Engineered antibodies and immunoconjugates and methods of use

Abstract

Provided herein are tetravalent antibodies and immunoconjugates, wherein the antibodies are conjugated to a payload. The antibodies can comprise a number of different architectures, with the paratopes formed, e.g., as Fabs or diabodies, positioned relative to one another and to the Fc region. Also provided are methods of making and using the above antibodies and immunoconjugates.

Description

Statement Regarding Federally Sponsored Research

[0001] none Cross-references to related applications

[0002] This application claims the priority date interests of U.S. Provisional Application No. 63 / 608,173, filed December 8, 2023; U.S. Provisional Application No. 63 / 609,776, filed December 13, 2023; U.S. Provisional Application No. 63 / 566,200, filed March 15, 2024; U.S. Provisional Application No. 63 / 566,201, filed March 15, 2024; U.S. Provisional Application No. 63 / 724,852, filed November 25, 2024; and U.S. Provisional Application No. 63 / 724,854, filed November 25, 2024, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0003] Antibody-drug conjugates (ADCs) represent a significant advance in cancer therapy. ADCs combine the specificity of monoclonal antibodies with the potency of cytotoxic drugs. By precisely targeting cancer cells, ADCs reduce damage to healthy tissues. In an ADC, a monoclonal antibody is engineered to bind to a specific antigen expressed on the surface of a cancer cell. Once bound, the ADC is internalized into the cancer cell, where the cytotoxic agent is released to induce cell death. This targeted delivery allows for the use of highly potent drugs that might be too toxic if delivered systemically. ADCs have shown promise in treating multiple cancers, including breast cancer, lung cancer, and leukemia. They represent personalized and targeted cancer therapy. Attached Figure Description

[0004] The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate exemplary embodiments and, together with the specification, further enable those skilled in the art to make and use these embodiments, as well as other embodiments that will be apparent to those skilled in the art. The invention will be described in more detail with reference to the following drawings, in which:

[0005] Figure 1 The form of Platform 1 antibody in the form of Fab-Fc-Fab (e.g., "IgG-Fab") is shown.

[0006] Figure 2 The platform 2 antibody in the form of a dual antibody-Fc-Fab is shown.

[0007] Figure 3 The form of Platform 3 antibody in the form of Fab-Fab-Fc (e.g., "Fab-IgG") is shown.

[0008] Figure 4The platform 4 antibody in Fab-dipeptide-Fc form is shown.

[0009] Figure 5 A- Figure 5 D shows four different configurations of the complementary site in a bispecific tetravalent form (in this case, the Fab-biantibody-Fc form). Figure 5 A: Symmetrical antibodies in which the complementary sites (complementary sites 1 and 2) of binding epitope 1 are located at the NW / NE position, and the complementary sites (complementary sites 3 and 4) of binding epitope 2 are located at the SW / SE position. Figure 5 B: Symmetrical antibodies in which the complementary sites (complementary sites 1 and 2) of epitope 2 are located at the NW / NE position, and the complementary sites (complementary sites 3 and 4) of epitope 1 are located at the SW / SE position. Figure 5 C: Asymmetric antibodies in which the complementary sites of epitope 1 (complementary sites 1 and 4) are located at the NW / SE position, and the complementary sites of epitope 2 (complementary sites 2 and 3) are located at the SW / NE position. Figure 5 D: Asymmetric antibodies in which the complementary sites (complementary sites 1 and 3) of epitope 1 are located at the NW / SW position, and the complementary sites (complementary sites 2 and 4) of epitope 2 are located at the NE / SE position.

[0010] Figure 6 A- Figure 6 C shows three different configurations of complementary sites in the form of Fab-biantibody-Fc trispecific and quadrispecific forms. Figure 6 A: The complementary bit of combination table 1 is located at the NW / NE position, the complementary bit of combination table 2 is located at the SE position, and the complementary bit of combination table 3 is located at the SW position. Figure 6 B: The complementary bit of combination table 1 is located at the NW position; the complementary bit of combination table 2 is located at the NE position, and the two complementary bits of combination table 3 are located at the SW / SE positions. Figure 6 C: The complementary bit of combination table 1 is located at the NW position, the complementary bit of combination table 2 is located at the NE position, the complementary bit of combination table 3 is located at the SW position, and the complementary bit of combination table 4 is located at the SE position.

[0011] Figure 7 A to Figure 7 C shows three different configurations of epitope binding to complementary sites in a bispecific tetravalent form (in this case, a biantibody-Fc-Fab form). Figure 7 A: A symmetrical antibody that binds to two complementary sites of epitope 1 at the NW / NE position and to two complementary sites of epitope 2 at the SW / SE position. Figure 7 B: A symmetrical antibody that binds to epitope 2 at the NW / NE position and to epitope 1 at the SW / SE position. Figure 7C: An asymmetric antibody in which the two complementary sites binding to epitope 1 are located at the NW / SW position, and the two complementary sites binding to epitope 2 are located at the NE / SE position.

[0012] Figure 8 A- Figure 8 C shows three different configurations of the complementary site in the form of biantibody-Fc-Fab trispecific and quadrispecific forms. Figure 8 A: The complementary bit of combination table 1 is located at the NW position, the complementary bit of combination table 2 is located at the NE position, and the two complementary bits of combination table 3 are located at the SW / SE positions. Figure 8 B: The two complementary bits of combination table 1 are located at the NW / NE position, the complementary bit of combination table 2 is located at the SE position, and the complementary bit of combination table 3 is located at the SW position. Figure 8 C: The complementary bit of combination table 1 is located at the NW position, the complementary bit of combination table 2 is located at the NE position, the complementary bit of combination table 3 is located at the SW position, and the complementary bit of combination table 4 is located at the SE position.

[0013] Figure 9 A- Figure 9 E shows that the bispecific tetravalent ADC demonstrates superior efficiency compared to the monospecific bivalent or tetravalent ADC.

[0014] Figure 10 A summary of CD30 / PD-L1 IgG1 binding agents is shown.

[0015] Figure 11 Different CD30-PD-L1 platform 1 builds are shown.

[0016] Figure 12 Different CD30-PD-L1 platform 2 and platform 3 builds are shown.

[0017] Figure 13 Different CD30-PD-L1 platform 4 builds are shown.

[0018] Figure 14 This document presents a summary of QC procedures for the production and analysis of multiple antibody biopharmaceuticals.

[0019] Figure 15 The binding of the CD30 / PD-L1 tetravalent antibody to Karpas 299 cells was shown by FACS analysis.

[0020] Figure 16 The efficacy of the CD30 / PD-L1 quadrivalent ADC was demonstrated in both CD30+VE and PD-L1+VE cells.

[0021] Figure 17The results showed that the CD30 / PD-L1 biologic exhibited functional activity comparable to that of clinical PD-L1 antibodies avelumab, atezolizumab, and BMS936559.

[0022] Figure 18 Size exclusion chromatography analysis of the trispecific HER2 / Trop2 platform 2 ADC is shown.

[0023] Figure 19 This study demonstrates a trispecific Her2-Trop2 platform ADC that exhibits superior efficacy compared to single-specific HER2 ADCs in A431 and MCF7 cells.

[0024] Figure 20 Different HER2 / Trop2 tetravalent constructs are shown (AB607 is trispecific).

[0025] Figure 21 The in vitro cellular efficacy of Platform 2 HER2 / Trop2 in SKOV3 cells is shown.

[0026] Figure 22 The in vitro cellular efficacy of Platform 4 HER2 / Trop2 in SKOV3 cells is shown.

[0027] Figure 23 The in vitro cellular efficacy of Platform 2 HER2 / Trop2 in JIMT1 cells is shown.

[0028] Figure 24 The in vitro cellular efficacy of Platform 4 HER2 / Trop2 in JIMT1 cells is shown.

[0029] Figure 25 The in vitro cellular efficacy of Platform 2 HER2 / Trop2 in OVCAR3 cells was demonstrated.

[0030] Figure 26 The in vitro cellular efficacy of Platform 4 HER2 / Trop2 in OVCAR3 cells was demonstrated.

[0031] Figure 27 A- Figure 27 C shows the analysis of three bispecific tetravalent ADCs containing binding sites for NaPi-2b and Muc16. Figure 27 A: Fab-biantibody-Fc form. Figure 27 B: Fab-biantibody-Fc form. Figure 27 C: Biantibody-Fc-Fab form. Data shows that the ADCs are assembled homogeneously. All ADCs exhibit a uniform 4 DAR via cysteine-engineered site-specific conjugation.

[0032] Figure 28 The effects of different ADCs on the cell viability of OVCAR3 cells were compared, and the 50% inhibitory concentration (IC50) values ​​were provided.

[0033] Figure 29 The effects of different ADCs on the viability of engineered OVCAR3-AV cells were compared. OVCAR3 cells are a cell line derived from human ovarian cancer, and OVCAR3-AV cells are a mixture of wild-type OVCAR3 cells and OVCAR3 cells that have been genetically modified to knock out Muc16 gene expression.

[0034] Figure 30 A- Figure 30 B compared the effects of different ADCs binding to Muc16 and NaPi-2b on Muc16 knockout OVCAR3 cells. Figure 30 A) and NaPi-2b knockout OVCAR3 cells ( Figure 30 The effects of B) on cell viability are shown, and a table with IC50 values ​​and DAR is provided.

[0035] Figure 31 A- Figure 31 Figure D shows cell viability assays of OVCAR3 WT and Muc16-KO cells with different ratios, comparing the effects of different ADCs binding Muc16 and NaPi-2b on cell viability of cell mixtures. OVCAR3-AV1 cells contained OVCAR3 Muc16 knockout and OVCAR3 wild-type cells in a ratio of 60:40. OVCAR3-AV2 cells contained OVCAR3 Muc16 knockout and OVCAR3 wild-type cells in a ratio of 70:30. OVCAR3-AV3 cells contained OVCAR3 Muc16 knockout and OVCAR3 wild-type cells in a ratio of 80:20. OVCAR3-AV4 cells contained OVCAR3 Muc16 knockout and OVCAR3 wild-type cells in a ratio of 90:10. Therefore, all OVCAR3-AV cell mixtures had constant expression levels of NaPi2b and varying expression levels of Muc16. This mimics the expression of both targets as seen in patient tumors. 31A: 60% Muc16 knockout, 40% OVCAR3 wild-type. 31B: 70% Muc16 knockout, 30% OVCAR3 wild-type. 31C: 80% Muc16 knockout, 20% OVCAR3 wild-type. 31D: 90% Muc16 knockout, 10% OVCAR3 wild-type. OVCAR3-AV cells had varying levels of Muc16 expression and constant NaPi-2b expression to mimic the expression of both targets in patient tumors. The IC50 values ​​for different cell ratios are shown in the table.

[0036] Figure 32 The effects of different ADCs on the cell viability of mixtures of OVCAR3-AV cells with varying expression levels of Muc16 and NaPi-2b were compared. (20% of cells were positive for both target antigens, while 60% were positive for NaPi-2b only, and 20% were positive for Muc-16 only.)

[0037] Figure 33A and Figure 33B The manufacturability of multiple antibody forms is shown. The Fab-biantibody-Fc and biantibody-Fc-Fab forms exhibit the highest yields. The antibody identified by the hash symbol has the lowest manufacturability.

[0038] Figures 34A-34C The in vivo efficacy of the ADC in an OVCAR3 ovarian xenograft tumor model using female CB.17 SCID mice is shown: A. Study design; tumor growth curves (B) and body weight changes (C) in mice after ADC treatment. The tetravalent bispecific ADC demonstrated superior in vivo efficacy compared to monospecific ADCs. No body weight changes were observed in any of the ADC-treated groups.

[0039] Figure 35 The effects of different ADCs on the cell viability of OVCAR3 cells were compared, and the 50% inhibitory concentration (IC50) values ​​were provided.

[0040] Figure 36A-Figure 3 6D illustrates the in vivo efficacy of ADC (40 nmol dose comparison) in the OVCAR3 ovarian xenograft tumor model: A. Study design; B. Tumor growth curve versus days; C. Tumor growth observed in multiple ADC treatment groups on day 32; D. p-values ​​observed between treatment groups on day 32. All groups were administered 40 nmol ADC (dose comparison at tumor sizes of 100–150 mm). 3 (Single dose on day 1).

[0041] Figures 37A-3 7D illustrates the in vivo efficacy of ADC (AB301 vs. AB309) in the OVCAR3 ovarian xenograft tumor model: A. Study design; B. Tumor growth curve versus day; C. Tumor growth observed in multiple ADC treatment groups on day 32; D. P-values ​​observed between treatment groups.

[0042] Figure 38A-Figure 38D illustrates the in vivo efficacy of ADC (AB312 vs. AB309) in the OVCAR3 ovarian xenograft tumor model: A. Study design; B. Tumor growth curves versus days; C. Tumor growth observed in multiple ADC treatment groups on day 32; D. P-values ​​observed between treatment groups.

[0043] Figure 39 The changes in body weight of mice after ADC treatment were compared.

[0044] Figure 40 The pharmacokinetic characterization of AB304 and AB309 antibodies in non-human primates is shown.

[0045] Figure 41 The effects of AB304 and AB306 ADCs on the cell viability of OVCAR3 cells were compared, and the 50% inhibitory concentration (IC50) values ​​were provided.

[0046] Figure 42 The effects of different ADCs on the cell viability of OVCAR3 cells were compared, and IC50 values ​​were provided.

[0047] Figure 43 The bispecific bivalent Her2 / Her2 ADC (AB105 ADC) demonstrated superior efficacy compared to the monospecific bivalent ADC (AB103 ADC) in both the high copy number Her2 cell line (SKBr) and the low copy number Her2 cell line (JIMT1).

[0048] Figure 44 Different Her2-PD-L1 platform 4 constructs with different spacer variants were described.

[0049] Figure 45 Different Her2-PD-L1 platform 2 and 4 constructs with different spacer variants are described.

[0050] Figure 46 Different multispecific Her2-PD-L1 platform 2 and 4 constructs with different spacer variants are shown.

[0051] Figure 47 Different bispecific Her2-Her2 platform 4 constructs with different spacer variants are described.

[0052] Figure 48 Different bispecific Her2-Her2 platform 2 constructs with different G4S spacer sizes were described.

[0053] Figure 49This study describes a CD30-PD-L1 multispecific quadrivalent ADC that exhibits excellent efficacy in Karpus 299-AV cells (a mixed cell population of CD30-KO and PD-L1 KO cells).

[0054] Figure 50 Different multispecific CD30-PD-L1 platform constructs 2 and 4 were described.

[0055] Figure 51 The in vitro cell potency of Platform 4 multispecific tetravalent (AB612 bispecific tetravalent) HER2 / Trop2 in SKOV3 and JMIT1 cells was demonstrated.

[0056] Figure 52 This study demonstrates multispecific tetravalent ADCs (AB616 and AB618 ADCs) that exhibit superior potency compared to monospecific or bispecific bivalent ADCs in SKOV3 cells. All complementary sites of the AB616 and AB618 ADCs are functional.

[0057] Figure 53 It describes that all complementary sites of AB616 and AB618 ADCs are functional in the NCI-N87 cell line.

[0058] Figure 54 Different multispecific Her2-Trop2 platform 2 and 4 constructs with two different Trop2 complementary sites (Trop2-17487 and H1D3) were described. Summary of the Invention

[0059] This document provides a tetravalent antibody architecture that can be monospecific, bispecific, trispecific, or tetraspecific. The complementary site can be formed by a Fab region, a bispecific antibody, and an scFv region. In the case of multispecific antibodies, the complementary site binding to a specific epitope can be located at any of the four positions in the tetravalent antibody. Some embodiments contain two identical heavy chains and two identical light chains. In other embodiments, the antibody can have an asymmetric heavy chain. In other cases, the complementary sites binding to different epitopes can have the same light chain variable region and different heavy chain variable regions. The antibodies of this disclosure exhibit excellent manufacturability and potency. The antibodies can be formulated as immunoconjugates (e.g., with toxic or detectable payloads) for therapeutic and diagnostic uses. Detailed Implementation I. Antibody

[0060] An "antibody" is a protein or protein complex containing a framework region from an immunoglobulin gene that binds to a target epitope. The framework region of an immunoglobulin gene refers to a relatively conserved sequence within the variable regions of the immunoglobulin (Ig) heavy and light chains. Therefore, the term "antibody" includes a complete tetrameric antibody comprising two immunoglobulin heavy chains and two immunoglobulin light chains. The term also includes any binding fragment of the tetrameric antibody. The term also includes engineered antibodies in which multiple immunoglobulin domains are rearranged or added to the immunoglobulin chain, for example to produce an antibody with more than two binding sites, as described herein. Antibodies described herein may have names prefixed with "AB". Such antibody names may be given as "AB101 antibody". Depending on the context, these names may refer to antibody-drug conjugates. For example, an ADC of AB101 may be referred to as "AB101-ADC". A. Antibody structure

[0061] An exemplary antibody structure is a complete tetrameric antibody. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a “light” chain (approximately 25 kD) and a “heavy” chain (approximately 50–70 kD). The antibody can be (i) any of the five major classes of immunoglobulins based on its identity through its heavy chain constant domains – α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM), or (ii) its subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The light chain can be λ or κ. In a complete antibody, the light chain comprises a light chain variable (VL) region and a light chain constant (CL) region from the N-terminus to the C-terminus. In a complete antibody, the heavy chain comprises a heavy chain variable (VH) region, a heavy chain constant 1 (CH1) region, a hinge region, a heavy chain constant 2 (CH2) region, and a heavy chain constant 3 (CH3) region from the N-terminus to the C-terminus. Two light chains associate with the heavy chain portion containing the VH and CH1 regions to form two Fab (fragment antigen-binding) regions. The light and heavy chains are linked by covalent bonds (e.g., disulfide bonds) and non-covalent interactions. The combination of the VL and VH regions forms the antigen-binding domain of the antibody, also known as the "complementary site." The combination of the hinge region, CH2 region, and CH3 region is collectively referred to as the Fc ("crystallizable fragment") region.

[0062] In antibody formation, different chains or parts of chains interact with each other to form higher-order structures. In this way, it can be said that antibody chains or parts thereof associate to form these structures. For example, but not limited to, heavy and light chains can associate to form complementary sites. Two heavy chains can associate to form an Fc dimer. The variable regions of the heavy and light chains in an scFv molecule or a biantibody can associate to form one or more complementary sites. Association can be achieved through a variety of covalent and non-covalent interactions. These interactions include, but are not limited to, disulfide bonds (which can link heavy chains to each other and light chains to heavy chains), as well as hydrogen bonds, hydrophobic interactions, and van der Waals forces. Association between chains or their parts can occur spontaneously, leading to the self-assembly of higher-order structures.

[0063] Complementary sites can be formed through the association of multiple light chain variable regions and heavy chain variable regions in various forms, as discussed in more detail herein. For example, the association of a light chain with a polypeptide containing a heavy chain variable region and a heavy chain constant 1 region forms a Fab containing a complementary site. Single chains containing both heavy chain variable regions and light chain variable regions can associate to form a scFv containing a complementary site. Two chains, each containing both heavy chain variable regions and light chain variable regions, can associate to form a biantibody containing two complementary sites. As used herein, the phrases “Fab containing a complementary site,” “scFv containing a complementary site,” and “biantibody containing two complementary sites” (etc.) can be equivalently expressed as “complementary site contained in Fab,” “complementary site contained in scFv,” and “two complementary sites contained in biantibody.”

[0064] Both the light and heavy chains of an antibody have variable regions consisting of a framework region and a complementarity-determining region (“CDR”) (also known as a “hypervariant” region). Each variable region contains three CDRs, labeled CDR1, CDR2, and CDR3. They typically have a length between approximately seven and 25 amino acids. CDRs play a role in antigen recognition. The framework region intersperses between the CDRs and provides structural integrity, contributing to the correct conformation of the CDRs for antigen binding.

[0065] The phrase "CDR sequence set" refers to the three heavy chain and / or three light chain CDRs of a specific antibody. "Light chain" CDR sequence set refers to light chain CDR sequences. "Heavy chain" CDR sequence set refers to heavy chain CDR sequences. "Complete" CDR sequence set refers to both heavy chain and light chain CDR sequences. CDRs can be predicted based on, for example, IMGT sequence alignment (international Immunogenetics information system).

[0066] The homeostatic region interacts with other immune cells in the body. Between the Fab and Fc regions of IgG, IgD, and IgA lies the hinge region, which provides flexibility to hinge antigen binding. B. Antibody Nomenclature

[0067] Antibodies can exist in natural or genetically modified forms, such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, transplanted, and in vitro produced antibodies.

[0068] As used herein, the term "monoclonal antibody" refers to a clonal formulation or composition of an antibody that has a single binding specificity and affinity for a given epitope on an antigen ("monoclonal antibody composition"). "Polyclonal antibody" refers to a formulation or composition of an antibody that produces antibodies against a single antigen but with different binding specificities and affinities ("polyclonal antibody composition").

[0069] As used herein, the term "chimeric antibody" refers to an antibody having amino acid sequences derived from two or more species. In one embodiment, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capability, while the constant regions contain amino acid sequences derived from another species (typically in the subject receiving the therapy, such as a human) to avoid triggering an immune response.

[0070] As used herein, the term "humanized antibody" refers to a chimeric antibody in which a CDR derived from the VH and VL regions of a nonhuman antibody having the desired specificity, affinity, and capability is transplanted into a human frame sequence. In one embodiment, the frame residues of the humanized antibody are modified to improve and optimize antibody specificity, affinity, and capability. Humanization (i.e., the replacement of the corresponding sequence of the human antibody with a nonhuman CDR sequence) can be performed according to, for example, the methods described in U.S. Patent Nos. 5,545,806; 5,569,825; 5,633,425; 5,661,016; Riechmann et al., Nature 332:323-327 (1988); Marks et al., Bio / Technology 10:779-783 (1992); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996).

[0071] As used herein, the term "human antibody" refers to an antibody produced by a human being or an antibody having an amino acid sequence corresponding to the human-produced antibody prepared by any technique known in the art.

[0072] As used herein, the term "immunoassay" refers to a method for detecting an analyte by detecting the binding between the analyte and an antibody that recognizes the analyte.

[0073] The amino acid sequences of antibody drugs can be found, for example, at go.drugbank.com / drugs and opig.stats.ox.ac.uk / webapps / sabdab-sabpred / and opig.stats.ox.ac.uk / webapps / sabdab-sabpred / therasabdab / , Raybould et al. Nucleic Acids Res. 48:D383-D388, (2020).

