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 CD137, compositions constituting the same, and uses thereof in delaying and / or preventing tumor growth.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to PCT / CN2022 / 085838 filed on April 8, 2023, the entire text of which is incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains an XML formatted sequence listing submitted electronically and the entire text of the sequence listing is incorporated herein by reference. An electronic copy of the sequence listing created on April 6, 2023 and named 123687_WO003_SL.xml is 488,908 bytes in size. Background Art

[0005] CD28 is a co-stimulatory signal molecule on T cells, and plays a key role in its activation, proliferation and survival. CD28 co-stimulatory signal molecule activation has the potential to enhance anti-tumor T cell immunity. However, systemic T cell activation therapy targeting CD28 has caused harmful cytokine storms and multiple organ failure (Suntharalingam et al., N Engl J Med. (2006) 355 (10): 1018-28). Therefore, it is necessary to improve CD28 targeted therapy to avoid serious autoimmune adverse events associated with systemic CD28 activation. Summary of the invention

[0006] The present invention provides novel binding molecules targeting tumor-associated antigens (e.g., B7-H3, HER2, TROP2 or PD-L1) and CD28, and pharmaceutical compositions comprising one or more such binding molecules, and the use of such binding molecules and pharmaceutical compositions to treat cancer. Compared to currently available cancer treatments (including antibody treatments), the binding molecules of the present invention are expected to provide better clinical responses.

[0007] In some aspects, the present invention provides an antigen binding protein (e.g., an antibody or an antigen binding fragment thereof) comprising a CD28 binding portion, wherein the CD28 binding portion binds to human CD28 and cross-reacts with cynomolgus macaque and mouse CD28. In some embodiments, the CD28 binding portion binds to a CD28 antigen epitope comprising amino acid residues 51-122 of human CD28 (SEQ ID NO: 1). In a specific embodiment, the CD28 antigen epitope comprises amino acid residues 51, 52, 54, 55, 98-101, 110-111, 113-114, and 118-122 of SEQ ID NO: 1.

[0008] In some embodiments, the present invention provides an antigen binding protein (e.g., an antibody or an antigen binding fragment thereof) comprising a CD28 binding portion that binds to human CD28, wherein the CD28 binding portion comprises an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), and V H and V L The invention comprises the heavy chain complementary determining regions (CDRs) 1-3 and the light chain CDRs 1-3 as shown, which are SEQ ID NOs: 5-10, SEQ ID NOs: 15, 6, 16, 17-19, SEQ ID NOs: 24, 6, 25, 26-28, SEQ ID NOs: 33, 6, 35-38, SEQ ID NOs: 43, 6, 44, 45, 9 and 46, SEQ ID NOs: 33, 51-53, 300 and 10, SEQ ID NOs: 24, 58, 59, 60, 300 and 61, SEQ ID NOs: 66-69, 300 and 70, SEQ ID NOs: 24, 6, 75, 76, 18 and 28, SEQ ID NOs: 24, 58, 81, 82, 27 and 83, SEQ ID NOs: 24, 58, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 NOs:88-91, 300 and 70, respectively, SEQ ID NOs:24, 96-98, 9 and 70, respectively, SEQ ID NOs:103-106, 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, or SEQ ID NOs:15, 155, 16, 156, 27 and 70, respectively. NOs:161, 6, 162, 163, 300 and 164.

[0009] In some embodiments, the CD28 binding portion comprises V as shown H and V L, SEQ ID NOs:11 and 12, respectively, SEQ ID NOs:20 and 21, respectively, SEQ ID NOs:29 and 30, respectively, SEQ ID NOs:39 and 40, respectively, SEQ ID NOs:47 and 48, respectively, SEQ ID NOs:54 and 55, respectively, SEQ ID NOs:62 and 63, respectively, SEQ ID NOs:71 and 72, respectively, SEQ ID NOs:77 and 78, respectively, SEQ ID NOs:84 and 85, respectively, SEQ ID NOs:92 and 93, respectively, SEQ ID NOs:99 and 100, respectively, SEQ ID NOs:107 and 108, respectively, SEQ ID NOs:115 and 116, respectively, SEQ ID NOs:122 and 123, respectively, SEQ ID NOs:130 and 131, respectively, SEQ ID NOs:137 and 138, respectively, SEQ ID NOs:144 and 145, respectively. NOs:151 and 152, respectively, are SEQ ID NOs:157 and 158, respectively, are SEQ ID NOs:165 and 166, respectively, are SEQ ID NOs:362 and 363, respectively, are SEQ ID NOs:364 and 365, respectively, are SEQ ID NOs:366 and 367, respectively, are SEQ ID NOs:368 and 369, respectively, are SEQ ID NOs:370 and 371, respectively, are SEQ ID NOs:372 and 373, respectively, are SEQ ID NOs:374 and 375, respectively, are SEQ ID NOs:376 and 12, respectively, are SEQ ID NOs:377 and 378, respectively, are SEQ ID NOs:379 and 380, respectively, are SEQ ID NOs:381 and 12, respectively, are SEQ ID NOs:382 and 12, respectively, are SEQ ID NOs:383 and 12, respectively, are SEQ ID NOs:384 and 12, respectively, are SEQ ID NOs:385 and 12, respectively, or are SEQ ID NOs:386 and 12, respectively. ID NOs:386 and 12.

[0010] In some embodiments, the CD28 binding portion comprises the heavy chain (HC) and light chain (LC) shown as 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: 134 and 135, respectively. 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.

[0011] In some aspects, the present invention also provides a pharmaceutical composition comprising an antigen binding protein (e.g., an antibody or antigen binding fragment thereof described herein) and a pharmaceutically 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 can be a prokaryotic cell or a eukaryotic cell (e.g., a mammalian cell).

[0012] In some aspects, the present invention also provides a method of producing an antigen binding protein (eg, an antibody or antigen binding fragment thereof described herein), comprising culturing a host cell under conditions that allow expression of the antigen binding protein, and isolating the antigen binding protein from the culture medium.

[0013] In some aspects, the present invention also provides a method for treating cancer in a patient in need, 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 agent. In a further embodiment, the other anti-cancer therapeutic agent is a bispecific antibody (bsAb) targeting CD3 and a tumor-associated antigen (TAA). In a specific embodiment, TAA is B7-H3, HER2 or TROP2. In some embodiments, the other anti-cancer therapeutic agent is an immune checkpoint inhibitor, optionally, an anti-PD-1, anti-CTLA4 or anti-PD-L1 antibody.

[0014] Also provided herein are antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof) of the present invention, or pharmaceutical compositions for treating cancer in patients in need thereof; the use of antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof) of the present invention in the manufacture of medicaments for treating cancer in patients in need thereof; and articles of manufacture (e.g., kits) containing one or more administration units of antigen-binding proteins (e.g., antibodies or fragments thereof) of the present invention.

[0015] Other features, purposes and advantages of the present invention are apparent in the following detailed description. However, it should be appreciated that the detailed description, while indicating embodiments and aspects of the present invention, is provided only in an illustrative and non-restrictive manner. According to the detailed description, those skilled in the art will clearly understand the various changes and modifications within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 To show the binding affinity of a panel of anti-CD28 antibodies to recombinant CD28 (human and mouse) protein.

[0017] Figure 2 To show the binding of anti-CD28 IgG to human CD3+ T cells.

[0018] Figure 3 A panel of IgGs were tested for ligand blocking assays with human CD28-CD80 (upper right and left panels) and CTLA-4-CD80 (bottom panel).

[0019] Figure 4A As a test of T cell proliferation, it was shown that IgG did not induce systemic T cell activation compared to the CD28 superagonist TAC2386 (TGN1412).

[0020] Figure 4B To display the (CTG) Readout of T cell activation co-stimulation assay - proliferation.

[0021] Figure 4CTo demonstrate T cell activation, costimulation was used to measure the release of IFN-γ.

[0022] Figure 5A To show activation and proliferation assay of T cells using OKT3 anti-CD28 test antibody by CTG readout - Proliferation.

[0023] Figure 5B To demonstrate T cell activation and proliferation, IL-2 release was measured.

[0024] Figure 6 To show the binding of anti-HER2 antibodies and bispecific antibodies (BsAb) to SK-OV3 cells. A table containing the plotted values ​​is also shown.

[0025] Figure 7 To show the Jurkat-NFκB luciferase reporter assay measuring the maximum signal value and EC when anti-HER2×CD28 BsAb (TY27566 or TY27807) is combined with a fixed concentration of anti-HER2×CD3 BsAb (TY25238), and anti-HER2×CD3 BsAb is combined with a fixed concentration of anti-HER2×CD28 BsAb. 50 Values ​​for the transduction stimulation effect on NFκB signaling. A table containing the plotted values ​​is also shown.

[0026] Figure 8 Figure 2 shows the killing assay of MCF-7 cells by CD28 BsAb and CD3 BsAb with the same or different HER2 epitopes and a table containing the plotted values.

[0027] Fig. 9 is a set of assays showing measurement of concentration-dependent binding activity of anti-TROP2 bispecific antibodies on tumor cell lines.

[0028] Fig. 10A To show the maximum signal value and EC of the bispecific antibody in Jurkat-NFκB luciferase reporter assay in H292 cells 50 Values ​​for the effects of stimulation of NFκB signaling. A table containing the plotted values ​​is also shown.

[0029] Fig. 10B To show the maximum signal value and EC of the bispecific antibody in Jurkat-NFκB luciferase reporter assay in H292 cells 50 Values ​​for the effects of stimulation of NFκB signaling. A table containing the plotted values ​​is also shown.

[0030] Fig.11 A set of flow cytometric images showing the co-expression of PD-L1 and B7H3 on MDA-MB-231 cells.

[0031] Fig.12 To show the binding assay of B7H3 IgG and B7H3xCD28 bsAb to MDA-MB-231 cells. A table containing the plotted values ​​is also shown.

[0032] Fig.13A and 13B To demonstrate the one-way MLR assay to test the blocking mAb activity of B7H3xCD28 bsAb in combination with anti-PD-1 or anti-PD-L1 on naive human T cells, the activation activity of IL-2 secretion ( Fig.13A ) and IFN-γ secretion ( Fig. 13B )Measurement.

[0033] Fig.14 To show in vitro assays, the tumor killing activity of anti-CD3-based or anti-CD28-based bsAbs targeting HER2 or their combination was measured on the MCF-7 tumor cell line. A table containing the plotted values ​​is also shown.

[0034] Fig.15 To show in vitro assays, the tumor killing activity of anti-CD3-based or anti-CD28-based HER2-targeted bsAbs or combinations thereof was measured on the EMT6-HER2 tumor cell line. A table containing the plotted values ​​is also shown.

[0035] Fig.16 For one panel showing assays measuring systemic cytokine release of IL-6 and IFN-γ (upper left and right panels), and measuring the percentage of CD3+ T cells to total CD45+ T cells (bottom panel), WT mice were treated with TCE.

[0036] Fig.17 In vivo efficacy studies and graphs showing HER2xCD3 bsAb and B7H3xCD28 bsAb alone or in combination in the SK-OV3+PBMC xenograft tumor model.

[0037] Fig.18 This is a set of studies showing the in vivo efficacy of B7H3xCD28 or HER2xCD28 bsAb in the EMT6-HER2 model.

[0038] Fig.19 ELISA measurements showing the shielding efficiency of anti-CD28 activatable antibodies binding to recombinant human CD28.

[0039] Fig. 20 ELISA measurements showing the shielding efficiency of anti-CD28 activatable antibodies binding to recombinant human CD28.

[0040] Fig.21 (SEQ ID NOs: 359-361) is a table showing the different binding residues of human and mouse CD28.

[0041] Fig.22A To show the binding of anti-PD-L1 × CD28 bsAbs TY29815 and TY30413 to human CD28.

[0042] Fig. 22B To show the binding of anti-PD-L1 × CD28 bsAbs TY29815, TY30406, TY30410, and TY30413 to mouse CD28.

[0043] Fig.23A To show the binding of anti-HER2×CD28 bsAbs TY27566, TY28652, TY28653, TY28654, TY28655, TY28656, TY28657, TY28658, TY28659, TY28660, and TY28661 to human CD28.

[0044] Fig. 23B To show the binding of anti-HER2 × CD28 bsAbs TY29109, TY29306, TY29307, TY29308, TY29309, TY29310, TY29311, TY29312, TY29313, TY29314, and TY29315 to human CD28.

[0045] Fig.24A To show the binding of anti-B7H3×CD28 bsAbs TY29021, TY30120, TY30121 and TY30123 to human CD28.

[0046] Fig. 24B To show the binding of anti-B7H3×CD28 bsAbs TY29021, TY30120, TY30121, and TY30123 to mouse CD28.

[0047] Fig.25 To show the mouse T cell binding assay, the maximum signal, EC 50 The binding effect of the values ​​and AUC values ​​on T cells. A table containing the plotted values ​​is also shown. DETAILED DESCRIPTION

[0048] I. Definitions

[0049] Before describing the present disclosure in detail, it is to be understood that the present disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0050] As used herein, the term "about" refers to the usual error range for the corresponding value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments related to that value or parameter itself.

[0051] It should be understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0052] As used herein, the term "and / or", phrases such as "A and / or B" are intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or", phrases such as "A, B and / or C" are intended to cover 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 (alone); B (alone); and C (alone).

[0053] The term "antibody" encompasses 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), multispecific antibodies (e.g., bispecific antibodies, including masked bispecific antibodies), and antibody fragments (e.g., single-chain variable fragments or scFvs), so long as they exhibit the desired biological activity (e.g., the ability to bind to a target antigen with the desired specificity and affinity).

[0054] The term "antibody" encompasses a variety of antibody structures, including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, shielded 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 full-length human antibodies.

[0055] In some embodiments, the term "antibody" refers to an antigen binding protein (i.e., immunoglobulin) having 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 an 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 has a variable region (also called a variable domain) (abbreviated herein as V H ), followed by the constant region. The heavy chain constant region consists of three domains: C H 1. C H 2 and C H 3. Each light chain has a variable region (also called variable domain) at the N-terminus (abbreviated herein as V L), followed by a constant region at the other end. The light chain constant region consists of one domain, C L . V L With V H Compare and C L The first constant domain of the heavy chain (C H 1) Compare. H and V L together form a single antigen binding site.

[0056] V H and V L Can be further subdivided into complementary determining region (CDR) and framework region (FW). CDR has the highest sequence variability and / or participates in antigen recognition. CDR and FR are distributed in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. CDR also contains "specificity determining residues" or "SDRs", which are residues that contact antigens. SDRs are contained in the region of CDR called abbreviated-CDR or a-CDR. Exemplary a-CDRs (a-LCDR1, a-LCDR2, a-LCDR3, a-HCDR1, a-HCDR2 and a-HCDR3) each appear at 31-34, 50-55, 89-96 of the light chain, and 31-35, 50-58 and 95-102 amino acid residues of the heavy chain. See Almagro and Fransson, Front.Biosci. (2008) 13: 1619-33. Unless otherwise indicated, variable region residues are numbered herein according to Kabat et al., J Biol Chem. (1977) 252:6609-16; Kabat et al., U. Sept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991).

[0057] Table 1 below provides exemplary CDR definitions according to various algorithms known in the art.

[0058] Table 1. CDR definitions

[0059] <![CDATA[Kabat 1 ]]> <![CDATA[Chothia 2 ]]> <![CDATA[MacCallum 3 ]]> <![CDATA[IMGT 4 ]]> <![CDATA[AHo 5 ]]> HCDR1 31-35 26-32 30-35 27-38 25-40 HCDR2 50-65 53-55 47-58 56-65 58-77 HCDR3 95-102 96-101 93-101 105-117 109-137 LCDR1 24-34 26-32 30-36 27-38 25-40 LCDR2 50-56 50-52 46-55 56-65 58-77 LCDR3 89-97 91-96 89-96 105-117 109-137

[0060] 1 The residue numbering follows the nomenclature of Kabat et al., supra.

[0061] 2The residue numbering follows the nomenclature of Chothia et al., J. Mol. Biol. (1987) 196:901-917; Al-Lazikani B. et al., J. Mol. Biol., (1997) 273:927-948.

[0062] 3 Residue numbering follows the nomenclature of MacCallum et al., J. Mol. Biol. (1996) 262:732-745; Abhinandan and Martin, Mol. Immunol., (2008) 45:3832-3839.

[0063] 4 Residue numbering follows the nomenclature of Lefranc MP et al., Dev. Comp. Immunol., (2003) 27:55-77; and Honegger and Plückthun, J. Mol. Biol., (2001) 309:657-670.

[0064] 5 Residue numbering follows the nomenclature of Honegger and Plückthun, J. Mol. Biol., (2001) 309:657-670.

[0065] The 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 sequence of their constant domains. H ) amino acid sequence, antibodies can be designated as different classes or isotypes. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. IgG class antibodies can be further classified into four subclasses IgG by the γ heavy chain Y1-Y4, respectively 1 IgG 2 IgG 3 and IgG 4 .

[0066] The terms "antigen-binding fragment" or "antigen-binding portion" are used interchangeably herein and refer to a portion of an antibody that retains the ability to bind to the antibody antigen. Examples of "antigen-binding fragments" of an antibody include, but are not limited to, (i) a Fab fragment consisting of V L 、V H , C L and C H1 Monovalent fragments consisting of domains, obtained by papain digestion; (ii) F(ab′) 2A fragment comprising two Fab fragments linked by a disulfide bond in the hinge region, obtained by pepsin digestion; (iii) a V fragment of a single arm of the antibody L and V H (iv) a single-chain Fv (scFv) fragment, which comprises the V domains of an antibody fused to each other. H and V L domain; and (v) a single-chain Fab (scFab) fragment comprising a V L 、V H , C L and C H1 A single polypeptide chain containing a domain.

[0067] The term "shielded antibody" refers to an antibody (including multispecific antibodies) or an antigen-binding fragment thereof comprising a shielding peptide that interferes with, hinders, reduces (ability), prevents, inhibits binding to its target, or competes with the antigen-binding domain of the antibody for binding to its target. Shielded antibodies can be produced by connecting a shielding peptide to the antigen-binding domain of an antibody. In some embodiments, the shielded antibody or its antigen-binding fragment exhibits a first binding affinity to the target in an unactivated state (e.g., inhibited or shielded by a shielding peptide), and exhibits a second binding affinity to the target in an activated state (e.g., not inhibited or shielded by a shielding peptide (e.g., a shielding peptide cleaved from an antibody)), wherein the second binding affinity is greater than the first binding affinity. Shielded antibodies can be produced by connecting a shielding peptide comprising an activatable portion (e.g., a cleavable site or "LU" of a linking unit) to the antigen-binding domain of an antibody. In some embodiments, the shielded antibody, or its shielded antigen-binding fragment is a multispecific antibody, which comprises a binding domain with specificity for T cell surface molecules (e.g., CD28, CD3) and tumor cell surface antigens (e.g., HER2, B7H3, TROP2, etc.). In some embodiments, the shielded antibody is bivalent and has a monovalent shielding on one of the two binding domains. In some embodiments, the shielded antibody is bivalent and has shielding on each of the two binding domains. For example, for a monovalent shielded antibody, one binding domain of the antibody is shielded by fusion or conjugation of a shielding peptide. For bispecific antibodies, one or two binding domains may be shielded by specific but different shielding peptides. An unactivated bispecific antibody that simultaneously targets cancer cells and T cells may have a binding site that shields two binding domains to inhibit (or minimize) binding to antigen-expressing cancer cells and T cells. However, in the activated state, the shielding portion is cleaved to allow the antibody to bind to tumor antigens and T cell surface molecules (e.g., CD28) in the tumor microenvironment (TME). In this case, the activated bispecific antibody selectively engages T cells and kills cancer cells expressing the targeted tumor antigen.

[0068] The term "shielding peptide" refers to a peptide that inhibits the binding of an antigen-binding domain to its target antigen, and generally comprises a shielding unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus. The C-terminus of the shielding peptide is generally connected to the V-terminus of the antigen-binding domain. H or V L In some embodiments, the shielding peptide or a portion thereof interferes with or inhibits the binding of the antigen binding domain to its target so effectively that the binding of the antigen binding domain to its target is extremely low and / or below the detection limit (e.g., no detectable binding in an ELISA or flow cytometry assay). The shielded antibody or polypeptide used herein may comprise one or more linkers, e.g., within LU, between MU and LU, between LU and V H or V L Between or V H and between the Fc hinge region.

[0069] The LU of the shielding peptide may include at least one cleavable site. The cleavage site generally includes a cleavable amino acid sequence, for example, used as a substrate for an enzyme and / or cysteine-cysteine ​​pair that can form a reducible disulfide bond. Thus, when the terms "cleavage", "cleavable", "cleaved" and the like are used in conjunction with the cleavage site, those terms encompass, for example, enzymatic cleavage by a protease, and the interruption of the reduction of a disulfide bond between a cysteine-cysteine ​​pair by a disulfide bond that can be generated by exposure to a reducing agent. The amino acid sequence of the cleavage site may overlap with the MU or be contained within the MU. The shielded antibody or shielded polypeptide may include a cleavage site configured to mediate antibody or polypeptide activation. For example, when the cleavage site of the activatable antibody is intact (e.g., not cleaved by the corresponding enzyme, and / or containing an unreduced cysteine-cysteine ​​disulfide bond), the shielding peptide or a portion thereof may interfere with or inhibit the antigen-binding domain from binding to its target. In some embodiments, the LU of the shielding peptide does not include a cleavable site.

