WO2019094265A1 - Molécules de liaison à un polypeptide pd1 - Google Patents

Molécules de liaison à un polypeptide pd1 Download PDF

Info

Publication number
WO2019094265A1
WO2019094265A1 PCT/US2018/058824 US2018058824W WO2019094265A1 WO 2019094265 A1 WO2019094265 A1 WO 2019094265A1 US 2018058824 W US2018058824 W US 2018058824W WO 2019094265 A1 WO2019094265 A1 WO 2019094265A1
Authority
WO
WIPO (PCT)
Prior art keywords
pdl
pbm
amino acid
seq
acid sequence
Prior art date
Application number
PCT/US2018/058824
Other languages
English (en)
Inventor
Scott Mccauley
Original Assignee
Armo Biosciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Armo Biosciences, Inc. filed Critical Armo Biosciences, Inc.
Publication of WO2019094265A1 publication Critical patent/WO2019094265A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • 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

  • PD-1 is a 288 amino acid protein expressed in B, T and myeloid derived cells.
  • PD-1 The cDNA of PD-1 was isolated in 1992 from a murine T cell hybridoma and a hematopoietic progenitor cell line undergoing apoptosis. It has become clear over the last 24 years that PD-l 's primary function is to attenuate immune responses. For example, PD-l 's role in controlling the immune system was revealed by genetic ablation studies where PD-1 deficiency resulted in multiple autoimmune phenotypes in different murine backgrounds. PD-1 has been shown to negatively regulate responses to antigenic stimulation. This was elucidated using allogeneic T cells expressing transgenic T cell receptors. In the absence of PD-1, these T cells exhibited enhanced responses to alloantigen stimulation.
  • PD-1 binds to both PD-L1 (B7-H1) and PD-L2 (B7-DC).
  • B7-H1 PD-L1
  • B7-DC PD-L2
  • the interaction of PD-1 with its ligands mediates its inhibitory effects.
  • PD-L2 expression is limited to professional antigen presenting cells, whereas PD-L1 is expressed over a much broader tissue distribution. Interactions between PD-L1 and PD-L2 are thought to maintain peripheral tolerance.
  • OPDIVO® nivolumab, BristolMyers Squibb, Princeton NJ
  • KEYTRUDA® pembrolizumab, Merck & Co., Kenilworth NJ
  • OPDIVO® was the first mAb targeting PD-1 to show significant clinical activity in unresectable or metastatic melanomas, non-small-cell lung carcinoma (NSCLC), and metastatic renal cell carcinoma.
  • NSCLC non-small-cell lung carcinoma
  • ORR objective response rates
  • KEYTRUDA® has exhibited similar efficacy and safety to OPDIVO® in a Phase 1 clinical trial and is now an FDA-approved second-line drug for the treatment of melanoma.
  • the present disclosure provides polypeptide binding molecules that bind to
  • PD-1 Programmed Cell Death-l 's
  • PD1 Programmed Cell Death Ligand-1
  • PD-L2 Programmed Cell Death Ligand-2
  • compositions useful in the preparation of such polypeptide binding molecules including nucleic acid sequences encoding said polypeptide binding molecules and recombinant cell lines expressing said polypeptide binding molecules
  • present disclosure further provides pharmaceutical formulations comprising the molecules of the present invention.
  • the present disclosure further provides methods of use of such polypeptide binding molecules in the treatment of various disorders and diseases.
  • the present disclosure further provides recombinant nucleic acid sequences encoding such polypeptide binding molecules and cells transformed therewith.
  • the present disclosure also provides methods of use of the molecules of the present disclosure in the diagnosis, prevention and treatment of various disorders or conditions in subjects including but not limited to the treatment of neoplastic disorders including but not limited to cancer.
  • Figure 1 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 2 of the present disclosure demonstrating that AMOOOl 's inhibition of PD-1 :PD-L1 is comparable to that of both
  • OPDIVO® nivolumab
  • KEYTRUDA® pembrolizumab
  • Figure 2 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 4 of the present disclosure demonstrating that AMOOOl blocks PDL1 binding
  • Figure 3 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 5 of the present disclosure demonstrating AMOOOl binding specificity for human, cynomolgus, and murine PD-1
  • Figure 4 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 5 of the present disclosure demonstrating AMOOOl binding specificity for Rat PD-1
  • Figure 5 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 5 of the present disclosure demonstrating AMOOOl binding specificity for Canine PD-1.
  • Figure 6 of the attached drawings provides an illustration of a gel stained with
  • Figure 7 of the attached drawings provides comparative binding data of AMOOOl and nivolumab to glycosylated and deglycosylated forms of the human and mouse PD-1 proteins under reaction conditions as provided in Example 7.
  • Figure 8 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 6 of the present disclosure demonstrating AM0001 blocks PD-1 binding to PD-L1 and PD-L2
  • Figure 9 of the attached drawings provides a graphical representation of the results of studies performed in substantial accordance with Example 7 of the present disclosure demonstrating the ability of AM0001 to inhibit of endogenous PD1 function in vitro.
  • Activity The term "activity,” in reference to an PDl-PBM ("PD-1 Polypeptide
  • Binding Molecule of the present disclosure, includes, but is not limited to, epitope or antigen binding affinity, ability to neutralize or antagonize a bioactivitv of PD-1 in vivo or in vitro and/or in vitro stability of an immunoglobulin (e.g. antibody) and the immunogenic properties of the immunoglobulin (e.g., antibody), e.g., when administered to a human subject.
  • an immunoglobulin e.g. antibody
  • the immunogenic properties of the immunoglobulin e.g., antibody
  • aforementioned properties or characteristics can be observed or measured using art-recognized techniques including, but not limited to, ELISA, competitive ELISA, surface plasm on resonance analysis, in receptor binding and immunohistochemistry with tissue sections from different sources including human, primate, or any other source as the need may be.
  • affinity refers to the degree of specific binding of a PBM (e.g., immunoglobulin) to its target and is measured by the binding kinetics expressed as K d , a ratio of the dissociation constant between the PDl-PBM and the extracellular domain of PDl (K 0 ff) and the association constant between the PBM and the extracellular domain of PDl (Kon).
  • K d a ratio of the dissociation constant between the PDl-PBM and the extracellular domain of PDl
  • Kon association constant between the PBM and the extracellular domain of PDl
  • high affinity is used in reference to PDl-PBMs having a Kd ⁇ 10e-07 , alternatively Kd ⁇ 10e-08, alternatively Kd ⁇ 10e-09 as determined by surface plasmon resonance (e.g. Biacore).
  • PDl-PBM of the present disclosure have a Kd for cynomologous monkey PD-1 of about 100 pM or less at 25 C 'C. In further embodiments, PDl-PBMs of the present disclosure have a binding affinity for human and cynomologous PD-1 of about 10 pM or less at 25°C and a binding affinity for cynomologous monkey PD-1 of about 100 pM or less at 25°C,
  • “activity comparable to”, “comparable effect”, “effect comparable to”, are understood as relative terms that can be viewed quantitatively and/or qualitatively.
  • one result to another result e.g., one result to a reference standard ⁇ “comparable” frequently means that one result deviates from a reference standard by less than 35%, by less than 30%, by less than 25%, by less than 20%, by less than 15%, by less than 10%, by less than 7%, by less than 5%, by less than 4%, by less than 3%, by less than 2%, or by less than 1%.
  • one result is comparable to a reference standard if it deviates by less than 15%, by less than 10%, or by less than 5% from the reference standard.
  • enriched refers to a sample is non-naturally manipulated so that component (e.g. a polypeptide) is present in: a) a greater concentration (e.g., at least 3 -fold greater, at least 4-fold greater, at least 8-fold greater, at least 64-fold greater, or more) than the concentration of the component in the starting sample, or b) a concentration greater than the environment in which the component was made.
  • component e.g. a polypeptide
  • Inhibit/Neutralize The term “inhibit” or “neutralize” as used herein with respect to a bioactivity of an PD1-PBM of the present disclosure means the ability to substantially antagonize, inhibit prevent, restrain, slow, disrupt, eliminate, stop, reduce or reverse a bioactivity of PD-1 (e.g., as measured in Example 3 herein).
  • Isolated The term “isolated” is used in reference to a polypeptide or nucleic acid of interest that, if naturally occurring, is in an environment different from that in which it can naturally occur. "Isolated” is meant to include polypeptides or nucleic acids that are within samples that are substantially enriched for the polypeptide or nucleic acid of interest and/or in which the polypeptide or nucleic acid of interest is partially or substantially purified. Where the polypeptide or nucleic acid is not naturally occurring, “isolated” indicates that the polypeptide or nucleic acid has been separated from an environment in which it was made by either synthetic or recombinant means.
  • CDRs As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services,
  • Residue numbering follows the nomenclature of Kabat et al., supra Residue numbering follows the nomenclature of Chothia et al., supra Residue numbering follows the nomenclature of MacCallum et al., supra
  • Kabat Numbering refers to a system of numbering amino acid residues which are more variable (e.g., hypervariable) than other amino acid residues in the heavy and light chain regions of
  • Modulator is meant to refer broadly agents that directly or indirectly increase or decrease the function or activity of an PDl-PBM. Examples of modulators include chemical compounds or physical phenomena (e.g., radiation). The terms “modulate”, “modulation” refer to the effect of a modulator.
  • N-Terminal/C-Terminal As used herein in the context of the structure of a polypeptide, "N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxyl terminus”) refer to the extreme amino and carboxyl ends of the polypeptide, respectively, while the terms “N-terminal” and “C-terminal” refer to relative positions in the amino acid sequence of the polypeptide toward the N-terminus and the C-terminus, respectively, and can include the residues at the N-terminus and C-terminus, respectively. "Immediately N-terminal" or
  • immediate C-terminal refers to a position of a first amino acid residue relative to a second amino acid residue where the first and second amino acid residues are covalently bound to provide a contiguous amino acid sequence.
  • nucleic acid As used herein the terms “nucleic acid,” “nucleic acid”, “nucleic acid molecule”, “polynucleotide” and the like are used interchangeably herein to refer to a phosphodiester-linked polymeric form of nucleotides of any length, either deoxy-ribonucleotides or ribonucleotides, or analogs thereof.
  • Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers and the like.
  • PD-1 As used herein, the term “PD-1 "(or “PD1”) refers to the 288 amino acid polypeptide having the amino acid sequence:
  • Numbering of amino acid residues in PD-1 refers to the full length polypeptide shown in SEQ ID NO: 9.
  • Amino acids 1-20 of SEQ ID NO: 9 define a signal sequence that is removed during translational processing resulting in the "mature PD1" molecule comprising amino acids 25-288 of SEQ ID NO: 9.
  • Amino acids 168-190 of SEQ ID NO: 9 define the transmembrane domain and resides 191-288 define the cytoplasmic domain.
  • the term PD-1 includes naturally occurring variants including the naturally occurring variant with the substitution of Alanine to Valine at position 215.
  • Amino acids 25 -167 define the 150 amino acid extracellular domain of PD-1 having the amino acid sequence:
  • the extracellular domain possesses four glycosylation sites at resides 49, 58, 74 and 116 and a disulfide bond exists between resides 54 and 123.
  • PD1 Receptor(s) As used herein, the term PD 1 receptor refers to either of the group consisting of B7-H1/PD-L1 (hereinafter “PD-L1”) and B7-DC/PD-L2. hereinafter “PD- L2").
  • PD-L1 B7-H1/PD-L1
  • PD- L2 B7-DC/PD-L2.
  • Polypeptide The terms "polypeptide,” “peptide,” and “protein”, used
  • a polypeptide may include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones.
  • Polypeptide includes but is not limited to fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without N-terminus methionine residues; fusion proteins with immunologically tagged proteins.
