Multispecific antibodies and uses thereof

Abstract

Disclosed are multispecific antibodies comprising an antibody or antigen binding fragment targeting CD3, an antibody or antigen binding fragment targeting 4-1BB, and another antibody or antigen binding fragment specific for a tumor associated antigen, such as WT1 presented by HLA-A2, expressed in a variety of tumor cancers. Such multispecific antibodies have enhanced efficacy and reduced toxicity, and are thus particularly suitable for treating diseases such as cancer.

Description

Background Technology

[0001] T-cell engagers are multifunctional molecules that recruit a patient's own T cells to malignant cells, thereby generating cytotoxic activity against the malignant cells. Common designs of T-cell engagers include anti-CD3 antibodies and another antibody specific to tumor-associated antigens (TAAs). Current T-cell engagers exhibit limited T-cell-mediated responses, particularly in poorly immunogenic tumors. Furthermore, T-cell agonist antibodies (such as CD3 antibodies) are known to cause serious side effects, such as cytokine release syndrome (CRS). Therefore, developing suitable T-cell engagers presents multiple challenges, including adequate tumor targeting, minimization of systemic activation, and in vitro and in vivo stability.

[0002] Wilms' blastoma gene 1 (WT1) was initially identified as an oncogene involved in the development of wilted blastoma. The oncogenic properties of WT1 have been confirmed in various hematologic malignancies and solid tumors. On the other hand, WT1 is rarely expressed in normal adult tissues. WT1-positive tumors include tumors of the stomach, prostate, biliary system, and urinary system, as well as malignant melanomas. Among these, glioblastoma, some soft tissue sarcomas, osteosarcomas, and cutaneous malignant melanomas exhibit extremely strong cytoplasmic staining compared to other tumors. WT1 is an intracellular protein located in the nucleus or cytoplasm. Intracellular proteins can be degraded by the proteasome, processed, and presented on the cell surface as a major histocompatibility complex (MHC) I epitope, and recognized by the T cell receptor (TCR). For WT1, the peptide RMFPNAPYL (WT1...) RMF ) and VLDFAPPGA (WT1 VLD HLA-A2 can be presented on the cell surface to trigger T cell recognition.

[0003] Currently, WT1 is considered a molecular target for various immunotherapies for malignant tumors, and can exert its effects through three classes of immunotherapies: vaccines, cell therapy, and T-cell adjuvants. Galinpepimut-S is a vaccine containing a heterologous peptide of nephroblastoma (WT1), which, as an immunotherapy, stimulates CD8. + and CD4 +T-cell therapy, designed to trigger an immune response, is intended for the treatment of acute myeloid leukemia (AML) in first remission, metastatic AML, malignant pleural mesothelioma (MPM), multiple myeloma (MM), chronic myeloid leukemia (CML), acute leukemia (AL), and other advanced cancers (including ovarian cancer, colorectal cancer, small cell lung cancer, and triple-negative breast cancer). In a trial report for mesothelioma patients, the study showed that patients receiving the WT1 cancer vaccine had a longer median overall survival (OS) of 39 months, compared to only 18 months in the control group. Additionally, the median progression-free survival (PFS) of the WT1 cancer vaccine was 11.5 months, compared to 5.5 months in the control group. Data also indicated that the drug exhibited a very good safety profile, with no grade 3 / 4 toxicities or dose-limiting toxicities. Regarding T-cell therapy, NexImmune reported results from a Phase 1 clinical trial of NEXI-001. Patients treated with NEXI-001 after lymphocyte-depleted chemotherapy showed increased CD8 counts. + and CD4 + Rapid reconstitution of all T-cell subtypes was achieved. To date, NEXI-001 has maintained a favorable tolerability profile across all dose levels, with no grade 3 or higher treatment-related SAEs occurring. Two patients experienced grade 2 CRS, which resolved within 24 hours of receiving tocilizumab therapy. No cases of ICANS occurred. All these data suggest that WT1 is a promising target for immunotherapy. Summary of the Invention

[0004] This disclosure provides a multispecific antibody comprising: (a) an anti-CD3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, the light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 21, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 22, 27, 28 or 29, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 23, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 24, VLCDR2 comprises the amino acid sequence of SEQ ID NO: 25, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 26; (b) an anti-4-1BB antibody or an antigen-binding fragment thereof; and (c) an anti-WT1 antibody or an antigen-binding fragment thereof.

[0005] In some embodiments, the VH and VL of the anti-CD3 antibody or its antigen-binding fragment respectively comprise the amino acid sequences of (1) SEQ ID NO: 19 and 20, (2) SEQ ID NO: 30 and 20, (3) SEQ ID NO: 31 and 20, (4) SEQ ID NO: 32 and 33 or (5) SEQ ID NO: 34 and 33. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment is a single-chain fragment (scFv).

[0006] In some embodiments, the anti-4-1BB antibody is a single-domain antibody (sdAb). In some embodiments, the sdAb comprises CDR1, CDR2, and CDR3, which respectively comprise the amino acid sequences of SEQ ID NO: 35, 36, and 37. In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the sdAb is fused to the C-terminus of the Fc fragment in a multispecific antibody.

[0007] In some embodiments, an anti-CD3 antibody or its antigen-binding fragment is fused to the N-terminus of one strand of the Fc fragment.

[0008] In some embodiments, the anti-WT1 antibody or its antigen-binding fragment is fused to the N-terminus of the second chain of the Fc fragment.

[0009] In some embodiments, the multispecific antibody comprises a second anti-WT1 antibody or an antigen-binding fragment thereof fused to the N-terminus of an anti-CD3 antibody or an antigen-binding fragment thereof.

[0010] In some embodiments, the anti-WT1 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprises VL CDR1, VLCDR2, and VL CDR3. The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 13-18. In some embodiments, the VH and VL of the anti-WT1 antibody or its antigen-binding fragment each comprise the amino acid sequences of SEQ ID NO: 1 and 2, respectively.

[0011] In one embodiment, a multispecific antibody is also provided, comprising (a) an anti-WT1 antibody or an antigen-binding fragment thereof, the anti-WT1 antibody or the antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, the light chain variable region comprising VL CDR1, VLCDR2 and VL CDR3, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 13-18; (b) an anti-CD3 antibody or an antigen-binding fragment thereof; and (c) an anti-4-1BB antibody or an antigen-binding fragment thereof.

[0012] In some embodiments, the VH and VL of the anti-WT1 antibody or its antigen-binding fragment contain the amino acid sequences of SEQ ID NO: 1 and 2, respectively.

[0013] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment comprises VH CDR1, VH CDR1 and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1, VH CDR1, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 comprise (1) SEQ ID NO: 21, 22, 23, 24, 25 and 26; (2) SEQ ID NO: 21, 27, 23, 24, 25 and 26; (3) SEQ ID NO: 21, 28, 23, 24, 25 and 26; or (4) SEQ ID NO: 21, 29, 23, 24, 25 and 26.

[0014] In some embodiments, the VH and VL of the anti-CD3 antibody or its antigen-binding fragment respectively contain the amino acid sequences of (1) SEQ ID NO: 19 and 20, (2) SEQ ID NO: 30 and 20, (3) SEQ ID NO: 31 and 20, (4) SEQ ID NO: 32 and 33 or (5) SEQ ID NO: 34 and 33.

[0015] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment is a single-chain fragment (scFv).

[0016] In some embodiments, the anti-4-1BB antibody is a single-domain antibody (sdAb). In some embodiments, the sdAb comprises CDR1, CDR2, and CDR3, which respectively comprise the amino acid sequences of SEQ ID NO: 35, 36, and 37. In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 38.

[0017] In some embodiments, the sdAb is fused to the C-terminus of the Fc fragment of a multispecific antibody. In some embodiments, an anti-CD3 antibody or its antigen-binding fragment is fused to the N-terminus of one strand of the Fc fragment. In some embodiments, an anti-WT1 antibody or its antigen-binding fragment is fused to the N-terminus of a second strand of the Fc fragment.

[0018] In some embodiments, the multispecific antibody comprises a second anti-WT1 antibody or an antigen-binding fragment thereof fused to the N-terminus of an anti-CD3 antibody or an antigen-binding fragment thereof.

[0019] It also provides pharmaceutical compositions comprising multispecific antibodies and pharmaceutically acceptable carriers, one or more polynucleotides encoding multispecific antibodies, vectors comprising one or more polynucleotides, and host cells comprising one or more polynucleotides or vectors.