[0074] As used herein, the term "polypeptide" refers to a molecule having a sequence of natural and / or non-natural amino acids linked by peptide bonds. The term "peptide" refers to a short polypeptide, typically no more than 30 amino acids in length. The amino acid sequence of a polypeptide is called its "primary structure." The term "protein" refers to a polypeptide having secondary, tertiary, and / or quaternary structures (e.g., structures stable by hydrogen bonds, secondary structures, and relationships between structures formed by more than one protein). Proteins can be further modified by other attached motifs such as carbohydrates (glycoproteins), lipids (lipoproteins), phosphate groups (phosphoproteins), etc.

[0075] As used in this article, an amino acid sequence consists only of the amino acids in that sequence.

[0076] As used herein, if the first amino acid sequence (1) contains the second amino sequence and (2) is no more than 1, 2, or 3 amino acids longer than the second amino acid sequence, then the first amino acid sequence is "substantially composed" of the second amino acid sequence.

[0077] As used herein, if a second amino acid sequence contains a first amino acid sequence, then the first amino acid sequence is a “fragment” of the second amino acid sequence. In some embodiments, the first amino acid sequence, which is a fragment of the second amino acid sequence, may have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fewer amino acids than the second amino acid sequence.

[0078] As used herein, a “functional equivalent” of a reference amino acid sequence is a sequence that is different from the reference sequence but contains minor alterations (e.g., insertion, deletion, or substitution of one or more amino acids). The functionally equivalent sequence retains the function (e.g., antigen binding, immunogenicity) of the reference sequence to which it is equivalent. If the functionally equivalent amino acid sequence contains substitutions of one or more amino acids relative to the reference sequence, these are typically conserved amino acid substitutions.

[0079] Regarding antibodies, the term "crosslinking" refers to the attachment of an antibody to a solid or semi-solid matrix (e.g., agarose, beads, microtiter plates) or another protein or antibody. For example, antibodies can be polymerized to produce antibody complexes having multiple (more than two) antigen-binding sites. Antibodies can be polymerized by expressing the antibody as a high-binding-valent isoform (e.g., IgA or IgM, which typically form complexes of 2 or 5 antibodies, respectively). Antibody polymerization can also be performed using crosslinking agents containing reactive groups capable of linking proteins (e.g., carbodiimides, NHS esters, etc.). Methods and compositions for crosslinking antibodies to a matrix are described, for example, in the Abcam and New England Biolab catalogues and websites (available at abcam.com and neb.com). Crosslinking agent compounds having multiple reactive groups are described, for example, in the Thermo Fisher Scientific catalogue and website (available at piercenet.com). C. Antibody binding

[0080] The specificity of antibody binding can be measured by comparing the dissociation constant of the complex between the antibody (or other targeting moiety) and its target with the dissociation constant (K0) of the complex between the antibody and the non-target molecule. d The larger (higher) K is represented by ) . d K describes lower affinity interactions. d Conversely, smaller (lower) K d K describes higher affinity interactions or tighter binding. d To give just one example, antibodies specifically bind to the target K. d It can be femtomolar, picomolar, nanomolar, or micromolar, and the antibody binds to K, which is unrelated to the material. d It can be millimolecular or higher. Binding affinity can be in the nanomolar range (K). d = 10 -7 M to 10 -9 M), Pimoll range (K) d = 10 -10 M to 10 -12 M), or femtomolar range (K) d = 10 -13 M to 10 -15 M) inside.

[0081] As used in this article, if the antibody is less than 10 -7 M (i.e., K in the nanomolar range) d If it binds to an antigen or epitope, then it "specifically binds" to the antigen or epitope or is "specific to the antigen or epitope". This includes, for example, less than 1 × 10⁻⁶. -7 M, 1×10-8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M or 1×10 -12 Any of M. Typically, specific binding is characterized by binding to the antigen with sufficient affinity, such that the antibody can be used as a diagnostic agent for detecting the antigen or epitope and / or as a therapeutic agent targeting the antigen or epitope.

[0082] The term "binding" in relation to cell type (e.g., antibodies binding to cancer cells) typically indicates that an agent binds to the majority of cells in a pure population of those cells. For example, an antibody binding to a given cell type typically binds to at least two-thirds of the cells in a specified cell population (e.g., 67%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the cell population). In some cases, antibody-peptide binding can be determined by comparing the binding of the antibody to cells presenting the peptide with the binding (or lack thereof) of the antibody to cells not expressing the peptide. Those skilled in the art will recognize that some variations will occur depending on the method used to determine binding and / or the threshold. The affinity of an antibody for a target can be determined according to methods known in the art (e.g., as reviewed in Ernst et al., Determination of Equilibrium Dissociation Constants, Therapeutic Monoclonal Antibodies (Wiley & Sons ed. 2009)).

[0083] As used herein, the term "greater affinity" refers to the relative degree of antibody binding, where antibody X binds more strongly to target Y and / or has a lower dissociation constant compared to target Z, and in this context, antibody X has a greater affinity for target Y compared to Z. Similarly, the term "less affinity" refers to the degree of antibody binding, where antibody X binds less strongly to target Y and / or has a higher dissociation constant compared to target Z, and in this context, antibody X has a lesser affinity for target Y compared to Z. The binding affinity between an antibody and its target antigen can be expressed as K. A equals 1 / K d K d equals k off / k on k can be measured using surface plasmon resonance techniques (e.g., using a molecular affinity screening system (MASS-1) (Sierra Sensors GmbH, Hamburg, Germany)). on and k offvalue.

[0084] Antagonists, or blocking antibodies, are antibodies that partially or completely block, inhibit, or neutralize the biological activity associated with a target antigen relative to their activity under similar physiological conditions in the absence of the antibody. Antagonists can be competitive, non-competitive, or irreversible. Competitive antagonists bind to the same site on the natural ligand or receptor used in the natural ligand-receptor interaction or conformally bind by inducing alterations to prevent the normal binding of the natural ligand. Non-competitive antagonists bind to a different site on the ligand or receptor used in the natural ligand-receptor interaction, but increase the Kk of the interaction. d Or it may reduce the signal generated by the interaction. Irreversible inhibitors cause, for example, covalent modifications to the receptor or the natural ligand, which prevents the natural ligand from binding to the receptor.

[0085] As used herein, the term “affinity” refers to the overall stability of the binding complex between an antibody and a target antigen. It is governed by three factors: (i) the intrinsic affinity of the antibody for the antigen, (2) the binding valence of the antibody, and (3) the geometric arrangement of the interacting components. Affinity is the strength of the interaction between an antibody and a single target, while affinity is the cumulative strength of multiple affinities.

[0086] As used herein, if an antibody binds to a first antigen with a greater affinity than to a second antigen, then the antibody “preferentially binds” to the first antigen relative to the second antigen. Preferential binding can be any of at least 2, 5, 9, 10, 20, 30, 40, 50, 100, 500, or 1000 times the affinity.

[0087] As used herein, when an antibody reduces or prevents the interaction between the ligand and the receptor, the antibody “blocks” or “antagonizes” the binding of the ligand to the receptor. In an embodiment, the measured reduction level can be any one of at least 5%, 10%, 25%, 50%, 80%, 90%, 95%, 97.5%, 99%, 99.5%, or 99.9% of the (control) ligand-receptor complex (i.e., the ligand-receptor complex in the absence of the antibody).

[0088] The term "capture" regarding antibody targets (e.g., antigens, analytes, immune complexes) typically indicates that an antibody binds to the majority of the antibody target in a pure population (assuming an appropriate molar ratio). For example, an antibody binding to a given antibody target typically binds to at least two-thirds of the antibody target in solution (e.g., at least 67%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the target). Those skilled in the art will recognize that some variations will occur depending on the method used to determine binding and / or the threshold. D. Antibody form

[0089] The following is a non-exhaustive list of different antibody forms that all retain antigen-binding activity: (1) Intact immunoglobulins (also known as “intact” antibodies) (two light chains and two heavy chains, such as tetramers, such as fragments of IgG, IgA, IgD, IgE and IgM). (2) Immunoglobulin polypeptides (light chain or heavy chain). (3) Antibody fragments, including rearranged binding regions. These include, for example: • Fv (a monovalent or divalent variable region segment, and may only cover the variable region (e.g., V) L and / or V H ), •Fab(V) L C L V H C H ), •F(ab')2、 •Fv(V) L V H ), •scFv (single-chain Fv) (containing V linked by spacers (e.g., peptide spacers) L and V H (peptides) •(scFv)2 (optionally bispecific) •sc(Fv)2 (optionally bispecific) • Miniature antibody (sc(Fv)2 fused with CH3 domain) • Biantibody (a non-covalent dimer of variable heavy chain (VH) and variable light chain (VL) regions linked by peptide spacers) (It should be understood that although the VH and VL regions on the first chain can be in any order, the order of these regions on the second chain is the reverse of the order of these regions on the first chain.) • Triantibody: trivalent sc(Fv)3 or trispecific sc(Fv)3. • The antibody fragment further includes Fd (the heavy chain portion contained in the Fab fragment) and a single-domain antibody. A single-domain antibody (sdAb) is a variable domain of either the heavy or light chain generated through recombination. (4) Multispecific antibodies (containing at least two complementary sites that bind to different epitopes). (5) Multivalent antibodies (antibodies with two or more complementary sites, including bivalent, trivalent and tetravalent antibodies). (6) Fusion proteins, which contain the binding portion of an immunoglobulin fused with another amino acid sequence (such as fluorescent protein).

[0090] Any of these forms can be single-specific or multi-specific, such as dual-specific, tri-specific, or tetra-specific. E. Antibody symmetry

[0091] Antibodies containing two heavy chains can typically be symmetrical or asymmetrical. Symmetrical antibodies have mirror-image portions. Typically, this involves antibodies with identical heavy chains. Asymmetrical antibodies are not formatted as mirror images. In this case, the heavy chains are typically different.

[0092] Asymmetric antibodies typically have two distinct heavy chains. Often, complementary sites at the same relative positions will bind different epitopes. The challenge in generating such antibodies is ensuring that the two distinct heavy chains pair correctly and selectively without mismatch. Mismatch can result in a mixture of monospecific and bispecific antibodies. These chains can also unstablely associate with each other. The pestle-and-mortar (“KIH”) technique addresses this problem. KIH involves engineering a CH3 domain to create a “pestle” in the first heavy chain and a “mortar” in the second heavy chain to facilitate heterodimerization between the first and second heavy chains. In asymmetric antibodies as disclosed herein, the complementary sites can be in either orientation, either on the chain containing the pestle or on the chain containing the mortar.

[0093] In this method, a "pestle" variant can be obtained by replacing a small amino acid (e.g., threonine) with a larger amino acid (e.g., tyrosine) in the CH3 domain of an antibody (e.g., T366Y). The pestle is designed to insert into a "mortar" in the CH3 domain of another antibody. In one embodiment, a "mortar" is generated by replacing a larger residue (e.g., tyrosine) with a smaller residue (e.g., threonine), as in Y407T.

[0094] When protein domains or subdomains interact, the pestle is a large side chain protruding into the opposing (“mortis”) domain, where the pestle aligns with the smaller side chain that makes this intrusion possible. In this method, heterodimerization of the pestle and mortis variants is expected due to the pestle's insertion into a suitably designed mortis on the CH3 domain of the mating body. The pestle is constructed by replacing the smaller side chain with the largest side chain, either tyrosine or tryptophan. Mortises of the same or similar size as the pestle are produced by replacing the large side chain with a smaller side chain (in this case, alanine or threonine). Thus, the two heavy chains of the pestle variant cannot homologously associate due to side chain conflict, and homologous association of the two mortis variants is less favorable due to the absence of stable side chain interactions. Subsequently, a disulfide bond is introduced near the C-terminus of the CH3 domain to further stabilize the assembled bispecific antibody. See, for example, U.S. Patent No. 7,183,076, which is incorporated herein by reference. F. Spacer section

[0095] The engineered antibodies disclosed herein comprise functional portions attached to each other. This includes, for example, variable regions attached to each other in the form of scFv or biantibodies, different complementary site configurations attached to each other (e.g., Fab, biantibodies, scFv), or complementary sites attached to the amino or carboxyl groups of the Fc region. These regions or portions can be attached via spacer portions. For example, for a tetravalent antibody of Platform 4, the C-terminal portion of the CH1 region of the heavy chain is attached to the N-terminal portion of the biantibody region. Depending on the form of the biantibody region, the N-terminus of the biantibody can be a VL or VH region. The C-terminus of the biantibody region can also be a VH or VL region. It is attached to the N-terminal region of the Fc region. The boundaries of these regions can be determined by the sequences described herein and antibody sequences well known in the art.

[0096] The spacer can be, for example, 0 (no spacer) to 50 amino acids, for example, 1 to 50 amino acid length. The spacer can have, for example, any of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. For example, a polypeptide spacer can have any of the following: 1 to 50, 5 to 50, 1 to 30, 1 to 25, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 15, 20, 1-10, or 1-5 amino acids. In one embodiment, the spacer is a monomer or polymer of the sequence (GGGGS)n (SEQ ID NO: 170), (GGGGA)n (SEQ ID NO: 171), or (GGGGG)n (SEQ ID NO: 173), wherein n is any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Embodiments of the spacer have the sequence GGGGSGGGGSEPKSSDKTHT (SEQ ID NO: 173) (e.g., to link a biantibody region to an Fc region). Another embodiment of the spacer has the sequence GGGSGGGSGGGSGGSGSTG (SEQ ID NO: 174) (e.g., to link an Fc region to a Fab region). Other exemplary spacers have the sequences: LEDKTHTKVEPKSS (SEQ ID NO: 175), SGSETPGTSESATPESGGG (SEQ ID NO: 176), or GTTAASGSSGGSSSGA (SEQ ID NO: 177).

[0097] As used herein, regions or portions of engineered antibodies are “directly” linked if there is no spacer (0 amino acids) connecting them. Regions or portions of engineered antibodies are “substantially directly” linked (“substantially without a spacer”) if they are linked by a spacer of one or two amino acids. The antibodies of Platform 2 comprise a biantibody region attached to the Fc region and an Fc region attached to the Fab region. In some embodiments, these regions are directly or substantially directly attached (i.e., 0, 1, or 2 amino acids). That is, the C-terminus of the variable region of the biantibody is directly attached to the N-terminus of CH2 of the Fc region, and the C-terminus of the Fc region is directly attached to the N-terminus of the light chain variable region. The reference to attachment to the CH2 region considers attachment to the hinge linked to CH2.

[0098] Platform 4's antibody includes a Fab region attached to the biantibody region and a biantibody region attached to the Fc region.

[0099] Tables 3 and 4 show the spacers between the functional parts of engineered antibodies. “G4S” refers to the sequence “GGGGS” (SEQ ID NO: 170). The number indicates the number of repetitions. Thus, for example, “(G4S)2” refers to the sequence GGGGSGGGGS (SEQ ID NO: 178). The number “0” indicates no spacer or a spacer with 0 amino acids. In any of these antibodies, the spacer (G4S)n can be replaced by (G4A)n, (G5)n, or an amino acid sequence containing an equal number of amino acids, which does not need to be an exact multiple of 5, but can be any one of (X5)n + 1, 2, 3, or 4, where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. II. Multivalent Antibody Platform Architecture

[0100] "Multivalent" antibodies have more than one antigen-binding site or complementary site. For example, an antibody with two binding sites is bivalent, an antibody with three binding sites is trivalent, and an antibody with four binding sites is tetravalent.

[0101] This article considers various forms of multispecific multivalent antibodies. Variables include binding valence, the form of complementary sites, and the arrangement of binding sites for specific epitopes.

[0102] Multivalent antibodies can have multiple binding sites that bind to the same epitope and one or more binding sites that bind to different epitopes. Naturally occurring antibodies (such as IgG molecules) are typically monospecific and bivalent. An antibody is "monospecific" if all its antigen-binding sites bind to the same epitope. Multispecific antibodies have at least two different antigen-binding sites that each bind to a different epitope or antigen. Therefore, for example, if a bivalent antibody has antigen-binding sites that each bind to a different epitope, the antibody is bispecific. Trivalent antibodies can be monospecific, bispecific, or trispecific. Quadrivalent antibodies can be monospecific, bispecific, trispecific, or tetraspecific.

[0103] In describing antibodies herein, unless otherwise explicitly stated, the orientation of the multiple functional portions of the heavy or light chain is described from the amino terminus to the carboxyl terminus.

[0104] In antibodies with more than one light chain associated with the heavy chain, there is the question of whether the complementary sites formed by these associations bind to the same epitope or different epitopes. For example, in Figure 5 A and Figure 5 In the antibody schematically shown in B, complementary sites 1 and 2 bind to the same epitope. However, in Figure 5 C and Figure 5 In the antibody schematically shown in D, complementary sites 1 and 2 bind to different epitopes. When complementary sites bind to the same epitope and the antibody is symmetrical, a single light chain and a single heavy chain expressed in a cell can assemble to form an antibody. When complementary sites bind to different epitopes and the antibody is asymmetrical, antibody assembly can be performed using the following methods. In one method, each different light chain / heavy chain pair can be expressed in different cells, and the antibody can be assembled in vitro. In another method, each heavy chain variable region pairs with a common light chain variable region. Such methods are well known in the art. See, for example, U.S. Patent 8,642,745 (“Method for making multi-specific antibodies having heteromultimeric and common components”); U.S. Patent 11,033,009 (“Transgenic chicken for production of antibodies having a common light chain”); U.S. Patent 9,303,081 (“Recombinant production of mixtures of antibodies”); and U.S. Patent 11,325,982 (“Biparatopic and multiparatopic antibodies with common light chain and method of use”).

[0105] Biantibodies consist of a pair of chains that associate to form two complementary sites. (See example) Figure 2 and Figure 4 Each chain contains VL and VH regions connected to each other. This combination is called the "biantibody region". These regions can be arranged in any order, provided that the regions of each partner chain are in the order consistent with the formation of the biantibody. In some antibody architectures, such as Figure 5 A, Figure 5 B and Figure 6 As shown in Figure B, the two complementary sites of the biantibody bind to the same epitope. In this configuration, each biantibody region can contain VL and VH regions targeting the same epitope. In other architectures, such as... Figure 5 C Figure 5 D、 Figure 6 A and Figure 6As shown in C; complementary sites bind to different epitopes. In this configuration, the biantibody region of the first chain includes a light chain variable region or a heavy chain variable region that binds to the first epitope, and a heavy chain variable region or a light chain variable region that binds to the second epitope, respectively. In this way, each variable region can associate with an appropriate variable region on the biantibody region of the other chain.

[0106] It should be understood that references to the “first,” “second,” “third,” and “fourth” regions or portions of an antibody are for naming purposes only and do not necessarily indicate specific locations of these regions or portions within the antibody, such as specific locations on a compass dial, or equivalence between different antibody forms. A. Targeted epitope

[0107] The antibodies of this disclosure can target any epitope for which antibodies can be prepared. This includes, but is not limited to, any cell surface marker, such as any cell surface marker on cancer cells or other pathological cells. Functional portions of antibodies targeting any epitope, such as their light chain and variable regions, heavy chain portions, or regions containing heavy chain variable regions and heavy chain constant 1 regions, as well as the Fc portion of the antibody, can be assembled into the antibody architecture described herein. Thus, for example, portions of different antibodies targeting different epitopes can be incorporated as Fab, biantibodies, or scFv into the tetravalent bispecific antibodies of this disclosure. Exemplary antibodies of this disclosure include complementary sites that specifically bind to the epitopes HER2 (epitope 1), HER2 (epitope 2), CD30, PD-L1 (epitope 1), PD-L1 (epitope 2), PD-L1 (epitope 3), PD-L1 (epitope 4), and Trop2.

[0108] In one embodiment, the antibody of this disclosure comprises complementary sites to two different epitopes of HER2. In some embodiments, one of these epitopes is an epitope that binds to the antibody trastuzumab. In some embodiments, one of these epitopes is an epitope that binds to the antibody pertuzumab.

[0109] In another embodiment, the antibody of this disclosure comprises at least one complementary site that binds to CD30 and at least one complementary site that binds to an epitope of PD-L1. Alternatively, the antibody may comprise a complementary site that binds to CD30, a complementary site that binds to a first epitope of PD-L1, and at least one complementary site that binds to a second epitope of PD-L1.

[0110] In another embodiment, the antibody of this disclosure comprises at least one complementary site for binding to Trop2 and at least one complementary site for binding to an epitope of HER2. Alternatively, the antibody may comprise a complementary site for binding to Trop2, a complementary site for binding to a first epitope of HER2, and a complementary site for binding to a second epitope of HER2.

[0111] In another embodiment, the antibody of this disclosure comprises at least one complementary site that binds to Muc16 and at least one complementary site that binds to an epitope of NaPi2b.

[0112] In another embodiment, the antibody of this disclosure comprises complementary sites that bind to different epitopes of Her2 and at least one complementary site that binds to an epitope of PD-L1.

[0113] In another embodiment, the antibody of this disclosure comprises complementary sites that bind to different epitopes of PD-L1 and at least one complementary site that binds to an epitope of CD30.

[0114] In another embodiment, the antibody of this disclosure comprises complementary sites that bind to different epitopes of Her2 and at least one complementary site that binds to an epitope of Trop2.

[0115] In another embodiment, the antibody of this disclosure comprises complementary sites that bind to different epitopes of Her2 and complementary sites that bind to different epitopes of PD-L1.

[0116] In another embodiment, the antibody of this disclosure comprises complementary sites for binding to different epitopes of Her2, complementary sites for binding to epitopes of PD-L1, and complementary sites for binding to epitopes of Trop2.

[0117] Exemplary sequences of antibodies that bind to such epitopes are presented in Table 2. B. Tetravalent multispecific (e.g., bispecific, trispecific, and tetraspecific) antibodies

[0118] Bispecific tetravalent antibodies can have two complementary sites targeting the first epitope and two complementary sites targeting the second epitope; or three complementary sites targeting the first epitope and one complementary site targeting the second epitope. Epitopes can be on different antigen molecules (i.e., the antibody binds to two different molecules), or epitopes can be different epitopes on the same antigen. The complementary sites can be arranged in any of the four compass positions consistent with the bispecific nature of the antibody.

[0119] The four complementary sites of a tetravalent antibody can be described as being located on a compass scale as "northwest," "northeast," "southeast," and "southwest." Antibodies typically have an asymmetrical shape, which can be described as including an "east" side (first heavy chain), a "west" side (second heavy chain), a "north" side (towards the N-terminus), and a "south" side (towards the C-terminus). For example, in a tetravalent antibody, the binding site location can be characterized as "northeast," "northwest," "southeast," and "southwest." Therefore, two binding sites that bind the same epitope can be associated as "northwest-northeast," "southwest-southeast," "northeast-southeast," "northwest-southwest," "northwest-southeast," and "northeast-southwest." See, for example... Figure 5 A- Figure 5 D.

