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    1/1 abstract â € œIsolated antibody or antigen-binding fragment thereof, isolated nucleic acid, host cell, method of testing a tissue sample removed from a human for expression of pd -l1, kit, and, antibody composition comprising a mixture of antibody moleculesâ € ”the present disclosure provides isolated antibodies that specifically bind to human pd-l1, as well as binding fragments§ antigen antigen of such antibodies, and kits comprising anti-pd-11 antibodies or binding fragments and a set of reagents to detect an antibody complex, or antigen binding fragment thereof, bound in human pd-l1. the antibodies and antigen-binding fragments of this disclosure are used for immunohistochemical detection of human pd-11 expression in tissue samples. nucleic acid molecules encoding the antibodies and antigen-binding fragments of this disclosure are also provided, as well as expression vectors and host cells for expression of these.
    公开号:BR112015014833B1
    申请号:R112015014833-6
    申请日:2013-12-18
    公开日:2020-06-16
    发明作者:Robert H. Pierce；Patrícia Bourne；Linda Liang；Michael Bigler
    申请人:Merck Sharp & Dohme Corp.；
    IPC主号:
    专利说明:

    ISOLATED ANTIBODY OR BINDING FRAGMENT, NUCLEIC ACID, IN VITRO METHOD, KIT AND ANTIBODY COMPOSITION
    FIELD OF THE INVENTION
    [001] The present invention relates to antibodies with specific sequences that bind to human programmed death ligand 1 (PD-L1) and are used to detect expression of PD-L1 in human tissue samples by immunohistochemical analysis ( IHC). The invention also concerns specific IHC assays that employ these anti-human PD-L1 antibodies.
    BACKGROUND OF THE INVENTION
    [002] PD-L1 is a cell surface glycoprotein that is one of the two known ligands for programmed death 1 (PD-1), which is recognized as having an important role in immune regulation and in maintaining peripheral tolerance. Expression of PD-L1 has been observed on the surface of a variety of immune cells, including naive lymphocytes and activated B and T cells, monocytes and dendritic cells (Id.). In addition, PD-L1 mRNA is expressed by non-lymphoid tissues including vascular endothelial cells, epithelial cells, muscle cells, and amygdala and placental tissue. See, for example, Keir, M.E. et al., Annu Rev Immunol. 26: 677-704 (2008); Sharp A.H. et al., Nature Immunol. 8: 239-245 (2007); Okazaki T and Honjo T, Internat. immunol. 19: 813-824 (2007).
    [003] PD-L1 expression has also been observed in a variety of human cancers, and the interaction of PD-L1 expressed in tumor cells with PD-1 can induce tumor-specific T cell display or apoptosis. In large sample sets, for example, of ovarian, kidney, colorectal, pancreatic, liver and melanoma cancers, PD-L1 expression was found to correlate with poor prognosis and low overall survival regardless of subsequent treatment. Monoclonal anti-PD-1 antibodies
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    2/68 that block binding of PD-L1 to PD-1 have been shown to have antitumor activity against a variety of tumor types, with early human clinical data suggesting that patients whose tumors express PD-L1 are more likely to respond to anti- PD-1. See, for example, Iwai et al., PNAS 99: 1229312297 (2002); Ohigashi et al., Clin Cancer Res 11: 2947-2953 (2005); Ghebeh et al., Neoplasia 8: 190-198 (2006); Hamanishi, J et al., PNAS 104: 3360-3365 (2007); Yang et al., Invest Ophthalmol Vis Sci. 49 (6): 2518-2525 (2008); Gao et al., Clin Cancer Res 15: 971-979 (2009); Brahmer J.R. et al., J Clin Oncol. 28: 31673175 (2010).
    [004] A recent report describes the comparison of 15 anti-human PD-L1 antibodies for use in detecting hPD-L1 expression in formalin-fixed paraffin-embedded human melanoma (FFPE) samples (Gadiot, J., et al., Cancer 117 (10): 2192-2201 (2011)). The utility criteria evaluated in this comparison were: (1) ability to stain paraffin embedded tissues, (2) low background color production, and (3) blocked binding to PD-L1 by pre-incubation with a PDL1 fusion protein . The authors concluded that Ab # 4059, a polyclonal rabbit anti-human antibody (obtained from ProSci, Poway, CA USA), was the only anti-human PD-L1 antibody of the 15 tested that accepted all of these criteria (Id. At 2195, 2 to column).
    SUMMARY OF THE INVENTION
    [005] The present invention relates to anti-human PD-L1 monoclonal antibodies, which produce an IHC staining pattern on FFPE tonsil tissue that the inventors here believe to be more immunologically relevant than that produced by Ab # 4059 ProSci . As described in the Examples below, the inventors observed that this ProSci antibody (PRS4059, Sigma-Aldrich lot 40590604) stained all strains
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    3/68 hematopoietic in the amygdala with the same intensity, while two antibodies of the present invention, 22C3 and 20C3, selectively stained cells of the tonsillar crypt epithelium and follicular CD68 + myeloid, which are morphologically consistent with macrophages. Furthermore, 22C3 and 20C3 demonstrate a consistent difference in intensity between these two discrete cell populations, with the intensity of staining in the crypt epithelium being much greater than in follicular macrophages. All three antibodies (PRS4059, 22C3 and 20C3) are neutralized with pre-incubation with the PD-L1 antigen, indicating that the reactivity is mediated by the antigen binding domain (CDRs). Thus, the invention also concerns the use of the antibodies of the present invention in detecting expression of PD-L1 on the surface of human cells, including in IHC analyzes to detect PDL1 in sections of FFPE tissue.
    [006] In one aspect, the invention provides an isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human PDL1. The isolated monoclonal antibody, or antigen binding fragment thereof, comprises three CDRL1, CDRL2 and CDRL3 light chain CDRs and three CDRH1, CDRH2 and CDRH3 heavy chain CDRs.
    [007] CDRL1 is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 21, a variant of SEQ ID NO: 9, and a variant of SEQ ID NO: 21. CDRL2 is selected from the group consisting of SEQ ID NO: 2 and a variant of SEQ ID NO: 2. CDRL3 is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 22, a variant of SEQ ID NO: 10, and a variant of SEQ ID NO: 22.
    [008] CDRH1 is selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 26, SEQ ID NO: 27, a variant of SEQ ID NO: 14, a variant of SEQ ID NO: 15, a variant of SEQ ID NO: 26, and a
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    4/68 variant of SEQ ID NO: 27. CDRH2 is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 28, a variant of SEQ ID NOs: 16, and a variant of SEQ ID NO: 28. CDRH3 is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 29, a variant of SEQ ID NOs: 17, and a variant of SEQ ID NO: 29.
    [009] In antibodies and antigen binding fragments of the invention, a variant CDR sequence (light chain or heavy chain) is identical to the reference sequence, except that it has one or two conservative amino acid substitutions in the reference sequence and, preferably , has only a conservative amino acid substitution in the reference sequence. In preferred embodiments, a maximum of two of the three light chain CDRs are a variant sequence, and a maximum of two of the three heavy chain CDRs are a variant sequence. In more preferred embodiments, only three, two or one of the six CDRs are variant sequences.
    [0010] In a preferred embodiment, the three light chain CDRs are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 and the three heavy chain CDRs are SEQ ID NO: 5, SEQ ID NO : 6 and SEQ ID NO: 7.
    [0011] In another preferred embodiment, the three light chain CDRs are SEQ ID NO: 9, SEQ ID NO: 2, and SEQ ID NO: 10 and the three heavy chain CDRs are SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17.
    [0012] In yet another preferred embodiment, the three light chain CDRs are SEQ ID NO: 21, SEQ ID NO: 2 and SEQ ID NO: 22 and the three heavy chain CDRs are SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 29.
    [0013] Some antibodies and antigen binding fragments of the invention comprise a light chain variable region and a heavy chain variable region. The light chain variable region is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 13, a variant of SEQ ID NO: 13, SEQ ID
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    NO: 25 and a variant of SEQ ID NO: 25, and the heavy chain variable region is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 20, a variant of SEQ ID NO: 20, SEQ ID NO: 32 and a variant of SEQ ID NO: 32. In such embodiments, a light chain variable region variant sequence is identical to the reference sequence except with up to five conservative amino acid substitutions in the framework region (i.e., outside the CDRs), and preferably has less than four, three or two substitutions conservative amino acid in the structure region. Similarly, a variable heavy chain variable region sequence is identical to the reference sequence except with up to 17 conservative amino acid substitutions in the structure region (i.e., outside the CDRs), and preferably has less than ten, nine, eight, seven , six or five conservative amino acid substitutions in the structure region.
    [0014] In a preferred antibody or antigen-binding fragment of the invention, the light chain variable region is SEQ ID NO: 13 and the heavy chain variable region is SEQ ID NO: 20.
    Another preferred antibody or antigen binding fragment of the invention comprises a light chain variable region of SEQ ID NO: 25 and a heavy chain variable region of SEQ ID NO: 32.
    [0016] In yet another embodiment, the antibody or binding fragment of the invention comprises a light chain variable region of SEQ ID NO: 25 and a heavy chain variable region of SEQ ID NO: 32, where X in SEQ ID NO: 32 is pE.
    [0017] Still in an additional embodiment, the antibody or binding fragment of the invention comprises a light chain variable region of SEQ ID NO: 25 and a heavy chain variable region of SEQ ID NO: 32, where X in SEQ ID NO: 32 is Q.
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    [0018] In all of the above antibody modalities, the isolated antibody can be a full-length antibody of any immunoglobulin class, including IgM, IgG, IgD, IgA and IgE. Preferably, the antibody is an IgG antibody, such as IgG1, IgG2, IgG3 or IgG4. In one embodiment, the antibody comprises a mouse IgG1 constant region.
    [0019] Particularly preferred antibodies are monoclonal antibodies 20C3 and 22C3, which are IgG1 antibodies expressed by hybridomas MEB037.20C3 and MEB037.22C3, respectively.
    [0020] The invention also provides an isolated monoclonal antibody, or an antigen-binding fragment thereof, which specifically binds to human PD-L1 and blocks binding to human PD-L1 of 20C3 or 22C3, or an antibody reference comprising SEQ ID NO: 25 and SEQ ID NO: 32. In a preferred embodiment, an antibody or antigen-binding fragment of the invention blocks binding to the human PD-L1 of each of 20C3 and 22C3, or of each of (a) a reference antibody comprising SEQ ID NO: 13 and SEQ ID NO: 20 and (b) a reference antibody comprising SEQ ID NO: 25 and SEQ ID NO: 32.
    [0021] The invention also provides an antibody composition, which comprises any of the antibodies or antibody fragments described above in a formulation. A suitable formulation comprises 20 mM sodium acetate and 9% sucrose at pH 5.0. In a preferred embodiment, the composition comprises a mixture of antibody molecules, in which a majority (i.e., more than any of 60%, 65%, 70%, 80%, 85%, 90% or 95%) of the molecules of the antibody in the mixture comprise SEQ ID NO: 25 and SEQ ID NO: 32, where X in SEQ ID NO: 32 is pE, and the rest of the antibody molecules in the mixture comprise SEQ ID NO: 25 and SEQ ID NO: 32 , where X in SEQ ID NO: 32 is Q.
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    [0022] In any of the above embodiments, the antigen-binding fragment is a Fab fragment, a Fab 'fragment, a (Fab') 2 fragment.
    [0023] In any of the foregoing embodiments, the antibody or antigen-binding fragment may additionally comprise a detectable marker.
    [0024] The invention also provides an isolated nucleic acid that encodes any of the variable regions of the antibody disclosed above. In a preferred embodiment, the nucleic acid comprises one or both of SEQ ID NO: 33 and SEQ ID NO: 34. In another preferred embodiment, the nucleic acid comprises one or both of SEQ ID NO: 35 and SEQ ID NO: 36. In any of these embodiments, the isolated nucleic acid is preferably an expression vector.
    [0025] The invention also relates to a host cell comprising an expression vector that encodes any of the variable regions of the antibody disclosed above. Preferably, the expression vector comprises SEQ ID NO: 33 and SEQ ID NO: 34 or comprises SEQ ID NO: 35 and SEQ ID NO: 36.
