WO2017024515A1 - Novel anti-pd-1 antibodies - Google Patents

Novel anti-pd-1 antibodies Download PDF

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Publication number
WO2017024515A1
WO2017024515A1 PCT/CN2015/086594 CN2015086594W WO2017024515A1 WO 2017024515 A1 WO2017024515 A1 WO 2017024515A1 CN 2015086594 W CN2015086594 W CN 2015086594W WO 2017024515 A1 WO2017024515 A1 WO 2017024515A1
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seq
antibody
variable region
chain variable
antigen
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PCT/CN2015/086594
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French (fr)
Inventor
Yong Zheng
Jing Li
Zhisheng CHEN
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Wuxi Biologics (Cayman) Inc.
Open Monoclonal Technology, Inc.
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Priority to PCT/CN2015/086594 priority Critical patent/WO2017024515A1/en
Priority to KR1020177036687A priority patent/KR102055396B1/en
Priority to MX2018001644A priority patent/MX2018001644A/en
Priority to BR112018002824A priority patent/BR112018002824A2/en
Priority to UAA201802340A priority patent/UA124379C2/en
Priority to EA201890468A priority patent/EA201890468A1/en
Priority to RU2018108048A priority patent/RU2729830C2/en
Priority to PCT/CN2016/094624 priority patent/WO2017025051A1/en
Priority to PE2018000225A priority patent/PE20181018A1/en
Priority to RU2020124273A priority patent/RU2020124273A/en
Priority to US15/751,236 priority patent/US11008391B2/en
Priority to MA042626A priority patent/MA42626A/en
Priority to EP16834675.7A priority patent/EP3334763A4/en
Priority to JP2018526985A priority patent/JP6883579B2/en
Priority to IL257062A priority patent/IL257062B/en
Priority to CA2993276A priority patent/CA2993276A1/en
Priority to IL293385A priority patent/IL293385A/en
Priority to MYPI2018700283A priority patent/MY187739A/en
Priority to AU2016305697A priority patent/AU2016305697B2/en
Priority to SG10201914109VA priority patent/SG10201914109VA/en
Publication of WO2017024515A1 publication Critical patent/WO2017024515A1/en
Priority to PH12018500183A priority patent/PH12018500183A1/en
Priority to CONC2018/0000992A priority patent/CO2018000992A2/en
Priority to MX2022006447A priority patent/MX2022006447A/en
Priority to CL2018000370A priority patent/CL2018000370A1/en
Priority to SA521422116A priority patent/SA521422116B1/en
Priority to SA518390903A priority patent/SA518390903B1/en
Priority to ECIEPI201818842A priority patent/ECSP18018842A/en
Priority to JP2020200034A priority patent/JP2021036914A/en
Priority to US17/239,866 priority patent/US11643465B2/en
Priority to JP2022131714A priority patent/JP2022161997A/en
Priority to AU2023200891A priority patent/AU2023200891A1/en
Priority to US18/186,793 priority patent/US20230348602A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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 [IGs], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure generally relates to novel anti-PD-1 antibodies.
  • lambrolizumab is a fully human monoclonal IgG4 antibody that acts against PD-1, which grabbed the FDA's new breakthrough designation after impressive IB data came through for skin cancer.
  • the results from a phase IB study have shown an objective anti-tumor response in 51%of 85 cancer patients, and a complete response in 9%of patients.
  • Roche's experimental MPDL3280A demonstrated an ability to shrink tumors in 29 of 140 (21%) advanced cancer patients with various tumor sizes.
  • the present disclosure provides novel monoclonal anti-PD-1 antibodies (in particular fully human antibodies) , polynucleotides encoding the same, and methods of using the same.
  • the present disclosure provides isolated monoclonal antibodies or antigen binding fragments thereof, which are capable of specifically binding to human PD-1 at a Kd value no more than 10 -8 M (e.g. no more than ⁇ 9x10 -9 M, ⁇ 8x10 -9 M, ⁇ 7x10 -9 M, ⁇ 6x10 -9 M, ⁇ 5x10 -9 M, ⁇ 4x10 -9 M, ⁇ 3x10 -9 M, ⁇ 2x10 -9 M, or ⁇ 10 -9 M) as measured by plasmon resonance binding assay.
  • a Kd value no more than 10 -8 M e.g. no more than ⁇ 9x10 -9 M, ⁇ 8x10 -9 M, ⁇ 7x10 -9 M, ⁇ 6x10 -9 M, ⁇ 5x10 -9 M, ⁇ 4x10 -9 M, ⁇ 3x10 -9 M, ⁇ 2x10 -9 M, or ⁇ 10 -9 M
  • the antibodies or antigen binding fragments thereof bind to monkey PD-1 at an EC50 of no more than 100 nM or no more than 10nM (e.g. no more than 50nM, 40nM, 30nM, 20nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM, ) .
  • the antibodies and antigen-binding fragments thereof do not bind to mouse PD-1 but bind to monkey PD-1 with a binding affinity similar to that of human PD-1.
  • the antibodies or antigen binding fragments thereof potently inhibit binding of human or monkey PD-1 to its ligand (e.g. PD-L1 or PD-L2) , at an IC50 of no more than 100 nM (e.g. no more than 50nM, 40nM, 30nM, 20nM, 10 nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1 nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, or 0.1nM) .
  • the EC50 or IC50 is measured by fluorescence-activated cell sorting (FACS) analysis.
  • the antibodies or antigen binding fragments thereof have substantially reduced effector function. In certain embodiments, the antibodies or antigen binding fragments thereof do not mediate ADCC or CDC or both.
  • the antibodies or antigen binding fragments thereof provided herein comprise a heavy chain CDR sequences selected from the group consisting of:SEQ ID NOs: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35 and 37.
  • the antibodies or an antigen binding fragments thereof provided herein comprise a light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 7, 9, 11, 17, 19, 27, 29, 31, 39, 41, and 43.
  • the antibodies or antigen binding fragments thereof provided herein comprise at least one, two, three, four, five or six CDRs selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 7, 9, and 11; or selected from the group consisting of:SEQ ID NOs: 13, 15, 5, 7, 17 and 11; or selected from the group consisting of: SEQ ID NOs: 1, 15, 5, 7, 17 and 19; or selected from the group consisting of: SEQ ID NOs: 21, 23, 25, 27, 29 and 31; or selected from the group consisting of: SEQ ID NOs: 33, 35, 37, 39, 41 and 43.
  • the antibodies or antigen binding fragments thereof provided herein comprise a heavy chain variable region selected from the group consisting of:
  • the antibodies or antigen binding fragments thereof provided herein comprise a light chain variable region selected from the group consisting of:
  • the antibodies or antigen binding fragments thereof provided herein comprise:
  • the antibodies or antigen binding fragments thereof provided herein comprise a heavy chain variable region selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57 and SEQ ID NO: 61.
  • the antibodies or antigen binding fragments provided herein comprise a light chain variable region selected from the group consisting of: SEQ ID NO:47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, and SEQ ID NO: 63.
  • the antibodies or antigen binding fragments thereof provided herein comprise:
  • the antibodies provided herein include, for example, 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb.
  • the antibodies or antigen binding fragments thereof provided herein compete for the same epitope with antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, or 1.153.7 hAb.
  • the antibodies or antigen binding fragments thereof are capable of blocking binding of human PD-1 to its ligand and thereby providing at least one of the following activities:
  • the antibodies provided herein are a monoclonal antibody, fully human antibody, humanized antibody, chimeric antibody, recombinant antibody, bispecific antibody, labeled antibody, bivalent antibody, or anti-idiotypic antibody.
  • the antibodies or antigen binding fragments thereof are fully human monoclonal antibodies, optionally produced by a transgenic rat, for example, a transgenic rat having inactivated endogenous expression of rat immunoglobulin genesand carrying recombinant human immunoglobulin loci having J-locu deletion and a C-kappa mutation.
  • the antigen-binding fragments thereof provided herein are a camelized single domain antibody, a diabody, a scFv, an scFv dimer, a BsFv, a dsFv, a (dsFv) 2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F (ab') 2, a ds diabody, a nanobody, a domain antibody, or a bivalent domain antibody.
  • the antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin constant region.
  • the antibodies or antigen-binding fragments thereof provided herein further comprise a conjugate.
  • the conjugate can be a detectable label, a pharmacokinetic modifying moiety, or a purification moiety.
  • the present disclosure provides isolated polynucleotides encoding the antibodies or antigen binding fragments thereof provided herein.
  • polynucleotides are provided that encode the amino acid sequences of the antibodies or antigen-binding fragments disclosed herein.
  • vectors are provided that comprise these polynucleotides, and in certain other embodiments, host cells are provided that comprise these vectors.
  • methods are provided for expressing one or more of the antibodies or antigen-binding fragments disclosed herein by culturing these host cells under conditions in which the antibodies or antigen-binding fragments encoded by the polynucleotides are expressed from a vector.
  • the polynucleotides provided herein are operably associated with a promoter such as a SV40 promoter in a vector.
  • a promoter such as a SV40 promoter in a vector.
  • host cells comprising the vectors provided herein are Chinese hamster ovary cell, or 293F cell.
  • kits comprising the antibody or antigen-binding fragment thereof.
  • the present disclosure provides methods of treating a condition associated with PD-1 in an individual, comprising: administering to the individual a therapeutically effective amount of antibody or antigen-binding fragment thereof provided herein.
  • the individual has been identified as having a disorder or a condition likely to respond to a PD-1 antagonist.
  • the individual has been identified as positive for presence or upregulated level of the PD-L1 in a test biological sample from the individual.
  • the present disclosure provides pharmaceutical compositions comprising the antibody or antigen-binding fragment thereof provided herein and one or more pharmaceutically acceptable carriers.
  • the pharmaceutical carriers may be, for example, diluents, antioxidants, adjuvants, excipients, or non-toxic auxiliary substances.
  • the present disclosure provides methods of treating a condition in a subject that would benefit from upregulation of immune response, comprising administering an effective amount of the antibody or antigen-binding fragment thereof provided herein to the subject.
  • the subject has upregulated expression of PD-L1, or has been identified as positive for expression of PD-L1.
  • the condition is cancer or chronic viral infection.
  • Figure 1 presents the binding of fully human anti-PD-1 antibodies to PD-1 expressing CHO cell as measured by FACS analysis.
  • Figure 2 presents the binding of fully human PD-1 antibodies to PD-1 expressing CHO cell with EC50 about 2nM as measured by FACS analysis.
  • Figure 3 is the binding of fully human anti-PD-1 antibody to PD-1 expressed on activated CD4+T cell as measured by FACS analysis.
  • Figure 4 shows that the fully human anti-PD-1 antibodies blocked the binding of PD-L1 to PD-1 transfected CHO cells with IC50 of about 3-8 nM as measured by FACS analysis.
  • Figure 5 shows that the fully human anti-PD-1 antibodies specifically bind to PD-1, but do not bind family members CD28 and CTLA4, as measured by FACS analysis.
  • Figure 6 shows that the fully human anti-PD-1 antibodies against PD-1 bind to cynomolgus monkey PD-1 but not murine PD-1.
  • Figure 7 is the full kinetics of binding affinity of PD-1 antibodies to human PD-1 ranging from 3.76E-9 to 1.76E-10 mol/L as determined by surface plasmon resonance.
  • Figure 8 illustrates the effect of fully human anti-PD-1 antibodies on IL-2 production in mixed lymphocyte reaction (MLR) .
  • FIG. 9 illustrates the effect of fully human anti-PD-1 antibodies on IFN ⁇ production in MLR.
  • FIG. 10 shows that fully human anti-PD-1 antibodies promoted T cell proliferation in MLR.
  • Figure 11 shows that fully human PD-1 antibodies promoted T cell proliferation in specific T cell response.
  • Figure 12 shows that anti-PD-1 antibodies reversed Treg’s suppressive function.
  • Figure 13 shows that the anti-PD-1 antibodies lacked ADCC on activated T cells
  • Figure 14 shows that the anti-PD-1 antibodies lacked CDC on activated T cells.
  • antibody as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a specific antigen.
  • a native intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , and mammalian light chains are classified as ⁇ or ⁇ .
  • the antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding.
  • Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variables region in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light (L) chain CDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs including HCDR1, HCDR2, HCDR3) .
  • CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A. M., J. Mol.
  • the three CDRs are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ heavy chains, respectively.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • antigen-binding fragment refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure.
  • antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F (ab') 2 , an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody.
  • an antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds.
  • an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
  • Fab with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.
  • Fab' refers to a Fab fragment that includes a portion of the hinge region.
  • F (ab') 2 refers to a dimer of Fab’ .
  • Fc with regard to an antibody refers to that portion of the antibody consisting of the second and third constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding.
  • the Fc portion of the antibody is responsible for various effector functions such as ADCC, and CDC, but does not function in antigen binding.
  • Fv with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen binding site.
  • An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988) ) .
  • Single-chain Fv-Fc antibody or “scFv-Fc” refers to an engineered antibody consisting of a scFv connected to the Fc region of an antibody.
  • “Camelized single domain antibody, ” “heavy chain antibody, ” or “HCAb” refers to an antibody that contains two V H domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2) : 25-38 (1999) ; Muyldermans S., J Biotechnol. Jun; 74 (4) : 277-302 (2001) ; WO94/04678; WO94/25591; U.S. Patent No. 6,005,079) .
  • Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas) .
  • VHH domain The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21 (13) : 3490-8. Epub 2007 Jun 15 (2007) ) .
  • a “nanobody” refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.
  • “Diabodies” include small antibody fragments with two antigen-binding sites, wherein the fragments comprise a V H domain connected to a V L domain in the same polypeptide chain (V H -V L or V H -V L ) (see, e.g., Holliger P. et al., Proc Natl Acad Sci U S A. Jul 15; 90 (14) : 6444-8 (1993) ; EP404097; WO93/11161) .
  • the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites.
  • the antigen–binding sites may target the same of different antigens (or epitopes) .
  • a “domain antibody” refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain.
  • two or more V H domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody.
  • the two V H domains of a bivalent domain antibody may target the same or different antigens.
  • a “ (dsFv) 2 ” comprises three peptide chains: two V H moieties linked by a peptide linker and bound by disulfide bridges to two V L moieties.
  • a “bispecific ds diabody” comprises V H1 -V L2 (linked by a peptide linker) bound to V L1 -V H2 (also linked by a peptide linker) via a disulfide bridge between V H1 and V L1 .
  • a “bispecific dsFv” or dsFv-dsFv' “comprises three peptide chains: a V H1 -V H2 moiety wherein the heavy chains are linked by a peptide linker (e.g., a long flexible linker) and bound to V L1 and V L2 moieties, respectively, via disulfide bridges, wherein each disulfide paired heavy and light chain has a different antigen specificity.
  • a peptide linker e.g., a long flexible linker
  • an “scFv dimer” is a bivalent diabody or bivalent ScFv (BsFv) comprising V H -V L (linked by a peptide linker) dimerized with another V H -V L moiety such that V H 's of one moiety coordinate with the V L 's of the other moiety and form two binding sites which can target the same antigens (or eptipoes) or different antigens (or eptipoes) .
  • an “scFv dimer” is a bispecific diabody comprising V H1 -V L2 (linked by a peptide linker) associated with V L1 -V H2 (also linked by a peptide linker) such that V H1 and V L1 coordinate and V H2 and V L2 coordinate and each coordinated pair has a different antigen specificity.
  • a fully human antibody as used herein, with reference to antibody or antigen-binding fragment, means that the antibody or the antigen-binding fragment has or consists of amino acid sequence (s) corresponding to that of an antibody produced by a human or a human immune cell, or derived from a non-human source such as a transgenic non-human animal that utilizes human antibody repertoires or other human antibody-encoding sequences.
  • a fully human antibody does not comprise amino acid residues (in particular antigen-binding residues) derived from a non-human antibody.
  • humanized as used herein, with reference to antibody or antigen-binding fragment, means that the antibody or the antigen-binding fragment comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human.
  • a humanized antibody or antigen-binding fragment is useful as human therapeutics in certain embodiments because it has reduced immunogenicity in human.
  • the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.
  • the humanized antibody or antigen-binding fragment is composed of substantially all human sequences except for the CDR sequences which are non-human.
  • the FR regions derived from human may comprise the same amino acid sequence as the human antibody from which it is derived, or it may comprise some amino acid changes, for example, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 changes of amino acid. In some embodiments, such change in amino acid could be present in heavy chain FR regions only, in light chain FR regions only, or in both chains.
  • the humanized antibodies comprise human FR1-3 and human JH and J ⁇ .
  • chimeric means an antibody or antigen-binding fragment, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species.
  • a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human species, such as from mouse.
  • PD-1 refers programmed cell death protein, which belongs to the superfamily of immunoglobulin and functions as coinhibitory receptor to negatively regulate the immune system.
  • PD-1 is a member of the CD28/CTLA-4 family, and has two known ligands including PD-L1 and PD-L2.
  • Representative amino acid sequence of human PD-1 is disclosed under the NCBI accession number: NP_005009.2, and the representative nucleic acid sequence encoding the human PD-1 is shown under the NCBI accession number: NM_005018.2.
  • PD-L1 refers to programmed cell death ligand 1 (PD-L1, see, for example, Freeman et al. (2000) J. Exp. Med. 192: 1027) .
  • Representative amino acid sequence of human PD-L1 is disclosed under the NCBI accession number: NP_054862.1, and the representative nucleic acid sequence encoding the human PD-L1 is shown under the NCBI accession number: NM_014143.3.
  • PD-L1 is expressed in placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumor or cancer cells.
  • PD-L1 binds to its receptor PD-1 or B7-1, which is expressed on activated T cells, B cells and myeloid cells.
  • the binding of PD-L1 and its receptor induces signal transduction to suppress TCR-mediated activation of cytokine production and T cell proliferation.
  • PD-L1 plays a major role in suppressing immune system during particular events such as pregnancy, autoimmune diseases, tissue allografts, and is believed to allow tumor or cancer cells to circumvent the immunological checkpoint and evade the immune response.
  • Anti-PD-1 antibody refers to an antibody that is capable of specific binding to PD-1 (e.g. human or monkey PD-1) with an affinity which is sufficient to provide for diagnostic and/or therapeutic use.
  • the term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen.
  • the antibodies or antigen-binding fragments provided herein specifically bind human and/or monkey PD-1 with a binding affinity (K D ) of ⁇ 10 -6 M (e.g., ⁇ 5x10 -7 M, ⁇ 2x10 -7 M, ⁇ 10 -7 M, ⁇ 5x10 -8 M, ⁇ 2x10 -8 M, ⁇ 10 -8 M, ⁇ 5x10 -9 M, ⁇ 2x10 -9 M, ⁇ 10 -9 M, 10 -10 M) .
  • K D refers to the ratio of the dissociation rate to the association rate (k off /k on ) , may be determined using surface plasmon resonance methods for example using instrument such as Biacore.
  • the ability to “block binding” or “compete for the same epitope” as used herein refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g. human PD-1 and an anti-PD-1 antibody) to any detectable degree.
  • an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50%. In certain embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.
  • epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies may bind the same epitope within an antigen if they exhibit competitive binding for the antigen. For example, if an antibody or antigen-binding fragment as disclosed herein blocks binding of the exemplary antibodies such as 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb to human PD-1, then the antibody or antigen-binding fragment may be considered to bind the same epitope as those exemplary antibodies.