  • Recombinant As used herein, the term recombinant to refer to polypeptides generated using recombinant DNA technology. The techniques and protocols for recombinant DNA technology are well known in the art.
  • response refers to a change in biochemical or physiological behavior, e.g., concentration, density, adhesion, or migration within a biological compartment, rate of gene expression, or state of differentiation, where the change is correlated with activation, stimulation, or treatment, or with internal mechanisms.
  • concentration e.g., concentration, density, adhesion, or migration within a biological compartment, rate of gene expression, or state of differentiation, where the change is correlated with activation, stimulation, or treatment, or with internal mechanisms.
  • activation refers to cell activation as regulated by internal mechanisms, as well as by external or environmental factors; whereas the terms “inhibition”, “down-regulation” and the like refer to the opposite effects.
  • binding domain refers to the situation in which one member of a specific binding pair does not significantly bind to molecules other than its specific binding partner(s) as measured by techniques well known in the including but not limited to competition ELISA, BIACORE® assays and/or KINEXA® assays.
  • the term is also applied where the antigen-binding domain of an PBM of the present disclosure is specific for a particular epitope that may be present on multiple antigens, in which case the antigen- binding domain of the PBM specifically binds to the various antigens carrying the epitope.
  • epitope refers to that portion of a molecule capable of being recognized by and bound by a PBM at one or more of the PBM's binding domains.
  • substantially pure is used to refer a sample wherein a component (e.g., a polypeptide) makes up greater than about 50% of the total content of the composition, and typically greater than about 60% of the total polypeptide content.
  • a component e.g., a polypeptide
  • the contribution of the liquid phase is not calculated with respect to the calculation of percentage of total content of the composition.
  • a substantially pure aqueous solution of an agent may consist primarily of water but the water is not factored in when calculating the percentage purity.
  • substantially pure refers to compositions in which at least 75%, at least 85%, at least 90%) or more of the total composition is the component of interest. In some cases, the polypeptide will make up greater than about 90%, or greater than about 95% of the total content of the composition.
  • Subject/Patient refers to a mammal, such as a human suffering from a neoplastic condition.
  • a subject is further characterized with a disease or disorder or condition that would benefit from a decreased level of PD-1 activity.
  • mammalian subjects include but are not limited to members of the superfamilies Cercopithecoidea and Hominoidea, in particular members of the family Hominidae including human beings.
  • the term subject also includes members of the families Canidae (including Canis familiaris), Felidae (including Felinae and species of the genus Felis, in particular members of specifically including Felis catus), Equidae (specifically including species of the genus Equus such as domesticated horses), and Bovidae (including species of the tribe Bovini such as Bos taurus).
  • Canidae including Canis familiaris
  • Felidae including Felinae and species of the genus Felis, in particular members of specifically including Felis catus
  • Equidae specifically including species of the genus Equus such as domesticated horses
  • Bovidae including species of the tribe Bovini such as Bos taurus.
  • CDR polypeptide CDR sequences useful for the preparation of PDl-PBMs.
  • the terms "complementary determining region” and its abbreviation "CDR" are those sequences typically provided in immunoglobulin molecules that determine the binding properties used herein to refer to the hypervariable domains of variable domains of immunoglobulins. Systematic identification of residues included in the CDRs have been developed by Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda). The numbering of these CDR positions herein is provided according to Kabat numbering
  • the present disclosure provides the CDRs useful in the preparation of PDl-PBMs as defined using the Kabat system in accordance with the present disclosure in Table 2 below:
  • the present disclosure further provides a PD-1 polypeptide binding molecule
  • PDl-PBM comprising one or more polypeptide sequences selected from the group consisting of SEQ IDs#l-6.
  • the term PDl Polypeptide Binding Molecule refers to a polypeptide binding molecule comprising at least one or more of the CDRs SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 SEQ ID NO: 4, SEQ ID NO: 5 and/or SEQ ID NO: 6, wherein the PDl-PBM specifically binds to the extracellular domain of a mammalian PDl and substantially inhibits the binding of the extracellular domain of a mammalian PDl at least one PDl receptor.
  • PBM Polypeptide Binding Molecule
  • immunoglobulins including but not limited to mammalian immunoglobulin classes IgGl, IgG2, IgG3 and IgG4 and immunoglobulin derivatives including but not limited to IgG(l-4)deltaCH2, F(ab') 2 , Fab, ScFv, VH, VL, tetrabodies, triabodies, diabodies, dsFv, F(ab') 3 , scFv-Fc and (scFv) 2 .
  • immunoglobulins including but not limited to mammalian immunoglobulin classes IgGl, IgG2, IgG3 and IgG4
  • immunoglobulin derivatives including but not limited to IgG(l-4)deltaCH2, F(ab') 2 , Fab, ScFv, VH, VL, tetrabodies, triabodies, diabodies, dsFv, F(ab
  • PBMs of the present disclosure include monoclonal immunoglobulins (e.g., monoclonal antibodies). While a monoclonal immunoglobulin (e.g., monoclonal antibody) can be produced using hybridoma production technology, other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries).
  • monoclonal immunoglobulin e.g., monoclonal antibody
  • other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries).
  • the PDl-PBM is an immunoglobulin that specifically binds the extracellular domain of a mammalian PDl having an affinity for the extracellular domain of a mammalian PDl less than 10e-12 and substantially inhibits the binding extracellular domain of said mammalian PDl to bind to at least one PDl receptor.
  • immunoglobulin of "IgG” is used herein refers to a polypeptide consisting of subsequences substantially encoded by all or part of the recognized immunoglobulin
  • the immunoglobulin genes include the kappa ( ), lambda ( ⁇ ), and heavy chain loci that comprise myriad variable region genes, and the constant region genes rau ( ⁇ ), delta ( ⁇ ), gamma ( ⁇ ), sigma ( ⁇ ), and alpha (a).
  • the conventional mammalian immunoglobulin structure e.g., human, as well as many other higher mammals
  • Light chain polypeptides are classified as kappa and lambda light chains.
  • Heavy chain polypeptides are classified as rau, delta, gamma, alpha, or epsilon, and define the immunoglobulin's (e.g., antibody's) isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgG has several subclasses, including, but not limited to IgGl , IgG2, IgG3, and IgG4.
  • IgM has subclasses, including, but not limited to, IgMl and IgM2.
  • IgA has several subclasses, including but not limited to IgAl and IgA2.
  • isotype as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • the known human immunoglobulin isotypes are IgGl , IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD, and IgE.
  • IgG includes IgG fusions, where the IgG fusion contains sequences from two or more IgG molecules.
  • the PDl -PBMs of the present disclosure may comprise sequences belonging to the IgG (including IgGl, IgG2, IgG3, IgG4 and fusions of any combination), IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG, IgM (and fusions of any combination) classes of antibodies, the IgG class being an embodiment of particular interest.
  • each of the light and heavy chains of an immunoglobulin is comprised of two distinct regions, referred to in the art as the variable and constant regions.
  • the human IgG heavy chain is composed of four immunoglobulin domains linked from N- to C- terminus in the order VH-CH1-CH2-CH3, referring to the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 respectively (also referred to as VH-Cy 1 -Cy2-Oy3 , referring to the heavy chain variable domain, constant gamma 1 domain, constant gamma 2 domain, and constant gamma 3 domain
  • the human IgG light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order VL-CL, referring to the light chain variable domain and the light chain constant domain respectively.
  • the carboxy-terminal portion of each heavy chain defines a constant region primarily responsible for effector function.
  • variable region as used herein is meant the region of an antibody that comprises one or more Ig domains substantially encoded by any of the VL (including VK and ⁇ ) and/or VH genes that make up the light chain (including kappa and lambda) and heavy chain immunoglobulin genetic loci respectively.
  • VL including VK and ⁇
  • VH variable region polypeptides
  • the present disclosure provides variable region polypeptides (SEQ ID NO: 7 and SEQ ID NO: 8) useful in the preparation of PDl-PBMs of the present disclosure as provided in Table 3 below:
  • VL and VH A light or heavy chain variable region (VL and VH) is composed of "framework"
  • CDRs complementarity determining regions
  • VH CDR1, VH CDR2, VH CDR3, VL CDR1 , VL CDR2, and VL CDRS six CDRs, three per each heavy and light chain, designated VH CDR1, VH CDR2, VH CDR3, VL CDR1 , VL CDR2, and VL CDRS, respectively.
  • the framework regions FR 1, FR2, FR3 and FR4 are separated by the CDRs (CDR 1 , CDR2 and CDR3).
  • the extent of the framework region and CDRs have been precisely defined, for example as in Kabat (see “Sequences of Proteins of Immunological Interest," E. Kabat et al., U.S. Department of Health and Human Services, (1983)).
  • the framework regions of an antibody serves to position and align the CDRs, which are primarily responsible for binding to an antigen and provides a stable scaffold upon which substantial antigen binding diversity (the CDRs) can be explored by the immune system to obtain specificity for a broad array of antigens.
  • immunoglobulin derivative refers to a PBM derived from an immunoglobulin.
  • derived from in the context of a PBM derived from an immunoglobulin
  • immunoglobulin refers to both physical isolation of elements from an immunoglobulin as well as sequence information derived from an isolated immunoglobulin. Consequently, for example, an immunoglobulin derivative may be wholly synthetic but based on sequence information derived from an isolated naturally occurring or engineered immunoglobulin and be considered "derived from" the immunoglobulin.
  • Immunoglobulin derivatives include antigen-binding fragments of an antibody, as well as polypeptides having the antigen-binding components of an antibody (e.g., single chain antibodies, where the heavy and light chains of the antibody or joined via a linker).
  • immunoglobulin derivatives include IgG(l-4)deltaCH2, F(ab') 2 , Fab, ScFv, VH, VL, tetrabodies, triabodies, diabodies, dsFv, F(ab') 3 , scFv-Fc and (scFv) 2 molecules.
  • the term F(atv)2 refers to a bivalent PBM comprising two linked Fab fragments.
  • Fab refers to a PBM consisting of VL, VH, CL and CHI domains of an immunoglobulin.
  • Fv refers to a PBM consisting of the VL and VH domains of a single immunoglobulin parent.
  • ScFv refers to a single chain pBM comprising the light and heavy chain variable regions (LCVR and HC VR) with a linker sequence. (See, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol . 1 13, Rosenburg and Moore eds., Springer- Verlag, New York, pp 269-315, 1994). ScFv molecules may be joined to a CH3 domain to create scFv-Fc molecules also referred to in the art as "minibodies,"
  • VH refers to a polypeptide chain PBM corresponding substantially to the heavy chain variable region of the parent antibody.
  • VL refers to a polypeptide chain PBM corresponding substantially to the light chain variable region of the parent antibody.
  • VH and/or VL polypeptides may be joined by linker peptides to create multivalent compounds commonly referred to diabodies (bivalent), triabodies (trivalent) and tetrabodies (quadravalent).
  • immunoglobulins and immunoglobulin derivatives may be derived from germline sequences (i.e. set of sequences that compose the natural genetic repertoire of antibodies and their associated alleles) or may synthetic by combining various domains from different immunoglobulin molecules to engineer a PBM having specific combinations of properties.
  • germline sequences i.e. set of sequences that compose the natural genetic repertoire of antibodies and their associated alleles
