WO2020150282A1 - Anticorps monoclonaux spécifiques de pd-l1 pour le traitement et le diagnostic de maladies - Google Patents

Anticorps monoclonaux spécifiques de pd-l1 pour le traitement et le diagnostic de maladies Download PDF

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WO2020150282A1
WO2020150282A1 PCT/US2020/013571 US2020013571W WO2020150282A1 WO 2020150282 A1 WO2020150282 A1 WO 2020150282A1 US 2020013571 W US2020013571 W US 2020013571W WO 2020150282 A1 WO2020150282 A1 WO 2020150282A1
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seq
acid sequence
amino acid
antibody
set forth
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PCT/US2020/013571
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Kurt SHANEBECK
Lei Liu
Lan Yang
Yuefeng Lu
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Askgene Pharma Inc.
Jiangsu Aosaikang Pharmaceutical Co., Ltd.
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Publication of WO2020150282A1 publication Critical patent/WO2020150282A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present application relates to antibodies for therapeutic use, and more specifically, to anti-PD-L1 antibodies and methods of treating diseases such as cancer, infectious diseases and neurodegenerative diseases.
  • Programmed cell death protein 1 also referred to as Programmed Death-1 or PD-1 is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression.
  • PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1 , and BTLA.
  • Two cell surface glycoprotein ligands for PD-1 have been identified, Programmed Death Ligand-1 (PD-L1 ) and Programmed Death Ligand-2 (PD-L2), that are expressed on antigen-presenting cells as well as many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et ai, J. Exp. Med. 192(7): 1027-34 (2000); Latchman et ai, Nat Immunol 2:261-8 (2001 )).
  • PD-1 primarily functions in peripheral tissues where activated T-cells may encounter the immunosuppressive PD-L1 (also called B7-H1 or CD274) and PD-L2 (B7-DC) ligands expressed by tumor and/or stromal cells (Flies et ai, Yale J Biol Med 84:409-21 (201 1 ); Topalian et ai, Curr Opln Immuno 24:1-6 (2012)).
  • PD-L1 also called B7-H1 or CD274
  • B7-DC PD-L2 ligands expressed by tumor and/or stromal cells
  • PD-L1 expression correlated inversely with intraepithelial CD8+ T-lymphocyte count, suggesting that PD- L1 on tumor cells may suppress antitumor CD8+ T cells (Hamanishi et ai, Proc Natl Acad Sci USA 104 (9): 3360-3365 (2007)).
  • PD-L1 has also been implicated in infectious disease, in particular chronic infectious disease.
  • Cytotoxic CD8 T lymphocytes (CTLs) play a pivotal role in the control of infection. Activated CTLs, however, often lose effector function during chronic infection.
  • PD-1 receptor and its ligand PD-L1 of the B7/CD28 family function as a T cell co-inhibitory pathway and are emerging as major regulators converting effector CTLs into exhausted CTLs during chronic infection with human immunodeficiency virus, hepatitis B virus, hepatitis C virus, herpes virus, and other bacterial, protozoan, and viral pathogens capable of establishing chronic infections.
  • Such bacterial and protozoal pathogens can include E.
  • PD1/PD-L1 is thus a target for developing effective prophylactic and therapeutic vaccination against chronic bacterial and viral infections (see, e.g., Hofmeyer et ai, Journal of Biomedicine and Biotechnology, vol. 201 1 , Article ID 451694, 9 pages, doi:10.1 155/201 1/451694).
  • immune checkpoints may be targeted therapeutically in neurodegenerative disease such as Alzheimer’s disease using antibodies to PD-L1 (see, e.g., Baruch et ai, Nature Medicine, January 2016, doi: 10.1038/nm.4022).