    [0026] The invention also provides a method of analyzing a human tissue sample that has been removed from a human for expression of PD-L1. The method of analysis comprises placing the tissue sample in contact with a PD-L1 binding reagent under conditions that allow specific binding of the PD-L1 binding reagent to the human PD-L1, removing PD-L1 binding reagent unbound, and detecting the presence or absence of bound PD-L1 binding agent. In a preferred embodiment, the method comprises further quantifying the amount of bound reagent. The binding reagent is any of the monoclonal antibody or antigen binding fragments described above.
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    Preferably, the binding reagent is an antibody that comprises SEQ ID NO: 13 and SEQ ID NO: 20, or comprises SEQ ID NO: 25 and SEQ ID NO: 32. In a preferred embodiment, the binding reagent is an antibody composition comprising a mixture of antibody molecules comprising SEQ ID NO: 25 and SEQ ID NO: 32, wherein a majority of the molecules (i.e., more than any of 60 %, 65%, 70%, 80%, 85%, 90% or 95%) have pE in the X position in SEQ ID NO: 32 and the rest of the molecules have Q in the X position in SEQ ID NO: 32.
    [0027] In another aspect, the invention provides a kit for analyzing a human tissue sample for expression of PD-L1. The kit comprises a PD-L1 binding agent and a set of reagents to detect a complex comprising the human PD-L1 binding agent. The PD-L1 binding agent is any monoclonal antibody or antigen-binding fragment described above that specifically binds to human PD-L1. Preferably, the antibody or binding fragment comprises SEQ ID NO: 13 and SEQ ID NO: 20, or comprises SEQ ID NO: 25 and SEQ ID NO: 32. In a preferred embodiment, the binding reagent is an antibody composition comprising a mixture of antibody molecules comprising SEQ ID NO: 25 and SEQ ID NO: 32, wherein a majority of the molecules (i.e., more than any of 60 %, 65%, 70%, 80%, 85%, 90% or 95%) have pE in the X position in SEQ ID NO: 32 and the rest of the molecules have Q in the X position in SEQ ID NO: 32.
    BRIEF DESCRIPTION OF THE DRAWINGS
    [0028] Figure 1 shows the nucleotide sequences for antibody variable light and heavy chain cDNA prepared from total RNA isolated from hybridoma MEB037.20C3 and the predicted amino acid sequences encoded hereby (bold font), with square brackets indicating
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    9/68 nucleotide and amino acid sequences for the leader peptide and underlines indicating the CDR sequences.
    [0029] Figure 2 shows the nucleotide sequences for antibody variable light and heavy chain cDNA prepared from total RNA isolated from hybridoma MEB037.22C3 and the predicted amino acid sequences encoded hereby (bold font), with square brackets indicating nucleotide and amino acid sequences for the leader peptide and underlines indicating the CDR sequences.
    [0030] Figure 3 shows the amino acid sequences aligned for the mature variable regions of light and heavy chains of antibodies 20C3 and 22C3, with bold font indicating the positions where the sequences vary, underlining indicating the CDR sequences, as defined by Kabat numbering system, and brackets indicating the CDR1 heavy chain as defined by the Chothia numbering system.
    [0031] Figure 4 shows staining of sections of amygdala produced by immunohistochemical analysis using the commercially available antibody PRS4059 (Fig. 4A) or the antibody of the invention 22C3 (Fig. 4B), with the sections on the right side of Figs. 4A and 4B showing the results after pre-incubation with a PD-L1-IgG1 fusion protein (R&D Systems), which competes with anti-human PD-L1 antibodies for binding to human PD-L1.
    [0032] Figure 5 shows photographs of sections of adjacent FFPE tonsil tissue in which human PD-L1 protein and in situ hybridization mRNA (ISH) expression were analyzed by IHC analysis using 22C33 antibody (Fig. 5A) and in situ hybridization (ISH) (Fig. 5B), respectively, and which demonstrate differential staining between two exclusive cell populations: crypt epithelium (Fig. 5A, left extended view and Fig. 5B, side view)
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    10/68 top) and follicular macrophages (Fig. 5A, right extended view and Fig. 5B, right from the base).
    [0033] Figure 6 illustrates the results of a flow cytometric evaluation of the binding of various anti-human PD-L1 antibodies and an isotope control antibody in HT144 cells, which have been known to be negative for hPD-L1 expression by analysis mRNA (qPCR) (Fig. 6A) and LOX melanoma cells, which have been known to express high levels of PD-L1 hRNAm (qPCR) (Fig. 6B).
    [0034] Figure 7 shows the IHC staining produced by the 22C3 antibody in FFPE cell precipitates from modified CHO cell lines (Fig. 7A) and human cell lines (Fig. 7B, top panel), and demonstrates that the staining intensity correlates well with mRNA of hPD-L1 expression levels measured in the same human cell lines (Fig. 7B, background panel).
    [0035] Figure 8 illustrates the selective binding and relative affinity of 22C3 and 20C3 antibodies to hPD-L1, with the graphs showing the results of a cell-based ELISA experiment in which cells that do not express hPDL1 (Fig. 8A), express hPDL -1 (Figs. 8B and 8C), or express human PD-L2 (Fig. 8D) were incubated with the indicated primary antibody at the indicated concentrations, and then binding the detected primary antibody with a secondary goat anti-human IgG antibody, as described in the Examples.
    [0036] Figure 9 shows the results of antibody binding competition analyzes showing that 22C3 and 20C3 antibodies bind non-identical but overlapping epitopes.
    [0037] Figure 10 illustrates the results of a semi-quantitative visual reading score of the ICH staining intensity of 22C3 of FFPE samples
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    11/68 of the indicated types of tumor, with the extension of staining increasing with an increase in the score numbers.
    [0038] Figure 11 illustrates that human PD-L1 expression detected with 22C3 antibody in an IHC analysis correlates with melanoma patients' response to therapy with an anti-human PD-1 antibody (MK-3475), showing images representative in Fig. 11A of 22C3 IHC stain interpreted as positive, negative or equivocal for hPD-L1 expression and Fig. 11B showing the number of patients who had a positive or negative response that were scored as positive or negative (including scored patients) equivocal) for expression of hPD-L1 by IHC analysis.
    DETAILED DESCRIPTION OF THE INVENTION
    [0039] Abbreviations. The following abbreviations will be used throughout the detailed description and examples of the invention:
    ADCC Antibody-dependent cellular cytotoxicity
    CDC Complement-dependent cytotoxicity
    CDR Complementary determining region in variable immunoglobulin regions, defined using the Kabat numbering system, unless otherwise indicated
    CHO Chinese hamster ovary
    Clothia An antibody numbering system described in Al-Lazikani et al., JMB 273: 927-948 (1997)
    EC50 Concentration that results in 50% efficiency or bonding
    ELISA Enzyme linked immunosorbent analysis
    Formalin fixed FFPE, embedded in paraffin
    FR Antibody structure region: the immunoglobulin variable regions excluding the CDR regions.
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    HRP horseradish peroxidase
    IFN interferon
    IC50 Concentration that results in 50% display
    IgG Immunoglobulin G
    Kabat An immunoglobulin numbering and alignment system initially presented by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) MAb or Mab or MAb Antibody monoclonal
    MES 2- (N-morpholino) ethanesulfonic acid
    MOA Mechanism of Action
    NHS Normal human serum
    PCR pE pyroglutamate polymerase chain reaction
    PK Pharmacokinetics
    SEB etafilococos B enterotoxin
    TT Tetanus toxoid
    Region V The segment of IgG chains that is variable in sequence between different antibodies. It extends to 109 Kabat residues in the light chain and 113 in the heavy chain.
    VH immunoglobulin heavy chain variable region
    VK kappa light chain variable region of immunoglobulin
    DEFINITIONS
    [0040] In order for the invention to be more easily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all
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    13/68 other technical and scientific terms used here are commonly understood by those versed in the technique to which this invention belongs.
    [0041] As used herein, including the appended claims, singular forms of words such as ", one", and ", a" include their corresponding plural references, unless the context clearly dictates otherwise.
    [0042] Activation in the way it applies to cells or receptors refers to the activation or treatment of a cell or receptor with a ligand, unless otherwise indicated or explicitly by context. Ligand encompasses natural and synthetic ligands, for example, cytokines, cytokine variants, analogs, muteins, and antibody-derived binding compounds. Ligand also encompasses small molecules, for example, cytokine peptide mimetics and antibody peptide mimetics. Activation can refer to cell activation regulated by internal mechanisms, as well as external or environmental factors.
    [0043] Activity of a molecule can describe or refer to the binding of the molecule in a ligand or in a receptor, for catalytic activity; the ability to stimulate gene expression or signaling, differentiation, or cell maturation; antigenic activity, for the modulation of activities of other molecules, and the like. Activity of a molecule can also refer to the activity of modulating and maintaining cell-to-cell interactions, for example, adhesion, or activity of maintaining a cell structure, for example, cell membranes or cytoskeleton. Activity can also mean specific activity, for example, concentration in a biological compartment [catalytic activity] / [mg protein], or [immunological activity] / [mg protein], or the like. Activity can refer to the modulation of components of innate or adaptive immune systems.
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    [0044] Administration and treatment ", as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of a pharmaceutical, therapeutic, exogenous diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell includes contact of a reagent with the cell, as well as contact of a reagent with a fluid, where the fluid is in contact with the cell. Administration and treatment also means in vitro and ex vivo treatments, for example, of a cell, by a reagent, diagnostic, binding compound, or by another cell. The term subject includes any organism, preferably an animal, more preferably a mammal (for example, rat, mouse, dog, cat, rabbit) and preferably a human.
    [0045] Treating or treating means administering a therapeutic agent, such as a composition containing any of the antibodies or antigen binding fragments of the present invention, internally or externally to a subject or patient with one or more symptoms of disease, or suspected of having a disease, for which the agent has therapeutic activity. Typically, the agent is administered in an effective amount to alleviate one or more symptoms of disease in the treated subject or population, either by inducing regression or by inhibiting the progression of such symptom (s) by any clinically measurable degree. The amount of a therapeutic agent that is effective in alleviating any symptom of a particular disease (also referred to as the therapeutically effective amount) can vary according to factors such as the condition of the disease, age, and the patient's weight, and the ability to the drug elicits a desired response in the subject. You can assess whether a symptom of illness has been alleviated by any clinical measurement typically used by doctors or other health care providers,
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    15/68 to assess the severity or progression of this symptom. Although a modality of the present invention (for example, a treatment method or article of manufacture) may not be effective in alleviating the target disease symptom (s) in each subject, it could alleviate the (s) target disease symptom (s) in a statistically significant number of subjects determined by any statistical test known in the art such as Student's t test, chi 2 test, U- test according to Mann and Whitney, the Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test.
    [0046] Treatment ”as applied to a human, veterinary, or research subject, refers to therapeutic treatment, as well as research and diagnostic applications. Treatment as it applies to a human, veterinary, or research subject, or cell, tissue, or organ, encompasses contact of the antibodies or antigen binding fragments of the present invention with a human or animal subject, a cell, tissue, physiological compartment, or physiological fluid.
    Anti-PD-L1 Antibodies
    [0047] Antibody 20C3 is the antibody produced by the hybridoma subclone MEB037.20C3.116.
    [0048] Antibody 22C3 is the antibody produced by hybridoma subclone MEB037.22C3.138, and corresponds to the S414R allotype of a mouse IgG1. The N-terminal residue of the mature 22C3 heavy chain is both glutamine and pyroglutamate (pE), which is a common post-translational modification that is often seen in monoclonal antibodies when the genetic sequence codes for an N-terminal glutamine in the mature heavy or light chain.
    [0049] The antibodies and antigen binding fragments of the present invention bind in the mature form of human PD-L1 (which does not have the
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    16/68 presecretory leader sequence, also referred to as leader peptide) that is expressed on the surface of certain human cells. The terms PD-L1 and mature PD-L1 are used interchangeably here, and it should be understood that they mean the same molecule unless otherwise indicated or easily apparent from the context. A PD-L1 human mature molecule consisting of amino acids 19-290 of the following sequence (SEQ ID NO: 37): MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEME DKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGG ADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTT TNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILG AILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET.
    [0050] The extracellular domain of mature human PD-L1 consists of the following sequence (SEQ ID NO: 38):
    FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQH SSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKIN QRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRIN TTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERT.
