WO2022105751A1 - PROTÉINE DE FUSION TGFβRII - Google Patents
PROTÉINE DE FUSION TGFβRII Download PDFInfo
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- WO2022105751A1 WO2022105751A1 PCT/CN2021/130930 CN2021130930W WO2022105751A1 WO 2022105751 A1 WO2022105751 A1 WO 2022105751A1 CN 2021130930 W CN2021130930 W CN 2021130930W WO 2022105751 A1 WO2022105751 A1 WO 2022105751A1
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- A—HUMAN NECESSITIES
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/495—Transforming growth factor [TGF]
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Definitions
- the present invention relates to novel fusion proteins comprising PD-L1 antibody and TGF ⁇ receptor. Furthermore, the present invention relates to nucleic acids encoding the fusion proteins or fragments thereof and host cells comprising the same, and related uses. Furthermore, the present invention relates to the therapeutic use of these fusion proteins or fragments thereof.
- Programmed death ligand 1 is a protein involved in suppressing immune system responses during chronic infection, pregnancy, tissue allotransplantation, autoimmune disease and cancer. PD-L1 regulates immune responses by binding to an inhibitory receptor called programmed death 1 (PD-1) expressed on the surface of T cells, B cells and monocytes. PD-L1 also negatively regulates T cell function through interaction with another receptor, B7.1 (also known as B7-1 or CD80). The formation of PD-L1/PD-1 and PD-L1/B7.1 complexes negatively regulates T-cell receptor signaling, leading to subsequent downregulation of T-cell activation and suppression of antitumor immune activity. PD-L1 is overexpressed in many cancers. PD-L1 overexpression in tumor cells promotes tumor invasion and is often associated with poor prognosis.
- PD-L1 Programmed death ligand 1
- TGF- ⁇ Transforming growth factor- ⁇
- TGF[beta] has three mammalian isoforms, TGF[beta]-1, TGF[beta]-2 and TGF[beta]-3, each with distinct functions in vivo. All 3 TGF ⁇ use the same receptor signaling system. Binding of TGF ⁇ to TGF ⁇ RII is a critical step in the initiation of activation of the TGF ⁇ signaling pathway, which leads to phosphorylation of Smad2 and translocation of the activated Smad2/Smad4 complex to the nucleus to regulate gene expression. Therefore, blocking the binding of human TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3 to human TGF ⁇ RII can inhibit angiogenesis and inhibit tumor cell growth, which is one of the research directions of cancer treatment.
- the molecule in the WO 2015/118175 A2 patent has a relatively low affinity for the anti-PD-L1 end, and there is a problem of fragmentation in the protein expression and purification; while the WO 2018/205985 A1 patent solves the problem of molecular fragmentation, it is carried out at the N-terminus of TGF ⁇ RII. truncation, but this truncation affects the affinity and blocking activity of the molecule for TGF ⁇ .
- the present invention relates to a novel fusion protein molecule comprising PD-L1 and TGF ⁇ RII, which has high affinity for PD-L1 to ensure a high occupancy ratio for PD-L1 and for PD-1/PD-L1 interaction Efficient blocking of the action, high binding activity to TGF ⁇ , and high stability at the same time.
- the present invention relates to the following specific embodiments:
- a fusion protein molecule comprising
- the anti-PD-L1 antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3 respectively shown in the following amino acid sequences: SEQ ID NOs: 1, 2 and 3, and respectively as shown in the following amino acid sequences LCDR1, LCDR2 and LCDR3: SEQ ID NOs: 8, 9 and 10;
- fusion protein molecule of embodiment 1 or 2 wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises:
- amino acid sequence set forth in SEQ ID NO:4 comprising the amino acid sequence set forth in SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or A VH consisting of the amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 11 and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% therewith or an amino acid sequence of 99% identity or a VL consisting of said amino acid sequence.
- amino acid sequence comprising or consisting of an amino acid sequence having no more than 20 amino acid changes compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 5;
- (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12; or
- amino acid sequence comprising or consisting of an amino acid sequence having no more than 20 amino acid changes compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 12.
- a fusion protein molecule comprising
- chain A anti-PD-L1 antibody light chain
- Chain B an anti-PD-L1 antibody heavy chain directly linked at the C-terminus to the ECD of TGF ⁇ RII or to a C-terminally truncated ECD;
- the anti-PD-L1 antibody light chain comprises LCDR1, LCDR2 and LCDR3 as respectively shown in the following amino acid sequences: SEQ ID NO: 8, 9 and 10
- the anti-PD-L1 antibody heavy chain comprises respectively as shown in the following amino acid sequences HCDR1, HCDR2, HCDR3: SEQ ID NOs: 1, 2 and 3.
- the anti-PD-L1 antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, An amino acid sequence of 96%, 97%, 98% or 99% identity or a VH consisting of said amino acid sequence
- the anti-PD-L1 antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 11 and has at least 90% therewith , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or VL consisting of said amino acid sequences.
- Chain A comprises or is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 14 a specific amino acid sequence or consist of said amino acid sequence;
- Chain B comprises or is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:7 a specific amino acid sequence or consist of said amino acid sequence.