  • the present invention provides an antibody which binds to human PD-L1 protein, the antibody selected from the group consisting of:
  • CDR1 containing at least the amino acid sequence as set forth in SEQ ID NO: 7 heavy chain CDR2 containing at least the amino acid sequence as set forth in SEQ ID NO: 8
  • heavy chain CDR3 containing at least the amino acid sequence as set forth in SEQ ID NO: 9 and a light chain variable region comprising light chain CDR1 containing at least the amino acid sequence as set forth in SEQ ID NO: 10, light chain CDR2 containing at least the amino acid sequence as set forth in SEQ I D NO: 1 1 , and light chain CDR3 containing at least the amino acid sequence as set forth in SEQ ID NO: 12;
  • an antibody comprising a heavy chain variable region comprising heavy chain CDR1 containing at least the amino acid sequence as set forth in SEQ ID NO: 13, heavy chain CDR2 containing at least the amino acid sequence as set forth in SEQ ID NO: 14, and heavy chain CDR3 containing at least the amino acid sequence as set forth in SEQ ID NO: 15, and a light chain variable region comprising light chain CDR1 containing at least the amino acid sequence as set forth in SEQ ID NO: 16, light chain CDR2 containing at least the amino acid sequence as set forth in SEQ I D NO: 17, and light chain CDR3 containing at least the amino acid sequence as set forth in SEQ ID NO: 18;
  • an antibody comprising a heavy chain variable region comprising heavy chain CDR1 containing at least the amino acid sequence as set forth in SEQ ID NO: 13, heavy chain CDR2 containing at least the amino acid sequence as set forth in SEQ ID NO: 14, and heavy chain CDR3 containing at least the amino acid sequence as set forth in SEQ ID NO: 19, and a light chain variable region comprising light chain CDR1 containing at least the amino acid sequence as set forth in SEQ ID NO: 16, light chain CDR2 containing at least the amino acid sequence as set forth in SEQ ID NO: 17, and light chain CDR3 containing at least the amino acid sequence as set forth in SEQ ID NO: 18; and (4) an antibody comprising a heavy chain variable region comprising heavy chain
  • CDR1 containing at least the amino acid sequence as set forth in SEQ ID NO: 7 heavy chain CDR2 containing at least the amino acid sequence as set forth in SEQ ID NO: 8
  • heavy chain CDR3 containing at least the amino acid sequence as set forth in SEQ ID NO: 20 and a light chain variable region comprising light chain CDR1 containing at least the amino acid sequence as set forth in SEQ ID NO: 10, light chain CDR2 containing at least the amino acid sequence as set forth in SEQ ID NO: 1 1 , and light chain CDR3 containing at least the amino acid sequence as set forth in SEQ ID NO: 12.
  • the CDR domains are identified using Chothia numbering method.
  • the antibody is humanized.
  • the CDR domains of the antibody have one, two, three, four or five amino acids mutated, deleted or added.
  • the antibody binds to human PD-L1 protein, comprising a heavy chain variable domain selected from the group consisting of SEQ ID NO: 1 , 2, 3, and 4.
  • the antibody binds to human PD-L1 protein comprising a light chain variable domain selected from the group consisting of SEQ ID NO: 5 and 6.
  • the present invention provides a humanized antibody which binds to human PD-L1 protein comprising a heavy chain variable domain selected from the group consisting of SEQ ID NO: 21 , 22, 23, 24, 25, and 26.
  • the present invention provides a humanized antibody which binds to human PD-L1 protein comprising a light chain variable domain selected from the group consisting of SEQ ID NO: 27, 28, 29, and 30. [016] In another aspect, the present invention provides a humanized antibody which binds to human PD-L1 protein comprising a heavy chain variable domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to NO: 21 , 22, 23, 24, 25, or 26.
  • the present invention provides a humanized antibody which binds to human PD-L1 protein comprising a light chain variable domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 27, 28, 29, or 30.
  • the present invention provides a humanized antibody which binds to human PD-L1 protein comprising a heavy chain variable domain having at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 21 , 22, 23, 24, 25, or 26, and a light chain variable domain having at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 27, 28, 29, or 30.
  • the humanized antibody is a bispecific PD-L1 antibody, which further comprises one or more binding domains, which bind to human VEGF, FAP, TGF-Beta, TIGIT, CD39, or CD73.
  • the present invention provides a humanized antibody which binds to human PD-L1 protein comprising a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 31-38, or 39, and a light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 40, 41 , 42, or 43.
  • the present invention provides a nucleic acid sequence which encodes a humanized human PD-L1 antibody heavy chain, having an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 31-38, or 39.
  • the present invention provides a nucleic acid sequence which encodes a humanized human PD-L1 antibody light chain, having an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 40, 41 , 42, or 43.
  • the present invention provides a pharmaceutical composition comprising an antibody as described above.
  • the present invention provides a method of treating cancer, the method comprising the step of administering a pharmaceutical composition as described above to a subject in need thereof, wherein the cancer is selected from the group consisting of kidney, breast, lung, kidney, bladder, urinary tract, urethra, penis, vulva, vagina, cervical, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, esophagus, and liver cancer.
  • the present invention provides a method of treating an infectious disease, the method comprising the step of administering a pharmaceutical composition as described above to a subject in need thereof, wherein the infectious disease is a bacterial or viral disease.
  • the infectious disease is a chronic infectious disease.
  • the viral disease is selected from the group consisting of hepatitis B virus (HBV), hepatitis C virus (HCV) or human immunodeficiency virus (HIV).
  • the present invention provides a method of treating a neurodegenerative disease, the method comprising the step of administering a pharmaceutical composition as described above to a subject in need thereof.
  • the neurodegenerative disease is Alzheimer’s disease.
  • Figures 1A, 1 B, 1 C, 1 D, 1 E and 1 F show the results of hybridoma clones APDL1 Bs- M1 through APDL1 BS-M30.
  • Figures 1 G and 1 H show the results of hybridoma clones APDL1 Ba-M1 through APDL1 Ba-M10.
  • Figure 11 shows the results of hybridoma clones APDL1 BN-M1 through APDL1 BN-M5.
  • 1 1 1 H2 vO is an internal control, which is a monoclonal antibody against human PD-L1 derived from rabbit B cell cloning.