    [0051] As used herein, an anti-human PD-L1 antibody or an anti-hPD-L1 antibody refers to an antibody that specifically binds to human PD-L1. An antibody that specifically binds to human PD-L1, "or an antibody that specifically binds to a polypeptide comprising the amino acid sequence of human PD-L1," is an antibody that exhibits preferential binding to human PD-L1 compared to other antigens, but this specificity does not require absolute binding specificity. An anti-hPD-L1 antibody is considered specific for human PD-L1 if its binding determines the presence of human PD-L1 in
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    17/68 a sample, for example, without producing unwanted results such as false positives in an IHC diagnostic analysis. The degree of specificity required for an anti-hPD-L1 antibody of the invention may depend on the intended use of the antibody, and whether any rate is defined by its suitability for use for an intended purpose. The antibody, or binding fragment thereof, of the invention binds to the human PD-L1 with an affinity that is at least twice as high, preferably at least ten times as high, more preferably at least 20 times as high, and most of all preferably at least 100 times greater than the affinity with any non-PD-L1 protein. As used herein, an antibody is said to specifically bind to a polypeptide comprising a given sequence, for example, mature human PD-L1 (in this case amino acids 19-290 of SEQ ID NO: 37), if it binds to polypeptides comprising this sequence, but does not bind to proteins that do not have this sequence. For example, an antibody that specifically binds to a polypeptide comprising 19-290 of SEQ ID NO: 37 can bind to a FLAG®-tagged form of 19-290 of SEQ ID NO: 37 but will not bind to other FLAG®-tagged proteins .
    [0052] As used herein, the term antibody refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), humanized, fully human antibodies, chimeric antibodies and camelized single domain antibodies. Parental antibodies are antibodies obtained by exposing an immune system to an antigen before modifying the antibodies for their intended use, such as
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    18/68 humanizing an antibody for use as a therapeutic human antibody.
    [0053] As used herein, unless otherwise indicated, antibody fragment or antigen binding fragment refers to antibody antigen binding fragments, i.e., antibody fragments that retain the ability to specifically bind in the antigen bound by the full-length antibody, for example, fragments that retain one or more CDR regions. Examples of antibody binding fragments include, but are not limited to, Fab, Fab ', F fragments (ab% and Fv; diabodies; linear antibodies; single chain antibody molecules, for example, sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.
    [0054] A Fab fragment is comprised of a light chain and the CH1 variable regions and a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A Fab fragment can be the papain cleavage product of an antibody.
    [0055] An "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are grouped by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
    A Fab 'fragment contains a light chain and a portion or fragment of a heavy chain containing the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, in such a way that an interchain disulfide bond can be formed between the two heavy chains of two Fab 'fragments to form an F (ab') 2 molecule.
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    An F (ab ') 2 fragment contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. An F (ab ') 2 fragment thus is composed of two Fab' fragments which are grouped by a disulfide bond between the two heavy chains. An F (ab ') 2 fragment can be the pepsin cleavage product of an antibody.
    [0058] The Fv region comprises variable regions of both heavy and light chains, but constant regions are missing.
    [0059] The term Fv single chain antibody or scFv refers to antibody fragments comprising the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. In general, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allow the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun (1994) The Pharmacology of Antybodies monoclonals, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also, publication of international patent application No. WO 88/01649 and U.S. Pat. We. 4,946,778 and 5,260,203.
    An antibody in the domain is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some cases, two or more VH regions are covalently joined with a peptide linker to create an antibody from the divalent domain. The two VH regions of an antibody in the bivalent domain can target the same or different antigens.
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    [0061] A divalent antibody comprises two antigen binding sites. In some cases, the two binding sites have the same antigen specificities. However, bivalent antibodies can be bispecific (See below).
    [0062] In certain embodiments, monoclonal antibodies here also include camelized single domain antibodies. See, for example, Muyldermans et al. (2001) Trends Biochem. Sci. 26: 230; Reichmann et al. (1999) J. Immunol. Methods 231: 25; WO 94/04678; WO 94/25591; U.S. Patent No. 6,005,079). In one embodiment, the present invention provides single domain antibodies comprising two VH domains modified in such a way that single domain antibodies are formed.
    [0063] As used herein, the term diabody refers to small antibody fragments with two antigen binding sites, whose fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). 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 and create two antigen binding sites. Diabodies are more fully described, for example, in EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of modified antibody variants in general See Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.
    [0064] Typically, an antibody or antigen binding fragment of the invention retains at least 10% of its human PD-L1 binding activity (when compared to the parental antibody) when this activity is expressed on a molar basis. Preferably, an antibody or fragment of
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    21/68 binding to the antigen of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of human PD-L1 as the parent antibody. It is also intended that an antibody or antigen-binding fragment of the invention may include conservative or non-conservative amino acid substitutions (referred to as conservative variants or variants with conserved antibody function) that do not substantially alter their biological activity.
    [0065] Isolated antibody refers to the state of purification and in such a context it means that the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cell fragments and growth media. In general, the term isolated is not intended to refer to a complete absence of such material, or to an absence of water, buffers, or salts, unless they are present in quantities that substantially interfere with the experimental or therapeutic use of the compound. connection as described here.
    [0066] The term monoclonal antibody, as used herein, refers to a population of substantially homogeneous antibodies, that is, the antibody molecules comprising the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in smaller quantities. In contrast, conventional (polyclonal) antibody preparations typically include a plurality of different antibodies with different amino acid sequences in their variable domains, particularly their CDRs, which are often specific for different epitopes. The monoclonal modifier indicates the character of the antibody being obtained from a population of substantially homogeneous antibodies, and should not be interpreted as requiring production of the antibody by any particular method. Per
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    22/68 example, monoclonal antibodies to be used according to the present invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or can be produced by recombinant DNA methods (See, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 116: 731.
    [0067] In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair with a light chain (about 25 kDa) and a heavy chain (about 50-70 kDa). The aminoterminal portion of each chain includes a variable region of about 100 to 110 or more amino acids basically responsible for antigen recognition. The carboxyterminal portion of the heavy chain can define a constant region basically responsible for the effector function. Typically, human light chains are classified as kappa and lambda light chains. In addition, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable and constant regions are joined by a J region of about 12 or more amino acids, with the heavy chain also including a D region of about 10 more amino acids. See, in general, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).
    [0068] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites, in general, are the same.
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    [0069] Typically, the variable domains of both heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), located in regions of relatively conserved structure (FR). CDRs are normally aligned by structure regions, allowing for attachment to a specific epitope. In general, from terminal N to terminal C, variable domains of both light and heavy chains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The designation of amino acids in each domain is, in general, according to the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al .; National Institutes of Health, Bethesda, Md .; 5th ed .; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat, et al., (1977) J. Biol. Chem. 252: 6609-6616; Chothia, et al., (1987) J Mol. Biol. 196: 901-917 or Chothia, et al., (1989) Nature 342: 878-883.
    [0070] As used herein, the term hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from a complementarity determining region or CDR (i.e., CDRL1, CDRL2 and CDRL3 in the light chain variable domain and CDRH1, CDRH2 and CDRH3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Defining the CDR regions of an antibody per sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining an antibody's CDR regions by structure). As used herein, the terms frame residues or FR refer to residues from variable domains other than the residues of the hypervariable region defined here as CDR residues.
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    [0071] Homology refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences when they are ideally aligned. When a position in both compared sequences is occupied by the same amino acid monomer base or subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous in that position. The homology percentage is the number of homologous positions shared by the two sequences divided by the total number of compared positions x100. For example, if 8 out of 10 of the positions in two strings are matched or homologous when the strings are ideally aligned, then the two strings are 80% homologous. In general, the comparison is made when two strings are aligned to give maximum percentage of homology. For example, the comparison can be performed by a BLAST algorithm in which the parameters of the algorithm are selected to give the greatest match between the respective sequences with respect to the entire length of the respective reference sequences.
    [0072] Isolated nucleic acid molecule ”means a DNA or RNA of genomic origin, mRNA, cDNA, or synthetic or some combination thereof that is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature , or is linked to a polynucleotide to which it is not bound in nature. For the purposes of this invention, it should be understood that a nucleic acid molecule comprising a particular nucleotide sequence does not comprise intact chromosomes. Isolated nucleic acid molecules comprising specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even twenty or more other proteins or portions or fragments thereof, or may include
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    25/68 operably linked regulatory sequences that control expression of the coding region of the cited nucleic acid sequences, and / or may include vector sequences.
    [0073] The term control sequences refers to the DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.
    [0074] A nucleic acid is operationally linked when it is placed in a functional relationship with another sequence of nucleic acids. For example, DNA for a pre-sequence or secretory leader is operably linked in DNA to a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operationally linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked in a coding sequence if it is positioned to facilitate translation. In general, operationally linked ”means that the DNA sequences being linked are contiguous and, in the case of a secretarial leader, contiguous and in the reading phase. However, breeders do not need to be contiguous. Connection is made by connection at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or ligands are used according to conventional practice.
    [0075] As used herein, the expressions cell, "cell line" and cell culture are used interchangeably and all such designations include
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    26/68 progeny. Thus, the transforming words and transformed cells include the primary subject cell and cultures derived from it, without considering the number of transfers. It should also be understood that not all progenies will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progenies that have the same biological function or activity classified in the originally transformed cell are included. Where distinct designations are targeted, they will be clear from the context.
    [0076] As used herein, polymerase chain reaction or PCR refers to a procedure or technique in which specific nucleic acid, RNA and / or DNA sequences are amplified as described, for example, in US Patent No. 4,683 .195. In general, sequence information from the ends of the region of interest, or beyond, is used to design primer oligonucleotides. These primer oligonucleotides will be identical or similar in sequence opposite the strands of the template to be amplified. The nucleotides at the 5 'terminus of the two primers can match the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences of total genomic DNA, and total cellular RNA transcribed cDNA, bacteriophage or plasmid sequences, etc. See in general Mullis et al. (1987) Cold Spring Harbor Symp. Quant. Biol. 51: 263; Erlich, ed., (1989) PCR Technology (Stockton Press, NY) As used herein, PCR is considered an example, but not the only one, of a nucleic acid polymerase reaction method to amplify a test sample of the acid nucleic acid comprising the use of a nucleic acid known as a primer oligonucleotide and a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid.
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    [0077] As used herein, germline sequence refers to a sequence of non-rearranged immunoglobulin DNA sequences. Any suitable source of non-rearranged immunoglobulin sequences can be used. Human germline sequences can be obtained, for example, from germline databases on the JOINSOLVER® website for the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the Unidadeed States National Institutes of Health. Mouse germline sequences can be obtained, for example, as described in Giudicelli et al. (2005) Nucleic Acids Res. 33: D256-D261.
    Physical and Functional Properties of Exemplary Anti-PD-L1 Antibodies
    [0078] The present invention provides isolated anti-PD-L1 antibodies and methods of using the antibodies or antigen binding fragments thereof to detect expression of PD-L1 on the cell surface. Examples of anti-PD-L1 antibodies of the invention include, but are not limited to: 20C3 and 22C3 antibodies (See Figures 1 and 2). The 20C3 and 22C3 antibodies bind non-identical, but adjacent, epitopes (See Example 2 and Figure 9), indicating that the CDRs of these two antibodies can be mixed to derive additional antibodies that specifically bind to PD-L1 in one or both of these epitopes. Thus, the isolated antibody or antigen-binding fragment of the same that binds human PD-L1 can comprise three of the light chain complementarity determining regions (CDRs) and three of the heavy chain CDRs shown in Tables 1 to 3 below.
