PSMA-steap1 dual variable domain immunoglobulin (DVD-ig) molecule and drug conjugate

Abstract

To provide a novel therapy in which a binder of prostatic cancer antigen is combined in order to enhance a cover range, alleviate resistivity, and accelerate continuous response of a tumor.SOLUTION: Provided is a dual variable domain immunoglobulin (DVD-Ig) molecule that binds to a human STEAP1 and a human PSMA, and a drug conjugate thereof, and a method of using them.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 604,884, filed Nov. 30, 2023, and U.S. Provisional Application No. 63 / 550,451, filed Feb. 6, 2024, the contents of which are hereby incorporated by reference in their entirety.

[0002] Sequence Listing This application includes an electronically submitted sequence listing (ABV21624USL2_ST26.xml, size: 27,909 bytes, created on Nov. 26, 2024), the contents of which are hereby incorporated by reference in their entirety.

Background Art

[0003] Prostate cancer is the second most commonly diagnosed cancer in men worldwide and the fifth leading cause of cancer death. Currently, there is no curative treatment available for metastatic prostate cancer.

[0004] PSMA (prostate-specific membrane antigen) and STEAP1 (six-transmembrane epithelial antigen of the prostate-1) are well-characterized antigens that are overexpressed in metastatic prostate cancer and underexpressed in normal tissues.

[0005] PSMA plays a role in folate metabolism, which may contribute to the progression of prostate cancer. The expression of PSMA increases in metastatic and hormone-resistant tumors and correlates with tumor malignancy, stage, recurrence, and castration resistance.

[0006] STEAP1 is an ion channel or ion transporter that regulates the concentration of small molecules, ions, and nutrients. STEAP1 functions in cell communication and cell adhesion processes. STEAP1 is overexpressed in advanced and metastatic tumors and is associated with invasive cell growth and invasion.

[0007] Antibody-drug conjugates (ADCs) are a rapidly growing class of anti-cancer agents that combine the cell surface targeting properties of antibodies with a potent cytotoxic drug conjugated using a chemical linker. The advantage of ADCs is the ability to deliver a toxic payload to tumors and reduce the toxicity associated with systemic exposure to normal tissues. Summary of the Invention Problems to be Solved by the Invention

[0008] There is a need for a novel therapy that combines binders of prostate cancer antigens to enhance tumor coverage, mitigate resistance, and promote a sustained response. Means for Solving the Problems

[0009] (Brief Summary of the Invention) In some embodiments, the present disclosure has the following structure:

[0010]

Chemical Formula

[0011] In some embodiments, the first heavy chain variable domain (VH1) comprises the amino acid sequence of SEQ ID NO: 2, the second heavy chain variable domain (VH2) comprises the amino acid sequence of SEQ ID NO: 7, the first light chain variable domain (VL1) comprises the amino acid sequence of SEQ ID NO: 12, and the second light chain variable domain (VL2) comprises the amino acid sequence of SEQ ID NO: 17.

[0012] In some embodiments, the first peptide linker (L1) comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 6). In some embodiments, the second peptide linker (L2) comprises the amino acid sequence GGSGGGGSG (SEQ ID NO: 16). In some embodiments, the IgG1 heavy chain constant region (CH1CH2CH3) comprises the amino acid sequence of SEQ ID NO: 21 or 23. In some embodiments, the kappa light chain constant region (Cκ) comprises the amino acid sequence of SEQ ID NO: 22.

[0013] In some embodiments, each of the heavy chains comprises the amino acid sequence of SEQ ID NO: 1 or 24, and each light chain comprises the amino acid sequence of SEQ ID NO: 11.

[0014] In some embodiments, the first heavy chain and the second heavy chain are connected by two disulfide bridges, and the two disulfide bridges are between the cysteine at position 226 of the first heavy chain and the cysteine at position 226 of the second heavy chain, and between the cysteine at position 229 of the first heavy chain and the cysteine at position 229 of the second heavy chain, according to EU numbering.

[0015] In some embodiments, the Top1i linker-drug is conjugated to the cysteine at position C220 of the first and second heavy chains of the DVD-Ig according to EU numbering.

[0016] In some embodiments, n is 1. In some embodiments, n is 2.

[0017] In some aspects, the present disclosure provides the following structure:

[0018]

Chemical formula

Brief Description of the Drawings

[0019]

Figure 1

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 3E

Figure 3F

Figure 4A

Figure 4B

Figure 5A

Figure 5B

Figure 6A

Figure 6B

Figure 6C

Figure 7A

Figure 7B

Mode for Carrying Out the Invention

[0020] In some embodiments, novel DVD-Ig molecules that bind to both human STEAP1 and human PSMA, and drug conjugates of said DVD-Ig molecules are described herein.

[0021] PSMA and STEAP1 are well-characterized antigens that are overexpressed in metastatic prostate cancer and underexpressed in normal tissues. Both PSMA and STEAP1 are expressed at varying intensities within prostate cancer cells. Such intratumoral heterogeneity can potentially impede the success of prostate cancer treatment. In the present disclosure, the DVD-Ig platform is utilized for the dual targeting of PSMA and STEAP1, which increases both the breadth and depth of the response compared to single-specificity ADCs. Targeting both antigens, as compared to single-specificity ADCs that target a single antigen, either PSMA or STEAP1, confers several advantages: (1) dual targeting has a broader range of efficacy against tumor cells and captures a larger patient population, (2) the DVD-Ig molecule conjugated to the cytotoxic drug (DVD-Ig DC) demonstrates enhanced internalization and activity compared to either single-specificity ADC, and (3) compared to the administration of a combination of two single-specificity ADCs, the DVD-Ig DC reduces the total dosage of the agents required.

[0022] DVD-Ig is a symmetric molecule having two identical light chains and two identical heavy chains, and thus has a manufacturing process that is simplified compared to the four-chain asymmetric form that has the problem of chain mispairing and requires downstream purification. However, it is difficult to identify DVD-Igs that are worthy of being drug candidates with the desired potency, physicochemical properties, efficacy, and manufacturability. The first challenge associated with its design is that the binding affinity of the inner variable regions to their target may be reduced or lost. This is due to steric hindrance and can sometimes be alleviated by the choice of linker between the variable regions. The second challenge is that the expression levels of some DVD-Igs may be lower than those of the parental IgG from which they are derived. Thirdly, drug-like properties such as thermal stability, aggregation, or the ability to achieve the high concentrations required for formulation may be poor in some molecules. The DVD-Igs described in the present disclosure have good expression levels, as well as strong binding specificity for both human STEAP and human PSMA, and strong induction of cancer cell killing.

[0023] To obtain DVD-Igs, 200 molecules were designed and cloned for expression. 149 molecules were successfully expressed, and 122 molecules had sufficient yields for characterization. 45 molecules had dual binding activity, and 20 were selected for further characterization. Lead molecules were identified for further development because they showed good cell killing ability and acceptable drug-like properties.

[0024] Definitions The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0025] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to mean "A and B", "A or B", "A", or "B".

[0026] "Binding to human STEAP1 or human PSMA" refers to a molecule that can bind to human STEAP1 or human PSMA with sufficient affinity to be useful as a therapeutic agent in targeting human STEAP or human PSMA.