  • “1.7.3 hAb” as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 45, light chain variable region of SEQ ID NO: 47, and a human constant region of IgG4 isotype.
  • hAb refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 49, light chain variable region of SEQ ID NO: 51, and a human constant region of IgG4 isotype.
  • 1.103.11 hAb refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 53, light chain variable region of SEQ ID NO: 55, and a human constant region of IgG4 isotype.
  • 1.139.15 hAb refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 57, light chain variable region of SEQ ID NO: 59, and a human constant region of IgG4 isotype.
  • “1.153.7 hAb” as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 61, light chain variable region of SEQ ID NO: 63, and a human constant region of IgG4 isotype.
  • a “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties.
  • conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g. Met, Ala, Val, Leu, and Ile) , among residues with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn and Gln) , among residues with acidic side chains (e.g. Asp, Glu) , among amino acids with basic side chains (e.g. His, Lys, and Arg) , or among residues with aromatic side chains (e.g. Trp, Tyr, and Phe) .
  • conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.
  • Percent (%) sequence identity with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F.
  • T cell as used herein includes CD4 + T cells, CD8 + T cells, T helper 1 type T cells, T helper 2 type T cells, T helper 17 type T cells and inhibitory T cells.
  • effector functions refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as C1 complex and Fc receptor.
  • exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to Fc receptor on an effector cell; and phagocytosis.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Cancer or “cancerous condition” as used herein refers to any medical condition mediated by neoplastic or malignant cell growth, proliferation, or metastasis, and includes both solid cancers and non-solid cancers such as leukemia.
  • Tumor refers to a solid mass of neoplastic and/or malignant cells.
  • Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.
  • “treating” or “treatment” may refer to inhibiting or slowing neoplastic or malignant cell growth, proliferation, or metastasis, preventing or delaying the development of neoplastic or malignant cell growth, proliferation, or metastasis, or some combination thereof.
  • “treating” or “treatment” includes eradicating all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.
  • an “isolated” substance has been altered by the hand of man from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated, ” but the same polynucleotide or polypeptide is “isolated” if it has been sufficiently separated from the coexisting materials of its natural state so as to exist in a substantially pure state.
  • the antibodies and antigen-binding fragments have a purity of at least 90%, 93%, 95%, 96%, 97%, 98%, 99%as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) , or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC) .
  • electrophoretic methods such as SDS-PAGE, isoelectric focusing, capillary electrophoresis
  • chromatographic methods such as ion exchange chromatography or reverse phase HPLC
  • vector refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein.
  • a vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell.
  • vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • a vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication.
  • a vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.
  • host cell refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.
  • a “disease associated with or related to PD-1” as used herein refers to any condition that is caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of PD-1 (e.g. a human PD-1) .
  • a therapeutically effective amount refers to the dosage or concentration of a drug effective to treat a disease or condition associated with human PD-1.
  • a therapeutically effective amount is the dosage or concentration of the antibody or antigen-binding fragment capable of eradicating all or part of a tumor, inhibiting or slowing tumor growth, inhibiting growth or proliferation of cells mediating a cancerous condition, inhibiting tumor cell metastasis, ameliorating any symptom or marker associated with a tumor or cancerous condition, preventing or delaying the development of a tumor or cancerous condition, or some combination thereof.
  • pharmaceutically acceptable indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
  • the present disclosure provides anti-PD-1 antibodies and the antigen-binding fragments thereof.
  • PD-1 also called as CD279
  • CD279 is known as a key immune-checkpoint receptor expressed by activated T cells, which mediates immunosuppression.
  • PD-1 ligand 1 (PD-L1) is a 40 kDa transmembrane protein expressed on various tumor cells, stromal cells or both, and binds to PD-1. Inhibition of the interaction between PD-1 and PD-L1 can enhance T-cell responses and thus mediates anti-cancer activity.
  • the present disclosure provides exemplary fully human monoclonal antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb, whose CDR sequences are shown in the below Table 1, and heavy or light chain variable region sequences are also shown below.
  • V segment IGHV4-39*01
  • V segment IGLV2-14*01
  • hAb-VH (20951-VH) : (SEQ ID NO: 49 for amino acid and SEQ ID NO: 50 for nucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 13, 15, 5 are amino acid sequences and SEQ ID NO: 14, 16, 6 are nucleic acid sequences, respectively:
  • V segment IGHV4-39*01
  • hAb-VL (21526-VL) : (SEQ ID NO: 51 for amino acid and SEQ ID NO: 52 for nucleic acid) with light chain CDRs 1-3: SEQ ID NOs: 7, 17, 11 are amino acid sequences and SEQ ID NO: 8, 18, 12 are nucleic acid sequences, respectively:
  • V segment IGLV2-14*01
  • V segment IGHV4-39*01
  • hAb-VL (21038-VL) : (SEQ ID NO: 55 for amino acid and SEQ ID NO: 56 for nucleic acid) with light chain CDRs 1-3: SEQ ID NOs: 7, 17, 19 are amino acid sequences and SEQ ID NO: 8, 18, 20 are nucleic acid sequences, respectively:
  • V segment IGLV2-14*01
  • hAb-VH (23521-VH) (SEQ ID NO: 57 for amino acid and SEQ ID NO: 58 for nucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 21, 23, 25 are amino acid sequences and SEQ ID NO: 22, 24, 26 are nucleic acid sequences, respectively:
  • V segment IGHV4-39*01
  • V segment IGLV2-18*02
  • V segment IGLV3-9*01
  • the anti-PD-1 antibodies and the antigen-binding fragments thereof comprise a heavy chain CDR sequences selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35 and 37. In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof comprise a light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 7, 9, 11, 17, 19, 27, 29, 31, 39, 41, and 43.
  • the anti-PD-1 antibodies and the antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of: a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5; a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 5;aheavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 15, and/or SEQ ID NO: 5; a heavy chain variable region comprising SEQ ID NO: 21, SEQ ID NO: 23, and/or SEQ ID NO: 25; and a heavy chain variable region comprising SEQ ID NO: 33, SEQ ID NO: 35, and/or SEQ ID NO: 37.
  • the anti-PD-1 antibodies and the antigen-binding fragments thereof comprise a light chain variable region selected from the group consisting of: a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9, and/or SEQ ID NO: 11; a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 11; a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 19; a light chain variable region comprising SEQ ID NO: 27, SEQ ID NO: 29, and/or SEQ ID NO: 31; and a light chain variable region comprising SEQ ID NO: 39, SEQ ID NO: 41, and/or SEQ ID NO: 43.
  • the anti-PD-1 antibodies and the antigen-binding fragments thereof comprising: a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5; and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9, and/or SEQ ID NO: 11; b) a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 5; and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 11; c) aheavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 15, and/or SEQ ID NO: 5; and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 19; d) a heavy chain variable region comprising SEQ ID NO: 21, SEQ ID NO: 23, and/or SEQ ID NO: 25 and a light chain variable
  • the CDR sequences provided in Table 1 can be modified to contain one or more substitutions of amino acids, so as to provide for an improved biological activity such as improved binding affinity to human PD-1.
  • a library of antibody variants such as Fab or scFv variants
  • computer software can be used to virtually simulate the binding of the antibodies to human PD-1, and identify the amino acid residues on the antibodies which form the binding interface. Such residues may be either avoided in the substitution so as to prevent reduction in binding affinity, or targeted for substitution to provide for a stronger binding.
  • at least one (or all) of the substitution (s) in the CDR sequences is conservative substitution.
  • the antibodies and the antigen-binding fragments thereof comprise one or more CDR sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 1, and in the meantime retain the binding affinity to human PD-1 at a level similar to or even higher than its parental antibody having substantially the same sequence except that the corresponding CDR sequence is in 100%sequence identity to that (or those) listed in Table 1.
  • the anti-PD-1 antibodies and the antigen-binding fragments thereof are fully human.
  • the fully human antibodies do not have the issues of immunogenicity in human and/or reduced binding affinity as often observed with humanized antibodies.
  • the fully human anti-PD-1 antibodies and the antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, and a homologous sequence thereof having at least 80% (e.g.
  • sequence identity at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity; and/or a light chain variable region selected from the group consisting of: SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, and a homologous sequence thereof having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity.
  • Fully human antibodies retain the binding affinity to human PD-1, preferably at a level similar to one of the exemplary antibodies: 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb.
  • the fully human anti-PD-1 antibodies and the antigen-binding fragments thereof comprise: a) a heavy chain variable region comprising SEQ ID NO: 45; and a light chain variable region comprising SEQ ID NO: 47; b) a heavy chain variable region comprising SEQ ID NO: 49; and a light chain variable region comprising SEQ ID NO: 51; c) a heavy chain variable region comprising SEQ ID NO: 53; and a light chain variable region comprising SEQ ID NO: 55; d) a heavy chain variable region comprising SEQ ID NO: 57; and a light chain variable region comprising SEQ ID NO: 59; or e) a heavy chain variable region comprising SEQ ID NO: 61; and a light chain variable region comprising SEQ ID NO: 63.
  • antibodies and the antigen-binding fragments that compete for the same epitope with the anti-PD-1 antibodies and the antigen-binding fragments thereof provided herein.
  • the antibodies block binding of 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, or 1.153.7 hAb to human or monkey PD-1, for example, at an IC 50 value (i.e. 50%inhibition concentration) of below10 -6 M, below 10 -7 M, below 10 -7.5 M, below 10 -8 M, below 10 -8.5 M, below 10 -9 M, or below 10 -10 M.
  • the IC 50 values are determined based on a competition assay such as ELISA assays, radioligand competition binding assays, and FACS analysis.
  • the anti-PD-1 antibodies and the antigen-binding fragments thereof provided herein are capable of specifically binding to human PD-1 with a binding affinity (Kd) of ⁇ 10 -6 M (e.g., ⁇ 5x10 -7 M, ⁇ 2x10 -7 M, ⁇ 10 -7 M, ⁇ 5x10 -8 M, ⁇ 2x10 -8 M, ⁇ 10 -8 M, ⁇ 5x10 -9 M, ⁇ 2x10 -9 M, ⁇ 10 -9 M, 10 -10 M) as measured by plasmon resonance binding assay.
  • Kd binding affinity
  • the binding affinity can be represented by K D value, which is calculated as the ratio of dissociation rate to association rate (k off /k on ) when the binding between the antigen and the antigen-binding molecule reaches equilibrium.
  • the antigen-binding affinity e.g. K D
  • K D can be appropriately determined using suitable methods known in the art, including, for example, plasmon resonance binding assay using instruments such as Biacore (see, for example, Murphy, M. et al, Current protocols in protein science, Chapter 19, unit 19.14, 2006) .
  • the antibodies and the fragments thereof provided herein binds to human PD-1 with an EC 50 (i.e. 50%binding concentration) of 0.1nM-100nM (e.g. 0.1nM-50nM, 0.1nM-30nM, 0.1nM-20nM, 0.1nM-10nM, or 0.1nM-1nM) .
  • Binding of the antibodies to human PD-1 can be measured by methods known in the art, for example, sandwich assay such as ELISA, Western Blot, FACS or other binding assay.
  • the test antibody i.e.
  • first antibody is allowed to bind to immobilized human PD-1 or cells expressing human PD-1, after washing away the unbound antibody, a labeled secondary antibody is introduced which can bind to and thus allow detection of the bound first antibody.
  • the detection can be conducted with a microplate reader when immobilized PD-1 is used, or by using FACS analysis when cells expressing human PD-1 are used.
  • the antibodies and the fragments thereof provided herein binds to human PD-1 with an EC 50 (i.e. 50%effective concentration) of 1nM to 10nM, or 1nM to 5nM as measured by FACS analysis.
  • the antibodies and the fragments thereof provided herein inhibit the binding of human PD-1 to its ligand at an IC 50 of 0.2nM-100nM (e.g. 0.2nM-50nM, 0.2nM-30nM, 0.2nM-20nM, 0.2nM-10nM, or 1nM-10nM) , as measured in a competition assay.
  • 0.2nM-100nM e.g. 0.2nM-50nM, 0.2nM-30nM, 0.2nM-20nM, 0.2nM-10nM, or 1nM-10nM
  • the antibodies and the fragments thereof provided herein block binding of human PD-1 to its ligand and thereby providing biological activity including, for example, inducing cytokine production from the activated T cells (such as CD4 + T cells and CD8 + T cells) , inducing proliferation of activated T cells (such as CD4+ T cells and CD8 + T cells) , and reversing T reg’s suppressive function.
  • cytokines include IL-2 and IFN ⁇ .
  • IL-2 refers to interleukin 2, a type of cytokine signaling molecule in the immune system that regulates the activities of white blood cells (e.g. leukocytes) .
  • Interferon gamma is a cytokine that is produced by natural killer (NK) , NK T cells, CD4 + and CD8 + T cells, which is a critical activator of macrophages and inducer of major histocompatibility complex (MHC) molecule expression.
  • NK natural killer
  • MHC major histocompatibility complex
  • the cytokine production can be determined using methods known in the art, for example, by ELISA. Methods can also be used to detect proliferation of T cells, including [ 3 H] thymidine incorporation assay.
  • the anti-PD-1 antibodies and the antigen-binding fragments thereof are specific for PD-1.
  • the antibodies and antigen-binding fragments thereof do not bind to CD28 and/or CTLA-4.
  • the binding affinity with CD28 and/or CTLA-4 is less than 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%of that with PD-1.
  • the antibodies and antigen-binding fragments thereof bind to monkey PD-1 at an EC50 of no more than 100nM, for example, no more than or about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, or 0.01nM, as measured by ELISA. In certain embodiments, the antibodies and antigen-binding fragments thereof bind to monkey PD-1 at an EC50 of about 1 nM -10nM.
  • the antibodies and antigen-binding fragments thereof do not bind to mouse PD-1 but bind to monkey PD-1 with a binding affinity similar to that of human PD-1.
  • binding of the exemplary antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb to mouse PD-1 is not detectable in conventional binding assays such as ELISA, or FACS analysis, whereas the binding of these antibodies to monkey PD-1 is at a similar affinity or EC50 value to that of human PD-1 as measured by ELISA or FACS.
  • the anti-PD-1 antibodies and the antigen-binding fragments thereof has reduced or depleted effector function. In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof have a constant region of IgG4 isotype, which has reduced or depleted effector function. Effector functions such as ADCC and CDC can lead to cytotoxicity to cells expressing PD-1. Many cells such as T cells normally express PD-1. In order to avoid potential unwanted toxicity to those normal cells, certain embodiments of the antibodies and antigen-binding fragments provided herein can possess reduced or even depleted effector functions.
  • ADCC or CDC activities for example, Fc receptor binding assay, C1q binding assay, and cell lysis assay, and can be readily selected by people in the art.
  • ADCC or CDC antibodies with reduced or depleted effector functions such as ADCC or CDC would cause no or minimal cytotoxicity to PD-1-expressing cells, for example those T cells, and therefore spare them from unwanted side effects, whereas in the meantime, blocking of PD-1 would boost immune system for the treatment of conditions such as cancer or chronic infection.
  • the anti-PD-1 antibodies and antigen-binding fragments thereofprovided herein have reduced side effects.
  • the antibodies and antigen-binding fragments thereof provided herein can have fully human IgG sequence and therefore reduced immunogenicity than a humanized antibody counterpart.
  • the antibodies and antigen-binding fragments thereof provided herein can be in IgG4 format to eliminate ADCC and CDC.
  • the anti-PD-1 antibodies and antigen-binding fragments thereof provided herein are advantageous in that they can be used in combination with immunogenic agents, such as tumor cells, purified tumor antigen, and cells transfected with genes encoding immune stimulating cytokines, tumor vaccines.
  • immunogenic agents such as tumor cells, purified tumor antigen, and cells transfected with genes encoding immune stimulating cytokines, tumor vaccines.
  • the anti-PD-1 antibodies and antigen-binding fragments thereof can be included in combination therapies, including standard chemo-and radio-therapies, target based small molecule therapies, emerging other immune checkpoint modulator therapies.
  • the antibodies and antigen-binding fragments thereof can be used as the base of antibody-drug conjugates, bispecific or multivalent antibodies.
  • the anti-PD-1 antibodies or antigen-binding fragments thereof provided herein can be a monoclonal antibody, polyclonal antibody, fully human antibody, humanized antibody, chimeric antibody, recombinant antibody, bispecific antibody, labeled antibody, bivalent antibody, or anti-idiotypic antibody.
  • a recombinant antibody is an antibody prepared in vitro using recombinant methods rather than in animals.
  • a bispecific or bivalent antibody is an artificial antibody having fragments of two different monoclonal antibodies and can bind two different antigens.
  • An antibody or antigen-binding fragment thereof that is “bivalent” comprises two antigen-binding sites. The two antigen binding sites may bind to the same antigen, or they may each bind to a different antigen, in which case the antibody or antigen-binding fragment is characterized as “bispecific. ”
  • the anti-PD-1 antibodies or antigen-binding fragments thereof provided herein are fully human antibodies.
  • the fully human antibodies are prepared using recombinant methods. For example, transgenic animal such as a mouse can be made to carry transgenes or transchromosomes of human immunoglobulin genes, and therefore capable of producing fully human antibodies after immunization with proper antigen such as human PD-1.
  • Fully human antibodies can be isolated from such transgenic animal, or alternatively, can be made by hybridoma technology by fusing the spleen cells of the transgenic animal with an immortal cell line to generate hybridoma cells secreting the fully human antibodies.
  • Exemplary transgenic animals include, without limitation, OmniRat, whose endogenous expression of rat immunoglobulin genes are inactivated and at the same time engineered to contain functional recombinant human immunoglobulin loci; OmniMouse, whose endogenous expression of mouse immunoglobulin genes are inactivated and at the same time engineered to contain recombinant human immunoglobulin loci having J-locus deletion and a C-kappa mutation; OmniFlic, which is a transgenic rat whose endogenous expression of rat immunoglobulin genes are inactivated and at the same time engineered to contain recombinant human immunoglobulin loci having a single common, rearranged VkJk light chain and functional heavy chain.
  • OmniRat whose endogenous expression of rat immunoglobulin genes are inactivated and at the same time engineered to contain functional recombinant human immunoglobulin loci
  • OmniMouse whose endogenous expression of mouse immunoglobulin genes are inactivated and
  • the anti-PD-1 antibodies and the antigen-binding fragments thereof is a camelized single domain antibody, a diabody, a scFv, an scFv dimer, a BsFv, a dsFv, a (dsFv) 2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F (ab') 2, a ds diabody, a nanobody, a domain antibody, or a bivalent domain antibody.
  • the anti-PD-1 antibodies and the antigen-binding fragments thereof further comprise an immunoglobulin constant region.
  • an immunoglobulin constant region comprises a heavy chain and/or a light chain constant region.
  • the heavy chain constant region comprises CH1, CH1-CH2, or CH1-CH3 regions.
  • the constant region may further comprise one or more modifications to confer desirable properties. For example, the constant region may be modified to reduce or deplete one or more effector functions, to improve FcRn receptor binding, or to introduce one or more cysteine residues.
  • the anti-PD-1 antibodies and the antigen-binding fragments thereof further comprise a conjugate.
  • a variety of conjugates may be linked to the antibodies or antigen-binding fragments provided herein (see, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J. M. Cruse and R. E.Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) .