    Table 1. Characteristics of monoclonal antibody MEB037.20C3 Characteristic ofAntibody Amino Acid Sequence SEQIDAT THE Light Chain
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    CDRL1 KSSQSLLNSRTRKNYLA 9 CDRL2 WASTRES 2 CDRL3 QQSYDVVT 10 Leader String MDSQAQVLILLLLWVSGTFG 11 Variable region MDSQAQVLILLLLWVSGTFGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQSYDVVTFGAGTKLELK 12 Variable RegionMature DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQSYDVVTFGAGTKLELK 13 Sequence ofDNA Coding the Variable Region ATGGATTCACAG GCCCAGGTTCTTATATTG CTGC TGCTATG GGTATCTGGTACCTTTG GGGACATTGT GATGTCACAATCTCCATCCTCCCTGGCTGTGTCA GCAGGAGAGAAGGTCACTATGAG CTGCAAATCC AGTCAGAGTCTGCTCAACAGTAGAACCCGAAAG AACTACTTGGCTTGGTACCAG CAGAAACCAGGG CAGTCTCCTAAACTGCTGATCTACTGG GCATCCA CTAGGGAATCTGGGGTCCCTGATCGCTTCACAG GCAGTGGATCTGGGACAGATTTCACTCTCACCAT CAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTA TTACTGCCAGCAATCTTATGATGTGGTCACGTTC GGTGCTGGGACCAAG CTGGAGCTGAAA 33 Heavy Chain CDRH1 KabatDef'n SYWMH 14
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    CDRH1 ChothiaDef'n GYIFTSYWMH 15 CDRH2 YINPSSDYNEYSEKFMD 16 CDRH3 SGWLVHGDYYFDY 17 Leader String MERHWIFLFLFSVTAGVHS 18 Variable Region MERHWIFLFLFSVTAGVHSQVQVQQSGAELAEPGASVKMSCKASGYIFTSYWMHWLKQRPGQGLEWIGYINPSSDYN EYSEKFMDKATLTADKASTTAYMQLISLTSEDSAVYYCARSGWLVHGDYYFDYWGQGTTLTVSS 19 Variable RegionMature QVQVQQSGAELAEPGASVKMSCKASGYIFTSYWMHWLKQRPGQGLEWIGYINPSSDYNEYSEKFMDKATLTADKASTTAYMQLISLTSEDSAVYYCARSGWLVHGDYYFDYWGQGTTLTVSS 20 Sequence ofDNA Coding the Variable Region ATGGAAAGGCACTGGATCTTTCTCTTCCTGIIIIC AGTAACTGCAGGTGTCCACTCCCAGGTCCAGGTT CAG CAGTCTGG GGCTGAACTGGCAGAACCTGG G GCCTCAGTGAAGATGTCCTGCAAG GCCTCTGGCT ACATCTTTACTAGCTACTGGATGCACTG GCTAAA GCAGAGGCCTGGACAGGGTCTGGAATGGATTG GATACATTAATCCCAGCAGTGATTATAATGAATA CAGTGAGAAATTCATGGACAAGGCCACATTGAC TGCAGACAAAGCCTCCACCACAGCCTACATGCAA CTGATCAGCCTGACATCTGAGGACTCTGCAGTCT ATTACTGTGCAAGATCGGGATGGTTAGTACATG GAGACTATTAIII IGACTACTGGGGCCAAGGCAC CACTCTCACAGTCTCCTCA 34
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    Table 2. Characteristics of MEB037.22C3 monoclonal antibody Characteristic ofAntibody Amino Acid Sequence SEQIDAT THE Light Chain CDRL1 KSSQSLLHTSTRKNYLA 21 CDRL2 WASTRES 2 CDRL3 KQSYDVVT 22 Leader String MDSQAQVLILLLLWVSGTCG 23 Variable Region MDSQAQVLILLLLWVSGTCGDIVMSQSPSSLAVSAGEKVTMTCKSSQSLLHTSTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYDVVTFGAGTKLELK 24 Variable RegionMature DIVMSQSPSSLAVSAGEKVTMTCKSSQSLLHTSTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYDVVTFGAGTKLELK 25 Sequence ofDNA Coding the Variable Region ATGGATTCACAG GCCCAGGTTCTTATATTG CTGC TGCTATGGGTATCTGGTACCTGTGGGGACATTGT GATGTCACAGTCTCCCTCCTCCCTGGCTGTGTCA GCAGGAGAGAAGGTCACTATGACCTGCAAATCCAGTCAGAGTCTGCTCCACACTAGCACCCGAAAGAACTACTTGGCTTGGTACCAG CAGAAACCAGGG CAGTCTCCTAAACTGCTGATCTATTG GGCATCCA CTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCAT CAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTA 35
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    TTACTGCAAACAATCTTATGATGTGGTCACGTTCGGTGCTGGGACCAAG CTGGAGCTGAAAHeavy Chain CDRH1 KabatDef'n SYWIH 26 CDRH1 ChothiaDef'n GTTFTSYWIH 27 CDRH2 YINPSSGYHEYNQKFID 28 CDRH3 SGWLIHGDYYFDF 29 Leader String MERHWIFLFLFSVTAGVHS 30 Variable Region MERHWIFLFLFSVTAGVHSQVHLQQSGAELAKPG ASVKMSCKASGYTFTSYWIHWIKQRPGQGLEWIG YIN PSSGYHEYN QKFIDKATLTADRSSSTAYMH LTS LTSEDSAVYYCARSGWLIHGDYG 31 Variable regionMature XVHLQQSGAELAKPGASVKMSCKASGYTFTSYWIHWIKQRPGQGLEWIGYIN PSSGYHEYNQKFIDKATLTADRSSSTAYMHLTSLTSEDSAVYYCARSGWLIHGDYYFDFWGQGTTLTVSS,where X = Q or pE 32 Sequence ofDNA Coding the Variable Region ATGGAAAGGCACTGGATCTTTCTCTTCCTGIIIIC AGTAACTGCAGGTGTCCACTCCCAGGTCCACCTTCAG CAGTCTGG GGCTGAACTGGCAAAACCTGGG GCCTCAGTGAAGATGTCCTGCAAG GCTTCTGGCT ACACGTTTACTAGTTACTGGATACACTGGATAAA GCAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATATATGT 36
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    AATCAGAAATTCATTGACAAGGCCACATTGACTGCTGACAGATCCTCCAGCACAGCCTACATGCACCTGACCAGCCTGACGTCTGAAGACTCTGCAGTCTATTACTGTGCAAGATCGGGATGGTTAATACATGGAGACTACTACTTTGACTTCTGGG GCCAAGGCACCACTCTCACAGTCTCCTCATable 3. Invention Consensus Antibody Sequences Antibody Feature Amino Acid Sequence SEQIDAT THE Light Chain CDRL1 KSSQSLLX1X2X3TRKNYLA,where Xi = H or N, X2 = S or T, and X3 = R or S 1 CDRL2 WASTRES 2 CDRL3 X1QSYDVVT, where Xi = Q or K 3 Variable RegionMature DIVMSQSPSSLAVSAGEKVTMX1CKSSQSLLX2X3X4TRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCX5QSYDVVTFGAGTKLELK,where Xi = S or T, X2 = H or N, X3 = S or T, X4 = R or S, and X5 = Q or K 4 Heavy Chain CDRH1 SYWXH, where X = I or M 5 CDRH2 YIN PSSX1YX2EYX3X4KFX5D,where X1 = D or G,X2 = H or N, X3 = S or N, X4 = E or Q, andX5 = I or M 6
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    CDRH3 SGWLX1HGDYYFDX2, where Xi = I or V and X2 = F or Y 7 Variable RegionMature XVX1X2QQSGAELAX3PGASVKMSCKASGYIFTSYWX4 HWX5KQRPGQGLEWIGYINPSSX6YX7EYX8X9KFX10DKATLTADX11X12SX13TAYMX14LX15SLTSEDSAVYYCARSGWLX16HGDYYFDX17WGQGTTLTVSS,where X = Q or pE, X1 = H or Q, X2 = L or V,X3 = E or K, X4 = I or M, X5 = I or L,X6 = D or G, X7 = H or N, X8 = N or S,X9 = E or Q, X10 = I or M, X11 = K or R,X12 = One or S, X13 = S or T, X14 = H or Q,X15 = I or T, X16 = I or V, and X17 = F or Y 8
    [0079] Conservatively modified variants or conservative substitution refer to substitutions of amino acids in a protein with other amino acids with similar characteristics (for example, charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and stiffness, etc.) , in such a way that changes can often be made without altering the biological activity of the protein. Experienced in the art realize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (See, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub Co., p. 224 (4th Ed.)). Furthermore, structurally or functionally similar amino acid substitutions are less likely to disrupt biological activity. Exemplary conservative substitutions are shown in Table 4.
    TABLE 4. Exemplary Conservative Amino Acid Substitutions
    Original waste Conservative substitution
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    Original waste Conservative substitution Wing (A) Gly; To be Arg (R) Lys; His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C) To be; Allah Gln (Q) Asn Glu (E) Asp; Gln Gly (G) Allah His (H) Asn; Gln Ile (I) Read; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M) Read; Ile; Tyr Phe (F) Tyr; Met; Read Pro (P) Allah Being (S) Thr Thr (T) To be Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Read
    [0080] Variants of conservative function of the antibodies of the invention are also contemplated by the present invention. Conservative function variants ”, as used herein, refer to antibodies or fragments in which one or more amino acid residues have been changed without changing a desired property, such as an affinity and / or antigen specificity. Such variants include, but are not limited to, substitution of an amino acid
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    35/68 with one with similar properties, such as the conservative amino acid substitutions in Table 4.
    [0081] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof that specifically binds PD-L1 and has VL domains and VH domains and shares 100% sequence homology for light and heavy chain CDRs from Tables 1 or 2, and at least 90%, 92%, 94%, 96%, 98% or 99% sequence homology for the mature light and heavy chain variable regions of Tables 1 or 2.
    Nucleic acids
    [0082] The present invention also provides nucleic acids that encode the immunoglobulin chains of anti-PD-L1 antibodies and antigen binding fragments disclosed herein. For example, the present invention includes nucleic acids that encode the amino acids described in Tables 1, 2 and 3, as well as nucleic acids that hybridize to them.
    [0083] In general, nucleic acids hybridize under strict low, moderate or high conditions, and encode antibodies that maintain the ability to specifically bind to PD-L1. A first nucleic acid molecule is hybridizable to a second nucleic acid molecule when a single stripe form of the first nucleic acid molecule can annular to the second nucleic acid molecule under the appropriate conditions of temperature and ionic concentration of the solution (See Sambrook, et al., supra). The conditions of temperature and ionic concentration determine the stringency of the hybridization. Typical low stringency hybridization conditions include 55 ° C, 5X SSC, 0.1% SDS and without formamide; or 30% formamide, 5X SSC, 0.5% SDS at 42 ° C. Typical moderate stringency hybridization conditions are 40% formamide, with 5X or 6X SSC and 0.1% SDS at 42 ° C. High stringency hybridization conditions are 50% formamide, 5X or 6X SSC at 42 ° C
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    36/68 or, optionally, at a higher temperature (for example, 57 ° C, 59 ° C, 60 ° C, 62 ° C, 63 ° C, 65 ° C or 68 ° C). In general, SSC is 0.15M NaC1 and 0.015M Na citrate. Hybridization requires that the two nucleic acids contain complementary sequences, although, depending on the stringency of hybridization, imperfect pairings between bases are possible. The appropriate rigor to hybridize nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the stringency to which nucleic acids can hybridize. For hybrids greater than 100 nucleotides in length, equations for calculating the melting temperature have been derived (See Sambrook, et al., Supra, 9.50-9.51). For hybridization with smaller nucleic acids, for example, oligonucleotides, the position of imperfect pairings becomes more important, and the length of the oligonucleotide determines its specificity (See Sambrook, et al., Supra, 11.7-11.8).
    [0084] The following references refer to the BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F., et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W., et al., (1993) Nature Genet. 3: 266-272; Madden, T.L., et al., (1996) Meth. Enzymol. 266: 131141; Altschul, S.F., et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J., et al., (1997) Genome Res. 7: 649-656; Wootton, J.C., et al., (1993) Comput. Chem. 17: 149-163; Hancock, J.M. et al., (1994) Comput. Appl. Biosci. 10: 67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O., et al., A model of evolutionary change in proteins. in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M.O. Dayhoff (ed.), Pp. 345-352, Natl. Biomed. Found., Washington, DC; Schwartz, R.M., et al., Matrices for detecting distant relationships. in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl.
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    3. M.O. Dayhoff (ed.), Pp. 353-358, Natl. Biomed. Found., Washington, DC; Altschul, S.F., (1991) J. Mol. Biol. 219: 555-565; States, D.J., et al., (1991) Methods 3: 66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919; Altschul, S.F., et al., (1993) J. Mol. Evol. 36: 290-300;
    ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87: 2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22: 2022-2039; and Altschul, S.F.