[0070] The term "shielding efficiency" refers to the efficiency of a shielding peptide in inhibiting the binding of an antigen binding domain to a target antigen. The shielding efficiency can be measured by the difference or ratio of the binding affinity of a shielded antibody or shielded polypeptide comprising an antigen binding domain relative to the binding affinity of an unshielded antibody or unshielded polypeptide comprising an antigen binding domain (e.g., a shielding peptide cleaved from an antibody). For example, the shielding efficiency can be measured by the EC of an inactive state of a shielded antibody (e.g., inhibiting, shielded and / or uncleaved) binding to a target antigen. 50 or K D divided by the EC of the unmasked antibody in its activated state (e.g., uninhibited, unmasked, and / or cleaved) bound to the target antigen 50 or K D, or the parent antibody (e.g., not linked to a shielding peptide) bound to the target antigen 50 or K D EC can be measured by ELISA or Jurkat NFAT reporter assay. 50 Value, such as described in U.S. Patent Publication No. US2021 / 0207126A1. D The value can be measured by, for example, using surface plasmon resonance.

[0071] The term "epitope" refers to the part of an antigen that is combined with an antibody (or its antigen-binding fragment). An epitope can be formed by continuous or discontinuous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed by continuous amino acids are usually retained when exposed to a denaturing solvent, while epitopes formed by tertiary folding are usually lost when treated with a denaturing solvent. An epitope can include different numbers of amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance, deuterium hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or all methods used in combination with antigens and computer modeling of their complex structures with binding antibodies and variants thereof. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by G.E. Morris (1996). Once the desired epitope of an antigen is determined, antibodies against the epitope can be generated, for example, using the techniques described herein. The generation and characterization of antibodies can also clarify information about the desired epitope. With this information, antibodies that bind to the same epitope can then be competitively screened. A way to achieve this is to perform cross-competition studies to find antibodies that compete with each other for binding, i.e., antibodies that compete for binding to the antigen. A high throughput method for "binning" antibodies based on their cross-competition is described in PCT Publication No. WO 03 / 48731.

[0072] The term "germline" refers to the nucleotide sequences of antibody genes and gene fragments as they are passed from parents to offspring through germ cells. Germline sequences differ from nucleotide sequences encoding antibodies in mature B cells, which have been altered by recombination and hypermutation events during B cell maturation.

[0073] The term "glycosylation site" refers to an amino acid residue that is recognized by a eukaryotic cell as a sugar residue attachment position. The amino acids to which carbohydrates (e.g., oligosaccharides) are attached are typically asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. A specific attachment site is typically represented by an amino acid sequence, 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 act as attachment sites between sugar molecules and amino acid residues. N-linked sugars are linked via amino groups; O-linked sugars are linked via hydroxyl groups. The term "glycan occupancy" refers to the presence of a carbohydrate moiety connected to a glycosylation site (i.e., the glycan site is occupied). In the case of at least two potential glycosylation sites on a polypeptide, the carbohydrate moiety may not be occupied (0-glycan site occupied), one site occupied (1-glycan site occupied), or two sites (2-glycan site occupied).

[0074] The term "host cell" refers to a cell system that can be engineered to produce a target protein, protein fragment or peptide. Host cells include but are not limited to cultured cells, such as mammalian cultured cells such as CHO, BHK, NSO, SP2 / 0, YB2 / 0 derived from rodents (rat, mouse, guinea pig or hamster); human cells, such as HEK293F cells, HEK293T cells; or human tissue or hybridoma cells, yeast cells, insect cells (such as S2 cells), bacterial cells (such as Escherichia coli (E. coli) cells) and cells contained in transgenic animals or cultured tissues. The term not only covers specific subject cells, but also covers the progeny of such cells. Because some modifications may occur in succession due to mutations or environmental influences, the progeny may not be the same as the parent cell, but is still included in the scope of the term "host cell".

[0075] A "human antibody" is an antibody having an amino acid sequence corresponding to that produced by a human or human cell or derived from a non-human source utilizing a human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0076] The term "humanized antibody" refers to a chimeric antibody comprising amino acid residues derived from human antibody sequences. A humanized antibody may comprise some or all CDRs from a non-human animal or synthetic antibody, while the framework and constant regions of the antibody comprise amino acid residues derived from human antibody sequences.

[0077] The term "exemplary antibody" refers to any of the antibodies described herein. These antibodies can be in any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each of the antibodies identified above encompasses antibodies with the same V L Area and V H In addition, antibodies in the IgG category may be in any subclass (e.g., IgG 1 IgG 2 IgG 3 and IgG 4 Thus, each antibody identified above in the IgG subclass encompasses antibodies in all four subclasses with the same V L Area and V H The amino acid sequences of the heavy chain constant regions of human antibodies in five classes and in four IgG subclasses are known in the art.

[0078] An "isolated" antibody or binding molecule is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). The term "isolated" is used interchangeably with the term "pure" or "pure" in conjunction with a nucleic acid. a ” refers to the association rate constant for a specific antibody-antigen interaction, while the term “k d ” refers to the dissociation rate constant for a specific antibody-antigen interaction.

[0079] The term "K D ” refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. It is represented by k d With k a The ratio (i.e., k d / k a ) and expressed as molar concentration (M). K D Used as a measure of the affinity of the binding of an antibody to its binding partner. D The smaller the K, the tighter 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 more tightly to a particular antigen than an antibody with a micromolar (μM) dissociation constant. D The K value can be determined using methods well established in the art. D The method is by using ELISA. For example, the detection procedure using ELISA.

[0080] The term "mammal" refers to any animal species of the class Mammalia. Examples of mammals include: humans; laboratory animals such as rats, mice, hamsters, rabbits, non-human primates, and guinea pigs; domestic animals such as cats, dogs, cows, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, elephants, etc.

[0081] The terms "prevent" or "preventing" with respect to a disease condition in a mammal means preventing or delaying the onset of the disease, or preventing the appearance of clinical or subclinical symptoms thereof.

[0082] As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. The amino acid sequence identity of a polypeptide can be routinely determined using known computer programs such as Bestfit, FASTA or BLAST (see, e.g., 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., Nucelic Acids Res. (1997) 25: 3389-3402). When Bestfit or any other sequence alignment program is used to determine whether a particular sequence has, for example, 95% identity with a reference amino acid sequence, the parameters are set so that the percentage of identity is calculated over the full length of the reference amino acid sequence and the interval of homology of the total number of amino acid residues in the reference sequence is allowed to be up to 5%. This above-described method of determining percent identity between polypeptides is applicable to all proteins, fragments or variants thereof disclosed herein.

[0083] As used herein, the term "binding", "specific binding" or "specific for..." refers to a measurable and reproducible interaction such as binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules including biomolecules. 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 greater affinity, avidity, more readily and / or with a longer duration than it binds to other targets. In one embodiment, the degree 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, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved between proteins from different species. In another embodiment, specific binding may include but does not require exclusive binding. For example, if the masked anti-CD28 antibodies described herein bind to the EC of human CD28 50 Lower than its EC for binding to different antigens in in vitro assays 50 If the amount of the masked anti-CD28 antibody is less than 10%, the masked anti-CD28 antibody described herein is said to selectively bind to human CD137.

[0084] The term "treat, treating or treatment" with respect to a disease condition in a mammal refers to causing a desired or beneficial effect in a mammal suffering from the disease condition. The 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 the number and / or activity of immune cells, etc.), or a stop or inhibition of further development of the disease, condition or illness. In the context of treating cancer in a mammal, the desired or beneficial effect may include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of cancer recurrence, reduction of cancer-related pain, or improvement in mammal survival. The effect may be subjective or objective. For example, if the mammal is a human, then the human may record improved energy or vitality or reduced pain as improved subjective symptoms or responses to therapy. Alternatively, a clinician may notice a reduction in tumor size or tumor load based on physical examination, laboratory parameters, tumor markers or imaging findings. For response to treatment, some laboratory signs that clinicians can observe include standardization of tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. In addition, clinicians can observe a reduction in detectable tumor markers. Alternatively, other tests can be used to assess objective improvement, such as sonograms, nuclear magnetic resonance tests, and positron emission tests.

[0085] The term "vector" refers to a nucleic acid molecule capable of transporting foreign nucleic acid molecules. Foreign nucleic acid molecules are connected to carrier nucleic acid molecules by recombinant techniques such as connection or recombination. This allows breeding, selection, further manipulation or expression of foreign nucleic acid molecules in host cells or organisms. The vector can be a plasmid, a phage, a transposon, a cosmid, a chromosome, a virus or a virion. One type of vector can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome (e.g., non-episomal mammalian vectors). Another type of vector can replicate autonomously in the host cell it introduces (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Another specific type of vector capable of directing the expression of the expressible foreign nucleic acid to which they are operatively connected is generally referred to as an "expression vector". An expression vector generally has a control sequence that drives the expression of the expressible foreign nucleic acid. The simpler vectors referred to as "transcription vectors" can only be transcribed but not translated: they can be replicated but not expressed in target cells. The term "vector" encompasses all types of vectors, regardless of their function. Vectors capable of directing the expression of expressible nucleic acids to which they are operably linked are generally referred to as "expression vectors." Other examples of "vectors" may include display vectors (e.g., vectors that direct the expression and display of encoded polypeptides on the surface of viruses or cells (such as bacterial cells, yeast cells, insect cells, and / or mammalian cells)).

[0086] As used herein, "subject", "patient" or "individual" may refer to a human or a non-human animal. "Non-human animal" may refer to any animal that is not classified as a human, such as domestic animals, farm animals or zoo animals, sports animals, pet animals (such as dogs, horses, cats, cows, etc.), and animals used in research. Research animals may refer 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.) without limitation. In some embodiments, the subject, patient or individual is a human.

[0087] "Effective amount" refers to at least the following quantity: at the necessary dosage and for the necessary period, the quantity effectively achieves one or more desired or indicated effects including treatment or prevention results. The effective amount can be provided by one or more administrations. For the purposes of this disclosure, the effective amount of an antibody, drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventive or therapeutic treatment. As understood in clinical situations, the 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 as administered in the form of a monotherapy or combination therapy). Therefore, an "effective amount" can be considered in the context of administering one or more therapeutic agents, and if in conjunction with one or more other agents, a desirable result can be achieved or achieved, then a single agent can be considered to be given in an effective amount.

[0088] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0089] II. Antibodies

[0090] Certain aspects of the invention relate to monospecific antibodies (e.g., traditional, non-masked monospecific antibodies), multispecific antibodies (e.g., non-masked multispecific antibodies), masked antibodies (e.g., activatable monospecific or multispecific antibodies), antigen-binding fragments thereof, or derivatives of such antibodies.

[0091] A. Fc region and C H 3 domains

[0092] In some embodiments, the antibodies described herein (e.g., multispecific antibodies) 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 belongs to an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region belongs to an isotype selected from κ and λ. In some embodiments, the antibodies comprise a human IgG constant region. In some embodiments, the antibodies comprise a human IgG constant region. 4 In some embodiments, the antibody comprises a human IgG 1 In some such embodiments, the antibody is in the human IgG 4 The constant region contains the S228P mutation.

[0093] Whether effector function is desired may depend on the particular processing method intended for the antibody. In some embodiments, when effector function is desired, an antibody comprising human IgG is selected. 1 Heavy chain constant region or human IgG 3 In some embodiments, when effector function is not required, antibodies comprising human IgG 4 or IgG2 In some embodiments, the antibody comprises a human IgG containing one or more mutations that reduce effector function. 1 Heavy chain constant region. In some embodiments, the antibody comprises an IgG containing an N297A substitution 1 Heavy chain constant region.

[0094] The multispecific antibodies described herein (including activatable multispecific antibodies) may comprise C H 3 domains, which have one or more engineered disulfide bonds, one or more engineered (e.g., rearranged or reversed) salt bridges, or a combination thereof. Unless otherwise indicated, all amino acid residue numbering herein is based on Eu numbering, and amino acid substitutions are made in wild-type (or naturally occurring) C H The corresponding amino acid positions in the 3 domain sequence are relative to the wild-type (or naturally occurring) sequence. It should be understood that the mutations or substitutions described herein are applicable to all IgG subclasses and allotypes. IgG allotypes have been 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 relative to the wild-type C of IgG1. H 3 domain sequence, such as IgG1 allotype G1m, 1(a), 2(x), 3(f), or 17(z). In some embodiments, the amino acid mutations or substitutions described herein are relative to IgG 4 The wild-type C H 3 domain sequence. For example, relative to a human IgG 1 Allotype of wild-type C H The D356K substitution in the 3 domain (Uniprot number P01857) is equivalent to the second human IgG 1 Allotype of wild-type C H 3 domains or human IgG 4 The wild-type C H E356K substitution in domain 3. H The 3 domain mutations are shown in Tables 2 and 3. In some embodiments, the amino acid mutations or substitutions described herein are relative to the wild-type Fc region sequence, such as IgG 1 Fc region or IgG 4 Fc region. C with mutation H The 3 sequence is described in WO 2021 / 148006, which is incorporated herein by reference in its entirety. In the following table and other parts of the specification, C H 3 The apostrophe in the mutation annotation indicates the second C HFor example, in N390C-S400'C, the S400C mutation is located at the second C H 3 domains.

[0095] Table 2. Fc mutations

[0096]

[0097] Table 3. Fc mutation ID

[0098]

[0099]

[0100] In some embodiments, a multispecific antibody (eg, an activatable multispecific antibody) comprises a first C H C390 in the 3 domain and the second C H Between C400 in the 3 domain, the first C H C392 in the 3 domain and the second C H 3 domain, or between C397 and the first C H C392 in the 3 domain and the second C H In some embodiments, the engineered disulfide bond between C400 in the wild-type C H Compared to the 3 domains, multispecific antibodies (eg, activatable multispecific antibodies) contain a rearranged salt bridge network, such as at the first C H 357 and 411 in the 3 domain and the second C H 3 domain between positions 351 and 370 (e.g., E357K:T411K-L351'D:K370'D), or at the first C H 357 and 364 in the 3 domain and the second C H 3 domain between positions 351 and 370 (e.g., E357K:S364K-L351'D:K370'D). In some embodiments, the wild-type C H Compared with the 3 domains, multispecific antibodies have H 356 in the 3 domain and the second C H The 439 positions in the 3 domains contain an inverted salt bridge (e.g., D356-K439'). H The 3-mutated antibodies can have high productivity, excellent stability (eg, resistance to aggregation and precipitation at high temperatures or due to freeze-thaw cycles), and potent activity.

[0101] In some embodiments, a multispecific antibody (eg, an activatable multispecific antibody) comprises C H3 domains, which have one or more engineered residues that promote heterodimer formation as described herein. Also contemplated herein are a plurality of C H 3 domains and a first polypeptide comprising a second engineered C H A heteromultimer formed by a second polypeptide having 3 domains and a heterodimer.

[0102] In some embodiments, a multispecific antibody (eg, 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 the first C H The 3 domain contains a cysteine ​​residue at position 400 and the second C H 3 domain comprises a cysteine ​​residue at position 390; or ii) the first C 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 the first C H The 3 domain contains a cysteine ​​residue at position 397 and the second C H 3 domain comprises a cysteine ​​residue at position 392; or iii) the first C 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 the first C H The 3 domain contains a cysteine ​​residue at position 400 and the second C H The 3 domain comprises a cysteine ​​residue at position 392; and wherein the amino acid residue numbering is based on Eu numbering.

[0103] In some embodiments, a multispecific antibody (eg, an activatable multispecific antibody) comprises: a first C H The first polypeptide of the 3 domain and the second polypeptide containing H 3 domains, wherein: i) a first C H The 3 domain also contains a positively charged residue at position 357 and a second C H The 3 domain also contains a negatively charged residue at position 351, or the first C H The 3 domain also contains a negatively charged residue at position 351 and a second C H 3 domain further comprises a positively charged residue at position 357; or ii) the first C H The 3 domain also contains a positively charged residue at position 411 and a second C HThe 3 domain also contains a negatively charged residue at position 370, or the first C H The 3 domain also contains a negatively charged residue at position 370 and a second C H 3 domain further comprises a positively charged residue at position 411; or iii) the first C H The 3 domain also contains a positively charged residue at position 364 and a second C H The 3 domain also contains a negatively charged residue at position 370, or the first C H The 3 domain also contains a negatively charged residue at position 370 and a second C H The 3 domain further comprises a positively charged residue at position 364; or a combination of i) and ii), or a combination of i) and iii), and wherein the amino acid residue numbering is based on Eu numbering.

[0104] In some embodiments, the first C H The 3 domain also contains a positively charged residue at position 356 and a second C H The 3 domain also contains a negatively charged residue at position 439, or the first C H The 3 domain also contains a negatively charged residue at position 439 and a second C H The 3 domain also contains a positively charged residue at position 356, and wherein the amino acid residue numbering is based on Eu numbering.

[0105] In some embodiments, a multispecific antibody (eg, an activatable multispecific antibody) comprises: a first C H The first polypeptide of the 3 domain and the second polypeptide containing H 3 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 the first C H The 3 domain contains a cysteine ​​residue at position 400 and the second C H 3 domain comprises a cysteine ​​residue at position 390; or ii) the first C 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 the first C H The 3 domain contains a cysteine ​​residue at position 397 and the second C H 3 domain comprises a cysteine ​​residue at position 392; or iii) the first C H The 3 domain contains a cysteine ​​residue at position 392 and a second C HThe 3 domain contains a cysteine ​​residue at position 400, or the first C H The 3 domain contains a cysteine ​​residue at position 400 and the second C H 3 domain comprises a cysteine ​​residue at position 392; and wherein: a) the first C H The 3 domain also contains a positively charged residue at position 357 and a second C H The 3 domain also contains a negatively charged residue at position 351, or the first C H The 3 domain also contains a negatively charged residue at position 351 and a second C H The 3 domain further comprises a positively charged residue at position 357; or b) the first C H The 3 domain also contains a positively charged residue at position 411 and a second C H The 3 domain also contains a negatively charged residue at position 370, or the first C H The 3 domain also contains a negatively charged residue at position 370 and a second C H 3 domain further comprises a positively charged residue at position 411; or c) the first C H The 3 domain also contains a positively charged residue at position 364 and a second C H The 3 domain also contains a negatively charged residue at position 370, or the first C H The 3 domain also contains a negatively charged residue at position 370 and a second C H The 3 domain further comprises a positively charged residue at position 364; or a combination of a) and b), or a combination of a) and c); wherein the amino acid residue numbering is based on Eu numbering.

[0106] In some embodiments, the first C H The 3 domain also contains a positively charged residue at position 356 and a second C H The 3 domain also contains a negatively charged residue at position 439, or the first C H The 3 domain also contains a negatively charged residue at position 439 and a second C H The 3 domain also contains a positively charged residue at position 356, and wherein the amino acid residue numbering is based on Eu numbering.

[0107] C H The C3 domain can be derived from any naturally occurring immunoglobulin molecule. H 3 domains derived from IgG 1 Molecule, IgG 2 Molecule, IgG 3 Molecules or IgG 4 In some embodiments, CH 3 domains are human C H 3 domains. In some embodiments, C H 3 domains derived from human IgG 1 molecular.

[0108] In some embodiments, a multispecific antibody (eg, an activatable multispecific antibody) comprises: a first C H The first polypeptide of the 3 domain and the second polypeptide containing H 3 domains, wherein: i) a first C H The 3 domain contains a N390C substitution and the second C H 3 domain contains S400C substitution, or the first C H The 3 domain contains an S400C substitution and the second C H 3 domain comprises a N390C substitution; or ii) the first C H The 3 domain contains a K392C substitution and the second C H 3 domain contains a V397C substitution, or the first C H The 3 domain contains a V397C substitution and the second C H 3 domain comprises a K392C substitution; or iii) the first C H The 3 domain contains a K392C substitution and the second C H 3 domain contains S400C substitution, or the first C H The 3 domain contains an S400C substitution and the second C H The 3 domain contains the K392C substitution.

[0109] In some embodiments, a multispecific antibody (eg, an activatable multispecific antibody) comprises: a first C H The first polypeptide of the 3 domain and the second polypeptide containing H 3 domains, wherein: i) a first C H The 3 domain contains E357K and T411K substitutions and the second C H 3 domain contains L351D and K370D substitutions, or the first C H The 3 domain contains L351D and K370D substitutions and the second C H 3 domain contains E357K and T411K substitutions; or ii) the first C H The 3 domain contains E357K and S364K substitutions and the second C H 3 domain contains L351D and K370D substitutions, or the first C H The 3 domain contains L351D and K370D substitutions and the second C H3 domain comprises E357K and S364K substitutions; or iii) the first C H The 3 domain contains D356K, E357K and S364K substitutions and the second C H 3 domain contains L351D, K370D and K439D substitutions, or the first C H The 3 domain contains L351D, K370D and K439D substitutions and the second C H The 3 domain contains D356K, E357K and S364K substitutions.

[0110] In some embodiments, a multispecific antibody (eg, an activatable multispecific antibody) comprises: a first C H The first polypeptide of the 3 domain and the second polypeptide containing H The second polypeptide of the 3 domain, wherein the first C H The 3 domain contains E357K, S364K and N390C substitutions and the second C H 3 domain contains 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 E357K, S364K and N390C substitutions.

[0111] In some embodiments, a multispecific antibody (eg, an activatable multispecific antibody) comprises: a first C H The first polypeptide of the 3 domain and the second polypeptide containing H The second polypeptide of the 3 domain, wherein the first C H The 3 domain contains E357K, S364K and S400C substitutions and the second C H 3 domain contains L351D, K370D and N390C substitutions, or the first C H The 3 domain contains L351D, K370D and N390C substitutions and the second C H The 3 domain contains E357K, S364K and S400C substitutions.

[0112] In some embodiments, a multispecific antibody (eg, an activatable multispecific antibody) comprises: a first C H The first polypeptide of the 3 domain and the second polypeptide containing H The second polypeptide of the 3 domain, wherein the first C H The 3 domain contains D356K, E357K, S364K and S400C substitutions and the second C H 3 domains contain L351D, K370D, N390C and K439D substitutions, or the first C HThe 3 domain contains L351D, K370D, N390C and K439D substitutions and the second C H The 3 domain contains D356K, E357K, S364K and S400C substitutions.

[0113] In some embodiments, a multispecific antibody (eg, an activatable multispecific antibody) comprises: a first C H The first polypeptide of the 3 domain and the second polypeptide containing H The second polypeptide of the 3 domain, wherein the first C H The 3 domain contains D356K, E357K, S364K and N390C substitutions and the second C H 3 domains contain 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.