[0020] Another embodiment provides a method for treating a disease or condition in a subject of need, the method comprising administering to the subject an effective amount of a multispecific antibody, a pharmaceutical composition, one or more polynucleotides, a carrier, or a host cell. Yet another embodiment provides the use of the multispecific antibody, pharmaceutical composition, one or more polynucleotides, a carrier, or a host cell in the preparation of a medicament for treating a disease or condition.

[0021] In some embodiments, the disease or condition is cancer. In some embodiments, cancer is selected from the group consisting of: ovarian cancer, prostate cancer, urinary tract cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, and thyroid cancer. Attached Figure Description

[0022] Figure 1 The results showed that WT1-09 exhibited higher affinity compared to RG-6007.

[0023] Figure 2 This demonstrates that WT1-09 does not bind to off-target peptides.

[0024] Figure 3The on-target (AB) and off-target (CE) binding of WT1-09 and RG-6007 are shown. HLA-A02 phenotype and WT1 mRNA levels in different cell lines are shown in (F). WT1-09 exhibits HLA-A02-dependent affinity for WT1.

[0025] Figure 4 The 1+1 and 2+1 forms of the WT1-CD3-4-1BB trispecific antibody were demonstrated.

[0026] Figure 5 Showing WT1-09 to CD34 + Hematopoietic stem cells showed lower affinity than RG-6007 (A). WT1-TOPA did not induce HSC-dependent tumor cell lysis (B).

[0027] Figure 6 The study showed that WT1-TOPA can induce cell lysis in different tumor cell lines.

[0028] Figure 7 It was shown that when incubated with the SW620 tumor cell line, WT1-TOPA can induce the release of IL-2 and IFN-γ from PBMCs.

[0029] Figure 8 CD3-NFAT (A) and 41BB-NFκB (B) of WT1-TOPA with different CD3 or 41BB sequences are shown.

[0030] Figure 9 Cell lysis activity of WT1-TOPA with different CD3 sequences was demonstrated.

[0031] Figure 10 The results showed that when treated with PBMCs alone, WT1-TOPA exhibited less IL-6 secretion than RG-6007.

[0032] Figure 11 The results show that WT1-TOPA exhibits superior sustained lethality compared to RG-6007.

[0033] Figure 12 It was shown that WT1-TOPA treatment resulted in less CD4(A) or CD8(B) T cell depletion compared to RG-6007.

[0034] Figure 13 The study showed that WT1-TOPA molecules resulted in a significant reduction in tumor size compared to RG-6007 (A). RG-6007 treatment induced activation of both CD4 and CD8 T cells, leading to cell death, which promoted tumor growth (BC). Detailed Implementation

[0035] definition

[0036] It should be noted that the term "a / an" refers to one or more of the same entity; for example, "an antibody" should be understood to represent one or more antibodies. Therefore, the terms "a / an," "one or more," and "at least one" are used interchangeably in this document.

[0037] As used herein, "antibody" or "antigen-binding moiety" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody as well as any antigen-binding fragment or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing at least a portion of an immunoglobulin molecule having the biological activity of binding an antigen. Examples of such molecules include, but are not limited to, the complementarity-determining region (CDR) of the heavy or light chain or its ligand-binding portion, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the frame (FR) region, or any portion thereof, or at least a portion of the binding protein.

[0038] Full-length antibodies consist of two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains may be referred to as “VH” and “VL”, respectively. The variable regions in both chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (including the light chain (LC) CDRs of LC-CDR1, LC-CDR2, and LC-CDR3, and the heavy chain (HC) CDRs of HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibody-antigen binding fragments disclosed herein can be defined or identified using the following conventions: Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three core-residue junctions (CDRs) of either the heavy or light chain lie between flanking segments called framework regions (FRs). These framework regions are more conserved than the CDRs and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further subdivided into subclasses, such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain).

[0039] As used herein, the term "half-antibody" refers to an immunoglobulin heavy chain associated with an immunoglobulin light chain. Those skilled in the art will readily understand that a half-antibody may encompass a fragment thereof and may also have an antigen-binding domain composed of a single variable domain, such as those derived from camelids.

[0040] As used herein, the term "single-chain half antibody" refers to a single-chain polypeptide comprising a VL domain, optionally a CL domain, a tether, a VH domain, optionally a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the domains are positioned relative to each other in the N-terminal to C-terminal direction as follows: VL-tether-VH-hinge-CH2-CH3, VL-tether-VH-partial hinge-CH2-CH3, VL-tether-VH-hinge variant-CH2-CH3, or VL-CL-tether-VH-CH1-hinge-CH2-CH3.

[0041] An antibody expressing a "single-domain antibody" (sdAb) or "single-variable-domain (SVD) antibody" generally refers to an antibody in which a single variable domain (VH or VL) is capable of conferring antigen binding ability. In other words, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single-domain antibodies include those derived from camelids (lambs and camels) and cartilaginous fish (e.g., nurse sharks), as well as those derived from human and mouse antibodies through recombinant methods (Nature [Nature] (1989) 341:544-546; Dev Comp Immunol [Developmental and Comparative Immunology] (2006) 30:43-56; Trend Biochem Sci [Trends in Biochemistry] (2001) 26:230-235; Trends Biotechnol [Trends in Biotechnology] (2003):21:484-490; WO 2005 / 035572; WO 03 / 035694; Febs Lett [Circular of the Federation of European Biochemical Societies] (1994) 339:285-290; WO00 / 29004; WO 02 / 051870). When sdAb contains only heavy chains, it is interchangeable with “VHH” or “single heavy chain variable domain antibody” or “nanobody”.

[0042] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spikelers, and dimeric antibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that exerts its antibody effect by binding to a specific antigen to form a complex.

[0043] In the context of antibodies, "Fab" refers to a monovalent antigen-binding fragment of the antibody, which consists of a light chain (including both variable and constant regions) linked by disulfide bonds to the variable region and a first constant region of a heavy chain. Fab can be obtained by digesting the antibody with papain at residues proximal to the N-terminus of the disulfide bonds between the heavy chains in the hinge region.

[0044] “Fab” refers to a Fab fragment that contains a portion of the hinge region. It can be obtained by digesting an antibody with pepsin at residues near the C-terminus of the disulfide bond between the heavy chains in the hinge region. Therefore, a small number of residues in the hinge region (including one or more cysteine ​​residues) are different from Fab.

[0045] "F(ab)2" refers to the dimer of Fab', which contains two light chains and a portion of two heavy chains.

[0046] "Single-chain variable fragment" or "scFv" refers to the immunoglobulin heavy chain (V). H ) and light chains (V L ( ) Fusion proteins with variable regions. In some respects, these regions are linked by short linker peptides of ten to approximately 25 amino acids. The linker peptides may be enriched with glycine to provide flexibility, and serine or threonine to improve solubility, and may link V H N-terminus and V L The C-terminus is removed, and vice versa. Despite the removal of the constant region and the introduction of a linker, this protein retains the specificity of the original immunoglobulin. scFv molecules are known in the art and are described, for example, in U.S. Patent 5,892,019.

[0047] The term antibody encompasses a wide range of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chains can be classified as γ, μ, α, δ, or ε (gamma, mu, alpha, delta, epsilon), including several subclasses (e.g., γ1-γ4). It is the properties of this chain that determine the "class" of the antibody, such as IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes) For exampleIgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to confer functional specificity. In view of this disclosure, those skilled in the art will readily identify these classes and modified versions of isotypes, which are accordingly covered within the scope of this disclosure. All immunoglobulin classes are obviously covered within the scope of this disclosure, and the following discussion will generally refer to immunoglobulin molecules of the IgG class. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of 53,000–70,000 Daltons. These four chains are typically linked by disulfide bonds in a “Y” configuration, wherein the light chain begins at the opening of the “Y”, wraps around the heavy chain, and extends into the variable region.

[0048] The antibodies, antigen-binding moieties, variants, or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primate-derived, or chimeric antibodies, single-chain antibodies, and epitope-binding fragments. For example Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VK or VH domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including...) For example Anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0049] Light chains are classified as K or λ (kappa, lambda). Each heavy chain class can bind to either a κ or λ light chain. Generally, the light and heavy chains are covalently bonded to each other, and when the immunoglobulin is produced by hybridoma cells, B cells, or genetically engineered host cells, the "tail" portions of the two heavy chains are bonded to each other via covalent disulfide bonds or non-covalent bonds. In this heavy chain, the amino acid sequence extends from the N-terminus at the Y-configuration fork to the C-terminus at the bottom of each chain.

