RU2812915C2 - Antibodies to programmed cell death protein 1 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following are presented: antibodies and their antigen-binding fragments that bind to PD-1, a transgenic rodent producing an antibody or an antigen-binding fragment, a nucleic acid encoding an antibody or an antigen-binding fragment, a cell for producing an antibody or an antigen-binding fragment. Also the following is disclosed: a pharmaceutical composition for inducing, stimulating, or enhancing an immune response, a method for inducing, stimulating, or enhancing an immune response in a patient, a method of treating cancer in a patient, a method of reducing tumor burden in a patient, and a method of treating an infection in a patient.

EFFECT: invention is aimed at producing new antibodies that immunospecifically bind to PD-1 and induce an immune response that activates the proliferation or activity of immune cells.

27 cl, 5 tbl, 14 dwg, 8 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit and priority to U.S. Provisional Applications No. 62/555156, filed September 7, 2017, No. 62/624843, filed February 1, 2018, and No. 62/657323, filed April 13, 2018, all of which are incorporated by links in full.

LINK TO LIST OF SEQUENCES

The sequence listing, filed on September 6, 2018, as a text file entitled "064466.071 sequence listing_ST25.txt", created on August 20, 2018, and having a size of 55.2 kilobytes, is incorporated herein by reference in accordance with § 1.52(e). (5) 37 CFR.

TECHNICAL FIELD

This invention generally relates to immunomodulation and antibodies that specifically bind to PD-1, and methods of using them.

BACKGROUND OF THE ART

The programmed cell death receptor protein 1 (PD-1)/programmed cell death receptor protein ligand 1 (PD-L1) pathway has demonstrated promising clinical efficacy as a target in cancer immunotherapy. Current antibodies that specifically target either PD-1 or PD-L1 can block this interaction and stimulate an immune response against cancer cells. Successful clinical trials using anti-PD-1 monoclonal antibodies and other immune checkpoint inhibitors have opened new perspectives in cancer immunology. At the same time, the failure of a large subgroup of cancer patients to respond to new immunotherapies has led to increased exploration of combination therapies and predictive biomarkers (Iwai, Y., et al., Journal of Biomedical Science, 24:26 (2017)).

It is therefore an object of this invention to provide compositions and methods for modulating signal transduction involving PD-1.

Another object of the present invention is to provide antibodies and antigen-binding fragments thereof that specifically bind to PD-1 and modulate signal transduction involving PD-1.

Another object of the present invention is to provide compositions and methods for treating cancer.

Another object of the present invention is to provide compositions and methods for treating infections.

BRIEF DESCRIPTION OF THE INVENTION

Provided are antibodies and antigen-binding fragments thereof that specifically bind to PD-1, preferably human or murine PD-1, and induce or stimulate an immune response that activates the proliferation or activity of immune cells. In one embodiment, the disclosed antibodies and antigen binding fragments thereof specifically bind to PD-1 expressed on immune cells. Binding of the disclosed antibodies and antigen-binding fragments thereof to PD-1 on immune cells causes activation of a signal to be transmitted to the immune cell, for example, a signal that enhances or stimulates the production of cytokines and/or activation of immune cell proliferation. Immune cells that express PD-1 include, but are not limited to, B and T cells, as well as cells of myeloid lineage (Riley, J., Immunol Rev. 229(1):114-125 (2009)). In one embodiment, the immune cell is a T cell, preferably a CD8+ T cell.

In another embodiment, a method of stimulating, activating, or enhancing an adaptive immune response in a subject in need thereof is provided by administering to the subject an effective amount of disclosed anti-PD-1 antibodies or antigen-binding fragments thereof for the purpose of inducing, enhancing, or stimulating an adaptive immune response in the subject.

In one embodiment, an antibody or antigen binding fragment thereof is provided having heavy chain complementarity determining regions (CDRs) having amino acid sequences according to SEQ ID NOs: 6, 7 and 8, and light chain CDRs having amino acid sequences according to SEQ ID NO:12, 13 and 14, wherein the antibody or antigen-binding fragment thereof binds PD-1.

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to to SEQ ID NO:4 or 5.

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to to SEQ ID NO:10 or 11.

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to to SEQ ID NO:4 or 5, and a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:10 or 11.

In one embodiment, a transgenic animal is provided that is engineered to express any of the disclosed antibodies or antigen binding fragments thereof. In one embodiment, the animal is a mouse.

In one embodiment, a nucleic acid encoding a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID is provided NO:4 or 5.

In one embodiment, a nucleic acid encoding a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID is provided NO:10 or 11.

In one embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain CDR having the amino acid sequences of SEQ ID NOs: 18, 19 and 20, and a light chain CDR having the amino acid sequences of SEQ ID NO: 24, 13 and 25.

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to to SEQ ID NO:16 or 17.

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to to SEQ ID NO:22 or 23.

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to to SEQ ID NO:16 or 17, and a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:22 or 23.

In one embodiment, a nucleic acid encoding a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID is provided NO:16 or 17.

In one embodiment, a nucleic acid encoding a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID is provided NO:22 or 23.

In one embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain CDR having the amino acid sequences of SEQ ID NOs: 29, 30 and 31, and a light chain CDR having the amino acid sequences of SEQ ID NO: 35, 36 and 37.

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to to SEQ ID NO:27 or 28.

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to to SEQ ID NO:33 or 34.

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to to SEQ ID NO:27 or 28, and a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:33 or 34.

In one embodiment, a nucleic acid encoding a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID is provided NO:27 or 28.

In one embodiment, a nucleic acid encoding a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID is provided NO:33 or 34.

In one embodiment, an antibody or antigen binding fragment thereof is provided comprising three light chain CDRs with amino acid sequences selected from the group consisting of SEQ ID NO: 12, 13, 14, 24, 25, 35, 36, or 37.

In another embodiment, an antibody or antigen binding fragment thereof is provided comprising three heavy chain CDRs with amino acid sequences selected from the group consisting of SEQ ID NO: 6, 7, 8, 18, 19, 20, 29, 30, or 31.

In another embodiment, an antibody or antigen binding fragment thereof is provided comprising three light chain CDRs with amino acid sequences selected from the group consisting of SEQ ID NO: 12, 13, 14, 24, 25, 35, 36 or 37, and three CDRs heavy chain with amino acid sequences selected from the group consisting of SEQ ID NO: 6, 7, 8, 18, 19, 20, 29, 30 or 31.

In one embodiment, an antibody or epitope-binding fragment or fusion protein thereof is provided that immunospecifically binds to SEQ ID NO:38. In one embodiment, the antibody binds to SEQ ID NO:38 on PD-1. In one embodiment, the antibody binds to PD-1 expressed on the surface of an immune cell and induces or stimulates a signal involving PD-1 that activates or stimulates the immune cell. In one embodiment, the immune cell that is activated or stimulated is a T cell, such as a CD8+ T cell.

In some embodiments, the antibody or antigen binding fragment thereof is human, murine, chimeric, humanized, monoclonal, bispecific, trispecific, or multispecific.

In one embodiment, a pharmaceutical composition is provided containing one or more of the disclosed antibodies or antigen binding fragments thereof. In some embodiments, the pharmaceutical composition comprises a second therapeutic agent and/or a pharmaceutically acceptable excipient. An exemplary second therapeutic agent includes cyclophosphamide.

In one embodiment, a method is provided for inducing, activating, or enhancing an immune response in a subject in need thereof by administering to the subject an effective amount of one or more disclosed antibodies or antigen-binding fragments thereof for the purpose of inducing, stimulating, or enhancing an immune response in the subject.

In one embodiment, a method of treating cancer in a subject in need thereof is provided by administering to the subject an effective amount of one or more disclosed antibodies or antigen binding fragments thereof for the purpose of treating cancer in the subject.

In one embodiment, a method is provided for reducing a tumor burden in a subject in need thereof by administering to the subject an effective amount of one or more disclosed antibodies or antigen binding fragments thereof to reduce the tumor burden in the subject.

In one embodiment, a method is provided for treating an infection in a subject in need thereof by administering to the subject an effective amount of one or more disclosed antibodies or antigen binding fragments thereof for the purpose of treating an infection in the subject.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

In FIG. 1 is a graph showing the kinetics of interaction as a function of time between monoclonal antibody 4G9 and human PD-1. The graph shows human PD-1 concentration curves at 0, 125, 250, 500, 500, and 1000 nM.

In FIG. 2 is a graph showing the kinetics of interaction as a function of time between monoclonal antibody 4G9 and mouse PD-1. The graph shows mouse PD-1 concentration curves at 0, 62.5, 125, 500, 500, and 1000 nM.

In FIG. 3 is a graph showing the kinetics of interaction as a function of time between monoclonal antibody 4C12 and human PD-1. The graph shows human PD-1 concentration curves at 0, 125, 250, 500, 500, and 1000 nM.

In FIG. 4 is a graph showing the kinetics of interaction as a function of time between monoclonal antibody 5C2 and human PD-1. The graph shows mouse PD-1 concentration curves at 0 and 1000 nM.

In FIG. 5 is a graph showing the kinetics of interaction as a function of time between monoclonal antibody 5C2 and mouse PD-1. The graph shows human PD-1 concentration curves at 0, 62.5, 125, 250, 500, 500, and 1000 nM.

In FIG. 6A is a flow cytometry histogram of EL4 cells stained with isotype control antibody or commercial anti-PD-1 antibody J43. In FIG. 6B shows a flow cytometry histogram of EL4 cells stained with secondary antibody alone or with antibodies 4G9, 5C2, and 4C12. In FIG. 6C shows a flow cytometry histogram of El4 cells stained with secondary antibody alone or with 4G9 antibody. In FIG. 6D shows a flow cytometry histogram of El4 cells stained with secondary antibody alone or with 5C2 antibody. In FIG. 6E is a flow cytometry histogram of El4 cells stained with secondary antibody alone or with 4C12 antibody. In FIG. 6F is a bar graph showing the binding of various purified anti-mouse PD-1 antibodies to EL4 cells.

In FIG. 7A and 7B are bar graphs demonstrating the concentration of IFNγ (FIG. 7A) or IL-2 in the supernatant from CD4 T cells treated with various antibodies. The X-axis represents treatment group and the Y-axis represents concentration (ng/mL). In FIG. 7C is a bar graph showing the concentration of IFNγ in the supernatant from human CD4 T cells treated with 4G9 or 5C2 antibodies. The X-axis represents treatment group and the Y-axis represents concentration (ng/mL).

In FIG. 8 is a bar graph showing the levels of intracellular pAKT staining in mouse CD4 T cells treated with various antibodies. The X axis represents the processing group, and the Y axis represents the MFI AKT (S473).

In FIG. 9 is a Western blot showing the heavy chain and light chain of IgG in various antibodies.

In FIG. 10 is a bar graph showing the binding of antibodies 4G9 and 5C2 to human PD-1-Fc. The X-axis represents the antibody concentration and the Y-axis represents the OD450.

In FIG. 11A and 11B depict flow cytometry histograms demonstrating binding of anti-PD-1 antibodies 4G9 and 5C2 in CD4 T cells from PD-1 KO mice (Fig. 11A) or PD-1 WT mice (Fig. 11B).

In FIG. 12 is a line graph demonstrating the expression of pS6 in CD4 T cells treated with 4G9, 5C2, commercial AT-1, commercial AT-2, or left untreated. The X-axis represents the total protein concentration (μg/mL) and the Y-axis represents the OD450.

In FIG. 13A is a schematic illustration showing the experimental design of the TC-1 tumor experiments. In FIG. 13B is a line graph showing the average tumor volume (cm3) over time (day) for TC-1 tumor bearing mice treated with E7 Vax, 4G9, RMP 1-14, E7 Vax+RMP 1-14, E7 Vax+ 4G9 or left without processing. The X axis represents time (days) and the Y axis represents the average tumor volume (cm ³ ). In FIG. 13C is a line graph showing percentage survival over time for mice bearing the TC-1 tumor treated with E7 Vax, 4G9, RMP 1-14, E7 Vax+RMP 1-14, E7 Vax+4G9, or left untreated. In FIG. 13D is a line graph showing the average tumor volume (cm3) over time (day) for TC-1 tumor bearing mice treated with E7 Vax, 4C12, 5C2, RMP 1-14, E7 Vax+RMP 1-14, E7 Vax+4C12, E7 Vax+5C2 or left without processing.

In FIG. 14A is a schematic illustration of the experimental design of the TC-1 tumor experiments. In FIG. 14B is a line graph showing the average tumor volume (cm3) over time (day) for TC-1 tumor bearing mice treated with E7 Vax, 4G9, RMP 1-14, J43, E7 Vax+4G9, E7 Vax+RMP 1-14, E7 Vax+J43 or left without processing. The X-axis represents time (days) and the Y-axis represents the average tumor volume (cm3). In FIG. 14C is a line graph showing percentage survival over time for TC-1 tumor bearing mice treated with E7 Vax, 4G9, 4C12, 5C2, RMP 1-14, J43, E7 Vax+4G9, E7 Vax+4C12, E7 Vax +5C2, E7 Vax+RMP 1-14, E7 Vax+J43 or left without processing. The X-axis represents time (days) and the Y-axis represents the percentage of survival.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

A molecule as used herein is considered to be capable of “immunospecifically binding” to a second molecule if such binding demonstrates the specificity and affinity of the antibody for its cognate antigen. Antibodies are said to be capable of “immunospecific binding” to a target region or conformation (“epitope”) of an antigen if such binding involves the antigen recognition site of an immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind to other antigens with lower affinity if the other antigen has some sequence similarity or conformational similarity that is recognized by the antigen recognition site, as determined, for example, by immunoassays, BIACORE® assays or other assays known in the art, however, will not bind to a completely unrelated antigen. Preferably, however, the antibodies (and their antigen-binding fragments) do not cross-react with other antigens. Antibodies can also bind to other molecules in a manner that is not immunospecific, such as Fc receptors, by binding domains in other regions/domains of the molecule that do not include the antigen recognition site, such as the Fc region.

A molecule as used herein is considered to “specifically bind” to a second molecule if such binding demonstrates the specificity and affinity of the receptor for its cognate antigen. A molecule may be capable of physiologically binding to more than one other molecule.

The term "antibody" as used herein is intended to mean an immunoglobulin molecule that has a "variable region" antigen recognition site. The term "variable region" is intended to delineate such an immunoglobulin domain from domains that are commonly shared by antibodies (such as the Fc domain of antibodies). The variable region contains a "hypervariable region", the residues of which are responsible for binding to the antigen. The hypervariable region contains amino acid residues from the “complementarity determining region” or “CDR” (i.e., typically residues at positions about 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain light chain and residues at positions approximately 27-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabatet al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and/or such residues from the “hypervariable loop” (i.e., residues at positions 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in heavy chain variable domain; Chothia and Lesk, 1987J. Mol. Biol.196:901-917). “Framework region” residues or “FR” residues are those variable domain residues that differ from the hypervariable region residues as defined herein. The term antibody includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camellized antibodies (see, for example, Muyldermanset al., 2001,Trends Biochem. Sci. 26:230; Nuttallet al., 2000,Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999J. Immunol. Meth. 231:25; international publication nos. WO 94/04678 and WO 94/25591; US Pat. No. 6,005,079), single chain Fv (scFv) (see e.g. Pluckthun inThe Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994)), single-chain disulfide-linked Fv (sdFv) antibodies, intrabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id and anti-anti-Id antibodies disclosed herein ). In particular, such antibodies include immunoglobulin molecules of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass.

As used herein, the term "antigen binding fragment" of an antibody refers to one or more portions of an antibody that contain complementarity determining regions ("CDRs"), and optionally framework residues, that include a "variable region" antigen recognition site, and exhibit the ability to bind immunospecifically with antigen. Such fragments include Fab', F(ab') ₂ , Fv, single chain fragment (ScFv) and mutant forms thereof, naturally occurring variants and fusion proteins comprising an antibody "variable region" antigen recognition site and a heterologous protein (eg, a toxin, antigen recognition site for another antigen, enzyme, receptor or receptor ligand, etc.).