[0114] In some embodiments, a multispecific antibody (eg, an activatable multispecific antibody) comprises an engineered C H The Fc region can be derived from any suitable Fc subclass, including but not limited to IgG 1 IgG 2 IgG 3 and IgG 4 Subcategory.

[0115] B. Cysteine ​​mutation

[0116] In some embodiments, a multispecific antibody described herein (eg, an activatable multispecific antibody described herein) comprises a first C H The first polypeptide of the 3 domain and the second polypeptide containing H The second polypeptide of the 3 domain, wherein the first C H The 3 domain contains a first engineered cysteine ​​residue and a second C H The 3 domain comprises a second engineered cysteine ​​residue, wherein the first engineered cysteine ​​residue and the second cysteine ​​residue form a disulfide bond.

[0117] In some embodiments, the first C H The 3 domain contains a C at position 390 and a second C H 3 domain contains a C at position 400, or the first C H The 3 domain contains a C at position 400 and a second C HThe 3 domain comprises a C at position 390. In some embodiments, the first C H The 3 domain contains a N390C substitution and the second C H 3 domain contains S400C substitution, or the first C H The 3 domain contains an S400C substitution and the second C H Domain 3 contains the N390C substitution.

[0118] In some embodiments, the first C H The 3 domain contains a C at position 392 and a second C H The 3 domain contains a C at position 397, or the first C H The 3 domain contains a C at position 397 and a second C H The 3 domain comprises a C at position 392. In some embodiments, the first C H The 3 domain contains a K392C substitution and the second C H 3 domain contains a V397C substitution, or the first C H The 3 domain contains a V397C substitution and the second C H The 3 domain contains the K392C substitution.

[0119] In some embodiments, the first C H The 3 domain contains a C at position 392 and a second C H 3 domain contains a C at position 400, or the first C H The 3 domain contains a C at position 400 and a second C H The 3 domain comprises a C at position 392. In some embodiments, the first C H The 3 domain contains a K392C substitution and the second C H 3 domain contains S400C substitution, or the first C H The 3 domain contains an S400C substitution and the second C H The 3 domain contains the K392C substitution.

[0120] C. Salt bridge mutation

[0121] In some embodiments, a multispecific antibody described herein (eg, an activatable multispecific antibody described herein) comprises a first C H The first polypeptide of the 3 domain and the second polypeptide containing H The second polypeptide of the 3 domain, wherein the first C H The 3 domain contains engineered positively charged residues and the second C HThe 3 domain contains an engineered negatively charged residue, wherein the engineered positively charged residue and the engineered negatively charged residue form a salt bridge. H 3 domains, the engineered salt bridge can be H In some embodiments, the engineered positively charged residues replace the wild-type C H In some embodiments, the engineered positively charged residues replace the wild-type C H The positively charged residues in the 3 domains were replaced with negatively charged residues. Rearrangement and reversal of salt bridges can allow the inclusion of engineered C H The isoelectric points (PI) of the heterodimers and homodimers of the 3-domain are altered, thereby allowing for better separation of heterodimers from homodimers during the purification process.

[0122] In some embodiments, the first C H The 3 domain contains a positively charged residue at position 357 and the second C H The 3 domain contains a negatively charged residue at position 351, or the first C H The 3 domain contains a negatively charged residue at position 351 and the second C H The 3 domain comprises a positively charged residue at position 357. In some embodiments, the first C H Domain 3 contains a K at position 357 and a second C H 3 domain contains a D at position 351, or the first C H The 3 domain contains a D at position 351 and a second C H The 3 domain comprises a K at position 357. In some embodiments, the first C H Domain 3 contains a K at position 357 and a second C H 3 domain contains an E at position 351, or the first C H The 3 domain contains an E at position 351 and a second C H The 3 domain comprises a K at position 357. In some embodiments, the first C H The 3 domain contains an R at position 357 and a second C H 3 domain contains a D at position 351, or the first C H The 3 domain contains a D at position 351 and a second C H The 3 domain comprises an R at position 357. In some embodiments, the first C HThe 3 domain contains an R at position 357 and a second C H 3 domain contains an E at position 351, or the first C H The 3 domain contains an E at position 351 and a second C H The 3 domain comprises an R at position 357. In some embodiments, the first C H The 3 domain contains the E357K substitution and the second C H 3 domain contains L351D substitution, or the first C H The 3 domain contains the L351D substitution and the second C H The 3 domain contains the E357K substitution.

[0123] In some embodiments, the first C H The 3 domain contains a positively charged residue at position 411 and the second C H The 3 domain contains a negatively charged residue at position 370, or the first C H The 3 domain contains a negatively charged residue at position 370 and the second C H The 3 domain comprises a positively charged residue at position 411. In some embodiments, the first C H The 3 domain contains a K at position 411 and a second C H 3 domain contains a D at position 370, or the first C H The 3 domain contains a D at position 370 and a second C H The 3 domain comprises a K at position 411. In some embodiments, the first C H The 3 domain contains a K at position 411 and a second C H 3 domain contains an E at position 370, or the first C H The 3 domain contains an E at position 370 and a second C H The 3 domain comprises a K at position 411. In some embodiments, the first C H The 3 domain contains an R at position 411 and a second C H 3 domain contains a D at position 370, or the first C H Domain 3 contains a D at position 370 and a second C H 3 domain comprises an R at position 411. In some embodiments, the first C H The 3 domain contains an R at position 411 and a second C H 3 domain contains an E at position 370, or the first C H The 3 domain contains an E at position 370 and a second C H 3 domain comprises an R at position 411. In some embodiments, the first C HThe 3 domain contains a T411K substitution and the second C H 3 domain contains K370D substitution, or the first C H The 3 domain contains a K370D substitution and the second C H Domain 3 contains a T411K substitution.

[0124] In some embodiments, the first C H The 3 domain contains a positively charged residue at position 364 and the second C H The 3 domain contains a negatively charged residue at position 370, or the first C H The 3 domain contains a negatively charged residue at position 370 and the second C H The 3 domain comprises a positively charged residue at position 364. In some embodiments, the first C H The 3 domain contains a K at position 364 and a second C H 3 domain contains a D at position 370, or the first C H Domain 3 contains a D at position 370 and a second C H The 3 domain comprises a K at position 364. In some embodiments, the first C H The 3 domain contains a K at position 364 and a second C H 3 domain contains an E at position 370, or the first C H The 3 domain contains an E at position 370 and a second C H The 3 domain comprises a K at position 364. In some embodiments, the first C H The 3 domain contains an R at position 364 and a second C H 3 domain contains a D at position 370, or the first C H Domain 3 contains a D at position 370 and a second C H 3 domain comprises an R at position 364. In some embodiments, the first C H The 3 domain contains an R at position 364 and a second C H 3 domain contains an E at position 370, or the first C H The 3 domain contains an E at position 370 and a second C H 3 domain comprises an R at position 364. In some embodiments, the first C H The 3 domain contains an S364K substitution and the second C H 3 domain contains K370D substitution, or the first C H The 3 domain contains a K370D substitution and the second C H Domain 3 contains the S364K substitution.

[0125] In some embodiments, the first C H The 3 domain contains a positively charged residue at position 356 and the second C H The 3 domain contains a negatively charged residue at position 439, or the first C H The 3 domain contains a negatively charged residue at position 439 and the second C H The 3 domain comprises a positively charged residue at position 356. In some embodiments, the first C H Domain 3 contains a K at position 356 and a second C H 3 domain contains a D at position 439, or the first C H The 3 domain contains a D at position 439 and a second C H The 3 domain comprises a K at position 356. In some embodiments, the first C H Domain 3 contains a K at position 356 and a second C H 3 domain contains an E at position 439, or the first C H The 3 domain contains an E at position 439 and a second C H The 3 domain comprises a K at position 356. In some embodiments, the first C H The 3 domain contains an R at position 356 and a second C H 3 domain contains a D at position 439, or the first C H The 3 domain contains a D at position 439 and a second C H The 3 domain comprises an R at position 356. In some embodiments, the first C H The 3 domain contains an R at position 356 and a second C H 3 domain contains an E at position 439, or the first C H The 3 domain contains an E at position 439 and a second C H The 3 domain comprises an R at position 356. In some embodiments, the first C H The 3 domain contains a D356K substitution and the second C H 3 domain contains K439D substitution, or the first C H The 3 domain contains a K439D substitution and the second C H Domain 3 contains the D356K substitution.

[0126] Any of the engineered salt bridges described herein can be combined with each other. In some embodiments, the first C H The 3 domain contains a positively charged residue at position 357 and a positively charged residue at position 411 and the second C HThe 3 domain contains a negatively charged residue at position 351 and a negatively charged residue at position 370, or the first C H The 3 domain contains a negatively charged residue at position 351 and a negatively charged residue at position 370 and the second C H The 3 domain comprises a positively charged residue at position 357 and a positively charged residue at position 411. In some embodiments, the first C H The 3 domain contains E357K and T411K substitutions and the second C H 3 domain contains L351D and K370D substitutions, or the first C H The 3 domain contains L351D and K370D substitutions and the second C H The 3 domain contains E357K and T411K substitutions.

[0127] In some embodiments, the first C H The 3 domain contains a positively charged residue at position 357 and a positively charged residue at position 364 and the second C H The 3 domain contains a negatively charged residue at position 351 and a negatively charged residue at position 370, or the first C H The 3 domain contains a negatively charged residue at position 351 and a negatively charged residue at position 370 and the second C H The 3 domain comprises a positively charged residue at position 357 and a positively charged residue at position 364. In some embodiments, the first C H The 3 domain contains E357K and S364K substitutions and the second C H 3 domain contains L351D and K370D substitutions, or the first C H The 3 domain contains L351D and K370D substitutions and the second C H The 3 domain contains E357K and S364K substitutions.

[0128] In some embodiments, the first C H The 3 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 3 domain comprises a negatively charged residue at position 351, a negatively charged residue at position 370, and a negatively charged residue at position 439, or the first C 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 the second C HThe 3 domain comprises a positively charged residue at position 356, a positively charged residue at position 357, and a positively charged residue at position 364. In some embodiments, the first C H The 3 domain contains D356K, E357K and S364K substitutions and the second C H 3 domain contains L351D, K370D and K439D substitutions, or the first C H The 3 domain contains L351D, K370D and K439D substitutions and the second C H The 3 domain contains D356K, E357K and S364K substitutions.

[0129] D. Other mutations

[0130] The C H The 3 domain or Fc region may also comprise an engineered disulfide bond and / or salt bridge as listed in Table 4 below.

[0131] Table 4. Exemplary Fc mutations

[0132]

[0133]

[0134] In some embodiments, the first C H The 3 domain also 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 3 domain contains a C at position 399 and a second C H The 3 domain comprises a C at position 392. In some embodiments, the first C H The 3 domain also contains a K392C substitution and the second C H The 3 domain also contains a D399C substitution, or the first C H The 3 domain also contains a D399C substitution and the second C H The 3 domain also contains a K392C substitution.

[0135] In some embodiments, the first C H The 3 domain also contains a C at position 394 and a second C H 3 domain contains a C at position 354, or the first C H The 3 domain contains a C at position 354 and a second C H The 3 domain comprises a C at position 394. In some embodiments, the first C H The 3 domain also contains a Y394C substitution and the second C H The 3 domain also contains an S354C substitution, or the first CH The 3 domain also contains an S354C substitution and the second C H The 3 domain also contains a Y394C substitution.

[0136] In some embodiments, the first C H The 3 domain also 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 3 domain contains a C at position 349 and a second C H The 3 domain comprises a C at position 356. In some embodiments, the first C H The 3 domain also contains a D356C substitution and the second C H The 3 domain also contains a Y349C substitution, or the first C H The 3 domain also contains a Y349C substitution and the second C H The 3 domain also contains a D356C substitution.

[0137] In some embodiments, the first C H The 3 domain also contains K392D and K409D substitutions and the second C H The 3 domain also contains D356K and D399K substitutions, or the first C H The 3 domain also contains D356K and D399K substitutions and the second C H The 3 domain also contains K392D and K409D substitutions.

[0138] In some embodiments, the first C H The 3 domain also contains L368D and K370S substitutions and the second C H The 3 domain also contains E357Q and S364K substitutions, or the first C H The 3 domain also contains E357Q and S364K substitutions and the second C H The 3 domain also contains L368D and K370S substitutions.

[0139] In some embodiments, the first C H The 3 domain also contains L351K and T366K substitutions and the second C H The 3 domain also contains L351D and L368E substitutions, or the first C H The 3 domain also contains L351D and L368E substitutions and the second C H The 3 domain also contains L351K and T366K substitutions.

[0140] In some embodiments, the first C H The 3 domain also contains P395K, P396K and V397K substitutions and the second CH 3 domain contains T394D, P395D and P396D substitutions, or the first C H The 3 domain also contains T394D, P395D and P396D substitutions and the second C H The 3 domain also contains P395K, P396K and V397K substitutions.

[0141] In some embodiments, the first C H The 3 domain also contains F405E, Y407E and K409E substitutions and the second C H 3 domain contains F405K and Y407K substitutions, or the first C H The 3 domain also contains F405K and Y407K substitutions and the second C H The 3 domain also contains F405E, Y407E and K409E substitutions.

[0142] In some embodiments, the first C H The 3 domain also contains T336S, L368A and Y407V substitutions and the second C H The 3 domain also contains a T366W substitution, or the first C H The 3 domain also contains a T366W substitution and the second C H The 3 domain also contains T336S, L368A and Y407V substitutions.

[0143] In some embodiments, the first C H The 3 domain contains L368V and Y407V substitutions and the second C H 3 domain contains a T366W substitution, or the first C H The 3 domain contains a T366W substitution and the second C H Domain 3 contains L368V and Y407V substitutions.

[0144] III. CD28 Binding Molecules

[0145] The present disclosure 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 for epitope binding and for complementary determining regions (CDRs), variable regions (V L 、V H ) and IgG (e.g., IgG 4 )Antibodies described by specific amino acid sequences of their light and heavy chains.

[0146] In some embodiments, the antibody or antigen-binding fragment thereof 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 can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assay, EMSA, etc. In some embodiments, the ability of an antibody or antigen-binding fragment to bind to a target antigen is measured by ELISA or RED96 measurement (see, e.g., Example 3 below).

[0147] In some embodiments, the antibody or antigen-binding fragment has a K of about 500 nM or less (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.). D Binds to human CD28. In some embodiments, the antibody or antigen-binding fragment binds to human CD28 with a K of about 100 nM or less. D Binds to human CD28. In some embodiments, the antibody or antigen-binding fragment binds to human CD28 with a K of about 50 nM or less. D Binds to human CD28. Measures the K of antibodies or antigen-binding fragments D The method can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assay, EMSA, etc. In some embodiments, K D pass RED96 system measurement (see, e.g., Example 3 below).

[0148] A. Anti-CD28 Antibody

[0149] In some embodiments, the disclosure provides an isolated monoclonal antibody that binds to human CD28 at an epitope represented by amino acid residues 33-37, 80-83, 92-96, and 100-104 of SEQ ID NO: 1. In a specific embodiment, the disclosure provides an isolated antibody that binds 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 antibody has a K of 10 nM or less. D Binds to human CD28, e.g., by RED96 system measurement. In certain embodiments, in addition to binding to human antigen epitopes, the antibodies of the present disclosure may cross-react with at least one non-human species selected from cynomolgus macaques, mice, rats and dogs. In certain embodiments, the antibodies of the present disclosure have the advantage of cross-species binding to mice, humans and monkeys, but the benchmark controls TAC2386 and TAC2387 of the present disclosure do not have species cross-reactivity of this range. In specific embodiments, the benchmark controls TAC2386 and TAC2387 bind to human antigen epitopes but do not bind to mouse antigen epitopes (see Table 7 herein). The species cross-reactivity of the disclosed antibodies can also provide additional advantages of using mice to simulate antibody safety, activity and function. Therefore, compared to TAC2386 and TAC2387 of the present disclosure, it is easier to perform animal modeling using the antibodies of the present disclosure.

[0150] In a specific embodiment, the isolated anti-CD28 monoclonal antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 6, and HCDR3 of SEQ ID NO: 7, and LCDR1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10. In a specific embodiment, 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 a specific embodiment, the isolated monoclonal antibody comprises a heavy chain of SEQ ID NO: 13 and a light chain of SEQ ID NO: 14.

[0151] The CD28 antibody described herein may be of any class, such as IgG, IgM, IgE, IgA or IgD. The CD28 antibody is preferably of the IgG class, such as IgG 1 IgG 2 IgG 3 or IgG 4Anti-CD28 antibodies can be converted from one class or subclass to another class or subclass using methods known in the art. An exemplary method for producing an antibody of a desired class or subclass comprises the following steps: isolating nucleic acid encoding an anti-CD28 antibody heavy chain and nucleic acid encoding a CD28 antibody light chain, isolating nucleic acid encoding a V H The sequence of the zone, V H The sequence is linked to a sequence encoding a heavy chain constant region of the desired class or subclass, the light chain gene and heavy chain construct are expressed in cells, and CD28 antibodies are collected.

[0152] The CD28 antibodies described herein may be of any class, such as IgG, IgM, IgE, IgA or IgD. Preferably, the anti-CD28 antibodies are of the IgG class, such as IgG 1 IgG 2 IgG 3 or IgG 4 Subclass. Anti-CD28 antibodies can be converted from one class or subclass to another class or subclass using methods known in the art. An exemplary method for producing an antibody belonging to a desired class or subclass comprises the following steps: isolating nucleic acid encoding the heavy chain of the anti-CD28 antibody and nucleic acid encoding the light chain of the CD28 antibody; isolating nucleic acid encoding V H The sequence of the region; V H The sequence is linked to a sequence encoding a heavy chain constant region of the desired class or subclass; the light chain gene and heavy chain construct are expressed in cells; and CD28 antibodies are collected.

[0153] Furthermore, the antibodies provided by the present disclosure may be monoclonal or polyclonal, but are preferably monoclonal.

[0154] Antibodies of the present disclosure can be produced by techniques known in the art, including conventional monoclonal antibody methods, e.g., standard somatic cell hybridization techniques (see, e.g., Kohler and Milstein, Nature (1975) 256:495, viral or oncogenic transformation of B lymphocytes, or recombinant antibody techniques, as described in detail below.

[0155] Hybridoma production is an extremely well established procedure. A common animal system for preparing hybridomas is the murine system. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. One well-known method that can be used to prepare the human CD28 antibodies provided by the present disclosure involves the use of a XenoMouse TM Animal System. XenoMouse TMThe mouse is an engineered mouse strain that contains large fragments of human immunoglobulin heavy and light chain loci and is defective in mouse antibody production. 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 can be a fragment of CD28, such as the extracellular domain of CD28, especially a CD28 extracellular domain fragment comprising amino acid residues 34, 34, 36 and 37, 80-83, 92 and 93, 95 and 96, and 100-104 of SEQ ID NO: 1. The immunization of the animal can 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. Patent No. 5,994,619. The CD28 antigen can be administered with an adjuvant to stimulate an immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulatory complex). After the animal is immunized with the CD28 antigen, an antibody-producing immortalized cell line is prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and lymph nodes and / or spleen B cells are immortalized. Methods for immortalizing cells include, but are not limited to, transferring them with oncogenes, infecting them with oncoviruses, culturing them under conditions that select for immortalized cells, subjecting them to carcinogenic or mutagenic compounds, fusing them with immortalized cells such as myeloma cells, and inactivating tumor suppressor genes. See, for example, Harlow and Lane, supra. If fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secreting cell line). Immortalized cells are screened using CD28, a portion thereof, or cells expressing CD28. CD28 antibody-producing cells such as hybridomas are selected, cloned, and further screened for desirable features, including robust growth, high antibody production, and desirable antibody characteristics, as discussed further below. Hybridomas can be amplified in vivo in isogenic animals of the same species, in animals lacking an immune system such as nude mice, or in vitro in cell culture. Methods for selecting, cloning, and amplifying hybridomas are well known to those of ordinary skill in the art.

[0156] Antibodies disclosed herein can also be prepared using phage display or yeast display methods. The 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.

[0157] B. Antigen Binding Fragment

[0158] In some other aspects, the present disclosure provides antigen-binding fragments of any of the CD28 antibodies provided by the present disclosure.

[0159] The antigen-binding fragment may comprise any sequence of an antibody. In some embodiments, the antigen-binding fragment comprises (1) a light chain of an anti-CD28 antibody; (2) a heavy chain of a CD28 antibody; (3) a variable region from a light chain of an anti-CD28 antibody; (4) a variable region from a heavy chain of a CD28 antibody; (5) one or more CDRs (two, three, four, five, or six CDRs) of an anti-CD28 antibody; or (6) an amino acid sequence of three CDRs from a light chain of an anti-CD28 antibody and three CDRs from a heavy chain of an anti-CD28 antibody.

[0160] In other specific embodiments, the antigen-binding fragment of an anti-CD28 antibody comprises: (i) a Fab fragment, which is a V L 、V H , C L and C H 1 domain-consisting monovalent fragment; (ii) F(ab') 2 A fragment which is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) a fragment consisting of V H and C H (iv) a single-arm V fragment of the antibody; L and V H(v) dAb fragments (Ward et al., Nature (1989) 341: 544-546), which are composed of V H (vi) separated CDRs, and (vii) single-chain antibodies (scFv), which are V L The V region is connected to the antibody H Bird et al., Science (1988) 242: 423-426 and Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85: 5879-5883.