[0050] Both the light and heavy chains are divided into regions with structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains (VK) of the light chain and the variable domains (VH) of the heavy chain determine antigen recognition and specificity. Conversely, the constant domains (CK) of the light chain and the constant domains (CH1, CH2, or CH3) of the heavy chain confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the farther the constant domain is from the antibody's antigen-binding site or N-terminus, the higher its number. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0051] As described above, the variable region enables antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, a subset of the antibody's VK and VH domains, or complementarity-determining regions (CDRs), binds to form the variable region defining a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites located at the ends of each arm of the Y-configuration. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each VH and VK chain. In some cases, such as certain immunoglobulin molecules (complete immunoglobulin molecules) derived from camel species or engineered based on camel immunoglobulins, may consist only of heavy chains, lacking light chains. See, for example Hamers-Casterman et al. , Nature [Nature] 363:446-448 (1993).

[0052] In naturally occurring antibodies, each antigen-binding domain contains six "complementarity-determining regions" or "CDRs," which are short, discontinuous sequences of amino acids precisely positioned to form the antigen-binding domain when the antibody assumes its three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain (called "framework" regions) exhibit low intermolecular variability. Framework regions primarily adopt a β-sheet conformation, and CDRs form loops that connect β-sheet structures and, in some cases, form part of a β-sheet. Thus, the framework regions act as a scaffold that positions the CDRs in the correct orientation through interchain, non-covalent interactions. The antigen-binding domain formed by these positioned CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its homologous epitope. For any given heavy or light chain variable region, those skilled in the art can readily identify the amino acids containing the CDR and framework regions, respectively, as they have been precisely defined (see "Sequences of Proteins of Immunological Interest," Kabat, E et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI.Biol [Journal of Molecular Biology], 196:901-917 (1987)).

[0053] Where a term used and / or accepted in the art has two or more definitions, the definition of the term as used herein is intended to include all such meanings unless expressly stated otherwise. A specific example is the use of the term “complementarity-determining region” (“CDR”) to describe discontinuous antigen-binding sites found in the variable regions of both heavy-chain and light-chain polypeptides. This specific region has been described in the following literature: Kabat et al., US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al., J. MoI.Biol[Journal of Molecular Biology]. 196:901-917 (1987), these references are incorporated herein by reference in their full text. The CDR definition by Kabat and Chothia (when compared with each other) includes overlaps or subsets of amino acid residues. However, the application of any definition referring to a CDR of an antibody or its variants is intended to be within the scope of the terminology defined and used herein. For comparison, the appropriate amino acid residues covering a CDR as defined in the aforementioned cited references are shown in the table below. The exact number of residues covering a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which residues contain a particular CDR based on the amino acid sequence of the variable region of the antibody.

[0054]

[0055] Kabat et al. also defined a variable domain sequence numbering system applicable to any antibody. Those skilled in the art can explicitly assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data outside of the sequence itself. As used herein, "Kabat number" refers to the numbering system described in the following reference: Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0056] In addition to the table above, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins approximately at amino acid 31 (approximately 9 residues after the first cysteine ​​residue), consists of approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, consists of approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins approximately at the thirty-third amino acid residue after the end of CDR-H2; consists of 3-25 amino acids; and ends at the sequence WGXG, where X is any amino acid. CDR-L1 begins approximately at residue 24 (after the cysteine ​​residue); consists of approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins approximately at the sixteenth residue after the end of CDR-L1, consists of approximately 7 residues. CDR-L3 begins at approximately the thirtieth residue after CDR-L2 (i.e., after the cysteine ​​residue); it consists of approximately 7-11 residues and ends at sequence F or WGXG, where X is any amino acid.

[0057] The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be derived from condricthoids (e.g., from sharks).

[0058] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide used in this disclosure may comprise a polypeptide chain containing a CH1 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH3 domain; or a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide disclosed herein comprises a polypeptide chain containing a CH3 domain. Furthermore, antibodies used in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant regions can be modified so that they differ from naturally occurring immunoglobulin molecules in their amino acid sequence.

[0059] The heavy chain constant regions of antibodies disclosed in this article may originate from different immunoglobulin molecules. For example, the heavy chain constant regions of peptides may contain components derived from IgG. l The molecule contains the CH1 domain and a hinge region derived from the IgG3 molecule. In another example, the heavy chain constant region may contain a portion derived from IgG. l The molecules and portions are derived from the hinge region of the IgG3 molecule. In another example, the heavy chain portion may contain portions derived from IgG. l The molecules and part of the chimeric hinges are derived from IgG4 molecules.

[0060] As used herein, the term "light chain constant region" includes an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region comprises at least one of a constant κ domain or a constant λ domain.

[0061] A "light chain-heavy chain pair" refers to a combination of light and heavy chains that can form a dimer through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0062] As previously mentioned, the subunit structures and three-dimensional conformations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the N-terminal variable domain of the immunoglobulin heavy chain, and the term "CH1 domain" includes the first (closest to the N-terminus) constant region domain of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located at the N-terminus of the hinge region of the immunoglobulin heavy chain molecule.

[0063] The “CH1 domain” (also known as the “C1” of the “H1” domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0064] As used herein, the term "hinge region" refers to the portion of a heavy-chain molecule that links the CH1 domain to the CH2 domain; in IgG, this region corresponds to Glu216 to Pro230 of human IgG1, according to the Burton EU numbering system. Molec.Immunol. [Molecular Immunology], 22:161-206 (1985)). The hinge region of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues forming the inter-heavy chain SS bond in the same position. This hinge region is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol [Journal of Immunology] 161:4083(1998)).

[0065] As used herein, the term "CH2 domain" encompasses the extended portion of the heavy chain molecule, for example, from approximately residues 244 to 360 of the antibody using conventional numbering schemes (Kabat numbering system, residues 244 to 360; and EU numbering system, residues 231 to 340; see Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). The CH2 domain is unique because it does not pair tightly with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. There is also ample evidence that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

[0066] The “CH3 domain” (also known as the “C3 domain”) contains a string of residues in the Fc region from the C-terminus to the CH2 domain (i.e. from approximately amino acid residue 341 to the C-terminus of the antibody sequence, typically amino acid residues 446 or 447 of IgG, EU numbering system).

[0067] The terms “Fc region,” “Fc domain,” or “crystallizable region fragment” used herein are used to define the C-terminal region of the immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the Fc region of the human IgG heavy chain is generally defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody production or purification or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Therefore, compositions of complete antibodies can comprise antibody populations with all K447 residues removed, antibody populations without K447 residue removal, and antibody populations containing a mixture of antibodies with and without K447 residues. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0068] As used herein, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group, which can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by disulfide bonds, with the two heavy chains connected by two disulfide bonds at positions 239 and 242 using the Kabat numbering system (positions 226 or 229 using the EU numbering system).

[0069] As used herein, the term "chimeric antibody" will be considered to mean any antibody in which the immune-reactive region or site is derived from or derived from a first species, and the constant region (which, according to this disclosure, may be whole, partial, or modified) is derived from a second species. In some embodiments, the target-binding region or site will be derived from a non-human source (e.g., mouse or primate), and the constant region will be of human origin.

[0070] This article uses the term "humanized antibody" to describe an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences has been modified to be more "human-like," i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody or a variant, derivative, analog, or fragment thereof that immunely and specifically binds to a target antigen and contains a frame (FR) region having substantially the amino acid sequence of a human antibody and a complementarity-determining region (CDR) having substantially the amino acid sequence of a human antibody. As used herein, in the context of CDR, the term "substantially" means a CDR having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical amino acid sequence to the CDR of a non-human antibody. Humanized antibodies substantially comprise all of at least one, typically two, variable domains (Fab, Fab', F(ab')2, Fv), all or substantially all of the CDR regions within the variable domains corresponding to the CDR regions of non-human immunoglobulins (i.e., donor antibodies), and all or substantially all of the frame regions being frame regions of human immunoglobulin common sequences. In embodiments, humanized antibodies also comprise at least a portion of the immunoglobulin constant region (Fc) (typically the constant region of human immunoglobulins). In some embodiments, humanized antibodies comprise a light chain and at least a variable domain of the heavy chain. The antibody may also comprise CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, humanized antibodies comprise only the humanized light chain. In some embodiments, humanized antibodies comprise only the humanized heavy chain. In certain embodiments, humanized antibodies comprise only the humanized variable domain of the light chain and / or the humanized heavy chain.