  • Figure 3 Binding kinetics of APDL1 BS-M7, M8, M9, M10, M1 1 , M12, M13, and M14 with human PD-L1 , as measured using Octet.
  • Figure 4 Blockade reporter assay results with antibody purified from hybridoma clones APDL1 BN-M1 through APDL1 BN-M5.
  • 1 1 1 H2 v1 is an internal control, which is a monoclonal antibody against human PD-L1 derived from rabbit B cell cloning.
  • Figure 5 Blockade reporter assay results with antibody purified from hybridoma clones APDL1 BS-M1 through APDL1 Bs-M6.
  • 1 1 1 H2 vO, v1 and v1.6 are internal controls, which are monoclonal antibodies against human PD-L1 derived from rabbit B cell cloning.
  • Figure 6 Blockade reporter assay results with antibody purified from hybridoma clones APDL1 BS-M7 through APDL1 Bs-M14.
  • 1 1 1 H2 vO and v1 are internal controls, which are monoclonal antibodies against human PD-L1 derived from rabbit B cell cloning.
  • APDL1 Bn-M2 (same as APDL1 BN) was included for comparison.
  • Figure 7 Binding of M2 with human (7A), mouse (7B) and monkey PD-L1 (7C), as measured by ELISA. 1 1 1 H2 and 80H8 are internal controls. Tecentriq is a commercialized anti-PD-L1 antibody, used as an external control.
  • FIG. 8 Binding of chimeric antibodies M1 , M3, and M5 with mouse (8A), human (8C) and monkey (8D) PD-L1 , as measured by ELISA. 1 1 1 H2 v22 and 1-6 (57H7) are internal controls. Binding kinetics (EC50) are listed in Figure 8B.
  • Figure 9 Blockade reporter assay results with chimeric M2 antibody purified from transient expression.
  • 1 1 1 H2 is an internal control.
  • Tecentriq is a commercialized anti-PD-L1 antibody, used as an external control.
  • FIG. 10 (A) Blockade reporter assay results with humanized M2 antibodies purified from transient expression.
  • JR1.35.1-6 and JR1.36.1-6 are purified humanized M2 antibodies comprising various humanized heavy chains and humanized light chains
  • ASKB1296 DS is an internal control.
  • Tecentriq is a commercialized anti-PD- L1 antibody, used as an external control.
  • the binding kinetics (EC50) of the respective heavy chains and light chains are listed in Figure 10(B).
  • Figure 1 Binding kinetics of humanized M2 antibodies JR1.35.1 and JR1.36.1 with human PD-L1 , as measured using Octet. 1 1 1 H2 is an internal control.
  • Figure 12 Binding of humanized M2 antibodies to cell surface PD-L1 on HEK293 cells stably transduced with human PD-L1 as detected by FACS.
  • Figure 12A and 12B show the binding of the antibodies JR1 .35.1-6.
  • Figure 12C and 12D show the binding of the antibodies JR1 .36.1 -6.
  • the present invention relates to compositions and methods for immunotherapy of a subject afflicted with diseases such as cancer, an infectious disease, or a neurodegenerative disease, e.g., Alzheimer’s disease, which methods comprise administering to the subject a composition comprising a therapeutically effective amount of an anti-PD-L1 antibody or portion thereof that potentiates an endogenous immune response, either stimulating the activation of the endogenous response or inhibiting the suppression of the endogenous response.
  • diseases such as cancer, an infectious disease, or a neurodegenerative disease, e.g., Alzheimer’s disease
  • antibodies designated APDL1 BN-M1 (or M1 ), APDL1 BN-M2 (or M2), APDL1 BN-M3 (or M3), APDL1 BN-M4 (or M4), and APDL1 BN-M5 (or M5) have the respective CDRs listed in Tables 4-7 below.
  • antibodies M1 , M2, M3, M4 and M5 have the respective light and heavy chain variable regions as listed in Tables 2 and 3 below.
  • humanized antibody M2 has the heavy and light chain as shown in Tables 8. The sequences of the heavy chain and the light chain of a humanized antibody of Clone M2 (ASKB1296M) are shown in Table 9.
  • the DNA sequences for the heavy chains and light chains forthe humanized antibodies are listed in Table 10.
  • the subject is selected as suitable for immunotherapy in a method comprising measuring the surface expression of PD-L1 in a test tissue sample obtained from a patient with cancer, infection, or a neurodegenerative disease of the tissue, for example, determining the proportion of cells in the test tissue sample that express PD-L1 on the cell surface, and selecting the patient for immunotherapy based on an assessment that PD-L1 is expressed on the surface of cells in the test tissue sample.
  • the “Programmed cell death protein 1 ,” “Programmed Death-1” or“PD-1 " receptor” refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is expressed predominantly on previously activated T cells in vivo, and binds to two ligands, PD-L1 and PD-L2.