  • synthetic by combining various domains from different immunoglobulin molecules to engineer a PBM having specific combinations of properties may be incorporated into any of a variety of framework regions to define synthetic VL and VH sequences.
  • the PDl-PBM is a human, humanized or deimmimized immunoglobulin molecule comprising one or more of the CDRs of SEQ ID NOS: 1, 2, 3, 4, 5, and 6.
  • the PDl-PBM is a human, humanized or deimmimized
  • immunoglobulin molecule e.g., antibody, antigen-binding fragment of an antibody having a VH having the CDRs of the heavy chain of SEQ ID NO: 8 (VH-09), a VL having the CDRs of the light chain of SEQ ID NO:7 (VL-09), or both.
  • the PDl-PBM is a human, humanized or deimmunized immunoglobulin molecule (e.g., antibody, antigen-binding fragment of an antibody) having a VH having CDRs of amino acid sequences of SEQ ID NO:4 (CDR-Hl ), SEQ ID NO: 5 (C DREE.) and SEQ ID NO:6 (CDR-H3).
  • CDR-Hl CDR-Hl
  • SEQ ID NO: 5 C DREE.
  • CDR-H3 SEQ ID NO:6
  • the PDl-PBM is a human, humanized or deimmunized immunoglobulin molecule (e.g., antibody, antigen-binding fragment of an antibody) having a VL having CDRs of amino acid sequences of SEQ ID NO: l (CDR-Ll), SEQ ID NO:2 (CDR-L2) and SEQ ID NO: (CDR-L3).
  • CDR-Ll CDR-Ll
  • CDR-L2 SEQ ID NO:2
  • CDR-L3 SEQ ID NO:
  • the PDl-PBM is a human, humanized or deimmunized immunoglobulin molecule (e.g., antibody, antigen-binding fragment of an antibody) having a VH having CDRs of amino acid sequences of SEQ ID NO:4 (CDR-Hl), SEQ ID NO: 5 (CDR-H2) and SEQ ID NO:6 (CDR-H3) and a VL having CDRs of amino acid sequences of SEQ ID NO: 1 (CDR-Ll), SEQ ID NO 2 (CDR-L2) and SEQ ID NO: 3 (CDR-L3).
  • a human, humanized or deimmunized immunoglobulin molecule e.g., antibody, antigen-binding fragment of an antibody having a VH having CDRs of amino acid sequences of SEQ ID NO:4 (CDR-Hl), SEQ ID NO: 5 (CDR-H2) and SEQ ID NO:6 (CDR-H3) and a VL having CDRs of amino acid sequences
  • humanized immunoglobulin is used herein to refer to an immunoglobulin comprising portions of immunoglobulins of different origin, wherein at least one portion comprises amino acid sequences of human origin and at least one portion is from a non-human origin (e.g., artificial origin (e.g., non-naturally occurring) or non-human origin).
  • a non-human origin e.g., artificial origin (e.g., non-naturally occurring) or non-human origin.
  • the immunoglobulin providing the CDR's is called the "donor” and the immunoglobulin providing the framework is called the "acceptor”.
  • deimmunized immunoglobulin refers to immunoglobulin which has one or more heavy chain framework regions, light chain framework regions, light chain constant regions, and/or heavy chain constant regions modified to reduce immunogenicity of the immunglobulin, e.g., to remove T cell epitopes that may induce an anti-immunoglobulin immune response (e.g., following administration to a human subject).
  • the PD1-PBM of the present disclosure incorporates the Fc region of an immunoglobulin.
  • Fc or “Fc region”, as used herein is meant the polypeptide comprising the constant region of an immunoglobulin excluding the first constant region immunoglobulin domain.
  • Fc refers to the C-terminal constant region
  • the Fc region may include the J chain.
  • the Fc region comprises immunoglobulin domains Cgamnia2 and Cgamma3 (Cy2 and Oy3) and the hinge between Cgammal (Oyl) and Cgamma2 (Cy2).
  • the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus.
  • an PD1-PBM is a mammalian immunoglobulin or "full length antibody” or “antibody” comprising variable and constant domains providing binding and effector functions.
  • a full length antibody comprises two light chains and two heavy chains, each light chain comprising a variable region and a constant region.
  • Constant regions may be derived from one or more IgG subclasses including IgGl, IgG2, IgG3 and igG4.
  • the constant and variable regions are "human" (i.e. possessing amino acid sequences characteristic of human immunoglobulins)
  • the present disclosure provides PDl-PBMs that specifically bind the extracellular domain of mature PD1 (SEQ ID NO: 10).
  • the present disclosure also provides PDl-PBMs (e.g., antibodies, antigen-binding antibody fragments) that specifically bind an epitope of PD-1, wherein the PD1-PBM competes for binding with an antibody that comprises light chain CDRs of an antibody light chain variable region comprising amino acid sequence SEQ ID NO: 7 and heavy chain CDRs of an antibody heavy chain variable region comprising amino acid sequence SEQ ID NO:8.
  • PDl-PBMs e.g., antibodies, antigen-binding antibody fragments
  • the present disclosure also provides PDl-PBMs that specifically bind an epitope of PD-1, wherein the PD1- PBM competes for binding with an antibody having i) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of GFTFSSYAMS (SEQ ID NO: 4), a CDR2 comprising the amino acid sequence of DISGGGGTTYYADSVKG (SEQ ID NO:5), and a CDR3 comprising the amino acid sequence of SGTVVTDFDY (SEQ ID NO:6), and ii) a light chain variable region comprising a CDR1 comprising the amino acid sequence of
  • GGNSIGSYSVH (SEQ ID NO: 1)
  • CDR2 comprising the amino acid sequence of DDSDRPS
  • CDR3 comprising the amino acid sequence of QVWDTSSYWV (SEQ ID NO: 3).
  • a PD1-PBM "competes for binding" with a reference anti-PD-1 antibody where specific binding of the PD1-PBM to PD-1 inhibits binding of the reference anti- PD-1 antibody to PD-1 or vice versa.
  • PDl-PBMs compete for binding with a reference anti-PD-1 antibody when, for example, the PD1-PBM binds the same or overlapping epitope bound by the reference anti-PD-1 antibody.
  • Competitive binding assays can be conducted under physiological conditions to detect specific binding to an extracellular region of PD-1, e.g., as presented on a surface of a PD-1 expressing cell, using conventional methods.
  • PDl-PBMs that compete for binding with a reference anti-PD-1 antibody can provide for an activity of the reference anti-PD- 1 antibody, e.g., inhibition of binding of one or both of PD-L1 (B7-H1) and PD-L2 (B7-DC) to cell-surface PD-1.
  • the present disclosure also provides PDl-PBMs (e.g., antibodies, antigen-binding antibody fragments) that specifically bind an epitope of PD-1 bound by with an antibody that comprises light chain CDRs of an antibody light chain variable region comprising amino acid sequence SEQ ID NO:7 and heavy chain CDRs of an antibody heavy chain variable region comprising amino acid sequence SEQ ID NO:8.
  • the present disclosure also provides PD l- PBMs that specifically bind an epitope of PD-1 bound by an antibody having i) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of GFTFSSYAMS (SEQ ID NO: 4), a CDR2 comprising the amino acid sequence of DISGGGGTTYYADSVKG (SEQ ID NO:5), and a CDR3 comprising the amino acid sequence of SGTVVTDFDY (SEQ ID NO:6), and ii) a light chain variable region comprising a CDR1 comprising the amino acid sequence of GGNSIGSYSVH (SEQ ID NO: 1), a CDR2 comprising the amino acid sequence of DDSDRPS (SEQ ID NO: 2), and a CDR3 comprising the amino acid sequence of
  • the PD1-PBM is AM0001 : a monoclonal antibody with a lambda 2 light chain and an IgG4 with a serine to proline substitution at position 228 (S228P) to provide a "hinge-stabilized" heavy chain, characterized by VL and VH CDRs having amino acid sequences corresponding to SEQ ID NOS: 1- 6 as set out in Table 2 hereinabove, a light chain variable region characterized by the sequence of SEQ ID NO: 7 and a heavy chain variable region characterized by the amino acid sequence of SEQ ID NO:8.
  • the AM0001 antibody is characterized as having a binding affinity (Kd) for human and
  • cynomologous monkey PD-1 of about 10 pM or less at 25° C (as determined by biolayer micrometry).
  • AMOOO l may be prepared in substantial accordance with the teaching of Example 1 herein.
  • the binding affinity of M9 and AMOOOl, measured by bio-layer interferometry (BLI, Octet Red 96, Forte Bio, Menlo Park, CA), are shown in Table 4 below.
  • AMOOOl are provided below:
  • the PD1 -PBMs of the present disclosure may be conjugated or operably linked to another therapeutic compound.
  • the therapeutic compound may be a cytotoxic agent, a chemotherapeutic agent, a toxin, a radioisotope, a cytokine, or other therapeutically active agent.
  • the PDl-PBMs of the present disclosure may be conjugated to a protein or molecule for utilization in tumor pretargeting or prodrug therapy.
  • the PDl-PBMs of the present disclosure may be linked to one of a variety of non-proteinaceous polymers, for example e.g., polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the present disclosure further provides a method for detecting PD-1 protein in a biological sample said method comprising contacting incubating a PDl-PBM of the disclosure with the biological sample under conditions and for a time sufficient to permit said PDl -PBM to bind to PD-1 protein, and detecting said binding.
  • the present disclosure further provides an imaging agent said imaging agent comprising a PD l-PBM molecule in stable association (e.g. covalent, coordinate covended) with an imaging agent.
  • imaging agent is used to describe any of a variety of compounds a signature that facilitates identification, tracing and/or localization of the PDl-PBM (or its metabolites) using diagnostic procedures.
  • imaging agents include but are not limited to fluorescent compounds, radioactive compounds, compounds opaque to imaging methods (e.g. X-ray, ultrasound).
  • the PDl-PBMs of the present disclosure may be prepared using conventional techniques well known to those of skill in the art. Weil established methods for antibody molecular biology, expression, purification, and screening are described in Antibody
  • the present disclosure further provides isolated nucleic acid molecules encoding the VH, VL, heavy chain and/or light chain of an antibody of the present disclosure: a vector comprising that nucleic acid, optionally operabiy-linked to control sequences recognized by a host ceil transformed with the vector; a host cell comprising that vector; a process for producing an antibody of the present disclosure comprising cuituring the host cell so that the nucleic acid is expressed and, optionally, recovering the antibody from the host cell culture medium.
  • a polynucleotide is "operably linked" when it is placed into a functional relationship with another polynucleotide.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
  • the heavy and light chains of the antibody can be provided on the same or different vectors, and may be expressed from the same or different promoters.
  • nucleic acids are created that encode the PD1 -PBMabs and that may then be cloned into host cells, expressed and assayed, if desired.
  • DNA is synthesized in accordance with well-known procedures. For example, a variety of methods that may find use in generating PDl-PBMabs herein are described in Molecular Cloning— A Laboratory Manual, 3 ld Ed. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001. Additionally, there are a variety of commercially available kits and methods for gene assembly, mutagenesis, vector subcloning, and the like, and useful in generating nucleic acids that encode PDl-PBMabs.
  • the present disclosure provides recombinant host cells (e.g., yeast cells, mammalian cells) containing a nucleic acid comprising a nucleotide sequence encoding a variable heavy chain polypeptide and a nucleic acid comprising a nucleotide sequence encoding a variable light chain polypeptide, wherein the variable heavy and light chains have the CDRS, or the full-length sequence, of a variable heavy and light chain polypeptides of a D l -PBM as disclosed herein.
  • the nucleic acids encoding the heavy and light chains of a PD1-PBM may be on the same or different expression constructs in the host ceil, and may optionally be integrated into the host ceil genome.
  • variable heavy chain comprises a CDR1 comprising the amino acid sequence of GFTFSSYAMS (SEQ ID NO: 4), a CDR2 comprising the amino acid sequence of DISGGGGTTYYADSVKG (SEQ ID NO:5), and a CDR3 comprising the amino acid sequence of SGTWTDFDY (SEQ ID NO:6).
  • variable light chain comprises a CDR1 comprising the amino acid sequence of
  • variable heavy chain comprises the amino acid sequence of SEQ ID NO: 8.
  • variable light chain comprises the amino acid sequence of SEQ ID NO: 7.
  • the present disclosure provides expression vectors having a nucleic acid comprising a nucleotide sequence encoding a variable heavy chain polypeptide, and/or a nucleic acid comprising a nucleotide sequence encoding a variable light chain, wherein the variable heavy and light chains have the CDRS, or the full-lengt sequence, of a variable heavy and light chain polypeptides of a PDl-PBM as disclosed herein.
