PSMA antibody and its use

Abstract

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

Description

[Technical Field]

[0001] Related applications This application claims the interests of priority patent application PCT / CN2022 / 134047, filed on 24 November 2022, and priority patent application PCT / CN2022 / 134163, filed on 24 November 2022, the disclosures thereof which are incorporated herein by reference to their entirety. [Background technology]

[0002] Sequence List The contents of the electronic sequence list (70351WO01 Seq List 16 Nov 2023.xml; size: 47 kilobytes; and creation date: November 16, 2023) are incorporated herein by reference to their full text. [Overview of the project]

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

[0004] [Figure 1-1] The SDS-PAGE and SEC-HPLC analyses of the exemplary PSMA antibody W305042 are shown. [Figure 1-2] The SDS-PAGE and SEC-HPLC analyses of the exemplary PSMA antibody W305042 are shown. [Figure 2]This shows the binding of antibodies (W305042, J591, and isotope control) to human PSMA by ELISA. [Figure 3] This shows the binding of antibodies (W305042, J591, and isotope control) to cynomolgus monkey PSMA by ELISA. [Figure 4] This shows the binding of antibodies (W305042, J591, and isotope control) to LNCaP cells by FACS. [Figure 5] This shows the binding of antibodies (W305042, J591, and isotope control) to cynomolgus monkey PSMA-expressing CHO cells as measured by FACS. [Figure 6] This shows the binding of antibodies (W305042 and isotope control) to mouse PSMA as measured by ELISA. [Figure 7] The DSF profile for W305042 is shown. [Figure 8] The HIC-HPLC profile for W305042 is shown. [Figure 9] This is a schematic diagram of the bispecific CD3×PSMA antibody W308051, where T3 represents the anti-CD3 arm, and the heavy chain variable domain of the anti-CD3 arm is fused with a modified human IgG4 TCRβ constant domain (represented by a gray rectangle) containing the S228P mutation, Fc null mutations (F234A, L235A) and knob mutations (S354C-T366W), and the hinge Fc region, as well as the VL of the anti-CD3 arm, which is fused with a modified TCRα constant domain (represented by another gray rectangle); and U5 represents the anti-PSMA arm, and the heavy chain variable domain of the anti-PSMA arm is fused with a human IgG4 hinge Fc region containing the S228P mutation, Fc null mutations (F234A, L235A) and hole mutations (Y349C-T366S-L368A-Y407V), and the VL of the anti-PSMA arm is fused with the CL domain. [Figure 10A] The results of SDS-PAGE (Figure 10A) and SEC-HPLC (Figure 10B) analysis of W308051 are shown. In Figure 10A, the lanes from left to right represent the protein marker, non-reducing antibody, reducing antibody, and protein marker, respectively. [Figure 10B] The results of SDS-PAGE (Figure 10A) and SEC-HPLC (Figure 10B) analysis of W308051 are shown. In Figure 10A, the lanes from left to right represent the protein marker, non-reducing antibody, reducing antibody, and protein marker, respectively. [Figure 11] This shows the binding of antibodies (W308051, AMG160, and isotope hIgG4 control) to human PSMA as measured by ELISA. [Figure 12-1] This shows the binding of antibodies (W308051, AMG340, AMG160, and isotope hIgG4 control) to human C4-2 (high PSMA expression), LNCaP (high PSMA expression), 22Rv1 (low PSMA expression), and PC-3 cells (PSMA negative) as measured by FACS. [Figure 12-2] This shows the binding of antibodies (W308051, AMG340, AMG160, and isotope hIgG4 control) to human C4-2 (high PSMA expression), LNCaP (high PSMA expression), 22Rv1 (low PSMA expression), and PC-3 cells (PSMA negative) as measured by FACS. [Figure 13] This shows the binding of antibodies (W308051, AMG340, AMG160, and isotope hIgG4 control) to CD3-positive Jurcut cells and primary human T cells as measured by FACS. [Figure 14A] Figure 14A shows the binding of antibodies (W308051, AMG160, and isotope hIgG4 control) to cynomolgus monkey PSMA (Figure 14B) as measured by ELISA. [Figure 14B] Figure 14A shows the binding of antibodies (W308051, AMG160, and isotope hIgG4 control) to cynomolgus monkey PSMA (Figure 14B) as measured by ELISA. [Figure 15] This shows the binding of antibodies (W308051, AMG340, AMG160, and isotope hIgG4 control) to cynomolgus monkey PSMA-positive cells as measured by FACS. [Figure 16-1]The T cell cytotoxicity of C4-2 cells, LNCaP cells, and PC-3 cells was demonstrated when co-cultured with CD3+ T cells and incubated with antibodies W308051, AMG340, AMG160, and the isotope hIgG4 control. [Figure 16-2] The T cell cytotoxicity of C4-2 cells, LNCaP cells, and PC-3 cells was demonstrated when co-cultured with CD3+ T cells and incubated with antibodies W308051, AMG340, AMG160, and the isotope hIgG4 control. [Figure 17] This shows cytokine release from C4-2 and PC-3 cells co-cultured with CD3+ T cells and incubated with antibodies W308051, AMG340, AMG160, and the isotope hIgG4 control. [Figure 18-1] This shows cytokine release from C4-2 cells co-cultured with PBMCs and incubated with antibodies W308051, AMG340, AMG160, and the isotope hIgG4 control. [Figure 18-2] This shows cytokine release from C4-2 cells co-cultured with PBMCs and incubated with antibodies W308051, AMG340, AMG160, and the isotope hIgG4 control. [Figure 19] This shows the thermal stability of antibody W308051 as measured by DSF. [Figure 20] The results for antibody W308051, measured by hydrophobic interaction chromatography (HIC-HPLC), are shown. [Figure 21] This shows the mean serum concentration of antibody W308051 in pharmacokinetic studies. [Figure 22A] The in vivo efficacy of antibodies (W308051, AMG340, and AMG160) in the NPG-hPBMC model is shown: (Figure 22A) Tumor growth curve; and (Figure 22B) Body weight of tumor-bearing mice. [Figure 22B]The in vivo efficacy of antibodies (W308051, AMG340, and AMG160) in the NPG-hPBMC model is shown: (Figure 22A) Tumor growth curve; and (Figure 22B) Body weight of tumor-bearing mice. Detailed description of the invention

[0005] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure have the meanings generally understood by those skilled in the art. The singular forms “a,” “an,” and “the” may include multiple references unless the context clearly indicates otherwise. For example, a reference to “a protein” includes multiple proteins, and a reference to “a cell” includes a mixture of cells, and so on. In this application, the use of “or” means “and / or” unless otherwise specified. Furthermore, the use of the term “comprising,” as well as other forms such as “comprises” and “comprised,” is non-limiting. In addition, the scope provided herein and in the appended claims includes all points at the endpoints and between the endpoints of both scopes.

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

[0007] As used herein, the term "antibody" or "Ab" is used in a broad sense and encompasses various antibody constructs including polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies). Intact natural antibodies are generally Y-shaped tetrameric proteins comprising two heavy (H) and two light (L) polypeptide chains linked by disulfide covalent bonds and non-covalent interactions. Light chains of antibodies can be classified into κ and λ light chains. Heavy chains can be classified into μ, δ, γ, α and ε, which define the antibody's isotype as IgM, IgD, IgG, IgA and IgE, respectively. In both light and heavy chains, the variable region is linked to the constant region via a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region / domain (V H ) and a heavy chain constant region / domain (C H ). The heavy chain constant region consists of three domains (C H 1, C H 2 and C H 3). Each light chain consists of a light chain variable region / domain (V L ) and a light chain constant region / domain (C L ). V H and V L regions can be further divided into hypervariable regions (called complementarity determining regions, CDRs), which are interspersed between relatively conserved regions (called framework regions, FRs). Each V H and V L consists of 3 CDRs and 4 FRs in the following order: from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (V H and V L ) of each heavy / light chain pair each form an antigen binding site. The antibody may be an antibody of different antibody isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

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

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

[0010] As used herein, the term “variable domain” in relation to an antibody refers to an antibody variable region or fragment thereof comprising one or more CDRs. A variable domain may comprise a complete variable region (such as an HCVR or LCVR), but it may also comprise fewer regions than a complete variable region, yet still retain the ability to bind to an antigen or form an antigen-binding site.

[0011] As used herein, the term “antigen-binding moiety” refers to an antibody fragment formed from an antibody portion comprising one or more CDRs, or to another antibody fragment that binds to an antigen but does not contain a complete native antibody structure. Examples of antigen-binding moieties 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 moiety can bind to the same antigen to which a parent antibody binds. In some cases, an antigen-binding moiety may comprise one or more CDRs from a particular human antibody grafted onto a framework region from one or more different human antibodies.

[0012] In relation to antibodies, "Fab" refers to the portion of the antibody consisting of a single light chain (both variable and constant regions) that associates the variable and first constant regions of a single heavy chain via disulfide bonds. In some cases, both the light and heavy chain constant regions are replaced by the TCR constant region.

[0013] "F(ab')2" represents the dimer of Fab'.

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

[0015] The "Fc" portion of an antibody represents the part of the antibody consisting of the second (CH2) and third (CH3) constant regions of the primary heavy chain, which are linked to the second and third constant regions of the secondary double chain by disulfide bonds. The Fc portion of an antibody contributes to various effector functions such as ADCC and CDC, but does not function in antigen binding.

[0016] In antibodies, the "hinge region" includes the heavy chain molecule portion that connects the CH1 domain to the CH2 domain. This hinge region consists of approximately 25 amino acid residues, is flexible, and therefore allows the two N-terminal antigen-binding regions to move independently.

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

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

[0019] In relation to antibodies, "Fv" represents the smallest fragment of an antibody that possesses a complete antigen-binding site. An Fv fragment consists of a variable domain of a single heavy chain and a variable domain of a single light chain conjugated to it. Several Fv designs are available, including dsFv, where the association between the two domains is enhanced by an introduced disulfide bond; scFv can be formed using a peptide linker to conjugate the two domains as a single polypeptide. Fv constructs have also been fabricated containing the corresponding variable domain of an immunoglobulin heavy or light chain and the variable domain of an immunoglobulin heavy or light chain associated with a constant domain. Fv can be polymerized, and diabodies and triabodies are also formed.

[0020] A single-chain Fv antibody, or "scFv," refers to a recombinant antibody consisting of a light chain variable region and a heavy chain variable region that are interconnected directly or by a peptide linker sequence.

[0021] In some cases, the "scFv dimer" is a part of V H The part is V of the other part LAnother V is formed to work in conjunction with the other part to form a two-binding site that can target the same antigen (or epitope) or a different antigen (or epitope). H -V L Partial and dimerized V H -V L It is a divalent diabody or divalent ScFv(BsFv) comprising (linked by a peptide linker).

[0022] In some cases, the "scFv dimer" is V H1 and V L1 They work together, V H2 and V L2 V L1 -V H2 V (linked by a peptide linker) and associated with H1 -V L2 It is a double specific deabody comprising (linked by a peptide linker).

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

[0024] "dsFv" represents a disulfide-stabilized Fv fragment in which the bond between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some cases, "(dsFv)2" or "(dsFv-dsFv')" represents a 3-peptide chain linked by a peptide linker (e.g., a long-chain flexible linker) and disulfide bridged, respectively, resulting in 2V L 2V coupled to the part H It comprises a portion thereof. In some cases, dsFv-dsFv' is bispecific, with each disulfide-paired heavy and light chain having different antigen specificities.

[0025] "Additive IgG" refers to a fusion protein that has a Fab arm fused to IgG, forming the bispecific (Fab)2-Fc format. This can generate "IgG-Fab" or "Fab-IgG" which have a Fab fused to the C-terminus or N-terminus of an IgG molecule, with or without a connector. In some cases, additional IgG can be further modified into the IgG-Fab4 format.

[0026] As used herein, the terms “anti-CD3 antibody” or “CD3 antibody” refer to an antibody as defined herein that can bind to CD3, for example, human CD3, to produce a possible therapeutic effect.

