Anti-CD28 antibodies and methods of use thereof

Abstract

The present disclosure relates to anti-CD28 antibodies and antigen-binding fragments thereof that bind to human CD28, compositions comprising same, and uses thereof to retard and / or prevent tumor growth.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to PCT / CN2022 / 085838, filed April 8, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The electronic copy of the sequence listing, created on April 6, 2023, and named 123687_WO003_SL.xml, is 488,908 bytes in size. [Background technology]

[0003] CD28 is a costimulatory signal molecule on T cells and plays a critical role in T cell activation, proliferation, and survival. Activation of CD28 costimulatory signals may enhance antitumor T cell immunity. However, therapies targeting CD28 for systemic T cell activation cause harmful cytokine storm and multiple organ failure (Suntharalingam et al., N Engl J Med. (2006) 355 (10) : 1018-28). Therefore, improved CD28-targeted therapies that avoid the severe autoimmune adverse events associated with systemic CD28 activation are needed. Summary of the Invention

[0004] The present invention provides novel binding molecules (e.g., antibodies) that target tumor-associated antigens (e.g., B7-H3, HER2, TROP2, or PD-L1) and CD28, as well as pharmaceutical compositions comprising one or more of these binding molecules, and the use of these binding molecules and pharmaceutical compositions for the treatment of cancer.Compared to currently available cancer treatments, including antibody therapy, it is believed that the binding molecules of the present disclosure may provide superior clinical responses.

[0005] In some aspects, the disclosure provides antigen binding proteins, such as antibodies or antigen-binding fragments thereof, that comprise a CD28 binding portion, where the CD28 binding portion binds to human CD28 and is cross-reactive with cynomolgus monkey and mouse CD28. In some embodiments, the CD28 binding portion binds to a CD28 epitope that includes amino acid residues 51-122 of SEQ ID NO:1 of human CD28. In certain embodiments, the CD28 epitope includes amino acid residues 51, 52, 54, 55, 98-101, 110-111, 113-114, and 118-122 of SEQ ID NO:1.

[0006] In some embodiments, the present disclosure provides antigen binding proteins, such as antibodies or antigen-binding fragments thereof, that comprise a CD28 binding portion that binds to human CD28, wherein the CD28 binding portion comprises an antibody heavy chain variable domain (V H ) and antibody light chain variable domain (V L ), including V H and V LSEQ ID NOs: 5 to 10, 15, 6, 16, 17 to 19, 24, 6, 25, 26 to 28, 33, 6, 35 to 38, 43, 6, 44, 45, 9 and 46, 33, 51 to 53, 300 and 10, 24, 58, 59, 60, 300 and 61, 66 to 69, 300 and 70, 24, 6, 75, 76, 18 and 28, 24, 58, 81, 82, 27 and 83, 88 to 91, 300 and 10, respectively, and 70, SEQ ID NOs: 24, 96 to 98, 9 and 70, respectively, SEQ ID NOs: 103 to 106, 18 and 83, respectively, SEQ ID NOs: 111, 6, 112, 113, 18 and 114, respectively, SEQ ID NOs: 15, 6, 119, 120, 9 and 121, respectively, SEQ ID NOs: 126, 67, 127, 128, 18 and 129, respectively, SEQ ID NOs: 134, 6, 135, 136, 27 and 83, respectively, SEQ ID NOs: 43, 58, 141, 142, 300 and 143, respectively, SEQ ID NOs: 148, 6, 149, 150, 300 and 83, respectively, SEQ ID NOs: 15, 155, 16, 156, 27 and 70, respectively, or SEQ ID NOs: 161, 6, 162, 163, 300 and 164.

[0007] In some embodiments, the CD28 binding moiety is SEQ ID NO:11 and 12, respectively, SEQ ID NO:20 and 21, respectively, SEQ ID NO:29 and 30, respectively, SEQ ID NO:39 and 40, respectively, SEQ ID NO:47 and 48, respectively, SEQ ID NO:54 and 55, respectively, SEQ ID NO:62 and 63, SEQ ID NO:71 and 72, respectively, SEQ ID NO:77 and 78, respectively, SEQ ID NO:84 and 85, respectively, SEQ ID NO:92 and 93, respectively, SEQ ID NO:99 and 100, respectively, SEQ ID NO:107 and 108, respectively, SEQ ID NO:115 and 116, respectively, SEQ ID NO:122 and 123, respectively, SEQ ID NO:130 and 131, respectively, SEQ ID NO:137 and 138, respectively, SEQ ID NO:144 and 145, respectively, SEQ ID NO:146 and 147, respectively, SEQ ID NO:149 and 150, respectively, SEQ ID NO:151 and 152, respectively, SEQ ID NO:153 and 154, respectively, SEQ ID NO:155 and 156, respectively, SEQ ID NO:157 and 158, respectively, SEQ ID NO:158 and 159, respectively, SEQ ID NO:160 and 161, respectively, SEQ ID NO:162 and 163, respectively, SEQ ID NO:163 and 164, respectively, SEQ ID NO:165 and 166, respectively, SEQ ID NO:167 and 168, respectively, SEQ ID NO:169 and 170, respectively, SEQ ID NO:171 and 172, respectively, SEQ ID NO:173 and 1 51 and 152, SEQ ID NOs: 157 and 158, respectively, SEQ ID NOs: 165 and 166, respectively, SEQ ID NOs: 362 and 363, respectively, SEQ ID NOs: 364 and 365, respectively, SEQ ID NOs: 366 and 367, respectively, SEQ ID NOs: 368 and 369, respectively, SEQ ID NOs: 370 and 371, respectively, SEQ ID NOs: 372 and 373, respectively, SEQ ID NOs: 374 and 375, respectively, SEQ ID NOs: 376 and 12, respectively, SEQ ID NOs: 377 and 378, respectively, SEQ ID NOs: 379 and 380, respectively, SEQ ID NOs: 381 and 12, respectively, SEQ ID NOs: 382 and 12, respectively, SEQ ID NOs: 383 and 12, respectively, SEQ ID NOs: 384 and 12, respectively, SEQ ID NOs: 385 and 12, respectively, or SEQ ID NOs: 386 and 12, respectively. H and V L Includes.

[0008] In some embodiments, the CD28 binding protein comprises a heavy chain (HC) and a light chain (LC) as set forth below: SEQ ID NOs: 13 and 14, respectively; SEQ ID NOs: 22 and 23, respectively; SEQ ID NOs: 31 and 32, respectively; SEQ ID NOs: 41 and 42, respectively; SEQ ID NOs: 49 and 50, respectively; SEQ ID NOs: 56 and 57, respectively; SEQ ID NOs: 64 and 65, respectively; SEQ ID NOs: 73 and 74, respectively; SEQ ID NOs: 79 and 80, respectively; SEQ ID NOs: 86 and 87, respectively; SEQ ID NOs: 94 and 95, respectively; SEQ ID NOs: 101 and 102, respectively; SEQ ID NOs: 109 and 110, respectively; SEQ ID NOs: 117 and 118, respectively; SEQ ID NOs: 124 and 125, respectively; SEQ ID NOs: 141 and 142, respectively; SEQ ID NOs: 132 and 133, respectively; SEQ ID NOs: 139 and 140, respectively; SEQ ID NOs: 146 and 147, respectively; SEQ ID NOs: 153 and 154, respectively; SEQ ID NOs: 159 and 160, respectively; or SEQ ID NOs: 167 and 168, respectively.

[0009] In some aspects, the present disclosure also provides pharmaceutical compositions comprising an antigen binding protein, such as an antibody or antigen-binding fragment thereof described herein, and a pharma- ceutically acceptable carrier, a nucleic acid molecule encoding the antigen binding protein, an expression vector comprising the nucleic acid molecule, and a host cell comprising the vector, wherein the host cell may be a prokaryotic cell or a eukaryotic cell, such as a mammalian cell.

[0010] In some aspects, the disclosure also provides a method of producing an antigen binding protein, such as an antibody or antigen-binding fragment thereof, described herein, comprising culturing host cells under conditions that allow for expression of the antigen binding protein, and isolating the antigen binding protein from the culture.

[0011] In some aspects, the disclosure also provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an antigen binding protein, such as an antibody or antigen binding fragment described herein. In some embodiments, the method further comprises administering to the patient another anti-cancer therapeutic. In further embodiments, the additional anti-cancer therapeutic is a bispecific antibody (bsAb) targeting CD3 and a tumor-associated antigen (TAA). In certain embodiments, the TAA is HER2, B7-H3, or TROP-2. In some embodiments, the additional anti-cancer therapeutic is an immune checkpoint inhibitor, optionally an anti-PD-1, anti-CTLA-4, or anti-PD-L1 antibody.

[0012] Also provided herein are the present antigen binding proteins, such as antibodies or antigen-binding fragments thereof, or pharmaceutical compositions, for use in treating cancer in a patient in need thereof; the use of the present antigen binding proteins, such as antibodies or antigen-binding fragments thereof, for the manufacture of a medicament for treating cancer in a patient in need thereof; and the use of an article of manufacture (e.g., a kit) comprising one or more dosage units of the present antigen binding proteins, such as antibodies or fragments thereof.

[0013] Other features, objects, and advantages of the present invention will be apparent in the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the present invention, is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief description of the drawings]

[0014] [Figure 1] 1 is a panel of graphs showing binding affinity assays of anti-CD28 antibodies to recombinant CD28 (human and mouse) proteins. [Diagram 2] 1 is a graph showing that anti-CD28 IgG binds to human CD3+ T cells. [Diagram 3]FIG. 1 is a panel of graphs showing ligand blocking assays of IgG against human CD28-CD80 (top right and top left graphs) and the CTLA-4 CD80 pair (bottom graph). [Figure 4A] FIG. 13 is a graph showing a T cell proliferation assay demonstrating that IgG does not induce systemic T cell activation compared to the CD28 superagonist TAC2386 (TGN1412). [Figure 4B] FIG. 1 is a graph showing T cell activation costimulation assay proliferation with CellTiter-Glo® (CTG) readout. [Figure 4C] Graph showing T cell activation costimulation assay for IFN-γ release. [Figure 5A] T cell activation and proliferation assay using anti-CD28 test antibodies with OKT3 - proliferation with CTG readout. [Figure 5B] 1 is a graph showing T cell activation and proliferation assays for IL-2 release. [Figure 6] Graph showing binding of anti-HER2 antibodies and bispecific antibodies (BsAbs) to SK-OV3 cells. A table with the plotted values ​​is also shown. [Figure 7] 1 is a graph showing a Jurkat-NFκB luciferase reporter gene assay measuring the NFκB signaling stimulating effect in terms of maximum signal and EC50 values ​​of anti-HER2×CD28 BsAb (TY27566 or TY27807) combined with a fixed concentration of anti-HER2×CD3 BsAb (TY25238) and anti-HER2×CD3 BsAb combined with a fixed concentration of anti-HER2×CD28 BsAb. A table with the plotted values ​​is also shown. [Figure 8] FIG. 1 is a graph showing killing assays of CD28 BsAbs and CD3 BsAbs with the same or different HER2 epitopes on MCF-7 cells and a table with plotted values. [Figure 9] 1 is a panel of graphs showing an assay measuring the concentration-dependent binding activity of anti-TROP2 bispecific antibodies to tumor cell lines. [Figure 10A] Graph showing Jurkat-NFκB luciferase reporter assay measuring NFκB signaling stimulation effect in terms of maximum signal and EC50 values ​​of bispecific antibodies on H292 cells. A table with plotted values ​​is also shown. [Figure 10B] Graph showing Jurkat-NFκB luciferase reporter assay measuring NFκB signaling stimulation effect in terms of maximum signal and EC50 values ​​of bispecific antibodies on H292 cells. A table with plotted values ​​is also shown. [Figure 11] 1 is a panel of flow cytometry plots showing co-expression of PD-L1 and B7H3 in MDA-MB-231 cells. [Figure 12] Graph showing binding assay of B7H3 IgG and B7H3xCD28 bsAb to MDA-MB-231 cells. A table with plotted values ​​is also shown. [Figure 13A] and 13B are graphs showing a single-cell MLR assay to test the activity of B7H3xCD28 bsAb in combination with anti-PD-1 or anti-PD-L1 blocking mAbs on primary human T cell activation as measured by IL-2 secretion (FIG. 13A) and IFN-γ secretion (FIG. 13B). [Figure 14] Graph showing an in vitro assay measuring the tumor killing activity of anti-CD3-based, or anti-CD28-based, HER2-targeted bsAbs, or their combination, against MCF-7 tumor cell line. A table with plotted values ​​is also shown. [Figure 15] Graph showing an in vitro assay measuring the tumor killing activity of anti-CD3-based, or anti-CD28-based, HER2-targeted bsAbs, or their combination, against the EMT6-HER2 tumor cell line. A table with plotted values ​​is also shown. [Figure 16]1 is a panel of graphs showing assays measuring systemic cytokine release of IL-6 and IFN-γ (top left and right graphs, respectively) and the percentage of CD3+ T cells relative to total CD45+ T cells (bottom graph) in WT mice treated with TCE. [Figure 17] In vivo efficacy studies and graphs of HER2xCD3 bsAb and B7H3xCD28 bsAb alone or in combination in a SK-OV3+ PBMC xenograft tumor model are shown. [Figure 18] 1 is a panel of graphs showing in vivo efficacy studies of B7H3xCD28 or HER2xCD28 bsAb in the EMT6-HER2 model. [Figure 19] FIG. 1 is a graph showing ELISA measurements of the masking efficiency of anti-CD28 activating antibodies binding to recombinant human CD28. [Figure 20] FIG. 1 is a graph showing ELISA measurements of the masking efficiency of anti-CD28 activating antibodies binding to recombinant human CD28. [Figure 21] (SEQ ID NOs: 359-361) is a table showing the different binding residues of human and mouse CD28. [Figure 22A] 23 is a graph showing the binding of anti-PD-L1xCD28 bsAbs TY29815 and TY30413 to human CD28. [Figure 22B] Figure 11 is a graph showing the binding of anti-PD-L1 x CD28 bsAbs TY29815, TY30406, TY30410, and TY30413 to mouse CD28. [Figure 23A] 13 is a graph showing binding of anti-HER2xCD28 bsAbs TY27566, TY28652, TY28653, TY28654, TY28655, TY28656, TY28657, TY28658, TY28659, TY28660, and TY28661 to human CD28. [Figure 23B]13 is a graph showing binding of anti-HER2xCD28 bsAbs TY29109, TY29306, TY29307, TY29308, TY29309, TY29310, TY29311, TY29312, TY29313, TY29314, and TY29315 to human CD28. [Figure 24A] 1 is a graph showing binding of anti-B7H3xCD28 bsAbs TY29021, TY30120, TY30121, and TY30123 to human CD28. [Figure 24B] 13 is a graph showing binding of anti-B7H3xCD28 bsAbs TY29021, TY30120, TY30121, and TY30123 to mouse CD28. [Diagram 25] FIG. 13 is a graph showing a mouse T cell binding assay measuring T cell binding efficacy in terms of maximum signal, EC50, and AUC values ​​for anti-B7H3xCD28 bsAbs TY29021, TY30120, TY30121, and TY30123. A table with the plotted values ​​is also shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] I. Definition Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0016] As used herein, the term "about" refers to a normal range of error for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0017] It is to be understood that the aspects and embodiments of the present disclosure described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0018] As used herein, the term "and / or" is intended to mean that phrases such as "A and / or B" include both A and B, A or B, A (single), and B (single). Similarly, as used herein, the term "and / or" is intended to mean that phrases such as "A, B, and / or C" include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0019] The term "antibody" includes various antibody structures, including, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, masked antibodies (e.g., activatable or non-activatable antibodies), multispecific antibodies (e.g., bispecific antibodies, masked bispecific antibodies, etc.), and antibody fragments (e.g., single-chain variable fragments or scFvs), provided that the antibody exhibits the desired biological activity (e.g., the ability to bind a target antigen with the desired specificity and affinity).

[0020] The term "antibody" includes a variety of antibody structures, including, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, masked antibodies (e.g., activatable or non-activatable antibodies), and multispecific antibodies (e.g., bispecific antibodies). The term "antibody" also includes, but is not limited to, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0021] In some embodiments, the term "antibody" refers to an antigen-binding protein (i.e., an immunoglobulin) with a basic four polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each heavy chain contains a variable region (also called a variable domain) at the N-terminus (referred to herein as V H The heavy chain constant region is divided into three domains, C H 1. C H 2 and C H Each light chain comprises a variable region (also called a variable domain) at the N-terminus (herein referred to as V L The light chain constant region consists of one domain, C L It consists of. V L is V H Aligned with C L is the first constant domain of the heavy chain (C H 1) Align with V H and V L together form a single antigen-binding site.

[0022] V H and V Lare further subdivided into complementarity determining regions (CDRs) and framework regions (FRs). CDRs are the most variable in sequence and are involved in antigen recognition. CDRs and FRs are interspersed in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. CDRs also contain "specificity determining residues" or "SDRs", which are residues that contact the antigen. SDRs are contained within regions of CDRs called truncated CDRs or a-CDRs. Representative a-CDRs (a-LCDR1, a-LCDR2, a-LCDR3, a-HCDR1, a-HCDR2, and a-HCDR3) are located at amino acid residues 31-34, 50-55, 89-96 in the light chain, and 31-35, 50-58, and 95-102 in the heavy chain, respectively. Almagro and Fransson, Front Biosci. (2008) 13: 1619-33. Unless otherwise indicated, variable domain residues are numbered herein according to Kabat et al., J Biol Chem. (1977) 252: 6609-16; Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991).

[0023] Table I below provides exemplary CDR definitions according to various algorithms known in the art. [Table 1]

[0024] L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Antibodies are made up of their heavy chains (C HDepending on the amino acid sequence of the constant domain of IgA, antibodies can be assigned to different classes or isotypes. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, each with heavy chains designated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu). The IgG class of antibodies is further divided into four subclasses, IgG ... 1 , IgG 2 , IgG 3 , and IgG 4 can be further classified into:

[0025] The terms "antigen-binding fragment" or "antigen-binding portion," as used interchangeably herein, refer to a portion of an antibody that retains the ability of the antibody to bind to an antigen. Examples of "antigen-binding fragments" of antibodies include, but are not limited to, (i) Fab fragments, V fragments obtained by papain digestion, L , V H , C L and C H1 (ii) a monovalent fragment consisting of the F(ab') domain; 2 (iii) a V fragment of a single arm of an antibody; a bivalent fragment containing two Fab fragments linked by a disulfide bond at the hinge region obtained by pepsin digestion; L and V H (iv) an Fv fragment consisting of the V domains of an antibody fused together; H and V L (v) V L , V H , C L and C H1 A single-chain Fab (scFab) fragment contains a single polypeptide containing the domain.

[0026] The term "masked antibody" refers to an antibody (including a multispecific antibody) or antigen-binding fragment thereof that includes a masking peptide that prevents, inhibits, reduces, prevents, inhibits, or competes with the ability of the antigen-binding domain of the antibody to bind to its target. A masked antibody may be generated by attaching a masking peptide to the antigen-binding domain of the antibody. In some embodiments, a masked antibody or antigen-binding fragment thereof exhibits a first binding affinity to the target when in an inactivated state (e.g., inhibited or masked by the masking peptide) and a second binding affinity to the target when in an activated state (e.g., not inhibited or masked by the masking peptide (e.g., the masking peptide has been cleaved from the antibody)), the second binding affinity being greater than the first binding affinity. A masked antibody may be generated by attaching a masking peptide that includes an activatable moiety (e.g., a cleavable site in a linking unit, or "LU") to the antigen-binding domain of the antibody. In some embodiments, the masked antibody, or masked antigen-binding fragment thereof, is a multispecific antibody that includes a binding domain specific for a T cell surface molecule (e.g., CD28, CD3) and a binding domain specific for a tumor cell surface antigen (e.g., HER2, B7H3, TROP2, etc.). In some embodiments, the masked antibody is bivalent and has a single mask at one of the two binding domains. In some embodiments, the masked antibody is bivalent and has a mask at each of the two binding domains. For example, in the case of a single masked antibody, one of the binding domains of the antibody is masked by a fused or attached masking peptide. In the case of a bispecific antibody, one or both binding domains may be masked by a specific but different masking peptide. In an inactivating bispecific antibody that targets both cancer cells and T cells, the binding sites of both binding domains may be masked to inhibit (or minimize) binding to cancer cells and T cells expressing the antigen. However, in the activated state, the mask is cleaved, allowing antibodies to bind to both tumor antigens and T cell surface molecules (e.g., CD28) within the tumor microenvironment (TME).In this case, the activated bispecific antibody selectively binds to T cells and kills cancer cells expressing the target tumor antigen.

[0027] A "masking peptide" refers to a peptide that inhibits the antigen-binding domain from binding to a target antigen, and typically includes, from the N-terminus to the C-terminus, a masking unit (MU) and a linkage unit (LU). The C-terminus of the masking peptide is usually linked to the N-terminus. H or V L In some embodiments, the masking peptide or portion thereof prevents or inhibits binding of the antigen-binding domain to its target so efficiently that binding of the antigen-binding domain to its target is extremely low and / or below the limit of detection (e.g., binding is not detectable by ELISA or flow cytometry assays). The masked antibodies or polypeptides described herein can be, for example, within LU, between MU and LU, between LU and V H or V L Between or V H and the hinge region of Fc.

[0028] The LU of the masking peptide may include at least one cleavable site. A cleavage site generally includes a cleavable amino acid sequence, e.g., an amino acid sequence that serves as a substrate for an enzyme, and / or a cysteine-cysteine ​​pair that can form a reducible disulfide bond. Thus, when terms such as "cleavage," "cleavable," "cleaved," and the like are used in connection with a cleavage site, these terms include not only enzymatic cleavage, e.g., by a protease, but also disruption of the disulfide bond between the cysteine-cysteine ​​pair by reduction of the disulfide bond, which may result from exposure to a reducing agent. The amino acid sequence of the cleavage site may overlap with or be contained within the MU. A masked antibody or masked polypeptide may include a cleavage site configured to mediate activation of the antibody or polypeptide. For example, if the cleavage site of an activatable antibody is intact (e.g., includes a cysteine-cysteine ​​disulfide bond that is not cleaved and / or reduced by the corresponding enzyme), the masking peptide, or a portion thereof, may interfere with or inhibit binding of the antigen-binding domain to its target. In some embodiments, the LU of the masking peptide does not contain a cleavable site.