  • the nucleic acids encoding the heavy and light chains of a PDl-PBM may be on the same or different expression vectors, and the nucleic acids encoding the variable heavy and variable light chains may be operably linked to the same or different promoters.
  • variable heavy chain comprises a CDR1 comprising the amino acid sequence of GFTFSSYAMS (SEQ ID NO: 4), a CDR2 comprising the amino acid sequence of DIS GGGGTTYYAD S VKG (SEQ ID NO: 5), and a CDRS comprising the amino acid sequence of SGTVVTDFDY (SEQ ID NO: 6).
  • variable light chain comprises a CDR1 comprising the amino acid sequence of
  • variable heavy chain comprises the amino acid sequence of SEQ ID NO: 8.
  • variable light chain comprises the amino acid sequence of SEQ ID NO: 7.
  • the nucleic acid sequence encoding the PDl-PBMs are typically incorporated into a vector for expression which are then used to transform a host cell to effect expression of the protein in the host cell.
  • the term "vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been operably linked including, but not limited to, plasmids and viral vectors. Certain vectors are capable of autonomous replication in a host ceil into which they are introduced while other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby, are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Exemplary vectors are well known in the art.
  • the nature of the vector depends on the nature of the host cell used for expression.
  • the expressions "cell,” “host cell,” and “cell culture” are used interchangeably and include an individual cell or cell culture that is a recipient of any isolated polynucleotide of the present disclosure or any recombinant vector(s) comprising a nucleotide sequence encoding a HCVR, LCVR or antibody of the present disclosure.
  • a host cell includes ceils genetically modified (e.g., transformed, transduced or infected) with a polynucleotide (e.g., in one or more recombinant vectors) for expression of a PD1-PB of the present disclosure or a light chain or heavy chain thereof.
  • the PDl-PBMs may be expressed in a mammalian expression system, including systems in which the expression constructs are introduced into the mammalian cells using virus.
  • a variety of mammalian cells may be used for such recombinant expression including but not limited to NIH3T3, CHO, COS, HEK293, PER C.6 and HeLa cells.
  • PDl -PBMs may be expressed in bacterial ceils. Bacterial expression systems are well known in the art, and include Escherichia coli (E, coli.
  • the PDl -PBMs may be produced in insect cells (e.g.
  • the PDl-PBMs may be produced by chemical synthesis methods.
  • transgenic expression systems both animal (e.g. cow, sheep or goat milk, embryonated hen's eggs, whole insect larvae, etc.) and plant (e.g. corn, tobacco, duckweed, etc.). The conditions appropriate for expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
  • the PDl -PBMs may be operably linked to a tag sequence joined via a linker sequence.
  • the linker sequence will comprise a small number of amino acids, typically less than ten, and are selected to be conformationally flexible and resistant to degradation such as GGGGS.
  • a tag sequence may be a targeting or signal sequence that directs the recombinantly express PDl-PBMs fusion protein to a desired cellular location or to the extracellular media.
  • the tag may also be a sequence that that facilitates purification and/or screening such as polyhistidme tags (His-tags), GST fusions, MBP fusions and epitope tags such as myc tags, flag-tags, and the like.
  • His-tags polyhistidme tags
  • GST fusions GST fusions
  • MBP fusions MBP fusions
  • epitope tags such as myc tags, flag-tags, and the like.
  • a tag may be covalent!y conjugated to the PD1 -PBM by traditional chemical means
  • the recombinantly expressed PDl-PBMs are purified or isolated after expression using conventional chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed ⁇ pha.se, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC.
  • Purification may also be achieved by methods also include electrophoretic, immunological, dialysis, and filtration techniques, in conjunction with protein concentration, are also useful.
  • the nucleic acid sequences encoding the heavy and light chains are as set out below:
  • the present disclosure provides a pharmaceutical composition comprising an PDl-PBM of the present disclosure and a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical composition comprises a homogeneous or substantially homogeneous population of a PDl-PBM of the present disclosure and a pharmaceutically acceptable carrier or diluent.
  • the present disclosure further provides the use of a PDl-PBM of the present disclosure for use in the preparation of a medicament.
  • a PD l-PBM of the present disclosure may be provided in a pharmaceutical formulation suitable for administration to a subject.
  • a PDl-PBM of the present disclosure may be administered to a subject directly but is more commonly provided in a pharmaceutical formulation comprising one or more pharmaceutically acceptable diluents, carriers, and/or excipients such as dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonic! ty agents, and/or stabilizing agents.
  • Said compositions can be designed in accordance with conventional techniques disclosed in, e.g., Biological Drug Products: Development and Strategies (201.3) Wang and Singh. Eds.
  • the formulation may be provided in a lyophilized powder form.
  • the formulation is typically provided in a kit of parts comprising vial containing the lyophilized formulation of the PDl- PBM and a quantity of reconstitution buffer.
  • the formulation may be provided in a dual chamber pre-filled deliver ⁇ ' device which facilitates reconstitution of the lyophilized PDl- PBM formulation with a buffer solution and delivery by either intravenous, intratumoral, intradermal, intraperitoneal, intracranial, intraocular, subcutaneous, and/or intramuscular routes of parenteral administration.
  • the present disclosure provides a method of treating or preventing a neoplastic disease in a subject by the administration of a therapeutically effective amount of a PDl-PBM.
  • the compositions of the present disclosure are useful in the treatment of mammalian subjects.
  • treat means “treat”, “treating”, treatment” are used interchangeably to refer to the administration of a compound of the present disclosure for treatment of a disease or condition in a subject to a subject suffering from a disease, disorder or condition, or a symptom thereof to temporarily or permanently eliminate, reduce, suppress, mitigate, or ameliorate a cause or symptom of such disease, disorder, or condition but does not necessarily indicate a total elimination of all disorder symptoms.
  • treatment includes the suspension or slowing the progression of the disease, disorder or condition or inhibiting progression thereof to a harmful or otherwise undesired state.
  • neoplastic disease refers to disorders or conditions in a subject arising from cellular hyper-proliferation or unregulated (or dysregulated) cell replication.
  • the term neoplastic disease refers to disorders arising from the presence of neoplasms in the subject. Neoplasms may be classified as: (1) benign (2) pre-malignant (or "pre-cancerous”); and (3) malignant (or "cancerous”). Examples benign neoplasms amenable to treatment using the compositions and methods of the present disclosure include but are not limited to adenomas, fibromas, hemangiomas, and lipomas.
  • pre-malignant neoplasms amenable to treatment using the compositions and methods of the present disclosure include but are not limited to hyperplasia, atypia, metaplasia, and dysplasia.
  • malignant neoplasms amenable to treatment using the compositions and methods of the present disclosure include but are not limited to carcinomas (cancers arising from epithelia tissues such as the skin or tissues that line internal organs), leukemias, lymphomas, and sarcomas typically derived from from bone fat, muscle, blood vessels or connective tissues).
  • neoplastic diseases amenable to treatment with the compositions and methods of the present disclosure include breast cancers; sarcomas (including but not limited to osteosarcomas and angiosarcomas) , and fibrosarcomas), leukemias, lymphomas, genitourinary cancers (including but not limited to ovarian, urethral, bladder, and prostate cancers); gastrointestinal cancers (including but not limited to colon esophageal and stomach cancers); lung cancers; myelomas; pancreatic cancers; liver cancers; kidney cancers; endocrine cancers; skin cancers; and brain or central and peripheral nervous (CNS) system tumors, malignant or benign, including gliomas and neuroblastomas, astrocytomas, myelodysplastic disorders; cervical carcinoma-in-situ; intestinal polyposes; oral leukoplakias; histiocytoses, hyperprofroliferative scars including keloid scars, he
  • hyperkeratoses and papulosquamous eruptions including arthritis are also included.
  • viral induced neoplasms such as warts and EBV induced disease (i.e., infectious mononucleosis), scar formation, hyperproliferative vascular disease including intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion and the like.
  • administration refers to contact of a subject, cell, tissue, organ, or biological fluid with a PD1-PBM, e.g., a pharmaceutical formulation comprising a PD1-PBM.
  • administration includes contact (in vitro or ex vivo) of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell.
  • the compositions of the present disclosure are administered by intravenous infusion.
  • therapeutically effective amount refers to the quantity of a therapeutic or prophylactic agent that when administered to a subject suffering from a disease, disorder of condition that provides a detectable, positive objective effect or subjective positive effect (e.g., a subject's feeling of well-being) on any symptom, aspect, or characteristic of the disease, disorder or condition.
  • a therapeutically effective amount can be ascertained by measuring observable physiological parameters (e.g., body temperature, blood pressure, etc.) or through diagnostic analysis of the subject (e.g. x-ray, ultrasound, CT-scan, analysis of bodily fluids).
  • compositions of the present disclosure are capable to routine determination by the skilled physician based on readily identifiable indicia such as age, weight, sex, general health, blood pressure, additional conditions (e.g. diabetes, metabolic syndrome), disease type, disease progression, tumor burden as well as the therapeutic modality such as whether the compositions are being administered in combination with other the co-administration of other agent which may lead to incompatibilities or cross-reactions.
  • compositions of the present disclosure a therapeutically effective amount in the treatment of a neoplastic disease is a dose of approximately 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg or 50 mg/kg provided daily for a period of 5 days by intravenous infusion.
  • compositions of the present disclosure may be administered in combination with additional anti -neoplastic therapeutic agents or therapies.
  • additional antineoplastic therapeutic agents include immune checkpoint inhibitors, immunomodulatory compounds, chemotherapeutic agents and physical methods.
  • immune checkpoint inhibitors include PD1 antagonists (e.g. anti-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1 antagonists (e.g. anti-PD1 antagonists (e.g. anti-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD1-PD
  • PD1 antibodies PD1 antibodies
  • PDL1 antagonists e.g. anti-PDLl antibodies
  • PDL2 antagonists e.g. anti- PDL2 antibodies
  • CTLA-4 antagonists e.g. anti-CTLA-4 antibodies
  • IDO inhibitors 2B4 (also known as CD244) receptor antagonists
  • B7 family inhibitory !igands such B7-H3 (also known as CD276), B7-H4 (also known as B7-S1, B7x and VCTN1), and LAG3 antagonists (e.g. anti- LAG3 antibodies).
  • Exemplary anti-PDl antibodies useful for use in combination with the compositions of the present disclosure include but are not limited to nivolumab (Bristol-Myers Squibb), and lambrolizumab (Merck)).
  • Exemplary anti -CTLA-4 antibodies useful for use in combination with the compositions of the present disclosure include but are not limited to ipilimumab.
  • compositions of the present disclosure include but are not limited to BMS-936559 (Bristol- Myers Squibb), MPDL3280A (Genentech/Roche) and MEDI4736 (Medlmmune).
  • Exemplary anti-PDL2 antibodies useful for use in combination with the compositions of the present disclosure include but are not limited to AMP -224 (Amplimmune/GlaxoSmithKline)).
  • Examples of a LAG-3 antagonists includes but is not limited to ⁇ 321 (ImmuFact).
  • B7-H3 also known as CD276