[0027] The terms “CD3” and “CD3 protein” are used interchangeably herein. The CD3 protein is present in virtually all T cells. The CD3-TCR complex modulates T cell function in both innate and adaptive immune responses, as well as in cellular and humoral immune functions. These include the elimination of pathogenic microorganisms and the control of tumor growth through broad cytotoxic effects. The CD3 T cell coreceptor is a protein complex consisting of four distinct chains: CD3γ chain, CD3δ chain, and two CD3ε chains. The four chains are associated with the molecule known as the T cell receptor (TCR) and the ζ chain, which generates an activation signal in T lymphocytes. The TCR, ζ chain, and CD3 molecule constitute the TCR complex, with the TCR as a subunit recognizing and binding to antigens, and the CD3 as a subunit transposing antigenic stimuli to signaling pathways and ultimately modulating T cell activity. The term “CD3” may include human CD3, as well as its variants, isoforms, and species homologs. Therefore, the antibodies or their antigen-binding moieties defined and disclosed herein may also bind to CD3 from species other than human, for example, cynomolgus monkey CD3.

[0028] As used herein, the term "human CD3" refers to human-derived CD3, including its complete amino acid sequence.

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

[0030] As used herein, the term “anti-PSMA antibody” refers to an antibody that specifically binds to PSMA. “Anti-PSMA antibodies” may include monovalent antibodies having single specificity. Exemplary anti-PSMA antibodies are described elsewhere in this specification.

[0031] The term "prostate-specific membrane antigen (PSMA)" refers to a type II membrane glycoprotein consisting of 750 amino acids that possesses folate hydrolase and NAALADase enzyme activity.

[0032] As used herein, the term “bivalent” refers to an antibody or antigen-binding fragment having two antigen-binding sites; the term “monovalent” refers to an antibody or antigen-binding fragment having only one single antigen-binding site; the term “polyvalent” refers to an antibody or antigen-binding fragment having multiple antigen-binding sites; the term “monovalent” refers to an antibody or antigen-binding fragment having only one single antigen-binding site. In some cases, an antibody or its antigen-binding portion is bivalent.

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

[0034] As used herein, “Het-mAb” is an IgG-like molecule having four distinct chains; a double chain and two light chains, which can target any two different epitopes on the same or different targets. These chains contain one pair of mutations in the Fc portion of the molecule that promote heavy chain dimerization and one pair of mutations on the Fab portion that promote correct heavy / light chain pairing to form a κ / κ or λ / κ subtype bispecific mAb.

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

[0036] When 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,” and “CD3 × PSMA antibody” all refer to bispecific antibodies that specifically bind to CD3 and PSMA, regardless of the order in which the targets are first mentioned.

[0037] As used herein, the terms “monoclonal antibody” or “mAb” refer to a formulation of an antibody molecule in a single-molecule composition. Monoclonal antibodies exhibit single-binding specificity and affinity for a specific epitope.

[0038] As used herein, the term “human antibody” is intended to include antibodies having a variable region in which both the framework region and the CDR region are derived from a human germline immunoglobulin sequence. Furthermore, if the antibody includes a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of this disclosure may include amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., random or site-directed mutagenic mutations in vitro or mutations induced by somatic mutations in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germ cells of another mammalian species, such as mouse, is grafted onto a human framework sequence.

[0039] The term "humanized antibody" is intended to refer to an antibody in which a CDR sequence derived from germ cells of another mammalian species, such as a mouse, is grafted onto a human framework sequence. Further modifications to the framework region may be performed within the human framework sequence.

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

[0041] 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 with an immunoglobulin gene of another species, an antibody isolated from a recombinant combinatorial antibody library, or an antibody prepared, expressed, produced, or isolated by other means, including splicing an immunoglobulin gene sequence to another DNA sequence.

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

[0043] The term “operatably linked” or “operatably linked” describes the parallel arrangement of two or more biological sequences of interest, with or without spacers or linkers, such that they are in a relationship that enables them to function in the intended manner. When used in reference to polypeptides, the polypeptide sequence is intended to mean that it is linked in such a way that the linked product has the desired biological function. For example, an antibody variable region may be operatably linked to a constant region to provide a stable product with antigen-binding activity. The term may also be used in reference to polynucleotides. As an example, when a polynucleotide encoding a polypeptide is operatably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), the polynucleotide sequence is intended to mean that it is linked in such a way that it enables the regulated expression of the polypeptide from the polynucleotide.

[0044] As used herein, the term “epitope” refers to a portion of an antigen to which an immunoglobulin or antibody specifically binds. “Epitope” is also known as an “antigenic determinant.” Epitopes, or antigenic determinants, generally consist of chemically active surface groups of molecules such as amino acids, carbohydrates, or sugar side chains, and generally possess a specific three-dimensional structure and specific charge properties. For example, an epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique conformation that may be “linear” or “conformal.” In a linear epitope, all interaction sites and interacting molecules (e.g., antibodies) between proteins are linearly located along the primary amino acid sequence of the protein. In a conformal epitope, the interaction sites span amino acid residues that are distant from each other in the protein. For example, testing for 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 these cross-competitions.

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

[0046] K D This is the ratio (k) of the dissociation rate to the association rate, which can be determined by surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (FACS, etc.). off / k on Used to represent ). In some cases, K D The value can be appropriately determined using flow cytometry.

[0047] When used in relation to amino acid sequences (e.g., peptides, polypeptides, or proteins), the terms “fusion” or “fused” refer to the joining of two or more amino acid sequences to a single amino acid sequence that does not exist in nature, for example, by chemical bonding or recombinant means. A fused amino acid sequence may be produced by genetic recombination of a two-coding polynucleotide sequence, and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

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

[0049] As used herein, the term “identity” refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing their sequences. “Identity Percentage” means the percentage of identical residues between amino acids and nucleotides in the molecules being compared, calculated based on the smallest size of the molecules being compared. For these calculations, any gaps in the alignment can be addressed by specific mathematical models or computer programs (i.e., “algorithms”).

[0050] As used herein, the term “immunogenicity” refers to the ability of an organism to stimulate the formation of specific antibodies or sensitized lymphocytes. This term describes not only the properties of an antigen that stimulate specific immune cells to be activated, proliferate, and differentiate to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also the specific immune response in which antibodies or sensitized T lymphocytes may be formed in the immune system of the organism after it has been stimulated by the antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce an immune response in a host depends on three factors: the properties of the antigen, the host's reactivity, and the means of immunization.

[0051] As used herein, the term “substitution” in relation to amino acid residues refers to the innate or induced substitution of one or more amino acids with another amino acid in a peptide, polypeptide, or protein. Substitutions in polypeptides may result in a decrease, enhancement, or elimination of the polypeptide’s function.

[0052] As used herein, the terms “mutation” or “mutated” in relation to amino acid residues refer to the substitution, insertion, or addition of an amino acid residue.

[0053] The native "T cell receptor" or native "TCR" is a heterodimeric T cell surface protein that forms a complex with an immutable CD3 chain that can mediate signal transduction. The TCR belongs to the immunoglobulin superfamily and is similar to a semi-antibody having a single heavy chain and a single light chain. The native TCR has an extracellular portion, a transmembrane portion, and an intracellular portion. The extracellular domain of the TCR has a membrane-proximal constant region and a membrane-distal variable region. In some cases, bispecific antibodies consist of a soluble chimeric protein having the variable domain of the antibody and the constant domain of the TCR, where subunits of the TCR constant domain (such as the α and β domains) are linked by recombinant disulfide bonds.

[0054] As used herein, the term "Ka" is intended to represent the association rate of a particular antibody-antigen interaction, while the term "Kd" is intended to represent the dissociation rate of a particular antibody-antigen interaction. The Kd value for an antibody can be determined using methods well established in the art. D The term "Kd" is intended to represent the dissociation coefficient of a specific antibody-antigen interaction, obtained from the ratio of Kd to Ka (i.e., Kd / Ka), and is intended to be expressed as molar concentration (M). Exemplary methods for determining the Kd of an antibody involve using surface plasmon resonance, such as with biosensor systems like the BIACORE system.

[0055] As used herein, the term "high affinity" for IgG antibodies means 1 × 10⁶ against the target antigen. -7 M or less, for example, 5 × 10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less, or 1 × 10 -9 K below M D This represents an antibody that possesses the characteristic feature.

[0056] The term "EC" is also referred to as "50% effective concentration" when used herein. 50 " represents the concentration of the drug, antibody, or toxin that elicits an intermediate response between baseline and maximum after a specific exposure time. In relation to this application, EC 50 It is expressed in units of "nM".

[0057] As used herein, the term “competing for binding” refers to the interaction of two antibodies in their binding to a binding target. The first antibody competes with the second antibody for binding if the binding of the first antibody to its congeneral epitope is detectedly reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Alternatively, the binding of the second antibody to its epitope may also be detectedly reduced in the presence of the first antibody, but this is not necessarily required. That is, the first antibody can inhibit the binding of the second antibody to its epitope if the second antibody does not inhibit the binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of other antibodies to its congeneral epitopes, then, to the same, greater, or lesser degree, the antibodies are said to “cross-compete” with each other for the binding of their respective epitopes.

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

[0059] As used herein, the term “isolated” refers to a state obtained from its natural state by artificial means. This is possible because, if a particular “isolated” substance or component exists naturally, its natural environment is altered, or the substance is isolated from its natural environment, or both. For example, a particular unisolated polynucleotide or polypeptide exists naturally in the body of a particular living animal, and a highly pure version of the same polynucleotide or polypeptide isolated from such a natural state is called an isolated polynucleotide or polypeptide. The term “isolated” does not exclude mixtures of artificial or synthetic substances, nor other impurities that do not affect the activity of the isolated substance.

[0060] As used herein, the term “isolated antibody” is intended to refer to an antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to the CD3 / PSMA protein substantially does not contain antibodies that specifically bind to antigen proteins other than CD3 / PSMA). However, an isolated antibody that specifically binds to the human CD3 / PSMA protein may have cross-reactivity to other antigens, such as CD3 / PSMA proteins from other species. Furthermore, an isolated antibody may substantially not contain other cellular material and / or chemicals.

[0061] As used herein, the term “vector” refers to a nucleic acid vehicle that may have a polynucleotide inserted therein. A vector is called an expression vector if it enables the expression of a protein encoded by the polynucleotide inserted therein. A vector may have a carried genetic material element expressed in a host cell by transformation, transduction, or transfection of the host cell. A vector may be a plasmid, phage, cosmid, artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC); a phage such as λ phage or M13 phage; and an animal virus. 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, and pavopaviruses (such as SV40). A vector may consist of multiple elements for controlling expression, including, but are not limited to, a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, a vector may include a replication starting point.

[0062] As used herein, the term “host cell” refers to a cell line that can be manipulated to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2 / 0, and 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 specific target cells but also their offspring. Certain modifications may occur in later generations due to mutation or environmental influences, and such offspring may not be identical to the parent cells, but are still included within the scope of the term “host cell.”

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

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

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

[0066] As used herein, the terms “subject” or “individual” or “animal” or “patient” refer to human or non-human animals, including mammals or primates, that require diagnosis, prognosis, improvement, prevention, and / or treatment of a disease or condition. Mammal subjects include humans, domesticated animals, farm animals, and zoo animals, sports animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, and bears.

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

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

[0069] The term "complement-dependent cell injury" or "CDC" refers to the degradation of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) conjugated to these congener antigens. CDC may be performed to assess complement activation.

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

[0071] When used herein in connection with the treatment of a disease, the terms “treatment,” “to treat,” or “treated” generally refer to treatments and therapies, whether in humans or animals, in which several desired therapeutic effects are achieved, such as inhibition of disease progression, including slowing the rate of progression, cessation of progression, alleviation of the disease, improvement of the disease, and cure of the disease. Treatment as a preventive measure (i.e., prophylaxis, prevention) is also included. With respect to cancer, “treatment” may mean weakening or slowing tumor or malignant cell growth, proliferation or metastasis, or some combination thereof. With respect to tumors, “treatment” includes the removal of all or part of the tumor, inhibition or slowing of tumor growth and metastasis, prevention or delay of tumor development, or some combination thereof.

[0072] As used herein, the term “effective dose” refers to an amount or dose of an active compound or substance, or a composition containing an active compound, that is effective in obtaining several desired therapeutic effects when administered according to a desired therapeutic regimen, and that is commensurate with a reasonable risk-benefit ratio. For example, as used in connection with the treatment of CD3 / PSMA-related diseases or conditions, “effective dose” refers to an amount or concentration of an antibody or its antigen-binding moiety that is effective in treating the aforementioned disease or condition.