[0029] The term "masking efficiency" refers to the efficiency with which a masking peptide inhibits binding of an antigen-binding domain to a target antigen. Masking efficiency can be measured as the difference or ratio between the binding affinity of a masked antibody or masked polypeptide comprising the antigen-binding domain and the binding affinity of an unmasked antibody or unmasked polypeptide comprising the antigen-binding domain (e.g., where the masking peptide has been cleaved from the antibody). For example, masking efficiency can be measured as the EC of a masked antibody binding to a target antigen in an inactivated (e.g., inhibited, masked, and / or uncleaved) state. 50 or K D is the EC of an unmasked antibody that binds to a target antigen in an activated (e.g., uninhibited, unmasked, and / or cleaved) state. 50 or K Dor the EC of the parent antibody (e.g., not linked to a masking peptide) that binds to the target antigen 50 or K D It can be measured by dividing by EC. 50 The value can be measured by ELISA assay or Jurkat NFAT reporter assay, for example, as described in U.S. Patent No. 6,363,950. App. Pub. No. US2021 / 0207126A1. D The value can be measured, for example, using surface plasmon resonance.

[0030] The term "epitope" refers to a portion of an antigen to which an antibody (or an antigen-binding fragment thereof) binds. Epitopes can be formed from both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include a variable number of amino acids in unique spatial conformations. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography, 2D nuclear magnetic resonance, deuterium and hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or any method used in combination with a computational model of the antigen and its complex structure with its bound antibody and its variants (see, for example, Epitop Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)). Once the desired epitope of an antigen is determined, antibodies against that epitope can be generated, for example, using the techniques described herein. Antibody generation and characterization can also elucidate information about the desired epitope. From this information, it is possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to perform cross-competition studies to find antibodies that competitively bind to each other, i.e., the antibodies compete for binding to the antigen. A high-throughput process for "binning" antibodies based on their cross-competition is described in PCT Publication No. WO03 / 48731.

[0031] The term "germline" refers to the nucleotide sequences of antibody genes and gene segments as passed from parents to offspring via reproductive cells. Germline sequences are distinguished from the nucleotide sequences encoding antibodies in mature B cells that have been altered by recombination and hypermutation events during B cell maturation.

[0032] The term "glycosylation site" refers to an amino acid residue that is recognized by a eukaryotic cell as a location for the attachment of a sugar residue. The amino acids to which carbohydrates, such as oligosaccharides, are attached are typically asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. The particular attachment site is typically signaled by a sequence of amino acids, referred to herein as a "glycosylation site sequence." The glycosylation site sequence for N-linked glycosylation is -Asn-X-Ser- or -Asn-X-Thr-, where X can be any conventional amino acid except proline. The terms "N-linked" and "O-linked" refer to chemical groups that serve as attachment sites between a sugar molecule and an amino acid residue. N-linked sugars are attached through an amino group. O-linked sugars are attached through a hydroxyl group. The term "glycan occupancy" refers to the presence of a carbohydrate moiety linked to a glycosylation site (i.e., the glycan site is occupied). When there are at least two potential glycosylation sites on a polypeptide, either zero (0-glycan site occupation), one (1-glycan site occupation), or both (2-glycan site occupation) sites can be occupied by carbohydrate moieties.

[0033] The term "host cell" refers to a cell line that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, e.g., mammalian cultured cells derived from rodents (rat, mouse, guinea pig, or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; human cells (e.g., HEK293F cells, HEK293T cells; or human tissue or hybridoma cells, yeast cells, insect cells (e.g., S2 cells), bacterial cells (e.g., E. coli cells), as well as cells contained within transgenic animals or cultured tissues. This term encompasses not only the particular subject cell, but also the progeny of such cells. Since certain modifications may occur in subsequent generations due to either mutations or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell".

[0034] A "human antibody" is one that possesses the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire, or an amino acid sequence that corresponds to other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0035] The term "humanized antibody" refers to a chimeric antibody that contains amino acid residues derived from human antibody sequences. A humanized antibody may contain some or all of the CDRs from a non-human animal or synthetic antibody, but the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences.

[0036] The term "exemplary antibody" refers to any one of the antibodies described herein. These antibodies can be of any class (e.g., IgA, IgD, IgE, IgG, and IgM). These antibodies can be of any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each of the antibodies identified above can be of any class (e.g., IgA, IgD, IgE, IgG, and IgM). L and V H It includes all five classes of antibodies that have the same amino acid sequence for the region. Furthermore, the IgG class of antibodies includes any subclass (e.g., IgG 1 , IgG 2 , IgG 3 , and IgG 4 ). Thus, each of the above-identified antibodies of the IgG subclass may be L and V H It includes antibodies of all four subclasses that have the same amino acid sequence for that region. The amino acid sequences of the heavy chain constant regions of human antibodies in the five classes as well as the four IgG subclasses are known in the art.

[0037] An "isolated" antibody or binding molecule is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007). a The term "k" refers to the association rate constant of a particular antibody-antigen interaction. d The term "dissociation rate constant" refers to the dissociation rate constant of a particular antibody-antigen interaction.

[0038] "K D The term "equilibrium dissociation constant" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. This is k d vs. k a The ratio of (i.e., k d / k a ) and expressed as molar concentration (M). D is used as a measure of the affinity of binding of an antibody to a binding partner. D The smaller the K, the stronger the antibody binds, or the higher the affinity between the antibody and the antigen. For example, an antibody with a nanomolar (nM) dissociation constant will bind to a particular antigen more strongly than an antibody with a micromolar (μM) dissociation constant. D Values ​​can be determined using methods well established in the art. D One way to determine is by using an ELISA. For example, an ELISA-based assay procedure.

[0039] The term "mammal" refers to any animal species of the mammalian class. Examples of mammals include humans; laboratory animals such as rats, mice, hamsters, rabbits, non-human primates, and guinea pigs; farm animals such as cats, dogs, cows, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, and elephants.

[0040] The terms "prevent" or "prophylaxis" in reference to a particular disease state in a mammal refer to preventing or delaying the onset of the disease or preventing the manifestation of its clinical or subclinical symptoms.

[0041] As used herein, "sequence identity" between two polypeptide sequences refers to the percentage of amino acids that are identical between sequences.The amino acid sequence identity of polypeptides can be determined conventionally using known computer programs such as Bestfit, FASTA, or BLAST (see, for example, Pearson, Methods Enzymol. (1990) 183:63-98; Pearson, Methods Mol.Biol. (2000) 132:185-219; Altschul et al., J.Mol.Biol. (1990) 215:403-10; Altschul et al., Nucleic Acids Res. (1997) 25:3389-3402). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference amino acid sequence, parameters are set such that the percentage of identity is calculated over the entire length of the reference amino acid sequence, allowing a difference in homology of up to 5% of the total number of amino acid residues in the reference sequence. This aforementioned method of determining the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants thereof disclosed herein.

[0042] As used herein, the terms "bind", "bind to", "specifically bind", "specifically bind to" or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or for longer than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding. For example, the masked anti-CD28 antibodies described herein are capable of detecting ECs that bind to different antigens in in vitro assays. 50 Less than 10 percent of EC 50 When an antibody binds to human CD28 at a specific concentration, it is said to selectively bind to human CD28.

[0043] The terms "treat", "treating" or "treatment" refer to a particular disease state in a mammal, and to causing a desired or beneficial effect in a mammal having a disease state. A desired or beneficial effect may include a reduction in the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and / or metastasis, or other effects mediated by immune cell numbers and / or activity, etc.), or preventing or inhibiting further progression of the disease, condition, or disorder. In the context of treating cancer in a mammal, a desired or beneficial effect may include inhibiting further growth or metastasis of cancer cells, killing cancer cells, inhibiting recurrence of cancer, reducing pain associated with cancer, or improving the survival of the mammal. The effect may be either subjective or objective. For example, if the mammal is a human, the human may perceive improved vitality or survival, or reduced pain, as subjective symptoms of improvement or response to treatment. Alternatively, the clinician may perceive a reduction in tumor size or burden based on physical examination, clinical laboratory values, tumor markers, or x-ray findings. Some clinical signs that a clinician may observe regarding a therapeutic response include normalization of laboratory values ​​such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. In addition, a clinician may observe a decrease in detectable tumor markers. Alternatively, other tests, such as ultrasound imaging, nuclear magnetic resonance imaging, and positron emission tomography, may be used to assess objective improvement.

[0044] The term "vector" refers to a nucleic acid molecule capable of transporting a foreign nucleic acid molecule. The foreign nucleic acid molecule is linked to the vector nucleic acid molecule by recombinant techniques such as ligation or recombination. This allows the foreign nucleic acid molecule to be propagated, selected, further manipulated, or expressed in a host cell or organism. The vector can be a plasmid, phage, transposon, cosmid, chromosome, virus, or virion. Some types of vectors can be integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome (e.g., non-episomal mammalian vectors). Other types of vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Another specific type of vector capable of directing the expression of an expressible foreign nucleic acid to which they are operably linked is usually referred to as an "expression vector". Expression vectors generally have control sequences that drive the expression of an expressible foreign nucleic acid. Simpler vectors, known as "transcription vectors", are capable of only transcription but not translation, and they can replicate but not express in target cells. The term "vector" encompasses all types of vectors, regardless of their function. Vectors capable of directing the expression of an expressible nucleic acid to which they are operatively linked are commonly referred to as "expressible vectors." Other examples of "vectors" can include display vectors (e.g., vectors that direct the expression and display of an encoded polypeptide on the surface of a virus or a cell, such as a bacterial cell, yeast cell, insect cell, and / or mammalian cell).

[0045] As used herein, a "subject," "patient," or "individual" may refer to a human or non-human animal. A "non-human animal" may refer to any animal not classified as a human, such as farm animals, livestock, or zoo animals, sport animals, pet animals (e.g., dogs, horses, cats, cows, etc.), and animals used in research. A research animal may refer to, but is not limited to, nematodes, arthropods, vertebrates, mammals, frogs, rodents (e.g., mice or rats), fish (e.g., zebrafish or pufferfish), birds (e.g., chickens), dogs, cats, and non-human primates (e.g., rhesus monkeys, cynomolgus monkeys, chimpanzees, etc.). In some embodiments, the subject, patient, or individual is a human.

[0046] "Effective amount" refers to at least an amount effective at the dosage and for the period of time necessary to achieve one or more desired or indicated effects, including therapeutic or prophylactic results. An effective amount may be provided in one or more administrations. For purposes of this disclosure, an effective amount of an antibody, drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition (e.g., an effective amount when administered as a monotherapy or combination therapy). Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if a desired result can or is achieved when combined with one or more other agents.

[0047] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0048] II. Antibodies Certain aspects of the present disclosure relate to monospecific antibodies (e.g., conventional unmasked monospecific antibodies), multispecific antibodies (e.g., unmasked multispecific antibodies), masked antibodies (e.g., activatable monospecific or multispecific antibodies), antigen-binding fragments thereof, or derivatives of such antibodies.

[0049] A. Fc Region and C H 3. Domain In some embodiments, the antibodies (e.g., multispecific antibodies) described herein comprise one or more antibody constant regions, such as a human heavy chain constant region and / or a human light chain constant region. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from kappa and lambda. In some embodiments, the antibody comprises a human IgG constant region. In some embodiments, the antibody comprises a human IgG 4 In some embodiments, the antibody comprises a human IgG 1 In some such embodiments, the antibody comprises a human IgG heavy chain constant region. 4 Contains the S228P mutation in the constant region.

[0050] Whether effector functions are desirable may depend on the particular treatment method desired for the antibody. In some embodiments, when effector functions are desirable, human IgG 1 Heavy chain constant region or human IgG 3 In some embodiments, an antibody is selected that contains a heavy chain constant region that is human IgG if effector function is undesirable. 4 or IgG 2 In some embodiments, the antibody is selected to comprise a heavy chain constant region of a human IgG1 that contains one or more mutations that reduce effector function. 1 In some embodiments, the antibody comprises an IgG heavy chain constant region comprising an N297A substitution. 1 Contains the heavy chain constant region.

[0051] The multispecific antibodies (including activatable multispecific antibodies) described herein can be any combination of C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C31, C42, C53, C64, C75, C86, C97, C10, C11, C12, C13, C14, C15, C26, C16, C17, C18, C21, C19, C22, C31, C42, C53, C64, C75, C86, C97, C10, C11, C12, C13, C21, C14, C15, C16, C17, C22, C18, C19, C23 H Unless otherwise stated, all amino acid residue numbering herein is based on EU numbering and amino acid substitutions are based on wild-type (or naturally occurring) C domains. H with respect to the wild-type (or naturally occurring) sequence at the corresponding amino acid position of the three-domain sequence. It is understood that the mutations or substitutions described herein are applicable to all IgG subclasses and allotypes. IgG allotypes are described, for example, in Jefferis and Lefranc mAbs (2009) 1:4,1-7, which is incorporated herein by reference in its entirety. In some embodiments, the amino acid mutations or substitutions described herein are made to the wild-type C of IgG1, e.g., IgG1 allotypes G1m, 1(a), 2(x), 3(f), or 17(z). H In some embodiments, the amino acid mutations or substitutions described herein are relative to the IgG 4 Wild-type C H Related to the three domain sequence. For example, one human IgG 1 Wild-type C of allotype (Uniprot number P01857) H The D356K substitution in the 3 domain results in a second human IgG 1 Allotype wild type C H 3 domains, or human IgG 4 Wild-type C H 3 domain. H The three domain mutations are shown in Tables 2 and 3. In some embodiments, the amino acid mutations or substitutions described herein are made in a wild-type Fc region sequence, e.g., an IgG 1 Fc region or IgG 4 Mutations in the Fc region HThe C3 sequences are described in WO 2021 / 148006, the disclosure of which is incorporated herein by reference in its entirety. In the tables below and elsewhere in the specification, H 3 The apostrophe in the mutation annotation is the second C H The residues in the 3 domain are shown. For example, in N390C-S400'C, the S400C mutation is located at the second C H It is located in 3 domains. [Table 2] [Table 3]

[0052] In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises a first C H 3 domain C390 and the second C H Between the three domains of C400, the first C H 3 domain C392 and the second C H Between the three domains C397 or the first C H 3 domain C392 and the second C H In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises an engineered disulfide bond between the C400 of the first C3 domain. H 3 domains, positions 357 and 411 and the second C H Between positions 351 and 370 of the first C3 domain (e.g., E357K:T411K-L351'D:K370'D) or H 3 domains, positions 357 and 364 and the second C H Between positions 351 and 370 of the three domains (e.g., E357K:S364K-L351'D:K370'D), H In some embodiments, the multispecific antibody (e.g., the activatable multispecific antibody) comprises a rearranged salt bridge network compared to the wild-type C3 domain. H Compared to the 3 domains, the first C H Position 356 of the 3rd domain and the second CH Contains an inverted salt bridge between position 439 of the three domains (e.g., D356-K439'). H Multispecific antibodies having three domains may have high yields, good stability (eg, resistance to aggregation and precipitation due to high temperature or freeze-thaw cycles), and potent activity.

[0053] In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises a C 3 -binding domain having one or more engineered residues that promote heterodimer formation as described herein. H The first engineered C H A first polypeptide comprising three domains and a second engineered C H Heteromultimers comprising multiple heterodimers formed with a second polypeptide comprising the three domains are also contemplated herein.

[0054] In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises: i) a first C H The 3 domain contains a cysteine ​​(C) residue at position 390 and a second C H The 3 domain contains a cysteine ​​residue at position 400 or H The 3 domain contains a cysteine ​​residue at position 400 and a second C H ii) the first C3 domain contains a cysteine ​​residue at position 390; or H The 3 domain contains a cysteine ​​residue at position 392 and a second C H The 3 domain contains a cysteine ​​residue at position 397 or H The 3 domain contains a cysteine ​​residue at position 397 and a second C H or iii) the first C3 domain contains a cysteine ​​residue at position 392; H The 3 domain contains a cysteine ​​residue at position 392 and a second C H The 3 domain contains a cysteine ​​residue at position 400 or H The 3 domain contains a cysteine ​​residue at position 400 and a second C HThe 3 domain contains a cysteine ​​residue at position 392; amino acid residue numbering is based on Eu numbering.

[0055] In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises a first C H a first polypeptide comprising a C3 domain and a second polypeptide comprising a C H and a second polypeptide comprising three domains, wherein i) a first C H The C3 domain further contains a positively charged residue at position 357 and a second C H The C3 domain further comprises a negatively charged residue at position 351 or H The C3 domain further contains a negatively charged residue at position 351 and a second C H ii) the first C3 domain further comprises a positively charged residue at position 357; or H The C3 domain further contains a positively charged residue at position 411 and a second C H The 3 domain further comprises a negatively charged residue at position 370 or H The 3 domain further contains a negatively charged residue at position 370 and a second C H or iii) the first C3 domain further comprises a positively charged residue at position 411; H The C3 domain further contains a positively charged residue at position 364 and a second C H The 3 domain further comprises a negatively charged residue at position 370 or H The 3 domain further contains a negatively charged residue at position 370 and a second C H The 3 domains further comprise a positively charged residue at position 364; or a combination of i) and ii), or a combination of i) and iii), where the amino acid residue numbering is based on Eu numbering.

[0056] In some embodiments, the first C H The C3 domain further contains a positively charged residue at position 356 and a second C H The C3 domain further comprises a negatively charged residue at position 439 or HThe C3 domain further contains a negatively charged residue at position 439 and a second C H The 3 domain further contains a positively charged residue at position 356, and the amino acid residue numbering is based on Eu numbering.

[0057] In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises a first C H a first polypeptide comprising a C3 domain and a second polypeptide comprising a C H and a second polypeptide comprising three domains, wherein i) a first C H The 3 domain contains a cysteine ​​(C) residue at position 390 and a second C H The 3 domain contains a cysteine ​​residue at position 400 or H The 3 domain contains a cysteine ​​residue at position 400 and a second C H ii) the first C3 domain contains a cysteine ​​residue at position 390; or H The 3 domain contains a cysteine ​​residue at position 392 and a second C H The 3 domain contains a cysteine ​​residue at position 397 or H The 3 domain contains a cysteine ​​residue at position 397 and a second C H or iii) the first C3 domain contains a cysteine ​​residue at position 392; H The 3 domain contains a cysteine ​​residue at position 392 and a second C H The 3 domain contains a cysteine ​​residue at position 400 or H The 3 domain contains a cysteine ​​residue at position 400 and a second C H The three domains contain a cysteine ​​residue at position 392; H The C3 domain further contains a positively charged residue at position 357 and a second C H The C3 domain further comprises a negatively charged residue at position 351 or H The C3 domain further contains a negatively charged residue at position 351 and a second C H or b) the first C3 domain further comprises a positively charged residue at position 357; H The C3 domain further contains a positively charged residue at position 411 and a second CH The 3 domain further comprises a negatively charged residue at position 370 or H The 3 domain further contains a negatively charged residue at position 370 and a second C H or c) the first C3 domain further comprises a positively charged residue at position 411; H The C3 domain further contains a positively charged residue at position 364 and a second C H The 3 domain further comprises a negatively charged residue at position 370 or H The 3 domain further contains a negatively charged residue at position 370 and a second C H The 3 domains further comprise a positively charged residue at position 364; or a combination of a) and b), or a combination of a) and c), where the amino acid residue numbering is based on Eu numbering.

[0058] In some embodiments, the first C H The C3 domain further contains a positively charged residue at position 356 and a second C H The C3 domain further comprises a negatively charged residue at position 439 or H The C3 domain further contains a negatively charged residue at position 439 and a second C H The 3 domain further contains a positively charged residue at position 356, and the amino acid residue numbering is based on Eu numbering.

[0059] C H The C3 domain may be derived from any naturally occurring immunoglobulin molecule. H 3 domains, IgG 1 molecule, IgG 2 molecule, IgG 3 molecule, or IgG 4 In some embodiments, the C H The three domains are human C H In some embodiments, the C H 3 domains, human IgG 1 It comes from the molecule.

[0060] In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises a first C H a first polypeptide comprising a C3 domain, and a second polypeptide comprising a C H a second polypeptide comprising three domains, wherein: i) a first C H The 3 domain contains a N390C substitution and a second C H The 3 domain contains an S400C substitution or the first C H The 3 domain contains an S400C substitution and a second C H ii) the first C3 domain contains a N390C substitution; or H The 3 domain contains a K392C substitution and the second C H The first C3 domain contains a V397C substitution or H The 3 domain contains a V397C substitution and the second C H or iii) the first C3 domain contains a K392C substitution; H The 3 domain contains a K392C substitution and the second C H The 3 domain contains an S400C substitution or the first C H The 3 domain contains an S400C substitution and a second C H The 3 domain contains a K392C substitution.

[0061] In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises a first C H a first polypeptide comprising a C3 domain, and a second polypeptide comprising a C H a second polypeptide comprising three domains, wherein: i) a first C H The third domain contains the E357K and T411K substitutions, and the second C H The first C3 domain contains the L351D and K370D substitutions or H The third domain contains the L351D and K370D substitutions, and the second C H ii) the first C3 domain contains E357K and T411K substitutions; or H The third domain contains the E357K and S364K substitutions, and the second C HThe first C3 domain contains the L351D and K370D substitutions or H The third domain contains the L351D and K370D substitutions, and the second C H or iii) the first C3 domain contains E357K and S364K substitutions; H The C3 domain contains D356K, E357K, and S364K substitutions, and the second C H The first C3 domain contains L351D, K370D, and K439D substitutions, or H The C3 domain contains L351D, K370D, and K439D substitutions, and the second C H The 3 domain contains D356K, E357K, and S364K substitutions.

[0062] In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises a first C H a first polypeptide comprising a C3 domain, and a second polypeptide comprising a C H a second polypeptide comprising a C3 domain, H The 3 domain contains E357K, S364K and N390C substitutions, and the second C H The 3 domain contains the L351D, K370D and S400C substitutions or the first C H The 3 domain contains L351D, K370D and S400C substitutions, and the second C H The 3 domain contains the E357K, S364K and N390C substitutions.

[0063] In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises a first C H a first polypeptide comprising a C3 domain, and a second polypeptide comprising a C H a second polypeptide comprising a C3 domain, H The 3 domain contains E357K, S364K and S400C substitutions, and the second C H The first C3 domain contains the substitutions L351D, K370D and N390C, or H The third domain contains the L351D, K370D and N390C substitutions, and the second C HThe 3 domain contains the E357K, S364K and S400C substitutions.

[0064] In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises a first C H a first polypeptide comprising a C3 domain, and a second polypeptide comprising a C H a second polypeptide comprising a C3 domain, H The 3 domain contains D356K, E357K, S364K and S400C substitutions, and the second C H The first C3 domain contains the substitutions L351D, K370D, N390C and K439D, or H The third domain contains L351D, K370D, N390C and K439D substitutions, and the second C H The 3 domain contains D356K, E357K, S364K and S400C substitutions.

[0065] In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises a first C H a first polypeptide comprising a C3 domain, and a second polypeptide comprising a C H a second polypeptide comprising a first C3 domain; H The 3 domain contains D356K, E357K, S364K and N390C substitutions, and the second C H The 3 domain contains L351D, K370D, K439D and S400C substitutions, or the first C H The 3 domain contains L351D, K370D, K439D and S400C substitutions, and the second C H The 3 domain contains D356K, E357K, S364K and N390C substitutions.