  • B7-H4 also known as B7-S1, B7x and VCTN1
  • B7-H3 also known as CD276
  • B7-H4 also known as B7-S1, B7x and VCTN1
  • B7-H3 also known as CD276
  • B7-H4 also known as B7-S1, B7x and VCTN1
  • B7-H3 also known as CD276
  • B7-H4 also known as B7-S1, B7x and VCTN1
  • B7-H3 also known as CD276
  • B7-H4 also known as B7-S1, B7x and VCTN1
  • B7-H3 also known as CD276
  • B7-H4 also known as B7-S1, B7x and VCTN1
  • B7-H3 also known as CD276
  • B7-H4 also known as B7-S1, B7x and VCTN1
  • Exemplary irn.munomodulat.ory agents useful for use in combination with the compositions of the present disclosure include but are not limited to tumor vaccines, IL-10, IL- 12, pegyiated IL-10, TIM3 antagonists (e.g. anti-TIM3 antibodies), gaiectin 9 antagonists, adenosine A2a receptor A2aR (A2a.R) antagonists that block binding by adenosine or by adenosine analogs.
  • TIM3 antagonists e.g. anti-TIM3 antibodies
  • gaiectin 9 antagonists e.g. anti-TIM3 antibodies
  • gaiectin 9 antagonists e.g. anti-TIM3 antibodies
  • gaiectin 9 antagonists e.g. anti-TIM3 antibodies
  • gaiectin 9 antagonists e.g. anti-TIM3 antibodies
  • gaiectin 9 antagonists e.g. anti-TIM3 antibodies
  • IL-10 is an example of an immumodulatory agent particularly useful in combination with the PD1 -PBMs of the present disclosure.
  • the terms "IL-10”, “IL-10 polypeptide(s), “IL-10 molecule(s)”, “IL-10 agent(s)” are used interchangeably herein to refer to an isolated naturally occurring form of an IL-10 molecule, with or without its signal peptide, including analogs, variants, muteins and active fragments thereof.
  • Naturally occurring mammalian forms of IL-10 are well known in the art and include but are not limited to human IL-10 ("hIL-10", NP_000563), murine IL-10 ("mIL-10", NP_034678), rat IL-10 ("rIL-10”, accession NP_036986.2; GI 148747382); bovine IL-10 ("bIL-10", accession NP_776513.1; GI 41386772); equine IL-10 ("eIL-10", accession NP_001009327.1; GI 57164347); canine IL-10 ("cIL-10", accession ABY86619.1; GI 166244598); and rabbit IL-10 ("rbIL-10", accession AAC23839.1; GI 3242896).
  • Naturally occurring IL-10 species are biologically active (interact with and activate the IL-10 receptor) as a homodimer each subunit containing a mature IL-10 polypeptide and a signal peptide.
  • Human IL-10 in its biologically active form exists as a homodimer with a molecular mass of 37kDa, comprising two 18.5kDa monomers (see, e.g., US Patent No. 6,217,857).
  • Each hIL-10 monomer is expressed as a single polypeptide sequence of 178 amino acids, the N-terminal 18 residues comprising a signal peptide which is cleaved during post-translational processing to yield a mature 160 amino acid monomer.
  • Each monomer contains two cysteine residues that form an intra-chain disulfide bond.
  • Two mature IL-10 monomers associate non-covalently to form the fully active IL-10 dimer.
  • the abbreviation may be preceded by an "r” to denote recombinantly produced as in "rhIL-10” referring to a human IL-10 peptide produced by recombinant DNA technology.
  • active fragments refers to polypeptide sequences containing contiguous amino acid residues derived from the mature IL-10 that retain IL-10 activity.
  • the length of contiguous amino acid residues of a peptide or a polypeptide subsequence varies depending on the specific naturally-occurring amino acid sequence from which the subsequence is derived.
  • active fragments can be from about 20 amino acids to about 40 amino acids, from about 40 amino acids to about 60 amino acids, from about 60 amino acids to about 80 amino acids, from about 80 amino acids to about 100 amino acids, from about 100 amino acids to about 120 amino acids, from about 120 amino acids to about 140 amino acids, from about 140 amino acids to about 150 amino acids, from about 150 amino acids to about 155 amino acids, from about 155 amino acids up to the full-length peptide or polypeptides.
  • IL-10 analogs refers to polypeptides comprising an amino acid sequence having at least 75%, at least 80%>, at least 85%>, at least 90%, at least 95%, at least 98%>, or at least 99%, amino acid sequence identity to a contiguous stretch of at least 20 amino acids, 40 amino acids, 60 amino acids, 80 amino acids, 100 amino acids, 120 amino acids, 140 amino acids, 150 amino acids, 155 amino acids, from about 155 amino acids up to the full-length an IL- 10 polypeptide and exhibiting at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the activity of the IL-10 polypeptide Methods of alignment of sequences for comparison are well-known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
  • IL-10 includes IL-10 variants.
  • variant encompasses naturally-occurring variants and non-naturally-occurring variants.
  • Naturally- occurring variants include homologs (polypeptides and nucleic acids that differ in amino acid or nucleotide sequence, respectively, from one species to another), and allelic variants
  • Non-naturally-occurring variants include polypeptides and nucleic acids that comprise a change in amino acid or nucleotide sequence, respectively, where the change in sequence is artificially introduced (e.g., muteins); for example, the change is generated in the laboratory by human intervention ("hand of man”).
  • mutein refers broadly to mutated recombinant proteins that usually carry single or multiple amino acid substitutions and are frequently derived from cloned genes that have been subjected to site-directed or random mutagenesis, or from completely synthetic genes.
  • IL-10 variants include IL-10 molecules comprising conservative amino acid substitutions.
  • conservative amino acid substitution refers to substitutions that preserve the activity of the protein by replacing an amino acid(s) in the protein with an amino acid with a side chain of similar acidity, basicity, charge, polarity, or size of the side chain.
  • Conservative amino acid substitutions generally entail substitution of amino acid residues within the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E.
  • Guidance for substitutions, insertions, or deletions can be based on alignments of amino acid sequences of different variant proteins or proteins from different species.
  • the present disclosure contemplates having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 usually no more than 20, 10, or 5 amino acid substitutions, where the substitution is usually a conservative amino acid substitution.
  • IL-10 variant incorporates one or more linkages other than peptide bonds, e.g., at least two adjacent amino acids are joined via a linkage other than an amide bond.
  • linkages other than peptide bonds e.g., at least two adjacent amino acids are joined via a linkage other than an amide bond.
  • one or more amide bonds within the backbone of IL-10 can be substituted.
  • One or more amide linkages in IL-10 can also be replaced by, for example, a reduced isostere pseudopeptide bond. See Couder et al. (1993) Int. J. Peptide Protein Res. 41 : 181-184.
  • IL-10 variants may comprise one or more non-conservative amino acid substitutions.
  • non-conservative amino acid substitutions include but are not limited to:
  • [00104] a) substitution of alkyl-substituted hydrophobic amino acids, including alanine, leucine, isoleucine, valine, norleucine, (S)-2-aminobutyric acid, (S)-cyclohexylalanine or other simple alpha-amino acids substituted by an aliphatic side chain from CI -CIO carbons including branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions;
  • amino acids containing basic side chains including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, including alkyl, alkenyl, or aryl -substituted (from CI -CIO branched, linear, or cyclic) derivatives of the previous amino acids, whether the substituent is on the heteroatoms such as the alpha nitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon, in the pro-R position.
  • amino acids containing basic side chains including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, including alkyl, alkenyl, or aryl -substituted (from CI -CIO branched, linear, or cyclic) derivatives of the previous amino acids, whether the substituent is on the heteroatoms such as the alpha nitrogen, or the distal nitrogen or nitrogen
  • g) substitution of an amino with a cysteine residue or a cysteine analog can be introduced into an IL-10 polypeptide to provide for linkage to another peptide via a disulfide linkage or to provide for cyclization of the IL-10 polypeptide.
  • Methods of introducing a cysteine or cysteine analog are known in the art; see, e.g., U.S. Patent No. 8,067,532.
  • IL-10 variants may also comprise one or more naturally occurring non-genetically encoded L-amino acids, synthetic L-amino acids, or D-enantiomers of an amino acid.
  • IL-10 can comprise only D-amino acids.
  • an IL-10 polypeptide can comprise one or more of the following residues: hydroxyproline, ⁇ -alanine, o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid, m-aminomethylbenzoic acid, 2,3- diaminopropionic acid, a-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine, citrulline, t-butylalanine, tbutyl glycine, N-methylisoleucine, phenyl glycine, cyclohexylalanine, norleucine, naphthylalanine, pyridylalanine 3-benzothienyl alanine, 4-chlorophenylalanine, 2- fluorophenylalanine, 3fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-
  • homoarginine N-acetyl lysine, 2,4diamino butyric acid, rho-aminophenylalanine, N- methylvaline, homocysteine, homoserine, samino hexanoic acid, co-aminohexanoic acid, co- aminoheptanoic acid, co-aminooctanoic acid, coaminodecanoic acid, co-aminotetradecanoic acid, cyclohexylalanine, ⁇ , ⁇ -diaminobutyric acid, a,pdiaminopropionic acid, ⁇ -amino valeric acid, and 2,3-diaminobutyric acid. Additional modifications
  • IL-10 variant includes a cyclized IL-10 polypeptide.
  • Cyclization may be effected by the introduction of one or more cysteines or cysteine analogs into an IL-10 polypeptide, where the introduced cysteine or cysteine analog can form a disulfide bond with a second introduced cysteine or cysteine analog.
  • Other means of cyclization include introduction of an oxime linker or a lanthionine linker; see, e.g., U.S. Patent No. 8,044,175. Any combination of amino acids (or non-amino acid moieties) that can form a cyclizing bond can be used and/or introduced.
  • a cyclizing bond can be generated with any combination of amino acids (or with an amino acid and -(CH2)n-CO- or -(CH2)n-C6H4-CO-) with functional groups which allow for the introduction of a bridge.
  • Some examples are disulfides, disulfide mimetics such as the -(CH2)n- carba bridge, thioacetal, thioether bridges (cystathionine or lanthionine) and bridges containing esters and ethers.
  • n can be any integer, but is frequently less than ten.
  • Other modifications to facilitate cyclization include, for example, an N-alkyl (or aryl) substitution ( ⁇ ), or backbone crosslinking to construct lactams and other cyclic structures.
  • IL-10 variant includes a retroinverso analog of IL-10 (see, e.g., Sela and Zisman (1997) FASEB J. 11 :449).
  • Retro-inverso peptide analogs are isomers of linear polypeptides in which the direction of the amino acid sequence is reversed (retro) and the chirality, D- or L-, of one or more amino acids therein is inverted (inverso), e.g., using D-amino acids rather than L-amino acids.
  • inverso e.g., using D-amino acids rather than L-amino acids.
  • IL-10 variant also includes an IL-10 molecules comprising a "Protein
  • a PTD is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic molecule that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane.
  • a PTD is covalently linked to the amino or carboxy terminus of an IL-10 polypeptide.
  • Exemplary protein transduction domains include, but are not limited to, a minimal decapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT); a polyarginine sequence comprising a number of arginine residues sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm.
  • a minimal decapeptide protein transduction domain corresponding to residues 47-57 of HIV-1 TAT
  • a polyarginine sequence comprising a number of arginine residues sufficient to direct entry into a cell
  • VP22 domain Zender et al. (2002) Cancer Gene
  • RKKRRQRRR (SEQ ID NO: 15); an arginine homopolymer of from 3 arginine residues to 50 arginine residues;
  • exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: 16); RKKRRQRR (SEQ ID NO: 17); YARAAARQARA (SEQ ID NO: 18); THRLPRRRRRR (SEQ ID NO: 19); and GGRRARRRRRR (SEQ ID NO: 20).
  • the carboxyl group can also be esterified with primary, secondary or tertiary alcohols including but not limited to, methanol, branched or unbranched Cl-C6-alkyl alcohols, e.g., ethyl alcohol or tert-butanol.
  • the carboxyl group can also be amidated with primary or secondary amines such as ammonia, branched or unbranched C1 -C6- alkylamines or C1-C6 di-alkylamines, e.g., methylamine or dimethylamine.
