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WO2025128264A1 - Anticorps monoclonal anti-pd-1 et ses méthodes d'utilisation - Google Patents

Anticorps monoclonal anti-pd-1 et ses méthodes d'utilisation Download PDF

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Publication number
WO2025128264A1
WO2025128264A1 PCT/US2024/055652 US2024055652W WO2025128264A1 WO 2025128264 A1 WO2025128264 A1 WO 2025128264A1 US 2024055652 W US2024055652 W US 2024055652W WO 2025128264 A1 WO2025128264 A1 WO 2025128264A1
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Prior art keywords
seq
heavy chain
composition
initial
complementarity determining
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Inventor
Ko-Chung Lin
Yi-Te YO
Shih-Long YAN
Xu Xu
Ming-Tang Chen
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PharmaEssentia Corp
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PharmaEssentia Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .xml format in ST.26 format.
  • the .xml file contains a sequence listing entitled “026-PCT- SEQ-Listing.xml” created on November 8, 2024 and having a size of 23,060 bytes and 24,576 bytes on disk.
  • the sequence listing contained in this .xml file is part of the specification and is herein incorporated by reference in its entirety.
  • the protein Programmed Death 1 (PD-1) is known in the art. Briefly, it is an inhibitory member of the CD28 family of receptors, that also includes CD28, CTLA-4, ICOS and BTLA. PD-1 is expressed on activated B cells, T cells, macrophages, and myeloid cells. The initial members of the family, CD28 and ICOS, were discovered by functional effects on augmenting T cell proliferation following the addition of monoclonal antibodies.
  • the PD-1 gene is a 50-55 kDa type I transmembrane protein that is part of the Ig gene superfamily.
  • PD-1 contains a membrane proximal immunoreceptor tyrosine inhibitory motif (ITIM) and a membrane distal tyrosine-based switch motif (ITSM).
  • ITIM membrane proximal immunoreceptor tyrosine inhibitory motif
  • ITMS membrane distal tyrosine-based switch motif
  • PD-1 lacks the MYPPPY motif that is critical for B7-1 and B7-2 binding.
  • Two ligands for PD-1 have been identified.
  • PD-L1 and PD-L2 that have been shown to downregulate T cell activation upon binding to PD-1.
  • Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1, but do not bind to other CD28 family members.
  • One ligand for PD-1, PD-L1 is abundant in a variety of human cancers.
  • the interaction between PD-1 and PD-L1 results in a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and immune evasion by the cancerous cells.
  • Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD- Ll, and the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well.
  • PD-1 has been found to play a role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type 1 diabetes, and rheumatoid arthritis.
  • GVHD graft-versus-host disease
  • the present disclosure relates to an isolated antibody or an antigen-binding portion thereof that preferentially binds PD-1, including a heavy chain complementarity determining region one (VHCDR1) including the amino acid sequence as recited in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 21, a heavy chain complementarity determining region two (VHCDR2) including the amino acid sequence as recited in SEQ ID NO: 10, and/or a heavy chain complementarity determining region three (VHCDR3) including the amino acid sequence as recited in SEQ ID NO: 11 or SEQ ID NO: 20; and a light chain complementarity determining region one (VLCDR1) including the amino acid sequence as recited in SEQ ID NO: 12, a light chain complementarity determining region two (VLCDR2) including the amino acid sequence as recited in SEQ ID NO: 13, and/or a light chain complementarity determining region three (VLCDR3) including the amino acid sequence
  • this disclosure is directed to an isolated antibody or an antigen-binding portion thereof that preferentially binds PD-1 including a heavy chain variable region (VH) comprising SEQ ID NO: 5 or SEQ ID NO:22; and a light chain variable region (VL) comprising SEQ ID NO: 6.
  • VH heavy chain variable region
  • VL light chain variable region
  • the disclosure is drawn to an isolated antibody or an antigen-binding portion thereof preferentially binds PD-1, comprising: a heavy chain complementarity determining region one (VHCDR1), a heavy chain complementarity determining region two (VHCDR2), and/or a heavy chain complementarity determining region three (VHCDR3) of or obtained from (e.g., determined, derived or copied from) a heavy chain variable region amino acid sequence as recited in SEQ ID NO: 5; and a light chain complementarity determining region one (VLCDR1), a light chain complementarity determining region two (VLCDR2), and/or a light chain complementarity determining region three (VLCDR3 obtained from (e.g., determined, derived or copied from) a light chain variable region amino acid sequence as recited in SEQ ID NO: 6.
  • VHCDR1 heavy chain complementarity determining region one
  • VHCDR2 heavy chain complementarity determining region two
  • VHCDR3 light chain complementarity
  • said isolated antibody or an antigen-binding portion thereof that preferentially binds PD-1 further comprises a heavy chain constant region (HCCR) including the amino acid sequence recited in SEQ ID NO: 7; and wherein the HCCR optionally include one, two, or three conservatively modified amino acid substitutions.
  • HCCR heavy chain constant region
  • aspect 2 includes that aspect 2’s said isolated antibody or an antigen-binding portion thereof further comprises a light chain constant region (LCCR) including the amino acid sequence of SEQ ID No: 8; and wherein the LCCR optionally includes one, two, or three conservatively modified amino acid substitutions.
  • LCCR light chain constant region
  • aspect 2 includes aspect 2’s isolated antibody or an antigen -binding portion thereof further comprising a heavy chain constant region (HCCR) including the amino acid sequence recited in SEQ ID NO: 7, and wherein said HCCR optionally includes one, two, or three conservatively modified amino acid substitutions; and wherein said isolated antibody or an antigen-binding portion thereof further comprises a light chain constant region (LCCR) including the amino acid sequence of SEQ ID No: 8, wherein the LCCR optionally includes one, two, or three conservatively modified amino acid substitutions.
  • HCCR heavy chain constant region
  • LCCR light chain constant region
  • the antibody or the antigen-binding portion’s VH further comprises a heavy chain signal peptide (HCSP) including the amino acids recited in SEQ ID NO: 15 on the N terminus of the VH.
  • HCSP heavy chain signal peptide
  • the antibody or the antigen-binding portion thereof of the present disclosure includes a VL, wherein said VL further comprises a light chain signal peptide (LCSP) including the amino acids recited in SEQ ID NO: 16 on the N terminus of the VL; optionally, the antibody or the antigen-binding portion thereof further includes a VH, which further comprises a heavy chain signal peptide (HCSP) including the amino acids recited in SEQ ID NO: 15 on the N terminus of the VH.
  • LCSP light chain signal peptide
  • HCSP heavy chain signal peptide
  • the present disclosure provides antibody or the antigen-binding portion that is a human version or humanized from a different species, and can be IgGl, IgG2, IgG3 or IgG4.
  • the present disclosure includes a pharmaceutical composition for treating, suppressing and/or inhibiting a solid tumor comprising (i) the antibody or the antigen-binding portion thereof disclosed herein; and (ii) a pharmaceutically accepted carrier.
  • a polynucleotide molecule encoding the antibody or the antigen-binding portion of the present disclosure is contemplated and made, which, in an embodiment, can be an expression vector.
  • such expression vector encodes a light chain signal peptide (LCSP) and/or a heavy chain signal peptide (HCSP) but does not comprise an intron.
  • LCSP light chain signal peptide
  • HCSP heavy chain signal peptide
  • the expression vector can be utilized in a host cell, such as E. coli.
  • the present disclosure relates to a method of preventing, treating and/or suppressing a tumor in a subj ect, comprising administering an effective amount of the antibody or the antigen-binding portion to the subject.
  • tumors can include Hepatocellular carcinoma, or solid tumor comprising melanoma and/or renal cell carcinoma (RCC).
  • the present disclosure is directed to a method of regulating the activity of one or more T cells in vitro, ex vivo, and/or in vivo in a subject, the method comprises: contacting the T cell with an antibody or an antigen-binding portion thereof which preferentially binds to human programmed death receptor 1 (hPD-1), wherein the antibody blocks and/or regulates the binding of human PD-L1 and/or human PD-L2 to hPD-1, and wherein said antibody or an antigen-binding portion thereof includes a heavy chain variable region (VH) comprising SEQ ID NO: 5 or SEQ ID NO:22; and a light chain variable region (VL) comprising SEQ ID NO: 6.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding portion thereof can further comprise a heavy chain constant region (HCCR) including the amino acid sequence recited in SEQ ID NO: 7, and the HCCR optionally includes one, two, or three conservatively modified amino acid substitutions and/or where the antibody or antigen-binding portion thereof further comprises a light chain constant region (LCCR) including the amino acid sequence of SEQ ID No: 8, and wherein the LCCR optionally includes one, two, or three conservatively modified amino acid substitutions.
  • HCCR heavy chain constant region
  • LCCR light chain constant region
  • the method of disclosed herein can regulate T cells such as one or more natural killer cells, one or more cytotoxic T cells, one or more helper T cells, one or more monocyte cells, one or more memory T cells, and/or one or more regulatory T cells.
  • T cells such as one or more natural killer cells, one or more cytotoxic T cells, one or more helper T cells, one or more monocyte cells, one or more memory T cells, and/or one or more regulatory T cells.
  • Another further aspect of the present disclosure includes a process for producing an antibody or an antigen-binding portion vector construct comprising culturing the host cell that contains an expression vector of interest under conditions allowing the expression of said antibody or antigen-binding portion construct; and purifying and/or recovering the produced antibody or antigen-binding portion vector construct from the culture.
  • the antibody or an antigen-binding portion thereof for use in the treatment of solid tumor can optionally comprise another therapeutic agent.
  • Such optional agent can be Pl 101 and/or another antihuman PD1 monoclonal antibody for treatment of solid tumor, including but not limited to RCC and/or melanoma.
  • the present disclosure includes a combination for use in the treating, suppressing, preventing and/or inhibiting of a solid tumor in a subject, wherein the combination comprises an antibody or an antigen-binding portion thereof; and another therapeutic agent, wherein the antibody or an antigen-binding portion thereof includes a heavy chain variable region (VH) comprising SEQ ID NO: 5 or SEQ ID NO:22; and a light chain variable region (VL) comprising SEQ ID NO: 6.
  • VH heavy chain variable region
  • VL light chain variable region
  • the first composition comprises an anti-PDl monoclonal antibody (P1801) and the second composition comprises (Pl 101).