[0073] When used herein in relation to specific disease conditions in mammals, the terms “prevention” or “prevention” mean preventing or delaying the onset of a disease, or preventing the manifestation of its clinical or subclinical symptoms.

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

[0075] As used herein, the term “pharmaceutically acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmaceutically and / or physiologically compatible with the subject and activator, and includes, but is not limited to, pH adjusters, surfactants, adjuvants and ionic 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 enhancers include, but are not limited to, sodium chloride.

[0076] As used herein, the term “adjuvant” refers to a nonspecific immunostimulant that, when delivered to an organism with or prior to an antigen, can enhance or alter the type of immune response to an antigen in an organism. Examples of adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund’s adjuvants (e.g., complete and incomplete Freund’s adjuvants), Corynebacterium parvum, lipopolysaccharides, cytokines, and similar substances.

[0077] Antibodies and their antigen-binding moieties The antibodies disclosed herein can bind to human PSMA and have the following properties: (a) 1 × 10 -8 K below M D To bind to human PSMA containing the following: (b) CD4 + Inducing the production of cytokines (e.g., IL-2 or IFN-γ) within T cells; (c) First Generation Human CD4 + To promote the proliferation of T cells; (d) Primary human CD4 in the presence of Treg cells + To promote the proliferation of T effector cells; (e) To bind to human or rhesus monkey PSMA, respectively; or (f) Not cross-reactive with human CD40, CD137, and CD271. It has one or more of the following.

[0078] Binding to PSMA can be evaluated using ELISA. Binding specificity can be determined by monitoring the binding of the antibody to cells expressing the PSMA protein, for example, by flow cytometry. For example, it can be tested by a flow cytometry assay in which the antibody reacts with human PSMA-expressing cell lines, such as CHO cells, that have been transfected to express PSMA on the surface of these cells. Binding dynamics (e.g., K dThe binding of antibodies containing (value) can be tested using the BIACORE binding assay. Other suitable binding assays include, for example, ELISA assays using recombinant PSMA protein. For example, the antibody may contain 1 × 10⁶ -8 M or less, 1×10 -9 M or less, 5×10 -10 M or less, 2×10 -10 M or less, 1×10 -10 M or less, 5×10 -11 M or less, 3×10 -11 M or less, or 2 × 10 -11 K below M D It can bind to human PSMA that has [a specific characteristic].

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

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

[0081] The protein sequences disclosed herein can be further used as "query sequences" to search public databases to identify, for example, related sequences. Such searches can be performed using the XBLAST program (version 2.0). A BLAST protein search can be performed using the XBLAST program, score=50, and word length=3 to obtain amino acid sequences that are homologous to antibody molecules in this disclosure. To obtain gapped alignments for comparison purposes, gapped BLAST can be used. When using the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

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

[0083] In some cases, the CDR of an isolated antibody or its antigen-binding moiety may contain two or fewer conservative substitutions of amino acids, or one or fewer amino acids. As used herein, the term “conservative substitution” refers to an amino acid substitution that does not adversely affect or alter the essential properties of a protein / polypeptide comprising an amino acid sequence. For example, conservative substitutions may be introduced by site-directed mutagenesis and PCR-mediated mutagenesis. Examples of conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, for example, a residue that is physically or functionally similar to the corresponding amino acid residue (having chemical properties including similar size, shape, charge, and ability to form covalent or hydrogen bonds). Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with alkaline side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with non-loading side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, corresponding amino acid residues can be substituted with other amino acid residues from the same side chain family.

[0084] In some cases, the primary antigen-binding and secondary antigen-binding regions of a bispecific antibody may associate with each other through knob-into-hole interactions.

[0085] In some cases, the primary and / or secondary antigen-binding moieties are divalent. The term "divalent" indicates the presence of two binding sites in each antigen-binding molecule. This can provide stronger binding to the antigen or epitope than a monovalent counterpart. In some cases, in a divalent antigen-binding moiety, the primary and secondary valencies of the binding sites are structurally identical (i.e., they have the same sequence).

[0086] In some cases, the antibodies and their antigen-binding fragments provided herein are bispecific. In some cases, the bispecific antibodies and their antigen-binding moieties provided herein have primary specificity to PSMA and secondary specificity to a second antigen different from PSMA, and their inhibition may yield a synergistic effect rather than blocking a single antigen alone.

[0087] In some cases, secondary specificity is against tumor-associated antigens or their epitopes. The term “tumor-associated antigen” refers to a target antigen that is expressed by tumor cells but may also be expressed by related cells (or healthy cells) before they become tumors. In some cases, tumor-associated antigens may only be presented by tumor cells and not by normal, i.e., non-tumor cells. In some cases, tumor-associated antigens may be exclusively expressed on tumor cells or may exhibit tumor-specific mutations compared to non-tumor cells. In some cases, tumor-associated antigens may be found in both tumor and non-tumor cells, but when compared to non-tumor cells, they may be overexpressed on tumor cells or antibody binding may be available in tumor cells due to the smaller, more compact structure of tumor tissue compared to non-tumor tissue. In some cases, tumor-associated antigens are present in the vascular system of the tumor.

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

[0089] In some cases, this disclosure includes a bispecific antibody or its antigen-binding moiety comprising an antigen-binding site that specifically binds to CD3 and an antigen-binding site that specifically binds to PSMA. Such an antibody may be referred to herein, for example, as an “anti-CD3 / anti-PSMA” or “anti-CD3 / PSMA” or “anti-CD3×PSMA” or “CD3×PSMA” bispecific antibody, or other similar terms.

[0090] The bispecific antibodies of this disclosure bind to human CD3 and human PSMA with high affinity. The binding of the antibodies of this disclosure to CD3 or PSMA can be evaluated using one or more techniques well established in the art, such as ELISA. The binding specificity of the antibodies of this disclosure can be determined by monitoring the binding of the antibodies to cells expressing CD3 protein or PSMA protein, for example, by flow cytometry. For example, it can be tested by a flow cytometry assay in which the antibodies react with human CD3-expressing cell lines, such as CHO cells, that have been transfected to express CD3 on the surface of these cells. In some cases, binding dynamics (e.g., K) may be used. D The binding of antibodies containing the specified value can be tested using the BIACORE binding assay. Other suitable binding assays include, for example, ELISA or FACS assays using recombinant CD3 protein.

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

[0092] In some cases, bispecific antibodies or their antigen-binding moieties possess the following characteristics: (a) High affinity and specific simultaneous binding to human CD3 and PSMA proteins; (b) Specific binding to human CD3 and / or cynomolgus monkey CD3 protein; (c) Specifically binding to human, mouse, and / or cynomolgus monkey PSMA proteins; (d) Compared to anti-CD3 antibodies, anti-PSMA antibodies, combinations thereof, and other bispecific antibodies targeting CD3 and PSMA, they can induce potent T cell activation in the presence of PSMA-expressing tumor cells; (e) It provides excellent thermal stability and is stable in human serum; and (f) To provide superior antitumor effects compared to anti-CD3 antibodies, anti-PSMA antibodies, combinations thereof, and other bispecific antibodies targeting CD3 and PSMA. It has one or more of the following.

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

[0094] antigen-binding moiety that specifically binds to PSMA The antigen-binding moieties provided herein specifically bind to PSMA and are also referred to herein as PSMA-binding moieties.

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

[0096] In some cases, the antigen-binding portion is: A heavy chain CDR1 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, or an amino acid sequence different from SEQ ID NO: 1 due to the addition, deletion, or substitution of two or fewer amino acids, A heavy chain CDR2 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 2, or an amino acid sequence different from SEQ ID NO: 2 due to the addition, deletion, or substitution of two or fewer amino acids, A heavy chain CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 3, or an amino acid sequence different from SEQ ID NO: 3 due to the addition, deletion, or substitution of one or fewer amino acids, A light chain CDR1 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 4, or an amino acid sequence different from SEQ ID NO: 4 due to the addition, deletion, or substitution of two or fewer amino acids, A light chain CDR2 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 5, or an amino acid sequence different from SEQ ID NO: 5 due to the addition, deletion, or substitution of one or fewer amino acids, A light chain CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 6, or an amino acid sequence different from SEQ ID NO: 6 due to the addition, deletion, or substitution of one or fewer amino acids, It consists of including.

[0097] In some cases, the antigen-binding portion is: a) A heavy chain CDR1 comprising the amino acid sequence represented by Sequence ID No. 1, b) A heavy chain CDR2 comprising the amino acid sequence represented by Sequence ID No. 2, c) A heavy chain CDR3 comprising the amino acid sequence represented by Sequence ID No. 3, d) A light chain CDR1 comprising the amino acid sequence represented by Sequence ID No. 4, e) A light chain CDR2 comprising the amino acid sequence represented by Sequence ID No. 5, f) A light chain CDR3 comprising the amino acid sequence represented by Sequence ID No. 6, It consists of including.

[0098] In some cases, the antigen-binding portion is: a) A heavy chain CDR1 consisting of the amino acid sequence represented by Sequence ID No. 1, b) A heavy chain CDR2 consisting of the amino acid sequence represented by Sequence ID No. 2, c) A heavy chain CDR3 consisting of the amino acid sequence represented by Sequence ID No. 3, d) A light chain CDR1 consisting of the amino acid sequence represented by Sequence ID No. 4, e) A light chain CDR2 consisting of the amino acid sequence represented by Sequence ID No. 5, f) A light chain CDR3 consisting of the amino acid sequence represented by Sequence ID No. 6, It consists of including.

[0099] In some cases, the heavy chain variable region of the antigen-binding portion is: (i) The amino acid sequence of Sequence ID No. 15, and (ii) an amino acid sequence (including, for example, the CDR disclosed above) that is at least 85% identical to SEQ ID NO: 15, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and at the same time maintains binding specificity to PSMA; or (iii) an amino acid sequence having the addition, deletion and / or substitution of one or more amino acids (e.g., 1-18, 1-15, 1-10, or 1-5) compared to SEQ ID NO: 15, while simultaneously maintaining binding specificity to PSMA (e.g., including the CDR disclosed above), It consists of including.

[0100] In some cases, the variable region of the light chain at the antigen-binding site is: (i) The amino acid sequence of Sequence ID No. 16, and (ii) an amino acid sequence (including, for example, the CDR disclosed above) that is at least 85% identical to SEQ ID NO: 16, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and at the same time maintains binding specificity to PSMA; or (iii) an amino acid sequence having the addition, deletion and / or substitution of one or more amino acids (e.g., 1-16, 1-15, 1-10, or 1-5) compared to SEQ ID NO: 16, while simultaneously maintaining binding specificity to PSMA (e.g., including the CDR disclosed above), It consists of including.

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

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

[0103] In some cases, the antigen-binding portion consists of two polypeptide chains: i) A double helix comprising an amino acid sequence having at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19, and simultaneously maintaining binding specificity with PSMA (e.g., including the CDR and / or variable region disclosed above); and ii) A second light chain comprising an amino acid sequence having at least 85%, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and at the same time maintaining binding specificity with PSMA (including, for example, the CDR and / or variable region disclosed above), It consists of including.

[0104] In some cases, the antigen-binding portion consists of two polypeptide chains: i) The double helix represented by Sequence ID 19; and ii) The second light chain represented by Sequence ID No. 20, It consists of including.

[0105] In some cases, the antigen-binding portion consists of two polypeptide chains: i) The double helix represented by Sequence ID 19; and ii) The second light chain represented by Sequence ID No. 20, It consists of.

[0106] In some cases, the antigen-binding portion consists of two polypeptide chains: i) A double helix comprising an amino acid sequence having at least 85%, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 31, and at the same time maintaining binding specificity with PSMA (including, for example, the CDR and / or variable region disclosed above); and ii) A second light chain comprising an amino acid sequence having at least 85%, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 32, and at the same time maintaining binding specificity with PSMA (including, for example, the CDR and / or variable region disclosed above), It consists of including.

[0107] In some cases, the antigen-binding portion consists of two polypeptide chains: i) The double hemisphere represented by Sequence ID No. 31; and ii) The second light chain represented by Sequence ID No. 32, It consists of including.

[0108] In some cases, the antigen-binding portion consists of two polypeptide chains: i) The double hemisphere represented by Sequence ID No. 31; and ii) The second light chain represented by Sequence ID No. 32, It consists of.