  • primary or secondary amines such as ammonia, branched or unbranched C1 -C6- alkylamines or C1-C6 di-alkylamines, e.g., methylamine or dimethylamine.
  • the term variant includes modifications to the amino group of the amino acid
  • the amino group can be present in a form protected by amino- protecting groups conventionally used in peptide chemistry, such as those provided above (e.g., Fmoc, Benzyloxy-carbonyl (Z), Boc, and Alloc).
  • Alkyl residues can be straight- chained, branched or cyclic (e.g., ethyl, isopropyl and cyclohexyl, respectively).
  • pegylated IL-10 and “PEG-IL-10” refer to IL-10 to which one or more polyethylene glycol (PEG) molecules covalently attached to at least one amino acid residue of IL-10.
  • PEG polyethylene glycol
  • hPEG-IL-10 refers to pegylated human IL-10.
  • the biological activity of PEG-IL-10 is typically measured by assessing the levels of inflammatory cytokines (e.g., T Fa or IFNy) in the serum of subjects challenged with a bacterial antigen (lipopolysaccharide (LPS)) and treated with PEG-IL-10, as described in U.S. Pat. No. 7,052,686.
  • cytokines e.g., T Fa or IFNy
  • LPS lipopolysaccharide
  • the terms "monopegylated IL-10" and “mono-PEG-IL-10” are used interchangeably herein to refer to a PEG-IL10 where one polyethylene glycol molecule is covalently attached to a single amino acid residue of one subunit of the IL-10 dimer.
  • di-PEG-IL-10 dipegylated IL-10
  • di-PEG-IL-10 di-PEG-IL-10
  • PEG-ILIO where at least one polyethylene glycol molecule is attached to a single residue on each subunit of the IL-10 dimer.
  • Strategies for pegylation of polypeptides known in the art may be employed in the preparation of PEG-IL10. Pegylation of various molecules is discussed in, for example, U.S. Pat. Nos. 5,252,714; 5,643,575; 5,919,455; 5,932,462; and 5,985,263. Methods for the preparation of PEG-IL10 species are described in United States Patent Nos. 7,052,686 and 8,691,205
  • the PEG component of a PEG-IL-10 may have an average molecular weight greater than about 5kDa, greater than about lOkDa, greater than about 15kDa, greater than about 20kDa, greater than about 30kDa, greater than about 40kDa, or greater than about 50kDa.
  • the average molecular weight of the PEG is from about 5kDa to about lOkDa, from about 5kDa to about 15kDa, from about 5kDa to about 20kDa, from about lOkDa to about 15kDa, from about lOkDa to about 20kDa, from about lOkDa to about 25kDa or from about lOkDa to about 30kDa.
  • the average molecular weight of the PEG is generally from about 5kDa to about 20kDa. It should be noted that PEG not typically provided as a homogenous preparation of a single PEG species.
  • PEG preparations typically provide PEG molecules having molecular weights over a range as understood by those in the art, a PEG preparation having an average molecular weight of lOkDa is understood by those of skill in the art as comprising multiple PEG species that, on average, have a molecular weight of approximately lOkDa daltons.
  • the PEG can be linear or branched. Branched PEG derivatives, "star-PEGs" and multi -armed PEGs are contemplated by the present disclosure.
  • the PEG molecule may be covalently attached directly to the IL-10 molecule or may be attached via a polypeptide linker.
  • Suitable linkers include “flexible linkers” which are generally of sufficient length to permit some movement between the modified polypeptide sequences and the linked components and molecules.
  • the linker molecules are generally about 6-50 atoms long.
  • the linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof.
  • Suitable linkers can be readily selected and can be of any suitable length, including but not limited to 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50 or more than 50 amino acids.
  • linkers useful in the practice of the present disclosure include glycine polymers (G)n, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers and combinations thereof, where m, n, and o are each independently selected from an integer of at least 1 to 20, e.g., 1-18, 216, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8,9, or 10.
  • PEG can be bound to a polypeptide of the present disclosure via a terminal reactive group (a "spacer") which mediates a bond between the free amino or carboxyl groups of one or more of the polypeptide sequences and polyethylene glycol.
  • the PEG having the spacer which can be bound to the free amino group includes N-hydroxysuccinylimide polyethylene glycol, which can be prepared by activating succinic acid ester of polyethylene glycol with N- hydroxysuccinylimide.
  • Another activated polyethylene glycol which can be bound to a free amino group is 2,4-bis(0-methoxypolyethyleneglycol)-6-chloro-s-triazine, which can be prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride.
  • the activated polyethylene glycol which is bound to the free carboxyl group includes
  • compositions of conjugates wherein the PEGs have different n values and thus the various different PEGs are present in specific ratios.
  • Such compositions can be produced by reaction conditions and purification methods known in the art and provided herein.
  • conjugates having, for example, the desired number of PEGs attached, purified free from unmodified protein sequences and from conjugates having other numbers of PEGs attached.
  • the conjugation reaction can be carried out in solution at a pH of from 5 to 10, at temperature from 4°C to room temperature, for 30 minutes to 20 hours, utilizing a molar ratio of reagent to protein of from 4: 1 to 30: 1.
  • Reaction conditions can be selected to direct the reaction towards producing predominantly a desired degree of substitution.
  • high temperature, neutral to high pH e.g., pH>7
  • longer reaction time tend to increase the number of PEGs attached.
  • Various means known in the art can be used to terminate the reaction.
  • the reaction is terminated by acidifying the reaction mixture and freezing at, e.g., 20°C.
  • chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alky! sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;
  • ethyl enimines and methyl amelamines including altretamine, triethyienemelamine,
  • trietylenephosphoramide, triethy!enethiophosphaoramide and trimethylolomelamime nitrogen mustards such as chlorambucil, chlornaphazine, cholophospharnide, estramustine, ifosfamide, mechlorethanime, mech!orethamine oxide hydrochloride, melphalan, novembichin, phenesteiine, predmmustine, trofosfamide, uracil mustard; mtrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinoniycin, authramycin, azaserine, bleomycins, cactinomycin, caiiclieamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinoniycin, daun
  • elfiptinium acetate etoglucid; gallium nitrate; hydroxyurea; lentinan, lonidamine; niitoguazone, mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide, procarbazine; razoxane; si ofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethyiamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitoiactol; pipobroman; gacytosine; arabinoside (Ara ⁇ C); cyclophosphamide, thiotepa; ta oids, e.g., paclitaxel and doxetaxe
  • methotrexate platinum and platinum coordination complexes such as cisplatin and carbopiatin; vinblastine; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine;
  • vinoreibine naveibine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPTI 1 ; topoisomerase inhibitors; difluorom ethyl ornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluorom ethyl ornithine
  • Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxyiamoxifen, trioxifene, keoxifene, onapri stone, and toremifene; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • combination therapy comprises administration of a hormone or related hormonal agent.
  • compositions of the present disclosure may be administered in combination with anti -neoplastic physical methods such radiotherapy, cryotherapy, hyperthermic therapy, surgery, laser ablation, proton therapy and the like.
  • Kits with unit doses of a subject antibody e.g. in oral or injectable doses, are provided.
  • kits in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the antibody in treating pathological condition of interest. Examples of compounds and unit doses are those described herein above.
  • OmniRat transgenic rats which produce antibodies containing fully human light chains and VH domains, with rat CHI, CH2, and CH3 domains.
  • Conditioned media from the hybridomas was first screened by FACS for binding to human PD-1 recombinantly expressed on the surface of CHO cells. Selected candidates were further screened via FACS for blockade of a PE-labeled PD-Ll-Fc to human PD-1 using the same cell line.
  • the hybridomas from the top 10 candidates were used to express the mAbs and generate purified protein which was further screened by for PD-1 binding in an ELISA format as well as reconfirmation of binding to the human PD-1 CHO cell line.
  • the VH and VL domains of the top 10 candidates were also DNA sequenced.
  • One of the mAbs, M9 exhibited superior binding kinetics to PD-1 compared to the others.
  • AMOOOl is the fully human version of M9 and retains all of the binding and inhibitory characteristics of M
  • PD-l/PD-Ll Blockade Bioassay is a bioluminescent cell-based assay that can be used to measure the potency and stability of antibodies and other biologies designed to block the PD-l/PD-Ll interaction.
  • the assay consists of two genetically engineered cell lines: PD- 1 Effector Cells, which are Jurkat T cells expressing human PD-1 and a luciferase reporter driven by an NFAT response element (NFAT-RE), and; PD-L1 aAPC/CHO-Kl Cells, which are CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate cognate TCRs in an antigen- dependent manner.
  • PD- 1 Effector Cells which are Jurkat T cells expressing human PD-1 and a luciferase reporter driven by an NFAT response element (NFAT-RE)
  • PD-L1 aAPC/CHO-Kl Cells which are CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate cognate TCRs in an antigen- dependent manner.
  • M9 and AMOOOl exhibit an ECso of 0.35 - 0.5 ⁇ g/mL respectively, measured using the Promega immune check point bioassay in substantial accordance with the instructions provided by the manufacturer.
  • This bioassay was prequalified by Promega according to the ICH guidelines and shows the precision, accuracy, and linearity required for routine use in potency and stability studies (PD-1 Technical Manual).
  • the bioassay consists of two genetically engineered cell lines, PD-1 Effector Cells, (Jurkat) and PD-L1 (aAPC/CHO-Kl) antigen presenting cells.
  • AMOOOl binding specificity was tested in an ELISA format by binding to human, cynomolgus monkey, murine, rat, and canine PD-l-Fc fusion proteins, with detection by anti- human IgG:HRP.
  • the results of these studies are provided in Figures 3, 4, and 5 of the attached drawings.
  • AMOOOl binds to both human and cynomologus monkey ( Figure 3) but not murine ( Figure 3), rat ( Figure 4), or canine ( Figure 5) PD-1.
  • the coated plate was then probed with AMOOOl or nivolumab (lOmg/ml, serially diluted 1 :2 in PBS down 16 wells to a final volume of lOOul/well) for one hour at room temperature with rocking.
  • AMOOOl or nivolumab (lOmg/ml, serially diluted 1 :2 in PBS down 16 wells to a final volume of lOOul/well) for one hour at room temperature with rocking.
  • Binding to the glycosylated and deglycosyalted PD1-FC was detected using a goat anti-human IgG:HRP (Abeam, 1 : 10,000 in PBS, lOOul/well, 1 hour at room termperature, with rocking), and developed with 1-Step Ultra TMB-ELISA solution (Therm oFisher, Catalog number: 34028), lOOul/well for approximately 3 minutes at room temperature. The reaction was quenched by the addition lOOul/well of Stop Solution (Life Technologies). The plates were read on a Molecular Devices SpectraMax 340PC plate reader (A450nm, Automix on). The results are presented in Figure 7 of the attached drawings.
  • pre-incubation of AMOOOl with PD-1 prior to exposure to PD-L1 or PD-L2 bound to the sensor blocks the interaction with PD-L1 or PD-L2, respectively.
  • Example 8 AMOOOl Blockade of endogenous PD-1 function
  • AMOOOl 's capacity to inhibit endogenous PD-1 function was assessed in the context of normal peripheral blood mononuclear cells response to staphylococcal enterotoxin B (SEB).
  • SEB staphylococcal enterotoxin B
  • a modified assay using whole blood instead of peripheral blood mononuclear cells (PBMC) was used. Briefly, whole blood was diluted 10-fold with complete Roswell Park Memorial Institute (RPMI) medium, and exposed to SEB with or without AMOOOl . After 3 to 4 days incubation, the cells were pelleted by centrifugation and the supernatant was measured by ELISA for IL-2.
  • RPMI Roswell Park Memorial Institute