[0120] The specific sequences of these configurations are provided in Table 2. 1. Platform 1: Fab-Fc-Fab antibody

[0121] A schematic diagram of an antibody with a Platform 1 configuration of Fab-Fc-Fab is presented in Figure 1 The antibody comprises a tetravalent antibody containing, for example, one or more payloads conjugated thereto, wherein the antibody comprises a first light chain, a second light chain, a third light chain, and a fourth light chain, as well as a first heavy chain and a second heavy chain, and includes, from the N-terminus to the C-terminus: a) a first pair of complementary sites contained in the Fab region; b) an Fc region; and c) a second pair of complementary sites contained in the Fab region. Fab directly attached to Fc (Fab-Fc) is typically referred to as "IgG" (or other immunoglobulin forms), but Fab can be attached via spacers.

[0122] By way of description, the antibody comprises a first light chain, a second light chain, a third light chain, and a fourth light chain, as well as a first heavy chain and a second heavy chain, wherein: a) the first light chain comprises a first light chain variable (VL) region and a first light chain constant (CL) region from the N-terminus to the C-terminus; b) the second light chain comprises a second VL region and a second CL region from the N-terminus to the C-terminus; c) the third light chain comprises a third light chain variable (VL) region and a third light chain constant (CL) region from the N-terminus to the C-terminus; d) the fourth light chain comprises a fourth VL region and a fourth CL region from the N-terminus to the C-terminus; e) the first heavy chain comprises, from the N-terminus to the C-terminus: (i) a first heavy chain portion containing a first heavy chain variable (VH) region and a first heavy chain constant 1 (CH1) region; (ii) a first Fc region containing a first heavy chain constant 2 (CH2) region and a first heavy chain constant 3 (CH3) region; (iii) a second heavy chain portion containing a second heavy chain variable (VH) region and a second heavy chain constant 1 (CH1) region; and f) The second heavy chain, from the N-terminus to the C-terminus, comprises: (i) a third heavy chain portion containing a third heavy chain variable (VH) region and a third heavy chain constant 1 (CH1) region; (ii) a second Fc region containing a second heavy chain constant 2 (CH2) region and a second heavy chain constant 3 (CH3) region; and (iii) a fourth heavy chain portion containing a fourth heavy chain variable (VH) region and a fourth heavy chain constant 1 (CH1) region; and wherein: the first light chain and the first heavy chain portion form a first Fab portion containing a first complementary position; the second light chain and the second heavy chain portion form a second Fab portion containing a second complementary position; the third light chain and the third heavy chain portion form a third Fab portion containing a third complementary position; and the fourth light chain and the fourth heavy chain portion form a fourth Fab portion containing a fourth complementary position. 2. Platform 2: Bispecific antibody-Fc-Fab antibody

[0123] A schematic diagram of an antibody with a platform 2 configuration featuring a dual-antibody-Fc-Fab structure is presented. Figure 2 The antibody comprises a tetravalent antibody, which includes, for example, one or more payloads conjugated thereto, wherein the antibody comprises a first light chain and a second light chain, as well as a first heavy chain and a second heavy chain, and comprises, from the N-terminus to the C-terminus: a) a first pair of complementary sites contained in the biantibody; b) an Fc region; and c) a second pair of complementary sites contained in the Fab region.

[0124] By way of description, the antibody comprises a first light chain and a second light chain, as well as a first heavy chain and a second heavy chain, wherein: a) the first light chain comprises a first light chain variable (VL) region and a first light chain constant (CL) region from the N-terminus to the C-terminus; b) the second light chain comprises a second VL region and a second CL region from the N-terminus to the C-terminus; c) the first heavy chain comprises, from the N-terminus to the C-terminus: (i) a first biantibody region containing a first VH region and a third VL region in any order; (ii) a first Fc region containing a first heavy chain constant 2 (CH2) region and a first heavy chain constant 3 (CH3) region; (iii) a first heavy chain portion containing a second heavy chain variable (VH) region and a first heavy chain constant 1 (CH1) region; d) the second heavy chain comprises, from the N-terminus to the C-terminus: (i) a second biantibody region containing a third VH region and a fourth VL region in any order; and (ii) a second Fc region containing a second heavy chain constant 2 (CH2) region and a second heavy chain constant 3 (CH3) region; (iii) The second heavy chain portion contains a fourth heavy chain variable (VH) region and a second heavy chain constant 1 (CH1) region; wherein: the first light chain and the first heavy chain portion form a first Fab portion containing a first complementary site; and the second light chain and the second heavy chain portion form a second Fab portion containing a second complementary site; the first biantibody region and the second biantibody region form a biantibody, the biantibody forming a third complementary site containing a third VL region and a third VH region and a fourth complementary site containing a fourth VL region and a first VH region. 3. Platform 3: Fab-Fab-Fc antibody

[0125] A schematic diagram of an antibody with a platform 3 configuration of Fab-Fab-Fc is presented in Figure 3 The antibody comprises a tetravalent antibody, which includes, for example, one or more payloads conjugated thereto, wherein the antibody comprises a first light chain, a second light chain, a third light chain, and a fourth light chain, as well as a first heavy chain and a second heavy chain, and comprises, from the N-terminus to the C-terminus: a) a first pair of complementary sites contained in the Fab region; b) a second pair of complementary sites contained in the Fab region; and c) an Fc region.

[0126] By way of description, the antibody comprises a first light chain, a second light chain, a third light chain, and a fourth light chain, as well as a first heavy chain and a second heavy chain, wherein: a) the first light chain comprises a first light chain variable (VL) region and a first light chain constant (CL) region from the N-terminus to the C-terminus; b) the second light chain comprises a second VL region and a second CL region from the N-terminus to the C-terminus; c) the third light chain comprises a third light chain variable (VL) region and a third light chain constant (CL) region from the N-terminus to the C-terminus; d) the fourth light chain comprises a fourth VL region and a fourth CL region from the N-terminus to the C-terminus; e) the first heavy chain comprises, from the N-terminus to the C-terminus: (i) a first heavy chain portion containing a first heavy chain variable (VH) region and a first heavy chain constant 1 (CH1) region; (ii) a second heavy chain portion containing a second heavy chain variable (VH) region and a second heavy chain constant 1 (CH1) region; and (iii) a first Fc region containing a first heavy chain constant 2 (CH2) region and a first heavy chain constant 3 (CH3) region; f) The second heavy chain, from the N-end to the C-end, comprises: (i) a third heavy chain portion containing a third heavy chain variable (VH) region and a third heavy chain constant 1 (CH1) region; (ii) a fourth heavy chain portion containing a fourth heavy chain variable (VH) region and a fourth heavy chain constant 1 (CH1) region; and (iii) a second Fc region containing a second heavy chain constant 2 (CH2) region and a second heavy chain constant 3 (CH3) region; wherein: the first light chain and the first heavy chain portion form a first Fab portion containing a first complementary position; and the second light chain and the second heavy chain portion form a second Fab portion containing a second complementary position; the third light chain and the third heavy chain portion form a third Fab portion containing a third complementary position; and the fourth light chain and the fourth heavy chain portion form a fourth Fab portion containing a fourth complementary position. 4. Platform 4: Fab-Dual Antibody-Fc Platform

[0127] A schematic diagram of an antibody with a platform 4 configuration featuring Fab-biantibody-Fc is presented. Figure 4 The antibody comprises a tetravalent antibody, the tetravalent antibody comprising, for example, one or more payloads conjugated thereto, wherein the antibody comprises a first light chain and a second light chain, and a first heavy chain and a second heavy chain, and comprises, from the N-terminus to the C-terminus: a) a first pair of complementary sites contained in the Fab region; b) a second pair of complementary sites contained in the biantibody; and c) an Fc region.

[0128] By way of description, the antibody comprises a first light chain and a second light chain, wherein: a) the first light chain comprises a first light chain variable (VL) region and a first light chain constant (CL) region from the N-terminus to the C-terminus; b) the second light chain comprises a second VL region and a second CL region from the N-terminus to the C-terminus; c) the first heavy chain comprises, from the N-terminus to the C-terminus: (i) a first heavy chain portion containing a first heavy chain variable (VH) region and a first heavy chain constant 1 (CH1) region; (ii) a first biantibody region containing a second VH region and a third VL region in any order; and (iii) a first Fc region containing a first heavy chain constant 2 (CH2) region and a first heavy chain constant 3 (CH3) region; d) the second heavy chain comprises, from the N-terminus to the C-terminus: (i) a second heavy chain portion containing a third VH region and a second CH1 region; (ii) a second biantibody region containing a fourth VH region and a fourth VL region in any order; and (iii) A second Fc region containing a second CH2 region and a second CH3 region; wherein: the first light chain and the first heavy chain portion form a first Fab portion containing a first complementary site; and the second light chain and the second heavy chain portion form a second Fab portion containing a second complementary site; the first biantibody region and the second biantibody region form a biantibody, wherein the biantibody forms a third complementary site containing a fourth VL region and a second VH region and a fourth complementary site containing a third VL region and a fourth VH region.

[0129] These regions can be directly (without spacers) or substantially directly attached to each other's spacers. C. Specific targets

[0130] Consider the following exemplary targeting strategies. Antibodies incorporating such targeting strategies may be in ADC or non-ADC form. 1.Her2 / Her2

[0131] Her2 is an antigen expressed on many different types of cancer cells, particularly breast and ovarian cancer. Antibodies such as trastuzumab and pertuzumab target this antigen.

[0132] The tetravalent monospecific antibody disclosed herein contains first, second, third, and fourth complementary sites of the same epitope against HER2.

[0133] The tetravalent bispecific antibody disclosed herein includes first and second complementary sites targeting the same first epitope of HER2 and third and fourth complementary sites targeting the same second epitope of HER2. In one embodiment, the complementary sites targeting the same epitope may be arranged at the NW / NE and SW / SE positions. In another embodiment, the complementary sites targeting the same epitope may be arranged at the NW / SW and NE / SE positions.

[0134] In some embodiments, there are no spacers or substantially no spacers between the Fab region and the biantibody, and between the biantibody and the Fc region. In another embodiment, there is a spacer of about 15 amino acids between the Fab region and the biantibody, and a spacer of about 10 amino acids between the biantibody and the Fc region. 2.CD30 / PD-L1

[0135] The tetravalent bispecific antibody disclosed herein includes first and second complementary sites against CD30 and third and fourth complementary sites against PD-L1. In another embodiment, the antibody includes first, second, and third complementary sites against CD30 and a fourth complementary site against PD-L1. In yet another embodiment, the antibody includes first, second, and third complementary sites against PD-L1 and a fourth complementary site against CD30.

[0136] CD30 is a cell membrane protein primarily found on activated T and B cells, playing a role in regulating cell proliferation and death. It is particularly associated with Hodgkin's lymphoma and some non-Hodgkin's lymphomas, as well as other lymphocytic proliferative disorders. In these cancers, CD30 is typically overexpressed on the surface of malignant cells. This overexpression makes CD30 a valuable therapeutic target.

[0137] PD-L1 (programmed death-ligand 1) is a checkpoint inhibitor that can be targeted in anticancer therapies. Targeting this marker can be used to treat some cancers such as melanoma, non-small cell lung cancer, and certain types of bladder, kidney, and head and neck cancers. 3. Her2 / Trop2

[0138] The tetravalent bispecific antibody disclosed herein includes first and second complementary sites against Her2 and third and fourth complementary sites against Trop2. In another embodiment, the antibody includes first, second, and third complementary sites against Her2 and a fourth complementary site against Trop2. In yet another embodiment, the antibody includes first, second, and third complementary sites against Trop2 and a fourth complementary site against Her2.

[0139] TROP2 (also known as EpCAM (epithelial cell adhesion molecule)) is a transmembrane glycoprotein primarily expressed on the surface of epithelial cells and plays a role in cell adhesion, migration, and proliferation. Its significance in oncology stems from its overexpression in various human cancers, including breast, colon, prostate, and ovarian cancers. This overexpression is typically associated with increased tumor aggressiveness, poor prognosis, and reduced patient survival, making TROP2 a valuable biomarker for cancer diagnosis and prognosis. Furthermore, the persistent overexpression of TROP2 on the surface of many cancer cells compared to normal tissues makes it an attractive target for cancer therapy, highlighting its importance as both a cancer marker and a therapeutic target.

[0140] In another embodiment, the tetravalent antibody comprises first and second complementary sites to the same epitope targeting HER2, and third and fourth complementary sites to the same epitope targeting Trop2. The complementary sites to the HER2-binding epitope can be located at NW / NE, SW / SE, or NW / SW positions.

[0141] In another embodiment, the tetravalent antibody comprises a first complementary site to a first epitope of Her2, a second complementary site to a second epitope of Her2, and third and fourth complementary sites to the same epitope of Trop2. The two complementary sites to the same epitope of Trop2 may be located at the NE / SE position. 4. Muc16 / NaPi2b

[0142] The tetravalent bispecific antibody disclosed herein includes first and second complementary sites against Muc16 and third and fourth complementary sites against NaPi-2b. In another embodiment, the antibody includes first, second, and third complementary sites against Muc16 and a fourth complementary site against NaPi-2b. In yet another embodiment, the antibody includes first, second, and third complementary sites against NaPi-2b and a fourth complementary site against Muc16.

[0143] Cell surface-associated mucin-16 (also known as Muc16 and CA125) is a member of the mucin family of proteins. These are large glycoproteins that play a role in the formation of mucus, which acts as a protective barrier in epithelial tissues. Muc16 is a membrane-associated mucin with a single transmembrane domain. Muc16 is primarily found in the epithelial cells of the respiratory, digestive, and reproductive tracts.

[0144] Aberrant overexpression of Muc16 has been observed in several human malignancies, including ovarian cancer, pancreatic cancer, breast cancer, and lung cancer. Muc16 is also believed to be involved in intercellular interactions that lead to tumor cell metastasis.

[0145] Elevated levels of Muc16 in serum are commonly used as a biomarker for diagnosing and monitoring certain types of cancer, particularly ovarian cancer. Elevated Muc16 expression is also associated with small cell lung cancer and oat cell lung cancer.

[0146] Muc16 has UniProt accession number Q8WXI7. The protein is encoded by the Muc16 gene located on chromosome 19p13.2.

[0147] Antibodies that specifically bind to Muc16 are known to include, for example, sofostouzumab. Antibodies that specifically bind to Muc16 are also described in U.S. Patents US RE 47194 (Genentech 11D10); 11,453,721, 10,759,869, 10,738,130, and 7,202,346. The CDR of any of these antibodies may be used in the bispecific antibodies described in this disclosure.

[0148] Sodium-dependent phosphate transporter 2b (also known as NaPi-2b, SLC34A2, NaPiIIb, and Npt2) is a member of the sodium / phosphate cotransporter family. It is a cell surface sodium-dependent phosphate transporter that regulates phosphate homeostasis. NaPi-2b is primarily expressed in alveolar type II cells and the small intestine, where it promotes the active transmembrane transport of phosphate ions bound to sodium ions.

[0149] NaPi-2b is a lineage marker expressed in 80%-90% of epithelial ovarian cancers. It has been used as a target for therapeutic antibodies.

[0150] The UniProt accession number for NaPi-2b is O95436. This protein is encoded by the SLC34A2 gene located on chromosome 4p15.2.

[0151] Known antibodies that specifically bind to NaPi-2b include, for example, lifatozumab and epifibitumab (see, for example, url drugs.ncats.io / substance / 15OY7NA275). Antibodies that specifically bind to NaPi-2b are also described in U.S. Patent 11,407,825. The CDR of any of these antibodies may be used in the bispecific antibodies described in this disclosure.

[0152] In another embodiment, the tetravalent antibody comprises first and second complementary sites to the same epitope targeting HER2, and third and fourth complementary sites to the same epitope targeting Trop2. The complementary sites to the HER2-binding epitope can be located at NW / NE, SW / SE, or NW / SW positions.

[0153] In another embodiment, the tetravalent antibody comprises a first complementary site to a first epitope of Her2, a second complementary site to a second epitope of Her2, and third and fourth complementary sites to the same epitope of Trop2. The two complementary sites to the same epitope of Trop2 may be located at the NE / SE position. 5. Her2 / Her2 / PD-L1

[0154] The trispecific tetravalent antibody disclosed herein includes a first complementary site to a first epitope of Her2, a second complementary site to a second epitope of Her2, a third complementary site to an epitope of PD-L1, and a fourth complementary site to the same epitope of PD-L1.

[0155] Antibodies can have any of the four platform architectures disclosed herein, particularly Platform 2 and Platform 4.

[0156] In one implementation, the complementary site for the first epitope of Her2 is located at the NE position, the complementary site for the second epitope of Her2 is located at the NW position, and the two complementary sites for PD-L1 are located at the SW and SE positions. This is an asymmetric antibody, and it should be understood that the club-and-socket structure can be oriented east-west or west-east.

[0157] In some embodiments, there are no spacers or substantially no spacers between the Fab region and the biantibody, and between the biantibody and the Fc region. In another embodiment, there is a spacer of about 15 amino acids between the Fab region and the biantibody, and a spacer of about 10 amino acids between the biantibody and the Fc region.

[0158] The antibodies in this implementation scheme include, for example, AB113, AB114, AB115, AB116, AB117, AB118, AB119, AB120, AB121, AB122, and AB123. 6. PD-L1 / PD-L1 / CD30

[0159] The trispecific tetravalent antibody disclosed herein includes a first complementary site to a first epitope of PD-L1, a second complementary site to a second epitope of PD-L1, a third complementary site to an epitope of CD30, and a fourth complementary site to the same epitope of CD30.

[0160] Antibodies can have any of the four platform architectures disclosed herein, particularly Platform 2 and Platform 4.

[0161] In one implementation, the complementary site for the same CD30 epitope is not located at the NW / SE or NE / SW position. This is an asymmetric antibody, and it should be understood that the club-and-mortar structure can be east-west oriented or west-east oriented.

[0162] In some embodiments, there are no spacers or substantially no spacers between the Fab region and the biantibody region, and between the biantibody region and the Fc region. In another embodiment, there is a spacer of about 15 amino acids between the Fab region and the biantibody region, and a spacer of about 10 amino acids between the biantibody region and the Fc region.

[0163] The antibodies used in this implementation include, for example, AB205-AB216, AB219, AB220, AB221, AB222, and AB623. 7. Her2 / Her2 / Trop2

[0164] The trispecific tetravalent antibody disclosed herein includes a first complementary site to a first epitope of Her2, a second complementary site to a first epitope of Her2, a third complementary site to a second different epitope of Her2, and a fourth complementary site to Trop2.

[0165] Antibodies can have any of the four platform architectures disclosed herein, particularly Platform 2 and Platform 4.

[0166] In one implementation, the complementary sites for the same Her2 epitope and the complementary sites for the same CD30 epitope are not located at NW / SE or NE / SW positions. This is an asymmetric antibody, and it should be understood that the club-and-socket structure can be east-west oriented or west-east oriented.

[0167] In some embodiments, there are no spacers or substantially no spacers between the Fab region and the biantibody region, and between the biantibody and the Fc region. In another embodiment, there is a spacer of about 15 amino acids between the Fab region and the biantibody, and a spacer of about 10 amino acids between the biantibody and the Fc region.

[0168] The antibodies used in this implementation include, for example, AB616, AB617, AB618, AB619, AB620, AB622, AB623, AB624, and AB625. 8. Her2 / Her2 / PD-L1 / PD-L1

[0169] The tetraspecific tetravalent antibody disclosed herein includes a first complementary site to a first epitope of Her2, a second complementary site to a second epitope of Her2, a third complementary site to a first epitope of PD-L1, and a fourth complementary site to a second epitope of PD-L1.

[0170] Antibodies can have any of the four platform architectures disclosed herein, particularly Platform 2 and Platform 4.

[0171] In one embodiment, the complementary site for the first epitope of Her2 is located at the SW position, the complementary site for the second epitope of Her2 is located at the SE position, the first complementary site for PD-L1 is located at the NW position, and the second complementary site for PD-L1 is located at the NE position. In one embodiment, this antibody has a platform 2 form. This is an asymmetric antibody, and it should be understood that the club-and-socket structure can be east-west oriented or west-east oriented.

[0172] In another embodiment, the complementary site for the first epitope of Her2 is located at the NW position, the complementary site for the second epitope of Her2 is located at the NE position, the first complementary site for PD-L1 is located at the SW position, and the second complementary site for PD-L1 is located at the SE position. In one embodiment, this antibody has a platform 4 form. This is an asymmetric antibody, and it should be understood that the mortise and tenon structure can be east-west oriented or west-east oriented.

[0173] In some embodiments, there is no spacer or substantially no spacer between the biantibody region and the Fc region. In another embodiment, there is a spacer of about 10 amino acids between the biantibody region and the Fc region. In another embodiment, there is no spacer or substantially no spacer between the Fc region and the Fab region. In another embodiment, there is a spacer of about 20 amino acids between the Fc region and the Fab region. In another embodiment, there is no spacer or substantially no spacer between the Fab region and the biantibody region. In another embodiment, there is a spacer of about 20 amino acids between the Fab region and the biantibody region.

[0174] The antibodies used in this implementation include, for example, AB124, AB125, AB126, and AB127. 9. Her2 / Her2 / PD-L1 / Trop2

[0175] The tetraspecific tetravalent antibody disclosed herein includes a first complementary site to a first epitope of Her2, a second complementary site to a second epitope of Her2, a third complementary site to an epitope of PD-L1, and a fourth complementary site to an epitope of PD-L1.

[0176] Antibodies can have any of the four platform architectures disclosed herein, particularly Platform 2 and Platform 4.

[0177] In one embodiment, the complementary site for the first epitope of Her2 is located at the SW position, the complementary site for the second epitope of Her2 is located at the SE position, the complementary site for PD-L1 is located at the NW position, and the complementary site for Trop2 is located at the NE position. In one embodiment, this antibody has a platform 2 form. This is an asymmetric antibody, and it should be understood that the platform structure can be east-west oriented or west-east oriented.

[0178] In some embodiments, a spacer of approximately 10 amino acids exists between the biantibody region and the Fc region. In another embodiment, a spacer of approximately 20 amino acids exists between the Fc region and the Fab region.

[0179] The antibodies used in this implementation include, for example, AB611. 10. CDR sequence set

[0180] An exemplary set of CDR sequences is provided in the sequences disclosed herein, wherein they are indicated by underscores.

