of the antigen-binding portion comprises:

[0227] (i) The amino acid sequence of SEQ ID NO: 13, and

[0228] (ii) An amino acid sequence that is at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13 and that simultaneously retains the binding specificity for CD3 (e.g., contains the CDRs disclosed above); or

[0229] (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 amino acids) compared to SEQ ID NO: 13 and that simultaneously retains the binding specificity for CD3 (e.g., contains the CDRs disclosed above).

[0230] In some instances, the light chain variable region of the antigen-binding portion comprises:

[0231] (i) The amino acid sequence of SEQ ID NO: 14, and

[0232] (ii) An amino acid sequence that is at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14 and that simultaneously retains the binding specificity for CD3 (e.g., contains the CDRs disclosed above); or

[0233] (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 amino acids) compared to SEQ ID NO: 14 and that simultaneously retains the binding specificity for CD3 (e.g., contains the CDRs disclosed above).

[0234] In some instances, 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 comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0235] In some instances, the antigen-binding portion comprises two polypeptide chains:

[0236] i) A first heavy chain comprising an amino acid sequence having at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to SEQ ID NO: 17, and simultaneously maintaining the binding specificity to CD3 (e.g., containing the CDRs and / or variable regions disclosed above); and

[0237] ii) A first light chain comprising an amino acid sequence having at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity to SEQ ID NO: 18, and simultaneously maintaining the binding specificity to CD3 (e.g., containing the CDRs and / or variable regions disclosed above).

[0238] In some instances, the antigen-binding portion comprises two polypeptide chains:

[0239] i) A first heavy chain represented by SEQ ID NO: 17; and

[0240] ii) A first light chain represented by SEQ ID NO: 18.

[0241] In some instances, the antigen-binding portion consists of two polypeptide chains:

[0242] i) A first heavy chain represented by SEQ ID NO: 17; and

[0243] ii) A first light chain represented by SEQ ID NO: 18.

[0244] In some aspects, the heavy chain variable region of the antigen-binding portion is operably linked to a C1 and hinge-Fc region, such as a human IgG Fc region, particularly a human IgG4 or IgG1 Fc region, e.g., a human IgG4 Fc region containing an S228P mutation, an Fc null mutation (F234A L235A), a pestle mutation (S354C-T366W), and / or a mortar mutation (Y349C-T366S-L368A-Y407V). In some instances, the VH region is fused to a modified TCR β constant domain and hinge-Fc region of human IgG4 having an S228P mutation, an Fc null mutation (F234A L235A), and a pestle mutation (S354C-T366W). In some instances, the VH region is fused to a hinge-Fc region of human IgG4 having an S228P mutation, an Fc null mutation (F234A L235A), and a mortar mutation (Y349C-T366S-L368A-Y407V).

[0245] TCR constant region

[0246] The human TCR β-chain constant region has two different variants, called TRBC1 and TRBC2 (IMGT nomenclature). In the present disclosure, the NCBI accession number for the sequence of the wild-type TCR β domain is A0A5B9. The modified TCR β constant domain in the present disclosure is:

[0247] DLKNVFPPEVAVFEPSECEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR (SEQ ID NO: 29).

[0248] In some instances, 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 instances, C1 comprises or consists of the modified TCR β constant region represented by SEQ ID NO: 29.

[0249] The human TCR α-chain constant region is called TRAC, and the NCBI accession number is P01848. The modified TCR α constant domain in the present disclosure is:

[0250] PDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFCS (SEQ ID NO: 30).

[0251] In some instances, the second T cell receptor (TCR) constant region (C2) comprises a modified TCRα constant region comprising the amino acid sequence of SEQ ID NO: 30, and in some instances, C2 comprises or consists of the modified TCR α constant region represented by SEQ ID NO: 30.

[0252] In the present disclosure, the first and second TCR constant regions of the polypeptide complex provided herein are capable of forming a dimer that comprises at least one non-native interchain bond between the TCR constant regions that stabilizes the dimer.

[0253] As used herein, the term "dimer" refers to an associated structure formed by two molecules (e.g., polypeptides or proteins) via covalent or non-covalent interactions. A homodimer or homodimerization is formed by two identical molecules, and a heterodimer or heterodimerization is formed by two different molecules. The dimer formed by the first and second TCR constant regions is a heterodimer.

[0254] An interchain bond is formed between an amino acid residue on one TCR constant region and another amino acid residue on another TCR constant region. In some instances, a non-natural interchain bond can be any bond or interaction capable of associating two TCR constant regions into a dimer. Examples of suitable non-natural interchain bonds include disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridges, or hydrophobic-hydrophilic interactions, a knob-into-hole, or combinations thereof.

[0255] A "disulfide bond" refers to a covalent bond having the structure R-S-S-R'. The amino acid cysteine contains a thiol group that can form a disulfide bond with a second thiol group (e.g., from another cysteine residue). A disulfide bond can be formed between the thiol groups of two cysteine residues located on two separate polypeptide chains, thereby forming an interchain bridge or interchain bond.

[0256] As used herein, a "non-natural" interchain bond refers to an interchain bond not found in the natural association of the native counterpart TCR constant regions. For example, a non-natural interchain bond can be formed between a mutant amino acid residue and a native amino acid residue, each located on a corresponding TCR constant region; or alternatively, between two mutant amino acid residues located on TCR constant regions, respectively. In some instances, at least one non-natural interchain bond is formed between a first mutant residue contained in a first TCR constant region of a polypeptide complex and a second mutant residue contained in a second TCR constant region.

[0257] As used herein, the term "contact interface" refers to a specific region on a polypeptide where polypeptides interact / associate with each other. The contact interface contains one or more amino acid residues capable of interacting with the corresponding amino acid residues that make contact or bind when the interaction occurs. The amino acid residues in the contact interface may or may not be in a contiguous sequence. For example, when the interface is three-dimensional, the amino acid residues within the interface can be separated at different positions in the linear sequence.

[0258] Generation of hybridomas producing antibodies

[0259] To generate hybridomas producing the antibodies disclosed herein (e.g., human monoclonal antibodies), splenocytes and / or lymph node cells from immunized mice can be isolated and fused with a suitable immortalized cell line (e.g., a murine myeloma cell line). The resulting hybridomas can be screened for the production of antigen-specific antibodies.

[0260] Generation of antibody-producing transfectomas

[0261] The antibodies of the present disclosure can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods. In some instances, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. As used herein, the term "operably linked" is intended to mean that the antibody gene is linked into the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene.

[0262] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. In some instances, the variable regions are used to generate full-length antibody genes of any antibody isotype by inserting the variable regions into expression vectors that already encode the heavy chain constant region and the light chain constant region of the desired isotype such that the heavy chain variable domain is operably linked to the CH segment within the vector and the light chain variable domain is operably linked to the CL segment within 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 in-frame with 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).

[0263] To express the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass the various techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. The antibodies of the present invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells, which can assemble and secrete properly folded and immunologically active antibodies.

[0264] Mammalian host cells for expressing the recombinant antibodies of the present invention include Chinese hamster ovary (CHO cells), NSO myeloma cells, COS cells, and SP2 cells. When the recombinant expression vector encoding the antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a time sufficient to allow expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are growing. The antibody can be recovered from the culture medium using standard protein purification methods.

[0265] Generation of bispecific antibodies

[0266] The bispecific antibodies and antigen-binding fragments provided herein can be prepared by any suitable method. For example, two immunoglobulin heavy chain-light chain pairs can be co-expressed in a host cell to recombinantly produce a bispecific antibody, which is then purified by affinity chromatography.

[0267] Recombinant methods can also be used, in which sequences encoding the heavy chain variable domains of two specificities are separately fused to immunoglobulin constant domain sequences and then inserted into an expression vector, which is co-transfected with an expression vector of 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.

[0268] In another method, leucine zipper peptides from Fos and Jun proteins can be genetically fused to the Fab’ portions of two different antibodies. The linked antibodies are reduced to four half-antibodies (i.e., monomers) in the hinge region and then re-oxidized to form heterodimers.

[0269] Two antigen-binding sites can also be conjugated or cross-linked to form a bispecific antibody or antigen-binding fragment. For example, one antibody can be conjugated with biotin, while another antibody is conjugated with avidin, and the strong association between biotin and avidin will complex the two antibodies together to form a bispecific antibody.

[0270] Bispecific antigen-binding fragments can be generated from bispecific antibodies, for example, by proteolytic cleavage or by chemical ligation. For example, antigen-binding fragments of an antibody (e.g., Fab 5 ) can be prepared and converted into Fab'-thiol derivatives, which are then mixed and reacted with another converted Fab 5 derivative with different antigen specificity to form a bispecific antigen-binding fragment.

[0271] Nucleic acid molecules encoding the antibodies of the present disclosure

[0272] In some aspects, the present disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding bispecific antibodies or antigen-binding portions as disclosed herein. For example, the nucleic acid sequences comprise any combination of heavy chain or light chain sequences of SEQ ID NOs: 35 to 38 (such as SEQ ID NO:35 and 36 or SEQ ID NO: 37 and 38). For example, the nucleic acid sequence can encode the heavy chain and / or light chain of a bispecific antibody. For example, the nucleic acid sequence comprises all the sequences of SEQ ID NOs: 35 to 38.

[0273] An isolated nucleic acid molecule encoding the heavy chain variable region of a CD3-binding portion can comprise a nucleic acid sequence selected from:

[0274] (A) A nucleic acid sequence encoding a heavy chain variable region as shown in SEQ ID NO: 13;

[0275] (B) A nucleic acid sequence as shown in SEQ ID NO: 21; or

[0276] (C) A nucleic acid sequence that hybridizes under highly stringent conditions to the complementary strand of the nucleic acid sequence of (A) or (B).

[0277] The isolated nucleic acid molecule encoding the light chain variable region of the CD3-binding portion may comprise a nucleic acid sequence selected from the following:

[0278] (A) A nucleic acid sequence encoding a light chain variable region as shown in SEQ ID NO: 14;

[0279] (B) A nucleic acid sequence as shown in SEQ ID NO: 22; or

[0280] (C) A nucleic acid sequence that hybridizes under highly stringent conditions to the complementary strand of the nucleic acid sequence of (A) or (B).

