WO2023193239A1 - Anti-cd28 antibodies and methods of use thereof - Google Patents

Anti-cd28 antibodies and methods of use thereof Download PDF

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WO2023193239A1
WO2023193239A1 PCT/CN2022/085838 CN2022085838W WO2023193239A1 WO 2023193239 A1 WO2023193239 A1 WO 2023193239A1 CN 2022085838 W CN2022085838 W CN 2022085838W WO 2023193239 A1 WO2023193239 A1 WO 2023193239A1
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seq
nos
antigen
antibody
fragment
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French (fr)
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Peter Peizhi Luo
Fangyong Du
Yan Li
Guizhong Liu
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Priority to PCT/CN2022/085838 priority Critical patent/WO2023193239A1/en
Priority to JP2024559144A priority patent/JP2025511713A/ja
Priority to CN202380044740.2A priority patent/CN120112552A/zh
Priority to PCT/CN2023/087277 priority patent/WO2023193817A1/en
Priority to AU2023250992A priority patent/AU2023250992A1/en
Priority to TW112113303A priority patent/TW202409082A/zh
Priority to KR1020247037360A priority patent/KR20250008811A/ko
Priority to US18/855,066 priority patent/US20250257133A1/en
Priority to CA3255415A priority patent/CA3255415A1/en
Priority to IL316065A priority patent/IL316065A/en
Priority to EP23723108.9A priority patent/EP4504787A1/en
Publication of WO2023193239A1 publication Critical patent/WO2023193239A1/en
Priority to MX2024012474A priority patent/MX2024012474A/es
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    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
    • A61K47/6879—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • A—HUMAN NECESSITIES
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    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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    • C07K2317/522—CH1 domain
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
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    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • CD28 is a key costimulatory signal with constitutive expression on T cells for the activation, proliferation, and survival of T cells.
  • Tumor-targeted activation of the CD28 costimulatory signal has the potential to enhance specific T cell responses towards neoantigen-presenting tumor cells.
  • therapies targeting CD28 for systemic T cell activation have caused severe cytokine storm and multiorgan failure ( Suntharalingam et al., N Engl J Med. (2006) 355 (10) : 1018-28) . Accordingly, there is a need for improved therapies targeting CD28 to avoid serious autoimmune adverse events associated with the non-targeted CD28 stimulation in systemic T cell activation.
  • the present invention is directed to novel binding molecules targeting B7-H3, HER2, TROP2, and/or CD28, as well as pharmaceutical compositions comprising one or more of these antibodies, and use of the antibodies and pharmaceutical compositions for treating cancer.
  • the binding molecules of the invention may provide a superior clinical response.
  • the present disclosure provides an antigen-binding protein, or an antigen-binding fragment thereof, comprising an CD28 binding portion, wherein the CD28 binding portion binds human CD28 and is cross-reactive with cynomolgus monkey and mouse CD28.
  • the CD28 binding portion binds to a CD28 epitope comprising amino acid residues 51-122 of human CD28 (SEQ ID NO: 1) .
  • 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.
  • the present disclosure provides an antigen-binding protein or fragment thereof comprising a CD28 binding portion that binds human CD28, wherein the CD28 binding portion comprises an antibody heavy chain variable domain (V H ) and an antibody light chain variable domain (V L ) , and wherein the V H and V L comprises heavy chain complementarity-determining regions (CDRs) 1-3 and light chain CDR1-3 set forth in 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,
  • the CD28 binding portion comprises V H and V L set forth in 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:
  • the CD28 binding protein comprises an HC and an LC set forth in 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:
  • the present disclosure also provides an antigen-binding protein or fragment thereof comprising pharmaceutical composition and a pharmaceutically acceptable carrier; a nucleic acid molecule or nucleic acid molecule (s) encoding the antigen-binding protein or fragment thereof; an expression vector or vectors comprising the nucleic acid molecule or nucleic acid molecule (s) ; and a host cell comprising the vector (s) , wherein the host cell may be a prokaryotic cell or an eukaryotic cell such as a mammalian cell.
  • the present disclosure also provides method of producing the antigen-binding protein or fragment thereof of any one of the preceding claims, comprising culturing the host cell under conditions that allow expression of the antigen-binding protein or fragment thereof, and isolating the antigen-binding protein or fragment thereof from the culture.
  • the present disclosure also provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the antigen-binding protein or fragment thereof.
  • the method further comprises administering to the patient another anti-cancer therapeutic.
  • the additional anti-cancer therapeutic is a bispecific antibody targeting CD3 and a tumor antigen, optionally wherein the tumor antigen is the same as or different from the TAA.
  • the TAA is B7-H3, HER2, or TROP2.
  • the additional anti-cancer therapeutics is an immune checkpoint inhibitor, optionally an anti-PD-1, anti-CTLA-4, or anti-PD-L1 antibody.
  • FIG. 1 is a panel of graphs showing a binding affinity assay of anti-CD28 antibodies to recombinant CD28 (human and mouse) protein.
  • FIG. 2 is a graph showing binding of anti-CD28 IgGs to human CD3+ T cells.
  • FIG. 3 is a panel of graphs showing ligand blocking assays of IgGs for human CD28-CD80 (top right and top left graphs) and CTLA-4 CD80 pairs (bottom graph) .
  • FIG. 5B is a graph that shows a T cell activation and proliferation assay for IL-2 release.
  • FIG. 10A is a graph showing a Jurkat-NF ⁇ B Luciferase reporter assay measuring NF ⁇ B signaling stimulatory effects in terms of maximum signal and EC 50 values of bispecific antibodies on H292 cells. Also shown is a table containing plotted values.
  • FIG. 11 is a panel of flow cytometry plots showing the co-expression of PD-L1 and B7H3 on MDA-MB-231 cells.
  • FIGs. 13A and 13B are graphs showing a one-way MLR assay to test the activity of B7H3xCD28 bsAb in combination with anti-PD-1 or anti-PD-L1 blocking mAbs on primary human T cell activation, as measured by IL-2 secretion (FIG. 13A) and IFN- ⁇ secretion (FIG. 13B) .
  • FIG. 14 is a graph showing an in vitro assay that measures tumor killing activity of anti-CD3-based, or anti-CD28-based HER2-targeted bsAbs or their combinations on the MCF-7 tumor cell line. Also shown is a table containing plotted values.
  • FIG. 15 is a graph showing an in vitro assay that measures tumor killing activity of anti-CD3-based, or anti-CD28-based HER2-targeted bsAbs or their combinations on the EMT6-HER2 tumor cell line. Also shown is a table containing plotted values.
  • FIG. 16 is panel of graphs showing an assay to measure systemic cytokine release of IL-6 and IFN- ⁇ (top left and right graphs, respectively) , and to measure CD3+ T cells percentage of total CD45+T cells (bottom graph) , in WT mice treated with TCEs.
  • FIG. 17 shows an in vivo efficacy study and graph of HER2xCD3 bsAb and B7H3xCD28 bsAb mono or in combination in SK-OV3+ PBMC xenograft tumor model.
  • FIG. 18 is a panel of graphs showing an in vivo efficacy study of B7H3xCD28 or HER2xCD28 bsAb in EMT6-HER2 model.
  • FIG. 21 is table showing the different binding residues from human and mouse CD28.
  • a and/or B is intended to include both A and B; A or B; A (alone) ; and B (alone) .
  • the term “and/or” as used herein a phrase such as “A, B, and/or C” is intended to encompass 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) .
  • antibody encompasses various antibody structures, including but not limited to monoclonal antibodies (including full length monoclonal antibodies) , polyclonal antibodies, masked antibodies (e.g., activatable or non-activatable antibodies) , multi-specific antibodies (e.g., bispecific antibodies, including masked bispecific antibodies) , and antibody fragments (e.g., a single-chain variable fragment or scFv) so long as they exhibit the desired biological activity (e.g., the ability to bind a target antigen with desired specificity and affinity) .
  • monoclonal antibodies including full length monoclonal antibodies
  • polyclonal antibodies e.g., masked antibodies (e.g., activatable or non-activatable antibodies)
  • multi-specific antibodies e.g., bispecific antibodies, including masked bispecific antibodies
  • antibody fragments e.g., a single-chain variable fragment or scFv
  • antibody encompasses various antibody structures, including but not limited to monoclonal antibodies (including full length monoclonal antibodies) , polyclonal antibodies, masked antibodies (e.g., activatable or non-activatable antibodies) , and multi-specific antibodies (e.g., bispecific antibodies) .
  • antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • 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, at the N-terminus, a variable region (also known as variable domain) (abbreviated herein as V H ) followed by a constant region.
  • the heavy chain constant region is comprised of three domains, C H 1, C H 2 and C H 3.
  • Each light chain has, at the N-terminus, a variable region (also known as variable domain) (abbreviated herein as V L ) followed by a constant region at its other end.
  • the light chain constant region is comprised of one domain, C L .
  • the V L is aligned with the V H and the C L is aligned with the first constant domain of the heavy chain (C H 1) .
  • the pairing of a V H and V L together forms a single antigen-binding site.
  • the V H and V L can be further subdivided into complementarity-determining regions (CDRs) and framework regions (FRs) .
  • CDRs are of highest sequence variability and/or involved in antigen recognition.
  • CDRs and FRs intersperse in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • CDRs also comprise “specificity determining residues, ” or “SDRs, ” which are residues that contact the antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.
  • Exemplary a-CDRs (a-LCDR1, a-LCDR2, a-LCDR3, a-HCDR1, a-HCDR2, and a-HCDR3) occur at amino acid residues 31-34, 50-55, 89-96 of the light chain, and 31-35, 50-58, and 95-102 of the heavy chain, respectively. See Almagro and Fransson, Front Biosci. (2008) 13: 1619-33) . Unless otherwise indicated, residues in the variable domain are numbered herein according to Kabat et al., J Biol Chem. (1977) 252: 6609-16; Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991) .
  • the L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
  • antibodies can be assigned to different classes or isotypes. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated ⁇ (alpha) , ⁇ (delta) , ⁇ (epsilon) , ⁇ (gamma) , and ⁇ (mu) , respectively.
  • the IgG class of antibody can be further classified into four subclasses IgG 1 , IgG 2 , IgG 3 , and IgG 4 by the gamma heavy chains, Y1-Y4, respectively.
  • antigen-binding fragment or “antigen-binding portion, ” used herein interchangeably, refers to parts of an antibody that retain the ability to bind to the antigen of the antibody.
  • antigen-binding fragments include, but are not limited to, (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H1 domains, obtainable by papain digestion; (ii) a F (ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, obtainable by pepsin digestion; (iii) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (iv) a single chain Fv (scFv) fragment comprising the V H and V L domains of an antibody that are fused to each other; and (v) a single chain Fab (scFab) fragment comprising a single polypeptide
  • a masked antibody exhibits a first binding affinity to a target when in an inactivated state (e.g., inhibited or masked by a masking peptide) , and exhibits a second binding affinity to the target in an activated state (e.g., uninhibited or unmasked by the masking peptide (e.g., the masking peptide is cleaved from the antibody) ) , where the second binding affinity is greater than the first binding affinity.
  • a masked antibody may be generated by linking a masking peptide comprising an activatable component (e.g., a cleavable site within a linkage unit, or “LU” ) to the antigen binding domain of an antibody.
  • the masked antibody, or an masked antigen-binding fragment thereof is a multi-specific antibody comprising a binding domain that is specific for a T-cell surface molecule (e.g., CD28, CD3) and a binding domain that is specific for tumor cell surface antigen (e.g., HER2, B7H3, TROP2, etc. ) .
  • the masked antibody is bivalent and has a single mask on one of the two binding domains.
  • the masked antibody is bivalent and has a mask on each of the two binding domains.
  • one of the binding domains of the antibody is masked by a fused or conjugated masking peptide.
  • one or both binding domains may be masked by specific but different masking peptides.
  • An unactivated bispecific antibody that targets both cancer cells and T cells may have the binding sites of both binding domains masked to inhibit (or minimize) binding to antigen-expressing cancer cells and T cells.
  • the masks are cleaved off to allow binding of the antibody to both the tumor antigen and the T-cell surface molecule (e.g., CD28) in the tumor microenvironment (TME) .
  • the activated bispecific antibody selectively engages T cells to kill target tumor antigen-expressing cancer cells.
  • a “masking peptide” refers to a peptide which inhibits binding of an antigen binding domain to its target antigen, and typically comprises, from N terminus to C terminus, a masking unit (MU) and a linkage unit (LU) .
  • the C terminus of the masking peptide is typically linked to the N terminus of the V H or the V L of the antigen-binding domain.
  • the masking peptide, or a portion thereof interferes with or inhibits binding of the antigen binding domain to its target so efficiently that binding of the antigen-binding domain to its target is extremely low and/or below the limit of detection (e.g., binding cannot be detected in an ELISA or flow cytometry assay) .
  • the masked antibodies or polypeptides described herein may comprise one or more linkers, e.g., within the LU, disposed between MU and LU, LU and V H or V L , or V H and hinge region of an Fc.
  • the LU of the masking peptide may comprise at least one cleavable site.
  • a cleavage site generally includes an amino acid sequence that is cleavable, for example, serves as the substrate for an enzyme and/or a cysteine-cysteine pair capable of forming a reducible disulfide bond.
  • the terms "cleavage, " “cleavable, " “cleaved” and the like are used in connection with a cleavage site, the terms encompass enzymatic cleavage, e.g., by a protease, as well as disruption of a disulfide bond between a cysteine-cysteine pair via reduction of the disulfide bond that can result from exposure to a reducing agent.
  • Masked antibodies or masked polypeptides may comprise a cleavage site configured to mediate activation of the antibody or the polypeptide.