[0161] In some embodiments, the anti-CD28 antibodies or antibody fragments of the present disclosure comprise 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 the amino acid sequence set forth in any one 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. H In some embodiments, the anti-CD28 antibodies or antibody fragments of the present disclosure 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 the amino acid sequence set forth in any one 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. LIn some embodiments, the anti-CD28 antibodies or antibody fragments of the present disclosure comprise a 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 one 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 of the present disclosure comprise a 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 one of SEQ ID NOs: 6, 51, 58, 67, 89, 96, 104, and 155. In some embodiments, the anti-CD28 antibodies or antibody fragments of the present disclosure comprise a 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 the amino acid sequence set forth in any one of 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 antibodies or antibody fragments of the present disclosure comprise a 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 the amino acid sequence set forth in any one 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. In some embodiments, the anti-CD28 antibodies or antibody fragments of the present disclosure comprise a 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 one of SEQ ID NOs: 9, 18, 27, 37, and 300. In some embodiments, the anti-CD28 antibodies or antibody fragments of the present disclosure comprise a 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 one of SEQ ID NOs: 10, 19, 28, 38, 46, 61, 70, 83, 114, 121, 129, 143, and 164.

[0162] In some embodiments, an antibody of the present disclosure comprises 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 an amino acid sequence as set forth in any one 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.

[0163] In some embodiments, the antibodies of the present disclosure 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 one 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.

[0164] IV. Multispecific Antibodies

[0165] Also provided are multispecific antibodies, which correspond to the activatable multispecific antibodies or shielded multispecific antibodies described herein. One aspect of the present application provides activatable 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 the off-target tumor effect, traditional TCE molecules are associated with high cytotoxicity, including toxicity to the central nervous system (CNS) and cytokine storms. Therefore, there is a need in the art for antibodies that can bind to T cells and target cells (such as tumor cells with enhanced specificity and reduced side effects).

[0166] In some embodiments, the multispecific antibody is a bispecific (bsAb). In some embodiments, the multispecific antibody is a trispecific (tsAb).

[0167] In certain embodiments, the bispecific antibodies of the present disclosure are specific for CD28 on the surface of T cells. In some embodiments, the multispecific antibodies are tumor-associated antigen (TAA) xCD28 bispecific antibodies that specifically bind to TAA and CD28. In some embodiments, the antibodies of the present disclosure are IgG antibodies, for example, comprising an IgG Fc region (e.g., a human IgG Fc region).

[0168] In some embodiments, the multispecific antibody binds to CD28 on the surface of a T cell. 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 shielding or cleavable moieties. In some embodiments, the multispecific antibody is obtained after cleavage of one or more cleavable moieties.

[0169] 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) x CD3 bispecific antibody that specifically binds to TAA and CD3. In some embodiments, the multispecific antibody specifically binds to CD3 with weak affinity, such as EC measured by ELISA. 50 In some embodiments, the multispecific antibody comprises 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 any shielding moiety or cleavable moiety. In some embodiments, the multispecific antibody is obtained after cleavage of one or more cleavable moieties.

[0170] In some embodiments, a multispecific antibody is provided comprising: a) a first antigen-binding fragment comprising 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 1; and b) a second antigen-binding fragment comprising VH2 and VH2 of an anti-CD3 antibody that specifically binds to CD3 L 2, wherein the first and / or second antigen binding fragment is fused to the first and / or second shielding peptide (MP1 / MP2). In some embodiments, a multispecific antibody is provided comprising: a) a first antigen binding fragment comprising 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 1; and b) a second antigen-binding fragment comprising VH2 and VH2 of an anti-CD28 antibody that specifically binds to CD28 L 2, wherein the first and / or second antigen binding fragment is fused to a first and / or second shielding peptide (MP1 / MP2).

[0171] 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 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 scFv, which comprises V from N-terminus to C-terminus. L2. Optional linker and VH2. In some embodiments, the first antigen binding fragment is Fab and the second antigen binding fragment is Fab. In some embodiments, the first antigen binding fragment is Fab and the second antigen binding fragment is scFv.

[0172] The C H Antibodies that have 3-domain disulfide bonds and / or salt bridges may also comprise one or more knob-into-hole residues. "Knob-into-hole" or "KIH" refers to a method known in the art for making bispecific antibodies, also known as the "protuberance-into-cavity" method (e.g., see U.S. Pat. No. 5,731,168). In this method, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each comprise 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 can be engineered so that a "knob" or "protuberance" (these terms are used interchangeably herein) located in the interface of one immunoglobulin polypeptide corresponds to a "hole" or "cavity" (these terms are used interchangeably herein) located in the interface of another immunoglobulin polypeptide. In some embodiments, the hole and the knob are of the same or similar size and are appropriately positioned so that when the two interfaces interact, the knob of one interface can be positioned in the corresponding hole of the other interface. Without wishing to be bound by theory, it is believed that this stabilizes heteromultimers and favors the formation of heteromultimers over other species (e.g., homomultimers). In some embodiments, the KIH method is used in combination with the engineered disulfide bonds and / or salt bridges described herein to promote heteromultimerization of two different immunoglobulin polypeptides, which results in a bispecific antibody comprising two immunoglobulin polypeptides with binding specificities for different epitopes. In some embodiments, the C of the activatable multispecific antibody described herein H The 3 domain does not contain KIH residues.

[0173] 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:

[0174] (i) The first polypeptide comprises a structure represented by the following formula:

[0175] V H 1-C H 1- Hinge-C H 2-First C H 3;

[0176] (ii) the second polypeptide comprises a structure represented by the following formula:

[0177] scFv-hinge-C H 2-a second CH3; and

[0178] (iii) the third polypeptide comprises a structure represented by the following formula:

[0179] V L 1-C L ;

[0180] in:

[0181] V L 1 is the first immunoglobulin light chain variable domain;

[0182] V H 1 is the first immunoglobulin heavy chain variable domain;

[0183] scFv is a single-chain variable fragment that contains the second immunoglobulin light chain variable domain (V L 2) and the second immunoglobulin heavy chain variable domain (V H 2);

[0184] C L is the immunoglobulin light chain constant domain;

[0185] C H 1 is immunoglobulin heavy chain constant domain 1;

[0186] C H 2 is immunoglobulin heavy chain constant domain 2; and

[0187] The hinge is connected to C H 1 domain and C H 2 domain immunoglobulin hinge region;

[0188] Where V L 1 is associated with VH1 to form a first Fv that specifically binds to a tumor antigen (e.g., B7-H3, HER2, TROP2, or PD-L1); and wherein the scFv specifically binds to CD28. In some embodiments, the scFv is determined by ELISA at a half-maximal binding antibody concentration (EC) ranging from 0.1 nM to 1000 nM. 50 ) binds to CD28 (e.g., as described in Example 3). In some embodiments, the scFv is expressed by The RED96 assay was performed at a half-maximal binding antibody concentration (EC 50) binds to CD28 (e.g., as described in Example 3). In certain embodiments, the scFv binds to CD28 with a dissociation constant (Kd) of less than 10 nM.

[0189] 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:

[0190] (i) The first polypeptide comprises a structure represented by the following formula:

[0191] V H 1-C H 1- Hinge-C H 2-first CH3;

[0192] (ii) the second polypeptide comprises a structure represented by the following formula:

[0193] V H 2-C H 1- Hinge-C H 2-second CH3;

[0194] (iii) the third polypeptide comprises a structure represented by the following formula:

[0195] V L 1-C L ;and

[0196] (iv) the fourth polypeptide comprises a structure represented by the following formula:

[0197] V L 2-C L ;

[0198] in:

[0199] V L 1 is the first immunoglobulin light chain variable domain;

[0200] V H 1 is the first immunoglobulin heavy chain variable domain;

[0201] V L 2 is a second immunoglobulin light chain variable domain;

[0202] V H 2 is a second immunoglobulin heavy chain variable domain;

[0203] C L is the immunoglobulin light chain constant domain;

[0204] C H 1 is immunoglobulin heavy chain constant domain 1;

[0205] C H 2 is immunoglobulin heavy chain constant domain 2; and

[0206] The hinge is connected to C H 1 domain and C H 2 domain immunoglobulin hinge region;

[0207] Where V L 1 and V H 1 to form a first Fv that specifically binds to a tumor antigen (e.g., B7-H3, HER2, TROP2, or PD-L1); and wherein V L 2 and V H The scFvs are bound to each other by binding to form a second Fv that specifically binds to CD28. In some embodiments, the scFvs are detected by ELISA at a half-maximal binding antibody concentration (EC) ranging from 0.1 nM to 1000 nM. 50 ) binds to CD28 (e.g., as described in Example 3). In some embodiments, the second Fv is The RED96 assay was performed at a half-maximal binding antibody concentration (EC 50 ) binds to CD28 (e.g., as described in Example 3). In certain embodiments, the second Fv binds to CD28 with a dissociation constant (Kd) of less than 10 nM.

[0208] In some embodiments, a multispecific antibody is provided, comprising:

[0209] a) a first antigen-binding fragment comprising a V domain 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 1; and

[0210] b) a second antigen-binding fragment comprising a V fragment 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 the first shielding peptide (MP1).

[0211] In some embodiments, a multispecific antibody is provided, comprising:

[0212] a) a first antigen-binding fragment comprising a V domain 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 1, wherein the first antigen binding fragment is fused to a first shielding peptide (MP1); and

[0213] b) a second antigen-binding fragment comprising a V fragment 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 shielding peptide (MP2).

[0214] 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 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 scFv, which comprises V from N-terminus to C-terminus. L 2. Optional connectors and V H 2.

[0215] In some embodiments, a multispecific antibody is provided, comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein:

[0216] (i) The first polypeptide comprises a structure represented by the following formula:

[0217] V H 1-C H 1- Hinge-C H 2-First C H 3;

[0218] (ii) the second polypeptide comprises a structure represented by the following formula:

[0219] MP1-V L 2-V H 2-Hinge-C H 2-Second C H 3; and

[0220] (iii) the third polypeptide comprises a structure represented by the following formula:

[0221] V L 1-C L ;

[0222] in:

[0223] V L 1 is the first immunoglobulin light chain variable domain;

[0224] V H 1 is the first immunoglobulin heavy chain variable domain;

[0225] V L 2 is a second immunoglobulin light chain variable domain;

[0226] V H2 is a second immunoglobulin heavy chain variable domain;

[0227] C L is the immunoglobulin light chain constant domain;

[0228] C H 1 is immunoglobulin heavy chain constant domain 1;

[0229] C H 2 is immunoglobulin heavy chain constant domain 2;

[0230] First C H 3 is the first immunoglobulin heavy chain constant domain 3;

[0231] Second C H 3 is the second immunoglobulin heavy chain constant domain 3;

[0232] The hinge is connected to C H 1 domain and C H 2 domain immunoglobulin hinge region;

[0233] MP1 is the first shielding peptide; MP1 comprises an N-terminal unit (NU), a shielding unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; the LU of the shielding peptide may not comprise a cleavage site, or may comprise at least one cleavage site.

[0234] In some embodiments, a multispecific antibody is provided, comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein:

[0235] (i) The first polypeptide comprises a structure represented by the following formula:

[0236] V H 1-C H 1- Hinge-C H 2-First C H 3;

[0237] (ii) the second polypeptide comprises a structure represented by the following formula:

[0238] MP2-V L 2-V H 2-Hinge-C H 2-Second C H 3; and

[0239] (iii) the third polypeptide comprises a structure represented by the following formula:

[0240] MP1-V L 1-C L ;

[0241] in:

[0242] V L 1 is the first immunoglobulin light chain variable domain;

[0243] V H 1 is the first immunoglobulin heavy chain variable domain;

[0244] V L 2 is a second immunoglobulin light chain variable domain;

[0245] V H 2 is a second immunoglobulin heavy chain variable domain;

[0246] C L is the immunoglobulin light chain constant domain;

[0247] C H 1 is immunoglobulin heavy chain constant domain 1;

[0248] C H 2 is immunoglobulin heavy chain constant domain 2;

[0249] First C H 3 is the first immunoglobulin heavy chain constant domain 3;

[0250] Second C H 3 is the second immunoglobulin heavy chain constant domain 3;

[0251] The hinge is connected to C H 1 domain and C H 2 domain immunoglobulin hinge region;

[0252] MP1 is a shielding peptide; MP1 comprises an N-terminal unit (NU), a shielding unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; the LU of the shielding peptide may not contain, contain at least one or more cleavage sites.

[0253] MP2 is a shielding peptide; MP2 comprises an N-terminal unit (NU), a shielding unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; the LU of the shielding peptide may not comprise a cleavage site, or may comprise at least one cleavage site.

[0254] In some embodiments, the bispecific antibody binds to a first and a second target, wherein the first target is CD28, and the bispecific antibody comprises a 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; a 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 a HCDR3 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 162; and a HCDR4 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 NOs:8, 17, 26, 36, 45, 53, 60, 76, 82, 91, 98, 106, 113, 120, 128, 136, 142, 150, 156 and 163; LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:9, 18, 27, 37 and 300; and 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 wherein 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.

[0255] In some embodiments, the bispecific antibody binds to a first and a second target, wherein the first target is CD28 and the second target is B7H3, wherein 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 wherein 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 a specific embodiment, the bispecific antibody binds to a first and a second target, wherein the first target is CD28 and the second target is B7H3, wherein 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.

[0256] In some embodiments, the bispecific antibody binds to a first and a second target, wherein the first target is CD28 and the second target is HER2, wherein the bispecific antibody comprises a CD28 binding portion comprising the amino acid sequence set forth in SEQ ID NO: 172, and wherein 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 a specific embodiment, the bispecific antibody binds to a first and a second target, wherein the first target is CD28 and the second target is HER2, wherein 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.

[0257] In some embodiments, the bispecific antibody binds to a first and a second target, wherein the first target is CD28 and the second target is HER2, wherein the bispecific antibody comprises a CD28 binding portion comprising the amino acid sequence set forth in SEQ ID NOs: 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 a specific embodiment, the bispecific antibody binds to a first and a second target, wherein the first target is CD28 and the second target is HER2, wherein 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.

[0258] In some embodiments, the bispecific antibody binds to a first and a second target, wherein the first target is human CD28 and the second target is TROP2, wherein 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, and wherein 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 a specific embodiment, the bispecific antibody binds to a first and a second target, wherein the first target is CD28 and the second target is TROP2, wherein 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.

[0259] In certain embodiments, the bispecific antibody binds to a first and a second target, wherein the first target is CD3 and the second target is B7-H3, and wherein the bispecific antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 177 or the scFv fusion polypeptide shown in SEQ ID NO: 299, a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 176, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 175.

[0260] In certain embodiments, the bispecific antibody binds to a first and a second target, wherein the first target is CD3 and the second target is TROP2.

[0261] In certain embodiments, the bispecific antibody binds to a first and a second target, wherein the first target is human CD28, and wherein 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.

[0262] V. Masked Antibodies

[0263] In some embodiments, the present disclosure provides shielded antibodies, which may be shielded monoclonal antibodies or multispecific (e.g., bispecific) antibodies directed against a specific target. In some embodiments, the shielded antibodies provided herein comprise full-length antibody light chains, e.g., kappa or lambda light chains. Additionally or alternatively, in some embodiments, the antibodies comprise full-length antibody heavy chains. The heavy chains of the antibodies may be of any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chains are of the IgG class, such as IgG 1 IgG 2 IgG 3 or IgG 4 Subclass. The antibody heavy chain can be converted from one class or subclass to another class or subclass using methods known in the art. In some embodiments, the masked antibody is or comprises a full-length antibody containing an Fc region (e.g., a human Fc region or a variant thereof). In some embodiments, the human Fc region is a human IgG 1 Fc region, human IgG 2 Fc region, human IgG 4 Fc region, or a variant of any of the foregoing. In some embodiments, the Fc region variant comprises one or more amino acid substitutions, insertions, or deletions relative to the wild-type human Fc region from which the Fc region variant is derived. In some embodiments, the masked antibody comprises human IgG 1 Fc region variants. In some embodiments, IgG 1 The Fc variant comprises one or more amino acid substitutions that increase the affinity of the Fc variant to FcγRIIb. In some embodiments, human IgG 1 The Fc region variant comprises 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, wherein the 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 1Fc region variants comprise a member selected from the group consisting of 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; G23 7D, P238D, P271G and A330R; E233D, H268D, P238D, P271G and A330R; G237D, H268D, P238D, P271G and A330R; and substitutions from the group consisting of E233D, G237D, P238D, H268D, P271G and A330R, wherein the amino acid numbering is according to the EU index. The foregoing substitutions are described in Mimoto et al. Protein Eng Des Sel. (2013) 26(10):589-98. Additionally or alternatively, in some embodiments, human IgG 1 The Fc region variant comprises an S2657A substitution (see Buschor et al. Int Arch Allergy Immunol. (2014) 163(3):206-14), wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, human IgG 1 Fc region variants comprise T437R and / or K248E substitutions (see Zhang et al. MAbs. (2017) 9(7): 1129-1142), wherein the amino acid numbering is according to the EU index. In some embodiments, the masked antibody comprises human IgG 4 Fc region variants. In some embodiments, IgG 4 The Fc region variant comprises one or more amino acid substitutions that increase the affinity of the Fc variant to FcγRIIb. 4 The Fc region variant comprises 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, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, human IgG 4Fc region variants comprise a member selected from the group consisting of 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; G23 7D, P238D, P271G and A330R; E233D, H268D, P238D, P271G and A330R; G237D, H268D, P238D, P271G and A330R; and substitutions from the group consisting of E233D, G237D, P238D, H268D, P271G and A330R, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, human IgG 4 The Fc region variant comprises an S2657A substitution, wherein the amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, human IgG 1 The Fc region variants comprise T437R and / or K248E substitutions, wherein the amino acid numbering is according to the EU index.

[0264] In some embodiments, the masked antibodies described herein further comprise human IgG 1 The domain or its variant contains one or more substitution mutations. In some embodiments, IgG 1 Variants include those selected from the group consisting of 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, H2 68D, P238D and P271G; G237D, P238D, P271G and A330R; E233D, H268D, 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 numbering is according to the EU index. In some embodiments, the masked antibodies described herein further comprise human IgG 4 The domain or its variant contains one or more substitution mutations. In some embodiments, IgG 4Variants include those selected from the group consisting of 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, H2 68D, P238D and P271G; G237D, P238D, P271G and A330R; E233D, H268D, 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 numbering is according to the EU index.

[0265] In some embodiments, the term "shielded antibody" refers to an antibody fragment, for example, a shielded antigen-binding fragment of a shielded anti-CD28 antibody. In some embodiments, the antibody fragment is or comprises Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv (see Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883), (scFv) 2 , linear antibodies, single-chain antibodies, minibodies or diabodies.

[0266] In some embodiments, the masked anti-CD28 antibodies described herein cross-react with CD28 from different species, thereby allowing the masked anti-CD28 antibodies to be used in preclinical and clinical studies. In some embodiments, the masked anti-CD28 antibodies described herein are activated (e.g., after the masked antibodies are activated by cleavage (e.g., protease cleavage)) in conjunction with two or more of human CD28, cynomolgus CD28, murine (mouse) CD28, and / or rat CD28. In some embodiments, the masked anti-CD28 antibodies are activated (e.g., after the masked antibodies are activated by cleavage (e.g., protease cleavage)) in conjunction with human CD28, cynomolgus CD28, murine (mouse) CD28, and rat CD28.

[0267] In some embodiments, the masked anti-CD28 antibodies described herein are context-dependent (e.g., activated (can only bind to its target) in certain contexts (such as in a protease-rich tumor microenvironment)). In some embodiments, the masked anti-CD28 antibodies described herein provide improved safety over more traditional non-shielded antibodies (e.g., showing reduced toxicity, not inducing significant changes in the weight of many organs, not changing liver histopathology, hematology and / or blood biochemistry, etc.). In some embodiments, the masked anti-CD28 antibodies described herein exhibit similar pharmacokinetic properties to traditional non-shielded anti-CD28 antibodies (e.g., having similar in vivo half-lives). In some embodiments, the masked anti-CD28 antibodies described herein exhibit improved pharmacokinetic properties (e.g., having longer in vivo half-lives) compared to more traditional non-shielded anti-CD28 antibodies.

[0268] In some embodiments, the antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ) forms an antigen binding domain (ABD) that binds to hCD28. In some embodiments, the shielding unit (MU) of the masked anti-CD28 antibodies described herein binds to the ABD and binds to hCD28 compared to a corresponding anti-CD28 antibody lacking the MU and / or reduces or inhibits the binding of the masked anti-CD28 antibody to hCD28 compared to the binding of the ABD to hCD28.

[0269] In some embodiments, the shielding unit has a shielding efficiency of 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 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, etc., including any ranges between these values) prior to removal of the MU from the shielded antibodies provided herein. For example, in some embodiments, the shielding efficiency of a masked anti-CD28 antibody is measured as the difference in affinity of a masked anti-CD28 antibody comprising a shielding unit (MU) to hCD28 (e.g., before activation of the masked antibody) relative to an anti-CD28 antibody lacking the MU to hCD28. In another example, the shielding efficiency is measured as the difference in affinity of a masked anti-CD28 antibody comprising a MU to hCD28 (e.g., before activation of the masked antibody by cleavage (e.g., protease cleavage)) relative to an unshielded anti-CD28 antibody (e.g., after activation of the masked antibody by cleavage (e.g., protease cleavage)). In some embodiments, the shielding efficiency can be measured by the EC of a masked antibody comprising a MU (e.g., before activation). 50 divided by the EC of the corresponding antibody specific for the same target lacking the masking peptide or the masking unit 50 In some embodiments, EC 50 Measured by ELISA. In some embodiments, the shielding unit (MU) of the shielded antibody binds to the ABD and prevents the shielded polypeptide from binding to its target. In specific embodiments, the target is CD28. In other embodiments, the target is CD3, B7-H3, HER2, or TROP2.

[0270] In some embodiments, the affinity of the shielded antibodies of the present disclosure 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 foregoing values) when the shielding unit is removed from the antibody (e.g., after activation by treatment with one or more proteases that cleave within the linking unit). In some embodiments, the EC of the masked antibodies described herein is 50 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 foregoing values) is reduced after activation by treatment with one or more proteases that cleave within the linking unit (e.g., as measured by ELISA or FACS assay).