[0281] The isolated nucleic acid molecule encoding the heavy chain variable region of the PSMA-binding portion may comprise a nucleic acid sequence selected from the following:

[0282] (A) A nucleic acid sequence encoding a heavy chain variable region as shown in SEQ ID NO: 15;

[0283] (B) A nucleic acid sequence as shown in SEQ ID NO: 23; or

[0284] (C) A nucleic acid sequence that hybridizes under highly stringent conditions to the complementary strand of the nucleic acid sequence of (A) or (B).

[0285] The isolated nucleic acid molecule encoding the light chain variable region of the PSMA-binding portion may comprise a nucleic acid sequence selected from the following:

[0286] (A) A nucleic acid sequence encoding a light chain variable region as shown in SEQ ID NO: 16;

[0287] (B) A nucleic acid sequence as shown in SEQ ID NO: 24; or

[0288] (C) A nucleic acid sequence that hybridizes under highly stringent conditions to the complementary strand of the nucleic acid sequence of (A) or (B).

[0289] In some instances, the present disclosure provides an isolated nucleotide sequence encoding the heavy chain of the CD3-binding portion, wherein the isolated nucleotide sequence encoding the heavy chain of the CD3-binding portion comprises the following or consists of the following:

[0290] A nucleic acid sequence encoding a heavy chain as set forth in SEQ ID NO: 17;

[0291] (B) A nucleic acid sequence as set forth in SEQ ID NO: 25 or 35; or

[0292] (C) A nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of the nucleic acid sequence of (A) or (B).

[0293] In some instances, the present disclosure provides an isolated nucleotide sequence encoding a light chain of a CD3 binding portion, wherein the isolated nucleotide sequence encoding the light chain of the CD3 binding portion comprises or consists of:

[0294] (A) A nucleic acid sequence encoding a light chain as set forth in SEQ ID NO: 18;

[0295] (B) A nucleic acid sequence as set forth in SEQ ID NO: 26 or 36; or

[0296] (C) A nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of the nucleic acid sequence of (A) or (B).

[0297] In some instances, the present disclosure provides an isolated nucleotide sequence encoding a heavy chain of a PSMA binding portion, wherein the isolated nucleotide sequence encoding the heavy chain of the PSMA binding portion comprises or consists of:

[0298] (A) A nucleic acid sequence encoding a heavy chain as set forth in SEQ ID NO: 19;

[0299] (B) A nucleic acid sequence as set forth in SEQ ID NO: 27 or 37; or

[0300] (C) A nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of the nucleic acid sequence of (A) or (B).

[0301] In some instances, the present disclosure provides an isolated nucleotide sequence encoding a light chain of a PSMA binding portion, wherein the isolated nucleotide sequence encoding the light chain of the PSMA binding portion comprises or consists of:

[0302] (A) A nucleic acid sequence encoding a light chain as set forth in SEQ ID NO: 20;

[0303] (B) A nucleic acid sequence as set forth in SEQ ID NO: 28 or 38; or

[0304] (C) A nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of the nucleic acid sequence of (A) or (B).

[0305] In some instances, the present disclosure provides an isolated nucleotide sequence encoding the heavy chain of a PSMA-binding portion, wherein the isolated nucleotide sequence encoding the heavy chain of the PSMA-binding portion comprises or consists of the following:

[0306] (A) The nucleic acid sequence of SEQ ID NO: 33;

[0307] (B) A nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 31; or

[0308] (C) A nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of the nucleic acid sequence of (A) or (B).

[0309] In some instances, the present disclosure provides an isolated nucleotide sequence encoding the light chain of a PSMA-binding portion, wherein the isolated nucleotide sequence encoding the light chain of the PSMA-binding portion comprises or consists of the following:

[0310] (A) The nucleic acid sequence of SEQ ID NO: 34;

[0311] (B) A nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 32; or

[0312] (C) A nucleic acid sequence that hybridizes under high stringency conditions to the complementary strand of the nucleic acid sequence of (A) or (B).

[0313] In some aspects, the present disclosure relates to a vector comprising a nucleic acid sequence as disclosed herein. In some instances, the expression vector further comprises a nucleotide sequence encoding a constant region of a bispecific antibody.

[0314] In the context of the present disclosure, a vector can be any suitable vector, including chromosomal, episomal, and synthetic nucleic acid vectors (nucleic acid sequences containing a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, bacteriophage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and bacteriophage DNA, and viral nucleic acid (RNA or DNA) vectors. In some instances, the nucleic acid encoding the CD3 or PSMA antibody is contained in a naked DNA or RNA vector, including, for example, linear expression elements, compacted nucleic acid vectors, plasmid vectors such as pBR322, pUC 19 / 18, or pUC 118 / 119, "midge" minimal size nucleic acid vectors, or nucleic acid vector constructs precipitated as, for example, CaP04 precipitates.

[0315] In some instances, the vector is suitable for expressing an anti-CD3 antibody and / or an anti-PSMA antibody in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors, pET vectors (Novagen, Madison WI), etc.). The vector can also alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.

[0316] The vector can also be or alternatively be a vector suitable for expression in mammalian cells, for example, a vector containing glutamine synthetase as a selection marker.

[0317] The nucleic acid and / or vector can also contain a nucleic acid sequence encoding a secretion / localization sequence that can target a polypeptide (such as a nascent polypeptide chain) to the periplasmic space or the cell culture medium. Such sequences can include a secretion leader peptide or a signal peptide.

[0318] The vector can contain any suitable promoter, enhancer, and other elements that promote expression or be associated therewith. Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter / enhancer and the RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, origins of replication of plasmid products in Escherichia coli, antibiotic resistance genes as selection markers, and / or convenient cloning sites (e.g., multiple cloning sites). The nucleic acid can also contain an inducible promoter as opposed to a constitutive promoter such as CMV IE.

[0319] In a further aspect, the present disclosure relates to a host cell comprising the vector specified above.

[0320] Accordingly, the present disclosure also relates to a recombinant eukaryotic or prokaryotic host cell that produces the bispecific antibody of the present disclosure, such as a transfectoma.

[0321] The CD3-specific antibody can be expressed in a recombinant eukaryotic or prokaryotic host cell (such as a transfectoma) that produces the antibody of the present disclosure as defined herein or the bispecific antibody of the present disclosure as defined herein. The PSMA-specific antibody can likewise be expressed in a recombinant eukaryotic or prokaryotic host cell (such as a transfectoma) that produces the antibody of the present disclosure as defined herein or the bispecific antibody of the present disclosure as defined herein.

[0322] Examples of host cells include yeast, bacteria, plant, and mammalian cells such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NSO cells or lymphocytes. For example, in some instances, the host cell can comprise a first and a second nucleic acid construct stably integrated into the cell genome. In some instances, the present disclosure provides cells comprising non-integrated nucleic acids such as plasmids, cosmids, phagemids, or linear expression elements, which comprise the first and second nucleic acid constructs as specified above.

[0323] In a 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, wherein the animal or plant produces the bispecific antibodies of the present disclosure.

[0324] In a further aspect, the present disclosure relates to hybridomas producing antibodies for the bispecific antibodies of the present disclosure as defined herein. In a 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, wherein the animal or plant produces an antibody or a bispecific antibody for the bispecific antibodies of the present disclosure.

[0325] In one aspect, the present disclosure relates to an expression vector comprising:

[0326] (i) a nucleic acid sequence encoding a heavy chain variable region of a first antigen-binding portion and / or a heavy chain variable region of a second antigen-binding portion according to any one of the examples or embodiments disclosed herein, optionally further encoding a CH1 domain or a CL domain;

[0327] (ii) a nucleic acid sequence encoding a light chain variable region of a first antigen-binding portion and / or a light chain variable region of a second antigen-binding portion according to any one of the examples or embodiments disclosed herein;

[0328] (iii) a nucleic acid sequence encoding a modified TCR β constant domain or a modified TCR α constant domain;

[0329] (iv) a nucleic acid sequence encoding an Fc region;

[0330] (v) a nucleic acid sequence encoding a linker; or

[0331] (vi) a combination of at least two of the above.

[0332] In one aspect, the present disclosure relates to a nucleic acid construct of one or more amino acid sequences as shown in the coding sequence listing.

[0333] In one aspect, the present disclosure relates to methods for generating bispecific antibodies according to any of the examples or embodiments disclosed herein, which comprise the steps of: culturing a host cell as disclosed herein, the host cell comprising one expression vector or more than one expression vector as disclosed herein that expresses a bispecific antibody as disclosed herein, and purifying the antibody from the culture medium. In one aspect, the present disclosure relates to a host cell comprising an expression vector as defined above. In some examples, the host cell is a recombinant eukaryotic, recombinant prokaryotic or recombinant microbial host cell.

[0334] Pharmaceutical composition

[0335] In some aspects, the present disclosure relates to a pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof as disclosed herein and a pharmaceutically acceptable carrier.

[0336] Components of the composition

[0337] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the present disclosure may also be administered in combination therapy with, for example, another immunostimulant, anticancer agent, antiviral agent or vaccine such that the anti-CD3 / anti-PSMA bispecific antibody enhances the immune response against the vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, multiple other combinations of these components or more.

[0338] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, emulsifying agents or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoethanol, mercaptoacetic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene and / or propyl galactoside. As disclosed in the present disclosure, the compositions disclosed in a solvent containing an antibody or antigen-binding fragment of the present disclosure include one or more antioxidants such as methionine, and the reducing antibody or its antigen-binding portion can be oxidized. Oxidation reduction can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending the shelf life. In some examples, the present disclosure provides a composition comprising 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 an antibody or its antigen-binding portion is mixed with one or more antioxidants such as methionine such that the antibody or its antigen-binding portion can prevent oxidation to extend its shelf life and / or increase its activity.