[0071] As used herein, the term "epitope" refers to a specific atom or amino acid group on an antigen to which an antibody or antibody moiety binds. If two antibodies or antibody moiety competitively bind to an antigen, they can bind to the same epitope within the antigen.

[0072] "Specific binding" or "specific to" generally refers to an antibody binding to an epitope via its antigen-binding domain, and this binding requires a certain complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds more easily to a particular epitope via its antigen-binding domain than to a random, unrelated epitope. This article uses the term "specificity" to identify the relative affinity of a particular antibody for a particular epitope. For example, antibody "A" can be considered more specific to a given epitope than antibody "B," or antibody "A" can be said to have a higher specificity for binding to epitope "C" than to its specificity for binding to related epitope "D."

[0073] As used herein, the term "treatment" refers to both therapeutic treatment and preventative or preventative measures aimed at preventing or slowing (alleviating) undesirable physiological changes or impairments, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization (i.e., non-deterioration) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and palliative care (whether partial or complete), whether detectable or undetectable. "Treatment" can also refer to extended survival compared to expected survival without treatment. Those in need of treatment include those with pre-existing conditions or impairments, those susceptible to conditions or impairments, or those whose conditions or impairments require prevention.

[0074] The terms "subject," "individual," "animal," "patient," or "mammal" refer to any subject who requires diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammal subjects include humans, domestic animals, farm and zoo animals, racing animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

[0075] As used herein, phrases such as “to a patient in need of treatment” or “subject in need of treatment” include subjects who will benefit from the administration of the antibodies or compositions disclosed herein for purposes such as detection, diagnostic procedures and / or treatment, such as mammalian subjects.

[0076] T-cell adaptors targeting WT1

[0077] T-cell adaptors are molecules that bind to both T cells and tumor cells, thus recruiting T cells to target and kill tumor cells. In some instances, such T-cell adaptors may further include an anti-4-1BB component.

[0078] For example, RG-6007, developed by Hoffmann-La Roche, is a bispecific T-cell antibody targeting the HLA-A2-WT1 complex on tumor cells and the CD3-like T-cell receptor (TCR) on T cells, intended for the treatment of acute myeloid leukemia (AML). In vitro data showed that, compared with WT1-TCB alone, in the presence of NIH-3T3 cells expressing low or high levels of FAP, WT1-TCB-mediated lysis of allogeneic healthy donor T cells was significantly increased when combined with FAP-4-1BBL.

[0079] This article describes the development of a method targeting WT1 / HLA-A presented on tumor cells. A novel anti-WT1 antibody, WT1-09, is derived from the 02 complex. Compared to the WT1 binding unit of the reference antibody RG-6007, WT1-09 exhibits better binding specificity, for example, it does not bind to MED13L and PIGQ peptides (Example 2).

[0080] Therefore, according to one embodiment of this disclosure, an antibody or antigen-binding fragment thereof specific to WT1 is provided. In some embodiments, the antibody or antigen-binding fragment thereof binds to HLA-A 02 WT1 presented. In some embodiments, the antibody or its antigen-binding fragment binds to RMFPNAPYL (SEQ ID NO: 3). In some embodiments, the antibody or its antigen-binding fragment does not bind to SEQ ID NO: 4 or 5.

[0081] In some embodiments, the anti-WT1 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region has VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region has VL CDR1, VLCDR2, and VL CDR3. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 14, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 15, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 16, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 17, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 18.

[0082] Table A. Anti-WT1 antibodies

[0083] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2.

[0084] It also offers a combination of WT1 / HLA-A and WT1-09 as a competitor. The antibody or antigen-binding fragment of SEQ ID NO: 3. In some embodiments, the binding is a binding with SEQ ID NO: 3.

[0085] The inventors of this invention designed and prepared a T-cell connective with a novel WT1 antibody sequence. They employed methods such as... Figure 4 The 1+1 or 2+1 form is shown, and it is called T cell pluripotent precision agonist antibody or TOPAbody (or simply TOPA).

[0086] 1+1 form ( Figure 4 A) This includes a single VH / VL pair of anti-WT1 antibody and a single-chain fragment (scFv) targeting CD3, both fused to the N-terminus of the Fc fragment. Additionally, two anti-4-1BB nanobodies are used at the C-terminus of the Fc fragment. Unlike the 1+1 configuration, as... Figure 4 As shown in B, the 2+1 form comprises two VH / VL pairs targeting WT1. One of the VH / VL pairs, as in the 1+1 form, is directly fused to the Fc; however, the other is fused to the N-terminus of the anti-CD3 scFv. TOPObody incorporating one or more anti-WT1 units is referred to herein as WT1-TOPA.

[0087] These newly developed WT1-TOPA modules exhibit excellent security features because they do not cause interference with CD34. + Hematopoietic stem cell toxicity (Example 4). On the other hand, they target multiple types of HLA-A02. + / WT1 + Cancer cells exhibit potent T-cell cytotoxicity (Example 5).

[0088] When compared with RG-6007, WT1-TOPA is more potent in T cell activation (Examples 6 and 9). Equally important, WT1-TOPA induces fewer T cell exhaustion markers than RG-6007, and is therefore safer than RG-6007 (Example 9).

[0089] Therefore, one embodiment of this disclosure provides a multispecific antibody comprising the anti-WT1 antibody or its antigen-binding fragment thereof, the anti-CD3 antibody or its antigen-binding fragment thereof, and the anti-4-1BB antibody or its antigen-binding fragment thereof.

[0090] In some embodiments, the anti-CD3 antibody or fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region has VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region has VL CDR1, VL CDR2, and VLCDR3. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 21, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 22, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 23, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 24, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 25, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 26.

[0091] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 20. A representative anti-CD3 antibody having such a sequence is 155z16. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 33. A representative anti-CD3 antibody having such a sequence is 155z05. It is noteworthy that they have the same CDR but differ in the frame region.

[0092] Several derivatives of 155z16 and 155z05 were also prepared and tested. In particular, several mutant versions of HCDR2 with excellent performance were tested. These include SEQ ID NO: 27 (PSG version), SEQ ID NO: 28 (ASG version), and SEQ ID NO: 29 (TG version) (see Table B).

[0093] Table B. Anti-CD3 antibodies

[0094] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 21, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 27, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 23, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 24, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 25, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 26.

[0095] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 21, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 28, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 23, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 24, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 25, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 26.

[0096] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 21, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 29, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 23, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 24, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 25, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 26.

[0097] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 19, and VL comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 30, and VL comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 31, and VL comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 32, and VL comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 34, and VL comprises the amino acid sequence of SEQ ID NO: 33.

[0098] In some embodiments, the anti-4-1BB antibody in the multispecific antibody is a single-domain antibody (sdAb), also known as a nanobody. In some embodiments, the sdAb comprises CDR1, CDR2, and CDR3. In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 35, CDR2 comprises the amino acid sequence of SEQ ID NO: 36, and CDR3 comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 38.

[0099] Table C. Anti-4-1BB antibodies

[0100] In some embodiments, at least one, two, three, four, five, or six of the above VH CDR1, CDR2, and CDR3 are modified by the addition, deletion, substitution, or combination thereof of one, two, or three amino acids.

[0101] The CDR, heavy chain variable region, light chain variable region, or single heavy chain variable domain disclosed herein may be further modified. In some embodiments, the modified heavy chain variable region, light chain variable region, or single heavy chain variable domain retains at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity and is still able to bind to the target site.

[0102] WT1-TOPA can take different forms. In some embodiments, anti-4-1BB sdAbs (preferably two of them) are fused to the C-terminus of an Fc fragment. In some embodiments, an anti-CD3 fragment (preferably scFv) is fused to the N-terminus of one of the Fc chains. In some embodiments, an anti-WT1 fragment (e.g., a VH-VL pair, optionally having a CH1-CL pair) is fused to the N-terminus of another Fc chain. In some embodiments, a second copy of the anti-WT1 fragment is used for the N-terminus of the anti-CD3 scFv.