As used herein, the term “fragment” refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

The term "binding molecule" as used herein is intended to refer to molecules that specifically interact and bind to a specific target. The target may be a biological or small (chemical) molecule. The target target may define an antigen or an antigenic fragment. Examples of binding molecules include, but are not limited to, antibodies (including monoclonal antibodies, bispecific antibodies, as well as antibody fragments), fusion proteins, and other antigen-binding molecules known to those skilled in the art.

As used herein, the term “modulate” refers to the ability to change an effect, outcome, or activity (eg, signal transmission). Such modulation may be agonistic or antagonistic. Antagonistic modulation may be partial (ie, facilitating but not eliminating) or it may completely eliminate such activity (ie, neutralizing). Modulation may involve internalization of the receptor following antibody binding or reduction of receptor expression on the target cell. Agonistic modulation may enhance or otherwise enhance or enhance activity (eg, signal transduction). In yet another further embodiment, such modulation may change the nature of the interaction between the ligand and its cognate receptor so as to measure the nature of the evoked signal transduction. For example, molecules may, by binding to a ligand or receptor, alter the ability of such molecules to bind to other ligands or receptors and thereby alter their overall activity. Preferably, such modulation will provide at least a 10% change in measured immune system activity, more preferably at least a 50% change in such activity, or at least a 2-fold, 5-fold, 10-fold, or even more preferably at least a 100-fold change in such activity.

The term “substantially” when used in the context of binding or exhibiting an effect is intended to mean that the observed effect is physiologically or therapeutically significant. Thus, for example, a molecule is capable of significantly blocking the activity of a ligand or receptor if the degree of blockade is physiologically or therapeutically significant (e.g., if such degree is greater than 60% full value, greater than 70% full value, greater than 75% full value, greater than 80% full value value, more than 85% of the full value, more than 90% of the full value, more than 95% of the full value or more than 97% of the full value). Similarly, a molecule is considered to have substantially the same immunospecificity and/or characteristic as another molecule if such immunospecificity and characteristic is more than 60% identical, more than 70% identical, more than 75% identical, more than 80 % identical, more than 85% identical, more than 90% identical, more than 95% identical, or more than 97% identical.

As used herein, “costimulatory” signals include positive costimulatory signals (eg, signals that lead to increased activity) and negative costimulatory signals (eg, signals that lead to inhibition of activity).

As used herein, the term “derivative” refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to the same target of the parent or reference antibody, but which differs in amino acid sequence from the parent or reference antibody or antigen-binding fragment thereof by including one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications with respect to the parent or reference antibody or antigen binding fragment thereof. Preferably, such derivatives will have substantially the same immunospecificity and/or characteristics, or the same immunospecificity and characteristics, as the parent or reference antibody or antigen binding fragment thereof. Amino acid substitutions or additions of such derivatives may include naturally occurring (ie, DNA-encoded) or non-naturally occurring amino acid residues. The term "derivative" includes, for example, chimeric or humanized variants, as well as variants having altered CH1 regions, hinge regions, CH2, CH3 or CH4 regions, to produce, for example, antibodies, etc. having different Fc regions that exhibit enhanced or reduced effector or binding characteristics.

As used herein, the term “chimeric antibody” is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules, for example, antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region.

As used herein, the term “humanized antibody” refers to an immunoglobulin comprising a human immunoglobulin framework region and one or more non-human (typically murine or rat) immunoglobulin CDRs. The non-human immunoglobulin representing the CDR is called "donor", and the human immunoglobulin representing the framework is called "acceptor". Constant regions may not be present, however, if present, they should be substantially identical to the constant regions of human immunoglobulins, ie, at least about 85-99%, preferably about 95% or more identical. Thus, all portions of the humanized immunoglobulin, with the possible exception of the CDR, are substantially identical to the corresponding portions of naturally occurring human immunoglobulin sequences. A humanized antibody is an antibody comprising a humanized immunoglobulin light chain and a humanized immunoglobulin heavy chain. For example, a humanized antibody will not include a typical chimeric antibody because, for example, the entire variable region of the chimeric antibody is non-human.

As used herein, the term “endogenous concentration” refers to the level at which a molecule is expressed in its native state (eg, in the absence of expression vectors or recombinant promoters) by a cell (where the cell may be a normal cell, a cancer cell, or an infected cell).

As used herein, the terms “treat,” “therapeutic,” “cure,” and “therapeutic use” refer to the elimination, amelioration, or normalization of one or more acute symptoms of a disease or disorder by an anti-PD-1 antibody or an antigenic fragment thereof.

As used herein, a “therapeutically effective amount” refers to an amount of a therapeutic agent that is effective to mediate clinically significant elimination, amelioration, or normalization of such symptoms. An effect is clinically significant if its magnitude is sufficient to influence the health or prognosis of the recipient subject. A therapeutically effective amount may refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of a disease, such as delaying or minimizing the spread of cancer. A therapeutically effective amount may also refer to an amount of a therapeutic agent that provides a therapeutic effect in treating or controlling a disease.

As used herein, the term “prophylactic agent” refers to an agent that can be used to prevent a disorder or disease before any symptoms of such disorder or disease have become apparent. A "prophylactically effective" amount is an amount of a prophylactic agent sufficient to mediate such protection. A prophylactically effective amount may also refer to an amount of a prophylactic agent that provides a prophylactic effect in preventing a disease.

As used herein, the term “cancer” refers to a neoplasm or tumor formed as a result of abnormal, uncontrolled cell growth. Cancer as used herein explicitly includes leukemias and lymphomas. The term "cancer" refers to a disease involving cells that have the potential to metastasize to distant sites and exhibit phenotypic features that differ from those of non-cancerous cells, for example, the formation of colonies in a three-dimensional substrate such as soft agar, or the formation of tubular networks or web-like matrices in a three-dimensional basement membrane or extracellular matrix preparation. Non-cancerous cells do not form colonies in soft agar and form distinct spherical structures in the three-dimensional basement membrane or extracellular matrix preparations.

As used herein, “immune cell” refers to any cell of hematopoietic origin, including, but not limited to, T cells, B cells, monocytes, dendritic cells and macrophages.

As used herein, “valency” refers to the number of binding sites available per molecule.

As used herein, the term "immunological" or "immune" response is the development of a beneficial humoral (antibody-mediated) and/or cellular (mediated by antigen-specific T cells or their secretion products) response directed against the peptide in the recipient patient. Such a response may be an active response induced by administration of an immunogen or a passive response induced by administration of an antibody or primed T cells. The cellular immune response is caused by the presentation of polypeptide epitopes in association with MHC class I or II molecules to activate antigen-specific CD4+ T helper cells and/or CD8+ cytotoxic T cells. The response may also include activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils, or other components of the innate immune system. The presence of a cell-mediated immunological response can be determined using proliferation assays (CD4+ T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contribution of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be characterized by separately isolating antibodies and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

As used herein, an “immunogenic agent” or “immunogen” is capable of inducing an immunological response against itself when administered to a mammal, optionally in combination with an excipient.

As used herein, the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein and refer to mammals, including, but not limited to, humans, rodents such as mice and rats, and others. laboratory animals.

As used herein, the term “polypeptide” refers to an amino acid chain of any length, regardless of modification (eg, phosphorylation or glycosylation). The term polypeptide includes proteins and their fragments. Polypeptides may be "exogenous", meaning that they are "heterologous", ie, foreign to the host cell to be used, such as a human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein as sequences of amino acid residues. These sequences are written from left to right in the direction from the amino to the carboxy terminus. According to standard nomenclature, amino acid residue sequences are designated using either a three-letter or a single-letter code, specified as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D ), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L ), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W) , tyrosine (Tyr, Y) and valine (Val, V).

As used herein, the term “variant” refers to a polypeptide or polynucleotide that differs from the reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference, polypeptide. Typically, the differences are limited such that the sequences of the reference polypeptide and the variant are generally very similar and identical in many areas. The variant and the reference polypeptide may differ in amino acid sequence due to one or more modifications (eg, substitutions, additions and/or deletions). The substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A polypeptide variant may occur naturally, such as an allelic variant, or may be a variant that is not known to occur in nature.

Modifications and changes can be made to the structure of the polypeptides of this disclosure and it is still possible to obtain a molecule having similar characteristics as the polypeptide (eg, a conservative amino acid substitution). For example, certain amino acids can be replaced by other amino acids in the sequence without noticeable loss of activity. Since it is the interactive ability and nature of the polypeptide that determines the biological functional activity of that polypeptide, certain amino acid substitutions can be made in the polypeptide sequence and still produce a polypeptide with similar properties.

When making such substitutions, the hydropathic index of amino acids can be taken into account. The importance of the amino acid hydropathic index in mediating the biological function of a polypeptide is generally understood in the art. It is known that certain amino acids can be replaced by other amino acids having a similar index or hydropathicity score and still result in the formation of a polypeptide with similar biological activity. Each amino acid was assigned a hydropathic index based on its hydrophobicity and charging characteristics. These indices are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The relative hydropathic nature of an amino acid is thought to determine the secondary structure of the resulting peptide, which in turn determines interactions with other molecules such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be replaced by another amino acid having a similar hydropathic index and still produce a functionally equivalent polypeptide. With such changes, substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more preferred.

Substitution of such amino acids can also be made on the basis of hydrophilicity, in particular if the polypeptide or peptide with equivalent biological function obtained in this manner is intended to be used in immunological embodiments. The following hydrophilicity values were assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparigine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is believed that an amino acid can be replaced by another having a similar hydrophilicity value and a biologically equivalent, and in particular an immunologically equivalent polypeptide can still be obtained. For such changes, substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more preferred.

As stated above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account the above characteristics are well known to those skilled in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), ( Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe) and (Val: Ile, Leu). Thus, embodiments of this disclosure provide biological equivalents of the polypeptide set forth above. In particular, embodiments of polypeptides may include variants that are about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of the polypeptide of interest .

The term "percentage (%) sequence identity" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to nucleotides or amino acids in a reference nucleic acid sequence, after sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining percent sequence identity can be achieved in a variety of ways that are within the skill of the person skilled in the art, for example, using publicly available software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign software ( DNASTAR). Suitable parameters for measuring alignment, including any alignments necessary to achieve maximum alignment relative to the overall size of the sequences to be compared, can be determined using known methods.

For the purposes of this document, the % sequence identity of a specific nucleotide or amino acid sequence C to, with, or against a specific nucleotide sequence D (which may alternatively be paraphrased as a specific sequence C that has or contains a specific % sequence identity to, with, or against a specific sequence D) is calculated as follows:

100 multiplied by the fraction W/Z,

where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program when aligning C and D in that program, and where Z is the total number of nucleotides or amino acids in D. It will be understood that if the length sequences C is not equal to the length of sequence D, the % identity of sequences C with respect to D will not be equal to the % identity of sequences D with respect to C.

As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers such as phosphate-buffered saline, water and emulsions such as oil/water or water/oil emulsion, and various types of wetting agents.

As used herein, the terms “antigenic determinant” and “epitope” are used interchangeably and refer to the structure recognized by the antibody.

As used herein, a “conformational epitope” is an epitope that contains contiguous stretches of the amino acid sequence of an antigen. Antibodies bind a conformational epitope based on the three-dimensional surface properties, shape, or tertiary structure of the antigen.

As used herein, a “linear epitope” is an epitope that is formed by a contiguous sequence of amino acids from an antigen. Linear epitopes typically contain from about 5 to about 10 contiguous amino acid residues. Antibodies bind a linear epitope based on the primary sequence of the antigen.

As used herein, the term “paratope,” also called “antigen-binding site,” is the portion of an antibody that recognizes and binds to an antigen.

II. Compositions

Antibodies and their antigen-binding fragments are proposed that immunospecifically bind to PD-1. In contrast to the current paradigm that PD-1 exclusively stimulates a suppressive immune response (Riley, J., Immunol Rev. 229(1):114-125 (2009)), the disclosed antibodies and their antigen-binding fragments immunospecifically bind to PD-1. 1 and cause the delivery of an activating signal to the immune cell, which activates the immune cell instead of suppressing the immune cell.

A. Programmed cell death receptor protein 1 (PD-1)

The disclosed antibodies and antigen-binding fragments thereof immunospecifically bind to PD-1. Antibodies and antigen binding fragments thereof can bind to PD-1 having, for example, the amino acid sequences set forth below.

The amino acid sequences of human PD-1 and mouse PD-1 are known in the art, and include, for example,

human PD-1

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTG QPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO:1), Accession No.: AJS10360, which is specifically incorporated by reference in its entirety;

PD-1 mouse

MWVRQVPWSFTWAVLQLSWQSGWLLEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSSPNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPEGRFQGMVIGIMSALVGIPVLLLLAWALAVFCSTSMSEARGAGSKDDTLK EEPSAAPVPSVAYEELDFQGREKTPELPTACVHTEYATIVFTEGLGASAMGRRGSADGLQGPRPPRHEDGHCSWPL

(SEQ ID NO:2) UniProtKB - Q02242 (PDCD1_MOUSE), which is specifically incorporated by reference in its entirety.

B. Antibody compositions

The disclosed anti-PD-1 antibodies and antigen-binding fragments thereof include whole immunoglobulin (i.e., intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of the antibody. In some embodiments, the disclosed antibody comprises both a light chain and at least a heavy chain variable domain of the antibody. In other embodiments, such molecules may further comprise one or more of CH1 regions, hinge regions, CH2, CH3 and CH4 heavy chain regions (especially CH1 regions and hinge regions, or CH1 regions, hinge regions and CH2 -regions, or CH1 regions, hinge regions, CH2 and CH3 regions). The antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG ₁ , IgG ₂ , IgG ₃ and IgG ₄ . In some embodiments, the constant domain is a complement fixing constant domain, wherein the antibody preferably exhibits cytotoxic activity and the class is typically IgG ₁ . In other embodiments, in which such cytotoxic activity is not necessary, the constant domain may be an IgG ₂ or IgG ₄ class. An antibody may contain sequences from more than one class or isotype, and the selection of specific constant domains to optimize desired effector functions is within the skill of the art.

Variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is generally not distributed evenly across the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both the light chain and heavy chain variable domains. The most highly conserved regions of the variable domains are called the framework (FR). Each of the variable domains of the native heavy and light chains contains four FR regions, mostly adopting a beta sheet configuration, connected by three CDRs that form loops that integrate the beta sheet structure and in some cases form part of it. CDRs on each chain are held in close proximity to each other by FR regions and, together with CDRs from the other chain, participate in the formation of the antigen-binding site of antibodies.

In some embodiments, anti-PD-1 antibodies are provided that have biological activity. Fragments, whether or not linked to other sequences, may contain insertions, deletions, substitutions, or other selected modifications of certain regions or certain amino acid residues, provided that the activity of the fragment is not substantially altered or impaired compared to an unmodified antibody or antibody fragment.

In another embodiment, single chain antibodies specific for PD-1 are provided. Methods for producing single chain antibodies are well known to those skilled in the art. A single-chain antibody can be produced by fusing the heavy and light chain variable domains together using a short peptide linker, thereby restoring the antigen-binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is linked to the N-terminus of another variable domain by a 15-25 amino acid peptide or linker have been developed without significantly affecting antigen binding or binding specificity. The linker is selected to ensure that the heavy chain and light chain are linked to each other in their respective conformational orientations.

In another embodiment, divalent single chain variable fragments (di-scFv) are provided, which can be developed by linking two scFvs. This can be accomplished by producing a single peptide chain with two VH and two VL regions forming tandem scFvs. ScFvs can also be constructed using linker peptides that are too short for the two variable regions to fold together (about five amino acids), allowing scFv dimerization. This type is known as diatel. Diabodies have been demonstrated to have dissociation constants up to 40 times lower than the corresponding scFvs, meaning that they have a much higher affinity for their target. Even shorter linkers (one or two amino acids) lead to the formation of trimers (triabody or triobody). Tetrabodies were also obtained. They exhibit even higher affinity for their targets compared to diabodies.