[0339] For further illustration, pharmaceutically acceptable carriers can include, for example, aqueous media such as sodium chloride injection, Ringer's injection, isosmotic dextrose injection, sterile water injection or dextrose and lactated Ringer's injection, non-aqueous media such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, osmotic agents such as sodium chloride or dextrose, buffering agents such as phosphoric or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. The antimicrobial agents used as carriers can be added to the pharmaceutical composition in a multi-dose container, which includes phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin.

[0340] Administration, Formulation and Dosage

[0341] The pharmaceutical compositions of the present disclosure can be administered to a subject in need thereof by a variety of routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or otherwise by implantation or inhalation. The subject compositions can be formulated into preparations in solid, semi-solid, liquid or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. The appropriate formulation and route of administration can be selected according to the intended application and treatment regimen.

[0342] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups or inhalants and their controlled release forms.

[0343] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), wherein the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant body fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of suitable isotonic carriers for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, the specific dosing regimen, including dose, timing, and repetition, will depend on the particular individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0344] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferating or tumorigenic cells, maintaining such a reduction in tumorous cells, reducing the proliferation of tumorous cells, or delaying the development of metastasis. In some instances, the dose of the administration can be adjusted or attenuated to manage potential side effects and / or toxicities. Alternatively, a sustained continuous release formulation of the subject therapeutic composition may be appropriate.

[0345] In some instances, the antibodies or antigen-binding portions thereof of the present disclosure can be administered in a suitable range, which can include from about 5 μg / kg body weight to about 100 mg / kg body weight per dose; from about 50 μg / kg body weight to about 5 mg / kg body weight per dose; from about 100 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges can include from about 100 μg / kg body weight to about 20 mg / kg body weight per dose and from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some instances, the dose is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, at least about 10 mg / kg body weight per dose.

[0346] In some instances, a treatment course involving the antibodies or antigen-binding portions thereof of the present disclosure will include multiple doses of the selected pharmaceutical product over a period of weeks or months. More specifically, the antibodies or antigen-binding portions thereof of the present disclosure can be administered once daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it should be understood that the dose or the interval can be changed based on patient response and clinical practice.

[0347] For the therapeutic compositions disclosed in an individual who has received one or more administrations, the dosage and regimen can also be determined empirically. For example, an individual can be administered increasing doses of the therapeutic composition produced as described herein. In some instances, the dose can be gradually increased or decreased or attenuated based on empirically determined or observed side effects or toxicities. To evaluate the efficacy of the selected composition, markers of a particular disease, disorder, or condition can be tracked as described above. For cancer, these include directly measuring tumor size via palpation or visual observation, indirectly measuring tumor size via x-ray or other imaging techniques; improvement evaluated by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor markers (e.g., PSA for prostate cancer) or tumorigenic antigens identified according to the methods described herein, alleviation of pain or paralysis; improvement of speech, vision, breathing, or other disabilities associated with the tumor; increased appetite; or improvement of quality of life measured by accepted tests or extended survival. It will be apparent to those skilled in the art that the dosage will vary depending on the individual, the type of tumor condition, the stage of the tumor condition, whether the tumor condition has begun to metastasize to other locations in the individual, and past and concurrent treatments.

[0348] Compatible formulations for parenteral administration (e.g., intravenous injection) will contain the antibody or antigen-binding portion thereof as disclosed herein at a concentration of from about 10 μg / ml to about 100 mg / ml. In some instances, the concentration of the antibody or antigen-binding portion thereof will include 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 instances, the ADC concentration includes 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.

[0349] Medical Use / Method

[0350] In some aspects, the present disclosure provides methods of treating a disorder in a subject, which include administering to a subject in need thereof (e.g., a mammal, such as a human) a therapeutically effective amount of an antibody or an antigen-binding portion thereof as disclosed herein. For example, the disorder is cancer.

[0351] A variety of cancers involving PSMA, whether malignant or benign, and whether primary or secondary, can be treated or prevented by the methods provided by the present disclosure. The cancer can be a solid cancer. Examples of these cancers include lung cancer, such as bronchogenic carcinoma (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (non-cancerous), and sarcoma (cancerous); kidney cancer; breast cancer; gastric cancer; colorectal cancer; glioblastoma; prostate cancer; pancreatic cancer; or ovarian cancer. In some instances, the cancer is prostate cancer, particularly metastatic castration-resistant prostate cancer (mCRPC).

[0352] In combination with chemotherapy

[0353] The antibody or an antigen-binding portion thereof can be used in combination with an anti-cancer agent, a cytotoxic agent, or a chemotherapeutic agent.

[0354] The term "anti-cancer agent" or "anti-proliferative agent" means any agent that can be used to treat a cell proliferative disorder (e.g., cancer), and includes but is not limited to cytotoxic agents, cytostatic agents, anti-angiogenic agents, cytoreductive agents, chemotherapeutic agents, radiotherapy and radiotherapy agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents, and immunotherapeutic agents. It should be understood that in some of the instances described above, these anti-cancer agents can include conjugates and can be conjugated with the disclosed site-specific antibodies prior to administration. More specifically, in some instances, a selected anti-cancer agent will be linked to an unpaired cysteine of an engineered antibody to provide an engineered conjugate as described herein. Accordingly, these engineered conjugates are expressly contemplated within the scope of the present disclosure. In some instances, the disclosed anti-cancer agents will be co-administered with a site-specific conjugate comprising different therapeutic agents as described above.

[0355] As used herein, the term "cytotoxic agent" means a substance that is toxic to cells and reduces or inhibits cell function and / or causes cell destruction. In some instances, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxins, staphylococcal enterotoxin A), fungi (e.g., α-sarcin, restrictocin), plants (e.g., abrin, ricin, modeccin, viscotoxin, pokeweed antiviral protein, saporin, gelonin, momordin, trichosanthin, hordotoxin, aleuritin, crotin, PAP (PAPI, PAPII, and PAP-S), momordica inhibitor, curcin, croton toxin, chlorambu officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, neomycin, and trichothecene toxins), or small molecule toxins or enzymatically active toxins from animals (e.g., cytotoxic ribonucleases such as extracellular pancreatic ribonuclease; deoxyribonuclease I, including fragments and / or variants thereof).

[0356] For the purposes of this disclosure, "chemotherapeutic agent" encompasses compounds (e.g., cytotoxic agents or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. These chemical agents typically target intracellular processes that are essential for cell growth or division and are thus particularly effective against cancer cells, which typically grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. In general, chemotherapeutic agents can include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may be cancerous or generate tumorigenic progeny (e.g., TICs). Such agents are typically administered in combination and are generally most effective, for example, in regimens such as CHOP or FOLFIRI.

[0357] Examples of anti-cancer agents (either as components of site-specific conjugates or in an unconjugated state) that can be used in combination with the site-specific constructs of the present disclosure include, but are not limited to, abiraterone, apalutamide, bicalutamide, alkylating agents, alkyl sulfonates, aziridines, ethyleneimines, and methylmelamines, annonaceous acetogenins, camptothecin, bryostatin, callystatin, CC-1065, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;Antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, antiadrenal agents, folic acid supplements such as leucovorin, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichloroethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine (“Ara-C”); cyclophosphamide; thiotepa;Taxanes, chlorambucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE vinorelbine; mitoxantrone; teniposide; edatrexate; daunorubicin; aminopterin; capecitabine; ibandronate; irinotecan (Camptosar, CPT-11), topoisomerase inhibitor RFS 2000; difluoromethylornithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; PKC-α, Raf, H-Ras, EGFR, and VEGF-A inhibitors that reduce cell proliferation, and any pharmaceutically acceptable salts, acids, or derivatives thereof. The definition also includes antihormonal agents for modulating or inhibiting the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit the aromatase that regulates estrogen production in the adrenal gland, and antiandrogens; and troxacitabine (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 inhibitor; ABARELIX rmRH; vinorelbine and esperamicin and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.;

[0358] Used in combination with radiotherapy

[0359] The present disclosure also provides combinations of antibodies or antigen-binding portions thereof with radiotherapy (i.e., any mechanism for locally causing DNA damage within tumor cells, such as γ-radiation, X-rays, UV radiation, microwaves, electron emission, etc.). Combinatorial therapies using the directed delivery of radioisotopes to tumor cells are also contemplated, and the disclosed conjugates can be used in combination with targeted anticancer agents or other targeting modalities. Generally, radiotherapy is administered in pulses over a period of about 1 to about 2 weeks. Radiotherapy can be administered to a subject with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple consecutive doses.

[0360] Drug packages and kits

[0361] Also provided are pharmaceutical packages and kits that include one or more containers that contain one or more doses of an antibody or an antigen-binding portion thereof. In some instances, unit doses are provided, where a unit dose contains a predetermined amount of a composition that includes, for example, an antibody or an antigen-binding portion thereof, with or without one or more additional agents. In some instances, such unit doses are supplied in the form of single-use prefilled syringes for injection. In some instances, the composition included in the unit dose can include saline, sucrose, etc.; buffers such as phosphate, etc.; and / or be formulated within a stable and effective pH range. Optionally, in some instances, the conjugate composition can be provided as a lyophilized powder that can be reconstituted after the addition of a suitable liquid (e.g., sterile water or saline solution). In some instances, the composition includes one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the enclosed conjugate composition is for the treatment of a selected oncological disease condition.

[0362] The present disclosure also provides kits for generating single-dose or multi-dose administration units of a site-specific conjugate and optionally one or more anti-cancer agents. The kits include a container and a label or package on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials such as glass or plastic and contains a pharmaceutically effective amount of the disclosed conjugate in conjugated or unconjugated form. In some instances, the container includes a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be penetrated by a subcutaneous injection needle). These kits will generally contain a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container and optionally one or more anti-cancer agents in the same or a different container. The kits can also contain other pharmaceutically acceptable formulations for diagnostic or combination therapies. For example, in addition to the antibodies or antigen-binding portions thereof of the present disclosure, these kits can contain any one or more of a range of anti-cancer agents, such as chemotherapeutic or radiotherapeutic drugs; anti-angiogenic agents; anti-metastatic agents; targeted anti-cancer agents; cytotoxic agents; and / or other anti-cancer agents.