  • the cleavage site of an activatable antibody e.g., uncleaved by a corresponding enzyme, and/or containing an unreduced cysteine-cysteine disulfide bond
  • the masking peptide, or a portion thereof may interfere with or inhibit binding of the antigen binding domain to its target.
  • the LU of the masking peptide does not comprise a cleavable site.
  • masking efficiency refers to the efficiency with which the masking peptide inhibits binding of the antigen binding domain to the target antigen.
  • Masking efficiency may be measured as the difference in or the ratio of the binding affinity of a masked antibody or masked polypeptide comprising an antigen binding domain and the binding affinity of an unmasked antibody or unmasked polypeptide comprising an antigen binding domain (e.g., the masking peptide is cleaved from the antibody) .
  • the masking efficiency may be measured by dividing the EC 50 or K D of a masked antibody for binding a target antigen in its inactivated (e.g., inhibited, masked, and/or uncleaved) state, relative to the EC 50 or K D of the unmasked antibody to bind to the target antigen in its activated (e.g., uninhibited, unmasked, and/or cleaved) state, or relative to EC 50 or K D of the parental antibody (e.g., not linked to a masking peptide) to bind to the target antigen.
  • the EC 50 values may be measured in an ELISA assay, or a Jurkat NFAT reporter assay, for example, as described in U.S. Pat. App. Pub. No. US2021/0207126 A1.
  • the K D values may be measured by, for example, using surface plasmon resonance.
  • epitope refers to a part of an antigen to which an antibody (or antigen-binding fragment thereof) binds.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope can include various numbers of amino acids in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance, deuterium and hydrogen exchange in combination with mass spectrometry, or site-directed mutagenesis, or all methods used in combination with computational modeling of antigen and its complex structure with its binding antibody and its variants (see e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996) ) .
  • antibodies to that epitope can be generated, e.g., using the techniques described herein. The generation and characterization of antibodies may also elucidate information about desirable epitopes.
  • germline refers to the nucleotide sequences of the antibody genes and gene segments as they are passed from parents to offspring via the germ cells.
  • the germline sequence is distinguished from the nucleotide sequences encoding antibodies in mature B cells which have been altered by recombination and hypermutation events during the course of B cell maturation.
  • glycosylation sites refers to amino acid residues which are recognized by a eukaryotic cell as locations for the attachment of sugar residues.
  • the amino acids where carbohydrate, such as oligosaccharide, is attached are typically asparagine (N-linkage) , serine (O-linkage) , and threonine (O-linkage) residues.
  • the specific site of attachment is typically signaled by a sequence of amino acids, referred to herein as a “glycosylation site sequence” .
  • the glycosylation site sequence for N-linked glycosylation is: -Asn-X-Ser-or -Asn-X-Thr-, where X may be any of the conventional amino acids, other than proline.
  • host cell refers to a cellular system which can be engineered to generate proteins, protein fragments, or peptides of interest.
  • Host cells include, without limitation, cultured cells, e.g., mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; human cells (e.g., HEK293F cells, HEK293T cells; or human tissues or hybridoma cells, yeast cells, insect cells (e.g., S2 cells) , bacterial cells (e.g., E. coli cells) and cells comprised within a transgenic animal or cultured tissue.
  • cultured cells e.g., mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2/0, YB2/0
  • the term encompasses not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell. ”
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • humanized antibody refers to a chimeric antibody that contains amino acid residues derived from human antibody sequences.
  • a humanized antibody may contain some or all of the CDRs from a non-human animal or synthetic antibody while the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences.
  • exemplary antibody refers to any one of the antibodies described herein. These antibodies may be in any class (e.g., IgA, IgD, IgE, IgG, and IgM) . Thus, each antibody identified above encompasses antibodies in all five classes that have the same amino acid sequences for the V L and V H regions. Further, the antibodies in the IgG class 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 that have the same amino acid sequences for the V L and V H regions. The amino acid sequences of the heavy chain constant regions of human antibodies in the five classes, as well as in the four IgG subclasses, are known in the art.
  • an “isolated” antibody or binding molecule is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95%or 99%purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF) , capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) .
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF) , capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • K D refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. It is obtained from the ratio of k d to k a (i.e., k d /k a ) and is expressed as a molar concentration (M) . K D is used as a measure for the affinity of an antibody’s binding to its binding partner. The smaller the K D , the more tightly bound the antibody is, or the higher the affinity between antibody and the antigen. For example, an antibody with a nanomolar (nM) dissociation constant binds more tightly to a particular antigen than an antibody with a micromolar ( ⁇ M) dissociation constant. K D values for antibodies can be determined using methods well established in the art. One method for determining the K D of an antibody is by using an ELISA. For example, an assay procedure using an ELISA.
  • mammal refers to any animal species of the Mammalia class. 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, cattle, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, elephants, and the like.
  • prevent or “preventing, ” with reference to a certain disease condition in a mammal, refers to preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms thereof.
  • sequence identity between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences.
  • the amino acid sequence identity of polypeptides can be determined conventionally 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., Nucleic Acids Res. (1997) 25: 3389-3402) .
  • the parameters are set such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5%of the total number of amino acid residues in the reference sequence are allowed.
  • This aforementioned method in determining the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants thereof disclosed herein.
  • the term “binds, ” “binds to, ” “specifically binds” “specifically binds to” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
  • an antibody that binds to or specifically binds to a target is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets.
  • the extent of binding of an antibody to an unrelated target is less than about 10%of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA) .
  • an antibody that specifically binds to a target has a dissociation constant (Kd) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
  • Kd dissociation constant
  • an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species.
  • specific binding can include, but does not require exclusive binding.
  • treat, ” “treating, ” or “treatment, ” with reference to a certain disease condition in a mammal refers causing a desirable or beneficial effect in the mammal having the disease condition.
  • the desirable or beneficial effect may include reduced frequency or severity of one or more symptoms of the disease (i.e., tumor growth and/or metastasis, or other effect mediated by the numbers and/or activity of immune cells, and the like) , or arrest or inhibition of further development of the disease, condition, or disorder.
  • the desirable or beneficial effect may include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of reoccurrence of cancer, reduction of pain associated with the cancer, or improved survival of the mammal.
  • vector refers to a nucleic acid molecule capable of transporting a foreign nucleic acid molecule.
  • the foreign nucleic acid molecule is linked to the vector nucleic acid molecule by a recombinant technique, such as ligation or recombination. This allows the foreign nucleic acid molecule to be multiplied, selected, further manipulated or expressed in a host cell or organism.
  • a vector can be a plasmid, phage, transposon, cosmid, chromosome, virus, or virion.
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors) .
  • Another type of vector is capable of autonomous replication in a host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) .
  • Another specific type of vector capable of directing the expression of expressible foreign nucleic acids to which they are operatively linked is commonly referred to as “expression vectors. ”
  • Expression vectors generally have control sequences that drive expression of the expressible foreign nucleic acids.
  • vectors Simpler vectors, known as “transcription vectors, ” are only capable of being transcribed but not translated: they can be replicated in a target cell but not expressed.
  • 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 operatively linked are commonly referred to “expression vectors. ” Other examples of “vectors” may include display vectors (e.g., vectors that direct expression and display of an encoded polypeptide on the surface of a virus or cell (such as a bacterial cell, yeast cell, insect cell, and/or mammalian cell) .
  • a “subject” , “patient” , or “individual” may refer to a human or a non-human animal.
  • a “non-human animal” may refer to any animal not classified as a human, such as domestic, farm, or zoo animals, sports, pet animals (such as dogs, horses, cats, cows, etc. ) , as well as animals used in research.
  • an “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve one or more desired or indicated effects, including a therapeutic or prophylactic result.
  • An effective amount can be provided in one or more administrations.
  • an effective amount of antibody, drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
  • an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition (e.g., an effective amount as administered as a monotherapy or combination therapy) .
  • an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
  • monospecific antibodies e.g., traditional, non-masked monospecific antibodies
  • multi-specific antibodies e.g., non-masked multi-specific antibodies
  • masked antibodies e.g., activatable monospecific or multi-specific antibodies
  • antigen-binding fragments thereof or derivatives of such antibodies.
  • the antibody (e.g., multi-specific antibody) described herein comprises one or more antibody constant regions, such as human heavy chain constant regions and/or human light chain constant regions.
  • the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD.
  • the human light chain constant region is of an isotype selected from ⁇ and ⁇ .
  • the antibody comprises a human IgG constant region.
  • the antibody comprises a human IgG 4 heavy chain constant region.
  • the antibody comprises a human IgG 1 heavy chain constant region.
  • the antibody comprises an S228P mutation in the human IgG 4 constant region.
  • effector function is desirable may depend on the particular method of treatment intended for an antibody.
  • an antibody comprising a human IgG 1 heavy chain constant region or a human IgG 3 heavy chain constant region is selected.
  • an antibody comprising a human IgG 4 or IgG 2 heavy chain constant region is selected.
  • the antibody comprises a human IgG 1 heavy chain constant region comprising one or more mutations that reduces effector function.
  • the antibody comprises an IgG 1 heavy chain constant region comprising an N297A substitution.
  • the amino acid mutations or substitutions described herein are relative to a wildtype C H 3 domain sequence of an IgG1, 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 a wildtype C H 3 domain sequence of an IgG 4 .
  • a D356K substitution relative to a wildtype C H 3 domain of one human IgG 1 allotype is equivalent to an E356K substitution relative to a wildtype C H 3 domain of a second human IgG 1 allotype, or a wildtype C H 3 domain of a human IgG 4 .
  • Exemplary C H 3 domain mutations are shown in Tables 2 and 3.
  • the amino acid mutations or substitutions described herein are relative to a wildtype Fc region sequence, e.g., an IgG 1 Fc region or an IgG 4 Fc region.
  • the multi-specific antibody (e.g., the activatable multi-specific antibody) comprises an engineered disulfide bond between C390 in a first C H 3 domain and C400 in a second C H 3 domain, between C392 in a first C H 3 domain and C397 in a second C H 3 domain, or between C392 in a first C H 3 domain and C400 in a second C H 3 domain.
  • the multi-specific antibody (e.g., the activatable multi-specific antibody) comprises a rearranged salt-bridge network as compared to wildtype C H 3 domains, e.g., among positions 357 and 411 in a first C H 3 domain and positions 351 and 370 in a second C H 3 domain (e.g., E357K: T411K-L351’D: K370’D) , or among positions 357 and 364 in a first C H 3 domain and positions 351 and 370 in a second C H 3 domain (e.g., E357K: S364K-L351’D: K370’D) .
  • wildtype C H 3 domains e.g., among positions 357 and 411 in a first C H 3 domain and positions 351 and 370 in a second C H 3 domain (e.g., E357K: T411K-L351’D: K370’D) , or among positions 357 and 364 in a first C
  • the multi-specific antibody (e.g., the activatable multi-specific antibody) comprises C H 3 domains having one or more engineered residues, which promote heterodimer formation as described herein.
  • Heteromultimers comprising multiple heterodimers formed by a first polypeptide comprising a first engineered C H 3 domain and a second polypeptide comprising a second engineered C H 3 domain are also contemplated herein.
  • the multi-specific antibody (e.g., the activatable multi-specific antibody) comprises: a first polypeptide comprising a first C H 3 domain and a second polypeptide comprising a second C H 3 domain, wherein: i) the first C H 3 domain further comprises a positively charged residue at position 357 and the second C H 3 domain further comprises a negatively charged residue at position 351, or the first C H 3 domain further comprises a negatively charged residue at position 351 and the second C H 3 domain further comprises a positively charged residue at position 357; or ii) the first C H 3 domain further comprises a positively charged residue at position 411 and the second C H 3 domain further comprises a negatively charged residue at position 370, or the first C H 3 domain further comprises a negatively charged residue at position 370 and the second C H 3 domain further comprises a positively charged residue at position 411; or iii) the first C H 3 domain further comprises a positively charged residue at position 364 and the second C H 3 domain further comprises a negatively charged residue at position
  • the first C H 3 domain further comprises a positively charged residue at position 356 and the second C H 3 domain further comprises a negatively charged residue at position 439, or the first C H 3 domain further comprises a negatively charged residue at position 439 and the second C H 3 domain further comprises a positively charged residue at position 356, and wherein the amino acid residue numbering is based on Eu numbering.
  • the multi-specific antibody (e.g., the activatable multi-specific antibody) comprises: a first polypeptide comprising a first C H 3 domain and a second polypeptide comprising a second C H 3 domain, wherein: i) the first C H 3 domain comprises a cysteine (C) residue at position 390 and the second C H 3 domain comprises a cysteine residue at position 400, or the first C H 3 domain comprises 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 3 domain comprises a cysteine residue at position 392 and the second C H 3 domain comprises a cysteine residue at position 397, or the first C H 3 domain comprises 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 3 domain comprises a cysteine residue at position 392 and the second C H 3 domain comprises a cysteine residue at position at position 3
  • first C H 3 domain further comprises a positively charged residue at position 356 and the second C H 3 domain further comprises a negatively charged residue at position 439, or first C H 3 domain further comprises a negatively charged residue at position 439 and the second C H 3 domain further comprises a positively charged residue at position 356, and wherein the amino acid residue numbering is based on Eu numbering.
  • the multi-specific antibody (e.g., the activatable multi-specific antibody) comprises: a first polypeptide comprising a first C H 3 domain and a second polypeptide comprising a second C H 3 domain, wherein: i) the first C H 3 domain comprises N390C substitution and the second C H 3 domain comprises S400C substitution, or the first C H 3 domain comprises S400C substitution and the second C H 3 domain comprises N390C substitution; or ii) the first C H 3 domain comprises K392C substitution and the second C H 3 domain comprises V397C substitution, or the first C H 3 domain comprises V397C substitution and the second C H 3 domain comprises K392C substitution; or iii) the first C H 3 domain comprises K392C substitution and the second C H 3 domain comprises S400C substitution, or the first C H 3 domain comprises S400C substitution and the second C H 3 domain comprises K392C substitution.