[0181] Exemplary CDR sequence sets for anti-her2 antibodies include: AB101 (Her2 10423) AB102 (Her2 11053).

[0182] Exemplary CDR sequence sets for anti-CD30 antibodies include: AB217 (CD30 18436).

[0183] Exemplary CDR sequence sets for anti-PD-L1 antibodies include: AB202 (PD-L1 18608) AB203 (PD-L1 18446) AB204 (PD-L1 18463) AB218 (PD-L1 18465-V1) AB223 (PD-L1 18446-V1).

[0184] Exemplary CDR sequence sets for anti-Trop2 antibodies include: AB601 (TROP2 17487) AB621 (TROP2 17487-H1D3).

[0185] Exemplary CDR sequence sets for anti-muc16 antibodies include: AB305.

[0186] Exemplary CDR sequence sets for anti-NaPi-2b antibodies include: AB304.

[0187] It should be understood that the antibody platform disclosed herein may include a set of CDR sequences of a target, said sequence set being derived from any antibody known to bind to that target. 11. Light chain, heavy chain region, biantibody region, and Fc region

[0188] The tetrameric antibodies disclosed herein comprise functional elements including a light chain, a heavy chain region, a biantibody region, and an Fc region. Examples of each of these functional elements are provided as portions of the sequences disclosed in Table 1 and can be identified from these sequences by examination (e.g., by sequence ends and spacer subsequences). It is contemplated herein that sequences of these elements be incorporated into the subject matter of an antibody containing sequences of functional elements not provided herein. For example, an antibody may comprise sequences of the light chain and heavy chain regions for binding epitopes of Her2, as provided herein, and additionally comprise an Fc portion containing, for example, cysteine ​​substitutions not provided in the sequences of Table 1. III. Antibody conjugates

[0189] Antibodies conjugated to a chemical entity (i.e., the "payload") are referred to herein as "antibody conjugates" or "immunoconjugates." Conjugation can be achieved through covalent coupling or non-covalent interactions (e.g., ionic bonds, van der Waals bonds, electrostatic bonds, or hydrogen bonds). The chemical entity conjugated to the antibody may also be referred to as a part. Chemical parts or payloads include, but are not limited to, drugs, radionuclides, biotin, RNA, antibiotics, proteins, and detectable parts (e.g., fluorophores).

[0190] As used herein, the term “antibody-drug conjugate” or “ADC” refers to an immunoconjugate in which an antibody is conjugated with a drug (such as a cytotoxic drug or an immunostimulant). Cytotoxic drugs include, but are not limited to, maytansine, DM-1, DM-4, ​​auristatin, monomethylauristatin E, monomethylauristatin F, sea haretoxin, microtubule-lysin, eribulin, cantharidin, benzodiazepine, indolino-benzodiazepine, isoquino-benzodiazepine, pyrrolo-benzodiazepine, α-amaminine, trichothecene, camptothecin derivatives (SN-38, essanotecan, belotecone, DXd, topotecan, samotecan), pyroxine, DGN549, CC1065, cazithromycin, N-acetylcazithromycin, enediyne antibiotics, taxanes, doxorubicin derivatives, anthracyclines and stereoisomers, azanofide, and the isosteres, analogs, heterodimers, homodimers, or derivatives of the aforementioned substances. Cytotoxic drugs may also include any of the drugs in Colombo et al. Cancer Discov (2024) 14(11): 2089-2108.

[0191] As used herein, the term "labeled antibody" refers to an antibody that binds to a detectable label, enabling the detection of the presence of a target (e.g., a molecule) by detecting the presence of the detectable label bound to the target. As used herein, the term "detectable label" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Examples of detectable labels are described herein and include, but are not limited to, colorimetric labels, fluorescent labels, chemiluminescent labels, enzyme labels, and radiolabels. For the purposes of this disclosure, a detectable label may also be a portion that does not itself generate a signal (e.g., biotin), but binds to a second portion capable of generating a signal (e.g., labeled avidin or streptavidin).

[0192] The term "small molecule" refers to organic or inorganic molecules with a size of up to about 5,000 Da, up to about 2,000 Da, or up to about 1,000 Da.

[0193] "Potential" refers to the IC50 value of a compound in killing cells (e.g., in a culture). 50 .

[0194] Many conjugation methods are known in the art. In some embodiments, the payload is attached to the antibody via a spacer. These include, but are not limited to, conjugation via cysteine ​​residues, as well as conjugation via lysine, arginine, or tyrosine residues. In some embodiments, conjugation involves covalent binding via a linker. A. Cysteine ​​residue conjugation

[0195] Cysteine ​​residue conjugation involves targeting the thiol (-SH) group of cysteine ​​residues in an antibody. This can be achieved, for example, by reducing an existing disulfide bond to expose a free thiol group or by engineering additional cysteine ​​residues into the antibody. The exposed thiol group is reactive and can conjugate with, for example, maleimide-functionalized drugs or labels to form stable thioether bonds. B. Lysine residue conjugation

[0196] Lysine residue conjugation involves targeting the amine group (-NH2) on the side chain of the lysine residue. This can be accomplished, for example, by using an N-hydroxysuccinimide (NHS) ester, which reacts with the amine group to form a stable amide bond. C. Site-specific conjugation method

[0197] Any of these site-selective conjugation methods can be used to conjugate the linker-payload to a given antibody. These site-selective conjugation methods are broadly classified into eight categories: cysteine ​​engineering, non-natural amino acid engineering, conjugation with natural cysteine, peptide tagging, glycan modification, enzymatic modification, disulfide rebridging, and conjugation with natural lysine (for further details, see, for example, Walsh SJ et al., “Site-selective modification strategies in antibody-drug conjugates,” Chem Soc Rev. 2021;50:1305-53. doi:10.1039 / d0cs00310g. PMID: 33290462). D. Connector Chemistry

[0198] The payload is typically conjugated to the antibody via a adapter. Adapters include cleavable and non-cleavable adapters.

[0199] Connectors can also be conjugated using non-natural amino acids, disulfide bond rebridging, peptide tags, polysaccharide modification, or enzymatic modification methods to generate site-specific immunoconjugates. E. Drug to Antibody Ratio

[0200] The drug-to-antibody ratio, or DAR, refers to the average number of payload portions attached to the antibody in the composition. DAR varies with the number of conjugable sites on the antibody. When the disulfide bonds of the antibody are reduced, producing reactive thiol groups, this can allow a DAR between 1 and 12. However, introducing cysteine ​​residues into the immunoglobulin chain can significantly increase the possible DAR. The DAR of an antibody composition can be, for example, any one of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more. F. Cysteine-substituted antibodies

[0201] Cysteine-substituted antibodies are antibodies containing at least one immunoglobulin amino acid residue in the light or heavy chain constant region, wherein the immunoglobulin amino acid residue in the light or heavy chain constant region has been replaced by a cysteine ​​residue not present in naturally occurring antibodies. These are sometimes referred to as “thiomabs” (Junutula et al., Nature Biotechnology, 2008:26(8):925-32, doi: 10.1038 / nbt.1480). Non-naturally occurring substitutions are non-isotype substitutions. In some embodiments, the substituted residues in the heavy chain constant region may include residues A118C, T155C, S157C, S239C, V266C, H285C, R301C, V303C, T307C, G316C, Y436C, and L441C. These sites are based on EU numbers, as known in the art and described at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html and in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969. Substituted residues in the light chain constant region may include residue V205C (Kabat number, Kabat, EA et al., Sequences of proteins of immunological interest. 5th ed. - USDepartment of Health and Human Services, NIH Publication No. 91-3242, pp. 662, 680, 689 (1991)). In some embodiments, the constant region belongs to isotype IgG1, IgG2, IgG3, or IgG4. IV. Nucleic acids and recombinant cells in the preparation method

[0202] This document provides nucleic acid molecules encoding immunoglobulin chains of antibodies of this disclosure. The antibodies of this disclosure may comprise a variety of different polypeptides assembled into the antibody. For example, an antibody may comprise two identical heavy chains and two identical light chains. Alternatively, an antibody may comprise two different heavy chains. Further combinations of different polypeptides are possible, such as two different heavy chains and one or two different light chains. Therefore, this document further provides nucleic acid molecules or collections of nucleic acid molecules that encode polypeptides in a combined manner (the polypeptides, upon assembly, produce the antibodies of this disclosure). A. Nucleic acid molecules

[0203] The antibodies described herein can be prepared by expressing polypeptides from nucleic acid molecules that encode them. Nucleic acids containing nucleotide sequences encoding heavy and light chain molecules can be incorporated into recombinant DNA molecules (such as expression vectors), transfected into cells, expressed by cells in cultures, harvested, and purified. Such methods are well known in the fields of molecular biology, cell biology, and biopharmaceutical manufacturing.

[0204] If a nucleic acid molecule contains a nucleotide sequence that produces a polypeptide during transcription and / or translation, then the nucleic acid molecule encodes a polypeptide. This includes, but is not limited to, continuous nucleotide sequences encoding a polypeptide and non-continuous nucleotide sequences encoding a polypeptide. Non-continuous nucleotide sequences encoding a polypeptide include, for example, nucleotide sequences containing both introns and exons. Such non-coding sequences are spliced ​​from messenger RNA. B. Expression Components and Carriers

[0205] Nucleic acid molecules encoding immunoglobulin molecules can be incorporated into expression constructs for expression. An expression construct is a polynucleotide containing an expression control sequence operatively linked to the heteronucleotide sequence to be expressed (i.e., a sequence to which the expression control sequence is not normally linked in nature).

[0206] Expression control sequences are nucleotide sequences that regulate transcription and / or translation of nucleotide sequences operatively linked to them. Expression control sequences include promoters, enhancers, repressors (transcriptional regulatory sequences), and ribosome binding sites (translational regulatory sequences). Appropriate regulation can originate from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes.

[0207] When expression control sequences function in a cell to regulate the transcription of nucleotide sequences, the nucleotide sequences are "operably linked" to the expression control sequences. This includes initiating the transcription of nucleotide sequences through interactions between polymerases and transcriptional regulatory sequences, such as promoters.

[0208] Promoters typically used in eukaryotic expression systems include, but are not limited to, the following: CMV (cytomegalovirus) promoter, EF-1α (elongation factor-1α) promoter, SV40 (simian virus 40) promoter, PGK (phosphoglycerate kinase) promoter, CAG promoter, AOX1 (alcohol oxidase 1) promoter, and GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter. Some promoters used in yeast (such as Saccharomyces cerevisiae) include the ADH (alcohol dehydrogenase) promoter, GAL1, and GAL10.

[0209] An expression vector is a polynucleotide comprising an expression construct and a sequence sufficient to replicate in a host cell or be inserted into a host chromosome. The vector may contain any intermediate medium for a nucleic acid molecule that enables the nucleic acid molecule to be introduced, for example, into prokaryotic and / or eukaryotic cells and / or integrated into the genome. Exemplary vectors for transfecting cells include, for example, plasmids, viral vectors (e.g., retroviruses, lentiviruses, adenoviruses, lactopolyvacuoviruses (e.g., SV40, polyomaviruses), parvoviruses (e.g., adeno-associated virus), or herpes simplex virus), bacterial artificial chromosomes, and yeast artificial chromosomes. As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material (typically a circular DNA double strand) that can replicate independently of chromosomal DNA. The vector should be compatible with the host cell used. Other sequences, such as origins of replication, DNA restriction sites, enhancers, and sequences conferring transcriptional induction, may be incorporated into the expression vector.

[0210] The recombinant expression vector may also contain a marker gene that helps select host cells transformed, infected, or transfected with the vector for expression of the antibodies described herein.

[0211] Recombinant expression vectors may also contain expression cassettes encoding a fusion moiety that provides enhanced expression or stability of the recombinant peptide; increased solubility of the recombinant peptide; or aids in the purification of the target recombinant peptide by acting as a ligand in affinity purification, including, for example, tags and labels described herein (e.g., (His)6 tags) (SEQ ID NO: 179). The recombinant peptide and its fusion moiety are collectively referred to as “fusion proteins.” Furthermore, proteolytic cleavage sites may be added to the fusion protein to allow the recombinant peptide to be separated from the fusion moiety after purification of the fusion protein. Typical fusion expression vectors include pGEX (AmradCorp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Massachusetts), and pRIT5 (Pharmacia, Piscataway, New Jersey), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A to the recombinant protein, respectively. C. Recombinant cells

[0212] The antibodies disclosed herein can be prepared by culturing cells engineered to express nucleic acid constructs encoding immunoglobulin peptides.

[0213] Recombinant host cells can be produced using any cell suitable for producing peptides (e.g., suitable for producing antibodies). For example, to introduce nucleic acids (e.g., vectors) into cells, cells can be transfected, transformed, or infected, depending on the vector used.

[0214] Methods for transfecting cells with recombinant DNA molecules are known in the art and include, for example, electroporation, liposomes and exosomes, as well as transfection via viral vectors.

[0215] Suitable host cells include a variety of prokaryotic and eukaryotic host cells. For example, the proteins described herein can be expressed in mammalian cells (e.g., CHO, 293); insect cells (e.g., Sf9 cells or high five (BTI-Tn-5B1-4) cells); yeast cells (e.g., Pichia pastoris or Saccharomyces cerevisiae); bacterial cells (e.g., Escherichia coli); or plant cells (e.g., Arabidopsis).

[0216] Among other possibilities, suitable mammalian cells include, in particular, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 / 0 cells, human embryonic kidney HEK 293 cells, or Per.C6 cells. Suitable expression vectors for guiding expression in mammalian cells typically include promoters (e.g., derived from viral genomes such as polyomavirus, adenovirus 2, cytomegalovirus, and simian virus 40) and other transcriptional and translational control sequences. Examples of mammalian expression vectors include pCDM8 and pMT2PC.

[0217] Suitable yeast and fungal host cells for antibody expression include, but are not limited to, several species of *Saccharomyces cerevisiae*, *Schizosaccharomyces pombe*, *Pichia*, *Kluyveromyces*, and *Aspergillus*. Examples of vectors used for expression in *Saccharomyces cerevisiae* include pYepSec1, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, California). Protocols for transforming yeasts and fungi are well known to those skilled in the art. D. Antibody production

[0218] The transformed / transfected cells are grown under suitable conditions that allow them to express recombinant antibodies.

[0219] The number of different immunoglobulin chains that must be expressed in the cell to produce antibodies depends on the antibody conformation. Generally, fewer different chains lead to more efficient antibody assembly.

[0220] Depending on the architecture, the antibodies described herein may contain one or two different heavy chains. Antibodies with identical heavy chains require only one heavy chain to be produced in the cell, and they will associate naturally. If the antibodies contain different heavy chains, two polypeptides are produced, and the club-and-mortar arrangement can promote proper assembly of the antibody, preventing the association of two "club" chains or two "mortar" chains.

[0221] The light chain will also associate with its respective heavy chain. When the antibody contains two identical heavy chains, only one light chain needs to be expressed. However, in some asymmetric antibodies, two different light chains may be required to associate with the asymmetric heavy chain. In some cases, however, two different complementary sites may be generated targeting different epitopes containing different heavy chain regions and the same light chain.

[0222] For cells secreting recombinant antibody chains, the culture medium containing immunoglobulin molecules is harvested, and the antibody is purified from the culture medium using protein A or protein G chromatography, which specifically binds to the Fc region of the antibody. In some embodiments, recombinant cells containing light and / or heavy chains are lysed, and the immunoglobulin chains are purified from the lysate using standard biochemical procedures.

[0223] If necessary, the purified antibody can then be conjugated with the type of payload described herein using the methods described herein. V. Pharmaceutical Composition

[0224] This document further provides a composition comprising the antibodies, immunoconjugates, nucleic acid molecules, vectors, or recombinant cells described herein, optionally having a suitable diluent, such as a pharmaceutically acceptable vector. The composition may, for example, comprise one or more antibodies or immunoconjugates.

[0225] In one embodiment, the pharmaceutical composition comprises an antibody or immunoconjugate, as described herein, formulated in a pharmaceutically acceptable carrier. The composition is typically formulated for intravenous injection. Therefore, the carrier can be an aqueous carrier, such as sterile water or a saline solution. The composition may further contain components that stabilize the antibody molecules. These may include, for example, sugars and polyols that facilitate lyophilization. Mannitol may also be used as a tension modifier and loosening agent in lyophilized formulations. Surfactants (such as polysorbate 20 or polysorbate 80) are also commonly used in monoclonal antibody formulations. Buffers (such as phosphates, histidines, and citrates) are frequently used in such formulations.

[0226] The compositions described herein can be prepared by methods known per se for preparing pharmaceutically acceptable compositions that can be administered to subjects, such that an effective amount of the active substance is combined with a pharmaceutically acceptable carrier in the mixture.

[0227] The term “pharmaceutical acceptable” refers to a carrier that is compatible with the other components of a pharmaceutical composition and can be safely administered to a subject. This term is used synonymously with “physiologically acceptable” and “pharmacologically acceptable.” According to this disclosure, pharmaceutical compositions and techniques for their preparation and use are known to those skilled in the art. A detailed list of suitable pharmacological compositions and their administration techniques can be found in the following articles: Remington's Pharmaceutical Sciences, 17th edition, 1985; Brunton et al., “Goodman and Gilman's The Pharmacological Basis of Therapeutics,” McGraw-Hill, 2005; University of the Sciences in Philadelphia (ed.), “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (ed.), “Remington: The Principles of Pharmacy Practice,” Lippincott Williams & Wilkins, 2008.

[0228] Pharmaceutically acceptable carriers are generally sterile, at least for human use. Pharmaceutical compositions typically contain agents for buffering and preservation of the drug and may include buffers and carriers for appropriate delivery, depending on the route of administration. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological (0.9%) saline, phosphate-buffered saline (PBS), Hank's balanced salt solution (HBSS), and various electrolyte solutions such as PlasmaLyte ATM (Baxter).

[0229] Suitable diluents for peptides (including antibodies and / or cells) include, but are not limited to, saline solutions, pH buffer solutions, and glycerol solutions, or other solutions suitable for freezing peptides and / or cells.

[0230] Suitable diluents for nucleic acids include, but are not limited to, water and saline solutions.

[0231] Pharmaceutical compositions include, but are not limited to, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, antibacterial agents, and solutes that make the composition substantially compatible with the tissues or blood of the intended recipient. Other components that may be present in such compositions include, for example, water, surfactants (such as Tween), alcohols, polyols, glycerol, and vegetable oils. Exemplary injectable solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions. The compositions may be provided, for example, and not by limitation, as lyophilized powders, which are reconstituted with sterile water or saline prior to administration to a patient.

[0232] Pharmaceutical compositions may comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include compositions that are substantially chemically inert and non-toxic, and that do not interfere with the efficacy of the bioactivity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3-dioleoyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), dioleoylphosphatidylethanolamine (DOPE), and liposomes. Such compositions should contain a therapeutically effective amount of the compound, optionally with a suitable amount of carrier, to provide a form for direct administration to a patient.

[0233] The composition may be in the form of pharmaceutically acceptable salts, including, but not limited to, those pharmaceutically acceptable salts having free amino groups formed from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid and tartaric acid, as well as those pharmaceutically acceptable salts formed from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, iron hydroxide, isopropylamine, triethylamine and 2-ethylaminoethanol.

[0234] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient administered to an individual at each administration. For the purposes of this invention, a dose may refer to the concentration of an antibody or related component, such as the amount of a therapeutic agent or the dose of a radiolabel. A dose will vary depending on many factors, including the frequency of administration; individual size and tolerability; severity of the condition; risk of side effects; route of administration; and imaging modality for detecting the label (if present). Those skilled in the art will recognize that the dose can be modified based on the foregoing factors and / or on treatment progress. The term “dosage form” refers to a specific form of the medicine and depends on the route of administration. For example, a dosage form may be a liquid, such as an injectable saline solution. VI. How to Use A. Treatment methods 1. Disease

[0235] This document provides a method for treating a subject's disease, the method comprising administering to the subject an effective amount of a nucleic acid, antibody, or immunoconjugate of this disclosure. An "effective amount" is an amount relative to the absence of a compound sufficient to enable an antibody or immunoconjugate to achieve its intended purpose. The intended purpose may be, for example, killing pathological cells in vitro or in vivo, or producing a therapeutic benefit to the subject. An example of an "effective amount" is an amount sufficient to induce treatment, prevention, or reduction of one or more symptoms of a disease, which may also be referred to as a "therapeutic effective amount." "Reduction" or "reducing" of symptoms means the elimination of symptoms or a reduction in the severity or frequency of symptoms. A "preventive effective amount" of a substance (e.g., an antibody) is an amount of the substance that, when administered to a subject, would prevent or delay the onset or recurrence of a disease, pathology, or symptom, or reduce the likelihood of such occurrence or recurrence. An effective amount does not necessarily occur with a single dose and may only occur after a series of doses. Therefore, an effective amount may be administered in one or more administrations. The exact amount will depend on the therapeutic purpose and will be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 1–3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th ed., 2003, edited by Gennaro, Lippincott, Williams & Wilkins).

[0236] For any antibody or ADC described herein, a preliminary estimate or determination of the therapeutically effective dose can be made using cell culture assays. The target concentration will be the concentration of the antibody or ADC that achieves the desired result (e.g., slowing cell growth), as measured using methods described herein or known in the art. As is well known in the art, a therapeutically effective dose for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve concentrations that have been found to be effective in animals. The dose in humans can be adjusted by monitoring efficacy and adjusting the dose up or down, as described above. Adjusting the dose based on the methods described above and others to achieve maximum efficacy in humans is entirely within the capabilities of a person skilled in the art.

[0237] Treatable cancers include, but are not limited to, solid tumors such as carcinomas, sarcomas, and adenocarcinomas; and lymphomas such as leukemia and lymphoma. Examples of different types of cancer include, but are not limited to, lung cancer (e.g., non-small cell lung cancer or NSCLC), breast cancer, ovarian cancer, prostate cancer, colorectal cancer, bladder cancer, leukemia, liver cancer (i.e., hepatocarcinoma), kidney cancer (i.e., renal cell carcinoma), thyroid cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, stomach cancer, kidney cancer, central nervous system cancers, skin cancer, glioblastoma, and melanoma.

[0238] It is anticipated that cancer cells expressing two epitopes targeted by the ADC of this disclosure exist in a mixed population, where different cells express each of these biomarkers to varying degrees. Not wishing to be theoretically limited, it is expected that bispecific ADC antibodies will be more effective against such populations compared to ADC antibodies targeting only a single target. 2. Application route

[0239] Pharmaceutical compositions containing antibodies disclosed herein may be administered to subjects via any suitable route of administration.