[0363] More specifically, the kit can have a single container containing the disclosed antibody or antigen-binding portion thereof, with or without additional components, or they can have separate containers for each desired reagent. In the case of providing combination therapeutic agents for conjugation, the single solution can be combined in molar equivalents or premixed with one component in excess of the other. Optionally, the conjugate of the kit and any optional anti-cancer agent can be maintained separately in different containers prior to administration to a patient. The kit can also include a second / third container device 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 solutions.

[0364] When the components of the kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution, e.g., a sterile aqueous solution or a saline solution. However, the components of the kit can be provided as dry powders. When the reagent or component is provided as a dry powder, the dry powder can be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent can also be provided in a separate container.

[0365] As briefly noted above, the kit can also contain means for administering the antibody or antigen-binding portion thereof and any optional components to a patient, e.g., one or more needles, I.V. bags or syringes, or even a dropper, pipette, or other similar device through which the formulation can be injected or introduced into an animal or administered to an affected area of the body. The kits of the present disclosure generally also include means for containing vials and the like, as well as other components for commercial sale that are subject to strict limitations, e.g., an injection or blow molded plastic container in which the desired vials and other equipment are placed and held.

[0366] Exemplary antibody

[0367] An 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 covers all residues defined by both IMGT and Kabat.

[0368] Amino acid sequences of the W308051 CDRs

[0369]

[0370] Amino acid sequences of the W308051 variable regions

[0371]

[0372] Note: VH and VL are in the format of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus, where FR is in bold and CDR is underlined.

[0373] Amino acid sequences of the heavy and light chains of W308051

[0374]

[0375]

[0376] Note: The modified TCR β constant region and the modified TCR α constant region in the anti-CD3 arm are highlighted in italics.

[0377] Nucleotide sequences of the variable regions of W308051

[0378]

[0379]

[0380] Nucleotide sequences of the heavy and light chains of W308051

[0381]

[0382]

[0383]

[0384] Note: The nucleotide sequences of the variable regions are shown in capital letters.

[0385] Codon-optimized nucleotide sequences of the heavy and light chains of W308051

[0386]

[0387]

[0388]

[0389] Another illustrative antibody is a fully human anti-PSMA monoclonal antibody named W305042-1.135.2-uIgG1L or WBP305042. CDR numbering is defined using IMGT+Kabat, covering all residues defined by both IMGT and Kabat.

[0390] CDR amino acid sequences of WBP305042

[0391]

[0392] Variable region amino acid sequence of WBP305042

[0393]

[0394] Variable region nucleotide sequence of WBP305042

[0395]

[0396] Heavy and light chains of WBP305042

[0397]

[0398]

[0399]

[0400] Exemplary embodiments

[0401] Embodiment 1: An isolated antibody or antigen-binding portion thereof that comprises 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.

[0402] Embodiment 2: The isolated antibody or antigen-binding portion thereof according to Embodiment 1, wherein the PSMA-binding portion comprises a heavy chain variable region and a light chain variable region, the 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 the 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.

[0403] Embodiment 3: The isolated antibody or antigen-binding portion thereof according to 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.

[0404] Embodiment 4: The isolated antibody or antigen-binding portion thereof according to 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.

[0405] Embodiment 5: The isolated antibody or antigen-binding portion thereof according to Embodiment 3 or 4, wherein the CD3-binding portion comprises a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region comprising the sequence of SEQ ID NO: 13 or the amino acid sequence encoded by SEQ ID NO: 21, and the light-chain variable region comprising the sequence of SEQ ID NO: 14 or the amino acid sequence encoded by SEQ ID NO: 22.

[0406] Embodiment 6: The isolated antibody or antigen-binding portion thereof according to Embodiment 5, wherein the CD3-binding portion comprises a heavy-chain TCR β constant region and a light-chain TCR β constant region, the heavy-chain TCR β constant region comprising the sequence of SEQ ID NO: 29, and the light-chain TCR β constant region comprising the sequence of SEQ ID NO: 30.

[0407] Embodiment 7: The isolated antibody or antigen-binding portion thereof according to Embodiment 6, wherein the CD3-binding portion comprises a heavy chain and a light chain, the heavy chain comprising the sequence of SEQ ID NO: 17, and the light chain comprising the sequence of SEQ ID NO: 18.

[0408] Embodiment 8: The isolated antibody or antigen-binding portion thereof according to any one of Embodiments 1-7, wherein the PSMA-binding portion comprises a heavy chain and a light chain, the heavy chain comprising the sequence of SEQ ID NO: 19, and the light chain comprising the sequence of SEQ ID NO: 20.

[0409] Embodiment 9: The isolated antibody or antigen-binding portion thereof according to Embodiment 1 or 2, wherein the PSMA-binding portion comprises a heavy chain and a light chain, the heavy chain comprising the sequence of SEQ ID NO: 31 or the amino acid sequence encoded by SEQ ID NO: 33, and the light chain comprising the sequence of SEQ ID NO: 32 or the amino acid sequence encoded by SEQ ID NO: 34.

[0410] Embodiment 10: The isolated antibody or antigen-binding portion thereof according to any one of Embodiments 1-9, which is a monoclonal antibody, chimeric antibody or humanized antibody.

[0411] Embodiment 11: The isolated antibody or antigen-binding portion thereof according to Embodiment 10, which is a monoclonal antibody.

[0412] Embodiment 12: The isolated antibody or antigen-binding portion thereof according to Embodiment 11, which is a human monoclonal antibody.

[0413] Embodiment 13: The isolated antibody or antigen-binding portion thereof according to any one of Embodiments 1-12, which is fused to a constant region of IgG, optionally human IgG, optionally human IgG1 or human IgG4.

[0414] Embodiment 14: A pharmaceutical composition comprising the isolated antibody or antigen-binding portion thereof according to any one of Embodiments 1-13 and a pharmaceutically acceptable carrier.

[0415] Embodiment 15: A conjugate comprising the isolated antibody or antigen-binding portion thereof according to any one of Embodiments 1-13 and one or more moieties conjugated to the isolated antibody or antigen-binding portion thereof.

[0416] Embodiment 16: An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the isolated antibody or antigen-binding portion thereof according to any one of Embodiments 1-13, optionally wherein the nucleic acid sequence comprises any combination of the sequences of SEQ ID NOs: 35 to 38.

[0417] Embodiment 17: A vector comprising the nucleic acid molecule according to Embodiment 16.

[0418] Embodiment 18: A host cell comprising the isolated nucleic acid molecule according to Embodiment 16 or the vector according to Embodiment 17.

[0419] Embodiment 19: A method for preparing the antibody or antigen-binding portion thereof according to any one of Embodiments 1-14, the method comprising: a) expressing the antibody or antigen-binding portion thereof in the host cell according to Embodiment 18; and b) isolating the antibody or antigen-binding portion thereof from the host cell.

[0420] Embodiment 20: A method for inhibiting the growth or metastasis of tumor cells 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 according to any one of Embodiments 1-13 or the pharmaceutical composition according to Embodiment 14.

[0421] Embodiment 21: A method for 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 according to any one of Embodiments 1-13 or the pharmaceutical composition according to Embodiment 14.

[0422] Embodiment 22: A method for treating or preventing a proliferative disorder, 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 according to any one of Embodiments 1-13 or the pharmaceutical composition according to Embodiment 14.

[0423] Embodiment 23: The method according to Embodiment 22, wherein the method treats a proliferative disorder, and the proliferative disorder is cancer, optionally prostate cancer, lung cancer, bronchiogenic carcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, adenocarcinoma, alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (non-cancerous), sarcoma, kidney cancer, breast cancer, gastric cancer, colorectal cancer, glioblastoma, pancreatic cancer, ovarian cancer, or metastatic castration-resistant prostate cancer (mCRPC).

[0424] Embodiment 24: The method according to Embodiment 23, wherein the cancer is prostate cancer.

[0425] Embodiment 25: The method according to any one of Embodiments 20-24, wherein the isolated antibody or antigen-binding portion thereof according to any one of Embodiments 1-13 or the pharmaceutical composition according to Embodiment 14 is administered in combination with a chemotherapeutic agent, radiotherapy, and / or another cancer immunotherapy.

[0426] Embodiment 26: A kit for treating or diagnosing a proliferative disorder, an immune disorder, or an infection, the kit comprising a container containing at least one of the isolated antibodies or antigen-binding portions thereof according to any one of Embodiments 1-13. Examples

[0427] Generally, the present invention will be more readily understood by the following examples, which are provided by way of illustration and are not intended to limit the present invention. The examples are not intended to represent that the following experiments are all or only the experiments conducted.

[0428] The commercially available materials used in Examples 1-4 are listed in the following table.

[0429]

[0430]

[0431] Preparation of Materials for Example 1

[0432] 1.1 Establishment of stable cell line

[0433] According to the user's instructions, generate the cell line WBP3xx042-FlpinCHO.cPro1.B7 (Cyno PSMA+ CHO cells) expressing cynomolgus macaque PSMA using Flp-in CHO cells (Thermo, R75807) transfected with the plasmid encoding the full-length cynomolgus macaque PSMA (XM_005579322.1, NCBI).

[0434] 1.2 Generation of reference antibody

[0435] Synthesize the DNA sequence encoding the J591 antibody (W3XX042-BMK1) according to Sequences 21&22 from WO 02 / 098897 A2 in Genewiz (Suzhou, China), and then subclone it into the modified pcDNA3.3 expression vector (Thermo).

[0436] Co-transfect the plasmids encoding the heavy and light chains into Expi293 cells. Culture the cells for 5 days and collect the supernatant for protein purification using a protein A column (GE Healthcare, 175438). Analyze the obtained antibody by SDS-PAGE and SEC-HPLC, and then store it at -80 °C.