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

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

[0111] In some cases, the antigen-binding portion is: a) A heavy chain CDR1 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 7, or an amino acid sequence different from SEQ ID NO: 7 due to the addition, deletion, or substitution of two or fewer amino acids, b) A heavy chain CDR2 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 8, or an amino acid sequence different from SEQ ID NO: 8 due to the addition, deletion, or substitution of two or fewer amino acids, c) A heavy chain CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 9, or an amino acid sequence different from SEQ ID NO: 9 due to the addition, deletion, or substitution of two or fewer amino acids, d) A light chain CDR1 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 10, or an amino acid sequence different from SEQ ID NO: 10 due to the addition, deletion, or substitution of two or fewer amino acids, e) A light chain CDR2 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 11, or an amino acid sequence different from SEQ ID NO: 11 due to the addition, deletion, or substitution of one or fewer amino acids, f) A light chain CDR3 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 12, or an amino acid sequence different from SEQ ID NO: 12 due to the addition, deletion, or substitution of one or fewer amino acids, It consists of including.

[0112] In some cases, the antigen-binding portion is: a) A heavy chain CDR1 comprising the amino acid sequence represented by Sequence ID No. 7, b) A heavy chain CDR2 comprising the amino acid sequence represented by Sequence ID No. 8, c) A heavy chain CDR3 comprising the amino acid sequence represented by Sequence ID No. 9, d) A light chain CDR1 comprising the amino acid sequence represented by Sequence ID No. 10, e) A light chain CDR2 comprising the amino acid sequence represented by Sequence ID No. 11, f) A light chain CDR3 comprising the amino acid sequence represented by Sequence ID No. 12, It consists of including.

[0113] In some cases, the antigen-binding portion is: a) A heavy chain CDR1 consisting of the amino acid sequence represented by Sequence ID No. 7, b) A heavy chain CDR2 consisting of the amino acid sequence represented by Sequence ID No. 8, c) A heavy chain CDR3 consisting of the amino acid sequence represented by Sequence ID No. 9, d) A light chain CDR1 consisting of the amino acid sequence represented by Sequence ID No. 10, e) A light chain CDR2 consisting of the amino acid sequence represented by Sequence ID No. 11, f) A light chain CDR3 consisting of the amino acid sequence represented by Sequence ID No. 12, It consists of including.

[0114] In some cases, the heavy chain variable region of the antigen-binding portion is: (i) The amino acid sequence of Sequence ID No. 13, and (ii) an amino acid sequence (including, for example, the CDR disclosed above) that is at least 85% identical to SEQ ID NO: 13, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and at the same time maintains binding specificity to CD3; or (iii) an amino acid sequence having the addition, deletion, and / or substitution of one or more amino acids (e.g., 1-18, 1-15, 1-10, or 1-5) compared to SEQ ID NO: 13, while simultaneously maintaining binding specificity to CD3 (e.g., including the CDR disclosed above). It consists of including.

[0115] In some cases, the variable region of the light chain at the antigen-binding site is: (i) The amino acid sequence of Sequence ID No. 14, and (ii) an amino acid sequence (including, for example, the CDR disclosed above) that is at least 85% identical to SEQ ID NO: 14, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and at the same time maintains binding specificity to CD3; or (iii) an amino acid sequence having the addition, deletion and / or substitution of one or more amino acids (e.g., 1-17, 1-15, 1-10, or 1-5) compared to SEQ ID NO: 14, while simultaneously maintaining binding specificity to CD3 (e.g., including the CDR disclosed above), It consists of including.

[0116] In some cases, 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.

[0117] In some cases, the antigen-binding portion consists of two polypeptide chains: i) A single heavy chain comprising an amino acid sequence having at least 85%, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 17, and at the same time maintaining binding specificity with CD3 (including, for example, the CDR and / or variable region disclosed above); and ii) A first light chain comprising an amino acid sequence having at least 85%, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 18, and at the same time maintaining binding specificity to CD3 (including, for example, the CDR and / or variable region disclosed above), It consists of including.

[0118] In some cases, the antigen-binding portion consists of two polypeptide chains: i) Single heavy chain represented by Sequence ID No. 17; and ii) The first light chain represented by Sequence ID No. 18, It consists of including.

[0119] In some cases, the antigen-binding portion consists of two polypeptide chains: i) Single heavy chain represented by Sequence ID No. 17; and ii) The first light chain represented by Sequence ID No. 18, It consists of.

[0120] In some embodiments, the heavy chain variable region of the antigen-binding moiety is operably linked to human IgG Fc regions, particularly human IgG4 or IgG1 Fc regions, such as human IgG4 Fc regions containing the S228P mutation, Fc null mutations (F234A L235A), knob mutations (S354C-T366W), and / or hole mutations (Y349C-T366S-L368A-Y407V), and C1 and hinged Fc regions. In some cases, the VH region is fused to the hinged Fc region of human IgG4 having a modified TCRβ constant domain as well as the S228P mutation, Fc null mutation (F234A L235A), and knob mutation (S354C-T366W). In some cases, the VH region is fused with the hinge Fc region of human IgG4 having the S228P mutation, Fc null mutation (F234A L235A), and hole mutation (Y349C-T366S-L368A-Y407V).

[0121] TCR steady state The human TCRβ chain constant region has two distinct variants known as TRBC1 and TRBC2 (IMGT nomenclature). In this disclosure, the sequence of the wild-type TCRβ domain has the NCBI accession number A0A5B9. The modified TCRβ constant domain in this disclosure is: [ka] That is the case.

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

[0123] The human TCRα chain constant region is known as TRAC and has the NCBI accession number P01848. The modified TCRα constant domain of this disclosure is: [ka] That is the case.

[0124] In some cases, 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 cases, C2 comprises or comprises the modified TCRα constant region represented by SEQ ID NO: 30.

[0125] In this disclosure, the first and second TCR constant regions of the polypeptide complex provided herein can form a dimer comprising at least one unnatural interchain bond between the TCR constant regions that can stabilize the dimer.

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

[0127] Interchain bonds are formed between one amino acid residue on a TCR constant region and another amino acid residue on another TCR constant region. In some cases, unnatural interchain bonds can be any bond or interaction that can associate two TCR constant regions into a dimer. Suitable examples of unnatural interchain bonds include disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridges, or hydrophilic-hydrophobic interactions, knob-into-holes, or combinations thereof.

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

[0129] As used herein, “unnatural” interchain links refer to interchain links not found in the natural association of the natural counterpart TCR constant regions. For example, unnatural interchain links can be formed between a mutant amino acid residue and a natural amino acid residue, each present on the respective TCR constant region; or between two mutant amino acid residues, each present on the TCR constant region. In some cases, at least one unnatural interchain link is formed between a first mutant residue in the first TCR constant region of a polypeptide complex and a second mutant residue in the second TCR constant region.

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

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

[0132] Creation of antibody-producing transfectomas The antibodies of this disclosure can also be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection methods. In some cases, DNA encoding a portion or full-length light and heavy chain obtained by standard molecular biochemistry techniques is inserted into one or more expression vectors so that the gene is operably ligated to transcriptional and translational regulatory sequences. In this context, the term “operably ligated” is intended to mean ligating the antibody gene to the vector so that the transcriptional and translational regulatory sequences within the vector perform these desired functions of regulating the transcription and translation of the antibody gene.

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

[0134] For the expression of light and heavy chains, expression vectors encoding the light and heavy chains are transfected into host cells using standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and similar methods. It is possible to express the antibodies of the present invention in either prokaryotic or eukaryotic host cells capable of assembling and secreting properly folded and immunoactive antibodies, for example, in mammalian host cells.

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

[0136] Production of bispecific antibodies The bispecific antibodies and antigen-binding fragments provided herein can be prepared using any suitable method, for example, by co-expressing two immunoglobulin heavy-light chain pairs in host cells to produce bispecific antibodies by recombinant method, which can then be purified by affinity chromatography.

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

[0138] Alternatively, leucine zipper peptides from Fos and Jun proteins can be linked to the Fab' moieties of two different antibodies via gene fusion. The linked antibodies are then reduced to four half-antibodies (i.e., monomers) in the hinge region, and subsequently reoxidized to produce heterodimers.

[0139] Two antigen-binding sites may be joined or tolerantly linked to produce a bispecific antibody or antigen-binding fragment. For example, one antibody can be bound to biotin, while the other antibody can be bound to avidin; the strong association of biotin and avidin will complex the two antibodies to produce a bispecific antibody.

[0140] Bispecific antigen-binding fragments may be prepared from bispecific antibodies, for example, by cleavage through proteolysis or by chemical bonding. For example, antigen-binding fragments of antibodies (e.g., Fab 5 Prepare ) and convert it to a Fab'-thiol derivative, then another converted Fab with different antigen specificity 5 The derivative may be reacted with the derivative to produce a bispecific antigen-binding fragment.

[0141] nucleic acid molecules encoding antibodies in this disclosure In some embodiments, the Disclosure relates to an isolated nucleic acid molecule, wherein the isolated nucleic acid molecule comprises a nucleic acid sequence encoding a bispecific antibody or antigen-binding moiety as disclosed herein, for example, a combination of the heavy chain or light chain sequences of SEQ ID NOs. 35 and 36, or SEQ ID NOs. 37 and 38. For example, the nucleic acid sequence may encode the heavy chain and / or light chain of a bispecific antibody, and for example, the nucleic acid sequence may comprise all sequences of SEQ ID NOs. 35 to 38.

[0142] The isolated nucleic acid molecule encoding the heavy chain variable region of the CD3 binding site is: (A) Nucleic acid sequence encoding the heavy chain variable region described in Sequence ID No. 13; (B) Nucleic acid sequence described in Sequence ID No. 21; and (C) Nucleic acid sequences hybridized with the complementary strand of nucleic acid sequence (A) or (B) under high stringency conditions. It may include nucleic acid sequences selected from the following.

[0143] The isolated nucleic acid molecule encoding the light chain variable region of the CD3 binding site is: (A) A nucleic acid sequence encoding the light chain variable region described in Sequence ID No. 14; (B) Nucleic acid sequence described in Sequence ID No. 22; and (C) Nucleic acid sequences hybridized with the complementary strand of nucleic acid sequence (A) or (B) under high stringency conditions. It may include nucleic acid sequences selected from the following.

[0144] The isolated nucleic acid molecule encoding the heavy chain variable region of the PSMA binding site is: (A) Nucleic acid sequence encoding the heavy chain variable region described in Sequence ID No. 15; (B) Nucleic acid sequence described in Sequence ID No. 23; and (C) Nucleic acid sequences hybridized with the complementary strand of nucleic acid sequence (A) or (B) under high stringency conditions. It may include nucleic acid sequences selected from the following.

[0145] The isolated nucleic acid molecule encoding the light chain variable region of the PSMA binding site is: (A) A nucleic acid sequence encoding the light chain variable region described in Sequence ID No. 16; (B) Nucleic acid sequence described in Sequence ID No. 24; and (C) Nucleic acid sequences hybridized with the complementary strand of nucleic acid sequence (A) or (B) under high stringency conditions. It may include nucleic acid sequences selected from the following.