  • conjugates may be linked to the antibodies or antigen-binding fragments by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods.
  • the antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugates.
  • a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate.
  • the antibodies may be linked to a conjugate indirectly, or through another conjugate.
  • the antibody or antigen-binding fragments may be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin.
  • the conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purification moiety, or a cytotoxic moiety.
  • detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or ⁇ -D-galactosidase) , radioisotopes (e.g.
  • the conjugate can be a pharmacokinetic modifying moiety such as PEG which helps increase half-life of the antibody.
  • conjugate can be a purification moiety such as a magnetic bead.
  • a “cytotoxic moiety” can be any agent that is detrimental to cells or that can damage or kill cells.
  • cytotoxic moiety examples include, without limitation, 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, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine) , alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lo
  • the present disclosure provides isolated polynucleotides that encode the anti-PD-1 antibodies and the antigen-binding fragments thereof.
  • the isolated polynucleotides comprise one or more nucleotide sequences as shown in Table 1, which encodes the CDR sequences provided in Table 1.
  • the isolated polynucleotides encodes a heavy chain variable region and comprise a sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, and a homologous sequence thereof having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity.
  • the isolated polynucleotides encodes a light chain variable region and comprise a sequence selected from the group consisting of: SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, and a homologous sequence thereof having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity.
  • the percentage identity is due to genetic code degeneracy, while the encoded protein sequence remains unchanged.
  • the isolated polynucleotide that encodes the anti-PD-1 antibodies and the antigen-binding fragments thereof can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art.
  • the antibody may be produced by homologous recombination known in the art.
  • DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) . Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g. SV40, CMV, EF-1 ⁇ ) , and a transcription termination sequence.
  • a signal sequence e.g. a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g. SV40, CMV, EF-1 ⁇ ) , and a transcription termination sequence.
  • the vector system includes mammalian, bacterial, yeast systems, etc, and comprises plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2 etc, and other laboratorial and commercially available vectors.
  • Suitable vectors may include, plasmid, or viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) .
  • Vectors comprising the polynucleotide sequence encoding the antibody or antigen-binding fragment can be introduced to a host cell for cloning or gene expression.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-PD-1 antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12, 424) , K. bulgaricus (ATCC 16, 045) , K. wickeramii (ATCC 24, 178) , K.
  • waltii ATCC 56, 500
  • K. drosophilarum ATCC 36, 906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402, 226)
  • Pichia pastoris EP 183, 070
  • Candida Trichoderma reesia
  • Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalis
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated antibodies or antigen-fragment provided here are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruiffly) , and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • vertebrate cells have been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36: 59 (1977) ) ; baby hamster kidney cells (BHK, ATCC CCL 10) ; Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.
  • mice sertoli cells TM4, Mather, Biol. Reprod. 23: 243-251 (1980) ) ; monkey kidney cells (CV1 ATCC CCL 70) ; African green monkey kidney cells (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cells (HELA, ATCC CCL 2) ; canine kidney cells (MDCK, ATCC CCL 34) ; buffalo rat liver cells (BRL 3A, ATCC CRL 1442) ; human lung cells (W138, ATCC CCL 75) ; human liver cells (Hep G2, HB 8065) ; mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982) ) ; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) .
  • the host cell is 2
  • Host cells are transformed with the above-described expression or cloning vectors for anti-PD-1 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce the antibodies or antigen-binding fragments provided herein may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma) , Minimal Essential Medium (MEM) , (Sigma) , RPMI-1640 (Sigma) , and Dulbecco's Modified Eagle's Medium (DMEM) , Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleotides (such as adenosine and thymidine) , antibiotics (such as GENTAMYCIN TM drug) , trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the antibody prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human . gamma. 1, . gamma. 2, or . gamma. 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) .
  • Protein G is recommended for all mouse isotypes and for human . gamma. 3 (Guss et al., EMBO J. 5: 1567 1575 (1986) ) .
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a CH3 domain
  • the Bakerbond ABX. TM. resin J.T. Baker, Phillipsburg, N.J. ) is useful for purification.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt) .
  • kits comprising the anti-PD-1 antibodies or the antigen-binding fragments thereof.
  • the kits are useful for detecting the presence or level of PD-1 in a biological sample.
  • the biological sample can comprise a cell or a tissue.
  • the kit comprises an anti-PD-1 antibody or the antigen-binding fragment thereof which is conjugated with a detectable label.
  • the kit comprises an unlabeled anti-PD-1 antibody or antigen-binding fragment, and further comprises a secondary labeled antibody which is capable of binding to the unlabeled anti-PD-1 antibody.
  • the kit may further comprise an instruction of use, and a package that separates each of the components in the kit.
  • the anti-PD-1 antibody or the antigen-binding fragment thereof are associated with a substrate or a device useful in a sandwich assay such as ELISA, or in an immunographic assay.
  • a substrate or a device useful in a sandwich assay such as ELISA, or in an immunographic assay.
  • Useful substrate or device can be, for example, microtiter plate and test strip.
  • the present disclosure further provides pharmaceutical compositions comprising the anti-PD-1 antibodies or the antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers.
  • Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
  • Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins.
  • Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate.
  • compositions that comprise one or more antibodies or antigen-binding fragments as disclosed herein and one or more antioxidants such as methionine. Further provided are methods for preventing oxidation of, extending the shelf-life of, and/or improving the efficacy of an antibody or antigen-binding fragment as provided herein by mixing the antibody or antigen-binding fragment with one or more antioxidants such as methionine.
  • pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (
  • Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
  • compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the pharmaceutical compositions are formulated into an injectable composition.
  • the injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion.
  • Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions.
  • the solutions may be either aqueous or nonaqueous.
  • unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
  • a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent.
  • the solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH.
  • the resulting solution will be apportioned into vials for lyophilization.
  • Each vial can contain a single dosage or multiple dosages of the anti-PD-1 antibody or antigen-binding fragment thereof or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing.
  • the lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
  • Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration.
  • the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.
  • Therapeutic methods comprising: administering a therapeutically effective amount of the antibody or antigen-binding fragment as provided herein to a subject in need thereof, thereby treating or preventing a condition or a disorder associated with related to PD-1.
  • methods are provided to treat a condition in a subject that would benefit from upregulation of immune response, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment as provided herein to a subject in need thereof.
  • an antibody or antigen-binding fragment as provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of tumor development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.
  • an antibody or antigen-binding fragment as provided herein may be administered at a therapeutically effective dosage of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg) .
  • the antibody or antigen-binding fragment is administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less.
  • the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) .
  • a single dose may be administered, or several divided doses may be administered over time.
  • the antibodies and antigen-binding fragments disclosed herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.
  • parenteral e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection
  • non-parenteral e.g., oral, intranasal, intraocular, sublingual, rectal, or topical routes.
  • Conditions and disorders associated with PD-1 can be immune related disease or disorder.
  • the PD-1 associated conditions and disorders include tumors and cancers, for example, non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas, mycoses fungoids, merkel cell cancer, and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL) , primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL)
  • the tumors and cancers are metastatic, especially metastatic tumors expressing PD-L1.
  • the PD-1 associated conditions and disorders include autoimmune diseases, such as systemic lupus erythematosus (SLE) , psoriasis, systemic scleroderma, autoimmune diabetes and the like.
  • the PD-1 associated conditions and disorders include infectious disease such as chronic viral infection, for example, viral infection of hepatitis B, hepatitis C, herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type 2, human papilloma virus, adenovirus, Kaposi West sarcoma associated herpes virus epidemics, thin ring virus (Torquetenovirus) , JC virus or BK virus.
  • infectious disease such as chronic viral infection, for example, viral infection of hepatitis B, hepatitis C, herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type 2, human papilloma virus, adenovirus, Kaposi West sarcoma associated herpes virus epidemics, thin ring virus (Torquetenovirus) , JC virus or BK virus.
  • infectious disease such
  • the present disclosure further provides methods of using the anti-PD-1 antibodies or the antigen-binding fragments thereof.
  • the present disclosure provides methods of treating a condition or a disorder associated with related to PD-1 in an individual, comprising administering a therapeutically effective amount of the anti-PD-1 antibody or antigen-binding fragment thereof.
  • the individual has been identified as having a disorder or condition likely to respond to a PD-1 antagonist.
  • the presence or level of PD-L1 on an interested biological sample can be indicative of whether the individual from whom the biological sample is derived could likely respond to a PD-1 antagonist.
  • Various methods can be used to determine the presence or level of PD-L1 in a test biological sample from the individual.
  • the test biological sample can be exposed to anti-PD-L1 antibody or antigen-binding fragment thereof, which binds to and detects the expressed PD-L1 protein.
  • PD-L1 can also be detected at nucleic acid expression level, using methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, and the like.
  • the test sample is derived from a cancer cell or tissue, or tumor infiltrating immune cells.
  • presence or upregulated level of the PD-L1 in the test biological sample indicates likelihood of responsiveness.
  • upregulated refers to an overall increase of no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%or greater, in the protein level of PD-L1 in the test sample as detected using the antibodies or antigen-binding fragments provided herein, as compared to the PD-L1 protein level in a reference sample as detected using the same antibody.
  • the reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from whom the test sample is obtained.
  • the reference sample can be a non-diseased sample adjacent to or in the neighborhood of the test sample (e.g. tumor) .
  • the antibodies or antigen-binding fragments disclosed herein may be administered alone or in combination with one or more additional therapeutic means or agents.
  • the antibodies or antigen-binding fragments disclosed herein may be administered in combination with chemotherapy, radiation therapy, surgery for the treatment of cancer (e.g., tumorectomy) , one or more anti-emetics or other treatments for complications arising from chemotherapy, or any other therapeutic agent for use in the treatment of cancer or any medical disorder mediated by PD-1.
  • an antibody or antigen-binding fragment as disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments the antibody or antigen-binding fragment and the additional therapeutic agent (s) may be administered as part of the same pharmaceutical composition.
  • an antibody or antigen-binding fragment administered “in combination” with another therapeutic agent does not have to be administered simultaneously with or in the same composition as the agent.
  • An antibody or antigen-binding fragment administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the antibody or antigen-binding fragment and second agent are administered via different routes.
  • additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians'Desk Reference 2003 (Physicians'Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002) ) or protocols well known in the art.
  • the therapeutic agents can induce or boost immune response against cancer.
  • a tumor vaccine can be used to induce immune response to certain tumor or cancer.
  • Cytokine therapy can also be used to enhance tumor antigen presentation to the immune system.
  • examples of cytokine therapy include, without limitation, interferons such as interferon- ⁇ , - ⁇ , and – ⁇ , colony stimulating factors such as macrophage-CSF, granulocyte macrophage CSF, and granulocyte-CSF, interleukins such IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12, tumor necrosis factors such as TNF- ⁇ and TNF- ⁇ .
  • interferons such as interferon- ⁇ , - ⁇ , and – ⁇
  • colony stimulating factors such as macrophage-CSF, granulocyte macrophage CSF, and granulocyte-CSF
  • agents that inactivate immunosuppressive targets can also be used, for example, TGF-beta inhibitors, IL-10 inihibitors, and Fas ligand inhibitors.
  • TGF-beta inhibitors IL-10 inihibitors
  • Fas ligand inhibitors Another group of agents include those that activate immune responsiveness to tumor or cancer cells, for example, those enhance T cell activation (e.g. agonist of T cell costimulatory molecules such as CTLA-4, ICOS and OX-40) , and those enhance dendritic cell function and antigen presentation.
  • Immunogen generation DNAs encoding PD-1 and PD-L1 ECD or full length were synthesized and inserted into the expression vector pcDNA3.3. Max-prep the plasmid DNAs and the inserted DNA sequences were verified by sequencing. Fusion proteins PD-1 ECD and PD-L1 ECD containing various tags, including human Fc, mouse Fc and His tags, were obtained by transfection of human PD-1 ECD gene into CHO-S or HEK293 cells. After 5 days, supernatants harvested from the culture of transiently transfected cells were used for protein purification. The fusion proteins were purified and quantitated for usage of immunization and screening.
  • CHO-K1, 293F or Ba/F3 cells were transfected with pCND3.3 expression vector containing full-length PD-1 or PD-L1 using Lipofectamine 2000 Transfection kit according to manufacturer’s protocol.
  • the transfected cells were cultured in medium containing Blasticidin or G418 for selection. Overtime this will select the cells that have stably incorporated PD-1 or PD-L1 genes into their genomic DNAs. Meanwhile the cells were checked for interested genes PD-1 and PD-L1 expression. Once the expression verified, single clones of interested were picked up by limited dilution and scaled up to large volumes. The established monoclonal cell lines then were maintained in medium containing lower dose of antibiotics Blasticidin or G418.
  • OMT-rats obtained from Open Monoclonal Technology, Inc., Palo Alto, US
  • 8-10 weeks of age were immunized with 10 ⁇ g of human PD-1 ECD protein in TiterMax in footpad for first boost, repeat the immunization every 3 days with PD-1 ECD protein in Aluminium.
  • Bleed rats every two weeks for serum collection and antibody titers were measured by ELISA or FACS assay.
  • ELISA assay was used as first screen method to test the binding of hybridoma supernatants to PD-1 protein. Briefly, Plates (Nunc) were coated with soluble protein of human PD-1 extracellular domain at 1 ⁇ g/ml overnight at 4 °C. After blocking and washing, the hybridoma supernatants were transferred to the coated plates and incubate at room temperature for 1 h. The plates were then washed and subsequently incubated with secondary antibody goat anti rat IgG1 HRP (Bethyl) and goat anti rat IgG2b HRP (Bethyl) for 45 min. After washing, TMB substrate was added and the interaction was stopped by 2M HCl.
  • the absorbance at 450 nm was read using a microplate reader (Molecular Device) .
  • FACS analysis was performed on PD-1 transfected CHO-S cell line.
  • CHO-S cells expressing human PD-1 were transferred in to 96-well U-bottom plates (BD) at a density of 1x10 6 cells/ml.
  • the hybridoma supernatants were then transferred to the plates and incubated for 1 h at 4 °C.
  • the secondary antibody goat anti rat FITC Jackson Immunoresearch Lab
  • FIG. 1 shows the binding of anti-human PD-1 antibodies to PD-1 expressing CHO cell.
  • the CHO cells transfected with full-length human PD-1 were stained with antibodies against human PD-1 from rat hybridoma, followed by 2nd antibody staining with FITC conjugated goat anti-rat-lgG Fc and analyzed by FACS. The data show that the antibodies specifically bind to PD-1 expressed on CHO cells.
  • human CD4+T cell were generated from PBMC cultured in IL-2 and OKT3 for 3 days and were stained with the antibodies against human PD-1. Binding of the antibodies to the PD-1 on the T cells were analyzed by FACS. As shown in Figure 3, FACS analysis showed that the antibodies specially bind to native PD-1 expressed on CD4+T cells.
  • CHO-S cells expressing human PD-1 were transferred in to 96-well U-bottom plates (BD) at a density of 1x10 6 cells/ml.
  • Antibodies were serially diluted in wash buffer (1XPBS/1%BSA) and incubated with the cells at 4 °C for 1 h. After washing, human Fc fusion-human PD-L1 protein was added and incubated at 4 °C for 1 h.
  • the secondary antibody goat anti human IgG Fc FITC antibody (no cross-reactivity to rat IgG Fc, Jackson Immunoresearch Lab) was incubated with cells at 4 °C in the dark for 1 h. The cells were then washed and resuspended in 1XPBS/1%BSA or fixed with 4%paraformldehyde, and analyzed by flow cytometery (BD) .
  • Hybridoma subcloning once specific binding and blocking were verified through first and confirmation screen, the positive hybridoma cell lines can be used for subcloning. Briefly, for each hybridoma cell line, cells were counted and diluted to give 5 cells/well, 1 cell/well and 0.5 cell/well in cloning medium. Plate 200 ⁇ l/well into 96-well plates, one plate at 5 cells/well, one plate at 1 cell/well and four plates at 0.5 cell/well. Place all plates in incubator at 37 °C, 5%CO 2 . Incubate until all the cell lines can be checked by ELISA assay.
  • EXAMPLE 2 Antibody hybridoma cell sequence and fully human antibody characterization
  • RNAs were isolated from monoclonal hybridoma cells with Trizol reagent. The VH and VL of PD-1 antibodies were amplified as following protocol: briefly, RNA is first reverse transcribed into cDNA using a reverse transcriptase as described here, Reaction system (20 ⁇ l) :
  • Step1 Step2 Step3 Step4 Temperature 25 37 85 4 Time 10min 120min 5min ⁇
  • the resulting cDNA is used as templates for subsequent PCR amplification using primers specific for interested genes.
  • the PCR reaction was done as following procedure;
  • VH and VL of PD-1 antibodies were amplified as described above.
  • the PCR reactions were purified with PCR clean-up kit and the VL and pCI vector were digested with restriction enzymes Pme I and BssH II at 37 °C for 2 hours. Run the reactions in 1%agarose and do gel extraction with kit according to manufacturer’s instruction. Ligation of digested VL and pCI vector as following procedures:
  • the mixture was incubated at 16 °C for 30 minutes. 10 ⁇ l of the reactions was used for tansformation and clone growth. Confirmed clones were used for the extraction of the plasmid pCI-VL DNA.
  • the pCI-VL vector and VH fragment were then digested with Xbal and Sal I and the purified digested VH and vector were ligated with T4 DNA ligase 30minutes at 16 °C. Once the sequence of inserted VL and VH were verified by sequencing, the expression vector containing whole IgG of fully human PD-1 antibody was used for transient transfection and stable cell line development.
  • SPR surface Plasmon resonance
  • the affinities of antibodies against PD-1 for recombinant human PD-1 were from 3.76E-9 to 1.76E-10 mol/L.
  • FIG. 1 shows the binding of the fully human PD-1 antibodies (i.e. 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb) to PD-1 expressing CHO cell.
  • Fully human antibodies against human PD-1 were used to stain the PD-1 transfected CHO cells and the FACS analysis show that fully human PD-1 antibodies specially bind to PD-1 with EC 50 about 2nmol/L.
  • CHO cells expressing human PD-1 were incubated with different concentrations of the antibodies against PD-1, then the mouse Fc-tagged human PD-L1 was added to the cells.
  • the binding of human PD-L1 to PD-1 expressing cell was detected by using FITC-conjugated goat anti-mouse IgG, followed by the FACS analysis. As shown in Figure 4, antibodies against PD-1 blocked the binding of PD-L1 to PD-1 transfected CHO cells.
  • Figure 5 shows that CHO cells transfected with PD-1, CD28 and 293F transfected with CTLA4 were stained with antibodies against PD-1 and analyzed by FACS. The result demonstrates PD-1 antibodies bind specifically to PD-1, but not to CD28 and CTLA4 of PD-1 family.
  • the binding epitope of PD-1 antibodies was binned against benchmark antibody A and B by FACS.
  • CHO cells expressing human PD-1 at the cell surface were incubated with benchmark antibody A or B at concentration of 10 ⁇ g/ml for 1 hour.
  • the cells were washed and the PD-1 antibodies of the disclosure were added and incubated for 1 hour.
  • the second antibody anti-rat IgG-FITC were added and incubated for 1 hour at 4 °C.
  • the cells were then washed once and resuspended in 1XPBS/1%BSA, and analyzed by flow cytometery (BD) .
