Antibody or antigen-binding fragment and epitope or fusion protein, nucleic acid, pharmaceutical composition, and, uses of an antibody or antigen-binding fragment or a pharmaceutical composition to prepare a medicine to induce, promote or enhance an immune response, to treat cancer, to reduce the tumor burden and to treat an infection in a subject in need

Abstract

Antibodies and their antigen-binding fragments are provided that bind immunospecifically to pd-1, preferentially human or mouse pd-1, and induce or promote an immune response that activates the proliferation or activity of immune cells. Contrary to the existing paradigm that pd-1 exclusively promotes a suppressive immune response, the released antibodies and their antigen-binding fragments bind immunospecifically to pd-1 and cause an activation signal to be delivered to the immune cell that activates the immune cell, rather than suppressing the immune cell. In one embodiment, the released antibodies and their antigen-binding fragments bind specifically to pd-1 expressed on immune cells.The binding of antibodies and their antigen-binding fragments to PD-1 on immune cells causes an activation signal to be transmitted to the immune cell, for example, a signal that increases or promotes cytokine production and / or the activation of immune cell proliferation. Immune cells that express PD-1 include, but are not limited to, BET cells, as well as cells derived from myeloid lineages. In one embodiment, the immune cell is a T cell, preferably a CD8+ T cell.

Description

/ 98 ANTIBODY OR ANTIGEN-BINDING FRAGMENT OR EPITOPE OR FUSION PROTEIN, NUCLEIC ACID, PHARMACEUTICAL COMPOSITION, AND, USES OF AN ANTIBODY OR ANTIGEN-BINDING FRAGMENT OR A PHARMACEUTICAL COMPOSITION TO PREPARE A MEDICINAL MEDICINE TO INDUCE, PROMOTE OR ENHANCE AN IMMUNE RESPONSE, TO TREAT CANCER, TO REDUCE TUMOR LOAD AND TO TREAT AN INFECTION IN A SUBJECT IN NEED CROSS-REFERENCE TO RELATED REQUESTS

[001] This request claims benefit of and priority over Requests Provisional USN 62 / 555,156 filed on September 7, 2017, 62 / 624,843 filed on February 1, 2018, and 62 / 657,323 filed on April 13, 2018, are all incorporated by reference in their entirety. REFERENCE TO THE LISTING OF SEQUENCES

[002] The Sequence Listing submitted on September 6, 2018, as a text file named 064466.071 sequence listing_ST25.txt created on August 21, 2018, and with a size of 55.2 kilobytes, is incorporated by reference pursuant to 37 CFR § 1.52(e)(5). TECHNICAL FIELD OF THE INVENTION

[003] The invention relates generally to immunomodulation and antibodies that bind specifically to PD-1 and methods of using them. FUNDAMENTALS OF THE INVENTION

[004] The programmed cell death receptor protein (PD1) / programmed cell death receptor protein ligand (PD-L1) pathway has shown promising clinical success as a cancer immunotherapy target. Current antibodies targeting PD-1 or PD-L1 may block this Petition 870200039602, dated 03 / 26 / 2020, page 9 / 113 / 98 interaction and increase the immune response against cancer cells. Successful clinical trials with monoclonal antibodies against PD-1 and other immune checkpoint inhibitors have opened new avenues in cancer immunology. However, the failure of a large subset of cancer patients to respond to new immunotherapies has led to intensified research on combination therapies and predictive biomarkers (Iwai, Y., et al., Journal of Biomedical Science, 24:26 (2017)).

[005] Thus, it is an objective of the invention to provide compositions and methods for modulating the PD-1 signal transduction.

[006] Another objective of the invention is to provide antibodies and their antigen-binding fragments that bind specifically to PD-1 and modulate PD-1 signal transduction.

[007] Another object of the invention is to provide compositions and methods for the treatment of cancer.

[008] Another object of the invention is to provide compositions and methods for the treatment of infections. SUMMARY OF THE INVENTION

[009] Antibodies and their antigen-binding fragments are provided that bind immunospecifically to PD-1, preferentially human or mouse PD-1, and induce or promote an immune response that activates the proliferation or activity of immune cells. In one embodiment, the released antibodies and their antigen-binding fragments bind specifically to PD-1 expressed on immune cells. The binding of the released antibodies and their antigen-binding fragments to PD-1 on immune cells causes an activation signal to be transmitted to the immune cell, for example, a signal that increases or promotes cytokine production and / or activation of immune cell proliferation. Immune cells expressing PD-1 include, but are not limited to, B and T cells, as well as cells derived from myeloid lineages (Riley, J., Immunol Rev. 229). Petition 870200039602, dated 03 / 26 / 2020, page 10 / 113 / 98 (1):114-125 (2009)). In one embodiment, the immune cell is a T cell, preferably a CD8+ T cell.

[0010] Another embodiment provides a method for stimulating, promoting, or enhancing an adaptive immune response in a subject in need, by administering to the subject an effective amount of the released anti-PD-1 antibodies or their antigen-binding fragment to induce, enhance, or promote an adaptive immune response in the subject.

[0011] One embodiment provides an antibody or its antigen-binding fragment possessing heavy chain complementarity-determining regions (CDRs) with amino acid sequences according to SEQ ID NOs:6, 7 and 8 and light chain CDRs with amino acid sequences according to SEQ ID NOs:12, 13 and 14, wherein the antibody or its antigen-binding fragment binds immunospecifically to PD-1.

[0012] An embodiment provides an antibody or its antigen-binding fragment having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4 or 5.

[0013] An embodiment provides an antibody or its antigen-binding fragment having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 10 or 11.

[0014] One embodiment provides an antibody or its antigen-binding fragment possessing a heavy chain with sequence identity of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% with 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 with SEQ ID NO: 10 or 11.

[0015] One modality provides a manipulated transgenic animal Petition 870200039602, dated 03 / 26 / 2020, p. 11 / 113 / 98 to express any of the disclosed antibodies or their antigen-binding fragments. In one embodiment, the animal is a mouse.

[0016] One embodiment provides a nucleic acid encoding a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4 or 5.

[0017] One embodiment provides a nucleic acid encoding a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 10 or 11.

[0018] One embodiment provides an antibody or its antigen-binding fragment that has heavy chain CDRs with amino acid sequences according to SEQ ID NOs: 18, 19 and 20, and light chain CDRs with amino acid sequences according to SEQ ID NOs: 24, 13 and 25.

[0019] One embodiment provides an antibody or its antigen-binding fragment having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 16 or 17.

[0020] One embodiment provides an antibody or its antigen-binding fragment having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 22 or 23.

[0021] One embodiment provides an antibody or its antigen-binding fragment having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with 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 with SEQ ID NO: 22 or 23.

[0022] One embodiment provides a nucleic acid encoding a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% Petition 870200039602, dated 03 / 26 / 2020, p. 12 / 113 / 98 or 100% sequence identity with SEQ ID NO: 16 or 17.

[0023] One embodiment provides a nucleic acid encoding a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 22 or 23.

[0024] One embodiment provides an antibody or its antigen-binding fragment that has heavy chain CDRs with amino acid sequences according to SEQ ID NOs: 29, 30 and 31, and light chain CDRs with amino acid sequences according to SEQ ID NOs: 35, 36 and 37.

[0025] One embodiment provides an antibody or its antigen-binding fragment having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 27 or 28.

[0026] An embodiment provides an antibody or its antigen-binding fragment having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 33 or 34.

[0027] One embodiment provides an antibody or its antigen-binding fragment having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with 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 with SEQ ID NO: 33 or 34.

[0028] One embodiment provides a nucleic acid encoding a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 27 or 28.

[0029] One embodiment provides a nucleic acid encoding a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 33 or 34. Petition 870200039602, dated 03 / 26 / 2020, p. 13 / 113 / 98

[0030] One embodiment provides an antibody or its antigen-binding fragment containing three light chain CDRs with amino acid sequences that are selected from the group consisting of the SEQ ID NOs: 12, 13, 14, 24, 25, 35, 36 or 37.

[0031] Another embodiment provides an antibody or its antigen-binding fragment containing three heavy chain CDRs with amino acid sequences that are selected from the group consisting of the SEQ ID NOs: 6, 7, 8, 18, 19, 20, 29, 30 or 31.

[0032] Another embodiment provides an antibody or its antigen-binding fragment containing three light chain CDRs with amino acid sequences that are selected from the group consisting of SEQ ID NOs: 12, 13, 14, 24, 25, 35, 36 or 37, and three heavy chain CDRs with amino acid sequences that are selected from the group consisting of SEQ ID NOs: 6, 7, 8, 18, 19, 20, 29, 30 or 31.

[0033] One embodiment provides an antibody or its epitope-binding fragment or a fusion protein that binds immunospecifically to SEQ ID NO:38. In one embodiment, the antibody binds to SEQ ID NO:38 on PD-1. In another embodiment, the antibody binds to PD-1 expressed on the surface of an immune cell and induces or promotes a signal through PD-1 that activates or stimulates the immune cell. In one embodiment, the immune cell that is activated or stimulated is a T cell, for example, a CD8+ T cell.

[0034] In some embodiments, the antibody or its antigen-binding fragment is human, mouse, chimeric, humanized, monoclonal, bispecific, trispecific, or multispecific.

[0035] An embodiment provides a pharmaceutical composition including one or more of the disclosed antibodies or their antigen-binding fragments. In some embodiments, the pharmaceutical compositions include a second therapeutic agent and / or an excipient. Petition 870200039602, dated 03 / 26 / 2020, page 14 / 113 / 98 pharmaceutically acceptable. A second exemplary therapeutic agent includes cyclophosphamide.

[0036] A modality provides a method of inducing, promoting, or enhancing an immune response in a subject in need by administering to the subject an effective amount of one or more of the antibodies or their antigen-binding fragments released to induce, promote, or enhance an immune response in the subject.

[0037] One embodiment provides a method for treating cancer in a subject in need by administering to the subject an effective amount of one or more of the disclosed antibodies or their antigen-binding fragments to treat the cancer in the subject.

[0038] One embodiment provides a method for reducing the tumor burden in a subject in need by administering to the subject an effective amount of one or more of the disclosed antibodies or their antigen-binding fragments to reduce the tumor burden in the subject.

[0039] An embodiment provides a method for treating an infection in a subject in need by administering to the subject an effective amount of one or more of the disclosed antibodies or their antigen-binding fragments to treat the infection in the subject. BRIEF DESCRIPTION OF THE FIGURES

[0040] Figure 1 is a graph showing the interaction kinetics as a function of time between the monoclonal antibody 4G9 and human PD-1. The graph shows traces of human PD-1 concentrations at 0, 125, 250, 500, 500 and 1000 nM.

[0041] Figure 2 is a graph showing the interaction kinetics as a function of time between the 4G9 monoclonal antibody and mouse PD-1. The graph shows traces of mouse PD-1 concentrations at 0, 62.5, 125, 500, 500 and 1000 nM.

[0042] Figure 3 is a graph showing the interaction kinetics in Petition 870200039602, dated 03 / 26 / 2020, page 15 / 113 / 98 function of time between the monoclonal antibody 4C12 and human PD-1. The graph shows traces of human PD-1 concentrations at 0, 125, 250, 500, 500 and 1000 nM.

[0043] Figure 4 is a graph showing the interaction kinetics as a function of time between the monoclonal antibody 5C2 and human PD-1. The graph shows traces of mouse PD-1 concentrations at 0 and 1000 nM.

[0044] Figure 5 is a graph showing the interaction kinetics as a function of time between the 5G2 monoclonal antibody and mouse PD-1. The graph shows traces of human PD-1 concentrations at 0, 62.5, 125, 250, 500, 500, and 1000 nM.

[0045] Figure 6A is a flow cytometry histogram of EL4 cells stained with an isotype control antibody or commercial J43 antiPD-1 antibody. Figure 6B is a flow cytometry histogram of EL4 cells stained with secondary antibody only or antibodies 4G9, 5C2, and 4C12. Figure 6C is a flow cytometry histogram of EL4 cells stained with secondary antibody only or antibody 4G9. Figure 6D is a flow cytometry histogram of EL4 cells stained with secondary antibody only or antibody 5C2. Figure 6E is a flow cytometry histogram of EL4 cells stained with secondary antibody only or antibody 4C12. Figure 6F is a bar graph showing the binding of various purified mouse PD-1 antibodies to EL4 cells.

[0046] Figures 7A and 7B are bar graphs showing the concentration of IFNγ (Fig. 7A) or IL-2 in the supernatant of CD4 T cells treated with various antibodies. The X-axis is the treatment group and the Y-axis is the concentration (ng / ml). Figure 7C is a bar graph showing the concentration of IFNγ in the supernatant of human CD4 T cells treated with 4G9 or 5C2 antibodies. The X-axis represents the treatment group and the Petition 870200039602, dated 03 / 26 / 2020, p. 16 / 113 / 98 Y-axis represents the concentration (ng / mL).

[0047] Figure 8 is a bar graph showing the intracellular pAKT staining levels in mouse CD4 T cells treated with various antibodies. The X-axis represents the treatment group and the Y-axis represents the pAKT MFI (S473).

[0048] Figure 9 is a Western blot showing the heavy chain and light chain of IgG in the various antibodies.

[0049] Figure 10 is a bar graph showing the binding of antibodies 4G9 and 5C2 to human PD-1-Fc. The X-axis represents antibody concentration and the Y-axis represents OD450.

[0050] Figures 11A and 11B are flow cytometry histograms showing the binding of antibodies 4G9 and 5C2 to PD-1 on CD4 T cells from PD-1 KO mice (Fig. 11A) or PD-1 WT mice (Fig. 11B).

[0051] Figure 12 is a line graph showing the expression of pS6 in CD4 T cells treated with 4G9, 5C2, commercial Ab-1, commercial Ab-2, or untreated. The X-axis represents the total protein concentration (pg / ml) and the Y-axis represents OD450.

[0052] Figure 13A is a schematic illustration showing the experimental design for the TC-1 tumor experiments. Figure 13B is a line graph showing the mean tumor volume (cm3) over time (days) for TC-1 tumor-bearing mice treated with E7 Vax, 4G9, RMP 1-14, E7 Vax + RMP 1-14, E7 Vax + 4G9, or untreated. The X-axis represents time (days) and the Y-axis represents mean tumor volume (cm3). Figure 13C is a line graph showing the percentage of survival over time for TC-1 tumor-bearing mice treated with E7 Vax, 4G9, RMP 1-14, E7 Vax + RMP 1-14, E7 Vax + 4G9, or untreated. Figure 13D is a line graph showing the mean tumor volume (cm3) over time (days) for mice bearing TC-1 tumors treated with E7 Vax, 4C12, 5C2, RMP 1-14, E7 Vax + RMP. Petition 870200039602, dated 03 / 26 / 2020, p. 17 / 113 / 98 1-14, E7 Vax + 4C12, E7 Vax + 5C2 or untreated.

[0053] Figure 14A is a schematic illustration showing the experimental design for the TC-1 tumor experiments. Figure 14B is a line graph showing the mean tumor volume (cm3) over time (days) 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 untreated. The X-axis represents time (days) and the Y-axis represents the mean tumor volume (cm3). Figure 14C is a line graph showing the percentage of survival over time of mice bearing TC-1 tumors 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 untreated. The X-axis represents time (days) and the Y-axis represents the percentage of survival. DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0054] As used in this document, a molecule is said to be capable of immunospecific binding to a second molecule if that binding exhibits the specificity and affinity of an antibody to its cognate antigen. Antibodies are said to be capable of immunospecific binding to a target region or conformation (epitope) of an antigen if that binding involves the antigen recognition site of the immunoglobulin molecule. An antibody that binds immunospecifically to a specific antigen may bind to other antigens with lower affinity if the other antigen has some sequence or conformational similarity that is recognized by the antigen recognition site, as determined by, for example, immunoassays, BIACORE® assays, or other assays known in the art, but would not bind to a completely unrelated antigen. Preferably, however, the antibodies (and their antigen-binding fragments) will not react Petition 870200039602, dated 03 / 26 / 2020, page 18 / 113 / 98 cross-linking with other antigens. Antibodies can also bind to other molecules in a non-immunospecific manner, such as to FcR receptors, by virtue of binding domains in other regions / domains of the molecule that do not involve the antigen recognition site, such as the Fc region.