In another embodiment, a monoclonal antibody specific for PD-1 is provided that induces an activating signal against immune cells. The monoclonal antibody may be produced from a substantially homogeneous population of antibodies, i.e., the individual antibodies in the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Monoclonal antibodies include "chimeric" antibodies in which part of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) is identical or is homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, so long as they exhibit the necessary antagonistic activity.

1. Chimeric and humanized antibodies

In another embodiment, chimeric anti-PD-1 antibodies and antigen binding fragments thereof are provided, including also providing one or more of the disclosed sequences and functional variants thereof, that bind to PD-1 and cause an activating signal to be transmitted to a PD-expressing immune cell -1.

Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229:1202; Oi et al. , 1986, BioTechniques 4:214; Gillies et al. , 1989, J. Immunol. Methods 125:191-202, and US Patent Nos. 6311415, 5807715, 4816567 and 4816397. Chimeric antibodies containing one or more CDRs from non-human species and framework regions from a human immunoglobulin molecule can be prepared using a variety of techniques known in the art. in the art, including, for example, CDR embedding (EP 239400, international publication no. WO 91/09967; and US patent Nos. 5225539, 5530101 and 5585089), venation and transfer (EP 592106; EP 519596; Padlan, 1991 , Molecular Immunology 28(4/5):489-498; Studnicka et al. , 1994, Protein Engineering 7:805; and Roguska et al. , 1994, Proc. Natl. Acad. Sci. USA 91:969) and permutations chains (US Patent No. 5565332).

The disclosed anti-PD-1 antibodies or antigen binding fragments thereof may be human or humanized antibodies or antigen binding fragments thereof. Many non-human antibodies (eg, antibodies derived from mice, rats, or rabbits) are naturally antigenic in humans and thus elicit unwanted immune responses when administered to humans. Thus, the use of human or humanized antibodies in methods helps reduce the likelihood that an antibody administered to a human will cause an undesirable immune response.

Transgenic animals (eg mice) that are capable of producing the full repertoire of human antibodies when immunized in the absence of endogenous immunoglobulin production can be used. For example, it has been described that homozygous deletion of the antibody heavy chain binding region gene (J(H)) in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of a set of human germline immunoglobulin genes into such germline mutant mice will result in the production of human antibodies upon antigen stimulation.

Optionally, antibodies are obtained from other species and “humanized” for administration to humans. Humanized forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding antibody sequences) that contain the minimal sequence originating from from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient antibody are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and ability. In some cases, human immunoglobulin Fv framework region residues are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. Typically, a humanized antibody will include substantially all of at least one, and typically two, variable domains, all or substantially all of the CDR regions corresponding to those of non-human immunoglobulin and all or substantially all of the FR regions originate from the human immunoglobulin consensus sequence. The humanized antibody will optimally also contain at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art, see, for example, European Patent Nos. EP 239400, EP 592106 and EP 519596; international publications WO 91/09967 and WO 93/17105; US Patent Nos. 5225539, 5530101, 5565332, 5585089, 5766886 and 6407213; and Padlan, 1991, Molecular Immunology 28(4/5):489498; Studnicka et al. , 1994, Protein Engineering 7(6):805-814; Roguska et al. , 1994, PNAS 91:969973; Tan et al. , 2002, J. Immunol . 169:1119-1125; Caldas et al. , 2000, Protein Eng . 13:353-360; Morea et al. , 2000, Methods 20:26779; Baca et al. , 1997, J. Biol. Chem . 272:1067810684; Roguska et al. , 1996, Protein Eng . 9:895904; Couto et al. , 1995, Cancer Res . 55 (23 Supp):5973s5977s; Couto et al. , 1995, Cancer Res . 55:171722; Sandhu 1994 Gene 150:40910; Pedersen et al. , 1994, J. Mol. Biol . 235:959-973; Jones et al. , 1986, Nature 321:522-525; Reichmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596).

Typically, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as “imported” residues, which are typically derived from an “imported” variable domain. Antibody humanization techniques typically involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of the antibody molecule. Humanization can essentially be accomplished by replacing rodent CDRs or CDR sequences with corresponding human antibody sequences. Accordingly, a humanized form of a non-human antibody (or fragment thereof) is a chimeric antibody or fragment in which a substantially less-than-intact human variable domain has been replaced by a corresponding sequence from a non-human species. In practical use, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues have been replaced with residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy chains, to be used in the production of humanized antibodies can be very important to reduce antigenicity. According to the "best fit" method, the variable domain sequence of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to that of the rodent is then adopted as the human framework region (FR) of the immunized antibody. Another method uses a specific framework region derived from the consensus sequence of all human antibodies of a specific subset of light or heavy chains. The same framework region can be used for several different humanized antibodies.

In addition, it is important that the antibodies are humanized while maintaining high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be obtained by analyzing the parent sequences and various conceptual humanized products using spatial models of the parent and humanized sequences. Spatial models of immunoglobulins are publicly available and known to those skilled in the art. Computer programs are available that illustrate and demonstrate the possible spatial conformational structures of some candidate immunoglobulin sequences. Examination of these images facilitates analysis of the likely role of residues in the functioning of the candidate immunoglobulin sequence, ie, analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this regard, FR residues can be selected and combined from the consensus and import sequence such that a desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, CDR residues directly and most substantially influence antigen binding.

A human, humanized, or chimeric antibody derivative may contain substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of non-human immunoglobulin (i.e. , donor antibody) and all or substantially all of the framework regions correspond to those of the human immunoglobulin consensus sequence. Such antibodies may also include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The constant domains of such antibodies can be selected based on the intended function of the antibody, in particular the effector function that may be required. In some embodiments, the constant domains of such antibodies are or may include human IgA, IgD, IgE, IgG, or IgM domains. In a particular embodiment, human IgG constant domains, especially the IgG1 and IgG3 isotypes, are used if the humanized antibody derivative is to be used for therapeutic use and antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity are required. In alternative embodiments, the IgG2 and IgG4 isotypes are used if the antibody is intended to be used for therapeutic purposes and the effector function of the antibodies is not required. Constant Fc domains containing one or more amino acid modifications that alter the effector functions of antibodies, such as those, are disclosed in US Patent Application Publication Nos. 2005/0037000 and 2005/0064514.

The framework regions and CDR regions of a humanized antibody do not need to exactly match the original sequences, for example, the donor CDR or consensus framework region can be mutated by substitution, insertion or deletion of at least one residue such that the CDR or framework residue at that site is not matched neither the consensus region nor the donor antibody. In some embodiments, such mutations are not significant. Typically, at least 75% of the residues of the humanized antibody will match those of the original framework region (FR) and CDR sequences, more often 90% and most often 95%. Humanized antibodies can be produced using a number of techniques known in the art, including, but not limited to, CDR insertion (European Patent No. 239400; International Publication No. WO 91/09967; and US Patent Nos. 5225539, 5530101 and 5585089), veining and transfer (European patents nos. 592106 and 519596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al. , 1994, Protein Engineering 7(6):805-814, and Roguska et al. , 1994, Proc. Natl. Acad. Sci. 91:969973-973), strand permutation (US Patent No. 5565332), as well as techniques disclosed, for example, in US Patent Nos. 6407213, 5766886, 5585089 , International Publication No. WO 9317105, Tan et al. , 2002, J. Immunol . 169:1119-25, Caldas et al. , 2000, Protein Eng . 13:353-60, Morea et al. , 2000, Methods 20:267-79, Baca et al. , 1997, J. Biol. Chem . 272:10678-84, Roguska et al. , 1996, Protein Eng . 9:895-904, Couto et al. , 1995, Cancer Res . 55 (23 Supp):5973s5977s, Couto et al. , 1995, Cancer Res . 55:171722, Sandhu 1994, Gene 150:40910, Pedersen et al. , 1994, J. Mol. Biol . 235:95973, Jones et al. , 1986, Nature 321:522-525, Riechmann et al. , 1988, Nature 332:323, and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596.

Often, framework residues in framework regions will be replaced by a corresponding residue from the CDR of the donor antibody to alter, preferably enhance, binding to the antigen. These framework substitutions are identified using methods well known in the art, for example, by modeling the interactions of CDRs and framework residues to identify framework residues important for antigen binding, and by comparing sequences to identify unusual framework regions at certain positions. (See, for example, Queen et al. , US patent No. 5585089; US publications No. 2004/0049014 and 2003/0229208; US patents No. 6350861; 6180370; 5693762; 5693761; 5585089 and 5530101, and RieCHmann et al. 1988, Nature 332:323).

Human, chimeric or humanized derivatives of the disclosed mouse anti-human Siglec-15 antibodies can be used for in vivo methods in humans. Mouse antibodies or antibodies of other species can be advantageously used for many applications (eg, in vitro or in situ detection assays, in vivo emergency use, etc.). Such a human or humanized antibody may contain amino acid substitutions, deletions or additions in one or more non-human CDRs. A derivative of a humanized antibody may have substantially the same binding, stronger binding, or weaker binding compared to a non-derivative humanized antibody. In specific embodiments, one, two, three, four, or five amino acid residues of the CDR have been replaced, deleted, or added (ie, mutated). Fully human antibodies are particularly preferred for the therapeutic treatment of human subjects.

Such human antibodies can be produced by a number of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences (see US Pat. Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741). Such human antibodies can be produced using transgenic mice that are unable to express functional endogenous immunoglobulins, but which can express human immunoglobulin genes.

For example, human immunoglobulin heavy and light chain gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region and diversity region can be introduced into mouse embryonic stem cells, in addition to the human heavy and light chain genes. The mouse immunoglobulin heavy and light chain genes can be rendered nonfunctional individually or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the _JH region prevents the production of endogenous antibodies. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then crossed to produce homozygous offspring that express human antibodies. Transgenic mice are immunized using standard techniques with a selected antigen, for example all or part of a polypeptide. Monoclonal antibodies directed against an antigen can be obtained from immunized transgenic mice using standard hybridoma production techniques (see, for example, US Pat. No. 5,916,771). Human immunoglobulin transgenes found in transgenic mice are rearranged during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, therapeutically useful IgG, IgA, IgM and IgE antibodies can be obtained using this technique. For a description of this procedure for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol . 13:65-93, which is incorporated herein by reference in its entirety). For a detailed description of this procedure for the production of human antibodies and human monoclonal antibodies and protocols for the production of such antibodies, see, for example, international publications Nos. WO 98/24893, WO 96/34096 and WO 96/33735; and US Patent Nos. 5413923, 5625126, 5633425, 5569825, 5661016, 5545806, 5814318 and 5939598, which are incorporated herein by reference in their entirety. In addition, companies can produce human antibodies directed against a selected antigen using techniques similar to those described above.

DNA sequences encoding human acceptor framework sequences include, but are not limited to, the FR segments from the human VH germline segment VH1-18 and JH6 and the human VL germline segment VK-A26 and JK4. In a specific embodiment, one or more of the CDRs are introduced into the framework regions using standard recombinant DNA techniques. The framework regions may be naturally occurring or be consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al. , 1998, “ Structural Determinants In The Sequences Of Immunoglobulin Variable Domain ,” J. Mol. Biol . 278: 457-479 regarding the list of human frame regions).

C. Antibody sequences

1. 4C12 heavy chain sequences

In one embodiment, a murine monoclonal antibody or antigen binding fragment thereof isolated from a 4C12 hybridoma is provided.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain encoded by a nucleic acid of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to:

ATGAGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCC ACTCCCAGGTCCAAC

TGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGGTGTCCTGCAAGGCTTCTGG

CTACACCTTCACC AGCTACTGGATGCAC TGGGTGAAGCAGAGGCCTGGCCAAGGCCTTGAGTGGATT

GGA AGGATTCATCCTTCTGATAGTGATACTAACTACAATCAAAAGTTCAAGGGC AAGGCCACATTGA

CTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGT

CTATTACTGTGCACCC TATGGTAACTACGCCTCCGGGTTTGCTTAC TGGGGCCAAGGGACTCTGGTC

ACTGTCTCTGCA GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAA

CTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTG

GAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACT

CTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCCAGACCGTCACCTGCAACGTTGCCC

ACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCAT

ATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAAGCCCAAGGATGTGCTCACCATT

ACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCA

GCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGAAACCCCGGGAGGAGCAGATCAACAG

CACTTTCCGTTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAA

TGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAACCAAAGGCAGAC

CGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCT

GACCTGCATGATAACAAACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCA

GCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGC

TCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCT

GCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA TGA (SEQ ID NO:3).

The underlined sequences correspond to complementarity determining regions (CDRs). A sequence with a double underscore corresponds to a constant region. Sequences with dotted underlining correspond to the leader sequence.

The nucleic acid may be in a vector, such as an expression vector. The nucleic acid may be extrachromosomal or inserted into the chromosome of a host cell, such as a Chinese hamster ovary cell.

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a heavy chain with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to:

MRWSCIILFLVATATGVHS QVQLQQPGAELVKPGASVKVSCKASGYTFT SYWMH WVKQRPGQGLEWI

G RIHPSDSDTNYNQKFKG KATLTVDKSSSTAYMQLSSLTSEDSAVYYCAP YGNYASGFAY WGQGTLV

TVSA AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT

LSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTI

TLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFK

CRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQP

AENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:4).

A single underscore matches the leader sequence. Double underscore corresponds to CDR, and dotted underscore corresponds to constant region.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain without a leader sequence with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with respect to:

QVQLQQPGAELVKPGASVKVSCKASGYTFT SYWMH WVKQRPGQGLEWIG RIHPSDSDTNYNQKFKG KATLTVDKSSSTAYMQLSSLTSEDSAVYYCAP YGNYASGFAY WGQGTLVTVSA AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSS TVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQ WNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:5).

A double underscore corresponds to a CDR, and a broken underscore corresponds to a constant region.

The amino acid sequence of CDR1 heavy chain 4C12 is

SYWMH (SEQ ID NO:6).

The amino acid sequence CDR2 of the heavy chain 4C12 is

RIHPSDSDTNYNQKFKG (SEQ ID NO:7).

The amino acid sequence CDR3 of the heavy chain 4C12 is

YGNYASGFAY (SEQ ID NO:8).

In one embodiment, an antibody or antigen binding fragment thereof having a heavy chain of SEQ ID NO:5 or 5 is provided.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain encoded by a nucleic acid of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:3.

In one embodiment, an antibody is provided having three different CDRs selected from the group consisting of SEQ ID NOs: 6, 7, and 8.

2. 4C12 light chain sequences

In another embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain encoded by a nucleic acid of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to:

ATGGGCATCAAGATGGAGTCACAGATTCAGGCATTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTG

ACGGA GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCAT

CACCTGC AAGGCCAGTCAGGATGTGAGTACTGCTGTAGCC TGGTATCAACAAAAACCAGGGCAATCT

CCTAAACTACTGATTTAC TGGGCATCCACCCGGCACACT GGAGTCCCTGATCGCTTCACAGGCAGTG

GATCTGGGACAGATTATACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCACTTTATTACTG

T CAGCAACATTATAGCACTCCGTGGACG TTCGGTGGAGGCACCAAGCTGGAAATCAAA CGGGCTGAT

GCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCG

TGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACG

ACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGC

ACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGA

CATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT TAG (SEQ ID NO:9).

The dotted underline represents the leader sequence. A single underscore represents a CDR. The double underscore represents a constant region.

The nucleic acid may be in a vector, such as an expression vector. The nucleic acid may be extrachromosomal or inserted into the chromosome of a host cell, such as a Chinese hamster ovary cell.

In another embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain having an amino acid sequence of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to:

MGIKMESQIQAFVFVFLWLSGVDG DIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVA WYQQKPGQS

PKLLIY WASTRHT GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC QQHYSTPWT FGGGTKLEIK RAD

AAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSS

TLTLTKDEYERHNSYTCEATHKTSSTSPIVKSFNRNEC (SEQ ID NO:10).

The underscore represents the leader sequence. The double underscore represents a CDR. The dotted underline represents a constant region.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a light chain without a leader sequence having an amino acid sequence of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with respect to:

DIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVA WYQQKPGQSPKLLIY WASTRHT GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC QQHYSTPWT FGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSY TCEATHKTSSTSPIVKSFNRNEC (SEQ ID NO:11).