  • BD flow cytometery
  • Human dendritic cells (DC) and CD4 + T, CD8 + T and total cells used in above allo-MLR were generated from the PBMC as following procedures: Human monocytes were purified from PBMC by negative selection using human monocyte enrichment cocktail kit according to the instructions of the manufacturer (StemCell Meylan) . Briefly, PBMC were isolated from blood of healthy donor using a Ficoll-Paque gradient. Cells were washed twice with PBS, then resuspended at 1X10 8 cells/ml in isolation buffer, and incubated with the monocyte enrichment Ab mixture at 4°C for 30 min. The cells were washed and subsequently incubated with magnetic colloid at 4°C for 30 min.
  • monocytes passed through the MACS column and were collected.
  • monocytes were cultured in RPMI 1640 medium containing 10%FCS and antibiotics with GM-CSF (PeproTech, Rocky Hill, NJ; 800 U/ml) and IL-4 (PeproTech; 500 U/ml) at concentration of 2X10 6 cells/ml. Half the medium was replaced every other day with GM-CSF-and IL-4-containing medium.
  • Mature DCs were generated by stimulating iDCs with LPS (026: B6; Sigma-Aldrich, St. Louis, MO; 1 ⁇ g/ml) on day 5 for an additional 24 h.
  • CD4 + T, CD8 + T and total T cells were purified by negative selection by incubating PBMC with human CD4 + T , CD8 + T and total T cell enrichment mixture and magnetic colloid according to the manufacturer’s instructions (Stemsep) .
  • Human CD4 + T Cells were stimulated with allogenenic DCs in the presence or absence of PD-1 antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb.
  • the proliferation of CD4 + T cells were assessed by [ 3 H] thymidine incorporation.
  • Figure 10 showed that 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb enhanced concentration dependent T cell proliferation.
  • the cultures were conducted in the presence or absence of human PD-1 antibodies and benchmark antibodies. After 5 days, the supernatants from each culture were harvested for cytokine IFN ⁇ measurement. The level of IFN ⁇ in supernatants was measured by ELISA assay. Briefly, Coat Maxisorp plates with anti-human IFN-gamma mAb diluted in coating buffer (0.75 ⁇ g/ml; i.e. a1/1360 dilution) , 50 ⁇ l/well (i.e. for a full 96-well plate add 3.7 ⁇ l of antibody to 5 ml of coating buffer) and incubated overnight at 4 °C. Block spare protein binding capacity by adding 200 ⁇ l/well of blocking buffer for 2 hours.
  • Figure 9 shows that human CD4 + T Cells were stimulated with allogenenic DCs in the presence or absence of antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb.
  • the level of IFN ⁇ was measured by ELISA.
  • the result showed the fully human PD-1 antibodies increased IFN ⁇ secretion in a dose manner.
  • Figure 8 shows that human CD4 + T Cells were stimulated with allogenenic DCs in the presence or absence of lead antibodies or control Ab.
  • the level of IL-2 was measured by ELISA. The results showed antibodies against PD-1 increased IL-2 secretion in a dose-dependent manner.
  • PD-1 antibodies enhanced concentration dependent CMV + -CD4 + T cell proliferation stimulated with CMV pp65 peptide-loaded autologous DC.
  • Tregs regulatory T cell suppressive function
  • CD4 + CD25 + regulatory T cell are associated with tumors because increased numbers of Tregs were found in patients with multiple cancers and is associated with a poorer prognosis.
  • CD4 + CD25 + and CD4 + CD25 - T cells were separated using specific anti-CD25 microbeads (Miltenyi Biotec, Auburn, CA) and positive or negative selection, respectively.
  • CD4 + T cells were purified by negative selection by incubating PBMC with human CD4 + T cell enrichment mixture and magnetic colloid according to the manufacturer’s instructions (Stemsep) .
  • CD4 + T cells were then resuspended in MACS buffer, incubated with CD25 + microbeads on ice for 30 min, washed, and loaded on the column.
  • CD4 + CD25 - T cells which did not bind to the column, were collected from the flow-through and washed before use.
  • CD4 + CD25 + T cells were subsequently retrieved from the column and washed before use.
  • Tregs were cultured with CD4 + CD25 - T cells and DCs (Treg: Teff 1: 1ratio) in the presence or absence of human PD-1 antibodies at a concentration of 10 ⁇ g/ml. Either no antibody or isotype antibody was used as negative control.
  • the supernatants from the cultures were taken on day 5 for cytokines detection by ELISA and the cell proliferation was measured by adding [ 3 H] thymidine at a concentration of 1uCi/well and incubated for further 18 hours. [ 3 H] thymidine incorporation was measured by scintillation counting.
  • the PD-1 antibodies abrogated Treg’s suppressive function and restored responding T cell proliferation and IFN ⁇ secretion.
  • ADCC/CDC assay to minimize the undesired toxicity on healthy PD-1 + cells, the selected anti-PD-1 fully human antibodies were confirmed to have no ADCC and CDC function.
  • ADCC Activated T cells expressing high levels of cell surface PD-1 were used as target cells and were pre-incubated with various concentrations of fully human antibodies in 96-well plates for 30min, then IL-2-activated PBMCs (used as a source of natural killer (NK) cells, i.e. the effector cells) were added at the effector/target ratio of 50: 1. The plates were incubated for 6 hours at 37°C in a 5%CO 2 incubator. Target cell lysis was determined by cytotoxicity detection kit (Roche) . Optical density was measured by Molecular Devices SpectraMax M5e Plate Reader. Results showed that, the tested fully human antibodies against PD-1 did not mediate ADCC (Figure 13) .
  • CDC target cells (activated T cell) , diluted human serum complement (Quidel-A112) and various concentrations of fully human PD-1 antibodies were mixed in a 96-well plate. The plate was incubated for 4 h at 37°C in a 5%CO 2 incubator. Target cell lysis was determined by CellTiter glo (Promega-G7573) . Rituxan (Roche) and human B lymphoma cell line Raji (CD20 positive) were used as positive control. The data showed that PD-1 antibodies did not mediated CDC ( Figure 14) .

Abstract

The present disclosure provides monoclonal antibodies against protein programmed cell death 1 (PD-1), which can block the binding of PD-1 ligands to PD-1, and therefore block the inhibitory function of PD-1 ligands on PD-1 expressing T cells. The antibodies of disclosure provide very potent agents for the treatment of multiple cancers via modulating human immune function.

Description

NOVEL ANTI-PD-1 ANTIBODIES FIELD OF THE INVENTION
The present disclosure generally relates to novel anti-PD-1 antibodies.
BACKGOROUND
Increasing evidences from preclinical and clinical results have shown that targeting immune checkpoints is becoming the most promising approach to treat patients with cancers. Programmed cell death 1, one of immune-checkpoint proteins, play a major role in limiting the activity of T cells that provide a major immune resistance mechanism by which tumor cells escaped immune surveillance. The interaction of PD-1 expressed on activated T cells, and PD-L1 expressed on tumor cells negatively regulate immune response and damp anti-tumor immunity. Expression of PD-L1 on tumors is correlated with reduced survival in esophageal, pancreatic and other types of cancers, highlighting this pathway as a new promising target for tumor immunotherapy. Multiple agents targeting PD-1 pathway have been developed by pharmaceutical companies, such as Bristol-Myers Squibb (BMS) , Merck, Roche and GlaxoSmithKline (GSK) . Data from clinical trials demonstrated early evidence of durable clinical activity and an encouraging safety profile in patients with various tumor types. Nivolumab, a PD-1 drug developed by BMS, is being put at center stage of the next-generation field. Now in 6 late-stage studies, the treatment spurred tumor shrinkage in three of 5 cancer groups studied, including 18％of 72 lung cancer patients, close to a third of 98 melanoma patients and 27％of 33 patients with kidney cancer. Developed by Merck, lambrolizumab is a fully human monoclonal IgG4 antibody that acts against PD-1, which grabbed the FDA's new breakthrough designation after impressive IB data came through for skin cancer. The results from a phase IB study have shown an objective anti-tumor response in 51％of 85 cancer patients, and a complete response in 9％of patients. Roche's experimental MPDL3280A demonstrated an ability to shrink tumors in 29 of 140 (21％) advanced cancer patients with various tumor sizes.
However, the existing therapies may not be all satisfactory and therefore new anti-PD-1 antibodies are still needed.
BRIEF SUMMARY OF THE INVENTION
The present disclosure provides novel monoclonal anti-PD-1 antibodies (in particular fully human antibodies) , polynucleotides encoding the same, and methods of using the same.
In one aspect, the present disclosure provides isolated monoclonal antibodies or antigen binding fragments thereof, which are capable of specifically binding to human PD-1 at a Kd value no more than 10-8 M (e.g. no more than ≤9x10-9 M, ≤8x10-9 M, ≤7x10-9 M, ≤6x10-9 M, ≤5x10-9 M, ≤4x10-9 M, ≤3x10-9 M, ≤2x10-9 M, or ≤10-9 M) as measured by plasmon resonance binding assay.
In certain embodiments, the antibodies or antigen binding fragments thereof bind to monkey PD-1 at an EC50 of no more than 100 nM or no more than 10nM (e.g. no more than 50nM, 40nM, 30nM, 20nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM, ) . In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind to mouse PD-1 but bind to monkey PD-1 with a binding affinity similar to that of human PD-1. In certain embodiments, the antibodies or antigen binding fragments thereof potently inhibit binding of human or monkey PD-1 to its ligand (e.g. PD-L1 or PD-L2) , at an IC50 of no more than 100 nM (e.g. no more than 50nM, 40nM, 30nM, 20nM, 10 nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1 nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, or 0.1nM) . In certain embodiments, the EC50 or IC50 is measured by fluorescence-activated cell sorting (FACS) analysis.
In certain embodiments, the antibodies or antigen binding fragments thereof have substantially reduced effector function. In certain embodiments, the antibodies or antigen binding fragments thereof do not mediate ADCC or CDC or both.
In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise a heavy chain CDR sequences selected from the group consisting of:SEQ ID NOs: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35 and 37.
In one aspect, the antibodies or an antigen binding fragments thereof provided herein comprise a light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 7, 9, 11, 17, 19, 27, 29, 31, 39, 41, and 43.
In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise at least one, two, three, four, five or six CDRs selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 7, 9, and 11； or selected from the group consisting  of:SEQ ID NOs: 13, 15, 5, 7, 17 and 11； or selected from the group consisting of: SEQ ID NOs: 1, 15, 5, 7, 17 and 19； or selected from the group consisting of: SEQ ID NOs: 21, 23, 25, 27, 29 and 31； or selected from the group consisting of: SEQ ID NOs: 33, 35, 37, 39, 41 and 43.
In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise a heavy chain variable region selected from the group consisting of:
a)a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5；
b)a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 5；
c)a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 15, and/or SEQ ID NO: 5；
d)a heavy chain variable region comprising SEQ ID NO: 21, SEQ ID NO: 23, and/or SEQ ID NO: 25； and
e)a heavy chain variable region comprising SEQ ID NO: 33, SEQ ID NO: 35, and/or SEQ ID NO: 37.
In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise a light chain variable region selected from the group consisting of:
a)a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9, and/or SEQ ID NO: 11；
b)a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 11；
c)a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 19；
d)a light chain variable region comprising SEQ ID NO: 27, SEQ ID NO: 29, and/or SEQ ID NO: 31； and
e)a light chain variable region comprising SEQ ID NO: 39, SEQ ID NO: 41, and/or SEQ ID NO: 43.
In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise:
a)a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5； and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO:  9, and/or SEQ ID NO: 11；
b)a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 5； and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO:17, and/or SEQ ID NO: 11；
c)a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 15, and/or SEQ ID NO: 5； and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO:17, and/or SEQ ID NO: 19；
d)a heavy chain variable region comprising SEQ ID NO: 21, SEQ ID NO: 23, and/or SEQ ID NO: 25 and a light chain variable region comprising SEQ ID NO: 27, SEQ ID NO: 29, and/or SEQ ID NO: 31； or
e)a heavy chain variable region comprising SEQ ID NO: 33, SEQ ID NO: 35, and/or SEQ ID NO: 37； and a light chain variable region comprising SEQ ID NO: 39, SEQ ID NO: 41, and/or SEQ ID NO: 43.
In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise a heavy chain variable region selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57 and SEQ ID NO: 61.
In certain embodiments, the antibodies or antigen binding fragments provided herein comprise a light chain variable region selected from the group consisting of: SEQ ID NO:47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, and SEQ ID NO: 63.
In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise:
a)a heavy chain variable region comprising SEQ ID NO: 45； and a light chain variable region comprising SEQ ID NO: 47；
b)a heavy chain variable region comprising SEQ ID NO: 49； and a light chain variable region comprising SEQ ID NO: 51；
c)a heavy chain variable region comprising SEQ ID NO: 53； and a light chain variable region comprising SEQ ID NO: 55；
d)a heavy chain variable region comprising SEQ ID NO: 57； and a light chain variable region comprising SEQ ID NO: 59； or
e)a heavy chain variable region comprising SEQ ID NO: 61； and a light chain variable region comprising SEQ ID NO: 63.
In certain embodiments, the antibodies provided herein include, for example, 1.7.3  hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb.
In certain embodiments, the antibodies or antigen binding fragments thereof provided herein compete for the same epitope with antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, or 1.153.7 hAb.
In certain embodiments, the antibodies or antigen binding fragments thereof are capable of blocking binding of human PD-1 to its ligand and thereby providing at least one of the following activities:
a)inducing production of IL-2 in CD4+T cells；
b)inducing production of IFNγ in CD4+T cells；
c)inducing proliferation of CD4+T cells and
d)reversing T reg’s suppressive function.
In certain embodiments, the antibodies provided herein are a monoclonal antibody, fully human antibody, humanized antibody, chimeric antibody, recombinant antibody, bispecific antibody, labeled antibody, bivalent antibody, or anti-idiotypic antibody. In certain embodiments, the antibodies or antigen binding fragments thereof are fully human monoclonal antibodies, optionally produced by a transgenic rat, for example, a transgenic rat having inactivated endogenous expression of rat immunoglobulin genesand carrying recombinant human immunoglobulin loci having J-locu deletion and a C-kappa mutation.
In certain embodiments, the antigen-binding fragments thereof provided herein are a camelized single domain antibody, a diabody, a scFv, an scFv dimer, a BsFv, a dsFv, a (dsFv) 2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F (ab') 2, a ds diabody, a nanobody, a domain antibody, or a bivalent domain antibody.
In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin constant region.
In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein, further comprise a conjugate.
In certain embodiments, the conjugate can be a detectable label, a pharmacokinetic modifying moiety, or a purification moiety.
In another aspect, the present disclosure provides isolated polynucleotides encoding the antibodies or antigen binding fragments thereof provided herein. In certain embodiments, polynucleotides are provided that encode the amino acid sequences of the antibodies or  antigen-binding fragments disclosed herein. In certain other embodiments, vectors are provided that comprise these polynucleotides, and in certain other embodiments, host cells are provided that comprise these vectors. In certain embodiments, methods are provided for expressing one or more of the antibodies or antigen-binding fragments disclosed herein by culturing these host cells under conditions in which the antibodies or antigen-binding fragments encoded by the polynucleotides are expressed from a vector. In certain embodiments, the polynucleotides provided herein are operably associated with a promoter such as a SV40 promoter in a vector. In certain embodiments, host cells comprising the vectors provided herein are Chinese hamster ovary cell, or 293F cell.
In another aspect, the present disclosure provides kits comprising the antibody or antigen-binding fragment thereof.
In another aspect, the present disclosure provides methods of treating a condition associated with PD-1 in an individual, comprising: administering to the individual a therapeutically effective amount of antibody or antigen-binding fragment thereof provided herein. In certain embodiments, the individual has been identified as having a disorder or a condition likely to respond to a PD-1 antagonist. In certain embodiments, the individual has been identified as positive for presence or upregulated level of the PD-L1 in a test biological sample from the individual.
In another aspect, the present disclosure provides pharmaceutical compositions comprising the antibody or antigen-binding fragment thereof provided herein and one or more pharmaceutically acceptable carriers. In certain of these embodiments, the pharmaceutical carriers may be, for example, diluents, antioxidants, adjuvants, excipients, or non-toxic auxiliary substances.
In another aspect, the present disclosure provides methods of treating a condition in a subject that would benefit from upregulation of immune response, comprising administering an effective amount of the antibody or antigen-binding fragment thereof provided herein to the subject. In certain embodiments, the subject has upregulated expression of PD-L1, or has been identified as positive for expression of PD-L1.
Use of the antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a condition that would benefit from upregulation of immune response. In certain embodiments, the condition is cancer or chronic viral infection.  BRIEF DESCFRIPTION OF FIGURES
Figure 1 presents the binding of fully human anti-PD-1 antibodies to PD-1 expressing CHO cell as measured by FACS analysis.
Figure 2 presents the binding of fully human PD-1 antibodies to PD-1 expressing CHO cell with EC50 about 2nM as measured by FACS analysis.
Figure 3 is the binding of fully human anti-PD-1 antibody to PD-1 expressed on activated CD4+T cell as measured by FACS analysis.
Figure 4 shows that the fully human anti-PD-1 antibodies blocked the binding of PD-L1 to PD-1 transfected CHO cells with IC50 of about 3-8 nM as measured by FACS analysis.
Figure 5 shows that the fully human anti-PD-1 antibodies specifically bind to PD-1, but do not bind family members CD28 and CTLA4, as measured by FACS analysis.
Figure 6 shows that the fully human anti-PD-1 antibodies against PD-1 bind to cynomolgus monkey PD-1 but not murine PD-1.
Figure 7 is the full kinetics of binding affinity of PD-1 antibodies to human PD-1 ranging from 3.76E-9 to 1.76E-10 mol/L as determined by surface plasmon resonance.
Figure 8 illustrates the effect of fully human anti-PD-1 antibodies on IL-2 production in mixed lymphocyte reaction (MLR) .
Figure 9 illustrates the effect of fully human anti-PD-1 antibodies on IFNγ production in MLR.
Figure 10 shows that fully human anti-PD-1 antibodies promoted T cell proliferation in MLR.
Figure 11 shows that fully human PD-1 antibodies promoted T cell proliferation in specific T cell response.
Figure 12 shows that anti-PD-1 antibodies reversed Treg’s suppressive function.
Figure 13 shows that the anti-PD-1 antibodies lacked ADCC on activated T cells
Figure 14 shows that the anti-PD-1 antibodies lacked CDC on activated T cells.
DETAILED DESCRIPTION OF THE INVENTION
The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.
Definitions
The term “antibody” as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a specific antigen. A native intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. The antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variables region in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light (L) chain CDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs including HCDR1, HCDR2, HCDR3) . CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A. M., J. Mol. Biol., 273 (4) , 927 (1997) ； Chothia, C. et al., J Mol Biol. Dec 5； 186 (3) : 651-63 (1985) ； Chothia, C. and Lesk, A. M., J. Mol. Biol., 196, 901 (1987) ； Chothia, C. et al., Nature. Dec 21-28；342 (6252) : 877-83 (1989) ； Kabat E. A. et al., National Institutes of Health, Bethesda, Md. (1991) ) . The three CDRs are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The  five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain) , IgG2 (γ2 heavy chain) , IgG3 (γ3 heavy chain) , IgG4 (γ4 heavy chain) , IgA1 (α1 heavy chain) , or IgA2 (α2 heavy chain) .