  • the term "PD-1 " as used herein includes human PD-1 (hPD-1 ), variants, isoforms, and species homologs of hPD-1 , and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under Genebank Accession No. U64863.
  • P-L1 Programmed Death Ligand-1
  • PD-L1 is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulate T cell activation and cytokine secretion upon binding to PD-1.
  • the term "PD-L1 " as used herein includes human PD-L1 (hPD-L1 ), variants, isoforms, and species homologs of hPD-L1 , and analogs having at least one common epitope with hPD-L1.
  • the complete hPD-L1 sequence can be found under Genebank Accession No. Q9NZQ7.
  • the anti-PD-L1 antibody of the invention designated M1 , M2, M3, M4 and M5 may comprise a heavy chain CDR and a light chain CDR, wherein the heavy chain CDR comprises a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the respective CDRs listed in Tables 4-7 below, and wherein the light chain CDR comprises a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the respective CDRs listed in Tables 4-7 below.
  • the anti-PD-L1 antibody of the invention designated M1 , M2, M3, M4, and M5 may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the respective heavy chain variable regions listed in Table 2 below, and wherein the light chain variable region comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the respective light chain variable regions listed in Table 3 below.
  • Humanized anti-PD-L1 antibodies of M2 may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the respective heavy chain variable regions listed in Table 8, and wherein the light chain variable region comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the respective light chain variable regions listed in Table 8 below.
  • Humanized anti-PD-L1 antibodies of M2 may comprise a heavy chain domain and a light chain domain, wherein the heavy chain comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the respective heavy chain variable regions listed in Table 8 below, and wherein the light chain comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the respective light chain domain listed in Table 9 below.
  • Antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N- terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
  • the CDR regions provided by the invention may be used to construct an anti- PD-L1 binding protein, including without limitation, an antibody, a scFv, a triabody, a diabody, a minibody, and the like.
  • an anti-PD-L1 protein of the invention will comprise at least one CDR region from Tables 4-7 listed below or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the CDR regions listed in Tables 4-7.
  • Anti-PD-L1 binding proteins may comprise, for example, a CDR-H1 , a CDR-H2, a CDR-H3, a CDR-L1 , a CDR-L2, a
  • an anti-PD-L1 binding protein may comprise all three CDR-H sequences of an antibody provided herein, all three CDR-L sequences of an antibody provided herein, or both.
  • Anti-PD-L1 CDR sequences may be used on an antibody backbone, or fragment thereof, and likewise may include humanized antibodies, or antibodies containing humanized sequences.
  • the CDR regions may be defined using the Kabat definition, the Chothia definition, the AbM definition, the contact definition, or any other suitable CDR numbering system.
  • the invention provides antibodies (e.g., diabodies, minibodies, triabodies) or fragments thereof having the CDRs of Tables 4-7 or a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the CDRs of Tables 4-7.
  • the diabodies possess the light and heavy chain of Tables 2 and 3 or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences of Tables 2 and 3.
  • Bispecific antibodies can be generated by chemical cross-linking or by the hybrid hybridoma technology.
  • bispecific antibody molecules can be produced by recombinant techniques. Dimerization can be promoted by reducing the length of the linker joining the VH and the VL domain from about 15 amino acids, routinely used to produce scFv fragments, to about 5 amino acids. These linkers favor intrachain assembly of the VH and VL domains. Any suitable short linker can be used. Thus, two fragments assemble into a dimeric molecule. Further reduction of the linker length to 0-2 amino acids can generate trimeric (triabodies) or tetrameric (tetrabodies) molecules.
  • a "chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background.
  • Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein.
  • polyclonal antibodies can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the selected antigen and not with other proteins.
  • This selection may be achieved by subtracting out antibodies that cross-react with other molecules.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre protein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e. , about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection.
  • sequences are then said to be “substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • Cancer refers to human cancers and carcinomas, sarcomas, adenocarcinomas, etc., including solid tumors, kidney, breast, lung, kidney, bladder, urinary tract, urethra, penis, vulva, vagina, cervical, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, esophagus, and liver cancer.
  • one or more cancer therapies e.g., chemotherapy, radiation therapy, immunotherapy, surgery, or hormone therapy can be co-administered further with the antibody of the invention.
  • the contacting of the patient with the antibody or antibody fragment can be by administering the antibody to the patient intravenously, intraperitoneally, intramuscularly, intratumorally, or intradermally.
  • the antibody is co-administered with a cancer therapy agent.
  • recombinant refers to a polypeptide produced through a biological host, selected from a mammalian expression system, an insect cell expression system, a yeast expression system, and a bacterial expression system.
  • formulation refers to the antibodies disclosed herein and excipients combined together which can be administered and has the ability to bind to the corresponding receptors and initiate a signal transduction pathway resulting in the desired activity.
  • the formulation can optionally comprise other agents.
  • the present specification also provides a pharmaceutical composition for the administration to a subject.
  • the pharmaceutical composition disclosed herein may further include a pharmaceutically acceptable carrier, excipient, or diluent.