[0027] The term "CDR" refers to the Complementarity Determining Regions within the immunoglobulin variable region sequences. There are three CDRs, called CDR1, CDR2, and CDR3, in the variable region of each of the heavy and light chains. The term "CDR set" refers to a group of the three CDRs present in a single variable region that can bind to an antigen. The exact boundaries of these CDRs have been defined with variations according to different systems. The system described by Kabat (Kabat et al., (1987) Sequences of Proteins of Immunological Interest, 4th ed., US Govt. Printing Off. No. 165 - 492; and Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 913 - 242) not only provides a clear residue numbering system applicable to any variable region of an antibody or binding protein, but also provides the exact residue boundaries that define the three CDRs in each heavy or light chain sequence. These CDRs are sometimes referred to as Kabat CDRs. Chothia et al. (Chothia and Lesk (1987) J. Mol. Biol. 196: 901 - 917; Chothia et al., (1989) Nature 342: 877 - 883) found that despite the great diversity at the amino acid sequence level, certain small portions within the Kabat CDRs adopt nearly identical peptide backbone structures. These small portions are called L1, L2, and L3 or H1, H2, and H3, where "L" indicates the light chain region and "H" indicates the heavy chain region respectively. These regions are sometimes referred to as Chothia CDRs and have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs overlapping with the Kabat CDRs are described by Padlan (1995) FASEB J. 9: 133 - 139 and MacCallum (1996) J. Mol. Biol. 262(5): 732 - 45).Still other definitions of CDR boundaries may not strictly follow one of the systems described herein, yet still overlap with Kabat CDRs, provided that they may be shortened or extended in light of predictions or experimental findings that certain residues or groups of residues, or entire CDRs, do not significantly affect antigen binding. In some embodiments, the CDRs of the DVD-Ig of the present disclosure are defined by EU numbering.

[0028] The terms “Kabat numbering,” “Kabat definition,” and “Kabat designations” are used interchangeably herein. These terms, as recognized in the art, refer to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or binding protein or antigen-binding portion thereof (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391, and Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable regions are in the ranges of amino acids 31-35 of CDR1, 50-65 of CDR2, and 95-102 of CDR3. For the light chain variable region, the hypervariable regions are in the ranges of amino acids 24-34 of CDR1, 50-56 of CDR2, and 89-97 of CDR3.

[0029] In some embodiments, the constant region (heavy chain constant region and / or light chain constant region) sequences are identified using EU numbering. The term "EU numbering" as recognized in the art refers to a system of numbering amino acid residues based on Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, pp. 78-85 (1969). The numbering schemes disclosed herein, including EU numbering and Kabat numbering, are based on conventional antibody structures. Since DVD-Ig has an extra variable domain (i.e., an external variable region) connected to the N-terminus of the internal variable domain via a flexible linker, one of ordinary skill in the art will understand that the exact positions of the amino acids based on the DVD-Ig structure will differ from both EU numbering and Kabat numbering. However, one of ordinary skill in the art will be able to readily identify the corresponding amino acids within the DVD-Ig sequence based on the numbering schemes disclosed herein (e.g., EU numbering and Kabat numbering). One of ordinary skill in the art can also identify the positions of the DVD-Ig amino acid sequence by counting from the processed N-terminus, which does not conform to any numbering scheme.

[0030] The terms "Fc" or "Fc region" are well-known terms to those of ordinary skill in the art and are involved in complement activation, Clq binding, C3 activation, and Fc receptor binding.

[0031] The term "hinge region" refers to a stretch of flexible amino acids in the central portion of the heavy chain of an immunoglobulin antibody that links these two chains by disulfide bonds. The "variable domain" or "variable region" (light chain variable domain / region (VL), heavy chain variable domain / region (VH)), as used herein, refers to each of the light and heavy chain pairs that are directly involved in binding of the antibody to its target. The variable human light and heavy chain domains have the same general structure, and each domain contains at least one complementarity-determining region (CDR), preferably three CDRs, which play a particularly important role in the binding specificity / affinity of the antibody.

[0032] DVD-Ig molecule Dual variable domain immunoglobulin (DVD-Ig) combines the target binding domains of two monoclonal antibodies (mAbs) via a flexible linker, resulting in a tetravalent IgG-like molecule. DVD-Ig is a symmetric bispecific antibody-like biological agent with a 2+2 antigen-binding stoichiometry. In some embodiments, in the linear representation of the mature sequence, DID-Ig comprises an external variable domain (VH1 or VL1) connected via a linker (L) to an internal variable domain (VH2 or VL2), and has a standard heavy chain (HC) or light chain (LC) constant region at the C-terminus.

[0033] In some embodiments, the DVD-Ig provided herein comprises an external variable domain (VH1 and VL1) that binds to human STEAP1 and an internal variable domain (VH2 and VL2) that binds to human PSMA. A diagram showing the design of a DVD-Ig molecule according to one embodiment of the invention is shown in FIG. 1.

[0034] For example, in some aspects, the present disclosure provides a DVD-Ig that binds to human STEAP1 and human PSMA and comprises two identical heavy chains and two identical light chains, each heavy chain comprising a) a first heavy chain variable domain (VH1) comprising VH1-CDR1 (SEQ ID NO: 3), VH1-CDR2 (SEQ ID NO: 4), and VH1-CDR3 (SEQ ID NO: 5), b) a second heavy chain variable domain (VH2) comprising VH2-CDR1 (SEQ ID NO: 8), VH2-CDR2 (SEQ ID NO: 9), and VH2-CDR3 (SEQ ID NO: 10), and c) an IgG1 heavy chain constant region (CH1CH2CH3) wherein VH1 and VH2 are connected via a first peptide linker (L1), and each heavy chain is in the form of VH1-L1-VH2-CH1CH2CH3, each light chain comprising a) a first light chain variable domain (VL1) comprising VL1-CDR1 (SEQ ID NO: 13), VL1-CDR2 (SEQ ID NO: 14), and VL1-CDR3 (SEQ ID NO: 15), b) A second light chain variable domain (VL2) comprising VL2-CDR1 (SEQ ID NO: 18), VL2-CDR2 (SEQ ID NO: 19), and VL2-CDR3 (SEQ ID NO: 20), and c) A kappa light chain constant region (Cκ) comprising, VL1 and VL2 are connected via a second peptide linker (L2), and each light chain is in the form of VL1-L2-VL2-Cκ, The first and second heavy chains form a dimeric Fc region, providing a DVD-Ig.

[0035] In some embodiments, the first heavy chain variable domain (VH1) comprises the amino acid sequence of SEQ ID NO: 2, the second heavy chain variable domain (VH2) comprises the amino acid sequence of SEQ ID NO: 7, the first light chain variable domain (VL1) comprises the amino acid sequence of SEQ ID NO: 12, and the second light chain variable domain (VL2) comprises the amino acid sequence of SEQ ID NO: 17.

[0036] In some embodiments, each heavy chain comprises the amino acid sequence of SEQ ID NO: 1 or 24, and each light chain comprises the amino acid sequence of SEQ ID NO: 11.

[0037] A flexible glycine-serine rich linker is preferred for the DVD-Ig. Examples of GS linkers for the DVD-Ig of the present disclosure include, but are not limited to, those listed in the following table. In some embodiments, the linker (e.g., the linker connecting VH1 and VH2, or L1) comprises GGGGSGGGGS (SEQ ID NO: 6). In some embodiments, the linker (e.g., the linker connecting VL1 and VL2, or L2) comprises GGSGGGGSG (SEQ ID NO: 16).

[0038]

Table 1

[0039] In some embodiments, the kappa light chain constant region (Cκ) is human Cκ or derived from the human Cκ region. In some embodiments, the LC cysteine that normally forms an interchain disulfide bond with C220 of the heavy chain (HC) is mutated to alanine, which creates a mismatched cysteine for linker-drug conjugation in a convenient site-specific manner at position C220 of the HC. For example, in some embodiments, the kappa light chain constant region (Cκ) is derived from human Cκ and includes an amino acid substitution of C214A according to the EU numbering of the amino acid numbers of the antibody chain.

[0040] In some embodiments, the kappa light chain constant region (Cκ) includes the amino acid sequence of SEQ ID NO: 22.

[0041] In one embodiment, the CH2CH3 of the first and second heavy chains forms a dimeric Fc region. In one embodiment, the Fc region of the present invention is derived from a human. In one embodiment, the Fc region of the present invention is a human IgG1 Fc region or derived from the human IgG1 Fc region.