  • the multi-specific antibody (e.g., the activatable multi-specific antibody) comprises: a first polypeptide comprising a first C H 3 domain and a second polypeptide comprising a second C H 3 domain, wherein: i) the first C H 3 domain comprises E357K and T411K substitutions and the second C H 3 domain comprises L351D and K370D substitutions, or the first C H 3 domain comprises L351D and K370D substitutions and the second C H 3 domain comprises E357K and T411K substitutions; or ii) the first C H 3 domain comprises E357K and S364K substitutions and the second C H 3 domain comprises L351D and K370D substitutions, or the first C H 3 domain comprises L351D and K370D substitutions and the second C H 3 domain comprises E357K and S364K substitutions; or iii) the first C H 3 domain comprises D356K, E357K, and S364K substitutions and the second C H
  • the multi-specific antibody (e.g., the activatable multi-specific antibody) comprises: a first polypeptide comprising a first C H 3 domain and a second polypeptide comprising a second C H 3 domain, wherein the first C H 3 domain comprises E357K, S364K, and N390C substitutions and the second C H 3 domain comprises L351D, K370D, and S400C substitutions, or the first C H 3 domain comprises L351D, K370D, and S400C substitutions and the second C H 3 domain comprises E357K, S364K, and N390C substitutions.
  • the multi-specific antibody (e.g., the activatable multi-specific antibody) comprises: a first polypeptide comprising a first C H 3 domain and a second polypeptide comprising a second C H 3 domain, wherein the first C H 3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second C H 3 domain comprises L351D, K370D, K439D, and S400C substitutions, or the first C H 3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second C H 3 domain comprises D356K, E357K, S364K, and N390C substitutions.
  • the multi-specific antibody (e.g., the activatable multi-specific antibody) comprises an IgG Fc region that comprises the engineered C H 3 domains.
  • the Fc region may be derived from any suitable Fc subclasses, including, but not limited to, IgG 1 , IgG 2 , IgG 3 , and IgG 4 subclasses.
  • the multi-specific antibodies described herein comprise a first polypeptide comprising a first C H 3 domain and a second polypeptide comprising a second C H 3 domain, wherein the first C H 3 domain comprises a first engineered cysteine residue and the second C H 3 domain comprises a second engineered cysteine residue, wherein the first engineered cysteine residue and the second cysteine residue form a disulfide bond.
  • the first C H 3 domain comprises a C at position 390 and the second C H 3 domain comprises a C at position 400, or the first C H 3 domain comprises a C at position 400 and the second C H 3 domain comprises a C at position 390.
  • the first C H 3 domain comprises N390C substitution and the second C H 3 domain comprises S400C substitution, or the first C H 3 domain comprises S400C substitution and the second C H 3 domain comprises N390C substitution.
  • the first C H 3 domain comprises a C at position 392 and the second C H 3 domain comprises a C at position 397, or the first C H 3 domain comprises a C at position 397 and the second C H 3 domain comprises a C at position 392.
  • the first C H 3 domain comprises K392C substitution and the second C H 3 domain comprises V397C substitution, or the first C H 3 domain comprises V397C substitution and the second C H 3 domain comprises K392C substitution.
  • the multi-specific antibodies described herein comprise a first polypeptide comprising a first C H 3 domain and a second polypeptide comprising a second C H 3 domain, wherein the first C H 3 domain comprises an engineered positively charged residue and the second C H 3 domain comprises an engineered negatively charged residue, wherein the engineered positively charged residue and the engineered negatively charged residue form a salt bridge.
  • the engineered salt bridge may introduce new salt bridges between the C H 3 domains, rearrange a salt-bridge network among two or more amino acid residues, or reverse the charges on the amino acid residues forming the salt bridge (i.e., “inverse” a salt bridge) with respect to wildtype C H 3 domains.
  • the engineered positively charged residue substitutes a negatively charged residue in a wildtype C H 3 domain with a positively charged residue. In some embodiments, the engineered negatively charged residue substitutes a positively charged residue in a wildtype C H 3 domain with a negatively charged residue.
  • the rearranged and inversed salt bridges may result in changes in the isoelectric points (PI) of the heterodimer and the homodimer comprising the engineered C H 3 domains, thereby allowing better separation of the heterodimer from the homodimer in a purification process.
  • the first C H 3 domain comprises a positively charged residue at position 357 and the second C H 3 domain comprises a negatively charged residue at position 351, or the first C H 3 domain comprises a negatively charged residue at position 351 and the second C H 3 domain comprises a positively charged residue at position 357.
  • the first C H 3 domain comprises a K at position 357 and the second C H 3 domain comprises a D at position 351, or the first C H 3 domain comprises a D at position 351 and the second C H 3 domain comprises a K at position 357.
  • the first C H 3 domain comprises a K at position 357 and the second C H 3 domain comprises an E at position 351, or the first C H 3 domain comprises an E at position 351 and the second C H 3 domain comprises a K at position 357.
  • the first C H 3 domain comprises an R at position 357 and the second C H 3 domain comprises a D at position 351, or the first C H 3 domain comprises a D at position 351 and the second C H 3 domain comprises an R at position 357.
  • the first C H 3 domain comprises an R at position 357 and the second C H 3 domain comprises an E at position 351, or the first C H 3 domain comprises an E at position 351 and the second C H 3 domain comprises an R at position 357.
  • the first C H 3 domain comprises E357K substitution and the second C H 3 domain comprises L351D substitution, or the first C H 3 domain comprises L351D substitution and the second C H 3 domain comprises E357K substitution.
  • the first C H 3 domain comprises a positively charged residue at position 411 and the second C H 3 domain comprises a negatively charged residue at position 370, or the first C H 3 domain comprises a negatively charged residue at position 370 and the second C H 3 domain comprises a positively charged residue at position 411.
  • the first C H 3 domain comprises a K at position 411 and the second C H 3 domain comprises a D at position 370, or the first C H 3 domain comprises a D at position 370 and the second C H 3 domain comprises a K at position 411.
  • the first C H 3 domain comprises a K at position 411 and the second C H 3 domain comprises an E at position 370, or the first C H 3 domain comprises an E at position 370 and the second C H 3 domain comprises a K at position 411.
  • the first C H 3 domain comprises an R at position 411 and the second C H 3 domain comprises a D at position 370, or the first C H 3 domain comprises a D at position 370 and the second C H 3 domain comprises an R at position 411.
  • the first C H 3 domain comprises an R at position 411 and the second C H 3 domain comprises an E at position 370, or the first C H 3 domain comprises an E at position 370 and the second C H 3 domain comprises an R at position 411.
  • the first C H 3 domain comprises T411K substitution and the second C H 3 domain comprises K370D substitution, or the first C H 3 domain comprises K370D substitution and the second C H 3 domain comprises T411K substitution.
  • the first C H 3 domain comprises a K at position 364 and the second C H 3 domain comprises an E at position 370, or the first C H 3 domain comprises an E at position 370 and the second C H 3 domain comprises a K at position 364.
  • the first C H 3 domain comprises an R at position 364 and the second C H 3 domain comprises a D at position 370, or the first C H 3 domain comprises a D at position 370 and the second C H 3 domain comprises an R at position 364.
  • the first C H 3 domain comprises an R at position 364 and the second C H 3 domain comprises an E at position 370, or the first C H 3 domain comprises an E at position 370 and the second C H 3 domain comprises an R at position 364.
  • the first C H 3 domain comprises S364K substitution and the second C H 3 domain comprises K370D substitution, or the first C H 3 domain comprises K370D substitution and the second C H 3 domain comprises S364K substitution.
  • the first C H 3 domain comprises a positively charged residue at position 356 and the second C H 3 domain comprises a negatively charged residue at position 439, or the first C H 3 domain comprises a negatively charged residue at position 439 and the second C H 3 domain comprises a positively charged residue at position 356.
  • the first C H 3 domain comprises a K at position 356 and the second C H 3 domain comprises a D at position 439, or the first C H 3 domain comprises a D at position 439 and the second C H 3 domain comprises a K at position 356.
  • the first C H 3 domain comprises a K at position 356 and the second C H 3 domain comprises an E at position 439, or the first C H 3 domain comprises an E at position 439 and the second C H 3 domain comprises a K at position 356.
  • the first C H 3 domain comprises an R at position 356 and the second C H 3 domain comprises a D at position 439, or the first C H 3 domain comprises a D at position 439 and the second C H 3 domain comprises an R at position 356.
  • the first C H 3 domain comprises an R at position 356 and the second C H 3 domain comprises an E at position 439, or the first C H 3 domain comprises an E at position 439 and the second C H 3 domain comprises an R at position 356.
  • the first C H 3 domain comprises D356K substitution and the second C H 3 domain comprises K439D substitution, or the first C H 3 domain comprises K439D substitution and the second C H 3 domain comprises D356K substitution.
  • the first C H 3 domain comprises a positively charged residue at position 357 and a positively charged residue at position 411
  • the second C H 3 domain comprises a negatively charged residue at position 351 and a negatively charged residue at position 370
  • the first C H 3 domain comprises a negatively charged residue at position 351 and a negatively charged residue at position 370
  • the second C H 3 domain comprises a positively charged residue at position 357 and a positively charged residue at position 411.
  • the first C H 3 domain comprises E357K and T411K substitutions
  • the second C H 3 domain comprises L351D and K370D substitutions
  • the first C H 3 domain comprises L351D and K370D substitutions
  • the second C H 3 domain comprises E357K and T411K substitutions.
  • the first C H 3 domain comprises a positively charged residue at position 357 and a positively charged residue at position 364, and the second C H 3 domain comprises a negatively charged residue at position 351 and a negatively charged residue at position 370, or the first C H 3 domain comprises a negatively charged residue at position 351 and a negatively charged residue at position 370, and the second C H 3 domain comprises a positively charged residue at position 357 and a positively charged residue at position 364.
  • the first C H 3 domain comprises E357K and S364K substitutions
  • the second C H 3 domain comprises L351D and K370D substitutions
  • the first C H 3 domain comprises L351D and K370D substitutions
  • the second C H 3 domain comprises E357K and S364K substitutions.
  • the first C H 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 and the second C H 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 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 and the second C H 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.
  • the C H 3 domains or the Fc regions described herein may further comprise engineered disulfide bonds and/or salt bridges listed in Table 4 below.
  • the first C H 3 domain further comprises a C at position 392 and the second C H 3 domain comprises a C at position 399, or the first C H 3 domain comprises a C at position 399 and the second C H 3 domain comprises a C at position 392.
  • the first C H 3 domain further comprises K392C substitution and the second C H 3 domain further comprises D399C substitution, or the first C H 3 domain further comprises D399C substitution and the second C H 3 domain further comprises K392C substitution.
  • the first C H 3 domain further comprises a C at position 394 and the second C H 3 domain comprises a C at position 354, or the first C H 3 domain comprises a C at position 354 and the second C H 3 domain comprises a C at position 394.
  • the first C H 3 domain further comprises Y394C substitution and the second C H 3 domain further comprises S354C substitution, or the first C H 3 domain further comprises S354C substitution and the second C H 3 domain further comprises Y394C substitution.
  • the first C H 3 domain further comprises a C at position 356 and the second C H 3 domain comprises a C at position 349, or the first C H 3 domain comprises a C at position 349 and the second C H 3 domain comprises a C at position 356.
  • the first C H 3 domain further comprises D356C substitution and the second C H 3 domain further comprises Y349C substitution, or the first C H 3 domain further comprises Y349C substitution and the second C H 3 domain further comprises D356C substitution.
  • the first C H 3 domain further comprises K392D and K409D substitutions and the second C H 3 domain further comprises D356K and D399K substitutions, or the first C H 3 domain further comprises D356K and D399K substitutions and the second C H 3 domain further comprises K392D and K409D substitutions.
  • the first C H 3 domain further comprises L368D and K370S substitutions and the second C H 3 domain further comprises E357Q and S364K substitutions, or the first C H 3 domain further comprises E357Q and S364K substitutions and the second C H 3 domain further comprises L368D and K370S substitutions.
  • the first C H 3 domain further comprises L351K and T366K substitutions and the second C H 3 domain further comprises L351D and L368E substitutions, or the first C H 3 domain further comprises L351D and L368E substitutions and the second C H 3 domain further comprises L351K and T366K substitutions.
  • the first C H 3 domain further comprises P395K, P396K, and V397K substitutions and the second C H 3 domain comprises T394D, P395D, and P396D substitutions, or the first C H 3 domain further comprises T394D, P395D, and P396D substitutions and the second C H 3 domain further comprises P395K, P396K, and V397K substitutions.
  • the first C H 3 domain further comprises F405E, Y407E, and K409E substitutions and the second C H 3 domain comprises F405K and Y407K substitutions, or the first C H 3 domain further comprises F405K and Y407K substitutions and the second C H 3 domain further comprises F405E, Y407E and K409E substitutions.
  • the first C H 3 domain further comprises T336S, L368A, and Y407V substitutions and the second C H 3 domain further comprises T366W substitution, or the first C H 3 domain further comprises T366W substitution and the second C H 3 domain further comprises T336S, L368A, and Y407V substitutions.
  • the first C H 3 domain comprises L368V and Y407V substitutions and the second C H 3 domain comprises T366W substitution, or the first C H 3 domain comprises T366W substitution and the second C H 3 domain comprises L368V and Y407V substitutions.
  • the present disclosure provides isolated binding molecules that bind to human CD28, including anti-CD28 antibodies and anti-CD28 antigen-binding fragments thereof.