The double underscore represents a CDR. The dotted underline represents a constant region.

The amino acid sequence of light chain CDR1 4C12 is

KASQDVSTAVA (SEQ ID NO:12).

The amino acid sequence CDR2 of the light chain 4C12 is

WASTRHT (SEQ ID NO:13).

The amino acid sequence CDR3 of the light chain 4C12 is

QQHYSTPWT (SEQ ID NO:14).

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to to SEQ ID NO:10 or 11.

In another embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain encoded by a nucleic acid of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:9.

In one embodiment, an antibody is provided having three different CDRs selected from the group consisting of SEQ ID NOs: 12, 13 and 14.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:4 or 5, and a light chain with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:10 or 11, or a combination of light and heavy chains thereof.

In another embodiment, an antibody or antigen binding fragment thereof is provided having three different heavy chain CDRs with an amino acid selected from the group consisting of SEQ ID NOs: 6, 7 and 8, and three different light chain CDRs with amino acids selected from the group consisting of from SEQ ID NO: 12, 13 and 14.

3. 2B5 heavy chain sequences

In one embodiment, a murine monoclonal antibody or antigen binding fragment thereof isolated from a 2B5 hybridoma is provided.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain encoded by a nucleic acid of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to:

ATGGAATGGAGCAGAGTCTTTATCTTTCTCTATCAGTAACTGCAGGTGTTCACTCC CAGGTCCAGC

TGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGACTTCAGTGAAGGTGTCCTGCAAGGCTTCTGG

ATACGCCTTCACT AATTACTTGATAGAG TGGGTAAAGCAGAGGCCTGGACAGGGCCTTGAGTGGATT

GGA GTGATTAATCCTGGAAGTGGTGGTACTAACTACAATGAGAAGTTC AAGGGCAAGGCAACACTGA

CTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGT

CTATTTCTGTGCAAGATCAGC TCAGGCCCCTGACTAC TGGGGCCAAGGCACCACTCTCACAGTCTCC

TCAGAGAGTCAGTCCTTCCCAAATGTCTTCCCCCTCGTCTCCTGCGAGAGCCCCCTGTCTGATAAGA

ATCTGGTGGCCATGGGCTGCCTGGCCCGGGACTTCCTGCCCAGCACCATTTCCTTCACCTGGAACTA

CCAGAACAACACTGAAGTCATCCAGGGTATCAGAACCTTCCCAACACTGAGGACAGGGGGCAAGTAC

CTAGCCACCTCGCAGGTGTTGCTGTCTCCCAAGAGCATCCTTGAAGGTTCAGATGAATACCTGGTAT

GCAAAATCCACTACGGAGGCAAAAACAAAGATCTGCATGTGCCCATTCCAGCTGTCGCAGAGATGAA

CCCCAATGTAAATGTGTTCGTCCCACCACGGGATGGCTTCTCTGGCCCTGCACCACGCAAGTCTAAA

CTCATCTGCGAGGCCACGAACTTCACTCCAAAACCGATCACAGTATCCTGGCTAAAGGATGGGAAGC

TCGTGGAATCTGGCTTCACCACAGATCCGGTGACCATCGAGAACAAAGGATCCACACCCCAAACCTA

CAAGGTCATAAGCACACTTACCATCTCTGAAATCGACTGGCTGAACCTGAATGTGTACACCTGCCGT

GTGGATCACAGGGGTCTCACCTTCTTGAAGAACGTGTCCTCACATGTGCTGCCAGTCCCTCCACAG

ACATCCTAACCTTCACCATCCCCCCCTCCTTTGCCGACATCTTCCTCAGCAAGTCCGCTAACCTGAC

CTGTCTGGTCTCAAACCTGGCAACCTATGAAACCCTGAATATCTCCTGGGCTTCTCAAAGTGGTGAA

CCACTGGAAACCAAAATTAAAATCATGGAAAGCCATCCCAATGGCACCTTCAGTGCTAAGGGTGTGG

CTAGTGTTTGTGTGGAAGACTGGAATAACAGGAAGGAATTTGTGTGTACTGTGACTCACAGGGATCT

GCCTTCACCACAGAAGAAATTCATCTCAAAACCCAATGAGGTGCACAAACATCCACCTGCTGTGTAC

CTGCTGCCACCAGCTCGTGAGCAACTGAACCTGAGGGAGTCAGCCACAGTCACCTGCCTGGTGAAGG

GCTTCTCTCCTGCAGACATCAGTGTGCAGTGGCTTCAGAGAGGGCAACTCTTGCCCCAAGAGAAGTA

TGTGACCAGTGCCCCGATGCCAGAGCCTGGGGCCCCAGGCTTCTACTTTACCCACAGCATCCTGACT

GTGACAGAGGAGGAATGGAACTCCGGAGAGACCTATACCTGTGTTGTAGGCCACGAGGCCCTGCCAC

ACCTGGTGACCGAGAGGACCGTGGACAAGTCCACTGGTAAACCCAACTGTACAATGTCTCCCTGAT

CATGTCTGACACAGGCGGCACCTGCTATTGA (SEQ ID NO:15).

The underlined sequences correspond to complementarity determining regions (CDRs). A sequence with a double underscore corresponds to a constant region. Sequences with dotted underlining correspond to the leader sequence.

The nucleic acid may be in a vector, such as an expression vector. The nucleic acid may be extrachromosomal or inserted into the chromosome of a host cell, such as a Chinese hamster ovary cell.

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a heavy chain with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to:

MEWSRVFIFLLSVTAGVHS QVQLQQSGAELVRPGTSVKVSCKASGYAFT NYLIE WVKQRPGQGLEWI

G VINPGSGGTNYNEKFKG KATLTADKSSSTAYMQLSSLTSEDSAVYFCAR SAQAPDY WGQGTTLTVS

S ESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKY

LATSQVLLSPKSILEGSDEYLVCKIHYGGKNKDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSK

LICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCR

VDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTTCLVSNLATYETLNISWASQSGE

PLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVY

LLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILT

VTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY (SEQ ID NO:16).

A single underscore matches the leader sequence. Double underscore corresponds to CDR, and dotted underscore corresponds to constant region.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain without a leader sequence with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with respect to:

QVQLQQSGAELVRPGTSVKVSCKASGYAFT NYLIE WVKQRPGQGLEWIG VINPGSGGTNYNEKFKG KATLTADKSSSTAYMQLSSLTSEDSAVYFCAR SAQAPDY WGQGTTLTVSS ESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNKDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTC AASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLV TERTVDKSTGKPTLYNVSLIMSDTGGTCY (SEQ ID NO:17).

Double underscore corresponds to CDR, and dotted underscore corresponds to constant region.

The amino acid sequence of CDR1 heavy chain 2B5 is

NYLIE (SEQ ID NO:18).

The amino acid sequence of CDR2 heavy chain 2B5 is

VINPGSGGTNYNEKFKG (SEQ ID NO:19).

The amino acid sequence of CDR3 heavy chain 2B5 is

SAQAPDY (SEQ ID NO:20).

In one embodiment, an antibody or antigen binding fragment thereof having a heavy chain according to SEQ ID NO:16 or 17 is provided.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain encoded by a nucleic acid of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:15.

In one embodiment, an antibody is provided having three different CDRs selected from the group consisting of SEQ ID NOs: 18, 19 and 20.

4. 2B5 light chain sequences

In another embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain encoded by a nucleic acid of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to:

ATGGGCATCAAGATGGAGACACATTCTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTG

AAGGA GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCAT

CACCTGC AAGGCCAGTCAGGATGTGGGTACTGCTGTAGCC TGGTATCAACAGAAACCAGGGCAATCT

CCTAAACTACTGATTTAC TGGGCATCCACCCGGCACACT GGAGTCCCTGATCGCTTCACAGGCAGTG

GATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGCAGATTATTTCTG

TCAGCAATATAGCAGCTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGAT

GCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCG

TGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACG

ACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGC

ACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGA

CATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT TAG (SEQ ID NO:21).

The dotted underline represents the leader sequence. A single underscore represents a CDR. The double underscore represents a constant region.

The nucleic acid may be in a vector, such as an expression vector. The nucleic acid may be extrachromosomal or inserted into the chromosome of a host cell, such as a Chinese hamster ovary cell.

In another embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain having an amino acid sequence of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to:

MGIKMETHSQVFVYMLLWLSGVEG DIVMTQSHKFMSTSVGDRVSITC KASQDVGTAVA WYQQKPGQS

PKLLIY WASTRHT GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC QQYSSYPFT FGSGTKLEIK RAD

AAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSS

TLTLTKDEYERHNSYTCEATHKTSSTSPIVKSFNRNEC (SEQ ID NO:22).

The underscore represents the leader sequence. The double underscore represents a CDR. The dotted underline represents a constant region.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a light chain without a leader sequence having an amino acid sequence of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with respect to:

DIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVA WYQQKPGQSPKLLIY WASTRHT GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC QQHYSTPWT FGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSY TCEATHKTSSTSPIVKSFNRNEC (SEQ ID NO:23).

The double underscore represents a CDR. The dotted underline represents a constant region.

The amino acid sequence of light chain CDR1 2B5 is

KASQDVSTAVA (SEQ ID NO:24).

The amino acid sequence of light chain CDR2 2B5 is

WASTRHT (SEQ ID NO:13).

The amino acid sequence of light chain CDR3 2B5 is

QQHYSTPWT (SEQ ID NO:25).

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to to SEQ ID NO:22 or 23.

In another embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain encoded by a nucleic acid of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:21.

In one embodiment, an antibody is provided having three different CDRs selected from the group consisting of SEQ ID NOs: 24, 13 and 25.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:16 or 17, and a light chain with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:22 or 23, or a combination of light and heavy chains thereof.

In another embodiment, an antibody or antigen binding fragment thereof is provided having three different heavy chain CDRs with an amino acid selected from the group consisting of SEQ ID NOs: 18, 19 and 20, and three different light chain CDRs with amino acids selected from the group consisting of from SEQ ID NO: 24, 13 and 25.

6. 4G9 heavy chain sequences

In one embodiment, a murine monoclonal antibody or antigen binding fragment thereof isolated from a 4G9 hybridoma is provided.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain encoded by a nucleic acid of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to:

ATGGGTTGGCTGTGGAACTTGCTATTCCTGATGGCAGCTGCCCAAAGTGCCCAAGCA CAGATCCAGT

TGGTACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGG

GTATACCTTCACA ACCTATGGAATGACC TGGGTGAAACAGGCTCCAGGAAAGGGTTTTAAAGTGGATG

GGC TGGATAAACACCACTCTGGAGTGCCAACATATGCTGATGACTTCAAGGGA CGGTTTGCCTTCT

CTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTAC

ATATTTTCTGTGCAAGA GGGGGACGGGGGTTTGCTTAC TGGGGCCAAGGGACTCTGGTCACTGTCTCT

GCAGCCAAAACAACACCCCCATCAGTCTATCCACTGGCCCCTGGGTGTGGAGATACAACTGGTTCCT

CTGTGACTCTGGGATGCCTGGTCAAGGGCTACTTCCCTGAGTCAGTGACTGTGACTTGGAACTCTGG

ATCCCTGTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACTCTACACTATGAGCAGC

TCAGTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGACCGTCACCTGCAGCGTTGCTCACCCAGCCA

GCAGCACCACGGTGGACAAAAAACTTGAGCCCAGCGGCCCATTTCAACAATCAACCCCTGTCCTCC

ATGCAAGGATGTCACAAATGCCCAGCTCCTAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCT

CCAAATATCAAGGATGTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTGGTGGATGTGA

GCGAGGATGACCCAGACGTCCGGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGAC

ACAAACCCATAGAGAGGATTACAACAGTACTATCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAG

GACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGA

GAACCATCTCAAAATTAAAGGGCTAGTCAGAGCTCCCACAAGTATACATCTTGCCGCCACCAGCAGA

GCAGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATCAGT

GTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACCGCACCAGTCCTGGACTCTG

ACGGTTCTTACTTCATATACAGCAAGCTCGATATAAAAACAAGCAAGTGGGAGAAAACAGATTCCTT

CTCATGCAACGTGAGACACGAGGTCTGAAAAATTACTACCTGAAGAAGACCATCTCCCGGTCTCCG

GGTAAATGA (SEQ ID NO:26).

The underlined sequences correspond to complementarity determining regions (CDRs). A sequence with a double underscore corresponds to a constant region. Sequences with dotted underlining correspond to the leader sequence.

The nucleic acid may be in a vector, such as an expression vector. The nucleic acid may be extrachromosomal or inserted into the chromosome of a host cell, such as a Chinese hamster ovary cell.

In one embodiment, an antibody or antigen-binding fragment thereof is provided having a heavy chain with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to:

MGWLWNLLFLMAAAQSAQA QIQLVQSGPELKKPGETVKISCKASGYTF TTYGMT WVKQAPGKGLKWM G WINTYSGVPTYADDFKG RFAFSLETSASTAYLQINNLKNEDTATYFCAR GGRGFAY WGQGTLVTVS

A AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSSVHTFPALLQSGLYTMSS

SVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFP

PNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQ

DWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDIS

VEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSP

GK (SEQ ID NO:27).

A single underscore matches the leader sequence. Double underscore corresponds to CDR, and dotted underscore corresponds to constant region.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain without a leader sequence with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with respect to:

QIQLVQSGPELKKPGETVKISCKASGYTF TTYGMT WVKQAPGKGLKWMG WINTYSGVPTYADDFKG RFAFSLETSASTAYLQINNLKNEDTATYFCAR GGRGFAY WGQGTLVTVSA AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVA HPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLD SDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSP

GK (SEQ ID NO:28).

Double underscore corresponds to CDR, and dotted underscore corresponds to constant region.

The amino acid sequence of CDR1 heavy chain 4G9 is

TYGMT (SEQ ID NO:29).

The amino acid sequence of CDR2 heavy chain 4G9 is

WINTYSGVPTYADDFKG (SEQ ID NO:30).

GGRGFAY (SEQ ID NO:31).

In one embodiment, a 4G9 antibody or antigen binding fragment thereof having a heavy chain according to SEQ ID NO:27 or 28 is provided.

In another embodiment, a 4G9 antibody or antigen binding fragment thereof is provided having a heavy chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100 % sequence identity to SEQ ID NO:26.

In one embodiment, a 4G9 antibody is provided having three different CDRs selected from the group consisting of SEQ ID NOs: 29, 30 and 31.

7. 4G9 light chain sequences

In another embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain encoded by a nucleic acid of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to:

ATGGTATCCACACCTCAGTTCCTTGTATTTTTGCTTTTCTGGATTCCAGCCTCCAGAGGT GACATCT

TGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGC AGGGC

CAGTCAGAGCATTGGCACAAGCATACAC TGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTC

ATAAAG TATGCTTCTGAGTCTATCTCT GGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAG

ATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGT CAACAAAGTAA

TAGCTGGCCGTACACG TTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACT

GTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGA

ACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGT

CCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTG

ACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCAC

CCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG (SEQ ID NO:32).

The dotted underline represents the leader sequence. A single underscore represents a CDR. The double underscore represents a constant region.

The nucleic acid may be in a vector, such as an expression vector. The nucleic acid may be extrachromosomal or inserted into the chromosome of a host cell, such as a Chinese hamster ovary cell.

In another embodiment, an antibody or antigen-binding fragment thereof is provided having a light chain having an amino acid sequence of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to:

MVSTPQFLVFLLFWIPASRG DILLTQSPAILSVSPGERVSFSC RASQSIGTSIH WYQQRTNGSPRLLIK YASESIS GIPSRFSGSGGTDFTLSINSVESEDIADYYC QQSNSWPYT FGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSST LTLTKDEYERHNSYTCEATHKTSSTSPIVKSFNRNEC (SEQ ID NO:33).