[0240] Intravenous administration involves delivering a drug composition directly into the bloodstream, which facilitates rapid systemic distribution and enables immediate therapeutic action. Intravenous administration can include bolus injection or slow infusion, depending on the pharmacokinetic characteristics required for the therapeutic indication.

[0241] Intraperitoneal administration involves introducing a drug composition into the peritoneal cavity. This mode of administration is particularly useful for diseases located in the abdominal region because it provides direct access to the pathological site.

[0242] The drug composition can be administered subcutaneously, that is, into the adipose tissue beneath the skin.

[0243] In intramuscular administration, the drug composition is injected into muscle tissue. In some cancers, the drug composition can be applied directly to the tumor.

[0244] For diseases of the central nervous system, the composition can be administered intrathecally or intraventricularly, for example, into the spinal canal or ventricles. 3. Treatment Plan

[0245] This disclosure describes a cancer treatment regimen tailored to the patient's specific cancer, using the antibody or ADC described herein. However, exemplary regimens for existing treatments include the following: Administration of the antibody or ADC at a dose between 1 mg and 12 mg per kilogram of body weight. After the initial dose, the drug may be administered approximately weekly or approximately every three weeks for five or more cycles, for example, until symptoms are relieved, adequately alleviated, completely alleviated, or until side effects or toxicity become uncontrollable or the drug no longer provides therapeutic benefit. B. Methods of killing cells

[0246] The antibodies and immunoconjugates of this disclosure can be used to kill or label cells in vitro or in vivo. The methods involve contacting cells with the antibodies or immunoconjugates of this disclosure (e.g., by introducing the antibody or immunoconjugate into a subject in need) and allowing the antibody to bind to target cells. The effectiveness of killing cells can vary with the ability to kill target cells without killing non-target cells in a mixed cell population. The ability to kill cells can also be compared to the ability of a control (e.g., a control antibody), wherein a greater ability of the test antibody to kill cells compared to a control antibody indicates that the test antibody is effective in killing those cells.

[0247] In vitro contact of cells derived from cancer cell lines with the compositions disclosed herein can be used to determine the toxicity of the compositions to the cells.

[0248] In vivo contact with immunoconjugates labeled with radioactive isotopes can be used to image the binding site, and thus the location of malignant cells. Detection methods include gamma camera imaging, single-photon emission coupled-to-computed tomography (SPECT), and positron emission tomography (PET). C. Detection Method

[0249] The antibodies of this disclosure can also be used to detect the presence in a sample or subject of cells expressing an epitope targeted by the antibody of this disclosure. The method involves contacting a sample with the antibody of this disclosure or providing the subject with the antibody of this disclosure, and detecting the binding between the antibody and the cells.

[0250] Binding can be detected by providing an antibody with a detectable marker and detecting the marker.

[0251] Detection methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (including fluorescence activated cell sorting (FACS)), surface plasmon resonance, microscopy, and immunoprecipitation.

[0252] In vivo detection methods include, for example, positron emission tomography (PET), in which antibodies are conjugated with radioactive isotopes; and magnetic resonance imaging (MRI), in which antibodies are conjugated with MRI contrast agents.

[0253] This type of method can also be used for the diagnosis of malignant tumors. Detection of a higher amount of cell epitopes targeted by the antibodies described in this disclosure indicates the presence of malignant cells. Exemplary Implementation

[0254] Exemplary embodiments of this disclosure include, but are not limited to:

[0255] 1. An antibody, such as an immunoconjugate, comprising a tetravalent antibody containing one or more payloads conjugated thereto, wherein the antibody comprises a first light chain, a second light chain, a third light chain, and a fourth light chain, as well as a first heavy chain and a second heavy chain, and comprises, from the N-terminus to the C-terminus: a) The first pair of complementary bits contained in the Fab region; b) Fc region; and c) The second pair of complementary bits contained in the Fab region.

[0256] 2. An antibody, such as an immunoconjugate, comprising a payload conjugated thereto, the antibody comprising a first light chain, a second light chain, a third light chain, and a fourth light chain, and a first heavy chain and a second heavy chain, wherein: a) The first light chain includes a first light chain variable (VL) region and a first light chain constant (CL) region from the N-terminus to the C-terminus; b) The second light chain includes a second VL region and a second CL region from the N-terminus to the C-terminus; c) The third light chain includes a variable (VL) region and a constant (CL) region from the N-terminus to the C-terminus; d) The fourth light chain includes a fourth VL region and a fourth CL region from the N-terminus to the C-terminus; e) The first heavy chain from the N-terminus to the C-terminus comprises: (i) The first heavy chain portion containing the first heavy chain variable (VH) region and the first heavy chain constant 1 (CH1) region; (ii) A first Fc region containing a first heavy chain constant 2 (CH2) region and a first heavy chain constant 3 (CH3) region; (iii) The second heavy chain portion containing the second heavy chain variable (VH) region and the second heavy chain constant 1 (CH1) region; and f) The second heavy chain from the N-terminus to the C-terminus comprises: (i) The third heavy chain portion containing the third heavy chain variable (VH) region and the third heavy chain constant 1 (CH1) region; (ii) A second Fc region containing a second heavy chain constant 2 (CH2) region and a second heavy chain constant 3 (CH3) region; (iii) The fourth heavy chain portion containing the fourth heavy chain variable (VH) region and the fourth heavy chain constant 1 (CH1) region; and in: The first light chain and the first heavy chain portion form a first Fab portion containing a first complementary site; and The second light chain and the second heavy chain portion form a second Fab portion containing a second complementary site; The third light chain and the third heavy chain portion form a third Fab portion containing a third complementary site; and The fourth light chain and the fourth heavy chain portion form a fourth Fab portion containing a fourth complementary site.

[0257] 3. An antibody, such as an immunoconjugate, comprising a tetravalent antibody containing one or more payloads conjugated thereto, wherein the antibody comprises a first light chain and a second light chain, and a first heavy chain and a second heavy chain, and comprises, from the N-terminus to the C-terminus: a) The first pair of complementary sites contained in the biantibody; b) Fc region; and c) The second pair of complementary bits contained in the Fab region.

[0258] 4. An antibody, such as an immunoconjugate, comprising a payload conjugated to the antibody, the antibody comprising a first light chain and a second light chain, and a first heavy chain and a second heavy chain, wherein: a) The first light chain includes a first light chain variable (VL) region and a first light chain constant (CL) region from the N-terminus to the C-terminus; b) The second light chain includes a second VL region and a second CL region from the N-terminus to the C-terminus; c) The first heavy chain from the N-terminus to the C-terminus comprises: (i) A first biantibody region containing a first VH region and a third VL region in any order; and (ii) A first Fc region containing a first heavy chain constant 2 (CH2) region and a first heavy chain constant 3 (CH3) region; and (iii) The first heavy chain portion containing the second heavy chain variable (VH) region and the first heavy chain constant 1 (CH1) region; d) The second heavy chain from the N-terminus to the C-terminus comprises: (i) A second biantibody region containing a third VH region and a fourth VL region in any order; and (ii) A second Fc region containing a second heavy chain constant 2 (CH2) region and a second heavy chain constant 3 (CH3) region; and (iii) The second heavy chain portion containing the fourth heavy chain variable (VH) region and the second heavy chain constant 1 (CH1) region; in: The first light chain and the first heavy chain portion form a first Fab portion containing a first complementary site; and The second light chain and the second heavy chain portion form a second Fab portion containing a second complementary site; and The first biantibody region and the second biantibody region form a biantibody, and the biantibody forms a third complementary site including the third VL region and the third VH region, and a fourth complementary site including the fourth VL region and the first VH region.

[0259] 5. An antibody, such as an immunoconjugate, comprising a tetravalent antibody containing one or more payloads conjugated thereto, wherein the antibody comprises a first light chain, a second light chain, a third light chain, and a fourth light chain, as well as a first heavy chain and a second heavy chain, and comprises, from the N-terminus to the C-terminus: a) The first pair of complementary bits contained in the Fab region; b) The second pair of complementary sites contained in the Fab region; and c) Fc region.

[0260] 6. An antibody, such as an immunoconjugate, comprising a payload conjugated thereto, the antibody comprising a first light chain, a second light chain, a third light chain, and a fourth light chain, and a first heavy chain and a second heavy chain, wherein: a) The first light chain includes a first light chain variable (VL) region and a first light chain constant (CL) region from the N-terminus to the C-terminus; b) The second light chain includes a second VL region and a second CL region from the N-terminus to the C-terminus; c) The third light chain includes a variable (VL) region and a constant (CL) region from the N-terminus to the C-terminus; d) The fourth light chain includes a fourth VL region and a fourth CL region from the N-terminus to the C-terminus; e) The first heavy chain from the N-terminus to the C-terminus comprises: (i) The first heavy chain portion containing the first heavy chain variable (VH) region and the first heavy chain constant 1 (CH1) region; (ii) The second heavy chain portion containing the second heavy chain variable (VH) region and the second heavy chain constant 1 (CH1) region; and (iii) A first Fc region containing a first heavy chain constant 2 (CH2) region and a first heavy chain constant 3 (CH3) region; f) The second heavy chain from the N-terminus to the C-terminus comprises: (i) The third heavy chain portion containing the third heavy chain variable (VH) region and the third heavy chain constant 1 (CH1) region; (ii) The fourth heavy chain portion containing the fourth heavy chain variable (VH) region and the fourth heavy chain constant 1 (CH1) region; and (iii) A second Fc region containing a second heavy chain constant 2 (CH2) region and a second heavy chain constant 3 (CH3) region; in: The first light chain and the first heavy chain portion form a first Fab portion containing a first complementary site; and The second light chain and the second heavy chain portion form a second Fab portion containing a second complementary site; The third light chain and the third heavy chain portion form a third Fab portion containing a third complementary site; and The fourth light chain and the fourth heavy chain portion form a fourth Fab portion containing a fourth complementary site.

[0261] 7. An antibody, such as an immunoconjugate, comprising a tetravalent antibody containing one or more payloads conjugated thereto, wherein the antibody comprises a first light chain and a second light chain, and a first heavy chain and a second heavy chain, and comprises, from the N-terminus to the C-terminus: a) The first pair of complementary bits contained in the Fab region; b) The second pair of complementary sites contained in the biantibody; and c) Fc region.

[0262] 8. An antibody, such as an immunoconjugate, comprising a payload conjugated to the antibody, the antibody comprising a first light chain and a second light chain, and a first heavy chain and a second heavy chain, wherein: a) The first light chain includes a first light chain variable (VL) region and a first light chain constant (CL) region from the N-terminus to the C-terminus; b) The second light chain includes a second VL region and a second CL region from the N-terminus to the C-terminus; c) The first heavy chain from the N-terminus to the C-terminus comprises: (i) The first heavy chain portion containing the first heavy chain variable (VH) region and the first heavy chain constant 1 (CH1) region; (ii) A first biantibody region containing a second VH region and a third VL region in any order; and (iii) A first Fc region containing a first heavy chain constant 2 (CH2) region and a first heavy chain constant 3 (CH3) region; and d) The second heavy chain from the N-terminus to the C-terminus comprises: (i) The second heavy chain portion containing the third VH region and the second CH1 region; (ii) A second biantibody region containing a fourth VH region and a fourth VL region in any order; and (iii) A second Fc region containing a second CH2 region and a second CH3 region; in: The first light chain and the first heavy chain portion form a first Fab portion containing a first complementary site; and The second light chain and the second heavy chain portion form a second Fab portion containing a second complementary site; The first biantibody region and the second biantibody region form a biantibody, and the biantibody forms a third complementary site containing the fourth VL region and the second VH region, and a fourth complementary site containing the third VL region and the fourth VH region.

[0263] 9. An antibody or immunoconjugate according to any one of embodiments 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site that binds to a first epitope of Her2 and at least one complementary site that binds to a second epitope of Her2.

[0264] 10. An antibody or immunoconjugate according to any one of embodiments 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site that binds to CD30 and at least one complementary site that binds to an epitope of PD-L1.

[0265] 11. An antibody or immunoconjugate according to any one of embodiments 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site binding to a first epitope of Her2, at least one complementary site binding to PD-L1, and optionally at least one complementary site binding to a second epitope of Her2.

[0266] 12. An antibody or immunoconjugate according to any one of embodiments 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site for binding to CD30, at least one complementary site for binding to a first epitope of PD-L1, and at least one complementary site for binding to a second epitope of PD-L1.

[0267] 13. An antibody or immunoconjugate according to any one of embodiments 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site for binding to Trop2, at least one complementary site for binding to a first epitope of Her2, at least one complementary site for binding to a second epitope of Her2, and a complementary site for binding to PD-L1.

[0268] 14. An antibody or immunoconjugate according to any one of embodiments 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site binding to Trop2, at least one complementary site binding to a first epitope of Her2, and optionally at least one complementary site binding to a second epitope of Her2.

[0269] 14bis. An antibody or immunoconjugate according to any one of embodiments 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site that binds to Muc16 and at least one complementary site that binds to an epitope of NaPi-2b.

[0270] 15. An antibody or immunoconjugate according to any one of embodiments 1 to 8, wherein the antibody or immunoconjugate is a monospecific antibody, wherein all complementary sites specifically bind to the same epitope.

[0271] 16. An antibody or immunoconjugate according to any one of embodiments 1 to 8, wherein the antibody or immunoconjugate is a bispecific antibody, wherein at least one of the complementary sites specifically binds a first epitope, and at least another of the complementary sites binds a second distinct epitope.

[0272] 17. The antibody or immunoconjugate according to embodiment 15, wherein two of the complementary sites specifically bind to a first epitope and two of the complementary sites specifically bind to a second different epitope; or three of the complementary sites bind to the same first epitope and one of the complementary sites specifically binds to a second different epitope.

[0273] 18. The antibody or immunoconjugate according to embodiment 15, wherein the first complementary site and the second complementary site bind to the same first epitope, and the third complementary site and the fourth complementary site bind to the same second epitope; or the first complementary site and the third complementary site bind to the same first epitope, and the second complementary site and the fourth complementary site bind to the same second epitope.

[0274] 19. The antibody or immunoconjugate according to embodiment 15, wherein two of the complementary sites bind to the same first epitope of Her2, and two of the complementary sites bind to the same second epitope of Her2, for example, the first and second complementary sites bind to the same first epitope and the third and fourth complementary sites bind to the same second epitope; or, the first and third complementary sites bind to the same first epitope, and the second and fourth complementary sites bind to the same second epitope.

[0275] 20. The antibody or immunoconjugate according to embodiment 18, wherein the first epitope is an epitope bound by trastuzumab, and the second epitope is an epitope bound by pertuzumab.

[0276] 21. The antibody or immunoconjugate according to embodiment 15, wherein one of the complementary sites binds CD30, one of the complementary sites binds a first epitope of Her2, and both of the complementary sites bind the same second epitope of Her2.

[0277] 22. An antibody or immunoconjugate according to any one of embodiments 1 to 8, wherein the antibody or immunoconjugate is a trispecific antibody, wherein at least one of the complementary sites binds to a first epitope, at least one of the complementary sites binds to a second different epitope, and at least one of the complementary sites binds to a third different epitope, for example, two of the complementary sites bind to the same first epitope, one of the complementary sites binds to a second different epitope, and one of the complementary sites binds to a third epitope that is different from the first and second epitopes.

[0278] 23. The antibody or immunoconjugate according to embodiment 21, wherein the first complementary site and the second complementary site bind to the same epitope; the first complementary site and the third complementary site bind to the same epitope; or the second complementary site and the third complementary site bind to the same epitope.

[0279] 24. An antibody or immunoconjugate according to any one of embodiments 1-21, wherein at least two complementary sites bind to different epitopes of the same antigen.

[0280] 25. An antibody or immunoconjugate according to any one of embodiments 1-23, wherein two of the complementary sites bind to different epitopes of the same first antigen, and two of the complementary sites bind to different epitopes of the same second antigen.

[0281] 26. The antibody or immunoconjugate according to embodiment 21, wherein: The first complementary bit and the second complementary bit are each combined with the table position of CD30; The third complementary bit combines with the first tabletop of PD-L1; and The fourth complementary position is combined with the second epitope of PD-L1.

[0282] 27. The antibody or immunoconjugate according to embodiment 21, wherein: The first complementary bit and the second complementary bit are each combined with the tabletop of Trop2; The third complementary position combines with the first epitope of Her2; and The fourth complementary position is combined with the second epitope of Her2.

[0283] 28. The antibody or immunoconjugate according to embodiment 1 or 3, wherein the antibody or immunoconjugate is a tetraspecific antibody, wherein each of the complementary sites binds to a different epitope.

[0284] 29. An antibody or immunoconjugate according to any one of embodiments 1-27, wherein the first heavy chain and the second heavy chain have different amino acid sequences and are associated by a mortar and pestle structure.

[0285] 30. The antibody or immunoconjugate according to embodiment 28, wherein the Fc region comprises two distinct heavy chains, at least one of the two distinct heavy chains comprising an amino acid modification to form complementarity between the two distinct heavy chains, thereby increasing the likelihood of forming a heterodimer of the distinct heavy chains and decreasing the likelihood of forming a homodimer of the same heavy chains.

[0286] 31. An antibody or immunoconjugate according to any one of embodiments 1-30, wherein the heavy chain and light chain are produced by cells genetically engineered to produce two heavy chains and one light chain.

[0287] 32. An antibody or immunoconjugate according to any one of embodiments 1-30, wherein all light chain variable regions have the same amino acid sequence.

[0288] 33. An antibody or immunoconjugate according to any one of embodiments 1-30, wherein the two light chains comprise a light chain variable region having the same amino acid sequence.

[0289] 34. An antibody or immunoconjugate according to any one of embodiments 1-30, wherein the first light chain and the second light chain have the same amino acid sequence.

[0290] 35. An antibody or immunoconjugate according to any one of embodiments 1-30, wherein the first heavy chain and the second heavy chain have the same amino acid sequence.

[0291] 36. An antibody or immunoconjugate according to any one of embodiments 1-35, wherein the antibody comprises: a) A biantibody region optionally attached to the Fc region via spacer 1, and an Fc region attached to the heavy chain portion via spacer 2; b) The heavy chain portion optionally attached to the biantibody region via spacer 3, and the biantibody region optionally attached to the Fc region via spacer 4; c) The Fc region attached to the heavy chain portion via spacer 5; or d) A heavy chain attached to another heavy chain portion via spacer 6.

[0292] 37. The antibody or immunoconjugate according to embodiment 36, wherein spacers 1-6 independently comprise between 0 and 50 amino acids.

[0293] 38. The antibody or immunoconjugate according to embodiment 37, wherein spacers 1-6 are independently selected from monomers or polymers of the sequence (GGGGS)n (SEQ ID NO: 170), (GGGGA)n (SEQ ID NO: 171), or (GGGGG) (SEQ ID NO: 172), wherein n is any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0294] 39. The antibody or immunoconjugate according to embodiment 38, wherein spacers 1-6 are independently selected from GGGGSGGGGSEPKSSDKTHT (SEQ ID NO: 173), GGGSGGGSGGGSGGGSGSTG (SEQ ID NO: 174), LEDKTHTKVEPKSS (SEQ ID NO: 175), SGSETPGTSESATPESGGG (SEQ ID NO: 176), and GTTAASGSSGGSSSGA (SEQ ID NO: 177).

[0295] 40. The antibody or immunoconjugate according to embodiment 38, wherein the antibody or immunoconjugate comprises a biantibody region optionally attached to the Fc region via spacer 1, and an Fc region attached to the heavy chain portion via spacer 2; wherein spacer 1 and spacer 2, if present, are selected from the following pair:

[0296] 41. The antibody or immunoconjugate according to embodiment 40, wherein spacer 1 and spacer 2, if present, are selected from the following pair:

[0297] 42. The antibody or immunoconjugate according to embodiment 36, wherein the antibody or immunoconjugate comprises (i) a heavy chain portion optionally attached to a biantibody region via spacer 3, and a biantibody region optionally attached to an Fc region via spacer 4, wherein spacer 3 and spacer 4, if present, are selected from the following pair:

[0298] 43. The antibody or immunoconjugate according to embodiment 42, wherein spacer 3 and spacer 4, if present, are selected from the following pair:

[0299] 44. An antibody or immunoconjugate according to any one of embodiments 1-43, wherein the payload is conjugated by a reduced disulfide bond.

[0300] 45. An antibody or immunoconjugate according to any one of embodiments 1-43, wherein the antibody or immunoconjugate comprises a plurality of payload molecules conjugated to the antibody.

[0301] 46. ​​An antibody or immunoconjugate according to any one of embodiments 1-43, wherein the antibody is a thioantibody comprising at least one substituted cysteine ​​residue in the heavy or light chain, wherein the payload is conjugated by the substituted cysteine ​​residue.

[0302] 47. The antibody or immunoconjugate according to embodiment 46, wherein the thioantibody comprises amino acid substitutions: (a) HC-A118C and LC-V205C; and / or (b) HC-S157C, HC-S239C and HC-V266C (EU numbers) (c) T155C, H285C, R301C, V303C, T307C, G316C, Y436C and L441C (EU numbers).

[0303] 48. An antibody or immunoconjugate according to any one of embodiments 1-47, wherein the payload comprises a cytotoxic agent, a radioactive isotope, a fluorescent moiety, or an enzyme.

[0304] 49. According to embodiment 48, the antibody or immunoconjugate, wherein the payload comprises a cytotoxic agent selected from: maytansine, DM-1, DM-4, ​​auristatin, monomethylaurestatin E, monomethylaurestatin F, saliton toxin, microtubule-lysin, eribulin, novozymin, benzodiazepine, indolino-benzodiazepine, isoquino-benzodiazepine, pyrrolo-benzodiazepine, α-amaminine, trichothecene, camptothecin derivatives (SN-38, essanotecan, belotecone, DXd, topotecan, samotecan), pyroxine, DGN549, CC1065, cazithromycin, N-acetylcazithromycin, enediyne antibiotics, taxanes, doxorubicin derivatives, anthracyclines and stereoisomers, azanofide, and isosteres, analogs, heterodimers, homodimers, or derivatives of the foregoing.

[0305] 50. An antibody or immunoconjugate according to any one of embodiments 1-49, wherein the antibody comprises at least one light chain, heavy chain, or full CDR sequence set of the antibody chain in Appendix 1.