  • the binding molecules include antibodies described with reference to epitope binding and antibodies described with reference to specific amino acid sequences of complementarity determining regions (CDR) , variable regions (V L , V H ) , and IgG (e.g., IgG 4 ) light and heavy chains.
  • CDR complementarity determining regions
  • V L , V H variable regions
  • IgG e.g., IgG 4
  • the antibodies or the antigen-binding fragments thereof bind to one or more amino acid residues within amino acid residues 34-108 of SEQ ID NO: 1. In some embodiments, the antibodies or antigen-binding fragments bind to one or more amino acid residues within amino acid residues 51-122 of SEQ ID NO: 1. In some embodiments, the antibodies or antigen-binding fragments bind 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 of measuring an antibody or antigen-binding fragment's ability to bind a target antigen may be carried out using any method known in the art, including for example, by surface plasmon resonance, an ELISA, isothermal titration calorimetry, a filter binding assay, an EMSA, etc.
  • the ability of the antibody or antigen-binding fragment to bind a target antigen is measured by ELISA or RED96 (see, e.g., Example 3 below) .
  • the antibodies or antigen-binding fragments bind to human CD28 with a K D 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.
  • a K D 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 n
  • the antibodies or antigen-binding fragments bind to human CD28 with a K D of about 100 nM or less. In some embodiments, the antibodies or antigen-binding fragments bind to human CD28 with a K D of about 50 nM or less. Methods of measuring the K D of an antibody or antigen-binding fragment may be carried out using any method known in the art, including for example, by surface plasmon resonance, an ELISA, isothermal titration calorimetry, a filter binding assay, an EMSA, etc. In some embodiments, the K D is measured by RED96 Systems (See, e.g., Example 3 below) .
  • the present disclosure provides an isolated monoclonal antibody that binds to human CD28 at an epitope within amino acid residues 33-37, 80-83, 92-96, and 100-104 of SEQ ID NO: 1.
  • the present 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.
  • the antibody in some embodiments, binds human CD28 with a K D of 10 nM or less as measured by RED96 Systems.
  • the antibody disclosed herein in addition to binding human epitopes, is cross-reactive (exhibits cross-species binding features) with at least one non-human species selected from the list consisting of cynomolgus monkey, mouse, rat and dog.
  • the antibody disclosed herein has the advantage of cross-species binding to mouse, humans and monkeys, whereas the benchmark controls TAC2386 and TAC2387 disclosed herein do not have this range of species cross-reactivity.
  • the benchmark controls TAC2386 and TAC2387 bind human epitopes but not mouse epitopes (see Table 7 herein) .
  • the species cross reactivity of the antibody disclosed herein also provides the added advantage of being able to use a mouse to model the antibody’s safety, activity, and function. Hence, compared to TAC2386 and TAC2387 disclosed herein, it is easier to do animal modeling with the antibody disclosed herein.
  • the isolated anti-CD28 monoclonal antibody comprises a HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 6, and HCDR3 of SEQ ID NO: 7, and a LCDR1 of SEQ ID NO: 8, a LCDR2 of SEQ ID NO: 9 and a LCDR3 of SEQ ID NO: 10.
  • the isolated monoclonal antibody comprises heavy chain variable region of a SEQ ID NO: 11 and light chain variable region of SEQ ID NO: 12.
  • the isolated monoclonal antibody comprises a heavy chain of SEQ ID NO: 13 and light chain of SEQ ID NO: 14.
  • An exemplary method for producing an antibody in a desired class or subclass comprises the steps of isolating a nucleic acid encoding a heavy chain of an anti-CD28 antibody and a nucleic acid encoding a light chain of a CD28 antibody, isolating the sequence encoding the V H region, ligating the V H sequence to a sequence encoding a heavy chain constant region of the desired class or subclass, expressing the light chain gene and the heavy chain construct in a cell, and collecting the CD28 antibody.
  • the anti-CD28 antibodies described herein can be in any class, such as IgG, IgM, IgE, IgA, or IgD. It is preferred that the anti-CD28 antibodies are in the IgG class, such as IgG 1 , IgG 2 , IgG 3 , or IgG 4 subclass.
  • An anti-CD28 antibody 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 in a desired class or subclass comprises the steps of isolating a nucleic acid encoding a heavy chain of an anti-CD28 antibody and a nucleic acid encoding a light chain of an anti-CD28 antibody, isolating the sequence encoding the V H region, ligating the V H sequence to a sequence encoding a heavy chain constant region of the desired class or subclass, expressing the light chain gene and the heavy chain construct in a cell, and collecting the CD28 antibody.
  • Antibodies of the present disclosure can be produced by techniques known in the art, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique (see e.g., Kohler and Milstein, Nature (1975) 256: 495) , viral or oncogenic transformation of B lymphocytes, or recombinant antibody technologies as described in detail herein below.
  • conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique (see e.g., Kohler and Milstein, Nature (1975) 256: 495) , viral or oncogenic transformation of B lymphocytes, or recombinant antibody technologies as described in detail herein below.
  • Hybridoma production is a very well-established procedure.
  • the common animal system for preparing hybridomas is the murine system. Immunization protocols and techniques for isolation of immunized splenocytes 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 may be used for making human CD28 antibodies provided by the present disclosure involves the use of a XenoMouse TM animal system.
  • XenoMouse TM mice are engineered mouse strains that comprise large fragments of human immunoglobulin heavy chain and light chain loci and are deficient in mouse antibody production.
  • the animal is immunized with an CD28 antigen.
  • the CD28 antigen is isolated and/or purified CD28, preferably CD28. It may be a fragment of CD28, such as the extracellular domain of CD28, particularly a CD28 extracellular domain fragment comprising amino acid resides 33, 34, 36 and 37, 80-83, 92 and 93, 95 and 96, and 100-104 of SEQ ID NO: 1.
  • Immunization of animals can be carried out by any method known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990.
  • the CD28 antigen may be administered with an adjuvant to stimulate the immune response.
  • adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes) .
  • lymph node and/or splenic B cells are immortalized.
  • Methods of immortalizing cells include, but are not limited to, transferring them with oncogenes, inflecting them with the oncogenic virus cultivating them under conditions that select for immortalized cells, subjecting them to carcinogenic or mutating compounds, fusing them with an immortalized cell, e.g., a myeloma cell, and inactivating a tumor suppressor gene. See, e.g., Harlow and Lane, supra. If fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin polypeptides (a non-secretory cell line) .
  • Antibodies of the disclosure can also be prepared using phage display or yeast display methods.
  • 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.
  • the present disclosure provides antigen-binding fragments of any of the CD28 antibodies provided by the present disclosure.
  • the antigen-binding fragment may comprise any sequences of the antibody.
  • the antigen-binding fragment comprises the amino acid sequence of: (1) a light chain of an anti-CD28 antibody; (2) a heavy chain of a CD28 antibody; (3) a variable region from the light chain of an anti-CD28 antibody; (4) a variable region from the heavy chain of a CD28 antibody; (5) one or more CDRs (two, three, four, five, or six CDRs) of an anti-CD28 antibody; or (6) three CDRs from the light chain and three CDRs from the heavy chain of an anti-CD28 antibody.
  • the antigen-binding fragments of an anti-CD28 antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the V L , V H , C L and C H 1 domains; (ii) a F (ab′) 2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H 1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., Nature (1989) 341: 544-546) , which consists of a V H domain; (vi) an isolated CDR, and (vii) single chain antibody (scFv) , which is a polypeptide comprising a V L region of an antibody linked to a V H region of an antibody. Bird et al.,
  • the anti-CD28 antibody or antibody fragment disclosed herein comprises a V H 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 an amino acid sequence as set forth in any 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.
  • the anti-CD28 antibody or antibody fragment disclosed herein comprises a V L 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 an amino acid sequence as set forth in any 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.
  • the anti-CD28 antibody or antibody fragment disclosed herein comprises an HCDR1 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to an amino acid sequence as set forth in any of SEQ ID NOs: 5, 15, 24, 33, 43, 66, 88, 103, 111, 126, 134, 148, and 161.
  • the anti-CD28 antibody or antibody fragment disclosed herein comprises an HCDR2 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to an amino acid sequence as set forth in any of SEQ ID NOs: 6, 51, 58, 67, 89, 96, 104, and 155.
  • the anti-CD28 antibody or antibody fragment disclosed herein comprises an HCDR3 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to an amino acid sequence as set forth in any of SEQ ID NOs: 7, 16, 25, 35, 44, 52, 59, 81, 90, 97, 105, 112, 119, 127, 135, 141, 149, and 162.
  • the anti-CD28 antibody or antibody fragment disclosed herein comprises an LCDR1 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to an amino acid sequence as set forth in any 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.
  • the anti-CD28 antibody or antibody fragment disclosed herein comprises an LCDR2 amino acid sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to an amino acid sequence as set forth in any of SEQ ID NOs: 9, 18, 27, 37, and 300.
  • the antibody disclosed herein comprises 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 an amino acid sequence as set forth in any of SEQ ID NOs: 14, 23, 32, 42, 50, 57, 65, 74, 80, 87, 95, 102, 110, 118, 125, 142, 133, 140, 147, 154, 160, and 168.
  • the multi-specific antibody is bispecific (bsAb) . In some embodiments, the multi-specific antibody is trispecific (tsAb) .
  • the multi-specific antibody binds to CD28 on the surface of T cells.
  • the multi-specific antibody is a tumor-associated antigen (TAA) xCD28 bispecific antibody that specifically binds to the TAA and CD28.
  • TAA tumor-associated antigen
  • the multi-specific antibody does not comprise any masking moiety or cleavable moiety.
  • the multi-specific antibody is obtained upon cleavage of the cleavable moiety or cleavable moieties.
  • the multi-specific antibody binds to CD3 on the surface of T cells.
  • the multi-specific antibody is a tumor-associated antigen (TAA) xCD3 bispecific antibody that specifically binds to the TAA and CD3.
  • TAA tumor-associated antigen
  • the multi-specific antibody specifically binds CD3 with a weak affinity, e.g., an EC 50 of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay, and/or a Kd of at least 50 nM.
  • the multi-specific antibody does not comprise any masking moiety or cleavable moiety.
  • the multi-specific antibody is obtained upon cleavage of the cleavable moiety or cleavable moieties.
  • a multi-specific antibody comprising: a) a first antigen-binding fragment comprising a VH1 and a V L 1 of an antibody that specifically binds a target antigen (e.g., a tumor antigen, such as B7-H3, HER2, or TROP2) ; and b) a second antigen-binding fragment comprising a VH2 and a V L 2 of an anti-CD3 antibody that specifically binds CD3, wherein the first and/or second antigen-binding fragment is fused to a first and/or second masking peptide (MP1/MP2) .
  • a target antigen e.g., a tumor antigen, such as B7-H3, HER2, or TROP2
  • a target antigen e.g., a tumor antigen, such as B7-H3, HER2, or TROP2
  • a target antigen e.g., a tumor antigen, such as B7-H3, HER2, or TROP2
  • the first antigen-binding fragment is selected from the group consisting of a Fab, a Fv, a scFab and a scFv. In some embodiments, the first antigen-binding fragment is a Fab. In some embodiments, the second antigen-binding fragment is selected from the group consisting of a Fab, a Fv, a scFab and a scFv. In some embodiments, the second antigen-binding fragment is a scFv comprising, from N-terminus to C-terminus, V L 2, an optional linker, and VH2. In some embodiments, the first antigen-binding fragment is a Fab and the second antigen-binding fragment is a Fab. In some embodiments, the first antigen-binding fragment is a Fab and the second antigen-binding fragment is a scFv.
  • a bispecific antibody targeting CD28 and a tumor antigen comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • V L 1-C L V L 1-C L ;
  • V L 1 is a first immunoglobulin light chain variable domain
  • V H 1 is a first immunoglobulin heavy chain variable domain
  • scFv is a single-chain variable fragment comprising a second immunoglobulin light chain variable domain (V L 2) and a second immunoglobulin heavy chain variable domain (V H 2) ;
  • C L is an immunoglobulin light chain constant domain
  • C H 1 is an immunoglobulin heavy chain constant domain 1;
  • C H 2 is an immunoglobulin heavy chain constant domain 2;
  • hinge is an immunoglobulin hinge region connecting the C H 1 and C H 2 domains
  • V L 1 and VH1 associate to form a first Fv that specifically binds the tumor antigen (e.g., B7-H3, HER2, or TROP2) ; and wherein the scFv specifically binds CD28.
  • the scFv binds CD28 with half-maximal binding at a concentration of antibody (EC 50 ) that is at less than 10 nM (e.g., between 1 nM and 0.1 pM) as determined by an RED96 assay (e.g., as described in Example 3) .
  • the scFv binds CD28 with a dissociation constant (Kd) of less than 10 nM.
  • a bispecific antibody targeting CD28 and a tumor antigen comprising a first polypeptide, a second polypeptide, a third polypeptide and a fourth polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • the fourth polypeptide comprises a structure represented by the formula:
  • V H 1 is a first immunoglobulin heavy chain variable domain
  • V L 2 is a second immunoglobulin light chain variable domain
  • V H 2 is a second immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H 1 is an immunoglobulin heavy chain constant domain 1;
  • C H 2 is an immunoglobulin heavy chain constant domain 2;
  • hinge is an immunoglobulin hinge region connecting the C H 1 and C H 2 domains
  • V L 1 and V H 1 associate to form a first Fv that specifically binds the tumor antigen (e.g., B7-H3, HER2, or TROP2) ; and wherein V L 2 and V H 2 associate to form a second Fv that specifically binds CD28.