The term “antigen-binding fragment” as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F (ab') 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
“Fab” with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.
“Fab'” refers to a Fab fragment that includes a portion of the hinge region.
“F (ab') 2” refers to a dimer of Fab’ .
“Fc” with regard to an antibody refers to that portion of the antibody consisting of the second and third constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding. The Fc portion of the antibody is responsible for various effector functions such as ADCC, and CDC, but does not function in antigen binding.
“Fv” with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.
“Single-chain Fv antibody” or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another  directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988) ) .
“Single-chain Fv-Fc antibody” or “scFv-Fc” refers to an engineered antibody consisting of a scFv connected to the Fc region of an antibody.
“Camelized single domain antibody, ” “heavy chain antibody, ” or “HCAb” refers to an antibody that contains two VH domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10； 231 (1-2) : 25-38 (1999) ； Muyldermans S., J Biotechnol. Jun； 74 (4) : 277-302 (2001) ； WO94/04678； WO94/25591； U.S. Patent No. 6,005,079) . Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas) . Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3； 363 (6428) : 446-8 (1993) ； Nguyen VK. et al. “Heavy-chain antibodies in Camelidae； a case of evolutionary innovation, ” Immunogenetics. Apr； 54 (1) : 39-47 (2002) ； Nguyen VK. et al. Immunology. May； 109 (1) : 93-101 (2003) ) . The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov； 21 (13) : 3490-8. Epub 2007 Jun 15 (2007) ) .
A “nanobody” refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.
“Diabodies” include small antibody fragments with two antigen-binding sites, wherein the fragments comprise a VH domain connected to a VL domain in the same polypeptide chain (VH-VL or VH-VL) (see, e.g., Holliger P. et al., Proc Natl Acad Sci U S A. Jul 15； 90 (14) : 6444-8 (1993) ； EP404097； WO93/11161) . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. The antigen–binding sites may target the same of different antigens (or epitopes) .
A “domain antibody” refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. In certain instances, two or more VH domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.
In certain embodiments, a “ (dsFv) 2” comprises three peptide chains: two VH moieties linked by a peptide linker and bound by disulfide bridges to two VL moieties.
In certain embodiments, a “bispecific ds diabody” comprises VH1-VL2 (linked by a peptide linker) bound to VL1-VH2 (also linked by a peptide linker) via a disulfide bridge between VH1 and VL1.
In certain embodiments, a “bispecific dsFv” or dsFv-dsFv' “comprises three peptide chains: a VH1-VH2 moiety wherein the heavy chains are linked by a peptide linker (e.g., a long flexible linker) and bound to VL1 and VL2 moieties, respectively, via disulfide bridges, wherein each disulfide paired heavy and light chain has a different antigen specificity.
In certain embodiments, an “scFv dimer” is a bivalent diabody or bivalent ScFv (BsFv) comprising VH-VL (linked by a peptide linker) dimerized with another VH-VL moiety such that VH's of one moiety coordinate with the VL's of the other moiety and form two binding sites which can target the same antigens (or eptipoes) or different antigens (or eptipoes) . In other embodiments, an “scFv dimer” is a bispecific diabody comprising VH1-VL2 (linked by a peptide linker) associated with VL1-VH2 (also linked by a peptide linker) such that VH1 and VL1 coordinate and VH2 and VL2 coordinate and each coordinated pair has a different antigen specificity.
The term “fully human” as used herein, with reference to antibody or antigen-binding fragment, means that the antibody or the antigen-binding fragment has or consists of amino acid sequence (s) corresponding to that of an antibody produced by a human or a human immune cell, or derived from a non-human source such as a transgenic non-human animal that utilizes human antibody repertoires or other human antibody-encoding sequences. In certain embodiments, a fully human antibody does not comprise amino acid residues (in particular antigen-binding residues) derived from a non-human antibody.
The term “humanized” as used herein, with reference to antibody or antigen-binding fragment, means that the antibody or the antigen-binding fragment comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human. A humanized antibody or antigen-binding fragment is useful as human therapeutics in certain embodiments because it has reduced immunogenicity in human. In some embodiments, the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster. In some embodiments, the humanized antibody or antigen-binding fragment is composed of substantially all human  sequences except for the CDR sequences which are non-human. In some embodiments, the FR regions derived from human may comprise the same amino acid sequence as the human antibody from which it is derived, or it may comprise some amino acid changes, for example, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 changes of amino acid. In some embodiments, such change in amino acid could be present in heavy chain FR regions only, in light chain FR regions only, or in both chains. In some preferable embodiments, the humanized antibodies comprise human FR1-3 and human JH and Jκ.
The term “chimeric” as used herein, means an antibody or antigen-binding fragment, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human species, such as from mouse.
“PD-1” as used herein refers programmed cell death protein, which belongs to the superfamily of immunoglobulin and functions as coinhibitory receptor to negatively regulate the immune system. PD-1 is a member of the CD28/CTLA-4 family, and has two known ligands including PD-L1 and PD-L2. Representative amino acid sequence of human PD-1 is disclosed under the NCBI accession number: NP_005009.2, and the representative nucleic acid sequence encoding the human PD-1 is shown under the NCBI accession number: NM_005018.2.
“PD-L1” as used herein refers to programmed cell death ligand 1 (PD-L1, see, for example, Freeman et al. (2000) J. Exp. Med. 192: 1027) . Representative amino acid sequence of human PD-L1 is disclosed under the NCBI accession number: NP_054862.1, and the representative nucleic acid sequence encoding the human PD-L1 is shown under the NCBI accession number: NM_014143.3. PD-L1 is expressed in placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumor or cancer cells. PD-L1 binds to its receptor PD-1 or B7-1, which is expressed on activated T cells, B cells and myeloid cells. The binding of PD-L1 and its receptor induces signal transduction to suppress TCR-mediated activation of cytokine production and T cell proliferation. Accordingly, PD-L1 plays a major role in suppressing immune system during particular events such as pregnancy, autoimmune diseases, tissue allografts, and is believed to allow tumor or cancer cells to circumvent the immunological checkpoint and evade the immune response.
“Anti-PD-1 antibody” as used herein refers to an antibody that is capable of specific binding to PD-1 (e.g. human or monkey PD-1) with an affinity which is sufficient to provide for diagnostic and/or therapeutic use.
The term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically bind human and/or monkey PD-1 with a binding affinity (KD) of ≤10-6 M (e.g., ≤5x10-7 M, ≤2x10-7 M, ≤10-7 M, ≤5x10-8 M, ≤2x10-8 M, ≤10-8 M, ≤5x10-9 M, ≤2x10-9 M, ≤10-9 M, 10-10 M) . KD as used herein refers to the ratio of the dissociation rate to the association rate (koff/kon) , may be determined using surface plasmon resonance methods for example using instrument such as Biacore.
The ability to “block binding” or “compete for the same epitope” as used herein refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g. human PD-1 and an anti-PD-1 antibody) to any detectable degree. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50％. In certain embodiments, this inhibition may be greater than 60％, greater than 70％, greater than 80％, or greater than 90％.
The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies may bind the same epitope within an antigen if they exhibit competitive binding for the antigen. For example, if an antibody or antigen-binding fragment as disclosed herein blocks binding of the exemplary antibodies such as 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb to human PD-1, then the antibody or antigen-binding fragment may be considered to bind the same epitope as those exemplary antibodies.
“1.7.3 hAb” as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 45, light chain variable region of SEQ ID NO: 47, and a human constant region of IgG4 isotype.
“1.49.9 hAb” as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 49, light chain variable region of SEQ ID NO: 51, and a human constant region of IgG4 isotype.
“1.103.11 hAb” as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 53, light chain variable region of SEQ ID NO: 55, and a human constant region of IgG4 isotype.
“1.139.15 hAb” as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 57, light chain variable region of SEQ ID NO: 59, and a human constant region of IgG4 isotype.
“1.153.7 hAb” as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 61, light chain variable region of SEQ ID NO: 63, and a human constant region of IgG4 isotype.
A “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g. Met, Ala, Val, Leu, and Ile) , among residues with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn and Gln) , among residues with acidic side chains (e.g. Asp, Glu) , among amino acids with basic side chains (e.g. His, Lys, and Arg) , or among residues with aromatic side chains (e.g. Trp, Tyr, and Phe) . As known in the art, conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.
“Percent (％) sequence identity” with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F. et al, J. Mol. Biol., 215: 403–410 (1990) ； Stephen F. et al, Nucleic Acids Res., 25: 3389–3402 (1997) ) , ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D. G. et al, Methods in Enzymology, 266: 383-402 (1996) ； Larkin M.A. et al, Bioinformatics (Oxford, England) , 23 (21) : 2947-8 (2007) ) , and  ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm.
“T cell” as used herein includes CD4+ T cells, CD8+ T cells, T helper 1 type T cells, T helper 2 type T cells, T helper 17 type T cells and inhibitory T cells.
“Effector functions” as used herein refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as C1 complex and Fc receptor. Exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and C1q on the C1 complex； antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to Fc receptor on an effector cell； and phagocytosis.
“Cancer” or “cancerous condition” as used herein refers to any medical condition mediated by neoplastic or malignant cell growth, proliferation, or metastasis, and includes both solid cancers and non-solid cancers such as leukemia. “Tumor” as used herein refers to a solid mass of neoplastic and/or malignant cells.
“Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof. With regard to cancer, “treating” or “treatment” may refer to inhibiting or slowing neoplastic or malignant cell growth, proliferation, or metastasis, preventing or delaying the development of neoplastic or malignant cell growth, proliferation, or metastasis, or some combination thereof. With regard to a tumor, “treating” or “treatment” includes eradicating all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.
An “isolated” substance has been altered by the hand of man from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated, ” but the same polynucleotide or polypeptide is “isolated” if it has been sufficiently separated from the coexisting materials of its natural state so as to exist in a substantially pure state. In certain embodiments, the  antibodies and antigen-binding fragments have a purity of at least 90％, 93％, 95％, 96％, 97％, 98％, 99％as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) , or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC) .
The term “vector” as used herein refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) . A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.
The phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.
A “disease associated with or related to PD-1” as used herein refers to any condition that is caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of PD-1 (e.g. a human PD-1) .
The term “therapeutically effective amount” or “effective dosage” as used herein refers to the dosage or concentration of a drug effective to treat a disease or condition associated with human PD-1. For example, with regard to the use of the antibodies or antigen-binding fragments disclosed herein to treat cancer, a therapeutically effective amount is the dosage or concentration of the antibody or antigen-binding fragment capable of eradicating all or part of a tumor, inhibiting or slowing tumor growth, inhibiting growth or proliferation of cells mediating a cancerous condition, inhibiting tumor cell metastasis, ameliorating any symptom or marker associated with a tumor or cancerous condition,  preventing or delaying the development of a tumor or cancerous condition, or some combination thereof.
The term “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
Anti-PD-1 antibody
In one aspect, the present disclosure provides anti-PD-1 antibodies and the antigen-binding fragments thereof. PD-1, also called as CD279, is known as a key immune-checkpoint receptor expressed by activated T cells, which mediates immunosuppression. PD-1 ligand 1 (PD-L1) is a 40 kDa transmembrane protein expressed on various tumor cells, stromal cells or both, and binds to PD-1. Inhibition of the interaction between PD-1 and PD-L1 can enhance T-cell responses and thus mediates anti-cancer activity.
In certain embodiments, the present disclosure provides exemplary fully human monoclonal antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb, whose CDR sequences are shown in the below Table 1, and heavy or light chain variable region sequences are also shown below.
Table 1
Figure PCTCN2015086594-appb-000001
Figure PCTCN2015086594-appb-000002
Figure PCTCN2015086594-appb-000003
1.7.3 hAb-VH (23466-VH) : (SEQ ID NO: 45 for amino acid and SEQ ID NO: 46 for nucleic acid) with heavy chain CDRs1-3: SEQ ID NOs: 1, 3, 5 are amino acid sequences and SEQ ID NO: 2, 4, 6 are nucleic acid sequences, respectively:
V segment: IGHV4-39*01
D segment: IGHD1-26*01
J segment: IGHJ4*02
Figure PCTCN2015086594-appb-000004
Figure PCTCN2015086594-appb-000005
1.7.3 hAb-VL (23195-VL) : (SEQ ID NO: 47 for amino acid and SEQ ID NO: 48 for nucleic acid) with light chain CDRs1-3: SEQ ID NOs: 7, 9, 11 are amino acid sequences and SEQ ID NO: 8, 10, 12 are nucleic acid sequences, respectively:
V segment: IGLV2-14*01
J segment: IGLJ3*02
Figure PCTCN2015086594-appb-000006
Figure PCTCN2015086594-appb-000007
1.49.9 hAb-VH (20951-VH) : (SEQ ID NO: 49 for amino acid and SEQ ID NO: 50 for nucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 13, 15, 5 are amino acid sequences and SEQ ID NO: 14, 16, 6 are nucleic acid sequences, respectively:
V segment: IGHV4-39*01
D segment: IGHD1-26*01
J segment: IGHJ4*02
Figure PCTCN2015086594-appb-000008
Figure PCTCN2015086594-appb-000009
1.49.9 hAb-VL (21526-VL) : (SEQ ID NO: 51 for amino acid and SEQ ID NO: 52 for nucleic acid) with light chain CDRs 1-3: SEQ ID NOs: 7, 17, 11 are amino acid sequences and SEQ ID NO: 8, 18, 12 are nucleic acid sequences, respectively:
V segment: IGLV2-14*01
J segment: IGLJ3*02
Figure PCTCN2015086594-appb-000010
Figure PCTCN2015086594-appb-000011
1.103.11 hAb-VH (20975-VH) : (SEQ ID NO: 53 for amino acid and SEQ ID NO:54 for nucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 1, 15, 5 are amino acid sequences and SEQ ID NO: 2, 16, 6 are nucleic acid sequences, respectively:
V segment: IGHV4-39*01
D segment: IGHD1-26*01
J segment: IGHJ4*02
Figure PCTCN2015086594-appb-000012
Figure PCTCN2015086594-appb-000013
1.103.11 hAb-VL (21038-VL) : (SEQ ID NO: 55 for amino acid and SEQ ID NO: 56 for nucleic acid) with light chain CDRs 1-3: SEQ ID NOs: 7, 17, 19 are amino acid sequences and SEQ ID NO: 8, 18, 20 are nucleic acid sequences, respectively:
V segment: IGLV2-14*01
J segment: IGLJ3*02
Figure PCTCN2015086594-appb-000014
Figure PCTCN2015086594-appb-000015
1.139.15 hAb-VH (23521-VH) (SEQ ID NO: 57 for amino acid and SEQ ID NO: 58 for nucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 21, 23, 25 are amino acid sequences and SEQ ID NO: 22, 24, 26 are nucleic acid sequences, respectively:
V segment: IGHV4-39*01
D segment: IGHD6-13*01
J segment: IGHJ4*02
Figure PCTCN2015086594-appb-000016
Figure PCTCN2015086594-appb-000017
1.139.15 hAb-VL (22895-VL) (SEQ ID NO: 59 for amino acid and SEQ ID NO: 60 for nucleic acid) with light chain CDRs 1-3: SEQ ID NOs: 27, 29, 31 are amino acid sequences and SEQ ID NO: 28, 30, 32 are nucleic acid sequences, respectively:
V segment: IGLV2-18*02
J segment: IGLJ3*02
Figure PCTCN2015086594-appb-000018
1.153.7 hAb-VH (20942-VH) : (SEQ ID NO: 61 for amino acid and SEQ ID NO: 62 for nucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 33, 35, 37 are amino acid sequences and SEQ ID NO: 34, 36, 38 are nucleic acid sequences, respectively:
V segment: IGHV3-23*01
D segment: IGHD7-27*01
J segment: IGHJ4*02
Figure PCTCN2015086594-appb-000019
1.153.7 hAb-VL (21110-VL) (SEQ ID NO: 63 for amino acid and SEQ ID NO: 64 for nucleic acid) with light chain CDRs 1-3: SEQ ID NOs: 39, 41, 43 are amino acid sequences and SEQ ID NO: 40, 42, 44 are nucleic acid sequences, respectively:
V segment: IGLV3-9*01
J segment: IGLJ3*02
Figure PCTCN2015086594-appb-000020
In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof comprise a heavy chain CDR sequences selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35 and 37. In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof comprise a light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 7, 9, 11, 17, 19, 27, 29, 31, 39, 41, and 43.
In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of: a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5； a  heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 5；aheavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 15, and/or SEQ ID NO: 5； a heavy chain variable region comprising SEQ ID NO: 21, SEQ ID NO: 23, and/or SEQ ID NO: 25； and a heavy chain variable region comprising SEQ ID NO: 33, SEQ ID NO: 35, and/or SEQ ID NO: 37.
In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof comprise a light chain variable region selected from the group consisting of: a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9, and/or SEQ ID NO: 11； a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 11； a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 19； a light chain variable region comprising SEQ ID NO: 27, SEQ ID NO: 29, and/or SEQ ID NO: 31； and a light chain variable region comprising SEQ ID NO: 39, SEQ ID NO: 41, and/or SEQ ID NO: 43.
In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof comprising: a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5； and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9, and/or SEQ ID NO: 11； b) a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 5； and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 11； c) aheavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 15, and/or SEQ ID NO: 5； and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 19； d) a heavy chain variable region comprising SEQ ID NO: 21, SEQ ID NO: 23, and/or SEQ ID NO: 25 and a light chain variable region comprising SEQ ID NO: 27, SEQ ID NO: 29, and/or SEQ ID NO: 31； or e) a heavy chain variable region comprising SEQ ID NO: 33, SEQ ID NO: 35, and/or SEQ ID NO: 37； and a light chain variable region comprising SEQ ID NO: 39, SEQ ID NO: 41, and/or SEQ ID NO: 43.
A skilled artisan will understand that the CDR sequences provided in Table 1 can be modified to contain one or more substitutions of amino acids, so as to provide for an improved biological activity such as improved binding affinity to human PD-1. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed with phage display technology, and then screened for the binding affinity to human PD-1. For another example, computer software can be used to virtually simulate the binding of the antibodies to human PD-1, and identify the amino acid residues on the antibodies which form  the binding interface. Such residues may be either avoided in the substitution so as to prevent reduction in binding affinity, or targeted for substitution to provide for a stronger binding. In certain embodiments, at least one (or all) of the substitution (s) in the CDR sequences is conservative substitution.
In certain embodiments, the antibodies and the antigen-binding fragments thereof comprise one or more CDR sequences having at least 80％ (e.g. at least 85％, 88％, 90％, 91％, 92％, 93％, 94％, 95％, 96％, 97％, 98％, 99％) sequence identity to that (or those) listed in Table 1, and in the meantime retain the binding affinity to human PD-1 at a level similar to or even higher than its parental antibody having substantially the same sequence except that the corresponding CDR sequence is in 100％sequence identity to that (or those) listed in Table 1.
In certain embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof are fully human. The fully human antibodies do not have the issues of immunogenicity in human and/or reduced binding affinity as often observed with humanized antibodies.