[0066] In some embodiments, a multispecific antibody (e.g., an activatable multispecific antibody) comprises an engineered C H The Fc region includes, but is not limited to, an IgG Fc region that includes three domains. 1 , IgG 2 , IgG 3 , and IgG 4The antibody may be derived from any suitable Fc subclass, including any suitable Fc subclass.

[0067] B. Cysteine ​​Mutations In some embodiments, a multispecific antibody described herein (e.g., an activatable multispecific antibody described herein) comprises a first C H a first polypeptide comprising a C3 domain, and a second polypeptide comprising a C H a second polypeptide comprising a first C3 domain; H The C3 domain contains a first engineered cysteine ​​residue and a second C H The 3 domain includes a second engineered cysteine ​​residue, and the first engineered cysteine ​​residue and the second cysteine ​​residue form a disulfide bond.

[0068] In some embodiments, the first C H The third domain contains a C at position 390 and a second C H 3 domains contain a C at position 400 or the first C H 3 domains contain a C at position 400 and a second C H In some embodiments, the first C H The 3 domain contains a N390C substitution and a second C H The 3 domain contains an S400C substitution or the first C H The 3 domain contains an S400C substitution and a second C H The 3 domain contains the N390C substitution.

[0069] In some embodiments, the first C H The third domain contains a C at position 392 and a second C H 3 domain contains a C at position 397 or the first C H The third domain contains a C at position 397 and a second C H In some embodiments, the first C H The 3 domain contains a K392C substitution and the second C H The first C3 domain contains a V397C substitution or HThe 3 domain contains a V397C substitution and the second C H The 3 domain contains a K392C substitution.

[0070] In some embodiments, the first C H The third domain contains a C at position 392 and a second C H 3 domains contain a C at position 400 or the first C H 3 domains contain a C at position 400 and a second C H In some embodiments, the first C H The 3 domain contains a K392C substitution and the second C H The 3 domain contains an S400C substitution or the first C H The 3 domain contains an S400C substitution and a second C H The 3 domain contains a K392C substitution.

[0071] C. Salt bridge mutation In some embodiments, a multispecific antibody described herein (e.g., an activatable multispecific antibody described herein) comprises a first C H A first polypeptide comprising three domains and a second polypeptide comprising three domains. H a second polypeptide comprising a first C3 domain; H The 3 domain contains engineered positively charged residues, and the second C H The 3 domain contains an engineered negatively charged residue, an engineered positively charged residue and an engineered negatively charged residue form a salt bridge. The engineered salt bridge is H Introducing new salt bridges between the three domains, rearranging the salt bridge network between two or more amino acid residues, or H For the C3 domain, the charge of the amino acid residues forming the salt bridge may be reversed (i.e., the salt bridge is "flipped"). In some embodiments, the engineered positively charged residues are H In some embodiments, the engineered negatively charged residues are replaced with positively charged residues in the wild-type C3 domain. H The positively charged residues in the C3 domain are replaced with negatively charged residues. The rearranged and inverted salt bridges are engineeredH This may result in a change in the isoelectric point (PI) of the heterodimers and homodimers containing the three domains, thereby allowing for better separation of the heterodimers from the homodimers during the purification process.

[0072] In some embodiments, the first CH3 domain comprises a positively charged residue at position 357 and the second C H The 3 domain contains a negatively charged residue at position 351 or H The 3 domain contains a negatively charged residue at position 351 and a second C H In some embodiments, the first C3 domain contains a positively charged residue at position 357. H The 3 domain contains a K at position 357 and a second C H The 3 domain contains a D at position 351 or the first C H The third domain contains a D at position 351 and a second C H In some embodiments, the first C3 domain contains a K at position 357. H The 3 domain contains a K at position 357 and a second C H The 3 domain contains either an E at position 351 or H The third domain contains an E at position 351 and a second C H In some embodiments, the first C3 domain contains a K at position 357. H The third domain contains an R at position 357 and a second C H The 3 domain contains a D at position 351 or the first C H The third domain contains a D at position 351 and a second C H In some embodiments, the first C3 domain contains an R at position 357. H The third domain contains an R at position 357 and a second C H The 3 domain contains either an E at position 351 or H The third domain contains an E at position 351 and a second C H In some embodiments, the first C3 domain contains an R at position 357. H The 3 domain contains an E357K substitution and a second C H The first C3 domain contains a L351D substitution orH The 3 domain contains a L351D substitution and a second C H The 3 domain contains the E357K substitution.

[0073] In some embodiments, the first C H The 3 domain contains a positively charged residue at position 411 and a second C H The 3 domain contains a negatively charged residue at position 370 or H The 3 domain contains a negatively charged residue at position 370 and a second C H In some embodiments, the first C3 domain contains a positively charged residue at position 411. H The 3 domain contains a K at position 411 and a second C H The 3 domain contains a D at position 370 or the first C H The third domain contains a D at position 370 and a second C H In some embodiments, the first C3 domain contains a K at position 411. H The 3 domain contains a K at position 411 and a second C H The 3 domain contains either an E at position 370 or H The third domain contains an E at position 370 and a second C H In some embodiments, the first C3 domain contains a K at position 411. H The third domain contains an R at position 411 and a second C H The 3 domain contains a D at position 370 or the first C H The third domain contains a D at position 370 and a second C H In some embodiments, the first C3 domain contains an R at position 411. H The third domain contains an R at position 411 and a second C H The 3 domain contains either an E at position 370 or H The third domain contains an E at position 370 and a second C H In some embodiments, the first C3 domain contains an R at position 411. H The 3 domain contains a T411K substitution and a second C H The first C3 domain contains a K370D substitution or HThe 3 domain contains a K370D substitution and a second C H The 3 domain contains a T411K substitution.

[0074] In some embodiments, the first C H The 3 domain contains a positively charged residue at position 364 and a second C H The 3 domain contains a negatively charged residue at position 370 or H The 3 domain contains a negatively charged residue at position 370 and a second C H In some embodiments, the first C3 domain contains a positively charged residue at position 364. H The 3 domain contains a K at position 364 and a second C H The 3 domain contains a D at position 370 or the first C H The third domain contains a D at position 370 and a second C H In some embodiments, the first C3 domain contains a K at position 364. H The 3 domain contains a K at position 364 and a second C H The 3 domain contains either an E at position 370 or H The third domain contains an E at position 370 and a second C H In some embodiments, the first C3 domain contains a K at position 364. H The third domain contains an R at position 364 and a second C H The 3 domain contains a D at position 370 or the first C H The third domain contains a D at position 370 and a second C H In some embodiments, the first C3 domain contains an R at position 364. H The third domain contains an R at position 364 and a second C H The 3 domain contains either an E at position 370 or H The third domain contains an E at position 370 and a second C H In some embodiments, the first C3 domain contains an R at position 364. H The 3 domain contains a S364K substitution and a second C H The first C3 domain contains a K370D substitution or H The 3 domain contains a K370D substitution and a second CH The 3 domain contains the S364K substitution.

[0075] In some embodiments, the first C H The 3 domain contains a positively charged residue at position 356 and a second C H The 3 domain contains a negatively charged residue at position 439 or H The 3 domain contains a negatively charged residue at position 439 and a second C H In some embodiments, the first C3 domain contains a positively charged residue at position 356. H The 3 domain contains a K at position 356 and a second C H 3 domain contains D at position 439 or the first C H The third domain contains a D at position 439 and a second C H In some embodiments, the first C3 domain contains a K at position 356. H The 3 domain contains a K at position 356 and a second C H 3 domain contains an E at position 439 or H The third domain contains an E at position 439 and a second C H In some embodiments, the first C3 domain contains a K at position 356. H The third domain contains an R at position 356 and a second C H 3 domain contains D at position 439 or the first C H The third domain contains a D at position 439 and a second C H In some embodiments, the first C3 domain contains an R at position 356. H The third domain contains an R at position 356 and a second C H 3 domain contains an E at position 439 or H The third domain contains an E at position 439 and a second C H In some embodiments, the first C3 domain contains an R at position 356. H The 3 domain contains a D356K substitution and a second C H The first C3 domain contains a K439D substitution or H The 3 domain contains a K439D substitution and the second C HThe 3 domain contains a D356K substitution.

[0076] Any of the engineered salt bridges described herein may be combined with one another. In some embodiments, the first C H The 3 domain contains a positively charged residue at position 357, a positively charged residue at position 411, and a second C H The 3 domain contains a negatively charged residue at position 351 and a negatively charged residue at position 370, or H The 3 domain contains a negatively charged residue at position 351, a negatively charged residue at position 370, and a second C H The C3 domain contains a positively charged residue at position 357 and a positively charged residue at position 411. H The third domain contains the E357K and T411K substitutions, and the second C H The first C3 domain contains the L351D and K370D substitutions or H The third domain contains the L351D and K370D substitutions, and the second C H The 3 domain contains the E357K and T411K substitutions.

[0077] In some embodiments, the first C H The 3 domain contains a positively charged residue at position 357, a positively charged residue at position 364, and a second C H The 3 domain contains a negatively charged residue at position 351 and a negatively charged residue at position 370, or H The 3 domain contains a negatively charged residue at position 351, a negatively charged residue at position 370, and a second C H The C3 domain contains a positively charged residue at position 357 and a positively charged residue at position 364. H The third domain contains the E357K and S364K substitutions, and the second C H The first C3 domain contains the L351D and K370D substitutions or H The third domain contains the L351D and K370D substitutions, and the second C H The 3 domain contains the E357K and S364K substitutions.

[0078] In some embodiments, the first C HThe third domain contains a positively charged residue at position 356, a positively charged residue at position 357, and a positively charged residue at position 364, and a second C H The first C3 domain contains a negatively charged residue at position 351, a negatively charged residue at position 370, and a negatively charged residue at position 439, or H The 3 domain contains a negatively charged residue at position 351, a negatively charged residue at position 370, and a negatively charged residue at position 439, and a second C H The C3 domain comprises a positively charged residue at position 356, a positively charged residue at position 357, and a positively charged residue at position 364. H The C3 domain contains D356K, E357K, and S364K substitutions, and the second C H The first C3 domain contains L351D, K370D, and K439D substitutions, or H The C3 domain contains L351D, K370D, and K439D substitutions, and the second C H The 3 domain contains D356K, E357K, and S364K substitutions.

[0079] D. Other mutations C as described herein H The three domains or Fc region may further comprise engineered disulfide bonds and / or salt bridges as listed in Table B below. [Table 4]

[0080] In some embodiments, the first C H The 3 domain further contains a C at position 392 and a second C H 3 domain contains a C at position 399 or the first C H The third domain contains a C at position 399 and a second C H In some embodiments, the first C H The 3 domain further contains a K392C substitution, and a second C H The first C3 domain further comprises a D399C substitution or H The C3 domain further comprises a D399C substitution, HThe 3 domain further contains a K392C substitution.

[0081] In some embodiments, the first C H The 3 domain further contains a C at position 394 and a second C H The 3 domain contains a C at position 354 or the first C H The third domain contains a C at position 354 and a second C H In some embodiments, the first C H The 3 domain further contains a Y394C substitution, H The first C3 domain further comprises a S354C substitution or H The 3 domain further contains a S354C substitution, and a second C H The 3 domain further contains a Y394C substitution.

[0082] In some embodiments, the first C H The 3 domain further contains a C at position 356 and a second C H 3 domain contains a C at position 349 or the first C H The third domain contains a C at position 349 and a second C H In some embodiments, the first C H The C3 domain further comprises a D356C substitution, H The first C3 domain further comprises a Y349C substitution or H The 3 domain further comprises a Y349C substitution, H The 3 domain further contains a D356C substitution.

[0083] In some embodiments, the first C H The C3 domain further contains K392D and K409D substitutions, H The first C3 domain further comprises D356K and D399K substitutions, or H The C3 domain further contains D356K and D399K substitutions, and the second C H The 3 domain further contains K392D and K409D substitutions.

[0084] In some embodiments, the first C H The C3 domain further contains L368D and K370S substitutions, and the second C H The first C3 domain further comprises E357Q and S364K substitutions, or H The C3 domain further contains E357Q and S364K substitutions, and the second C H The 3 domain further contains the L368D and K370S substitutions.

[0085] In some embodiments, the first C H The C3 domain further contains L351K and T366K substitutions, and the second C H The first C3 domain further comprises L351D and L368E substitutions, or H The C3 domain further comprises L351D and L368E substitutions, and the second C H The 3 domain further contains the L351K and T366K substitutions.

[0086] In some embodiments, the first C H The C3 domain further comprises P395K, P396K, and V397K substitutions, and the second C H The first C3 domain contains T394D, P395D, and P396D substitutions, or H The C3 domain further comprises T394D, P395D, and P396D substitutions, and the second C H The three domains further contain P395K, P396K, and V397K substitutions.

[0087] In some embodiments, the first C H The C3 domain further comprises F405E, Y407E, and K409E substitutions, and the second C H The first C3 domain contains F405K and Y407K substitutions, or H The C3 domain further contains F405K and Y407K substitutions, and the second C H The 3 domain further contains F405E, Y407E, and K409E substitutions.

[0088] In some embodiments, the first C H The C3 domain further contains T336S, L368A, and Y407V substitutions, and the second C H The first C3 domain further comprises a T366W substitution or H The C3 domain further comprises a T366W substitution, H The three domains further contain the following substitutions: T336S, L368A, and Y407V.

[0089] In some embodiments, the first C H The C3 domain contains the L368V and Y407V substitutions, and the second C H The first C3 domain contains a T366W substitution or H The 3 domain contains a T366W substitution and a second C H Domain 3 contains the L368V and Y407V substitutions.

[0090] III. CD28 binding molecules The present invention provides isolated binding molecules that bind to human CD28, including anti-CD28 antibodies and anti-CD28 antigen-binding fragments thereof. In some embodiments, the binding molecules include antibodies described with reference to epitope binding, and complementary determining regions (CDRs), variable regions (Vs), and fusion proteins. L , V H ), as well as IgG (e.g., IgG 4 ) Antibodies described with reference to specific amino acid sequences of their light and heavy chains.

[0091] In some embodiments, the antibody or antigen-binding fragment binds to one or more amino acid residues within amino acid residues 34-108 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment binds to one or more amino acid residues within amino acid residues 51-122 of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment binds to one or more amino acid residues selected from the group consisting of amino acid residues 51, 52, 54, 55, 98-101, 110-111, 113-114, and 118-122 of SEQ ID NO:1. Methods for measuring the ability of an antibody or antigen-binding fragment to bind to a target antigen may be performed using any method known in the art, including, for example, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, and the like. In some embodiments, the ability of an antibody or antigen-binding fragment to bind to a target antigen is measured by ELISA or Octet® RED96 (see, e.g., Example 3 below).

[0092] In some embodiments, the antibody or antigen-binding fragment has a K of about 500 nM or less. D (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.). In some embodiments, the antibody or antigen-binding fragment binds to human CD28 with a K of about 100 nM or less. D In some embodiments, the antibody or antigen-binding fragment binds to human CD28 with a K of about 50 nM or less. D and binds to human CD28. The K D Methods for measuring K may be performed using any method known in the art, including, for example, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, and the like. In some embodiments, KD is measured by an Octet® RED96 system (see, for example, Example 3 below).

[0093] A. Anti-CD28 antibody In some embodiments, the disclosure provides isolated monoclonal antibodies that bind to human CD28 at an epitope within amino acid residues 33-37, 80-83, 92-96, and 100-104 of SEQ ID NO: 1. In certain embodiments, the disclosure provides isolated antibodies that bind to human CD28 at an epitope represented by amino acid residues 33, 34, 36 and 37, 80-83, 92 and 93, 95 and 96, and 100-104 of SEQ ID NO: 1. In some embodiments, the antibodies have a K of 10 nM or less as measured by Octet® RED96 Systems. D In certain embodiments, in addition to binding to a human epitope, the antibodies disclosed herein are cross-reactive (exhibit cross-species binding properties) with at least one non-human species selected from the list consisting of cynomolgus monkey, mouse, rat, and dog. In certain embodiments, the antibodies disclosed herein have the advantage of cross-species binding to mouse, human, and monkey, while the benchmark controls TAC2386 and TAC2387 disclosed herein do not have this range of cross-species cross-reactivity. In certain embodiments, the benchmark controls TAC2386 and TAC2387 bind to a human epitope but not to a mouse epitope (see Table 7 herein). The species cross-reactivity of the antibodies disclosed herein also provides the added advantage that mice can be used to simulate the safety, activity, and function of the antibodies. Thus, animal modeling is easier with the antibodies disclosed herein compared to TAC2386 and TAC2387 disclosed herein.

[0094] In certain embodiments, the isolated anti-CD28 monoclonal antibody comprises an HCDR1 of SEQ ID NO:5, an HCDR2 of SEQ ID NO:6, and an HCDR3 of SEQ ID NO:7, and an LCDR1 of SEQ ID NO:8, an LCDR2 of SEQ ID NO:9, and an LCDR3 of SEQ ID NO:10. In certain embodiments, the isolated monoclonal antibody comprises a heavy chain variable region of SEQ ID NO:11 and a light chain variable region of SEQ ID NO:12. In certain embodiments, the isolated monoclonal antibody comprises a heavy chain of SEQ ID NO:13 and a light chain of SEQ ID NO:14.

[0095] The CD28 antibodies described herein can be of any class, such as IgG, IgM, IgE, IgA, or IgD. 1 , IgG 2 , IgG 3 , or IgG 4 Preferably, the anti-CD28 antibody is of the IgG class, such as V subclass. Anti-CD28 antibodies can be converted from one class or subclass to another using methods known in the art. An exemplary method for producing an antibody of a desired class or subclass is to isolate nucleic acid encoding an anti-CD28 antibody heavy chain and nucleic acid encoding an anti-CD28 antibody light chain, H The coding sequence for the region was isolated and H The method includes the steps of linking the sequence to a sequence encoding a heavy chain constant region of the desired class or subclass, expressing the light chain gene and the heavy chain construct in a cell, and harvesting the CD28 antibody.

[0096] The CD28 antibodies described herein can be of any class, such as IgG, IgM, IgE, IgA, or IgD. 1 , IgG 2 , IgG 3 , or IgG 4Preferably, the anti-CD28 antibody is of the IgG class, such as V subclass. Anti-CD28 antibodies can be converted from one class or subclass to another using methods known in the art. An exemplary method for producing an antibody of a desired class or subclass is to isolate nucleic acid encoding an anti-CD28 antibody heavy chain and nucleic acid encoding an anti-CD28 antibody light chain, H The coding sequence for the region was isolated and H The method includes the steps of linking the sequence to a sequence encoding a heavy chain constant region of the desired class or subclass, expressing the light chain gene and the heavy chain construct in a cell, and harvesting the CD28 antibody.

[0097] Furthermore, the antibodies provided by the present disclosure can be monoclonal or polyclonal, but are preferably monoclonal.

[0098] The antibodies of the present disclosure can be produced by techniques known in the art, including conventional monoclonal antibody methodology, such as standard somatic cell hybridization techniques (see, e.g., Kohler and Milstein, Nature (1975) 256:495, viral or oncogene transformation of B lymphocytes, or recombinant antibody techniques, as described in detail herein below.

[0099] Hybridoma production is a very well-established procedure. A common animal system for preparing hybridomas is the mouse system. Immunization protocols and techniques for isolation of immune splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known. One well-known method that can be used to generate human CD28 antibodies provided by the present disclosure includes the use of the XenoMouse™ animal system. XenoMouse™ mice are genetically engineered mouse strains that contain large fragments of human immunoglobulin heavy and light chain loci and are deficient in the production of mouse antibodies. See, for example, Green et al., Nature Genetics (1994) 7:13-21 and WO2003 / 040170. The animal is immunized with a CD28 antigen. The CD28 antigen is isolated and / or purified CD28, preferably CD28. It may be a fragment of CD28, such as the extracellular domain of CD28, in particular a CD28 extracellular domain fragment comprising amino acid residues 33, 34, 36 and 37, 80-83, 92 and 93, 95 and 96, and 100-104 of SEQ ID NO: 1. Immunization of animals may be performed by any method known in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing non-human animals, such as mice, rats, sheep, goats, pigs, cattle, and horses, are well known in the art. See, for example, Harlow and Lane, supra, and U.S. Pat. No. 5,994,619. The CD28 antigen may be administered with an adjuvant to stimulate the immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulating complex). After immunization of animals with CD28 antigen, antibody-producing immortalized cell lines are prepared from cells isolated from the immunized animals. After immunization, the animals are sacrificed and lymph node and / or splenic B cells are immortalized.Methods for immortalizing cells include, but are not limited to, introducing oncogenes into them, infecting them with tumor viruses, culturing them under conditions that select for immortalized cells, subjecting them to oncogenic or mutating compounds, fusing with immortalized cells, e.g., myeloma cells, and inactivating tumor suppressor genes. See, e.g., Harlow and Lane, supra. When fusion with myeloma cells is used, it is preferred that the myeloma cells do not secrete immunoglobulin polypeptides (non-secretory cell lines). Immortalized cells are screened using CD28, a portion thereof, or cells expressing CD28. CD28 antibody-producing cells, e.g., hybridomas, are selected, cloned, and further screened for desirable properties, including robust proliferation, high antibody production, and desirable antibody properties, as discussed further below. Hybridomas can be grown in vivo in syngeneic animals, animals lacking an immune system, e.g., nude mice, or in cell culture in vitro. Methods for selecting, cloning, and growing hybridomas are well known to those skilled in the art.

[0100] The antibodies of the present disclosure can also be prepared using phage display or yeast display methods. Such display methods for isolating human antibodies are established in the art, such as Knappik, et al., "Fully Synthetic Human Combinatorial Antibody Libraries (HuCAL) Based on Modular Consensus Frameworks and CDRs Randomized with Trinucleotides" J. Mol. Biol. (2000) 296, 57-86; and Feldhaus, et al., "Flow-cytometric isolation of human antibodies from a non-immune Saccharomyces cerevisiae surface display library" Nat Biotechnol (2003) 21:163-170.

[0101] B. Antigen-binding fragments In some other aspects, the present disclosure provides antigen-binding fragments of any of the CD28 antibodies provided by the present disclosure.

[0102] The antigen-binding fragment may comprise any sequence of an antibody. In some embodiments, the antigen-binding fragment comprises the following amino acid sequences: (1) a light chain of an anti-CD28 antibody; (2) a heavy chain of a CD28 antibody; (3) a variable region from the light chain of an anti-CD28 antibody; (4) a variable region from the heavy chain of a CD28 antibody; (5) one or more CDRs (2, 3, 4, 5, or 6 CDRs) of an anti-CD28 antibody; or (6) three CDRs from the light chain and three CDRs from the heavy chain of an anti-CD28 antibody.