The underscore represents the leader sequence. The double underscore represents a CDR. The dotted underline represents a constant region.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a light chain without a leader sequence having an amino acid sequence of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with respect to:

DILLTQSPAILSVSPGERVSFSC RASQSIGTSIH WYQQRTNGSPRLLIK YASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQSNSWPYT FGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHK TSTSPIVKSFNRNEC (SEQ ID NO:34).

The double underscore represents a CDR. The dotted underline represents a constant region.

The amino acid sequence of light chain CDR1 4G9 is

RASQSIGTSIH (SEQ ID NO:35).

The amino acid sequence of CDR2 light chain 4G9 is

YASESIS (SEQ ID NO:36).

The amino acid sequence CDR3 of the 4G9 light chain is

QQSNWPYT (SEQ ID NO:37).

In one embodiment, a 4G9 antibody or antigen binding fragment thereof is provided having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to in relation to SEQ ID NO:33 or 34.

In another embodiment, a 4G9 antibody or antigen binding fragment thereof is provided having a light chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100 % sequence identity to SEQ ID NO:32.

In one embodiment, a 4G9 antibody is provided having three different CDRs selected from the group consisting of SEQ ID NOs: 35, 36 and 37.

In another embodiment, an antibody or antigen binding fragment thereof is provided having a heavy chain with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:27 or 28, and a light chain with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:33 or 34, or a combination of light and heavy chains thereof.

In another embodiment, a 4G9 antibody or antigen binding fragment thereof is provided having three different heavy chain CDRs with an amino acid selected from the group consisting of SEQ ID NOs: 29, 30 and 31, and three different light chain CDRs with amino acids selected from the group consisting of SEQ ID NO:35, 36 and 37.

D. PD-1 activating epitope

Disclosed herein are epitope-specific fragments that bind PD-1. In one embodiment, the disclosed binding fragments immunospecifically bind to PD-1 and activate PD-1-mediated signaling.

The disclosed PD-1 epitope is formed by amino acids 96-110 of SEQ ID NO:1 and has the following amino acid sequence:

TYLCGAISLAPKAQI (SEQ ID NO:38).

1. Antibodies

In one embodiment, an antibody or epitope-binding fragment thereof is provided that immunospecifically binds to SEQ ID NO:38 on the surface of an immune cell and activates the immune cell. In one embodiment, the preferred immune cell is a T cell, more specifically a CD8 ⁺ T cell. In another embodiment, the antibody or epitope-binding fragment thereof immunospecifically binds SEQ ID NO:38 to PD-1 on the surface of an immune cell and stimulates or induces an activating signal through PD-1 to activate the immune cell.

The epitope-specific antibody may be a monoclonal antibody, a humanized antibody, a human antibody, a murine antibody, a chimeric antibody, or a fragment thereof. Exemplary antibodies and methods for preparing them are discussed below.

2. Fusion protein

In one embodiment, the epitope-specific PD-1 binding fragment is a fusion protein. All or a portion of one or more of the disclosed PD-1 epitope antibodies or epitope-binding fragments can bind to other polypeptides to form fusion proteins. Fusion polypeptides have a first fusion partner, including all or a portion of one or more of the disclosed PD-1 epitope antibodies or epitope-binding fragments, fused to a second polypeptide directly or via a linker peptide sequence that is fused to the second polypeptide. Fusion proteins optionally contain a domain that functions to dimerize two or more fusion proteins. The peptide/polypeptide linker domain may be either a separate domain or alternatively contained within one of the other domains (the first polypeptide or the second polypeptide) of the fusion protein. Likewise, the domain that functions to dimerize or multimerize the fusion proteins may be either a separate domain or alternatively contained in one of the other domains (first polypeptide, second polypeptide, or peptide/polypeptide linker domain) of the fusion protein. In one embodiment, the dimerization/multimerization domain and the peptide/polypeptide linker domain are the same.

The fusion proteins disclosed herein are represented by Formula I:

NR ₁ -R ₂ -R ₃ -C,

where "N" represents the N-terminus of the fusion protein, "C" represents the C-terminus of the fusion protein, "R ₁ " represents all or part of one of the disclosed antibodies to a PD-1 epitope or epitope-binding fragments or a functional variant or fragment thereof " _R2 " represents an optional peptide/polypeptide linker domain and " _R3 " represents a second polypeptide. Alternatively, R ₃ is all or part of one of the disclosed anti-PD-1 epitope antibodies or epitope-binding fragments or a functional variant or fragment thereof, and R ₁ is a second polypeptide.

Dimerization or multimerization can occur between or within one or more fusion proteins involving dimerization or multimerization domains. Alternatively, dimerization or multimerization of the fusion proteins can occur through chemical cross-linking. The dimers or multimers that are formed may be homodimeric/homomultimeric or heterodimeric/heteromultimeric.

3. Aptamers

In some embodiments, the epitope-specific binding moiety is an aptamer. Aptamers are molecules that interact with a target molecule, preferably in a specific manner. In one embodiment, the aptamer binds to SEQ ID NO:38 on the surface of an immune cell and stimulates or induces an activating signal through PD-1 to activate the immune cell. Typically, aptamers are small nucleic acids, ranging in length from 15-50 bases, that are folded into specific secondary and tertiary structures, such as stem loops or G-quartets. Aptamers can bind to proteins, cells, small organic molecules, or peptides. Aptamers can bind small molecules such as ATP and theophylline, as well as large molecules such as reverse transcriptase and thrombin. Aptamers can bind very strongly with a Kd value for the target molecule of less than 10 ^-12 M. Preferably, aptamers bind the target molecule with a Kd value of less than 10 ^-6 , 10 ^-8 , 10 ^-10 or 10 ^-12 . Aptamers can bind a target molecule with a very high degree of specificity. For example, aptamers have been isolated that have a greater than 10,000-fold difference in binding affinities for a target molecule and another molecule that differs at only one position of the molecule. Preferably, the aptamer has a Kd value for the target molecule that is at least 10, 100, 1000, 10,000, or 100,000 times less than the Kd value for the background binding molecule. When comparing a polypeptide, it is preferable that the background molecule is another polypeptide. Illustrative examples of how to prepare and use aptamers to bind a number of different target molecules are known in the art.

4. Small molecules

In some embodiments, the epitope-binding moiety may be a small molecule. The term "small molecule" generally refers to small inorganic compounds having a molecular weight of greater than about 100 and less than about 2500 daltons, preferably from 100 to 2000, more preferably from about 100 to about 1250, more preferably from about 100 to about 1000 , more preferably from about 100 to about 750, more preferably from about 200 to about 500 daltons. Small molecule agonists of the PD-1 activating epitope can be identified using standard screening methods. In some embodiments, the screening assay may include random screening of large libraries of test compounds. Assays may include determinations of PD-1-induced immune response or T cell activation.

E. Pharmaceutical compositions

Pharmaceutical compositions containing the disclosed antibodies and their antigen-binding fragments are proposed. Pharmaceutical compositions containing antibodies and antigen-binding fragments thereof may be administered by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection.

In some in vivo approaches, the compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein, the term “effective amount” or “therapeutically effective amount” means a dose sufficient to treat, inhibit, or normalize one or more symptoms of the disorder being treated, or to otherwise provide a desired pharmacological and/or physiological effect. The exact dosage will vary depending on a number of factors, such as subject-specific variables (eg, age, immune system status, etc.), disease, and treatment being administered.

With respect to the disclosed antibodies and antigen binding fragments thereof, as more research is conducted, information regarding dosage levels for the treatment of various conditions will become available and one skilled in the art, taking into account the therapeutic context, age and general health of the recipient, will be able to determine the appropriate dosage . The dose chosen depends on the desired therapeutic effect, the route of administration and the duration of treatment required. For the disclosed antibodies and antigen binding fragments thereof, dosage levels ranging from 0.001 to 20 mg/kg body weight are typically administered to mammals. Typically, in the case of intravenous injection or infusion, the dose may be lower.

In certain embodiments, the antibodies or antigen-binding fragments thereof are administered locally, for example, by injection directly into the site to be treated. Typically, the injection causes an increased local concentration of the immunomodulatory agent composition that is higher than the concentration that can be achieved by systemic administration. The immunomodulatory agent compositions can be combined with a matrix, as described above, to help create an increased local concentration of the polypeptide compositions by reducing the passive diffusion of the polypeptides outside the site being treated.

1. Formulations for parenteral administration

In some embodiments, compositions containing the disclosed antibodies and antigen-binding moieties are administered in an aqueous solution by parenteral injection. The composition may also be in the form of a suspension or emulsion. Generally provided are pharmaceutical compositions containing effective amounts of an antibody or antigen-binding fragment thereof, and optionally containing pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, excipients and/or carriers. Such compositions optionally contain one or more of the following: diluents, sterile water, buffered saline with varying buffer content (eg, Tris-HCl, acetate, phosphate), pH and ionic strength; and excipients such as detergents and solubilizing agents (eg TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (eg ascorbic acid, sodium metabisulfite) and preservatives (eg thimerasol, benzyl alcohol) and bulking agents (for example, lactose, mannitol). Examples of non-aqueous solvents and carriers are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin and injectable organic esters such as ethyl oleate. The formulations may be lyophilized or re-dissolved/re-suspended immediately prior to use. The composition can be sterilized, for example, by filtration through a bacteria-retaining filter, by including sterilizing agents in the compositions, by irradiating the compositions, or by heating the compositions.

2. Formulations for oral administration

In some embodiments, the antibody compositions are formulated for oral delivery. Oral antibody formulations are resistant to proteolysis and can deliver a higher proportion of immunoreactive antibody locally to the gastrointestinal tract to treat infections or promote absorption of antibodies to treat or prevent systemic pathological conditions (Reilly, RM, et al. Clin Pharmacokinet., 32(4):313-23 (1997); Victoria S Jasion and Bruce P Burnett, Nutr J.; 14: 22 (2015); and Philippart, M., et al., Drug Res (Stuttg) 66(03) :113-120 (2016)).

Solid dosage forms for oral administration are generally described in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) Chapter 89. Solid dosage forms include tablets, capsules, lozenges, lozenges, wafers, wafers, pellets, powders or granules, or incorporation of a substance into microparticulate polymer formulations. , such as polylactic acid, polyglycolic acid, etc. into liposomes. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed compound. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042), pp. 1435-1712, which is incorporated herein by reference. The compositions may be prepared in liquid form or may be in dry powder (eg, lyophilized) form. Encapsulation in liposomes or protenoids can be used for formulation. Liposome encapsulation can be used, and the liposomes can be derivatized with various polymers (eg, US Pat. No. 5,013,556). See also Marshall, K. in: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979. Typically, the formulation will contain the peptide (or chemically modified forms thereof) and inert ingredients that protect the peptide in the gastric environment and release it biologically active substance into the intestines.

Antibodies and their antigen-binding fragments can be chemically modified to make oral delivery of the derivative effective. Typically, the intended chemical modification is the addition of at least one fragment to the molecule of the component itself, the fragment allowing entry into the bloodstream from the stomach or intestines or entry directly into the intestinal mucosa. In addition, it is necessary to increase the overall stability of the component or components and increase the circulation time in the body. PEGylation is an exemplary chemical modification for pharmaceutical use. Other moieties that may be used include: propylene glycol, ethylene glycol-propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane and poly-1,3,6-thioxocane [see eg, Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York, NY) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem . 4:185-189].

In another embodiment, liquid dosage forms for oral administration are provided, including pharmaceutically acceptable emulsions, solutions, suspensions and syrups, which may contain other components, including inert solvents; auxiliary substances such as wetting agents, emulsifying and suspending agents; and sweeteners, flavors and flavorings.

For oral formulations, the site of release may be the stomach, small intestine (duodenum, jejunum or ileum) or large intestine. In some embodiments, the release will eliminate the deleterious effects of the gastric environment, either by protecting the agent (or derivative) or by releasing the agent (or derivative) outside the gastric environment, such as in the intestine. To ensure complete resistance to gastric acid, a coating is required that is impervious to at least a pH value of 5.0. Examples of more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™, cellulose acetate phthalate (CAP) , Eudragit L™, Eudragit S™ and Shellac™. These coatings can be used as mixed films.

3. Polymer matrices with controlled delivery

The antibodies and antigen binding fragments thereof disclosed herein may also be administered in controlled release formulations. Antibodies and their antigen-binding fragments can be included in an inert matrix that allows release either by diffusion mechanisms or by washout mechanisms, such as gum. Slowly degrading matrices may also be included. Another form of controlled release is based on the Oros therapeutic system (Alza Corp.), i.e., the antibody or antigen-binding fragment is enclosed in a semi-permeable membrane that allows water to enter and the drug to be expelled through one small hole as a result of osmotic effects.

Controlled-release polymer articles can be formulated for sustained systemic release following implantation of a polymer article (rod, cylinder, film, disk) or injection (microparticle). The matrix may be in the form of microparticles such as microspheres, in which the agent is dispersed in a solid polymer matrix, or microcapsules, in which the core consists of a material other than the polymer shell and the peptide is dispersed or suspended in the core, which may be liquid or solid by it's nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin plate or film ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.

Either biodegradable or biodegradable matrices can be used to deliver fusion polypeptides or nucleic acids encoding fusion polypeptides, although in some embodiments, biodegradable matrices are preferred. They may be natural or synthetic polymers, however synthetic polymers are preferred in some embodiments due to superior degradation and release profiles. The polymer is selected based on the period of time over which release is required. In some cases, linear release may be most suitable, although in other cases, pulsed release or "scale release" may provide more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% by weight of water) and may optionally be cross-linked with multivalent ions or polymers.

Matrices can be prepared by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Biodegradable microspheres can be prepared using any methods developed to produce microspheres for drug delivery purposes, for example, as described by Mathiowitz and Langer, J. Controlled Release , 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers , 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci ., 35:755-774 (1988).

Devices can be developed for local release to target the site of implantation or injection, which will typically deliver a dose that is much lower than that for whole body treatment or systemic delivery. They can be implanted or injected subcutaneously, into muscle, fatty tissue, or ingested.

III. Methods of obtaining

A. Methods for producing antibodies

The disclosed antibodies may be produced in cell culture, phage, or in a variety of animals, including, but not limited to, cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees , great apes. In one embodiment, the various animals may be transgenic animals genetically engineered to produce human or humanized antibodies. Thus, in one embodiment, the antibody is a mammalian antibody. Phage techniques can be used to isolate the original antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are standard and well known in the art. In one embodiment, the antibody is generated using recombinant agents known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector containing a DNA sequence encoding the antibody. One or more vectors can be used to transfect a DNA sequence expressing at least one VL and one VH region into a host cell. Illustrative descriptions of recombinant agents for producing and producing antibodies include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et al., Monoclonal Antibodies (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principles And Practice (Academic Press, 1993); Current Protocols In Immunology (John Wiley & Sons, most recent edition).

The disclosed antibodies may be modified by recombinant means to increase the effectiveness of the antibody in mediating the desired function. Thus, within the scope of this invention, antibodies can be modified by substitutions using recombinant agents. Typically, replacements will be conservative replacements. For example, at least one amino acid in the constant region of an antibody can be replaced with another residue. See, for example, US Patent No. 5624821, US Patent No. 6194551, Application No. 9958572; and Angal, et al., Mol. Immunol. 30:105-08 (1993). Amino acid modification includes deletions, additions, and substitutions of amino acids. In some cases, such changes are made to reduce undesirable activities, such as complement-dependent cytotoxicity. Often antibodies are labeled by binding, either covalently or non-covalently, to a substance that provides a detectable signal. A wide variety of labels and conjugation techniques are known and widely described in both the scientific and patent literature. These antibodies can be screened for binding to proteins and polypeptides. See, for example, Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

For example, appropriate antibodies with the desired biological activities can be determined using in vitro assays, including, but not limited to: proliferation, migration, adhesion, growth on soft agar, angiogenesis, intercellular communication, apoptosis, transport, signaling and in vivo assays such as tumor growth inhibition. The antibodies provided herein may also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for their ability to bind to a specific antigen without inhibiting receptor binding or biological activity of the antigen. As neutralizing antibodies, antibodies may be useful in competitive binding assays.