[0271] In some embodiments, when a shielding unit is bound to the ABD of a shielded antibody described herein, the K of the antibody for its target is D is about the K of the antibody when the shielding unit is removed from the ABD of the shielded anti-CD28 antibody (e.g., after treatment with a protease that cleaves within the linking unit). D In some embodiments, when the shielding unit is bound to the ABD of a shielded antibody described herein, the K of the antibody to its target is 2 times (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 foregoing values). D Approximately the K of the corresponding antibody for the same target specificity lacking the shielding peptide or shielding unit Dtwice (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 foregoing values).

[0272] In some embodiments, the shielding unit sterically hinders the shielded binding polypeptide from binding to its target and / or allosterically hinders the shielded binding polypeptide from binding to its target.

[0273] In some embodiments, the dissociation constant of the shielding unit for a shielded antibody described herein (e.g., anti-CD28) to ABD is higher than the dissociation constant of the shielded antibody to its target (e.g., hCD28; when the shielded antibody is in an activated form, e.g., after treatment with a protease). In some embodiments, the dissociation constant of the shielding unit for the ABD of a shielded antibody described herein (e.g., anti-CD28) is about 2 (e.g., 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 foregoing values) times greater than the dissociation constant of the shielded antibody for its target (e.g., hCD28; when the shielded antibody is in an activated form, such as after treatment with a protease). In some embodiments, the dissociation constant of the shielding unit for the shielded antibody described herein (e.g., anti-CD28) to the ABD is approximately equal to the dissociation constant of the shielded antibody to its target (e.g., hCD28; when the shielded antibody is in an activated form, such as after treatment with a protease). In some embodiments, when the shielding antibody has not been activated (e.g., after treatment with one or more proteases that are cleaved within the linking unit), the shielding unit (MU) binds to the ABD of the shielded antibody described herein (e.g., anti-CD28) and prevents the antibody from binding to its target (e.g., hCD28). 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., substitution of the shielding unit (MU)), which results in the shielding peptide no longer preventing the polypeptide from binding to its target.

[0274] A. One-armed shielded antibody

[0275] In some embodiments, a shielded monoclonal antibody is provided, comprising a shielding peptide (MP) and an antibody that binds CD28, wherein the antibody comprises a heavy chain variable region (V H ) and light chain variable region (V L ), where MP is connected to VL The N-terminus of the antibody, wherein the MP comprises a shielding unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus, wherein the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215-248; and wherein the antibody V H The antibody V region comprises a 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; a HCDR2 amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 51, 58, 67, 89, 96, 104 and 155; and a 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; and an antibody V L The region comprises a 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; a LCDR2 amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 18, 27, 37 and 300; and a 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.

[0276] In some embodiments, a shielded monoclonal antibody is provided, comprising a shielding peptide (MP) and an antibody that binds CD28, wherein the antibody comprises a heavy chain variable region (V H ) and light chain variable region (V L ), where MP is connected to V LN-terminus of the present invention, wherein the MP comprises a shielding unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus, wherein the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215-248; and 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 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 shielded monoclonal antibody is provided, comprising a shielding peptide (MP) and an antibody that binds to human CD28, wherein the antibody comprises a heavy chain and a light chain, wherein the MP is linked to the N-terminus of the LC, wherein the MP comprises a shielding unit (MU) from the N-terminus to the C-terminus, 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 comprises an 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 wherein the antibody LC comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: The amino acid sequence of the group consisting of NOs: 14, 23, 32, 42, 50, 57, 65, 74, 80, 87, 95, 102, 110, 118, 125, 142, 133, 140, 147, 154, 160 and 168.

[0277] B. One-armed shielded multispecific antibodies

[0278] In some embodiments, a shielded bispecific monoclonal antibody specific for a first and a second target is also provided, wherein the first target is CD28, wherein the antibody comprises a shielding peptide (MP) and a CD28 binding portion, wherein the antibody comprises a heavy chain variable region (V H ) and light chain variable region (V L ), where MP is connected to V L The N-terminus of the present invention, wherein the MP comprises a shielding unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus, wherein the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215-248, wherein the CD28 binding portion comprises V HA region comprising HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 6, and HCDR3 of SEQ ID NO: 7, and V L A region comprising LDR1 of SEQ ID NO:8, LCDR2 of SEQ ID NO:9 and LCDR3 of SEQ ID NO:10, and wherein the second target is B7-H3, HER2 or TROP2 protein.

[0279] In some embodiments, a shielded bispecific monoclonal antibody specific for a first and a second target is also provided, wherein the first target is CD28, wherein the antibody comprises a shielding peptide (MP) and a CD28 binding portion, wherein the antibody comprises a heavy chain variable region (V H ) and light chain variable region (V L ), where MP is connected to V L The N-terminus of the present invention, wherein the MP comprises a shielding unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus, wherein the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215-248, and wherein the CD28 binding portion 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 The invention relates to a light chain variable region of an amino acid sequence of the group consisting of NOs: 12, 21, 30, 40, 48, 55, 63, 72, 78, 85, 93, 100, 108, 116, 123, 131, 138, 145, 152, 158 and 166, and wherein the second target is B7-H3, HER2 or TROP2 protein.

[0280] C. Dual-arm masked multispecific antibodies

[0281] In certain embodiments, shielded bispecific monoclonal antibodies specific for a first and a second target are also provided, wherein the first target is CD28 and the second target is a tumor-associated antigen selected from HER2, B7-H3 and TROP2, wherein the antibody comprises two shielding peptides (MPs).

[0282] VI. Shielding Peptides

[0283] In some embodiments, the MP further comprises an N-terminal unit. In some embodiments, the N-terminal unit is between about 1 and 10 amino acids in length. In some embodiments, the N-terminal unit comprises SEQ ID NO: 210. In some embodiments, the LU comprises at least one first cleavage site (CS 1)(e.g., a first protease cleavage site). In some embodiments, the LU further comprises a second cleavage site (CS 2). In some embodiments, the first and / or second cleavage site is a protease cleavage site. In some embodiments, the first and second cleavage sites 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 (e.g., a protease known to co-localize with a target of a polypeptide comprising a cleavage site) can be used, including, for example, by urokinase-type plasminogen activator (uPA); matrix metalloproteinases (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-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., ADAM 8, ADAM 9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, 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 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 ​​proteases (e.g., Cruzipain, legumin and / or Otubain-2); KLKs (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and / or KLK14); metalloproteases (e.g., methyldopa, enkephalin, PSMA and / or BMP-1); serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase and / or coagulation factor proteases (such as FVIIa, FIXa, FXa, FXla, FXIIa)); elastase; granzyme B; guanidinobenzoate esterase; HtrA1; human neutrophil elastase; lactoferrin; marapsin; NS3 / 4A; PACE4; tPA; tryptase;Protease cleavage sites recognized and / or cleaved by type II transmembrane serine proteases (TTSPs) such as DESC1, DPP-4, FAP, Hepsin, Interstitial proteinase-2, MT-SP1 / Interstitial proteinase, TMPRSS2, TMPRSS3 and / or TMPRSS4. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from the group consisting of 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 the group consisting of uPA, MMP-2, MMP-9, and / or TEV protease. ;

[0284] In some embodiments, LU further comprises a first linker (L 1 In some embodiments, the first linker (L 1 ) is the first cleavage site (CS 1 ) (e.g., a first protease cleavage site). In some embodiments, LU comprises from N-terminus to C-terminus (CS 1 )-L 1 In some embodiments, LU further comprises a second linker (L 2 ). In some embodiments, L 2 In some embodiments, LU comprises (C S) from N-terminus to C-terminus. 1 )-L 1 -(CS 2 )-L 2 In some embodiments, L 1 and L 2Any suitable linker (e.g., a flexible linker) known in the art includes, but is not limited to, for example, glycine polymers (G) n, wherein 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, wherein 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; and the like. The linker sequence can be of 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 a 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.

[0285] 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 shielding peptide (MP) comprises a structure of (MU)-(LU) from the N-terminus to the C-terminus, wherein LU comprises (CS 1 )-L 1 or (CS 1 )-L 1 -(CS 2 )-L 2 In some embodiments, the shielding peptide of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215-248.

[0286] In some embodiments, the masking peptide (MP) comprises the MU set forth in any one of SEQ ID NOs: 178-211 and the LU set forth in SEQ ID NOs: 213 or 214. In some embodiments, the MP comprises the sequence set forth in any one of SEQ ID NOs: 215-248.

[0287] VII. Antibody Production

[0288] Another aspect of the present disclosure provides one or more isolated nucleic acid molecules comprising a nucleotide sequence encoding an amino acid sequence of an anti-CD28 antibody described herein, including a shielded anti-CD28 antibody. In some embodiments, one or more isolated nucleic acid molecules are provided, comprising a nucleotide sequence encoding an amino acid sequence of a multispecific antibody described herein, including a shielded multispecific antibody. The amino acid sequence encoded by the nucleotide sequence may be any portion of an antibody described herein (such as a CDR, a sequence comprising one, two or three CDRs, a heavy chain variable region, a light chain variable region), or may be a full-length heavy chain or a full-length light chain. The nucleic acid of the present disclosure may be, for example, DNA or RNA, and may or may not contain an intron sequence. Typically, the nucleic acid is a cDNA molecule.

[0289] In some embodiments, the present invention provides an isolated nucleic acid molecule comprising or consisting of a nucleotide sequence encoding an amino acid sequence, e.g., a heavy chain variable region and / or a light chain variable region of an antibody described herein, or, e.g., a full-length heavy chain or full-length light chain of an antibody described herein.

[0290] Any suitable molecular biology technique (eg, PCR amplification or cDNA cloning technique) can be used to obtain the nucleic acid of the present invention. For the antibodies described herein obtained from the screening of the library, the nucleic acid encoding the antibody can be recovered from the library.

[0291] By encoding V H The DNA is operably linked to a heavy chain constant region encoding H 1. C H 2 and C H 3) and encode V H The isolated DNA of the heavy chain constant region is converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained by standard PCR amplification. The heavy chain constant region can be IgG 1 IgG 2 IgG 3 IgG 4 , IgA, IgE, IgM or IgD constant region, but most preferably IgG without ADCC effect 4 or IgG 2 Constant region. IgG 4 The constant region sequence can be any of the various alleles or allotypes known to exist in different individuals. These allotypes represent IgG4 The naturally occurring amino acid substitutions in the constant region. H The DNA is operably linked to the heavy chain C H 1 constant region of another DNA molecule.

[0292] By encoding V L The DNA is operably linked to the DNA encoding the light chain constant region C L Another DNA molecule encoding V L The isolated DNA of the region is converted into a full-length light chain gene (and a Fab light chain gene). The sequence of human light chain constant region genes is known in the art (see, for example, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH publication No. 91-3242), and the DNA fragments covering these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0293] To generate scFv genes, the V H and V L The DNA fragment is operably linked to a flexible linker encoding an amino acid sequence (Gly 4 -Ser) 3 Another fragment of V H and V L The sequence can be expressed as a V L and V H The invention relates to a continuous single-chain protein containing a region of the leukemia / lysine peptide (see, e.g., Bird et al., Science (1988) 242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85:5879-5883; and McCafferty et al., Nature (1990) 348:552-554).

[0294] The present disclosure further provides vectors comprising one or more nucleic acid molecules described herein. In some embodiments, the vector is an expression vector that can be used to express an antibody described herein or an antigen-binding fragment of such an antibody. In some embodiments, vectors are provided herein, wherein the first vector comprises a polynucleotide sequence encoding a heavy chain variable region as described herein, and the second vector comprises a polynucleotide sequence encoding a light chain variable region as described herein. In some embodiments, a single vector comprises polynucleotides encoding a heavy chain variable region as described herein and a light chain variable region as described herein.

[0295] To express the binding molecules of the present disclosure, DNA encoding part or full-length light chain and heavy chain is inserted into expression vector so that DNA molecule is operably connected to transcription and translation control sequence. In this case, the term "operably connected" means that antibody gene is connected to vector so that transcription and translation control sequence in the vector play their predetermined function of regulating transcription and translation of DNA molecule. Expression vector and expression control sequence are selected to be compatible with the expression host cell used. Antibody light chain gene and antibody heavy chain gene can be inserted into separate vector, or more generally, two genes can be inserted into the same expression vector. Antibody gene is inserted into expression vector by any suitable method (such as connection of complementary restriction sites on antibody gene fragment and vector, or connection of DNA based on homologous recombination). The light chain variable region and heavy chain variable region of antibody described herein can be used to create full-length antibody gene of any antibody isotype and subclass in the following manner: insert them into expression vector of heavy chain constant region and light chain constant region encoding desired isotype and subclass so that V H The segment is operably linked to one or more C H segment, and V L The segment is operably linked to the C L Segment. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector so that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0296] In addition to the antibody chain gene, the expression vector of the present disclosure generally also carries a regulatory sequence for controlling the expression of the antibody chain gene in the 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 gene. The regulatory sequence is 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, may depend on factors such as the selection of the host cell to be transformed, the desired protein expression level, etc. Examples of regulatory sequences for mammalian host cell expression include viral elements that guide high-level 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 polyomavirus. Alternatively, non-viral regulatory sequences such as ubiquitin promoters or β-globin promoters may be used. In addition, regulatory elements are composed of sequences from different 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).

[0297] In addition to antibody chain genes and regulatory sequences, expression vectors may also carry additional sequences, such as enhancer components, transcription termination sequences, sequences (e.g., replication origins) and selectable marker genes that regulate the replication of the vector in the host cell. Selectable marker genes may help select host cells into which vectors have been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017, all completed by Axel et al.). For example, typically, selectable marker genes confer resistance to drugs such as G418, hygromycin or methotrexate to host cells into which vectors have been introduced. Selectable marker genes include dihydrofolate reductase (DHFR) genes (used in dhfr-host cells in conjunction with methotrexate selection / amplification) and neo genes (selected for G418).

[0298] For the expression of light and heavy chains, one or more expression vectors encoding heavy and light chains are transfected into host cells 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. Although it is possible to express the antibodies described herein in prokaryotic or eukaryotic host cells, it is most typical to express antibodies in eukaryotic cells, such as mammalian host cells.

[0299] The present disclosure also provides a host cell containing a nucleic acid molecule or a vector provided by the present disclosure. 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, and can be a prokaryotic cell such as a bacterial cell. The introduction of a recombinant nucleic acid construct into a host cell can be achieved by calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, or phage infection.

[0300] Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0301] Mammalian host cells for expressing the binding molecules of the present disclosure include, for example, Chinese hamster ovary (CHO) cells (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA (1980) 77: 4216-20, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol. (1982) 159: 601-21), NS0 myeloma cells, COS cells, and Sp2 cells. In particular, for use with NS0 myeloma or CHO cells, another expression system is the GS (glutamine synthetase) gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP338841.

[0302] The antibodies (or antigen-binding fragments thereof) disclosed herein can be produced using any method known in the art. Exemplary techniques for antibody production are described in U.S. Pat. No. 4,816,567; however, this exemplary technique is provided for illustrative purposes only and is not intended to be limiting. When 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 to allow the antibody to be expressed in the host cell or secreted into the culture medium in which the host cell is grown. Therefore, in some embodiments, a method for producing an antibody described herein is provided, the method comprising culturing a host cell comprising one or more nucleic acids or vectors encoding the antibody (e.g., provided above) under suitable conditions for expressing the antibody. In some embodiments, the method further comprises recovering the antibody from the host cell (or host cell culture medium). Any suitable protein purification method can be used to recover the antibody from the culture medium.

[0303] VIII. Pharmaceutical Compositions

[0304] 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 pharmaceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0305] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering a polypeptide (e.g., an antibody described herein). The carrier may be an anti-adhesive, a binding agent, a coating agent, a disintegrant, a filler or a diluent, a preservative (such as an antioxidant, an antibacterial agent or an antifungal agent), a sweetener, an absorption delaying agent, a wetting agent, an emulsifier, a buffer and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) dextrose, vegetable oils (such as olive oil), saline, buffers, buffered saline and isotonic agents, such as sugars, polyols, sorbitol and sodium chloride.

[0306] The composition can be in any suitable form, such as liquid, semi-solid and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable solutions and insoluble solutions), microemulsions, liposomes, dispersions or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules and powders. A specific form of the composition suitable for delivering the antibodies described herein is a sterile liquid for injection or infusion, such as a solution, suspension or dispersion. A sterile solution can be prepared by incorporating the antibody into a suitable carrier in the desired amount, followed by sterile microfiltration. Dispersions can be prepared by incorporating the antibody into a sterile vehicle containing a basic dispersion medium and other carriers. In the case of a sterile powder for the preparation of a sterile liquid, the preparation method includes vacuum drying and freeze drying (lyophilization) to obtain a powder of the active ingredient plus any other desired ingredients from a solution previously sterile filtered. The various dosage forms of the composition can be prepared by conventional techniques known in the art.

[0307] The relative amount of antibody included in the composition will vary depending on many factors, such as the specific polypeptide and carrier used, the dosage form, and the desired release and pharmacodynamic characteristics. The amount of antibody in a single dosage form will generally be that amount that produces a therapeutic effect, but may also be a lesser amount. Typically, this amount will be in the range of about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30%, relative to the total weight of the dosage form.

[0308] In addition to the antibodies described herein, one or more additional therapeutic agents may be included in the composition. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiotherapy 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 alkylation platinum derivatives, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 Antibodies, anti-CD1d antibodies, anti-TIM3 antibodies, anti-SLAMF7 antibodies, anti-KIR receptor antibodies, anti-OX40 antibodies, anti-HER2 antibodies, anti-ErbB-2 antibodies, anti-EGFR antibodies, cetuximab, rituximab, trastuzumab, pembrolizumab, radiotherapy, single dose radiation, fractionated radiation, focal radiation, whole organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptors, T cells of adoptive transfer, anticancer vaccines and oncolytic viruses. The suitable amount of the additional therapeutic agent included in the composition can be easily selected by those skilled in the art, and will vary depending on many factors, such as the specific agent and carrier used, dosage form, and required release and pharmacodynamic characteristics. The amount of the additional therapeutic agent included in the single dosage form will generally be the amount of the agent that produces the therapeutic effect, but it may also be a smaller amount.

[0309] The antibodies described herein can be further modified. In some embodiments, these antibodies are linked to additional molecular entities. Examples of additional molecular entities include pharmaceutical preparations, peptides or proteins, detection agents or labels, and antibodies.

[0310] 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 radioactive isotopes. Specific examples of cytotoxic agents include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, d-actinomycin ... D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologues.Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan The invention relates to the treatment of leukemia and leukemia with the aid of a radioactive isotope. The radioactive isotope may be conjugated to an antibody for diagnostic or therapeutic use, for example, iodine. The radioactive isotope may be conjugated to an antibody for diagnostic or therapeutic use, for example, iodine. 131 ,indium 111 ,yttrium 90 and Lutetium 177Methods for linking polypeptides to pharmaceutical agents are known in the art, such as using various linker technologies. Examples of linker types include hydrazones, thioethers, esters, disulfide, and peptide-containing linkers. For further discussion of linkers and methods for attaching therapeutic agents to antibodies, see, e.g., Saito et al., Adv. Drug Deliv. Rev. (2003) 55:199-15; Trail et al., Cancer Immunol. Immunother. (2003) 52:328-37; Payne, Cancer Cell (2003) 3:207-12; Allen, Nat. Rev. Cancer (2002) 2:750-63; Pastan and Kreitman, Curr. Opin. Investig. Drugs (2002) 3:1089-91; Senter and Springer Adv. Drug Deliv. Rev. (2001) 53:247-64.

[0311] Any of the antibodies and / or compositions (eg, pharmaceutical compositions) described herein can be used in the preparation of a medicament (eg, an agent for treating or delaying progression of cancer in a subject in need thereof).

[0312] IX. Treatment Methods

[0313] The antibodies and pharmaceutical compositions described herein can be used for therapeutic purposes, such as treating cancer or enhancing the efficacy of other cancer therapies. Therefore, in some embodiments, the disclosure provides methods of using antibodies or pharmaceutical compositions described herein. In some embodiments, the disclosure provides a method of treating a subject (e.g., a human subject) cancer, comprising administering an effective amount of an antibody described herein to the subject. In some embodiments, the cancer is breast cancer, liver cancer, or colorectal cancer, gastric cancer, ovarian cancer, lung cancer, pancreatic cancer, or renal cancer.

[0314] In the practice of treatment methods, the shielded anti-CD28 antibodies described herein can be administered alone, for example, as a monotherapy or in combination with one or more additional therapeutic agents or therapies. Therefore, on the other hand, the present disclosure provides a combination therapy comprising a binding molecule in combination with one or more additional therapies or therapeutic agents 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 antibodies described herein. In some embodiments, the present invention provides a method for treating cancer in a subject (e.g., a human subject), comprising administering to the subject an effective amount of an antibody described herein (e.g., an anti-CD28 antibody or a multispecific antibody targeting CD28 and one or more other targets) and an effective amount of an anti-PD-1 antibody. In some embodiments, the present invention provides a method for treating cancer in a subject (e.g., a human subject), comprising administering to the subject an effective amount of an antibody described herein (e.g., an anti-CD28 antibody or a multispecific antibody targeting 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.

[0315] X. Kits and Products

[0316] In some embodiments, a kit is provided, comprising one or more antibodies described herein (e.g., anti-CD28 antibodies or multispecific antibodies targeting CD28 and one or more other targets). In some embodiments, the kit also includes a package insert containing instructions for using the antibodies described herein. In some embodiments, the article or kit comprises a container comprising one or more shielded antibodies or compositions described herein. In certain embodiments, the article or kit comprises a container comprising nucleic acids encoding one (or more) shielded antibodies described herein. In some embodiments, the kit includes cells of a cell line producing antibodies described herein. In some embodiments, the kit includes one or more CD28 (e.g., human CD28, cynomolgus monkey CD28, mouse CD28, rat CD28, or any fragment of the foregoing) or CD28 + Positive Controls for Cells. In some embodiments, the kit includes a negative control, such as a surface or solution that contains little or no CD28, or cells that do not express CD28.