[0103] In multispecific antibodies, one or more linkers may optionally be included between adjacent fragments. Linkers within the scope of this disclosure are characterized by amino acid content, length, rigidity, and secondary structure. Linkers within the scope of this disclosure separate a functional peptide from another functional peptide and allow for proper folding and function of each domain. In this way, linkers can be customized according to a specific functional peptide and other functional peptides. According to one aspect, functional independence of structural and fusion (heterologous) domains is maximized through appropriate linkers to limit spatial interference between domains during bacterial cell export and assembly.

[0104] The linkers within the scope of this disclosure include amino acid residues. Amino acid residues can be any naturally occurring amino acid residue. Amino acid residues can also be synthetic amino acids known to those skilled in the art. Representative amino acids that can be used as linkers include glycine, alanine, valine, leucine, isoleucine, serine, cysteine, selenocysteine, threonine, methionine, proline, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, aspartic acid, glutamic acid, asparagine, and glutamine.

[0105] In some embodiments, the length of the connector is from about 3 to about 50 amino acids, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, or 48 amino acids.

[0106] In some embodiments, the linker sequence comprises at least 50% glycine or serine residues, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more glycine or serine residues. In some embodiments, the linker sequence consists of glycine and serine residues.

[0107] Typically, two identical Fc regions form a homodimer. However, two distinct Fc regions can form a heterodimer, where one or both individual chains abruptly change, via, for example, a kilo-hole structure (KIH), disulfide bonds (-SS-), or through hydrophobic, electrostatic, hydrophilic interactions, or increased flexibility. Fc region pairing can be achieved through heterodimer formation via kilo-hole structures (KIH), hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility.

[0108] In some embodiments, the Fc domain provided herein contains a pestle mutation, and the paired Fc domain contains a mortar mutation, or vice versa.

[0109] As used herein, the term "mortar and pestle structure" or "KIH" technology refers to a technique that directs two peptides to pair together in vitro or in vivo by introducing a protrusion (mortar) into one peptide and a cavity (pothole) into the other peptide at the interface where two peptides interact. For example, KIHs can be introduced into the Fc:Fc binding interface, CL:CH1 interface, or VH / VL interface of an antibody (see, for example, US 2011 / 0287009, US 2007 / 0178552, WO 96 / 027011, WO 98 / 050431, Zhu et al., 1997, Protein Science 6:781-788, and WO 2012 / 106587). In some embodiments, KIHs drive the pairing of two different heavy chains together during the manufacture of multispecific antibodies. For example, a multispecific antibody with KIH in its Fc region can further include a single variable domain linked to each Fc region, or further include different heavy chain variable domains paired with similar or different light chain variable domains. KIH technology can also be used to pair two different receptor extracellular domains together, or to pair any other polypeptide sequences containing different target recognition sequences (e.g., including affinity bodies, peptide bodies, and other Fc fusions).

[0110] As used herein, the term "pallet mutation" refers to a mutation that introduces a protrusion (pallet) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a mortar mutation.

[0111] As used herein, the term "pothole mutation" refers to a mutation that introduces a cavity (pothole) into the polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a pothole mutation.

[0112] In some embodiments, the club-shaped mutation in the Fc domain includes T366W (EU number). In some embodiments, the mortar-shaped mutation in the Fc domain includes T366S, L368A, and Y407V (EU numbers).

[0113] In some embodiments, the Fc domain and the paired Fc domain form a heterodimer with a mortar and pestle structure (KIH): the Fc domain of SEQ ID NO: 98 (or a variant thereof having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) may be paired with the paired Fc domain of SEQ ID NO: 99 (or a variant thereof having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity).

[0114] In some embodiments, the Fc region is an Fc domain, i.e., an Fc region having some or all of the effector functions, including, for example, complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) functions. In some embodiments, the Fc domain is derived from IgG1 or IgG3.

[0115] In some embodiments, one or more amino acid modifications may be introduced into the Fc domain to create an Fc domain variant. The Fc domain variant may comprise a human Fc domain sequence (e.g., derived from the human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions. In some embodiments, the Fc domain variant alters one or more functional and / or pharmacokinetic properties of the antibody.

[0116] The Fc region can also be engineered to enhance or eliminate effector functions. IgG antibodies can induce direct antitumor activity through Fc-mediated effector functions, which are involved in other immune cell or killing mechanisms. As used herein, “effector function” or “antibody effector function” refers to the biological activity attributable to the binding of the antibody’s Fc region to its effectors, such as the C1 complex and Fc receptors (FcγRIIa or FcγRIIIa)). Exemplary effector functions include: complement-dependent cytotoxicity (CDC) induced by the interaction of antibody and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the antibody’s Fc region to Fc receptors on effector cells; and antibody-dependent cell-mediated phagocytosis (ADCP), in which nonspecific cytotoxic cells expressing the Fcγ receptor (FcγR) recognize antibodies bound to target cells and subsequently induce phagocytosis of the target cells.

[0117] In some embodiments, the Fc region provided herein preserves or improves effector functionality, such as ADCC and / or CDC.

[0118] Of the four IgG subclasses, IgG1 and IgG3 induce the strongest Fc effector functions. However, due to the longest half-life and greater stability of IgG1 than IgG3, most therapeutic antibodies with Fc-mediated functions are IgG1 isotypes.

[0119] Both IgG2 and IgG4 isoforms exhibit significantly lower binding affinity for FcγR. Recent evidence suggests that the IgG2 isoform is not entirely devoid of effector function, while the IgG4 isoform can undergo in vivo Fab arm exchange, producing bispecific antibodies and off-target effects.

[0120] In some embodiments, the Fc domain is derived from human IgG1. In some embodiments, the human IgG1-derived Fc domain does not contain the L234A mutation and / or the L235A mutation. In some embodiments, the human IgG1-derived Fc domain contains the L234A mutation and / or the L235A mutation. In some embodiments, the Fc domain is derived from human IgG3. In some embodiments, the Fc domain is derived from human IgG2 or IgG4. In some embodiments, the Fc domain is derived from human IgG4. In some embodiments, the human IgG4-derived Fc domain contains the S228P, F234A, and / or L235A mutation. In some embodiments, the human IgG4-derived Fc domain does not contain the S228P, F234A, and / or L235A mutation.

[0121] In some embodiments, alterations in the Fc domain result in changes (i.e., improvements or reductions) in C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol., 164:4178-4184 (2000).

[0122] In some embodiments, the multispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 6, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain comprising the amino acid sequence of SEQ ID NO: 8.

[0123] In some embodiments, the multispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 6, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 8.

[0124] In some embodiments, the multispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 6, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 10, and a light chain comprising the amino acid sequence of SEQ ID NO: 8.

[0125] In some embodiments, the multispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 6, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 11, and a light chain comprising the amino acid sequence of SEQ ID NO: 8.

[0126] In some embodiments, the multispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 6, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain comprising the amino acid sequence of SEQ ID NO: 8.

[0127] In some embodiments, the multispecific construct comprises a variant Fc domain containing one or more amino acid substitutions that alter the half-life and / or the binding to the neonatal Fc receptor (FcRn). Antibodies with an extended half-life and improved binding to the neonatal Fc receptor (FcRn) (responsible for transferring maternal IgG to the fetus) (Guyer et al.) J. Immunol [Journal of Immunology] 117:587 (1976) and Kim et al., J. Immunol[Journal of Immunology] 24:249 (1994) describes this in US 2005 / 0014934A1 (Hinton et al.). Those antibodies contain an Fc region with one or more substitutions, thereby altering the binding of the Fc region to FcRn. Such Fc variants include those with substitutions (e.g., substitution of Fc region residue 434) on one or more Fc region residues (US Patent No. 7,371,826).

[0128] In some embodiments, the antibody comprises an amino acid sequence or one or more portions that do not normally associate with the antibody. Exemplary modifications are described in more detail below. For example, the antibody disclosed herein may comprise a flexible linker sequence or may be modified to add a functional portion (e.g., a PEG, drug, toxin, or marker).

[0129] The antibodies, variants, or derivatives disclosed herein include modified derivatives, i.e., those obtained by covalently attaching any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to the epitope. For example, but not limited to, antibodies can be modified by: glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization via known protecting / blocking groups, proteolytic cleavage, and linkage to cellular ligands or other proteins. Any of these chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Additionally, antibodies may contain one or more non-classical amino acids.