[0146] In some cases, the present disclosure is an isolated nucleotide sequence encoding the heavy chain of the CD3 binding moiety, wherein the isolated nucleotide sequence encoding the heavy chain of the CD3 binding moiety is: (A) Nucleic acid sequence encoding the heavy chain as described in Sequence ID No. 17; (B) Nucleic acid sequence described in Sequence ID No. 25 or 35; or (C) A nucleic acid sequence hybridized with the complementary strand of the nucleic acid sequence of (A) or (B) under high stringency conditions. It consists of including or comprising

[0147] In some cases, the present disclosure is an isolated nucleotide sequence encoding the light chain of the CD3 binding moiety, wherein the isolated nucleotide sequence encoding the light chain of the CD3 binding moiety is: (A) Nucleic acid sequence encoding the light chain as described in Sequence ID No. 18; (B) Nucleic acid sequence described in Sequence ID No. 26 or 36; or (C) A nucleic acid sequence hybridized with the complementary strand of the nucleic acid sequence of (A) or (B) under high stringency conditions. It consists of including or comprising

[0148] In some cases, the present disclosure is an isolated nucleotide sequence encoding the heavy chain of the PSMA binding moiety, wherein the isolated nucleotide sequence encoding the heavy chain of the PSMA binding moiety is: (A) Nucleic acid sequence encoding the heavy chain as described in Sequence ID No. 19; (B) Nucleic acid sequence described in Sequence ID No. 27 or 37; or (C) A nucleic acid sequence hybridized with the complementary strand of the nucleic acid sequence of (A) or (B) under high stringency conditions. It consists of including or comprising

[0149] In some cases, the present disclosure is an isolated nucleotide sequence encoding the light chain of the PSMA binding moiety, wherein the isolated nucleotide sequence encoding the light chain of the PSMA binding moiety is: (A) Nucleic acid sequence encoding the light chain as described in Sequence ID No. 20; (B) Nucleic acid sequence described in Sequence ID No. 28 or 38; or (C) A nucleic acid sequence hybridized with the complementary strand of the nucleic acid sequence of (A) or (B) under high stringency conditions. It consists of including or comprising

[0150] In some cases, the present disclosure is an isolated nucleotide sequence encoding the heavy chain of the PSMA binding moiety, wherein the isolated nucleotide sequence encoding the heavy chain of the PSMA binding moiety is: (A) Nucleic acid sequence of sequence number 33; (B) A nucleic acid sequence encoding the amino acid sequence described in Sequence ID No. 31; or (C) A nucleic acid sequence hybridized with the complementary strand of the nucleic acid sequence of (A) or (B) under high stringency conditions. It consists of including or comprising

[0151] In some cases, the present disclosure is an isolated nucleotide sequence encoding the light chain of the PSMA binding moiety, wherein the isolated nucleotide sequence encoding the light chain of the PSMA binding moiety is: (A) Nucleic acid sequence of sequence number 34; (B) A nucleic acid sequence encoding the amino acid sequence described in Sequence ID No. 32; or (C) A nucleic acid sequence hybridized with the complementary strand of the nucleic acid sequence of (A) or (B) under high stringency conditions. It consists of including or comprising

[0152] In some embodiments, this disclosure relates to vectors comprising nucleic acid sequences disclosed herein. In some cases, the expression vector further comprises a nucleotide sequence encoding the constant region of a bispecific antibody.

[0153] The vectors relating to this disclosure may be any suitable vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences comprising an appropriate set of expression regulatory elements). Examples of such vectors include SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In some cases, CD3 or PSMA antibody-coding nucleic acids may be contained in naked DNA or RNA, and examples include linear expression elements, compact nucleic acid vectors, plasmid vectors such as pBR322, pUC 19 / 18, or pUC 118 / 119, "midge" minimal-size nucleic acid vectors, or precipitated nucleic acid vector constructs such as CaP04 precipitated constructs.

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

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

[0156] The nucleic acid and / or vector may contain a nucleic acid sequence encoding a secretion / localization sequence that can target polypeptides, such as nascent polypeptide chains, into the pericellular lumen or cell culture medium. Examples of such sequences include secretion leaders or signal peptides.

[0157] The vector described above may contain or be associated with any suitable promoter, enhancer, and other expression-enhancing elements. Examples of such elements include potent expression promoters (e.g., human CMV IE promoter / enhancer, as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly(A) stop sequences, replication start sites for plasmid production in Escherichia coli (E. coli), antibiotic resistance genes as selectable markers, and / or convenient cloning sites (e.g., polylinkers). The nucleic acid may also contain an inducible promoter, as opposed to a constitutive promoter such as CMV IE.

[0158] In a further embodiment, this disclosure relates to a host cell comprising the vector defined in this specification above.

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

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

[0161] 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 cases, the host cells may comprise first and second nucleic acid constructs stably integrated into the cell genome. In some cases, the disclosure provides cells comprising linear expression elements comprising non-integrated nucleic acids such as plasmids, cosmids, phagemids, or the first and second nucleic acid constructs defined above.

[0162] In a further embodiment, the 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 Disclosure.

[0163] In a further embodiment, the Disclosure relates to a hybridoma that produces antibodies for use in the bispecific antibodies of the Disclosure as defined herein. In a further embodiment, the 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 antibodies for use in the bispecific antibodies or the bispecific antibodies of the Disclosure.

[0164] In one aspect, this disclosure is: (i) A nucleic acid sequence that encodes the heavy chain variable region of the first antigen-binding moiety and / or the heavy chain variable region of the second antigen-binding moiety, and optionally further encodes a CH1 domain or a CL domain, according to any one of the examples or embodiments disclosed herein; (ii) Nucleic acid sequences encoding the light chain variable region of the first antigen-binding moiety and / or the heavy chain variable region of the light chain variable region of the second antigen-binding moiety, according to any one of the examples or embodiments disclosed herein; (iii) Nucleic acid sequences encoding a modified TCRβ constant domain or a modified TCRα constant domain; (iv) Nucleic acid sequence encoding the Fc region; (v) Nucleic acid sequence encoding the linker; or (vi) at least two combinations of the above, This relates to an expression vector comprising the following:

[0165] In one aspect, the disclosure relates to nucleic acid constructs encoding one or more amino acid sequences listed in a sequence list.

[0166] In one aspect, the present disclosure relates to a method for producing a bispecific antibody according to any example or embodiment disclosed herein, the method comprising the steps of culturing a host cell disclosed herein comprising an expression vector or a plurality of expression vectors expressing the bispecific antibody disclosed herein, and purifying the antibody from the culture medium. In one aspect, the present disclosure relates to a host cell comprising the expression vector defined above. In some cases, the host cell is a recombinant eukaryotic host cell, a recombinant prokaryotic host cell, or a recombinant microbial host cell.

[0167] Pharmaceutical composition In some embodiments, this disclosure relates to pharmaceutical compositions comprising at least one antibody or its antigen-binding moiety as disclosed herein and a pharmaceutically acceptable carrier.

[0168] Composition components The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of this disclosure may also be administered in combination therapy with, for example, another immunostimulant, anticancer drug, antiviral drug, or vaccine, thereby enhancing the immune response to the vaccine with the anti-CD3 / anti-PSMA bispecific antibody. Examples of pharmaceutically acceptable carriers include pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspensions / dispersants, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, components, and many other various combinations.

[0169] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, fragrances, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene, and / or propylgalacte. As disclosed herein, in a solvent containing the antibody or antigen-binding fragment of this disclosure, the composition may contain one or more antioxidants, such as methionine, to oxidize the reducing antibody or its antigen-binding moiety. Redox oxidation can prevent or reduce binding affinity, thereby enhancing antibody stability and extending shelf life. In some cases, this disclosure provides one or more antibodies or their antigen-binding moieties and one or more antioxidants, such as methionine. This disclosure further provides various methods for mixing an antibody or its antigen-binding moiety with one or more antioxidants such as methionine, thereby preventing oxidation of the antibody or its antigen-binding moiety and extending their shelf life and / or increasing their activity.

[0170] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous media such as plant-derived fixative oils, cottonseed oil, corn oil, sesame oil, or peanut oil, or antimicrobial agents in bacteriostatic or fungiostatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphoric acid or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspensions and dispersants such as carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (Tween 80); and metal ion sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers may be added to pharmaceutical compositions in multi-dose containers containing phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methyl p-hydroxybenzoate and propyl esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients include, for example, water, physiological saline, dextrose, glycerol, or ethanol. Suitable non-toxic additives include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, dissolution accelerators, or sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0171] Administration, formulation, and dosage The pharmaceutical compositions of this disclosure may be administered to the target subject by various routes, including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, ventricular, intratracheal, intrabuccal, rectal, intraperitoneal, intradermal, topical, transdermal, and subarachnoid, or by other routes including implantation or inhalation. The compositions of the subject may be formulated into solid, semi-solid, liquid, or gaseous preparations, including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. The appropriate formulation and route of administration may be selected according to the intended application and treatment regimen.

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

[0173] Suitable formulations for parenteral administration (e.g., by injection) include aqueous or nonaqueous, isotonic, heat-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is provided dissolved, suspended, or otherwise (e.g., in liposomes or other microparticles). Such liquids may further contain other pharmaceutically acceptable components such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspensions, thickeners, and solutes that give the formulation isotonic with the blood (or other relevant body fluids) of the recipient of the interest. Examples of excipients include, for example, water, alcohol, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include sodium chloride injection, Ringer's solution, or Ringer's lactate injection. Similarly, specific administration regimens, including dose, timing, and repetition, will depend on the specific individual and their medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

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

[0175] In some cases, the antibody or antigen-binding portion thereof of the present disclosure may be administered within a suitable range, and examples of the above range include from about 5 μg / kg body weight to about 100 mg / kg body weight per administration; from about 50 μg / kg body weight to about 5 mg / kg body weight per administration; and from about 100 μg / kg body weight to about 10 mg / kg body weight per administration. Other ranges include from about 100 μg / kg body weight to about 20 mg / kg body weight per administration and from about 0.5 mg / kg body weight to about 20 mg / kg body weight per administration. In some cases, 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, or at least about 10 mg / kg body weight per administration.

[0176] In some cases, a course of treatment comprising the antibody or antigen-binding portion thereof of the present disclosure will comprise multiple doses of the selected drug product over a period of weeks or months. More specifically, the antibody or antigen-binding portion thereof of the present disclosure may be administered once daily, once every 2 days, once every 4 days, once weekly, once every 10 days, once every 2 weeks, once every 3 weeks, once monthly, once every 6 weeks, once every 2 months, once every 10 weeks, or once every 3 months. In this regard, it will be understood that the dose may be changed, or the interval may be adjusted based on the patient's response and clinical practice.

[0177] Dosages and regimens may be determined empirically for the therapeutic compositions disclosed herein for a subject administered one or more doses. For example, a subject may be administered escalating doses of a therapeutic composition manufactured as described herein. In some cases, the dosage may be gradually increased, or gradually decreased or reduced based on empirically determined or observed side effects or toxicity, respectively. To assess the efficacy of a selected composition, markers of a particular disease, disorder or condition can be monitored as previously described. For cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; improvement as assessed by direct tumor biopsy and microscopic examination of the tumor sample; measurement of indirect tumor markers (e.g., PSA for prostate cancer) or oncogenic antigens identified by the methods described herein, reduction in pain or anesthesia; improved speech, vision, breathing or other physical impairment associated with a tumor; increased appetite; or improved quality of life or prolonged survival as measured by accepted tests. 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 prior and concurrent therapies being used.

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

[0179] Medical Use / Methods In some embodiments, the Disclosure provides a method for treating a disorder in a subject, the method comprising administering a therapeutically effective amount of an antibody or its antigen-binding moiety disclosed herein to a subject in need of treatment (e.g., a mammal, e.g., a human). For example, the disorder is cancer.

[0180] Various cancers involving PSMA, whether malignant or benign, primary or secondary, may be treated or prevented using the methods provided herein. The cancers may be solid tumors. Examples of such cancers include lung cancers such as bronchogenic cancers (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondrolytic hamartoma (non-cancerous), and sarcoma (cancerous); kidney cancer; breast cancer; gastric cancer; colorectal cancer; glioblastoma; prostate cancer; pancreatic cancer; or ovarian cancer. In some cases, the cancer is prostate cancer, particularly metastatic castration-resistant prostate cancer (mCRPC).

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

[0182] The terms “anticancer agent” or “antiproliferative agent” mean a drug that can be used to treat cell proliferation disorders such as cancer, and include, but are not limited to, cytotoxic agents, cell proliferation inhibitors, angiogenesis inhibitors, tumor debulking agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and antimetastatic and immunotherapeutic agents. It will be understood that, in some cases as discussed above, such anticancer agents may include a conjugate and may be associated with a site-specific antibody disclosed prior to administration. More specifically, in some cases, a selected anticancer agent is linked to a non-paired cysteine ​​of an antibody engineered to provide the engineered conjugate described herein. Thus, such engineered conjugates are expressly intended to be within the scope of this disclosure. In some cases, the disclosed anticancer agents are given in combination with site-specific conjugates comprising the different therapeutic agents described above.