[0317] In certain embodiments, the article or kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, test tubes, etc. The container can be formed of a variety of materials, such as glass or plastic. The container holds an antibody described herein (or a composition comprising such an antibody) used herein, which, by itself or in combination with another composition, can be effectively used to treat, delay the progression, and / or prevent cancer in a subject (e.g., a human subject). The container can have a sterile access port (e.g., a container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). In some embodiments, the label or package insert indicates that the composition is used to treat breast cancer, liver cancer, or colorectal cancer in a subject (e.g., a human subject).

[0318] In addition, the article or kit may include (a) a first container containing a composition, wherein the composition includes an antibody (or an immunologically active fragment thereof) described herein; and (b) a second container containing a composition, wherein the composition includes an additional cytotoxic agent or an additional therapeutic agent. In some embodiments, the second container contains a composition comprising an anti-PD-1 antibody, and an article comprising a label or package insert indicating that the antibody and anti-PD-L1 are used to treat colon cancer in a subject in need (e.g., a human subject), for example, according to the methods provided herein. In some embodiments, the second container contains a composition comprising an anti-CTLA4 antibody (e.g., a shielded anti-CTLA-4 antibody), and an article comprising a label or package insert indicating that the antibody described herein and the anti-CTLA4 antibody (e.g., a shielded anti-CTLA-4 antibody) are used to treat colon cancer in a subject in need (e.g., a human subject), for example, according to the methods provided herein.

[0319] In addition, the article of manufacture may further comprise other containers comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0320] The previous written description is considered sufficient to enable those skilled in the art to implement the present disclosure. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. In fact, in addition to those modifications shown and described herein, various modifications of the present disclosure will become apparent to those skilled in the art based on the previous description and fall within the scope of the appended claims.

[0321] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present 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 the present disclosure. In the event of a conflict, the present specification (including definitions) shall prevail. In addition, unless the context otherwise requires, singular terms shall include pluralities, and plural terms shall include the singular. In the present specification and examples, the words "have" and "comprise", or variations such as "has", "having", "comprises" or "comprising" shall be understood to include the integer or integer group, but not any other integer or integer group. 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, to the extent that the references are expressly and individually indicated to be incorporated by reference in their entirety. Although many documents are cited herein, the citation does not constitute an admission that any of such documents constitutes part of the common general knowledge in the art. As used herein, the term "approximately" or "about" is applied to one or more values ​​of interest to refer to values ​​similar to the reference value. In certain embodiments, unless otherwise specified or seen from the context, the term refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the reference value.

[0322] According to the present disclosure, a back-reference in a dependent claim is a shorthand for direct and explicit disclosure of each claim combination indicated by the back-reference. Any compound disclosed herein can be used in any method of treatment herein, wherein the subject to be treated is defined anywhere herein. In addition, the headings herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any way.

[0323] For a better understanding of the present invention, the following examples are listed. Such examples are provided only for illustrative purposes and are not intended to limit the scope of the present invention in any way.

[0324] Example

[0325] Example 1: Generation of primary Fabs that specifically bind to human CD28

[0326] A proprietary phagemid library was used for screening against human CD28 antigen. A total of 3 or 4 rounds of screening were performed. After the last round of screening, the culture supernatant of individual clones was tested by ELISA to identify clones that specifically recognized human CD28 (e.g., primary hits). When the ELISA signal was at least twice the background, the clone was defined as positive. Positive clones were selected for sequencing, and the Fab corresponding to the unique hit was expressed in E. coli and affinity purified. Its affinity for human CD28 was determined by RED96 system (ForteBio) measurement.In brief, use dip and read anti-human IgG Fc capture (AHC) biosensor (ForteBio) to capture human or mouse CD28-Fc fusion protein, and dip into the hole containing purified Fab, these Fab are diluted 5 to 10 times in ForteBio dynamic buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% v / v Surfactant P20, pH 7.4).The acquired data are processed with Octet data analysis software 7.1 (ForteBio), and the kinetic data are fitted to 1: 1 Langmuir (Langmuir) binding model.

[0327] Primary Fab hits that were cross-reactive to human and mouse CD28 species were further characterized by ELISA, and from these primary hits, a panel of 46 unique sequence Fabs were converted to IgG with the core hinge mutation S241P (Kabat numbering scheme) 4 Isotype mAbs were selected for detailed biophysical and functional characterization (Table 5).

[0328] Example 2: IgG 4 Transformation and expression

[0329] The engineered human anti-CD28 IgG listed in Table 5 4 The heavy and light chains of the isotype mAbs were cloned into the mammalian expression vector pcDNA3.3 (ThermoFisher Scientific). The plasmid pairs carrying one heavy chain and one light chain were transiently transfected into HEK293 cells according to the manufacturer's instructions. After incubation, the supernatant was collected, centrifuged and clarified by filtration, and the IgG 4 Isotype mAb by MabSelect TM SuRe TM Protein A affinity chromatography (GE Healthcare) capture.

[0330] The mAb was eluted and neutralized, and the elution buffer was exchanged into recovery buffer (20 mM histidine, pH 5.5). Protein concentration was determined by UV-spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing and non-reducing conditions.

[0331] Table 5. Human anti-CD28 IgG 4 List of monoclonal antibodies

[0332]

[0333]

[0334] Example 3: Binding properties of anti-CD28 antibodies

[0335] pass The RED96 system (ForteBio), ELISA and CytoFlex flow cytometer (Beckman) measured the binding affinity of the anti-CD28 mAb panel to human, cynomolgus macaque and mouse CD28 and human CTLA4. Anti-CD28 antibodies TAC2386 (also known as TGN1412, as described in WO2006 / 050949A2) and TAC2387 (as described in WO2019 / 246514A2) were used as benchmark controls.

[0336] Binding affinity to human CD28 and CTLA4 by the Octet RED96 system

[0337] use The RED96 system (ForteBio) was used to evaluate the binding kinetics of the anti-CD28 mAb panel to human CD28 and CTLA4. 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 dipping and reading AHC biosensors (ForteBio). The sensors were then allowed to associate with His-tagged human CD28 and CTLA4 proteins (100 nM) for 300 seconds and in kinetic buffer for another 300 seconds. The association and dissociation curves were fitted to a 1:1 Langmuir binding model using Data Analysis Software 7.1 (ForteBio).

[0338] As shown in Table 6, The anti-CD28 test panel and benchmark mAbs exhibited high binding affinity (<10 nM) to human CD28 as measured by the RED96 system. In addition, test mAbs TY24773, TY24853, TY24854, TY24860, TY24865, and TY24871, as well as the benchmark control mAb, exhibited no detectable affinity to human CTLA4.

[0339] Table 6. Binding affinity to human CD28 and CTLA4

[0340]

[0341] ND: Not Detected

[0342] ELISA to measure affinity for human and mouse CD28

[0343] Recombinant human and mouse CD28-Fc was diluted to 2 μg / mL in PBS and coated on Nunc MaxiSorp at 4°C. TM Overnight on a high protein binding capacity 96-well ELISA plate (ThermoFisher Scientific). The plate was blocked with PBS supplemented with 3% skim milk for 1 hour at 37°C. After washing, 50 μL of 3-fold serial dilutions of the anti-CD28 test mAb group were added to each well. After incubation at 37°C for 1 hour, the plate was washed 4 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 plate was incubated at 37°C for 1 hour, washed four times, and then 50 μL of TMB substrate (3,3',5,5'-tetramethylbenzidine) solution was added to each well, and the plate was incubated at room temperature. After adding 50 μL of sulfuric acid stop solution to each well to stop the reaction, the absorbance at 450 nm was measured. EC was estimated by fitting the ELISA data to an asymmetric sigmoidal (four-parameter logistic equation) model using GraphPad Prism 7 (Windows version, GraphPad Software, La Jolla California USA, www.graphpad.com). 50 .

[0344] As shown in Table 7 and Figure 1 As shown, the anti-CD28 test mAb panel and the two benchmark controls exhibited similar affinity for human CD28. In addition, the test mAbs (except TY24890) bound to mouse CD28, while the two benchmark controls did not bind to mouse CD28.

[0345] Table 7. Binding affinity to recombinant CD28 measured by ELISA

[0346]

[0347] ND: Not Detected

[0348] Binding activity of anti-CD28 antibodies in Jurkat cells

[0349] Jurkat cells (clone E6-1) were cultured at 1.0 × 10 5 (50 μL / well) were seeded in 96-well plates and incubated with serially diluted benchmark positive controls, isotype negative control antibodies and a panel of anti-CD28 test mAbs (100, 20, 4, 0.8, 0.16 and 0.032 nM) in 2% fetal bovine serum / Dulbecco's PBS (FBS / DPBS) at 4°C for 30 minutes. Next, the cells were washed twice with DPBS and further incubated with APC-anti-human IgG Fc secondary antibody (1 μg / mL, 100 μL / well, Biolegend) at 4°C for 30 minutes. Finally, the cells were washed twice with DPBS and suspended in FACS buffer for flow cytometry analysis. For analysis, mean fluorescence intensity (MFI) values ​​were plotted versus concentration using FlowJo 10 software (FlowJo LLC), and the data were further fitted by four-parameter nonlinear regression of GraphPad Prism 7 (Windows version, GraphPad Software, La Jolla California USA, www.graphpad.com) to obtain EC 50 value.

[0350] As shown in Table 8, except for TY24775 and TY24867, all other anti-CD28 tested mAbs showed high binding activity with CD28 molecules on Jurkat cells (EC 50 Values ​​ranged from 0.38 to 10.42 nM). In addition, the measured binding activity was comparable to both benchmark controls.

[0351] Table 8. Binding affinity of CD28 expressed on Jurkat cells measured by flow cytometry

[0352] Ab ID <![CDATA[EC 50 (nM)]]> Ab ID <![CDATA[EC 50 (nM)]]> Ab ID <![CDATA[EC 50 (nM)]]> TAC2386 1.10 TY24863 2.60 TY24881 0.38 TAC2387 0.81 TY24864 0.54 TY24882 2.24 TY24772 0.74 TY24865 0.46 TY24883 5.92 TY24773 0.71 TY24866 0.70 TY24884 1.09 TY24774 3.35 TY24867 ND TY24885 5.77 TY24775 ND TY24868 2.08 TY24886 10.42 TY24776 1.83 TY24869 1.07 TY24887 3.17 TY24777 1.50 TY24870 3.95 TY24888 0.58 TY24853 0.43 TY24871 0.89 TY24889 2.32 TY24854 0.60 TY24872 1.07 TY24890 0.59 TY24855 0.61 TY24873 2.08 TY24891 0.62 TY24856 0.76 TY24874 1.65 TY24892 1.30 TY24857 0.75 TY24875 1.72 Isotype Control ND TY24858 0.70 TY24876 0.38 TY24859 0.97 TY24877 0.79 TY24860 0.78 TY24878 0.39 TY24861 2.25 TY24879 1.17 TY24862 2.82 TY24880 1.62

[0353] ND: Not Detected

[0354] In vitro binding of anti-CD28 antibodies to human T cells measured by flow cytometry

[0355] Using EasySep TM Human naive total T cell isolation kit (STEMCELL Technologies) was used to isolate human CD3 +T cells were isolated from cryopreserved peripheral blood mononuclear cells (PBMC). The isolated human T cells were cultured at 1.0×10 5 Cells / well were added to 96-well plates, and 100nM benchmark controls, isotype negative control antibodies and a group of anti-CD28 test mAbs were incubated in FACS buffer for 30 minutes on ice. Next, cells were washed three times with PBS and further incubated on ice with PE-labeled secondary antibodies for 30 minutes. Finally, cells were washed three times with PBS and resuspended in FACS buffer for flow cytometry analysis. For analysis, FlowJo 10 software (FlowJo LLC) was used to calculate MFI values, and geometric mean MFI values ​​and mAb graphs were drawn by GraphPad Prism 7 (Windows version, GraphPad Software, La Jolla California USA, www.graphpad.com).

[0356] like Figure 2 As shown, all tested mAbs, except TY24772, displayed comparable MFI values ​​compared to the benchmark controls TAC2386 and TAC2387. TY24772 bound approximately twice as much as the benchmark controls.

[0357] Example 4: Ligand competition using Elisa

[0358] The ability of the antibodies to block the binding of CD28 or CTLA4 to their natural ligand CD80 was tested by ELISA.

[0359] like Figure 3 As shown in Table 9, compared to the control anti-CTLA4 antibody TY21580, the benchmark controls TAC2386 and TAC2387 and all tested anti-CD28 mAbs blocked the binding of CD28 to CD80 but not the binding of CTLA4 to CD80.

[0360] Table 9. IC of anti-CD28 or anti-CTLA4 antibodies against human CD28-CD80 and CTLA4-CD80 pairs 50

[0361]

[0362]

[0363] Example 5: Evaluation of superagonists in dry-coated human T cell proliferation assays

[0364] pass Fluorescent cell viability assay (Promega) measures the level of lymphocyte proliferation induced by the benchmark controls TAC2386 and TAC2387, isotype negative control antibodies, and a panel of anti-CD28 test mAbs. In this assay, 100 nM test mAbs were added to 96-well microplates at 50 μL / well in triplicate and air-dried directly onto the well walls. Next, the microplates were washed twice with PBS. Cryopreserved PBMCs were recovered and the cell density was adjusted to 5.0 × 10 5 cells / mL, and 1.0×10 5 PBMC (200 μL / well) was added to the pre-coated microplate. The cells were incubated at 37°C and 5% CO 2 Incubate for 72 hours and then use Tests assess lymphocyte proliferation.

[0365] like Figure 4A As shown, the immobilized benchmark control TAC2386 (a known CD28 superagonist) induced lymphocyte proliferation at a level significantly higher than all tested mAbs. Compared with TAC2386, the benchmark control TAC2387 and anti-CD28 test mAbs (including TAC2387, TY24865, TY24866, TY24876, TY24878, TY24879 and TY24884) exhibited weaker superagonist activity in vitro.

[0366] Example 6: Effect of anti-CD28 antibodies on in vitro human T cell activation

[0367] T cell activation co-stimulation assay: IFN-γ release

[0368] The biological activity of anti-CD28 mAbs as agonistic T cell co-stimulators for in vitro activation of human T cells was measured by ELISA for IFN-γ cytokine secretion. Ultra-LEAF TM Purified anti-human CD28 antibody (Biolegend) was used as a positive control.

[0369] Using EasySep TM Human naive total T cell isolation kit (STEMCELL Technologies) was used to isolate human CD3 + T cells were isolated from cryopreserved PBMCs. The isolated cells were cultured in 96-well tissue culture plates (1.0×10 400 μL / well) pre-coated with a suboptimal concentration (10 nM, 50 μL / well) of anti-human CD3 antibody (OKT3) in the presence of serially diluted benchmark controls, isotype negative control antibodies, commercial anti-human CD28 positive control antibodies, and a panel of anti-CD28 test mAbs (0.1, 1, 10, and 100 nM). 5The cells were cultured at 37°C and 5% CO 2 The cells were incubated for 120 h and the cell supernatants were then collected for IFN-γ cytokine analysis by ELISA. Measurement of T cell proliferation.

[0370] like Figure 4B and 4C As shown, anti-CD28 mAbs exhibited concentration-dependent biological activities, including T cell proliferation and IFN-γ cytokine secretion, compared to isotype control antibodies. In general, the effects of anti-CD28 mAbs on human T cell activation were comparable or more potent than the two benchmark control antibodies.

[0371] T cell activation co-stimulation assay: IL-2 release

[0372] pass Fluorescent cell viability assay (Promega) measures T cell proliferation and ELISA measures IL-2 cytokine secretion. Anti-CD28 mAb is used as a co-stimulator of agonistic T cells to activate human T cells in vitro. TM Purified anti-human CD28 antibody (Biolegend) was used as a positive control.

[0373] Using EasySep TM Human T cells were isolated from fresh PBMCs of Asian donors using the Human Naive Total T Cell Isolation Kit (STEMCELL Technologies). The cells were cultured in 96-well tissue culture plates (1.0 × 10 50 μL) pre-coated with a suboptimal concentration (5 nM) of anti-human CD3 antibody (OKT3) in the presence of serially diluted benchmark control TAC2387, isotype negative control antibody, commercial anti-human CD28 positive control antibody, and a panel of anti-CD28 test mAbs. 5 The cells were cultured at 37°C and 5% CO 2 The cells were incubated for 72 hours and the cell supernatants were then collected for IL-2 cytokine analysis by ELISA and used The assay measures the level of T cell proliferation.

[0374] like Figure 5A As shown in Figures 1 to 4, anti-CD28 antibodies showed concentration-dependent biological activities, including T cell proliferation and IL-2 cytokine secretion, compared to isotype control antibodies. The effects of TY24859, TY24865, TY24866, and TY24890 on human T cell activation were comparable to the benchmark control TAC2387. The negative control group of anti-CD3 without pre-coating showed no detectable T cell proliferation and IL-2 cytokine secretion.

[0375] Example 7: Construction and functional characterization of anti-HER2×CD28 bispecific antibodies

[0376] Generation of anti-HER2 × CD28 bispecific antibodies

[0377] Using TYM13 Fc mutations (D or E356K:E357K:S364K:S400CL351'D:K370'D:N390'C:K439'D; according to IgG1 C H The heterodimeric bispecific framework was engineered using the Kabat numbering scheme of the 3 domains. The light chain-heavy chain half antibody and the single chain variable fragment (scFv)-Fc chain were combined to form a bispecific antibody (BsAb) with a hetero-Fc domain with the TYM13 mutation.

[0378] Plasmids encoding BsAb heavy chain, light chain and scFv-Fc chain were transiently transfected into mammalian cells. Seven days after transfection, cells containing bispecific antibodies were harvested by centrifugation at 14000g for 30 minutes, and the culture supernatant was filtered through a sterile filter (0.22 μm). TM SuRe TM Antibodies were purified by protein A affinity chromatography on prepacked columns (GE Healthcare) and subsequently buffer exchanged into 20 mM histidine, pH 5.5 buffer.

[0379] The TY24865 (high affinity CD28) and TY24865 mutant (low affinity CD28) frameworks were chosen to construct CD28 BsAbs. The constructs are described in Table 10.

[0380] Table 10. Anti-HER2×CD28 bispecific antibody design

[0381]

[0382] Binding to SK-OV-3 cells measured by FACS

[0383] Flow cytometry was used to measure the activity of anti-HER2×CD28 BsAb (TY27566), anti-HER2×CD3 BsAb (TY25238, also described in PCT / CN2021 / 076626, which is incorporated herein by reference in its entirety), and anti-HER2 mAb (TAC2319) or (TAC2320) concentration-dependent binding activity. (TAC2319) and (TAC2320) binds to different epitopes in the HER2 dimerization domain.

[0384] SK-OV-3 cells were cultured and 8.0×10 4 Cells / well were added to a 96-well plate and incubated with serially diluted test BsAb in 2% FBS / RPMI1640 buffer at 4°C for 60 minutes. Next, the cells were washed twice with DPBS and further incubated with secondary APC-anti-human IgG Fc antibody (1:400 dilution) at 4°C for 30 minutes. Finally, the 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 the data were further fitted by four-parameter nonlinear regression of GraphPad Prism 7 (Windows version, GraphPad Software, La Jolla California USA, www.graphpad.com) to obtain EC 50 Isotype antibody was used as negative control.

[0385] like Figure 6 As shown, compared with anti-HER2 mAb and (TAC2319) or (TAC2320), anti-HER2 × CD28 or anti-HER2 × CD3 BsAbs displayed very similar binding affinities to SK-OV-3 cells.

[0386] Stimulatory activity of T cell receptor and CD28 receptor signaling

[0387] The stimulatory activity of T cell receptor (TCR) and CD28 receptor signaling was evaluated by anti-HER2×CD3 BsAb, anti-HER2×CD28 BsAb or their combination. In this assay, simultaneous activation of TCR and CD28 leads to enhanced transcriptional activity of NFkB, which in turn induces reporter gene production. Isotype antibodies served as negative controls.

[0388] Jurkat-NFkB-Nluc effector (E) reporter cells (5×10 4 cells / well) and SK-OV-3 target (T) cells (1×10 4Serial dilutions of anti-HER2×CD3 BsAb or isotype control antibody with a fixed concentration of anti-HER2×CD28 BsAb (10 nM), or conversely, serial dilutions of anti-HER2xCD28 BsAb or isotype control antibody with a fixed concentration of anti-HER2×CD3 BsAb (0.01 nM) were added to the reporter cell system to evaluate their combined effects in stimulating downstream luciferase activity. The co-cultured cells were incubated at 37°C with 5% CO 2 Then, 100 μL Luciferase Assay System (Promega) reagent was added to the cells and the cells were lysed for 10 min. The supernatant (100 μL) was collected for use Luminescence measurement on the i3x Multimode Microplate Reader (Molecular Components).

[0389] like Figure 7 As shown, the combination of anti-HER2×CD28 BsAb (TY27566) with a fixed concentration of anti-HER2×CD3 BsAb (TY25238), and the combination of anti-HER2×CD3 BsAb with a fixed concentration of anti-HER2×CD28 BsAb, showed significant differences in the maximum signal value and EC 50 The values ​​showed synergistic or enhanced stimulatory effects.

[0390] Cytotoxicity of anti-HER2 × CD28 BsAb and anti-HER2 × CD3 BsAb with the same or different TAA epitopes

[0391] The in vitro cytotoxic activity of anti-HER2×CD3 BsAb (TY25238) alone or in combination with anti-HER2×CD28 BsAb (TY27566 or TY27807) on MCF-7 tumor cell line was measured using a lactate dehydrogenase (LDH) release assay. TY25238 binds to a different tumor-associated antigen (TAA) epitope of the HER2 dimerization domain than TY27566, but binds to the same TAA epitope as TY27807.