[0055] As used in this document, a molecule is said to bind physiologically specifically to a second molecule if that binding exhibits the specificity and affinity of a receptor to its cognate binding ligand. A molecule may be able to bind physiologically specifically to more than one other molecule.

[0056] As used in this document, the term antibody is intended to designate an immunoglobulin molecule that possesses a variable region antigen recognition site. The term variable region is intended to distinguish this immunoglobulin domain from domains that are widely shared by antibodies (such as an antibody Fc domain). The variable region includes a hypervariable region, whose residues are responsible for antigen binding. The hypervariable region includes amino acid residues of a Complementarity Determinant Region or CDR (i.e., typically approximately at residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the variable light chain domain and approximately at residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the variable heavy chain domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.(1991)) and / or the residues of a hypervariable loop (i.e., residues 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 the heavy chain variable domain, Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). The residues of the Framework Region or FR are the variable domain residues other than the residues of the hypervariable region, as defined herein. Petition 870200039602, dated 03 / 26 / 2020, p. 19 / 113 / 98 document. The term antibody includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (See, for example, Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25; International Publications No. WO 94 / 04678 and WO 94 / 25591; US ​​Patent 6,005,079), single-chain Fvs (scFv) (see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315) (1994)), single-chain antibodies, disulfide-linked Fvs (sdFv), intracorporeal and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id and antibodies to anti-anti-Id antibodies).In particular, these antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0057] As used in this document, the term antibody antigen-binding fragment refers to one or more parts of an antibody that contain the antibody's Complementarity Determinant Regions (CDRs) and, optionally, framework residues that include the antibody's variable region antigen recognition site and exhibit an ability to bind to the antigen immunospecifically. Such fragments include Fab', F(ab')2, Fv, single-chain (ScFv) and their mutants, naturally occurring variants, and fusion proteins, including the antibody's variable region antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc.).

[0058] As used in this document, the term fragment refers to a peptide or polypeptide including a sequence of Petition 870200039602, dated 03 / 26 / 2020, page. 20 / 113 / 98 amino acids 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.

[0059] The term binding molecule, as used in this document, is intended to refer to molecules that specifically interact with and bind to a specific target. The target may comprise a biological or small (chemical) molecule. The target molecule may define an antigen or an antigenic fragment. Examples of a binding molecule 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.

[0060] As used in this document, the term modulation refers to an ability to alter an effect, outcome, or activity (e.g., signal transduction). This modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (i.e., attenuates but does not abolish) or can completely abolish that activity (e.g., neutralizes). Modulation may include the internalization of a receptor after antibody binding or a reduction in receptor expression in Petition 870200039602, dated 03 / 26 / 2020, page 21 / 113 / 98 target cell. Agonistic modulation can intensify or otherwise increase or accentuate an activity (e.g., signal transduction). In a further embodiment, this modulation can alter the nature of the interaction between a ligand and its cognate receptor, so as to alter the nature of the induced signal transduction. For example, molecules may, by binding to the ligand or receptor, alter the ability of such molecules to bind to other ligands or receptors and thus alter their overall activity. Preferably, this modulation will provide a change of at least 10% in a measurable activity of the immune system, more preferably a change of at least 50% in that activity, or at least 2-fold, 5-fold, 10-fold, or even more preferably a change of at least 100-fold in that activity.

[0061] The term substantially, as used in the context of binding or exhibited effect, is intended to designate that the observed effect is physiologically or therapeutically relevant. Thus, for example, a molecule is capable of substantially blocking the activity of a ligand or receptor if the degree of blockade is physiologically or therapeutically relevant (e.g., if that degree is more than 60% complete, more than 70% complete, more than 75% complete, more than 80% complete, more than 85% complete, more than 90% complete, more than 95% complete, or more than 97% complete). Similarly, a molecule is said to possess substantially the same immunospecificity and / or characteristics as another molecule if these immunospecificities and characteristics are 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.

[0062] As used in this document, co-stimulatory signals encompass positive co-stimulatory signals (e.g., signals that result in increased activity) and co-stimulatory signals Petition 870200039602, dated 03 / 26 / 2020, page 22 / 113 / 98 negative (for example, signs that result in the inhibition of an activity).

[0063] As used in this document, the term derivative refers to an antibody or its antigen-binding fragment that binds immunospecifically to the same target as a parental or reference antibody, but which differs in amino acid sequence from the parental or reference antibody or its antigen-binding fragment by including one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications relative to the parental or reference antibody or its antigen-binding fragment. Preferably, such derivatives will have substantially the same immunospecificity and / or characteristics, or the same immunospecificity and characteristics as the parental or reference antibody or its antigen-binding fragment. Amino acid substitutions or additions of such derivatives may include amino acid residues that occur naturally (i.e., encoded by DNA) or that do not occur naturally.The term derivative encompasses, for example, chimeric or humanized variants, as well as variants with altered CH1, hinge, CH2, CH3, or CH4 regions, so as to form, for example, antibodies, etc., possessing variant Fc regions that exhibit enhanced or compromised effector or binding characteristics.

[0064] As used in this document, a chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, such as antibodies that have a variable region derived from a non-human antibody and a constant region from human immunoglobulin.

[0065] As used in this document, the term humanized antibody refers to an immunoglobulin that includes a human framework region and one or more CDRs from a non-human immunoglobulin (usually mouse or rat). The non-human immunoglobulin that provides the CDRs is called the donor and the human immunoglobulin Petition 870200039602, dated 03 / 26 / 2020, p. 23 / 113 / 98, which provides the framework, is called the acceptor. Constant regions do not need to be present, but if they are, they must be substantially identical to the constant regions of human immunoglobulin, that is, at least about 85-99%, preferably about 95% or more identical. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding parts of the natural human immunoglobulin sequences. A humanized antibody is an antibody that includes a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody because, for example, the entire variable region of a chimeric antibody is non-human.

[0066] As used in this document, the term endogenous concentration refers to the level at which a molecule is natively expressed (i.e., in the absence of recombinant expression vectors or promoters) by a cell (which cell may be a normal cell, a cancerous cell, or an infected cell).

[0067] As used in this document, the terms treat, treating, treatment and therapeutic use refer to the elimination, reduction or improvement of one or more symptoms of a disease or disorder exacerbated by anti-PD-1 antibodies or their antigen fragment.

[0068] As used in this document, a therapeutically effective amount refers to the amount of a therapeutic agent sufficient to mediate a clinically relevant elimination, reduction, or improvement of such symptoms. An effect is clinically relevant if its magnitude is sufficient to affect the health or prognosis of a recipient subject. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, for example, delaying or minimizing the spread of cancer. A therapeutically effective amount may also refer to the amount Petition 870200039602, dated 03 / 26 / 2020, page 24 / 113 / 98 of the therapeutic agent that provides a therapeutic benefit in the treatment or control of a disease.

[0069] As used in this document, the term prophylactic agent refers to an agent that can be used in the prevention of a disorder or disease before the detection of any symptoms of that disorder or disease. A prophylactically effective amount is the amount of prophylactic agent sufficient to mediate this protection. A prophylactically effective amount may also refer to the amount of prophylactic agent that provides a prophylactic benefit in preventing the disease.

[0070] As used in this document, the term cancer refers to a neoplasm or tumor resulting from abnormal uncontrolled cell growth. As used in this document, cancer explicitly includes leukemias and lymphomas. The term cancer refers to a disease involving cells that have the potential to metastasize to distal sites and exhibit phenotypic traits that differ from those of non-cancerous cells, for example, colony formation on 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 on soft agar and form distinct spherical structures in three-dimensional basement membrane or extracellular matrix preparations.

[0071] As used in this document, an immune cell refers to any cell of hematopoietic origin, including, without limitation, T cells, B cells, monocytes, dendritic cells, and macrophages.

[0072] As used in this document, valence refers to the number of binding sites available per molecule.

[0073] As used in this document, the terms immune response or immune response refer to the development of a humoral (antibody-mediated) response and / or a cellular (T cell-mediated) response. Petition 870200039602, dated 03 / 26 / 2020, page 25 / 113 / 98 antigen-specific or their secretion products) beneficial response directed against a peptide in a recipient patient. This response can be an active response induced by the administration of an immunogen or a passive response induced by the administration of an antibody or exposed (primed) T cells. A cellular immune response is triggered by the presentation of polypeptide epitopes in association with MHC Class I or Class II molecules to activate antigen-specific CD4+ helper T cells and / or CD8+ cytotoxic T cells. The response may also involve the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils, or other components of innate immunity. The presence of a cell-mediated immune response can be determined by proliferation assays (CD4+ T cells) or CTLs (cytotoxic T lymphocytes).The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

[0074] As used in this document, an immunogenic or immunogen agent is capable of inducing an immune response against itself upon administration to a mammal, optionally in conjunction with an adjuvant.

[0075] As used in this document, the terms individual, host, subject, and patient are used interchangeably in this document and refer to a mammal, including, without limitation, humans, rodents such as mice and rats, and other laboratory animals.

[0076] As used in this document, the term polypeptide refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or Petition 870200039602, dated 03 / 26 / 2020, page 26 / 113 / 98 glycosylation). The term polypeptide includes proteins and their fragments. Polypeptides can be exogenous, meaning they are heterologous, that is, foreign to the host cell used, such as the human polypeptide produced by a bacterial cell. Polypeptides are disclosed in this document as sequences of amino acid residues. These sequences are written from left to right in the direction from the amino terminal to the carboxy terminal.According to standard nomenclature, amino acid residue sequences are designated by a three-letter or single-letter code, as indicated below: 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).

[0077] As used in this document, the term variant refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, the differences are limited, so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may occur naturally, as an allelic variant, or it may be a variant not known to occur naturally.

[0078] Modifications and alterations can be made to the structure of the disseminated polypeptides and still obtain a molecule with characteristics Petition 870200039602, dated 03 / 26 / 2020, page 27 / 113 / 98 similar to those of the polypeptide (for example, a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without considerable loss of activity. As it is the interactive capacity and nature of a polypeptide that defines the biological functional activity of that polypeptide, certain amino acid sequence substitutions can be made in a polypeptide sequence and yet obtain a polypeptide with similar properties.

[0079] When making these changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function to a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted by other amino acids with a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid was assigned a hydropathic index based on its hydrophobicity and charge characteristics. These indices are: 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).

[0080] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted for another amino acid with a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferential, those within ± 1 are particularly preferential, and those within ± 0.5 are even more so. Petition 870200039602, dated 03 / 26 / 2020, page 28 / 113 / 98 particularly preferential.

[0081] The substitution of similar amino acids can also be done based on hydrophilicity, particularly where the biologically equivalent functional polypeptide or peptide thus created is intended for use in immunological modalities. The following hydrophilicity values ​​were assigned to the amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+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 understood that an amino acid can be replaced by another with a similar hydrophilicity value and still obtain a biologically equivalent and, in particular, an immunologically equivalent polypeptide.In such changes, the substitution of amino acids whose hydrophilicity values ​​are within ± 2 is preferential, those within ± 1 are particularly preferential, and those within ± 0.5 are even more particularly preferential.

[0082] As described above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account several of 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), (Dica: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). The forms of this disclosure therefore encompass functional or biological equivalents of a polypeptide, as established above. In particular, forms of polypeptides may include variants with approximately Petition 870200039602, dated 03 / 26 / 2020, page 29 / 113 / 98 of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the polypeptide of interest.

[0083] The term percentage (%) of sequence identity is defined as the percentage of nucleotides or amino acids in a candidate sequence that is identical to the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining percentage of sequence identity can be achieved in several ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. The appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment along the entire length of the sequences being compared, can be determined by known methods.

[0084] For the purposes described in this document, the % sequence identity of a given nucleotide or amino acid sequence C with or against a given nucleic acid sequence D (which may alternatively be expressed as a given sequence C having or comprising a certain % sequence identity with or against a given sequence D) is calculated as follows: 100 times the W / Z ratio, where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in the C and D alignment of that program, and where Z is the total number of nucleotides or amino acids in D. It will be noticed that when the length of sequence C is not equal to the length of sequence D, the % sequence identity from C to D will not be equal to the % sequence identity from D to C. Petition 870200039602, dated 03 / 26 / 2020, p. 30 / 113 / 98

[0085] As used in this document, a pharmaceutically acceptable carrier includes any of the standard pharmaceutical carriers, such as a phosphate-buffered saline solution, water and emulsions, such as an oil / water or water / oil emulsion and various types of wetting agents.

[0086] As used in this document, the terms antigenic determinant and epitope are used interchangeably and refer to the structure recognized by an antibody.

[0087] As used in this document, a conformational epitope is an epitope that includes discontinuous sections of the antigen's amino acid sequence. Antibodies bind to a conformational epitope based on 3-D surface features, shape, or tertiary structure of the antigen.

[0088] As used in this document, a linear epitope is an epitope formed by a continuous sequence of amino acids from the antigen. Linear epitopes typically include about 5 to about 10 continuous amino acid residues. Antibodies bind to a linear epitope based on the primary sequence of the antigen.

[0089] As used in this document, a paratope, also called an antigen-binding site, is a part of an antibody that recognizes and binds to an antigen. II. Compositions

[0090] Antibodies and their antigen-binding fragments are provided that bind immunospecifically to PD-1. Contrary to the existing paradigm, PD-1 exclusively promotes a suppressive immune response (Riley, J., Immunol Rev. 229(1):114-125 (2009)), the antibodies and their released antigen-binding fragments bind immunospecifically to PD-1 and cause an activation signal to be delivered to the immune cell that activates the immune cell, rather than suppressing the cell. Petition 870200039602, dated 03 / 26 / 2020, page 31 / 113 / 98 immune. A. Programmed Death Receptor Protein 1 (PD-1)

[0091] The antibodies and their antigen-binding fragments disclosed bind immunospecifically to PD-1. The antibodies and their antigen-binding fragments can bind to PD-1 by having, for example, the amino acid sequences provided below.

[0092] The amino acid sequences for human PD-1 and mouse PD-1 are known in the art and include, for example, human PD-1 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTE GDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDC RFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRA ELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWV LAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTP EPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGH CSWPL (SEQ ID NO: 1), Access: AJS10360 and which is specifically incorporated herein by reference in its entirety. Mouse PD-1 MWVRQVPWSFTWAVLQLSWQSGWLLEVPNGPWRSLTFYPAWLTVS EGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQ DARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGA ELVVTERILETSTRYPSPSPKPEGRFQGMVIGIMSALVGIPVLLLLAWA LAVFCSTSMSEARGAGSKDDTLKEEPSAAPVPSVAYEELDFQGREKTP ELPTACVHTEYATIVFTEGLGASAMGRRGSADGLQGPRPPRHEDGHC SWPL (SEQ ID NO: 2) UniProtKB - Q02242 (PDCD1_MOUSE) and which is specifically incorporated herein by reference in its entirety. B. Antibody Compositions Petition 870200039602, dated 03 / 26 / 2020, page 32 / 113 / 98

[0093] Disclosed anti-PD-1 antibodies or their antigen-binding fragments include whole immunoglobulin (i.e., an intact antibody) of any class, its fragments, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. In some embodiments, the disclosed antibody contains an antibody light chain as well as at least the variable domain of an antibody heavy chain. In other embodiments, these molecules may further include one or more of the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain (especially the CH1 and hinge regions, or the CH1, hinge and CH2 regions, or the CH1, hinge, CH2, and CH3 regions). The antibody may be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4.In some embodiments, the constant domain is a complement-binding constant domain, where cytotoxic activity is desired in the antibody and the class is typically IgG1. In other embodiments, where this cytotoxic activity is not desired, the constant domain may be of the IgG2 or IgG4 classes. The antibody may include sequences from more than one class or isotype, and the selection of particular constant domains to optimize the desired effector functions is within the common knowledge of the art.