  • the second Fv binds CD28 with half-maximal binding at a concentration of antibody (EC 50 ) that is less than 10 nM (e.g., between 1 nM and 0.1 pM) as determined by an RED96 assay (e.g., as described in Example 3) .
  • the second Fv binds CD28 with a dissociation constant (Kd) of less than 10 nM.
  • a multi-specific antibody comprising:
  • a first antigen-binding fragment comprising a V H 1 and a V L 1 of an antibody that specifically binds a target antigen (e.g., a tumor antigen, such as B7-H3, HER2, or TROP2) ; and
  • a target antigen e.g., a tumor antigen, such as B7-H3, HER2, or TROP2
  • a second antigen-binding fragment comprising a V H 2 and a V L 2 of an anti-CD28 antibody that specifically binds CD28, wherein the second antigen-binding fragment is fused to a first masking peptide (MP1) .
  • a multi-specific antibody comprising:
  • a) a first antigen-binding fragment comprising a V H 1 and a V L 1 of an antibody that specifically binds a target antigen (e.g., a tumor antigen, such as B7-H3, HER2, or TROP2) , wherein the first antigen-binding fragment is fused to a first masking peptide (MP1) ; and
  • a target antigen e.g., a tumor antigen, such as B7-H3, HER2, or TROP2
  • a second antigen-binding fragment comprising a V H 2 and a V L 2 of an anti-CD28 antibody that specifically binds CD28, wherein the second antigen-binding fragment is fused to a second masking peptide (MP2) .
  • MP2 second masking peptide
  • the first antigen-binding fragment is selected from the group consisting of a Fab, a Fv, a scFab and a scFv. In some embodiments, the first antigen-binding fragment is a Fab. In some embodiments, the second antigen-binding fragment is selected from the group consisting of a Fab, a Fv, a scFab and a scFv. In some embodiments, the second antigen-binding fragment is a scFv comprising, from N-terminus to C-terminus, V L 2, an optional linker, and V H 2.
  • a multi-specific antibody comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • V L 1-C L V L 1-C L ;
  • V L 1 is a first immunoglobulin light chain variable domain
  • V H 1 is a first immunoglobulin heavy chain variable domain
  • V L 2 is a second immunoglobulin light chain variable domain
  • V H 2 is a second immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H 1 is an immunoglobulin heavy chain constant domain 1;
  • C H 2 is an immunoglobulin heavy chain constant domain 2;
  • first C H 3 is a first immunoglobulin heavy chain constant domain 3;
  • second C H 3 is a second immunoglobulin heavy chain constant domain 3;
  • hinge is an immunoglobulin hinge region connecting the C H 1 and C H 2 domains
  • MP1 is a first masking peptide; MP1 comprises, from N-terminus to C-terminus, an N-terminal unit (NU) , a masking unit (MU) , and a linkage unit (LU) ; the LU of the masking peptide may not comprise a cleavage site, or comprise at least one cleavage site.
  • NU N-terminal unit
  • MU masking unit
  • LU linkage unit
  • a multi-specific antibody comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • V L 1 is a first immunoglobulin light chain variable domain
  • V L 2 is a second immunoglobulin light chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H 2 is an immunoglobulin heavy chain constant domain 2;
  • first C H 3 is a first immunoglobulin heavy chain constant domain 3;
  • second C H 3 is a second immunoglobulin heavy chain constant domain 3;
  • MP1 is a masking peptide; MP1 comprises, from N-terminus to C-terminus, an N-terminal unit (NU) , a masking unit (MU) and a linkage unit (LU) ; the LU of the masking peptide may comprise non, at least one or more cleavage site.
  • NU N-terminal unit
  • MU masking unit
  • LU linkage unit
  • MP2 is a masking peptide; MP2 comprises, from N-terminus to C-terminus, an N-terminal unit (NU) , a masking unit (MU) and a linkage unit (LU) ; the LU of the masking peptide may not comprise a cleavage site, or comprise at least one cleavage site.
  • the bispecific antibody binds to a first and second target, where the first target is CD28, and the bispecific antibody comprises an HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 15, 24, 33, 43, 66, 88, 103, 111, 126, 134, 148, and 161; an HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 51, 58, 67, 89, 96, 104, and 155; and an HCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 7, 16, 25, 35, 44, 52, 59, 81, 90, 97, 105, 112, 119, 127, 135, 141, 149, and 162; and an LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 17, 26, 36, 45, 53, 60, 76, 82, 91, 98, 106, 113, 120, 12
  • the bispecific antibody binds to a first and second target, where the first target is human CD28, and where 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.
  • the bispecific antibody binds to a first and second target, wherein the first target is CD28 and the second target is HER2, wherein the bispecific antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 172, a second heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 170, and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 169.
  • the bispecific antibody binds to a first and second target, wherein the first target is CD28 and the second target is HER2, wherein the bispecific antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 171, a second heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 170, and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 169.
  • the bispecific antibody binds to a first and 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 where the bispecific antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 174, and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 173.
  • the bispecific antibody binds to a first and second target, wherein the first target is CD28 and the second target is TROP2, wherein the bispecific antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 171, a second heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 174, and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 173.
  • the bispecific antibody binds to a first and second target, where the first target is CD3 and the second target is B7-H3, and where the bispecific antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 177 or an scFv fusion polypeptide set forth in SEQ ID NO: 299, a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 176, and a light chain comprising an amino acid sequence set forth in SEQ ID NO: 175.
  • the bispecific antibody binds to a first and second target, where the first target is CD3 and the second target is TROP2.
  • the bispecific antibody binds to a first and second target, where the first target is human CD28, and where 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.
  • 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,
  • the present disclosure provide a masked antibody which may be a masked monoclonal antibody to a specific target or a multi-specific (e.g., bispecific) antibody.
  • the masked antibody provided herein comprises a full length antibody light chain, e.g., a kappa or lambda light chain. Additionally or alternatively, in some embodiments, the antibody comprises a full-length antibody heavy chain.
  • the antibody heavy chain may be in any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is in the IgG class, such as IgG 1 , IgG 2 , IgG 3 , or IgG 4 subclass.
  • an antibody heavy chain described herein may be converted from one class or subclass to another class or subclass using methods known in the art.
  • the masked antibody is or comprises a full length antibody that comprises an Fc region, e.g., a human Fc region or a variant thereof.
  • the human Fc region is a human IgG 1 Fc region, a human IgG 2 Fc region, a human IgG 4 Fc region, or a variant of any one of the preceding.
  • the variant Fc region comprises one or more amino acid substitutions, insertions, or deletions relative to the wild type human Fc region from which the variant is derived.
  • the masked antibody comprises a variant of a human IgG 1 Fc region.
  • the IgG 1 Fc variant comprises one or more amino acid substitutions that increases the affinity of the Fc variant for Fc ⁇ RIIb.
  • the variant of the human IgG 1 Fc region comprises substitution (s) selected from the group consisting of: G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F, wherein 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 preceding substitutions are described in Chu et al. Mol Immunol. (2008) 45 (15) : 3926-33.
  • the variant of the human IgG 1 Fc region comprises substitution (s) 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.
  • the preceding substitutions are described in Mimoto et al. Protein Eng Des Sel. (2013) 26 (10) : 589-98.
  • the variant of the human IgG 1 Fc region comprises an S2657A substitution (see Buschor et al. Int Arch Allergy Immunol. (2014) 163 (3) : 206-14) , wherein amino acid numbering is according to the EU index. Additionally or alternatively, in some embodiments, the variant of the human IgG 1 Fc region comprises a T437R and/or a K248E substitution (see Zhang et al. MAbs. (2017) 9 (7) : 1129-1142) , wherein amino acid numbering is according to the EU index. In some embodiments, the masked antibody comprises a variant of a human IgG 4 Fc region.
  • the IgG 4 Fc variant comprises one or more amino acid substitutions that increases the affinity of the Fc variant for Fc ⁇ RIIb.
  • the variant of the human IgG 4 Fc region comprises substitution (s) selected from the group consisting of: G236D; L328F; S239D; S267E; G236D and S267E; S239D and S267E; S267E and L328S; and S267E and L328F, wherein amino acid numbering is according to the EU index.
  • the variant of the human IgG 4 Fc region comprises substitution (s) 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.
  • the variant of the human IgG 4 Fc region comprises an S2657A substitution, wherein amino acid numbering is according to the EU index.
  • the variant of the human IgG 1 Fc region comprises a T437R and/or a K248E substitution wherein amino acid numbering is according to the EU index.
  • the masked antibodies disclosed herein further comprise a human IgG 1 domain or a variant thereof that comprises one or more substitution mutation (s) .
  • the IgG 1 variant comprises substitution (s) 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.
  • the masked antibodies disclosed herein further comprise a human IgG 4 domain or a variant thereof that comprises one or more substitution mutation (s) .
  • the IgG 4 variant comprises substitution (s) 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.
  • the term “masked antibody” refers to an antibody fragment, e.g., a masked antigen-binding fragment of a masked anti-CD28 antibody.
  • the antibody fragment is or comprises a Fab, an Fab’, a Fab’-SH, a F (ab’) 2, an Fv, an scFv (see Bird et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883) , an (scFv) 2, a linear antibody, a single-chain antibody, a minibody, or a diabody.
  • a masked anti-CD28 antibody described herein cross-reacts with CD28 from different species, thus permitting the masked anti-CD28 antibody to be used in both preclinical and clinical studies.
  • a masked anti-CD28 antibody described herein binds to two or more of human CD28, cynomolgus CD28, murine (mouse) CD28, and/or rat CD28 following activation (i.e., after activation of the masked antibody via cleavage, e.g., protease cleavage) .
  • a masked anti-CD28 antibody binds human CD28, cynomolgus CD28, murine (mouse) CD28, and a rat CD28 following activation (i.e., after activation of the masked antibody via cleavage, e.g., protease cleavage) .
  • masked anti-CD28 antibodies described herein are context-dependent (e.g., are activated (are only capable of binding their targets) in certain contexts (such as in the protease-rich tumor microenvironment) ) .
  • the masked anti-CD28 antibodies described herein provide improved safety over more traditional, non-masked antibodies (e.g., show reduced toxicity, do not induce significant alterations to the weights of many organs, do not alter liver histopathology, hematology, and/or blood biochemistry, etc. ) .
  • the antibody heavy chain variable region (V H ) and the antibody light chain variable region (V L ) of a masked anti-CD28 antibody described herein form an antigen binding domain (ABD) that binds hCD28.
  • the masking unit (MU) of a masked anti-CD28 antibody described herein binds to the ABD of the and reduces or inhibits binding of the masked anti-CD28 antibody to hCD28, as compared to the binding of a corresponding anti-CD28 antibody lacking the MU to hCD28 and/or as compared to the binding of the ABD to hCD28.
  • the masking unit has a masking 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 1,000, at least about 1,100, at least about 1,200, at least about 1,300, at least about 1,400, at least about 1,500, etc., including any range in between these values) prior to removing the MU from a masked antibody provided herein.
  • 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,
  • the masking efficiency of a masked anti-CD28 antibody is measured as the difference in affinity of the masked anti- CD28 antibody comprising the masking unit (MU) for binding to hCD28 (i.e., before activation of the masked antibody) relative to the affinity of an anti-CD28 antibody lacking the MU for binding to hCD28.
  • MU masking unit
  • the masking efficiency is measured by dividing the EC 50 for target-binding of a masked antibody comprising an MU (i.e., before activation) by the EC 50 of a corresponding antibody specific for the same target that lacks the masking peptide or masking unit.
  • the EC 50 is measured by ELISA.
  • the masking unit (MU) of the masked antibody binds to the ABD, and prevents the masked polypeptide from binding to its target.
  • the target is CD28.
  • the target is CD3, B7-H3, HER2, or TROP2.
  • the affinity of a masked antibody of the present disclosure increases by at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3, 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 range in between the preceding values) when the masking unit is removed from the antibody (e.g., after activation by treatment with one or more proteases that cleave within the
  • the EC 50 of a masked antibody described herein decreases by at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3, 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 range in between the preceding values) after activation by treatment with one or more proteases that cleave within the linkage unit (e.g., as measured by an enzyme, e.
  • the K D of the antibody for its target is about 2 (e.g., about 2, about 2.5, about 3, about 3.5 about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more, including any range in between the preceding values) times greater than the K D of the antibody when the masking unit of the masked anti-CD28 antibody is removed from the ABD (such as after protease treatment to cleave within the linkage unit) .
  • the K D of the antibody for its target is about 2 (e.g., about 2, about 2.5, about 3, about 3.5 about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more, including any range in between the preceding values) times greater than the K D of a corresponding antibody that is specific to the same target but lacks a masking peptide or masking unit.
  • the masking unit sterically hinders binding of the masked binding polypeptide to its target and/or allosterically hinders binding of the masked binding polypeptide to its target.
  • the dissociation constant of the masking unit for the ABD of a masked antibody (e.g., anti-CD28) described herein is greater than the dissociation constant of the masked antibody for its target (e.g., hCD28; when the masked antibody is in active form, such as after protease treatment) .
  • the dissociation constant of the masking unit for the ABD of a masked antibody (e.g., anti-CD28) described herein is about 2 (e.g., about 2, about 2.5, about 3, about 3.5 about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more, including any range in between the preceding values) times greater than the dissociation constant of the masked antibody for its target (e.g., hCD28; when the masked antibody is in active form, such as after protease treatment) .
  • target e.g., hCD28; when the masked antibody is in active form, such as after protease treatment
  • the dissociation constant of the masking unit for the ABD of a masked antibody (e.g., anti-CD28) described herein is about equal to the dissociation constant of the masked antibody for its target (e.g., hCD28; when the masked antibody is in active form, such as after protease treatment) .