[0306] 51. The antibody or immunoconjugate according to embodiment 50, wherein: a) The CDR sequence set for Her2 binding, if available, is selected from the light chain, heavy chain, or full CDR sequence set of AB101 or the CDR sequence set of AB102. b) The set of CDR sequences used for CD30 binding, if available, is selected from the light chain, heavy chain, or full CDR sequence set of AB217; c) The CDR sequence set for PD-L1 binding, if present, is selected from the light chain, heavy chain, or full CDR sequence set of AB202, AB203, AB204, AB218, or AB223; d) The set of CDR sequences for Trop2 binding, if present, is selected from the light chain, heavy chain, or full CDR sequence set of AB601 or AB621.

[0307] 52. An antibody or immunoconjugate according to any one of embodiments 50-51, wherein: a) The light chain used for Her2 binding, if present, is selected from AB101, AB102, AB103, AB105, AB106, AB107, AB108, AB113, AB114, AB115, AB116, AB117, AB118, AB119, AB120, AB121, AB122, AB123, AB124, AB125, AB126, AB127, AB128, AB129, AB130, AB131, AB 132, AB133, AB134, AB135, AB136, AB137, AB138, AB139, AB140, AB141, AB142, AB143, AB144, AB607, AB608, A B609, AB610, AB611, AB612, AB613, AB614, AB616, AB617, AB618, AB619, AB620, AB622, AB623, AB624, AB625; b) The light chain used for Trop2 binding, if present, is selected from AB601, AB605, AB606, AB608, AB610, AB613, AB614, and AB626. c) The light chain used for PD-L1 binding, if present, is selected from AB205, AB206, AB207, AB208, AB210, AB211, AB212, AB213, AB216, AB220, AB222; or d) The light chain used for CD30 binding, if present, is selected from AB205, AB206, AB207, AB208, AB209, AB211, AB212, AB213, AB214, AB215, AB216, AB217, AB219, AB220, AB221, and AB222.

[0308] 53. An antibody or immunoconjugate according to any one of embodiments 50-52, wherein: a) The heavy chain used for Her2 binding, if present, is selected from AB101, AB102, AB103, AB105, AB106, AB107, AB108, AB113, AB114, AB115, AB116, AB117, AB118, AB119, AB120, AB121, AB122, AB123, AB124, AB125, AB126, AB127, AB128, AB129, AB130, AB131, AB 132, AB133, AB134, AB135, AB136, AB137, AB138, AB139, AB140, AB141, AB142, AB143, AB144, AB607, AB608, A B609, AB610, AB611, AB612, AB613, AB614, AB616, AB617, AB618, AB619, AB620, AB622, AB623, AB624, AB625; b) The heavy chain used for Trop2 binding, if present, is selected from AB601, AB605, AB606, AB608, AB610, AB613, AB614, and AB626. c) The heavy chain used for PD-L1 binding, if present, is selected from AB205, AB206, AB207, AB208, AB210, AB211, AB212, AB213, AB216, AB220, AB222; or d) The heavy chain used for CD30 binding, if present, is selected from AB205, AB206, AB207, AB208, AB209, AB211, AB212, AB213, AB214, AB215, AB216, AB217, AB219, AB220, AB221, and AB222.

[0309] 54. An antibody or immunoconjugate according to any one of embodiments 50-53, wherein: a) If present, the biantibody regions for binding different Her2 epitopes are selected from biantibody regions of AB106, AB128, AB129, AB130, AB131, AB132, AB133, AB134, AB135, AB136, AB137, AB138, AB139, AB140, AB141, AB142, AB143, AB144 or AB606; b) If present, the biantibody region for binding to the same Her2 epitope is selected from biantibody regions of AB107, AB108, AB608, and AB613. c) If present, the biantibody regions for binding different PD-L1 epitopes are selected from biantibody regions of AB209, AB214, AB215, AB124, AB125, AB126, AB127, AB219, and AB221. d) If present, the biantibody region for binding to the same CD30 epitope is selected from the biantibody region of AB210; e) The biantibody region for binding PD-L1 and CD30, if present, is selected from the biantibody regions of AB216, AB220, and AB222; or f) A biantibody region for binding to the same Trop2 epitope, if present, selected from AB607, AB609, or AB612. g) The biantibody region for binding Her2 and Trop2, if present, is selected from biantibody regions of AB610, AB614, AB616, AB617, AB618, AB619, AB620, AB622, AB623, AB624, and AB625. h) The biantibody region for binding PD-L1 and Trop2, if present, is selected from the biantibody region of AB611.

[0310] 55. An antibody or immunoconjugate according to any one of embodiments 50-54, wherein the antibody or immunoconjugate comprises an Fc region selected from Table 1.

[0311] 56. An antibody or immunoconjugate according to any one of embodiments 1-55, wherein the antibody or immunoconjugate comprises one or more antibody chains as shown in Table 1.

[0312] 57. A method for preparing an immunoconjugate, the method comprising: a) Provide an antibody according to any one of embodiments 1-56; and b) Conjugate one or more payloads to the antibody.

[0313] 58. The method according to embodiment 57, wherein at least one of the payloads is conjugated to the antibody via a linker.

[0314] 59. The method according to embodiment 57, wherein at least one of the payloads is conjugated to the antibody via a reduced disulfide bond.

[0315] 60. The method according to embodiment 57, wherein the antibody is a cysteine-substituted antibody (“thio-antibody”), and at least one cytotoxic payload is coupled to the antibody via the substituted cysteine.

[0316] 61. The method according to embodiment 57, wherein the drug-to-antibody ratio (“DAR”) of the immune conjugate is at least one of 1, 2, 3, 4, 6, 7, 8, 9, 10, 11 and 12.

[0317] 62. One or more nucleic acid molecules, said one or more nucleic acid molecules encoding the heavy chain and / or light chain of an antibody according to any one of embodiments 1-61.

[0318] 63. One or more nucleic acid molecules according to embodiment 62, wherein the one or more nucleic acid molecules encode one or more antibodies according to any one of embodiments 1-56.

[0319] 64. One or more nucleic acid molecules according to embodiment 61, wherein the one or more nucleic acid molecules encode both the light chain and the heavy chain of an antibody according to any one of embodiments 1-56.

[0320] 65. One or more nucleic acid molecules according to any one of embodiments 62-64, the one or more nucleic acid molecules further comprising one or more expression control sequences, the one or more expression control sequences being operatively linked to one or more nucleotide sequences encoding the one or more light chain variable regions and / or one or more heavy chain variable regions.

[0321] 66. One or more nucleic acid molecules according to any one of embodiments 61-64, wherein the one or more nucleic acid molecules are contained in one or more vectors, the vectors being selected, for example, from plasmids, viral vectors (e.g., retroviruses, lentiviruses and adenoviruses, and adeno-associated viruses or herpes simplex viruses), bacterial artificial chromosomes and yeast artificial chromosomes.

[0322] 67. A cell comprising one or more nucleic acid molecules according to any one of embodiments 61-65.

[0323] 68. The cells according to embodiment 56, wherein the cells are selected from mammalian cells (e.g., Chinese hamster ovary (CHO) cells, NSO cells, SP2 / 0 cells, and human embryonic kidney HEK 293 cells or Per.C6 cells); insect cells (e.g., Sf9 cells or high five (BTI-Tn-5B1-4) cells); yeast cells (e.g., Pichiapastoris or Saccharomyces cerevisiae); bacterial cells (e.g., Escherichia coli); and plant cells.

[0324] 69. A method for preparing an antibody, the method comprising: a) Provide one or more cells according to any one of embodiments 67-68; b) Expressing an immunoglobulin molecule encoded by the nucleotide sequence in one or more of the cells; and c) Recover the expressed immunoglobulin molecules.

[0325] 70. The method according to embodiment 69, wherein the antibody is an asymmetric antibody, and the cell comprises one or more nucleic acid molecules encoding a heavy chain comprising a pestle, a heavy chain comprising a mortar, and at least one light chain.

[0326] 71. The method according to embodiment 70, wherein the cell comprises a nucleotide sequence encoding only one light chain, wherein the light chain is configured to associate with different heavy chain regions to produce two complementary sites.

[0327] 72. The method according to embodiment 70, wherein the cell comprises a nucleic acid encoding two different light chains, wherein the first light chain is configured to associate with a first heavy chain region to generate a first complementary site that binds to a first epitope, and the second light chain is configured to associate with a second heavy chain region to generate a second complementary site that binds to a second different epitope.

[0328] 73. The method according to embodiment 70, the method comprising generating different immunoglobulin chains in different cells, recovering the immunoglobulin chains, and contacting the immunoglobulin chains, wherein the immunoglobulin chains are assembled into antibodies.

[0329] 74. The method according to embodiment 70, the method further comprising purifying the recovered antibody, for example using protein A or protein G.

[0330] 75. A pharmaceutical composition comprising an antibody or immunoconjugate according to any one of embodiments 1-45 and a pharmaceutically acceptable carrier, diluent or excipient.

[0331] 76. The pharmaceutical composition according to embodiment 75, wherein the pharmaceutical composition is formulated for intravenous injection.

[0332] 77. A method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of embodiments 64-65.

[0333] 78. The method according to embodiment 77, wherein the cancer is selected from ovarian cancer, breast cancer, lung cancer, gastric cancer, endometrial cancer, and pancreatic cancer.

[0334] 79. A method for detecting cells expressing an antigen comprising an epitope, the method comprising: a) contacting the cells with an antibody or immunoconjugate according to any one of embodiments 1-45 to allow binding between the antibody or immunoconjugate and the cells; and b) detecting the binding of the antibody or immunoconjugate to the cells.

[0335] 80. The method according to embodiment 79, the method comprising a) contacting the cells with an immunoconjugate comprising an antibody conjugated to a detectable portion; and b) detecting the detectable portion bound to the cells.

[0336] 81. A method of killing cells, the method comprising contacting cells with an antibody or immunoconjugate according to any one of embodiments 1-56, wherein one or more complementary sites of the antibody specifically bind to epitopes of the cells.

[0337] 82. The method according to embodiment 81, wherein the two different complementary sites of the antibody bind to two different epitopes of the cell.

[0338] 83. The method according to embodiment 82, wherein the epitope is contained in a cell surface antigen.

[0339] 84. Use of the antibody or immunoconjugate according to any one of embodiments 1-56 in the manufacture of a pharmaceutical product.

[0340] 85. Use of the antibody according to any one of embodiments 1-56 for the production of immunoconjugates. Example I. Example 1: Her2 / Her2 dual complementary site antibody

[0341] Figure 9 The production and activity of bispecific tetravalent antibodies targeting the first and second distinct epitopes of HER2 are illustrated. Monospecific (AB108) or bispecific (AB106 and AB107) tetravalent antibodies were produced in Fab-biantibody-Fc form (AB106) and biantibody-Fc-Fab form (AB107, AB108), wherein the first pair of complementary sites (10423) binds Her2 domain 4, and the second pair of complementary sites (11053) binds Her2 domain 2, with both complementary sites sharing a common light chain. All of these resulted in yields similar to the industry-standard trastuzumab (AB103) and exhibited a single peak on HPLC. All antibodies had engineered cysteine ​​residues at HC-A118C (EU number) and LC-V205C (Kabat number) to enable site-specific conjugation to the linker-payload, allowing for conjugation of the cytotoxic portion without disrupting naturally occurring disulfide bonds.

[0342] The following conjugation method was used for standard IgG or antibody-drug conjugates (ADCs) of the form of monomethylolpropionate (MMAE)-derived IgG or tetravalent Fab-biantibody-Fc / biantibody-Fc-Fab: The antibody was reduced with 40 molar equivalents of 10 mM tris(2-carboxyethyl)phosphine (TCEP) by incubation overnight at room temperature. The reduced sample was then dialyzed for 3.5 h at room temperature using a 10 kDa dialysis tube with 1x PBS buffer (pH 7.4). After dialysis, the sample was re-oxidized at room temperature with 30 molar equivalents of 10 mM dehydroascorbic acid (DHAA) for 3.5 h to allow for the formation of interchain disulfide bonds. Following re-oxidation, the antibody was conjugated with 10 molar equivalents of MC-vc-PAB-MMAE linker-payload by incubation overnight at room temperature (see, for example, Doronina SO et al., “Development of potent monoclonal antibody auristatin conjugates for cancer therapy,” Nat Biotechnol. 2003 Jul;21(7):778-84. doi:10.1038 / nbt832. Epub 2003 Jul 1. PMID: 12778055.), and the excess linker-payload was then dialyzed against ADC formulation buffer. The drug amount (DAR) of each antibody was quantified by analysis of the ADC samples on LC / MS.

[0343] Both bispecific tetravalent ADCs (AB106 and AB107) showed superior in vitro potency compared to the monospecific tetravalent ADC (AB108) and the standard bivalent trastuzumab ADC (AB103). Figure 9 C Figure 9 D and Figure 9 E shows the 50% inhibitory concentration (IC50) of the described antibody against high-copy (SKBr3) and low-copy (JIMT1) cell lines expressing the Her2 antigen. To determine the 50% inhibitory concentration (IC50) of the ADC... 50 To determine the ADC concentration, SKBr3 / JIMT1 cells were seeded at 5000 cells / well in 100 μL of cell culture medium in 96-well clear substrate plates and incubated overnight at 37ºC with 5% CO2. After overnight incubation, serially diluted ADC samples (50 μL) were added in duplicate to each well, resulting in a final ADC concentration ranging from 1000 ng / mL to 0.05 ng / mL. The plates were then incubated at 37ºC for 60–72 h. To measure cell viability, the plates were equilibrated at room temperature for 30 min. 100 μL of CellTiter-Glo luminescence solution (Promega, catalog number G7570) was added to each well, and the plates were incubated on a plate shaker (300 rpm) for 20 min. Luminescence was measured using a BioTek Synergy plate reader. IC50 was calculated using GraphPad software. 50 value. II. Example 2: CD30-PD-L1 Trispecific Antibody

[0344] Figure 10 Characterization of individual CD30 and PD-L1 binding agents (IgG) is shown. The biochemical affinity of all IgGs was characterized by analyzing the binding of all IgGs to their respective antigens using the OCTET system via biolayer interferometry (BLI).

[0345] CD30L Blocking Assay: The binding of recombinant CD30 to CD30L was measured using an ELISA. Briefly, 1 μg / ml of CD30-Fc fusion protein (813-CD, R&D Biosystems) diluted in PBS was coated onto a 384-well Maxisorp plate and incubated overnight at 4°C. The remaining binding sites were blocked for 1 hour at room temperature with 0.5% bovine serum albumin diluted in PBS. The wells were washed with PBS + 0.05% Tween-20 (6 x 80 μl). Twenty-five μL of the test antibody diluted to 1 μM in PBS + 0.05% Tween-20 was added to the wells at room temperature for 30 minutes, followed by the addition of 5 μl of His-labeled CD30L (1028-CL-050, R&D Biosystems) to a final assay concentration of 0.2 μg / ml (approximately EC75 concentration) for 20 minutes. Unbound proteins were removed by washing as described above, and the fixed proteins were detected in PBS + 0.5% BSA for 20 minutes using HRP-conjugated anti-his secondary antibody (A7058, Sigma). Unbound proteins were removed by washing as described above, and HRP was detected using TMB reagent. Absorbance measurements at 450 nm were determined using an Epoch plate reader (Biotek, inc.). Measurements were performed in triplicate, and data were analyzed using a GraphPad Prism.

[0346] PD1 / PD-L1 Functional Blockade Assay: PD-L1 inhibition was measured using the PD1-PD-L1 Blockade Bioassay (J1255, Promega). This thawing and use of the assay was based on the co-culture of PD1-expressing Jurkat T cells with the CHO-K1 cell line, which was engineered to contain the luciferase gene under the control of NFAT response elements, and the CHO-K1 cell line was engineered to express human PD-L1 and additional cell surface proteins that can activate T cell receptors in an antigen-independent manner. During co-culture, the PD1 / PD-L1 interaction inhibited TCR response activation, resulting in low levels of luciferase expression. This control was released after inhibition of the PD1 / PD-L1 interaction by the test antibody to allow TCR activation, leading to luciferase expression. Assays were performed according to the manufacturer's protocol to test the antibody's inhibition of PD-L1 at final assay concentrations of 50, 12.5, and 0.5 nM. Emissions were measured using a Biotek Synergy multimode plate reader (Biotek, Inc.). Measurements were performed in triplicate, and the data were analyzed in a GraphPad Prism.

[0347] As described in paragraph 000259, all IgGs were conjugated with the MC-vc-PAB-MMAE linker-payload, and their in vitro cell potency was tested to determine their IC50 value in the Karpas299 cell line (as described in paragraph 000260).

[0348] Figures 11-13 Several schematic diagrams are described, illustrating the design and production of multiple trispecific tetravalent antibodies targeting the first and second distinct epitopes of CD30 and PD-L1.

[0349] Figure 14 Generation and characterization of multiple trispecific tetravalent CD30 / PD-L1 antibodies.

[0350] Figure 15 Cell binding analysis of multiple trispecific tetravalent CD30 / PD-L1 antibodies was performed using FACS.

[0351] Figure 16 Monospecific CD30 (AB201) or PD-L1 (AB204) and CD30 / PD-L1 trispecific tetravalent CD30 / PD-L1 antibodies were conjugated with MC-vc-PAB-MMAE in four drugs (4DAR) for each antibody, and their cellular potency was determined using CD30+ve (CD30-expressing) or PD-L1+ve (PD-L1-expressing) cell lines as described in paragraph 000260. Unlike monospecific ADCs, only the CD30 / PD-L1 trispecific tetravalent ADC showed cellular potency in both cell lines.

[0352] Figure 17 All CD30 / PD-L1 trispecific tetravalent CD30 / PD-L1 antibodies showed comparable PD1 / PD-L1 functional blocking activity to avelumumab, atezolizumab, or BMS936559 (as described in paragraph 000263). III. Example 3: Her2 / Trop2 Trispecific Antibody

[0353] Figures 18-19The activity of trispecific tetravalent ADCs targeting the first and second distinct epitopes of Trop2 and HER2 is demonstrated. As described above, AB606 (a trispecific tetravalent Her2 / Trop2 antibody) and AB103 (a monospecific bivalent Her2 antibody) were conjugated to the MC-vc-PAB-MMAE linker-payload via engineered cysteine ​​residues (HC-A118C and LC-V205C) to generate 4DAR ADCs. These ADCs were tested in several cell lines (A431, HCC1569, and MCF7). The Her2 / Trop2 trispecific tetravalent ADCs showed consistently improved cellular potency compared to Her2 monospecific ADCs. Figures 20-26 Several bispecific or trispecific tetravalent Her2 / Trop2 antibodies (AB607-AB610 and AB612-AB614) were designed and generated, and conjugated with DAR4 to the MC-vc-PAB-MMAE linker-payload. Their cellular efficacy was tested in multiple cell lines expressing both Her2 and Trop2 antigens (SKOV3, JIMT1, and OVCAR3). Some showed superior cellular efficacy compared to AB103, and some showed superior cellular efficacy compared to AB601 (a monospecific ADC), but they consistently demonstrated target-dependent cellular efficacy in all tested cell lines. IV. Example 4: Muc16 / NaPi2b Bispecific Tetravalent Antibody

[0354] Figures 27-32 and Figure 42 The activity of the bispecific tetravalent antibody against Muc16 and NaPi2b was demonstrated.

[0355] Figure 27 A- Figure 27 C illustrates the generation of three bispecific tetravalent ADCs derived from monomethyl ozretamine E (MMAE) and analyzed as described above, wherein the ADCs contain binding sites for NaPi-2b and Muc16. Figure 27 The ADC of A (AB304-ADC; SEQ ID NO: 69 and SEQ ID NO: 70) is in the form of a Fab-biantibody-Fc. It contains a complementary site for binding NaPi-2b at the NW / NE position and a complementary site for binding Muc16 at the SW / SE position. Figure 27 The ADCs of B (AB305-ADC; SEQ ID NO: 71 and SEQ ID NO: 72) also exist in the form of Fab-biantibody-Fc. They contain a complementary site for binding Muc16 at the NW / NE position and a complementary site for binding NaPi-2b at the SW / SE position. Figure 27The C-type ADC (AB309-ADC) is in the form of a biantibody-Fc-Fab. It contains a complementary site for binding NaPi-2b at the NW / NE position and a complementary site for binding Muc16 at the SW / SE position. These ADCs contain cysteine-substituted antibodies and have a 4-DAR.

[0356] Figure 28 It shows Figure 27 The ADC described in the assay represents a 50% inhibitory concentration (IC50) against OVCAR3 cells (an ovarian cancer cell line). Cells used in the assay: NIH:OVCAR-3 (OVCAR3, ATCC# HTB-161™). To determine the 50% inhibitory concentration (IC50) of the ADC... 50 OVCAR3 cells were seeded at 5000 cells / well in 100 μL of cell culture medium in 96-well clear tissue culture plates and incubated overnight at 37ºC with 5% CO2. After overnight incubation, serially diluted (1:3 dilution) ADC samples (50 μL) were added in duplicate to each well, resulting in a final ADC concentration ranging from 1000 ng / mL to 0.05 ng / mL. The plates were then incubated at 37ºC with 5% CO2 for 60–72 h. To measure cell viability, the plates were equilibrated at room temperature for 30 min. 100 μL of CellTiter-Glo luminescence solution (Promega, catalog number G7570) was added to each well, and the plates were incubated on a plate shaker (300 rpm) for 20 min. Luminescence was measured using a BioTek Synergy plate reader. IC50 values ​​were calculated using GraphPad software. AB304-ADC demonstrated the best cell potency among all five ADCs because IC50... 50 The value was 0.03, compared to 0.6 for AB305-ADC or AB309-ADC, and 0.53 for the control monoclonal antibody lifatuzumab. Error bars are derived from replicate datasets of a single experiment.

[0357] Figure 29Compared to the ADC AB301-ADC (anti-Muc16 IgG ADC), the bispecific AB304-ADC (Fab-biantibody-Fc ADC; NaPi-2b NW / NE, Muc16 SW / SE) retained its cytotoxic potency against a subset of the Muc16-KO OVCAR3 cell lines. Here, engineered OVCAR3-AV cells were used to measure the ADC's cytotoxic potency. OVCAR3 cells with MUC-16 knockout (KO) were generated via CRISPR. Cell sorting was performed based on the fluorescence intensity of MUC-16 to enrich MUC-16 KO cells. The initial two rounds of MUC-16 enrichment were performed to generate OVCAR3-AV cells composed of both original OVCAR3 cells and MUC-16 KO cells. The cytotoxicity of the monospecific and bispecific ADCs was tested for dependence on the presence of the MUC-16 protein on the cell surface using these mixed pools of OVCAR3 and MUC-16 KO cells. As expected, in the mixed pool of MUC-16 KO cells, the potency of the monospecific MUC-16-targeting ADC AB302-ADC was significantly reduced, while the potency of the monospecific NaPi-2b-targeting ADC AB301-ADC and the bispecific ADC remained largely unaffected.