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des molécules de liaison à un polypeptide qui se lient à la protéine de mort cellulaire programmée 1 (PD-1) et inhibent l'interaction de PD-1 avec à la fois le ligand 1 de mort cellulaire programmée (PD-L1) et/ou le ligand 2 de mort cellulaire programmée (PD-L2). La présente invention concerne en outre des formulations pharmaceutiques comprenant les molécules de la présente invention. La présente invention concerne en outre des procédés d'utilisation de ces molécules de liaison à un polypeptide dans le traitement de divers troubles et maladies. La présente invention concerne en outre des séquences d'acide nucléique de recombinaison codant pour de telles molécules de liaison à un polypeptide et des cellules transformées par celles-ci. La présente invention concerne également des procédés d'utilisation des molécules de la présente invention dans le diagnostic, la prévention et le traitement de divers troubles ou états chez des sujets comprenant, mais s'y limiter, le traitement de troubles néoplasiques comprenant, mais sans s'y limiter, le cancer.
PCT/US2018/058824 2017-11-10 2018-11-02 Molécules de liaison à un polypeptide pd1 WO2019094265A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762584562P 2017-11-10 2017-11-10
US62/584,562 2017-11-10

Publications (1)

Publication Number Publication Date
WO2019094265A1 true WO2019094265A1 (fr) 2019-05-16