[0183] As used herein, the term “cytotoxic agent” means a substance that is toxic to cells, impairs or inhibits cellular function, and / or causes cell destruction. In some cases, such substances are naturally occurring molecules of biological origin. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas aeruginosa toxin and exotoxin, Staphylococcus enterotoxin A), fungi (e.g., α-sarcin, restrictosin), plants (e.g., abrin, lysine, modesin, biscamin, porkweed antiviral protein, saporin, geronin, momorizin, tricosatin, barley toxin, tungstenol protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitors, curcin, crotin, chlorambu officinalis inhibitors, geronin, mitegerin, restrictosin, phenomycin, neomycin, and trichothecenes) or animals (e.g., cytotoxic RNases such as extracellular pancreatic RNases; DNase I including fragments and / or variants thereof).

[0184] For the purposes of this disclosure, “chemotherapeutic agents” include chemical compounds (e.g., cytotoxic or cell proliferation inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemical agents often target intracellular processes necessary for cell growth or differentiation and are therefore particularly effective against cancer cells that grow and differentiate rapidly in general. For example, vincristine depolymerizes microtubules and thus inhibits cells from entering mitosis. In general, chemotherapeutic agents include chemical agents that inhibit or are designed to inhibit cancer cells or cells that are likely to become cancerous or produce oncogenic progeny. Such agents are often administered in combination, for example, in regimens such as CHOP or FOLFIRI, and are often most effective in this way.

[0185] Examples of anticancer agents (either site-specific complexes or non-complexes) that may be used in combination with the site-specific constructs of this disclosure include abiraterone, apalutamide, bicalutamide, alkylating agents, alkyl sulfons, aziridines, ethyleneimines and methylamelamines, acetogenins, camptothecin, bryostatin, calistatin, CC-1065, cryptophycins, dorastatin, duocalmycin, eloiterobin, pancratistatin, sarcodicin, spongstatin, nitrogen mustard, antibiotics, enediine antibiotics, dinemisin, bisphosphonates, esperamisin, chromoprotein enediine antibiotic chromophores, acrasinomycins, actinomycin, ausuramycin, azaserin, bleomycin, kakutinomycin, carabicin, carminomycin, cardinophilin, chromomycins, dactinomycin, daunorubicin, and Torubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, mycophenolic acids, nogaramycin, olibomycins, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin; antimetabolites, erlotin Nib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, anti-adrenal agents, folic acid supplements such as phloric acid, acegraton, aldofamide glycoside, aminolevulinic acid, enyluracil, amsacrin, bestrabusil, bisanthren, edatraxate, dehofamine, demecoltin, diazicon, elfornithine, eriptinium acetate, epotilon, etoglucide, gallium nitrate;Hydroxyurea, Lentinan, Ronidynin, Maytansinoids, Mitoguazone, Mitoxantrone, Mopidamol, Nitraeline, Pentostatin, Fenamet, Pirarubicin, Rosoxantrone, Podophyllic Acid, 2-Ethylhydrazide, Procarbazine, PSK Polysaccharide Complex (JHS Natural Products (Eugene, Oregon, USA): Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadiquan; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, beraclin A, loridine A and anguidin); Urethanes; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, Chlorambucil; Gemzaar gemcitabine; 6-Thiogunine; Mercaptopurine; Methotrexate; Platinum analogs, Vinblastine, Platinum; E Examples of pharmaceutically acceptable substances include, but are not limited to, toposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (camptosar, CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; PKC-α inhibitors, Raf, H-Ras, cell proliferation-reducing EGFR and VEGF-A, and any pharmaceutically acceptable salts, acids, or derivatives of any of the above. Furthermore, this definition includes anti-hormone agents that modulate or inhibit hormonal activity on tumors, such as anti-estrogens and selective estrogen receptor modulators; aromatase inhibitors that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal gland; and anti-androgens; as well as 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 derivatives, or any pharmaceutically acceptable salt, acid, or derivative thereof.

[0186] Combined use with radiation therapy This disclosure also provides combinations of an antibody or its antigen-binding moiety with radiotherapy (i.e., any mechanism for locally inducing DNA damage within tumor cells, such as gamma-ray radiation, X-rays, UV radiation, microwaves, electron radiation, and similar). Combination therapies using targeted delivery of radioisotopes to tumor cells are also intended, and the conjugates of this disclosure may be used in conjunction with targeted anticancer agents or other targeting means. Typically, radiotherapy is administered in pulses over a period of about 1 to 2 weeks. Radiotherapy may be administered to subjects with head and neck cancer for about 6 to 7 weeks. If necessary, radiotherapy may be administered as a single dose, multiple doses, or sequentially.

[0187] Pharmaceutical packs and kits Pharmaceutical packs and kits are also provided, comprising one or more containers and comprising one or more doses of an antibody or its antigen-binding moiety. In some cases, the unit dose is provided, comprising a predetermined amount of a composition comprising, for example, an antibody or its antigen-binding moiety, with or without one or more additional agents. In some cases, such unit doses are supplied in single-use prefill syringes for injection. In some cases, the composition contained in the unit dose comprises a buffer such as saline, sucrose, or the like; phosphate, or the like; and / or may be formulated within a stable and effective pH range. Alternatively, in some cases, the complex composition may be provided as a lyophilized powder that can be re-prepared upon addition of a suitable liquid, such as sterile water or saline. In some cases, the composition comprises, but is not limited to, one or more substances that inhibit protein aggregation, including sucrose and arginine. Labels on or associated with the containers indicate that the enclosed complex composition is for use in treating a selected neoplastic disease condition.

[0188] This disclosure also provides site-specific conjugates and, optionally, kits for obtaining single or multiple doses of one or more anticancer agents. The kit comprises a container and a label or accompanying information on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and others. The container may be molded from a variety of materials, such as glass or plastic, and may contain a pharmaceutically effective amount of the conjugate of this disclosure, either in a conjugate or unconjugated form. In some cases, the container may include a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured with a subcutaneous needle). Such a kit would, in general, comprise a pharmaceutically acceptable formulation of the manipulated conjugate and, optionally, one or more anticancer agents in the same or different containers, in a suitable container. The kit may also comprise other pharmaceutically acceptable formulations for either diagnostic or combination therapy. For example, in addition to the antibody or antigen-binding portion of the present disclosure, such kit may comprise one or more anticancer agents, such as chemotherapeutic agents or radiotherapy agents; angiogenesis inhibitors; antimetastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents.

[0189] More specifically, the kit may have a single container comprising the disclosed antibody or its antigen-binding moiety, with or without additional components, or it may have different containers for each desired drug. If combination therapy drugs are provided for conjugation, the single drugs may be pre-mixed either by mixing in molar equivalents or by mixing one component in excess of the others. Alternatively, the kit's conjugate and any desired anticancer drug may be kept separately in different containers before administration to the patient. The kit may also include second / third container means for containing sterile, pharmaceutically acceptable buffers or other diluents such as bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and dextrose solution.

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

[0191] As briefly indicated above, the kit may comprise means for administering the antibody or an antigen-binding portion thereof and any optional components to a patient, for example, one or more needles, an intravenous bag or syringe, or an eye dropper, a pipette, or other such device that can inject or introduce the formulation into an animal or apply it to a diseased region of the body. The kit of the present disclosure will generally also comprise a vial, or means for holding such an article, and other components in a sealed container for commercial sale, such as, for example, a blow-molded plastic container in which a syringe or desired vials and other devices are placed and held.

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

[0193] [Table 1]

[0194] [Table 2]

[0195] [Table 3]

[0196] [Table 4]

[0197] [Table 5] JPEG0007918357000008.jpg213170

[0198] [Table 6] JPEG0007918357000010.jpg196169

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

[0200] [Table 7]

[0201] [Table 8]

[0202] [Table 9]

[0203] [Table 10]

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

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

[0206] Embodiment 3: The isolated antibody or its antigen-binding portion, as described above, is a bispecific antibody or its antigen-binding portion, comprising a CD3-binding portion capable of binding to CD3, as described in Embodiment 1 or 2.

[0207] Embodiment 4: The isolated antibody or its antigen-binding portion 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.

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

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

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

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

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

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

[0214] Embodiment 11: The isolated antibody or its antigen-binding portion according to Embodiment 10, wherein the isolated antibody or its antigen-binding portion is a monoclonal antibody.

[0215] Embodiment 12: The isolated antibody or its antigen-binding portion according to Embodiment 11, wherein the isolated antibody or its antigen-binding portion is a monoclonal antibody.

[0216] Embodiment 13: The isolated antibody or its antigen-binding portion according to any one of Embodiments 1 to 12, wherein the isolated antibody or its antigen-binding portion is fused with a constant region of IgG, optionally human IgG, optionally human IgG1, or human IgG4.

[0217] Embodiment 14: A pharmaceutical composition comprising an isolated antibody or its antigen-binding moiety as described in any one of Embodiments 1 to 13 and a pharmaceutically acceptable carrier.

[0218] Embodiment 15: A complex comprising an isolated antibody or its antigen-binding portion as described in any one of Embodiments 1 to 13, and one or more portions bound to the isolated antibody or its antigen-binding portion.

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

[0220] Embodiment 17: A vector comprising the nucleic acid molecule described in Embodiment 16.

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

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

[0223] Embodiment 20: A method for inhibiting the proliferation or metastasis of tumor cells in a subject (e.g., a human subject), the method comprising administering to the subject an effective amount of an isolated antibody or its antigen-binding portion described in any one of Embodiments 1 to 13 or a pharmaceutical composition described in Embodiment 14.

[0224] 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 an isolated antibody or its antigen-binding portion described in any one of Embodiments 1 to 13 or a pharmaceutical composition described in Embodiment 14.

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

[0226] Embodiment 23: The method according to Embodiment 22, for treating a proliferative disorder which, if applicable, is cancer, prostate cancer, lung cancer, bronchogenic cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, adenocarcinoma, alveolar cell carcinoma, bronchial adenoma, chondropathic hamartoma (non-cancerous), sarcoma, renal cancer, breast cancer, gastric cancer, colorectal cancer, glioblastoma, pancreatic cancer, ovarian cancer, or metastatic castration-resistant prostate cancer.

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

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

[0229] Embodiment 26: A treatment or diagnostic kit for a proliferative disorder, an immune disorder, an inflammatory disease, or an infection, comprising a container containing at least one of the isolated antibodies or antigen-binding moieties described in any one of Embodiments 1 to 13. [Examples]

[0230] The present invention, as described above in general terms, will be readily understood by referring to the following examples, which are provided as examples and are not intended to limit the invention. The examples are not intended to indicate that the following experiments are all or only experiments in which the invention was performed.

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

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

[0233] 1.2 Manufacturing of benchmark antibodies The DNA sequence encoding the J591 antibody (W3XX042-BMK1) was synthesized in Genewiz (Suzhou, China) using sequences 21 and 22 from International Publication No. 02 / 098897(A2), and then subcloned into a modified pcDNA3.3 expression vector (Thermo).

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

[0235] Example 2. Antibody hybridoma production 2.1 Immunization and Cell Fusion Three OMT rats aged 6–8 weeks were alternately immunized with human PSMA ECD protein and cynomolgus monkey PSMA ECD protein. Serum antibody titers against the antigens 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 with blocking buffer (1×PBS / 2% BSA) at ambient temperature for 1 hour. Rat serum was serially diluted 3-fold in blocking buffer, starting with a 1:100 dilution, and incubated at ambient temperature for 1 hour. Wells without serum samples were used as negative controls. After washing the plates, the secondary antibody and goat anti-rat IgG-Fc-HRP (Bethyl) were incubated for 1 hour. After washing, TMB substrate was added, and the interaction was stopped with 2M HCl. Absorbance at 450 nm was read using a microplate reader (molecular device). For FACS, plates (96 wells) were pre-coated with 3 × 10⁴ LNCaP per well and cultured for 2 days in an incubator set to 37°C and 5% CO₂. The plates were blocked at ambient temperature for 1 hour using blocking buffer (1 × PBS / 5% milk). Then, 50 μL of hybridoma supernatant was added to the plates and incubated at ambient temperature for 1 hour. After washing the plates three times with PBS, they were incubated with secondary antibody and goat anti-rat Fc-Alexa647 (1:500) at ambient temperature for 1 hour. The mean fluorescence intensity (MFI) of the cells was measured by flow cytometry and analyzed by FLOWJO.

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

[0237] 2.2 High-throughput screening and IgG conversion of hybridoma supernatant A high-throughput screening method using the supernatant of hybridoma culture media includes primary screening by ELISA binding with human PSMA, and confirmatory screening by FACS / ELISA binding with human and cynomolgus monkey PSMA.