    Evaluating the statistical significance of multiplu distinct local alignments. in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York.
    [0085] In another embodiment, the invention provides an isolated nucleic acid, or nucleic acids, for example, DNA, which encodes at least one of the isolated anti-PD-L1 antibody polypeptide chains or antigen binding fragments described here . In some embodiments, the isolated nucleic acid encodes both a light and a heavy chain in a single nucleic acid molecule, and in other embodiments, the light and heavy chains are encoded in a separate nucleic acid molecule. In another embodiment, the nucleic acids additionally encode a signal sequence.
    [0086] The present invention also provides expression vectors comprising nucleic acids isolated from the invention, wherein the nucleic acid is operably linked in the control sequences that are recognized by a host cell when the host cell is transfected with the vector. Host cells are also provided comprising an expression vector of the present invention and methods for producing the antibody or antigen-binding fragment thereof disclosed herein comprising culturing a host cell that hosts a vector of
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    38/68 expression encoding the antibody or antigen-binding fragment in culture medium, and isolating the antigen or antigen-binding fragment from the host cell or culture medium.
    Epitope Binding
    [0087] The present invention additionally provides antibodies or antigen binding fragments thereof that block 20C3 or 22C3 antibody binding to human PD-L1 binding on the same 20C3 or 22C3 epitope, respectively. Such antibodies and binding fragments can be identified using any cross-blocking or competition analysis known in the art, including the Octet competition analyzes described in Example 2, followed by identification of the human PD-L1 epitope in which the cross-blocking antibody turns on. A first antibody is considered to cross-block the binding of a second antibody, if preligation of the target with the first antibody until saturation increases the concentration of second antibody required to achieve semi-maximum binding of the target 2, 3, 4, 5, 10, 20, 50 , 100, 200 times or more. The binding epitope for a cross-blocking antibody can be identified using techniques well known in the art.
    [0088] An epitope mapping technique like this is hydrogen / deuterium exchange coupled with proteolysis and mass spectrometry (HDXMS). This method is based on the precise measurement and comparison of the degree of deuterium incorporation by an antigen when incubated in heavy water (D2O) by itself and in the presence of its antibody at various intervals. Deuterium is exchanged with hydrogen in the protein's amide backbone in exposed areas while regions of the antigen bound in the antibody will be protected and show less or no exchange after analysis by
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    39/68 liquid chromatography - tandem mass spectrometry (LC-MS / MS) of proteolytic fragments.
    [0089] Based on the HDX-MS epitope mapping described in Example 3, the proposed mature human PD-L1 epitope to 22C3 antibody comprises residues in two discontinuous amino acid segments in the extracellular domain (SEQ ID NO: 38): 156 178 and 196 to 206. Additional epitope residues are likely to be present in the following segments in the extracellular domain (SEQ ID NO: 38): 3 to 9; 10 to 13; 88 to 93 and 135 to 147.
    [0090] Thus, in one embodiment, an antibody that blocks 22C3 antibody binding to human PD-L1 binding on the same 22C3 epitope binds to residues in a first amino acid segment 156 to 178 of SEQ ID NO: 38 and to residues in a second amino acid segment 196 to 206 of SEQ ID NO: 38, and in some embodiments it also bonds to residues in any, two, or three, or all four, of the following segments of SEQ ID NO: 38: amino acids 3 to 9; amino acids 10 to 13; amino acids 88 to 93 and amino acids 135 to 147.
    Methods of Producing Antibodies and Antigen-Binding Fragments
    [0091] Hybridoma cells that produce parental monoclonal anti-X antibodies (e.g., rodent) can be produced by methods that are commonly known in the art. These methods include, but are not limited to, the hybridoma technique originally developed by Kohler, et al., (1975) (Nature 256: 495-497), as well as the trioma technique (Hering, et al., (1988) Biomed Biochim. Acta. 47: 211-216 and Hagiwara, et al., (1993) Hum. Antibod. Hybridomas 4:15), the human B cell hybridoma technique (Kozbor, et al., (1983) Immunology Today 4:72 and Cote, et al., (1983) Proc. Natl. Acad. Sci. USA 80: 2026-2030), the EBV hybridoma technique (Cole, et al., In
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    Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), and electric field-based electrofusion using a large Cyto Pulse chamber cell fusion electroporator (Cyto Pulse Sciences, Inc., Glen Burnie, MD). Preferably, mouse splenocytes are isolated and fused with PEG or by electrofusion into a mouse myeloma cell line based on standard protocols.
    [0092] The resulting hybridomas can then be classified according to the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice can be fused to one sixth of the number of non-secretory mouse myeloma cells P3X63-Ag8.653 (ATCC, CRL 1580) with 50% PEG. Cells can be plated at approximately 2 x 10 5 cells / mL on a flat-based microtiter plate, followed by a two-week incubation in selective medium containing 20% Fetal Clone Serum, 18% 653 conditioned media, 5% origin (IGEN), 4 mM L-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units / mL penicillin, 50 mg / mL streptomycin, 50 mg / mL gentamicin and 1X HAT (Sigma; HAT is added 24 hours after fusion). After two weeks, cells can be grown in a medium in which HAT is replaced with HT. Individual wells can then be classified by ELISA for anti-X monoclonal IgG antibodies. Once extensive hybridoma growth occurs, the medium can be seen normally after 10-14 days. Antibody-secreting hybridomas can be replanted, re-classified, and if still positive for human IgG anti-X monoclonal antibodies, they can be subcloned at least twice limiting dilution.
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    [0093] The stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization. For example, about 1 gram of the 22C3 antibody can be produced and purified from the MEB037.22C3.138 mouse hybridoma cell line using the following procedure. Frozen MEB037.22C3.138 cells are thawed and fitted into a shake flask using hybridoma-free media with an additional 2 mM L-glutamine with or without 0.18% Pluronic F-68. The presence of Pluronic F-68 can improve the viability of the shake culture flask. Once the cells are completely adapted in the shake flask, a 20 liter production culture is performed in serum-free media in a WAVE bioreactor (GE Healthcare Life Sciences) with the addition of 10% efficient CHO CD Feed B (Invitrogen , Catalog # A10240-01). For cell expansion, a 1 liter culture is started in a small WAVE bag, and then the 1 L WAVE culture is expanded into a 20 L culture in the WAVE bioreactor. The 20-liter culture can be started at a cell density of 0.5 x 10 6 viable cells / mL, fed with 10% efficient CHO CD Feed B on Day 1, and pH adjusted daily with 1N Na COs. The cells are harvested after four days. Small samples can be collected daily for NOVA analysis.
    [0094] Anti-hPD-L1 antibodies of the invention can be purified from a hybridoma culture by the following process. The hybridoma culture is clarified by depth filtration using 1.2 micrometer of fiberglass and 0.2 micrometer of cellulose acetate filter. An equal volume of 2X ProSepA Buffer (100 mM boric acid, 5M NaCl, pH 8.5) is added to the clarified crop and the diluted crop is loaded onto the 170 ml bed volume protein A column. The column is washed with 5 column volumes (CV) of 1X ProSepA Buffer (50 mM Boric acid, 2.5 M NaCl, pH
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    8.5), then washed with 2CV of 1X PBS, and the anti-hPD-Ll antibody eluted with 5CV of Elution Buffer (0.1M Glycine, pH 3.0). The elution fractions containing IgG are combined and the pH neutralized by adding 1/10 volume of 1.0M Tris, pH buffer. The neutralized antibody composition is then filtered sterile using a disposable 10 kDa TFF cassette. The antibody can be formulated for storage by diafiltration against 10-liter formulation buffer (20 mM sodium acetate, 9% sucrose, pH 5.0) and using 20 volume changes. Using this protocol, 22C3 antibody at a concentration of about 5.0 mg / mL can be prepared and at least 98% pure by measurements of SDS-PAGE, SEC HPLC and C8 RPHPLC, with endotoxin levels less than 0 , 1 EU / mL and less than 0.02 EU / mg.
    [0095] The anti-PD-L1 antibodies disclosed here can also be produced recombinantly (for example, in an E. coli / T7 expression system in the manner discussed above). In this embodiment, nucleic acids that encode the antibody molecules of the invention (for example, VH or VL) can be inserted into a plasmid based on pE and expressed in the E. coli / T7 system. There are several methods by which to produce recombinant antibodies that are known in the art. An example of a method for recombinant antibody production is disclosed in U.S. Patent No. 4,816,567. Transformation can be by any known method of introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) in liposomes , biological injection and direct microinjection of DNA into the nuclei. Beyond
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    43/68 moreover, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461 and 4,959,455.
    [0096] Anti-PD-L1 antibodies can also be scaled by any of the methods presented in U.S. Patent No. 6,331,415.
    [0097] Mammalian cell lines available as hosts for expression of the antibodies or fragments disclosed herein are well known in the art, and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney cells (BHK), monkey kidney cells (COS), human hepatocellular carcinoma cells ( for example, Hep G2), A549 cells, 3T3 cells, HEK-293 cells and numerous other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through a determinant whose cell lines have high levels of expression. Other cell lines that can be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion or fragment thereof, the light chain and / or antigen-binding fragment thereof are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or, more preferably, secretion of the antibody in the culture medium in which the host cells are grown.
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    [0098] Antibodies can be recovered from the culture medium using standard protein purification methods. In addition, antibody expression of the invention (or other fractions thereof) from the production cell lines can be improved using numerous known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for improving expression under certain conditions. The GS system is discussed in whole or in part with European patent Nos. 0 216 846, 0 256 055 and 0 323 997 and European patent application No. 89303964.4.
    [0099] A polyclonal antibody is an antibody that has been produced between or in the presence of one or more other non-identical antibodies. In general, polyclonal antibodies are produced from collections of different B lymphocytes, for example, the B lymphocyte of an animal treated with an immunogen of interest, which produces a population of different antibodies that are all directed to the immunogen. Typically, polyclonal antibodies are obtained directly from an immunized animal, for example, spleen, serum or ascites fluid.
    The present invention further includes antibody fragments of the anti-PD-L1 antibodies disclosed herein. Antibody fragments include F (ab) 2 fragments, which can be produced by enzymatic cleavage of an IgG, for example, by pepsin. Fab fragments can be produced, for example, by reducing F (ab) 2 with dithiothreitol or mercaptoethylamine. A Fab fragment is a VL-CL chain attached to a VH-CH1 chain by a disulfide bond. An F (ab) 2 fragment is two Fab fragments that, in turn, are attached by two disulfide bonds. The Fab portion of an F (ab) 2 molecule includes a portion of the Fc region between the
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    45/68 which disulfide bonds are located. An Fv fragment is a Vl or Vh region.
    [00101] Immunoglobulins can be designed for different classes, depending on the amino acid sequences of the constant domain of their heavy chains. There are at least five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), for example, IgG-1, IgG-2, IgG-3 and IgG-4 ; IgA-1 and IgA-2. The invention comprises antibodies and antigen binding fragments of any such antibody class or subclass.
    [00102] In one embodiment, the antibody or antigen binding fragment comprises a heavy chain constant region, for example, a human constant region, such as a human heavy chain constant region γ1, γ2, γ3 or γ4 or a variant thereof. In another embodiment, the antibody or antigen binding fragment comprises a light chain constant region, for example, a human light chain constant region, such as a lambda or kappa human light chain region or variant thereof. For example, and not by way of limitation, the human heavy chain constant region can be γ1 and the human light chain constant region can be kappa. In an alternative embodiment, the antibody Fc region is γ4 with a Ser228Pro mutation (Schuurman, J et. Al., Mol. Immunol. 38: 1-8, 2001).
    [00103] In some modalities, different constant domains can be attached in humanized V1 and VH regions derived from the CDRs provided here. For example, if a particular targeted use of an antibody (or fragment) of the present invention were to look for altered effectors, a heavy chain constant domain other than human IgG1 can be used, or hybrid IgG1 / IgG4 can be used.
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    Antibody Modification
    [00104] In particular embodiments, it will be desirable to change certain amino acids containing side chains exposed to another amino acid residue in order to provide for greater chemical stability of the final antibody, as follows. Deamidation of asparagine can occur in the N-G or D-G sequences and result in the creation of an isoaspartic acid residue that introduces a link in the polypeptide chain and decreases its stability (effect of isoaspartic acid). In certain embodiments, the antibodies of the present invention do not contain asparagine isomerism sites.