  • the first composition comprises an isolated antibody or an antigen-binding portion thereof that preferentially binds PD- 1 , comprising: a heavy chain complementarity determining region one (VHCDR1) including the amino acid sequence as recited in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 21, aheavy chain complementarity determining region two (VHCDR2) including the amino acid sequence as recited in SEQ ID NO: 10, and/or a heavy chain complementarity determining region three (VHCDR3) including the amino acid sequence as recited in SEQ ID NO: 11, SEQ ID NO:20 or SEQ ID NO:23; and a light chain complementarity determining region one (VLCDR1) including the amino acid sequence as recited in SEQ ID NO: 12, a light chain complementarity determining region two (VLCDR2) including the amino acid sequence
  • the disclosure contemplates a first composition and a second composition for use in any of the methods of preventing, treating and/or suppressing a solid tumor in a subject described herein.
  • the first composition comprises the isolated antibody or an antigen-binding portion thereof described herein, an anti-PDl monoclonal antibody Pl 801, Pl 101, ipilimumab, pembrolizumab, Cemiplimab, or nivolumab.
  • Figure 1(d) denotes the light chain of P1801 with no light chain signal peptide (SEQ ID No:4).
  • Figure 1(f) denotes the full heavy chain variable region of P1801 (SEQ ID No:5).
  • Figure 1(g) denotes the full light chain variable region of P1801 (SEQ ID No:6).
  • Figure 1(h) denotes the heavy chain constant region of P1801 (SEQ ID No:7).
  • Figure l(i) denotes the light chain constant region of P1801 (SEQ ID No:8).
  • Figure 1 (j) denotes heavy chain CDR1 of P1801 (SEQ ID No:9).
  • Figure 1 (k) denotes heavy chain CDR2 of P1801 (SEQ ID No: 10).
  • Figure 1(1) denotes heavy chain CDR3 of P1801 (SEQ ID No: 11).
  • Figure l(m) denotes light chain CDR1 of P1801 (SEQ ID No: 12).
  • Figure l(n) denotes light chain CDR2 of P1801 (SEQ ID No: 13).
  • Figure l(o) denotes light chain CDR3 of P1801 (SEQ ID No: 14).
  • Figure l(p) denotes heavy chain signal peptide of P1801 (SEQ ID No: 15).
  • Figure l(q) denotes light chain signal peptide of P1801 (SEQ ID No: 16).
  • Figure l(r) denotes the hinge sequence is be located in heavy chains (SEQ ID No: 17).
  • Figure 2 denotes the chemical structure of P 1101.
  • IFN stands for human interferon-a2b.
  • mPEG stands for polyethylene glycol polymer, and each mPEG has a molecular weight from about 10KD to 30 KD, and/or each mPEG has a molecular weight from about 20 kD.
  • Figure 3 denotes competition ELISA for testing interfered capacity of selected murine anti-hPD-1 mAb (mP1801) as compared to commercially known anti-PDl mAb.
  • Figure 5 denotes another different embodiment of administration regimens of a combination of the molecules of present disclosure.
  • molecule 1 can be Pl 101 or an anti-PDl monoclonal antibody, such as Pl 801.
  • Molecule 2 can be Pl 101 or an anti-PDl monoclonal antibody, such as Pl 801, or vice versa.
  • Figure 7 denotes another embodiment of administration regimens of a combination of molecules disclosed in the present disclosure.
  • Figure 7(a) shows molecule 1 can be hIgG4 (as isotope control), Pl 101 or an anti-PDl monoclonal antibody such as Pl 801, Cemiplimab, Pembrolizumab or Novolumab administered individually.
  • Figure 7(b) shows a version of alternating between molecule 1 and 2, where molecule 1 can be mPHOl, molecule 2 can be P1801, and vice versa.
  • Figures 8(a) and 8(b) denote binding assay of Pl 801 as compared to various commercially available anti-PD 1 mAb against PD 1 antigen.
  • the Y-Axis is wavelength, and the x-axis is time in seconds.
  • the second line from the top is Pl 801 and is marked with an arrow.
  • the third line from the top is Pl 801 and is marked with an arrow.
  • Figure 9 denotes binding specificity of Pl 801.
  • P1801 specifically binds to PD-1, but not with PD-L1, PD-L2, CTLA4 or CD28 as those are flat and undetectable.
  • Figure 10 denotes the mean RCC tumor volume from 7 different groups as described in protocols in Example 9.
  • Figure 11 denotes actual RCC tumor measure from group 1 of the Example 9.
  • Figure 12 denotes actual RCC tumor measure from group 6 of the Example 9.
  • Figure 13 denotes five different cohort that is used for a subject to determine effect dosage of Pl 101 + P1801.
  • Figure 14 denotes further refinement from Figure 13 dosage cohort for effective dosage pf Pl 101 + Pl 801.
  • the top circle is for RCC and the bottom is for melanoma.
  • Figure 15 is another example of another cohort.
  • Figure 16 is an image generated with ChimeraX of the final 3D reconstruction of the PDl-AcroBio + PS 00066 + PS00067 at a nominal resolution of 2.9A.
  • Figure 17 is an image generated with ChimeraX of the final 2.9A map colored by the local resolution values calculated by cryoSPARC 4.4.
  • Figure 18 is an image of the structure of the P1801/PD1 complex solved by Cryo-Electron Microscopy.
  • Figure 19 is an image of the structure of the P1801/PL1/PD1 complex solved by Cryo-Electron Microscopy showing an overlay of P1801/PD1 and PD-L1/PD1.
  • Figure 20 is an image of the epitope mapping of the P1801 epitope.
  • Figure 21 is an image of the epitope mapping of the PD-L1 epitope.
  • Figure 22 is a model of the Pl 801 CDRs at the PD-1 binding interface.
  • isolated molecule as referring to a molecule (where the molecule is, for example, a polypeptide, a polynucleotide, or an antibody) that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same source, e.g., species, cell from which it is expressed, library, etc., (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • a molecule that is chemically synthesized, or expressed in a cellular system different from the system from which it naturally originates will be “isolated” from its naturally associated components.
  • a molecule also can be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art.
  • Molecule purity or homogeneity may be assayed by a number of means known in the art.
  • the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art.
  • higher resolution may be provided by using HPLC or other means well known in the art of purification.
  • an “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • the term encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen binding portion thereof that competes with the intact antibody for specific binding, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site.
  • Antigen binding portions include, for example, Fab, Fab’, F(ab’)2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), fragments including complementarity determining regions (CDRs), single chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the target polypeptide.
  • An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
  • the heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are known in the art.
  • variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination.
  • variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs) also known as hypervariable regions, and contribute to the formation of the antigen binding site of antibodies.
  • FRs framework regions
  • CDRs complementarity determining regions
  • variants of a subject variable region are desired, particularly with substitution in amino acid residues outside of a CDR region (i.e., in the framework region), appropriate amino acid substitution, preferably, conservative amino acid substitution, can be identified by comparing the subject variable region to the variable regions of other antibodies which contain CDR1 and CDR2 sequences in the same canonical class as the subject variable region.
  • definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and/or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the contact definition, and the conformational definition. In an embodiment, the present disclosure’s CDRs are determined by the Kabat definition.
  • the CDRs are determined by the Chothia definition.
  • the “EU index” or “EU numbering” system is based on the sequential numbering of the first human IgG sequenced (the EU antibody). Because the most common reference for this convention is the Kabat sequence manual, the EU index is sometimes erroneously used synonymously with the Kabat index. The EU index does not provide insertions and deletions, and thus in some cases comparisons of IgG positions across IgG subclass and species can be unclear, particularly in the hinge regions. Nonetheless, the convention has sufficed at enabling straightforward comparison between Fc regions in numerous Fc structure function studies.
  • CDRs formational definition of CDRs, refers to the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding.
  • a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches.
  • the methods used herein may utilize CDRs defined according to any of these approaches.
  • the CDRs may be defined in accordance with any of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), IMGT, Chothia (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), Martin, extended, AbM (AbM antibody model software using Oxford Molecular), contact, conformational definitions and/or any CDR determination method well known in the art.
  • CDRs are commonly considered as structured loops that are involved in antigen binding and exhibiting a hyper-variable amino acid composition. Defining a CDR based on antibody amino acid sequences can use different numbering schemes or utilize different definitions of CDR lengths. For example, the Kabat (and IMGT) CDR definitions are based on sequence alignments while the Chothia CDR definition better reflects the loop structure in antibodies' 3D architecture. The Kabat numbering scheme is often considered as the standard that is widely adopted for numbering antibody residues. The Chothia numbering scheme can be based on the alignment of antibody structures and ensures a better correspondence to the structural loops.
  • Chothia numbering scheme possesses that topologically aligned residues from different antibodies are localized at the same position number and that the Chothia CDR definition corresponds in most antibody sequences to the structural antigen-binding loops.
  • the CDRs defined on the hypervariable amino acids according to Kabat and based on loop topology in Chothia's nomenclature have for some CDRs shifted location and/or comprise deviating loop lengths.
  • the Martin numbering scheme focuses on the structural alignment of different framework regions of unconventional lengths.
  • the Martin numbering scheme highlighted residues that are absent in most sequences and structures and therefore define these as deletion positions and proposes a correction of the insertion point within the framework region 3 of the heavy chain domain.
  • the Martin numbering scheme uses the numbering software, ABnum, and provides a numbering scheme that consists of the Chothia numbering system corrected by the ABnum software, which integrates sequences from Kabat, IMGT, and the PDB databases.
  • IMGT numbering scheme is a reference in immunogenetics and immuno-informatics and provides a standardized numbering system for all the protein sequences of the immunoglobulin superfamily, including variable domains from antibody light and heavy chains as well as T cell receptor chains from different species.
  • IMGT numbering scheme is based on amino acid sequence alignment of the germ -line V genes. IMGT numbering scheme covers the entire variable domains and developed various tools to analyze the full-length sequences. Alternatively, or additionally, any other new method or methods not mentioned in the present disclosure can be used to determine CD Rs.
  • a “constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.
  • the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567.
  • the monoclonal antibodies may also be isolated from phage libraries generated using the techniques known in the art.
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.
  • An “antibody or an antigen-binding portion thereof preferentially binds PD-1 paratope” residue can have, for example, an atomic nucleus within 4 about A of an atomic nucleus of all isoforms.
  • the terms “Programmed Death 1,” “Programmed Cell Death 1,” “Protein PD-1,” “PD-1,” PD1,” “PDCD1,” “hPD-1” and “hPD-I” are all used interchangeably, and include variants, isoforms, species homologs of human PD-1, and analogs having at least one common epitope with PD-1.
  • the complete PD-1 sequence can be found under GenBank Accession No. U64863 and is incorporate herein by reference.