[0094] Variable domains differ in sequence between antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, variability is generally not uniformly distributed across antibody variable domains. It is typically concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions, both in the light chain and heavy chain variable domains. The most highly conserved parts of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, adopting in Petition 870200039602, dated 03 / 26 / 2020, page 33 / 113 / 98 largely a beta-sheet configuration, connected by three CDRs, which form loops that connect and, in some cases, form part of the beta-sheet structure. The CDRs in each chain are held together in the vicinity of the FR regions and, with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibodies.

[0095] Some embodiments provide fragments of anti-PD-1 antibodies that have bioactivity. The fragments, whether attached to other sequences or not, may include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment.

[0096] Another embodiment provides PD-1 specific single-chain antibodies. Methods for producing single-chain antibodies are well known to those skilled in the art. A single-chain antibody can be created by fusing the variable domains of the heavy and light chains using a short peptide linker, thus reconstituting an antigen-binding site in a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is attached to the N-terminus of the other variable domain via a peptide or peptide linker of 15 to 25 amino acids have been developed without significantly disrupting antigen binding or binding specificity. The peptide linker is chosen to allow the heavy chain and light chain to bind in their proper conformational orientation.

[0097] Another embodiment provides variable bivalent single-chain fragments (di-scFvs) that can be manipulated by linking two scFvs. This can be done by producing a single peptide chain with two VH regions and two VL regions, producing tandem scFvs. The ScFvs also Petition 870200039602, dated 03 / 26 / 2020, page 34 / 113 / 98, can be manipulated with peptide linkers that are too short for the two variable regions to fold (about five amino acids), forcing the scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40 times lower than the corresponding scFvs, meaning they have a much higher affinity for their target. Even shorter peptide linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity for their targets than diabodies.

[0098] Another embodiment provides a PD-1 specific monoclonal antibody that induces an activation signal for immune cells. The monoclonal antibody may be obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within 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 a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired antagonistic activity. 1. Chimeric and Humanized Antibodies

[0099] Another embodiment provides chimeric anti-PD-1 antibodies and their antigen-binding fragments, including one or more of the disclosed sequences, and also provides their functional variants that bind to PD-1 and cause an activation signal to be transmitted to an immune cell expressing PD-1. Petition 870200039602, dated 03 / 26 / 2020, p. 35 / 113 / 98

[00100] Methods for the production of 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 USN Patents 6,311,415, 5,807,715, 4,816,567 and 4,816,397. Chimeric antibodies, including one or more CDRs of a non-human species and structural regions of a human immunoglobulin molecule, can be produced using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; International Publication No. WO 91 / 09967; and USN patents 5,225,539, 5,530,101 and 5,585,089), coating or surface reassembly (EP 592,106; EP 519,596; 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 chain rearrangement. (US Patent 5,565,332).

[00101] The disclosed anti-PD-1 antibodies or their antigen-binding fragments may be human or humanized antibodies, or their antigen-binding fragments. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans and therefore may give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to decrease the chance of an antibody administered to a human eliciting an undesirable immune response.

[00102] Transgenic animals (e.g., mice) that are capable, after immunization, of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production can be used. For example, it has been described that homozygous deletion of the antibody heavy chain junction region (J(H)) gene in chimeric and germline mutant mice results in complete inhibition. Petition 870200039602, dated 03 / 26 / 2020, page 36 / 113 / 98 regarding the production of endogenous antibodies. The transfer of the human germline immunoglobulin gene array into these germline mutant mice will result in the production of human antibodies upon antigenic exposure.

[00103] Optionally, antibodies are generated in other species and humanized for administration in humans. Humanized forms of non-human antibodies (e.g., murine) are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding antibody subsequences) containing minimal sequences derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues of a complementarity-determining region (CDR) of the recipient antibody are replaced by residues of a CDR from a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired capability, affinity and specificity. In some cases, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the imported CDR or structural sequences. In general, the humanized antibody will contain substantially all of at least one, and typically two, variable domains, where all, or substantially all, of the CDR regions correspond to those of a non-human immunoglobulin and all, or substantially all, of the FR regions are those of a human immunoglobulin consensus sequence. Ideally, the humanized antibody will also contain at least a portion of an immunoglobulin constant (Fc) region, typically that of a human immunoglobulin.

[00104] Methods for humanizing non-human antibodies are well known in the art, see, for example, European Patents No. EP Petition 870200039602, dated 03 / 26 / 2020, p. 37 / 113 / 98 239,400, EP 592,106 and EP 519,596; Publicações Internacionais No. WO 91 / 09967 and WO 93 / 17105; Patentes USN 5,225,539, 5,530,101, 5,565,332, 5,585,089, 5,766,886 and 6,407,213; and Padlan, 1991, Molecular Immunology 28 (4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; Roguska et al., 1994, PNAS 91:969-973; Tan et al., 2002, J. Immunol. 169:1119-1125; Caldas et al., 2000, Protein Eng. 13:353-360; Morea et al., 2000, Methods 20:267-79; Baca et al., 1997, J. Biol. Chem. 272:1067810684; Roguska et al., 1996, Protein Eng. 9:895-904; Couto et al., 1995, Cancer Res. 55 (23 Supp):5973s-5977s; Couto et al., 1995, Cancer Res.55:1717-22; Sandhu, 1994, Gene 150:409-10; 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).

[00105] Generally, 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 import residues, which are usually taken from an import variable domain. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can be achieved essentially by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. Therefore, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or fragment in which substantially less than an intact human variable domain has been replaced with the corresponding sequence from a non-human species.In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are present. Petition 870200039602, dated 03 / 26 / 2020, page 38 / 113 / 98 replaced by residues from analogous sites in rodent antibodies.

[00106] The choice of human variable domains, both light and heavy, to be used in the production of humanized antibodies can be very important in reducing 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 closest to the rodent sequence is then accepted as the human framework (FR) for the humanized antibody. Another method uses a particular framework derived from the consensus sequence of all human antibodies from a specific subset of light or heavy chains. The same framework can be used for several different humanized antibodies.

[00107] It is also important that antibodies be humanized with high affinity retention for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analyzing parental sequences and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the probable role of residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen.Thus, RF residues can be selected and combined from the consensus and import sequence to achieve the desired antibody characteristic, such as higher affinity for the target antigen(s). In general, CDR residues are directly and... Petition 870200039602, dated 03 / 26 / 2020, page 39 / 113 / 98 most substantially involved in the influence of antigen binding.

[00108] A human, humanized, or chimeric antibody derivative may include substantially all of at least one and typically two variable domains in which all or substantially all CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all framework regions are those of a human immunoglobulin consensus sequence. Such antibodies may also include at least a portion of an immunoglobulin constant (Fc) region, typically that of a human immunoglobulin. The constant domains of such antibodies may be selected in relation to the proposed 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 one specific embodiment, constant domains of human IgG, especially of the IgG1 and IgG3 isotypes, are used when the humanized antibody derivative is intended for therapeutic use and antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity. In alternative embodiments, IgG2 and IgG4 isotypes are used when the antibody is intended for therapeutic purposes and antibody effector function is not required. Fc constant domains, including one or more amino acid modifications that alter antibody effector functions, such as those disclosed in USN Patent Application Publications 2005 / 0037000 and 2005 / 0064514.

[00109] The framework and CDR regions of a humanized antibody do not need to exactly match the parental sequences; for example, the donor CDR or consensus framework may be mutagenized by substitution, insertion, or deletion of at least one residue, so that the CDR or framework residue at that site does not match the consensus. Petition 870200039602, dated 03 / 26 / 2020, p. 40 / 113 / 98 or to the donor antibody. In some modalities, these mutations are not extensive. Typically, at least 75% of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences, more frequently 90% or more than 95%. Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and US Patents 5,225,539, 5,530,101 and 5,585,089), coating or surface reshaping (European Patents Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28 (4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814); chain rearrangement (US Patent No. 5,565,332) and techniques disclosed in, for example, US Patents Nos. 6,407,213, 5,666,886.5585,089, 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. Gene 150:409-10, Pedersen et al., 1994, J. Mol. Biol. 235:959-73, 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.

[00110] Frequently, the structural residues in the structural regions will be replaced by the corresponding residue of the CDR donor antibody to alter, for example, improve antigen binding.These framework substitutions are identified by well-known methods in the art, for example, by modeling the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, for example, Queen.) Petition 870200039602, dated 03 / 26 / 2020, p. 41 / 113 / 98 et al., USN Patent 5,585,089; USN Publication 2004 / 0049014 and 2003 / 0229208; USN Patents 6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101 and Riechmann et al., 1988, Nature 332:323).

[00111] Human, chimeric, or humanized derivatives of disclosed murine anti-human Siglec-15 antibodies can be used for in vivo methods in humans. Murine antibodies or antibodies from other species can be advantageously employed for many uses (e.g., in vitro or in situ detection assays, acute in vivo use, etc.). Such a human or humanized antibody may include substitutions, deletions, or additions of amino acid residues in one or more non-human CDRs. The humanized antibody derivative may have substantially the same binding, a stronger binding, or a weaker binding when compared to a non-derived humanized antibody. In specific embodiments, one, two, three, four, or five amino acid residues of the CDR have been substituted, deleted, or added (i.e., mutated). Completely human antibodies are particularly desirable for the therapeutic treatment of human subjects.

[00112] Such human antibodies can be produced by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences (see US Patents 4,444,887 and 4,716,111; and International Publications 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 can express human immunoglobulin genes.

[00113] For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or Petition 870200039602, dated 03 / 26 / 2020, page 42 / 113 / 98 by homologous recombination in 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 heavy and light chain immunoglobulin genes can be rendered inoperative separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then reared to produce homozygous offspring expressing human antibodies.Transgenic mice are immunized using conventional methodologies with a selected antigen, for example, all or a portion of a polypeptide. Monoclonal antibodies directed against the antigen can be obtained from immunized transgenic mice using conventional hybridoma technology (see, for example, US Patent 5,916,771). The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using this technique, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for the production of 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 discussion of this technology for the production of human antibodies and human monoclonal antibodies and protocols for the production of such antibodies, see, for example, International Publication Nos. WO 98 / 24893, WO 96 / 34096 and WO 96 / 33735; and US Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806. Petition 870200039602, dated 03 / 26 / 2020, page 43 / 113 / 98 5,814,318 and 5,939,598, which are incorporated by reference in this document in their entirety. In addition, companies may be contracted to supply human antibodies targeted against a selected antigen using technology similar to that described above.

[00114] DNA sequences encoding human acceptor framework sequences include, but are not limited to, FR segments from the human germline VH segment VH1-18 and JH6 and the human germline VK-A26 and JK4 VL segment. In a specific embodiment, one or more of the CDRs are inserted into the framework regions using routine recombinant DNA techniques. The framework regions may be naturally occurring or consensus framework regions and human framework regions (see, for example, Chothia et al., 1998, Structural Determinants In The Sequences Of Immunoglobulin Variable Domain, J. Mol. Biol. 278: 457-479 for a list of human framework regions). C. Antibody Sequences 1. 4C12 heavy chain sequences

[00115] One embodiment provides a murine monoclonal antibody or its antigen-binding fragment isolated from the 4C12 hybridoma.

[00116] Another embodiment provides an antibody or its antigen-binding fragment that has a heavy chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with: ATGAGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAG GTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGG TGAAGCCTGGGGCTTCAGTGAAGGGTGTCCTGCAAGGCTTCTGGCTA CACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGG CCAAGGCCTTGAGTGGATTGGAAGGATTCATCCTTCTGATAGTGAT Actaactacaatcaaaagttcaagggcaaggccacattgactgta Petition 870200039602, dated 03 / 26 / 2020, p. 44 / 113 / 98 GACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACA TCTGAGGACTCTGCGGTCTATTACTGTGCACCCTATGGTAACTACG CCTCCGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTC TGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGA TCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCA AGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATC CCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGAC CTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGC CCAGCCAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCA CCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGC CTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCC CCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCA CGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGT TCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGA AACCCCGGGAGGAGCAGATCAACAGCACTTTCCGTTCAGTCAGTG AACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCA AATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAA CCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACA CCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTC TGACCTGCATGATAACAAACTTCTTCCCTGAAGACATTACTGTGGA GTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCT CAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTG CTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAG CCTCTCCCACTCTCCTGGTAAATGA (SEQ ID NO:3).

[00117] Underlined sequences correspond to complementarity determining regions (CDRs). A sequence underlined twice corresponds to the constant region. Dashed underlined sequences correspond to the leader sequence. Petition 870200039602, dated 03 / 26 / 2020, p. 45 / 113 / 98

[00118] The nucleic acid may be in a vector, for example, an expression vector. The nucleic acid may be extrachromosomal or inserted into the chromosome of a host cell, for example, a Chinese Hamster Ovary cell.

[00119] An embodiment provides an antibody or its antigen-binding fragment that has a heavy chain with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with: MRWSCIILFLVATATGVHSQVQLQQPGAELVKPGASVKVSCKASGYT FTSYWMHWVKQRPGQGLEWIGRIHPSDSDTNYNQKFKGKATLTVDK SSSTAYMQLSSLTSEDSAVYYCAPYGNYASGFAYWGQGTLVTVSAA KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSS GVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDK KIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISK DDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLN GKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKV SLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKL NVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:4).

[00120] The single underline corresponds to the leader sequence. The double underline corresponds to CDRs and the dashed underline corresponds to the constant region.

[00121] Another embodiment provides an antibody or its antigen-binding fragment that has a heavy chain without the leader sequence with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with: QVQLQQPGAELVKPGASVKVSCKASGYTFTSYWMHWVKQRPGQGL EWIGRIHPSDSDTNYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAV YYCAPYGNYASGFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTN Petition 870200039602, dated 03 / 26 / 2020, page 46 / 113 / 98 SMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSV TVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSS VFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTA QTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQW NGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLH EGLHNHHTEKSLSHSPGK (SEQ ID NO:5).

[00122] The double underline corresponds to CDRs and the broken underline corresponds to the constant region.

[00123] The amino acid sequence for CDR1 of the 4C12 heavy chain is SYWMH (SEQ ID NO: 6).

[00124] The amino acid sequence for CDR2 of the 4C12 heavy chain is RIHPSDSDTNYNQKFKG (SEQ ID NO: 7).

[00125] The amino acid sequence for CDR3 of the 4C12 heavy chain is YGNYASGFAY (SEQ ID NO: 8).

[00126] One embodiment provides an antibody or its antigen-binding fragment that has a heavy chain according to SEQ ID NO: 4 or 5.

[00127] Another embodiment provides an antibody or its antigen-binding fragment that has a heavy chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 3.

[00128] One embodiment provides an antibody with three different CDRs selected from the group consisting of SEQ ID NO: 6, 7 and 8. 2. 4C12 light chain sequences

[00129] Another modality provides an antibody or a fragment thereof of Petition 870200039602, dated 03 / 26 / 2020, page. 47 / 113 / 98 antigen linkage that has a light chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with: ATGGGCATCAAGATGGAGTCACAGATTCAGGCATTTGTATTCGTGT TTCTCTGGTTGTCTGGTGTTGACGGAGACATTGTGATGACCCAGTC TCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCAC CTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCA ACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATC CACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAGATTATACTCTCACCATCAGCAGTGTGCAGGCTGAAGAC CTGGCACTTTATTACTGTCAGCAACATTATAGCACTCCGTGGACGT TCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCAC CAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGG AGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGAC ATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGC GTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTAC AGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGA CATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCA CCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG (SEQ ID NO:9).

[00130] The dashed underline represents the leading sequence. The single underline represents CDRs. The double underline represents the constant region.