  • the masking unit (MU) binds to the ABD of a masked antibody (e.g., anti-CD28) described herein and prevents the antibody from binding to its target (e.g., hCD28) only when the masked antibody has not been activated (e.g., by treatment with one or more proteases that cleave within the linkage unit) .
  • activation induces cleavage of the polypeptide within the cleavage site. In some embodiments, activation induces conformation changes in the polypeptide (e.g., displacement of the masking unit (MU) ) , leading to the masking peptide no longer preventing the polypeptide from binding to its target.
  • conformation changes in the polypeptide e.g., displacement of the masking unit (MU)
  • a masked monoclonal antibody comprising a masking peptide (MP) and an antibody that binds CD28, wherein the antibody comprises a heavy chain variable regions (V H ) and a light chain variable region (V L ) , wherein the MP is linked to an N-terminus of the V L , wherein the MP comprises, from N-terminus to C-terminus, a masking unit (MU) , and a linkage unit (LU) , wherein the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215-248, and wherein the antibody V H 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
  • a masked monoclonal antibody comprising a masking peptide (MP) and an antibody that binds CD28, wherein the antibody comprises a heavy chain variable regions (V H ) and a light chain variable region (V L ) , wherein the MP is linked to an N-terminus of the V L , wherein the MP comprises, from N-terminus to C-terminus, a masking unit (MU) , and a linkage unit (LU) , wherein the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215-248, 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:
  • a masked monoclonal antibody comprising a masking peptide (MP) and an antibody that binds human CD28, wherein the antibody comprises a heavy chain and a light chain, wherein the MP is linked to an N-terminus of the LC, wherein the MP comprises, from N-terminus to C-terminus, a masking unit (MU) , and a linkage unit (LU) , wherein the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215-248, wherein the antibody HC comprises an amino acid sequence selected from 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 the amino acid sequences selected from group consisting of SEQ ID NOs: 14, 23, 32, 42, 50, 57, 65, 74
  • a masked bispecific monoclonal antibody specific for a first and second target where the first target is CD28
  • the antibody comprises a masking peptide (MP) and a CD28 binding portion
  • the antibody comprises a heavy chain variable region (V H ) and a light chain variable region (V L )
  • the MP is linked to an N-terminus of the V L
  • the MP comprises, from N-terminus to C-terminus, a masking unit (MU) , and a linkage unit (LU)
  • the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215-248
  • the CD28 binding portion comprises a V H region comprising a HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 6, and HCDR3 of SEQ ID NO: 7, and a V L region comprising a LCDR1 of SEQ ID NO: 8, a LCDR2 of SEQ ID NO: 9
  • a masked bispecific monoclonal antibody specific for a first and second target where the first target is CD28
  • the antibody comprises a masking peptide (MP) and a CD28 binding portion
  • the antibody comprises a heavy chain variable region (V H ) and a light chain variable region (V L )
  • the MP is linked to an N-terminus of the V L
  • the MP comprises, from N-terminus to C-terminus, a masking unit (MU) , and a linkage unit (LU)
  • the MP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 215-248
  • 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
  • the first target is CD28
  • the antibody comprises a masking peptid
  • a masked bispecific monoclonal antibody specific for a first and second target where 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 masking peptides (MP) .
  • the MP further comprises a N-terminal unit.
  • the N-terminal unit is between about 1 and 10 amino acids in length.
  • the N-terminal unit comprises SEQ ID NO: 210.
  • the LU comprises at least a first cleavage site (CS 1 ) (e.g., a first protease cleavage site) .
  • the LU further comprises a second cleavage site (CS 2 ) .
  • the first and/or second cleavage site are a protease cleavage site.
  • the first and second cleavage sites are the same.
  • the first protease cleavage site is a cleavage site for a protease selected from uPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, TEV protease, plasmin, Thrombin, Factor X, PSA, PSMA, Cathepsin D, Cathepsin K, Cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE.
  • the first protease cleavage site is a cleavage site for a protease selected from uPA, MMP-2, MMP-9,
  • the LU further comprises a first linker (L 1 ) .
  • the first linker (L 1 ) is C-terminal to the first cleavage site (CS 1 ) (e.g., a first protease cleavage site) .
  • the LU comprises a structure, from N-terminus to C-terminus, of: (CS 1 ) -L 1 .
  • the LU further comprises a second linker (L 2 ) .
  • the L 2 is C-terminal to the second cleavage site.
  • Linker sequences may be of any length, such as from about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., 20 amino acid glycine polymers or glycine-serine polymers) , about 1 amino acid to about 15 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, etc.
  • 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.
  • the masking peptide comprises an MU set forth in any one of SEQ ID NOs: 178-211 and an LU set forth in SEQ ID NO: 213 or 214.
  • the MP comprises a sequence set forth in any one of SEQ ID NOs: 215-248.
  • Another aspect of the disclosure provides one or more isolated nucleic acid molecule (s) that comprises nucleotide sequence (s) encoding an amino acid sequence (s) of an anti-CD28 antibody described herein, including a masked anti-CD28 antibody.
  • nucleic acid molecule that comprises nucleotide sequence (s) encoding an amino acid sequence (s) of a multi-specific antibody described herein, including a masked multi-specific 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 variable region of a heavy chain, variable region of a light chain, or may be a full-length heavy chain or full length light chain.
  • a nucleic acid of the disclosure can be, for example, DNA or RNA, and may or may not contain intronic sequences. Typically, the nucleic acid is a cDNA molecule.
  • the disclosure provides an isolated nucleic acid molecule that comprises or consists of a nucleotide sequence encoding an amino acid sequence of, 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 and/or full length light chain of an antibody described herein.
  • the isolated DNA encoding the V H region can be converted to a full-length heavy chain gene by operatively linking the V H -encoding DNA to another DNA molecule encoding heavy chain constant regions (C H 1, C H 2 and C H 3) .
  • C H 1, C H 2 and C H 3 heavy chain constant regions
  • 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, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA, IgE, IgM or IgD constant region, but most preferably is an IgG 4 or IgG 2 constant region without ADCC effect.
  • the IgG 4 constant region sequence can be any of the various alleles or allotypes known to occur among different individuals. These allotypes represent naturally occurring amino acid substitution in the IgG 4 constant regions.
  • the V H -encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain C H 1 constant region.
  • the isolated DNA encoding the V L region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the V L -encoding DNA to another DNA molecule encoding the light chain constant region, C L .
  • the sequences of human light chain constant region genes are known in the art (see e.g., Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region.
  • the V H -and V L -encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly 4 -Ser) 3 , such that the V H and V L sequences can be expressed as a contiguous single-chain protein, with the V L and V H regions joined by the flexible linker (see e.g., Bird et al., Science (1988) 242: 423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85: 5879-83; and McCafferty et al., Nature (1990) 348: 552-554) .
  • a flexible linker e.g., encoding the amino acid sequence (Gly 4 -Ser) 3
  • the present disclosure further provides a vector that comprises one or more nucleic acid molecule (s) provided by the present disclosure.
  • the vector is an expression vector useful for the expression of an antibody described herein or an antigen binding fragment of such an antibody.
  • a first vector comprises a polynucleotide sequence encoding a heavy chain variable region as described herein
  • a second vector comprises a polynucleotide sequence encoding a light chain variable region as described herein.
  • a single vector comprises polynucleotides encoding a heavy chain variable region as described herein and a light chain variable region as described herein.
  • the antibody genes are inserted into the expression vector by any suitable methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or homologous recombination-based DNA ligation) .
  • the light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype and subclass by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype and subclass such that the V H segment is operatively linked to the C H segment (s) within the vector and the V L segment is operatively linked to the C L segment within the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein) .
  • the expression vectors of the disclosure typically carry regulatory sequences that control the expression of the antibody chain genes in a host cell.
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) ) . It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) , Simian Virus 40 (SV40) , adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • nonviral regulatory sequences may be used, such as the ubiquitin promoter or ⁇ -globin promoter.
  • regulatory elements 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) .
  • the expression vectors may carry additional sequences, such as enhancer element (s) , a transcription termination sequence (s) , sequence (s) that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker gene (s) .
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al. ) .
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection) .
  • DHFR dihydrofolate reductas
  • the expression vector (s) encoding the heavy and light chains is transfected into a host cell by any suitable techniques.
  • the various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • electroporation e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • expression of antibodies in eukaryotic cells e.g., mammalian host cells, is most typical.
  • the present disclosure further provides a host cell containing nucleic acid molecule (s) or vector (s) provided by the present disclosure.
  • the host cell can be virtually any cell for which expression vectors are available. It may be, for example, a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the recombinant nucleic acid construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, electroporation or phage infection.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • GS glucose synthetase
  • An antibody (or antigen binding fragment thereof) of the present disclosure may be produced by any means known in the art. Exemplary techniques for antibody production are in U.S. Patent No. 4,816,567; however these exemplary techniques are provided for illustrative purposes only and are not intended to be limiting.
  • nucleic acid (s) or expression vector (s) encoding an antibody described herein are introduced into a host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown.
  • a method of producing an antibody described herein comprises culturing a host cell comprising one or more nucleic acid (s) or vector (s) that encode the antibody (e.g., as provided above) under conditions suitable for expression of the antibody.
  • the method further comprises recovering the antibody from the host cell (or host cell culture medium) .
  • the antibody can be recovered from the culture medium using any suitable protein purification methods.
  • the present disclosure provides a composition comprising one or more of the antibodies described herein.
  • the composition is a pharmaceutical composition comprising an antibody described herein and a pharmaceutically acceptable carrier.
  • the compositions can be prepared by conventional methods known in the art.
  • pharmaceutically acceptable carrier refers to any inactive substance that is suitable for use in a formulation for the delivery of a polypeptide (e.g., an antibody described herein) .
  • a carrier may be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial, or antifungal agent) , sweetener, absorption delaying agent, wetting agent, emulsifying agent, buffer, and the like.
  • 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, buffer, buffered saline, and isotonic agents such as sugars, polyalcohols, sorbitol, and sodium chloride.
  • compositions may be in any suitable forms, such as liquid, semi-solid, and solid dosage forms.
  • liquid dosage forms include solution (e.g., injectable and infusible solutions) , microemulsion, liposome, dispersion, or suspension.
  • solid dosage forms include tablet, pill, capsule, microcapsule, and powder.
  • a particular form of the composition suitable for delivering an antibody described herein is a sterile liquid, such as a solution, suspension, or dispersion, for injection or infusion.
  • Sterile solutions can be prepared by incorporating the antibody in the required amount in an appropriate carrier, followed by sterilization microfiltration.
  • Dispersions may be prepared by incorporating the antibody into a sterile vehicle that contains a basic dispersion medium and other carriers.
  • compositions for the preparation of sterile liquid, methods of preparation include vacuum drying and freeze-drying (lyophilization) to yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • methods of preparation include vacuum drying and freeze-drying (lyophilization) to yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the various dosage forms of the compositions can be prepared by conventional techniques known in the art.
  • one or more additional therapeutic agents may be included in the composition.
  • the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, target therapies, radiation therapies, and chemotherapies.
  • the at least one additional therapeutic agent is selected from the group consisting of pomalyst, revlimid, lenalidomide, pomalidomide, thalidomide, a DNA-alkylating platinum-containing derivative, cisplatin, 5-fluorouracil, cyclophosphamide, an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CD20 antibody, an anti-CD40 antibody, an anti-DR5 antibody, an anti-CD1d antibody, an anti-TIM3 antibody, an anti-SLAMF7 antibody, an anti-KIR receptor antibody, an anti-OX40 antibody, an anti-HER2 antibody, an anti-ErbB-2 antibody, an anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, radiotherapy, single dose radiation, fractionated radiation, focal radiation, whole organ radiation, IL-12, IFN ⁇ , GM-CSF, a
  • the suitable amount of the additional therapeutic agent to be included in the composition can be readily selected by a person skilled in the art, and will vary depending on a number of factors, such as the particular agent and carriers used, dosage form, and desired release and pharmacodynamic characteristics.
  • the amount of the additional therapeutic agent included in a single dosage form will generally be that amount of the agent which produces a therapeutic effect, but may be a lesser amount as well.
  • the antibodies described herein may be further modified.
  • the antibodies are linked to an additional molecular entity.
  • additional molecular entities include pharmaceutical agents, peptides or proteins, detection agent or labels, and antibodies.
  • an antibody described herein is linked to a pharmaceutical agent.
  • pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents, and radioactive isotopes.
  • cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine) , alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU) , cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin) , anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin) , antibiotics (e.g., dactinomycin (formerly actinomycin) , bleomycin, mithramycin, and anthramycin (AMC) ) ,
  • radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine 131 , indium 111 , yttrium 90 and lutetium 177 .
  • Methods for linking a polypeptide to a pharmaceutical agent are known in the art, such as using various linker technologies. Examples of linker types include hydrazones, thioethers, esters, disulfides and peptide-containing linkers.
  • linkers and methods for linking therapeutic agents to antibodies see e.g., Saito et al., Adv. Drug Deliv. Rev. (2003) 55: 199-15; Trail, et al., Cancer Immunol. Immunother.
  • any of the antibodies and/or compositions (e.g., pharmaceutical compositions) described herein may be used in the preparation of a medicament (e.g., a medicament for use in treating or delaying progression of cancer in a subject in need thereof) .
  • the present disclosure provides methods of using the antibodies or pharmaceutical compositions described herein.
  • the present disclosure provides a method of treating cancer in a subject (e.g., a human subject) , comprising administering to the subject an effective amount of an antibody described herein.
  • the cancer is breast cancer, liver cancer, or colorectal cancer, gastric cancer, ovarian cancer, lung cancer, pancreatic cancer, or kidney cancer.