Antibodies that can be used in the disclosed compositions and methods include whole immunoglobulin (i.e., intact antibody) of any class, a fragment thereof, and synthetic proteins containing at least the antigen-binding variable domain of the antibody. Variable domains in antibodies differ in sequence and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is generally not distributed evenly across the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions, in both the light chain and heavy chain variable domains. The most highly conserved regions of the variable domains are called the framework (FR). Each of the variable domains of the native heavy and light chains contains four FR regions, mostly adopting a beta sheet configuration, connected by three CDRs that form loops that integrate the beta sheet structure and in some cases form part of it. CDRs on each chain are held in close proximity to each other by FR regions and, together with CDRs from the other chain, participate in the formation of the antigen-binding site of antibodies.

Also disclosed are antibody fragments that have biological activity. Fragments, whether or not linked to other sequences, contain insertions, deletions, substitutions, or other specified modifications of specified regions or specified amino acid residues, provided that the activity of the fragment is not substantially altered or impaired compared to an unmodified antibody or antibody fragment.

The techniques can also be adapted to produce single chain antibodies from the disclosed antibodies and antigen binding fragments thereof. Methods for producing single chain antibodies are well known to those skilled in the art. A single-chain antibody can be produced by fusing the heavy and light chain variable domains together using a short peptide linker, thereby restoring the antigen-binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is linked to the N-terminus of another variable domain by a 15-25 amino acid peptide or linker have been developed without significantly affecting antigen binding or binding specificity. The linker is selected to ensure that the heavy chain and light chain are linked to each other in their respective conformational orientations.

In one embodiment, divalent single chain variable fragments (di-scFv) are provided that can be developed by linking two scFvs. This can be accomplished by producing a single peptide chain with two VH and two VL regions forming tandem scFvs. ScFvs can also be constructed using linker peptides that are too short for the two variable regions to fold together (about five amino acids), allowing scFv dimerization. This type is known as diatel. Diabodies have been demonstrated to have dissociation constants up to 40 times lower than the corresponding scFvs, meaning that they have a much higher affinity for their target. Even shorter linkers (one or two amino acids) lead to the formation of trimers (triabody or triobody). Tetrabodies were also obtained. They exhibit even higher affinity for targets compared to diabodies.

In another embodiment, a monoclonal antibody is provided that is derived from a substantially homogeneous population of antibodies, i.e., the individual antibodies in the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Monoclonal antibodies include “chimeric” antibodies in which part of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) is identical or is homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, so long as they exhibit the necessary antagonistic activity.

Monoclonal antibodies can be produced by any procedure that results in the formation of monoclonal antibodies. In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to activate lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

The disclosed antibodies can also be produced using recombinant DNA methods. DNA encoding the disclosed antibodies can be readily isolated and/or sequenced using standard procedures (eg, by using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques.

Methods for producing antibodies using protein chemistry are also known in the art. One method of producing antibody proteins is by linking two or more peptides or polypeptides to each other using protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or Boc (tert-butylcarbonyl) chemistry. (Applied Biosystems, Inc., Foster City, California). One skilled in the art will readily appreciate that a peptide or polypeptide corresponding to an antibody, for example, can be synthesized using standard chemical reactions. For example, a peptide or polypeptide may be synthesized and not separated from its synthetic resin, while another antibody fragment may be synthesized and then separated from the resin, exposing a terminal group that is operably locked on the other fragment. Through peptide condensation reactions, the two moieties can be covalently linked via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody or fragment thereof. Alternatively, the peptide or polypeptide is independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides can be coupled to form an antibody or antigen binding fragment thereof using similar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segments promotes the binding of relatively short peptide fragments to form larger peptide fragments, polypeptides, or entire protein domains. Alternatively, native chemical ligation of synthetic peptides can be used to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-stage chemical reaction. The first step is the chemoselective reaction of an unprotected synthetic peptide and alpha-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to form a thioester-linked intermediate as the starting covalent product. Without changing the reaction conditions, this intermediate undergoes a spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.

B. Methods for preparing isolated nucleic acid molecules

In one embodiment, nucleic acids encoding the disclosed antibodies or antigen-binding fragments thereof are provided. The nucleic acids may encode the entire antibody or antigen-binding fragments thereof, or a light chain, a heavy chain, combinations thereof, or CDRs thereof.

Isolated nucleic acid molecules can be obtained using standard techniques, including, but not limited to, conventional molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain isolated nucleic acid encoding a variant polypeptide. PCR is a technique in which target nucleic acids are enzymatically amplified. Typically, sequence information starting at or beyond the ends of the region of interest can be used to design oligonucleotide primers that are identical in sequence to the opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers are typically 14 to 40 nucleotides in length, but can vary from 10 nucleotides to hundreds of nucleotides in length. General PCR procedures are described, for example, in PCR Primer: A Laboratory Manual , ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. Using RNA as a template source, reverse transcriptase can be used to synthesize the complementary strand of DNA (cDNA). Ligase chain reaction, strand displacement amplification, self-sustaining sequence replication, or nucleic acid sequence-based amplification may also be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292-1293.

Isolated nucleic acids can be synthesized chemically, either as a single nucleic acid molecule or as a series of oligonucleotides (eg, using amidophosphide technology to automatically synthesize DNA in the 3' to 5' direction). For example, one or more pairs of long oligonucleotides (eg, >100 nucleotides) can be synthesized to contain the desired sequence, with each pair containing a short complementarity segment (eg, about 15 nucleotides) such that a duplex is formed when the oligonucleotide the couple is annealed. DNA polymerase can be used to extend oligonucleotides, resulting in one double-stranded nucleic acid molecule per oligonucleotide pair, which can then be ligated into a vector. Isolated nucleic acids can also be obtained by mutagenesis. Nucleic acids encoding proteins can be mutated using standard techniques, including oligonucleotide-directed mutagenesis and/or site-directed mutagenesis using PCR. See Short Protocols in Molecular Biology . Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al, 1992.

IV. Methods of application

The disclosed antibodies and antigen binding fragments thereof can be used to modulate the immune response in a subject in need thereof. In one embodiment, a method is provided for activating PD-1 expressing immune cells, such as T cells, to proliferate or enhance the biological activity of the PD-1 expressing immune cells by administering the disclosed antibodies and antigen binding fragments thereof, optionally containing a second therapeutic agent .

A. Stimulation of immune responses

1. Therapeutic strategies

Methods are provided for inducing or enhancing an immune response in a subject. Typically, the methods involve administering to a subject an effective amount of one or more of the disclosed antibodies and antigen binding fragments thereof to bind immunospecifically to PD-1 and induce, stimulate, or enhance a stimulatory or activating signal involving PD-1 to activate an immune cell. The immune response may be, for example, the induction, stimulation or enhancement of T cell activation, secretion of cytokines by immune cells, T cell proliferation. The disclosed antibodies or antigen binding fragments thereof can be administered to a subject in need thereof in an effective amount to overcome T cell exhaustion and/or T cell anergy. Overcoming T-cell exhaustion or T-cell anergy can be determined by measuring T-cell function using known techniques.

The methods can be used in vivo or ex vivo to induce, stimulate or enhance an immune response.

In some embodiments, the antibody or antigen-binding fragment thereof, or a nucleic acid encoding the antibody or antigen-binding fragment thereof, is administered directly to the subject. In some embodiments, the antibody or antigen binding fragment thereof is contacted with cells (eg, immune cells) ex vivo and the treated cells are administered to a subject (eg, adoptive transfer). The antibody or antigen-binding fragment thereof may help enable a more robust immune response. The disclosed compositions are useful for stimulating or enhancing immune responses involving T cells inducing an activating signal involving PD-1 on immune cells.

2. Subjects to be treated

a. Cancer treatment

The disclosed antibodies and their compositions and methods can be used to treat cancer. Typically, the agents are used to stimulate or enhance an immune response against cancer in a subject by administering to the subject an amount of a disclosed antibody or antigen-binding fragment thereof that induces, stimulates, or enhances an activating signal involving PD-1. The method may reduce one or more symptoms of cancer.

Immune cells activated by the disclosed antibodies or fragments thereof can destroy cancer cells and reduce the tumor burden in a subject. The term “cancer cell” is intended to include pre-malignant and malignant cancer cells. In some embodiments, cancer refers to a benign tumor that has remained localized. In other embodiments, cancer refers to a malignant tumor that has invaded and destroyed adjacent body structures and spread to distant sites. In yet other embodiments, the cancer is associated with a specific cancer antigen (e.g., common carcinoma antigen (KS 1/4), ovarian carcinoma antigen (CA125), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CD19 , CD20, HER2/neu, etc.).

The methods and antibody compositions disclosed herein are useful in the treatment or prevention of a number of different types of cancer and other pathological proliferative diseases, including (but not limited to) the following: carcinomas, including carcinomas of the bladder, breast, intestines, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of the lymphoid cell line, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of the myeloid cell line, including acute and chronic myelogenous leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanomas, seminomas, teratocarcinomas, neuroblastomas and gliomas; tumors of the central and peripheral nervous system, including astrocytomas, neuroblastomas, gliomas and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid cancer and teratocarcinoma.

Cancers caused by disorders of apoptosis may also be treated using the disclosed methods and compositions. Such cancers may include, but are not limited to, follicular lymphomas, p53 mutation carcinomas, hormone-dependent breast, prostate and ovarian tumors, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndromes. In specific embodiments, cancer or dysproliferative changes (such as metaplasias and dysplasias) or hyperproliferative disorders are treated or prevented by these methods and compositions in the ovary, bladder, breast, intestine, lung, skin, pancreas, or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia is treated or prevented using the disclosed methods and compositions.

Specific cancers and related disorders that may be treated or prevented by the methods and compositions disclosed herein include, but are not limited to, leukemias, including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic leukemias, erythroleukemias and myelodysplastic syndrome, chronic leukemias such as, but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hair cell leukemia; polycythemia vera; lymphomas such as, but not limited to, Hodgkin's lymphoma or non-Hodgkin's lymphoma; lymphoma (eg, diffuse large B-cell lymphoma (DLBCL), anaplastic lymphoma kinase (ALK)-negative); diffuse large B-cell lymphoma (DLBCL), anaplastic lymphoma kinase (ALK)-positive; ALK+ anaplastic large cell lymphoma (ALCL), anaplastic lymphoma kinase (ALK) positive, acute myeloid lymphoma (AML)); multiple myelomas, such as, but not limited to, smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenström's macroglobulinemia; monoclonal gammopathy of unknown etiology; benign monoclonal gammopathy; heavy chain disease; Bone and connective tissue sarcomas such as, but not limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, giant cell sarcoma, bone fibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma , liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, including, but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, non-glial tumor, acoustic neurilemma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer, including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease and edematous-infiltrative mammary cancer; adrenal cancer, including, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, including, but not limited to, insulinoma, gastrinoma, glucagonoma, VIPoma, somatostatinoma and carcinoid and islet cell tumors; malignant tumors of the pituitary gland, including, but not limited to, Cushing's disease, prolactinoma, acromegaly and diabetes insipidus; malignant tumors of the eye, including, but not limited to, ocular melanoma such as iris melanoma, choroidal melanoma and ciliary body melanoma and retinoblastoma; malignant tumors of the vagina, including, but not limited to, squamous cell carcinoma, adenocarcinoma and melanoma; vulvar cancer, including, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma and Paget's disease; malignant tumors of the cervix, including, but not limited to, squamous cell carcinoma and adenocarcinoma; malignant tumors of the uterus, including, but not limited to, endometrial carcinoma and uterine sarcoma; malignant ovarian tumors, including, but not limited to, epithelial ovarian carcinoma, borderline tumor, germinal cell tumor and stromal tumor; esophageal malignancies, including, but not limited to, squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma and oat cell (small cell) carcinoma; malignant tumors of the stomach, including, but not limited to, adenocarcinoma, fungiform (polypoid), ulcerated, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma and carcinosarcoma; malignant intestinal tumors; malignant tumors of the rectum; malignant tumors of the liver, including, but not limited to, hepatocellular carcinoma and hepatoblastoma, malignant tumors of the gallbladder, including, but not limited to, adenocarcinoma; cholangiocarcinomas, including, but not limited to, papillary, nodular and diffuse; malignant lung tumors, including, but not limited to, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer; malignant testicular tumors, including, but not limited to, germinoma, seminoma, anaplastic, classical (typical), spermatocytic, non-seminoma, embryonal carcinoma, teratoma, carcinoma, choriocarcinoma (yolk sac tumor), malignant prostate tumors, including , but not limited to, adenocarcinoma, leiomyosarcoma and rhabdomyosarcoma; malignant tumors of the penis; malignant tumors of the oral cavity, including, but not limited to, squamous cell carcinoma; basal cell malignant tumors; malignant tumors of the salivary glands, including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma and adenoid cystic carcinoma; malignant tumors of the pharynx, including, but not limited to, squamous cell carcinoma and verrucous carcinoma; malignant skin tumors, including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; malignant kidney tumors, including, but not limited to, renal cell carcinoma, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/or ureter); Wilms tumor; malignant tumors of the bladder, including, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangiotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenosarcoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinomas (for a review of such disorders, see in Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America).

b. Treatment of infections

The disclosed antibody compositions and methods can be used to treat infections and infectious diseases. Typically, the agents are used to stimulate or enhance an immune response to an infection in a subject by administering to the subject an amount of one or more of the disclosed antibodies or antigen binding fragments thereof that convey an activating or stimulatory signal involving PD-1. The method may reduce one or more symptoms of the infection.

The infection or disease may be caused by a bacterium, virus, protozoan, helminth, or other microbial pathogen that enters intracellularly and is attacked, for example, by cytotoxic T lymphocytes.

The infection or disease can be acute or chronic. An acute infection is typically an infection of short duration. During acute microbial infection, immune cells begin to express immunomodulatory receptors. Accordingly, in some embodiments, the method includes enhancing an immune stimulatory response against an acute infection.

The infection may be caused by, for example, but not limited to, Candida albicans , Listeria monocytogenes , Streptococcus pyogenes , Streptococcus pneumoniae , Neisseria meningitidis , Staphylococcus aureus , Escherichia coli , Acinetobacter baumannii , Pseudomonas aeruginosa or Mycobacterium .

In some embodiments, the disclosed antibody compositions are used to treat chronic infections, such as infections in which T cell exhaustion or T cell anergy has occurred, causing the infection to persist in the host for an extended period of time.

Exemplary infections to be treated are chronic infections caused by hepatitis virus, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), herpes virus, Epstein-Barr virus, or human papillomavirus.

Since viral infections are cleared predominantly by T cells, increasing T cell activity would be therapeutically beneficial in situations where more rapid or thorough clearance of the infectious viral agent would be advantageous in an animal or human subject. Thus, the disclosed compositions can be administered for the treatment of local or systemic viral infections, including, but not limited to, immunodeficiency (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g. , human influenza A virus) and colds (eg human rhinovirus) and other viral infections caused by, for example, HTLV, hepatitis virus, respiratory syncytial virus, vaccinia virus and rabies virus. The molecules can be administered topically to treat viral skin diseases such as cold sores or herpes zoster or genital warts. The molecules can also be administered systemically to treat systemic viral diseases, including, but not limited to, AIDS, influenza, colds or encephalitis.

Illustrative infections that may be treated include, but are not limited to, infections caused by microorganisms including, but not limited to , Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium , Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, Yersinia, Cryptoc occus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mans oni.

Other microorganisms that may be treated by the disclosed compositions and methods include bacteria such as Klebsiella, Serratia, Pasteurella; pathogens associated with cholera, tetanus, botulism, anthrax, plague and Lyme disease; or fungal or parasitic pathogens such as Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix (schenkii), Blastomyces (dermatitidis), Paracoccidioides (brasiliensis), Coccidioides (immitis) and Histoplasma (capsulatuma), Entamoeba, histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondi, etc.), Sporothrix, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba, Histolytica, Balantidium, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium, Babesia or Trypanosoma, etc.