[00131] The nucleic acid may be in a vector, for example, an expression vector. The nucleic acid may be extrachromosomal or inserted into the chromosome of a host cell, for example, a Chinese Hamster Ovary cell.

[00132] Another embodiment provides an antibody or its antigen-binding fragment that has a light chain with a sequence of Petition 870200039602, dated 03 / 26 / 2020, p. 48 / 113 / 98 amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with: MGIKMESQIQAFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGT DYTLTISSVQAEDLALYYCQQHYSTPWTFGGGTKLEIKRADAAPTVSI FPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWT DQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRN EC (SEQ ID NO:10).

[00133] The underline represents the leading sequence. The double underline represents CDRs. The dashed underline represents the constant region.

[00134] Another embodiment provides an antibody or its antigen-binding fragment that has a light chain without the leader sequence with an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLL IYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYSTP WTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDI NVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHN SYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:11).

[00135] The double underline represents CDRs. The dashed underline represents the constant region.

[00136] The amino acid sequence for CDR1 of the 4C12 light chain is KASQDVSTAVA (SEQ ID NO:12).

[00137] The amino acid sequence for CDR2 of the 4C12 light chain is WASTRHT (SEQ ID NO:13).

[00138] The amino acid sequence for CDR3 of the 4C12 light chain is QQHYSTPWT (SEQ ID NO:14).

[00139] One embodiment provides an antibody or its antigen-binding fragment having a light chain with at least 50%, 60%, Petition 870200039602, dated 03 / 26 / 2020, page 49 / 113 / 98 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 10 or 11.

[00140] Another embodiment provides an antibody or its antigen-binding fragment that has a light chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 9.

[00141] One embodiment provides an antibody with three different CDRs selected from the group consisting of SEQ ID NO: 12, 13 and 14.

[00142] Another embodiment provides an antibody or its antigen-binding fragment that has a heavy chain with an amino acid sequence that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4 or 5 and a light chain with an amino acid sequence of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 10 or 11 or combinations of light and heavy chains.

[00143] Another embodiment provides an antibody or its antigen-binding fragment that has 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 SEQ ID NOs: 12, 13 and 14. 3. 2B5 heavy chain sequences

[00144] One embodiment provides a murine monoclonal antibody or its antigen-binding fragment isolated from hybridoma 2B5.

[00145] Another embodiment provides an antibody or its antigen-binding fragment that has a heavy chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with: ATGGAATGGAGCAGAGTCTTTATCTTTCTCCTATCAGTAACTGCAG GTGTTCACTCCCAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGT Petition 870200039602, dated 03 / 26 / 2020, p. 50 / 113 / 98 AAGGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATA CGCCTTCACTAATTACTTGATAGAGTGGGTAAAGCAGAGGCCTGG ACAGGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGG TACTAACTACAATGAGAAGTTCAAGGGCAAGGCAACACTGACTGC AGACAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGAC ATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCAGCTCAGGCC CCTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGAG AGTCAGTCCTTCCCAAATGTCTTCCCCCTCGTCTCCTGCGAGAGCC CCCTGTCTGATAAGAATCTGGTGGCCATGGGCTGCCTGGCCCGGG ACTTCCTGCCCAGCACCATTTCCTTCACCTGGAACTACCAGAACAA CACTGAAGTCATCCAGGGTATCAGAACCTTCCCAACACTGAGGAC AGGGGGCAAGTACCTAGCCACCTCGCAGGTGTTGCTGTCTCCCAA GAGCATCCTTGAAGGTTCAGATGAATACCTGGTATGCAAAATCCA CTACGGAGGCAAAAACAAAGATCTGCATGTGCCCATTCCAGCTGT CGCAGAGATGAACCCCAATGTAAATGTGTTCGTCCCACCACGGGA TGGCTTCTCTGGCCCTGCACCACGCAAGTCTAAACTCATCTGCGAG GCCACGAACTTCACTCCAAAACCGATCACAGTATCCTGGCTAAAG GATGGGAAGCTCGTGGAATCTGGCTTCACCACAGATCCGGTGACC ATCGAGAACAAAGGATCCACACCCCAAACCTACAAGGTCATAAGC ACACTTACCATCTCTGAAATCGACTGGCTGAACCTGAATGTGTACA CCTGCCGTGTGGATCACAGGGGTCTCACCTTCTTGAAGAACGTGTCCTCCACATGTGCTGCCAGTCCCTCCACAGACATCCTAACCTTCACC ATCCCCCCCTCCTTTGCCGACATCTTCCTCAGCAAGTCCGCTAACC TGACCTGTCTGGTCTCAAACCTGGCAACCTATGAAACCCTGAATAT CTCCTGGGCTTCTCAAAGTGGTGAACCACTGGAAACCAAAATTAA AATCATGGAAAGCCATCCCAATGGCACCTTCAGTGCTAAGGGTGT GGCTAGTGTTTGTGTGGAAGACTGGAATAACAGGAAGGAATTTGT GTGTACTGTGACTCACAGGGATCTGCCTTCACCACAGAAGAAATTC ATCTCAAAACCCAATGAGGTGCACAAACATCCACCTGCTGTGTAC Petição 870200039602, de 26 / 03 / 2020, pág. 51 / 113 / 98 CTGCTGCCACCAGCTCGTGAGCAACTGAACCTGAGGGAGTCAGCC ACAGTCACCTGCCTGGTGAAGGGCTTCTCTCCTGCAGACATCAGTG TGCAGTGGCTTCAGAGAGGGCAACTCTTGCCCCAAGAGAAGTATG TGACCAGTGCCCCGATGCCAGAGCCTGGGGCCCCAGGCTTCTACTT TACCCACAGCATCCTGACTGTGACAGAGGAGGAATGGAACTCCGG AGAGACCTATACCTGTGTTGTAGGCCACGAGGCCCTGCCACACCT GGTGACCGAGAGGACCGTGGACAAGTCCACTGGTAAACCCACACT GTACAATGTCTCCCTGATCATGTCTGACACAGGCGGCACCTGCTAT TGA_(SEQ ID NO:15).

[00146] Underlined sequences correspond to complementarity determining regions (CDRs). A sequence underlined twice corresponds to the constant region. Dashed underlined sequences correspond to the leader sequence.

[00147] The nucleic acid may be in a vector, for example, an expression vector. The nucleic acid may be extrachromosomal or inserted into the chromosome of a host cell, for example, a Chinese Hamster Ovary cell.

[00148] An embodiment provides an antibody or its antigen-binding fragment that has a heavy chain with an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with: MEWSRVFIFLLSVTAGVHSQVQLQQSGAELVRPGTSVKVSCKASGYA FTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKS SSTAYMQLSSLTSEDSAVYFCARSAQAPDYWGQGTTLTVSSESQSFPN VFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRT FPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNKDLHVPIP AVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKD GKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVD HRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLA Petition 870200039602, dated 03 / 26 / 2020, page 52 / 113 / 98 TYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNR KEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRES ATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYF THSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYN VSLIMSDTGGTCY (SEQ ID NO:16).

[00149] The single underline corresponds to the leader sequence. The double underline corresponds to CDRs and the dashed underline corresponds to the constant region.

[00150] Another embodiment provides an antibody or its antigen-binding fragment that has a heavy chain without the leader sequence with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with: QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLE WIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAV YFCARSAQAPDYWGQGTTLTVSSESQSFPNVFPLVSCESPLSDKNLVA MGÇLARDFLPSTISFTWNYQNNTEyIQGIRTFPTLRTGGKYLATSQyL LSPKSILEGSDEYLVCKIHYGGKNKDLHVPIPAVAEMNPNVNVFVPPR DGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIEN KGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAA SPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPL ETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQ KKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISV QWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGET YTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY (SEQ ID NO:17).

[00151] The double underline corresponds to CDRs and the dashed underline corresponds to the constant region.

[00152] The amino acid sequence for CDR1 of the 2B5 heavy chain is Petition 870200039602, dated 03 / 26 / 2020, p. 53 / 113 / 98 NYLIE (SEQ ID NO:18).

[00153] The amino acid sequence for CDR2 of the 2B5 heavy chain is VINPGSGGTNYNEKFKG (SEQ ID NO:19).

[00154] The amino acid sequence for CDR3 of the 2B5 heavy chain is SAQAPDY (SEQ ID NO:20).

[00155] One embodiment provides an antibody or its antigen-binding fragment that has a heavy chain according to SEQ ID NO: 16 or 17.

[00156] Another embodiment provides an antibody or its antigen-binding fragment that has a heavy chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 15.

[00157] One embodiment provides an antibody with three different CDRs selected from the group consisting of SEQ ID NO: 18, 19 and 20. 4. 2B5 light chain sequences

[00158] Another embodiment provides an antibody or its antigen-binding fragment that has a light chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with: ATGGGCATCAAGATGGAGACACATTCTCAGGTCTTTGTATACATGT TGCTGTGGTTGTCTGGTGTTGAAGGAGACATTGTGATGACCCAGTC TCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCAC CTGCAAGGCCAGTCAGGATGGTGGGTACTGCTGTAGCCTGGTATCA ACAGAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATC CACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACT TGGCAGATTATTTCTGTCAGCAATATAGCAGCTATCCATTCACGTT Petition 870200039602, dated 03 / 26 / 2020, page 54 / 113 / 98 CGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACC AACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGA GGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACA TCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCG TCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACA GCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGAC ATAACAGCTATACTGTGGAGGCCACTCACAAGACATCAACTTCAC CCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG (SEQ ID NO:21).

[00159] The dashed underline represents the leading sequence. The single underline represents CDRs. The double underline represents the constant region.

[00160] The nucleic acid may be in a vector, for example, an expression vector. The nucleic acid may be extrachromosomal or inserted into the chromosome of a host cell, for example, a Chinese Hamster Ovary cell.

[00161] Another embodiment provides an antibody or its antigen-binding fragment that has a light chain with an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with: MGIKMETHSQVFVYMLLWLSGVEGDIVMTQSHKFMSTSVGDRVSIT CKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSG TDFTLTISNVQSEDLADYFCQQYSSYPFTFGSGTKLEIKRADAAPTVSI FPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWT DQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRN EC (SEQ ID NO:22).

[00162] The underline represents the leading sequence. The double underline represents CDRs. The dashed underline represents the constant region.

[00163] Another embodiment provides an antibody or its antigen-binding fragment that has a light chain without the leader sequence with Petition 870200039602, dated 03 / 26 / 2020, p. 55 / 113 / 98 an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLL IYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYSTP WTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDI NVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHN SYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:23).

[00164] The double underline represents CDRs. The dashed underline represents the constant region.

[00165] The amino acid sequence for CDR1 of the 2B5 light chain is KASQDVSTAVA (SEQ ID NO:24).

[00166] The amino acid sequence for CDR2 of the 2B5 light chain is WASTRHT (SEQ ID NO:13).

[00167] The amino acid sequence for CDR3 of the 2B5 light chain is QQHYSTPWT (SEQ ID NO:25).

[00168] One embodiment provides an antibody or its antigen-binding fragment having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 22 or 23.

[00169] Another embodiment provides an antibody or its antigen-binding fragment that has a light chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 21.

[00170] One embodiment provides an antibody with three different CDRs selected from the group consisting of SEQ ID NO: 24, 13 and 25.

[00171] Another embodiment provides an antibody or its antigen-binding fragment that has a heavy chain with an amino acid sequence that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 16 or 17 and chain Petition 870200039602, dated 03 / 26 / 2020, page 56 / 113 / 98 light with an amino acid sequence of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 22 or 23 or combinations of light and heavy chains.

[00172] Another embodiment provides an antibody or its antigen-binding fragment that has 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 SEQ ID NOs: 24, 13 and 25. 6. 4G9 heavy chain sequences

[00173] One embodiment provides a murine monoclonal antibody or its antigen-binding fragment isolated from the 4G9 hybridoma.

[00174] Another embodiment provides an antibody or its antigen-binding fragment that has a heavy chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with: ATGGGTTGGCTGTGGAACTTGCTATTCCTGATGGCAGCTGCCCAAA GTGCCCAAGCACAGATCCAGTTGGTACAGTCTGGACCTGAGCTGA AGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGT ATACCTTCACAACCTATGGAATGACCTGGGTGAAACAGGCTCCAG GAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACTCTGGAG TGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTT GGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAA AAATGAGGACACGGCTACATATTTCTGTGCAAGAGGGGGACGGGG GTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCC AAAACAACACCCCCATCAGTCTATCCACTGGCCCCTGGGTGTGGA GATACAACTGGTTCCTCTGTGACTCTGGGATGCCTGGTCAAGGGCT ACTTCCCTGAGTCAGTGACTGTGACTTGGAACTCTGGATCCCTGTC CAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACTCTAC ACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCAAGTC Petição 870200039602, de 26 / 03 / 2020, pág. 57 / 113 / 98 AGACCGTCACCTGCAGCGTTGCTCACCCAGCCAGCAGCACCACGG TGGACAAAAAACTTGAGCCCAGCGGGCCCATTTCAACAATCAACC CCTGTCCTCCATGCAAGGAGTGTCACAAAATGCCCAGCTCCTAACCT CGAGGGTGGACCATCCGTCTTCATCTTCCCTCCAATCAAGGAT GTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTGGTGG ATGTGAGCGAGGATGACCCAGACGTCCGGATCAGCTGGTTTGTGA ACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAG GATTACAACAGTACTATCCGGGTGGTCAGTGCCCTCCCCAGCCA ACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACA ACAAAGACCTCCCATCACCCATCGAGAGAACCATCTCAAAAATTA AAGGGCTAGTCAGAGCTCCACAAGTATATCTTGCCGCCACCAG CAGAGCAGTTGTCCAGGAAAGATGTCAGTCTCACTTGGGTCGT GGGCTTCAACCCTGGAGACATCAGTGTGGAGTGGACCAGCAATGG GCATACAGAGGAGAACTACAAGGACACCGCACCAGTCCTGGACTC TGACGGTTCTTACTTCATATACAAGCAAGCTCGATAAAAAACAAGC AAGTGGGAGAAAACAGATTCCTTCATGCAACGTGAAGGGACACG GGTCTGAAAAATTACTACCTGAAGAAGACCATCTCCCGGTCTCCG GGTAAATGA (SEQ ID NO:26).

[00175] Underlined sequences correspond to complementarity determining regions (CDRs). A sequence underlined twice corresponds to the constant region. Dashed underlined sequences correspond to the leader sequence.

[00176] The nucleic acid may be in a vector, for example, an expression vector. The nucleic acid may be extrachromosomal or inserted into the chromosome of a host cell, for example, a Chinese Hamster Ovary cell.

[00177] One embodiment provides an antibody or its antigen-binding fragment that has a heavy chain with an amino acid sequence of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% Petition 870200039602, dated 03 / 26 / 2020, p. 58 / 113 / 98 or 100% sequence identity with: MGWLWNLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGY TFTTYGMTWVKQAPGKGLKWMGWINTYSGVPTYADDFKGRFAFSL ETSASTAYLQINNLKNEDTATYFCARGGRGFAYWGQGTLVTVSAAK TTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSV HTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLE PSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVT CVVVDVSEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALP IQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAE QLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGS YFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID NO:27).

[00178] The single underline corresponds to the leader sequence. The double underline corresponds to CDRs and the dashed underline corresponds to the constant region.

[00179] Another embodiment provides an antibody or its antigen-binding fragment that has a heavy chain without the leader sequence with an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with: QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMTWVKQAPGKGLK WMGWINTYSGVPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTAT YFCARGGRGFAYWGQGTLVTVSAAKTTPPSVYPLAPGCGDTTGSSVT LGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPS STWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAP NLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNN VEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKD LPSPIERTISKIKGLyRAPQyYILPPPAEQLSRKDVSLTÇLyyGFNPGDI SVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSC NVRHEGLKNYYLKKTISRSP Petition 870200039602, dated 03 / 26 / 2020, page 59 / 113 / 98 GK (SEQ ID NO:28).