[0437] Example 2 Antibody hybridoma generation

[0438] 2.1 Immunization and cell fusion

[0439] Three 6-8-week-old OMT rats 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, a 96-well plate (Nunc) was coated overnight at 4 °C with 100 μL of 2 μg / mL human PSMA antigen, and then blocked for 1 hour at ambient temperature with blocking buffer (1 X PBS / 2% BSA). Rat sera were serially diluted 3-fold starting at a 1:100 dilution in blocking buffer and incubated for 1 hour at ambient temperature. Wells without serum samples were used as negative controls. The plates were then washed, followed by incubation with a secondary antibody, goat anti-rat IgG-Fc-HRP (Bethyl), for 1 hour. After washing, TMB substrate was added, and the interaction was terminated with 2M HCl. Absorbance at 450 nm was read using a microplate reader (Molecular Device). For FACS, plates (96-well) were pre-coated with 3×104 LNCaP cells per well and cultured for 2 days in an incubator set at 37 °C and 5% CO2. The plates were blocked for 1 hour at ambient temperature with blocking buffer (1 x PBS / 5% milk). Then 50 μl of hybridoma supernatant was added to the plates and incubated for 1 hour at ambient temperature. The plates were washed 3 times with PBS, followed by incubation with a 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 by flow cytometry and analyzed by FLOWJO.

[0440] Lymph nodes and spleens from the 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 terminated with 100% FBS. The cells were washed and counted. According to the general electrofusion procedure, B cells were fused with Sp2 / 0 myeloma cells at a ratio of 1:1 in an electrofusion solution. The fused cells were resuspended in DMEM medium supplemented with 20% FBS and 1X HAT and then transferred to a 96-well plate. The fused cells were cultured in an incubator set at 37 °C and 5% CO2 for 10-14 days.

[0441] 2.2 High-throughput screening and IgG conversion of hybridoma supernatants

[0442] The procedure for high-throughput screening with hybridoma culture supernatants included a primary screen by ELISA binding to human PSMA and a confirmatory screen by FACS / ELISA binding to both human and cynomolgus monkey PSMA.

[0443] 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 antibody 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 products were then ligated into the pMD18-T vector, and 10 μl of the ligation product was transformed into Top 10 competent cells. The transformed cells were plated on 2xYT plates with carbocinin and incubated overnight at 37°C. A total of 12 positive colonies were randomly selected for DNA sequencing.

[0444] After sequence analysis and functional screening, candidates were selected for full-human antibody production. The DNA sequences of the variable domains of the candidates were synthesized and cloned into a modified pcDNA3.4 vector containing the human IgG1 Fc. After sequence confirmation, the expression vector containing the complete IgG of the full-human antibody was used for transient transfection to produce the antibody.

[0445] The purified IgG antibodies were further screened by ELIAS and FACS that bind to human and cynomolgus monkey PSMA. Antibody W305042 was designated as one of the antibodies.

[0446] Example 3 Construction and Purification of Full-Human Antibody Molecules

[0447] According to the manufacturer's instructions, the heavy-chain and light-chain expression plasmids of 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 a protein A column was used for protein purification. The antibody concentration was measured by NanoDrop. The protein purity was evaluated by SDS-PAGE and SEC-HPLC ( Figure 1 ). After purification, the yield of W305042 was 223.16 mg / L, and the purity evaluated by SEC-HPLC was 99.19%.

[0448] Example 4 Antibody Characterization

[0449] 4.1 Binding Ability Test by ELISA

[0450] The binding of the W305042 antibody to human PSMA was determined by ELISA. The plates were pre-coated overnight with 1 μg / mL THETM His-tag antibody in a refrigerator set at 4°C. After blocking for 1 hour with 200 μl of 1×PBS / 2% BSA, the plates were washed three times with 1×PBST, and then 0.5 μg / mL human PSMA ECD protein diluted in 1×PBS / 2% BSA was added to the plates at a volume of 50 μl / well and incubated for 1 hour at ambient temperature. After washing the plates three times with 1×PBST, various concentrations (serially diluted 4-fold from 100 nM to 0.095 pM in 1×PBS / 2% BSA) of the W305042 antibody, J591 (positive control), and human IgG isotype antibody (negative control) were added to the plates at a volume of 50 μL / well and incubated for 2 hours at ambient temperature. After washing the plates three times with 1×PBST, 50 μl / well of HRP-labeled goat anti-human IgG antibody (diluted 1:5000 in 1×PBS / 2% BSA) was added to the plates and incubated for 1 hour at ambient temperature. After washing the plates six times with 1×PBST, 50 μL / well of TMB substrate was added for color development for 4 - 8 minutes, and then the reaction was terminated by adding 50 μL / well of 2M HCl. Absorbance was read at 450 nm using a microplate reader. The binding EC50 was calculated by plotting the antibody concentration (x-axis) against the OD450 value (y-axis) using GraphPad Prism and performing the following analysis: non-linear regression (curve fitting) - log(agonist) vs. response - variable slope (four parameters).

[0451] The results of the antibody binding to human PSMA protein are shown in Figure 2 and Table 1. W305042 could effectively bind to human PSMA ECD protein with an EC50 of 0.017 nM, which was slightly higher than that of the reference antibody J591. The human IgG isotype antibody used as a negative control showed no significant binding to human PSMA protein. The results indicated that W305042 had good binding ability to human PSMA.

[0452] Table 1. Antibodies Binding to Human PSMA

[0453]

[0454] 4.2 Binding Ability Analysis by Flow Cytometry

[0455] Fluorescence-activated cell sorting (FACS) was used to detect the binding of the W305042 antibody to human PSMA. Briefly, 1×105 LNCaP cells per well were incubated with various concentrations of the W305042 antibody (serially diluted 4-fold from 100 nM to 0.095 pM with 1×PBS / 1% BSA) at a volume of 100 μL / well in a refrigerator set at 4°C for 1 hour. J591 was used as a positive control, and human IgG isotype antibody was used as a negative control. After washing the cells twice with 1×PBS / 1% BSA, Alexa fluor647-labeled goat anti-human antibody (diluted 1:500 with 1×PBS / 1% BSA) was added to the cells and incubated in the dark in a refrigerator set at 4°C for 0.5 hour. After washing the cells twice with 1×PBS / 1% BSA, the mean fluorescence intensity (MFI) of the cells was measured by flow cytometry and analyzed by FLOWJO. The binding EC50 was calculated by plotting the antibody concentration (x-axis) against the MFI (y-axis) using GraphPad Prism and analyzing as follows: non-linear regression (curve fitting) - log(agonist) vs. response - variable slope (four parameters).

[0456] The binding results of the antibody to LNCaP cells expressing human PSMA are shown in Figure 3 and Table 2. W305042 could effectively bind to LNCaP cells with an EC50 of 0.25 nM, which was comparable to the reference antibody J591. The human IgG isotype antibody used as a negative control showed no significant binding to LNCaP cells. The results indicated that W305042 had good binding ability to LNCaP cells.

[0457] Table 2. Antibodies binding to LNCaP

[0458]

[0459] 4.3 Orthologous gene (across species) binding test

[0460] 4.3.1 Binding to cynomolgus monkey PSMA was determined by ELISA

[0461] The binding of the W305042 antibody to cynomolgus monkey PSMA was determined by ELISA. The plates were pre-coated overnight with 1 μg / mL THETM His-tag antibody 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, and then 0.5 μg / mL cynomolgus monkey PSMA ECD protein diluted in 1×PBS / 2% BSA was added to the plates at a volume of 50 μl / well and incubated for 1 hour at ambient temperature. After washing the plates three times with 1×PBST, various concentrations (serially diluted 4-fold from 10 nM to 0.0095 pM in 1×PBS / 2% BSA) of the W305042 antibody, J591 (positive control), and human IgG isotype antibody (negative control) were added to the plates at a volume of 50 μL / well and incubated for 2 hours at ambient temperature. After washing the plates three times with 1×PBST, 50 μl / well of HRP-labeled goat anti-human IgG antibody (diluted 1:5000 in 1×PBS / 2% BSA) was added to the plates and incubated for 1 hour at ambient temperature. After washing the plates six times with 1×PBST, the reaction was developed by applying 50 μL / well of TMB substrate for 4 - 8 minutes and then terminated by adding 50 μL / well of 2M HCl. Absorbance was read at 450 nm using a microplate reader. The EC50 was determined as described above.

[0462] The results of the antibody binding to cynomolgus monkey PSMA protein are shown in Figure 4 and Table 3. W305042 can effectively bind to cynomolgus monkey PSMA ECD protein with an EC50 of 0.006 nM, which is comparable to the reference antibody J591. The human IgG isotype antibody used as a negative control showed no significant binding to cynomolgus monkey PSMA protein. The results indicate that W305042 has good binding ability to cynomolgus monkey PSMA.

[0463] Table 3. Antibodies Binding to Cynomolgus Monkey PSMA

[0464]

[0465] 4.3.2 Detection of Binding to Cynomolgus Monkey PSMA by FACS

[0466] The binding of the W305042 antibody to cynomolgus monkey PSMA was detected using fluorescence-activated cell sorting (FACS). This method can quantitatively analyze and identify specific molecules expressed on the surface of live cells. Before detection, unlabeled cells were used as a control to set the threshold, and the percentage change in the fluorescence intensity threshold in each group 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, 1×105 cells per well of W3xx042.FIpinCHO.cPro1.B7 were incubated with various concentrations of the W305042 antibody (serially diluted 4-fold from 100 nM to 6.1 pM with 1×PBS / 1% BSA) at a volume of 100 μL / well in a refrigerator set at 4°C for 1 hour. J591 was used as a positive control, and human IgG isotype antibody was used as a negative control. After washing the cells twice with 1×PBS / 1% BSA, Alexa fluor 647-labeled goat anti-human antibody (diluted 1:500 with 1×PBS / 1% BSA) was added to the cells and incubated in the dark in a refrigerator set at 4°C for 0.5 hour. After washing the cells twice with 1×PBS / 1% BSA, the mean fluorescence intensity (MFI) of the cells was measured by flow cytometry and analyzed by FLOWJO. The MFI and EC50 were determined as described above.