  • pharmaceutically acceptable means that the composition is sufficient to achieve the therapeutic effects without deleterious side effects, and may be readily determined depending on the type of the diseases, the patient's age, body weight, health conditions, gender, and drug sensitivity, administration route, administration mode, administration frequency, duration of treatment, drugs used in combination or coincident with the composition disclosed herein, and other factors known in medicine.
  • the pharmaceutical composition including the antibody disclosed herein may further include a pharmaceutically acceptable carrier.
  • the carrier may include, but is not limited to, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a colorant, and a flavorant.
  • the carrier may include a buffering agent, a preserving agent, an analgesic, a solubilizer, an isotonic agent, and a stabilizer.
  • the carrier may include a base, an excipient, a lubricant, and a preserving agent.
  • examples of the carrier, the excipient, and the diluent suitable for the pharmaceutical formulations include, without limitation, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils.
  • the pharmaceutical formulations may further include fillers, anti-coagulating agents, lubricants, humectants, flavorants, and antiseptics.
  • the total effective dose of the compositions disclosed herein may be administered to a patient in a single dose, or may be administered for a long period of time in multiple doses according to a fractionated treatment protocol.
  • the content of active ingredient may vary depending on the disease severity.
  • the total daily dose of the peptide disclosed herein may be approximately 0.0001 pg to 500 mg per 1 kg of body weight of a patient.
  • the effective dose of the peptide is determined considering various factors including patient's age, body weight, health conditions, gender, disease severity, diet, and secretion rate, in addition to administration route and treatment frequency of the pharmaceutical composition. In view of this, those skilled in the art may easily determine an effective dose suitable for the particular use of the pharmaceutical composition disclosed herein.
  • the pharmaceutical composition disclosed herein is not particularly limited to the formulation, and administration route and mode, as long as it shows suitable effects.
  • the pharmaceutical composition may be administered alone or in combination or coincident with other pharmaceutical formulations showing prophylactic or therapeutic efficacy.
  • the present specification provides a method for preventing or treating of cancer, infectious diseases or neurodegenerative diseases comprising the step of administering to a subject the chimeric protein or the pharmaceutical composition including the same.
  • the present specification provides a use of the therapeutic protein or the pharmaceutical composition including the same in the preparation of drugs for the prevention or treatment of cancer, a neurodegenerative or an infectious disease.
  • the dose of the composition may be administered daily, semi-weekly, weekly, bi-weekly, or monthly.
  • the period of treatment may be for a week, two weeks, a month, two months, four months, six months, eight months, a year, or longer.
  • the initial dose may be larger than a sustaining dose.
  • the dose ranges from a weekly dose of at least 0.01 mg/kg, at least 0.25 mg/kg, at least 0.3 mg/kg, at least 0.5 mg/kg, at least 0.75 mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, or at least 30 mg/kg
  • a weekly dose may be at most 1.5 mg/kg, at most 2 mg/kg, at most 2.5 mg/kg, at most 3 mg/kg, at most 4 mg/kg, at most 5 mg/kg, at most 6 mg/kg, at most 7 mg/kg, at most 8 mg/kg, at most 9 mg/kg, at most 10 mg/kg, at most 15 mg/kg, at most 20 mg/kg, at most
  • PD-L1 specific monoclonal antibodies were generated using the hybridoma approach using the antigen human PD-L1-his (Acrobiosystems cat. #PD1-H5221 ).
  • MAbs monoclonal antibodies
  • the initial screening used the culture supernatants of the hybridomas.
  • the supernatants were analyzed to with their activities in blocking the binding between PD- L1 and PD1 using ELISA (results not shown). They were also analyzed by their binding to PD-L1 expressing HEK 293 cells (data not shown).
  • Three groups of positive clones have been identified, with the APDLI Ba group with 10 clones (APDL1 Ba-M1 to M10), the APDL1 BN group with 5 clones (APDL1 BN-M1 to M5), and the APDLI Bs group with 30 clones (APDL1 Bs-M1 to M30).
  • FIG. 1A, 1 B, 1 C, 1 D, 1 E and 1 F show the results of hybridoma clones APDL1 BS-M1 through APDL1 Bs-M30.
  • Figures 1 G and 1 H show the results of hybridoma clones APDL1 Ba-M1 through APDL1 Ba-M10.
  • Figure 11 shows the results of hybridoma clones APDL1 BN-M1 through APDL1 BN-M5.
  • 1 1 1 H2 vO is an internal control, which is a monoclonal antibody against human PD-L1 derived from rabbit B cell cloning.
  • Example 2 Hybridoma supernatants were tested for binding to PD-L1 by ELISA.
  • Antigens tested include; human PD-L1-Fc (Acrobiosystems, cat. #PD1 -5257), human PD-L1-his, murine PD-L1-his. Plates were then blocked with PBS + 10% goat serum for 1 hour. After washing with deionized water, samples were added in PBS/10% goat serum and incubated for 1 hour. Plates were washed, and 100pL goat anti-rabbit IgG Fc-HRP (Jackson ImmunoResearch, cat. #1 1 1-035-046) was added at a 1 :5000 dilution in PBS/10% goat serum for 1 hour.