[00180] The double underline corresponds to CDRs and the dashed underline corresponds to the constant region.

[00181] The amino acid sequence for CDR1 of the 4G9 heavy chain is TYGMT (SEQ ID NO:29).

[00182] The amino acid sequence for CDR2 of the 4G9 heavy chain is WINTYSGVPTYADDFKG (SEQ ID NO:30).

[00183] The amino acid sequence for CDR3 of the 4G9 heavy chain is GGRGFAY (SEQ ID NO:31).

[00184] One embodiment provides a 4G9 antibody or its antigen-binding fragment that has a heavy chain according to SEQ ID NO: 27 or 28.

[00185] Another embodiment provides a 4G9 antibody or its antigen-binding fragment that has a heavy chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 26.

[00186] One embodiment provides a 4G9 antibody with three different CDRs selected from the group consisting of SEQ ID NO: 29, 30 and 31. 7. 4G9 light chain sequences

[00187] Another embodiment provides an antibody or its antigen-binding fragment that has a light chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with: ATGGTATCCACACCTCAGTTCCTTGTATTTTTGCTTTTCTGGATTCC AGCCTCCAGAGGTGACATCTTGCTGACTCAGTCTCCAGCCATCCTG TCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGT Petition 870200039602, dated 03 / 26 / 2020, page 60 / 113 / 98 CAGAGCATTGGCACAAGCATACACTGGTATCAGCAAAGAACAAAT GGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTG GGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTAC TCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTAC TGTCAACAAAGTAATAGCTGGCCGTACACACGTTCGGAGGGGGGACC AAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATC TTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCG TGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTG GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTG GACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCAC CCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAC CTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAG CTTCAACAGGAATGAGTGTTAG (SEQ ID NO:32).

[00188] The dashed underline represents the leading sequence. The single underline represents the CDRs. The double underline represents the constant region.

[00189] The nucleic acid may be in a vector, for example, an expression vector. The nucleic acid may be extrachromosomal or inserted into the chromosome of a host cell, for example, a Chinese Hamster Ovary cell.

[00190] Another embodiment provides an antibody or its antigen-binding fragment that has a light chain with an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with: MVSTPQFLVFLLFWIPASRGDILLTQSPAILSVSPGERVSFSCRASQSIG TSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESE DIADYYCQQSNSWPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGG ASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSM SSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID Petition 870200039602, dated 03 / 26 / 2020, page 61 / 113 / 98 NO:33).

[00191] The underline represents the leading sequence. The double underline represents CDRs. The dashed underline represents the constant region.

[00192] Another embodiment provides an antibody or its antigen-binding fragment that has a light chain without the leader sequence with an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with: DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKY ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPYTFGG GTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKW kidgserqnGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNEC (SEQ ID NO:34).

[00193] The double underline represents CDRs. The dashed underline represents the constant region.

[00194] The amino acid sequence for CDR1 of the 4G9 light chain is RASQSIGTSIH (SEQ ID NO:35).

[00195] The amino acid sequence for CDR2 of the 4G9 light chain is YASESIS (SEQ ID NO:36).

[00196] The amino acid sequence for CDR3 of the 4G9 light chain is QQSNSWPYT (SEQ ID NO: 37).

[00197] One embodiment provides a 4G9 antibody or its antigen-binding fragment having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 33 or 34.

[00198] Another embodiment provides a 4G9 antibody or its antigen-binding fragment that has a light chain encoded by a nucleic acid with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 32.

[00199] One modality provides a 4G9 antibody with three CDRs Petition 870200039602, dated 03 / 26 / 2020, page 62 / 113 / 98 different selected from the group consisting of SEQ ID NO: 35, 36 and 37.

[00200] Another embodiment provides an antibody or its antigen-binding fragment that has a heavy chain with an amino acid sequence that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 27 or 28 and a light chain with an amino acid sequence of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 33 or 34 or combinations of light and heavy chains.

[00201] Another embodiment provides a 4G9 antibody or its antigen-binding fragment that has 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 NOs: 35, 36, 37. D. PD-1 Activation Epitope

[00202] The epitope-specific PD-1 binding fractions are disclosed in this document. In one embodiment, the disclosed binding fractions bind immunospecifically to PD-1 and activate PD-1-mediated signal transduction.

[00203] The disclosed PD-1 epitope is formed by amino acids 96-110 of SEQ ID NO: 1 and has an amino acid sequence as follows, TYLCGAISLAPKAQI (SEQ ID NO:38). 1. Antibodies

[00204] One embodiment provides an antibody or its epitope-binding fragment that binds immunospecifically 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 its epitope-binding fragment binds immunospecifically to the SEQ ID NO: 38 of PD-1 on the surface Petition 870200039602, dated 03 / 26 / 2020, page 63 / 113 / 98 of an immune cell and promotes or induces an activation signal through PD-1 to activate the immune cell.

[00205] The epitope-specific antibody may be a monoclonal antibody, a humanized antibody, a human antibody, a mouse antibody, a chimeric antibody, or a fragment thereof. Exemplary antibodies and methods of their production are discussed below. 2. Fusion protein

[00206] In one embodiment, the epitope-specific PD-1 binding fragment is a fusion protein. Any part of one or more of the PD-1 epitope antibodies or epitope-binding fragments disclosed above can be coupled to other polypeptides to form fusion proteins. Fusion polypeptides have a first fusion partner, including all or part of one or more of the disclosed PD-1 epitope antibodies or epitope-binding fragments fused with a second polypeptide directly or through a linker peptide sequence that is fused to the second polypeptide. Fusion proteins optionally contain a domain that functions to dimerize or multimerize two or more fusion proteins. The peptide / polypeptide linker domain may be a separate domain or, alternatively, may be contained within one of the other domains (first polypeptide or second polypeptide) of the fusion protein.Similarly, the domain that functions to dimerize or multimerize fusion proteins may be a separate domain or, alternatively, may be contained within 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.

[00207] The fusion proteins disclosed in this document have the formula I: N-R1-R2-R3-C Petition 870200039602, dated 03 / 26 / 2020, page 64 / 113 / 98 where N represents the N-terminus of the fusion protein, C represents the C-terminus of the fusion protein, R1 is all or part of one of the PD-1 epitope antibodies or epitope-binding fragments disclosed, or a functional variant or fragment thereof, R2 is an optional peptide / polypeptide-binding domain, and R3 is a second polypeptide. Alternatively, R3 is all or part of one of the PD-1 epitope antibodies or epitope-binding fragments disclosed, or a functional variant or fragment thereof, and R1 is the second polypeptide.

[00208] Dimerization or multimerization can occur between or among two or more fusion proteins through dimerization or multimerization domains. Alternatively, dimerization or multimerization of fusion proteins can occur through chemical crosslinking. The dimers or multimers that are formed can be homodimeric / homomultimeric or heterodimeric / heteromultimeric. 3. Aptamers

[00209] In some embodiments, the epitope-specific binding portion is an aptamer. Aptamers are molecules that interact with the target molecule, preferentially in a specific manner. In one embodiment, the aptamer binds to SEQ ID NO:38 on the surface of an immune cell and promotes or induces an activation signal via PD-1 to activate the immune cell. Typically, aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loop or G-quartets. Aptamers can bind to proteins, cells, small organic molecules, or peptides. Aptamers can bind to small molecules such as ATP and theophylline, as well as large molecules such as reverse transcriptase and thrombin. Aptamers can bind very tightly to target molecules with Kd values ​​lower than 10⁻¹² M. It is preferable for aptamers to bind to target molecules with a Kd lower than 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹². Petition 870200039602, dated 03 / 26 / 2020, p. 65 / 113 / 98, can bind to the target molecule with a very high degree of specificity. For example, aptamers that have been isolated have a greater than 10,000-fold difference in binding affinities between the target molecule and another molecule that differs only at one position in the molecule. It is preferred that the aptamer have a Kd with the target molecule at least 10, 100, 1000, 10,000, or 100,000 times smaller than the Kd with a secondary binding molecule. It is preferred when making the comparison to a polypeptide, for example, that the secondary molecule be a different polypeptide. Representative examples of how to create and use aptamers to bind to a variety of different target molecules are known in the art. 4. Small molecules

[00210] In some embodiments, the epitope-specific binding moiety may be a small molecule. The term small molecule generally refers to small organic compounds with a molecular weight greater than about 100 and less than about 2,500 Daltons, preferably between 100 and 2000, more preferably between about 100 and about 1250, more preferably between about 100 and about 1000, more preferably between about 100 and about 750, most preferably between about 200 and about 500 Daltons. Small molecule agonists of the PD-1 activating epitope can be identified using routine 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

[00211] Pharmaceutical compositions including the disclosed antibodies and their antigen-binding fragments are provided. The pharmaceutical compositions containing the antibodies and their antigen-binding fragments may be for parenteral administration (injection). Petition 870200039602, dated 03 / 26 / 2020, page 66 / 113 / 98 intramuscular, intraperitoneal, intravenous (IV) or subcutaneous).

[00212] In some in vivo approaches, the compositions disclosed in this document are administered to a subject in a therapeutically effective amount. As used in this document, the term effective amount or therapeutically effective amount means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or otherwise provide a desired pharmacological and / or physiological effect. The precise dosage will vary according to a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being performed.

[00213] For disclosed antibodies and their antigen-binding fragments, as further studies are conducted, information will emerge regarding appropriate dosage levels for the treatment of various conditions in various patients, and a practitioner skilled in the art, considering the therapeutic context, age, and general health of the recipient, will be able to determine the appropriate dosage. The selected dosage depends on the desired therapeutic effect, the route of administration, and the desired duration of treatment. For disclosed antibodies and their antigen-binding fragments, dosage levels of 0.001 to 20 mg / kg body weight daily are generally administered to mammals. In general, for intravenous injection or infusion, the dosage may be lower.

[00214] In certain embodiments, antibodies and their antigen-binding fragments are administered locally, for example, by injection directly into a treatment site. Typically, local injection causes an increased local concentration of the immunomodulatory agent composition that is greater than that which can be achieved by systemic administration. The immunomodulatory agent compositions may be combined with a matrix as described above to aid in Petition 870200039602, dated 03 / 26 / 2020, page 67 / 113 / 98 creation of an increased localized concentration of polypeptide compositions, reducing the passive diffusion of polypeptides outside the site to be treated. 1. Formulations for parenteral administration

[00215] In some embodiments, the compositions containing the disclosed antibodies and antigen-binding fragments are administered in an aqueous solution by parenteral injection. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of an antibody or its antigen-binding fragment and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers.These compositions optionally include one or more of the following: diluents, sterile water, buffered saline solution with varying buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., timersol, benzyl alcohol), and bulking agents (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles 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 can be lyophilized and dissolved / suspended again immediately before use.The formulation can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. 2. Formulations for Oral Administration

[00216] In some embodiments, the antibody compositions are Petition 870200039602, dated 03 / 26 / 2020, page 68 / 113 / 98 formulated for oral delivery. Oral antibody dosage forms resist proteolysis and can deliver a larger fraction of immunoreactive antibody locally in the gastrointestinal tract for the treatment of infections or allow the absorption of antibodies for the treatment or prevention of systemic 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)).

[00217] Oral solid dosage forms are generally described in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton, Pa. 18042) in chapter 89. Solid dosage forms include tablets, capsules, pills, lozenges or pastilles, pellets, powders or granules, or incorporation of the material into particulate preparations of polymeric compounds, such as polylactic acid, polyglycolic acid, etc., or into liposomes. Such compositions may influence the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed substance. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, which are incorporated by reference herein. The compositions can be prepared in liquid form or as a dry powder (e.g., freeze-dried). Liposomal or proteinoid encapsulation can be used to formulate the compositions.Liposomal encapsulation can be used, and liposomes can be derivatized with various polymers (e.g., US Patent 5,013,556). See also Marshall, K. In: Modern Pharmaceutics Edited by G.S. Banker and C.T. Rhodes Chapter 10, 1979. In general, the formulation will include the peptide (or its chemically modified forms) and inert ingredients that protect the peptide in the stomach environment and the release of the biologically active material in the intestine.

[00218] Antibodies and their antigen-binding fragments can Petition 870200039602, dated 03 / 26 / 2020, page 69 / 113 / 98, to be chemically modified so that the oral delivery of the derivative is effective. Generally, the chemical modification contemplated is the attachment of at least one portion to the component molecule itself, where the portion allows absorption into the bloodstream from the stomach or intestine, or absorption directly into the intestinal mucosa. Increased overall stability of the component or components and increased circulation time in the body are also desired. PEGylation is an exemplary chemical modification for pharmaceutical use. Other moieties that can be used include: propylene glycol, ethylene glycol and propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane and poly-1,3,6-thioxocane [see, for example, 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].

[00219] Another embodiment provides liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions and syrups, which may contain other components, including inert diluents; adjuvants such as wetting agents, emulsifying and suspending agents; and sweetening, flavoring and perfuming agents.

[00220] For oral formulations, the release site may be the stomach, the small intestine (duodenum, jejunum, or ileum), or the large intestine. In some embodiments, the release will avoid the deleterious effects of the stomach environment, either by protecting the agent (or derivative) or by releasing the agent (or derivative) beyond the stomach environment, such as into the intestine. To ensure complete gastric resistance, a coating impermeable to at least pH 5.0 is essential. Examples of the most common inert ingredients used as enteric coatings are cellulose trimellitate acetate (CAT) and hydroxypropylmethylcellulose phthalate. Petition 870200039602, dated 03 / 26 / 2020, pp. 70 / 113 / 98 (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 blended films. 3. Controlled Delivery Polymer Matrices

[00221] The antibodies and their antigen-binding fragments disclosed in this document can also be administered in controlled-release formulations. The antibodies and their antigen-binding fragments can be incorporated into an inert matrix that allows release by diffusion or leaching mechanisms, for example, gums. Slow-degenerating matrices can also be incorporated into the formulation. Another form of controlled release is based on the Oros therapeutic system (Alza Corp.), i.e., the antibody or its antigen-binding fragment is embedded in a semipermeable membrane that allows water to enter and push the drug out through a single small opening due to osmotic effects.

[00222] Controlled-release polymeric devices can be made for long-term systemic release following implantation of a polymeric device (rod, cylinder, film, disc) or injection (microparticles). The matrix can be in the form of microparticles, such as microspheres, where the agent is dispersed within a matrix, or solid polymeric microcapsules, where the core is of a material different from the polymeric shell and the peptide is dispersed or suspended in the core, which can be liquid or solid in nature. Unless specifically defined in this document, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer can be molded as a thin plate or film, ranging from nanometers to four centimeters, a powder produced by milling or other standard techniques, or even a gel, such as a hydrogel. Petition 870200039602, dated 03 / 26 / 2020, page 71 / 113 / 98

[00223] Non-biodegradable or biodegradable matrices can be used for the delivery of fusion polypeptides or nucleic acids encoding fusion polypeptides, although in some embodiments biodegradable matrices are preferred. These can be natural or synthetic polymers, although synthetic polymers are preferred due to better characterization of degradation and release profiles. The polymer is selected based on the period during which release is desired. In some cases, linear release may be more useful, although in others, pulse release or bulk 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, optionally, may be crosslinked with multivalent ions or polymers.

[00224] The matrices can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, 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).

[00225] Devices can be formulated for local release to treat the implantation or injection area, which will typically deliver a dosage that is much lower than the dosage for whole-body treatment, or systemic delivery. These can be implanted or injected subcutaneously, into muscle, fat, or swallowed. III. Manufacturing Methods A. Antibody Manufacturing Methods

[00226] The disclosed antibodies can be generated in cell culture, in phages, or in various animals, including but not limited to cows, Petition 870200039602, dated 03 / 26 / 2020, pg. 72 / 113 / 98 rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes. In one embodiment, the various animals may be genetically engineered transgenic animals to produce human or humanized antibodies. Therefore, in one embodiment, the antibody is a mammalian antibody. Phage techniques may be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, a recombinant antibody may be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect the DNA sequence that expresses at least one VL region and one VH region in the host cell.Exemplary descriptions of recombinant means of generating 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).