[0467] The binding results of the antibody to engineered CHO cells expressing cynomolgus monkey PSMA are shown in Figure 5 and Table 4. W305042 can effectively bind to cynomolgus monkey PSMA+ CHO cells with an EC50 of 2.25 nM, which is comparable to the reference antibody J591. The human IgG isotype antibody used as a negative control showed no significant binding to cynomolgus monkey PSMA+ CHO cells. The results indicate that W305042 has good binding ability to cynomolgus monkey PSMA.

[0468] Table 4. Antibodies Binding to CHO Cells Expressing Cynomolgus Monkey PSMA

[0469]

[0470] 4.3.3 Binding to Mouse PSMA Measured by ELISA

[0471] Briefly, 2 μg / mL of mouse PSMA protein (His-tagged) was coated onto the wells of an ELISA plate with coating buffer and incubated in a refrigerator set at 4°C for 16 hours. After washing the plate once with 1x PBST, 200 μL / well of 2% BSA was added to the wells and incubated for one hour at ambient temperature. After washing three times with 1x PBST, various concentrations of antibodies (against W305042-1.135.2-uIgG1L and isotype control, serially diluted 6-fold from 100 nM to 0.357 pM) diluted in 2% BSA were added to the wells at a volume of 100 μL / well and incubated for one hour at ambient temperature. After washing three times with 1x PBST, 100 μL / well of the secondary antibody, goat anti-human IgG-Fc-HRP (1:5000 dilution) for W305042-1.135.2-uIgG1L and isotype control, was added to the wells and incubated for one hour at ambient temperature. After washing six times with 1x PBST, 100 µL / well of the TMB substrate solution was added to the wells and incubated in the dark for 5 minutes, then 100 μL / well of 2M HCL was added to the wells to terminate the reaction. Relative light units (RLU) were measured at OD450 and OD540 by SPECTRAMAX M5E. EC50 was determined as described above.

[0472] The results of antibody binding to mouse PSMA protein are shown in Figure 6 and Table 5. W305042 can effectively bind to mouse PSMA ECD protein with an EC50 of 0.026 nM. The human IgG isotype antibody used as a negative control showed no significant binding to mouse PSMA protein. The results indicate that W305042 has good binding ability to mouse PSMA.

[0473] Table 5. Antibodies binding to mouse PSMA

[0474]

[0475] 4.4 Binding affinity was tested by surface plasmon resonance (SPR)

[0476] The binding affinities of W305042 and J591 to human and cynomolgus monkey PSMA were detected by SPR assay using BIACORE 8K. Each antibody was captured on a CM5 sensor chip (Cytiva) immobilized with anti-human IgG Fc antibody. Different concentrations of human and cynomolgus monkey PSMA were injected onto the sensor chip at a flow rate of 30 μL / min for a binding 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).

[0477] Subtract the sensorgrams of the blank surface and the buffer channel from the test sensorgram. The experimental data were fitted by a 1:1 binding model. Molecular weights of 81.4 and 82.7 kDa were used to calculate the molar concentrations of human and cynomolgus monkey PSMA.

[0478] The association rate constant (ka), dissociation rate constant (kd), and affinity constant (KD) of the antibodies are listed in Table 6. The binding affinity of W305042 for human PSMA is higher than that of J591, and the binding affinity of W305042 for cynomolgus monkey PSMA is similar to that of J591.

[0479] Table 6. Kinetic affinity results of the antibodies

[0480]

[0481] 4.5 Developability testing

[0482] Thermal stability was determined by DSF

[0483] The Tm (melting temperature) of each antibody was studied using a QUANTSTUDIO 7 Flex real-time PCR system (Applied Biosystems). 19 μL of the antibody solution was mixed with 1 μL of 80X SYPRO Orange protein gel stain and transferred to a 96-well plate. The plate was sealed with an optical adhesive film and centrifuged at 3,000 rpm for 5 min 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 were collected. The negative derivative of the fluorescence change with respect to different temperatures was calculated, and the maximum value was defined as the melting temperature Tm. If the protein had multiple unfolding transitions, the first two Tm values were reported, named Tm1 and Tm2. Data collection and Tm calculation were performed automatically by the QUANTSTUDIO real-time PCR software (v1.3). W305042 showed good thermal stability, with Tm1 of 69.5 °C and Tm2 of 71.1 °C ( Figure 7 ).

[0484] Diffusion interaction parameter (kD) was determined by DLS

[0485] kD measurements were studied using a DYNAPRO Plate Reader III (Wyatt Technology). During sample preparation, the appearance of the sample was recorded during thawing, filtration, and concentration. Then, 7.5 μL of the sample solution was added to a 1536-well microplate. Data collection was performed by the DYNAMICS operating software (v7.8.1.3). Five acquisitions were made for each protein sample, with each acquisition time of 5 s. For each measurement, the diffusion coefficient was determined and plotted against the protein concentration. The kD value was calculated automatically by the software.

[0486] W305042 shows a high kD value and a monodisperse size distribution, indicating that W305042 has good solubility characteristics. See Table 7.

[0487] Table 7. Diffusion interaction parameter (kD) detected by DLS

[0488]

[0489] Hydrophobic interaction chromatography HPLC (HIC-HPLC)

[0490] The hydrophobic properties of the antibody were detected by an HPLC 1260 Infinity II system (Agilent TechnologicsTM) with a TSKgel butyl-NPR column. The sample was diluted in PBS buffer, and 20 μL of the diluted sample was injected into the column and separated at a flow rate of 0.5 ml / min for 61 min. The peak retention was detected by UV light at wavelengths of 280 nm and 230 nm. The retention time was analyzed by the HIC-HPLC analysis method to integrate all peak areas from 20 min to 40 min. The operating and analysis software was OPENLAB CDS Workstation (v2.6.0.691). The retention time of W305042 analyzed by HIC-HPLC was 24.57 min, indicating that W305042 has low hydrophobicity ( Figure 8 ).

[0491] Example 5 Preparation of materials, cell lines, and benchmark (BMK) antibodies

[0492] Information on commercially available materials used in the following examples is provided in the table below.

[0493]

[0494]

[0495] Generation of stable cell lines

[0496] According to the user's instructions, a cell line expressing cynomolgus macaque PSMA, WBP3xx042-FlpinCHO.cPro1.B7 (Cyno PSMA+CHO cells), was generated using Flp-in CHO cells (Thermo, R75807) transfected with a plasmid encoding full-length cynomolgus macaque PSMA (XM_005579322.1, NCBI).

[0497] Production of benchmark (BMK) antibodies

[0498] Synthesize the DNA sequence encoding the CD3xPSMA reference antibody, the DNA sequence of AMG 340 / TNB-585 (BMK1) according to SEQ ID No: 49&56&61 from WO 2021 / 222578A1, and then subclone it into the modified pcDNA3.3 expression vector (Thermo).

[0499] Synthesize the DNA sequence encoding the CD3xPSMA reference antibody, the DNA sequence of AMG 160 (BMK2) according to SEQ ID No: 382 from US 20170218079A1, and then subclone it into the modified pcDNA3.3 expression vector (Thermo).

[0500] Transfect the recombinant plasmid expressing the reference antibody into Expi293 cells. Culture the cells for 5 days and collect the supernatant for protein purification using a Protein A column (GE Healthcare, 175438) and / or an SEC column (Cytiva, 28990944). Analyze the obtained antibody by SDS-PAGE and HPLC-SEC, and then store it at -80 o °C.

[0501] Example 6 Generation of Bispecific Antibodies

[0502] Discover anti-CD3 monoclonal antibodies (WO2019057099A1) from immunized mice by hybridoma technology. Discover anti-PSMA monoclonal antibodies from immunized transgenic rats by hybridoma technology.

[0503] Use standard molecular biology protocols for the construction of bispecific antibodies. For the construction of the CD3xPSMA bispecific antibody W308051-T3U5.E17-61.uIgG4V322 (W308051), as Figure 9 shown, fuse the DNA sequence encoding the VH region of the anti-CD3 antibody with the modified TCRβ constant domain and hinge Fc region of human IgG4 with S228P mutation, Fc null mutation (F234A L235A) and pestle mutation ((S354C-T366W); fuse the DNA sequence encoding the VL region of the anti-CD3 antibody with the modified TCRα constant domain; fuse the DNA sequence encoding the VH region of the anti-PSMA antibody with the hinge-Fc region of human IgG4 with S228P mutation, Fc null mutation (F234A L235A) and mortar mutation (Y349C-T366S-L368A-Y407V); fuse the DNA sequence encoding the VL region of the anti-PSMA antibody with the CL domain. Then clone the coding region into the modified pcDNA3.3 expression vector.

[0504] The plasmid encoding the bispecific antibody was transfected into Expi293 cells at a scale of 1000 mL. The cells were cultured for 5 days, and the supernatant was collected for protein purification using a Protein A column (Cytiva, 17549802) and a CEX column (Cytiva, 17118001). The antibody concentration was detected by Nano Drop at 280 nm. The purity of the antibody was analyzed by SDS-PAGE and SEC-HPLC. The endotoxin level was determined using a PTS / MCS reagent card. The antibody was stored at -80 º °C.

[0505] As Figure 10A and 10B shown, the yield of W308051 was 79.22 mg / L, and the purity evaluated by SEC-HPLC was 98.18%.

[0506] Example 7 In Vitro Characterization

[0507] 7.1 Target Binding Measured by ELISA / FACS

[0508] Binding to human PSMA measured by ELISA

[0509] Briefly, 1 μg / mL human PSMA protein (His-tagged) was coated onto the wells of an ELISA plate with coating buffer at 4°C and incubated for 16 hours. After washing the plate once with 1xPBST, the plate was blocked for one hour by using 200 μL / well of 2% BSA. After washing the plate three times with 1xPBST, antibodies at various concentrations (serially diluted 5-fold 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 one 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 one hour. After washing six times with 1x PBST, 100 µL / well of TMB substrate solution was added to the wells and incubated in the dark for 5 minutes, then 100 µL / well of 2M HCL was added to the wells to terminate the reaction. The relative light units (RLU) were measured by SPECTRAMAX M5E at OD 450 and OD 540 The antibody concentration (x-axis) versus OD 450-540 (y-axis) was plotted using GraphPad Prism 7 software and analyzed as follows to calculate the binding EC 50 : Nonlinear regression (curve fitting) - log(agonist) vs. response - variable slope (four parameters).