    [00105] For example, an asparagine residue (Asn) can be changed to Gln or Ala to reduce the potential for isoaspartate formation in any of the Asn-Gly sequences, particularly without a CDR. A similar problem can occur with an Asp -Gly sequence. Reissner and Aswad (2003) Cell. Mol. Life Sci. 60: 1281. Isoaspartate formation can weaken or completely disengage an antibody from binding to its target antigen. See, Presta (2005) J. Allergy Clin. Immunol. 116: 731 to 734. In one embodiment, asparagine is changed to glutamine (Gln). It may also be desirable to alter an adjacent amino acid in an asparagine (Asn) or glutamine (Gln) residue to reduce the likelihood of deamidation, which occurs at higher rates when small amino acids occur adjacent to asparagine or glutamine. See, Bischoff & Kolbe (1994) J. Chromatog. 662: 261. Furthermore, any of the methionine residues (typically exposed solvent Met) in CDRs can be switched to Lys, Leu, Ala, or Phe in order to reduce the possibility that methionine sulfur will oxidize, which could reduce antigen binding affinity and it also contributes to molecular heterogeneity in the final antibody preparation. Id. In one embodiment, methionine is
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    47/68 changed to alanine (Ala). Additionally, in order to prevent or minimize potential scissile linkages of Asn-Pro peptide, it may be desirable to change any combination of Asn-Pro found in a CDR to Gln-Pro, AlaPro, or Asn-Ala. Antibodies with such substitutions are subsequently classified to ensure that the substitutions do not decrease the affinity or specificity of the antibody to human PD-L1, or other desired biological activity to unacceptable levels.
    TABLE 5 - Exemplary stabilizing CDR variants
    CDR residue Variant StringStabilizer Asn-Gly (N-G) Gln-Gly, Ala-Gly, or Asn-Ala (Q-G), (A-G), or (N-A) Asp-Gly (D-G) Glu-Gly, Ala-Gly or Asp-Ala (E-G), (A-G), or (D-A) Met (typically solventexposed) (M) Lys, Leu, Ala, or Phe (K), (L), (A), or (F) Asn(N) Gln or Ala(Q) or (A) Asn-Pro(N-P) Gln-Pro, Ala-Pro, or Asn-Ala (Q-P), (A-P), or (N-A)
    Conjugates of Antibody
    [00106] The anti-PD-L1 antibody molecules disclosed here can also be conjugated to a chemical fraction such as a radionucleotide or other detectable marker. Radionucleotides include 99Tc, 90 Y, 111 In, 32P, 14c 125i 3H 131I 11C 15O 13N 18F 35S 51Cr 57T o 226 R a 60Co 59Fe 57Se 152E .. 67CU,,,,,,,,,,,,,,, r, o, a, o , e, e, u ,, 217 Ci 211 At 212 Pb 47 Sc 109 Pd 234 Th and 40 K 157 Gd 55Mn 52 Tr and 56 Fe Markers, t,, c,,,,, n, r, and e. ark ores
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    48/68 fluorescents or chemiluminescents include fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, phytocyanin, o-phthalaldehyde, fluorescamine, 152Eu, dansila, umbeliferone, luciferin, luminal marker isoluminal marker, aromatic acridinium ester marker, imidazole marker, acridimium salt marker, oxalate ester marker, aequorin marker, 2,3-dihydrophthalazinediones, biotin / avidin, spin markers and stable free radicals .
    [00107] Any method known in the art to conjugate the antibody molecules with the various fractions can be employed, including those methods described by Hunter, et al., (1962) Nature 144: 945; David, et al., (1974) Biochemistry 13: 1014; Pain, et al., (1981) J. Immunol. Meth. 40: 219; and Nygren, J., (1982) Histochem. and Cytochem. 30: 407. Methods for conjugating antibodies are conventional and very well known in the art.
    Experimental and diagnostic uses
    [00108] The anti-PD-L1 antibodies and antibody fragments disclosed here can be used to specifically detect human PD-L1 expressed on the surface of a cell. The cell can be present in a tissue or serum sample obtained from a human subject and detection of PD-L1 expression is performed using any of a variety of in vitro analysis methods known in the art.
    [00109] For example, particular modalities include ELISA (enzyme linked immunosorbent analysis), which typically comprises the following steps:
    (a) coating a substrate (for example, surface of a microtiter plate well, for example, a plastic plate) with an antigen-binding fragment of the anti-PD-L1 antibody thereof;
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    49/68 (b) apply a sample to be tested for the presence of human PD-L1 in the substrate;
    (c) wash the plate, so that unbound material in the sample is removed;
    (d) applying detectably labeled antibodies (for example, enzyme-linked antibodies) which are also specific for human PD-L1;
    (e) washing the substrate so that the unbound labeled antibodies are removed;
    (f) if the labeled antibodies are bound to the enzyme, apply a chemical that is converted by the enzyme into a fluorescent signal; and (g) detecting the presence of the labeled antibody.
    [00110] In an additional embodiment, the labeled antibody is labeled with peroxidase which reacts with ABTS (for example, 2,2'-azino-bis (3-ethylbenzothioazoline-6-sulfonic acid)) or 3.3 ', 5.5' -tetramethylbenzidine to produce a color change that is detectable. Alternatively, the labeled antibody is labeled with a detectable radioisotope (e.g., 3 H) that can be detected by a scintillation counter in the presence of a scintillant.
    [00111] Anti-PD-L1 antibodies and antigen binding fragments thereof can be used in a Western blot or immunoprotein blot procedure. Such a procedure forms part of the present invention and includes for example:
    (1) placing a membrane or other solid substrate to be tested for the presence of human PD-L1 thereof, in contact with an antibody or antigen-binding fragment of the same invention. A membrane like this can take the form of a nitrocellulose or a vinyl-based membrane (for example, polyvinylidene fluoride (PVDF)) in which proteins to be
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    50/68 tested for the presence of X in a non-denaturing PAGE gel (polyacrylamide gel electrophoresis) or SDS-PAGE gel (sodium dodecyl sulfate polyacrylamide gel electrophoresis) were transferred (for example, after electrophoretic separation in the gel) . Prior to contacting the membrane with the anti-PD-L1 antibody or fragment, the membrane is optionally blocked, for example, with skimmed milk powder or the like in order to bind non-specific protein binding sites on the membrane;
    (2) wash the membrane one or more times to remove anti-PDL1 antibody or unbound fragment and other unbound substances; and (3) detecting the anti-PD-L1 antibody or ligated fragment.
    [00112] The bound antibody or fragment can be detected by incubating the antibody or bound fragment with a secondary antibody (an anti-immunoglobulin antibody) that is detectably labeled and then detecting the presence of the secondary antibody. The anti-PD-L1 antibodies and antigen-binding fragments thereof disclosed herein can also be used in immunohistochemical analysis (IHC), which can be performed using a variety of IHC formats known in the art, and are embodiments of the invention. . A typical HCI analysis uses a section of FFPE tissue of about 3-4 millimeters, and preferably 4 microns, mounted and dried on a microscope slide and comprises, for example, (1) subjecting the tissue section to dewaxing and hydration, placing the rehydrated tissue section in contact with an anti-PD-L1 antibody of the invention or antigen-binding fragment thereof; and (2) detecting the antiPD-L1 antibody or antigen-binding fragment thereof on the surface of one or more cells in the tissue. If the antibody or fragment itself is detectably labeled, it can be detected directly.
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    Alternatively, the antibody or fragment can be linked by a detectably labeled secondary antibody that is detected.
    [00113] A preferred HCI analysis employs the commercially available Dako EnVisionTM FLEX detection system, which is intended for use in conjunction with a Dako Autostainer instrument (Dako, an Agilent Technologies Company, Glostrup, Denmark). When this system is employed with the 22C3 antibody, or an antibody comprising the variable regions of the heavy and light chain of the 22C3 antibody, the HCI analysis can be performed as follows. Four micron sections of thick FFPE fabric mounted on the slides are air dried overnight, baked at 60 ° C for 45 minutes, dewaxed and rehydrated. After dewaxing, FFPE slides are subjected to heat-induced epitope recovery using EnVision ™ FLEX High pH Target Retrieval Solution at 97 ° C followed by 20 minutes at room temperature. The slides are then washed, stained with 22C3 at 2 μg / mL for 60 minutes, and then detected using Dako EnVision ™ FLEX reagents as follows: EnVision ™ FLEX + MS Linker (15 minutes), EnVision ™ FLEX / HRP (20 minutes ), EnVision ™ FLEX DAB (10 minutes), and DAB Enhencer (7 minutes) with intervening washing steps.
    [00114] Certain anti-PD-L1 antibodies and antigen binding fragments thereof disclosed herein can also be used for tumor imaging in vivo. Such a method may include injection of an anti-PD-L1 antibody or antigen-binding fragment from the same radiolabelled into the body of a human patient to be tested for the presence of a tumor associated with PD-L1 expression, followed by formation nuclear imaging of the patient's body to detect the presence of the antibody or labeled fragment, for example, at a loci comprising a high concentration of the antibody or fragment that is bound in the tumor.
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    [00115] Image formation techniques include SPECT image formation (computed tomography by single photon emission) or PET image formation (positron emission tomography). Markers include, for example, iodine-123 ( 123 I) and technetium-99m ( 99m Tc), for example, along with SPECT or 11 C, 13 N, 15 O or 18 F imaging, for example, along with PET or Indian-111 image (See, for example, Gordon et al., (2005) International Rev. Neurobiol. 67: 385-440).
    Detection Kits and Therapeutic Kits
    [00116] For the sake of convenience, an antibody or specific binding agent disclosed here can be supplied in a kit, that is, a packaged combination of reagents in predetermined quantities with instructions for performing the diagnostic analysis or detection. Where the antibody is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (for example, a precursor substrate that provides the detectable chromophore or fluorophore). In addition, other additives can be included such as stabilizers, buffers (for example, a blocking buffer or lysis buffer) and the like. The relative quantities of the various reagents can be varied widely to provide solution concentrations of the reagents that substantially optimize the sensitivity of the analysis. In particular, the reagents can be supplied in the form of dry powders, usually freeze-dried, including excipients which, upon dissolution, will provide a reagent solution of the appropriate concentration.
    [00117] Diagnostic or detection reagents and kits comprising one or more such reagents are also provided for use in a variety of detection assays, including, for example, immunoassays such as ELISA (sandwich type or competitive format). Kit components can be pre-attached to a solid support, or can be applied to the surface of a
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    53/68 solid support when the kit is used. In some embodiments, the signal generating device may come pre-associated with an antibody of the invention or may require combination with one or more components, for example, buffers, antibody-enzyme conjugates, enzyme substrates, or the like, before use. Kits can also include additional reagents, for example, blocking reagents to reduce non-specific binding to the surface of the solid phase, washing reagents, enzyme substrates, and the like. the surface of the solid phase can be in the form of a tube, a microsphere, a microtiter plate, a microsphere, or other materials suitable for immobilizing proteins, peptides, or polypeptides. In particular aspects, an enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is a component of the signal generating device. Such enzymes are well known in the art. Kits can comprise any of the capture agents and detection reagents described here. Optionally, the kit can also comprise instructions for carrying out the methods of the invention.
    [00118] The detection kits disclosed here can also be prepared that comprise at least one of the antibody or antigen binding fragment disclosed here and instructions for using the composition as a detection reagent. Containers for use in such kits can typically comprise at least one vial, test tube, vial, bottle, syringe or other suitable container, into which one or more of the detection composition (s) can be placed, and preferably suitably aliquoted. The kits disclosed here will also typically include a means to contain the vial (s) in close confinement for commercial sale, such as, for example, injection or blow molded plastic containers in which the vial (s)
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    54/68 desired vial (s) are repeated. Where a radio marker, chromogenic, fluorene, or other type of detectable marker or detection device is included in the kit, the marking agent can be supplied either in the same container as the detection composition itself, or alternatively it can be placed in a second separate container device in which this second composition can be placed and suitably aliquoted. Alternatively, the detection reagent can be prepared in a single container device, and in most cases, the kit will also typically include a device for containing the confined vial (s) for commercial sale and / or packaging and distribution convenient.