[0130] In some embodiments, antibodies may be conjugated with therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modulators, pharmaceuticals, or PEG.

[0131] Antibodies can be conjugated or fused with therapeutic agents, which may include detectable markers (such as radioactive markers), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutics or diagnostics, cytotoxic agents (which may be drugs or toxins), ultrasound enhancers, non-radioactive markers, combinations thereof, and other such agents known in the art.

[0132] Polynucleotides encoding antibodies and methods for preparing antibodies

[0133] This disclosure also provides isolated polynucleotide or nucleic acid molecules encoding antibodies, variants, or derivatives thereof. The polynucleotides disclosed herein may encode the entire heavy and light chain variable regions of antigen-binding polypeptides, variants, or derivatives on the same polynucleotide molecule or on separate polynucleotide molecules. Furthermore, the polynucleotides disclosed herein may encode portions of the heavy and light chain variable regions of antigen-binding polypeptides, variants, or derivatives on the same polynucleotide molecule or on separate polynucleotide molecules.

[0134] Humanized antibodies can be engineered to minimize unwanted immune responses to rodent anti-human antibodies, which limits the duration and effectiveness of therapeutic applications of these portions in human receptors. Humanized antibodies can have one or more amino acid residues introduced from a non-human source. These non-human residues are often referred to as “input” residues, and they are typically derived from the variable domain. Humanization can be performed by replacing the corresponding sequence of a human antibody with a hypervariable region sequence. Thus, such “humanized” antibodies are chimeric antibodies, in which substantially less than the complete human variable domain is replaced by a corresponding sequence from a non-human species. See, for example, U.S. Patent No. 4,816,567, the contents of which are incorporated herein by reference. Humanized antibodies can be human antibodies in which some of the hypervariable region residues, and possibly some FR residues, are replaced by residues at similar sites in rodent antibodies. The humanization or engineering of the antibodies of the present invention can be performed using any known method, such as, but not limited to, those described in U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

[0135] treat

[0136] As described herein, the antibodies, variants, or derivatives disclosed herein may be used for certain therapeutic applications.

[0137] This disclosure further relates to antibody-based therapies involving the administration of the disclosed antibodies to patients (such as animals, mammals, and humans) to treat one or more disorders or conditions described herein. The therapeutic compounds disclosed include, but are not limited to, the antibodies disclosed (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding the antibodies disclosed (including variants and derivatives thereof as described herein).

[0138] In some embodiments, a method for treating cancer in patients in need is provided. In one embodiment, the method involves administering an effective amount of the antibody disclosed herein to the patient.

[0139] In some embodiments, the use of the antibody disclosed herein is provided in the manufacture of a medicament for treating cancer in patients in need.

[0140] In some embodiments, antibodies of this disclosure are provided for use in treating cancer in patients in need.

[0141] Unless otherwise stated, the terms “cancer” and “tumor” are used interchangeably in this document. These terms specifically refer to, but are not limited to, cancers and tumors selected from the group comprising: basal cell carcinoma; bladder cancer; bone cancer, such as osteosarcoma; central nervous system tumors, such as cerebellar astrocytoma, brain astrocytoma / malignant glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medullary epithelioma, moderately differentiated pineal parenchymal tumor, primitive neuroectodermal tumor, pinealoblastoma, and spinal cord tumors; Burkitt lymphoma; breast cancer; cervical cancer; chronic myeloid leukemia; colon cancer; rectal cancer; colorectal cancer; esophageal cancer; Ewing family tumors; extrahepatic bile duct cancer; gallbladder cancer; gastrointestinal stromal tumor (GIST); glioma; Head and neck cancer; pancreatic islet cell tumors; Kaposi's sarcoma; leukemia; liver cancer; lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; T-cell lymphoma; mesothelioma; multiple myeloma / plasma cell tumor; myeloid leukemia; multiple myeloma; nasopharyngeal carcinoma; neuroblastoma; small cell lung cancer; non-small cell lung cancer; oropharyngeal carcinoma; osteosarcoma; ovarian cancer; pancreatic cancer; parathyroid carcinoma; penile cancer; pharyngeal cancer; pheochromocytoma; pituitary adenoma; prostate cancer; renal cell carcinoma; respiratory tract cancer; retinoblastoma; skin cancer (melanoma); small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck carcinoma; stomach / gastric cancer; testicular cancer; laryngeal cancer; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; urethral cancer; uterine cancer; vaginal cancer; vulvar cancer; and nephroblastoma.

[0142] The specific dosage and treatment regimen for any particular patient will depend on a number of factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health condition, sex, diet and timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of healthcare professionals regarding such factors is within the realm of ordinary technical skill in this field. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined using pharmacological and pharmacokinetic principles well known in the art.

[0143] Methods of administering antibodies and variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding peptides or compositions can be administered via any convenient route, such as by infusion or bolus injection, absorption through the epithelial or mucosal lining (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides disclosed herein can be administered orally, rectally, parenterally, intracerebrospinally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), buccally, or as oral or nasal sprays.

[0144] As used in this article, the term “parenteral” refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0145] Administration can be systemic or local. Additionally, it may be desirable to introduce the antibodies disclosed herein into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated, for example, by an intraventricular catheter attached to a reservoir (such as an Ommaya reservoir). Lung administration may also be employed, for example, by using an inhaler or nebulizer, and formulations containing nebulizing agents.

[0146] It may be desirable to apply the antigen-binding peptides or compositions disclosed herein topically to the area requiring treatment; this can be achieved, for example, but not limited to, local infusion during surgery, topical application (e.g., in conjunction with postoperative wound dressings), by injection, via catheter, via suppository, or via implantation, said implant being a porous, non-porous, or gel-like material, including membranes such as sialastic membranes or fibers. Preferably, when administering the proteins (including antibodies) disclosed herein, care must be taken to use materials that are not absorbed by the protein.

[0147] Composition

[0148] This disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0149] In certain embodiments, the term "pharmaceuticalally acceptable" means approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and more particularly in humans. Furthermore, "pharmaceuticalally acceptable carrier" generally refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0150] The term "carrier" refers to a diluent, adjuvant, excipient, or medium that is administered with a therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid carriers, especially for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetate, citrate, or phosphate. Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for regulating tension, such as sodium chloride or dextrose, are also considered. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated as suppositories containing conventional binders and carriers, such as triglycerides. Oral formulations may include standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide (preferably in purified form) and an appropriate carrier to provide a suitable form of administration to the patient. The formulation should be suitable for the mode of administration. Parenteral preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0151] In the embodiments, the composition is formulated according to conventional procedures to be a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also include a solubilizer and a local anesthetic, such as lidocaine, to reduce pain at the injection site. Generally, the ingredients are provided individually or in combination in unit dosage forms, for example, as lyophilized powders or anhydrous concentrates in sealed containers (such as ampoules or pouches), with the amount of active agent indicated. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0152] The compounds disclosed herein can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include salts that form with anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts that form with cations, such as salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0153] Example

[0154] Example 1: Preparation and affinity test of anti-WT1 antibody

[0155] This example tested a newly developed anti-WT1 antibody (WT1-09, with VH and VL sequences as shown in Table 1) that targets the WT1 / HLA-A02 complex presented on tumor cells.

[0156] The binding affinity of anti-WT1 antibodies to human WT-1 / HLA-A02:01 complex protein was tested using a capture method with Biacore. Antibodies were captured using a Protein A chip. Human WT1-His protein at a concentration of 1 μg / mL was injected onto the captured antibody at a flow rate of 30 μL / min for 3 min. Antigen association was allowed for 180 s and dissociation for 600 s. All experiments were performed on a Biacore 8K. Data analysis was performed using Biacore 8K evaluation software. The anti-WT1 fraction of RG-6007 (a bispecific antibody targeting WT1 / HLA-A02 and CD3 developed by Roche) was used as a reference. Figure 1 A- Figure 1 As shown in C, the binding affinity of WT1-09 is higher than that of RG-6007 against WT1 because WT1-09 dissociates from WT1 more slowly.

[0157] Table 1: WT1-09 Sequence

[0158] Example 2: WT1-09 has no cross-reactivity with off-target peptides.

[0159] A common problem with antibodies is cross-reactivity with peptide homologs expressed in normal tissues. For example, Ataie et al. (J Mol Biol. [Journal of Molecular Biology] (2016) 428:194-205) reported that the antibody ESK1 binds to homologous peptides derived from the proteins MED13L and PIGQ. The amino acid sequences of the MED13L, PIGQ, and WT1-RMF peptides are shown in Table 2.