  • the present disclosure provides a method of treating cancer in a subject (e.g., a human subject) that comprises administering to the subject an effective amount of an antibody described herein (e.g., an anti-CD28 antibody or multi-specific antibody that targets CD28 and one or more other targets) and an effective amount of an anti-PD-1 antibody.
  • the present disclosure provides a method of treating cancer in a subject (e.g., a human subject) that comprises administering to the subject an effective amount of an antibody described herein (e.g., an anti-CD28 antibody or multi-specific antibody that targets CD28 and one or more other targets) and an effective amount of an anti-CTLA4 antibody.
  • the anti-CTLA4 antibody is a masked anti-CTLA4 antibody.
  • kits comprising one or more antibodies described herein (e.g., an anti-CD28 antibody or multi-specific antibody that targets CD28 and one or more other targets) .
  • the kit further comprises a package insert comprising instructions for use of the antibodies described herein.
  • the article of manufacture or kit comprises a container containing one or more of the masked antibodies or compositions described herein.
  • the article of manufacture or kit comprises a container containing nucleic acid (s) encoding one (or more) of the masked antibodies described herein.
  • the kit includes a cell of cell line that produces an antibody described herein.
  • the kit includes one or more positive controls, for CD28 (e.g., human CD28, cynomolgus CD28, mouse CD28, rat CD28 or fragments of any of the preceding) or CD28 + cells.
  • the kit includes negative controls, for example a surface or solution that is substantially free of CD28, or a cell that does not express CD28.
  • the article of manufacture 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 containers may be formed from a variety of materials such as glass or plastic.
  • the container holds an antibody described herein (or a composition comprising such an antibody) , which is by itself or combined with another composition effective for treating, delaying progression of, and/or preventing cancer in a subject (e.g. a human subject) .
  • the container may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) .
  • the label or package insert indicates that the composition is used for treating breast cancer, liver cancer, or colorectal cancer in a subject (e.g., a human subject) .
  • the article of manufacture or kit may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody described herein (or immunologically active fragment thereof) ; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the second container contains a composition comprising an anti-PD-1 antibody
  • the article of manufacture comprises a the label or package insert indicates that the antibody and the anti-PD-L1 are for use in the treatment of colon cancer in a subject (e.g., human subject) in need thereof, e.g., according to a method provided herein.
  • the second container contains a composition comprising an anti-CTLA4 antibody (e.g., a masked anti-CTLA4 antibody)
  • the article of manufacture comprises a the label or package insert indicates that the antibody described herein and the anti-CTLA4 antibody (e.g., a masked anti-CTLA4 antibody) are for use in the treatment of colon cancer in a subject (e.g., human subject) in need thereof, e.g., according to a method provided herein.
  • the article of manufacture may further comprise an additional container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • a pharmaceutically-acceptable buffer such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer's solution and dextrose solution.
  • Proprietary phagemid libraries were employed to pan against human CD28 antigens. A total of three or four rounds of panning were conducted. After the final round of panning, the culture supernatants of individual clones were tested by ELISA to identify those that specifically recognized human CD28 (i.e., primary hits) . Clones were defined as positive when ELISA signals were at least twice that of background. The positive clones were picked to confirm sequence and the Fabs corresponding to the unique hits were expressed in in E. coli and affinity purified. Their affinities against human CD28 were measured by the RED96 Systems (ForteBio) .
  • AHC Dip and Read Anti-Human IgG Fc Capture
  • the primary Fabs hits were further characterized for human and mouse CD28 species cross-reactivity by ELISA and, from these primary hits, a panel of 46 unique sequence Fabs were then converted into IgG 4 isotype mAbs with the core hinge mutation S241P (Kabat numbering scheme) for detailed biophysical and functional characterization (Table 5) .
  • the heavy chains and light chains of the engineered human anti-CD28 IgG 4 isotype mAbs listed in Table 5 were cloned into the mammalian expression vector pcDNA3.3 (ThermoFisher Scientific) . Pairs of plasmids bearing one heavy and one light chain were transiently transfected into HEK293 cells following the manufacturer’s instructions. After incubation, the supernatants were harvested, cleared by centrifugation and filtration, and the IgG 4 isotype mAbs were captured by MabSelect TM SuRe TM protein A affinity chromatography (GE Healthcare) .
  • the mAbs were eluted and neutralized, and the eluate buffer exchanged into restore buffer (20 mM histidine, pH 5.5) . Protein concentrations were determined by UV spectrophotometry, and IgG purity was analyzed under denaturing, reducing, and non-reducing conditions by SDS-PAGE or SEC-HPLC.
  • the binding affinities of the panel of anti-CD28 mAbs to human, cynomolgus monkey, and mouse CD28 and human CTLA4 were measured by the RED96 Systems (ForteBio) , ELISA, and CytoFlex flow cytometry (Beckman) .
  • the anti-CD28 antibody TAC2386 also known as TGN1412 as described in Patent WO2006/050949A2
  • TAC2387 as described in Patent WO2019/246514A2 were used as a benchmark controls.
  • the RED96 Systems was used to assess the binding kinetics of the panel of anti-CD28 mAbs to human CD28 and CTLA4. Briefly, the mAbs were diluted to 15 ⁇ g/mL in kinetic buffer (PBS supplemented with 0.02%Tween 20 and 0.1%BSA) , and captured by Dip and Read AHC biosensors (ForteBio) in parallel. The sensors were then allowed to associate with His-tagged human CD28 and CTLA4 proteins (100 nM) for 300 seconds, and to dissociate in kinetic buffer for another 300 seconds. The association and dissociation curves were fitted to a 1: 1 Langmuir binding model using the Data Analysis Software Version 7.1 (ForteBio) .
  • the panel of anti-CD28 test and benchmark mAbs exhibited high binding affinity ( ⁇ 10 nM) to human CD28.
  • the test mAbs TY24773, TY24853, TY24854, TY24860, TY24865, and TY24871 and the benchmark control mAbs exhibited no detectable affinity to human CTLA4.
  • Recombinant human and mouse CD28-Fc were diluted to 2 ⁇ g/mL in PBS and coated on Nunc MaxiSorp TM high protein-binding capacity 96 well ELISA plates (ThermoFisher Scientific) at 4°C overnight. Plates were blocked with PBS supplemented with 3%non-fat milk at 37°C for 1 hour. After washing, 50 ⁇ L of 3-fold serial dilutions of a panel of anti-CD28 test mAbs were added to each well.
  • HRP horseradish peroxidase
  • Fab specific horseradish peroxidase 1: 6000 dilution
  • TMB substrate 3, 3', 5, 5'-tetramethylbenzidine
  • Absorbance at 450 nm was measured after the reactions were stopped by adding 50 ⁇ L of a sulfuric acid stop solution to each well.
  • the EC 50 was evaluated by fitting the ELISA data using the asymmetrical sigmoidal (4-parameter logistic equation) model of GraphPad Prism version 7 for Windows, GraphPad Software, La Jolla California USA, www. graphpad. com.
  • the panel of anti-CD28 test mAbs exhibited similar affinities to human CD28 as the two benchmark controls. Moreover, the test mAbs except TY24890 bound to mouse CD28 while the two benchmark controls did not.
  • Jurkat (clone E6-1) cells were seeded in 96-well plates at 1.0 ⁇ 10 5 (50 ⁇ L/well) and incubated with serially diluted benchmark positive controls, an isotype negative control antibody, and a panel of anti-CD28 test mAbs (100, 20, 4, 0.8, 0.16, and 0.032 nM) for 30 minutes at 4 °C in 2%fetal bovine serum/Dulbecco’s PBS (FBS/DPBS) . Next, the cells were washed twice with DPBS and further incubated with a APC-anti-human IgG Fc secondary antibody (1 ⁇ g/mL, 100 ⁇ L/well, Biolegend) for 30 minutes at 4°C.
  • a APC-anti-human IgG Fc secondary antibody (1 ⁇ g/mL, 100 ⁇ L/well, Biolegend
  • MFI Mean Fluorescence Intensity
  • Human CD3 + T cells were isolated from cryopreserved peripheral blood mononuclear cells (PBMCs) using the EasySep TM Human Pan T Cell Isolation Kit (STEMCELL Technologies) .
  • the isolated human T cells were added in 96-well plate at 1.0 ⁇ 10 5 cells/well and incubated with 100 nM of benchmark controls, an isotype negative control antibody, and a panel of anti-CD28 test mAbs for 30 minutes on ice in FACS buffer. Next, the cells were washed three times with PBS and further incubated with PE-labeled secondary antibody for 30 minutes on ice. Finally, the cells were washed three times with PBS and resuspended in FACS buffer for flow cytometry analysis.
  • MFI values were calculated using FlowJo 10 software (FlowJo LLC) and the geometric mean MFI values versus mAb were plotted using GraphPad Prism version 7 for Windows, GraphPad Software, La Jolla California USA, www. graphpad. com.
  • Antibodies were tested for their ability to block the binding of CD28 or CTLA4 to its natural ligand CD80 by ELISA.
  • the benchmark controls TAC2386 and TAC2387 and all of the tested anti-CD28 test mAbs blocked the binding of CD28 to CD80, while none blocked the binding of CTLA4 to CD80, compared with an anti-CTLA4 antibody TY21580 included as a control.
  • Cryopreserved PBMCs were restored and the cell densities were adjusted to 5.0 ⁇ 10 5 cells/mL with 10%FBS/RPMI1640 and 1.0 ⁇ 10 5 PBMCs (200 ⁇ L per well) were added to the pre-coated microplates. The cells were incubated at 37°C, 5%CO 2 for 72 hours and then the lymphocyte proliferation was assessed using the assay.
  • the level of lymphocyte proliferation induced by immobilized benchmark control TAC2386 was significantly higher than that of all the test mAbs.
  • the benchmark control TAC2387 and the anti-CD28 test mAbs including TAC2387, TY24865, TY24866, TY24876, TY24878, TY24879 and TY24884 exhibited weak super agonistic activity in vitro.
  • the biological activity of the anti-CD28 mAbs, as agonistic T cell co-stimulatory agents in activating human T cells in vitro was measured by IFN- ⁇ cytokine secretion using ELISA.
  • Ultra-LEAF TM Purified anti-human CD28 Antibody (Biolegend) was included as a positive control.
  • Human CD3 + T cells were isolated from cryopreserved PBMCs using the EasySep TM Human Pan T Cell Isolation Kit (STEMCELL Technologies) .
  • the isolated cells were cultured in 96-well tissue culture plates (1.0 ⁇ 10 5 per well) pre-coated with a suboptimal concentration (10 nM, 50 ⁇ L per well) of anti-human CD3 antibody (OKT3) , in the presence of serially diluted benchmark controls, an isotype negative control antibody, a commercial anti-human CD28 positive control antibody, and a panel of anti-CD28 test mAbs (0.1, 1, 10, and 100 nM) .
  • the cells were incubated at 37°C, 5%CO 2 for 120 hours and then cell supernatants were collected for IFN- ⁇ cytokine analysis by ELISA, with T cell proliferation measured by
  • the biological activity of the anti-CD28 mAbs as agonistic T cell co-stimulatory agents in activating human T cells in vitro was measured by T cell proliferation with the Luminescent Cell Viability Assay (Promega) and IL-2 cytokine secretion by ELISA. Ultra-LEAF TM Purified anti-human CD28 Antibody (Biolegend) was included as a positive control.
  • Human T cells were isolated from fresh PBMCs from Asian donor using the EasySep TM Human Pan T Cell Isolation Kit (STEMCELL Technologies) .
  • the cells were cultured in 96-well tissue culture plates (1.0 ⁇ 10 5 cells/well) pre-coated with a suboptimal concentration (5 nM) of anti-human CD3 antibody (OKT3) , in the presence of serially diluted benchmark control TAC2387, an isotype negative control antibody, a commercial anti-human CD28 positive control antibody, and a panel of anti-CD28 test mAbs.
  • the cells were incubated at 37°C, 5%CO 2 for 72 hours, and then the cell supernatants were collected for IL-2 cytokine analysis by ELISA and the level of T cell proliferation was measured using the assay.
  • the anti-CD28 antibodies showed concentration-dependent biological activity including T cell proliferation and IL-2 cytokine secretion.
  • the effects of TY24859, TY24865, TY24866, and TY24890 on human T cell activation were comparable to the benchmark control TAC2387.
  • the negative control groups without pre-coating anti-CD3 showed no detectable T cell proliferation and IL-2 cytokine secretion.
  • a heterodimeric bispecific scaffold was engineered using the TYM13 Fc mutant (D or E356K: E357K: S364K: S400C L351’D: K370’D: N390’C: K439’D; according to Kabat numbering scheme for an IgG1 C H 3 domain) .
  • a light chain-heavy chain half antibody and a single-chain fragment variable (scFv) -Fc chain were combined to form a bispecific antibody (BsAb) , with TYM13 mutations in the hetero-Fc domain.
  • Plasmids encoding the heavy chain, light chain, and scFv-Fc chain of BsAbs were transiently transfected into mammalian cells.
  • Bispecific antibody-containing cell culture supernatants were harvested 7 days after transfection by centrifugation at 14000 g for 30 minutes and were filtered through a sterile filter (0.22 ⁇ m) .
  • Antibodies were purified by protein A affinity chromatography using MabSelect TM SuRe TM prepacked columns (GE Healthcare) and were subsequently buffer exchanged in 20 mM histidine (pH 5.5) buffer.
  • TY24865 high affinity CD28
  • TY24865 mutant low affinity CD28
  • anti-HER2 ⁇ CD28 BsAb TY27566
  • anti-HER2 ⁇ CD3 BsAb TY25238, also described in PCT/CN2021/076626, which is incorporated by reference herein in its entirety
  • anti-HER2 mAbs for (TAC2319) or (TAC2320) were measured using flow cytometry.