    [00119] A device or apparatus for performing the detection or monitoring methods described here is also provided. Such an apparatus may include a chamber or tube into which the sample can be fed, a fluid handling system optionally including valves or pumps to direct sample flow through the device, optional filters to separate plasma or serum from blood, mixture for the addition of capture agents or detection reagents, and optionally a detection device for detecting the amount of detectable marker bound in the immunocomplex capture agent. The sample flow can be passive (for example, by capillary, hydrostatic, or other forces that do not require additional manipulation of the device once the sample is applied) or active (for example, by applying force generated by pumps mechanical, electro-osmotic pumps, centrifugal force, or higher air pressure), or by a combination of active and passive forces.
    [00120] Additional modalities also provide a processor, a computer-readable memory, and a routine stored in the computer-readable memory and adapted to run on the processor to perform
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    55/68 any of the methods described here. Examples of suitable computing systems, environments and / or configurations include personal computers, server computers, portable or manual devices, multiprocessor systems, microprocessor-based systems, television set-top boxes, consumer-programmable electronic equipment, network PCs , minicomputers, large computers, distributed computing environments that include any of the previous systems or devices, or any other system known in the art.
    GENERAL METHODS
    [00121] Standard methods in molecular biology are described Sambrook, Fritsch and Maniatis (1982 & 1989 2 nd Edition, 2001 3 rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.; Wu (1993) recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).
    [00122] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York ). Chemical analysis, chemical modification, post-transcational modification, production of fusion proteins, glycosylation of proteins are described (See, for example, Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons , Inc., New York;
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    Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antybodies , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (See, for example, Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).
    [00123] Monoclonal, polyclonal, and humanized antibodies can be prepared (See, for example, Sheperd and Dean (eds.) (2000) Monoclonal Antybody, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) ( 2001) Antybody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antybody A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139-243; Carpenter, et al. (2000) J Immunol. 165: 6205; He, et al. (1998) J. Immunol. 160: 1029; Tang et al. (1999) J. Biol. Chem. 274: 27371-27378; Baca et al. (1997) J Biol. Chem. 272: 10678-10684; Chotioa et al. (1989) Nature 342: 877-883; Foote and Winter (1992) J. Mol. Biol. 224: 487-499; US patent No. 6,329,511 ).
    [00124] An alternative for humanization is to use human antibody libraries displayed in phage or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14: 309314; Barbas (1995) Nature Medicine 1: 837- 839; Mendez et al. (1997) Nature Genetics 15: 146-156; Hoogenboom and Chames (2000) Immunol. Today 21: 371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold
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    Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnol. 17: 397-399).
    [00125] Antigen purification is not necessary for the generation of antibodies. Animals can be immunized with cells that carry the antigen of interest. Splenocytes can then be isolated from immunized animals, and the splenocytes can fuse with a myeloma cell line to produce a hybridoma (See, for example, Meyaard et al. (1997) Immunity 7: 283-290; Wright et al. (2000 ) Immunity 13: 233-242; Preston et al., Supra; Kaithamana et al. (1999) J. Immunol. 163: 5157-5164).
    [00126] Antibodies can be conjugated, for example, with small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are used for therapeutic purposes, diagnostics, kit composition or other purposes, and include antibodies coupled, for example, in dyes, radioisotopes, enzymes or metals, for example, colloidal gold (See, for example, Le Doussal et al. ( 1991) J. Immunol. 146: 169-175; Gibellini et al. (1998) J. Immunol. 160: 3891-3898; Hsing and Bishop (1999) J. Immunol.
    162: 2804-2811; Everts et al. (2002) J. Immunol. 168: 883-889).
    [00127] Methods for flow cytometry, including fluorescence-activated cell classification (FACS), are available (See, for example, Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2 nd ed .; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid initiator oligonucleotides and probes, polypeptides, and antibodies, for use, for example, as
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    58/68 diagnosis, are available (Molecular Probes (2003) Catalog, Molecular Probes, Inc., Eugene, OU; Sigma-Aldrich (2003) Catalog, St. Louis, MO).
    [00128] Standard methods of histology of the immune system are described (See, for example, Muller-Harmelink (ed.) (1986) Human Thymus:
    Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).
    [00129] Software packages and databases to determine, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments, are available (See, for example, GenBank, Vector NTI ® Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada); Menne, et al. (2000) Bioinformatics 16 : 741-742; Menne, et al. (2000) Bioinformatics Applications Note 16: 741-742; Wren, et al. (2002) Comput. Methods Programs Biomed. 68: 177-181; von Heijne (1983) Eur. J Biochem 133: 17-21; von Heijne (1986) Nucleic Acids Res. 14: 4683-4690).
    EXAMPLES
    Example 1. Generation and Classification of Anti-PD-L1 Hybridomas
    [00130] Balb / C mice were immunized with a human PD-L1-Fc fusion protein (R&D Systems® Catalog No. 156-B7-100) in adjuvant. This fusion protein contains the PD-L1 extracellular domain (Phe19-Thr239) fused to a human IgG1 fragment (Pro100-Lys 300). After 12 immunizations, lymphonds from two mice with high titers in human PD-L1 were harvested and an electrofusion was performed to generate two batches of hybridomas, which were given the lab names MEB033 and MEB037.
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    [00131] Supernatants from the hybridoma batches MEB033 and MEB037 were classified to identify hybridomas that produce antibodies to human PD-L1. The classification employed a protein-based ELISA for binding to the human hPD-L1-Fc protein; and cell-based ELISAs for binding to parental CHO cells and stable human PDO L1 CHO transformants into human CHO PD-L1 as a negative control. Supernatants from 88 MEB037 hybridoma clones and 23 MEB033 clones tested positive for the presence of anti-PD-L1 antibody (data not shown) and samples of them were tested for IHC reactivity in FFPE tissue sections from tonsils of normal human (data not shown).
    [00132] Of the 88 clones, only 11 clones of the MEB037 batch produced staining patterns of sufficient intensity and specifically apparent to warrant further evaluation, based on comparison with the staining patterns obtained with the commercially available anti-PD-L1 antibodies listed in Table below:
    TABLE 6 Commercially Available Antibody PD-L1 Antibodies
    Company Number ofCatalog Species Number ofLot eBioscience 14-5983 Mouse 14-5983-82 R&D AF156 Goat EE1010109111 US Biological 22 US Biological 22E Sigma PRS4059 Bunny 40590604
    [00133] When the inventors compared the various staining patterns obtained with the experimental antibodies with patterns obtained with these commercially available anti-human PD-L1 antibodies, they observed significant differences between the staining patterns, including the
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    60/68 staining location and cell types stained. In an attempt to explain this difference, the inventors conducted a number of additional experiments and found that none of these commercially available antibodies provided the combination of attributes required for use in IHC of PD-L1 expression in FFPE sections: (1) sensitivity - the ability to detect normal physiological expression in positive control tissues (for example, human amygdala) as well as expression in tumor tissue (for example, human melanoma samples); (2) specifically, the staining pattern must correlate with known anatomical / cellular distribution of PD-L1 and must be neutralized; and (3) robust, little to no variation in staining patterns when used to analyze duplicate sections of tissue.
    [00134] For example, the inventors observed that the antibody
    Sigma / ProSco PRS4059 showed multiple bands in the amygdala lysate, none of which could be confirmed to represent PD-L1, failed to stain LOX melanoma cell lines that were shown to be PD-L1 positive by flow cytometry, and failed to differentiate between cell lines positive and negative by IHC.
    [00135] Some of these data are shown in figure 4, in which immunohistochemical staining of tonsil sections identified 22C3 and 20C3 as two antibodies with unusual and useful immunohistochemical properties in the FFPE tissue, compared with the PRS4059 antibody identified by Gadiot et al (supra) as the only suitable candidate candidate among 15 anti-human PD-L1 antibodies to detect expression of PD-L1 in sections of FFPE tissue. In experiments carried out by the inventors, the Prosci antibody (PRS4059, lot 40590604, used at 0.4 mg / mL primary, followed by the rabbit polymer detection system (DAKO Envision) stained all of the
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    61/68 hematopoietic lines in the amygdala with equal intensity (Fig. 4A) while the 22C3 antibody selectively stained myeloid CD68 + cells in the crypt epithelium and amygdala follicular cells, which are morphologically consistent with macrophages (Fig. 4B). Substantially the same staining pattern was observed with 20C3 antibody (data not shown). Furthermore, 22C3 and 20C3 demonstrated a consistent difference in staining intensity between these two discrete cell populations with much larger crypt epithelium than follicular macrophages. All three antibodies could be neutralized by pre-incubation with the PD-L1 antigen, indicating that reactivity is mediated by the antigen binding domain (CDRs).
    Example 2. Quality assessment of anti-PD-L120C3 and 22C3 antibodies
    [00136] This example describes additional experiments that were conducted to evaluate the usefulness of antibodies 20C3 and 22C3 for use in IHC analyzes of FFPE tissue sections.
    [00137] An experiment evaluated the ability of these two antibodies to detect a human PD-L1 protein expression range (hPD-L1) in IHC analysis of the FFPE sections of the normal human amygdala, and representative images for 22C3 are shown in figure 5A. Immunohistochemical staining with 22C3 strongly marks the epithelium of the tonsillar crypt as well as demonstrating weak to moderate staining of a CD68 + follicular myeloid population (presumed macrophages). Both antibodies (20C3 data not shown) marked cells in a well-defined membrane / cell surface pattern in these two cell types. The convenience of expression of hPDL1 in amygdala (ie, restriction of IHC staining in the two cell populations (crypt epithelium and follicular macrophages) was corroborated by an independent methodology (in situ hybridization [ISH] for hPD-L1 mRNA) in
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    62/68 adjacent tonsil tissue from the FFPE sections. Furthermore, the differential expression of hPD-L1 protein assessed by IHC (crypt epithelium >> follicular macrophages) corresponds to the relative abundance of PD-L1 hRNAm observed with ISH.
    [00138] Another experiment evaluated the binding specificity of 20C3 and 22C3 for hPD-L1 expression cells. HT144 cells that were known to be negative for hPD-L1 expression by mRNA (qPCR) analysis and LOX melanoma cells known to express high levels of PD-L1 hRNAm (qPCR) were stained with 1 microgram / ml of mouse IgG purified from seven hybridomas generated in the experiments described in Example 1 above. An irrelevant isotype control mouse antibody was also used at the identical concentration. A secondary fluorescent-labeled anti-mouse antibody was used to detect the primary mouse antibodies. After staining and repeated washing, cells were analyzed by flow cytometry, with median fluorescent intensities calculated for the population (> 10,000 events collected). The results are shown in figure 6.
    [00139] 20C3 and 22C3 as well as other hPD-L1 antibodies were used as flow cytometric reagents to detect hPD-L1 from the cell surface. The significant right shift of the histogram curves of both 20C3 and 22C3 (Figure 6A) compared to the isotype control antibody curve reflects selective detection of hPD-L1 in the LOX hPD-L1-positive melanoma cell line. The median fluorescent intensities associated with these histograms and others from this analysis are shown in figure 6B. The selectivity of binding of 20C3 and 22C3 is further supported by the lack of significant binding (i.e., MFI of 22C3 and 20C3 comparable to the isotype) in the negative cell line, HT144. To
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    63/68 otherwise, the data in Fig. 6B show that both 20C3 and 22C3 produce at least 10 times more MFI compared to the isotype in the hPD-L1 positive LOX melanoma cell line. Thus, both 20C3 and 22C3 (in addition to clones 5F9, 7C8, 13D2 and 31D3) demonstrate selective binding in hPD-L1 expression cells by flow cytometric evaluation.
    [00140] Another experiment evaluated the ability of the 22C3 antibody to detect hPD-L1 expression in modified and human cell lines. Chinese hamster ovary (CHO) cell lines, which are negative for hPD-L1, were transfected with an expression vector encoding human PD-L1 to create a modified positive control cell line. As shown in Fig. 7B, immunohistochemical staining with 22C3 of formalin-fixed paraffin-embedded cell precipitates (FFPE) from the parental CHO cell line (negative control) and transfected CHO cell line (positive control) demonstrates positive and negative staining appropriate.