[00227] The disclosed antibodies can be modified by recombinant means to increase antibody efficacy in mediating the desired function. Thus, it is within the scope of the invention that antibodies can be modified by substitutions using recombinant means. Typically, the substitutions will be conservative substitutions. For example, at least one amino acid in the constant region of the antibody can be replaced by a different residue. See, for example, US Pat. 5,624,821, US Pat. 6,194,551, Application No. WO 9958572; and Angal et al., Mol. Immunol. 30:105-08 (1993). Modification of amino acids includes deletions, additions, and substitutions of amino acids. In Petition 870200039602, dated 03 / 26 / 2020, p. 73 / 113 / 98 In some cases, such changes are made to reduce undesirable activities, for example, complement-dependent cytotoxicity. Frequently, antibodies are labeled by the covalent or non-covalent attachment of a substance that provides a detectable signal. A wide variety of markers and conjugation techniques are known and widely reported in the scientific and patent literature. These antibodies can be tracked for binding to proteins and polypeptides. See, for example, Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

[00228] For example, suitable antibodies with desired biological activities can be identified using in vitro assays, including, but not limited to: proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and in vivo assays such as tumor growth inhibition. The antibodies provided in this document may also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for their ability to bind to the specific antigen without inhibiting the receptor-binding activity or biological activity of the antigen. As neutralizing antibodies, the antibodies may be useful in competitive binding assays.

[00229] Antibodies that may be used in the disclosed compositions and methods include whole immunoglobulin (i.e., an intact antibody) of any class, its fragments, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. The variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, the variability is generally not uniformly distributed across the variable domains of antibodies. It is typically concentrated in three segments called determinant regions. Petition 870200039602, dated 03 / 26 / 2020, page 74 / 113 / 98 of complementarity (CDRs) or hypervariable regions, both in the variable domains of the light and heavy chains. The most highly conserved parts of the variable domains are called the framework (FR). The variable domains of the native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops that connect and, in some cases, form part of the beta-sheet structure. The CDRs in each chain are held together in the vicinity of the FR regions and, with the CDRs of the other chain, contribute to the formation of the antigen-binding site of antibodies.

[00230] Bioactive antibody fragments are also disclosed. The fragments, whether attached to other sequences or not, may include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, provided that the fragment's activity is not significantly altered or impaired compared to the unmodified antibody or antibody fragment.

[00231] The techniques can also be adapted for the production of single-chain antibodies based on disclosed antibodies and their antigen-binding fragments. Methods for the production of single-chain antibodies are well known to those skilled in the art. A single-chain antibody can be created by fusing the variable domains of the heavy and light chains using a short peptide linker, thus reconstituting an antigen-binding site in a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is attached to the N-terminus of the other variable domain via a peptide or peptide linker of 15 to 25 amino acids have been developed without significantly disrupting antigen binding or binding specificity. The peptide linker is chosen to allow the Petition 870200039602, dated 03 / 26 / 2020, page 75 / 113 / 98 heavy chain and light chain link in their proper conformational orientation.

[00232] Another embodiment provides bivalent single-chain variable fragments (di-scFvs) that can be manipulated by linking two scFvs. This can be done by producing a single peptide chain with two VH regions and two VL regions, producing tandem scFvs. scFvs can also be manipulated with linker peptides that are too short for the two variable regions to fold (about five amino acids), forcing the scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40 times lower than the corresponding scFvs, meaning they have a much higher affinity for their target. Even shorter peptide linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity for their targets than diabodies.

[00233] Another embodiment provides a monoclonal antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies within 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 a portion 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 antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired antagonistic activity.

[00234] Monoclonal antibodies can be produced using Petition 870200039602, dated 03 / 26 / 2020, page 76 / 113 / 98 any procedure that produces monoclonal antibodies. In a hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that bind specifically to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[00235] Disclosed antibodies can also be produced by recombinant DNA methods. The DNA encoding the disclosed antibodies can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or fragments of active antibodies can also be generated and screened using phage display techniques.

[00236] Antibody production methods using protein chemistry are also known in the art. One method of producing proteins comprising antibodies is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). A person skilled in the art can quickly understand that a peptide or polypeptide corresponding to the antibody, for example, can be synthesized by standard chemical reactions. For example, one peptide or polypeptide can be synthesized and not cleaved from its synthesis resin, while the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thus exposing a terminal group that is functionally blocked in the other fragment.Through peptide condensation reactions, these two fragments can be joined. Petition 870200039602, dated 03 / 26 / 2020, page 77 / 113 / 98 covalently through a peptide bond at its carboxyl and amine terminals, respectively, to form an antibody or a fragment thereof. Alternatively, the peptide or polypeptide is independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides can be linked to form an antibody or its antigen-binding fragment through similar peptide condensation reactions.

[00237] For example, enzymatic linkage of cloned or synthetic peptide segments allows relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides, or entire protein domains. Alternatively, the native chemical linkage of synthetic peptides can be used to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction. The first step is the chemoselective reaction of an unprotected synthetic alpha-thioester peptide with another unprotected peptide segment containing an amino-terminal Cys residue to produce a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes a rapid and spontaneous intramolecular reaction to form a native peptide bond at the linkage site. B. Methods for the Production of Isolated Nucleic Acid Molecules

[00238] One embodiment provides nucleic acids that encode the disclosed antibodies or their antigen-binding fragments. The nucleic acids may encode the entire antibody or its antigen-binding fragments or a light chain, heavy chain, combinations thereof, or CDRs.

[00239] Isolated nucleic acid molecules can be produced by standard techniques, including, without limitation, common molecular cloning techniques and chemical synthesis of nucleic acids. For example, polymerase chain reaction (PCR) techniques can be used to obtain an acid Petition 870200039602, dated 03 / 26 / 2020, page 78 / 113 / 98 isolated nucleic acid encoding a variant polypeptide. PCR is a technique in which target nucleic acids are enzymatically amplified. Typically, sequence information from the ends of the region of interest or beyond can be used to design oligonucleotide primers that are sequence-identical to the opposite strands of the template to be amplified. PCR can be used to amplify specific DNA and RNA sequences, including sequences of total genomic DNA or total cellular RNA. Primers are typically 14 to 40 nucleotides long, but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.By using RNA as a template source, reverse transcriptase can be used to synthesize a complementary DNA strand (cDNA). Ligase chain reaction, strand displacement amplification, self-sustaining sequence replication, or nucleic acid sequence-based amplification can 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.

[00240] Isolated nucleic acids can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated DNA synthesis in the 3' to 5' direction). For example, one or more long oligonucleotide pairs (e.g., > 100 nucleotides) can be synthesized containing the desired sequence, with each pair containing a small complementarity segment (e.g., about 15 nucleotides), so that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase can be used to extend Petition 870200039602, dated 03 / 26 / 2020, pp. 79 / 113 / 98 oligonucleotides, resulting in a single double-stranded nucleic acid molecule per oligonucleotide pair, which can then be ligated to a vector. Isolated nucleic acids can also be obtained by mutagenesis. Nucleic acids encoding proteins can undergo mutation using standard techniques, including oligonucleotide-directed mutagenesis and / or site-directed mutagenesis via 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 Use

[00241] Released antibodies and their antigen-binding fragments can be used to modulate an immune response in an individual in need. One embodiment provides a method of activating immune cells expressing PD-1, for example, T cells, to proliferate or enhance the biological activity of PD-1-expressing immune cells by administering the released antibodies and their antigen fragments, optionally including a second therapeutic agent. A. Stimulation of the Immune Response 1. Therapeutic Strategies

[00242] 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 their antigen-binding fragments to bind immunospecifically to PD-1 and induce, promote, or potentiate a stimulatory or activating signal through PD-1 to activate the immune cell. The immune response may be, for example, inducing, promoting, or potentiating T cell activation, cytokine secretion by immune cells, T cell proliferation. The disclosed antibodies or their antigen-binding fragments may be administered to a subject in need thereof in an amount effective to overcome T cell exhaustion and / or T cell anergy. Petition 870200039602, dated 03 / 26 / 2020, page 80 / 113 / 98 overcoming T cell exhaustion or T cell anergy can be determined by measuring T cell function using known techniques.

[00243] The methods can be used in vivo or ex vivo to induce, promote or enhance a stimulating immune response.

[00244] In some embodiments, the antibody or its antigen-binding fragment, or nucleic acid encoding the antibody or its antigen-binding fragment, is administered directly to the subject. In some embodiments, the antibody or its antigen-binding fragment is brought into contact with cells (e.g., immune cells) ex vivo, and the treatment cells are administered to the subject (e.g., adoptive transfer). The antibody or its antigen-binding fragment may enable a more robust immune response. The disclosed compositions are useful for stimulating or potentiating immune responses involving T cells, causing an activation signal via PD-1 in immune cells. 2. Subjects to be treated a. Cancer Treatment

[00245] Disclosed antibodies and their compositions and methods can be used to treat cancer. Generally, the agents are used to stimulate or enhance an immune response to cancer in the subject by administering to the subject an amount of a disclosed antibody or its antigen-binding fragment that induces, promotes, or increases an activation signal via PD-1. The method can reduce one or more cancer symptoms.

[00246] Immune cells activated by released antibodies or their fragments can kill cancerous cells and reduce the tumor burden in a subject. The term cancerous cell should encompass both pre-malignant and malignant cancerous cells. In some modalities, cancer refers to a benign tumor that has remained localized. In other modalities, cancer refers to a malignant tumor that has invaded and destroyed Petition 870200039602, dated 03 / 26 / 2020, page 81 / 113 / 98 the neighboring body structures and spread to distant sites. In still other modalities, cancer is associated with a specific cancer antigen (e.g., pan-carcinoma antigen (KS 1 / 4), ovarian carcinoma antigen (CA125), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2 / neu, etc.).

[00247] The antibody methods and compositions disclosed in this document are useful in the treatment or prevention of a variety of cancers or other abnormal proliferative diseases, including (but not limited to) the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myeloid leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma, and glioma; Tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas;Mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoactanthoma, seminoma, follicular thyroid cancer, and teratocarcinoma.

[00248] Cancers caused by aberrations in apoptosis can also be treated by the methods and compositions disclosed. Such cancers may include, among others, follicular lymphomas, carcinomas with p53 mutations, hormone-dependent breast, prostate and ovarian tumors, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndromes. In specific modalities, malignancy or alterations Petition 870200039602, dated 03 / 26 / 2020, pp. 82 / 113 / 98 dysproliferative disorders (such as metaplasias and dysplasias) or hyperproliferative disorders are treated or prevented by the methods and compositions in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. In other specific modalities, sarcoma, melanoma, or leukemia is treated or prevented by the methods and compositions.

[00249] The specific cancers and related disorders that can be treated or prevented by the methods and compositions disclosed in this document 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, erythroleukemia and myelodysplastic syndrome, chronic leukemias such as, but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, ciliated cell leukemia; polycythemia vera; Lymphomas such as, but not limited to, Hodgkin's disease lymphomas or non-Hodgkin's disease lymphomas (e.g., diffuse anaplastic kinase (ALK)-negative lymphoma, large B-cell lymphoma (DLBCL); diffuse anaplastic kinase (ALK)-positive lymphoma; large B-cell lymphoma (DLBCL);anaplastic kinase (ALK)-positive lymphoma, ALK+ anaplastic large cell lymphoma (ALCL), acute myeloid lymphoma (AML); multiple myelomas, such as, but not limited to, latent multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; Bone and connective tissue sarcomas, such as, but not limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, bone fibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma; Petition 870200039602, dated 03 / 26 / 2020, page 83 / 113 / 98 neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors including, but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, non-glial tumor, acoustic neuroma, 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 inflammatory breast cancer; adrenal cancer, including, but not limited to, pheochromocytoma and adrenocortical carcinoma; Thyroid cancer, including 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, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancer, including but not limited to Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipidus; eye cancer, including but not limited to ocular melanoma, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer, 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; cervical cancer, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, including but not limited to endometrial carcinoma and uterine sarcoma;Ovarian cancer including, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancer including, but not limited to, squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, carcinoma; Petition 870200039602, dated 03 / 26 / 2020, page 84 / 113 / 98 verrucous and oat grain cell carcinoma (small cell); stomach cancer including, but not limited to, adenocarcinoma, fungal type (polypoid), ulceration, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma and carcinosarcoma; colon cancer; rectal cancer; liver cancer including, but not limited to, hepatocellular carcinoma and hepatoblastoma, gallbladder cancer including, but not limited to, adenocarcinoma; cholangiocarcinomas including, but not limited to, papillary, nodular and diffuse; lung cancer including, but not limited to, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer;Testicular cancers including, but not limited to, germ cell tumor, anaplastic seminoma, classic (typical), spermatocytic, non-seminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk sac tumor); prostate cancer including, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancers; oral cancers including, but not limited to, squamous cell carcinoma; basal cell carcinoma; salivary gland cancers including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancers including, but not limited to, squamous cell carcinoma and verrucous carcinoma; skin cancers including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, malignant lentigo melanoma, acral lentiginous melanoma;Kidney cancer including, but not limited to, renal cell carcinoma, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or uterus); Wilms' tumor; bladder cancer including, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma; Petition 870200039602, dated 03 / 26 / 2020, pp. 85 / 113 / 98 lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and adenocarcinomas (for a review of such disorders, see Fishman et al., 1985, Medicine, 2nd 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 USA, Inc., United States of America). b. Treatment of Infections

[00250] The disclosed antibody compositions and methods can be used to treat infections and infectious diseases. Generally, the agents are used to stimulate or enhance an immune response to an infection in the subject by administering to the subject an amount of one or more of the disclosed antibodies or their antigen-binding fragments that send an activation or stimulation signal via PD-1. The method can reduce one or more symptoms of the infection.

[00251] The infection or disease may be caused by a bacterium, virus, protozoan, helminth, or other microbial pathogen that enters intracellularly and is attacked, that is, by cytotoxic T lymphocytes.

[00252] Infection or disease can be acute or chronic. An acute infection is typically a short-lived infection. During an acute microbial infection, immune cells begin to express immunomodulatory receptors. Therefore, in some modalities, the method involves increasing a stimulatory immune response against an acute infection.

[00253] The infection may be caused, 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 Petition 870200039602, dated 03 / 26 / 2020, p. 86 / 113 / 98 Mycobacterium.

[00254] In some embodiments, the disclosed antibody compositions are used to treat chronic infections, for example, 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.

[00255] Exemplary infections to be treated are chronic infections caused by hepatitis viruses, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), herpes viruses, Epstein-Barr virus, or human papillomavirus.

[00256] Since viral infections are primarily cleared by T cells, an increase in T cell activity would be therapeutically useful in situations where faster or more complete clearance of an infectious viral agent would be beneficial to 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 the common cold (e.g., 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 herpes lesions 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, the common cold, or encephalitis.

[00257] Representative infections that can be treated include, but are not limited to, infections caused by microorganisms including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Petition 870200039602, dated 03 / 26 / 2020, p. 87 / 113 / 98 Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus, influenza tipo B (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B e C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, e Treponema, Vibrio, Yersinia, Cryptococcus 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 e Schistosoma mansoni.,

[00258] Other microorganisms that can be treated using the disclosed compositions and methods include bacteria, such as Klebsiella, Serratia, Pasteurella; pathogens associated with cholera, tetanus, botulism, anthrax, bubonic 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, Petition 870200039602, dated 03 / 26 / 2020, pages 88 / 113 / 98 Pneumocystis, Plasmodium, Babesia, or Trypanosoma, etc. V. Combined Therapies to Enhance Immune Responses

[00259] Released antibodies and their antigen-binding fragments and their compositions may 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 their antigen-binding fragments and the additional therapeutic agent are administered separately but simultaneously. The antibodies and their antigen-binding fragments and the additional therapeutic agent may also be administered as part of the same composition. In other embodiments, the antibodies and their antigen-binding fragments and the second therapeutic agent are administered separately and at different times, but as part of the same treatment regimen. The additional therapeutic agents may be administered before, after, or alternately with the administration of the released antibodies and their antigen-binding fragments.