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

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

[0240] Purified IgG antibodies were further screened by ELIAS and FACS conjugation with human and cynomolgus monkey PSMA. Antibody W305042 was selected as one of the antibodies.

[0241] Example 3. Construction and purification of a fully human antibody molecule. Heavy 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 used for protein purification using a protein A column. Antibody concentration was measured by NanoDrop. 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 by SEC-HPLC was 99.19%.

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

[0243] The results for antibodies binding to human PSMA protein are shown in Figure 2 and Table 1. W305042 was able to effectively bind to human PSMA ECD with an EC50 of 0.017 nM, which was slightly potent than the reference antibody J591. The human IgG isotype antibody used as a negative control did not show clear binding to human PSMA protein. The results suggest excellent binding ability of W305042 to human PSMA.

[0244] [Table 12]

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

[0246] The binding results of the antibody to human PSMA-expressing LNCaP cells are shown in Figure 3 and Table 2. W305042 was able to effectively bind to LNCaP cells at an EC50 of 0.25 nM, comparable to the reference antibody J591. The human IgG isotype antibody used as a negative control did not show clear binding to LNCaP cells. The results suggest superior binding ability of W305042 to LNCaP cells.

[0247] [Table 13]

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

[0249] The results for antibodies binding to cynomolgus monkey PSMA protein are shown in Figure 4 and Table 3. W305042 was able to effectively bind to cynomolgus monkey PSMA ECD protein at an EC50 of 0.006 nM, comparable to the reference antibody J591. The human IgG isotype antibody used as a negative control did not show clear binding to cynomolgus monkey PSMA protein. The results suggest the superior binding ability of W305042 to cynomolgus monkey PSMA.

[0250] [Table 14]

[0251] 4.3.2 Binding of cynomolgus monkey PSMA by FACS Fluorescence-activated cell classification (FACS) was used to detect the binding of the W305042 antibody to cynomolgus monkey PSMA. This method allows for the quantitative analysis and identification of specific molecules expressed on the surface of living cells. Unlabeled cells were used as a control to establish a threshold before detection, and the percentage change in each group that exceeded the fluorescence intensity threshold was analyzed. Engineered cells expressing cynomolgus monkey PSMA (W3xx042.FIpinCHO.cPro1.B7) were maintained in F-12 medium containing 10% FBS and 600 μg / mL hygromycin. Briefly, 1 × 10⁵ cells per well of W3xx042.FIpinCHO.cPro1.B7 were incubated with various concentrations of W305042 antibody (100 nM to 6.1 pM, 4-fold serial dilutions in 1 × PBS / 1% BSA) at a volume of 100 μL / well for 1 hour in a refrigerator set to 4°C. J591 was used as a positive control, and a human IgG isotype antibody was used as a negative control. After washing the cells twice with 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 the cells were incubated in the dark for 0.5 hours in a refrigerator set to 4°C. 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. MFI and EC50 were determined as described above.

[0252] The binding results of the antibody to engineered CHO cells expressing cynomolgus monkey PSMA are shown in Figure 5 and Table 4. W305042 effectively bound to cynomolgus monkey PSMA+CHO cells at an EC50 of 2.25 nM, comparable to the reference antibody J591. Human IgG isotype antibodies used as a negative control did not show clear binding to cynomolgus monkey PSMA+CHO cells. The results suggest superior binding ability of W305042 to cynomolgus monkey PSMA.

[0253] [Table 15]

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

[0255] The results for antibodies binding to mouse PSMA protein are shown in Figure 6 and Table 5. W305042 was able to effectively bind to mouse PSMA ECD protein at an EC50 of 0.026 nM. Human IgG isotype antibody, used as a negative control, did not show clear binding to mouse PSMA protein. The results suggest excellent binding ability of W305042 to mouse PSMA.

[0256] [Table 16]

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

[0258] The sensorgrams of the blank surface and buffer channels were subtracted from the test sensorgram. The experimental data were fitted using a 1:1 binding model. Molar concentrations of human and cynomolgus monkey PSMA were calculated using molecular weights of 81.4 and 82.7 kDa.

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

[0260] [Table 17]

[0261] 4.5 Feasibility Testing Thermal stability due to DSF The melting point (Tm) of each antibody was examined using a QUANTSTUDIO 7 Flex real-time PCR system (Applied Biosystems). 19 μL of antibody solution was mixed with 1 μL of 80 X SYPRO Orange protein detection gel stain and transferred to a 96-well plate. The plate was sealed with optical adhesive film and centrifuged at 3,000 rpm for 5 minutes to remove some 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 point (Tm). If a protein had multiple unfolding transitions, the first two Tm values ​​were reported and referred to as Tm1 and Tm2. Data collection and Tm calculation were performed automatically using QUANTSTUDIO real-time PCR software (v1.3). W305042 exhibited excellent thermal stability, with a Tm1 of 69.5°C and a Tm2 of 71.1°C (Figure 7).

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

[0263] W305042 exhibited a high kD value and monodisperse size distribution, demonstrating excellent solubility. See Table 7.

[0264] [Table 18]

[0265] Hydrophobic interaction chromatography (HIC-HPLC) The hydrophobic properties of the antibody were detected using an HPLC 1260 Infinity II system (Agilent Technologics™) with a TSK gel butyl-NPR column. The sample was diluted in PBS buffer, and 20 μL of the diluted sample was injected into the column and separated for 61 minutes at a flow rate of 0.5 ml / min. Peak retention was detected using UV light at wavelengths of 280 nm and 230 nm. Retention times were analyzed by HIC-HPLC, and the total peak area from 20 to 40 minutes was integrated. The operation and analysis software used was OPENLAB CDS workstation (v2.6.0.691). The retention time for W305042 by HIC-HPLC was 24.57 minutes, indicating that W305042 has low hydrophobicity (Figure 8).

[0266] Example 5. Preparation of materials, cell lines, and benchmark (BMK) antibody Information regarding the commercially available materials used in the following examples is shown in the table immediately below. [Table 19] JPEG0007918357000024.jpg208170

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

[0268] Production of benchmark (BMK) antibodies The DNA sequences encoding the CD3×PSMA reference antibody AMG340 / TNB-585(BMK1) were synthesized using Sequence IDs 49, 56, and 61 from International Publication No. 2021 / 222578(A1), and then subcloned into a modified pcDNA3.3 expression vector (Thermo).

[0269] The CD3×PSMA reference antibody and the DNA sequence encoding AMG160(BMK2) were synthesized using Sequence ID No. 382 from U.S. Patent Application Publication No. 2017 / 0218079(A1), and then subcloned into a modified pcDNA3.3 expression vector (Thermo).

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

[0271] Example 6. Preparation of bispecific antibodies Anti-CD3 monoclonal antibodies were discovered in mice immunized using hybridoma technology (International Publication No. 2019 / 057099(A1)). Anti-PSMA monoclonal antibodies were discovered in transgenic rats immunized using hybridoma technology.

[0272] The construction of the bispecific antibody was carried out using a standard molecular biology protocol. As illustrated in Figure 9, to construct the CD3×PSMA bispecific antibody W308051-T3U5.E17-61.uIgG4V322 (W308051), the DNA sequence encoding the VH region of the anti-CD3 antibody was fused with the modified TCRβ constant domain and hinge Fc region of human IgG4 having the S228P mutation, Fc null mutation (F234A L235A), and knob mutation (S354C-T366W); the DNA sequence encoding the VL region of the anti-CD3 antibody was fused with the modified TCRα constant domain; and the DNA sequence encoding the VH region of the anti-PSMA antibody was fused with the S228P mutation, Fc null mutation (F234A L235A). The hinge Fc region of human IgG4 containing the L235A mutation and the whole mutation (Y349C-T366S-L368A-Y407V) was fused with the CL domain; the DNA sequence encoding the VL region of the anti-PSMA antibody was then fused with the CL domain. The coding region was then cloned into a modified pcDNA3.3 expression vector.

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

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

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

[0276] The results are shown in Figure 11 and Table 8, where W308051 has an EC of 0.027 nM. 50 It bound to human PSMA protein, and this was more potent than the reference antibody AMG160.

[0277] [Table 20]

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

[0279] The results are shown in Figures 12-13 and Table 9. As shown in Figure 12 and Table 9, W308051 yielded EC values ​​of 1.48 nM, 0.58 nM, and 0.29 nM, respectively. 50 W308051 bound to human PSMA-positive C4-2 (high PSMA expression), LNCaP (high PSMA expression), and 22Rv1 (low PSMA expression) human tumor cells, demonstrating potency compared to AMG160 and AMG340. Simultaneously, W308051 did not bind to PSMA-negative PC-3 cell lines. As shown in Figure 13, W308051 bound to CD3-positive Jurcutt cells and primary human T cells with lower binding affinity than AMG160.

[0280] [Table 21]

[0281] 3.2 Cross-species binding measured by ELISA / FACS Binding of cynomolgus monkey and mouse PSMA as measured by ELISA In short, 1 μg / mL cynomolgus monkey PSMA protein (His-tagged) and 2 μg / mL mouse PSMA protein (His-tagged) were coated onto the wells of an ELISA plate in coating buffer at 4°C for 16 hours. After washing the plate once with 1×PBST, the plate was blocked for 1 hour with 200 μL / well 2% BSA. After washing three times with 1×PBST, antibodies of various concentrations diluted in 2% BSA (5-fold serial dilutions from 200 nM to 0.004 pM for W308051, AMG160, and isotype control for cynomolgus monkey PSMA; 6-fold serial dilutions from 100 nM to 0.357 pM for W308051, AMG160, and isotype control for mouse PSMA; and 6-fold serial dilutions from 500 nM to 1.786 pM for AMG340 for mouse PSMA) were added and incubated at ambient temperature for 1 hour. After washing three times with 1×PBST, goat anti-human IgG-Fc-HRP (1:5000 dilution) was added and incubated for 1 hour. After washing six times with 1×PBST, 100 μL of TMB substrate solution was added to each well and incubated in the dark for 5 minutes. Then, 100 μL / well of 2M HCl was added to each well to stop the reaction. Relative luminescence (RLU) was measured using SPECTRAMAX M5E. 450 and OD 540 Measured at EC. 50 The decision was made as described above.

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

[0283] [Table 22]

[0284] [Table 23]

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

[0286] As shown in Figure 15 and Table 12, W308051 has an EC of 3.20 nM. 50 It bound to cynomolgus monkey PSMA-positive cells, and this was more potent than AMG160 and AMG340.

[0287] [Table 24]

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

[0289] The sensorgrams of the blank surface and buffer channels were subtracted from the test sensorgram. The experimental data were fitted using a 1:1 coupling model. The molar concentrations of W308051, AMG340, and AMG160 were calculated using molecular weights of 147, 111, and 106 kDa, respectively.

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

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

[0292] Table 13 shows the complete kinetic affinity of W308051 to human PSMA and CD3. The affinity of W308051 to human PSMA and CD3 was 1.58 × 10⁻⁶, respectively. -11 M and 6.02×10-8 It was M.

[0293] [Table 25]

[0294] 7.4 T cell cytotoxicity and cytokine release The efficacy of a bispecific antibody in mediating tumor cell lysis by human T cells was evaluated using a CTG-based cytotoxicity assay. In short, 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 antibody was added to a 96-well plate. Effector cells (1 × 10⁶) were then lysed. 5 Cells (50 μL / well) and target cells (1 × 10⁶) 4 Cells (50 μL / well) were added to the corresponding wells (E / T ratio = 10:1). After 72 hours of incubation, the plates were washed once with DPBS, CTG solution (75 μL / well) was added, and incubated at ambient temperature for 10 minutes. Relative luminescence (RLU) signals were measured using an invasion reader. Cytotoxicity % was expressed as (1-(RLU)). サンプル -RLU エフェクター細胞のみ ) / (RLU エフェクター細胞+標的細胞 -RLU エフェクター細胞のみ Calculated as )) × 100%. Combined IC 50 This was calculated using GraphPad Prism7 software by plotting antibody concentration (x-axis) against cytotoxicity (y-axis) and analyzing it as a nonlinear regression (curve fit) - log(inhibitor) vs. response - variable slope (4 parameters).