  • agonist refers to a substance which promotes (i.e., induces, causes, enhances, or increases) the biological activity or effect of another molecule.
  • agonist encompasses substances which bind receptor, such as an antibody, and substances which promote receptor function without binding thereto (e.g., by activating an associated protein).
  • antagonist refers to a substance that prevents, blocks, inhibits, neutralizes, or reduces a biological activity or effect of another molecule, such as a receptor.
  • an “anti -PD-1 antibody” refers to an antibody that is able to inhibit PD-1 biological activity and/or downstream events(s) mediated by PD-1.
  • Anti -PD-1 antibodies encompass antibodies that block, antagonize, suppress or reduce (to any degree including significantly) PD-1 biological activity, including downstream events mediated by PD-1, such as PD-L1 binding and downstream signaling, PD-L2 binding and downstream signaling, inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL- 10, and inhibition of anti-tumor immune responses.
  • anti-PD-1 antibody encompasses all the previously identified terms, titles, and functional states and characteristics whereby PD-1 itself, a PD-1 biological activity, or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any degree.
  • an anti-PD-1 antibody binds PD-1 and upregulates an anti-tumor immune response.
  • anti-PD-1 antibodies are provided herein, such as Pl 801.
  • Pl 801 is first generated as a murine version, and can later be humanized for human use.
  • RMP 1-14 which is a known version of the murine anti-PD 1 monoclonal antibody can be used to demonstrate efficacy in mice models.
  • polypeptide “oligopeptide”, “peptide” and “protein” are used interchangeably herein to refer to chains of amino acids of any length.
  • the chain may be linear or branched, it may comprise modified amino acids, and/or may be interrupted by non-amino acids.
  • the terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • the polypeptides can occur as single chains or associated chains.
  • polynucleotide refers to chains of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the chain.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g.,
  • any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports.
  • the 5’ and 3’ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls may also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2’-O-methyl-, 2’-O-allyl, 2’- fluoro- or 2 ’-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • One or more phosphodiester linkages may be replaced by alternative linking groups.
  • linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (0)NR.2 (“amidate”), P(O)R, P(O)OR’, CO or CH2 (“formacetal”), in which each R or R’ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether ( — O — ) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
  • An antibody that “preferentially binds” or “specifically binds” (used interchangeably herein) to an epitope are terms understood in the art, and methods to determine such specific or preferential binding are also known in the art.
  • a molecule is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances.
  • an antibody that specifically or preferentially binds to a PD-1 epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other PD-1 epitopes or non-PD-1 epitopes. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.
  • substantially pure refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
  • a “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • a host cell includes cells transfected in vivo with a polynucleotide(s) of this disclosure.
  • One example is E. Coli.
  • any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • Fc receptor and “FcR” describe a receptor that binds to the Fc region of an antibody.
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. FcRs are reviewed and known in the art.
  • FcR also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus.
  • the term “compete”, as used herein with regard to an antibody means that a first antibody, or an antigen-binding portion thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody, or an antigen-binding portion thereof, such that the result of binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody.
  • the alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope.
  • each antibody detectably inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s).
  • Both competing and cross-competing antibodies are encompassed by the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing and/or cross-competing antibodies are encompassed and can be useful for the methods disclosed herein.
  • a “functional Fc region” possesses at least one effector function of a native sequence Fc region.
  • exemplary “effector functions” include C Iq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptor), etc.
  • Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays known in the art for evaluating such antibody effector functions.
  • a “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, yet retains at least one effector function of the native sequence Fc region.
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide.
  • the variant Fc region herein can possess at least about 80% sequence identity with a native sequence Fc region and/or with an Fc region of a parent polypeptide, at least about 90% sequence identity therewith, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least 99.5% sequence identity therewith.
  • dose refers to a quantity of active ingredient(s) intended to be administered to a subject each time.
  • dosage sometimes refers to plural form of the term dose.
  • Each dose and dosage can contain constant and/or variable quantity of active ingredient(s), which may, depending on the context, be different in each dose or dosage.
  • the unit dose or unit dosage may be in the form of a tablet, capsule, sachet, or liquid, etc.
  • a dose e.g., in the form of one or two separate tablets
  • dose and dosage can be used interchangeably.
  • an “effective dosage”, “effective dose” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired results.
  • an effective amount prevents, alleviates, ameliorates symptoms of disease, and/or prolongs the survival of the subject being treated.
  • beneficial or desired results include, but not limited to: eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • beneficial or desired results include, but not limited to clinical results such as reducing one or more symptoms of a disease such as, for example, cancer including, for example without limitation, solid tumors, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, and/or delaying the progression of the cancer in patients.
  • An effective dosage can be administered in one or more administrations.
  • an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
  • an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.
  • an “effective dosage” or “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
  • An “individual” or a “subject” is a mammal, non-limiting example is a human. Mammals also include, but are not limited to, farm animals (e.g., cows, pigs, horses, chickens, etc.), sport animals, pets, primates, horses, dogs, cats, mice and rats.
  • farm animals e.g., cows, pigs, horses, chickens, etc.
  • sport animals e.g., pets, primates, horses, dogs, cats, mice and rats.
  • vector or “expression vector” means a construct, which is capable of delivering, and expressing one or more gene(s) or sequence(s) of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
  • expression control sequence means a nucleic acid sequence that directs transcription of a nucleic acid.
  • An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer.
  • the expression control sequence is operably linked to the nucleic acid sequence to be transcribed.
  • “pharmaceutically acceptable carrier” or “pharmaceutical acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity or stability and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions comprising such carriers are formulated by known conventional methods.
  • PBS phosphate buffered saline
  • Compositions comprising such carriers are formulated by known conventional methods.
  • kon refers to the rate constant for association of an antibody to an antigen. Specifically, the rate constants (kon and koff) and equilibrium dissociation constants are measured using full- length antibodies and/or Fab antibody fragments (i.e., univalent) and PD-1.
  • Koff refers to the rate constant for dissociation of an antibody from the antibody/antigen complex.
  • immunomodulator refers to a substance capable of altering (e.g., inhibiting, decreasing, increasing, enhancing, or stimulating) the immune response (as defined herein) or the working of any component of the innate, humoral or cellular immune system of a host mammal.
  • the term “immune modulator” encompasses the “immune-effector-cell enhancer” as defined herein and the “immune- suppressive-cell inhibitor” as defined herein, as well as substance that affects other components of the immune system of a mammal.
  • immune response refers to any detectable response to a particular substance (such as an antigen or immunogen) by the immune system of a host mammal, such as innate immune responses (e.g., activation of Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells, such as generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids).
  • innate immune responses e.g., activation of Toll receptor signaling cascade
  • cell-mediated immune responses e.g., responses mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system
  • humoral immune responses e.g., responses mediated by B cells, such as generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids.
  • immunogenic refers to the ability of a substance to cause, elicit, stimulate, or induce an immune response, or to improve, enhance, increase or prolong a pre-existing immune response, against a particular antigen, whether alone or when linked to a carrier, in the presence or absence of an adjuvant.
  • immune-suppressive-cell inhibitor refers to a substance capable of reducing or suppressing the number or function of immune suppressive cells of a mammal.
  • immune suppressive cells include regulatory T cells (“T regs”), myeloid-derived suppressor cells, and tumor- associated macrophages.
  • intradermal administration refers to the delivery of the substance into the dermis layer of the skin of the mammal.
  • the skin of a mammal is composed of an epidermis layer, a dermis layer, and a subcutaneous layer.
  • the epidermis is the outer layer of the skin.
  • the dermis which is the middle layer of the skin, contains nerve endings, sweat glands and oil (sebaceous) glands, hair follicles, and blood vessels.
  • the subcutaneous layer is made up of fat and connective tissue that houses larger blood vessels and nerves.
  • “subcutaneous administration” refers to the administration of a substance into the subcutaneous layer
  • topical administration refers to the administration of a substance onto the surface of the skin.
  • preventing refers to (a) keeping a disorder from occurring or (b) delaying the onset of a disorder or onset of symptoms of a disorder.
  • vaccine-based immunotherapy regimen refers to a therapeutic regimen in which a vaccine is administered in combination with one or more immune modulators.
  • the vaccine and the immune modulators may be administered together in a single formulation or administered separately.
  • antigen-binding portion of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-1). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody.
  • isotype refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.
  • KD is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to KA (i.e., Kd/Ka) and is expressed as a molar concentration (M).
  • KD values for antibodies can be determined using methods established in the art.
  • a method for determining the KD of an antibody is by using surface plasmon resonance, such as using a biosensor system such as a Biacore (trademarked) system.
  • values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
  • a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0. 1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range and can be up to two decimals for such number.
  • conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar 1 side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • An “adverse event” as used herein is any unfavorable and generally unintended, even undesirable, sign (including an abnormal laboratory finding), symptom, or disease associated with the use of a medical treatment.
  • an adverse event may be associated with activation of the immune system or expansion of immune system cells (e.g., T cells) in response to a treatment.
  • a medical treatment may have one or more associated AEs and each AE may have the same or different level of severity.
  • Reference to methods capable of “altering adverse events” means a treatment regime that decreases the incidence and/or severity of one or more AEs associated with the use of a different treatment regime.
  • the term “drug” refers to any compound possessing a desired biological activity and a reactive functional group available for preparing the conjugate of the disclosure.
  • the biological activity includes activity for use in the diagnosis, cure, mitigation, treatment, or prevention of a disease in human or other animals.
  • the compound involved by the term “drug” include drugs identified in the official national pharmacopeia as well as e.g., official Homeopathic Pharmacopeia of the United States, or official National Formulary, or any supplements thereof.
  • Exemplary drugs are set forth in the Physician's Desk Reference (PDR) and in the Orange Book maintained by the U.S. Food and Drug Administration (FDA).
  • HCC refers to Hepatocellular carcinoma which is a cancer that starts in liver. It's different from “secondary” liver cancers, which have spread to the liver from other organs.
  • cytotoxic agents refers to substances that inhibit or block cell expression activity, cell function and/or result in cell destruction.
  • the term includes radioisotopes, chemotherapeutics, and toxins, such as small -molecular toxins or enzymatically active toxins (including fragments and/or variants thereof) derived from bacteria, fungi, plants or animals.
  • cytotoxic agents include, but are not limited to: Auristatins (for example, Auristatin E, Auristatin F, MMAE and MMAF), chlortetracycline, metotanol, ricin, ricin A-chain, cobustatin, dokamicin, Dorastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, ccl065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracnose diketone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, a-Sarcina, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, cro
  • Figure 1(a) denotes the full heavy chain of Pl 801 including three CD Rs and variable domain plus constant regions. It also includes heavy chain signal peptides, that will be eventually cleaved by the host cell. The full heavy chain sequence is denoted as SEQ ID NO: 1.