[0042] In some embodiments, the Fc region includes a first CH2CH3 region and a second CH2CH3 region, and the first CH2CH3 region is covalently bound to the second CH2CH3 region by two disulfide bridges. In some embodiments, the first heavy chain and the second heavy chain are connected by two disulfide bridges, and the two disulfide bridges are between the cysteine at position 226 of the first heavy chain and the cysteine at position 226 of the second heavy chain, and between the cysteine at position 229 of the first heavy chain and the cysteine at position 229 of the second heavy chain according to the EU numbering. For example, in some embodiments, the first heavy chain and the second heavy chain have the amino acid sequence of SEQ ID NO: 1 or 24, and the two disulfide bridges are between C362 of the first heavy chain of SEQ ID NO: 1 or 24 and C362 of the second heavy chain of SEQ ID NO: 1 or 24, and between C365 of the first heavy chain of SEQ ID NO: 1 or 24 and C365 of the second heavy chain of SEQ ID NO: 1 or 24.

[0043] In one embodiment, the Fc region of the present invention includes a hinge region. Various hinge regions can be used in the bispecific molecules of the present invention, for example, to optimize certain properties. In an exemplary example, human IgG 1 , IgG 2 , IgG 3 or IgG 4 One or more amino acid substitutions, insertions and / or deletions can be introduced into the hinge region to reduce the level or rate of fragmentation and / or aggregation.

[0044] In one embodiment, the Fc region of the present invention includes one or more mutations that modulate (e.g., reduce) IgG Fc (crystalline fragment) receptor and complement binding. In one embodiment, the Fc region of the present invention includes one or more mutations that modulate (e.g., reduce) the Fc receptor-based function of the Fc region. In one embodiment, the Fc region of the present invention includes one or more mutations that modulate (e.g., reduce) the effector function based on FcγR of the Fc region. In one embodiment, the Fc region of the present invention is derived from the human IgG1 Fc region and includes amino acid substitutions of L234A and / or L235A according to the EU numbering of the amino acid numbers of the antibody chain.

[0045] In one embodiment, the CH1CH2CH3 of the present invention includes the amino acid sequence of SEQ ID NO: 21 or 23.

[0046] In some embodiments, the polypeptide sequence of one or both heavy chains of the DVD-Ig molecules described herein lacks a C-terminal lysine, resulting in a C-terminal glycine residue. For example, in some embodiments, one or both heavy chains of the DVD-Ig molecules described herein include the amino acid sequence of SEQ ID NO: 24.

[0047] In some embodiments, the present disclosure provides a DVD-Ig that binds to human STEAP1 and human PSMA and includes two identical heavy chains and two identical light chains, each heavy chain including the amino acid sequence of SEQ ID NO: 1 or 24, each light chain including the amino acid sequence of SEQ ID NO: 11, the first heavy chain and the second heavy chain being connected by two disulfide bridges, the two disulfide bridges being between the cysteine at position 226 of the first heavy chain and the cysteine at position 226 of the second heavy chain and between the cysteine at position 229 of the first heavy chain and the cysteine at position 229 of the second heavy chain according to EU numbering.

[0048] DVD-Ig drug conjugate (DVD-Ig DC) In some embodiments, the DVD-Ig molecules described herein are conjugated to a drug, such as a topoisomerase 1 inhibitor (TOP1i). Topoisomerase 1 (TOP1) removes supercoils formed during DNA replication. A topoisomerase 1 inhibitor (TOP1i) can bind to the TOP1-DNA complex and stabilize it, inducing DNA strand breaks and apoptosis. In some embodiments, the DVD-Ig molecules described herein are conjugated to a topoisomerase 1 inhibitory drug (a "TOP1i drug") according to Structural Formula (I).

[0049]

Chemical formula

[0050] In some embodiments, the TOP1i drug is a compound according to Formula (I). In embodiments, the TOP1i drug is (7S)-14-(3-aminobicyclo[1.1.1]pentan-1-yl)-7-ethyl-7-hydroxy-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H,13H)-dione.

[0051] The TOP1i drugs contemplated herein can be conjugated to the DVD-Ig described herein as the DVD-Ig DC shown in Structural Formula (II).

[0052]

Chem.

[0053]

Chem.

[0054] The topoisomerase inhibitors described herein can be conjugated to the DVD-Ig described herein to form a DVD-Ig TOP1i drug conjugate. The DVD-Ig-drug conjugate can enhance the therapeutic efficacy of the DVD-Ig in treating a disease due to the ability of the one or more drug moieties to selectively deliver to a target tissue, such as a tumor expressing a tumor-associated antigen, e.g., human STEAP1 and / or human PSMA. Thus, in various embodiments, the present disclosure provides the DVD-Ig DC described herein for therapeutic use, e.g., in the treatment of prostate cancer.

[0055] In certain embodiments, the TOP1i drug is of Structural Formula (III):

[0056]

Chem.

[0057] In some embodiments of the DVD-Ig DC described herein, the TOP1i drug is conjugated to the DVD-Ig by a linker moiety. As will be appreciated by those skilled in the art, the linker connects the TOP1i drug to the DVD-Ig by forming a covalent bond with the TOP1i drug at one position and a covalent bond with the antibody at another position. The covalent bond is formed by a reaction between functional groups on the linker and functional groups on the TOP1i drug and the DVD-Ig. In some embodiments, the Top1i linker-drug is conjugated to cysteine at position C220 of the first and second heavy chains of the DVD-Ig described herein.

[0058] In some embodiments, the synthetic intermediate compound that can be used to form the DVD-Ig can contain a linker drug (LD) represented by Structural Formula (V)

[0059]

Chemical formula

[0060] (2S)-2-(2-Bromoacetamido)-N-[(2S)-1-({3-[(7S)-7-Ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide

[0061] The DVD-Ig DCs disclosed herein contain drug molecules linked to the DVD-Ig moiety in various stoichiometric molar ratios, depending on the conformation of the DVD-Ig and the method used, at least in part, to achieve conjugation.

[0062] The term "drug loading" or "performing drug loading" refers to the number of drug molecules per antibody in an individual DVD-Ig DC molecule. The number of TOP1i drugs linked to the DVD-Ig DC can vary and is limited by the number of available binding sites on the DVD-Ig. When contemplated for the DVD-Ig DC of the present invention, the linker links a single TOP1i drug to one DVD-Ig. In some embodiments, the DVD-Ig DCs described herein have an n of 1 or 2. In embodiments, n is 1. In embodiments, n is 2. In embodiments, the drug loading can include one type of drug molecule, or two types of drug molecules.

[0063] In some aspects, the present disclosure is of the following structure:

[0064] [Chemical formula] [wherein n is 1 or 2, and the DVD-Ig binds to human STEAP1 and human PSMA and comprises two identical heavy chains and two identical light chains, each heavy chain a) a first heavy chain variable domain (VH1) comprising VH1-CDR1 (SEQ ID NO: 3), VH1-CDR2 (SEQ ID NO: 4), and VH1-CDR3 (SEQ ID NO: 5), b) a second heavy chain variable domain (VH2) comprising VH2-CDR1 (SEQ ID NO: 8), VH2-CDR2 (SEQ ID NO: 9), and VH2-CDR3 (SEQ ID NO: 10), and c) an IgG1 heavy chain constant region (CH1CH2CH3) and comprises VH1 and VH2 are connected via a first peptide linker (L1), and each heavy chain is in the form of VH1-L1-VH2-CH1CH2CH3, each light chain a) a first light chain variable domain (VL1) comprising VL1-CDR1 (SEQ ID NO: 13), VL1-CDR2 (SEQ ID NO: 14), and VL1-CDR3 (SEQ ID NO: 15), b) A second light chain variable domain (VL2) comprising VL2-CDR1 (SEQ ID NO: 18), VL2-CDR2 (SEQ ID NO: 19), and VL2-CDR3 (SEQ ID NO: 20), and c) A kappa light chain constant region (Cκ) comprising, VL1 and VL2 are connected via a second peptide linker (L2), and each light chain is in the form of VL1-L2-VL2-Cκ, The first heavy chain and the second heavy chain form a dimeric Fc region to provide a dual variable domain immunoglobulin (DVD-Ig) drug conjugate.

[0065] In some embodiments, the first heavy chain variable domain (VH1) comprises the amino acid sequence of SEQ ID NO: 2, the second heavy chain variable domain (VH2) comprises the amino acid sequence of SEQ ID NO: 7, the first light chain variable domain (VL1) comprises the amino acid sequence of SEQ ID NO: 12, and the second light chain variable domain (VL2) comprises the amino acid sequence of SEQ ID NO: 17.