[0392] Human T cells were isolated from cryopreserved PBMCs. MCF-7 tumor target cells (1×10 4 Cells / well) were incubated with serially diluted anti-CD3 BsAb FG14127 alone or in combination with a fixed concentration (1 μg / mL) of two anti-CD28 BsAbs (TY27566 or TY27807) that bind to different TAA antigen epitopes for 30 minutes. 4cells / well) and incubated at 37°C, 5% CO 2 Incubate for 72 hours at 4 °C (E:T=2:1). A non-radioactive cytotoxicity assay (Promega) quantified cytotoxicity based on the killing of MCF-7 target cells and the release of LDH into the supernatant.

[0393] like Figure 8 As shown, as a single agent, anti-HER2 × CD3 BsAb (TY25238) elicited potent concentration-dependent cytotoxicity on MCF-7 target cells. The combination of anti-HER2 × CD28 BsAb (TY27566) and anti-HER2 × CD3 BsAb (TY25238) targeting different TAA antigen epitopes further enhanced the ECs with tumor killing activity in vitro. 50 However, the addition of anti-HER2×CD28 BsAb (TY27807) with the same TAA epitope significantly reduced the EC of MCF-7 tumor cells compared with anti-HER2×CD3 BsAb (TY25238) alone. 50 and maximum lysis. Isotype antibodies served as negative controls for single-drug testing.

[0394] Example 8: Construction and functional characterization of anti-TROP2×CD28 or anti-TROP2×CD3 BsAbs

[0395] Anti-tumor-related calcium signaling protein 2 (TROP2) x CD28 or anti-TROP2 x CD3 heterodimeric bispecific frameworks were designed using TYM13 Fc mutants as described in Example 7. The constructs of anti-TROP2 x CD28 BsAbs are described in Table 11.

[0396] The constructs of anti-TROP2×CD3 BsAbs are described in Table 12.

[0397] Table 11. Anti-TROP2×CD28 bispecific antibody design

[0398] BsAb ID Fab arm (anti-TROP2) scFv arm (anti-CD28) TY27571 TY25616 TY24865

[0399] Table 12. Anti-TROP2×CD3 bispecific antibody design

[0400] BsAb ID Fab arm (anti-TROP2) scFv arm (anti-CD3) TY25839 TY25616 TY24742

[0401] Binding of CD3- or CD28-based bispecifics to tumor cell lines with high, medium, or low TROP2 expression

[0402] Flow cytometry was used to measure the concentration-dependent binding activity of anti-TROP2 BsAb on different tumor cell lines with high, medium or low TROP2 expression. Isotype antibodies served as negative controls.

[0403] H292, NCI-N87 or HT29 cells were cultured and 1.0×10 5 Cells / well were seeded in 96-well plates and incubated with serially diluted test anti-TROP2 BsAb in 2% FBS / RPMI1640 buffer for 30 minutes at 4°C. Next, the cells were washed twice with DPBS and further incubated with secondary anti-human IgG antibodies (APC-anti-human IgG Fc antibody, 1:300 dilution for H292 cells; APC-F(ab')2 fragment goat anti-human IgG (H+L), 1:500 dilution for NCI-N87 and HT29 cells) for 30 minutes at 4°C. Finally, the 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 the data were further fitted by four-parameter nonlinear regression of GraphPad Prism 7 (Windows version) to obtain EC 50 value.

[0404] like Fig. 9 As shown in Table 13, TY25839 and TY27571 had similar TROP2 arm binding activities on three different tumor cell lines with high, medium or low TROP2 expression.

[0405] Table 13. EC of anti-TROP2 bispecific antibodies on tumor cell lines 50 value

[0406]

[0407] Stimulatory activity of TCR and CD28 receptor signaling

[0408] The stimulatory activity of TCR and CD28 receptor signaling was evaluated by anti-TROP2×CD3 BsAb, anti-TROP2×CD28 BsAb or their combination. Simultaneous activation of TCR and CD28 led to enhanced transcriptional activity of NFkB, which in turn induced the production of reporter genes.

[0409] Jurkat-NFkB-Nluc effector reporter cells (5×10 4Serial dilutions of anti-TROP2×CD3 BsAb and a fixed concentration of anti-TROP2×CD28 BsAb (5 nM), or conversely, serial dilutions of anti-TROP2xCD28 BsAb and a fixed concentration of anti-TROP2×CD3 BsAb (0.01 nM) were added to the reporter cell system to evaluate their combined activity in stimulating downstream luciferase activity. The co-cultured cells were incubated at 37°C, 5% CO 2 Then, 100 μL of Nano-Glo luciferase assay system (Promega) reagent was added to the cells and the cells were lysed for 10 minutes. The supernatant (100 μL) was collected for luminescence measurement using a SpectraMax i3x multi-mode microplate reader (Molecular Components).

[0410] As shown in Figure 10, single agents based on CD28 BsAbs exhibited very weak reporter gene activity. However, the combination of anti-TROP2 × CD3 and anti-TROP2 × CD28 BsAbs showed a significant difference in the maximum signal value or EC. 50 The values ​​showed synergistic or enhanced stimulatory effects.

[0411] Example 9: Construction and functional characterization of B7H3×CD28 bispecific antibody

[0412] B7H3×CD28 BsAb was constructed. The constructs are described in Table 14.

[0413] The B7H3×CD3 BsAb constructs are described in Table 15.

[0414] Table 14. B7H3×CD28 bispecific antibody design

[0415] BsAb ID Fab Arm (anti-B7H3) scFv arm (anti-CD28) TY27556 TY21601 TY24865

[0416] Table 15. B7H3×CD3 bispecific antibody design

[0417] BsAb ID Fab Arm (anti-B7H3) scFv arm (anti-CD3) TY26999 TY21601 Intermediate affinity CD3

[0418] Binding to MDA-MB-231 cells measured by FACS

[0419] Using flow cytometry ( Fig.11 ) Measurement of concentration-dependent binding activity of anti-B7H3×CD28 BsAb TY27556 ​​and its parent anti-B7H3 mAb TY21601 on MDA-MB-231 cells. Isotype antibody served as negative control.

[0420] MDA-MB-231 cells were cultured and cultured at 1.0 × 105 Cells / well were seeded in 96-well plates and incubated with serially diluted test BsAb in 2% FBS / RPMI1640 buffer at 4°C for 60 minutes. Next, the cells were washed twice with DPBS and further incubated with secondary APC-anti-human IgG Fc antibody (1:400 dilution) at 4°C for 30 minutes. Finally, the cells were washed twice with DPBS and resuspended in FACS buffer for flow cytometry analysis. For analysis, MFI values ​​were plotted versus concentration using FlowJo10 software (FlowJo LLC), and the data were further fitted by four-parameter nonlinear regression of GraphPad Prism 7 (Windows version) to obtain EC 50 value.

[0421] like Fig.12 As shown, anti-B7H3 mAb TY21601 exhibited a sub-nM (0.4525 nM) binding affinity to MDA-MB-231 target cells, and BsAb TY27556 ​​had an approximately 28-fold reduction in binding activity to target cells (12.84 nM).

[0422] B7H3xCD28 bispecific antibody enhances the ability of PD-1 or PD-L1 blockade to induce T cell activation in vitro

[0423] The effect of anti-PD-1 blocking mAb combined with B7H3xCD28 bispecific Ab on in vitro activation of naive human T cells was measured by ELISA with secretion of IFN-γ and IL-2. A modified mixed lymphocyte reaction (MLR) was used to mimic physiological PD-L1 expression and TCR / CD3 stimulation. To generate a one-way MLR assay, human T cells (1×10 5 cells / well) in the presence of different test antibodies (B7H3xCD28 or isotype control) alone or in combination with anti-PD-1 (pembrolizumab, ) or anti-PD-L1 (atezolizumab, ) were incubated with allogeneic MDA-MB-231 cells (E:T = 5:1). The cells were then incubated at 37°C, 5% CO 2 The cells were cultured in an incubator for 120 hours. IL-2 (72 hours) and IFN-γ (120 hours) cytokines secreted from the supernatant of activated T cells were quantified using Elisa kits.

[0424] like Figures 13A to 13BAs shown, in the MLR assay, the addition of 100nM PD-1 / PD-L1 mAb or titrated B7H3xCD28 resulted in no or only slight cytokine release. However, the combination of B7H3xCD28 with 100nM PD-1 / PD-L1 mAb significantly increased T cell activation compared to monotherapy. Such results demonstrate that the B7H3xCD28 bispecific can synergistically bind PD-1 / PD-L1 blockade to promote T cell activation in the presence of tumor cells, endogenously expressing PD-L1 and B7H3.

[0425] Co-stimulatory bispecific antibodies enhance T cell cytotoxicity against MCF-7 cells in vitro when bound bidirectionally

[0426] The in vitro tumor cell killing activity of anti-CD3-based, or anti-CD28-based BsAbs or their combination on the MCF-7 tumor cell line was measured using an LDH release cytotoxicity assay.

[0427] Human T cells were isolated from cryopreserved PBMCs. MCF-7 cells (1×10 4 Cells / well) were incubated with serial dilutions of anti-HER2×CD3 BsAb TY25238 or with a fixed concentration 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) for 30 minutes, and human T cells (2×10 4 cells / well) at 37°C, 5% CO 2 Incubate for 72 hours (E:T = 2:1). The non-radioactive cytotoxicity assay (Promega) quantified cytotoxicity based on the release of LDH in the supernatant of killed MCF-7 target cells.

[0428] like Fig.14 As shown, as a single agent, CD28-based BsAbs did not induce cytotoxicity on MCF-7 target cells. In contrast, as a single agent, anti-CD3 BsAb TY25238 showed a concentration-dependent effect, and BsAbs against HER2 induced the highest cytotoxicity, with 54.47% of MCF-7 cell lysis. The combination of anti-CD3 and anti-CD28 BsAbs further enhanced the in vitro tumor cell killing activity, as shown by EC 50 Compared with anti-CD3 BsAb TY25238 alone, the addition of anti-CD28 BsAb TY27566 with a high affinity CD28 arm significantly enhanced the binding of CD28 by about 10 times (EC 50However, no enhanced cytotoxicity was detected when combined with the anti-CD28 BsAb TY27881 with a low affinity CD28 arm.

[0429] Co-stimulatory bispecific antibodies enhance T cell cytotoxicity against EMT-6-HER2 cells in vitro when bound bidirectionally

[0430] 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.

[0431] Using EasySep TM Human T cells were isolated from fresh PBMCs using the Human Primary Total T Cell Isolation Kit (STEMCELL Technologies). EMT-6-HER2 (5 × 10 3 Target cells were incubated with serial dilutions of anti-HER2 × CD3 BsAb (TY25238) or with a fixed concentration of anti-B7H3 × CD3 BsAb TY27556 ​​(8 nM) for 30 min at 37 °C, or conversely, EMT-6-HER2 (5 × 10 3 Cells were incubated with serial dilutions of anti-B7H3×CD3 BsAb (TY26999) or with a fixed concentration of anti-HER2×CD28 BsAb TY27566 (8 or 0.8 nM) for 30 minutes. 4 cells / well) and cultured at 37°C, 5% CO 2 Incubate for 72 hours (E:T = 2:1). Non-radioactive cytotoxicity assay (Promega) quantified cytotoxicity based on the release of LDH in the supernatant of EMT-6-HER2 target cells. Isotype antibodies were used as negative controls for single-agent testing.

[0432] like Fig.15 As shown, as single agents, TY27556 ​​(anti-B7H3×CD28) or TY27566 (anti-HER2×CD28) did not show killing efficacy against EMT-6-HER2 cells. Compared with TY25238, the combination of TY25238 and TY27556 ​​resulted in 50 It was reduced by about 2 times, and the maximum lethality increased by about 3 times (from 17% to 50%).

[0433] Compared with TY26999, the combination of TY26999 and TY27566 resulted in EC 50It decreased by about 1.3 times, and the maximum lethality increased by about 2 times (from 23% to 47%).

[0434] Risk of systemic cytokine release in vivo with anti-CD3 or anti-CD28 based bispecific antibodies or their combination

[0435] In vivo systemic cytokine release (IL-6 and IFN-γ) in a BALB / c mouse model was measured using ELISA based on anti-mouse CD3 or anti-CD28 BsAbs alone or in combination.

[0436] BALB / c mice were randomly divided into four groups (3 mice 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, in which the CD3 arm was derived from the mouse-specific 145-2C11 clone, 2 mg / kg) or their combination (TY27556, 2 mg / kg and TY27042, 2 mg / kg). Serum and whole blood were collected from mice before and at different time points after injection (3.5 and 24 hours before administration). Systemic cytokine release risk was assessed by ELISA via IL-6 and IFN-γ. CD3 was measured at each time point by flow cytometry. + The percentage of T cells to total peripheral T cells.

[0437] like Fig.16 As shown, administration of anti-mCD3 or anti-B7H3×CD3 (TY27042) to mice 3.5 hours after test antibody treatment resulted in a significant reduction in cytokine release (IL-6 and IFN-γ). Anti-B7H3×CD28 (TY27556)-treated mice showed no detectable cytokine release after test antibody treatment. In addition, the combination of anti-B7H3×CD3 (TY27042) and anti-B7H3×CD28 (TY27556) did not show an enhanced risk of cytokine release compared with single-agent anti-B7H3×CD3 (TY27042). In the single-agent anti-mouse CD3, anti-B7H3×CD3 (TY27042) and anti-B7H3×CD28 (TY27556) groups and the combination of anti-B7H3×CD3 (TY27042) and anti-B7H3×CD28 (TY27556) groups, peripheral CD3 + T cells dropped sharply to almost zero. 3.5 hours after injection, only about 2% to 3% of CD3 + T cells remain in the peripheral blood.

[0438] Example 10: In vivo efficacy study

[0439] In vivo efficacy study of B7H3xCD28 BsAb monotherapy and combination therapy with HER2xCD3 BsAb in the SK-OV3 model

[0440] Immunodeficient M-NSG mice (n=8 / group, female, 7 to 8 weeks old) were transplanted with 5×10 6 7 days later, 2×10 6 SK-OV3 cells. The average tumor volume reached about 90 mm on day 8 after tumor inoculation. 3 Treatment was started at 5 mg / kg hIgG by intraperitoneal injection 1 Isotype control, 0.2 mg / kg anti-HER2×CD3 bispecific double-chain shielded antibody, 5 mg / kg anti-B7H3×CD28 bispecific antibody TY27556, or a combination of 0.2 mg / kg TY27151 and 5 mg / kg TY27556. TY27151 was previously described in PCT / CN2021 / 076626. The Ab was administered to mice twice a week for a total of five doses. Tumor growth was monitored twice a week and reported as mean tumor volume ± sem over time.

[0441] like Fig.17 As shown, the double-chain shielded anti-HER2×CD3 bispecific antibody TY27151 and the anti-B7H3×CD28 bispecific antibody TY27556 ​​showed a strong synergistic anti-tumor effect in the model.

[0442] In vivo efficacy of CD28 BsAb in the EMT6-HER2 mouse breast cancer syngeneic model

[0443] BALB / c mice (n=5 / group, female, 8 to 9 weeks old) were subcutaneously inoculated with 5×10 5 EMT-HER2 cells. The average tumor volume reached about 110 mm on the 7th day after tumor inoculation. 3 Treatment was started at 1:10 p.m. ...

[0444] like Fig.18 As shown in Figure 2, the anti-B7H3×CD28 bispecific antibody TY27556 ​​showed a dose-dependent anti-tumor effect in the model. Fig.18As shown, the anti-HER2×CD28 bispecific antibody TY27566 showed a strong anti-tumor effect.

[0445] Example 11: Method for Identifying Self-Blocking Peptides Masked by Anti-CD28 Antibodies

[0446] Generation of masked anti-CD28 antibodies

[0447] A screening system has been designed and implemented for effectively discovering masking parts that can effectively mask unmasked parent anti-CD28 antibodies with good developability. In this system, the target anti-CD28 scFv is first displayed on the surface of yeast and functionally bound to its CD28 antigen. Then the masking peptide (MP) from the improved MP peptide library is directly fused to the N-terminus of the light chain of the target anti-CD28 scFv, and a yeast library displaying the fusion protein on the surface of yeast is constructed. The yeast library then undergoes several rounds of FACS-based screening: 1) Yeast clones with low binding to the antigen are concentrated, 2) the concentrated yeast clones are treated with protease to remove the N-terminal MP, and 3) clones showing high binding to the antigen are selected. After 5 to 6 rounds of sorting, plasmids are extracted from such clones and the MP sequence is confirmed by DNA sequencing. In the presence of MP, the selected masked anti-CD28 antibody clones in the form of scFv show little binding to the antigen. However, when yeast cells were treated with tobacco etch virus nucleic acid content, an endopeptidase (TEV) to remove MPs, binding to the antigen increased dramatically. Incorporation of a TEV recognition site as a cleavage site into MPs, combined with the use of TEV protease to validate selected clones, significantly increased the success rate of MP selection.

[0448] To identify MP sequences, shuttle plasmids were extracted from selected yeast clones using a plasmid extraction kit (Generay) and transformed into active E. coli cells. Plasmids were prepared and the regions encoding the MPs were sequenced and aligned. As expected, such sequences can be divided into several groups, indicating the cleanup concentration through the sorting rounds. The masking efficiency of the selected MPs is shown in Table 16 below, and the sequence of each MP is shown in the sequence section below.

[0449] Converting the masked anti-CD28 scFv protein into IgG 4 Isotype mAb. Masked IgG 4 The mAb was engineered to include 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 displayed on the yeast surface. The heavy and light chains were cloned separately into the mammalian expression vector pCDNA3.3 (ThermoFisher Scientific). The V H and V LThe sequences are listed in the sequence section below.

[0450] Plasmid was transiently transfected into HEK293F cells. After 6 days, supernatant was collected, clarified by centrifugation and filtration, and IgG was purified with standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). IgG was eluted and neutralized, and buffer exchanged to 20mM histidine, pH 5.5 buffer. Protein concentration was determined by UV-spectrophotometry, and IgG purity was analyzed by SDS-PAGE or EC-HPLC under denaturation, reduction and non-reducing conditions. Protein concentration was measured by ultraviolet spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturation, reduction and non-reducing conditions. Importantly, the expression level of shielded antibodies in HEK293 cells was similar to or lower than their parent antibodies, and their purification yields after protein A resin were also similar, thus showing that the presence of shielding peptides and cleavage peptides did not have a significant negative effect on the expression of antibodies in mammalian cells.

[0451] Table 16. Masking peptides

[0452] IgG ID ELISA masking efficiency IgG ID ELISA masking efficiency IgG ID ELISA masking efficiency TY26142 3062 TY26152 1867 TY26162 ND TY26143 ND TY26153 1825 TY26163 ND TY26144 ND TY26154 ND TY26164 ND TY26145 1430 TY26155 368 TY26165 ND TY26146 2179 TY26156 670 TY26166 ND TY26147 4882 TY26157 ND TY26167 1427 TY26148 ND TY26158 887 TY26168 2567 TY26149 3841 TY26159 468 TY26169 1818 TY26150 1027 TY26160 1461 TY26170 ND TY26151 2476 TY26161 1484 TY26171 406

[0453] Measurement of shading efficiency

[0454] When the shielding efficiency was measured by ELISA, recombinant human CD28-Fc was diluted to 2 μg / mL in PBS and coated on the MaxiSorp TM High protein binding capacity 96-well ELISA plates (ThermoFisher Scientific) were incubated overnight. The plates were blocked with PBS supplemented with 3% skim milk for 1 hour at 37°C. After washing, 100 μL of 3-fold serial dilutions of anti-CD28 test mAb were added to each well. After incubation for 1 hour 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 at 37°C for 1 hour, washed four times, and then 50 μL of TMB substrate (3,3',5,5'-tetramethylbenzidine) solution was added to each well, and the plates were incubated at room temperature. After adding 50 μL of sulfuric acid stop solution to each well to stop the reaction, the absorbance at 450 nm was measured. GraphPadPrism 6 (Windows version, GraphPad Software, La Jolla California USA, www.graphpad.com ) was used to fit the ELISA data to evaluate EC 50 .

[0455] The shielding efficiency of the selected shielded anti-CD28 test mAb was determined by the EC of the bound shielded mAb. 50 Divided by the EC of the unmasked parent mAb (TY24865) 50 Calculated and listed in Table 17. Fig.19 As shown, all activatable mAbs showed drastically reduced binding to their antigens compared to the parent mAb, and the calculated shielding efficiencies ranged from 368 to above 4000. These results indicate that many of the MPs identified from the improved MP peptide library maintained their shielding efficiencies and as part of a full IgG molecule when expressed in mammalian cells.

[0456] Table 17. SEC purity and masking efficiency of masked anti-CD28 antibodies

[0457]

[0458] Optimization of selected anti-CD28 activatable antibodies

[0459] For the two lead masked anti-CD28 mAbs (TY26149 and TY26152), modifications were made on their MPs, including removal of some N-terminal residues and addition of an "S" amino acid residue between the "D" and "G" residues of the TY26149 sequence (bold and underlined residues of TY26149 in Table 18). Fig. 20 As shown, the expression and shielding efficiency of the novel shielded antibodies were not significantly affected.

[0460] Table 18. Modification of masking peptide sequences

[0461]

[0462] Example 12: Epitope Mapping

[0463] In order to determine the binding region of the test antibody at the amino acid residue level, a series of mutants of the extracellular domain of human CD28 were prepared (Table 19). HEK293F cells were transfected with these CD28 mutant plasmids. The binding of antibodies to human CD28 mutants was evaluated by flow cytometry analysis. The results are summarized in Table 19, together with the cross-reactivity of these antibodies with people, monkeys and mouse CD28 in the difference (differentiation) of interest. TY24865 cross-reacts with people, monkeys and mouse CD28, but TAC2386 and TAC2387 do not bind to mouse CD28. The mutant construct is intended to distinguish the epitope bound by TY24863 from the reference antibodies TAC2386 and TAC2387. Clearly, TY24865 retains the ability to bind to RE49AA, VY68AA, YS79AA and KT81AA, indicating that TY24865 does not bind to RE49, VY68, YS79 and KT81 residues, and these residues are located in the non-conserved regions of human and mouse CD28. However, TY24865 loses the ability to bind to FR51AA, SL54AA, YL98AA, QN100AA, YF110AA, KI113AA, YP118AA, PPP119AAA, PP120AA, PY121AA and Y122A mutations, indicating that its binding antigen epitopes are within these regions, such as amino acid residues 51, 52, 54, 55, 98-101, 110-111, 113-114, 118-122 of SEQ ID NO.: 1.