In some embodiments, the fully human anti-PD-1 antibodies and the antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, and a homologous sequence thereof having at least 80％ (e.g. at least 85％, 88％, 90％, 91％, 92％, 93％, 94％, 95％, 96％, 97％, 98％, 99％) sequence identity； and/or a light chain variable region selected from the group consisting of: SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, and a homologous sequence thereof having at least 80％ (e.g. at least 85％, 88％, 90％, 91％, 92％, 93％, 94％, 95％, 96％, 97％, 98％, 99％) sequence identity. Theses fully human antibodies retain the binding affinity to human PD-1, preferably at a level similar to one of the exemplary antibodies: 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb.
In some embodiments, the fully human anti-PD-1 antibodies and the antigen-binding fragments thereof comprise: a) a heavy chain variable region comprising SEQ ID NO: 45； and a light chain variable region comprising SEQ ID NO: 47； b) a heavy chain variable region comprising SEQ ID NO: 49； and a light chain variable region comprising SEQ ID NO: 51； c) a heavy chain variable region comprising SEQ ID NO: 53； and a light chain variable region comprising SEQ ID NO: 55； d) a heavy chain variable region comprising SEQ ID NO:  57； and a light chain variable region comprising SEQ ID NO: 59； or e) a heavy chain variable region comprising SEQ ID NO: 61； and a light chain variable region comprising SEQ ID NO: 63.
Also contemplated herein are antibodies and the antigen-binding fragments that compete for the same epitope with the anti-PD-1 antibodies and the antigen-binding fragments thereof provided herein. In certain embodiments, the antibodies block binding of 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, or 1.153.7 hAb to human or monkey PD-1, for example, at an IC50 value (i.e. 50％inhibition concentration) of below10-6 M, below 10-7 M, below 10-7.5 M, below 10-8 M, below 10-8.5 M, below 10-9 M, or below 10-10 M. The IC50 values are determined based on a competition assay such as ELISA assays, radioligand competition binding assays, and FACS analysis.
In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof provided herein are capable of specifically binding to human PD-1 with a binding affinity (Kd) of ≤10-6 M (e.g., ≤5x10-7 M, ≤2x10-7 M, ≤10-7 M, ≤5x10-8 M, ≤2x10-8 M, ≤10-8 M, ≤5x10-9 M, ≤2x10-9 M, ≤10-9 M, 10-10 M) as measured by plasmon resonance binding assay. The binding affinity can be represented by KD value, which is calculated as the ratio of dissociation rate to association rate (koff/kon) when the binding between the antigen and the antigen-binding molecule reaches equilibrium. The antigen-binding affinity (e.g. KD) can be appropriately determined using suitable methods known in the art, including, for example, plasmon resonance binding assay using instruments such as Biacore (see, for example, Murphy, M. et al, Current protocols in protein science, Chapter 19, unit 19.14, 2006) .
In certain embodiments, the antibodies and the fragments thereof provided herein binds to human PD-1 with an EC50 (i.e. 50％binding concentration) of 0.1nM-100nM (e.g. 0.1nM-50nM, 0.1nM-30nM, 0.1nM-20nM, 0.1nM-10nM, or 0.1nM-1nM) . Binding of the antibodies to human PD-1 can be measured by methods known in the art, for example, sandwich assay such as ELISA, Western Blot, FACS or other binding assay. In an illustrative example, the test antibody (i.e. first antibody) is allowed to bind to immobilized human PD-1 or cells expressing human PD-1, after washing away the unbound antibody, a labeled secondary antibody is introduced which can bind to and thus allow detection of the bound first antibody. The detection can be conducted with a microplate reader when immobilized PD-1 is used, or by using FACS analysis when cells expressing human PD-1 are used. In certain embodiments, the antibodies and the fragments thereof provided herein binds  to human PD-1 with an EC50 (i.e. 50％effective concentration) of 1nM to 10nM, or 1nM to 5nM as measured by FACS analysis.
In certain embodiments, the antibodies and the fragments thereof provided herein inhibit the binding of human PD-1 to its ligand at an IC50 of 0.2nM-100nM (e.g. 0.2nM-50nM, 0.2nM-30nM, 0.2nM-20nM, 0.2nM-10nM, or 1nM-10nM) , as measured in a competition assay.
In certain embodiments, the antibodies and the fragments thereof provided herein block binding of human PD-1 to its ligand and thereby providing biological activity including, for example, inducing cytokine production from the activated T cells (such as CD4+ T cells and CD8+ T cells) , inducing proliferation of activated T cells (such as CD4+ T cells and CD8+ T cells) , and reversing T reg’s suppressive function. Exemplary cytokines include IL-2 and IFNγ. The term “IL-2” refers to interleukin 2, a type of cytokine signaling molecule in the immune system that regulates the activities of white blood cells (e.g. leukocytes) . The term “Interferon gamma (IFNγ) ” is a cytokine that is produced by natural killer (NK) , NK T cells, CD4+ and CD8+T cells, which is a critical activator of macrophages and inducer of major histocompatibility complex (MHC) molecule expression. The cytokine production can be determined using methods known in the art, for example, by ELISA. Methods can also be used to detect proliferation of T cells, including [3H] thymidine incorporation assay.
The anti-PD-1 antibodies and the antigen-binding fragments thereof are specific for PD-1. In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind to CD28 and/or CTLA-4. For example, the binding affinity with CD28 and/or CTLA-4 is less than 15％, 10％, 9％, 8％, 7％, 6％, 5％, 4％, 3％, 2％, or 1％of that with PD-1.
In certain embodiments, the antibodies and antigen-binding fragments thereof bind to monkey PD-1 at an EC50 of no more than 100nM, for example, no more than or about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, or 0.01nM, as measured by ELISA. In certain embodiments, the antibodies and antigen-binding fragments thereof bind to monkey PD-1 at an EC50 of about 1 nM -10nM.
In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind to mouse PD-1 but bind to monkey PD-1 with a binding affinity similar to that of human PD-1. For example, binding of the exemplary antibodies 1.7.3 hAb, 1.49.9 hAb,  1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb to mouse PD-1 is not detectable in conventional binding assays such as ELISA, or FACS analysis, whereas the binding of these antibodies to monkey PD-1 is at a similar affinity or EC50 value to that of human PD-1 as measured by ELISA or FACS.
In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof has reduced or depleted effector function. In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof have a constant region of IgG4 isotype, which has reduced or depleted effector function. Effector functions such as ADCC and CDC can lead to cytotoxicity to cells expressing PD-1. Many cells such as T cells normally express PD-1. In order to avoid potential unwanted toxicity to those normal cells, certain embodiments of the antibodies and antigen-binding fragments provided herein can possess reduced or even depleted effector functions. Various assays are known to evaluate ADCC or CDC activities, for example, Fc receptor binding assay, C1q binding assay, and cell lysis assay, and can be readily selected by people in the art. Without wishing to be bound to theory, but it is believed that antibodies with reduced or depleted effector functions such as ADCC or CDC would cause no or minimal cytotoxicity to PD-1-expressing cells, for example those T cells, and therefore spare them from unwanted side effects, whereas in the meantime, blocking of PD-1 would boost immune system for the treatment of conditions such as cancer or chronic infection.
In certain embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereofprovided herein have reduced side effects. For example, the antibodies and antigen-binding fragments thereof provided herein can have fully human IgG sequence and therefore reduced immunogenicity than a humanized antibody counterpart. For another example, the antibodies and antigen-binding fragments thereof provided herein can be in IgG4 format to eliminate ADCC and CDC.
In certain embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereofprovided herein are advantageous in that they can be used in combination with immunogenic agents, such as tumor cells, purified tumor antigen, and cells transfected with genes encoding immune stimulating cytokines, tumor vaccines. In addition, the anti-PD-1 antibodies and antigen-binding fragments thereof can be included in combination therapies, including standard chemo-and radio-therapies, target based small molecule therapies, emerging other immune checkpoint modulator therapies. In certain embodiments, the  antibodies and antigen-binding fragments thereof can be used as the base of antibody-drug conjugates, bispecific or multivalent antibodies.
The anti-PD-1 antibodies or antigen-binding fragments thereof provided herein can be a monoclonal antibody, polyclonal antibody, fully human antibody, humanized antibody, chimeric antibody, recombinant antibody, bispecific antibody, labeled antibody, bivalent antibody, or anti-idiotypic antibody. A recombinant antibody is an antibody prepared in vitro using recombinant methods rather than in animals. A bispecific or bivalent antibody is an artificial antibody having fragments of two different monoclonal antibodies and can bind two different antigens. An antibody or antigen-binding fragment thereof that is “bivalent” comprises two antigen-binding sites. The two antigen binding sites may bind to the same antigen, or they may each bind to a different antigen, in which case the antibody or antigen-binding fragment is characterized as “bispecific. ”
In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof provided herein are fully human antibodies. In certain embodiments, the fully human antibodies are prepared using recombinant methods. For example, transgenic animal such as a mouse can be made to carry transgenes or transchromosomes of human immunoglobulin genes, and therefore capable of producing fully human antibodies after immunization with proper antigen such as human PD-1. Fully human antibodies can be isolated from such transgenic animal, or alternatively, can be made by hybridoma technology by fusing the spleen cells of the transgenic animal with an immortal cell line to generate hybridoma cells secreting the fully human antibodies. Exemplary transgenic animals include, without limitation, OmniRat, whose endogenous expression of rat immunoglobulin genes are inactivated and at the same time engineered to contain functional recombinant human immunoglobulin loci； OmniMouse, whose endogenous expression of mouse immunoglobulin genes are inactivated and at the same time engineered to contain recombinant human immunoglobulin loci having J-locus deletion and a C-kappa mutation； OmniFlic, which is a transgenic rat whose endogenous expression of rat immunoglobulin genes are inactivated and at the same time engineered to contain recombinant human immunoglobulin loci having a single common, rearranged VkJk light chain and functional heavy chain. Detailed information can be further found at: Osborn M. et al, Journal of Immunology, 2013, 190: 1481-90； Ma B. et al, Journal of Immunological Methods 400–401 (2013) 78-86； Geurts A. et al, Science, 2009, 325: 433； U.S. Pat. 8,907,157； EP patent 2152880B1； EP patent 2336329B1, all of which are incorporated herein by reference to its entirety. Other suitable  transgenic animals can also be used, for example, HuMab mice (see, for details, Lonberg, N. et al. Nature 368 (6474) : 856 859 (1994) ) , Xeno-Mouse (Mendez et al. Nat Genet., 1997, 15: 146–156) , TransChromo Mouse (Ishida et al. Cloning Stem Cells, 2002, 4: 91–102) and VelocImmune Mouse (Murphy et al. Proc Natl Acad Sci USA, 2014, 111: 5153–5158) , Kymouse (Lee et al. Nat Biotechnol, 2014, 32: 356–363) , and transgenic rabbit (Flisikowska et al. PLoS One, 2011, 6: e21045) .
In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof is a camelized single domain antibody, a diabody, a scFv, an scFv dimer, a BsFv, a dsFv, a (dsFv) 2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F (ab') 2, a ds diabody, a nanobody, a domain antibody, or a bivalent domain antibody.
In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof further comprise an immunoglobulin constant region. In some embodiments, an immunoglobulin constant region comprises a heavy chain and/or a light chain constant region. The heavy chain constant region comprises CH1, CH1-CH2, or CH1-CH3 regions. In some embodiments, the constant region may further comprise one or more modifications to confer desirable properties. For example, the constant region may be modified to reduce or deplete one or more effector functions, to improve FcRn receptor binding, or to introduce one or more cysteine residues.
In some embodiments, the anti-PD-1 antibodies and the antigen-binding fragments thereof further comprise a conjugate. It is contemplated that a variety of conjugates may be linked to the antibodies or antigen-binding fragments provided herein (see, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J. M. Cruse and R. E.Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) . These conjugates may be linked to the antibodies or antigen-binding fragments by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods. In certain embodiments, the antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugates. For example, such a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate. In certain embodiments, the antibodies may be linked to a conjugate indirectly, or through another conjugate. For example, the antibody or antigen-binding fragments may be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin. The conjugate can be a detectable label, a  pharmacokinetic modifying moiety, a purification moiety, or a cytotoxic moiety. Examples of detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or β-D-galactosidase) , radioisotopes (e.g. 123I, 124I, 125I, 131I, 35S, 3H, 111In, 112In, 14C, 64Cu, 67Cu, 86Y, 88Y, 90Y, 177Lu, 211At, 186Re, 188Re, 153Sm, 212Bi, and 32P, other lanthanides, luminescent labels) , chromophoric moiety, digoxigenin, biotin/avidin, a DNA molecule or gold for detection. In certain embodiments, the conjugate can be a pharmacokinetic modifying moiety such as PEG which helps increase half-life of the antibody. Other suitable polymers include, such as, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like. In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead. A “cytotoxic moiety” can be any agent that is detrimental to cells or that can damage or kill cells. Examples of cytotoxic moiety include, without limitation, 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, puromycin and analogs thereof, 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) ) , and anti-mitotic agents (e.g., vincristine and vinblastine) .
Polynucleotides and Recombinant Methods
The present disclosure provides isolated polynucleotides that encode the anti-PD-1 antibodies and the antigen-binding fragments thereof. In certain embodiments, the isolated polynucleotides comprise one or more nucleotide sequences as shown in Table 1, which encodes the CDR sequences provided in Table 1.
In some embodiments, the isolated polynucleotides encodes a heavy chain variable region and comprise a sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, and a homologous sequence  thereof having at least 80％ (e.g. at least 85％, 88％, 90％, 91％, 92％, 93％, 94％, 95％, 96％, 97％, 98％, 99％) sequence identity. In some embodiments, the isolated polynucleotides encodes a light chain variable region and comprise a sequence selected from the group consisting of: SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, and a homologous sequence thereof having at least 80％ (e.g. at least 85％, 88％, 90％, 91％, 92％, 93％, 94％, 95％, 96％, 97％, 98％, 99％) sequence identity. In certain embodiments, the percentage identity is due to genetic code degeneracy, while the encoded protein sequence remains unchanged.
The isolated polynucleotide that encodes the anti-PD-1 antibodies and the antigen-binding fragments thereof (e.g. including the sequences in Table 1) can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art. In another embodiment, the antibody may be produced by homologous recombination known in the art. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) . Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g. SV40, CMV, EF-1α) , and a transcription termination sequence.
In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc, and comprises plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2 etc, and other laboratorial and commercially available vectors. Suitable vectors may include, plasmid, or viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) .
Vectors comprising the polynucleotide sequence encoding the antibody or antigen-binding fragment can be introduced to a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and  Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-PD-1 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe； Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12, 424) , K. bulgaricus (ATCC 16, 045) , K. wickeramii (ATCC 24, 178) , K. waltii (ATCC 56, 500) , K. drosophilarum (ATCC 36, 906) , K. thermotolerans, and K. marxianus； yarrowia (EP 402, 226) ； Pichia pastoris (EP 183, 070) ； Candida； Trichoderma reesia (EP 244, 234) ； Neurospora crassa； Schwanniomyces such as Schwanniomyces occidentalis； and filamentous fungisuch as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
Suitable host cells for the expression of glycosylated antibodies or antigen-fragment provided here are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruiffly) , and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651) ； human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36: 59 (1977) ) ； baby hamster kidney cells (BHK, ATCC CCL 10) ； Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980) ) ； mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980) ) ； monkey kidney cells (CV1 ATCC CCL 70) ； African green monkey kidney cells (VERO-76, ATCC CRL-1587) ； human cervical carcinoma cells (HELA, ATCC CCL 2) ；  canine kidney cells (MDCK, ATCC CCL 34) ； buffalo rat liver cells (BRL 3A, ATCC CRL 1442) ； human lung cells (W138, ATCC CCL 75) ； human liver cells (Hep G2, HB 8065) ； mouse mammary tumor (MMT 060562, ATCC CCL51) ； TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982) ) ； MRC 5 cells； FS4 cells； and a human hepatoma line (Hep G2) . In some preferable embodiments, the host cell is 293F cell.
Host cells are transformed with the above-described expression or cloning vectors for anti-PD-1 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
The host cells used to produce the antibodies or antigen-binding fragments provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma) , Minimal Essential Medium (MEM) , (Sigma) , RPMI-1640 (Sigma) , and Dulbecco's Modified Eagle's Medium (DMEM) , Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44 (1979) , Barnes et al., Anal. Biochem. 102: 255 (1980) , U.S. Pat. No. 4,767,704； 4,657,866； 4,927,762； 4,560,655； or 5,122,469； WO 90/03430； WO 87/00195； or U.S. Pat. Re. 30, 985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleotides (such as adenosine and thymidine) , antibiotics (such as GENTAMYCINTM drug) , trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris  can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
The antibody prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human . gamma. 1, . gamma. 2, or . gamma. 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) . Protein G is recommended for all mouse isotypes and for human . gamma. 3 (Guss et al., EMBO J. 5: 1567 1575 (1986) ) . The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX. TM. resin (J.T. Baker, Phillipsburg, N.J. ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column) , chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
Following any preliminary purification step (s) , the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt) .
Kits
The present disclosure provides kits comprising the anti-PD-1 antibodies or the antigen-binding fragments thereof. In some embodiments, the kits are useful for detecting  the presence or level of PD-1 in a biological sample. The biological sample can comprise a cell or a tissue.
In some embodiments, the kit comprises an anti-PD-1 antibody or the antigen-binding fragment thereof which is conjugated with a detectable label. In certain other embodiments, the kit comprises an unlabeled anti-PD-1 antibody or antigen-binding fragment, and further comprises a secondary labeled antibody which is capable of binding to the unlabeled anti-PD-1 antibody. The kit may further comprise an instruction of use, and a package that separates each of the components in the kit.
In certain embodiments, the anti-PD-1 antibody or the antigen-binding fragment thereof are associated with a substrate or a device useful in a sandwich assay such as ELISA, or in an immunographic assay. Useful substrate or device can be, for example, microtiter plate and test strip.
Pharmaceutical Composition and Method of Treatment
The present disclosure further provides pharmaceutical compositions comprising the anti-PD-1 antibodies or the antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers.
Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, inclusion of one or more antioxidants such as methionine in a composition comprising an antibody or antigen-binding fragment and conjugates as provided herein decreases oxidation of the antibody or antigen-binding fragment. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf-life. Therefore, in certain  embodiments compositions are provided that comprise one or more antibodies or antigen-binding fragments as disclosed herein and one or more antioxidants such as methionine. Further provided are methods for preventing oxidation of, extending the shelf-life of, and/or improving the efficacy of an antibody or antigen-binding fragment as provided herein by mixing the antibody or antigen-binding fragment with one or more antioxidants such as methionine.
To further illustrate, pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid) , ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
The pharmaceutical compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
In embodiments, the pharmaceutical compositions are formulated into an injectable composition. The injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion. Preparations for injection  may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions. The solutions may be either aqueous or nonaqueous.
In certain embodiments, unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
In certain embodiments, a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides a desirable formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial can contain a single dosage or multiple dosages of the anti-PD-1 antibody or antigen-binding fragment thereof or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10％) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4 ℃ to room temperature.
Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.
Therapeutic methods are also provided, comprising: administering a therapeutically effective amount of the antibody or antigen-binding fragment as provided herein to a subject in need thereof, thereby treating or preventing a condition or a disorder associated with  related to PD-1. In another aspect, methods are provided to treat a condition in a subject that would benefit from upregulation of immune response, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment as provided herein to a subject in need thereof.