[0066] In some embodiments, each heavy chain comprises the amino acid sequence of SEQ ID NO: 1 or 24, and each light chain comprises the amino acid sequence of SEQ ID NO: 11.

[0067] In some embodiments, the first peptide linker (L1) comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 6). In some embodiments, the second peptide linker (L2) comprises the amino acid sequence GGSGGGGSG (SEQ ID NO: 16).

[0068] In some embodiments, the IgG1 heavy chain constant region (CH1CH2CH3) comprises the amino acid sequence of SEQ ID NO: 21 or 23. In some embodiments, the kappa light chain constant region (Cκ) comprises the amino acid sequence of SEQ ID NO: 22.

[0069] In some embodiments, the first heavy chain and the second heavy chain are connected by two disulfide bridges, which are between the cysteine at position 226 of the first heavy chain and the cysteine at position 226 of the second heavy chain, and between the cysteine at position 229 of the first heavy chain and the cysteine at position 229 of the second heavy chain. The amino acid positions are numbered using EU numbering. For example, in some embodiments, the first heavy chain and the second heavy chain have the amino acid sequence of SEQ ID NO: 1 or 24, and the two disulfide bridges are between C362 of the first heavy chain of SEQ ID NO: 1 or 24 and C362 of the second heavy chain of SEQ ID NO: 1 or 24, and between C365 of the first heavy chain of SEQ ID NO: 1 or 24 and C365 of the second heavy chain of SEQ ID NO: 1 or 24.

[0070] In some embodiments, the Top1i linker-drug is conjugated to the cysteine at position C220 of the first and second heavy chains of the DVD-Ig by EU numbering.

[0071] In some embodiments, n is 1.

[0072] In some embodiments, n is 2.

[0073] In some aspects, the present disclosure has the following structure:

[0074]

Chemical formula

[0075] Other compositions and uses In another aspect, the present disclosure relates to a pharmaceutical composition comprising a DVD-Ig molecule or a DVD-Ig DC described herein and a pharmaceutically acceptable excipient or carrier.

[0076] In another aspect, the present disclosure relates to a method of treating prostate cancer, the method comprising administering to a subject (e.g., a patient) in need thereof a DVD-Ig drug conjugate or a pharmaceutical composition thereof described herein.

[0077] In another aspect, the present disclosure relates to a nucleic acid molecule encoding a heavy chain and / or a light chain of a DVD-Ig molecule described herein.

[0078] In another aspect, the present disclosure relates to a vector comprising a nucleic acid molecule encoding a heavy chain and / or a light chain of a DVD-Ig molecule described herein.

[0079] In another aspect, the present disclosure relates to a host cell capable of producing a DVD-Ig molecule described herein.

Examples

[0080] The following examples are provided for illustrative purposes and not for limitation.

[0081] [Example 1] Preparation of STEAP1 DVD-Ig and PSMA DVD-Ig PSMA and STEAP1 are well-characterized antigens that are overexpressed in metastatic prostate cancer and underexpressed in normal tissues. Both PSMA and STEAP1 are expressed at various levels in most prostate tumors. To bind both antigens on tumor cells with various antigen expressions, DVD-Ig targeting PSMA and STEAP1 was prepared. DVD-Ig targeting both antigens can capture a larger patient population because it may be effective in patients with PSMA1 "high" tumors, STEAP1 "high" tumors, and double "moderate" expression tumors (Figure 2A).

[0082] To reduce the risks associated with the DVD-Ig format, multiple variable regions of PSMA and STEAP1 were identified and humanized for inclusion in a large panel of screening molecules. The variable regions were derived from mAbs with favorable expression and drug-like property profiles because it is thought that the weakness of the variable domains can be amplified when incorporated into a bispecific format. In particular, two well-behaved variable regions may interact with each other when placed in the DVD-Ig format, which may subsequently cause unfavorable properties. Therefore, a library of candidate DVD-Igs was designed and constructed, and screening was performed to identify DVD-Igs with favorable properties.

[0083] Library Design and Plasmid Construction The parental mAbs of the DVD-Ig molecules were generated using mouse (PSMA) and rat (STEAP1) hybridoma technologies, and then the hybridoma supernatants were screened for activity. The antibody heavy and light chain variable regions (VH and VL) of the selected hybridomas were cloned, expressed as chimeric antibodies, and subsequently further humanized using computer-aided high-throughput humanization design software developed internally at AbbVie. Each of the 10 selected humanized PSMA variable regions and each of the 10 selected humanized STEAP1 variable regions were combined in both orientations with linkers (GS10 on HC and GS9 on LC) to obtain a target library of 200 DVD-Ig molecules.

[0084] In a transient mammalian expression system, the DVD-Ig was expressed using two plasmids, one for the HC and one for the LC. The plasmids were constructed by overlap extension PCR and ligation. Briefly, each variable domain was amplified by PCR using primers to incorporate appropriate overhangs for the vector or linker. The PCR products were purified and combined for overlap extension PCR. Subsequently, the pHybE cloning vector with the purified insert and constant region was digested with EcoRI / SalI for the heavy chain (HC) and EcoRI / BsiWI for the light chain (LC). The pooled ligation reactions were transformed into DH5 alpha cells, and six-fold oversamples of the colonies were screened by colony PCR. This process was repeated for pools with low recovery rates.

[0085] For all DVD-Igs for which matching sequences were identified for both the HC plasmid and the LC plasmid, the bacterial cell cultures were rearrayed into 96-well plates and the plasmid DNA was purified for transfection.

[0086] High-throughput antibody production (HTAP) Using a 5 mL scale HTAP platform, DVD-Ig was expressed and purified in Expi293 cells (Thermo). This production scale generally yields approximately 100 mg of protein for screening. Using a Hamilton STAR robot, DNA was transfected from a Costar 3357 96-well plate containing HC and LC DNA in a 3:2 ratio into four 24-well plates using ExpiFectamine™ (Gibco, A29129). Cultures were fed on day 1. After 5 days of transfection, the supernatant containing DVD-Ig was harvested, repositioned, and filtered for purification. Protein A purification was performed in 96-well plate format on an Assaymap Bravo. The purified protein was screened by size exclusion chromatography (SEC) for binding to, internalization into, and stability against both PSMA and STEAP1.

[0087] Of the 200 DVD-Igs, 149 were successfully expressed and 122 DVD-Igs had sufficient yields for detailed characterization. 45 dual-target binders were identified by FACS binding assay and 20 were selected for further characterization (Figure 2B).

[0088] [Example 2] Assays and Characterization Initial drug-like properties, including the aggregation profile and thermal stability of the DVD, indicate that a significant proportion of the produced DVD-Igs do not meet the aggregation threshold of at least 90% monomer nor the DSF thermal stability threshold of T-onset > 55 °C.

[0089] To characterize HTAP DVD-Ig, an indirect cytotoxicity assay was used. LNCaP.FGC cells cultured in RPMI medium supplemented with 10% heat-inactivated FBS, and HEK293 cells engineered to express PSMA (HEK293-PSMA) and HEK293 cells engineered to express STEAP1 (HEK293-STEAP1) cultured in DMEM medium supplemented with 10% heat-inactivated FBS were counted at mid-log phase (viability > 90%), and 100 μl containing 1500 - 2500 cells was plated into each well of a white 96-well plate (Corning, 3610). After incubation at 37 °C for 24 h in a humidified incubator containing 5% CO 2 Two different dilutions (1:100, 1:1000) of small-scale hybridoma supernatants generated with HTAP were incubated for 20 min with goat anti-human IgG1-MMAF, DAR8 (final concentration 2 μg / mL) conjugated secondary antibody. Samples were added to each well and incubated for 5 days. Cell viability was evaluated according to the manufacturer's instructions using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7570), except that the plate was shaken for 15 min before reading. Luminescence was detected with a Perkin Elmer Victor X3 instrument.