V. Combination therapies to enhance immune responses

The disclosed antibodies and antigen binding fragments thereof and compositions thereof can be administered to a subject in need thereof, alone or in combination with one or more additional therapeutic agents. In some embodiments, the antibodies and antigen-binding fragments thereof and the additional therapeutic agent are administered separately but simultaneously. The antibodies and antigen binding fragments thereof and the additional therapeutic agent may also be administered as part of the same composition. In some embodiments, the antibodies and antigen-binding fragments thereof and the second therapeutic agent are administered separately and at different times, but as part of the same treatment regimen. Additional therapeutic agents may be administered before, after, or alternately with the administration of the disclosed antibodies and their antigen-binding fragments.

The subject may be administered the first therapeutic agent 1, 2, 3, 4, 5, 6 or more hours or 1, 2, 3, 4, 5, 6, 7 or more days before the administration of the second therapeutic agent. In some embodiments, the subject may be administered one or more doses of the first agent every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, or 48 days prior to the first administration of the second agent. Antibodies and antigen binding fragments thereof may constitute a first or second therapeutic agent.

Antibodies and antigen-binding fragments thereof and an additional therapeutic agent may also be administered as part of the treatment regimen. For example, if the first therapeutic agent may be administered to a subject on every fourth day, then the second therapeutic agent may be administered on the first, second, third or fourth day, or combinations thereof. The first therapeutic agent or the second therapeutic agent may be re-administered throughout the treatment regimen.

Illustrative additional therapeutic agents include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapy drugs, enzymes, antibiotics, antivirals (especially protease inhibitors alone or in combination with nucleosides for the treatment of HIV or hepatitis B or C) , antiparasitic drugs (for the treatment of helminths, protozoa), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and their bioactive fragments (including humanized, single-chain and chimeric antibodies), drugs based on antigens and vaccines (including adjuvants ), peptide drugs, anti-inflammatory drugs, ligands that bind to Toll-like receptors (including, but not limited to, CpG oligonucleotides) to activate the innate immune system, molecules that mobilize and adapt the immune system, other molecules, that activate or enhance the action of cytotoxic T lymphocytes, natural killer cells and T helper cells, as well as other molecules that deactivate or negatively regulate suppressor or regulatory T cells.

Additional therapeutic agents are selected based on the pathological condition, disorder or disease being treated. For example, an immunomodulatory agent may be administered in conjunction with one or more additional agents that function to enhance or activate the immune response or to weaken or inhibit the immune response.

A. Antimicrobials

In one embodiment, the antibodies and antigen-binding fragments thereof may be administered to a subject in combination with an antimicrobial agent, such as an antibiotic, an antifungal agent, an antiviral agent, an antiparasitic agent, or an essential oil. In another embodiment, the disclosed antibodies and antigen-binding fragments thereof may be used in a prophylactic or prophylactic role in the treatment and prevention of disease, as well as in the context of severe traumatic injuries such as a major burn, open fracture, accidental amputation, or other injuries.

In some embodiments, the subject is administered antibodies and antigen-binding fragments thereof and/or an antimicrobial agent during a hospital admission to prevent subsequent bacterial, fungal, or viral complications. An antibiotic can specifically target pathogens, and antibodies and their antigen-binding moieties can stimulate the immune system to mount an enhanced response to treat or prevent subsequent infection or disease.

1 . Chemotherapeutic agents

Antibodies and antigen-binding fragments thereof can be combined with one or more chemotherapeutic agents and proapoptotic agents. Exemplary chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed , pentostatin, procarbazine, raltitrexide, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof. Exemplary proapotonic agents include, but are not limited to, fludarabine, staurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), and combinations thereof.

2. Other immunomodulators

a. PD-1 antagonists

In some embodiments, the antibodies and antigen binding fragments thereof are co-administered with a PD-1 antagonist. Programmed cell death protein-1 (PD-1) is a member of the CD28 receptor family that delivers a negative immune response when induced on T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces T cell multiplication and/or the strength and/or duration of the T cell response. Suitable PD-1 antagonists are described in US Pat. Nos. 8,114,845, 8,609,089, and 8,709,416, which are specifically incorporated herein by reference in their entirety, and include compounds or agents that both bind to and block PD-1 ligand it to disrupt or inhibit ligand binding to the PD-1 receptor, or bind directly to and block the PD-1 receptor without inducing inhibitory signal transmission through the PD-1 receptor.

In some embodiments, a PD-1 receptor antagonist binds directly to the PD-1 receptor without causing inhibitory signal transmission, and also binds to a PD-1 receptor ligand for the purpose of attenuating or inhibiting ligand-mediated signaling through the PD-1 receptor. By reducing the number and/or content of ligands that bind to the PD-1 receptor and cause inhibitory signal transmission, fewer cells are attenuated by the negative signal delivered by PD-1 signaling, and a more robust immune response can be achieved.

PD-1 signaling is thought to be regulated by binding to a PD-1 ligand (such as B7-H1 or B7-DC) in close proximity to a peptide antigen presented by the major histocompatibility complex (MHC) (see, for example, Freeman, Proc. Natl. Acad. Sci. US A , 105:10275-10276 (2008). Thus, proteins, antibodies or small molecules that prevent coligation of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.

In some embodiments, PD-1 receptor antagonists are small molecule antagonists or antibodies that attenuate or disrupt PD-1 receptor signaling by binding to PD-1 ligands or to PD-1 itself, particularly in cases where PD coligation -1 to the TCR does not follow such binding, thereby causing no inhibitory signal transmission through the PD-1 receptor. Other PD-1 antagonists provided by the methods of this invention include antibodies that bind to PD-1 or PD-1 ligands and other antibodies.

Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following US patents: 7332582, 7488802, 7521051, 7524498, 7563869, 7981416, 8088905, 8287856, 8580247, 8728474, 87 79105, 9067999, 9073994, 9084776, 9205148, 9358289, 9387247, 9492539, 9492540, all of which are incorporated by reference in their entirety.

See also Berger et al., Clin. Cancer Res. , 14:30443051 (2008).

Exemplary anti-PD-L1 antibodies include, but are not limited to, those described in the following US Patent Nos. 8383796, 9102725, 9273135, 9393301 and 9580507, all of which are specifically incorporated herein by reference in their entirety .

For anti-B7-DC (also called anti-PD-L2) antibodies, see US Patent Nos: 7411051, 7052694, 7390888, 8188238 and 9255147.

Other exemplary PD-1 receptor antagonists include, but are not limited to, PD-L2 polypeptides, including homologues and variants thereof, as well as active fragments of any of the foregoing and fusion proteins that include any of them. In some embodiments, the fusion proteins include a solute region of B7-DC linked to the Fc region of an antibody, such as a human IgG, and does not include all or a portion of the transmembrane region of human B7-DC.

The PD-1 antagonist may also be a fragment of mammalian PD-L1, for example from a mouse or a primate such as a human, wherein the fragment binds to and blocks PD-1 but does not result in transmission of an inhibitory signal through PD-1. The fragments may also be part of a fusion protein, such as an Ig fusion protein.

Other useful PD-1 antagonist polypeptides include those that bind to PD-1 receptor ligands. They include PD-1 receptor protein or solubilized fragments thereof that bind to PD-1 ligands such as PD-L1 or B7-DC and prevent binding to the endogenous PD-1 receptor, thereby preventing transmission of the inhibitory signal. It has also been demonstrated that PD-L1 binds to the B7.1 protein (Butte et al., Immunity , Vol. 27, pp. 111-122, (2007)). Such fragments also include the solute portion of the PD-1 ECD protein, which contains mutations such as the A99L mutation, which enhances binding to natural ligands (Molnar et al., PNAS , 105:10483-10488 (2008)). B7-1 or solubilized fragments thereof that bind to the PD-L1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing transmission of the inhibitory signal, are also useful.

PD-1 and PD-L1 antisense nucleic acids, both DNA and RNA, as well as siRNA molecules, can also be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 on T cells, as well as the production of T cell ligands such as PD-L1 and/or PD-L2. For example, an siRNA (e.g., about 21 nucleotides in length that is specific for a gene encoding PD-1 or encoding a PD-1 ligand, and the oligonucleotides of which are readily available commercially) complexed with carriers such as polyethylenimine (see Cubillos-Ruiz et al., J Clin Invest 119(8): 2231-2244 (2009), is readily taken up by cells that express PD-1 as well as PD-1 ligands and attenuates the expression of these receptors and ligands in order to achieve a decrease in inhibitory signal transmission in T cells, thereby activating T cells.

b. CTLA4 antagonists

Other molecules useful in mediating the effects of T cells in the immune response are also provided as additional therapeutic agents. In some embodiments, the molecule is a CTLA4 antagonist, such as an anti-CTLA4 antagonist antibody. An example of an anti-CTLA4 antibody for use in the methods of this invention includes the antibody described in PCT/US2006/043690 (Fischkoff et al., WO/2007/056539).

Doses of anti-PD-1, anti-B7-H1 and anti-CTLA4 antibodies are known in the art and may range, for example, from 0.1 to 100 mg/kg or in shorter ranges from 1 to 50 mg/kg. kg or from 10 to 20 mg/kg. A suitable dose for a human subject may be from 5 to 15 mg/kg, with 10 mg/kg of antibody (eg, human anti-PD-1 antibody) being a particular embodiment.

Specific examples of an anti-CTLA4 antibody useful in the methods of this invention are ipilimumab, a human anti-CTLA4 antibody administered at a dose of, for example, about 10 mg/kg, and tremelimumub, a human anti-CTLA4 antibody administered at a dose of, for example, about 15 mg/kg. See also Sammartino, et al., Clinical Kidney Journal , 3(2):135–137 (2010), published online December 2009.

In other embodiments, the antagonist is a small molecule. A number of small organic molecules have been demonstrated to bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe et al., J. Biol. Chem. , 277:7363-7368 (2002). Such small organic molecules can be administered into alone or together with an anti-CTLA4 antibody to attenuate T cell inhibitory signal transmission.

3. Potentiating agents

In some embodiments, the additional therapeutic agents include a potentiating agent. The potentiating agent acts to enhance the effectiveness of the positive regulator of the immune response, possibly through more than one mechanism, although the exact mechanism of action is not necessary for the broad practical application of this invention.

In some embodiments, the potentiating agent is cyclophosphamide. Cyclophosphamide (CTX, Cytoxan ^® or Neosar ^® ) is an oxazophosphorine drug, and analogues include ifosfamide (IFO, Ifex), perfosfamide, trofosfamide (trofosfamide; ixotene) and pharmaceutically acceptable salts, solvates, prodrugs and their metabolites (application US Pat. No. 20070202077, which is incorporated in its entirety). Ifosfamide (MITOXANA ^® ) is a structural analogue of cyclophosphamide and is believed to have an identical or substantially similar mechanism of action to that of cyclophosphamide. Perfosfamide (4-hydroperoxycyclophosphamide) and trophosfamide are also alkylating agents that are structurally related to cyclophosphamide. For example, perfosfamide alkylates DNA, thereby inhibiting DNA replication and the synthesis of RNA and proteins. New oxazophosphorine derivatives have been developed and evaluated for increased selectivity and response with reduced host toxicity (Liang J, Huang M, Duan W, Yu XQ, Zhou S. Design of new oxazaphosphorine anticancer drugs. Curr Pharm Des. 2007;13(9 ):963-78. Review). These include mafosfamide (NSC 345842), glufosfamide (D19575, beta-D-glucosylphosphoramidiprit), S-(-)-bromophosphamide (CBM-11), NSC 612567 (aldophosphamide perhydrothiazine), and NSC 613060 (aldophosphamide thiazolidine). Mafosfamide is an oxazophosphorine analogue, which is a chemically stable 4-thioethanesulfonic acid salt of 4-hydroxy-CPA. Glufosfamide is an IFO derivative in which isophosphoramidiprit, an alkylating metabolite of IFO, is glycosidically linked to a beta-D-glucose molecule. Additional cyclophosphamide analogues are described in US Pat. No. 5,190,929 entitled “Cyclophosphamide Analogs Useful as Antineoplastic Agents,” which is incorporated herein by reference in its entirety.

Although CTX itself is nontoxic, some of its metabolites are cytotoxic alkylating agents that cause DNA cross-linking and, at higher doses, strand breaks. Many cells are resistant to CTX because they express high levels of the detoxifying enzyme aldehyde dehydrogenase (ALDH). CTX targets proliferating lymphocytes because lymphocytes (but not hepatopoietic stem cells) express only minor levels of ALDH and dividing cells are the most sensitive to DNA alkylation agents.

In one embodiment, low doses of CTX are used in combination with the disclosed antibodies and antigen binding fragments thereof. Low doses of CTX (<200 mg/kg) can have immunostimulatory effects, including stimulation of antitumor immune responses in humans and in mouse models of cancer (Brode & Cooke Crit Rev. Immunol. 28:109–126 (2008)). Such low doses are subtherapeutic and have no direct antitumor activity. In contrast, high doses of CTX inhibit the antitumor response. Several mechanisms may explain the role of CTX in potentiating the antitumor immune response: (a) depletion of CD4+CD25+FoxP3+ Tregs (and in particular proliferating Tregs, which may be particularly suppressive), (b) depletion of B cells; (c) induction of nitrite oxide (NO), leading to suppression of tumor cell growth; (d) mobilization and expansion of CD11b+Gr-1+ MDSCs. These primary effects have numerous secondary effects; for example, after Treg depletion, macrophages produce more IFN-γ and less IL-10. It has also been demonstrated that CTX induces type I IFN expression and activates homeostatic lymphocyte proliferation.

Treg depletion is most often cited as the mechanism by which CTX potentiates the antitumor response. This conclusion is based in part on the results of adaptive transfer experiments. In the AB1-HA tumor model, treatment with CTX on day 9 resulted in a 75% cure rate. Transfer of purified Tregs on day 12 almost completely inhibited the CTX response (van der Most et al. Cancer Immunol. Immunother. 58:1219-1228 (2009). A similar result was observed in the HHD2 tumor model: adaptive transfer of CD4+CD25+ Tregs after preliminary treatment with CTX abolished the therapeutic response to the vaccine (Taieb, J. J. Immunol. 176:2722-2729 (2006)).

Numerous human clinical studies have demonstrated that low-dose CTX is a safe, well-tolerated, and effective agent for activating antitumor responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47 :1-12 (1998)).

In one embodiment, the optimal dose of CTX to potentiate an antitumor immune response is one that reduces total T cell numbers by reducing Treg levels below the normal range, but which remains subtherapeutic (see Machiels et al. Cancer Res. 61:3689- 3697 (2001)).

In some embodiments, CTX is used as an immunopotentiating agent at a dose of 300 mg/m ² . In another embodiment, for an average male (6 feet, 170 pounds (78 kg) with a body surface area of 1.98 m ² ) of 300 mg/m ² , the dose of CTX is 8 mg/kg or 624 mg of total protein. In mouse models of cancer, efficacy has been demonstrated at doses ranging from 15-150 mg/kg, corresponding to 0.45-4.5 mg of total protein in a 30 g mouse (Machiels et al. Cancer Res. 61:3689- 3697 (2001), Hengst et al Cancer Res. 41:2163-2167 (1981), Hengst Cancer Res. 40:2135-2141 (1980)).

In the case of larger mammals, such as a primate, such as a human patient, such doses in mg/m ² can be used, however, single doses over a finite period of time can also be used. Such single doses may be administered on a daily basis for a finite period of time, such as up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days, or up to 20 days, or up to 25 days, all of which specifically provided by this invention. The same scheme can be used for other potentiating agents mentioned herein.

In other embodiments, the potentiating agent is an agent that reduces the activity and/or number of regulatory T lymphocytes (T-reg), such as sinitinib ( ^SUTENT® ), anti-TGFβ antibody, or imatinib ( ^GLEEVAC® ). Said treatment regimen may also include administration of an excipient.

Suitable potentiating agents also include mitosis inhibitors, such as paclitaxel, aromatase inhibitors (eg, letrozole), and angiogenesis inhibitors (VEGF inhibitors, eg, Avastin, VEGF-Trap) (see, for example, Li et al., Vascular endothelial growth factor blockade reduces intratumoral regulatory T cells and enhances the efficacy of a GM-CSF-secreting cancer immunotherapy. Clin Cancer Res. 2006 Nov 15; 12(22):6808-16.), anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists and antagonists IL-18.