  • Figure 1(b) denotes the full light chain of P1801 including three CDRs and variable domain plus constant regions. It also includes light chain signal peptides, that will be eventually cleaved by the host cell. The full light chain sequence is denoted as SEQ ID NO: 2.
  • Figure 1(c) denotes the heavy chain of P1801 with no heavy chain signal peptide (SEQ ID No:3).
  • Figure 1(d) denotes the light chain of P1801 with no light chain signal peptide (SEQ ID No:4).
  • Figure 1(f) denotes the full heavy chain variable region of P1801 (SEQ ID No:5).
  • Figure 1(g) denotes the full light chain variable region ofPI801 (SEQ ID No:6).
  • Figure 1(h) denotes the heavy chain constant region of Pl 801 (SEQ ID No:7).
  • Figure l(i) denotes the light chain constant region of P1801 (SEQ ID No:8).
  • Figure l(j) denotes heavy chain CDR1 of P1801 (SEQ ID No:9).
  • Figure l(k) denotes heavy chain CDR2 of P1801 (SEQ ID No: 10).
  • Figure 1(1) denotes heavy chain CDR3 of P1801 (SEQ ID No: 11).
  • Figure I(m) denotes light chain CDR1 of P1801 (SEQ ID No: 12).
  • Figure l(n) denotes light chain CDR2 of P1801 (SEQ ID No: 13).
  • Figure l(o) denotes light chain CDR3 of P1801 (SEQ ID No: 14).
  • Figure l(p) denotes heavy chain signal peptide of P1801 (SEQ ID No: 15).
  • Figure l(q) denotes light chain signal peptide of P1801 (SEQ ID No: 16).
  • Figure l(r) denotes the hinge sequence is be located in heavy chains (SEQ ID No: 17).
  • Figure 1 denotes an alternative heavy chain CDR1 of P1801 (SEQ ID No: 18).
  • Figure l(t) denotes an alternative heavy chain CDR1 of P1801 (SEQ ID No: 19).
  • Figure l(u) denotes an alternative heavy chain CDR1 of P1801 (SEQ ID No:20).
  • Figure l(v) denotes an alternative heavy chain CDR1 of P1801 (SEQ ID No:21).
  • Figure l(w) denotes an alternative heavy chain CDR2 of P1801 (SEQ ID No: 10).
  • Figure l(x) denotes an alternative heavy chain CDR3 of P1801 (SEQ ID No:23).
  • P 1801 refers to the anti-PD- 1 monoclonal antibody of the present disclosure as denoted in Figure 1.
  • Detailed amino sequences such as heavy/light chains and all CD Rs or alternative CD Rs are denoted in Figure 1(a) to l(o) and Figure 1(a) to l(o) plus Figure l(s), without signal peptides.
  • Figure 1 denotes the mapping of human P1801 with alternative CDR in Figure l(s).
  • the term “Pl 101”, “Ropeg”, and/or “pegylated human interferon-a2b” are used interchangeably.
  • Pl 101 is known in the art, for example, see U.S. Patent Numbers: US 8,143,214 B2, US 8,273,343, US 8,617,532, and/or US 8,106,160 B2, the content of all of which are incorporate herein in their entirety.
  • the chemical formula, method of manufacturing, and its uses are disclosed therein.
  • the interferon is the version that is functional in human subjects for Pl 101.
  • mPHOl refers to the same structure of pegylation and linker as Pl 101, except that the interferon is the mouse version which is functional in murine subjects.
  • Figure l(a) to l(x) show the various amino acid sequence embodiments of Pl 801.
  • Table 1 below shows molecular characteristics of P 1801.
  • the antibody of the present disclosure comprises a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and/or a light chain variable region from a particular germline light chain immunoglobulin gene.
  • the nucleic acid encoding Pl 801 and the vector expressing such amino acids contains nucleic acid molecules encoding signal peptides (SEQ ID No: 15 for heavy chain and SEQ ID No: 16 for light chain), where the signal peptide is located at the N terminus of heavy chain and/or light chain variable domain.
  • the nucleic acid encoding the light chain signal peptide does not include any intron.
  • nucleic acid molecules that encode the antibodies of the disclosure.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art.
  • DNA or RNA and may or may not contain intronic sequences.
  • the nucleic acid is an eDNA molecule.
  • Nucleic acids of the present disclosure can be obtained using standard molecular biology techniques.
  • hybridomas e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below
  • cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.
  • nucleic acid encoding the antibody can be recovered from the library.
  • VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene.
  • a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term “operatively linked”, as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in -frame.
  • the isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and CH3).
  • the sequences of human heavy chain constant region genes are known in the art, and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, particularly be IgGl or IgG4’s constant region.
  • the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CHI constant region.
  • the isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL.
  • the sequences of human light chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region, or kappa constant region.
  • VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker.
  • a flexible linker e.g., encoding the amino acid sequence (Gly4-Ser)3
  • the disclosure also provides methods of generating, selecting, and making anti-PD-1 antibodies.
  • the antibodies of this disclosure can be made by procedures known in the art. In some embodiments, antibodies may be made recombinantly and expressed using any method known in the art.
  • Cloning vectors may be constructed according to known techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, end product cloning vectors will generally have the ability to self-replicate, carry possess a single target for a particular restriction endonuclease, and/or can carry genes for a marker that can be used in selecting clones containing the vector.
  • Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other commercial cloning vectors are available from various vendors.
  • Expression vectors are further provided in this disclosure.
  • Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide according to the disclosure. It is implied that an expression vector must be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA.
  • Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT Publication No. WO 87/04462.
  • Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminators). For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.
  • the vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus).
  • electroporation employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances
  • microprojectile bombardment e.g., where the vector is an infectious agent such as vaccinia virus.
  • infection e.g., where the vector is an infectious agent such as vaccinia virus.
  • the choice of introducing vectors or polynucleotides depends on features of the host cell.
  • the disclosure herein also provides host cells comprising any of the polynucleotides described herein. Any host cells capable of over-expressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest.
  • mammalian host cells include but not limited to COS, HeLa, and CHO cells. See also PCT Publication No. WO 87/04462.
  • Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K. lactis).
  • the host cells express the cDNAs at a level of about 5-fold higher, 10-fold higher, or even 20-fold higher than that of the corresponding endogenous antibody or protein of interest, if present, in the host cells.
  • Pl 801 can be made by using Chinese hamster ovary (CHO) DG44 cells is with dhfir gene deficiency cell line(dhfr-) and comes from one of the CHO cell lineages.
  • Screening the host cells for a specific binding to PD- 1 or a PD- 1 domain is affected by an immunoassay or fluorescence activated cell sorting (FACS). A cell overexpressing the antibody or protein of interest can be identified.
  • FACS fluorescence activated cell sorting
  • An expression vector can be used to direct expression of an anti-PD-1 antibody.
  • One skilled in the art is familiar with administration of expression vectors to obtain expression of an exogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471, all ofthe contents are incorporated herein in reference in their entirety.
  • Administration of expression vectors includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration.
  • the expression vector is administered directly to the sympathetic trunk or ganglion, or into a coronary artery, atrium, ventricle, or pericardium.
  • Vectors can be transfected to host cell and stored in a Master Cell Bank.
  • a Master Cell Bank typically, one or two Master Cell Bank (MCB) vials are taken from a liquid nitrogen container. After thawing at about 37°C, the MCB vial is transferred into a 125 ml Tri-forest shake flask with pre-warmed Dynamis basic medium under a biosafety cabinet. The cells can be then cultured at about 37°C in an INFORS incubator with shaking.
  • MCB Master Cell Bank
  • Seed culture passage After about 72-96 hours of cell growth, the initial seed culture is transferred into a 500 ml Tri-forest shake flask with Dynamis basic medium and about 30ml cells under a biosafety cabinet. This culture passage can be repeated one or more times as needed.
  • cell medium can be inoculated into a SUB50 with a basic medium LC100B.
  • Cell density is determined by sampling the culture and measured using a Beckman Vi-Cell counter.
  • the pH of the fed-batch cell culture is controlled.
  • the dissolved oxygen (DO) level can be maintained at about 45%, while stirring.
  • the feeding medium and components can be individually added based on a fixed schedule.
  • Antifoam emulsion is added as needed.
  • the inoculation cell density can be about 1.5xl0 6 cells/ml. Once the cells reach the peak cell density, the cell culture is then harvested. After harvesting, the fed-batch culture can be filtrated one or more times.
  • a first molecule and a second molecule can be administered in an alternate fashion for one dose each, but with 1 or 2 days in-between the two administrations.
  • This regiment can be repeated for a fixed period (e.g., every 3, 4, 5, or 6 days), for two, three, four, or five times. Alternatively, or additionally, this can be followed by a single administration of either molecule at a single dosage with a fixed period (e.g., 3, 4, 5, or 6 days).
  • Figure 4(c) top illustrates such non-limiting example.
  • the solid arrows denote administration of Pl 101 to a subject
  • the dotted arrows denote administration of anti- PDl monoclonal antibody, such as Pl 801.
  • the solid arrows denote administration of anti-PDl monoclonal antibody, such as P1801 to a subject, and the dotted arrows denote administration of P 1101.
  • a first molecule and a second molecule can be administered in an alternate fashion for one dose each, but with 1 or 2 days in-between the two administrations. This regiment can be repeated for a fixed period (e.g., every 3, 4, 5, or 6 days), for two, three, four, or five times.
  • Figure 4(c) Bottom illustrates such non-limiting example.
  • the solid arrows denote administration of Pl 101 to a subject
  • the dotted arrows denote administration of anti-PDl monoclonal antibody, such as Pl 801.
  • the solid arrows denotes administration of anti-PDl monoclonal antibody, such as P1801 to a subject, and the dotted arrows denote administration of Pl 101.
  • two molecules are administered at the same time or on the same day, yet one molecule can be initially administered with a fewer amount of doses, follow by double, triple, or quadruple amount of doses in second, third, fourth, or 5th administrations, while a second molecule is administered with a constant amount of doses.
  • the vice versa can be done, where a first molecule is administered with a constant amount of doses, while the second molecule can be initially administered with a fewer amount of doses, follow by double, triple, or quadruple amount of doses in second, third, fourth, or 5th administrations.
  • the first molecule administration can be Pl 101
  • the second molecule can be anti-PDl monoclonal antibody, such as Pl 801.