[0358] Figure 30The monospecific ADC showed loss of potency against OVCAR3 cells in which the target of the monospecific ADC was knocked out. The bispecific tetravalent ADCs AB304-ADC and AB309-ADC retained potency against these cells. OVCAR3 cells with MUC-16 knockout (KO) and OVCAR3 cells with NaPi-2b KO were generated via CRISPR. The phenotypic KO status (impaired expression of MUC-16 or NaPi-2b protein on the cell surface) of KO cells was validated by FACS analysis using anti-MUC-16 and anti-NaPi-2b antibodies (2000 ng / mL), followed by secondary antibodies (1:1000). To enrich the MUC-16 KO and NaPi-2b KO populations, cells were sorted based on the fluorescence intensity of MUC-16 and NaPi-2b expression, respectively. MUC-16 was enriched three times to generate “MUC-16 KO” cells. Wild-type (WT) and MUC-16 KO cells were incubated with multiple concentrations of ADCs to evaluate cytotoxic efficacy. As expected, the cytotoxic efficacy of AB302-ADC (binding MUC-16) and AB301-ADC (binding NaPi-2b) was significantly reduced (more than 100-fold) when incubated with MUC-16 KO and NaPi-2b KO cells, respectively. Therefore, the cytotoxic efficacy of all bispecific multispecific ADCs is now comparable to that of monospecific ADCs when incubated with their respective KO cells. These data demonstrate that bispecific ADCs can kill cells expressing MUC-16 (with low / no NaPi-2b expression), cells expressing NaPi-2b (with low / no MUC-16 expression), and double-positive cells (expressing both MUC-16 and NaPi-2b). Co-expression of the two targets further enhanced the efficacy of the bispecific ADCs. IC50 values ​​of the ADCs in multiple cell types are shown.

[0359] Figure 31The bispecific tetravalent ADC AB304-ADC demonstrated greater potency against cell populations that were mixtures of cells with constant levels of NaPi-2b and varying levels of Muc16 expression, compared to the monovalent ADCs AB301-ADC (NaPi-2b) and AB302-ADC (MUC-16). To assess the potency of AB304-ADC against cells expressing normal or reduced amounts of MUC-16, the ADC was incubated with mixtures of WT and MUC-16 KO OVCAR3 cells in varying proportions. These conditions mimicked the composition of patient tumors, where cells may have varying levels of MUC-16 expression but constant levels of NaPi-2b expression. However, these experiments do not necessarily address the effects on tumors with varying antigen density levels on cell surfaces. As expected, and as measured by IC50, the cytotoxic potency of ADCs AB301-ADC (binding NaPi-2b) and AB304-ADC (binding NaPi-2b and MUC16) remained similar across all four test conditions, while the potency of AB302-ADC (binding MUC-16) decreased with decreasing proportions of WT cells expressing MUC16 in the cell mixture. AD shows the normalized cell viability (%) of ADCs at multiple concentrations in multiple mixtures of WT and KO cells. E. IC50 values ​​of AB301-ADC, AB302-ADC, and AB304-ADC under four test conditions. IC50 values ​​are derived from replicate experimental groups.

[0360] Figure 32 The bispecific tetravalent ADC AB304-ADC exhibited greater potency compared to a single monospecific ADC (AB301-ADC or AB302-ADC) or a combination thereof (AB301-ADC + AB302-ADC). AB001-ADC is a non-binding ADC without detectable activity. To investigate whether the cytotoxic potency of the bispecific ADCs AB304-ADC and AB309-ADC against OVCAR3 cells expressing normal or reduced levels of MUC16 was due to an additive or synergistic effect of these ADCs binding to their target antigens, the potency of these individual ADCs was compared to that of mixtures of two reference monospecific ADCs binding only NaPi-2b (AB301-ADC) or only MUC-16 (AB302-ADC). In the cell mixtures, 20% of the cells were positive for both target antigens, while 60% of the cells were positive for NaPi-2b only, and 20% of the cells were positive for MUC-16 only. This mixture serves as a model of heterogeneous tumors expressing different levels of two target antigens. Both bispecific ADCs demonstrated superior potency (lower IC50 values) compared to a mixture or combination of two control monospecific ADCs.

[0361] Figures 33A-33B The manufacturability of multiple antibody forms was demonstrated. The Fab-biantibody-Fc and biantibody-Fc-Fab forms exhibited the highest yields. The antibody identified by the hash symbol had the lowest manufacturability. All antibody molecules were expressed in Expi293 cells and purified from the clarified culture supernatant using protein A chromatography. The protein yield after protein A purification was assessed and used to calculate the expression titer (in milligrams of protein per liter of cell culture, as shown in Figure 33). The purified protein was analyzed by size exclusion chromatography (SEC) to assess homogeneity. Nonspecific IgG was used as an internal control in the assay.

[0362] Figures 34A-34C The efficacy of multiple ADCs was demonstrated in an OVCAR3 ovarian xenograft model using female CB.17 SCID mice. Tumor xenografting was initiated using human ovarian cancer cells maintained by serial subcutaneous transplantation in athymic nude mice. On the day of tumor implantation, each test mouse received a 4 mm subcutaneous implant in the right ventral region. 3 OVCAR3 fragments were used to monitor tumor growth until the average size approached 100 to 150 mm. 3 The target range is determined. The tumor is measured in two dimensions using calipers, and its volume is calculated. It can be assumed that 1 mg corresponds to 1 mm². 3 Tumor weight is estimated by the tumor volume. When establishing a palpable tumor, a size of 150 mm is considered. 3 Mice were randomized to each group (8 mice / group; range = 100-150 mm) based on the average tumor volume. 3 ), and then they were treated once intravenously with 15 nmol doses of AB301-ADC, AB302-ADC, AB304-ADC and AB309-ADC (day 1). Figure 34A Plot the mean tumor volume (±sem) over time. Figure 34B Both bispecific ADCs (AB304-ADC and AB309-ADC) demonstrated superior efficacy compared to single-specific ADCs (AB301-ADC and AB302-ADC). No changes in body weight were observed during the study duration. Figure 34C This indicates that all ADCs are safe at this dose. All ADCs have the same drug-to-antibody ratio (DAR) of 4.

[0363] Figure 35 The in vitro validation of different ADCs in the OVCAR3 cell line is shown, and these ADCs are designed for in vivo tumor model testing in the OVCAR3 xenograft model. OVCAR3 cells were exposed to the above... Figures 27-28 The normalized percentage of activity of the following different ADCs prepared and analyzed as described herein: AB001-ADC (non-binding ADC control), AB301-ADC, AB302-ADC, AB301-ADC+AB302-ADC (combination), AB304-ADC, and AB309-ADC or AB312-ADC (DMUC4064A (Genentech®)). Both bispecific ADCs (AB304-ADC and AB309-ADC) performed better than the monospecific ADCs (AB301-ADC, AB302-ADC) or their combinations (AB301-ADC+AB302-ADC) or the clinical comparator (AB312-ADC). The drug-to-antibody ratio (DAR) was the same for all ADCs, at 4.

[0364] Figure 36A-Figure 3 6D demonstrates the efficacy of multiple ADCs at a 40 nmol dose in an OVCAR3 ovarian xenograft model using female CB.17 SCID mice. The study was conducted as shown in Figure 34. All ADCs were administered intravenously at a single 40 nmol dose on day 1. Figure 36A The mean tumor volume (±sem) over time was plotted (Figure 36B). Differences in tumor growth between the different treatment groups observed on day 32 are shown in Figure 36C, and p-values ​​between the different dose groups are shown in Figure 36D. Both bispecific ADCs (AB304-ADC and AB309-ADC) demonstrated superior efficacy compared to monospecific ADCs (AB301-ADC and AB302-ADC) or combinations thereof (AB301-ADC + AB302-ADC) or the clinical comparator (AB312-ADC). The drug-to-antibody ratio (DAR) was the same for all ADCs, at 4.

[0365] Figures 37A-3 7D illustrates the efficacy of different doses of AB309-ADC compared to AB301-ADC. The study was conducted as shown in Figure 34. A single intravenous injection of AB309-ADC at doses of 10 nmol, 20 nmol, or 40 nmol was administered on day 1. Figure 37AThe mean tumor volume (±sem) over time was plotted (Figure 37B) and compared with a single 40 nmol dose of AB301-ADC. A 10 nmol dose of AB309-ADC showed similar efficacy to the 40 nmol dose of AB301-ADC, indicating an approximately 4-fold improvement in efficacy. Both 20 nmol and 40 nmol doses of AB309-ADC showed statistically significant improvements in efficacy compared to the 40 nmol AB301-ADC. The drug-to-antibody ratio (DAR) was the same for all ADCs, at 4.

[0366] Figure 38A-Figure 3 Figure 8D illustrates a comparison of the efficacy of different doses of AB309-ADC versus AB312-ADC. The study was conducted as shown in Figure 34. On day 1, a single intravenous dose of AB309-ADC (10 nmol, 20 nmol, or 40 nmol) or AB312-ADC (20 nmol, 40 nmol, or 80 nmol) was administered. Figure 38A Furthermore, plotting the mean tumor volume (±sem) over time (Figure 38B), the 10 nmol or 20 nmol doses of AB309-ADC showed similar efficacy to the 40 nmol or 80 nmol doses of AB312-ADC, indicating an approximately 4-fold improvement in the efficacy of AB309-ADC. Both the 20 nmol and 40 nmol doses of AB309-ADC showed statistically significant improvements in efficacy compared to the 40 nmol or 80 nmol doses of AB312-ADC. The drug-to-antibody ratio (DAR) was the same for all ADCs, at 4.

[0367] Figure 39 The results showed that no changes in body weight were observed during the study duration, indicating that all ADCs were safe within the tested dose range (10 nmol-80 nmol).

[0368] Figure 40The pharmacokinetic (PK) characteristics of antibodies AB304 and AB309 when administered to cynomolgus monkeys via a single intravenous (IV) (slow bolus) injection were characterized. Non-human primates (NHPs) were administered 3 mg / kg, and blood was collected at specified time intervals for serum chemistry. Serum concentrations of antibodies AB304 and AB309 were determined using a standard ELISA-based bioanalytical method and plotted against time intervals. No changes in body weight associated with either antibody AB304 or AB309 were observed. All animals survived to the end of the study. No clinical observations related to either antibody AB304 or AB309 were observed. In summary, administration of either antibody AB304 or AB309 via intravenous (slow bolus) injection at 3 mg / kg was well tolerated. The mean clearance (Cl) of antibodies AB304 and AB309 was 19.84 mL / day / kg and 13.37 mL / day / kg, respectively. These clearance rates are in the same range as those reported for anti-NaPi-2b antibodies (13.8 mL / day / kg) as described in Clin Cancer Res. 2015;21(22):5139-5150.

[0369] Figure 41 The validation of different ADCs in the OVCAR3 cell line is shown. Normalized viability percentages of OVCAR3 cells exposed to different ADCs (AB304 (quadrivalent bispecific ADC) or AB306 (bivalent bispecific ADC) prepared and analyzed as described in Figure 37 above, except for the following modifications). Cells were incubated with the ADCs for a short time (4 h instead of continuous exposure). After 4 h, the cells were washed and added to growth medium without ADC. The tetravalent bispecific ADC (AB304) showed a 4-fold improvement in potency compared to the monospecific ADC. The drug-to-antibody ratio (DAR) was the same for both ADCs, at 4.

[0370] Figure 42 Exposure to the above text is shown Figures 27-28 The normalized percentage of OVCAR3 cell viability for each of the three different antibody forms (AB301-ADC, AB303-ADC, and AB306-ADC) prepared and analyzed as described herein. Both bispecific ADCs (AB303-ADC and AB306-ADC) performed better than the single-specific ADC (AB301-ADC). V. Example 5: Her2 / Her2 / PD-L1 Bispecific Quadrivalent Antibody

[0371] Figure 43The superior potency of Her2 bicomplementary site AB105-ADC compared to the monospecific AB103-ADC was described. High-copy-number Her2-expressing (SKBr) and low-copy-number Her2-expressing (JIMT1) cell lines were incubated with the ADC for a short time (4 h), and the growth medium consisting of the ADC was removed and replaced with fresh growth medium. Cell viability was measured after 96 h. The Her2 bicomplementary site ADC showed a more significant improvement in potency when using the low-copy-number cell line. VI. Example 6: PD-L1 / PD-L1 / CD30 trispecific tetravalent antibody

[0372] Figure 49 The monospecific ADC showed loss of potency against Karpus 299 cells in which the target of the monospecific ADC was knocked out. The CD30-PD-L1 quadrivalent ADC and AB209-ADC retained potency against these cells. Karpus 299 cells with CD30 knockout (KO) and PD-L1 KO were generated via CRISPR. The phenotypic KO status (impaired expression of CD30 or PD-L1 protein on the cell surface) of KO cells was validated by FACS analysis using anti-CD30 and anti-PD-L1 antibodies. CD30 KO cells, PD-L1 KO cells, or mixed cell populations (CD30-KO:PD-L1-KO / 75:25 ratio) were incubated with multiple concentrations of ADC to assess cytotoxic potency. As expected, the cytotoxic potency of AB201-ADC (binding CD30) and AB204-ADC (binding PD-L1) was significantly reduced when incubated with CD30KO (100-fold) and PD-L1 cells (7-fold), respectively. Therefore, when incubated with their respective KO cells, the cytotoxic potency of the CD30-PD-L1 multispecific ADC is now comparable to that of the single-specific ADCs. In mixed cell populations, the multispecific ADC outperformed both single-specific ADCs. VII. Example 7: Her2 / Her2 / Trop2 trispecific tetravalent antibody

[0373] Figure 51 The in vitro cellular efficacy of Her2-Trop2 bispecific tetravalent ADC (AB612-ADC) or multispecific tetravalent ADC (AB616-618 ADC) in JIMT1 and SKOV3 cells was described.

[0374] Figure 52This study describes the superior potency of Her2-Trop2 multispecific tetravalent ADCs (AB616-ADC and AB618-ADC) in SKOV3 cells compared to both monospecific ADCs (Her2-binding AB103-ADC or Trop2-binding AB601-ADC) and bispecific ADCs (AB605-ADC). The activity of a single complementary site in the multispecific ADCs was demonstrated by adding excess unconjugated antibody. Trop2-17487 complementary site ADC activity was tested in the presence of excess Trop2-17487 + Her2-11053 antibody. Her-10423 complementary site ADC activity was tested in the presence of excess Trop2-17487 + Her2-11053 antibody. Her-11053 complementary site ADC activity was tested in the presence of excess Trop2-17487 + Her2-10423 antibody. Figure 53 The efficacy of Her2-Trop2 multispecific tetravalent ADCs (AB616-ADC and AB618-ADC) in NCI-N87 cells was described as similar or slightly superior to that of monospecific ADCs (Her2-binding AB103-ADC or Trop2-binding AB601-ADC) and bivalent bispecific ADCs (AB605-ADC). Figure 52 As described above, the activity of a single complementary site in a multispecific ADC was demonstrated by adding an excess of unconjugated antibody.

[0375] As used herein, unless otherwise stated, the following meanings apply. The words “can” and “may” are used in a permissive sense (i.e., meaning possible) rather than a mandatory sense (i.e., meaning mandatory). The words “include,” “including,” “includes,” etc., mean including but not limited to. The singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, although other terms and phrases (such as “one or more”) are used for one or more elements, a reference to “a” includes a combination of two or more elements. Thus, the phrase “at least one” includes “a”, “one or more”, and “multiple” is expected to use the term “multiple.” Unless otherwise stated, the term “or” is non-exclusive, i.e., encompasses both “and” and “or.” The term “any” between a modifier and a sequence means that the modifier modifies each member of the sequence. Therefore, for example, the phrase "at least 1, 2, or 3" means "at least 1, at least 2, or at least 3". The term "about" refers to a range of 5% plus or minus the stated value in the context of a particular usage. The term "consistently of" means including the listed elements and other elements that do not substantially affect the essential and novel features of the claimed combination.

[0376] It should be understood that the specification and drawings are not intended to limit the invention to the specific forms disclosed, but rather, the invention covers all modifications, equivalents, and substitutions falling within the spirit and scope of the invention as defined by the appended claims. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in light of this specification. Therefore, this specification and drawings are to be understood only as illustrative and intended to teach those skilled in the art the general manner of practicing the invention. It should be understood that the forms of the invention shown and described herein will be considered as examples of embodiments. Elements and materials may be substituted for those shown and described herein, components and processes may be reversed or omitted, and certain features of the invention may be used independently, all of which are as will be apparent to those skilled in the art after benefiting from this specification. Changes may be made to the elements described herein without departing from the spirit and scope of the invention as set forth in the following claims.

[0377] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent or patent application were explicitly and individually indicated to be incorporated by reference. Table 1: Engineered Antibodies The table shows bispecific antibodies for binding to dual targets (CD30 and PD-L1, HER2 and TROP2) as well as antibodies for binding to different epitopes on the same target (HER2). Elsewhere in this article, in Table 2 entitled “Antibody Sequences”, the amino acid sequences of the molecules listed in the “Antibody Name” column (AB101, AB102, etc.) are provided. Table 2: Antibody sequences: CDR: Underlined or underlined with an asterisk Cysteine ​​residues of thioantibodies: underline or add underline and hash symbol Constant structural domains in AB101 and AB102: italics

Claims

1. An antibody, such as an immunoconjugate, comprising a tetravalent antibody containing one or more payloads conjugated thereto, wherein the antibody comprises a first light chain, a second light chain, a third light chain, and a fourth light chain, as well as a first heavy chain and a second heavy chain, and comprises, from the N-terminus to the C-terminus: a) The first pair of complementary bits contained in the Fab region; b) Fc region; and c) The second pair of complementary bits contained in the Fab region.

2. An antibody, such as an immunoconjugate, comprising a payload conjugated thereto, the antibody comprising a first light chain, a second light chain, a third light chain, and a fourth light chain, and a first heavy chain and a second heavy chain, wherein: a) The first light chain includes a first light chain variable (VL) region and a first light chain constant (CL) region from the N-terminus to the C-terminus; b) The second light chain includes a second VL region and a second CL region from the N-terminus to the C-terminus; c) The third light chain includes a variable (VL) region and a constant (CL) region from the N-terminus to the C-terminus; d) The fourth light chain includes a fourth VL region and a fourth CL region from the N-terminus to the C-terminus; e) The first heavy chain from the N-terminus to the C-terminus comprises: (i) The first heavy chain portion containing the first heavy chain variable (VH) region and the first heavy chain constant 1 (CH1) region; (ii) A first Fc region containing a first heavy chain constant 2 (CH2) region and a first heavy chain constant 3 (CH3) region; (iii) The second heavy chain portion containing the second heavy chain variable (VH) region and the second heavy chain constant 1 (CH1) region; and f) The second heavy chain from the N-terminus to the C-terminus comprises: (i) The third heavy chain portion containing the third heavy chain variable (VH) region and the third heavy chain constant 1 (CH1) region; (ii) A second Fc region containing a second heavy chain constant 2 (CH2) region and a second heavy chain constant 3 (CH3) region; (iii) The fourth heavy chain portion containing the fourth heavy chain variable (VH) region and the fourth heavy chain constant 1 (CH1) region; and in: The first light chain and the first heavy chain portion form a first Fab portion containing a first complementary site; and The second light chain and the second heavy chain portion form a second Fab portion containing a second complementary site; The third light chain and the third heavy chain portion form a third Fab portion containing a third complementary site; and The fourth light chain and the fourth heavy chain portion form a fourth Fab portion containing a fourth complementary site.

3. An antibody, such as an immunoconjugate, comprising a tetravalent antibody containing one or more payloads conjugated thereto, wherein the antibody comprises a first light chain and a second light chain, and a first heavy chain and a second heavy chain, and comprises, from the N-terminus to the C-terminus: a) The first pair of complementary sites contained in the biantibody; b) Fc region; and c) The second pair of complementary bits contained in the Fab region.

4. An antibody, such as an immunoconjugate, comprising a payload conjugated to the antibody, the antibody comprising a first light chain and a second light chain, and a first heavy chain and a second heavy chain, wherein: a) The first light chain includes a first light chain variable (VL) region and a first light chain constant (CL) region from the N-terminus to the C-terminus; b) The second light chain includes a second VL region and a second CL region from the N-terminus to the C-terminus; c) The first heavy chain from the N-terminus to the C-terminus comprises: (i) A first biantibody region containing a first VH region and a third VL region in any order; and (ii) A first Fc region containing a first heavy chain constant 2 (CH2) region and a first heavy chain constant 3 (CH3) region; and (iii) The first heavy chain portion containing the second heavy chain variable (VH) region and the first heavy chain constant 1 (CH1) region; d) The second heavy chain, from the N-terminus to the C-terminus, comprises: (i) A second biantibody region containing a third VH region and a fourth VL region in any order; and (ii) A second Fc region containing a second heavy chain constant 2 (CH2) region and a second heavy chain constant 3 (CH3) region; and (iii) The second heavy chain portion containing the fourth heavy chain variable (VH) region and the second heavy chain constant 1 (CH1) region; in: The first light chain and the first heavy chain portion form a first Fab portion containing a first complementary site; and The second light chain and the second heavy chain portion form a second Fab portion containing a second complementary site; and The first biantibody region and the second biantibody region form a biantibody, wherein the biantibody forms a third complementary site including the third VL region and the third VH region and a fourth complementary site including the fourth VL region and the first VH region.

5. An antibody, such as an immunoconjugate, comprising a tetravalent antibody containing one or more payloads conjugated thereto, wherein the antibody comprises a first light chain, a second light chain, a third light chain, and a fourth light chain, as well as a first heavy chain and a second heavy chain, and comprises, from the N-terminus to the C-terminus: a) The first pair of complementary bits contained in the Fab region; b) The second pair of complementary sites contained in the Fab region; and c) Fc region.