[0090] To characterize HTAP DVD-Ig, a flow cytometry binding assay was also used. Specifically, HEK293-PSMA or HEK293-STEAP1 cells grown in T-75 flasks (Corning, 4430720) were harvested using 5 mL of enzyme-free cell dissociation buffer (Invitrogen, 13151-014), washed once with 5 mL of FACs buffer (BD Pharmingen, 554657), and subsequently resuspended in FACs buffer at a cell density of 2.5×10 6 cells / mL. Subsequently, 2.5×10 5Cells with a volume of 100 μL corresponding to per well were dispensed into a V-bottom 96-well plate. Subsequently, 100 μL of various concentrations of DVD-Ig were added, and the samples were incubated at 4 °C for 1 hour. The wells were washed twice with FACs buffer, and then resuspended in 100 μL of 1:100 anti-human IgG antibody (PE, Southern Biotech, 2040-09) together with 1 μL / mL of Far Red Live / Dead Stain (Invitrogen, L34974A) diluted with FACs buffer. After the plate was incubated at 4 °C for an additional 1 hour, it was washed twice with FACs buffer. Subsequently, the cells were resuspended in 100 μL of D-PBS (Sigma, D8537) / 1% formaldehyde and analyzed using a BD FACS Canto II flow cytometer. Subsequently, the data collected from the cytometer were analyzed using FlowJo flow cytometry analysis software.

[0091] Twenty kinds of DVD-Ig passed the initial production and binding screening. Table 3 provides an overview of the in vitro potency, flow cytometry binding of DVD-Ig to LNCaP.FGC, and HEK293 cells engineered to express PSMA and STEAP1.

[0092] [Table 2]

[0093] [Example 3] PSMA-STEAP1 DVD-Ig conjugated with MMAE showed cytotoxic activity in vitro.

[0094] Eight PSMA-STEAP1 DVD-Igs (clone 2, clone 4, clone 9, clone 11, clone 12, clone 13, clone 14, and clone 15) were conjugated to monomethyl auristatin E (MMAE) and evaluated in vitro in LNCaP.FGC, HEK293-STEAP1, and HEK293-PSMA cells. Anti-STEAP1-MMAE ADC and two equivalent anti-PSMA-MMAE ADCs were used as controls.

[0095] Cells (LNCaP.FGC, HEK293-PSMA, and HEK293-STEAP1) were counted (viability >90%) and 100 μg / L of medium containing 1500 - 2500 cells was plated into each well of a white 96-well plate (Corning, 3610). After incubation at 37 °C for 24 h in a humidified incubator containing 5% CO 2 2, the 50 μL of a 3-fold stock solution of the conjugate was added to each well and the cells were incubated for an additional 7 days. Samples were analyzed in triplicate. Cell viability was evaluated according to the manufacturer's instructions using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7570), except that the plate was shaken for 15 min before reading. Luminescence was detected with a Perkin Elmer Victor X3 instrument. Untreated control wells were included in each plate. Cells treated with AB095 DVD-Ig, an anti-tetanus toxoid DVD-Ig conjugated to MMAE, were also included as a control.

[0096] All eight PSMA-STEAP1 DVD-Igs conjugated to MMAE showed cytotoxic activity in vitro in LNCaP.FGC cells (Figure 3A), HEK293-PSMA (Figure 3B), and HEK-STEAP1 (Figure 3C). Their IC50s are shown in Table 5A below.

[0097] [Table 3]

[0098] The in vitro cytotoxic activity of PSMA-STEAP1 DVD-Ig clone 15 conjugated with MMAE (“DVD-MMAE”) was confirmed and is shown in FIGS. 3D-3F. FIG. 3D shows that the DVD-MMAE activity was comparable to that of PSMA-MMAE or STEAP1-MMAE in LNCaP.FGC cells expressing both targets. FIG. 3E shows that the DVD-MMAE activity was weaker than that of PSMA-MMAE in HEK293-PSMA cells, which was expected since PSMA is the internal variable domain of DVD-Ig. FIG. 3F shows that the DVD-MMAE activity was comparable to that of STEAP1-MMAE in HEK293-STEAP1 cells.

[0099] [Example 4] PSMA-STEAP1 DVD-Ig conjugated with MMAE showed cytotoxic activity in a xenograft model Seven out of eight MMAE-conjugated PSMA-STEAP1 DVD-Igs (clone 4, clone 9, clone 11, clone 12, clone 13, clone 14, and clone 15) that showed in vitro cytotoxic activity were further evaluated in an in vivo test. PSMA-STEAP1 DVD-Ig clone 2 conjugated with MMAE was not included in the in vivo test because of its high aggregation rate.

[0100] For both xenograft studies of LNCaP.FGC and 22Rv1, male NSG mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed at 7-8 per cage for each treatment group. Food and water were provided ad libitum. Mice were acclimated to the animal facility for at least one week before the start of the experiment. Mice were tested during the light phase of a 12-hour light:12-hour dark schedule (lights on at 06:00).

[0101] To generate xenografts from the LNCaP.FGC (ATCC) and 22Rv1 (ATCC) cell lines, 5×10 6Individual viable LNCaP.FGC and 22Rv1 cells were each subcutaneously inoculated into the right flank of male NSG mice. The injection volume was 0.1 mL and was composed of a 1:1 mixture with Matrigel (BD, Franklin Lakes, NJ). The tumor length (L) and width (W) were measured via an electronic caliper, and the tumor volume was calculated according to the following formula: V = L × W 2 / 2.

[0102] Mice were assigned to treatment groups such that the mean tumor volume within the group was approximately 175 - 250 mm 3 . The anti - PSMA - MMAE ADC at dose - 1 1X, the anti - STEAP1 - MMAE ADC at dose - 1 1X, and the various PSMA / STEAP - 1 DVD - Ig conjugated with dose - 1 1.33X of MMAE (clone 13 was administered at dose - 1 0.87X due to limited compound availability) were administered within 24 - 48 hours from the assignment to treatment groups based on tumor volume. The tumor volume was estimated twice a week after administration, and the body weight of the test groups was measured at the time of tumor measurement. Mice were euthanized when (1) the tumor volume reached 2,000 mm 3 , (2) when general efficacy parameters were firmly determined, or (3) when required due to other health problems of the mice.

[0103] The efficacy parameters were determined as follows.

[0104] TGI (Tumor Growth Inhibition) = 1 - (mean tumor volume of treatment group / mean tumor volume of treatment control group) × 100 based on the measured value on the day of the vehicle endpoint. The P - value was derived from a Student's t - test comparison of the treatment group versus the vehicle control group.

[0105] TGD (Tumor Growth Delay) = (T - C) / C × 100 (where T is the median time to the endpoint of the treatment group and C is the median time to the endpoint of the treatment control group). The P - value was derived from a Kaplan - Meier log - rank comparison of the treatment group versus the vehicle control group.

[0106] The efficacy parameters for all seven MMAE-conjugated PSMA-STEAP1 DVD-Ig clones tested in the LNCaP.FGC xenograft assay are shown in Table 5B below.

[0107] [Table 4]

[0108] PSMA-STEAP1 DVD-Ig clone 15 conjugated with MMAE ("DVD-MMAE") showed equivalent or superior activity compared to single-specificity ADCs ("PSMA-MMAE" and "STEAP1-MMAE") in 22Rv1 (Figure 4A) and LNCaP.FGC (Figure 4B) xenograft models. The surface expression of PSMA and STEAP1 is high in both 22Rv1 and LNCaP.FGC models.

[0109] [Example 5] Risk-Responsibility Design of Lead Candidates Based on the results of in vitro screening and in vivo tests, clone 15 was selected for further evaluation and manipulation. It showed good efficacy and acceptable drug-like properties, but a deamidation motif (20%) was observed in the PSMA-binding domain.

[0110] Rational design was carried out to identify and remove sequences with a high potential for risk responsibility. Mutant DVDs were prepared and evaluated by Biacore assay, and from the kinetics of binding to PSMA, the identification of NT as a non-destructive mutation distant from "NG" was obtained. Since no significant loss of binding occurred, NT was used at this position of HCDR2 in further versions of DVD-Ig. To facilitate conjugation of DAR2 or DAR6, the variable region was cloned onto the light chain containing a mutation from cysteine to alanine. This generates an unpaired cysteine at position C220 of the HC for linker-drug conjugation in an easy site-specific manner.