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

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

[0297] [Table 26]

[0298] [Table 27]

[0299] 7.5 Cytokine release from C4-2 cells co-cultured with PBMCs Briefly, 100 µL / well of antibodies (10-fold serial dilutions from 20 nM to 0.2 pM for W308051, AMG160, and the isotype control, and 10-fold serial dilutions from 500 nM to 5.0 pM for AMG340) were 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 20 hours of incubation, the supernatant was collected and stored at -80°C. Released human IFN-γ, IL-2, TNF-α, and IL-6 in the supernatant 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 supernatant was quantified via a standard curve. EC 50 was calculated using GraphPad Prism 7 software by plotting antibody concentration (x-axis) against percentage (y-axis) and analyzing as non-linear regression (curve fit) - log(agonist) vs. response - variable slope (4 parameters).

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

[0301]

Table 28

[0302] 7.6 Thermal stability measured by DSF T of each antibody mThe melting point (Tm) was measured using a QUANTSTUDIO 7 Flex Real-Time PCR System (Applied Biosystems). 19 μL of the antibody solution was mixed with 1 μL of 80 X SYPRO Orange gel staining reagent for protein detection, and transferred to a 96-well plate. The plate was sealed with Optical Adhesive Film, centrifuged at 3,000 rpm for 5 minutes to remove some air bubbles. The plate was heated from 26°C to 95°C at a rate of 0.9°C / min, and the obtained fluorescence data was collected. The negative derivative of fluorescence change with respect to different temperatures was calculated, and the maximum value was taken as the melting point T m . When a protein has multiple unfolding transitions, the first two Tm are reported, designated as T m 1 and T m 2. Data collection and T m calculation were automatically performed by QUANTSTUDIO Real-Time PCR Software (v1.3). As shown in Figure 19, W308051 exhibited excellent thermostability with a T m m of 62.9°C and a T m m of 69.3°C.

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

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

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

[0306] [Table 29]

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

[0308] As shown in Figure 21 and Table 18, for intravenous PK testing at 10 mg / kg using three ELISA settings (i.e., coated with Fc, PSMA, or CD3, and detected using Fc, CD3, or PSMA, respectively), WBP308051 showed mean serum clearances of 6.05, 8.07, and 7.51 mL / day / kg, respectively; mean half-lives of 177, 142, and 132 hours, respectively; volume of distribution (Vss) of 59.1, 65.8, and 58.8 mL / kg, respectively; and AUC0 at 30332, 24087, and 26745 hours / μg / mL, respectively. Similar results from three different ELISA settings suggest excellent in vivo stability of W308051.

[0309] [Table 30]

[0310] 8.2 In vivo efficacy in the NPG-hPBMC model The in vivo efficacy of WBP308051 was tested in an LNCaP xenograft model using male NPG mice with human PBMC rearrangements. 2 × 10 6 LNCaP tumor cells were transplanted (subcutaneously) into the right flank of NPG mice. Each mouse received 2 × 10⁶ cells via intraperitoneal injection. 6 I also received PBMC cell therapy. The tumor was approximately 105 mm in volume. 3 When tumor-bearing mice reached a certain level, they were randomly divided into nine groups. The nine groups of mice each received the following intraperitoneal injections twice a week for a total of six injections: medium-PBS; 0.30 mg / kg AMG340; 1.51 mg / kg AMG340; 0.057 mg / kg AMG160; 0.29 mg / kg AMG160; 1.44 mg / kg AMG160; 0.08 mg / kg W308051; 0.4 mg / kg W308051; 2 mg / kg W308051. Mouse body weight and tumor growth were measured twice a week. Tumor volume was calculated using the formula (1 / 2)·(length × width). 2The calculations were performed using ). All procedures regarding the handling, care, and treatment of animals in the study were carried out in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi Biologics' LARC, which are in accordance with the guidelines of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC).

[0311] The tumor growth curve results are shown in Figure 22A. The mean tumor volume in the PBS group was 1527.3 ± 216.73 mm² at 21 days post-treatment. 3 The results showed that treatment with AMG160 at 0.057, 0.29, and 1.44 mg / kg exhibited a potent antitumor effect compared to treatment with PBS, with tumor sizes of 86.63±10.42, 74.85±10.52, and 50.36±4.54 mm. 3 The mean tumor burdens were (TGI = 101.39, 102.21, and 103.93%). Treatment with AMG340 at 0.30 and 1.51 mg / kg showed an antitumor effect compared to PBS treatment, with mean tumor burdens of 1061.97±296.65 and 441.45±297.15 (TGI = 32.75 and 78.53%). Treatment with W308051 at 0.4 and 2 mg / kg showed a potent antitumor effect, with mean tumor burdens of 193.46±264.26 and 69.09±10.48 mm. 3 The mean tumor volume was (TGI = 93.86, 102.63%). However, treatment with W308051 at 0.08 showed no antitumor effect, with a mean tumor volume of 2022.29 ± 328.64 mm. 3 The average tumor volume was (TGI = -34.84%).

[0312] The mouse body weight is shown in Figure 22B, and slight weight loss was observed during the experiment, which may be due to the GVHD (graft-versus-host disease) effect in the human PBMC rearrangement model.

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

[0314] Those skilled in the art will further understand that this disclosure may be embodied in other specific forms without departing from its essence or central characteristics. It should be understood that other variations are intended to be within the scope of this disclosure, given that the foregoing description of this disclosure discloses only exemplary embodiments. Therefore, this disclosure is not limited to the specific embodiments described in detail herein. Rather, one should refer to the appended claims which indicate the scope and content of this disclosure.

Claims

1. An isolated bispecific antibody or its antigen-binding portion comprising a PSMA-binding portion capable of binding to prostate-specific membrane antigen (PSMA) and a CD3-binding portion capable of binding to CD3, The aforementioned PSMA coupling portion is: A heavy chain CDR1 comprising the sequence of sequence number 1, A heavy chain CDR2 comprising the sequence of sequence number 2, A heavy chain CDR3 comprising the sequence of sequence number 3, A light chain CDR1 comprising the sequence of sequence number 4, A light chain CDR2 comprising the sequence of sequence number 5, A light chain CDR3 comprising the sequence of sequence number 6, It includes, The aforementioned CD3 coupling portion is: A heavy chain CDR1 comprising the sequence of sequence number 7, A heavy chain CDR2 comprising the sequence of sequence number 8, A heavy chain CDR3 comprising the sequence of sequence number 9, A light chain CDR1 comprising the sequence of sequence number 10, A light chain CDR2 comprising the sequence of sequence number 11, A light chain CDR3 comprising the sequence of sequence number 12, An isolated bispecific antibody or its antigen-binding moiety comprising the above.

2. (a) The PSMA binding portion comprises a heavy chain variable region comprising an amino acid sequence encoded by the sequence of SEQ ID NO: 15 or SEQ ID NO: 23 and a light chain variable region comprising an amino acid sequence encoded by the sequence of SEQ ID NO: 16 or SEQ ID NO: 24, and / or (b) The CD3 binding portion comprises a heavy chain variable region comprising the amino acid sequence encoded by the sequence of SEQ ID NO: 13 or SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence encoded by the sequence of SEQ ID NO: 14 or SEQ ID NO:

22. The isolated bispecific antibody or its antigen-binding moiety according to claim 1.

3. The isolated bispecific antibody or its antigen-binding portion according to claim 1, wherein the CD3 binding portion comprises a heavy chain TCRβ constant region comprising the sequence of SEQ ID NO: 29 and a light chain TCRβ constant region comprising the sequence of SEQ ID NO:

30.

4. (a) The CD3 binding portion comprises a heavy chain comprising the sequence of sequence number 17 and a light chain comprising the sequence of sequence number 18, and / or (b) The PSMA binding portion comprises a heavy chain comprising the sequence of SEQ ID NO: 19 and a light chain comprising the sequence of SEQ ID NO:

20. The isolated bispecific antibody or its antigen-binding moiety according to claim 1.

5. The isolated bispecific antibody or its antigen-binding portion according to claim 1, wherein the isolated bispecific antibody or its antigen-binding portion is a monoclonal antibody, a chimeric antibody, or a humanized antibody.

6. The isolated bispecific antibody or its antigen-binding portion according to claim 1, wherein the isolated bispecific antibody or its antigen-binding portion is a human monoclonal antibody.

7. The isolated bispecific antibody or its antigen-binding portion is fused with the constant region of IgG, as described in claim 1.

8. The isolated bispecific antibody or its antigen-binding portion according to claim 1, wherein the isolated bispecific antibody or its antigen-binding portion is fused with the constant region of human IgG.

9. The isolated bispecific antibody or its antigen-binding portion according to claim 1, wherein the isolated bispecific antibody or its antigen-binding portion is fused with the constant region of human IgG1 or human IgG4.

10. A pharmaceutical composition comprising an isolated bispecific antibody or its antigen-binding moiety according to any one of claims 1 to 9.

11. A complex comprising an isolated bispecific antibody or its antigen-binding portion according to any one of claims 1 to 9 and one or more portions bound to the isolated bispecific antibody or its antigen-binding portion.

12. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a PSMA-binding heavy chain and a PSMA-binding light chain or their antigen-binding moiety of an isolated bispecific antibody according to any one of claims 1 to 9.

13. The isolated nucleic acid molecule according to claim 12, wherein the nucleic acid sequence comprises the sequence of SEQ ID NO: 37 and / or SEQ ID NO:

38.

14. The isolated nucleic acid molecule according to claim 12, wherein the nucleic acid sequence further comprises the sequence of SEQ ID NO: 35 and / or SEQ ID NO:

36.

15. The isolated nucleic acid molecule according to claim 12, wherein the nucleic acid sequence comprises all four sequences of sequence numbers 35 to 38.

16. A vector comprising the nucleic acid molecule described in claim 12.

17. A host cell comprising the isolated nucleic acid molecule described in claim 12.

18. A host cell comprising the vector described in claim 16.

19. A method for preparing an isolated bispecific antibody or its antigen-binding moiety according to any one of claims 1 to 9, wherein the method is: a) Expressing the antibody or its antigen-binding portion in the host cell of claim 17; and b) Isolating the antibody or its antigen-binding portion from the host cell, A method that includes the following:

20. A method for preparing an isolated bispecific antibody or its antigen-binding moiety according to any one of claims 1 to 9, wherein the method is: a) Expressing the antibody or its antigen-binding moiety in the host cell of claim 18; and b) Isolating the antibody or its antigen-binding portion from the host cell, A method that includes the following:

21. The pharmaceutical composition according to claim 10 for inhibiting the proliferation or metastasis of tumor cells.

22. A pharmaceutical composition according to claim 10 for regulating an immune response.

23. The pharmaceutical composition according to claim 10 for the treatment or prevention of proliferative disorders, autoimmune disorders, inflammatory disorders, or infectious disorders.

24. The pharmaceutical composition according to claim 23, wherein the proliferative disorder is cancer.

25. The pharmaceutical composition according to claim 24, wherein the cancer is prostate cancer, lung cancer, bronchogenic cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, adenocarcinoma, alveolar cell carcinoma, bronchial adenoma, chondropathic hamartoma (non-cancerous), sarcoma, kidney cancer, breast cancer, gastric cancer, colorectal cancer, glioblastoma, pancreatic cancer, ovarian cancer, or metastatic castration-resistant prostate cancer.

26. The pharmaceutical composition according to claim 24, wherein the cancer is prostate cancer.

27. The pharmaceutical composition according to claim 10, for administration in combination with a chemotherapy agent, radiation, and / or another cancer immunotherapy.

28. A kit for the treatment or diagnosis of a proliferative disorder, an immune disorder, or an infectious disease, comprising a container containing at least one isolated bispecific antibody or its antigen-binding moiety as described in any one of claims 1 to 9.

29. An isolated antibody or its antigen-binding moiety comprising a PSMA-binding moiety capable of binding to prostate-specific membrane antigen (PSMA), The aforementioned PSMA coupling portion is: A heavy chain CDR1 comprising the sequence of sequence number 1, A heavy chain CDR2 comprising the sequence of sequence number 2, A heavy chain CDR3 comprising the sequence of sequence number 3, A light chain CDR1 comprising the sequence of sequence number 4, A light chain CDR2 comprising the sequence of sequence number 5, A light chain CDR3 comprising the sequence of sequence number 6, An isolated antibody or its antigen-binding moiety comprising the above.

Images