  • the first molecule can be any anti-PDl monoclonal antibody, such as Pl 801, and the second molecule can be Pl 101.
  • a first molecule can be administered in a fixed dosage every day for the first 13 days, and the second molecule can start to be administered on day 6, with 5 more administrations, all being 3 days apart.
  • the first 4 administrations of the second molecules can have the same dosage, followed by the 5 th administrations having double the dosage than the first 4. Future the 6 th administration can have dosage that is quadrupled than the first 4 administrations.
  • Anon-limiting example can be shown in Figure 5.
  • the first molecule administration can be Pl 101
  • the second molecule can be anti- PDl monoclonal antibody, such as Pl 801.
  • the first molecule can be anti-PDl monoclonal antibody, such as Pl 801, and the second molecule can be Pl 101.
  • a first molecule can be administered either via i.p. or s.c. at 6, 7, or 8 days apart (QW x 4 from days 1-22), while the second molecules can be administered either via i.p. or s.c. at day 11, 13, 18, 20, 25 and 27 (BIW x2, day 11 to day 27).
  • the first molecule administration can be P 1101
  • the second molecule can be anti-PD 1 monoclonal antibody, such as Pl 801.
  • the first molecule can be anti-PDl monoclonal antibody, such as Pl 801, and the second molecule can be Pl 101.
  • the length between administration of a single dose of Pl 801 or a single dose of P 1101 to a subject can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, and/or 10 weeks or longer.
  • the length between administration of a single regiment of Pl 801 and a single regiment of Pl 101 to a subject can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, and/or 10 weeks or longer.
  • the use of anti-PDl monoclonal antibody in the present disclosure enhances activation of antigen specific CD4 + or CD8 + T cells, thereby enhance a subject’s immunity for treating, suppressing and/or preventing cancer, such as a solid tumor.
  • solid tumors include melanoma, Hepatocellular carcinoma (HCC), and/or kidney tumor (renal cell carcinoma or RCC).
  • P1801 and/or the combination of P1801 with another molecule can be used for the treatment of subjects with unresectable or metastatic melanoma.
  • the treatment can further optionally combine with pembrolizumab and/or nivolumab.
  • P1801 and/or the combination of P1801 with another molecule can be used for the adjuvant treatment of subjects with renal cell carcinoma (RCC) at intermediate-high or high risk of recurrence following nephrectomy or following nephrectomy and resection of metastatic lesions.
  • RCC renal cell carcinoma
  • the treatment can optionally further combine Cemiplimab, pembrolizumab and/or nivolumab.
  • Pl 801 or combined with another molecule (e.g., Pl 101) can be used to treat people with a type of skin cancer called melanoma that has spread or cannot be removed by surgery (advanced melanoma).
  • This therapy can further be optionally combined with Cemiplimab, nivolumab, pembrolizumab, and/or ipilimumab.
  • Cemiplimab Cemiplimab
  • nivolumab nivolumab
  • pembrolizumab e.g., ipilimumab
  • P1801, or combined with another molecule can be used to treat certain subjects with kidney cancer when their cancer has spread (advanced renal cell carcinoma) and the subject has not already had treatment for the advanced RCC.
  • This therapy can further be optionally combined with Cemiplimab, nivolumab, pembrolizumab, and/or ipilimumab.
  • P 1801 or combined with another molecule (e .g . , P 1101) can be used to treat people with kidney cancer (renal cell carcinoma) when the cancer has spread or grown after treatment with other cancer medications.
  • This therapy can further be optionally combined with Cemiplimab, pembrolizumab and/or nivolumab.
  • liver cancer hepatocellular carcinoma
  • P1801 or combined with another molecule (e.g., Pl 101, nivolumab, and/or ipilimumab), can be formulated as injections for intravenous (IV) use.
  • Pl 101 e.g., Pl 101, nivolumab, and/or ipilimumab
  • Pl 101 can be between about 50 to about 540 ug.
  • Non-limiting examples include sequential 6 doses of Pl 101 at 450 mcg, Q2W with P1801 at 0.3 mg/kg, Q2W; sequential 6 doses of P1101 at 450 mcg, Q2W with P1801 at 0.75 mg/kg, Q2W; sequential 6 doses of Pl 101 at 450 mcg, Q2W with P1801 at 1.5 mg/kg, Q2W; sequential 6 doses ofPHOl at 450 mcg, Q2W with Pl 801 at 2.0 mg/kg, Q2W; or sequential 6 doses ofPHOl at 450 mcg, Q2W with P1801 at 3.0 mg/kg, Q2W.
  • the treatment period can be during a 28-day, 35-day, 42-day, 49-day, or 56-day treatment cycle.
  • This example was performed to measure the binding affinity and inhibition ability of Pl 801 antibody to antigens.
  • the direct binding ELISA was performed to measure the binding specificity and cross reactivity.
  • the competitive ELISA and cell-based reporter gene assay were performed to measure the inhibition ability between P1801 PD1/PD-L1 and PD1/PD-L2.
  • Antigens were diluted to appropriate concentrations with IX DPBS, 50 pL/well overnight, and blocked with 1 % BSA, 200 pL/well for 2 hr.
  • the analytical method for a cell-based reporter gene assay for the inhibition measurement of PD- 1/PD- L1 of P1801 anti-PDl antibody was performed. Briefly, the PD-1 stably expressed Jurkat cell (Jurkat/PDl) expresses a luciferase reporter driven by NFAT response element (NFAT-RE) via TCR activation. However, while PD-1 on the Jurkat cell binds PD-L1, the interaction blocks the TCR-mediated luminescence. The luciferase is induced when the Jurkat cell is activated by TCR agonist and the PD-1/PD-L1 interaction is disrupted by anti-PD 1 ( Figure 9). More the anti-PDl blocks more interaction and induces more luciferase. The IC50 can be determined by the dose-response curve of test sample.
  • Table 4 shows the inhibitory Activity of Pl 801.
  • P1801 has a human PD 1 affinity (EC50) of 0.57 nM and a cynomolgus PD-1 affinity (EC50) of 0.44 nM. No significant difference was noted between the P1801 binding affinity of human and cynomolgus PD1. No cross reactivity was noted between Pl 801 and mouse-PD-1. The competition effect of human PD-L1/PD-L2 between P1801 and human PD-1 were observed both in competitive ELISA and cellbased assay. Pl 801 bound human PD-1 and cross-reacts with monkey PD-1, and binding efficacy (EC50) was not significantly different between human and monkey PD-1. Pl 801 did not cross-react with mouse PD-1.
  • Pl 801 inhibited the binding of PD-1 to PD-L1 and PD-L2 in a competitive binding ELISA and a cell-based reporter gene assay. Pl 801 reacts with human and Cynomogus PD-1, but not mouse PD-1.
  • P1801’s epitope binding position was compared with two existing commercially available anti-PDl mAb, namely pembrolizumab and nivolumab.
  • BLI technology was used to determine the biomolecular mutual effect.
  • This technique is an optical analytic approach analyzing the interference pattern of white light reverberated from two surfaces: an inner reference layer, and a layer of settled protein on the biosensor tip. A variation in the interference pattern is induced by any altering in the number of molecules combined with the biosensor tip. In this case, interactions can be gauged in real time, offering the capacity to supervise binding specificity, the ratio of association and dissociation, concentration, fidelity and accuracy.
  • P1801 exhibits response ranked at 0.0453 against nivolumab and 0.0426 nm against pembrolizumab. Therefore, the results exhibited P1801 is a partial blocker to PD1 as compared to the two commercially available anti-PDl mAb epitope binding sites, yet Pl 801 exhibits comparable other characteristics, which is surprising and unexpected.
  • This example was performed to measure the binding affinity of Pl 801 antibodies to antigens using Biacore 8K.
  • immobilization of FcRn and Clq onto CM5 sensor chip were performed.
  • the immobilization of FcRn and Clq was performed under 25 degrees Celsius while HBS EP was used as the running buffer.
  • the sensor chip surface of flow cells 1 and 2 were activated by freshly mixed 50 mmol/L N Hydroxysuccinimide (NHS) and 200 mmol/L 1 ethyl 3 —(3 dimethylaminopropyl) carbodii mide hydrochloride (EDC) for 200s (10 pL/min).
  • NHS N Hydroxysuccinimide
  • EDC dimethylaminopropyl carbodii mide hydrochloride
  • FcRn and Clq diluted in 30 mmol/L NaAC (pH 4.5) was injected into the flow cell 2 to achieve conjugation of appropriate Response Unit respectively, while flow cell 1 was set as blank.
  • the remaining active coupling sites on chip surface were blocked with 200 s injection of 1 mol/L ethanolamine hydrochloride.
  • the assay was performed at 25°C and the running buffer was HBS EP (PH 6.0). Diluted Pl 801 antibodies were injected over the surface of flow cell 1 and 2 as association phase, followed by injecting running buffer as dissociation phase. Next, affinity of human PD1 was measured.
  • the assay was performed at 25°C and the running buffer was HBS EP (PH 7.4). Diluted P1801 antibodies (2pg/ml) were injected over the surface as capture phase, human PD1 proteins were injected over the surface as association phase followed by injecting running buffer as dissociation phase. Finally, Pl 801 Fc-binding characterization was perfomed by SPR where kinetic data of P1801 antibodies to human PD1, FcRn and Clq were obtained through Affinity measurement. All the data was processed using the Biacore 8K Evaluation software version 1.1. Flow cell 1 and blank injection of buffer in each cycle were used as double reference for Response Units subtraction.
  • Pl 801 binds to human PD-1 with affinities (KD) of 2.50 and 3.13 nM with two drug lots, and Pl 801 binds to FcRn and Clq with an affinity (KD) that was comparable to pembrolizumab.
  • This example shows multi -cycle Kinetic Analysis of Human PD 1 , Cynomolgus PD 1 and Human CD64 to Anti-PDl Ab.
  • the ligand anti-PDl Ab was captured on the surface of a Sensor Chip Protein A.
  • the multi -cycle kinetic analysis of human PD1, cynomolgus PD1 and human CD64 to anti-PDl Ab was performed by using Biacore T200.
  • Ligand anti-PDl Ab capturing on the surface of a Sensor Chip Protein A.
  • Anti-PDl Ab was first diluted with lxHBS-EP+ to obtain a final concentration of 3 pg/ml and captured by flow cell 2 of a Sensor Chip Protein A.
  • the chip contains MabSelect SuRe ligand on the surface, allowing orientation-specific binding of the Fc region of an antibody.
  • the assay was performed by using the Kinetic / Affinity wizard.