[0103] In other particular embodiments, the antigen-binding fragment of the CD28 antibody comprises: (i) V L , V H , C L , and C H (ii) a Fab fragment, which is a monovalent fragment consisting of one domain; (ii) a F(ab') fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region. 2 Fragment; (iii) V H and C H (iv) a single-arm V of an antibody; L and V H (v) Fv fragment consisting of V domains; H (vi) isolated CDRs, and (vii) the V domain of an antibody. H V of antibody linked to domain L Single chain antibodies (scFv) are polypeptides containing a region, see Bird et al. Science (1988) 242:423-426 and Huston et al. Proc. Natl. Acad. Sci. USA (1988) 85:5879-5883, which are antigen-binding fragments.

[0104] In some embodiments, the anti-CD28 antibodies or antibody fragments disclosed herein have a V sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the amino acid sequences set forth in SEQ ID NOs: 11, 20, 29, 39, 47, 54, 62, 71, 77, 84, 92, 99, 107, 115, 122, 130, 137, 144, 151, 157, and 165. H In some embodiments, the anti-CD28 antibodies or antibody fragments disclosed herein comprise a V region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the amino acid sequences set forth in SEQ ID NOs: 12, 21, 30, 40, 48, 55, 63, 72, 78, 85, 93, 100, 108, 116, 123, 131, 138, 145, 152, 158, and 166. LIn some embodiments, the anti-CD28 antibodies or antibody fragments disclosed herein comprise an HCDR1 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 5, 15, 24, 33, 43, 66, 88, 103, 111, 126, 134, 148, and 161. In some embodiments, the anti-CD28 antibodies or antibody fragments disclosed herein comprise an HCDR2 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 6, 51, 58, 67, 89, 96, 104, and 155. In some embodiments, the anti-CD28 antibodies or antibody fragments disclosed herein comprise an HCDR3 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the amino acid sequences set forth in SEQ ID NOs: 7, 16, 25, 35, 44, 52, 59, 81, 90, 97, 105, 112, 119, 127, 135, 141, 149, and 162. In some embodiments, the anti-CD28 antibody or antibody fragment disclosed herein comprises an LCDR1 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the amino acid sequences set forth in SEQ ID NOs: 8, 17, 26, 36, 45, 53, 60, 76, 82, 91, 98, 106, 113, 120, 128, 136, 142, 150, 156, and 163.In some embodiments, the anti-CD28 antibodies or antibody fragments disclosed herein comprise an LCDR2 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 9, 18, 27, 37, and 300. In some embodiments, the anti-CD28 antibodies or antibody fragments disclosed herein comprise an LCDR3 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 10, 19, 28, 38, 46, 61, 70, 83, 114, 121, 129, 143, and 164.

[0105] In some embodiments, the antibodies disclosed herein comprise a heavy chain that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 13, 22, 31, 41, 49, 56, 64, 73, 79, 86, 94, 101, 109, 117, 124, 141, 132, 139, 146, 153, 159, and 167.

[0106] In some embodiments, the antibodies disclosed herein comprise a light chain that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 14, 23, 32, 42, 50, 57, 65, 74, 80, 87, 95, 102, 110, 118, 125, 142, 133, 140, 147, 154, 160, and 168.

[0107] IV. Multispecific antibodies Also provided are multispecific antibodies corresponding to the activatable multispecific antibodies or masked multispecific antibodies described herein. One aspect of the present application provides multispecific antibodies (including activatable bispecific T cell engager (TCE) molecules) that can bind to both T cells and target cells, such as tumor cells. Due to on-target and off-tumor effects, traditional TCE molecules are associated with high cytotoxicity, including toxicity to the central nervous system (CNS) and cytokine storm. Thus, there is a need for antibodies that can bind to T cells and target cells, such as tumor cells, with enhanced specificity and reduced side effects.

[0108] In some embodiments, the multispecific antibody is a bispecific antibody (bsAb). In some embodiments, the multispecific antibody is a trispecific antibody (tsAb).

[0109] In certain embodiments, the bispecific antibody of the present disclosure is specific for CD28 on the surface of T cells. In some embodiments, the multispecific antibody is a tumor-associated antigen (TAA)xCD28 bispecific antibody that specifically binds to TAA and CD28. In some embodiments, the antibody of the present disclosure is an IgG antibody, for example, comprising an IgG Fc region (e.g., a human IgG Fc region).

[0110] In some embodiments, the multispecific antibody binds to CD28 on the surface of T cells. In some embodiments, the multispecific antibody is a tumor associated antigen (TAA)xCD28 bispecific antibody that specifically binds to TAA and CD28. In some embodiments, the multispecific antibody does not include any masking moiety or a cleavable moiety. In some embodiments, the multispecific antibody is obtained by cleavage of a cleavable moiety or a cleavable moiety.

[0111] In some embodiments, the multispecific antibody binds to CD3 on the surface of T cells. In some embodiments, the multispecific antibody is a tumor associated antigen (TAA)xCD3 bispecific antibody that specifically binds to TAA and CD3. In some embodiments, the multispecific antibody has a weak affinity, e.g., an EC determined by an ELISA assay. 50 specifically binds to CD3 with a Kd of at least 10 nM (e.g., at least 100 nM) and / or a Kd of at least 50 nM. In some embodiments, the multispecific antibody does not comprise a masking moiety or a cleavable moiety. In some embodiments, the multispecific antibody is obtained by cleavage of a cleavable moiety or a cleavable moiety.

[0112] In some embodiments, a multispecific antibody is provided that includes: a) a VH1 and VH2 of an antibody that specifically binds to a target antigen (e.g., a tumor antigen such as B7-H3, HER2, or TROP2). L and b) a first antigen-binding fragment comprising VH2 and VH3 of an anti-CD3 antibody that specifically binds to CD3. L and a second antigen-binding fragment comprising a VH1 and VH2 of an antibody that specifically binds to a target antigen (e.g., a tumor antigen such as B7-H3, HER2, or TROP2). L and b) a first antigen-binding fragment comprising VH2 and VH3 of an anti-CD28 antibody that specifically binds to CD28. L a second antigen-binding fragment comprising 2, where the first and / or second antigen-binding fragment is fused to a first and / or second masking peptide (MP1 / MP2).

[0113] In some embodiments, the first antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab and scFv. In some embodiments, the first antigen-binding fragment is a Fab. In some embodiments, the second antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab and scFv. In some embodiments, the second antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab and scFv. In some embodiments, the second antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab and scFv, from the N-terminus to the C-terminus, L In some embodiments, the first antigen-binding fragment is a Fab and the second antigen-binding fragment is a Fab. In some embodiments, the first antigen-binding fragment is a Fab and the second antigen-binding fragment is an scFv.

[0114] The engineered C HAntibodies that contain three-domain disulfide bonds and / or salt bridges may further contain one or more knob-into-hole residues. "Knobs-into-hole" or "KIH" refers to an approach known in the art for making bispecific antibodies, also known as the "protuberance-into-cavity" approach (see, e.g., U.S. Pat. No. 5,731,168). In this approach, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each constitute an interface. The interface of one immunoglobulin polypeptide interacts with a corresponding interface on the other immunoglobulin polypeptide, thereby allowing the two immunoglobulin polypeptides to associate. These interfaces may be engineered such that a "knob" or "protuberance" (these terms may be used interchangeably herein) located at the interface of one immunoglobulin polypeptide corresponds to a "hole" or "cavity" (these terms may be used interchangeably herein) located at the interface of the other immunoglobulin polypeptide. In some embodiments, the holes are the same or similar size as the knobs and are appropriately positioned such that when the two interfaces interact, the knobs of one interface can be positioned into the corresponding holes of the other interface. Without wishing to be bound by theory, it is believed that this stabilizes the heteromultimer and promotes the formation of heteromultimers over other species, e.g., homomultimers. In some embodiments, the KIH approach is used in combination with engineered disulfide bonds and / or salt bridges described herein to promote heteromultimerization of two different immunoglobulin polypeptides to create bispecific antibodies comprising two immunoglobulin polypeptides with binding specificities for different epitopes. In some embodiments, the C of the activatable multispecific antibodies described herein is H The 3 domain does not contain a KIH residue.

[0115] In some embodiments, a bispecific antibody targeting CD28 and a tumor antigen (e.g., B7-H3, HER2, TROP2 or PD-L1) is provided, comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein: (i) the first polypeptide has the formula: H 1-C H 1-hinge-C H 2 - The first contains the structure represented by CH3; (ii) the second polypeptide has the formula: scFv-hinge-C H 2- The second CH3 structure; and (iii) the third polypeptide has the formula V L 1-C L Contains the structure represented by: Where: V L 1, the first immunoglobulin light chain variable domain; V H 1, the first immunoglobulin heavy chain variable domain; The scFv is a second immunoglobulin light chain variable domain (V L 2) and a second immunoglobulin heavy chain variable domain (V H 2) is a single-chain variable fragment containing; C L is the immunoglobulin light chain constant domain; C H 1 is immunoglobulin heavy chain constant domain 1; C H 2 is immunoglobulin heavy chain constant domain 2; hinge is C H 1 Domain and C H is the immunoglobulin hinge region that connects the two domains, V L In some embodiments, the scFv specifically binds to CD28, and VH1 and VH2 associate to form a first Fv that specifically binds to a tumor antigen (e.g., B7-H3, HER2, TROP2, or PD-L1), and the scFv specifically binds to CD28. In some embodiments, the scFv has an antibody binding activity (EC) in the range of 0.1 nM to 1000 nM as determined by ELISA. 50) with half-maximal binding (e.g., as described in Example 3). In some embodiments, the scFv binds to CD28 at an antibody concentration (EC ) of less than 10 nM (e.g., between 1 nM and 0.1 pM) as determined by an Octet® RED96 assay (e.g., as described in Example 3). 50 ) and binds to CD28 with half-maximal binding. In certain embodiments, the scFv binds to CD28 with a dissociation constant (Kd) of less than 10 nM.

[0116] In some embodiments, a bispecific antibody targeting CD28 and a tumor antigen (e.g., B7-H3, HER2, or TROP2) is provided, comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein: (i) the first polypeptide has the formula: H 1-C H 1-hinge-C H 2 - The first contains the structure represented by CH3; (ii) the second polypeptide has the formula: H 2-C H 1-hinge-C H 2 - Contains the structure represented by the second CH3; (iii) the third polypeptide has the formula V L 1-C L and (iv) the fourth polypeptide has the formula V L 2-C L Contains the structure represented by: Where: V L 1, the first immunoglobulin light chain variable domain; V H 1, the first immunoglobulin heavy chain variable domain; V L 2, the second immunoglobulin light chain variable domain; V H 2 is the second immunoglobulin heavy chain variable domain; C L is the immunoglobulin light chain constant domain; C H 1 is immunoglobulin heavy chain constant domain 1; C H 2 is immunoglobulin heavy chain constant domain 2; and The hinge is C H 1 Domain and C H an immunoglobulin hinge region connecting the two domains; V L 1 and V H 1 associate to form a first Fv that specifically binds to a tumor antigen (e.g., B7-H3, HER2, TROP2, or PD-L1), and V L 2 and V H 2 associate to form a second Fv that specifically binds CD28. In some embodiments, the scFv binds CD28 and has an antibody titer (EC) in the range of 0.1 nM to 1000 nM, as determined by an ELISA assay (e.g., as described in Example 3). 50 In some embodiments, the second Fv exhibits half-maximal binding at an antibody concentration (EC) of less than 10 nM (e.g., between 1 nM and 0.1 pM), as determined by an Octet® RED96 assay (e.g., as described in Example 3). 50 ) and binds to CD28 with half-maximal binding. In certain embodiments, the second Fv binds to CD28 with a dissociation constant (Kd) of less than 10 nM.

[0117] In some embodiments, a multispecific antibody is provided that comprises: a) V of an antibody that specifically binds to a target antigen (e.g., a tumor antigen such as B7-H3, HER2, or TROP2) H 1 and V L a first antigen-binding fragment comprising: b) V of an anti-CD28 antibody that specifically binds to CD28 H 2 and V L 2, wherein the second antigen-binding fragment is fused to a first masking peptide (MP1).

[0118] In some embodiments, a multispecific antibody is provided that comprises: a) V of an antibody that specifically binds to a target antigen (e.g., a tumor antigen such as B7-H3, HER2, or TROP2) H 1 and V L a first antigen-binding fragment comprising: b) V of an anti-CD28 antibody that specifically binds to CD28 H 2 and V L 2, wherein the second antigen-binding fragment is fused to a second masking peptide (MP2).

[0119] In some embodiments, the first antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab and scFv. In some embodiments, the first antigen-binding fragment is a Fab. In some embodiments, the second antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab and scFv. In some embodiments, the second antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab and scFv. In some embodiments, the second antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab and scFv, from the N-terminus to the C-terminus, L 2, an optional linker, and V H 2.

[0120] In some embodiments a multispecific antibody is provided comprising a first polypeptide, a second polypeptide, and a third polypeptide, (i) the first polypeptide has the formula: H 1-C H 1-hinge-C H 2-First C H Contains the structure represented by 3; (ii) the second polypeptide has the formula: MP1-V L 2-V H 2-hinge-C H 2- Second C H 3; and (iii) the third polypeptide has the formula: L 1-C LContains the structure represented by: Where: V L 1, the first immunoglobulin light chain variable domain; V H 1, the first immunoglobulin heavy chain variable domain; V L 2, the second immunoglobulin light chain variable domain; V H 2 is the second immunoglobulin heavy chain variable domain; C L is the immunoglobulin light chain constant domain; C H 1 is immunoglobulin heavy chain constant domain 1; C H 2 is immunoglobulin heavy chain constant domain 2; The first C H 3 is the first immunoglobulin heavy chain constant domain 3; The second C H 3, the second immunoglobulin heavy chain constant domain 3; The hinge is C H 1 Domain and C H the immunoglobulin hinge region that connects the two domains; MP1 is the first masking peptide, which comprises, from the N-terminus to the C-terminus, an N-terminal unit (NU), a masking unit (MU), and a linking unit (LU), where the LU of the masking peptide does not contain a cleavage site or contains at least one cleavage site.

[0121] In some embodiments a multispecific antibody is provided comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein: (i) the first polypeptide has the formula: H 1-C H 1-hinge-C H 2-First C H 3) contains a structure represented by the formula: (ii) the second polypeptide has the formula: MP2-V L 2-V H 2-hinge-C H2- Second C H 3; and (iii) the third polypeptide has the formula: MP1-V L 1-C L Contains the structure represented by: Where: V L 1, the first immunoglobulin light chain variable domain; V H 1 is the first immunoglobulin heavy chain variable domain; V L 2, the second immunoglobulin light chain variable domain; V H 2 is the second immunoglobulin heavy chain variable domain; C L is the immunoglobulin light chain constant domain; C H 1 is immunoglobulin heavy chain constant domain 1; C H 2 is immunoglobulin heavy chain constant domain 2; The first C H 3 is the first immunoglobulin heavy chain constant domain 3; The second C H 3, the second immunoglobulin heavy chain constant domain 3; The hinge is C H 1 Domain and C H the immunoglobulin hinge region that connects the two domains; MP1 is a masking peptide and comprises, from the N-terminus to the C-terminus, an N-terminal unit (NU), a masking unit (MU) and a linking unit (LU), and the LU of the masking peptide may contain no cleavage site or at least one or more cleavage sites. MP2 is a masking peptide, which consists of an N-terminal unit (NU), a masking unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; the LU of the masking peptide contains either zero or at least one cleavage site.

[0122] In some embodiments, the bispecific antibody binds to a first and a second target, the first target is CD28, and the bispecific antibody has an HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 15, 24, 33, 43, 66, 88, 103, 111, 126, 134, 148, and 161; an HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 51, 58, 67, 89, 96, 104, and 155; and an HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 16, 25, 35, 44, 52, 59, 81, 90, 97, 105, 112, 119, 127, 135, 141, 149, and and 162; and an LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 17, 26, 36, 45, 53, 60, 76, 82, 91, 98, 106, 113, 120, 128, 136, 142, 150, 156, and 163; an LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 18, 27, 37, and 300; and an LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 19, 28, 38, 46, 61, 70, 83, 114, 121, 129, 143, and 164. In some embodiments, the bispecific antibody binds to a first and a second target, wherein the first target is human CD28, and the bispecific antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 20, 29, 39, 47, 54, 62, 71, 77, 84, 92, 99, 107, 115, 122, 130, 137, 144, 151, 157, and 165, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 21, 30, 40, 48, 55, 63, 72, 78, 85, 93, 100, 108, 116, 123, 131, 138, 145, 152, 158, and 166.

[0123] In some embodiments, a bispecific antibody binds to a first and a second target, the first target is CD28 and the second target is B7H3, the bispecific antibody comprises a CD28 binding portion comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, and 171, and the bispecific antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 176 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 175. In certain embodiments, a bispecific antibody binds to a first and a second target, the first target is CD28 and the second target is B7H3, the bispecific antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 171, a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 1176, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 175.

[0124] In some embodiments, the bispecific antibody binds to a first and a second target, the first target is CD28 and the second target is HER2, the bispecific antibody comprises a CD28 binding portion comprising the amino acid sequence set forth in SEQ ID NO: 172, and the bispecific antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 170 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 169. In certain embodiments, the bispecific antibody binds to a first and a second target, the first target is CD28 and the second target is HER2, the bispecific antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 172, a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 170, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 169.

[0125] In some embodiments, the bispecific antibody binds to a first and a second target, the first target is CD28 and the second target is HER2, and the bispecific antibody comprises a CD28 binding portion comprising the amino acid sequence set forth in SEQ ID NO: 11, 12, 171, a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 170, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 169. In certain embodiments, the bispecific antibody binds to a first and a second target, the first target is CD28 and the second target is HER2, and the bispecific antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 171, a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 170, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 169.

[0126] In some embodiments, the bispecific antibody binds to a first and a second target, the first target is human CD28 and the second target is TROP2, the bispecific antibody comprises a CD28 binding portion comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, and 171, the bispecific antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 174 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 173. In certain embodiments, the bispecific antibody binds to a first and a second target, the first target is CD28 and the second target is TROP2, the bispecific antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 171, a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 174, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 173.

[0127] In certain embodiments, the bispecific antibody binds to a first and a second target, the first target is CD3 and the second target is B7-H3, and the bispecific antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 177 or an scFv fusion polypeptide set forth in SEQ ID NO: 299, a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 176, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 175.

[0128] In certain embodiments, the bispecific antibody binds to a first and a second target, the first target being CD3 and the second target being TROP2.

[0129] In some embodiments, the bispecific antibody binds to a first and a second target, wherein the first target is human CD28, and the bispecific antibody comprises a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 22, 31, 41, 49, 56, 64, 73, 79, 86, 94, 101, 109, 117, 124, 141, 132, 139, 146, 153, 159, and 167, and a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 23, 32, 42, 50, 57, 65, 74, 80, 87, 95, 102, 110, 118, 125, 142, 133, 140, 147, 154, 160, and 168.

[0130] V. Masked Antibodies In some embodiments, the present disclosure provides masked antibodies, which may be masked monoclonal antibodies against a specific target, or multispecific (e.g., bispecific) antibodies. In some embodiments, the masked antibodies provided herein comprise a full-length antibody light chain, e.g., a kappa light chain or a lambda light chain. Additionally, or alternatively, in some embodiments, the antibody comprises a full-length antibody heavy chain. The antibody heavy chain may be of any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain comprises an IgG 1 , IgG 2 , IgG 3 , or IgG 4 In some embodiments, the masked antibody is in the IgG class, such as in the IgG subclass. The antibody heavy chains described herein can be converted from one class or subclass to another class or subclass by methods known in the art. In some embodiments, the masked antibody is or comprises a full-length antibody comprising an Fc region, such as a human Fc region or a variant thereof. In some embodiments, the human Fc region is in the human IgG 1 Fc region, human IgG 2 Fc region, human IgG 4Fc region, or a variant of any of the foregoing. In some embodiments, the variant Fc region comprises one or more amino acid substitutions, insertions, or deletions compared to the wild-type human Fc region from which the variant is derived. In some embodiments, the masked antibody is a human IgG 1 In some embodiments, the IgG 1 The Fc variant comprises one or more amino acid substitutions that increase the affinity of the Fc variant for FcγRIIb. In some embodiments, the Fc variant comprises a human IgG 1 The variants of the Fc region include substitutions selected from the group consisting of G236D, L328F, S239D, S267E, G236D and S267E, S239D and S267E, S267E and L328S, and S267E and L328F, where amino acid numbering is according to the EU index. (See, e.g., Edelman et al., Proc Natl Acad Sci USA (1969) 63: 78-85). The foregoing substitutions are described in Chu et al. Mol Immunol. (2008) 45 (15): 3926-33. Additionally or alternatively, in some embodiments, human IgG 1 The Fc region variants are: E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D, and A330R; E233D, P231G, P238D, and A330R; G237D, H268D, P238D, and P271G; G237D, P238 and E233D, G237D, P238D, H268D, P238D, P271G, and A330R, where amino acid numbering is according to the EU index. The foregoing substitutions are described in Mimoto et al. Protein Engineering Selection. (2013) 26 (10): 589-98. Additionally or alternatively, in some embodiments, the human IgG 1The variant of the Fc region comprises a S2657A substitution (Buschor et al. Int Arch Allergy Immunol. (2014) 163 (3): 206-14), amino acid numbering according to the EU index. Additionally or alternatively, in some embodiments, a human IgG 1 Mutations in the Fc region include T437R and / or K248E substitutions (Zhang et al. MAbs. (2017) 9 (7): 1129-1142), amino acid numbering according to the EU index. In some embodiments, the masked antibody is a human IgG 4 In some embodiments, the IgG 4 The Fc variant comprises one or more amino acid substitutions that increase the affinity of the Fc variant for FcγRIIb. In some embodiments, the Fc variant comprises a human IgG 4 The variants of the Fc region comprise a substitution selected from the group consisting of G236D, L328F, S239D, S267E, G236D and S267E, S239D and S267E, S267E and L328S, and S267E and L328F, where amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the ... and S267E, S267E and L328S, and S267E and L328F, where amino acid numbering is according to the EU index. 4 The Fc region variants were E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D, and A330R; E233D, P231G, P238D, and A330R; G237D, H268D, P238D, and P271G; G237D, P238D, P27 1G, and A330R; E233D, H268D, P238D, P271G, and A330R; G237D, H268D, P238D, P271G, and A330R; and E233D, G237D, P238D, H268D, P271G, and A330R and K248E, where amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the human IgG 4The variant of the Fc region comprises a S2657A substitution, where amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the variant of the Fc region comprises a human IgG 1 The Fc region variants include T437R and / or K248E substitutions, where amino acid numbering is according to the EU index.