[0111] DVD-Ig1 was prepared by deamidation of VH2-CDR2 of clone 15, and its sequence is shown in Table 1 below.

[0112]

Table 5

[0113] [Example 6] Preparation of DVD-Ig1 TOP1i Drug Conjugate As shown in the above examples, DVD-Ig1 was identified based on its lack of expression, drug-like properties, in vitro bivalent binding ability, in vivo activity, and sequence instability. Subsequently, this was used to prepare a TOP1i drug conjugate.

[0114] The structure of the TOP1i drug-linker is shown below.

[0115]

Chemical formula

[0116] 509.9 mL of the antibody solution (11.34 mg / mL) in PBS was added to 2.0 mL of a 0.5 M, pH 7.5 EDTA solution. Subsequently, a solution of diphenyl-phosphino-4-benzoic acid (10 mM, 18.38 mL, 2.5 equivalents) in DMA was added, and the mixture was maintained at 4 °C overnight (18 hours) for reduction. 51.6 mL (20% v / v) of a 100 mM, pH 8.5 sodium bicarbonate buffer was added to the reduced antibody, followed by treatment with a drug-linker solution (10 mM, 11.4 mL, 4.0 equivalents relative to the antibody), and maintained at 4 °C for 60 minutes. The crude conjugate was purified to remove excess drug-linker and other small molecule reagents.

[0117] HIC analysis: Hydrophobic interaction chromatography was performed at 30 °C using a TSK-GEL (registered trademark) Ether-5PW column (TOSOH Bioscience, 2.0 mm ID × 7.5 cm, 10 μM). The ADC was loaded with 50% buffer A and eluted with 10% B for 1 minute followed by a gradient from 10% B to 100% B over 30 minutes. Mobile phase A was 25 mM sodium phosphate buffer, 1.5 M ammonium sulfate, pH 7.0, and mobile phase B was a mixture of 75% 25 mM sodium phosphate buffer pH 7.0 and 25% isopropyl alcohol, and the flow rate was 0.1 mL / min. The data are shown in Figure 5A.

[0118] Aggregation analysis: Size exclusion chromatography was performed using a Waters ACQUITY UPLC (registered trademark) Protein BEH SEC column (200 Å, 1.7 μm, 4.6 mm × 150 mm) with 100 mM sodium phosphate, 150 mM sodium chloride, 10% volume / volume acetonitrile, pH 7.0 at a flow rate of 0.4 mL / min. The absorbance peak area at 280 nm was determined for each of the high molecular weight eluate and monomer eluate by integration of the area under the curve. The percentage of aggregates in the conjugate sample was determined by dividing the absorbance peak area at 280 nm for the high molecular weight eluate by the sum of the absorbance peak areas at 280 mM for the high molecular weight eluate and monomer eluate and multiplying by 100%. The aggregation percentage was determined to be 2.7% (Figure 5B).

[0119] Mass spectrometry: LC-MS analysis was performed using an Agilent 1100 HPLC system connected to an Agilent LC / MSD TOF 6220 ESI mass spectrometer. The ADC was reduced with 25 mM (final concentration) of Bond-Breaker® TCEP solution (Thermo Scientific, Rockford, IL) and loaded onto a Protein Microtrap (Michrom Bioresorces, Auburn, CA) desalting cartridge, eluted at ambient temperature for 0.2 minutes with a gradient of 10% B to 75% B. Mobile phase A was H2O containing 0.1% formic acid, mobile phase B was acetonitrile containing 0.1% formic acid, and the flow rate was 0.2 mL / min. Electrospray ionization time-of-flight mass spectra of the co-eluting light and heavy chains were acquired using Agilent MassHunter® acquisition software. The deconvolution of the extracted intensity vs. m / z spectra was performed using the Maximum Entropy function of the MassHunterp software to determine the mass of each reduced antibody fragment. The DAR was calculated from the deconvoluted spectrum by summing the intensities of the naked and modified peaks for the light and heavy chains and normalizing by multiplying by the number of drugs attached to the intensity. The summed and normalized intensities were divided by the sum of the intensities, and the final average DAR value for the complete ADC was obtained from the sum of the results for two light chains and two heavy chains.

[0120] MS data: 35836 Dalton - light chain (LC, LC+0). 65144 Dalton - heavy chain (HC+0, des-K, G0F). 65827 Dalton - heavy chain + one drug (HC+1)

[0121]

Table 6

[0122] [Example 7] In Vitro Characterization of PSMA-STEAP1-Top1i The internal migration and in vitro cytotoxicity characteristics of the DVD-Ig1 drug conjugate were determined.

[0123] Internal migration assay Cells were maintained in a humidified 37 °C incubator containing 5% CO 2 Exponentially growing LNCaP.FGC cells were detached using 0.25% trypsin-EDTA (Sigma, T4049) and plated at 20,000 cells / well in 50 μl of cell culture medium in a 96-well plate (Corning, 3610). The next day, antibody conjugates (PSMA-STEAP1-Top1i, PSMA-Top1i, and STEAP1-Top1i) and FabFluor reagent (Sartorius, 4722) were mixed in a 1:3 molar ratio in the medium for 15 minutes. The labeled antibodies were serially diluted and added to each well in triplicate. The plate was placed in an Incucyte Zoom (Sartorius) for up to 24 hours for image collection. The collected images were processed and analyzed. The final data were exported to GraphPad Prism software for graphing.

[0124] Intracellular accumulation assay The average intracellular concentration of unconjugated Top1i was measured using an intracellular accumulation assay. Specifically, 2 million LNCaP.FGC cells in 40 mL of medium were plated in T150 flasks 24 hours prior to treatment. Each T150 flask was treated with 0 nM, 1 nM, 10 nM, or 30 nM of PSMA-STEAP1-Top1i, PSMA-Top1i, or STEAP1-Top1i. Samples were collected at 4, 6, and 24 hours. Two technical replicates were used at each time point. Cells without treatment (0 nM) were used as a control. At each time point, the cells were washed twice with PBS and detached with trypsin to prepare a single-cell suspension. The number of live cells and cell diameter were determined from the single-cell suspension to calculate the total cell volume (liters). LC-MS analysis was performed to determine the amount (nanomoles) of small molecules inside the cells. By LC-MS method, the total of cell-related molecules not covalently bound to any entity, i.e., the total of bound and unbound molecules inside the cells, was quantified. All molecules covalently bound to cell proteins / lipids were not extracted and measured. The Top1i compound has two forms: a closed-ring (lactone) form active at low pH and an open-ring (carboxylate) form inactive. When preparing samples for LC-MS analysis, since the equilibrium shifts to the active closed-ring form as the pH gets lower, the protein was precipitated with an acidic diluent. During the assay, the pH of the medium became acidic. In LC-MS bioanalysis, since most of the unconjugated payload was converted to the closed-ring state, the "total payload" (i.e., the open-ring and closed-ring forms) was measured regardless of their original forms present in the sample.

[0125] Subsequently, the average intracellular concentration (nanomoles) was calculated based on the total cell volume and the amount of small molecules inside the cells. Details of the additional calculations are shown below.

[0126] Nanomoles per sample = LCMS concentration of cell sample [nM] × sample volume [L]

[0127]

Number

[0128] [Number]

[0129] [Number]

[0130] Anti-proliferation assay LNCaP.FGC cells were dissociated and counted using 0.25% trypsin-EDTA (Sigma, T4049) (viability >90%). 100 μL of growth medium containing 2500 cells was plated into each well of a white 96-well plate (Corning, 3610). After incubation at 37 °C for 24 h in a humidified incubator containing 5% CO 2 , 50 μL of a 3× stock solution of the conjugate or payload was added to each well and the cells were incubated for an additional 7 days. Samples were analyzed in triplicate. Cell viability was evaluated according to the manufacturer's instructions using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7570), except that the plates were shaken for 15 min before reading. Luminescence was detected using a Perkin Elmer Victor X3 instrument. Untreated control wells were included in each plate. The percent of the control was calculated using the following formula: 100×(treated) / (control). The half-maximal inhibitory concentration (IC 50 ) value was calculated using GraphPad Prism software.