  • the flow path was 2-1, as the ligand was injected and captured in flow cell 2 and flow cell 1 acted as a reference.
  • Series of concentrations of analytes, human PD1, cynomolgus PD1 and human CD64 were injected, respectively, over the reference and the ligand surfaces consecutively as the association phase, with short dissociation phases in between by injecting running buffer.
  • regeneration solution was injected as the regeneration phase. All the resulting data were fitted to a 1: 1 binding model by using Biacore T200 Evaluation Software version 3. Flow cell 1 and blank injection of running buffer were used as double references for subtraction. Results are summarized below in Table 6.
  • Pl 801 binds to human PD-1 with an affinity (KD) of 1.48 nM, and a cynomolgus PD1 affinity (KD) ofl.62nM.
  • P1801 has a human CD64 (IgG receptor Fc gamma RI) affinity (KD) of 2.96xlO 10 M.
  • Example 5 in vivo therapeutic efficacy
  • This Example is to show the in vivo therapeutic efficacy of test compound Pl 801 in the treatment of the HuCell MC38-hPDLl model in female hPD-1 HuGEMM mice, as well as the effect of Pl 801 + mPHOl.
  • Each mouse will be inoculated subcutaneously in the right rear flank region with HuCell MC38 tumor cells (1 x 10 6 ) in 0.1 ml of PBS for tumor development. Treatment was initiated when tumors reached a mean volume of approximately 65mm 3 . Treatment with test compounds Pl 801 resulted in significant decrease in the mean tumor volume as compared to the vehicle group. 30 mice were enrolled in the study. All animals were randomly allocated to 3 different study groups.
  • the mean tumor size at randomization was approximately 65 mm 3 .
  • the date of tumor randomization is denoted as day 0.
  • the animals were checked daily for morbidity and mortality.
  • the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (body weight was measured twice per week after randomization), and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals.
  • Tumor weight was measured at the end of the study. Dosing as well as tumor and body weight measurement were conducted in a Laminar Flow Cabinet.
  • This example contains the results of Whole Blood Cytokine Response Assay of Pl 801.
  • the assay observed that IL-2 production of SEB1 -stimulated whole blood would be enhanced by Pl 801 in a dose dependent fashion.
  • Heparinized blood from healthy donor was pre-incubated with Pl 801 and subsequently treated with SEB to induce IL-2 release.
  • IL-2 inducement can in turn regulate and/or active T-cell responses, thereby suppressing and/or treating cancer.
  • IL-2 was quantified by ELISA.
  • Whole blood was diluted ten-fold with culture medium and pre-incubated in the absence (Non-SEB induction and untreated) or presence of test articles and NC (0.04, 0.2, 1, 5 and 25 pg/mL) for 60 minutes in a 96-well plate at 37°C, 5% CO2 incubator.
  • Whole blood cells were subsequently stimulated with SEB (0.1 pg/mL) except control wells (Non-SEB induction) and then incubated at 37°C, 5% CO2 incubator for 48 hours.
  • IL-2 was quantified by ELISA following the manufacturer’s instructions. Briefly, standard and supernatants were serially diluted with Calibrator Diluent RD5-5. Add 100 pL of Assay Diluent RD 1W to each well and subsequently add 100 pL of blank, standards (31.3 ⁇ 2000 pg/mL) and diluted supernatants (2-, 4-, 8- and 16-fold dilution) to indicated wells for two hours.
  • the IL-2 concentration of all supernatants read from the standard curve must be multiplied by the dilution factor.
  • C Acceptance criteria a. If the delta O.D. of supernatant is below the lowest standard (LLOQ3), the IL-2 value will be represented by the symbol “ ⁇ LLOQ” and excluded from subsequent calculation. If the RPD 4 calculated from the IL-2 values of two indicated dilutions are higher than 20%, the concentration of lower dilution will be reported. Conversely, the average of the concentration from both dilutions will be reported.
  • N 8 for each dose level.
  • IL -2 was not detected in whole blood without SEB stimulation. At concentrations of 0.2, 1, 5 and 25 pg/mL, there were statistically significant differences in IL-2 production between Pl 801 and human IgG4 (p values of 0.019, 0.001, 0.001 and 0.015, respectively). At concentrations of 0.04, 1, 5 and 25 pg/mL, there were not statistically significant differences in IL-2 production between Pl 801 and nivolumab (p values > 0.05). Specific fold change over IgG4 was up to 1.49 in Pl 801 treated group and was comparable to nivolumab. IL-2 production in SEB-stimulated whole blood can be enhanced by Pl 801 in a dose dependent manner.
  • This example evaluates the pharmacokinetics, anti-drug antibody (ADA), receptor occupancy (RO), and IL-2 release in Cynomolgus monkeys following a single intravenous infusion of P1801 at three dose levels. All cynomolgus monkeys assigned to the study were identified as healthy animals by regular blood tests before dosing. Actual body weights and dosing details following single intravenous injections to cynomolgus monkeys were measured. The weekly records of body weights were measured. Slight body weight changes due to blood collection and no abnormal was observed during the dosing to sample collection period.
  • ADA anti-drug antibody
  • RO receptor occupancy
  • IL-2 release analysis was collected for IL-2 release analysis from all surviving animals at predose (-7 day, -1 day), and 24 hours post dose, and 168 (Day 7), 672 (Day 28) and 1334 (Day 56) hours post dose.
  • IL-2 release from monkey white blood cells (predose, -7 day) was tested in naive monkeys treated ex vivo with 0.04, 0.2, 1, 5, or 25 pg/mL Pl 801, Nivolumab, or isotype (IgG4).
  • IgG4 treatment In the SEB stimulation study, treatment with P1801 and Nivolumab promoted IL-2 release in a dose-dependent manner, while IgG4 treatment only led to a slight induction of IL- 2 release that did not increase with increasing dose.
  • IL-2 release from monkey white blood cells was measured in Pl 801 -treated monkeys intravenously infused with a single dose of 1, 5, or 20 mg/kg Pl 801. Monkey whole blood was collected at five time points (D-l, DI, D7, D28 and D56) of the pharmacokinetics study. Time-dose dependent IL-2 release was observed in the 5 and 20 mg/kg groups, while no significant Pl 801 dose-dependent IL-2 release was observed. A summary is provided in Tables 9 and 10 below.
  • Human freshly isolated PBMCs were stimulated by the anti-CD3/CD28 coated Dynabeads for 2 days to induce PD-1 expression and was then incubated with Pl 801 at the indicated concentration.
  • Pl 801 bound PD-1+ T cells were detected by using a secondary fluorophore -conjugated antibody (anti-human IgG4 Fc antibody) and analyzed on a flow cytometer.
  • Human PBMCs were isolated from heparinized blood of healthy donors.
  • Mouse AntiHuman CD3 Antibody with Fluorophore Allophycocyanin (APC) were used.
  • T Cell Activator Dynabeads (trademarked) Human T-Activator CD3/CD28 were used.
  • Human PBMCs were isolated using Ficoll-Paque (trademark) PLUS from heparinized whole blood of healthy donors not more than 6 hours after its collection.
  • Human PBMC were stimulated to express PD-1 by Dynabeads (trademarked) Human T-Activator CD3/CD28 at 37°C, 5% CO2 incubator for 2 days. After two-day activation, suspend PBMC and remove Dynabeads by magnet rack (DynaMag) (trademaek).
  • PBMC peripheral blood mononuclear cells
  • PD-1+ % the percentage of CD3+ T cells bound with anti-PD-1 antibody and calculated to subtract untreated from treated by Overton method.
  • the Geo MFI is defined as the geometric mean fluorescence intensity of PE channel, and it represents the binding level of anti-PD-1 antibody to PD-1 on the surface of all CD3+ T cell. Data in the summary table were expressed as the means ⁇ SE3 of ten donors in eight independent experiments and analyzed by one-way AN OVA4 for Pl 801 and comparator. Results are summarized in the below Table 11.
  • Results show that the percent P 1801 binding to human PBMCs was dose-dependent.
  • the percent PD- 1 Positive CD3+ T Cells of Pl 801 was not significantly different from that of nivolumab at 0.1 and 1 pg/mL.
  • the PD-1 binding activity of Pl 801 showed dose dependent.
  • At high concentration of 1 pg/mL there were no statistically significant differences in Geo MFI between P1801 and nivolumab (p > 0.05).
  • the murine version of both molecules was used, namely mP 1101 where mice interferon was used instead of human version, and RMP 1-14, a known mouse anti-PDl mAb, was used as anti-PDl mAb.
  • the study design includes: randomization into study groups; daily dosing, 7 days a week for 3 weeks; post-treatment observation period based on implantation site; subcutaneous or orthotopic administrations. During dosing and observation period, animals were examined daily, 5 days a week; body weight measured x2 weekly and tumor volume measured x2 weekly, if subcutaneous implantation.
  • Table 12 shows the design summary similar to Figure 4(a) to 4(c) show the administration of the combination with each molecule’s frequency.
  • Q3D stands for one administration every 3 days. *6 means 6 time.
  • S.C. stands for Subcutaneous administration.
  • I.P. stands for Intraperitoneal injection.
  • ROA stands for route of administration.
  • the Renca (CL-00796) cells were maintained in vitro with DMEM medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. The cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation.
  • DMEM medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air.
  • the cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation.
  • each mouse was inoculated subcutaneously in the right lower flank region with Renca tumor cells (lx 10 6 ) in 0.1 ml of PBS for tumor development. Randomization started when the mean tumor size reached approximately 70 mm3. 56 mice were enrolled in the study. All animals were randomly allocated to 7 study groups, 8 mice in each group.
  • Randomization was performed based on “Matched distribution” method/ “Stratified” method (StudyDirector (trademarked) software, version 3.1.399.19). The date of randomization was denoted as day 0. The treatment was initiated on the same day of randomization (day 1) per study design.
  • Group 6 treated with mPHOl 0.1 mg/kg Q3D x 3 followed with Anti-PDl 12 mg/kg Q3D x 6 with TGI value of 63.67 % on day 22.
  • Group 7 treated with mPHOl 0.1 mg/kg Q3D x 6 followed with Anti-PDl 12 mg/kg Q3D x 6 with TGI value of 76.40 % on day 22.
  • MST median survival time
  • test agent mP 1101 was evaluated in murine Renca model in female BALB/c mice. Compare with vehicle group, single agent mPHOl 0.1 mg/kg (Group 3) and combination with Anti-PD 1 12 mg/kg (Group 04, 05, 06 and 07) showed significant anti-tumor efficacy (P ⁇ 0.001) In addition, single agent mPHOl 0.1 mg/kg (Group 3) and combination with Anti-PDl 12 mg/kg (Group 04, 06 and 07) prolonged the survival of the mice.