[00260] A first therapeutic agent may be administered to the subject 1, 2, 3, 4, 5, 6 or more hours, or 1, 2, 3, 4, 5, 6, 7 or more days before the administration of a second therapeutic agent. In some embodiments, one or more doses of the first agent may be administered to the subject every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35 or 48 days before the first administration of the second agent. Antibodies and their antigen-binding fragments may be the first or second therapeutic agent.

[00261] Antibodies and their antigen-binding fragments and the additional therapeutic agent may be administered as part of a therapeutic regimen. For example, if a first therapeutic agent may be administered to an individual every four days, 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 agent Petition 870200039602, dated 03 / 26 / 2020, pp. 89 / 113 / 98 therapeutic may be administered repeatedly throughout the treatment regimen.

[00262] Examples of additional therapeutic agents include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutics, enzymes, antibiotics, antivirals (especially protease inhibitors alone or in combination with nucleosides for the treatment of HIV or hepatitis B or C), antiparasitics (helminths, protozoa), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and their bioactive fragments (including humanized, single-chain and chimeric antibodies), antigen formulations 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 optimize the adaptive immune system, other molecules that activate or positively regulate the action of cytotoxic T lymphocytes,natural killer cells and helper T cells and other molecules that deactivate or negatively regulate the suppressor or regulator T cells.

[00263] Additional therapeutic agents are selected based on the condition, disorder, or disease being treated. For example, the immunomodulatory agent may be co-administered with one or more additional agents that work to increase or promote an immune response or to reduce or inhibit an immune response. A. Antimicrobials

[00264] In one embodiment, antibodies and their antigen-binding fragments can be administered to the subject in combination with an antimicrobial, such as an antibiotic, an antifungal, an antiviral, an antiparasitic, or an essential oil. In another embodiment, the released antibodies and their antigen-binding fragments can be Petition 870200039602, dated 03 / 26 / 2020, pp. 90 / 113 / 98 used in a preventive or prophylactic role in the treatment and prevention of diseases, and also in the context of severe traumatic injuries, such as severe burns, open bone fractures, accidental amputation or other wounds.

[00265] In some modalities, the subject receives antibodies and their antigen-binding fragments and / or the antimicrobial at the time of hospital admission to prevent further bacterial, fungal, or viral complications. The antibiotic may target pathogens, and the antibodies and their antigen-binding fragments may stimulate the immune system to provide an enhanced response to treat or prevent further infections or diseases. 1. Chemotherapeutic agents

[00266] Antibodies and their antigen-binding fragments can be combined with one or more chemotherapeutic agents and pro-apoptotic agents.Representative chemotherapeutic agents include, but are not limited to, ansacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, chrysantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mecaptopurine, mesna, methotrexate, mitomycin, mitoxntron, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafururacil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof.Representative pro-apoptotic agents include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ (2) and combinations thereof. Petition 870200039602, dated 03 / 26 / 2020, pp. 91 / 113 / 98 2. Other immunomodulators a. PD-1 antagonists

[00267] In some embodiments, antibodies and their antigen-binding fragments are co-administered with a PD-1 antagonist. Programmed Death-1 (PD-1) is a member of the CD28 family of receptors that deliver a negative immune response when induced in T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that decreases T cell multiplication and / or the resistance and / or duration of a T cell response. Suitable PD-1 antagonists are described in US Patents 8,114,845, 8,609,089, and 8,709,416, which are specifically incorporated by reference herein in their entirety, and include compounds or agents that bind to and block a PD-1 ligand to interfere with or inhibit ligand binding to the PD-1 receptor, or bind directly to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.

[00268] In some modalities, the PD-1 receptor antagonist binds directly to the PD-1 receptor without triggering inhibitory signal transduction and also binds to a PD-1 receptor ligand to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and trigger the transduction of an inhibitory signal, fewer cells are attenuated by the negative signal delivered by PD-1 signal transduction and a more robust immune response can be achieved.

[00269] PD-1 signaling is believed to be activated by binding to a PD-1 ligand (such as B7-H1 or B7-DC) in the vicinity of a peptide antigen presented by the major histocompatibility complex (MHC) (see, for example, Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)). Therefore, proteins, antibodies, or small molecules that prevent PD-1 and TCR binding on the membrane Petition 870200039602, dated 03 / 26 / 2020, pp. 92 / 113 / 98 T-cell antagonists are also useful PD-1 antagonists.

[00270] In some embodiments, PD-1 receptor antagonists are small molecule antagonists or antibodies that reduce or interfere with PD-1 receptor signal transduction by binding to PD-1 ligands or PD-1 itself, especially where PD-1 binding with TCR does not follow such binding, thus not triggering inhibitory signal transduction through the PD-1 receptor.

[00271] Other PD-1 antagonists contemplated by the methods of this invention include antibodies that bind to PD-1 or PD-1 ligands and other antibodies.

[00272] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following U.S. Patent Nos.: 7332582, 7488802, 7521051, 7524498, 7563869, 7981416, 8088905, 8287856, 8580247, 8728474, 8779105, 9067999, 9073994, 9084776, 9205148, 9358289, 9387247, 9492539, 9492540, all incorporated by reference in their entirety.

[00273] See also Berger et al., Clin. Cancer Res., 14: 30443051 (2008).

[00274] Exemplary anti-PD-L1 antibodies include, but are not limited to, those described in the following U.S. Patent Nos.: 8383796, 9102725, 9273135, 9393301, and 9580507, all of which are specifically incorporated by reference herein in their entirety.

[00275] For anti-B7-DC antibodies (also referred to as anti-PDL2), see US Pat. Nos.: 7,411,051, 7,052,694, 7,390,888, 8188238 and 9255147.

[00276] Other exemplary PD-1 receptor antagonists include, but are not limited to, PD-L2 polypeptides, including homologs and variants thereof, as well as active fragments of any of the foregoing and fusion proteins incorporating any of these. In a Petition 870200039602, dated 03 / 26 / 2020, page 93 / 113 / 98 preferred embodiment, the fusion protein comprises the soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.

[00277] The PD-1 antagonist may also be a fragment of a mammalian PD-L1, for example from a mouse or primate, such as a human, where the fragment binds to and blocks PD-1, but does not result in inhibitory signal transduction through PD-1. The fragments may also be part of a fusion protein, for example, an Ig fusion protein.

[00278] Other useful polypeptide PD-1 antagonists include those that bind to PD-1 receptor ligands. These include the PD-1 receptor protein, or its soluble fragments, which can bind to PD-1 ligands such as PD-L1 or B7-DC and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction. PD-L1 has also been shown to bind to protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)).These fragments also include the soluble ECD portion of the PD-1 protein that includes mutations, such as the A99L mutation, which increases binding to natural ligands (Molnar et al., PNAS, 105:10483-10488 (2008)). B7-1 or its soluble fragments, which can bind to the PD-L1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction, are also useful.

[00279] Antisense PD-1 and PD-L1 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 in T cells, as well as the production of T cell ligands, such as PD-L1 and / or PD-L2. For example, siRNA (e.g., about 21 nucleotides in length, which is specific to the gene encoding PD-1, or encoding a PD-1 ligand, and whose oligonucleotides can be readily acquired commercially) complexed with carriers, such as Petition 870200039602, dated 03 / 26 / 2020, page 94 / 113 / 98 polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8):2231-2244 (2009), are readily occupied by cells expressing PD-1, as well as PD-1 ligands, and reduce the expression of these receptors and ligands to achieve a decrease in inhibitory signal transduction in T cells, thereby activating T cells. b. CTLA4 antagonists

[00280] Other molecules useful in mediating the effects of T cells in an immune response are also contemplated as additional therapeutic agents. In some embodiments, the molecule is a CTLA4 antagonist, for example, an anti-CTLA4 antagonist antibody. An example of an anti-CTLA4 antibody contemplated for use in the methods of the invention includes an antibody as described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[00281] Dosages for anti-PD-1, anti-B7-H1 and anti-CTLA4 antibodies are known in the art and may be in the range of 0.1 to 100 mg / kg, with preferred lower ranges of 1 to 50 mg / kg and / or 10 to 20 mg / kg. An appropriate dose for a human subject may be between 5 and 15 mg / kg, with 10 mg / kg of antibody (e.g., human anti-PD-1 antibody) being a specific modality.

[00282] Specific examples of an anti-CTLA4 antibody useful in the methods of the invention are Ipilimumab, a human anti-CTLA4 antibody, administered at a dose of, for example, about 10 mg / kg, and Tremelimumab, 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 in December 2009.

[00283] In other embodiments, the antagonist is a small molecule. A number of small organic compounds have been shown to bind to the B7-1 ligand to prevent CTLA4 binding (see Erbe et al., J. Biol. Petition 870200039602, dated 03 / 26 / 2020, pages 95 / 113 / 98 Chem., 277:7363-7368 (2002). Such small organics could be administered alone or in conjunction with an anti-CTLA4 antibody to reduce inhibitory T cell signal transduction. 3. Enhancing Agents

[00284] In some embodiments, additional therapeutic agents include a potentiating agent. The potentiating agent acts to increase the efficacy of the positive regulator of the immune response, possibly by more than one mechanism, although the precise mechanism of action is not essential for the broad practice of the present invention.

[00285] In some embodiments, the potentiating agent is cyclophosphamide. Cyclophosphamide (CTX, Cytoxan® or Neosar®) is an oxazaosphorin drug, and analogues include ifosfamide (IFO, Ifex), perfosfamide, trofosfamide (trofosfamide; Ixoten) and their pharmaceutically acceptable salts, solvates, prodrugs and metabolites (US patent application 20070202077, which is incorporated in its entirety). Ifosfamide (MITOXANA®) is a structural analogue of cyclophosphamide, and its mechanism of action is considered identical or substantially similar to that of cyclophosphamide. Perfosfamide (4-hydroperoxycyclophosphamide) and trofosfamide are also alkylating agents, which are structurally related to cyclophosphamide. For example, perfosphamide alkylates DNA, thereby inhibiting DNA replication and the synthesis of RNA and proteins.New oxazaphosphorine derivatives have been designed and evaluated in an attempt to improve 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-glucosylisophosphoramide mustard), S-(-)-bromophosphamide (CBM-11), NSC 612567 (perhydrothiazine aldofosfamide), and NSC 613060 (thiazolidine aldofosfamide). Mafosfamide is an analog of oxazaphosphorine, which is a salt of... Petition 870200039602, dated 03 / 26 / 2020, pp. 96 / 113 / 98 chemically stable 4-hydroxy-CPA 4-thioethanesulfonic acid. Glufosphamide is an IFO derivative in which isophosphoramide mustard, the alkylating metabolite of IFO, is glycosidically linked to a beta-D-glucose molecule. Additional cyclophosphamide analogs are described in US Patent 5,190,929 entitled Cyclophosphamide analogs useful as antitumor agents, which is incorporated herein by reference in its entirety.

[00286] Although CTX itself is nontoxic, some of its metabolites are cytotoxic alkylating agents that induce 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, since lymphocytes (but not hematopoietic stem cells) express only low levels of ALDH, and cells in cycle are more sensitive to DNA alkylating agents.

[00287] In one embodiment, low doses of CTX are used in combination with the released antibodies and their antigen-binding fragments. Low doses of CTX (<200 mg / kg) may have immunostimulatory effects, including stimulation of antitumor immune responses in human and mouse cancer models (Brode & Cooke Crit Rev. Immunol. 28:109-126 (2008)). These 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+ Treg (and specifically proliferating Treg, which may be especially suppressive), (b) depletion of B lymphocytes; (c) induction of nitric oxide (NO), resulting in suppression of tumor cell growth; (d) mobilization and expansion of CD11b+Gr1+ MDSC. These primary effects have numerous secondary effects; for Petition 870200039602, dated 03 / 26 / 2020, pp. 97 / 113 / 98, for example, after Treg depletion, macrophages produce more IFN-γ and less IL-10. It has also been shown that CTX induces the expression of type I IFN and promotes homeostatic lymphocyte proliferation.

[00288] Treg depletion is most frequently cited as the mechanism by which CTX potentiates the antitumor immune response. This conclusion is based in part on the results of adoptive transfer experiments. In the AB1-HA tumor model, treatment with CTX on day 9 provides a 75% cure rate. Transfer of purified Treg 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: adoptive transfer of CD4+CD25+ Treg after pretreatment with CTX eliminated the therapeutic response to the vaccine (Taieb, JJ Immunol. 176:2722-2729 (2006)).

[00289] Numerous clinical trials in humans have demonstrated that low-dose CTX is a safe, well-tolerated, and effective agent for promoting antitumor immune responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47: 1-12 (1998)).

[00290] In one embodiment, the ideal dose for CTX to potentiate an antitumor immune response is that which decreases the overall T cell count by lowering Treg levels below the normal range, but is subtherapeutic (see Machiels et al. Cancer Res. 61:3689-3697 (2001)).

[00291] In some embodiments, CTX is used as an immunopotentiating agent at a dose of 300 mg / m2. In another embodiment, for an average man (6 feet, 170 pounds (78 kg) with a body surface area of ​​1.98 m2), 300 mg / m2, the CTX dose is 8 mg / kg, or 624 mg of total protein. In mouse cancer models, efficacy was observed at doses ranging from 15 to 150 mg / kg, which corresponds 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 Petition 870200039602, dated 03 / 26 / 2020, pp. 98 / 113 / 98 Cancer Res. 40:2135-2141 (1980)).

[00292] For larger mammals, such as a primate, such as a human patient, doses of mg / m2 can be used, but unit doses administered over a finite time interval can also be used. These unit doses can be administered daily 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 are specifically contemplated by the invention. The same regimen can be applied to the other potentiating agents mentioned in this document.

[00293] In other modalities, the potentiating agent is an agent that reduces the activity and / or number of regulatory T lymphocytes (T-regs), such as Sunitinib (SUTENT®), anti-TGFe or Imatinib (GLEEVAC®). The prescribed treatment regimen may also include the administration of an adjuvant.

[00294] Useful potentiating agents also include mitosis inhibitors such as paclitaxel, aromatase inhibitors (e.g., letrozole), and angiogenesis inhibitors (VEGF inhibitors, e.g., 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 IL-18 antagonists. VI Transgenic animals

[00295] One embodiment provides a transgenic animal that produces antibodies or their antigen-binding fragments possessing heavy chain CDRs with amino acid sequences according to SEQ ID NOs: 6, 7 and 8 and light chain CDRs with amino acids according to SEQ ID NOs: 12, 13 and 14. In one embodiment, the transgenic animal is a rodent, for example, a mouse.

[00296] Another option provides a transgenic animal that produces Petition 870200039602, dated 03 / 26 / 2020, pp. 99 / 113 / 98 antibodies or their antigen-binding fragments that possess heavy chain CDRs with amino acid sequences according to SEQ ID NOs: 18, 19 and 20 and three different light chain CDRs with amino acids selected from the group consisting of SEQ ID NOs: 24, 13 and 25. In one embodiment, the transgenic animal is a rodent, for example, a mouse.

[00297] One embodiment provides a transgenic animal that produces antibodies or their antigen-binding fragments possessing heavy chain CDRs with amino acid sequences according to SEQ ID NOs: 29, 30 and 31 and light chain CDRs with amino acids according to SEQ ID NOs: 35, 36 and 37. In one embodiment, the transgenic animal is a rodent, for example, a mouse.