VI. Transgenic animals

In one embodiment, a transgenic animal is provided that produces antibodies or antigen binding fragments thereof having a heavy chain CDR with the amino acid sequences according to SEQ ID NOs: 6, 7 and 8, and a light chain CDR with the amino acid sequences according to SEQ ID NO: 12, 13 and 14. In one embodiment, the transgenic animal is a rodent, such as a mouse.

In another embodiment, a transgenic animal is provided that produces antibodies or antigen binding fragments thereof having a heavy chain CDR with amino acid sequences in accordance with SEQ ID NOs: 18, 19 and 20, and three different light chain CDRs with amino acid sequences selected from the group, consisting of SEQ ID NOs: 24, 13 and 25. In one embodiment, the transgenic animal is a rodent, such as a mouse.

In another embodiment, a transgenic animal is provided that produces antibodies or antigen binding fragments thereof having a heavy chain CDR with amino acid sequences according to SEQ ID NOs: 29, 30 and 31, and a light chain CDR with amino acid sequences according to SEQ ID NO: 35 , 36 and 37. In one embodiment, the transgenic animal is a rodent, such as a mouse.

Methods for producing transgenic animals that produce antibodies are known in the art. See for example A. Jakobovits, Curr Opin Biotechnol., 6(5):561-6 (1995), and Brüggemann, M., et al., . Arch Immunol Ther Exp (Warsz)., 63(2):101-108 (2015); Jakobovits, A., et al., “From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice.” Nat Biotechnol. 25(10):1134-43 (2007); Lonberg N. (2005) “Human antibodies from transgenic animals.” Nat Biotechnol. 23(9):1117-25, and US Patent Nos. 9708635; 9686970; 9499838; 9445581; 9388446; 8835712; 8703485; 8232449; 7795494; and 5939598.

Examples

Example 1: Preparation of Anti-PD-1 Antibodies

results

Production of anti-PD-1 antibodies resulted in clones 4G9, 4C12, and 5C2, which were selected for characterization.

Example 2: Kinetics of interaction between anti-PD-1 antibodies and PD-1

Materials and methods

Antibodies from clones 4G9, 4C12 and 5C2 were characterized using the Biocore™ system available from GE. The analyte was mouse or human PD-1 and the ligand was an anti-PD-1 antibody. Analyte concentrations were 0, 62.5, 125, 250, 500, and 1000 nM as indicated.

results

In FIG. 1 and in Table. Figure 1 depicts an analysis of the interaction of 4G9 with human PD-1. The equilibrium association constant (K _A ) was 9.52×10 ⁵ (1/M). The equilibrium dissociation constant (K _D ) was 1.05×10 ^-6 (M).

Table 1 . Analysis of the interaction of 4G9 with human PD-1.

Ligand Analyst Ka (1/Ms) Kd (1/s) Rmax KA (1/M) KD(M) Conc. (Nm) Chi ² 4G9
580 OE human PD-1 But. But. 15.1 9.52×10 ⁵ 1.05x 10 ^-6 0
125
250
500
500
1000 0.464

In FIG. 2 and in Table. Figure 2 shows an analysis of the interaction of 4G9 with mouse PD-1. The equilibrium association constant (K _A ) was 1.94×10 ⁵ (1/M). The equilibrium dissociation constant (K _D ) was 5.15×10 ^-6 (M).

Table 2. Analysis of the interaction of 4G9 with mouse PD-1.

Ligand Analyst Ka (1/Ms) Kd (1/s) Rmax KA (1/M) KD(M) Conc. (Nm) Chi ² 4G9
580 OE human PD-1 2.00×10 ³ 0.0103 38.8 1.94×10 ⁵ 5.15x 10 ^-6 0
125
250
500
500
1000 0.112

In FIG. 3 and in Table. Figure 3 depicts an analysis of the interaction of 4C12 with human PD-1. The equilibrium association constant (K _A ) was 3.14×10 ⁶ (1/M). The equilibrium dissociation constant (K _D ) was 3.19×10 ^-7 (M).

Table 3 . Analysis of the interaction of 4C12 with human PD-1.

Ligand Analyst Ka (1/Ms) Kd (1/s) Rmax KA (1/M) KD(M) Conc. (Nm) Chi ² 4C12
425 OE human PD-1 But. But. 33.9 3.14×10 ⁶ 3.19x 10 ^-7 0
125
250
500
500
1000 2.33

In FIG. 4 and in Table. Figure 4 depicts an analysis of the interaction of 5C2 with human PD-1. The equilibrium association constant (K _A ) was 2.02×10 ⁵ (1/M). The equilibrium dissociation constant (K _D ) was 4.95×10 ^-6 (M).

Table 4. Analysis of the interaction of 5C2 with human PD-1.

Ligand Analyst Ka (1/Ms) Kd (1/s) Rmax KA (1/M) KD(M) Conc. (Nm) Chi ² 5C2
320 OE human PD-1 But. But. 28.4 2.02×10 ⁵ 4.95x 10 ^-6 0
125
250
500
500
1000 0.0263

In FIG. 5 and in Table. Figure 5 depicts an analysis of the interaction of 5C2 with mouse PD-1. The equilibrium association constant (K _A ) was 1.18×10 ⁶ (1/M). The equilibrium dissociation constant (K _D ) was 8.50×10 ^-7 (M).

Table 5. Analysis of the interaction of 5C2 with mouse PD-1.

Ligand Analyst Ka (1/Ms) Kd (1/s) Rmax KA (1/M) KD(M) Conc. (Nm) Chi ² 5C2
320 OE human PD-1 But. But. eleven 1.18×10 ⁶ 8.50x 10 ^-7 0
125
250
500
500
1000 0.107

Example 3: Anti-PD-1 Antibody Binding to EL4 Cells

Materials and methods

Mouse EL4 cells, which constitutively express PD-1, were used in a fluorescence-activated cell sorter to assess the binding of 4G9, 5C2, and 4C12 to the cells.

results

In FIG. 6A is a flow cytometry histogram showing that the commercial anti-PD-1 antibody binds to EL4 cells and the isotype control antibody does not bind to EL4 cells. In FIG. 6B is a flow cytometry histogram showing that 4G9, 5C2 and 4C12 bind to EL4 cells, but the secondary antibody alone does not bind. In FIG. 6C is a flow cytometry histogram showing that the anti-PD-1 antibody 4G9 binds to EL4 cells, but the secondary antibody alone does not. In FIG. 6D is a flow cytometry histogram showing that the anti-PD-1 antibody 5C2 binds to EL4 cells, but the secondary antibody alone does not. In FIG. 6E is a flow cytometry histogram showing that the anti-PD-1 antibody 4C12 binds to EL4 cells, but the secondary antibody alone does not.

Example 4: Agonist activity of anti-PD-1 antibodies

Materials and methods

Mouse CD4 T cells

Purified mouse CD4 T cells were stimulated with anti-CD3/anti-CD28 Ab for 48 hours, then anti-PD-1 Ab (10 μg/ml) was added to the culture along with protein A beads for an additional 48 hours. In some samples, anti-PD-L1 Abs were added to block the PD-1/PD-L1 interaction in order to analyze the agonistic effect of the tested Abs. The CBA assay was used to determine the concentrations of IFNγ and IL-2 in the supernatants.

Human CD4 T cells

Purified human CD4 T cells were stimulated with anti-CD3/anti-CD28 Ab for 48 hours, then anti-PD-1 Ab (1 or 10 μg/ml) was added to the culture along with protein A beads for an additional 48 hours. The CBA assay was used to determine IFNγ concentrations in the supernatants.

results

In FIG. 7A and 7B show that stimulated CD4 T cells treated with anti-PD-1 antibodies 5C2, 4C12 and 4G9 had higher concentrations of IFNγ and IL-2 in their supernatants compared to untreated cells or cells treated with anti-PD-L1 antibodies. In FIG. 7C shows that stimulated human CD4 T cells treated with anti-PD-1 antibodies 4G9 and 5C2 had a higher concentration of IFNγ supernatant compared to untreated cells or cells treated with an isotype control antibody.

Example 5: Hybridomas Increase Akt Phosphorylation

Materials and methods

Intracellular pAKT staining (S473) was used as a marker of T cell activation.

results

Hybridomas from mice immunized with peptide E increased Akt (S473) phosphorylation in mouse CD4 T cells (Figure 8).

Example 6: Characterization of Three Anti-PD-1 Antibodies

Materials and methods

Purified antibodies from hybridomas 5C2, 4C12 and 4G9 were characterized.

results

The isotypes for each of the three anti-PD-1 antibodies were determined. Both 5C2 and 4C12 belonged to the IgG1 isotype, and 4G9 was found to belong to the IgG2b isotype (Fig. 9). Sequencing of hybridomas showed 100% sequence identity in the case of antibodies 5C2 and 4C12.

Example 7: 4G9 and 5C2 specifically bind to human PD-1

Materials and methods

ELISA was used to determine the binding of 4G9 and 5C2 to human PD-1-Fc.

results

In FIG. 10 shows the ELISA results for assessing the binding of antibodies 4G9 and 5C2 to human PD-1-Fc. Both 4G9 and 5C2 bind specifically to human PD-1. In FIG. 11 A depicts a flow cytometry histogram demonstrating that 4G9 and 5C2 do not bind PD-1 KO CD4 T cells, while they do bind CD4 T cells from wild-type mice (Fig. 11 B).

Example 8: Signal transmission

Materials and methods

Mouse CD4 T cells were prestimulated for 48 hours to promote PD-1 expression and then treated with purified 4G9 and 5C2 antibodies. The pS6 concentration was determined using an ELISA kit (Cell Signaling Tech).

results

Unlike blocking antibodies (RMP1-14 and J43), 4G9 and 5C2 activate T cells through a pathway involving S6. Treatment of mouse CD4 T cells prestimulated for 48 hours (to promote PD-1 expression) with purified 4G9 and 5C2 antibodies resulted in a significant increase in pS6 within the linear range (ELISA kit, Cell Signaling Tech, * P < 0.05, * * P < 0.01, *** P < 0.001 compared with untreated). This phenomenon is due to direct activation rather than blockade of PD-1/PD-L1, since treatment with commercial AT-1 (RMP1-14) and AT-2 (J43), which block the PD-1/PD-L1 interaction, does not result in to an increase in pS6.

Example 8: In Vivo Efficacy Evaluation

Materials and methods

In FIG. 13A is a schematic illustration of the TC-1 tumor model used in this experiment. Briefly, mice were subcutaneously injected with TC-1 tumor cells on day 0. On day 10 (D10), day 17 (D17) and day 24 (D24) after tumor injection, mice were treated with vaccine (E7+PADRE+Quil A). Mice were treated with anti-PD-1 antibodies on day 10, day 14, day 17, day 21, day 24 and day 28.

results

Anti-PD-1 antibodies 4G9, 4C12 and 5C2 reduced tumor volume and increased survival when combined with the E7 vaccine (Figures 13B-13D).

Example 9: In Vivo Efficacy Evaluation - Anti-EpE Antibodies

Materials and methods

Antibodies against etipop E were generated and tested in the TC-1 tumor model as described in Example 8 above and in FIG. 14A.

results

Mice treated with 4G9, an antibody raised against the E epitope, exhibited significantly lower tumor volume and higher survival rates compared to traditional checkpoint inhibitors, anti-PD1 blocking antibodies RMP1-14 and J43 (Figures 14B-14C).

Claims (43)

1. An antibody or antigen-binding fragment thereof that specifically binds to PD-1, where the antibody or antigen-binding fragment contains three heavy chain CDRs (HCDR1, HCDR2 and HCD3) contained in the heavy chain variable region (HCVR) of the sequence SEQ ID NO:27 or SEQ ID NO:28; And three light chain CDRs (LCDR1, LCDR2 and LCD3) contained in the light chain variable region (LCVR) of the sequence SEQ ID NO:33 or SEQ ID NO:34. 2. An antibody or antigen-binding fragment thereof that specifically binds to PD-1, where the antibody or antigen-binding fragment contains Heavy chain CDR1 with sequence SEQ ID NO:29, Heavy chain CDR2 of SEQ ID NO:30, and Heavy chain CDR3 of SEQ ID NO:31, and Light chain CDR1 with sequence SEQ ID NO:35, Light chain CDR2 of SEQ ID NO:36, and Light chain CDR3 of SEQ ID NO:37. 3. An antibody or antigen binding fragment thereof as claimed in claim 1 or 2, comprising a heavy chain that is at least 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:27 or 28. 4. An antibody or antigen binding fragment thereof as claimed in claim 1 or 2, comprising a light chain that is at least 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:33 or 34. 5. Antibody or antigen-binding fragment thereof according to claim 1 or 2, containing a heavy chain that is at least 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:27 or 28, and a light chain that is at least 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:33 or 34. 6. A transgenic rodent producing an antibody or an antigen-binding fragment thereof according to any one of claims 1-5, containing a nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-5. 7. The transgenic rodent according to claim 6, wherein the rodent is a mouse. 8. Nucleic acid encoding an antibody or antigen-binding fragment according to any one of claims 1 to 5. 9. A cell for producing an antibody or antigen-binding fragment according to any one of claims 1 to 5, containing a nucleic acid according to claim 8. 10. The cell of claim 9, wherein the nucleic acid is expressed from a vector. 11. An antibody or antigen binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen binding fragment thereof is human, murine, chimeric, humanized or monoclonal. 12. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is human or chimeric. 13. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or an antigen-binding fragment thereof is a full-length immunoglobulin. 14. An antibody or antigen binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen binding fragment thereof is IgG, IgE, IgM, IgD, IgA or IgY. 15. An antibody or antigen binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen binding fragment thereof is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2. 16. An antibody or antigen binding fragment thereof according to any one of claims 1 to 5, wherein the antigen binding fragment is selected from the group consisting of Fab', F(ab') ₂ , Fv, single chain (ScFv), di-ScFv, diabody and tribody. 17. An antibody or antigen-binding fragment thereof according to any one of claims 1-5 or 11-16, which immunospecifically binds to TYLCGAISLAPKAQI (SEQ ID NO:38) PD-1 on the surface of an immune cell. 18. The antibody or antigen-binding fragment thereof according to claim 17, wherein the immune cell is a T cell. 19. The antibody or antigen-binding fragment thereof according to claim 18, wherein the T cell is a CD8+ T cell. 20. An antibody or antigen binding fragment thereof according to any one of claims 1 to 5 or claim 19, which immunospecifically binds to SEQ ID NO:38 on PD-1 expressed on the surface of an immune cell and induces or stimulates a signal involving PD-1 1, which activates or stimulates an immune cell. 21. A pharmaceutical composition for inducing, stimulating or enhancing an immune response, containing an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-5 or 11-19 and a pharmaceutically acceptable excipient. 22. The pharmaceutical composition according to claim 21, further containing a second therapeutic agent. 23. The pharmaceutical composition according to claim 22, wherein the second therapeutic agent is cyclophosphamide. 24. A method for inducing, stimulating or enhancing an immune response in a patient, including: administering to the patient an effective amount of an antibody or antigen-binding fragment according to any one of claims 1-5 or 11-19 or a pharmaceutical composition according to any one of claims 21-23 for inducing, stimulating or enhancing an immune response in the patient. 25. A method of treating cancer in a patient, comprising: administering to a patient an effective amount of an antibody or antigen binding fragment according to any one of claims 1-5 or 11-19, or a pharmaceutical composition according to any one of claims 21-23 for treating cancer in a patient. 26. A method for reducing the tumor load in a patient, including: administering to the patient an effective amount of an antibody or antigen-binding fragment according to any one of claims 1-5 or 11-19, or a pharmaceutical composition according to any one of claims 21-23 to reduce the tumor burden in the patient. 27. A method of treating an infection in a patient, including: administering to a patient an effective amount of an antibody or antigen binding fragment according to any one of claims 1-5 or 11-19 or a pharmaceutical composition according to any one of claims 21-23 for treating a chronic infection in a patient.