  • mammal subjects can be treated with Pl 801 in an effect amount to reduce, suppress and/or treat CRC, RCC and/or melanoma.
  • Pl 801 Each subject who has RCC or melanoma is treated.
  • Treatments with P1801 result in decrease in the mean tumor volume as compared to the vehicle control group.
  • 30 subjects are enrolled in the study. All subjects are randomly allocated to 3 different study groups.
  • the mean tumor size at randomization initially is approximately 65 mm 3 .
  • the date of tumor randomization is denoted as day 0. All subjects are checked daily.
  • the study design includes: randomization into study groups; daily dosing, 7 days a week for 3 weeks; post-treatment observation period based on implantation site; subcutaneous or orthotopic administrations. During dosing and observation period, human subjects are examined daily, 5 days a week; body weight measured x2 weekly and tumor volume measured x2 weekly, if subcutaneous implantation.
  • Table 13 shows the experimental design summary, and Figure 4(a) to 4(c) show the administration of the combination with each molecule’s frequency.
  • Q3D stands for one administration every 3 days. *6 means 6 time.
  • S.C. stands for Subcutaneous administration.
  • I.P stands for Intraperitoneal injection. Table 14
  • Results show that tumor size of RCC, CRC, and/or melanoma are reduced by all of the following: Pl 801 treatment along, Pl 101 treatment along, and the combination treatment of Pl 801 and Pl 101, in human subjects for treatment for RCC, CRC, and/or melanoma.
  • the screening will take place for subjects to meeting criteria such as age >18, who has solid tumor in RCC, melanoma, Hepatocellular carcinoma (HCC) and/or CRC, and some may be in advance phase.
  • the subject would also have a life expectancy of greater than or equal to 3 months, with normal thyroid function and negative for HBV, HCV and HIV.
  • the structure and 3D model of the P 1801/PD- 1 was determined using a high-resolution Cryo-Electron Microscopy to provide atomic -level resolution structures of Pl 801/PD- 1 to reveal the spatial arrangement of amino acids in the 3D structure and determination of the paratopes.
  • the selected particles were used in one round of ab initio reconstruction followed by four heterogenous refinements with three decoys and a non-uniform refinement.
  • the final 128 K particles generated a map with the highest resolution, and that map was selected for further refinement using CTF global refinement followed by local resolution refinement and reference -based motion correction.
  • the estimated resolution is 2.9 A based on gold standard Fourier shell correlation of 0.143 ( Figures 16-17).
  • the half-maps were further refined using DeepEMhancer a python package implemented in cryoSPARC. Models for the PD 1 (Alpha-fold) and two FABs were docked into the available density using ChimeraX.
  • variable regions of the FABs were then mutated according to the sequence provided by the client for the PS00066 and PS00067. The constant regions were not shown.
  • the resulting model underwent iterative rounds of manual rebuilding (using COOT) and computer-based refinement (Phenix). Glycans were built manually at position ASN 49 and ASN 58. The final model was validated against the half-maps and its quality assessed by MolProbity. The obtained structure provides the location of the protein-protein interface between the PD1 and PS00066/PS00067.
  • PDl-AcroBio + PS00066 + PS00067 was prepared by mixing sample PDl-AcroBio, sample PS00066, and sample PS00067, in a 1: 1: 1 molar ratio and incubating for 30 minutes rt, yielding a final protein concentration of 0.18 mg/mL forthe complex.
  • Sample PDl-AcroBio, sample PS00066, and sample PS00067, in a 1: 1: 1 molar ratio was diluted 17.5-fold in DPBS (provided by client), yielding a final sample concentration of 0.01 mg/mL prior to grid preparation and imaging.
  • DPBS provided by client
  • a 3 /./ 1 drop of sample suspension is applied to an EM grid that has been plasma-cleaned using a Gatan Solarus (Pleasanton, California). After blotting the sample away with filter paper, grids are plunge-frozen in liquid ethane. Grids are stored under liquid nitrogen until transferred to the transmission electron microscope for imaging.
  • Cryo-EM Imaging using FEI Titan Krios Microscope Electron microscopy is performed using an FEI Titan Krios (Hillsboro, Oregon) transmission electron microscope operated at 300kV and equipped with a Gatan Quantum 967 LS imaging filter, a Volta phase plate, and Gatan K3 Direct Detection Camera. Vitreous ice grids are clipped into cartridges, transferred into a cassette and then into the Krios autoloader, all while maintaining the grids at cryogenic temperature (below -170C 0 ). Automated data-collection is carried out using Leginon software, where high magnification movies are acquired by selecting targets at a lower magnification.
  • CryoSPARC (Cryo-EM Single Particle Analysis Reconstruction and Classification) was used to streamline cryo-EM data processing, provide capabilities such as particle picking, 2D classification, 3D reconstruction, and refinement. Initially, grids were screened and the data complied. From the data generated from the cryoSPARC 4.4 micrograph data a map was generated. Figure 16 is an image generated with ChimeraX of the final 3D reconstruction of the PDl-AcroBio + PS 00066 + PS00067 at a nominal resolution of 2.9A. The generated map with the highest resolution was selected for further refinement using CTF global refinement followed by local resolution refinement and reference -based motion correction. The estimated resolution is 2.9 A based on gold standard Fourier shell correlation of 0.143.
  • the obtained structure provided the location of the protein-protein interface between the PD1 and PS00066/PS00067.
  • the model illustrated the hydrophobic and electrostatic interactions between PD1 and PS00067.
  • the protein-protein contact appears to be primarily driven by hydrophobic interactions, with a cluster of aliphatic and aromatic residues such as Tyr 32, Pro 33, His 52, Tyr 54 (EPS00067), Tyr 95 (M:PS00067), and Pro 39, Ala 40, Leu 41, Leu 42, Vai 43, Thr 45, Thr 145 (PD1).
  • Arg 143 and Glu 141 (PD1) may be involved in hydrogen bonding with Glu 99 (I:PS00067) and Tyr 101 (M:PS00067) / His 104 (EPS00067), respectively.
  • Figure 18 is an image of the structure of the P 1801/PD 1 complex solved by Cryo-Electron Microscopy.
  • Figure 19 is an image of the structure of the P1801/PL1/PD1 complex solved by Cryo-Electron Microscopy showing an overlay of P 1801/PD 1 ad PD-L 1/PD 1.
  • Figure 20 is an image of the epitope mapping of the P 1801 epitope showing binding to T59, S60, E61, S62, V64*, P83, E84*, D85, R86, L128*, and A129 of the heavy chain and S87, P130*, K131*, and A132* of the light chain, wherein * denotes sharing between epitopes.
  • Figure 21 is an image of the epitope mapping of the PD-L1 epitope showing binding to V64*, N66, Y68, S73, N74, Q75, T76, D77, K78, E84*, G124, 1126, L128*, P130*, K131*, A132*, 1134, and E136, wherein * denotes sharing between epitopes.
  • Figure 22 is a model of the P1801 CDRs at the PD-1 binding interface.
  • Figure 23 is a graph of epitope binding anti-PD-1 Antibodies.
  • Figure 23 shows all antibodies can bind strongly to PD-1 in the presence of Pl 801, indicating their epitope is different.
  • ISIS-clonel9 was selected as non-competing Fa.
  • Peresolimab is a humanized immunoglobulin G1 monoclonal antibody that stimulates human PD-1.
  • the Pl 801 Variable Region Heavy chain sequence with CDRs delineated is EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATITGGGSYTYYP DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLNYFDYWGQGTLVTVSS (SEQ ID No:5).
  • VHCDR1 sequence SSYDMS SEQ ID No: 18
  • VHCDR2 sequence TITGGGSYTYYPDSVKG SEQ ID No: 10
  • VHCDR3 sequence PYLNYFDY SEQ ID No: 11
  • VLCDR1 sequence RASQSISNNLH SEQ ID No: 12
  • VLCDR2 sequence YASQSIS SEQ ID No: 13
  • VLCDR3 sequence QQSNSWPLT SEQ ID No: 14
  • the P 1801 Variable Region Heavy chain sequence with CDRs delineated is EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATITGGGSYTYYP DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLNYFDYWGQGTLVTVSS (SEQ ID No:5).
  • VHCDR1 sequence GFTFSSYDMS (SEQ ID No: 19); VHCDR2 sequence TITGGGSYTYYPDSVKG (SEQ ID No: 10); and VHCDR3 sequence ASPYLNYFDY (SEQ ID No:20).
  • the Pl 801 Variable Region light chain sequence with CDRs delineated is
  • VLCDR1 sequence RASQSISNNLH SEQ ID No: 12
  • VLCDR2 sequence YASQSIS SEQ ID No: 13
  • VLCDR3 sequence QQSNSWPLT SEQ ID No: 14
  • the Pl 801 Variable Region Heavy chain sequence with CDRs delineated illustrating the paratopes is EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATITGGGSYTYYP DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLNYFDYWGQGTLVTVSS (SEQ ID No:5).
  • VHCDR1 sequence GFTFSSYDMS (SEQ ID No: 19) can also be written as Gly Phe Thr Phe Ser Ser Tyr Asp Met Ser and numbered as Glyl Phe2 Thr3 Phe4 Ser5 Ser6 Tyr7 Asp8 Met9 SerlO.
  • the paratopes of the VHCDR1 sequence share Phe2 Ser5 Ser6 and Tyr7 over all paratopes.
  • Glyl Thr3 Phe4 Asp8 Met9 and SerlO may be individually substituted for any amino acid residue.
  • Glyl may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai;
  • Thr3 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Vai;
  • Phe4 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, lie, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Vai;
  • Asp8 may be independently substituted for Ala, Arg, Asn, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met
  • VHCDR2 sequence TITGGGSYTYYPDSVKG (SEQ ID No: 10) can be Thr He Thr Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Vai Lys Gly and numbered as Thr 11 He 12 Thrl3 Glyl4 Glyl5 Glyl6 Serl7 Tyrl8 Thr 19 Tyr20 Tyr21 Pro22 Asp23 Ser24 Val25 Lys26 Gly27.
  • the paratopes of the VHCDR2 sequence share Thrl3 Glyl4 Glyl5 Glyl6 Serl7 and Tyrl8 over all paratopes.
  • Thrll lie 12, Thrl9, Tyr20, Tyr21, Pro22, Asp23, Ser24, Val25, Lys26, and Gly27 may be individually substituted for any amino acid residue.