    [00141] Additional FFPE human cell lines precipitated (A375, HS578T and LOX melanoma) were stained with 22C3 and demonstrate a range of staining patterns and intensities, as shown in figure 7B. Staining with 22C3 was strong and uniform in Lox melanoma cells, but only showed only rare positive cells in A375 and HS578T cell pellets. Similar staining was observed with the 20C3 antibody (data not shown). The levels of hPD-L1 expression detected in these 3 cell lines by 22C3 in the IHC analysis correlated well with levels of PD-L1 mRNA in these cell lines as assessed using qPCR with ubiquitin mRNA as the baseline.
    [00142] The selective binding and relative affinity of 22C3 to hPD-L1 expression cells was evaluated in a cell-based ELISA experiment.
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    Several cell lines (hPDL1-CHOK1 cells, parental CHOK1 cells, hPDL2-CHOK1 cells, and LOX cells) were plated in the individual wells of collagen-coated 96-well plates and grown until confluence. Media was removed and replaced with fresh CHOK1 media (DMEM / F12 containing 10% BCS) containing a primary antibody with increasing concentrations between 1.4 and 3,000 ng / mL. The following primary antibodies were used: two different production batches each of the 20C3 and 22C3 antibody, with a mouse IgG1 isotype, an anti-PD-L1 antibody (BioLegend), and an anti-PD-L2 antibody serving as controls . The primary antibody was incubated for 1 hour at 37 ° C, washed 3X with PBS / 0.01% Tween 20 and the secondary antibody, goat anti-human IgG, Fc-specific HRP Fc conjugate (Southern Biotech, Cat # 1030 -05) was added at 1: 2,000 dilution in CHOK1 media. The secondary antibody was incubated for 1 hour at 37 ° C and washed 5X in the previous manner. The ELISA was developed using TMB, interrupted with 0.1N phosphoric acid, and the absorbance read at 450 nm with background subtraction at 650 nm. The results, which are shown in figure 8, demonstrate selective binding of 22C3 and 20C3 in cells expressing hPD-L1, with the affinity affinity of 22C3 binding being greater than 20C3 for both modified hPD-L1 CHOK1 cells and LOX cells.
    [00143] A similar ELISA experiment was carried out to assess whether both the 20C3 and the 22C3 antibody binds in mouse PD-L1. No significant antibody binding was observed in mouse PD-L1 (data not shown).
    [00144] Analyzes of antibody binding competition ("cross-blocking") were performed between different pairs of anti-hPD-L1 antibodies identified in the experiments described in Example 1. The analyzes employ
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    65/68 the ForteBio® Octet® platform, which is based on bi-layer interferometry. Briefly, this technique measures binding of an initial antibody (mAb1) to the surface of the biosensor tip as a wavelength shift (Δλ) due to the bound antibody increasing the optical thickness (Y axis) at the tip of the biosensor over time (X axis ). The tip consists of an anti-huIgG sensor to which hPD-L1-Fc is connected. The change in optical thickness upon binding of the anti-PD-L1 antibody is reflected in the upward sloping curve beginning at the first dotted red vertical line (See graphs in Figs. 9A, 9B and 9C), which represents the addition of a saturation concentration of mAb1 (10 micrograms / mL) in the solution. After leaving it in equilibrium (~ 1,000 seconds), a second antibody is injected into the analysis solution (indicated by the second, vertical red line dotted in Figs. 9A, 9B and 9C). Binding of the second mAb2, indicated by an additional excursion of the curve, suggests that the two antibodies bind in non-overlapping epitopes, while little to no excursion of the curve suggests that the two antibodies bind in overlapping or identical epitopes.
    [00145] In summary, the results demonstrate that 22C3 binding competes with additional binding of all other anti-hPDL-1 clones tested as mAb2, except for 5H9 (Fig. 9A). Similarly, 20C3 also failed to compete with 5H9 binding, but it shows an intermediate degree of additional binding with 22C3 as well as 4B7 (Fig. B). Taken together, these data indicate that 20C3 and 22C3 link overlapping epitopes.
    [00146] The ability of the 22C3 antibody to detect an expression range of hPD-L1 in different types of tumor was assessed by performing IHC analysis on the FFPE sections prepared from the following tumors: bladder, esophagus, head & neck, kidney, HCC, breast, lung, ovary and gastric. A preliminary 22C3 reactivity rating with these sections
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    66/68 of tumor tissue was performed using a semi-quantitative perception of the extent of staining. As shown in figure 10, 22C3 is capable of detecting a range of PD-L1 expression essentially without any coloring (Score = 0) the prominent, strong expression (Score = 4), demonstrating the usefulness of IHC analysis with 22C3 to types of a future tumor guide that may respond to the ban on immunosuppressive PD-1 / PDL1 interactions.
    [00147] The usefulness of the 22C3 antibody to stratify patients who are most likely to respond to therapy that blocks interaction between PD-1 and PD-L1, has been evaluated in studies using 22C3 immunohistochemical assessment of archival samples obtained from 18 patients with melanoma enrolled in a phase 1 (P001) experiment with MK-3475, a therapeutic anti-PD1 antibody being developed by Merck and Co., Inc. The cases were evaluated by two pathologists independently and designed as positive, negative or equivocal, and representative images describing these three categories are shown in Fig. 11A. Interpathological agreement in this set of samples (n = 18) was 100%.
    [00148] Clinical responses were assessed using immune related response criteria (irRC) and correlated with IHC results. For this analysis, equivocal cases were considered negative, which results in a sensitivity of analysis of 72% and a specificity of 86%. The results, which are shown in Fig. 11B, suggest that immunohistochemical staining of 22C3 in FFPE tissue will be useful as a patient selection biomarker.
    [00149] Based on the results of the experiments described above, the inventors here determined that the antibodies produced by two of the 88 experimental hybridomas - MEB037.20C3.138 and
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    MEB037.20C3.116 - had the combination of sensitivity, specificity and robustness requirement to be considered for development as candidate FFPE reactive IHC diagnostic reagents.
    Example 3. Mapping of the Human PD-L1 Epitope to the 22C3 Anti-PD-L1 Antibody
    [00150] HDX-MS epitope mapping was performed using 22C3 antibody and a PD-L1-His protein, which contained the extracellular domain of mature human PDL1 (SEQ ID NO: 38) fused to a histidine11mer tag. Segments 156 to 178 and 196 to 206 in the extracellular domain of human PD-L1 (SEQ ID NO: 38) showed strong protection (a difference in the average deuteration level of> 10%) by binding to 22C3 antibody. Furthermore, segments 3 to 9, 10 to 13, 88 to 93, and 135 to 147 showed significant yet marginal protection (a difference in the average deuteration level of 5% to 10%).
    [00151] All references cited here are incorporated by reference to the same extent as if each individual publication, database entry (for example, entries from the Genbank or GeneID strings), patent application, or patent, were specifically and individually indicated to be incorporated by reference. This declaration of incorporation by reference is endorsed by applicants, as per 37 C.F.R. §1.57 (b) (1), to relate to any individual publication, database entry (for example, entries from the Genbank or GeneID strings), patent application, or patent, each of which is clearly identified accordingly with 37 CFR §1.57 (b) (2), even if such a quote is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated incorporation statements by reference, if any, within the specification does not weaken in any way
    Petition 870190102584, of 10/11/2019, p. 80/86
    68/68 any this general declaration of incorporation by reference. Citation of references here is not intended as an admission that the reference is relevant prior art, nor does it constitute any admission of the content or data of these publications or documents.
    [00152] The present invention should not be limited in scope by the specific modalities described here. Certainly, several modifications of the invention in addition to those described here will be apparent to those skilled in the art from the description presented and the attached figures. Such modifications must be within the scope of the attached claims.
    [00153] The specification previously written is considered sufficient to allow those skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described here will become apparent to those skilled in the art from the previous description and are within the scope of the appended claims.
    权利要求:
    Claims (11)
    [1]
    1. Isolated antibody or antigen-binding fragment that specifically binds to a human PD-L1 and comprises three light chain CDRs, CDRL1, CDRL2 and CDRL3, and three heavy chain CDRs, CDRH1, CDRH2 and CDRH3, characterized by the fact that:
    (a) CDRL1 is SEQ ID NO: 9, CDRL2 is SEQ ID NO: 2, CDRL3 is SEQ ID NO: 10, CDRH1 is SEQ ID NO: 14 or 15, CDRH2 is SEQ ID NO: 16 and CDRH3 is SEQ ID NO : 17, or (b) CDRL1 is SEQ ID NO: 21, CDRL2 is SEQ ID NO: 2, CDRL3 is SEQ ID NO: 22, CDRH1 is SEQ ID NO: 26, CDRH2 is SEQ ID NO: 28 and CDRH3 is SEQ ID NO: 29.
    [2]
    2. Isolated antibody or antigen-binding fragment thereof according to claim 1, characterized by the fact that the three light chain CDRs are SEQ ID NO: 9, SEQ ID NO: 2 and SEQ ID NO: 10 and the three heavy chain CDRs are SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17.
    [3]
    3. Isolated antibody or antigen-binding fragment thereof according to claim 1, characterized by the fact that the three light chain CDRs are SEQ ID NO: 21, SEQ ID NO: 2 and SEQ ID NO: 22 and the three heavy chain CDRs are SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 29.
    [4]
    4. Isolated antibody or antigen-binding fragment thereof according to claim 1, characterized by the fact that it comprises a light chain variable region and a heavy chain variable region, in which:
    (a) the light chain variable region is SEQ ID NO: 13 and the heavy chain variable region is SEQ ID NO: 20;
    (b) the light chain variable region is SEQ ID NO: 25 and the heavy chain variable region is SEQ ID NO: 32, where X in SEQ ID NO: 32 is Q; or (c) the light chain variable region is SEQ ID NO: 25 and the heavy chain variable region is SEQ ID NO: 32, where X in SEQ ID NO: 32 is pE.
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    [5]
    5. Isolated nucleic acid, characterized by the fact that it encodes one or both of a variable region of the antibody light chain and a variable region of the antibody heavy chain, in which:
    (a) the nucleic acid encoding the variable regions of the yeast antibody chain is SEQ ID NO: 33 and the nucleic acid encoding the heavy chain variable region of the antibody is SEQ ID NO: 34; or (b) the nucleic acid encoding the yeast antibody variable chain region is SEQ ID NO: 35 and the nucleic acid encoding the antibody heavy chain variable region is SEQ ID NO: 36.
    [6]
    6. In vitro method of analyzing a tissue or cell sample removed from a human for expression of PD-L1, characterized by the fact that the method comprises:
    (a) bringing the tissue sample into contact with a PD-L1 binding reagent under conditions that allow specific binding of the PD-L1 binding reagent to human PD-L1, where the binding reagent comprises the antibody or fragment binding agent to the antigen defined in any one of claims 1 to 4, (b) removing the unbound PD-L1 binding reagent, and (c) detecting the presence or absence of bound PD-L1 binding agent.
    [7]
    Method according to claim 6, characterized in that it further comprises quantifying the amount of bound binding reagent.
    [8]
    Method according to claim 6 or 7, characterized in that the binding reagent comprises SEQ ID NO: 13 and SEQ ID NO: 20, or comprises SEQ ID NO: 25 and SEQ ID NO: 32.
    [9]
    9. Kit, characterized by the fact that it comprises the isolated antibody or antigen-binding fragment defined in any one of claims 1 to 4 and a set of reagents to detect a complex of
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    3/3 antibody or antigen-binding fragment bound to human PD-L1.
    [10]
    Kit according to claim 9, characterized in that the antibody or antibody-binding fragment comprises SEQ ID NO: 13 and SEQ ID NO: 20, or comprises SEQ ID NO: 25 and SEQ ID NO: 32 .
    [11]
    11. Antibody composition comprising a mixture of antibody molecules that specifically bind to human PD-L1, characterized by the fact that more than 60% of the antibody molecules in the mixture comprise a light chain variable region of SEQ ID NO: 25 and a heavy chain variable region of SEQ ID NO: 32, where X in SEQ ID NO: 32 is pE, and the rest of the antibody molecules in the mixture comprise a light chain variable region of SEQ ID NO: 25 and a heavy chain variable region of SEQ ID NO: 32, where X in SEQ ID NO: 32 is Q.
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    PCT/US2013/075932|WO2014100079A1|2012-12-21|2013-12-18|Antibodies that bind to human programmed death ligand 1 |
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