[0510] The results are as Figure 11As shown in Table 8, W308051 binds to human PSMA protein, with an EC 50 of 0.027 nM, which shows stronger binding potency than the reference antibody AMG 160.

[0511] Table 8. EC of antibodies against human PSMA 50

[0512]

[0513] NA: Not applicable.

[0514] Binding to human PSMA and CD3 measured by FACS

[0515] Four human prostate cancer cell lines, human C4-2 (with high PSMA expression), LNCaP (with high PSMA expression), 22Rv1 (with low PSMA expression), PC-3 (PSMA negative), as well as Jurkat 2B8 (CD3 positive) cells and primary T cells isolated from fresh PBMC (5×10 4 cells) were incubated with various concentrations of antibodies (for W308051, AMG160 and isotype control, serially diluted 5-fold from 200 nM to 2.56 pM, for AMG 340, serially diluted 5-fold from 500 nM to 6.40 pM) at 4°C for one hour. After washing twice with 1×PBS / 1% BSA / 0.1 mM EDTA, Alexa fluor 647-labeled goat anti-human IgG Fc (1:500 dilution) was added, and the plates were incubated in the dark at 4°C for half an hour. After washing twice with 1xPBS / 1% BSA / 0.1 mM EDTA, the cells were resuspended in 1xPBS / 1% BSA / 0.1 mM EDTA, and the mean fluorescence intensity (MFI) was measured by flow cytometry and analyzed by FLOWJO. The antibody concentration (x-axis) versus OD 450-540 (y-axis) was plotted using GraphPad Prism 7 software and analyzed as follows to calculate the binding EC 50 : Nonlinear regression (curve fitting) - log(agonist) vs. response - variable slope (four parameters).

[0516] The results are shown in Figures 12 - 13 and Table 9. As Figure 12 shown in and Table 9, W308051 has EC values of 1.48 nM, 0.58 nM and 0.29 nM respectively 50Binds to human PSMA-positive C4-2 (with high PSMA expression), LNCaP (with high PSMA expression), and 22Rv1 (with low PSMA expression) human tumor cells, indicating that W308051 is stronger than AMG 160 and AMG 340. Meanwhile, W308051 does not bind to the PSMA-negative PC-3 cell line. As Figure 13 shown, W308051 binds to CD3-positive Jurkat cells and primary human T cells with a lower binding affinity than AMG 160.

[0517] Table 9. Binding of antibodies to PSMA-positive and negative cells

[0518]

[0519] NA: Not applicable.

[0520] 3.2 Cross-species binding measured by ELISA / FACS

[0521] Binding to cyno and mouse PSMA measured by ELISA

[0522] Briefly, 1 μg / mL cynomolgus monkey PSMA protein (His-tag) and 2 μg / mL mouse PSMA protein (His-tag) were coated onto the wells of an ELISA plate in coating buffer at 4 °C for 16 hours. After washing the plate once with 1xPBST, the plate was blocked for one hour by using 200 μL / well of 2% BSA. After washing three times with 1xPBST, various concentrations of antibodies diluted in 2% BSA were added (for W308051, AMG 160, and the isotype control for cynomolgus monkey PSMA, serially diluted 5-fold from 200 nM to 0.004 pM, for W308051, AMG 160, and the isotype control for mouse PSMA, serially diluted 6-fold from 100 nM to 0.357 pM, and for AMG 340 against mouse PSMA, serially diluted 6-fold from 500 nM to 1.786 pM), and incubated for one hour at ambient temperature. After washing three times with 1xPBST, goat anti-human IgG-Fc-HRP (1:5000 dilution) was added and incubated for one hour. After washing six times with 1xPBST, 100 µL / well of TMB substrate solution was added to the wells and incubated in the dark for 5 minutes, then 100 µL / well of 2M HCL was added to the wells to terminate the reaction. Relative light units (RLU) were measured by SPECTRAMAX M5E at OD 450 and OD 540 . The EC 50 was determined as described above.

[0523] As shown in Tables 10 - 11 andFigure 14A and 14B As shown in 14B , W308051 cross-reacts with cynomolgus monkey and mouse PSMA. W308051 binds to cyno PSMA ECD protein, and the EC 50 is 0.045 nM, which is stronger than AMG 160. W308051 also binds to mouse ECD PSMA protein, and the EC 50 is 1.34 nM. However, AMG 160 and AMG 340 do not bind to mouse PSMA protein.

[0524] Table 10. Binding to cynomolgus monkey PSMA

[0525]

[0526] NA: Not applicable.

[0527] Table 11. Binding to mouse PSMA

[0528]

[0529] NA: Not applicable.

[0530] Binding to cynomolgus monkey PSMA-positive cells measured by FACS

[0531] WBP3xx042-FlpinCHO.cPro1.B7 (5×10 4 cells) was incubated with different concentrations of antibody for one hour at 4°C. After washing twice with 1×PBS / 1% BSA / 0.1 mM EDTA, a secondary antibody, Alexa fluor 647-labeled goat anti-human IgG Fc, was added, and the plate was incubated in the dark for half an hour at 4°C. After washing twice with 1xPBS / 1% BSA / 0.1 mM EDTA, the cells were resuspended in 1xPBS / 1% BSA / 0.1 mM EDTA. The MFI and EC 50 were determined as described above.

[0532] As Figure 15 shown in Figure 15 and Table 12, W308051 binds to cynomolgus monkey PSMA-positive cells, and the EC 50 is 3.20 nM, which is stronger than AMG160 and AMG 340.

[0533] Table 12. Binding to cynomolgus monkey PSMA-positive cells

[0534]

[0535] NA: Not applicable.

[0536] 7.3 Affinity Measured by SPR

[0537] The binding affinities of antibodies W308051, AMG 340, and AMG 160 to human PSMA were detected by SPR assay using a BIACORE 8K. Biotinylated human PSMA was captured on a streptavidin immobilized CM5 sensor chip (Cytiva). Different concentrations of the antibodies were injected onto the sensor chip at a flow rate of 30 μL / min in a single-cycle injection type for a 180 s binding phase, followed by a 600 - 3600 s dissociation. Then, in the last binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5).

[0538] The sensorgrams of the blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data were fitted by a 1:1 binding model. Molecular weights of 147 and 106 kDa were used to calculate the molar concentrations of W308051, AMG 340, and AMG 160, respectively.

[0539] The binding affinities of W308051, AMG 340, and AMG 160 to the human CD3δ and CD3ε heterodimer 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). Different concentrations of the human CD3δ & CD3ε heterodimer were injected onto the sensor chip at a flow rate of 30 μL / min for a 120 s binding phase, followed by a 240 s dissociation. After each binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5).

[0540] The sensorgrams of the blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data were fitted by a 1:1 binding model. A molecular weight of 31 kDa was used to calculate the molar concentration of the human CD3δ & Cd3ε heterodimer.

[0541] The complete kinetic affinities of W308051 to human PSMA and CD3 are shown in Table 13. The affinities of W308051 to human PSMA and CD3 are 1.58×10 -11 M and 6.02×10 -8 M, respectively.

[0542] Table 13. Affinities to Human PSMA and CD3 Measured by SPR

[0543]

[0544] 7.4 Cytotoxicity and Cytokine Release of T Cells

[0545] The efficacy of bispecific antibodies in mediating the lysis of tumor cells by human T cells was evaluated by a CTG-based cytotoxicity assay. Briefly, C4-2, LNCaP, and PC-3 cells were used as target cells, and human T cells isolated from fresh human PBMCs were used as effector cells. 150 μL / well of the antibody was added to a 96-well plate. Then, 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 incubation for 72 hours, the plate was washed once with DPBS, CTG solution (75 μL / well) was added, and the plate was incubated at ambient temperature for 10 minutes. The relative light unit (RLU) signal was measured by Invasionreader. The cytotoxicity % was calculated as follows: (1 - (RLU 样品 - RLU 仅效应细胞 ) / (RLU 效应子+靶细胞 - RLU 仅效应细胞 )) x 100%. The antibody concentration (x-axis) versus cytotoxicity (y-axis) was plotted using GraphPad Prism 7 software and analyzed as follows to calculate the IC 50 : Nonlinear regression (curve fit) - log(inhibitor) vs. response - variable slope (four parameters).

[0546] The levels of cytokines released in the supernatant were determined by an ELISA-based quantitative assay. Briefly, after incubation for 20 hours, 125 μL of the 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. The plate was 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 one hour, 50 μL of the standard or sample was added to each well and incubated at ambient temperature for two hours. After washing 3 times with 1×PBST, 50 μL of a biotin-conjugated detection antibody specific for human IFN-γ (1:250) and peroxidase-conjugated streptavidin (1:250) were added to each well and incubated at ambient temperature for one hour. After washing six times with 1×PBST, color development was carried out by adding 50 μL of TMB substrate, and then terminated by adding 50 μL of 2M HCL. The relative light unit (RLU) was measured by SPECTRAMAX M5E at OD 450 and OD 540 . The concentration of human IFN-γ in the supernatant was quantified according to the standard curve.

[0547] As Figures 16 - 17As shown in Table 14-15, the results demonstrated that W308051 induced effective 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.

[0548] Table 14. Cytotoxicity of T cells in co-cultures of C4-2, LNCaP, and PC-3 cells with CD3+ T cells

[0549]

[0550] NA: Not applicable.

[0551] Table 15. Cytokine release in co-cultures of C4-2 and PC-3 cells with CD3+ T cells

[0552]

[0553] NA: Not applicable.