[0160] To determine the specificity of WT1-09, this example loads these peptides onto T2 cells and analyzes the binding affinity of WT1-09 using flow cytometry-based binding assays. Figure 2 A- Figure 2 As shown in Figure C, WT1-09 did not bind to MED13L and PIGQ peptides, while RG-6007 showed significant binding at 300 nM and 100 nM.

[0161] Table 2: Antigen Sequences

[0162] Example 3: WT1-09 combination depends on HLA-A02 and WT1

[0163] To further confirm the specificity of the WT1 antibody, this example selected cells with different HLA-A types and WT1 expression levels for affinity measurements. Cell line information is shown in... Figure 3 In F. For example... Figure 3 A- Figure 3 As shown in Figure B, WT1-09 and RG-6007 exhibited high binding affinity in WT1 and HLA-A02 double-positive cell lines (SKM-1 and THP-1), with an EC50 of approximately 1 nM. Figure 3 C- Figure 3 As shown in E, WT1-09 and RG-6007 are associated with HLA-A02. + / WT1 - The binding of cell lines is negligible, and it is associated with HLA-A02. - / WT1 + Cell binding is even lower.

[0164] Example 4: Construction and safety testing of trispecific antibodies

[0165] In this example, a trispecific antibody containing the WT1-09 fragment and specific for CD3 and 4-1BB was prepared and tested.

[0166] The trispecific antibodies are in a configuration referred to herein as TOPAbody (T-cell pluripotent precision agonist antibody). Some representative configurations of these trispecific antibodies are shown in... Figure 4 A- Figure 4 B in. Figure 4 A illustrates a 1+1 form comprising a single VH / VL pair of anti-WT1 antibody and a single-chain fragment (scFv) targeting CD3 (both fused to the N-terminus of IgG1 Fc). Additionally, this form includes two anti-4-1BB nanobodies at the C-terminus of the Fc fragment. Unlike the 1+1 form, as shown... Figure 4As shown in B, the 2+1 form comprises two VH / VL pairs targeting WT1. One VH / VL pair, as in the 1+1 form, is directly fused to the Fc fragment; however, the other is fused to the N-terminus of the anti-CD3scFv fragment. Similar to the 1+1 form, the 2+1 form also includes two anti-4-1BB nanobodies fused to the C-terminus of the Fc fragment. The Fc portion in both conformations is engineered to silence effector function by replacing leucine (L) at positions 234 and 235 with alanine (A). Both forms are collectively referred to as WT1-TOPA. The relevant sequences are shown in Table 3.

[0167] According to reports, CD34 + Hematopoietic stem cells are WT1 positive. Therefore, if WT1-TOPAbody binds to them, it may be harmful (Mod Pathol. [Modern Pathology](2006) 19:804-814). HLA-A02 obtained from JUNX-Bio... + CD34 + HSC was used to test the binding activity of WT1-TOPA.

[0168] Data shows that WT1-09 has a lower affinity for CD34+HSC than RG-6007. Figure 5 A). To confirm that TOPA does not induce cytotoxicity against HSCs, CD34 was used... + HSC with 2×10 5 The density of cells / pores and 2×10 4 SKM-1 cells were seeded together in 96-well plates. Bispecific antibody RG-6007 and trispecific antibody WT1-TOPA were serially diluted 4-fold and added to the 96-well plates to a final concentration ranging from 100 nM to 0.0003 nM. After incubation at 37°C for 72 hours, the supernatant was collected, and the activity of lactate dehydrogenase (LDH) released from the cytosol of damaged cells was detected using the Cytotoxicity Detection Kit PLUS (Roche). Figure 5 As shown in B, WT1-TOPA and RG-6007 did not show cytotoxicity against HSCs.

[0169] Table 3: Sequence of WT1-TOPAbody

[0170] Example 5: T cells responding to different HLA-A02 + / WT1 + Cytotoxicity of cancer cell lines

[0171] To evaluate the HLA02-mediated targeting by WT1-TOPA + / WT1 + The cytotoxicity of tumor cells was assessed using the Cytotoxicity Detection Kit PLUS (Roche), which examined the activity of lactate dehydrogenase (LDH) released from the cytosol of damaged cells. Briefly, the cells were cultured at a density of 2 × 10⁶ cells / well in 96-well plates. 5 Human PBMCs were administered at 2 × 10⁶ cells / well. Target cells THP-1, SW620, and MDA-MB-231 were added at 2 × 10⁶ cells / well. 4 The antibody was seeded at a density of 10:1. Serial dilutions of the antibody were performed fourfold and added to 96-well assay plates to final concentrations ranging from 100 nM to 0.0003 nM. After incubation at 37°C for 72 hours, the supernatant was collected for further analysis. LDH was checked according to the manufacturer's protocol. The absorbance of the sample was measured at 492 nm using a plate reader. Figure 6 A- Figure 6 As shown in D, WT1-TOPA induces different HLA-A02... + / WT1 + Tumor cell lysis.

[0172] Example 6: T cell activation

[0173] WT1-TOPA-induced IL-2 and IFN-γ secretion was examined using LANCE (PerkinElmer). Briefly, cultures were performed in 96-well plates at a density of 2 × 10⁶. 5 Human PBMCs per cell / well. Target cells SW620 were loaded at 2 × 10⁻⁶ cells / well. 4 / well density seeding. Therefore, the E:T ratio was 10:1. The antibody was serially diluted 4-fold and added to 96-well plates to final concentrations ranging from 100 nM to 0.098 nM. After incubation at 37°C for 48 hours, the supernatant was collected for further analysis. IL-2 and IFN-γ were measured by TR-FRET assay, and then according to the manufacturer's protocol. Lance signal was detected using Envision. Dual emission from 615 nM (channel 1) and 665 nM (channel 2) was obtained. A standard curve (Ch1 / Ch2 ratio relative to standard concentration) was generated by plotting Lance counts. The data were analyzed using a nonlinear regression 4-parameter logistic equation. Figure 7 A- Figure 7 As shown in B, compared with the baseline RG-6007, WT1-TOPA effectively induced stronger T cell activation, which can be measured by pro-inflammatory cytokines.

[0174] Example 7: CD3 engineering of WT1-TOPA to enhance T cell activity

[0175] To further improve the activity and stability of WT1-TOPA, this example generated a series of WT1-TOPA with different anti-CD3 units. The complete sequences of these engineered WT1-TOPA are listed in Table 4.

[0176] Table 4: Complete sequence of WT1-TOPAbody

[0177] To evaluate the ability of WT1-TOPA to activate the CD3 or 4-1BB signaling pathways, Jurkat-CD3-NFAT or Jurkat-4-1BB-NFκB were used as reporter cell lines. The Jurkat-CD3-NFAT cell line was genetically modified with a CD3 downstream NFAT response element. The Jurkat-4-1BB-NFκB cell line was transfected with human 4-1BB and modified with a 4-1BB downstream NFκB response element. Luciferase expression was induced when the antibody activated the CD3-NFAT or 4-1BB-NFκB pathway. Briefly, both reporter cells and the HLA-A02 / WT1 transfected cell line HCT116-B-Tg were used at 2.5 × 10⁻⁶. 4 Cells / well were plated at a density of 100 nM / well in white 96-well plates. Test antibodies were serially diluted 4-fold and added to the white 96-well plates, starting at a final concentration of 100 nM. After incubation at 37°C for 6 hours, luminescence was obtained by adding luciferase substrate and measured using a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0178] like Figure 8 A- Figure 8 The results shown in B confirm that WT1 trispecific antibodies with different CD3 sequences induce downstream CD3 and 4-1BB signaling. To determine the cytotoxicity of these WT1-TOPAs, the activity of LDH released from lysed tumor cells was measured, as previously described. Figure 9 As shown, WT1-TOPA exhibits targeting HLA-A02. + / WT1 + Cytotoxicity of SKM-1 cells, although at different potency levels.