  • Thrll may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Vai
  • He 12 may be independently substituted for for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai
  • Thrl9 may be independently substituted for for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Va
  • VHCDR3 sequence ASPYLNYFDY (SEQ ID No:20) can be Ala Ser Pro Tyr Leu Asn Tyr Phe Asp Tyr and numbered as Ala30 Ser31 Pro32 Tyr33 Leu34 Asn35 Tyr36 Phe37 Asp38 Tyr39.
  • the paratopes of the VHCDR3 sequence share Ser31, Tyr33, Leu34, Asn35, Asp38, and Tyr39 over all paratopes.
  • Ala30, Pro32, Tyr36, and Phe37 may be individually substituted for any amino acid residue.
  • Ala30 may be independently substituted for Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai;
  • Pro32 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Vai;
  • Tyr36 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai; and
  • Phe37 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu,
  • VHCDR1 alternative sequence GFTFSYDMS (SEQ ID No:21) can also be written as Gly Phe Thr Phe Ser Tyr Asp Met Ser and numbered as Gly 1 Phe2 Thr3 Phe4 Ser5 Tyr6 Asp 7 Met8 Ser9.
  • the paratopes of the VHCDR1 sequence share Phe2, Ser5, and Tyr6 over all paratopes.
  • Glyl, Thr3, Phe4, Asp8, Met9, and SerlO may be individually substituted for any amino acid residue.
  • Glyl may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai;
  • Thr3 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Vai;
  • Phe4 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, lie, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Vai;
  • Asp7 may be independently substituted for Ala, Arg, Asn, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met
  • VHCDR2 sequence TITGGGSYTYYPDSVKG (SEQ ID No: 10) can be Thr lie Thr Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Vai Lys Gly and numbered as Thrl 1 lie 12 Thrl3 Glyl4 Glyl5 Glyl6 Serl7 Tyrl8 Thrl9 Tyr20 Tyr21 Pro22 Asp23 Ser24 Val25 Lys26 Gly27.
  • the paratopes of the VHCDR2 sequence share Thrl3, Glyl4, Glyl5, Glyl6, Serl7, and Tyrl8 over all paratopes.
  • Thrl l lie 12, Thr 19, Tyr20, Tyr21, Pro22, Asp2,3 Ser24, Val25, Lys26, and Gly27 may be individually substituted for any amino acid residue.
  • Thrll may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Vai
  • He 12 may be independently substituted for for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai
  • Thr 19 may be independently substituted for for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Vai
  • VHCDR3 sequence ASPYLNYFDY (SEQ ID No:20) can be Ala Ser Pro Tyr Leu Asn Tyr Phe Asp Tyr and numbered as Ala30 Ser31 Pro32 Tyr33 Leu34 Asn35 Tyr36 Phe37 Asp38 Tyr39.
  • the paratopes of the VHCDR3 sequence share Ser31, Tyr33, Leu34, Asn35, Asp38, and Tyr39 over all paratopes.
  • Ala30, Pro32, Tyr36, and Phe37 may be individually substituted for any amino acid residue.
  • Ala30 may be independently substituted for Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai;
  • Pro32 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Vai;
  • Tyr36 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai; and
  • Phe37 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu,
  • VHCDR3 sequence PYLNYFDY (SEQ ID No: 11) can be Pro Tyr Leu Asn Tyr Phe Asp Tyr and numbered as Pro32 Tyr33 Leu34 Asn35 Tyr36 Phe37 Asp38 Tyr39.
  • the paratopes of the VHCDR3 sequence share Tyr33, Leu34, Asn35, Asp38, and Tyr39 over all paratopes.
  • Pro32, Tyr36, and Phe37 may be individually substituted for any amino acid residue.
  • Tyr36 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai; and Phe37 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Vai.
  • VHCDR3 sequence SPYLNYFDY (SEQ ID No:23) can be Ser Pro Tyr Leu Asn Tyr Phe Asp Tyr and numbered as Ser31 Pro32 Tyr33 Leu34 Asn35 Tyr36 Phe37 Asp38 Tyr39.
  • the paratopes of the VHCDR3 sequence share Ser31, Tyr33, Leu34, Asn35, Asp38, and Tyr39 over all paratopes.
  • Pro32, Tyr36, and Phe37 may be individually substituted for any amino acid residue.
  • Pro32 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, lie, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Vai;
  • Tyr36 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai;
  • Phe37 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Vai.
  • the Pl 801 Variable Region light chain sequence with CDRs delineated illustrating the paratopes is
  • VLCDR1 sequence RASQSISNNLH (SEQ ID No: 12) can also be written as Arg Ala Ser Gin Ser He Ser Asn Asn Leu His and numbered as Argl Ala2 Ser3 Gln4 Ser5 Ile6 Ser7 Asn8 Asn9 Leu 10 Hisll.
  • the paratopes of the VLCDR1 sequence share Asn9 over all paratopes.
  • Argl, Ala2, Ser3, Gln4, Ser5, Ile6, Ser7, Asn8, LeulO, and Hisl 1 may be individually substituted for any amino acid residue.
  • Argl may be independently substituted for Ala, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai
  • Ala2 may be independently substituted for Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai
  • Ser3 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai
  • ln4 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Glx, Gly, His, lie, Leu, Lys, Met,
  • VLCDR2 sequence YASQSIS (SEQ ID No: 13) can be Tyr Ala Ser Gin Ser lie Ser and numbered as Tyrl2 Alal3 Serl4 Glnl5 Serl6 lie 17 Serl8.
  • the paratopes of the VLCDR2 sequence share Tyrl2 and Seri 8 over all paratopes.
  • Alal3, Serl4, Glnl5, Serl6, and lie 17 may be individually substituted for any amino acid residue.
  • Alal3 may be independently substituted for Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai
  • Serl4 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai
  • Gin 15 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Glx, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai
  • Seri 6 may be independently substituted for Ala, Arg, Asn, As
  • VLCDR3 sequence QQSNSWPLT (SEQ ID No: 14) can be Gin Gin Ser Asn Ser Trp Pro Leu Thr and numbered as Gln20 Gln21 Ser22 Asn23 Ser24 Trp25 Pro26 Leu27 Thr28.
  • the paratopes of the VLCDR3 sequence share Ser22, Asn23, and Trp25 over all paratopes.
  • Gln20, Gln21, Ser24, Pro26, Leu27, and Thr28 may be individually substituted for any amino acid residue.
  • Gln20 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai
  • Gln21 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai
  • Ser24 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai
  • Pro26 may be independently substituted for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Ly
  • amino acid residues presented in bold and underlined are constant over the various proteins.
  • the remaining amino acid residues in the sequences may be individually substituted for any amino acid residue, e.g., Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gin, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai.
  • the bolded and underlined residues in the CDR’s are maintained the remaining amino acid residues may be individually substituted while maintaining function.
  • the present disclosure envisions numerous variations in the sequence of the CDRs provided the bolded and underlined residues in the CDR’s are maintained with one or more of the remaining amino acid residues being substituted. Given this functional definition the current inventors contemplate a plethora of different CDR sequences characterized by the bolded and underlined residues in the CDR, one more or substituted amino acids and the retention of the function of the sequence. [0284] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • the skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

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Abstract

La présente divulgation concerne une méthode de prévention, de traitement et/ou de suppression d'une tumeur solide chez un sujet, la méthode comprenant les étapes : d'administration d'une première composition pendant une première période de traitement, la première période de traitement comprenant une première administration de composition initiale suivie d'une ou plusieurs premières administrations ultérieures espacées à un premier intervalle de 2 à 4 jours ; et d'administration d'une deuxième composition pendant une deuxième période de traitement, la deuxième période de traitement comprenant une deuxième administration de composition initiale suivie d'une ou plusieurs deuxièmes administrations ultérieures espacées à un deuxième intervalle de 2 à 4 jours.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190038763A1 (en) * 2016-01-28 2019-02-07 INSERM (Institute National de la Santé et de la Recherche Médicale) Methods and pharmaceutical composition for the treatment of cancer
US20200024350A1 (en) * 2016-12-07 2020-01-23 Agenus Inc. Antibodies and methods of use thereof
WO2023098798A1 (fr) * 2021-12-01 2023-06-08 正大天晴药业集团股份有限公司 Association de médicaments pour le traitement du cancer du poumon non à petites cellules
US20230212155A1 (en) * 2018-02-13 2023-07-06 Gilead Sciences, Inc. Pd-1/pd-l1 inhibitors

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SG11201502816YA (en) * 2012-10-12 2015-05-28 Agency Science Tech & Res Optimised heavy chain and light chain signal peptides for the production of recombinant antibody therapeutics
HRP20210122T1 (hr) * 2013-05-02 2021-04-16 Anaptysbio, Inc. Protutijela usmjerena protiv programirane smrti-1 (pd-1)
SG11201700496WA (en) * 2014-07-22 2017-02-27 Cb Therapeutics Inc Anti-pd-1 antibodies
ES2853823T3 (es) * 2014-09-30 2021-09-17 Intervet Int Bv Anticuerpos de PD-L1 que se unen a PD-L1 canino
CN108341871A (zh) * 2017-01-24 2018-07-31 三生国健药业（上海）股份有限公司 抗pd-1单克隆抗体及其制备方法和应用
US10981993B2 (en) * 2017-02-03 2021-04-20 Beijing Dongfang Biotech Co., Ltd. Anti-PD-1 monoclonal antibody and obtaining method therefor
TWI879716B (zh) * 2017-05-09 2025-04-11 美商提薩羅有限公司 治療癌症的組合療法
SG11202013170RA (en) * 2018-07-11 2021-01-28 Kahr Medical Ltd Pd1-4-1bbl variant fusion protein and methods of use thereof
WO2022026775A1 (fr) * 2020-07-30 2022-02-03 Prellis Biologics, Inc Compositions et procédés de ciblage de coronavirus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190038763A1 (en) * 2016-01-28 2019-02-07 INSERM (Institute National de la Santé et de la Recherche Médicale) Methods and pharmaceutical composition for the treatment of cancer
US20200024350A1 (en) * 2016-12-07 2020-01-23 Agenus Inc. Antibodies and methods of use thereof
US20230212155A1 (en) * 2018-02-13 2023-07-06 Gilead Sciences, Inc. Pd-1/pd-l1 inhibitors
WO2023098798A1 (fr) * 2021-12-01 2023-06-08 正大天晴药业集团股份有限公司 Association de médicaments pour le traitement du cancer du poumon non à petites cellules

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