[0131] In some embodiments, the masked antibodies disclosed herein further comprise human IgG 1 In some embodiments, the IgG 1 The mutants were: G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F; E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D, and A330R; E233D, P231G, P238D, and A330R; G237D, H268D, P238 In some embodiments, the masked antibodies disclosed herein comprise a substitution selected from the group consisting of: S2657A, S2657B, S2657C, T437R, K248E, and T437R and K248E, wherein the amino acid numbers are according to the EU index. In some embodiments, the masked antibodies disclosed herein comprise a substitution selected from the group consisting of: S2657A, S2657B, S2657C, T437R, K248E, and T437R and K248E, wherein the amino acid numbers are according to the EU index. In some embodiments, the masked antibodies disclosed herein comprise a substitution selected from the group consisting of: S2657A, S2657B, S2657C, T437R, K248E, and T437R and K248E, wherein the amino acid numbers are according to the EU index. 4 In some embodiments, the IgG 4The mutants were: G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F; E233D and P238D; G237D and P238D; H268D and P238D; P271G and P238D; A330R and P238D; E233D, P238D, and A330R; E233D, P231G, P238D, and A330R; G237D, H268D, P238 and E233D, G237D, P238D, P271G, and A330R; G237D, H268D, P238D, P271G, and A330R; and E233D, G237D, P238D, H268D, P271G, and A330R; S2657A; T437R; K248E; and T437R and K248E, wherein the amino acid numbers are according to the EU index.

[0132] In some embodiments, the term "masked antibody" refers to an antibody fragment, e.g., a masked antigen-binding fragment of a masked anti-CD28 antibody. In some embodiments, an antibody fragment includes Fab, Fab', Fab'-SH, F(ab') 2 , Fv, scFv (Bird et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883), (scFv) 2 , a linear antibody, a single chain antibody, a minibody, or a diabody.

[0133] In some embodiments, the masked anti-CD28 antibodies described herein cross-react with CD28 of different species, thus allowing the masked anti-CD28 antibodies to be used in both preclinical studies and clinical trials. In some embodiments, the masked anti-CD28 antibodies described herein bind to two or more of human CD28, cynomolgus monkey CD28, mouse CD28, and / or rat CD28 after activation (i.e., after activation by cleavage of the masked antibody, e.g., protease cleavage). In some embodiments, the masked anti-CD28 antibodies bind to human CD28, cynomolgus monkey CD28, mouse CD28, and rat CD28 after activation (i.e., after activation by cleavage of the masked antibody, e.g., protease cleavage).

[0134] In some embodiments, the masked anti-CD28 antibodies described herein are context-dependent (e.g., are only activated (capable of binding to a target) in certain contexts (such as a protease-rich tumor microenvironment). In some embodiments, the masked anti-CD28 antibodies described herein have improved safety (e.g., reduced toxicity, do not significantly change the weight of many organs, do not alter liver histopathology, hematology, and / or blood biochemistry, etc.) over more traditional unmasked antibodies. In some embodiments, the masked anti-CD28 antibodies described herein exhibit similar pharmacokinetic properties (e.g., have similar in vivo half-lives) as traditional unmasked anti-CD28 antibodies. In some embodiments, the masked anti-CD28 antibodies described herein exhibit improved pharmacokinetic properties (e.g., have longer in vivo half-lives) compared to more traditional unmasked anti-CD28 antibodies.

[0135] In some embodiments, the antibody heavy chain variable region (V) of the masked anti-CD28 antibody described herein H ) and the antibody light chain variable region (V L) form the antigen binding domain (ABD) that binds to hCD28. In some embodiments, the masking unit (MU) of the masked anti-CD28 antibodies described herein binds to the ABD of hCD28 and reduces or inhibits binding of the masked anti-CD28 antibody to hCD28 compared to binding of a corresponding anti-CD28 antibody lacking the MU to hCD28 and / or compared to binding of the ABD to hCD28.

[0136] In some embodiments, the masking units (MU) measure at least about 2.0 (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000, at least about 1,100, at least about 1,200, at least about 1,300, at least about 1,400, at least about 1,500, etc., including any ranges between these values) prior to removal of MU from a masked antibody provided herein. For example, in some embodiments, the masking efficiency of a masked anti-CD28 antibody is measured as the difference in binding affinity to hCD28 of a masked anti-CD28 antibody comprising a masking unit (MU) (i.e., before activation of the masked antibody) and an anti-CD28 antibody lacking MU. In a further example, the masking efficiency is measured as the difference in affinity to hCD28 of a masked anti-CD28 antibody comprising MU (i.e., before activation of the masked antibody by cleavage, e.g., protease cleavage) and an unmasked anti-CD28 antibody (i.e., after activation of the masked antibody by cleavage, e.g., protease cleavage). In some embodiments, the masking efficiency is measured as the EC for binding of a masked antibody comprising MU. 50(i.e., before activation) was compared with the EC of a corresponding antibody specific for the same target lacking the masking peptide or masking unit. 50 In some embodiments, the EC 50 is measured by ELISA. In some embodiments, the masking unit (MU) of the masked antibody binds to the ABD and prevents the masked polypeptide from binding to its target. In certain embodiments, the target is CD28. In other embodiments, the target is CD3, B7-H3, HER2, or TROP2.

[0137] In some embodiments, when the masking unit is removed from the antibody (e.g., after activation by treatment with one or more proteases that cleave within the linking unit), the inhibitory effect on the antibody is improved by at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold, or more (including any range between the aforementioned values)) compared to a corresponding antibody without the masking peptide or masking unit. In some embodiments, the EC 50 is increased by at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold, or more (including any ranges between the aforementioned values) after activation by treatment with one or more proteases that cleave within a binding unit, e.g., as measured by ELISA or FACS assay.

[0138] In some embodiments, when a masking unit is attached to the ABD of a masked antibody described herein, the K D is that if the masking unit of a masked anti-CD28 antibody is removed from the ABD (e.g., cleaved within the target by protease treatment), the K D about 2-fold (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more, including any range between the aforementioned values) greater than the K of the antibody against its target. D is the K of the corresponding antibody lacking the masking peptide or masking unit. D about twice (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more, including any ranges between the aforementioned values).

[0139] In some embodiments, the masking unit sterically hinders the binding of the masked binding polypeptide to its target and / or allosterically hinders the binding of the masked binding polypeptide to its target.

[0140] In some embodiments, the dissociation constant of a masking unit for the ABD of a masked antibody described herein (e.g., anti-CD28) is greater than the dissociation constant of the masked antibody for its target (e.g., hCD28; when the masked antibody is in an active form, such as after protease treatment). In some embodiments, the dissociation constant of a masking unit for the ABD of a masked antibody described herein (e.g., anti-CD28) is about 2-fold (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more, including any ranges between the aforementioned values) greater than the dissociation constant of the masked antibody for its target (e.g., hCD28; when the masked antibody is in an active form, such as after protease treatment). In some embodiments, the dissociation constant of the masking unit with respect to the ABD of a masked antibody (e.g., anti-CD28) described herein is approximately equal to the dissociation constant of the masked antibody with respect to its target (e.g., hCD28; when the masked antibody is in an activated form, such as after protease treatment). In some embodiments, the masking unit (MU) binds to the ABD of a masked antibody (e.g., anti-CD28) described herein and prevents the antibody from binding to its target (e.g., hCD28) only if the masked antibody is not activated (e.g., by treatment with one or more proteases that cleave within the binding unit). In some embodiments, activation induces cleavage of the polypeptide within the cleavage site. In some embodiments, activation induces a conformational change in the polypeptide (e.g., displacement of the masking unit (MU)) such that the masking peptide no longer prevents the polypeptide from binding to the target.

[0141] A. Single Masked Antibody In some embodiments, a masked monoclonal antibody is provided comprising a masking peptide (MP) and an antibody that binds to CD28, wherein the antibody comprises a heavy chain variable region (V H ) and the light chain variable region (VL ), where MP is V L and the MP comprises, from the N-terminus to the C-terminus, a masking unit (MU) and a linkage unit (LU), wherein the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215 to 248. H The region comprises an HCDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 15, 24, 33, 43, 66, 88, 103, 111, 126, 134, 148, and 161; an HCDR2 amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 51, 58, 67, 89, 96, 104, and 155; and an HCDR3 amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 16, 25, 35, 44, 52, 59, 81, 90, 97, 105, 112, 119, 127, 135, 141, 149, and 162; L The region comprises an LCDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 17, 26, 36, 45, 53, 60, 76, 82, 91, 98, 106, 113, 120, 128, 136, 142, 150, 156, and 163, an LCDR2 amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 18, 27, 37, and 300, and an LCDR3 amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 19, 28, 38, 46, 61, 70, 83, 114, 121, 129, 143, and 164.

[0142] In some embodiments, a masked monoclonal antibody is provided comprising a masking peptide (MP) and an antibody that binds to CD28, wherein the antibody comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), where MP is V Lwherein the MP comprises, from the N-terminus to the C-terminus, a masking unit (MU) and a linking unit (LU), and wherein the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215 to 248; wherein the antibody heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 20, 29, 39, 47, 54, 62, 71, 77, 84, 92, 99, 107, 115, 122, 130, 137, 144, 151, 157, and 165; and the antibody light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 21, 30, 40, 48, 55, 63, 72, 78, 85, 93, 100, 108, 116, 123, 131, 138, 145, 152, 158, and 166. In some embodiments, a masked monoclonal antibody is provided that comprises an antibody that binds to human CD28 and a masking peptide (MP), the antibody comprising a heavy chain and a light chain, wherein the MP is linked to the N-terminus of the LC, wherein the MP comprises, from the N-terminus to the C-terminus, a masking unit (MU) and a linking unit (LU), wherein the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215-248, wherein the antibody HC is selected from the group consisting of SEQ ID NOs: 13, 22, 31, 41, 49, 56, 64, 73, 79, 86, 94, 101, 109, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 600, 610, 620, 630, 640, 650, 660, 670, 680, 700, 710, 720, 730, 740, 750, 760, 770 17, 124, 141, 132, 139, 146, 153, 159, and 167, wherein antibody LC comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 23, 32, 42, 50, 57, 65, 74, 80, 87, 95, 102, 110, 118, 125, 142, 133, 140, 147, 154, 160, and 168.

[0143] B. Single-masked multispecific antibodies In some embodiments, a masked bispecific monoclonal antibody specific for a first and a second target is also provided, wherein the first target is CD28, and the antibody comprises a masking peptide (MP) and a CD28 binding portion, and the antibody comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), and MP is V Lthe MP comprises, from the N-terminus to the C-terminus, a masking unit (MU) and a linking unit (LU), the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215 to 248; the CD28 binding portion comprises an H region comprising HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 6, and HCDR3 of SEQ ID NO: 7; L The region includes an LCDR1 of SEQ ID NO:8, an LCDR2 of SEQ ID NO:9, and an LCDR3 of SEQ ID NO:10, and the second target is a B7-H3, HER2, or TROP2 protein.

[0144] In some embodiments, a masked bispecific monoclonal antibody specific for a first and a second target is also provided, wherein the first target is CD28, and the antibody comprises a masking peptide (MP) and a CD28 binding portion, and the antibody comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), and MP is V L and the MP comprises, from the N-terminus to the C-terminus, a masking unit (MU) and a linking unit (LU), and the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215 to 248. The CD28 binding portion is selected from SEQ ID NOs: 11, 20, 29, 39, 47, 54, 62, 71, 77, 84, 92, 99, 107, 115, 122, 130, 137, 144, 151, 157, and 16 and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 21, 30, 40, 48, 55, 63, 72, 78, 85, 93, 100, 108, 116, 123, 131, 138, 145, 152, 158, and 166, wherein the second target is a B7-H3, HER2, or TROP2 protein.

[0145] C. Double-masked multispecific antibodies In certain embodiments, a masked bispecific monoclonal antibody specific for a first and a second target is also provided, wherein the first target is CD28 and the second target is a tumor-associated antigen selected from HER2, B7-H3, and TROP2, and the antibody comprises two masking peptides (MPs).

[0146] VI. Masking peptides In some embodiments, the MP further comprises an N-terminal unit. In some embodiments, the N-terminal unit is about 1-10 amino acids in length. In some embodiments, the N-terminal unit is SEQ ID NO: 210. In some embodiments, the LU further comprises at least a first cleavage site (CS 1 ) (e.g., a first protease cleavage site). In some embodiments, the LU contains a second cleavage site (CS 2). In some embodiments, the first cleavage site and / or the second cleavage site are protease cleavage sites. In some embodiments, the first cleavage site and the second cleavage site are the same. In some embodiments, the first and second cleavage sites are different. Any suitable protease cleavage site that is recognized and / or cleaved by any protease known in the art may be used (e.g., a protease that is known to co-localize with a target of the polypeptide that includes the cleavage site), including, for example, a protease cleavage site recognized and / or cleaved by urokinase-type plasminogen activator (uPA); a matrix metalloproteinase (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19, MMP-20, MMP-21, MMP-22, MMP-23, MMP-24, MMP-25, MMP-26, MMP-27, MMP-28, MMP-29, MMP-30, MMP-31, MMP-32, MMP-33, MMP-34, MMP-35, MMP-36, MMP-37, MMP-38, MMP-39, MMP-40, MMP-41, MMP-42, MMP-43, MMP-44, MMP-45, MMP-46, MMP-47, MMP-48, MMP-49, MMP-49, MMP-40, MMP-41, MMP-42, MMP-43, MMP-44, MMP-45, MMP-46, MMP-4 , MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, and / or MMP-27; tobacco etch virus (TEV) protease; plasmin; thrombin; PSA; PSMA; ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE , ADAMDECI, ADAMTSI, ADAMTS4, and / or ADAMTS5); caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, and / or caspase-14); aspartic proteases (e.g., RACE and / or renin); aspartic acid proteases (e.g., cathepsins (e.g., cathepsin D and / or cathepsin E); cysteine ​​cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, and / or cathepsin X / Z / P); cysteine ​​proteinases (e.g., cruzipain, legumain, and / or otubain-2); KLKs (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and / or KLK14);metalloproteinases (e.g., meprin, neprilysin, PSMA, and / or BMP-1); serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase, and / or coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXla, FXIIa)); elastase; granzyme B; guanidinobenzoatase; HtrAl; human neutrophil elastase; lactoferrin; marapsin; NS3 / 4A; PACE4; tPA; tryptase; type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3, and / or TMPRSS4), and the like. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from uPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from uPA, MMP-2, MMP-9, and / or TEV protease. ;

[0147] In some embodiments, LU is a first linker (L 1 In some embodiments, the first linker (L 1 ) is the first cleavage site (CS 1 ) (e.g., the first protease cleavage site). In some embodiments, LU is N-terminal to C-terminally, (CS 1 )-L 1 In some embodiments, LU comprises a second linker (L 2 In some embodiments, L2 is C-terminal to the second cleavage site. In some embodiments, LU is N-terminal to C-terminally comprised of (CS 1 )-L 1 -(CS 2 )-L 2 In some embodiments, L 1 and L 2 is any suitable linker (e.g., flexible linker) known in the art, such as, for example, glycine polymers (G)n, where n is an integer of at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.); glycine-serine polymers (GS)n, where n is an integer of at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.), such as SEQ ID NOs: 249-257; glycine-alanine polymers; alanine-serine polymers, etc. The linker sequence may be any length, such as from about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., a 20 amino acid glycine polymer or glycine-serine polymer), from about 1 amino acid to about 15 amino acids, from about 3 amino acids to about 12 amino acids, from about 4 amino acids to about 10 amino acids, from about 5 amino acids to about 9 amino acids, from about 6 amino acids to about 8 amino acids, etc. In some embodiments, the linker is any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0148] In some embodiments, LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213 and 213. In some embodiments, the masking peptide (MP) comprises, from N-terminus to C-terminus, the structure (MU)-(LU), where LU is (CS 1 )-L 1 or (CS 1 )-L 1 -(CS 2 )-L 2In some embodiments, the masking peptide of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215-248.

[0149] In some embodiments, the masking peptide (MP) comprises an MU set forth in any one of SEQ ID NOs: 178-211 and an LU set forth in SEQ ID NO: 213 or 214. In some embodiments, the MP comprises a sequence set forth in any one of SEQ ID NOs: 215-248.

[0150] VII. Antibody Production Another aspect of the present disclosure provides one or more isolated nucleic acid molecules comprising a nucleotide sequence encoding the amino acid sequence of an anti-CD28 antibody described herein, including a masked anti-CD28 antibody. In some embodiments, one or more isolated nucleic acid molecules comprising a nucleotide sequence encoding the amino acid sequence of a multispecific antibody described herein (including a masked multispecific antibody) are provided herein. The amino acid sequence encoded by the nucleotide sequence can be any portion of an antibody described herein, such as a CDR, a sequence comprising one, two, or three CDRs, a variable region of a heavy chain, a variable region of a light chain, or a full-length heavy chain or a full-length light chain. The nucleic acid of the present disclosure can be, for example, DNA or RNA, and may or may not include intron sequences. Typically, the nucleic acid is a cDNA molecule. The nucleic acid of the present disclosure can be, for example, DNA or RNA, and may or may not include intron sequences. Typically, the nucleic acid is a cDNA molecule.

[0151] In some embodiments, the disclosure provides isolated nucleic acid molecules comprising or consisting of a nucleotide sequence encoding the amino acid sequence of, e.g., the heavy chain variable region and / or the light chain variable region of an antibody described herein, or, e.g., the full-length heavy chain and / or full-length light chain of an antibody described herein.

[0152] The nucleic acids of the invention can be obtained using any suitable molecular biology technique, such as PCR amplification or cDNA cloning techniques. In the case of antibodies, nucleic acids encoding the antibodies obtained by library screening can be recovered from the library.

[0153] V H The isolated DNA encoding the region can be prepared by synthesizing the VH-encoding DNA with the heavy chain constant region (C H 1. C H 2, and C H The heavy chain constant region can be converted to a full-length heavy chain gene by operably linking it to another DNA molecule encoding the heavy chain constant region of the IgG1A-IgG2A-IgG3A-IgG4A-IgG5A-IgG6A-IgG7A-IgG8A-IgG1A-IgG1B-IgG1B-IgG2B-IgG3B-IgG4B-IgG1B-IgG1B-IgG2B-IgG1 ...4B-IgG1B-IgG1B-IgG2B-IgG3B-IgG4B-IgG1B-IgG4B-IgG1B-IgG1B-IgG2B-IgG3B-IgG4B-IgG1B-IgG4B-IgG1B-IgG1B-IgG2B-IgG3B-IgG4B-IgG1B-IgG4B-IgG1B-IgG1B-IgG2B-IgG3B-IgG4B-IgG1B-IgG4B-IgG1B 1 , IgG 2 , IgG 3 , IgG 4 The constant region may be an IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG4 or IgG2 constant region, which has no ADCC effect. 4 The constant region sequences may be any of the various alleles, or allotypes, that are known to occur among different individuals. These allotypes are 4 represents naturally occurring amino acid substitutions in the constant region. For the Fab fragment heavy chain gene, V H The DNA encoding the heavy chain C H It may be operably linked to another DNA molecule encoding only one constant region.

[0154] V L The isolated DNA encoding the region is L The DNA encoding the light chain constant region, CL The light chain constant region can be converted into a full-length light chain gene by operably linking it to another DNA molecule encoding the light chain constant region. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0155] To generate the scFv gene, H and V L The DNA fragment encoding V H and V L The sequences are linked by a flexible linker L and V H A flexible linker, e.g., the amino acid sequence (Gly) is encoded so that the protein can be expressed as a continuous single-chain protein having a region. 4 -Ser) 3 (see, e.g., Bird et al., Science (1988) 242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85:5879-83; and McCafferty et al., Nature (1990) 348:552-554).

[0156] The present invention further provides vectors comprising one or more of the nucleic acid molecules provided by the present invention. In some embodiments, the vector is an expression vector useful for expressing an antibody described herein or an antigen-binding fragment of such an antibody. In some embodiments, vectors are provided herein, in which a first vector comprises a polynucleotide sequence encoding a heavy chain variable region described herein, and a second vector comprises a polynucleotide sequence encoding a light chain variable region described herein. In some embodiments, a single vector comprises polynucleotides encoding a heavy chain variable region described herein and a light chain variable region described herein.

[0157] To express the binding molecules of the present disclosure, DNA encoding partial or full-length light and heavy chains is inserted into an expression vector such that the DNA molecule is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" means that the antibody gene is ligated into a vector such that the transcriptional and translational control sequences in the vector perform their intended function of regulating the transcription and translation of the DNA molecule. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or both genes can be inserted into the same expression vector. The antibody gene is inserted into the expression vector by any suitable method (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or homologous recombination-based DNA ligation). The light and heavy chain variable regions of the antibodies described herein are used to ligate the V and VD chains into an expression vector already encoding heavy and light chain constant regions of the desired isotype and subclass. H The segment is C in the vector H operatively coupled to the segment(s), L The segment is C in the vector LBy inserting them operably linked to the segment, full-length antibody genes of any antibody isotype and subclass can be generated. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0158] In addition to the antibody chain genes, the expression vectors of the disclosure typically carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of the host cell to be transformed, the expression level of protein desired, and the like. Examples of regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the β-globin promoter can be used. Furthermore, regulatory elements are composed of sequences from various sources, such as the SR promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al. Mol. Cell. Biol. (1988) 8:466-72).

[0159] In addition to the antibody chain genes and regulatory sequences, expression vectors can carry additional sequences, such as enhancer elements, transcription termination sequences, sequences that regulate replication of the vector in a host cell (such as origins of replication), and selectable marker genes. The selectable marker gene facilitates selection of a host cell into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0160] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into a host cell by any suitable technique. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. The antibodies described herein can be expressed in either prokaryotic or eukaryotic host cells, although expression of the antibodies in eukaryotic cells, such as mammalian host cells, is most common.

[0161] The present invention further provides a host cell comprising the nucleic acid molecule or vector provided by the present invention. The host cell can be virtually any cell for which an expression vector is available. It can be, for example, a higher eukaryotic host cell such as a mammalian cell, a lower eukaryotic host cell such as a yeast cell, or a prokaryotic cell such as a bacterial cell. The introduction of the recombinant nucleic acid construct into the host cell can be achieved by calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, or phage infection.

[0162] Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0163] Mammalian host cells for expressing the binding molecules of the disclosure include, for example, Chinese hamster ovary (CHO) cells (including, for example, dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA (1980) 77:4216-20, used with DHFR, as described in Kaufman and Sharp, J. Mol. Biol. (1982) 159:601-21), NS0 myeloma cells, COS cells and Sp2 cells. Another expression system, particularly for use with NS0 myeloma or CHO cells, is the GS (glutamine synthetase) gene expression system disclosed in WO87 / 04462, WO89 / 01036, and EP338841.

[0164] The antibodies (or antigen-binding fragments thereof) of the present invention can be produced by any means known in the art. Exemplary techniques for antibody production are described in U.S. Pat. No. 4,816,567, but these exemplary techniques are provided for illustrative purposes only and are not limiting. Once a nucleic acid or expression vector encoding an antibody described herein is introduced into a host cell, the antibody is produced by culturing the host cell for a period of time sufficient for the antibody to be expressed within the host cell or secreted into the culture medium in which the host cell is grown. Thus, in some embodiments, a method of producing the antibodies described herein is provided, the method comprising culturing a host cell comprising one or more nucleic acids or vectors encoding an antibody (e.g., those provided above) under conditions suitable for expression of the antibody. In some embodiments, the method further comprises recovering the antibody from the host cell (or host cell culture medium). The antibody can be recovered from the culture medium using any suitable protein purification method.