[0554] 7.5 Cytokine release in co-cultures of C4-2 cells with PBMC

[0555] Briefly, 100 μL / well of antibody (for W308051, AMG160, and isotype control, serially diluted 10-fold from 20 nM to 0.2 pM, for AMG 340, serially diluted 10-fold from 500 nM to 5.0 pM) was added to 96-well plates. Then, effector cells (fresh human PBMC, 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 incubation for 20 hours, the supernatants were collected and stored at -80°C. Human IFN-γ, IL-2, TNF-α, and IL-6 released in the supernatants were measured by ELISA. Relative light units (RLU) were measured at OD 450 and OD 540 using a SPECTRAMAX M5E. The concentration of human cytokines in the supernatants was quantified according to the standard curves. Antibody concentration (x-axis) versus percentage (y-axis) was plotted using GraphPad Prism 7 software and analyzed as follows to calculate EC 50 : Nonlinear regression (curve fitting) - log(agonist) vs. response - variable slope (four parameters).

[0556] As Figure 18 shown in and Table 16, W308051 induced low release of a panel of cytokines in the PBMC and C4-2 cell co-culture assay.

[0557] Table 16. Cytokine Release in Co-culture of C4-2 Cells and PBMC

[0558]

[0559] 7.6 Thermal Stability Measured by DSF

[0560] The T m (melting temperature) of each antibody was studied using the QUANTSTUDIO 7 Flex real-time PCR system (Applied Biosystems). 19 μL of the antibody solution was mixed with 1 μL of 80X SYPRO Orange protein gel stain and transferred to a 96-well plate. The plate was sealed with an optical adhesive film and centrifuged at 3,000 rpm for 5 min 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 respect to different temperatures was calculated, and the maximum value was defined as the melting temperature T m . If the protein has multiple unfolding transitions, the first two T m s are reported, named T m 1 and T m 2. Data collection and T m calculation were automatically performed by the QUANTSTUDIO real-time PCR software (v1.3). As Figure 19 shown, W308051 exhibited good thermal stability, with T m 1 being 62.9 °C and T m 2 being 69.3 °C.

[0561] 7.7 Hydrophobic Interaction Chromatography HPLC (HIC-HPLC)

[0562] The hydrophobic properties of the antibody were detected by an HPLC 1260 Infinity II system (Agilent Technologies TM ) with a TSKgel butyl-NPR column. The sample was diluted in PBS buffer, and 20 μL of the diluted sample was injected into the column and separated at a flow rate of 0.5 ml / min for 61 min. Peaks were detected by UV light at wavelengths of 280 nm and 230 nm. The retention time was analyzed by the HIC-HPLC analysis method to integrate all peak areas from 20 min to 40 min. The operating and analysis software was OpenLab CDS Workstation (v2.6.0.691). As Figure 20As shown, the retention time of W308051 analyzed by HIC-HPLC was 25.15 min, indicating that W308051 has low hydrophobicity.

[0563] 3.8 Determination of the diffusion interaction parameter (kD) by DLS

[0564] The kD measurement was investigated using a DYNAPRO Plate Reader III (Wyatt Technology). During sample preparation, the appearance of the sample was recorded during thawing, filtration, and concentration. Then, 7.5 μL of the sample solution was added to a 1536-well microplate. Data collection was performed using DYNAMICS operating software (v7.8.1.3). Five acquisitions were made for each protein sample, with each acquisition lasting 5 s. For each measurement, the diffusion coefficient was determined and plotted against the protein concentration. The kD value was automatically calculated by the software.

[0565] As shown in Table 17, W308051 showed a high kD value and a monodisperse size distribution, indicating that W308051 has good solubility characteristics.

[0566] Table 17. Determination of the diffusion interaction parameter (kD) by DLS

[0567]

[0568] Example 8 In Vivo Characterization

[0569] 8.1 Rat PK Study

[0570] The preliminary pharmacokinetic study of WBP308051 was tested in female CD(SD)IGS rats by single intravenous bolus administration. Briefly, female CD(SD)IGS rats (Beijing Charles River) at 8 - 10 weeks of age were used in this study. Five animals, as a group, were administered 10 mg / kg of W308051 by single intravenous bolus injection. PK serum samples were collected, and the serum concentration of WBP308051 was determined by using 3 bioanalytical ELISA methods. In Method 1 (Fc+Fc), a 96-well ELISA plate was coated with goat anti-human IgG overnight at 4°C, and then serial diluted plasma samples were added. Biotinylated goat anti-human IgG Fc was used as the detection antibody. In Method 2 (PSMA+CD3), a 96-well ELISA plate was coated with human PSMA ECD protein overnight at 4°C, and then serial diluted plasma samples were added. Biotinylated 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 serial diluted plasma samples were added. Biotinylated 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). Non-compartmental pharmacokinetic analysis of the serum concentration of the WBP308051 lead antibody in rats was performed by using Phoenix WINNONLIN software (version 8.1, Pharsight, Mountain View, CA). The linear / log trapezoidal rule was applied to obtain PK parameters.

[0571] As Figure 21 shown in Table 18, for the 10 mg / kg IV PK study using 3 ELISA settings (i.e., coated with Fc, PSMA, or CD3 respectively and detected with Fc, CD3, or PSMA), the mean serum clearance rates of WBP308051 were 6.05, 8.07, and 7.51 mL / day / kg respectively; the mean half-lives were 177, 142, and 132 h respectively; the volume of distribution (Vss) were 59.1, 65.8, and 58.8 mL / kg respectively; and the AUC0-last were 30332, 24087, and 26745 h*μg / mL respectively. The similar results from 3 different ELISA settings indicated the good in vivo stability of W308051.

[0572] Table 18. Summary of Pharmacokinetic Parameters

[0573]

[0574] *When AUC_% Extrap_obs is greater than 20%, t ½ may be inaccurate.

[0575] 8.2 In vivo efficacy of the NPG-hPBMC model

[0576] The in vivo efficacy of WBP308051 was tested in an LNCaP xenograft model of male NPG mice reconstituted with human PBMC. 2x10^6 LNCaP tumor cells were implanted (s.c.) into the right flank of NPG mice. Each mouse also received 2x10^6 PBMC cells by i.p. injection. When the tumors reached approximately 105 mm in volume 3 , the tumor-bearing mice were randomly divided into 9 groups. The 9 groups of mice received subsequent intraperitoneal injections twice a week for a total of 6 injections: vehicle - PBS; 0.30 mg / kg of AMG340; 1.51 mg / kg of AMG 340; 0.057 mg / kg of AMG 160; 0.29 mg / kg of AMG 160; 1.44 mg / kg of AMG 160; 0.08 mg / kg of W308051; 0.4 mg / kg of W308051; 2 mg / kg of W308051. The body weight and tumor growth of the mice were measured twice a week. The tumor volume was calculated using the formula (½ (length × width 2 ). All procedures related to the handling, care, and treatment of animals in this study were conducted in accordance with the guidelines approved by the WuXi Biologics LARC Institutional Animal Care and Use Committee (IACUC), following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

[0577] The results of the tumor growth curves are shown in Figure 22A . The average tumor volume of the PBS group was 1527.3 ± 216.73 mm on day 21 after treatment 3 . Compared with the treatment with PBS, the treatments with 0.057, 0.29, 1.44 mg / kg of AMG160 showed strong anti-tumor effects, with average tumor volumes of 86.63 ± 10.42, 74.85 ± 10.52, 50.36 ± 4.54 mm 3 (TGI = 101.39, 102.21, 103.93%). Compared with the treatment with PBS, the treatments with 0.30 and 1.51 mg / kg of AMG 340 showed anti-tumor effects, with average tumor volumes of 1061.97 ± 296.65 and 441.45 ± 297.15 (TGI = 32.75, 78.53%). The treatments with 0.4 and 2 mg / kg of W308051 showed strong anti-tumor effects, with average tumor volumes of 193.46 ± 264.26, 69.09 ± 10.48 mm 3(TGI = 93.86, 102.63%). However, treatment with 0.08 mg / kg of W308051 did not show antitumor effects, with an average tumor volume of 2022.29 ± 328.64 mm 3 (TGI = -34.84%).

[0578] The body weights of the mice were as Figure 22B shown, and a slight weight loss was observed during the study, which may be due to the GVHD (graft-versus-host disease) effect in the human PBMC reconstitution model.

[0579] In summary, W308051 inhibits the growth of LNCaP cells in vivo in a dose-dependent manner. W308051 induces tumor growth inhibition equivalent to that of equimolar AMG 160 at high dose levels (0.40 and 2.00 mg / kg), and induces stronger tumor growth inhibition than equimolar AMG 340.

[0580] Those skilled in the art will further understand that the present disclosure may be embodied in other specific forms without departing from its spirit or core attributes. Given the foregoing description of the present disclosure only discloses its exemplary embodiments, it should be understood that other variations are considered to be within the scope of the present disclosure. Therefore, the present disclosure is not limited to the specific embodiments described in detail herein. Instead, reference should be made to the appended claims as an indication of the scope and content of the present disclosure.

Claims

1. An isolated antibody or antigen-binding portion thereof that comprises 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.

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

24.

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

4. The isolated antibody or antigen-binding portion thereof according to claim 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.

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

22.

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

30.

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

18.

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

20.

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

34.

10. An isolated antibody or antigen-binding portion thereof according to any one of claims 1-9, which is a monoclonal antibody, a chimeric antibody or a humanized antibody.

11. An isolated antibody or antigen-binding portion thereof according to claim 10, which is a monoclonal antibody.

12. An isolated antibody or antigen-binding portion thereof according to claim 11, which is a human monoclonal antibody.

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

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

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

16. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an isolated antibody or antigen-binding portion thereof according to any one of claims 1-13, 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. A host cell comprising the isolated nucleic acid molecule of claim 16 or the vector of claim 17.

19. A method for preparing an antibody or antigen-binding portion thereof according to any one of claims 1-13, the 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.

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

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

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

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

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

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

26. A kit for treating or diagnosing a proliferative disorder, an immune disorder or an infection, the kit comprising a container containing at least one of the isolated antibodies or antigen-binding portions thereof according to any one of claims 1-13.

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