[0131] Figures 6A and 6B show that the DVD-Ig1 conjugated to Top1i at the DAR2 site (“PSMA-STEAP1-Top1i”) trafficked internally faster and delivered more payload compared to either of the single-specificity ADCs (“PSMA-Top1i” or “STEAP1-Top1i”).

[0132] Figure 6B shows that intracellular payload accumulation for PSMA-STEAP1-Top1i was approximately two-fold higher than that of either the single-specificity ADC, PSMA-Top1i or STEAP-Top1i, in cells treated with 30 nM of the drug conjugate for 24 hours.

[0133] PSMA-STEAP1-Top1i also demonstrated superior anti-proliferative activity compared to either of the single-specificity ADCs, PSMA-Top1i or STEAP-Top1i (Figure 6C).

[0134] [Example 8] Characterization of PSMA-STEAP1-Top1i in vivo DVD-Ig1 conjugated to Top1i (“PSMA-STEAP1-Top1i”) demonstrated efficacy in prostate tumors expressing STEAP1 and PSMA in an in vivo xenograft model.

[0135] For the LNCaP.FGC xenograft study, male NSG mice were obtained from Jackson Laboratories (Bar Harbor, ME). Male CB17.SCID (Charles River Laboratories, Wilmington, MA) mice were used for the 22Rv1 xenograft study. Mice were housed at 7-8 per cage per treatment group. Food and water were provided ad libitum. Mice were acclimated to the animal facility for at least one week prior to the start of the experiment. Mice were tested during the light phase of a 12-hour light:12-hour dark schedule (lights on at 06:00).

[0136] To generate xenografts from the LNCaP.FGC (ATCC) and 22Rv1 (ATCC) cell lines, 5×10 6Individual viable LNCaP.FGC and 22Rv1 cells were subcutaneously inoculated into the right flank of male NSG mice and male CB17.SCID mice, respectively. The injection volume was 0.1 mL and consisted of a 1:1 mixture with Matrigel (BD, Franklin Lakes, NJ). The tumor length (L) and width (W) were measured via an electronic caliper, and the tumor volume was calculated according to the following formula: V = L × W 2 / 2.

[0137] Mice were assigned to treatment groups such that the average tumor volume within the group was approximately 175 - 250 mm 3 . For the LNCaP.FGC xenograft study, PSMA-Top1i at dose-2 (1×), STEAP1-Top1i at dose-2 (1×), and PSMA-STEAP1-Top1i at dose-2 (1.3×) were administered within 24 - 48 hours after randomization. For the LNCaP.FGC xenograft study, PSMA-Top1i at dose-3 (1×), STEAP1-Top1i at dose-3 (1×), and PSMA-STEAP1-Top1i at dose-3 (1.3×) were administered within 24 - 48 hours after randomization. The tumor volume was estimated twice a week after administration, and the body weight of the test group was measured at the time of tumor measurement. Mice were euthanized when (1) the tumor volume reached 2,000 mm 3 , (2) when general efficacy parameters were firmly determined, or (3) when other health problems in the mice were observed.

[0138] PSMA-STEAP1-Top1i was highly effective against LNCaP.FGC subcutaneous flank tumors when administered as a single dose at dose-2 (1.3×) (Figure 7B). Treatment of these cell-derived xenografts (CDX) resulted in tumor volume regression over a long period (more than 30 days to more than 100 days) in this model, which expressed both PSMA and STEAP1 surface molecules at high levels according to RNA expression analysis.

[0139] For 22Rv1, a CDX model that expresses both PSMA and STEAP1 at high levels based on RNA expression, PSMA-STEAP1-Top1i was highly effective even at dose -3 (1.3×) (Figure 7A). Drug conjugates targeting either PSMA (“PSMA-Top1i”) or STEAP1 (“STEAP1-Top1i”) were equally effective, and differences were only observed when the single-dose administration was well exceeded.

[0140] These data indicate that the DVD-Ig1 disclosed herein has activity comparable to or better than that of individual ADCs, and that single-dose administration of PSMA-STEAP1-Top1i supports sustained tumor regression.

[0141] Exemplary embodiments: Embodiment 1. The following structure:

[0142]

Chemical formula

[0143] Embodiment 2. The DVD-Ig drug conjugate according to Embodiment 1, wherein the first heavy chain variable domain (VH1) comprises the amino acid sequence of SEQ ID NO: 2, the second heavy chain variable domain (VH2) comprises the amino acid sequence of SEQ ID NO: 7, the first light chain variable domain (VL1) comprises the amino acid sequence of SEQ ID NO: 12, and the second light chain variable domain (VL2) comprises the amino acid sequence of SEQ ID NO: 17.

[0144] Embodiment 3. The DVD-Ig drug conjugate according to Embodiment 1 or 2, wherein the first peptide linker (L1) comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 6).

[0145] Embodiment 4. The DVD-Ig drug conjugate according to any one of Embodiments 1 to 3, wherein the second peptide linker (L2) comprises the amino acid sequence GGSGGGGSG (SEQ ID NO: 16).

[0146] Embodiment 5. The DVD-Ig drug conjugate according to any one of Embodiments 1 to 4, wherein the IgG1 heavy chain constant region (CH1CH2CH3) comprises the amino acid sequence of SEQ ID NO: 21 or 23.

[0147] Embodiment 6. The DVD-Ig drug conjugate according to any one of Embodiments 1 to 5, wherein the kappa light chain constant region (Cκ) contains the amino acid sequence of SEQ ID NO: 22.

[0148] Embodiment 7. The DVD-Ig drug conjugate according to any one of Embodiments 1 to 6, wherein each of the heavy chains contains the amino acid sequence of SEQ ID NO: 1 or 24, and each light chain contains the amino acid sequence of SEQ ID NO: 11.

[0149] Embodiment 8. The DVD-Ig drug conjugate according to any one of Embodiments 1 to 7, wherein the first heavy chain and the second heavy chain are connected by two disulfide bridges, and the two disulfide bridges are between the cysteine at position 226 of the first heavy chain and the cysteine at position 226 of the second heavy chain, and between the cysteine at position 229 of the first heavy chain and the cysteine at position 229 of the second heavy chain, according to EU numbering.

[0150] Embodiment 9. The DVD-Ig drug conjugate according to any one of Embodiments 1 to 8, wherein the Top1i linker-drug is conjugated to the cysteine at position C220 of the first and second heavy chains of the DVD-Ig according to EU numbering.

[0151] Embodiment 10. The DVD-Ig drug conjugate according to any one of Embodiments 1 to 9, wherein n is 1.

[0152] Embodiment 11. The DVD-Ig drug conjugate according to any one of Embodiments 1 to 9, wherein n is 2.

[0153] Embodiment 12. The following structure:

[0154]

Chemical formula

[0155] Embodiment 13. A DVD-Ig that binds to human STEAP1 and human PSMA and comprises two identical heavy chains and two identical light chains, wherein each heavy chain a) a first heavy chain variable domain (VH1) comprising VH1-CDR1 (SEQ ID NO: 3), VH1-CDR2 (SEQ ID NO: 4) and VH1-CDR3 (SEQ ID NO: 5), b) a second heavy chain variable domain (VH2) comprising VH2-CDR1 (SEQ ID NO: 8), VH2-CDR2 (SEQ ID NO: 9) and VH2-CDR3 (SEQ ID NO: 10), and c) an IgG1 heavy chain constant region (CH1CH2CH3) is included, wherein VH1 and VH2 are connected via a first peptide linker (L1), and each heavy chain is in the form of VH1-L1-VH2-CH1CH2CH3, wherein each light chain a) a first light chain variable domain (VL1) comprising VL1-CDR1 (SEQ ID NO: 13), VL1-CDR2 (SEQ ID NO: 14) and VL1-CDR3 (SEQ ID NO: 15), b) a second light chain variable domain (VL2) comprising VL2-CDR1 (SEQ ID NO: 18), VL2-CDR2 (SEQ ID NO: 19) and VL2-CDR3 (SEQ ID NO: 20), and c) a kappa light chain constant region (Cκ) is included, VL1 and VL2 are connected via a second peptide linker (L2), and each light chain has the form of VL1-L2-VL2-Cκ, The first heavy chain and the second heavy chain form a dimeric Fc region, DVD-Ig.