  • TMB substrate Thermo Scientific, cat. #P1134021
  • Development was stopped with 100pL 1 N H2SO4, and OD450 was measured using a microplate spectrophotometer.
  • Figure 7 shows the binding of M2 with human (7A), mouse (7B) and monkey
  • 1 1 1 H2 and 80H8 are internal controls.
  • Tecentriq is a commercialized anti-PD-L1 antibody, used as an external control.
  • Figure 8 shows the binding of M1 , M3, and M5 with mouse (8A), human (8C) and monkey (8D) PD- L1 , as measured by ELISA.
  • 1 1 1 H2 v22 and 1-6 (57H7) are internal controls. Binding kinetics for mouse PD-L1 are listed in Figure 8B. The results showed that M2 was able to bind to both human and monkey PD-L1.
  • Example 3. Competition ELISA Purified chimeric and humanized anti-PD-L1 antibodies were tested for their ability to block PD-L1 binding to the receptor PD1.
  • ELISA plates were coated with 100pL human PD-L1 -Fc at 1 pg/mL in Carbonate buffer pH 9.6 overnight at 4°C or for 1 hour at 37°C. Plates were washed twice with TBST (50 mM Tris HCI, pH 7.4, 150 mM NaCI, 0.1 % Tween). Plates were blocked with
  • Example 4 SEB Assay: Purified anti-PD-L1 antibodies were tested for their ability to enhance IL-2 secretion from whole blood treated with staphylococcus enterotoxin B (SEB).
  • SEB staphylococcus enterotoxin B
  • Heparinized whole blood is diluted 1 :5 with RPMI 1640 + gentamicin and SEB (List Biological, cat. #122) is added to 0.2pg/mL.
  • Serial 3-fold dilutions of PD-L1 antibodies are made starting at 10pg/mL (final concentration after addition of whole blood) in 100pL/well RPMI, gentamicin, and 1 % autologous plasma.
  • Diluted whole blood is then added at 100pL/well, and plates are incubated for 4 days at 37°C in 5% CO2. Supernatant is then collected for measurement of IL-2 secretion by ELISA.
  • IL-2 ELISA Plates were coated with 100pL mouse anti-human IL-2 (B-D Pharmingen, cat. #555051 ) at 2pg/mL in PBS overnight at 4°C or 1 hour at 37°C. Add 100pL/well PBS/10% goat serum to block. Incubate 1 hour. Plates were washed with deionized water. Samples and standards were added in 100pL/well PBS/10% goat serum and incubated for 1 hour. After washing with deionized water, 100pL/well anti human IL-2 biotin (B-D Pharmingen, cat. #5550400) was added at 1 pg/mL in PBS 10% goat serum, and incubated for 1 hour.
  • MLR Mixed lymphocyte reaction
  • PBMC dendritic cells
  • PBMC peripheral blood mononuclear cells
  • Buffy coats were diluted 1 :3 in PBS and layered over 15mL cushions of Lymphoprep in 50mL tubes and centrifuged at 2000 rpm for 25 minutes.
  • PBMC were collected from the gradient interface, and washed 3 times with PBS.
  • PBMC are then cultured in tissue culture flasks at approximately 1-2 x 10 6 cells/cm 2 in RPM I 1640 + 1 % fetal bovine serum. Incubate 1-1.5 hours at 37°C. Wash cells 2 times with serum free RPMI 1640 to remove non-adherent cells. Culture adherent cells in complete medium + 30ng/mL human GM-CSF (Prospec, cat. #cyt- 221 ) and 10ng/mL human IL-4 (Prospec, cyt-271 ) for 7 days.
  • Interferon-gamma ELISA Interferon-gamma ELISA. Plates were coated with 100pL mouse anti-human IFN-gamma (Biolegend, cat. #507502) at 1 pg/mL in PBS overnight at 4°C or 1 hour at 37°C. Add 100pL/well PBS/10% goat serum to block. Incubate 1 hour. Plates were washed with deionized water. Samples and standards were added in 100pL/well PBS/10% goat serum and incubated for 1 hour. After washing with deionized water, 100pL/well anti-human IFN-gamma biotin (Biolegend, cat. #5002504) was added at 1 pg/mL in PBS 10% goat serum, and incubated for 1 hour.
  • Example 5 Flow Cytometry: Binding of candidate anti-PD-L1 antibodies to cell surface expressed PD-L1 was measured using flow cytometry.
  • HEK293 cells were seeded in one well of a 6 well plate in complete medium and cultured overnight. The culture medium was removed, and 2 ml_ of fresh complete medium containing 8pg/ml_ polybrene (Santa Cruz Biotechnology, cat. #sc-134220) was added. Human PD-L1 lentiviral particles (G & P Biosciences, cat. #LTV-PD-L1-puro) were added in 0.5ml_. After overnight culture, PD-L1 + cells were selected in puromycin (Life T echnologies, cat. #A1 1 13803).