The therapeutically effective amount of an antibody or antigen-binding fragment as provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of tumor development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.
In certain embodiments, an antibody or antigen-binding fragment as provided herein may be administered at a therapeutically effective dosage of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg) . In certain of these embodiments, the antibody or antigen-binding fragment is administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. In certain embodiments, the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.
Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) . For example, a single dose may be administered, or several divided doses may be administered over time.
The antibodies and antigen-binding fragments disclosed herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or  intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.
Conditions and disorders associated with PD-1 can be immune related disease or disorder. In certain embodiments, the PD-1 associated conditions and disorders include tumors and cancers, for example, non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas, mycoses fungoids, merkel cell cancer, and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL) , primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL) , plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, neoplasm of the central nervous system (CNS) , such as primary CNS lymphoma, spinal axis tumor, brain stem glioma. In certain embodiments, the tumors and cancers are metastatic, especially metastatic tumors expressing PD-L1. In certain embodiments, the PD-1 associated conditions and disorders include autoimmune diseases, such as systemic lupus erythematosus (SLE) , psoriasis, systemic scleroderma, autoimmune diabetes and the like. In certain embodiments, the PD-1 associated conditions and disorders include infectious disease such as chronic viral infection, for example, viral infection of hepatitis B, hepatitis C, herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type 2, human papilloma virus, adenovirus, Kaposi West sarcoma associated herpes virus epidemics, thin ring virus (Torquetenovirus) , JC virus or BK virus.
Methods of Use
The present disclosure further provides methods of using the anti-PD-1 antibodies or the antigen-binding fragments thereof.
In some embodiments, the present disclosure provides methods of treating a condition or a disorder associated with related to PD-1 in an individual, comprising administering a therapeutically effective amount of the anti-PD-1 antibody or antigen-binding fragment thereof. In certain embodiments, the individual has been identified as having a disorder or condition likely to respond to a PD-1 antagonist.
The presence or level of PD-L1 on an interested biological sample can be indicative of whether the individual from whom the biological sample is derived could likely respond to a PD-1 antagonist. Various methods can be used to determine the presence or level of PD-L1 in a test biological sample from the individual. For example, the test biological sample can be exposed to anti-PD-L1 antibody or antigen-binding fragment thereof, which binds to and detects the expressed PD-L1 protein. Alternatively, PD-L1 can also be detected at nucleic acid expression level, using methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, and the like. In some embodiments, the test sample is derived from a cancer cell or tissue, or tumor infiltrating immune cells. In certain embodiments, presence or upregulated level of the PD-L1 in the test biological sample indicates likelihood of responsiveness. The term “upregulated” as used herein, refers to an overall increase of no less than 10％, 15％, 20％, 25％, 30％, 35％, 40％, 45％, 50％, 55％, 60％, 65％, 70％, 75％, 80％or greater, in the protein level of PD-L1 in the test sample as detected using the antibodies or antigen-binding fragments provided herein, as compared to the PD-L1 protein level in a reference sample as detected using the same antibody. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from whom the test sample is obtained. For example, the reference sample can be a non-diseased sample adjacent to or in the neighborhood of the test sample (e.g. tumor) .
The antibodies or antigen-binding fragments disclosed herein may be administered alone or in combination with one or more additional therapeutic means or agents. For example, the antibodies or antigen-binding fragments disclosed herein may be administered in combination with chemotherapy, radiation therapy, surgery for the treatment of cancer (e.g., tumorectomy) , one or more anti-emetics or other treatments for complications arising from chemotherapy, or any other therapeutic agent for use in the treatment of cancer or any medical disorder mediated by PD-1. In certain of these embodiments, an antibody or antigen-binding fragment as disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments the antibody or antigen-binding fragment and the additional therapeutic agent (s) may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment administered “in combination” with another therapeutic agent does not have to be administered simultaneously with or in the same composition as the agent. An antibody or  antigen-binding fragment administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the antibody or antigen-binding fragment and second agent are administered via different routes. Where possible, additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians'Desk Reference 2003 (Physicians'Desk Reference, 57th Ed； Medical Economics Company； ISBN: 1563634457； 57th edition (November 2002) ) or protocols well known in the art.
In certain embodiments, the therapeutic agents can induce or boost immune response against cancer. For example, a tumor vaccine can be used to induce immune response to certain tumor or cancer. Cytokine therapy can also be used to enhance tumor antigen presentation to the immune system. Examples of cytokine therapy include, without limitation, interferons such as interferon-α, -β, and –γ, colony stimulating factors such as macrophage-CSF, granulocyte macrophage CSF, and granulocyte-CSF, interleukins such IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12, tumor necrosis factors such as TNF-α and TNF-β. Agents that inactivate immunosuppressive targets can also be used, for example, TGF-beta inhibitors, IL-10 inihibitors, and Fas ligand inhibitors. Another group of agents include those that activate immune responsiveness to tumor or cancer cells, for example, those enhance T cell activation (e.g. agonist of T cell costimulatory molecules such as CTLA-4, ICOS and OX-40) , and those enhance dendritic cell function and antigen presentation.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.
EXAMPLE 1: Antibody hybridoma generation
1.1 Immunogen generation: DNAs encoding PD-1 and PD-L1 ECD or full length were synthesized and inserted into the expression vector pcDNA3.3. Max-prep the plasmid DNAs and the inserted DNA sequences were verified by sequencing. Fusion proteins PD-1 ECD and PD-L1 ECD containing various tags, including human Fc, mouse Fc and His tags, were obtained by transfection of human PD-1 ECD gene into CHO-S or HEK293 cells. After 5 days, supernatants harvested from the culture of transiently transfected cells were used for protein purification. The fusion proteins were purified and quantitated for usage of immunization and screening.
1.2 Stable cell lines establishment. In order to obtain tools for antibody screening and validation, we generated PD-1 and PD-L1 transfectant cell lines. Briefly, CHO-K1, 293F or Ba/F3 cells were transfected with pCND3.3 expression vector containing full-length PD-1 or PD-L1 using Lipofectamine 2000 Transfection kit according to manufacturer’s protocol. At 48-72 hours post transfection, the transfected cells were cultured in medium containing Blasticidin or G418 for selection. Overtime this will select the cells that have stably incorporated PD-1 or PD-L1 genes into their genomic DNAs. Meanwhile the cells were checked for interested genes PD-1 and PD-L1 expression. Once the expression verified, single clones of interested were picked up by limited dilution and scaled up to large volumes. The established monoclonal cell lines then were maintained in medium containing lower dose of antibiotics Blasticidin or G418.
1.3 Antibody hybridoma generation.
1.3.1 Immunization and cell fusion: OMT-rats (obtained from Open Monoclonal Technology, Inc., Palo Alto, US) , 8-10 weeks of age, were immunized with 10μg of human PD-1 ECD protein in TiterMax in footpad for first boost, repeat the immunization every 3 days with PD-1 ECD protein in Aluminium. Bleed rats every two weeks for serum collection and antibody titers were measured by ELISA or FACS assay. When the antibody titer reached sufficient high, rats were given a final boost without adjuvant (add 100μl 1XPBS instead) and cell fusion was performed as following: B lymphocytes isolated from lymph node of immunized OMT-rats were combined with myeloma cells (at 1: 1 ratio) . Cell mixture were washed and suspended with 5-10ml ECF solution. Add ECF solution to adjust the concentration to 2x106cells/ml. After electronic cell fusion, cell suspension from the fusion chamber was immediately transferred into a sterile tube containing more volume of medium. After incubation for more than 24 hours in a 37 ℃, the cell suspension was mixed and pipetted into 96-well plates (0.5x106cells/plate) . Cells were incubated at 37℃, 5％CO2.  When the clones were big enough, transfer 100μl supernatant from the 96-well plates to assay for antibody screening.
1.3.2 First and confirmation screen of hybridoma supernatants: ELISA assay was used as first screen method to test the binding of hybridoma supernatants to PD-1 protein. Briefly, Plates (Nunc) were coated with soluble protein of human PD-1 extracellular domain at 1 μg/ml overnight at 4 ℃. After blocking and washing, the hybridoma supernatants were transferred to the coated plates and incubate at room temperature for 1 h. The plates were then washed and subsequently incubated with secondary antibody goat anti rat IgG1 HRP (Bethyl) and goat anti rat IgG2b HRP (Bethyl) for 45 min. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device) . In order to confirm the native binding of PD-1 antibodies on conformational PD-1 molecules expressed on cell membrane, FACS analysis was performed on PD-1 transfected CHO-S cell line. CHO-S cells expressing human PD-1 were transferred in to 96-well U-bottom plates (BD) at a density of 1x106 cells/ml. The hybridoma supernatants were then transferred to the plates and incubated for 1 h at 4 ℃. After washing with 1XPBS/1％BSA, the secondary antibody goat anti rat FITC (Jackson Immunoresearch Lab) was applied and incubated with cells at 4 ℃ in the dark for 1 h. The cells were then washed and resuspended in 1XPBS/1％BSA or fixed with 4％paraformldehyde, and analyzed by flow cytometery (BD) . Antibody binding to parental CHO-S cell line was performed using the same method. Figure 1 shows the binding of anti-human PD-1 antibodies to PD-1 expressing CHO cell. The CHO cells transfected with full-length human PD-1 were stained with antibodies against human PD-1 from rat hybridoma, followed by 2nd antibody staining with FITC conjugated goat anti-rat-lgG Fc and analyzed by FACS. The data show that the antibodies specifically bind to PD-1 expressed on CHO cells.
To test the binding affinity of the antibodies to native PD-1 expressed on human CD4+T cells, human CD4+T cell were generated from PBMC cultured in IL-2 and OKT3 for 3 days and were stained with the antibodies against human PD-1. Binding of the antibodies to the PD-1 on the T cells were analyzed by FACS. As shown in Figure 3, FACS analysis showed that the antibodies specially bind to native PD-1 expressed on CD4+T cells.
Testing the blocking activity of antibodies was used as confirmation screen to select potential antibody hits. Selected antibodies were tested for the ability to block the binding of the ligand PD-L1 to PD-1 transfected CHO-S cells by FACS analysis. CHO-S cells  expressing human PD-1 were transferred in to 96-well U-bottom plates (BD) at a density of 1x106 cells/ml. Antibodies were serially diluted in wash buffer (1XPBS/1％BSA) and incubated with the cells at 4 ℃ for 1 h. After washing, human Fc fusion-human PD-L1 protein was added and incubated at 4 ℃ for 1 h. The secondary antibody goat anti human IgG Fc FITC antibody (no cross-reactivity to rat IgG Fc, Jackson Immunoresearch Lab) was incubated with cells at 4 ℃ in the dark for 1 h. The cells were then washed and resuspended in 1XPBS/1％BSA or fixed with 4％paraformldehyde, and analyzed by flow cytometery (BD) .
1.3.3 Hybridoma subcloning: once specific binding and blocking were verified through first and confirmation screen, the positive hybridoma cell lines can be used for subcloning. Briefly, for each hybridoma cell line, cells were counted and diluted to give 5 cells/well, 1 cell/well and 0.5 cell/well in cloning medium. Plate 200μl/well into 96-well plates, one plate at 5 cells/well, one plate at 1 cell/well and four plates at 0.5 cell/well. Place all plates in incubator at 37 ℃, 5％CO2. Incubate until all the cell lines can be checked by ELISA assay.
EXAMPLE 2: Antibody hybridoma cell sequence and fully human antibody characterization
2.1 Antibody hybridoma cell sequence: RNAs were isolated from monoclonal hybridoma cells with Trizol reagent. The VH and VL of PD-1 antibodies were amplified as following protocol: briefly, RNA is first reverse transcribed into cDNA using a reverse transcriptase as described here, Reaction system (20μl) :
Figure PCTCN2015086594-appb-000021
Nuclease-free H2O to 20.0μl
Reaction condition
  Step1 Step2 Step3 Step4
Temperature
25 37 85 4
Time 10min 120min 5min ∞
The resulting cDNA is used as templates for subsequent PCR amplification using primers specific for interested genes. The PCR reaction was done as following procedure；
Figure PCTCN2015086594-appb-000022
Reaction condition:
Figure PCTCN2015086594-appb-000023
Take 10μl of PCR reaction to do the ligation with pMD18-T vector. Do the transformation of Top10 competent cells with 10μl ligation products and Transfer the mixture onto the pre-warmed 2-YT+Cab plates follow the standard protocol, incubate overnight. Positive clones were checked by PCR using M13-48 and M13-47 primers followed by sequencing.
2.2 fully human antibody molecule construction: The VH and VL of PD-1 antibodies were amplified as described above. The PCR reactions were purified with PCR clean-up kit and the VL and pCI vector were digested with restriction enzymes Pme I and BssH II at 37 ℃ for 2 hours. Run the reactions in 1％agarose and do gel extraction with kit according to manufacturer’s instruction. Ligation of digested VL and pCI vector as following procedures:
Figure PCTCN2015086594-appb-000024
The mixture was incubated at 16 ℃ for 30 minutes. 10μl of the reactions was used for tansformation and clone growth. Confirmed clones were used for the extraction of the plasmid pCI-VL DNA. The pCI-VL vector and VH fragment were then digested with Xbal and Sal I and the purified digested VH and vector were ligated with T4 DNA ligase 30minutes at 16 ℃. Once the sequence of inserted VL and VH were verified by sequencing, the expression vector containing whole IgG of fully human PD-1 antibody was used for transient transfection and stable cell line development.
EXAMPLE 3: Fully human antibody characterization
3.1 Full kinetic binding affinity tested by surface Plasmon resonance (SPR) : Antibodies were characterized for affinity and binding kinetics to PD-1 by SPR assay using ProteOn XPR36 (Bio-Rad) . Protein A protein (Sigma) was immobilized to a GLM sensor chip (Bio-Rad) through amine coupling. Purified antibodies were flowed over the sensor chip and captured by the Protein A. The chip was rotated 90° and washed with running buffer (1XPBS/0.01％Tween20, Bio-Rad) until the baseline is stable. Five concentrations of human PD-1 and running buffer were flowed against the antibody flow cell at a flow rate of 100 μL/min for an association phase of 240s, followed by 600s dissociation. The chip was regenerated with pH 1.7 H3PO4 after each run. The association and dissociation curve was fit to a 1: 1 Langmiur binding model using ProteOn software.
As shown in Figure 7, using surface plasmon resonance, the affinities of antibodies against PD-1 for recombinant human PD-1 were from 3.76E-9 to 1.76E-10 mol/L.
3.2 Binding affinity of PD-1 antibodies to cell surface PD-1 molecules tested by flow cytometry (FACS) : Antibody binding affinity to cell surface PD-1 was performed by FACS analysis. CHO-S cells expressing human PD-1 were transferred in to 96-well U-bottom plates (BD) at a density of 5x105 cells/ml. Tested antibodies were 1: 2 serially diluted in wash buffer (1XPBS/1％BSA) and incubated with cells at 4 ℃ for 1 h. The secondary antibody goat anti-human IgG Fc FITC (3.0moles FITC per mole IgG, (Jackson Immunoresearch Lab) was added and incubated at 4 ℃ in the dark for 1 h. The cells were then washed once and resuspended in 1XPBS/1％BSA, and analyzed by flow cytometery (BD) . Fluorescence intensity will be converted to bound molecules/cell based on the quantitative beads QuantumTM MESF Kits, Bangs Laboratories, Inc. ) . KD was calculated using Graphpad Prism5. Figure 2 shows the binding of the fully human PD-1 antibodies (i.e. 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb) to PD-1 expressing CHO cell. Fully human antibodies against human PD-1 were used to stain the PD-1 transfected CHO cells and the FACS analysis show that fully human PD-1 antibodies specially bind to PD-1 with EC50 about 2nmol/L.
CHO cells expressing human PD-1 were incubated with different concentrations of the antibodies against PD-1, then the mouse Fc-tagged human PD-L1 was added to the cells. The binding of human PD-L1 to PD-1 expressing cell was detected by using FITC-conjugated goat anti-mouse IgG, followed by the FACS analysis. As shown in Figure 4, antibodies against PD-1 blocked the binding of PD-L1 to PD-1 transfected CHO cells.
3.3 Orthologue (cross-species) and homologue (cross-families) screen:
3.3.1 Cross-reactivity to cynomolgus PD-1 and murine PD-1: Cross-reactivity was measured by ELISA. Plates (Nunc) were coated with cynomolgus PD-1 (Sino Biological) and murine PD-1 (Sino biological) at 1 μg/ml overnight at 4 ℃. After blocking and washing, 1 μg/ml antibodies were added to the plates and incubated at room temperature for 1 h. The plates were then washed and subsequently incubated with secondary antibody goat anti rat IgG1 HRP (Bethyl) and goat anti rat IgG2b HRP (Bethyl) for 45 min. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device) .
The result of cross-species experiment demonstrates that antibodies against PD-1 bind to cynomolgus monkey PD-1 but not bind to murine PD-1 (Figure 6) .
3.3.2 Cross-reactivity to PD-1 family members CD28, CTLA4 and ICOS: to examine the cross-family binding activity of the fully human antibodies, cells lines that express PD-1, CD28, CTLA4 or ICOS were stained with the antibodies, followed by 2nd antibody staining with FITC conjugated goat anti-human IgG Fc. PD-1 expressing cells were used as positive control. Corresponding parental cell lines were used as negative controls. The stained cells were analyzed by using a BD Biosciences FACSCanto II and FlowJo Version software.
Figure 5 shows that CHO cells transfected with PD-1, CD28 and 293F transfected with CTLA4 were stained with antibodies against PD-1 and analyzed by FACS. The result demonstrates PD-1 antibodies bind specifically to PD-1, but not to CD28 and CTLA4 of PD-1 family.
3.4 Epitope binning test:
3.4.1 The binding epitope of PD-1 antibodies was binned against benchmark antibody A and B by SPR assay using ProteOn XPR36 (Bio-Rad. Benchmark antibodies A and B were immobilized on GLC sensor chip (Bio-Rad) through amine coupling. Human PD-1 solution was flowed over the antibody immobilized channels and captured by the benchmark antibodies. The chip was then rotated 90° and washed with running buffer until the baseline is stable. Selected antibodies were flowed over the sensor chip.
3.4.2 The binding epitope of PD-1 antibodies was binned against benchmark antibody A and B by FACS. CHO cells expressing human PD-1 at the cell surface were incubated with benchmark antibody A or B at concentration of 10μg/ml for 1 hour. The cells were washed and the PD-1 antibodies of the disclosure were added and incubated for 1 hour. The second antibody anti-rat IgG-FITC were added and incubated for 1 hour at 4 ℃. The cells were then washed once and resuspended in 1XPBS/1％BSA, and analyzed by flow cytometery (BD) .
The results of SPR assay and FACS for the binning test showed that the epitope on human PD-1 bound by the fully human PD-1 antibodies (i.e. 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb) was different from the existing PD-1 antibodies (i.e. benchmark antibody A and B) .