[0165] VIII. Pharmaceutical Compositions In some embodiments, the present disclosure provides a composition comprising one or more antibodies described herein. In some embodiments, the composition is a pharmaceutical composition comprising an antibody described herein and a pharma- ceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0166] The term "pharmaceutical acceptable carrier" refers to any inert substance suitable for use in a formulation for delivery of a polypeptide (e.g., an antibody described herein). Carriers can be anti-adherents, binders, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption retardants, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutical acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), glucose, vegetable oils (such as olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyalcohols, sorbitol, and sodium chloride.

[0167] The composition may be in any suitable form, such as liquid, semi-solid, and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable and infusible solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. A particular form of the composition suitable for delivery of the compositions described herein is a sterile liquid, such as a solution, suspension, or dispersion for injection or infusion. Sterile solutions can be prepared by incorporating the antibody in the required amount in a suitable carrier, followed by sterile microfiltration. Dispersions are prepared by incorporating the antibody in a sterile vehicle containing a basic dispersion medium and other carriers. In the case of sterile powders for the preparation of sterile liquids, the preparation method includes vacuum drying and freeze-drying (lyophilization) to obtain a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution. Various dosage forms of the composition can be prepared by conventional techniques known in the art.

[0168] The relative amount of antibody included in the composition will vary depending on many factors, such as the particular polypeptide and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of antibody in a single dosage form will generally be that amount that produces a therapeutic effect, but may be a lesser amount. Generally, this amount will range from about 0.01 percent to about 99 percent, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent, based on the total weight of the dosage form.

[0169] In addition to the antibodies described herein, one or more additional therapeutic agents may be included in the composition, in some embodiments, at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiation therapy, and chemotherapy. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of pomalyst, Revlimid, lenalidomide, pomalidomide, thalidomide, DNA alkylating platinum-containing derivatives, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, radiation therapy, single radiation, fractionated radiation, focal radiation, whole organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, adoptively transferred T cells, anti-cancer vaccines, and oncolytic viruses. Suitable amounts of additional therapeutic agents to be included in the compositions can be readily selected by one of skill in the art and will vary depending on many factors, such as the particular agent and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of additional therapeutic agent included in a single dosage form will generally be that amount of agent that provides a therapeutic effect, but may be a lesser amount.

[0170] The antibodies described herein may be further modified. In some embodiments, the antibodies are linked to additional molecular entities. Examples of additional molecular entities include pharmaceutical agents, peptides or proteins, detection agents or labels, antibodies, etc.

[0171] In some embodiments, the antibodies described herein are linked to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents, and radioisotopes. Specific examples of cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, corticine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclosporine, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include iodine. 131 ,indium 111 ,yttrium 90 , and lutetium 177Methods of linking a polypeptide to a pharmaceutical agent are known in the art, including, but not limited to, using a variety of linker technologies. Exemplary linker types include hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. For further discussion of linkers and methods for linking therapeutic agents to antibodies, see, e.g., Saito et al., Adv. Drug Deliv. Rev. (2003) 55:199-215; Trail, et al., Cancer Immunol. Immunother. (2003) 52:328-337; Payne, Cancer Cell (2003) 3:207-212; Allen, Nat. Rev. Cancer (2002) 2:750-763; Pastan and Kreitman, Curr. Opin. Investig. Drugs 3: (2002) 1089-1091; Senter and Springer Adv. Drug Deliv. Rev. (2001) 53:247-264.

[0172] Any of the antibodies and / or compositions (e.g., pharmaceutical compositions) described herein can be used in the preparation of a medicament (e.g., a medicament for use in treating cancer or delaying the progression of cancer in a subject in need thereof).

[0173] IX. Treatment method The antibodies and pharmaceutical compositions described herein are useful for therapeutic purposes, such as treating cancer or improving the effectiveness of other cancer treatments.Thus, in some embodiments, the present disclosure provides a method of using the antibodies or pharmaceutical compositions described herein.In some embodiments, the present disclosure provides a method of treating cancer in a subject (e.g., a human subject), comprising administering to the subject an effective amount of an antibody described herein.In some embodiments, the cancer is breast cancer, liver cancer, or colon cancer, gastric cancer, ovarian cancer, lung cancer, pancreatic cancer, or kidney cancer.

[0174] When carrying out the method of treatment, the masked anti-CD28 antibody described herein may be administered alone, i.e., as a monotherapy, or in combination with one or more additional therapeutic agents or therapies. Thus, in another aspect, the present disclosure provides a combination therapy comprising a binding molecule in combination with one or more additional therapeutic agents or therapies for separate, sequential, or simultaneous administration. In some embodiments, the term "additional therapy" refers to a therapy that does not use an antibody described herein as a therapeutic agent. In some embodiments, the term "additional therapeutic agent" refers to any therapeutic agent other than the antibody described herein. In some embodiments, the present disclosure provides a method of treating cancer in a subject (e.g., a human subject), the method comprising administering to the subject an effective amount of an antibody described herein (e.g., an anti-CD28 antibody or a multispecific antibody that targets CD28 and one or more other targets) and an effective amount of an anti-PD-1 antibody. In some embodiments, the disclosure provides a method of treating cancer in a subject (e.g., a human subject), the method comprising administering to the subject an effective amount of an antibody described herein (e.g., an anti-CD28 antibody or a multispecific antibody that targets CD28 and one or more other targets) and an effective amount of an anti-CTLA4 antibody. In some embodiments, the anti-CTLA4 antibody is a masked anti-CTLA4 antibody.

[0175] X. KITS AND PRODUCTS In some embodiments, a kit is provided that includes one or more antibodies described herein (e.g., an anti-CD28 antibody or a multispecific antibody targeting CD28 and one or more other targets). In some embodiments, the kit further includes a package insert that includes instructions for use of the antibodies described herein. In some embodiments, the article of manufacture or kit includes a container that includes one or more of the masked antibodies or compositions described herein. In certain embodiments, the article of manufacture or kit includes a container that includes a nucleic acid encoding one (or more) of the masked antibodies described herein. In some embodiments, the kit includes cells of a cell line that produces an antibody described herein. In some embodiments, the kit includes CD28 (e.g., human CD28, cynomolgus monkey CD28, mouse CD28, rat CD28, or a fragment of any of the foregoing) or CD28 + One or more positive controls for the cells are included. In some embodiments, the kit includes a negative control, such as, for example, a surface or solution that is substantially free of CD28, or cells that do not express CD28.

[0176] In certain embodiments, the article of manufacture or kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous fluid bags, test tubes, and the like. The containers are formed from a variety of materials, including glass and plastic. The container holds an antibody (or a composition comprising such an antibody) as described herein, which is effective, alone or in combination with other compositions, for treating, delaying the progression of, and / or preventing cancer in a subject (e.g., a human subject). The container may be provided with a sterile access port (e.g., the container may be a vial or an intravenous fluid bag with a stopper pierceable by an intravenous needle). In some embodiments, the label or package insert indicates that the composition is used to treat breast cancer, liver cancer, or colon cancer in a subject (e.g., a human subject).

[0177] Additionally, the article of manufacture or kit may include (a) a first container housing a composition comprising an antibody (or immunologically active fragment thereof) as described herein, and (b) a second container housing a composition, the composition comprising an additional cytotoxic or other therapeutic agent. In some embodiments, the second container housing a composition comprising an anti-PD-1 antibody, and the article of manufacture includes a label or package insert indicating that the antibody and anti-PD-L1 are used to treat, such as colon cancer, in a subject (e.g., a human subject) in need thereof, e.g., according to the methods provided herein. In some embodiments, the second container housing a composition comprising an anti-CTLA4 antibody (e.g., a masked anti-CTLA4 antibody), and the article of manufacture includes a label or package insert indicating that the antibody and anti-CTLA4 antibody (e.g., a masked anti-CTLA4 antibody) as described herein are used to treat, such as colon cancer, in a subject (e.g., a human subject) in need thereof, e.g., according to the methods provided herein.

[0178] In addition, the article of manufacture may further comprise additional containers containing pharma- ceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution, etc. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0179] The above description is deemed sufficient to enable those skilled in the art to practice the present disclosure. The following examples are presented for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Indeed, in addition to the modifications shown and described herein, various modifications of the present disclosure will become apparent to those skilled in the art from the above description and will fall within the scope of the appended claims.

[0180] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. In the event of a conflict, this specification, including definitions, will control. Further, unless otherwise required by context, singular terms include the plural and plural terms include the singular. Throughout this specification and the embodiments, the words "having" and "including" or variations such as "having," "having," "including," or "including" are understood to mean the inclusion of a specified integer or group of integers but not the exclusion of other integers or groups of integers. All references cited herein, including patent applications, patents and non-patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. Although numerous documents are cited herein, this citation does not acknowledge that any of these documents constitutes part of the common general knowledge in the art. As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, the term refers to a value within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction (greater or less) of the stated reference value, unless otherwise stated or clear from the context.

[0181] According to this disclosure, a back reference in a dependent claim is intended as a shorthand for directly and unambiguously disclosing each and every combination of the claims indicated by the back reference. Any compound disclosed herein can be used in any of the treatment methods herein, and the individual to be treated is as defined anywhere herein. Furthermore, the headings herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any way.

[0182] In order that the present invention may be better understood, the following examples are presented: These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. EXAMPLES

[0183] Example 1: Generation of primary Fabs that specifically bind to human CD28

[0184] A proprietary phagemid library was used to pan against human CD28 antigen. A total of 3–4 rounds of panning were performed. After the final round of panning, culture supernatants of individual clones were tested by ELISA to identify those that specifically recognized human CD28 (i.e., primary hits). Clones were defined as positive when the ELISA signal was at least twice the background. Positive clones were selected and sequence confirmed, and Fabs corresponding to unique hits were expressed in E. coli and affinity purified. Affinity to human CD28 was measured by Octet® RED96 Systems (ForteBio). Briefly, human or mouse CD28-Fc fusion proteins were captured using Dip and Read Anti-Human IgG Fc Capture (AHC) biosensors (ForteBio) and immersed into wells containing purified Fab diluted 5–10-fold in ForteBio kinetic buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% v / v surfactant P20, pH 7.4). Acquired data were processed with Octet Data Analysis Software Version 7.1 (ForteBio) and kinetic data were fitted to a 1:1 Langmuir binding model.

[0185] The primary Fab hits were further characterized for cross-reactivity with human and mouse CD28 species by ELISA, and from these primary hits, a panel of 46 unique sequence Fabs were identified, each of which contained an IgG1 Fab with the core hinge mutation S241P (Kabat numbering scheme). 4 They were converted into isotype mAbs and subjected to detailed biophysical and functional characterization (Table 5 ).

[0186] Example 2: IgG 4 Transformation and Expression

[0187] Engineered human anti-CD28 IgG as listed in Table 5 4The heavy and light chains of isotype mAbs were cloned into the mammalian expression vector pcDNA3.3 (ThermoFisher Scientific). A pair of plasmids carrying one heavy chain and one light chain was transiently transfected into HEK293 cells following the manufacturer's instructions. After incubation, the supernatant was collected and clarified by centrifugation and filtration, and IgG 4 MabSelect isotype mAbs TM SuRe TM Capture was performed by protein A affinity chromatography (GE Healthcare).

[0188] mAbs were eluted and neutralized, and the elution buffer was exchanged into renaturation buffer (20 mM histidine, pH 5.5). Protein concentration was measured by UV spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing, and non-reducing conditions. [Table 5-1] [Table 5-2]

[0189] Example 3: Binding characteristics of anti-CD28 antibodies

[0190] The binding affinity of the anti-CD28 mAbs panel to human, cynomolgus monkey, mouse CD28 and human CTLA4 was measured by Octet® RED96 Systems (ForteBio), ELISA, and CytoFlex flow cytometry (Beckman). The anti-CD28 antibodies TAC2386 (also known as TGN1412 as described in patent WO2006 / 050949A2) and TAC2387 (described in patent WO2019 / 246514A2) were used as benchmark controls.

[0191] Binding affinity to human CD28 and CTLA4 by the Octet RED96 system

[0192] The binding kinetics of a panel of anti-CD28 mAbs to human CD28 and CTLA4 were assessed using Octet® RED96 Systems (ForteBio). Briefly, mAbs were diluted to 15 μg / mL in kinetic buffer (PBS supplemented with 0.02% Tween 20 and 0.1% BSA) and captured in parallel by Dip and Read AHC biosensors (ForteBio). The sensors were then allowed to bind with His-tagged human CD28 and CTLA4 proteins (100 nM) for 300 s and dissociated in kinetic buffer for an additional 300 s. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using Octet® Data Analysis Software Version 7.1 (ForteBio).

[0193] As shown in Table 6, the panel of anti-CD28 test and benchmark mAbs demonstrated high binding affinity (<10 nM) for human CD28 as measured by the Octet® RED96 Systems. Additionally, test mAbs TY24773, TY24853, TY24854, TY24860, TY24865, and TY24871 as well as the benchmark control mAbs demonstrated no detectable affinity for human CTLA4. [Table 6]

[0194] Binding affinity to human and mouse CD28 using ELISA

[0195] Recombinant human and mouse CD28-Fc were diluted to 2 μg / mL in PBS and incubated with Nunc MaxiSorp TMHigh protein binding capacity 96-well ELISA plates (ThermoFisher Scientific) were coated and left overnight at 4 °C. The plates were blocked with PBS supplemented with 3% nonfat milk for 1 h at 37 °C. After washing, 50 μL of 3-fold serial dilutions of anti-CD28 test mAbs panel were added to each well. After 1 h incubation at 37 °C, the plates were washed four times and 100 μL of horseradish peroxidase (HRP)-conjugated anti-human IgG (Fab specific) (1:6000 dilution) secondary antibody was added to each well. The plates were incubated for 1 h at 37 °C and washed four times, after which 50 μL of TMB substrate (3,3',5,5'-tetramethylbenzidine) solution was added to each well and the plates were incubated at room temperature. The reaction was stopped by adding 50 μL of sulfuric acid stop solution to each well, after which the absorbance at 450 nm was measured. EC 50 was assessed by fitting the ELISA data with an asymmetric sigmoidal (four-parameter logistic equation) model in GraphPad Prism version 7 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com.

[0196] As shown in Table 7 and Figure 1, the panel of anti-CD28 test mAbs showed similar affinity for human CD28 as the two benchmark controls. Furthermore, all test mAbs, except for TY24890, bound to mouse CD28, whereas the two benchmark controls did not. [Table 7]

[0197] Binding activity of anti-CD28 antibodies to Jurkat cells

[0198] Jurkat (clone E6-1) cells were plated in a 96-well plate at 1.0 × 10 5(50 μL / well) and incubated with serially diluted benchmark positive control, isotype negative control antibodies, and anti-CD28 test mAbs panel (100, 20, 4, 0.8, 0.16, and 0.032 nM) in 2% fetal bovine serum / Dulbecco's PBS (FBS / DPBS) for 30 min at 4 °C. Cells were then washed twice with DPBS and further incubated with APC anti-human IgG Fc secondary antibody (1 μg / mL, 100 μL / well, Biolegend) for 30 min at 4 °C. Finally, cells were washed twice with DPBS and suspended in FACS buffer for flow cytometry analysis. For analysis, mean fluorescence intensity (MFI) values ​​versus concentration were plotted using FlowJo 10 software (FlowJo LLC), and data were further fitted using a four-parameter nonlinear regression, and ECs were analyzed with GraphPad Prism version 7 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com. 50 Got the value.

[0199] As shown in Table 8, all the anti-CD28 test mAbs except for TY24775 and TY24867 showed high binding activity (EC 50 The binding activity measured was comparable to that of two benchmark controls. [Table 8]

[0200] In vitro binding of anti-CD28 antibodies to human T cells by flow cytometry

[0201] Human CD3 + T cells are TMHuman T cells were isolated from cryopreserved peripheral blood mononuclear cells (PBMCs) using the Human Naive Pan T Cell Isolation Kit (STEMCELL Technologies). Isolated human T cells were plated in 96-well plates at 1.0 × 10 5 Cells were added at 100 nM / well and incubated with 100 nM of benchmark control, isotype negative control antibody, and anti-CD28 test mAbs panel in FACS buffer for 30 min on ice. Cells were then washed three times with PBS and further incubated with PE-conjugated secondary antibodies for 30 min on ice. Finally, cells were washed three times with PBS and resuspended in FACS buffer for flow cytometry analysis. For analysis, MFI values ​​were calculated using FlowJo 10 software (FlowJo LLC) and geometric mean MFI values ​​were plotted against mAbs using GraphPad Prism version 7 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com.

[0202] As shown in Figure 2, when compared to the benchmark controls TAC2386 and TAC2387, all test mAbs except TY24772 showed comparable MFI values, with binding of TY24772 being approximately twice that of the benchmark controls.

[0203] Example 4: Ligand competition using Elisa

[0204] Antibodies were tested for their ability to inhibit the binding of CD28 or CTLA4 to its natural ligand, CD80, by ELISA.

[0205] As shown in Figure 3 and Table 9, compared to the anti-CTLA4 antibody TY21580 included as a control, the benchmark controls TAC2386 and TAC2387 and all anti-CD28 test mAbs tested inhibited CD28 binding to CD80, but none inhibited CTLA4 binding to CD80. [Table 9]

[0206] Example 5: Evaluation of superagonism in a dry-coat human T-cell proliferation assay

[0207] The levels of lymphocyte proliferation induced by the benchmark controls TAC2386 and TAC2387, an isotype negative control antibody, and a panel of anti-CD28 test mAbs were measured by the CellTiter-Glo® Luminescent Cell Viability Assay (Promega). In this assay, 100 nM of test mAbs were added in triplicate to a 96-well microplate at 50 μL / well and allowed to air dry directly onto the walls of the wells. The microplates were then washed twice with PBS. Cryopreserved PBMCs were reconstituted and cultured in 10% FBS / RPMI1640 at a cell density of 5.0 × 10 5 Adjust to 1.0 x 10 cells / mL 5 PBMCs (200 μL per well) were added to the pre-coated microplates. The cells were incubated at 37°C and 5% CO 2 for 72 h, after which lymphocyte proliferation was assessed using the CellTiter-Glo® assay.

[0208] As shown in Figure 4A, the level of lymphocyte proliferation induced by immobilized benchmark control TAC2386 (a known CD28 superagonist) was significantly higher than all test mAbs. Compared to TAC2386, the benchmark control TAC2387 and the anti-CD28 test mAbs including TAC2387, TY24865, TY24866, TY24876, TY24878, TY24879, and TY24884 showed weak superagonist activity in vitro.

[0209] Example 6: Effect of anti-CD28 antibodies on human T cell activation in vitro

[0210] T cell activation costimulation assay: IFN-γ release

[0211] The biological activity of anti-CD28 mAb as an agonistic T cell costimulator in activating human T cells in vitro was measured by IFN-γ cytokine secretion using ELISA. Ultra-LEAF TM Purified anti-human CD28 antibody (Biolegend) was included as a positive control.

[0212] Human CD3 + T cells are TM Cells were isolated from cryopreserved PBMCs using the Human Naive Pan T Cell Isolation Kit (STEMCELL Technologies). Isolated cells were plated onto 96-well tissue culture plates (1.0 × 10 per well) pre-coated with a suboptimal concentration (10 nM, 50 μL per well) of anti-human CD3 antibody (OKT3) in the presence of serially diluted benchmark controls, isotype negative control antibodies, a commercial anti-human CD28 positive control antibody, and an anti-CD28 test mAb panel (0.1, 1, 10, and 100 nM). 5 The cells were cultured at 37°C and 5% CO 2The cells were cultured at 4°C for 120 h, after which cell supernatants were harvested for IFN-γ cytokine analysis by ELISA and T cell proliferation was measured by CellTiter-Glo®.

[0213] As shown in Figures 4B and 4C, compared with isotype control antibodies, anti-CD28 mAbs exhibited concentration-dependent biological activities, including T cell proliferation and IFN-γ cytokine secretion. In general, the effects of anti-CD28 mAbs on human T cell activation were as potent or more potent than the two benchmark control antibodies.

[0214] T cell activation costimulation assay: IL-2 release

[0215] The biological activity of anti-CD28 mAbs as agonistic T cell costimulators in the activation of human T cells in vitro was measured by T cell proliferation using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) and IL-2 cytokine secretion using ELISA. TM Purified anti-human CD28 antibody (Biolegend) was included as a positive control.

[0216] EasySep TM Human T cells were isolated from fresh PBMCs from Asian donors using the Human Naive Pan T Cell Isolation Kit (STEMCELL Technologies). Cells were plated in 96-well tissue culture plates (1.0 × 10 ) pre-coated with a suboptimal concentration (5 nM) of anti-human CD3 antibody (OKT3) in the presence of serially diluted benchmark control TAC2387, an isotype negative control antibody, a commercial anti-human CD28 positive control antibody, and a panel of anti-CD28 test mAbs. 5 The cells were incubated at 37°C and 5% CO 2The cells were incubated for 72 hours after which cell supernatants were harvested for IL-2 cytokine analysis by ELISA and levels of T cell proliferation were measured by CellTiter-Glo® assay.

[0217] As shown in Figure 5A-B, compared to the isotype control antibody, anti-CD28 antibody showed concentration-dependent biological activity including T cell proliferation and IL-2 cytokine secretion. The effects of TY24859, TY24865, TY24866, and TY24890 on human T cell activation were comparable to that of the benchmark control TAC2387. The negative control group without pre-coating with anti-CD3 showed no detectable T cell proliferation or IL-2 cytokine secretion.

[0218] Example 7: Construction and functional characterization of anti-HER2 x CD28 bispecific antibodies

[0219] Generation of anti-HER2 × CD28 bispecific antibodies

[0220] TYM13 Fc mutant (D or E356K:E357K:S364K:S400C L351'D:K370'D:N390'C:K439'D, IgG1 C H A heterodimeric bispecific scaffold was designed using a 3-domain (following the Kabat numbering scheme) with a light-heavy half-antibody and a single-chain fragment variable (scFv)-Fc chain to form a bispecific antibody (BsAb) and TOM13 mutations in the hetero-Fc domain.

[0221] Plasmids encoding the heavy, light and scFv-Fc chains of BsAbs were transiently transfected into mammalian cells. Cell culture supernatants containing bispecific antibodies were harvested 7 days after transfection by centrifugation at 14000 g for 30 min and filtered through a sterile filter (0.22 μm). Antibodies were purified using MabSelect TM SuRe TMIt was purified by protein A affinity chromatography using a prepacked column (GE Healthcare) and then buffer exchanged into 20 mM histidine (pH 5.5) buffer.