[0156] Embodiment 14. The first heavy chain variable domain (VH1) contains the amino acid sequence of SEQ ID NO: 2, the second heavy chain variable domain (VH2) contains the amino acid sequence of SEQ ID NO: 7, the first light chain variable domain (VL1) contains the amino acid sequence of SEQ ID NO: 12, and the second light chain variable domain (VL2) contains the amino acid sequence of SEQ ID NO: 17. The DVD-Ig according to Embodiment 13.

[0157] Embodiment 15. The first peptide linker (L1) contains the amino acid sequence GGGGSGGGGS (SEQ ID NO: 6). The DVD-Ig according to Embodiment 13 or 14.

[0158] Embodiment 16. The second peptide linker (L2) contains the amino acid sequence GGSGGGGSG (SEQ ID NO: 16). The DVD-Ig according to any one of Embodiments 13 to 15.

[0159] Embodiment 17. The IgG1 heavy chain constant region (CH1CH2CH3) contains the amino acid sequence of SEQ ID NO: 21 or 23. The DVD-Ig according to any one of Embodiments 13 to 16.

[0160] Embodiment 18. The kappa light chain constant region (Cκ) contains the amino acid sequence of SEQ ID NO: 22. The DVD-Ig according to any one of Embodiments 13 to 17.

[0161] Embodiment 19. Each of the heavy chains contains the amino acid sequence of SEQ ID NO: 1 or 24, and each light chain contains the amino acid sequence of SEQ ID NO: 11. The DVD-Ig according to any one of Embodiments 13 to 18.

[0162] Embodiment 20. The first heavy chain and the second heavy chain are connected by two disulfide bridges, and the two disulfide bridges are between the cysteine at position 226 of the first heavy chain and the cysteine at position 226 of the second heavy chain, and between the cysteine at position 229 of the first heavy chain and the cysteine at position 229 of the second heavy chain, according to EU numbering, of the DVD-Ig according to any one of Embodiments 13 to 19.

[0163] Embodiment 21. A DVD-Ig that binds to human STEAP1 and human PSMA and includes two identical heavy chains and two identical light chains, wherein each heavy chain includes the amino acid sequence of SEQ ID NO: 1 or 24, each light chain includes the amino acid sequence of SEQ ID NO: 11, the first heavy chain and the second heavy chain are connected by two disulfide bridges, and the two disulfide bridges are between the cysteine at position 226 of the first heavy chain and the cysteine at position 226 of the second heavy chain, and between the cysteine at position 229 of the first heavy chain and the cysteine at position 229 of the second heavy chain, according to EU numbering.

[0164] Embodiment 22. A nucleic acid encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 or 24 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 11.

[0165] Embodiment 23. A vector comprising the nucleic acid according to Embodiment 22.

[0166] Embodiment 24. A host cell comprising the nucleic acid according to Embodiment 22 or the vector according to Embodiment 23.

[0167] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

[0168] Although various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the invention.

Claims

1. The structure: 【Chemistry 1】 [In the formula, n is 1 or 2, DVD-Ig binds to human STEAP1 and human PSMA; DVD-binding protein comprises two identical heavy chains and two identical light chains, Each heavy chain is a) a first heavy chain variable domain (VH1) comprising VH1-CDR1 (SEQ ID NO: 3), VH1-CDR2 (SEQ ID NO: 4) and VH1-CDR3 (SEQ ID NO: 5); b) a second heavy chain variable domain (VH2) comprising VH2-CDR1 (SEQ ID NO: 8), VH2-CDR2 (SEQ ID NO: 9) and VH2-CDR3 (SEQ ID NO: 10), and c) IgG1 heavy chain constant region (CH1CH2CH3) wherein VH1 and VH2 are connected via a first peptide linker (L1), and each heavy chain is in the form VH1-L1-VH2-CH1CH2CH3; Each light chain is a) a first light chain variable domain (VL1) comprising VL1-CDR1 (SEQ ID NO: 13), VL1-CDR2 (SEQ ID NO: 14) and VL1-CDR3 (SEQ ID NO: 15); b) a second light chain variable domain (VL2) comprising VL2-CDR1 (SEQ ID NO: 18), VL2-CDR2 (SEQ ID NO: 19) and VL2-CDR3 (SEQ ID NO: 20), and c) Kappa light chain constant region (Cκ) Including, VL1 and VL2 are connected via a second peptide linker (L2), and each light chain is in the form VL1-L2-VL2-Cκ; The first and second heavy chains form a dimeric Fc region. A dual variable domain immunoglobulin (DVD-Ig) drug conjugate comprising:

2. the first heavy chain variable domain (VH1) comprises the amino acid sequence of SEQ ID NO:2; the second heavy chain variable domain (VH2) comprises the amino acid sequence of SEQ ID NO:7; the first light chain variable domain (VL1) comprises the amino acid sequence of SEQ ID NO: 12; The second light chain variable domain (VL2) comprises the amino acid sequence of SEQ ID NO: 17; 2. The DVD-Ig drug conjugate of claim 1.

3. 3. The DVD-binding protein of claim 1 or 2, wherein the first peptide linker (L1) comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO:6).

4. 4. The DVD-binding protein of claim 1, wherein the second peptide linker (L2) comprises the amino acid sequence GGSGGGGSG (SEQ ID NO: 16).

5. 5. The DVD-Ig drug conjugate of any one of claims 1 to 4, wherein the IgG1 heavy chain constant region (CH1CH2CH3) comprises the amino acid sequence of SEQ ID NO: 21 or 23.

6. 6. The DVD-binding protein of any one of claims 1 to 5, wherein the kappa light chain constant region (CK) comprises the amino acid sequence of SEQ ID NO:

22.

7. each of the heavy chains comprises the amino acid sequence of SEQ ID NO: 1 or 24; each light chain comprises the amino acid sequence of SEQ ID NO: 11; The DVD-Ig drug conjugate of any one of claims 1 to 6.

8. 8. The DVD-binding protein of any one of claims 1 to 7, wherein the first and second heavy chains are connected by two disulfide bridges, the two disulfide bridges being between a cysteine ​​at position 226 of the first heavy chain and a cysteine ​​at position 226 of the second heavy chain, and between a cysteine ​​at position 229 of the first heavy chain and a cysteine ​​at position 229 of the second heavy chain, according to the EU numbering.

9. 9. The DVD-binding protein of any one of claims 1-8, wherein the Top1i linker-drug is conjugated to a cysteine ​​at position C220 of the first and second heavy chains of the DVD-binding protein, according to EU numbering.

10. The DVD-binding protein of any one of claims 1 to 9, wherein n is 1.

11. The DVD-binding protein of any one of claims 1 to 9, wherein n is 2.

12. The structure: 【Chemistry 2】 wherein n is 2, the DVD-binding protein binds to human STEAP1 and human PSMA and comprises two identical heavy chains and two identical light chains; each of the heavy chains comprises the amino acid sequence of SEQ ID NO: 1 or 24; each light chain comprises the amino acid sequence of SEQ ID NO: 11; the first heavy chain and the second heavy chain are connected by two disulfide bridges, the two disulfide bridges being between cysteine ​​at position 226 of the first heavy chain and cysteine ​​at position 226 of the second heavy chain, and between cysteine ​​at position 229 of the first heavy chain and cysteine ​​at position 229 of the second heavy chain according to the EU numbering; Top1i linker-drug is conjugated to the cysteine ​​at position C220 of the first and second heavy chains of DVD-binding protein according to EU numbering. A dual variable domain immunoglobulin (DVD-Ig) drug conjugate comprising:

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