  • Figure 12A and 12B show the binding of the antibodies JR1.35.1-6 to the HEK293 cells expressing human PD-L1.
  • Figure 12C and 12D show the binding of the antibodies JR1.36.1-6 to the HEK293 cells expressing human PD-L1.
  • EC50 values of the bindings are also listed.
  • Example 6 The affinity measurement was conducted with Octet RED 96 (ForteBio) instrument at 30 degree Celsius. Briefly, anti-human IgG capture sensor (AHC from ForteBio cat #18-5060) was equilibrated with assay buffer (1x dilution of 10x Kinetics Buffer (ForteBio, Cat #18-5032). Test antibody samples were diluted to 3 pg/mL and allowed to bind to the sensors for 5 min. The sensors were then washed in assay buffer for 3 minutes, and PD-L1 ligand diluted at different concentrations were allowed to bind to the mAb coated on the sensors for 5 minutes. Afterwards, dissociation was followed for 10 minutes in the assay buffer. The sensors could be regenerated by washing in glycine buffer and assay buffer 3 times. The data were fitted with 1 : 1 binding model using the ForteBio software.
  • Figure 2 show the parameters of the binding kinetics of APDL1 BN-M1 , M2, M3,
  • FIG. 3 shows the parameters of the binding kinetics of APDL1 BS-M7, M8, M9, M10, M1 1 , M12, M13, and M14 with human PD-L1 , as measured using Octet.
  • the results showed that M2 had a binding affinity with human PD-L1 of about 4 nM.
  • Figure 1 1 show the parameters of the binding kinetics of the humanized M2 antibodies JR1.35.1 and JR1.36.1 with human PD-L1 , as measured using Octet. 1 1 1 H2 is an internal control. The results showed that JR1.35.1 and JR1.36.1 had binding affinity of about 13 and 7 nM respectively.
  • Example 7 PD1/PD-L1 blockade reporter assay : The ability of anti-PD-L1 antibodies to block PD-L1 mediated PD1 signaling was measured using two engineered cell lines. The first is a CHO-K1 cell line (CHO-K1/TCRA/PD-L1 , BPS Bioscience cat #60536) expressing both human PD-L1 and a T cell receptor activator. The second cell line (PD1/NFAT, BPS Bioscience cat # 60535) is a Jurkat T cell line expressing PD1 and an NFAT firefly luciferase reporter. The T cell receptor activator on the CHO-K1 cells will activate the Jurkat cells resulting in expression of the NFAT luciferase reporter. However, since the CHO-K1 cells also express PD-L1 , signaling via PD1 results in inhibition of NFAT activation. Blocking the PD-L1/PD1 interaction will restore NFAT activation and luciferase activity.
  • CHO-K1/TCRA/PD-L1 cells are seeded in 96 well flat bottom plates at 35,000 cells/well in 100mI_ assay medium (RPMI 1640, 10% Fetal Bovine serum, Non- essential amino acids, 2-mercaptoethanol, and gentamicin) in 96 well white walled, flat bottom plates. After overnight culture, the culture medium is removed and samples and standards are added at 2x concentration in 50pL/well. Plates are incubated 20 minutes, and 20,000 PD1/NFAT cells are added to each well in 50mI_. Plates are incubated 6 hours at 37°C.
  • 100mI_ assay medium RPMI 1640, 10% Fetal Bovine serum, Non- essential amino acids, 2-mercaptoethanol, and gentamicin
  • the PD1/PD-L1 blockade reporter assay had been used as the cell-based functional assay for the discovery of the PD-L1 antibodies.
  • the supernatants of the hybridomas from three groups of positive clones APDL1 Ba group (APDL1 Ba-M1 to M10), APDL1 BN group (APDL1 BN-M1 to M5), and APDLI Bs group (APDL1 Bs-M1 to M3Q) were tested by this functional assay.
  • the humanized M2 antibodies were expressed and purified.
  • the humanized antibodies were analyzed by the functional assay ( Figure 10A and 10B). A number of the humanized antibodies showed similar Emax and EC50 values as that of the internal and external controls.
  • Antibody variable regions of the selected clones were cloned using SMART technology following the protocol provided by the vendor (Akara, Kusatsu, Shiga, Japan).
  • the heavy chain variable domain sequences of the selected clones are shown in Table 2, the light chain variable domains in Table 3, and the CDR for each top candidate are provided Tables 4-7.
  • HEK293 cells were plated at 1.5 x 10 5 cells/well in 1 ml_ complete medium in a 24 well tissue culture plate, and cultured overnight. Transfection was performed using 500ng heavy chain DNA and 500ng light chain DNA with Lipofectamine 3000 (Life Technologies, cat. #L3000015) per manufacturer’s instructions. Supernatants were harvested after 3-5 days and assayed for binding activity by ELISA.