6. An antibody, such as an immunoconjugate, comprising a payload conjugated thereto, the antibody comprising a first light chain, a second light chain, a third light chain, and a fourth light chain, and a first heavy chain and a second heavy chain, wherein: a) The first light chain includes a first light chain variable (VL) region and a first light chain constant (CL) region from the N-terminus to the C-terminus; b) The second light chain includes a second VL region and a second CL region from the N-terminus to the C-terminus; c) The third light chain includes a variable (VL) region and a constant (CL) region from the N-terminus to the C-terminus; d) The fourth light chain includes a fourth VL region and a fourth CL region from the N-terminus to the C-terminus; e) The first heavy chain from the N-terminus to the C-terminus comprises: (i) The first heavy chain portion containing the first heavy chain variable (VH) region and the first heavy chain constant 1 (CH1) region; (ii) The second heavy chain portion containing the second heavy chain variable (VH) region and the second heavy chain constant 1 (CH1) region; and (iii) A first Fc region containing a first heavy chain constant 2 (CH2) region and a first heavy chain constant 3 (CH3) region; f) The second heavy chain from the N-terminus to the C-terminus comprises: (i) The third heavy chain portion containing the third heavy chain variable (VH) region and the third heavy chain constant 1 (CH1) region; (ii) The fourth heavy chain portion containing the fourth heavy chain variable (VH) region and the fourth heavy chain constant 1 (CH1) region; and (iii) A second Fc region containing a second heavy chain constant 2 (CH2) region and a second heavy chain constant 3 (CH3) region; in: The first light chain and the first heavy chain portion form a first Fab portion containing a first complementary site; and The second light chain and the second heavy chain portion form a second Fab portion containing a second complementary site; The third light chain and the third heavy chain portion form a third Fab portion containing a third complementary site; and The fourth light chain and the fourth heavy chain portion form a fourth Fab portion containing a fourth complementary site.

7. An antibody, such as an immunoconjugate, comprising a tetravalent antibody containing one or more payloads conjugated thereto, wherein the antibody comprises a first light chain and a second light chain, and a first heavy chain and a second heavy chain, and comprises, from the N-terminus to the C-terminus: a) The first pair of complementary bits contained in the Fab region; b) The second pair of complementary sites contained in the biantibody; and c) Fc region.

8. An antibody, such as an immunoconjugate, comprising a payload conjugated to the antibody, the antibody comprising a first light chain and a second light chain, and a first heavy chain and a second heavy chain, wherein: a) The first light chain includes a first light chain variable (VL) region and a first light chain constant (CL) region from the N-terminus to the C-terminus; b) The second light chain includes a second VL region and a second CL region from the N-terminus to the C-terminus; c) The first heavy chain from the N-terminus to the C-terminus comprises: (i) The first heavy chain portion containing the first heavy chain variable (VH) region and the first heavy chain constant 1 (CH1) region; (ii) A first biantibody region containing a second VH region and a third VL region in any order; and (iii) A first Fc region containing a first heavy chain constant 2 (CH2) region and a first heavy chain constant 3 (CH3) region; and d) The second heavy chain, from the N-terminus to the C-terminus, comprises: (i) The second heavy chain portion containing the third VH region and the second CH1 region; (ii) A second biantibody region containing a fourth VH region and a fourth VL region in any order; and (iii) A second Fc region containing a second CH2 region and a second CH3 region; in: The first light chain and the first heavy chain portion form a first Fab portion containing a first complementary site; and The second light chain and the second heavy chain portion form a second Fab portion containing a second complementary site; The first biantibody region and the second biantibody region form a biantibody, and the biantibody forms a third complementary site containing the fourth VL region and the second VH region, and a fourth complementary site containing the third VL region and the fourth VH region.

9. The antibody or immunoconjugate according to any one of claims 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site that binds to a first epitope of Her2 and at least one complementary site that binds to a second epitope of Her2.

10. The antibody or immunoconjugate according to any one of claims 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site that binds to CD30 and at least one complementary site that binds to an epitope of PD-L1.

11. The antibody or immunoconjugate according to any one of claims 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site binding to a first epitope of Her2, at least one complementary site binding to PD-L1, and optionally at least one complementary site binding to a second epitope of Her2.

12. The antibody or immunoconjugate according to any one of claims 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site for binding to CD30, at least one complementary site for binding to a first epitope of PD-L1, and at least one complementary site for binding to a second epitope of PD-L1.

13. The antibody or immunoconjugate according to any one of claims 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site binding to Trop2, at least one complementary site binding to a first epitope of Her2, at least one complementary site binding to a second epitope of Her2, and a complementary site binding to PD-L1.

14. The antibody or immunoconjugate according to any one of claims 1 to 8, wherein the antibody or immunoconjugate comprises at least one complementary site binding to Trop2, at least one complementary site binding to a first epitope of Her2, and optionally at least one complementary site binding to a second epitope of Her2.

15. The antibody or immunoconjugate according to any one of claims 1 to 8, wherein the antibody or immunoconjugate is a monospecific antibody, wherein all complementary sites specifically bind to the same epitope.

16. The antibody or immunoconjugate according to any one of claims 1 to 8, wherein the antibody or immunoconjugate is a bispecific antibody, wherein at least one of the complementary sites specifically binds to a first epitope, and at least another of the complementary sites binds to a second distinct epitope.

17. The antibody or immunoconjugate according to claim 15, wherein, Two of the complementary positions specifically bind to a first epitope, and two of the complementary positions specifically bind to a second different epitope; or three of the complementary positions bind to the same first epitope, and one of the complementary positions specifically binds to a second different epitope.

18. The antibody or immunoconjugate of claim 15, wherein the first complementary site and the second complementary site bind to the same first epitope, and the third complementary site and the fourth complementary site bind to the same second epitope; or the first complementary site and the third complementary site bind to the same first epitope, and the second complementary site and the fourth complementary site bind to the same second epitope.

19. The antibody or immunoconjugate of claim 15, wherein two of the complementary sites bind to the same first epitope of Her2, and two of the complementary sites bind to the same second epitope of Her2, for example, the first and second complementary sites bind to the same first epitope and the third and fourth complementary sites bind to the same second epitope; or, the first and third complementary sites bind to the same first epitope, and the second and fourth complementary sites bind to the same second epitope.

20. The antibody or immunoconjugate of claim 18, wherein the first epitope is an epitope bound by trastuzumab, and the second epitope is an epitope bound by pertuzumab.

21. The antibody or immunoconjugate of claim 15, wherein one of the complementary sites binds CD30, one of the complementary sites binds a first epitope of Her2, and both of the complementary sites bind the same second epitope of Her2.

22. The antibody or immunoconjugate according to any one of claims 1 to 8, wherein the antibody or immunoconjugate is a trispecific antibody, wherein at least one of the complementary sites binds to a first epitope, at least one of the complementary sites binds to a second different epitope, and at least one of the complementary sites binds to a third different epitope, for example, two of the complementary sites bind to the same first epitope, one of the complementary sites binds to a second different epitope, and one of the complementary sites binds to a third epitope that is different from the first epitope and the second epitope.

23. The antibody or immunoconjugate of claim 21, wherein the first complementary site and the second complementary site bind to the same epitope; the first complementary site and the third complementary site bind to the same epitope; or the second complementary site and the third complementary site bind to the same epitope.

24. The antibody or immunoconjugate according to any one of claims 1-21, wherein at least two complementary sites bind to different epitopes of the same antigen.

25. The antibody or immunoconjugate according to any one of claims 1-23, wherein two of the complementary sites bind to different epitopes of the same first antigen, and two of the complementary sites bind to different epitopes of the same second antigen.

26. The antibody or immunoconjugate according to claim 21, wherein: The first complementary bit and the second complementary bit are each combined with the table position of CD30; The third complementary bit combines with the first tabletop of PD-L1; and The fourth complementary position is combined with the second epitope of PD-L1.

27. The antibody or immunoconjugate according to claim 21, wherein: The first complementary bit and the second complementary bit are each combined with the tabletop of Trop2; The third complementary position combines with the first epitope of Her2; and The fourth complementary position is combined with the second epitope of Her2.

28. The antibody or immunoconjugate according to claim 1 or 3, wherein the antibody or immunoconjugate is a tetraspecific antibody, wherein each of the complementary sites binds to a different epitope.

29. The antibody or immunoconjugate according to any one of claims 1-27, wherein the first heavy chain and the second heavy chain have different amino acid sequences and are associated by a club-and-mortar structure.

30. The antibody or immunoconjugate of claim 28, wherein the Fc region comprises two distinct heavy chains, and at least one of the two distinct heavy chains comprises an amino acid modification to form complementarity between the two distinct heavy chains, thereby increasing the likelihood of forming a heterodimer of the distinct heavy chains and decreasing the likelihood of forming a homodimer of the same heavy chains.

31. The antibody or immunoconjugate according to any one of claims 1-30, wherein the heavy chain and light chain are produced by cells genetically engineered to produce two heavy chains and one light chain.

32. The antibody or immunoconjugate according to any one of claims 1-30, wherein all light chain variable regions have the same amino acid sequence.

33. The antibody or immunoconjugate according to any one of claims 1-30, wherein the two light chains comprise a light chain variable region having the same amino acid sequence.

34. The antibody or immunoconjugate according to any one of claims 1-30, wherein the first light chain and the second light chain have the same amino acid sequence.

35. The antibody or immunoconjugate according to any one of claims 1-30, wherein the first heavy chain and the second heavy chain have the same amino acid sequence.

36. The antibody or immunoconjugate according to any one of claims 1-35, wherein the antibody comprises: a) A biantibody region optionally attached to the Fc region via spacer 1, and an Fc region attached to the heavy chain portion via spacer 2; b) The heavy chain portion optionally attached to the biantibody region via spacer 3, and the biantibody region optionally attached to the Fc region via spacer 4; c) The Fc region attached to the heavy chain portion via spacer 5; or d) A heavy chain attached to another heavy chain portion via spacer 6.

37. The antibody or immunoconjugate according to claim 36, wherein spacers 1-6 independently comprise between 0 and 50 amino acids.

38. The antibody or immunoconjugate according to claim 37, wherein spacers 1-6 are independently selected from monomers or polymers of the sequence (GGGGS)n (SEQ ID NO: 170), (GGGGA)n (SEQ ID NO: 171), or (GGGGG) (SEQ ID NO: 172), wherein n is any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

39. The antibody or immunoconjugate according to claim 38, wherein spacers 1-6 are independently selected from GGGGSGGGGSEPKSSDKTHT (SEQ ID NO: 173), GGGSGGGSGGGSGGGSGSTG (SEQ ID NO: 174), LEDKTHTKVEPKSS (SEQ ID NO: 175), SGSETPGTSESATPESGGG (SEQ ID NO: 176), and GTTAASGSSGGSSSGA (SEQ ID NO: 177).

40. The antibody or immunoconjugate of claim 38, wherein the antibody or immunoconjugate comprises a biantibody region optionally attached to the Fc region via spacer 1, and an Fc region attached to the heavy chain portion via spacer 2; wherein spacer 1 and spacer 2, if present, are selected from the following pair: 。 41. The antibody or immunoconjugate according to claim 40, wherein spacer 1 and spacer 2, if present, are selected from the following pair: 。 42. The antibody or immunoconjugate of claim 36, wherein the antibody or immunoconjugate comprises (i) a heavy chain portion optionally attached to a biantibody region via spacer 3, and a biantibody region optionally attached to an Fc region via spacer 4, wherein spacer 3 and spacer 4, if present, are selected from the following pair: 。 43. The antibody or immunoconjugate according to claim 42, wherein spacer 3 and spacer 4, if present, are selected from the following pair: 。 44. The antibody or immunoconjugate according to any one of claims 1-43, wherein the payload is conjugated by a reduced disulfide bond.

45. The antibody or immunoconjugate according to any one of claims 1-43, wherein the antibody or immunoconjugate comprises a plurality of payload molecules conjugated to the antibody.

46. ​​The antibody or immunoconjugate according to any one of claims 1-43, wherein the antibody is a thioantibody comprising at least one substituted cysteine ​​residue in the heavy or light chain, wherein the payload is conjugated by the substituted cysteine ​​residue.

47. The antibody or immunoconjugate of claim 46, wherein the thioantibody comprises amino acid substitutions: (a) HC-A118C and LC-V205C; and / or (b) HC-S157C, HC-S239C and HC-V266C (EU numbers) (c) T155C, H285C, R301C, V303C, T307C, G316C, Y436C and L441C (EU numbers).

48. The antibody or immunoconjugate according to any one of claims 1-47, wherein the payload comprises a cytotoxic agent, a radioisotope, a fluorescent moiety, or an enzyme.

49. The antibody or immunoconjugate according to claim 48, wherein the payload comprises a cytotoxic agent selected from: maytansine, DM-1, DM-4, ​​auristatin, monomethylauristatin E, monomethylauristatin F, salicornin, microtubule-lysin, eribulin, novozymin, benzodiazepine, indolino-benzodiazepine, isoquino-benzodiazepine, pyrrolo-benzodiazepine, α-amaminine, trichothecene, camptothecin derivatives (SN-38, essanotecan, belotecone, DXd, topotecan, samotecan), pyroxine, DGN549, CC1065, cazithromycin, N-acetylcazithromycin, enediyne antibiotics, taxanes, doxorubicin derivatives, anthracyclines and stereoisomers, azanofide, and isosteres, analogs, heterodimers, homodimers, or derivatives of the foregoing.

50. The antibody or immunoconjugate according to any one of claims 1-49, wherein the antibody comprises at least one light chain, heavy chain, or complete CDR sequence set of the antibody chain in Appendix 1.

51. The antibody or immunoconjugate according to claim 50, wherein: a) The CDR sequence set for Her2 binding, if available, is selected from the light chain, heavy chain, or full CDR sequence set of AB101 or the CDR sequence set of AB102. b) The set of CDR sequences used for CD30 binding, if available, is selected from the light chain, heavy chain, or full CDR sequence set of AB217; c) The CDR sequence set for PD-L1 binding, if present, is selected from the light chain, heavy chain, or full CDR sequence set of AB202, AB203, AB204, AB218, or AB223; d) The set of CDR sequences for Trop2 binding, if present, is selected from the light chain, heavy chain, or full CDR sequence set of AB601 or AB621.

52. The antibody or immunoconjugate according to any one of claims 50-51, wherein: a) The light chain used for Her2 binding, if present, is selected from AB101, AB102, AB103, AB105, AB106, AB107, AB108, AB113, AB114, AB115, AB116, AB117, AB118, AB119, AB120, AB121, AB122, AB123, AB124, AB125, AB126, AB127, AB128, AB129, AB130, AB131, AB 132, AB133, AB134, AB135, AB136, AB137, AB138, AB139, AB140, AB141, AB142, AB143, AB144, AB607, AB608, A B609, AB610, AB611, AB612, AB613, AB614, AB616, AB617, AB618, AB619, AB620, AB622, AB623, AB624, AB625; b) The light chain used for Trop2 binding, if present, is selected from AB601, AB605, AB606, AB608, AB610, AB613, AB614, and AB626. c) The light chain used for PD-L1 binding, if present, is selected from AB205, AB206, AB207, AB208, AB210, AB211, AB212, AB213, AB216, AB220, AB222; or d) The light chain used for CD30 binding, if present, is selected from AB205, AB206, AB207, AB208, AB209, AB211, AB212, AB213, AB214, AB215, AB216, AB217, AB219, AB220, AB221, and AB222.

53. The antibody or immunoconjugate according to any one of claims 50-52, wherein: a) The heavy chain used for Her2 binding, if present, is selected from AB101, AB102, AB103, AB105, AB106, AB107, AB108, AB113, AB114, AB115, AB116, AB117, AB118, AB119, AB120, AB121, AB122, AB123, AB124, AB125, AB126, AB127, AB128, AB129, AB130, AB131, AB 132, AB133, AB134, AB135, AB136, AB137, AB138, AB139, AB140, AB141, AB142, AB143, AB144, AB607, AB608, A B609, AB610, AB611, AB612, AB613, AB614, AB616, AB617, AB618, AB619, AB620, AB622, AB623, AB624, AB625; b) The heavy chain used for Trop2 binding, if present, is selected from AB601, AB605, AB606, AB608, AB610, AB613, AB614, and AB626. c) The heavy chain used for PD-L1 binding, if present, is selected from AB205, AB206, AB207, AB208, AB210, AB211, AB212, AB213, AB216, AB220, AB222; or d) The heavy chain used for CD30 binding, if present, is selected from AB205, AB206, AB207, AB208, AB209, AB211, AB212, AB213, AB214, AB215, AB216, AB217, AB219, AB220, AB221, and AB222.

54. The antibody or immunoconjugate according to any one of claims 50-53, wherein: a) If present, the biantibody regions for binding different Her2 epitopes are selected from biantibody regions of AB106, AB128, AB129, AB130, AB131, AB132, AB133, AB134, AB135, AB136, AB137, AB138, AB139, AB140, AB141, AB142, AB143, AB144 or AB606; b) If present, the biantibody region for binding to the same Her2 epitope is selected from biantibody regions of AB107, AB108, AB608, and AB613. c) If present, the biantibody regions for binding different PD-L1 epitopes are selected from biantibody regions of AB209, AB214, AB215, AB124, AB125, AB126, AB127, AB219, and AB221. d) If present, the biantibody region for binding to the same CD30 epitope is selected from the biantibody region of AB210; e) The biantibody region for binding PD-L1 and CD30, if present, is selected from the biantibody regions of AB216, AB220, and AB222; or f) A biantibody region for binding to the same Trop2 epitope, if present, selected from AB607, AB609, or AB612. g) The biantibody region for binding Her2 and Trop2, if present, is selected from biantibody regions of AB610, AB614, AB616, AB617, AB618, AB619, AB620, AB622, AB623, AB624, and AB625. h) The biantibody region for binding PD-L1 and Trop2, if present, is selected from the biantibody region of AB611.

55. The antibody or immunoconjugate according to any one of claims 50-54, wherein the antibody or immunoconjugate comprises an Fc region selected from Table 1.

56. The antibody or immunoconjugate according to any one of claims 1-55, wherein the antibody or immunoconjugate comprises one or more antibody chains as shown in Table 1.

57. A method for preparing an immunoconjugate, the method comprising: a) Provide the antibody according to any one of claims 1-56; as well as b) Conjugate one or more payloads to the antibody.

58. The method of claim 57, wherein at least one of the payloads is conjugated to the antibody via a connector.

59. The method of claim 57, wherein at least one of the payloads is conjugated to the antibody via a reduced disulfide bond.

60. The method of claim 57, wherein the antibody is a cysteine-substituted antibody ("thioantibody"), and at least one cytotoxic payload is coupled to the antibody via the substituted cysteine.

61. The method of claim 57, wherein the drug-to-antibody ratio ("DAR") of the immunoconjugate is at least one of 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, and 12.

62. One or more nucleic acid molecules, said one or more nucleic acid molecules encoding the heavy chain and / or light chain of an antibody according to any one of claims 1-61.

63. One or more nucleic acid molecules according to claim 62, wherein the one or more nucleic acid molecules encode one or more antibodies according to any one of claims 1-56.

64. One or more nucleic acid molecules according to claim 61, wherein the one or more nucleic acid molecules encode both the light chain and the heavy chain of the antibody according to any one of claims 1-56.

65. One or more nucleic acid molecules according to any one of claims 62-64, wherein the one or more nucleic acid molecules further comprise one or more expression control sequences, the one or more expression control sequences being operatively linked to one or more nucleotide sequences encoding the one or more light chain variable regions and / or one or more heavy chain variable regions.

66. One or more nucleic acid molecules according to any one of claims 61-64, wherein the one or more nucleic acid molecules are contained in one or more vectors, said vectors being selected, for example, from plasmids, viral vectors (e.g., retroviruses, lentiviruses and adenoviruses, and adeno-associated viruses or herpes simplex viruses), bacterial artificial chromosomes and yeast artificial chromosomes.

67. A cell comprising one or more nucleic acid molecules according to any one of claims 61-65.

68. The cell of claim 56, wherein the cell is selected from mammalian cells (e.g., Chinese hamster ovary (CHO) cells, NSO cells, SP2 / 0 cells, and human embryonic kidney HEK 293 cells or Per.C6 cells); insect cells (e.g., Sf9 cells or high five (BTI-Tn-5B1-4) cells); yeast cells (e.g., Pichiapastoris or Saccharomyces cerevisiae); bacterial cells (e.g., Escherichia coli); and plant cells.

69. A method for preparing an antibody, the method comprising: a) Provide one or more cells according to any one of claims 67-68; b) Expressing an immunoglobulin molecule encoded by the nucleotide sequence in one or more of the cells; and c) Recover the expressed immunoglobulin molecules.

70. The method of claim 69, wherein the antibody is an asymmetric antibody, and the cell comprises one or more nucleic acid molecules encoding a heavy chain comprising a pestle, a heavy chain comprising a mortar, and at least one light chain.

71. The method of claim 70, wherein the cell comprises a nucleotide sequence encoding only one light chain, wherein the light chain is configured to associate with different heavy chain regions to produce two complementary sites.

72. The method of claim 70, wherein the cell comprises a nucleic acid encoding two different light chains, wherein the first light chain is configured to associate with a first heavy chain region to generate a first complementary site that binds to a first epitope, and the second light chain is configured to associate with a second heavy chain region to generate a second complementary site that binds to a second different epitope.

73. The method of claim 70, the method comprising generating different immunoglobulin chains in different cells, recovering the immunoglobulin chains, and contacting the immunoglobulin chains, wherein the immunoglobulin chains are assembled into antibodies.

74. The method of claim 70, further comprising purifying the recovered antibody, for example using protein A or protein G.

75. A pharmaceutical composition comprising an antibody or immunoconjugate according to any one of claims 1-45, and a pharmaceutically acceptable carrier, diluent, or excipient.

76. The pharmaceutical composition according to claim 75, wherein the pharmaceutical composition is formulated for intravenous injection.

77. A method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of claims 64-65.

78. The method of claim 77, wherein the cancer is selected from ovarian cancer, breast cancer, lung cancer, gastric cancer, endometrial cancer, and pancreatic cancer.

79. A method for detecting cells expressing an antigen containing an epitope, the method comprising: a) Contacting the cells with an antibody or immunoconjugate according to any one of claims 1-45 to allow binding between the antibody or immunoconjugate and the cells; and b) detecting the binding of the antibody or immunoconjugate to the cells.

80. The method of claim 79, the method comprising a) contacting the cells with an immunoconjugate comprising an antibody conjugated to a detectable portion; and b) detecting the detectable portion bound to the cells.

81. A method of killing cells, the method comprising contacting cells with an antibody or immunoconjugate according to any one of claims 1-56, wherein one or more complementary sites of the antibody specifically bind to epitopes of the cells.

82. The method of claim 81, wherein the two different complementary sites of the antibody bind to two different epitopes of the cell.

83. The method of claim 82, wherein the epitope is contained in a cell surface antigen.

84. Use of the antibody or immunoconjugate according to any one of claims 1-56 in the manufacture of a pharmaceutical product.

85. Use of the antibody according to any one of claims 1-56 for the production of immunoconjugates.

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