[0464] Table 19. Epitope mapping

[0465] Mutants TY24865 TAC2386 TAC2387 Hu_WT + + + Cyno_WT + + + Mouse_WT + - - Hu_RE49AA + + - Hu_FR51AA - - - Hu_SL54AA - - - Hu_VY68AA + + - Hu_Y69A + + - Hu_YS79AA + - + Hu_KT81AA + - + Hu_YL98AA - - - Hu_QN100AA - - - Hu_YF110AA - - - Hu_KI113AA - - - Hu_M117A + + - Hu_YP118AA - + - Hu_PPP119AAA - + - Hu_PP120AA - + - Hu_PY121AA - + - Hu_Y122A - + +

[0466] Example 13: TY24865 variants

[0467] Two approaches were used to generate TY24865 variants. Different point mutations were introduced into the YYYY sequence of TY24865 scFv, and new batches of TY24865 scFv variants were discovered by yeast-based maturation library screening. Anti-TAA × CD28 bispecific antibodies were constructed in the Fab-scFv-Fc format, and the CD28 affinity of these bsAbs was measured using ELISA.

[0468] Anti-PD-L1×CD28 bispecific antibodies were constructed in the form of Fab-scFv-Fc of anti-PD-L1 TY21421 Fab (see WO 2019 / 185035) and TY24865 scFv (VH-VL format) or its variants. The affinity of some of these bsAbs to CD28 is summarized in Table 20 and Fig.22A and 22BTY29815 is a wild-type bsAb, and TY30413 has similar affinity to TY29815, while other bsAbs have higher or lower affinities.

[0469] Table 20. Construction and characterization of anti-PD-L1×CD28 BsAbs with TY24865 variants

[0470]

[0471] Anti-HER2×CD28 bsAb or anti-B7-H3×CD28 bsAb were constructed using TY24865 scFv variants in VH-VL or VL-VH format. As shown in Table 21, these bsAbs showed normal expression and SEC purity. Fig.23A and 23B As shown in Table 22, the CD28 affinity of these bsAbs was measured by ELISA. These scFv variants showed variable affinities ranging from 0.954 to 417 nM, and the scFv in the VL-VH format had higher affinity than the scFv in the VH-VL format. Table 22 and Fig.24A and 24B Some variants showed high affinity for both humans and mice, and this affinity was further confirmed in mouse T cell binding assays ( Fig.25 ).

[0472] Table 21. Construction and characterization of anti-HER2×CD28 BsAbs with TY24865 variants

[0473]

[0474]

[0475] Table 22. Construction and characterization of anti-B7-H3×CD28 BsAbs with TY24865 variants

[0476]

[0477] The above non-limiting examples are provided for illustrative purposes only, so as to more fully understand the disclosed subject matter. These examples should not be construed as limiting any embodiments described in this specification, including those involving antibodies, pharmaceutical compositions, or methods and uses for treating cancer, neurodegenerative diseases or infectious diseases.

[0478] sequence

[0479] The sequences disclosed herein are listed in the table below and the SEQ ID NOs (SEQ) are shown in the left column.

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501] SEQ Flexible joints 249 GGGGS 250 GGGGT 251 SGGS 252 GGS 253 GGSGG 254 GSGSG 255 GSGGG 256 GGGSG 257 GSSSG

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

Claims

1. An antigen binding protein or antigen binding fragment thereof, comprising a CD28 binding portion, wherein the CD28 binding portion binds to human CD28 and cross-reacts with cynomolgus macaque and mouse CD28.

2. The antigen binding protein or fragment thereof of claim 1, wherein the CD28 binding portion binds to a CD28 epitope comprising amino acid residues 51-122 of human CD28 (SEQ ID NO: 1).

3. The antigen binding protein or fragment thereof of claim 2, wherein the CD28 epitope comprises amino acid residues 51, 52, 54, 55, 98-101, 110-111, 113-114 and 118-122 of SEQ ID NO:

1.

4. The antigen binding protein or fragment thereof according to any one of claims 1 to 3, wherein the CD28 binding portion comprises an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), and V H and V L comprising the heavy chain complementarity determining regions (CDRs) 1-3 and light chain CDRs 1-3 as indicated, SEQ ID NOs: 5-10, respectively, SEQ ID NOs: 15, 6, 16, 17-19, respectively, SEQ ID NOs: 24, 6, 25, 26-28, respectively, SEQ ID NOs: 33, 6, 35-38, respectively, SEQ ID NOs: 43, 6, 44, 45, 9 and 46, respectively, SEQ ID NOs: 33, 51-53, 300 and 10, respectively, SEQ ID NOs: 24, 58, 59, 60, 300 and 61, respectively, SEQ ID NOs: 66-69, 300 and 70, respectively, SEQ ID NOs: 24, 6, 75, 76, 18 and 28, respectively, SEQ ID NOs: 24, 58, 81, 82, 27 and 83, respectively, SEQ ID NOs: 88-91, 300 and 70, respectively, SEQ ID NOs: 24, 96-98, 9 and 70, respectively, SEQ ID NOs: 103-106, 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, SEQ ID NOs: 161, 6, 162, 163, 300 and 164, respectively, SEQ ID NOs: 5, 96, 307, 76, 308 and 46, respectively, SEQ ID NOs: 5, 96, 309, 310, 9 and 311, respectively, SEQ ID NOs: 43, 96, 312, 76, 9 and 28, respectively, SEQ ID NOs: 43, 96, 307, 310, 9 and 313, respectively, SEQ ID NOs: 314, 315, 309, 76, 9 and 313, respectively, SEQ ID NOs: 33, 316, 312, 8, 308 and 313, respectively, SEQ ID NOs: 314, 96, 317, 76, 308 and 318, respectively, SEQ ID NOs: 43, 316, 312, 8, 9 and 10, respectively, SEQ ID NOs: 43, 6, 319, 320, 9 and 10, respectively, SEQ ID NOs: 5, 67, 307, 310, 308 and 28, respectively, SEQ ID NOs: 43, 6, 321, 8, 9 and 10, respectively, SEQ ID NOs: 314, 316, 322, 8, 9 and 10, respectively, SEQ ID NOs: 33, 6, 321, 8, 9 and 10, respectively, SEQ ID NOs: 5, 6, 317, 8, 9 and 10, respectively, SEQ ID NOs: 5, 6, 323, 8, 9 and 10, respectively, or They are SEQ ID NOs: 5, 6, 324, 8, 9 and 10 respectively.

5. An antigen binding protein or antigen binding fragment thereof, comprising 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 ), and V H and V L comprising the heavy chain complementarity determining regions (CDRs) 1-3 and light chain CDRs 1-3 as indicated, SEQ ID NOs: 5-10, respectively, SEQ ID NOs: 15, 6, 16, 17-19, respectively, SEQ ID NOs: 24, 6, 25, 26-28, respectively, SEQ ID NOs: 33, 6, 35-38, respectively, SEQ ID NOs: 43, 6, 44, 45, 9 and 46, respectively, SEQ ID NOs: 33, 51-53, 300 and 10, respectively, SEQ ID NOs: 24, 58, 59, 60, 300 and 61, respectively, SEQ ID NOs: 66-69, 300 and 70, respectively, SEQ ID NOs: 24, 6, 75, 76, 18 and 28, respectively, SEQ ID NOs: 24, 58, 81, 82, 27 and 83, respectively, SEQ ID NOs: 88-91, 300 and 70, respectively, SEQ ID NOs: 24, 96-98, 9 and 70, respectively, SEQ ID NOs: 103-106, 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, SEQ ID NOs: 161, 6, 162, 163, 300 and 164, respectively, SEQ ID NOs: 5, 96, 307, 76, 308 and 46, respectively, SEQ ID NOs: 5, 96, 309, 310, 9 and 311, respectively, SEQ ID NOs: 43, 96, 312, 76, 9 and 28, respectively, SEQ ID NOs: 43, 96, 307, 310, 9 and 313, respectively, SEQ ID NOs: 314, 315, 309, 76, 9 and 313, respectively, SEQ ID NOs: 33, 316, 312, 8, 308 and 313, respectively, SEQ ID NOs: 314, 96, 317, 76, 308 and 318, respectively, SEQ ID NOs: 43, 316, 312, 8, 9 and 10, respectively, SEQ ID NOs: 43, 6, 319, 320, 9 and 10, respectively, SEQ ID NOs: 5, 67, 307, 310, 308 and 28, respectively, SEQ ID NOs: 43, 6, 321, 8, 9 and 10, respectively, SEQ ID NOs: 314, 316, 322, 8, 9 and 10, respectively, SEQ ID NOs: 33, 6, 321, 8, 9 and 10, respectively, SEQ ID NOs: 5, 6, 317, 8, 9 and 10, respectively, SEQ ID NOs: 5, 6, 323, 8, 9 and 10, respectively, or They are SEQ ID NOs: 5, 6, 324, 8, 9 and 10 respectively.

6. The antigen binding protein or fragment thereof of claim 4 or 5, wherein the CD28 binding portion comprises V H and V L , SEQ ID NOs: 11 and 12, respectively, SEQ ID NOs: 20 and 21, respectively, SEQ ID NOs: 29 and 30, respectively, SEQ ID NOs: 39 and 40, respectively, SEQ ID NOs: 47 and 48, respectively, SEQ ID NOs: 54 and 55, respectively, SEQ ID NOs: 62 and 63, respectively, SEQ ID NOs: 71 and 72, respectively, SEQ ID NOs: 77 and 78, respectively, SEQ ID NOs: 84 and 85, respectively, SEQ ID NOs: 92 and 93, respectively, SEQ ID NOs: 99 and 100, respectively, SEQ ID NOs: 107 and 108, respectively, SEQ ID NOs: 115 and 116, respectively, SEQ ID NOs: 122 and 123, respectively, SEQ ID NOs: 130 and 131, respectively, SEQ ID NOs: 137 and 138, respectively, SEQ ID NOs: 144 and 145, respectively, SEQ ID NOs: 151 and 152, respectively, 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.

7. The antigen-binding protein of claim 4 or 5, comprising the antibody heavy chain and the antibody light chain, 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.

8. The antigen binding protein or fragment thereof according to any one of claims 1 to 7, wherein the antigen binding protein or fragment thereof does not have super agonist activity.

9. The antigen binding protein or fragment thereof according to any one of claims 1 to 8, further comprising a second antigen binding portion targeting a tumor associated antigen (TAA).

10. The antigen binding protein or fragment thereof according to claim 9, wherein TAA is HER2, B7-H3 or TROP-2.

11. The antigen binding protein or fragment thereof of claim 10, comprising a HER2 binding portion comprising HCDR1-3 and LCDR1-3 as shown in SEQ ID NOs: 262-264 and 258-260, respectively, or V as shown in SEQ ID NOs: 265 and 261, respectively H and V L .

12. The antigen binding protein or fragment thereof of claim 10, comprising a B7-H3 binding portion comprising HCDR1-3 and LCDR1-3 as shown in SEQ ID NOs: 290-292 and 287, 18 and 288, respectively, or V as shown in SEQ ID NOs: 293 and 289, respectively H and V L .

13. The antigen binding protein or fragment thereof of claim 10, comprising a TROP-2 binding portion comprising HCDR1-3 and LCDR1-3 as shown in SEQ ID NOs: 280-282 and 277, 259 and 278, respectively, or V as shown in SEQ ID NOs: 283 and 279, respectively H and V L .

14. The antigen binding protein or fragment thereof according to any one of claims 1 to 13, wherein any one or both of the CD28 binding portion and the TAA binding portion are single chain Fv (scFv), Fv, scFab or Fab.

15. An antigen binding protein or fragment thereof as claimed in claim 11, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 169 and 170, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO: 171 or a scFv fusion polypeptide comprising SEQ ID NO:

266.

16. An antigen binding protein or fragment thereof as claimed in claim 11, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 169 and 170, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO: 172 or a scFv fusion polypeptide comprising SEQ ID NO:

266.

17. An antigen binding protein or fragment thereof as claimed in claim 11, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 271 and 276, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO: 171 or a scFv fusion polypeptide comprising SEQ ID NO:

266.

18. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

327.

19. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

333.

20. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

334.

21. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

335.

22. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

336.

23. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

337.

24. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

338.

25. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

339.

26. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

340.

27. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

341.

28. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

342.

29. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

343.

30. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

344.

31. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

345.

32. An antigen binding protein or fragment thereof as claimed in claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

346.

33. An antigen binding protein or fragment thereof as claimed in claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

347.

34. An antigen binding protein or fragment thereof as claimed in claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

348.

35. The antigen binding protein or fragment thereof of claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

349.

36. An antigen binding protein or fragment thereof as claimed in claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

350.

37. An antigen binding protein or fragment thereof as claimed in claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

351.

38. An antigen binding protein or fragment thereof as claimed in claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

352.

39. An antigen binding protein or fragment thereof as claimed in claim 10, wherein the HER2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 331 and 332, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

353.

40. An antigen binding protein or fragment thereof as described in claim 12, wherein the B7-H3 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 175 and 176, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO: 171 or a scFv fusion polypeptide comprising SEQ ID NO:

266.

41. An antigen binding protein or fragment thereof as described in claim 10, wherein the B7-H3 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 354 and 355, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

327.

42. An antigen binding protein or fragment thereof as described in claim 10, wherein the B7-H3 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 354 and 355, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

356.

43. An antigen binding protein or fragment thereof as described in claim 10, wherein the B7-H3 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 354 and 355, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

357.

44. An antigen binding protein or fragment thereof as described in claim 10, wherein the B7-H3 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 354 and 355, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

358.

45. An antigen binding protein or fragment thereof as claimed in claim 13, wherein the TROP2 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 173 and 174, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO: 171 or a scFv fusion polypeptide comprising SEQ ID NO:

266.

46. ​​An antigen binding protein or fragment thereof as claimed in any one of claims 1 to 45, further comprising another antigen binding portion that targets an immune checkpoint inhibitor.

47. The antigen binding protein of claim 46, wherein the immune checkpoint inhibitor is PD-L1.

48. An antigen binding protein or fragment thereof as claimed in claim 47, wherein the PD-L1 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 325 and 326, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

327.

49. An antigen binding protein or fragment thereof as claimed in claim 47, wherein the PD-L1 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 325 and 326, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

328.

50. The antigen binding protein or fragment thereof of claim 47, wherein the PD-L1 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 325 and 326, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

329.

51. The antigen binding protein or fragment thereof of claim 47, wherein the PD-L1 binding portion comprises an antibody light chain and an antibody heavy chain comprising SEQ ID NOs: 325 and 326, respectively, and wherein the CD28 binding portion comprises a heavy chain comprising SEQ ID NO:

330.

52. An antigen binding protein or fragment thereof as claimed in any one of claims 1 to 51, further comprising an Fc region.

53. The antigen binding protein or fragment thereof of claim 52, wherein the Fc region is of human IgG1 subclass.

54. The antigen binding protein or fragment thereof of claim 52, wherein the Fc region is of human IgG4 subclass.

55. An antigen binding protein or fragment thereof as claimed in any one of claims 52 to 54, wherein the Fc region has reduced or no effector function, reduced or no antibody dependent cellular cytotoxicity (ADCC) effect and / or reduced or no cross-linking effect.

56. The antigen binding protein or fragment thereof according to any one of claims 1 to 55, comprising a first CH3 domain and a second CH3 domain, in: i) the first CH3 domain comprises a cysteine ​​(C) residue at position 390 and the second CH3 domain comprises a cysteine ​​residue at position 400, or the first CH3 domain comprises a cysteine ​​residue at position 400 and the second CH3 domain comprises a cysteine ​​residue at position 390; or ii) the first CH3 domain comprises a cysteine ​​residue at position 392 and the second CH3 domain comprises a cysteine ​​residue at position 397, or the first CH3 domain comprises a cysteine ​​residue at position 397 and the second CH3 domain comprises a cysteine ​​residue at position 392; or iii) the first CH3 domain comprises a cysteine ​​residue at position 392 and the second CH3 domain comprises a cysteine ​​residue at position 400, or the first CH3 domain comprises a cysteine ​​residue at position 400 and the second CH3 domain comprises a cysteine ​​residue at position 392; and wherein the amino acid residue numbering is based on Eu numbering.

57. An antigen binding protein or fragment thereof as claimed in claim 56, in: i) the first CH3 domain further comprises a positively charged residue at position 357 and the second CH3 domain further comprises a negatively charged residue at position 351, or the first CH3 domain further comprises a negatively charged residue at position 351 and the second CH3 domain further comprises a positively charged residue at position 357; or ii) the first CH3 domain further comprises a positively charged residue at position 411 and the second CH3 domain further comprises a negatively charged residue at position 370, or the first CH3 domain further comprises a negatively charged residue at position 370 and the second CH3 domain further comprises a positively charged residue at position 411; or iii) the first CH3 domain further comprises a positively charged residue at position 364 and the second CH3 domain further comprises a negatively charged residue at position 370, or the first CH3 domain further comprises a negatively charged residue at position 370 and the second CH3 domain further comprises a positively charged residue at position 364; or a combination of i) and ii), or a combination of i) and iii); and wherein the amino acid residue numbering is based on Eu numbering.

58. An antigen binding protein or fragment thereof as claimed in claim 57, wherein the first CH3 domain comprises D / E356K, E357K, S364K and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C and K439D substitutions and the second CH3 domain comprises D / E356K, E357K, S364K and S400C substitutions (Eu numbering).

59. The antigen binding protein or fragment thereof of claim 58, wherein the CH3 domain further comprises a N297A substitution (Eu numbering).

60. An antigen-binding protein or fragment thereof as described in any one of claims 1 to 60, further comprising at least one shielding peptide, wherein the shielding peptide (MP) is linked to the N-terminus of VL, wherein the MP comprises a shielding unit (MU) and a linking unit (LU) with or without a cleavage site from the N-terminus to the C-terminus.

61. The antigen binding protein or fragment thereof of claim 60, wherein MU comprises a sequence selected from the group consisting of SEQ ID NOs: 173-206.

62. The antigen binding protein or fragment thereof of claim 61, wherein the MP further comprises an N-terminal unit (NU) linked to the N-terminus of the MU.

63. An antigen binding protein or fragment thereof as claimed in claim 62, wherein the N-terminal unit is about 1-10 amino acid residues in length.

64. The antigen binding protein or fragment thereof of claim 63, wherein the N-terminal unit comprises E or EVGSY.

65. An antigen binding protein or fragment thereof as described in any one of claims 60 to 64, wherein LU comprises a cleavage site.

66. An antigen binding protein or fragment thereof according to claim 65, wherein the first cleavage site is selected from the group consisting of protease cleavage sites of the following proteases: urokinase-type plasminogen activator / uPA, matrix metalloproteinase-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, Casparase-1, Casparase-2, Casparase-3, Casparase-4, Casparase-5, Casparase-6, Casparase-7, Casparase-8, Casparase-9, Casparase-10, Casparase-11, Casparase-12, Casparase-13, Casparase-14 and TACE.

67. An antigen binding protein or fragment thereof as described in any one of claims 60 to 66, wherein at least one shielding peptide is linked to a B7-H3, HER2 or TROP2 binding portion or a CD28 binding portion, optionally, wherein the antigen binding protein or fragment thereof comprises two shielding peptides that bind to a B7-H3, HER2 or TROP2 binding portion and a CD28 binding portion, respectively.

68. An antigen binding protein or fragment thereof as claimed in any one of claims 1 to 67, further comprising a conjugated therapeutic agent moiety, optionally wherein the therapeutic agent moiety is a radioactive moiety or a cytotoxic moiety.

69. A pharmaceutical composition comprising an antigen binding protein or fragment thereof as claimed in any one of claims 1 to 68 and a pharmaceutically acceptable carrier.

70. A nucleic acid molecule or a nucleic acid molecule encoding an antigen binding protein or fragment thereof as claimed in any one of claims 1 to 68.

71. A host cell comprising the nucleotide sequence of claim 70.

72. A method for producing an antigen binding protein or an antigen binding fragment thereof, include: Cultivating the host cell of claim 71 under conditions that allow expression of the antigen binding protein or fragment thereof, and The antigen binding protein or fragment thereof is isolated from the culture medium.

73. 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 or fragment thereof according to any one of claims 1 to 68 or a pharmaceutical composition according to claim 69.

74. The method of claim 73, further comprising administering to the patient an additional anti-cancer therapeutic agent.

75. The method of claim 74, wherein the additional anti-cancer therapeutic agent is a bispecific antibody targeting CD3 and a tumor antigen, optionally wherein the tumor antigen is the same as or different from the TAA.

76. An antigen binding protein or fragment thereof as claimed in claim 75, wherein the TAA is B7-H3, HER2 or TROP2.

77. The method of claim 74, wherein the additional anti-cancer therapeutic is an immune checkpoint inhibitor, optionally, an anti-PD-1, anti-CTLA-4 or anti-PD-L1 antibody.

78. The method of any one of claims 73 to 77, wherein the patient has a solid tumor or a hematological malignancy, optionally selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, kidney cancer, pancreatic cancer and colon cancer.

79. Use of an antigen binding protein or fragment thereof as claimed in any one of claims 1 to 68 for the manufacture of a medicament for the treatment of cancer, optionally, a method as claimed in any one of claims 73 to 78.

80. An antigen binding protein or fragment thereof according to any one of claims 1 to 68, or a pharmaceutical composition according to claim 69, for use in the treatment of cancer, optionally, according to a method according to any one of claims 73 to 78.

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