[0222] TY24865 (high affinity CD28) and TY24865 mutants (low affinity CD28) were selected to construct CD28 BsAbs using this scaffold. The components are described in Table 10. [Table 10]

[0223] Binding to SK-OV-3 cells by FACS

[0224] Concentration-dependent binding activity of anti-HER2xCD28 BsAb (TY27566), anti-HER2xCD3 BsAb (TY25238, also described in PCT / CN2021 / 076626, incorporated herein by reference in its entirety), and anti-HER2 mAbs to Perjeta® (TAC2319) or Herceptin® (TAC2320) was measured using flow cytometry. Perjeta® (TAC2319) binds to a different epitope in the HER2 dimerization domain than Herceptin® (TAC2320).

[0225] Culture SK-OV-3 cells at 8.0 x 10 in a 96-well plate. 4Cells were added at 1000 cells / well and incubated with test BsAbs serially diluted in 2% FBS / RPMI1640 buffer for 60 min at 4 °C. Next, cells were washed twice with DPBS and further reacted with secondary APC-anti-human IgG Fc antibody (1:400 dilution) for 30 min at 4 °C. Finally, cells were washed twice with DPBS and resuspended in FACS buffer for flow cytometry analysis. For analysis, MFI values ​​were plotted versus concentration using FlowJo 10 software (FlowJo LLC), and data were further fitted using a four-parameter nonlinear regression and EC was calculated using GraphPad Prism version 7 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com. 50 Values ​​were obtained. Isotype antibodies were included as negative controls.

[0226] As shown in Figure 6, anti-HER2×CD28 or anti-HER2×CD3 BsAbs showed very similar binding affinity to SK-OV-3 cells compared to Perjeta ® (TAC2319) or Herceptin ® (TAC2320) anti-HER2 mAbs.

[0227] Stimulatory effects on T cell receptor and CD28 receptor signaling

[0228] The stimulatory activity of anti-HER2×CD3 BsAb, anti-HER2×CD28 BsAb, or their combination on T cell receptor (TCR) and CD28 receptor signaling was assessed. In this assay, coactivation of TCR and CD28 enhances the transcriptional activity of NFkB, inducing the production of a reporter gene. Isotype antibodies were included as negative controls.

[0229] Jurkat-NFkB-Nluc effector (E) reporter cells (5 × 10 4 cells / well) were cultured with SK-OV-3 target (T) cells that endogenously express the HER2 antigen (1 × 104 Serially diluted anti-HER2×CD3 BsAbs or isotype control antibodies were added to the reporter cell system with a fixed concentration of anti-HER2×CD28 BsAb (10 nM), or conversely, serially diluted anti-HER2×CD28 BsAbs or isotype control antibodies were added to the reporter cell system with a fixed concentration of anti-HER2×CD3 BsAb (0.01 nM) to evaluate their combinatorial effects in stimulating downstream luciferase activity. Co-cultured cells were incubated at 37°C in 5% CO 2 The cells were incubated at 4°C for 6 h. Then, 100 μL of Nano-Glo® Luciferase Assay System (Promega) reagent was added to the cells and the cells were lysed for 10 min. The supernatant (100 μL) was collected and luminescence measurements were performed using a SpectraMax® i3x Multi-Mode Microplate Reader (Molecular Devices).

[0230] As shown in Figure 7, anti-HER2×CD28 BsAb (TY27566) combined with a fixed concentration of anti-HER2×CD28 BsAb (TY25238) and anti-HER2×CD3 BsAb combined with a fixed concentration of anti-HER2×CD28 BsAb showed the highest maximal signal and EC 50 The results showed a synergistic or enhanced stimulatory effect on the values.

[0231] Cytotoxicity of anti-HER2×CD28 BsAb and anti-HER2×CD3 BsAb with the same or different TAA epitopes

[0232] In vitro cytotoxic activity of anti-HER2 × CD3 BsAb (TY25238) alone or in combination with anti-HER2 × CD28 BsAb (TY27566 or TY27807) against MCF-7 tumor cell line was measured using lactate dehydrogenase (LDH) release assay. TY25238 binds to a tumor-associated antigen (TAA) epitope in the HER2 dimerization domain that is distinct from TY27566 but the same TAA epitope as TY27807.

[0233] Human T cells were isolated from cryopreserved PBMCs. Cultured MCF-7 tumor target cells (1 × 10 4 cells / well) were incubated for 30 min at 37 °C with serially diluted anti-CD3 BsAb FG14127 alone or in combination with a fixed concentration (1 μg / mL) of two anti-CD28 BsAbs against different TAA epitopes TY27566 or TY27807. Then, isolated human T effector cells (2 × 10 4 Add 100 cells / well and incubate at 37°C, 5% CO 2 (E:T = 2:1) for 72 h. Cytotoxicity, based on LDH release into the supernatant from killed MCF-7 target cells, was quantified using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega).

[0234] As shown in Figure 8, anti-HER2xCD3 BsAb (TY25238) as a single agent induced potent concentration-dependent cytotoxicity against MCF-7 target cells. The combination of anti-HER2xCD28 BsAb (TY27566) and anti-HER2xCD3 BsAb (TY25238) against different TAA epitopes enhanced EC 50 In terms of the HER2×CD3 BsAb (TY25238) alone, the addition of anti-HER2×CD28 BsAb (TY27807) with the same TAA epitope further enhanced the in vitro tumor killing activity. 50 and significantly reduced MCF-7 tumor cell lysis with respect to maximum lysis. Isotype antibodies were included as negative controls in single-agent assays.

[0235] Example 8: Construction and functional characterization of anti-TROP2xCD28 or anti-TROP2xCD3 BsAbs

[0236] Anti-tumor associated calcium signaling receptor 2 (TROP2) x CD28 or anti-TROP2 x CD3 heterodimeric bispecific scaffolds were designed using the TYM13 Fc variant as described in Example 7. The composition of the anti-TROP2 x CD28 BsAb is listed in Table 11.

[0237] The composition of the anti-TROP2 × CD3 BsAbs is listed in Table 12 . [Table 11] [Table 12]

[0238] CD3- or CD28-based bispecific binding to TROP2 high, medium, and low expressing tumor cell lines

[0239] Flow cytometry was used to measure the concentration-dependent binding activity of anti-TROP2 BsAbs against a range of tumor cell lines with high, intermediate or low TROP2 expression. Isotype antibodies were included as negative controls.

[0240] Culture H292, NCI-N87, or HT29 cells at 1.0 × 10 in 96-well plates. 5Cells / well were seeded and incubated with test anti-TROP2 BsAbs serially diluted in 2% FBS / RPMI1640 buffer for 30 min at 4 °C. Then, cells were washed twice with DPBS and further incubated with secondary anti-human IgG antibodies (APC-anti-human IgG Fc antibody for H292 cells, 1:300 dilution; APC-F(ab')2 fragment goat anti-human IgG (H+L), 1:500 dilution for NCI-N87 and HT29 cells) for 30 min at 4 °C. Finally, cells were washed twice with DPBS and resuspended in FACS buffer for flow cytometry analysis. For analysis, MFI values ​​vs. concentration were plotted using FlowJo 10 software (FlowJo LLC), and data were further fitted using a four-parameter nonlinear regression and ECs were calculated with GraphPad Prism version 7 for Windows. 50 Got the value.

[0241] As shown in Figure 9 and Table 13, the TROP2 arm binding activity of TY25839 and TY27571 was similar in three different tumor cell lines with high, intermediate, or low TROP2 expression. [Table 13]

[0242] Stimulatory effects on TCR and CD28 receptor signaling

[0243] The stimulatory activity of anti-TROP2 × CD3 BsAbs, anti-TROP2 × CD28 BsAbs, or the combination on TCR and CD28 receptor signaling was assessed. Coactivation of TCR and CD28 enhances the transcriptional activity of NFkB, inducing the production of a reporter gene.

[0244] Jurkat-NFkB-Nluc effector reporter cells (5x10 4The reporter cells (cells / well) were co-cultured with H292 target cells (E:T=5:1). Serially diluted anti-TROP2×CD3 BsAbs were added to the reporter cell system with a fixed concentration of anti-TROP2×CD28 BsAb (5 nM), or conversely, serially diluted anti-TROP2×CD28 BsAbs were added to the reporter cell system with a fixed concentration of anti-TROP2×CD3 BsAb (0.01 nM) to assess their combined activity in stimulating downstream luciferase activity. The co-cultured cells were incubated at 37°C in 5% CO 2 The cells were incubated at 4°C for 6 h. Then, 100 μL of Nano-Glo Luciferase Assay System (Promega) reagent was added to the cells and the cells were lysed for 10 min. The supernatant (100 μL) was harvested and luminescence was measured using a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices).

[0245] As shown in Figure 10, single-agent CD28-based BsAbs showed very weak reporter gene activity. However, the combination of anti-TROP2 x CD3 and anti-TROP2 x CD28 BsAbs showed no significant difference in maximal signal or EC 50 showed synergistic or enhanced stimulatory effects with respect to value.

[0246] Example 9: Construction and functional characterization of B7H3xCD28 bispecific antibodies

[0247] A B7H3×CD28 BsAb was constructed, the components of which are described in Table 14.

[0248] The composition of the B7H3×CD3 BsAbs is listed in Table 15. [Table 14] [Table 15]

[0249] Binding to MDA-MB-231 cells by FACS

[0250] The concentration-dependent binding activity of anti-B7H3 x CD28 BsAb TY27556 ​​and its parental anti-B7H3 mAb TY21601 to MDA-MB-231 cells was measured using flow cytometry (Figure 11). An isotype antibody was included as a negative control.

[0251] Culture MDA-MB-231 cells at 1.0 x 10 in a 96-well plate. 5 Cells / well were seeded and incubated with serially diluted test BsAbs in 2% FBS / RPMI1640 buffer for 60 min at 4 °C. Then, cells were washed twice with DPBS and further incubated with secondary APC anti-human IgG Fc antibody (1:400 dilution) for 30 min at 4 °C. Finally, cells were washed twice with DPBS and resuspended in FACS buffer for flow cytometry analysis. For analysis, MFI values ​​vs. concentration were plotted using FlowJo 10 software (FlowJo LLC), and data were further fitted using a four-parameter nonlinear regression and ECs were calculated using GraphPad Prism version 7 for Windows. 50 Got the value.

[0252] As shown in FIG. 12, anti-B7H3 mAb TY21601 exhibited sub-nM (0.4525 nM) binding affinity to MDA-MB-231 target cells, whereas the binding activity of BsAb TY27556 ​​to the target cells was reduced by approximately 28-fold (12.84 nM).

[0253] B7H3xCD28 bispecific antibody enhanced the ability of PD-1 or PD-L1 blockade to induce T cell activation in vitro

[0254] The effect of combining anti-PD-1 blocking mAbs with B7H3xCD28 bispecific Abs on primary human T cell activation in vitro was measured by IFN-γ and IL-2 secretion by ELISA. Here, we used a modified mixed lymphocyte reaction (MLR) that simulates physiological PD-L1 expression and TCR / CD3 stimulation. To generate the one-way MLR assay, human T cells (1 × 10 per well) from one healthy donor (D#XC11147W) were cultured in 1 well of 10 wells. 5 Cells) were incubated with allogeneic MDA-MB-231 cells (E:T=5:1) in the presence of different test antibodies (B7H3xCD28 or isotype control) alone or in combination with anti-PD-1 (pembrolizumab, Keytruda®) or anti-PD-L1 (atezolizumab, Tecentriq®). Cells were then incubated in an incubator at 37°C and 5% CO 2 The cells were co-cultured for 120 hours. The cytokine secretion of IL-2 (72 hours) and IFN-γ (120 hours) into the supernatant by activated T cells was quantified using an ELISA kit.

[0255] As shown in Figure 13A-13B, addition of 100 nM PD-1 / PD-L1 mAbs or titrated B7H3xCD28 did not result in any or only slight cytokine release in this MLR assay. However, the combination of B7H3xCD28 with 100 nM PD-1 / PD-L1 mAbs significantly increased T cell activation compared to monotherapy. These results suggest that the B7H3xCD28 bispecific antibody can bind synergistically with PD-1 / PD-L1 inhibitors to promote T cell activation in the presence of tumor cells that endogenously express PD-L1 and B7H3.

[0256] Costimulatory bispecific antibodies enhance in vitro T cytotoxicity against MCF-7 cells upon bidirectional engagement

[0257] The in vitro tumor cell killing activity of anti-CD3-based, or anti-CD28-based BsAbs, or their combination against MCF-7 tumor cell line was measured using an LDH release cytotoxicity assay.

[0258] Human T cells were isolated from cryopreserved PBMCs. MCF-7 cells (1 × 10 4 cells / well) were combined with serial dilutions of anti-HER2×CD3 BsAb TY25238, or fixed concentrations of high affinity CD28 arm anti-HER2×CD28 BsAb TY27566, anti-CD28×B7H3 TY27556, or low affinity CD28 arm anti-HER2×CD28 BsAb TY27881 (1 ug / mL or 10 ug / mL) and incubated at 37°C for 30 minutes with human T cells (2×10 4 Add 100 cells / well and incubate at 37℃ in 5% CO 2 and incubated for 72 h (E:T = 2:1). Cytotoxicity, based on LDH release into the supernatant from killed MCF-7 target cells, was quantified using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega).

[0259] As shown in Figure 14, CD28-based BsAbs as single agents did not induce cytotoxicity against MCF-7 target cells. In contrast, as a single agent, the anti-CD3 BsAb TY25238 showed a concentration-dependent effect, with the highest cytotoxicity induced by the BsAb against HER2, which lysed 54.47% of MCF-7 cells. The combination of anti-CD3 and anti-CD28 BsAbs significantly reduced EC 50 Addition of anti-CD28 BsAb TY27566, which is equipped with a high affinity CD28 arm, significantly enhanced tumor cell lysis by approximately 10-fold compared to treatment with anti-CD3 BsAb TY25238 alone, which further enhanced in vitro tumor cell killing activity as revealed by a reduction in EC 50 However, when combined with the anti-CD28 BsAb TY27881, which has a low affinity CD28 arm, no enhanced cytotoxicity was observed.

[0260] Costimulatory bispecific antibodies enhance in vitro T cytotoxicity against EMT-6-HER2 cells upon bidirectional engagement

[0261] The in vitro tumor cell killing activity of anti-CD3-based or anti-CD28-based BsAbs, or their combination on the EMT-6-HER2 tumor cell line, was measured using an LDH release cytotoxicity assay.

[0262] Human T cells are TM EMT-6-HER2 (5 × 10 3 cells / well) target cells were incubated for 30 min at 37 °C in combination with serial dilutions of anti-HER2 × CD3 BsAb (TY25238) or a fixed concentration of anti-B7H3 × CD28 BsAb TY27556 ​​(8 nM), and conversely, EMT-6-HER2 (5 × 10 3 (2 × 10 cells / well) cells were incubated with serial dilutions of anti-B7H3 × CD3 BsAb TY26999 or a fixed concentration of anti-HER2 × CD28 BsAb TY27566 (8 or 0.8 nM) for 30 min at 37 °C. Isolated human T effector cells (2 × 10 4 Add 100 cells / well and incubate at 37°C, 5% CO 2 The cells were incubated for 72 h at 4°C (E:T = 2:1). Cytotoxicity based on LDH release into the supernatant from killed EMT-6-HER2 target cells was quantified using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega). Isotype antibodies were included as negative controls for single-agent assays.

[0263] As shown in Figure 15, TY27556 ​​(anti-B7H3 x CD28) or TY27566 (anti-HER2 x CD28) did not demonstrate killing efficacy against EMT-6-HER2 cells as single agents. TY25238 in combination with TY27556 ​​reduced EC 50 This resulted in an approximately two-fold decrease in maximal mortality and an approximately three-fold increase in maximum mortality (from 17% to 50%).

[0264] TY26999 combined with TY27566 had a lower EC 50 This resulted in an approximately 1.3-fold decrease in mitochondrial function and an approximately 2-fold increase in maximum mortality (from 23% to 47%).

[0265] Risk of in vivo systemic cytokine release by anti-CD3 or anti-CD28 based bispecific antibodies or their combination

[0266] The risk of in vivo systemic cytokine release (IL-6 and IFN-γ) of anti-mouse CD3- or anti-CD28-based BsAbs alone or in combination in a BALB / c mouse model was measured using ELISA.

[0267] BALB / c mice were randomly divided into four groups (three per group) and injected with anti-mouse CD3 mAb (145-2C11 clone, 1 mg / kg), anti-B7H3×CD28 (TY27556, 2 mg / kg), anti-B7H3×CD3 (TY27042, 2 mg / kg, where the CD3 arm is derived from the mouse-specific 145-2C11 clone), or their combination (TY27556, 2 mg / kg and TY27042, 2 mg / kg). Mouse serum and whole blood were collected at various time points before and after injection (pre-dose, 3.5, and 24 hours). Systemic cytokine release risk was assessed with IL-6 and IFN-γ by ELISA. The percentage of total peripheral T cells that were CD3+ T cells was measured by flow cytometry at each time point.

[0268] As shown in Figure 16, mice administered anti-mCD3 or anti-B7H3×CD3 (TY27042) showed a significant induction of cytokine (IL-6 and IFN-γ) release 3.5 hours after test antibody treatment. Mice treated with anti-B7H3×CD28 (TY27556) showed no detectable cytokine release after test antibody treatment. Furthermore, the combination of anti-B7H3×CD3 (TY27042) and anti-B7H3×CD28 (TY27556) did not show an increased risk of cytokine release compared to anti-B7H3×CD3 (TY27042) alone. Peripheral CD3 +T cells were rapidly reduced to almost zero in the single-agent anti-CD3, anti-B7H3×CD3 (TY27042), anti-B7H3×CD28 (TY27556) groups, and the combined anti-B7H3×CD3 (TY27042) and anti-B7H3×CD28 (TY27556) groups. CD3 remaining in peripheral blood 3.5 hours after injection + Only about 2% to 3% were T cells.

[0269] Example 10: In vivo efficacy studies

[0270] In vivo efficacy study of B7H3xCD28 BsAb monotherapy or in combination with HER2xCD3 BsAb in the SK-OV3 model

[0271] Immunodeficient M-NSG mice (n = 8 per group, female, 7–8 weeks old) were injected intraperitoneally with 5 × 10 6 PBMCs were then transplanted. Seven days later, mice were transplanted with 2 × 10 6 SK-OV3 cells were inoculated subcutaneously. Treatment was initiated 8 days after tumor inoculation, when the mean tumor volume was approximately 90 mm 3 In mice, hIgG 1 Isotype control 5 mg / kg, anti-HER2×CD3 bispecific double masked antibody 0.2 mg / kg, anti-B7H3×CD28 bispecific antibody TY27556 ​​5 mg / kg, or a combination of TY27151 0.2 mg / kg and TY27556 ​​5 mg / kg were administered by intraperitoneal injection. TY27151 was previously described in PCT / CN2021 / 076626. Mice were administered these antibodies twice weekly for a total of five doses. Tumor growth was monitored twice weekly and reported as mean tumor volume ± sem over time.

[0272] As shown in FIG. 17, in this model, the double-masked anti-HER2×CD3 bispecific antibody TY27151 exhibited potent synergistic anti-tumor effects with the anti-B7H3×CD28 bispecific antibody TY27556.

[0273] In vivo efficacy study of CD28 BsAbs in an EMT6-HER2 mouse breast cancer syngeneic model

[0274] BALB / c mice (n = 5 per group, female, 8-9 weeks old) were injected with 5 × 10 5 EMT-HER2 cells were inoculated subcutaneously. Treatment was initiated 7 days after tumor inoculation, when the mean tumor volume was approximately 110 mm 3 Mice received vehicle, anti-B7H3×CD28 BsAb TY27556 ​​at 0.5 mg / kg and 0.05 mg / kg, or anti-HER2×CD28 bispecific antibody TY27566 at 0.2 mg / kg by intraperitoneal injection. Mice received these antibodies twice weekly for a total of four doses. Tumor growth was monitored twice weekly and reported as mean tumor volume ± sem over time.

[0275] As shown in Figure 18, the anti-B7H3xCD28 bispecific antibody TY27556 ​​exhibited a dose-dependent anti-tumor effect in this model. As shown in Figure 18, the anti-HER2xCD28 bispecific antibody TY27566 exhibited a strong anti-tumor effect.

[0276] Example 11: Methods for identifying self-blocking peptides for masked anti-CD28 antibodies

[0277] Generation of masked anti-CD28 antibodies

[0278] A screening system was designed and implemented to efficiently discover masking moieties that could effectively mask the unmasked parent anti-CD28 antibody with good developability. In this system, the target anti-CD28 scFv was first displayed on the surface of yeast and confirmed to be functional in binding to the CD28 antigen. Then, a masking peptide (MP) from the improved MP peptide library was directly fused to the N-terminus of the light chain of the target anti-CD28 scFv to construct a yeast library that displays the fusion protein on the yeast surface. The yeast library was then subjected to several rounds of FACS-based screening. 1) Yeast clones with low binding to the antigen were enriched, 2) the enriched yeast clones were treated with protease to remove the N-terminal MP, and 3) clones with high binding to the antigen were selected. After 5-6 rounds of selection, plasmids were extracted from these clones and the MP sequences were confirmed by DNA sequencing. The masked anti-CD28 antibody clones selected in scFv format showed little binding to the antigen in the presence of MP. However, treatment of yeast cells with tobacco etch virus nuclear inclusion alpha-endopeptidase (TEV) to remove MPs dramatically increased binding to the antigen. Incorporation of a TEV recognition site as a cleavage site in the MPs, combined with application of TEV protease to validate selected clones, significantly increased the success rate of MP selection.

[0279] To identify the MP sequences, shuttle plasmids were extracted from the selected yeast clones using a plasmid extraction kit (Generay) and transformed into competent E. coli cells. Plasmids were prepared and the MP-encoding regions were sequenced and aligned. As expected, these sequences fell into several groups, indicating clear enrichment through the rounds of sorting. The MPs selected for masking efficiency are shown in Table 16 below, and the sequences of each MP are shown in the sequence section below.

[0280] The masked anti-CD28 scFv protein is IgG 4Converted to isotype mAbs. Masked IgG 4 The mAbs were engineered to contain an MP with a single invariant matrix metalloproteinase (MMP) cleavage site fused to the N-terminus of the light chain in the same manner as it is displayed on the yeast surface. The heavy and light chains were cloned separately into the mammalian expression vector pCDNA3.3 (Thermo Fisher Scientific). The V of the parent anti-CD28 antibody H Array and V L The sequence (TY24865) is listed in the Sequences section below.