3.5 In vitro function of PD-1 antibodies tested by cell-based assays:
3.5.1 Effects of human PD-1 antibodies on T cells proliferation. An allogeneic response was used to test the effects of PD-1 antibodies on T lymphocytes proliferation. Primary dendritic cell (DC) -stimulated MLR was conducted in 96-well, U-bottom tissue culture plates in 200 μl of RPMI 1640 containing 10％FCS and antibiotics. DCs were mixed with 1X105 allogeneic total CD4+T cells at a ratio between 1: 10 and 1: 100 DC: T cells. Cultures were also conducted in the presence or the absence of neutralizing mAbs: human PD-1 antibodies and benchmark antibody A and B and used at 10 μg /ml. Assays were incubated for 5 days, and during the last 16 h [3H] thymidine was added at 1 uCi/well. [3H] thymidine incorporation was measured by scintillation counting, and proliferative responses were expressed as the mean [3H] thymidine incorporation (counts per minute) of triplicate wells. Counts due to DCs alone were routinely <1000 cpm. Results shown are representative examples of a minimum of five experiments performed.
Human dendritic cells (DC) and CD4+T, CD8+T and total cells used in above allo-MLR were generated from the PBMC as following procedures: Human monocytes were purified from PBMC by negative selection using human monocyte enrichment cocktail kit according to the instructions of the manufacturer (StemCell Meylan) . Briefly, PBMC were isolated from blood of healthy donor using a Ficoll-Paque gradient. Cells were washed twice with PBS, then resuspended at 1X108 cells/ml in isolation buffer, and incubated with the monocyte enrichment Ab mixture at 4℃ for 30 min. The cells were washed and subsequently incubated with magnetic colloid at 4℃ for 30 min. Unlabeled monocytes passed through the MACS column and were collected. To generate iDCs, monocytes were cultured in RPMI 1640 medium containing 10％FCS and antibiotics with GM-CSF (PeproTech, Rocky Hill, NJ； 800 U/ml) and IL-4 (PeproTech； 500 U/ml) at concentration of 2X106 cells/ml. Half the medium was replaced every other day with GM-CSF-and IL-4-containing medium. Mature DCs were generated by stimulating iDCs with LPS (026: B6； Sigma-Aldrich, St. Louis, MO； 1 μg/ml) on day 5 for an additional 24 h. CD4+T, CD8+T and total T cells, were purified by negative selection by incubating PBMC with human CD4+T , CD8+T and total T cell enrichment mixture and magnetic colloid according to the manufacturer’s instructions (Stemsep) .
Human CD4+ T Cells were stimulated with allogenenic DCs in the presence or absence of PD-1 antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb. The proliferation of CD4+ T cells were assessed by [3H] thymidine incorporation. Figure 10 showed that 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb enhanced concentration dependent T cell proliferation.
3.5.2 Effects of human PD-1 antibodies on cytokine IFNγsecretion in vitro: to directly assess the effect of human PD-1 antibodies blockade on cytokine IFNγproduction, we performed experiments on IFNγproduction in allo-MLR. Briefly, human CD4+T cells were purified from PBMC by negative selection with CD4+T cell enrichment cocktail kit according to the instruction of the manufacturer. Immature DCs were generated from monocytes by cultured in GM-CSF and IL-4 for 5 days and mature DCs were differentiated by stimulation with LPS at 1μg/ml for overnight. CD4+T cells were mixture with iDC/mDc at a ratio between 10: 1 and 100: 1 T: DC ratio. The cultures were conducted in the presence or absence of human PD-1 antibodies and benchmark antibodies. After 5 days, the supernatants from each culture were harvested for cytokine IFNγmeasurement. The level of IFNγin supernatants was measured by ELISA assay. Briefly, Coat Maxisorp plates with anti-human IFN-gamma mAb diluted in coating buffer (0.75μg/ml； i.e. a1/1360 dilution) , 50μl/well (i.e. for a full 96-well plate add 3.7μl of antibody to 5 ml of coating buffer) and incubated overnight at 4 ℃. Block spare protein binding capacity by adding 200μl/well of blocking buffer for 2 hours. Prepare dilutions of recombinant IFN-gamma to act as standards, two-fold dilutions from 8000pg/ml down to 125pg/ml, diluted in complete medium, plus complete medium alone. Wash plates and add standards and test supernatants (100μl/well) , incubate for 2-4 hours. The biotinylated anti-IFN-gamma mAb (1/1333) in blocking buffer was added followed by adding Extra-avidin Peroxidase. The reaction was developed by adding TMB substrate and stopped with 2M HCl. Measure absorbance at 450nm.
Figure 9 shows that human CD4+ T Cells were stimulated with allogenenic DCs in the presence or absence of antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb. The level of IFNγwas measured by ELISA. The result showed the fully human PD-1 antibodies increased IFNγsecretion in a dose manner.
3.5.3 Effects of human PD-1 antibodies on interleukin 2 (IL-2) production in vitro: CD4+T cells were mixture with iDC/mDc at a ratio between 10: 1 and 100: 1 T: DC ratio. The cultures were conducted in the presence or absence of human PD-1 antibodies and benchmark antibodies. After 5 days, the supernatants from each culture were harvested for cytokine measurement. The level of IL-2 in supernatants was measured by ELISA assay.
Figure 8 shows that human CD4+ T Cells were stimulated with allogenenic DCs in the presence or absence of lead antibodies or control Ab. The level of IL-2 was measured by ELISA. The results showed antibodies against PD-1 increased IL-2 secretion in a dose-dependent manner.
3.5.4 Effect of human PD-1 antibodies on cell proliferation and cytokine production by autologous antigen specific immune response: in this assay, the T cells and DCs were from a same donor. Briefly, CD4+T cell were purified from PBMC and cultured in the presence of CMV pp65 peptide and low dose of IL2 (20U/ml) , at the meantime, DCs were generated by culturing monocytes from the same donor’s PBMC in GM-CSF and IL-4. After 5 days, the CMV pp65 peptide treated CD4+T cells were co-cultured with DCs pulsed with pp65 peptide in the absence or presence of human PD-1 antibodies and benchmark antibodies (as control) . On day 5, 100μl of supernatants were taken from each of cultures for cytokine IFNγand IL-2 measurement. The level of IFNγand IL-2 production was detected by ELISA assay. The proliferation of specific T cells to CMVpp65 peptide-pulsed DCs were assessed by [3H] thymidine incorporation.
As shown in Figure 11, PD-1 antibodies enhanced concentration dependent CMV+-CD4+ T cell proliferation stimulated with CMV pp65 peptide-loaded autologous DC.
3.5.5 Effect of human PD-1 antibodies on regulatory T cell (Tregs) suppressive function: Tregs, a subpopulation of T cells, are a key immune modulator and play key roles in maintaining self-tolerance. CD4+CD25+ regulatory T cell are associated with tumors because increased numbers of Tregs were found in patients with multiple cancers and is associated with a poorer prognosis. To directly assess the effect of human PD-1 antibodies on immune suppressive response, we performed experiment on Tregs. CD4+CD25+ and CD4+CD25-T cells were separated using specific anti-CD25 microbeads (Miltenyi Biotec, Auburn, CA) and positive or negative selection, respectively. Initially, CD4+ T cells were purified by negative selection by incubating PBMC with human CD4+T cell enrichment mixture and magnetic colloid according to the manufacturer’s instructions (Stemsep) . CD4+T cells were then resuspended in MACS buffer, incubated with CD25+microbeads on ice for 30 min, washed, and loaded on the column. CD4+CD25-T cells, which did not bind to the column, were collected from the flow-through and washed before use. CD4+CD25+T cells were subsequently retrieved from the column and washed before use. Tregs were cultured with CD4+CD25-T cells and DCs (Treg: Teff 1: 1ratio) in the presence or absence of human PD-1 antibodies at a concentration of 10μg/ml. Either no antibody or isotype antibody was used as  negative control. The supernatants from the cultures were taken on day 5 for cytokines detection by ELISA and the cell proliferation was measured by adding [3H] thymidine at a concentration of 1uCi/well and incubated for further 18 hours. [3H] thymidine incorporation was measured by scintillation counting. As shown in Figure 12, the PD-1 antibodies abrogated Treg’s suppressive function and restored responding T cell proliferation and IFNγ secretion.
3.6 ADCC/CDC assay: to minimize the undesired toxicity on healthy PD-1+ cells, the selected anti-PD-1 fully human antibodies were confirmed to have no ADCC and CDC function.
3.6.1 ADCC: Activated T cells expressing high levels of cell surface PD-1 were used as target cells and were pre-incubated with various concentrations of fully human antibodies in 96-well plates for 30min, then IL-2-activated PBMCs (used as a source of natural killer (NK) cells, i.e. the effector cells) were added at the effector/target ratio of 50: 1. The plates were incubated for 6 hours at 37℃ in a 5％CO2 incubator. Target cell lysis was determined by cytotoxicity detection kit (Roche) . Optical density was measured by Molecular Devices SpectraMax M5e Plate Reader. Results showed that, the tested fully human antibodies against PD-1 did not mediate ADCC (Figure 13) .
3.6.2 CDC: target cells (activated T cell) , diluted human serum complement (Quidel-A112) and various concentrations of fully human PD-1 antibodies were mixed in a 96-well plate. The plate was incubated for 4 h at 37℃ in a 5％CO2 incubator. Target cell lysis was determined by CellTiter glo (Promega-G7573) . Rituxan (Roche) and human B lymphoma cell line Raji (CD20 positive) were used as positive control. The data showed that PD-1 antibodies did not mediated CDC (Figure 14) .
While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments) , it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

Claims (33)

  1. An isolated antibody or an antigen binding fragment thereof, comprising a heavy chain CDR sequences selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35 and 37.
  2. The antibody or an antigen binding fragment thereof of claim 1, comprising a light chain CDR sequences selected from the group consisting of: 7, 9, 11, 17, 19, 27, 29, 31, 39, 41, and 43.
  3. The antibody or an antigen binding fragment thereof of any of the preceding claims, comprising a heavy chain variable region selected from the group consisting of:
    a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5；
    b) a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 5；
    c) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 15, and/or SEQ ID NO: 5；
    d) a heavy chain variable region comprising SEQ ID NO: 21, SEQ ID NO: 23, and/or SEQ ID NO: 25； and
    e) a heavy chain variable region comprising SEQ ID NO: 33, SEQ ID NO: 35, and/or SEQ ID NO: 37.
  4. The antibody or an antigen binding fragment thereof of any of the preceding claims, comprising a light chain variable region selected from the group consisting of:
    a) a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9, and/or SEQ ID NO: 11；
    b) a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 11；
    c) a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 19；
    d) a light chain variable region comprising SEQ ID NO: 27, SEQ ID NO: 29, and/or SEQ ID NO: 31； and
    e) a light chain variable region comprising SEQ ID NO: 39, SEQ ID NO: 41, and/or SEQ ID NO: 43.
  5. The antibody or an antigen binding fragment thereof of any of the preceding claims, comprising:
    a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5； and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9, and/or SEQ ID NO: 11；
    b) a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 5； and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 11；
    c) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 15, and/or SEQ ID NO: 5； and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 19；
    d) a heavy chain variable region comprising SEQ ID NO: 21, SEQ ID NO: 23, and/or SEQ ID NO: 25 and a light chain variable region comprising SEQ ID NO: 27, SEQ ID NO: 29, and/or SEQ ID NO: 31； or
    e) a heavy chain variable region comprising SEQ ID NO: 33, SEQ ID NO: 35, and/or SEQ ID NO: 37； and a light chain variable region comprising SEQ ID NO: 39, SEQ ID NO: 41, and/or SEQ ID NO: 43.
  6. The antibody or an antigen binding fragment thereof of any of the preceding claims, comprising a heavy chain variable region selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57 and SEQ ID NO: 61.
  7. The antibody or an antigen binding fragment thereof of any of the preceding claims, comprising a light chain variable region selected from the group consisting of: SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, and SEQ ID NO: 63.
  8. The antibody or an antigen binding fragment thereof of any of the preceding claims, comprising:
    a) a heavy chain variable region comprising SEQ ID NO: 45； and a light chain variable region comprising SEQ ID NO: 47；
    b) a heavy chain variable region comprising SEQ ID NO: 49； and a light chain variable region comprising SEQ ID NO: 51；
    c) a heavy chain variable region comprising SEQ ID NO: 53； and a light chain variable region comprising SEQ ID NO: 55；
    d) a heavy chain variable region comprising SEQ ID NO: 57； and a light chain variable region comprising SEQ ID NO: 59； or
    e) a heavy chain variable region comprising SEQ ID NO: 61； and a light chain variable region comprising SEQ ID NO: 63.
  9. The antibody or an antigen binding fragment thereof of any of the preceding claims,  capable of specifically binding to human PD-1 at an Kd value no more than 10-8 M as measured by plasmon resonance binding assay.
  10. The antibody or an antigen binding fragment thereof of any of the preceding claims, which binds to monkey PD-1 at an EC50 of no more than 100nM, or no more than 10nM, and/or does not bind to mouse PD-1.
  11. The antibody or an antigen binding fragment thereof of any of the preceding claims, capable of inhibiting binding of human or monkey PD-1 to its ligand at an IC50 of no more than 100 nM.
  12. The antibody or an antigen binding fragment thereof of any of the preceding claims, which does not substantially bind to CD28 or CTLA4.
  13. The antibody or an antigen binding fragment thereof of any of the preceding claims, which does not mediate ADCC or CDC or both.
  14. The antibody or an antigen binding fragment thereof of any of the preceding claims, which is a fully human monoclonal antibody.
  15. The antibody or an antigen binding fragment thereof of claim 14, wherein the fully human monoclonal antibody is produced by a transgenic rat.
  16. An antibody or an antigen binding fragment thereof, which competes for the same epitope with the antibody or the antigen binding fragment thereof of any of the preceding claims.
  17. The antibody or an antigen binding fragment thereof of any of the preceding claims, capable of blocking binding of human PD-1 to its ligand and thereby providing at least one of the following activities:
    a) inducing production of IL-2 in CD4+T cells；
    b) inducing production of IFNγ in CD4+T cells；
    c) inducing proliferation of CD4+T cells； and
    d) reversing T reg’s suppressive function.
  18. The antibody or antigen-binding fragment thereof of any of the preceding claims, which is a camelized single domain antibody, a diabody, a scFv, an scFv dimer, a BsFv, a dsFv, a (dsFv) 2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F (ab') 2, a ds diabody, a nanobody, a domain antibody, or a bivalent domain antibody.
  19. The antibody or antigen-binding fragment thereof of any of the preceding claims, further comprising an immunoglobulin constant region.
  20. The antibody or antigen-binding fragment thereof of any of the preceding claims, further comprising a conjugate.
  21. An isolated polynucleotide encoding the antibody or an antigen binding fragment thereof of claims 1-19.
  22. A vector comprising the isolated polynucleotide of claim 21.
  23. A host cell comprising the vector of claim 22.
  24. A method of expressing the antibody or antigen-binding fragment thereof of any of claims 1-19, comprising culturing the host cell of claim 23 under the condition at which the polynucleotide of claim 21 is expressed.
  25. A kit comprising the antibody or antigen-binding fragment thereof of any of claims 1-20.
  26. A method of treating a condition associated with PD-1 in an individual, comprising: administering a therapeutically effective amount of antibody or antigen-binding fragment thereof of any of claims 1-20 to the individual.
  27. The method of claim 26, wherein the individual has been identified as having a disorder or a condition likely to respond to a PD-1 antagonist.
  28. The method of claim 27, wherein the individual has been identified as positive for presence or upregulated level of the PD-L1 in a test biological sample from the individual.
  29. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any of claims 1-20 and one or more pharmaceutically acceptable carriers.
  30. A method of treating a condition in a subject that would benefit from upregulation of immune response, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any of claims 1-20 to the subject.
  31. The method of claim 30, wherein the subject has upregulated expression of PD-L1.
  32. Use of the antibody or antigen-binding fragment thereof of any of claims 1-20 in the manufacture of a medicament for treating a condition that would benefit from upregulation of immune response.
  33. The use of claim 32, wherein the condition is cancer or chronic viral infection.
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PCT/CN2015/086594 WO2017024515A1 (en) 2015-08-11 2015-08-11 Novel anti-pd-1 antibodies
IL293385A IL293385A (en) 2015-08-11 2016-08-11 Novel anti-pd-1 antibodies
CA2993276A CA2993276A1 (en) 2015-08-11 2016-08-11 Novel anti-pd-1 antibodies
BR112018002824A BR112018002824A2 (en) 2015-08-11 2016-08-11 antibody, polynucleotide, vector, cell, methods to express the antibody, to treat a condition associated with pd-1 and to treat a condition in a subject, kit, pharmaceutical composition and use of the antibody
UAA201802340A UA124379C2 (en) 2015-08-11 2016-08-11 Novel anti-pd-1 antibodies
EA201890468A EA201890468A1 (en) 2015-08-11 2016-08-11 NEW ANTIBODIES AGAINST PD-1 PROTEIN
RU2018108048A RU2729830C2 (en) 2015-08-11 2016-08-11 Novel anti-pd-1 antibodies
MYPI2018700283A MY187739A (en) 2015-08-11 2016-08-11 Novel anti-pd-1 antibodies
PE2018000225A PE20181018A1 (en) 2015-08-11 2016-08-11 NOVELTY ANTI-PD-1 ANTIBODIES
RU2020124273A RU2020124273A (en) 2015-08-11 2016-08-11 NEW ANTI-PD-1 ANTIBODIES
US15/751,236 US11008391B2 (en) 2015-08-11 2016-08-11 Anti-PD-1 antibodies
MA042626A MA42626A (en) 2015-08-11 2016-08-11 NEW ANTI-PD-1 ANTIBODIES
MX2018001644A MX2018001644A (en) 2015-08-11 2016-08-11 Novel anti-pd-1 antibodies.
JP2018526985A JP6883579B2 (en) 2015-08-11 2016-08-11 New anti-PD-1 antibody
IL257062A IL257062B (en) 2015-08-11 2016-08-11 Novel anti-pd-1 antibodies
KR1020177036687A KR102055396B1 (en) 2015-08-11 2016-08-11 Novel Anti-PD-1 Antibodies
EP16834675.7A EP3334763A4 (en) 2015-08-11 2016-08-11 Novel anti-pd-1 antibodies
PCT/CN2016/094624 WO2017025051A1 (en) 2015-08-11 2016-08-11 Novel anti-pd-1 antibodies
AU2016305697A AU2016305697B2 (en) 2015-08-11 2016-08-11 Novel anti-PD-1 antibodies
SG10201914109VA SG10201914109VA (en) 2015-08-11 2016-08-11 Novel anti-pd-1 antibodies
PH12018500183A PH12018500183A1 (en) 2015-08-11 2018-01-24 Novel anti-pd-1 antibodies
CONC2018/0000992A CO2018000992A2 (en) 2015-08-11 2018-01-30 Novel anti-PD-1 antibodies
MX2022006447A MX2022006447A (en) 2015-08-11 2018-02-08 Novel anti-pd-1 antibodies.
CL2018000370A CL2018000370A1 (en) 2015-08-11 2018-02-09 New anti-pd-1 antibodies
SA518390903A SA518390903B1 (en) 2015-08-11 2018-02-11 Novel anti-PD-1 antibodies
SA521422116A SA521422116B1 (en) 2015-08-11 2018-02-11 Novel anti-PD-1 antibodies
ECIEPI201818842A ECSP18018842A (en) 2015-08-11 2018-03-09 NEW ANTI-PD-1 ANTIBODIES
JP2020200034A JP2021036914A (en) 2015-08-11 2020-12-02 Novel anti-pd-1 antibodies
US17/239,866 US11643465B2 (en) 2015-08-11 2021-04-26 Anti-PD-1 antibodies
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