[0179] Example 8: No significant IL6 induction

[0180] To investigate the potential of WT1-TOPA in inducing cytokine release syndrome following intravenous administration, high concentrations of the antibody were added to PBMCs, and the levels of IL-6 (a major driver of cytokine release syndrome) in the supernatant culture medium were measured. The culture density was 2 × 10⁶ wells in 96-well plates. 5 Human PBMCs were analyzed using 100 cells / well. Antibodies were serially diluted 4-fold and added to 96-well plates to final concentrations ranging from 400 nM to 1.563 nM. After incubation at 37°C for 72 hours, the supernatant was collected, and cytokines were detected using the Human Th1 / Th2 Cytokine Kit II from BD Flow Cytometry Microsphere Array (CBA) kit. Figure 10 The results showed that WT1-TOPA induced only a negligible increase in IL-6 production, far less than that RG-6007 and the CD3 antibody OKT3.

[0181] Example 9: WT1-TOPA induces long-term activation of T cells

[0182] The inclusion of 4-1BB signaling in the WT1-TOPA molecule was considered to induce stronger and longer T cell activation and generation compared to T cell connectives containing only CD3 antibodies (activation signal 1 only). To test this, this study examined the continuous killing of tumor cells mediated by WT1-TOPA and compared it to RG6007. The assay protocol involved seeding 2 × 10⁶ cells per well in a 96-well plate. 5 One PBMC and 2×10 4 Target cells were selected, resulting in an E:T ratio of 10:1. Target cells OVCAR3 were modified to express luciferase. The final antibody concentration was 50 nM, and assays were repeated 6–8 times. For each round of kill assays, PBMCs from replicate samples were collected after incubation at 37°C for 72–96 hours and temporarily stored for the next round of kill assays. Luminescence was obtained by adding luciferase substrate and measured using a microplate reader to calculate antibody-mediated kill, compared with a control group. At the start of each kill round, the number of live PBMCs was statistically adjusted to ensure the same number of PBMCs in each group and to maintain the E:T ratio at 10:1. The procedure was repeated 5 times. Figure 11 The results showed that T cells stimulated with the WT1-CD3-4-1BB trispecific antibody were more potent than those stimulated with RG-6007 during a 16-day incubation period, especially in 3-5 rounds of tumor killing, indicating that WT1-CD3-4-1BB can induce a durable response in T cells.

[0183] To further elucidate the underlying mechanisms, this case study analyzed T cell exhaustion markers induced by the RG-6007 group and the WT1 trispecific antibody. Figure 12 A- Figure 12 As shown in B, the T exhaustion analysis showed that, compared to the WT1-TOPAbody group, all exhaustion markers (including 2B4, PD-1, etc.) in cytotoxic T cells and helper T cells were significantly upregulated in the RG-6007 group.

[0184] Example 10: WT1-TOPA showed a superior tumor inhibition rate compared to RG-6007.

[0185] This study tested the in vivo efficacy of the WT1-TOPAbody molecule in syngeneic B-Tg (CAG-WT1 peptide-HLA-A02) MC38 cell tumors. HLA02 / WT1-expressing B-Tg (CAG-WT1 peptide-HLA-A02) MC38 cells were seeded into CD3 / 4-1BB / HLA-A2.1 humanized mice. The tumors were screened when the average tumor size reached 100 mm. 3 Animals were given 1.5 mpk or 10 mpk CT01-3-09-155-16-B31Z2-t31-hIgG1LALA, 2.0 mpk CT01-3-09-155-5-B31Z2-t311-hIgG1LALA, or 2.0 mpk RG-6007 twice a week for three consecutive weeks.

[0186] Figure 13 The results in A showed that, at the end of the experiment, all treatments with WT1-TOPAbody molecules had significant tumor inhibition, with a TGI of 51.4% for CT01-3-09-155-5-B31Z2-t311-hIgG1LALA, and TGIs of 63.3% and 95.9% for 1.5 mg / kg and 10 mg / kg CT01-3-09-155-16-B31Z2-t31-hIgG1LALA, respectively. Unexpectedly, at the experimental endpoint, the tumor growth inhibition rate in the RG-6007 treatment group was higher than that in the PBS group, and FACS measurements showed that this effect was accompanied by RG-6007-mediated CD8+ inhibition. + and CD4 + T cells were significantly reduced ( Figure 13 B- Figure 13 C).

[0187]

[0188] The scope of this disclosure is not limited to the particular embodiments described, which are intended as a single illustration of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover such modifications and variations, provided they fall within the scope of the appended claims and their equivalents.

[0189] All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

[0190] This invention has been described according to specific embodiments discovered or proposed by the inventors to encompass preferred modes of carrying out the invention. Those skilled in the art will understand that, based on this disclosure, many modifications and alterations can be made to the specific embodiments of the examples without departing from the intended scope of the invention. For example, the basic DNA sequence can be altered without affecting the protein sequence due to codon redundancy. Furthermore, the protein structure can be altered without affecting the type or number of biological functions for reasons of biological equivalence. All such modifications are intended to be included within the scope of the appended claims.

Claims

1. A multispecific antibody comprising: (a) An anti-CD3 antibody or its antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 21, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 22, 27, 28, or 29, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 23, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 24, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 25, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:

26. (b) Anti-4-1BB antibody or its antigen-binding fragment, and, (c) Anti-WT1 antibody or its antigen-binding fragment.

2. The multispecific antibody as claimed in claim 1, wherein the VH and VL of the anti-CD3 antibody or its antigen-binding fragment respectively comprise the amino acid sequences of (1) SEQ ID NO: 19 and 20, (2) SEQ ID NO: 30 and 20, (3) SEQ ID NO: 31 and 20, (4) SEQ ID NO: 32 and 33 or (5) SEQ ID NO: 34 and 33.

3. The multispecific antibody as claimed in any of the preceding claims, wherein the anti-CD3 antibody or its antigen-binding fragment is a single-chain fragment (scFv).

4. The multispecific antibody as described in any of the preceding claims, wherein the anti-4-1BB antibody is a single-domain antibody (sdAb).

5. The multispecific antibody of claim 4, wherein the sdAb comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 35, 36 and 37, respectively.

6. The multispecific antibody of claim 5, wherein the sdAb comprises the amino acid sequence of SEQ ID NO:

38.

7. The multispecific antibody according to any one of claims 4-6, wherein the multispecific antibody further comprises an Fc fragment, and wherein the sdAb is fused to the C-terminus of the Fc fragment in the multispecific antibody.

8. The multispecific antibody of claim 7, wherein the anti-CD3 antibody or its antigen-binding fragment is fused to the N-terminus of one strand of the Fc fragment.

9. The multispecific antibody of claim 7 or 8, wherein the anti-WT1 antibody or its antigen-binding fragment is fused to the N-terminus of the second chain of the Fc fragment.

10. The multispecific antibody of claim 9, wherein the multispecific antibody comprises a second anti-WT1 antibody or antigen-binding fragment fused to the N-terminus of the anti-CD3 antibody or its antigen-binding fragment.

11. The multispecific antibody as claimed in any of the preceding claims, wherein the anti-WT1 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, the light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 13-18.

12. The multispecific antibody of claim 11, wherein the VH and VL of the anti-WT1 antibody or its antigen-binding fragment respectively comprise the amino acid sequences of SEQ ID NO: 1 and 2.

13. A multispecific antibody comprising: (a) An anti-WT1 antibody or its antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 13-18; (b) Anti-CD3 antibody or its antigen-binding fragment; and, (c) Anti-4-1BB antibody or its antigen-binding fragment.

14. A pharmaceutical composition comprising a multispecific antibody as described in any one of claims 1-13, and a pharmaceutically acceptable carrier.

15. One or more polynucleotides encoding a multispecific antibody as described in any one of claims 1-13.

16. A vector comprising one or more polynucleotides as described in claim 15.

17. A host cell comprising one or more polynucleotides as described in claim 13 or a vector as described in claim 16.

18. A method of treating a disease or condition in a subject in need, the method comprising administering to the subject an effective amount of a multispecific antibody as described in any one of claims 1-13, a pharmaceutical composition as described in claim 14, one or more polynucleotides as described in claim 15, a carrier as described in claim 16, or a host cell as described in claim 17.

19. Use of the multispecific antibody of any one of claims 1-13, the pharmaceutical composition of claim 14, one or more polynucleotides of claim 15, the carrier of claim 16, or the host cell of claim 17 for the preparation of a medicament for treating a disease or condition.

20. The method of claim 18 or the use of claim 19, wherein the disease or condition is cancer.

21. The method or use of claim 20, wherein the cancer is selected from the group consisting of: ovarian cancer, prostate cancer, urinary tract cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, and thyroid cancer.

Images