  • TAC2319 binds to a different epitope of the HER2 dimerization domain than (TAC2320) .
  • SK-OV-3 cells were cultured and added to 96-well plates at 8.0 ⁇ 10 4 cells/well and incubated with serially diluted test BsAbs for 60 minutes at 4°C in 2%FBS/RPMI1640 buffer. Next, the cells were washed twice with DPBS and further incubated with a secondary APC- anti-human IgG Fc antibody (1: 400 dilution) for 30 minutes at 4°C. Finally, the cells were washed twice with DPBS and resuspended in FACS buffer for flow cytometry analysis.
  • anti-HER2 ⁇ CD28 or anti-HER2 ⁇ CD3 BsAbs showed very similar binding affinity to SK-OV-3 cells, compared with anti-HER2 mAbs for (TAC2319) or (TAC2320) .
  • TCR T Cell Receptor
  • CD28 receptor signaling by the anti-HER2 ⁇ CD3 BsAb, the anti-HER2 ⁇ CD28 BsAb, or their combination were evaluated.
  • the simultaneous TCR and CD28 activation leads to enhanced transcriptional activity of NFkB, which in turn induces the production of the reporter gene.
  • Isotype antibodies were included as negative controls.
  • Serially diluted anti-HER2 ⁇ CD3 BsAb or isotype control antibody with a fixed concentration of anti-HER2 ⁇ CD28 BsAb (10 nM) or conversely serially diluted anti-HER2 ⁇ CD28 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 effect in stimulating downstream luciferase activity.
  • the co-cultured cells were incubated at 37°C, 5%CO 2 for 6 hours. Then, 100 ⁇ L of Luciferase Assay System (Promega) reagent was added to the cells, and the cells were lysed for 10 minutes. Supernatants (100 ⁇ L) were collected for luminescence measurements using a i3x Multi-Mode Microplate Reader (Molecular Devices) .
  • Luciferase Assay System Promega
  • anti-HER2 ⁇ CD28 BsAb (TY27566) in combination with a fixed concentration of anti-HER2 ⁇ CD3 BsAb (TY25238)
  • anti-HER2 ⁇ CD3 BsAb in combination with a fixed concentration of anti-HER2 ⁇ CD28 BsAb, exhibited synergistic or enhanced stimulatory effects in terms of maximum signal and EC 50 values.
  • TY252338 binds to a different tumor-associated antigen (TAA) epitope of the HER2 dimerization domain than TY27566 but to the same TAA epitope as TY27807.
  • TAA tumor-associated antigen
  • anti-HER2 ⁇ CD3 BsAb As shown in FIG. 8, as a single agent, anti-HER2 ⁇ CD3 BsAb (TY25238) elicited potent concentration-dependent cytotoxicity on MCF-7 target cells.
  • the combination of the anti-HER2 ⁇ CD28 BsAb (TY27566) and anti-HER2 ⁇ CD3 BsAb (TY25238) against different TAA epitopes further enhanced the in vitro tumor killing activity in terms of EC 50 .
  • anti-HER2 ⁇ CD3 BsAb TY25238) alone
  • the addition of anti-HER2 ⁇ CD28 BsAb (TY27807) with the same TAA epitope significantly decreased the MCF-7 tumor cell lysis in terms of EC 50 and maximum lysis.
  • An isotype antibody was included as a negative control for the single agent assay.
  • An anti-tumor-associated calcium signal transducer 2 (TROP2) ⁇ CD28 or anti-
  • TROP2 ⁇ CD3 heterodimeric bispecific scaffold was designed using the TYM13 Fc mutant as described in Example 7.
  • the constructs of anti-TROP2 ⁇ CD28 BsAb are described in Table 11.
  • concentration-dependent binding activity of anti-TROP2 BsAbs on different tumor cell lines with high, medium, or low TROP2 expression was measured using flow cytometry. Isotype antibodies were included as negative controls.
  • H292, NCI-N87, or HT29 cells were cultured and seeded in 96-well plates at 1.0 ⁇ 10 5 cells/well and incubated with serially diluted test anti-TROP2 BsAbs for 30 minutes at 4°C in 2%FBS/RPMI1640 buffer. 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 cell; 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.
  • API-anti-human IgG Fc antibody 1: 300 dilution for H292 cell
  • APC-F (ab’ ) 2 fragment goat anti-human IgG (H+L) 1: 500 dilution for NCI-N87 and HT29 cells
  • the TROP2 arm binding activities were similar between TY25839 and TY27571 on three different tumor cell lines with high, medium, or low TROP2 expression.
  • TCR and CD28 receptor signaling were evaluated.
  • the simultaneous TCR and CD28 activation leads to enhanced transcriptional activity of NFkB, which in turn induces the production of the reporter gene.
  • Serially diluted anti-TROP2 ⁇ CD3 BsAbs with fixed concentration of anti-TROP2 ⁇ CD28 BsAb (5 nM) , or conversely serially diluted anti-TROP2 ⁇ CD28 BsAbs with 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 for 6 hours.
  • a B7H3 ⁇ CD28 BsAb was constructed. The constructs are described in Table 14.
  • MDA-MB-231 cells were cultured and seeded in 96-well plates at 1.0 ⁇ 10 5 cells/well and incubated with serially diluted test BsAbs for 60 minutes at 4°C in 2% FBS/RPMI1640 buffer. Next, the cells were washed twice with DPBS and further incubated with secondary APC-anti-human IgG Fc antibody (1: 400 dilution) 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, the MFI values versus concentrations were plotted using FlowJo 10 software (FlowJo LLC) and the data were further fitted with four-parameter non-linear regression to obtain EC 50 values by GraphPad Prism version 7 for Windows.
  • the anti-B7H3 mAb TY21601 showed sub-nM (0.4525 nM) binding affinity for MDA-MB-231 target cells and the binding activity of the BsAb TY27556 to target cells was reduced by about 28-fold (12.84 nM) .
  • B7H3xCD28 bispecific antibody enhanced the ability of PD-1 or PD-L1 blockade to induce T cell activation in vitro
  • the cells were then co-cultured at 37°C, 5%CO 2 in the incubator for 120 h.
  • Co-stimulatory bispecific antibodies enhance in vitro T cell cytotoxicity against MCF-7 cells upon bidirectional binding
  • CD28-based BsAbs elicited no cytotoxicity on MCF-7 target cells.
  • the anti-CD3 BsAb TY25238 showed concentration-dependent effects and the highest cytotoxicity was elicited with BsAb directed against HER2 with 54.47%of MCF-7 cells lysed.
  • the combination of anti-CD3 and anti-CD28 BsAbs further enhanced the in vitro tumor cell killing activity as manifested by EC 50 reduction.
  • the addition of anti-CD28 BsAb TY27566 with a high affinity CD28 arm considerably enhanced tumor cell lysis by about 10-fold (EC 50 ) .
  • anti-CD28 BsAb TY27881 with a low affinity CD28 arm, no enhanced cytotoxicity was observed.
  • Co-stimulatory bispecific antibodies enhance in vitro T cell cytotoxicity against EMT-6-HER2 cells upon bidirectional binding
  • EMT-6-HER2 (5 ⁇ 10 3 cells/well) target cells were incubated with serially diluted anti-HER2 ⁇ CD3 BsAb (TY25238) or in combination with fixed concentrations of anti-B7H3 ⁇ CD28 BsAb TY27556 (8 nM) for 30 minutes at 37 °C, or conversely EMT-6-HER2 (5 ⁇ 10 3 cells/well) cells were incubated with serially diluted anti-B7H3 ⁇ CD3 BsAbs TY26999 or combination with fixed concentrations of anti-HER2 ⁇ CD28 BsAb TY27566 (8 or 0.8 nM) for 30 minutes at 37°C.
  • TY27556 anti-B7H3 ⁇ CD28
  • TY27566 anti-HER2 ⁇ CD28
  • TY25238 in combination with TY27556 resulted in about 2-fold decrease of EC 50 , and about 3-fold increase of maximal killing (from 17%to 50%) , compared with TY25238.
  • TY26999 combined with TY27566 resulted in about 1.3-fold decrease of EC 50 and about 2-fold increase of maximal killing (from 23%to 47%) , compared with TY26999.
  • mice were randomly divided into four groups (3 mice per group) , and were injected with an anti-mouse CD3 mAb (145-2C11 clone, 1 mg/kg) , anti-B7H3 ⁇ CD28 (TY27556, 2 mg/kg) , anti-B7H3 ⁇ CD3 (TY27042, in which CD3 arm was derived from mouse specific 145-2C11 clone, 2 mg/kg) or the combination (TY27556, 2 mg/kg with TY27042, 2 mg/kg) , respectively.
  • the mouse serum and whole blood were collected at various time points before and post injection (pre-dose, 3.5, and 24 hours) .
  • the systemic cytokine release risk was assessed with IL-6 and IFN- ⁇ by ELISA.
  • the percentage of total peripheral T cells that were CD3 + T cells was determined by flow cytometry at each time point.
  • mice administered anti-mCD3 or anti-B7H3 ⁇ CD3 lead to a significant induction of cytokine release (IL-6 and IFN- ⁇ ) at 3.5h after test antibody treatment.
  • Mice treated with anti-B7H3 ⁇ CD28 showed no detectable cytokine release after test antibody treatment.
  • anti-B7H3 ⁇ CD3 combined with anti-B7H3 ⁇ CD28 (TY27556) showed no increased cytokine release risk above that found with the single agent anti-B7H3 ⁇ CD3 (TY27042) .
  • the mice were administered with hIgG 1 isotype control at 5 mg/kg, anti-HER2 ⁇ CD3 bispecific double masked antibody at 0.2 mg/kg, anti-B7H3 ⁇ CD28 bispecific antibody TY27556 at 5 mg/kg, or TY27151 at 0.2 mg/kg in combination with TY27556 at 5 mg/kg by i.p. injection.
  • the double masked anti-HER2 ⁇ CD3 bispecific antibody TY27151 showed strong synergistic anti-tumor effect with the anti-B7H3 ⁇ CD28 bispecific antibody TY27556 in this model.
  • the mice were administered Vehicle, anti-B7H3 ⁇ CD28 BsAb TY27556 at 0.5 mg/kg and 0.05 mg/kg, or anti-HER2 ⁇ CD28 bispecific antibody TY27566 at 0.2 mg/kg by i.p. injection.
  • the mice were administered these Abs twice per week for a total of four doses. Tumor growth was monitored twice a week and reported as the mean tumor volume ⁇ s.e.m. over time.
  • anti-B7H3 ⁇ CD28 bispecific antibody TY27556 showed dose dependent anti-tumor effect in this model.
  • anti-HER2 ⁇ CD28 bispecific antibody TY27566 showed strong anti-tumor effect.
  • Example 11 Methods of Identifying Self-Blocking Peptides for Masked Anti-CD28 Antibodies
  • a screening system has been designed and executed for efficient discovery of masking moieties that can effectively mask a non-masked parental anti-CD28 antibody with good developability.
  • the target anti-CD28 scFv was first displayed on the surface of yeast and confirmed to be functional in binding to its CD28 antigen.
  • masking peptides (MP) from an improved MP peptide library were directly fused to the N-terminus of the light chain of the target anti-CD28 scFv, and a yeast library was constructed that displayed the fusion protein on the yeast surface.
  • Pairs of plasmids were transiently transfected into HEK293F cells. After six days, the supernatants were harvested, cleared by centrifugation and filtration, and IgGs were purified with standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare) . The IgGs were eluted and neutralized, and buffer exchanged into 20 mM histidine, pH 5.5 buffer. Protein concentrations were determined by UV-spectrophotometry, and IgG purity was analyzed under denaturing, reducing and non-reducing conditions by SDS-PAGE or SEC-HPLC.
  • recombinant human CD28-Fc was diluted to 2 ⁇ g/mL in PBS and coated onto a MaxiSorp TM high protein-binding capacity 96 well ELISA plate (ThermoFisher Scientific) at 4°C overnight. Plates were blocked with PBS supplemented with 3%non-fat milk at 37°C for 1 hour. After washing, 100 ⁇ L of 3-fold serial dilutions of anti-CD28 test mAbs were added to each well.
  • HRP horseradish peroxidase
  • Fab specific horseradish peroxidase 1: 6000 dilution
  • TMB substrate 3, 3', 5, 5'-tetramethylbenzidine
  • Absorbance at 450 nm was measured after the reactions were stopped with 50 ⁇ L sulfuric acid stop solution per well.
  • the EC 50 was evaluated by fitting the ELISA data using the sigmoidal (four-parameter logistic equation) model of GraphPad Prism version 6 for Windows, GraphPad Software, La Jolla California USA, www. graphpad. com.
  • TY26149 and TY26152 were modified on their MPs, including removing some N-terminal residues, and adding an “S” amino acid residue between residues “D” and “G” of the TY26149 sequence (bolded and underlined residues of TY26149 in Table 18) . As shown in FIG. 20, the expression and masking efficiency of the new masked antibodies were not significantly influenced.
  • TY24865 kept the binding ability to RE49AA, VY68AA, YS79AA and KT81AA, which indicated TY24865 does not bind to residues RE49, VY68, YS79 and KT81, and these residues are in un-conserved region of human and mouse CD28.
  • TY24865 lost the binding ability to FR51AA, SL54AA, YL98AA, QN100AA, YF110AA, KI113AA, YP118AA, PPP119AAA, PP120AA, PY121AA, Y122A mutations, indicating that their binding epitopes are within these regions, e.g., amino acid residues 51, 52, 54, 55, 98-101, 110-111, 113-114, 118-122 of SEQ ID NO.: 1.

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KR1020247037360A KR20250008811A (ko) 2022-04-08 2023-04-10 항-cd28 항체 및 이의 사용 방법
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