  • C67BL/6 mice transplanted with colon cancer cell line MC-38 are used to evaluate the efficacy of PD-L1 antibody ASKB1296M in comparison with the marketed PD-L1 antibody Tecentriq (positive control).
  • the cancer cells are transplanted subcutaneously on Day 0.
  • the dosing of PD-L1 antibodies or negative control human IgG were started on Day 1.
  • the dosages and dosing frequency are listed on Table 1.
  • the inhibition of the tumor formation and the sizes of the tumors formed are measured to assess the efficacy of the PD-L1 antibodies.
  • Table 1 Animal study groups, dosages and dosing schedule.
  • Antibody candidate M2 was selected for humanization.
  • Several humanized variants for each candidate were designed based on sequence analysis, and then tested experimentally by creating the humanized mAb expression constructs and making the humanized mAb proteins from transient expression system.
  • the humanized sequences of the variable domains are provided in Tables 8.
  • the sequences of the heavy chain and the light chain of a humanized antibody of Clone M2 (ASKB1296M) are shown in Table 9.
  • Table 8 Protein Sequences for Humanized M2 Variable Domains.
  • Table 9 Protein Sequences for Humanized PD-L1 antibodies.
  • CHOZN-GS-/- cells and pCGS3 expression vector from Sigma are used. DNA sequences encoding the genes of one of the humanized antibodies are cloned into the pCGS3 expression vectors. These DNA constructs then are linearized and introduced into CHOZN-GS-/- cells by electroporation. The transfected cells are selected by medium without L-glutamine. The survived cells are subcloned by ClonePix or limiting dilution and analyzed for the protein expression levels using ELISA or Bio-Layer Interferometry technology.
  • expression plasmid constructs containing DNA sequences encoding the genes of one of the humanized antibodies were introduced into HEK-293 cells transiently by using polyethylenimine (PEI).
  • PEI polyethylenimine
  • the transfected cells were treated by alproic acid (VPA) 24 hours post transfection to enhance protein expression.
  • the supernatants were harvested on day 6 and the antibodies were purified.
  • the cells are seeded at approximately 0.5 million per ml in the 1 L bioreactors. The cells are fed and cultured for approximately 10-14 days. The supernatants are harvested and the antibodies are purified.
  • the purification of the humanized antibody involved the Protein A affinity column followed by an anion exchange chromatography operated in the flow-through mode. It was further followed by a mixed mode chromatography.
  • the purified antibody was formulated in a formulation containing 10 mM Acetic acid, 7% sucrose, 0.01 % polysorbate-80, pH of approximately 5. It was stored at 2-8°C or -80°C until use.

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Abstract

La présente invention concerne des compositions et des méthodes d'immunothérapie pour un sujet souffrant de maladies telles que le cancer, une maladie infectieuse ou une maladie neurodégénérative, ces méthodes consistant à administrer au sujet une composition comprenant une quantité thérapeutiquement efficace d'un anticorps anti-PD-L1 ou d'une partie de celui-ci qui potentialise une réponse immunitaire endogène, soit par stimulation de l'activation de la réponse endogène, soit par inhibition de la suppression de la réponse endogène.
PCT/US2020/013571 2019-01-18 2020-01-14 Anticorps monoclonaux spécifiques de pd-l1 pour le traitement et le diagnostic de maladies WO2020150282A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130302346A1 (en) * 2010-11-24 2013-11-14 Lexicon Pharmaceuticals, Inc. Antibodies that bind notum pectinacetylesterase
WO2016111645A1 (fr) * 2015-01-09 2016-07-14 Agency For Science, Technology And Research Anticorps anti-pd-l1
US20170198045A1 (en) * 2014-05-29 2017-07-13 Macrogenics, Inc. Tri-Specific Binding Molecules and Methods of Use Thereof
US20170320960A1 (en) * 2014-09-05 2017-11-09 Abbvie Stemcentrx Llc Novel anti-mfi2 antibodies and methods of use
WO2017218435A1 (fr) * 2016-06-13 2017-12-21 Askgene Pharma Inc. Anticorps monoclonaux spécifiques de pd-l1 pour le traitement et le diagnostic de maladies

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130302346A1 (en) * 2010-11-24 2013-11-14 Lexicon Pharmaceuticals, Inc. Antibodies that bind notum pectinacetylesterase
US20170198045A1 (en) * 2014-05-29 2017-07-13 Macrogenics, Inc. Tri-Specific Binding Molecules and Methods of Use Thereof
US20170320960A1 (en) * 2014-09-05 2017-11-09 Abbvie Stemcentrx Llc Novel anti-mfi2 antibodies and methods of use
WO2016111645A1 (fr) * 2015-01-09 2016-07-14 Agency For Science, Technology And Research Anticorps anti-pd-l1
WO2017218435A1 (fr) * 2016-06-13 2017-12-21 Askgene Pharma Inc. Anticorps monoclonaux spécifiques de pd-l1 pour le traitement et le diagnostic de maladies

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