[0281] The plasmid pairs were transiently transfected into HEK293F cells. After 6 days, the supernatants were harvested and clarified by centrifugation and filtration, and IgG was purified by standard Protein A affinity chromatography (MabSelect SuRe, GE Healthcare). IgG was eluted, neutralized, and buffer exchanged into 20 mM histidine, pH 5.5 buffer. Protein concentration was measured by UV spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing, and non-reducing conditions. Importantly, expression levels of the masked antibodies in HEK293 cells were comparable or lower than those of the parental antibodies, and purification yields after Protein A resin were similar. This suggests that the presence of the masking and cleavage peptides does not have a significant adverse effect on antibody expression in mammalian cells. [Table 16]

[0282] Measurement of masking efficiency

[0283] For the measurement of masking efficiency by ELISA, recombinant human CD28-Fc was diluted to 2 μg / mL in PBS and incubated with MaxiSorp TMHigh protein binding capacity 96-well ELISA plates (ThermoFisher Scientific) were coated overnight at 4°C. The plates were blocked with PBS supplemented with 3% nonfat milk for 1 h at 37°C. After washing, 100 μL of 3-fold serial dilutions of anti-CD28 test mAbs were added to each well. After 1 h incubation at 37°C, the plates were washed four times and 100 μL of horseradish peroxidase (HRP)-conjugated anti-human IgG (Fab specific) (1:6000 dilution) secondary antibody was added to each well. The plates were incubated for 1 h at 37°C and washed four times. After this, 50 μL of TMB substrate (3,3',5,5'-tetramethylbenzidine) solution was added to each well and the plates were incubated at room temperature. The reaction was stopped with 50 μL of sulfuric acid stop solution per well, after which the absorbance at 450 nm was measured. EC 50 was assessed by fitting the ELISA data using a sigmoidal (four-parameter logistic equation) model in GraphPad Prism version 6 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com.

[0284] The masking efficiency of selected masked anti-CD28 test mAbs was determined by the EC 50 EC of the unmasked parent mAb (TY24865) 50 The masking efficiencies were calculated by dividing by and are shown in Table 17. As shown in Figure 19, compared to the parental mAb, all of the activatable mAbs had dramatically reduced binding to the antigen, with calculated masking efficiencies ranging from 368 to over 4000. These results indicate that multiple MPs identified from the improved MP peptide library maintain their masking efficiency when expressed in mammalian cells and when expressed as part of a complete IgG molecule. [Table 17]

[0285] Optimization of selected anti-CD28 activating antibodies

[0286] For the two lead masked anti-CD28 mAbs, TY26149 and TY26152, the MP was modified, including the deletion of several N-terminal residues and the addition of an "S" amino acid residue between residues "D" and "G" in the TY26149 sequence (bold underlined residues for TY26149 in Table 18). As shown in Figure 20, the expression and masking efficiency of the new masked antibodies were not significantly affected. [Table 18]

[0287] Example 12: Epitope Mapping

[0288] To determine the binding region of the tested antibodies at the amino acid residue level, a series of mutations (Table 19) were made in the extracellular domain of human CD28. These CD28 mutant plasmids were used to transfect HEK293F cells. Binding of the antibodies to human CD28 mutants was evaluated by flow cytometry analysis. The results are summarized in Table 19 along with the cross-reactivity of these antibodies with human, monkey and mouse CD28 in interesting differences. TY24865 cross-reacts with human, monkey and mouse CD28, whereas TAC2386 and TAC2387 do not bind to mouse CD28. The mutant constructs were aimed at distinguishing the epitope by TY24865 and the reference antibodies by TAC2386 and TAC2387. TY24865 clearly exhibits the binding ability to RE49AA, VY68AA, YS79AA, and KT81AA, indicating that TY24865 does not bind to residues RE49, VY68, YS79, and KT81, which are in the non-conserved regions of human and mouse CD28, whereas TY24865 loses the binding ability to FR51AA, SL54AA, YL98AA, QN100AA, YF110AA, KI113AA, YP118AA, PPP119AAA, PP120AA, PY121AA, and Y122A mutations, indicating that its binding epitope is within these regions, e.g., amino acid residues 51, 52, 54, 55, 98-101, 110-111, 113-114, and 118-122 of SEQ ID NO:1. [Table 19]

[0289] Example 13: Mutants of TY24865

[0290] Two approaches were taken to generate variants of TY24865: various point mutations were introduced into the YYYY sequence of TY24865 scFv, and a new batch of TY24865 scFv variants was discovered through yeast-based maturation library screening. Anti-TAA × CD28 bispecific antibodies were constructed in a Fab-scFv-Fc format, and the CD28 affinity of these bsAbs was measured using ELISA.

[0291] Anti-PD-L1 x CD28 bispecific antibodies were constructed in a Fab-scFv-Fc format using anti-PD-L1 TY21421 Fab (see WO 2019 / 185035) and TY24865 scFv (VH-VL format) or its variants. The affinities of these bsAbs to CD28 are shown in Table 20 and Figures 22A and 22B; TY29815 is the wild type bsAb, TY30413 has similar affinity to TY29815, while the other bsAbs have higher or lower affinity. [Table 20]

[0292] Anti-HER2×CD28 bsAbs or anti-B7-H3×CD28 bsAbs were constructed using TY24865 scFv variants in VH-VL or VL-VH format. As shown in Table 21, these bsAbs show normal titers and SEC purity. The CD28 affinity of these bsAbs was measured by ELISA and is shown in Figure 23A and 23B. These scFv variants show various affinities ranging from 0.954 to 417 nM, with the scFv in VL-VH format showing higher affinity than the scFv in VH-VL format. Some variants in Table 22 and Figure 24A and 24B show high affinity for human and mouse, and these affinities were further confirmed in a mouse T cell binding assay (Figure 25). [Table 21] [Table 22]

[0293] The above non-limiting examples are provided for illustrative purposes only to facilitate a more complete understanding of the disclosed subject matter, and should not be construed as limiting any of the embodiments described herein, including those relating to antibodies, pharmaceutical compositions, or methods and uses for treating cancer, neurodegenerative diseases, or infectious diseases.

[0294] array

[0295] The table below lists the sequences disclosed herein with the sequence numbers (SEQ) listed in the left column.

[0296] [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] [Table 23-5] [Table 23-6] [Table 23-7] [Table 23-8] [Table 23-9] [Table 23-10]

Table 23-11

Table 23-12

Table 23-13

Table 23-14

Table 23-15

Table 23-16

Table 23-17

Table 23-18

Table 23-19

Table 23-20

Table 23-21

Table 23-22

Table 23-23

Table 23-24

Table 23-25

Table 23-26

Table 23-27

Table 23-28

Table 23-29

Table 23-30

Table 23-31

Table 23-32

Table 23-33

Table 23-34

Table 23-35

Table 23-36

Table 23-37

Table 23-38

Table 23-39

Table 23-40

Table 23-41

Table 23-42

Table 23-43

Claims

1. An antigen-binding protein or antigen-binding fragment containing a CD28 binding portion, wherein the CD28 binding portion is (i) Amino acid residues 51-122 of SEQ ID NO: 1 of human CD28, or (ii) Amino acid residues 51, 52, 54, 55, 98-101, 110-111, 113-114, and 118-122 of SEQ ID NO: 1 It binds to the CD28 epitope, Optionally, an antigen-binding protein or fragment thereof, wherein the CD28-binding portion is cross-reactive with cynomolgus monkey and mouse CD28 and / or does not possess superagonist activity.

2. The CD28 binding portion is the antibody heavy chain variable domain (V H ) and antibody light chain variable domain (V L ) including V H and V L However, including the heavy chain complementarity determining regions (CDRs) 1-3 and light chain CDRs 1-3 described below, Sequence numbers 5-10, Sequence numbers 15, 6, 16, 17-19, respectively Sequence numbers 24, 6, 25, 26-28, respectively. Sequence numbers 33, 6, 35-38, respectively. Sequence numbers 43, 6, 44, 45, 9, and 46, respectively. Sequence numbers 33, 51-53, 300, and 10, respectively. Sequence numbers 24, 58, 59, 60, 300, and 61, respectively. Sequence numbers 66-69, 300, and 70, respectively. Sequence numbers 24, 6, 75, 76, 18, and 28, respectively. Sequence numbers 24, 58, 81, 82, 27, and 83, respectively. Sequence numbers 88-91, 300, and 70, respectively. Sequence numbers 24, 96-98, 9, and 70, respectively. Sequence numbers 103-106, 18, and 83, respectively. Sequence numbers 111, 6, 112, 113, 18, and 114, respectively. Sequence numbers 15, 6, 119, 120, 9, and 121, respectively. Sequence numbers 126, 67, 127, 128, 18, and 129, respectively. Sequence numbers 134, 6, 135, 136, 27, and 83, respectively. Sequence numbers 43, 58, 141, 142, 300, and 143, respectively. Sequence numbers 148, 6, 149, 150, 300, and 83, respectively. Sequence numbers 15, 155, 16, 156, 27, and 70, respectively. Sequence numbers 161, 6, 162, 163, 300, and 164, respectively. Sequence numbers 5, 96, 307, 76, 308, and 46, respectively. Sequence numbers 5, 96, 309, 310, 9, and 311, respectively. Sequence numbers 43, 96, 312, 76, 9, and 28, respectively. Sequence numbers 43, 96, 307, 310, 9, and 313, respectively. Sequence numbers 314, 315, 309, 76, 9, and 313, respectively. Sequence numbers 33, 316, 312, 8, 308, and 313, respectively. Sequence numbers 314, 96, 317, 76, 308, and 318, respectively. Sequence numbers 43, 316, 312, 8, 9, and 10, respectively. Sequence numbers 43, 6, 319, 320, 9, and 10, respectively. Sequence IDs 5, 67, 307, 310, 308, and 28, respectively. Sequence numbers 43, 6, 321, 8, 9, and 10, respectively. Sequence numbers 314, 316, 322, 8, 9, and 10, respectively. Sequence numbers 33, 6, 321, 8, 9, and 10, respectively. Sequence numbers 5, 6, 317, 8, 9, and 10, respectively. Sequence numbers 5, 6, 323, 8, 9, and 10, respectively, or Sequence numbers 5, 6, 324, 8, 9, and 10, respectively. The antigen-binding protein or fragment thereof according to claim 1.

3. The aforementioned V H and V L but, Sequence IDs 11 and 12, respectively Sequence IDs 20 and 21, respectively Sequence IDs 29 and 30, respectively Sequence IDs 39 and 40, respectively Sequence IDs 47 and 48, respectively Sequence IDs 54 and 55, respectively Sequence IDs 62 and 63, respectively Sequence IDs 71 and 72, respectively Sequence IDs 77 and 78, respectively Sequence IDs 84 and 85, respectively Sequence IDs 92 and 93, respectively Sequence IDs 99 and 100, respectively Sequence IDs 107 and 108, respectively Sequence IDs 115 and 116, respectively Sequence IDs 122 and 123, respectively Sequence IDs 130 and 131, respectively Sequence IDs 137 and 138, respectively Sequence IDs 144 and 145, respectively Sequence IDs 151 and 152, respectively Sequence IDs 157 and 158, respectively Sequence IDs 165 and 166, respectively Sequence IDs 362 and 363, respectively Sequence IDs 364 and 365, respectively Sequence IDs 366 and 367, respectively Sequence IDs 368 and 369, respectively Sequence IDs 370 and 371, respectively Sequence IDs 372 and 373, respectively Sequence IDs 374 and 375, respectively Sequence IDs 376 and 12, respectively. Sequence IDs 377 and 378, respectively Sequence IDs 379 and 380, respectively Sequence IDs 381 and 12, respectively. Sequence IDs 382 and 12, respectively. Sequence IDs 383 and 12, respectively. Sequence IDs 384 and 12, respectively. Sequence IDs 385 and 12, respectively, or Sequence IDs 386 and 12, respectively. The antigen-binding protein or fragment thereof according to claim 2, comprising:

4. Sequence IDs 13 and 14, respectively Sequence IDs 22 and 23, respectively Sequence IDs 31 and 32, respectively Sequence IDs 41 and 42, respectively Sequence IDs 49 and 50, respectively Sequence IDs 56 and 57, respectively Sequence IDs 64 and 65, respectively. Sequence IDs 73 and 74, respectively Sequence IDs 79 and 80, respectively Sequence IDs 86 and 87, respectively Sequence IDs 94 and 95, respectively Sequence IDs 101 and 102, respectively Sequence IDs 109 and 110, respectively Sequence IDs 117 and 118, respectively Sequence IDs 124 and 125, respectively Sequence IDs 132 and 133, respectively Sequence IDs 139 and 140, respectively Sequence IDs 146 and 147, respectively Sequence IDs 153 and 154, respectively Sequence IDs 159 and 160, respectively, or Sequence IDs 167 and 168, respectively The antigen-binding protein according to claim 2, comprising an antibody heavy chain and an antibody light chain.

5. The antigen-binding protein or fragment according to any one of claims 1 to 4, further comprising a second antigen-binding moiety that targets the tumor-associated antigen (TAA), optionally the TAA being HER2, B7-H3, or TROP-2, and optionally one or each of the CD28-binding moiety and the TAA-binding moiety being a single-chain Fv (scFv), Fv, scFab, or Fab.

6. The TAA coupling portion, a) HCDR1-3 and LCDR1-3 as described in SEQ ID NOs. 262-264 and 258-260 respectively, or V as described in SEQ ID NOs. 265 and 261 respectively. H and V L The HER2 bond portion including b) an HCDR1-3 and LCDR1-3 described in SEQ ID NOs: 290 to 292 and 287, 18, and 288, respectively, or a V and a V described in SEQ ID NOs: 293 and 289, respectively H and V L comprising a B7-H3 binding portion, or c) HCDs described in Sequence IDs 280-282 and 277, 259, and 278, respectively. V as described in R1-3 and LCDR1-3, or sequence numbers 283 and 279, respectively. H and V L TROP-2 binding portion including The antigen-binding protein or fragment thereof according to claim 5.

7. The TAA bond portion is a HER2 bond portion that includes the following: An antibody light chain and an antibody heavy chain, respectively, comprising SEQ ID NOs: 169 and 170, where the CD28 binding portion comprises a heavy chain comprising SEQ ID NOs: 171 or an scFv fusion polypeptide comprising SEQ ID NOs: 266; An antibody light chain and an antibody heavy chain, each containing SEQ ID NOs: 169 and 170, respectively, wherein the CD28 binding portion comprises a heavy chain containing SEQ ID NOs: 172 or an scFv fusion polypeptide containing SEQ ID NOs: 266; Antibody light chain and antibody heavy chain, respectively, comprising SEQ ID NOs. 271 and 276, where the CD28 binding portion comprises a heavy chain comprising SEQ ID NOs. 171 or an scFv fusion polypeptide comprising SEQ ID NOs. 266; An antibody light chain and an antibody heavy chain, each containing SEQ ID NOs. 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains SEQ ID NO. 327; Antibody light chain and antibody heavy chain, respectively, containing SEQ ID NOs. 331 and 332, where the CD28 binding portion includes the heavy chain containing SEQ ID NO. 333; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 334; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 335; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 336; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 337; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 338; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 339; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 340; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion includes the heavy chain containing sequence number 341; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 342; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 343; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 344; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 345; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 346; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 347; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 348; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 349; An antibody light chain and an antibody heavy chain, each containing sequence numbers 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 350; Antibody light chain and antibody heavy chain, respectively, containing SEQ ID NOs. 331 and 332, where the CD28 binding portion includes the heavy chain containing SEQ ID NO. 351; An antibody light chain and an antibody heavy chain, each containing SEQ ID NOs. 331 and 332, respectively, wherein the CD28 binding portion of the heavy chain contains SEQ ID NO. 352; or Antibody light chain and antibody heavy chain, respectively, containing SEQ ID NOs. 331 and 332, where the CD28 binding portion includes the heavy chain containing SEQ ID NO.

353. The antigen-binding protein or fragment thereof according to claim 6.

8. The TAA bond portion is a B7-H3 bond portion that includes the following: Antibody light chain and antibody heavy chain, respectively, comprising SEQ ID NOs: 175 and 176, where the CD28 binding portion comprises a heavy chain comprising SEQ ID NOs: 171 or an scFv fusion polypeptide comprising SEQ ID NOs: 266; An antibody light chain and an antibody heavy chain, each containing sequence numbers 354 and 355, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 327; An antibody light chain and an antibody heavy chain, each containing sequence numbers 354 and 355, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 356; An antibody light chain and an antibody heavy chain, each containing SEQ ID NOs. 354 and 355, respectively, wherein the CD28 binding portion of the heavy chain contains SEQ ID NO. 357; or Antibody light chain and antibody heavy chain, respectively, containing SEQ ID NOs. 354 and 355, where the CD28 binding portion includes the heavy chain containing SEQ ID NO.

358. The antigen-binding protein or fragment thereof according to claim 6.

9. The antigen-binding protein or fragment thereof according to claim 6, wherein the TAA binding portion is a TROP2 binding portion comprising an antibody light chain and an antibody heavy chain, respectively, comprising SEQ ID NOs. 173 and 174, and the CD28 binding portion comprises a heavy chain comprising SEQ ID NO. 171 or an scFv fusion polypeptide comprising SEQ ID NO.

266.

10. The present invention further comprises another antigen-binding moiety that targets the aforementioned immune checkpoint inhibitor, optionally wherein the immune checkpoint inhibitor is PD-L1, and further optionally wherein the PD-L1-binding moiety comprises the following: Antibody light chain and antibody heavy chain, respectively, containing SEQ ID NOs. 325 and 326, where the CD28 binding portion includes the heavy chain containing SEQ ID NO. 327; An antibody light chain and an antibody heavy chain, each containing sequence numbers 325 and 326, respectively, wherein the CD28 binding portion of the heavy chain contains sequence number 328; An antibody light chain and an antibody heavy chain, each containing SEQ ID NOs. 325 and 326, respectively, wherein the CD28 binding portion of the heavy chain contains SEQ ID NO. 329; or Antibody light chain and antibody heavy chain, respectively, containing SEQ ID NOs. 325 and 326, where the CD28 binding portion includes the heavy chain containing SEQ ID NO.

330. The antigen-binding protein or fragment thereof according to any one of claims 1 to 4.

11. Furthermore, it includes an Fc region, and optionally, the Fc region is of the human IgG1 subclass or the human IgG4 subclass, and / or The Fc region has a reduced effector function effect, or does not have an effector function effect, has a reduced antibody-dependent cell-mediated cytotoxicity (ADCC) effect, or does not have an antibody-dependent cell-mediated cytotoxicity (ADCC) effect, and / or has a reduced crosslinking effect, or does not have a crosslinking effect. The antigen-binding protein or fragment thereof according to any one of claims 1 to 4, wherein the Fc domain optionally has an N297A substitution (EU numbering).

12. The antigen-binding protein or its fragment comprises a first CH3 domain and a second CH3 domain, where: i) The first CH3 domain contains a cysteine ​​(C) residue at position 390 and the second CH3 domain contains a cysteine ​​residue at position 400, or the first CH3 domain contains a cysteine ​​residue at position 400 and the second CH3 domain contains a cysteine ​​residue at position 390; or ii) The first CH3 domain contains a cysteine ​​residue at position 392 and the second CH3 domain contains a cysteine ​​residue at position 397, or the first CH3 domain contains a cysteine ​​residue at position 397 and the second CH3 domain contains a cysteine ​​residue at position 392; or iii) The first CH3 domain contains a cysteine ​​residue at position 392 and the second CH3 domain contains a cysteine ​​residue at position 400, or the first CH3 domain contains a cysteine ​​residue at position 400 and the second CH3 domain contains a cysteine ​​residue at position 392; The amino acid residue numbering is based on EU numbering; Optionally, iv) The first CH3 domain further contains a positively charged residue at position 357 and the second CH3 domain further contains a negatively charged residue at position 351, or the first CH3 domain further contains a negatively charged residue at position 351 and the second CH3 domain further contains a positively charged residue at position 357; or v) The first CH3 domain further contains a positively charged residue at position 411 and the second CH3 domain further contains a negatively charged residue at position 370, or the first CH3 domain further contains a negatively charged residue at position 370 and the second CH3 domain further contains a positively charged residue at position 411; or vi) The first CH3 domain further contains a positively charged residue at position 364 and the second CH3 domain further contains a negatively charged residue at position 370, or the first CH3 domain further contains a negatively charged residue at position 370 and the second CH3 domain further contains a positively charged residue at position 364; or a combination of i) and ii), or a combination of i) and iii), wherein the amino acid residue numbering is based on EU numbering; Furthermore, optionally, the first CH3 domain may include D / E356K, E357K, S364K and S400C substitutions, and the second CH3 domain may include L351D, K370D, N390C and K439D substitutions (Eu numbering), or The first CH3 domain includes L351D, K370D, N390C and K439D substitutions, and the second CH3 domain includes D / E356K, E357K, S364K and S400C substitutions (Eu numbering). The antigen-binding protein or fragment thereof according to any one of claims 1 to 4.

13. Furthermore, it comprises at least one masking peptide, the at least one masking peptide (MP) being linked to the N-terminus of VL, and the MP comprising a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus, with or without cleavage sites. Optionally, the MU includes an array selected from sequence numbers 173 to 206; Furthermore, optionally, the MP further includes an N-terminal unit (NU) bonded to the N-terminus of the MU. The N-terminal unit may optionally be approximately 1 to 10 amino acid residues in length. Furthermore, optionally, the N-terminal unit includes E or EVGSY; The LU includes the cut portion, Optionally, the cleavage site is urokinase-type plasminogen activator / uPA, The protease cleavage site of a protease selected from rixmetalloprotease-1 / MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus protease / TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE; Furthermore, optionally, at least one masking peptide is linked to a B7-H3, HER2, or TROP2 binding site or a CD28 binding site, and optionally, the antigen-binding protein or its fragment contains two masking peptides linked to a B7-H3, HER2, or TROP2 binding site and a CD28 binding site, respectively. The antigen-binding protein or fragment thereof according to any one of claims 1 to 4.

14. The antigen-binding protein or fragment according to any one of claims 1 to 4, further comprising the bound therapeutic site, wherein optionally the therapeutic site is a radioactive portion or a cytotoxic portion.

15. A pharmaceutical composition comprising an antigen-binding protein or a fragment thereof according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

16. One or more nucleic acid molecules encoding an antigen-binding protein or a fragment thereof, as described in any one of claims 1 to 4.

17. A host cell containing the nucleic acid molecule described in claim 16.

18. A method for producing an antigen-binding protein or an antigen-binding fragment thereof, The host cells according to claim 17 are cultured under conditions that enable the expression of an antigen-binding protein or a fragment thereof, and optionally further Isolating antigen-binding proteins or their fragments from cultures. Methods that include...

19. The pharmaceutical composition according to claim 15 for treating cancer in patients who require cancer treatment.

20. The pharmaceutical composition according to claim 19, wherein the patient is administered another anticancer drug, optionally the additional anticancer drug being a bispecific antibody targeting CD3 and a tumor antigen, optionally the tumor antigen being the same as or different from the TAA, and further optionally the TAA being B7-H3, HER2, or TROP2; or an immune checkpoint inhibitor, optionally an anti-PD-1, anti-CTLA-4, or anti-PD-L1 antibody; and further optionally the patient having a solid tumor or hematological malignancy optionally selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, kidney cancer, pancreatic cancer, and colon cancer.

Images