Family

ID=66437987

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/058824 WO2019094265A1 (fr) 2017-11-10 2018-11-02 Molécules de liaison à un polypeptide pd1

Country Status (1)

Country Link
WO (1) WO2019094265A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8735553B1 (en) * 2013-09-13 2014-05-27 Beigene, Ltd. Anti-PD1 antibodies and their use as therapeutics and diagnostics
US20160051672A1 (en) * 2014-06-17 2016-02-25 Medimmune Limited Methods of treatment with antagonists against pd-1 and pd-l1 in combination with radiation therapy
US20160159905A1 (en) * 2014-12-09 2016-06-09 Rinat Neuroscience Corp. Anti-pd-1 antibodies and methods of use thereof
US9493565B2 (en) * 2009-11-24 2016-11-15 Medimmune Limited Targeted binding agents against B7-H1
WO2017020858A1 (fr) * 2015-08-06 2017-02-09 Wuxi Biologics (Shanghai) Co. Ltd. Nouveaux anticorps anti-pd-l1
WO2017055443A1 (fr) * 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anticorps anti-pd1 et méthodes d'utilisation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9493565B2 (en) * 2009-11-24 2016-11-15 Medimmune Limited Targeted binding agents against B7-H1
US8735553B1 (en) * 2013-09-13 2014-05-27 Beigene, Ltd. Anti-PD1 antibodies and their use as therapeutics and diagnostics
US20160051672A1 (en) * 2014-06-17 2016-02-25 Medimmune Limited Methods of treatment with antagonists against pd-1 and pd-l1 in combination with radiation therapy
US20160159905A1 (en) * 2014-12-09 2016-06-09 Rinat Neuroscience Corp. Anti-pd-1 antibodies and methods of use thereof
WO2017020858A1 (fr) * 2015-08-06 2017-02-09 Wuxi Biologics (Shanghai) Co. Ltd. Nouveaux anticorps anti-pd-l1
WO2017055443A1 (fr) * 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anticorps anti-pd1 et méthodes d'utilisation

Similar Documents

Publication Publication Date Title
RU2754058C2 (ru) Антитело к cd73 человека
EP3176181B1 (fr) Anticorps monoclonal anti-ctla4 ou fragment de celui-ci se liant à l'antigène, composition médicinale et son utilisation
RU2627185C1 (ru) Новые связывающие молекулы с противоопухолевой активностью
US20190382497A1 (en) Antibodies Directed Against CD127
WO2016142768A1 (fr) Anticorpd anti-ror2
US20240108690A1 (en) Compositions and methods of use of interleukin-10 in combination with immune check-point pathway inhibitors
KR20240027854A (ko) Tgf-β-수용체 엑토도메인 융합 분자 및 그의 용도
US20240209101A1 (en) Compositions and methods related to il27 receptor binding
KR20220127867A (ko) 편향된 il2 뮤테인 방법 및 조성물
JP2017505614A (ja) Ｐ９７融合タンパク質
JP2005504005A (ja) ＥｒｂＢ界面ペプチド擬態およびその使用方法
CN113474362A (zh) 对cd44特异性的抗体
ES2911663T3 (es) Anticuerpos humanos anti-IFN-alfa
KR20230065259A (ko) Il10 수용체 결합 분자 및 사용 방법
TR201906781T4 (tr) Anti-transglutaminaz 2 antikorları.
CN114901310A (zh) 使用il-2受体激动剂和免疫检查点抑制剂的组合疗法
JP7097465B2 (ja) キメラ抗原受容体細胞療法と併用するｉｌ－１０剤の組成およびその使用方法
KR20220127866A (ko) Il2 뮤테인
CN112930195A (zh) 抗人Fn14抗体
WO2019094265A1 (fr) Molécules de liaison à un polypeptide pd1
KR20230158058A (ko) 시알산-결합 ig-유사 렉틴 15에 특이적인 항체 및 이의 용도
JP2020508636A (ja) ＩＦＮ−γ誘導性制御性Ｔ細胞転換性抗癌（ＩＲＴＣＡ）抗体およびその使用
KR20230060527A (ko) Pd-1 폴리펩티드 변이체
WO2019244934A1 (fr) Nouvel anticorps anti-pad2
KR20130057959A (ko) 항 ｈｅｒ2 단일클론항체와 ｉｌ-2를 포함하는 융합 단일클론 항체 및 이를 포함하는 암치료용 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18875366

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18875366

Country of ref document: EP

Kind code of ref document: A1