[00298] 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 Patents No. 9,708,635; 9,686,970; 9,499,838; 9,445,581; 9,388,446; 8,835,712; 8,703,485; 8,232,449; 7,795,494; and 5,939,598. Examples Example 1: Production of anti-PD-1 antibodies Results

[00299] The production of anti-PD-1 antibodies yielded clones 4G9, 4C12, and 5C2, which were selected for characterization. Example 2: Kinetics of the interaction between anti-PD-1 antibodies and PD-1. Materials and methods. Petition 870200039602, dated 03 / 26 / 2020, pages 100 / 113 / 98

[00300] Antibodies from clones 4G9, 4C12, and 5C2 were characterized using a Biocore™ system available from GE. The analyte was mouse or human PD-1 and the ligand was anti-PD-1 antibody. Analyte concentrations were 0, 62.5, 125, 250, 500, and 1000 nM, when indicated. Results

[00301] Figure 1 and Table 1 show the interaction analysis of 4G9 with human PD-1. The equilibrium association constant (KA) was 9.52 x 10⁵ (1 / M). The equilibrium dissociation constant (KD) was 1.05 x 10⁻⁶ (M). Table 1. Interaction analysis of 4G9 with human PD-1. Ligand Analyte Ka (1 / Ms) Kd (1 / s) Rmax KA (1 / M) KD (M) Conc (Nm) Qui2 4G9 580 RU human PD-1 NA NA 15.1 9.52x105 1.05x 10-6 0 125 250 500 500 1000 0.464

[00302] Figure 2 and Table 2 show the interaction analysis of 4G9 with mouse PD-1. The equilibrium association constant (KA) was 1.94 x 10⁵ (1 / M). The equilibrium dissociation constant (KD) was 5.15 x 10⁻⁶ (M). Table 2. Interaction analysis of 4G9 with mouse PD-1. Ligand Analyte Ka (1 / Ms) Kd (1 / s) Rmax KA (1 / M) KD (M) Conc (Nm) Chi2 4G9 580 RU Human PD-1 2.00x103 0.0103 38.8 1.94x105 5.15x 10-6 0 125 250 500 500 1000 0.112

[00303] Figure 3 and Table 3 show the interaction analysis of 4C12 with human PD-1. The equilibrium association constant (KA) was 3.14 x 106(1 / M). The equilibrium dissociation constant (KD) was 3.19 x 10-7(M). Table 3. Interaction analysis of 4G12 with human PD-1. _____________ Ligand Analyte Ka (1 / Ms) Kd (1 / s) Rmax KA (1 / M) KD (M) Çone (Nm) Qui2~| Petition 870200039602, dated 03 / 26 / 2020, pages 101 / 113 / 98 4C12 425 RU human PD-1 NA NA 33.9 3.14x106 3.19x10-7 0 125 250 500 500 1000 2.33

[00304] Figure 4 and Table 4 show the interaction analysis of 5C2 with human PD-1. The equilibrium association constant (KA) was 2.02 x 105(1 / M). The equilibrium dissociation constant (KD) was 4.95 x 10-6(M). Table 4. Interaction analysis of 5G2 with human PD-1. Ligand Analyte Ka (1 / Ms) Kd (1 / s) Rmax KA (1 / M) KD (M) Conc (Nm) Qui2 5C2 320 RU human PD-1 NA NA 28.4 2.02x105 4.95x 10-6 0 125 250 500 500 1000 0.0263

[00305] Figure 5 and Table 5 show the interaction analysis of 5C2 with mouse PD-1. The equilibrium association constant (KA) was 1.18 x 106(1 / M). The equilibrium dissociation constant (KD) was 8.50 x 10-7(M). Table 5. Interaction analysis of 5G2 with mouse PD-1. Ligand Analyte Ka (1 / Ms) Kd (1 / s) Rmax KA (1 / M) KD (M) Conc (Nm) Qui2 5C2 320 RU Human PD-1 NA NA 11 1.18x106 8.50x 10-7 0 125 250 500 500 1000 0.107 Example 3: Binding of anti-PD-1 antibodies to EL4 cells Materials and methods

[00306] Murine EL4 cells constitutively expressing PD-1 were used in the fluorescence-activated cell sorter to assess the binding of 4G9, 5C2, and 4C12 to the cells. Results

[00307] Figure 6A is a flow cytometry histogram showing that the commercial anti-PD-1 binds to EL4 cells and the control isotype antibody does not bind to EL4 cells. Figure 6B is a flow cytometry histogram showing that 4G9, 5C2, and 4C12 bind to EL4 cells and Petition 870200039602, dated 03 / 26 / 2020, pp. 102 / 113 / 98, the secondary antibody alone does not. Figure 6C is a flow cytometry histogram showing that the 4G9 anti-PD-1 antibody binds to EL4 cells and the secondary antibody alone does not. Figure 6D is a flow cytometry histogram showing that the 5C2 anti-PD-1 antibody binds to EL4 cells and the secondary antibody alone does not. Figure 6E is a flow cytometry histogram showing that the 4C12 anti-PD-1 antibody binds to EL4 cells and the secondary antibody alone does not. Example 4: Agonistic activity of anti-PD-1 antibodies Materials and methods Mouse CD4 T cells

[00308] Purified mouse CD4 T cells were stimulated with anti-CD3 / anti-CD28 Ab for 48 hours, then anti-PD-1 Abs (10 µg / ml) was added to the culture along with protein A beads for another 48 hours. In some samples, anti-PD-L1 Ab was added to block the PD-1 / PD-L1 interaction to dissect the agonist effect of the test Abs. The CBA assay was used to detect IFNγ and IL-2 concentrations in supernatants. Human CD4 T cells

[00309] Purified mouse CD4 T cells were stimulated with anti-CD3 / anti-CD28 Ab for 48 hours, then anti-PD-1 Abs (1 or 10 µg / ml) were added to the culture along with protein A beads for another 48 hours. The CBA assay was used to detect IFN concentrations in supernatants. Results

[00310] Figures 7A and 7B show that stimulated CD4 T cells treated with anti-PD-1 antibodies 5C2, 4C12, and 4G9 exhibited higher concentrations of IFNγ and IL-2 in the supernatants compared to untreated cells or cells treated with anti-PD-L1 antibodies. Figure 7C shows that stimulated human CD4 T cells Petition 870200039602, dated 03 / 26 / 2020, pp. 103 / 113 / 98 treated with anti-PD-1 antibodies 4G9 and 5C2 had a higher concentration of IFNγ supernatant compared to untreated cells or cells treated with isotype control antibody. Example 5: Hybridomas increase Akt phosphorylation. Materials and methods

[00311] Intracellular pAKT staining (S473) was used as a marker for T cell activation. Results

[00312] Hybridomas from mice immunized with E-peptide increased Akt (S473) phosphorylation in mouse CD4 T cells (Figure 8). Example 6: Characterization of three anti-PD-1 antibodies Materials and methods

[00313] Antibodies purified from hybridomas 5C2, 4C12 and 4G9 were characterized. Results

[00314] The isotypes for each of the three anti-PD-1 antibodies were determined. 5C2 and 4C12 were both IgG1 isotypes and 4G9 was considered an IgG2b isotype (Figure 9). Hybridoma sequencing showed 100% sequence identity for antibodies 5C2 and 4C12. Example 7: 4G9 and 5C2 bind specifically to human PD-1. Materials and methods:

[00315] An ELISA assay was used to determine the binding of 4G9 and 5C2 to human PD-1-Fc. Results:

[00316] Figure 10 shows the results of an ELISA assay to evaluate the binding of 4G9 and 5C2 antibodies to human PD-1-Fc. 4G9 and 5C2 bind specifically to human PD-1. Figure 11A is a flow cytometry histogram showing that 4G9 and 5C2 do not bind to PD-1 T cells. Petition 870200039602, dated 03 / 26 / 2020, pp. 104 / 113 / 98 KO CD4, while binding to CD4 T cells from wild-type mice (Figure 11B). Example 8: Signage Materials and methods:

[00317] Mouse CD4 T cells were pre-stimulated for 48 hours to ensure 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:

[00318] Unlike blocking antibodies (RMP1-14 and J43), 4G9 and 5C2 activate T cells via the S6 pathway. Treatment of mouse CD4 T cells pre-stimulated for 48h (to ensure PD-1 expression) with purified 4G9 and 5C2 antibodies led to a significant increase in pS6 within the linear range (Cell Signaling Tech ELISA kit, *P <0.05, **P0.01, ***P0.001 compared to untreated). This phenomenon is due to direct activation, and not to PD-1 / PD-L1 blockade, since treatment with commercial Ab-1 (RMP1-14) and Ab-2 (J43) which blocks the PD-1 / PD-L1 interaction does not result in an increase in pS6. Example 8: In vivo efficacy assessment Materials and methods:

[00319] Figure 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 days 10 (D10), 17 (D17), and 24 (D24) after tumor injection, mice were treated with vaccine (E7 + PADRE + Quil A). Mice were treated with anti-PD-1 antibodies on days 10, 14, 17, 21, 24, and 28. Results:

[00320] The anti-PD-1 antibodies 4G9, 4C12, and 5C2 reduced tumor volume and increased survival when combined with the E7 vaccine. Petition 870200039602, dated 03 / 26 / 2020, pp. 105 / 113 / 98 (Figure 13B-13D). Example 9: In vivo efficacy assessment - Anti-EpE antibodies Materials and methods:

[00321] Antibodies against the E epitope were generated and tested in the TC-1 tumor model as described in Example 8 above and Figure 14A. Results:

[00322] Mice treated with 4G9, an antibody generated against the E epitope, exhibited significantly lower tumor volume and higher survival rate when compared to traditional checkpoint inhibitors, anti-PD1 blocking antibodies RMP1-14 and J43 (Figure 14B-14C). Petition 870200039602, dated 03 / 26 / 2020, pages 106 / 113

Claims

1 / 5 CLAIMS 1. Isolated antibody or its antigen-binding fragment that specifically binds to PD-1, characterized in that it comprises three heavy chain complementarity-determining regions (HCDRs) (HCDR1, HCDR2 and HCDR3) contained within any of the heavy chain variable regions (HCVR) with amino acid sequences according to SEQ ID NOs: 4 or 5, and three light chain CDRs (LCDR1, LCDR2 and LCDR3), contained within any of the light chain variable region (LCVR) sequences with amino acid sequences according to SEQ ID NOs: 10 or 11.

2. Antibody or its antigen-binding fragment according to claim 1, characterized in that it comprises: (a) an HCDR1 domain with an amino acid sequence of SEQ ID NO:6; (b) an HCDR2 domain with an amino acid sequence of SEQ ID NO:7; (c) an HCDR3 domain with an amino acid sequence of SEQ ID NO:8; (d) an LCDR1 domain with an amino acid sequence of SEQ ID NO:12; (e) an LCDR2 domain with an amino acid sequence of SEQ ID NO:13; and (f) an LCDR3 domain with an amino acid sequence of SEQ ID NO:

14.

3. Nucleic acid or combination of nucleic acids, characterized in that it encodes the antibody or antigen-binding fragment as defined in any of claims 1 to 2, comprising a nucleic acid sequence according to SEQ ID NO:3 and a nucleic acid sequence according to Petition 870260063552, dated 06 / 26 / 2026, page 12 / 24 2 / 5 SEQ ID NO:

9.

4. Isolated antibody or its antigen-binding fragment that specifically binds to PD-1, characterized in that it has three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any of the heavy chain variable regions (HCVR) with amino acid sequences according to SEQ ID NOs: 16 or 17, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any of the light chain variable region (LCVR) sequences with amino acid sequences according to SEQ ID NOs: 22 or 23.

5. Antibody or its antigen-binding fragment according to claim 4, characterized in that it comprises: (a) an HCDR1 domain with an amino acid sequence of SEQ ID NO:18; (b) an HCDR2 domain with an amino acid sequence of SEQ ID NO:19; (c) an HCDR3 domain with an amino acid sequence of SEQ ID NO:20; (d) an LCDR1 domain with an amino acid sequence of SEQ ID NO:24; (e) an LCDR2 domain with an amino acid sequence of SEQ ID NO:13; and (f) an LCDR3 domain with an amino acid sequence of SEQ ID NO:

25.

6. Nucleic acid or combination of nucleic acids, characterized in that it encodes the antibody or antigen-binding fragment as defined in any one of claims 4 to 5, comprising a nucleic acid sequence according to SEQ ID NO:15 and a nucleic acid sequence according to SEQ ID NO:

21. Petition 870260063552, dated 06 / 26 / 2026, p. 13 / 24 3 / 5 7. Isolated antibody or its antigen-binding fragment that specifically binds to PD-1, characterized in that it comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any of the heavy chain variable regions (HCVR) with amino acid sequences according to SEQ ID NOs: 27 or 28, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any of the light chain variable region (LCVR) sequences with amino acid sequences according to SEQ ID NOs: 33 or 34.

8. Antibody or its antigen-binding fragment according to claim 7, characterized in that it comprises: (a) an HCDR1 domain with an amino acid sequence of SEQ ID NO:29; (b) an HCDR2 domain with an amino acid sequence of SEQ ID NO:30; (c) an HCDR3 domain with an amino acid sequence of SEQ ID NO:31; (d) an LCDR1 domain with an amino acid sequence of SEQ ID NO:35; (e) an LCDR2 domain with an amino acid sequence of SEQ ID NO:36; and (f) an LCDR3 domain with an amino acid sequence of SEQ ID NO:

37.

9. Nucleic acid or combination of nucleic acids, characterized in that it encodes the antibody or antigen-binding fragment as defined in any one of claims 7 to 8, comprising a nucleic acid sequence according to SEQ ID NO:26 and a nucleic acid sequence according to SEQ ID NO:

32. Petition 870260063552, dated 06 / 26 / 2026, p. 14 / 24 4 / 5 10. An antibody or its antigen-binding fragment according to any one of claims 1 to 2, or 4 to 9, characterized in that the antibody or its antigen-binding fragment is human, mouse, chimeric, humanized, monoclonal, bispecific, trispecific or multispecific.

11. Antibody or its antigen-binding fragment according to claim 10, characterized in that the immune cell is a T cell.

12. Antibody or its antigen-binding fragment according to claim 11, characterized in that the T cell is a CD8+ T cell.

13. Antibody or its antigen-binding fragment according to claim 10, characterized in that it binds immunospecifically to SEQ ID NO: 38 on PD-1 expressed on the surface of an immune cell and induces or promotes a signal through PD-1 that activates or stimulates the immune cell.

14. Pharmaceutical composition, characterized in that it comprises the antibody or its antigen-binding fragment as defined in any one of claims 1 to 2, 4 to 9, 10 or 13.

15. Pharmaceutical composition according to claim 14, characterized in that it further comprises a second therapeutic agent.

16. Pharmaceutical composition according to claim 14 or 15, characterized in that it further comprises a pharmaceutically acceptable excipient.

17. Pharmaceutical composition according to claim 15, characterized in that the second therapeutic agent comprises a chemotherapeutic agent.

18. Use of an effective amount of an antibody or antigen-binding fragment as defined in any of claims 1 to 2, 4 to 9, 10 or 13, or of the pharmaceutical composition as defined in any of claims 14 to 17, characterized in that it is for preparing a medicament to induce, promote or enhance an immune response in a subject in need.

19. Use of an effective amount of an antibody or antigen-binding fragment as defined in any one of claims 1 to 2, 4 to 9, 10 or 13, or the pharmaceutical composition as defined in any one of claims 14 to 17, characterized in that it is for preparing a medicament to treat cancer in a subject in need.

20. Use of an effective amount of an antibody or antigen-binding fragment as defined in any one of claims 1 to 2, 4 to 9, 10 or 13, or of the pharmaceutical composition as defined in any one of claims 14 to 17, characterized in that it is for preparing a medicament to reduce the tumor burden in a subject in need.

21. Use of an effective quantity of an antibody or antigen-binding fragment as defined in any one of claims 1 to 2, 4 to 9, 10 or 13, or the pharmaceutical composition as defined in any one of claims 14 to 17, characterized in that it is for preparing a medicament to treat an infection in a subject in need. Petition 870260063552, dated 06 / 26 / 2026, pp. 16 / 24

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