- amino acid sequence comprising or consisting of an amino acid sequence having no more than 5, 4, 3, 2, 1 amino acid changes compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 41.
- (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 42 or 43; or
- amino acid sequence of amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions of amino acids) of or consisting of said amino acid sequences.
- the antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single chain antibody (eg, scFv), (Fab') 2 , single domain antibodies such as VHHs, dAbs (domain antibodies) or linear antibodies.
- a host cell comprising nucleic acids encoding chain A and chain B of the fusion protein molecule of any one of embodiments 6-12, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from CHO -S cells or HEK293 cells or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
- a method of making a molecule of a fusion protein comprising culturing the host cell of embodiment 16 under conditions suitable for expressing nucleic acids encoding strand A and strand B of the fusion protein molecule of any one of embodiments 6-12 , optionally isolating the antibody or antigen-binding fragment thereof, optionally the method further comprising recovering the fusion protein molecule from the host cell.
- a pharmaceutical composition comprising the fusion protein molecule of any one of embodiments 1 to 13, and optionally one or more other therapeutic agents such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small Molecular drugs or immunomodulators, and optionally pharmaceutical excipients.
- a pharmaceutical combination comprising the fusion protein molecule of any one of embodiments 1 to 13, and one or more other therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immune regulator.
- therapeutic agents such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immune regulator.
- a method of preventing or treating a tumor in a subject comprising administering to the subject an effective amount of the fusion protein molecule of any one of embodiments 1 to 13, or the pharmaceutical composition of embodiment 18, or the pharmaceutical combination of embodiment 19.
- the cancer has elevated levels (eg nucleic acid or protein levels) of TGF ⁇ and/or PD-L1 (or PD-1 or PD-L2) ), for example the cancer is colon cancer.
- the method further comprises administering to the patient one or more therapies, such as a therapeutic modality and/or other therapeutic agents, preferably the therapeutic modality includes surgery and/or radiation therapy,
- a therapeutic modality includes surgery and/or radiation therapy
- the other therapeutic agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulatory agents.
- FIG. 1A shows the NMR structure of TGF ⁇ RII
- FIG. 1B shows a schematic diagram of the fusion protein of the present invention.
- Figure 2 shows that the fusion protein molecule PTR2, together with two control molecules, M7824 and SHR1701, blocks the signal generated by SMAD2 reporter cells upon stimulation of TGF ⁇ .
- Figure 3 shows the fusion protein molecule PTR2, with two control molecules M7824 and SHR1701, to block PD-1 binding to PD-L1 to restore the signal intensity of PD-1 overexpressing reporter cells.
- Figure 4 shows the reversal of TGF ⁇ 1 inhibition of T cell proliferation by the fusion protein molecule PTR2, with two control molecules, M7824 and SHR1701.
- Figure 5 shows the activation of the T-cell factor IFN- ⁇ by the fusion protein molecule PTR2, with two control molecules, M7824 and SHR1701, and by anti-PD-L1 mAb.
- Figure 6 shows the anti-tumor activity of the fusion protein molecule PTR2, with two control molecules M7824 and SHR1701, and anti-PD-L1 mAb.
- Figure 7 shows the restoration of the signal intensity of PD-1 overexpressing reporter cells by blocking the binding of PD-1 to PD-L1 by the fusion protein molecule PTR2.
- Figure 8 shows the effect of the fusion protein molecule PTR2 on blocking the signal generated by SMAD2 reporter cells stimulated by TGF[beta].
- the term “comprising” or “comprising” means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.
- the terms “comprising” or “comprising” are used, unless otherwise indicated, combinations of the stated elements, integers or steps are also encompassed.
- reference to an antibody variable region that "comprises” a particular sequence is also intended to encompass antibody variable regions that consist of that particular sequence.
- programmed cell death 1 ligand 1 refers to those derived from Any native PD-L1 from any vertebrate source, including mammals, such as primates (eg, humans) and rodents (eg, mice and rats).
- the term encompasses "full-length”, unprocessed PD-L1 as well as any form of PD-L1 produced by processing in a cell.
- PD-L1 can exist as a transmembrane protein or as a soluble protein.
- the term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants.
- the basic structure of PD-L1 includes four domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain. Additional information on the human PD-L1 gene, including genomic DNA sequence, can be found under NCBI Gene ID No. 29126. Additional information on the mouse PD-L1 gene, including genomic DNA sequence, can be found under NCBI Gene ID No. 60533. Amino acid sequences of exemplary full-length human PD-L1 proteins can be found, for example, under NCBI Accession No. NP_001254653 or UniProt Accession No. Q9NZQ7, while exemplary full-length mouse PD-L1 proteins can be found, for example, under NCBI Accession No. NP_068693 or Uniprot Accession No. Q9EP73. L1 protein sequence.
- anti-PD-L1 antibody refers to an antibody that is capable of binding PD with sufficient affinity -L1 protein or a fragment thereof.
- the anti-PD-L1 antibody binds to a non-PD-L1 protein to a degree that is less than about 10%, about 20%, about 30%, about 40%, about 50% of the binding of the antibody to PD-L1 , about 60%, about 70%, about 80%, or about 90% or more, as measured, for example, by radioimmunoassay (RIA) or bioluminescence interferometry or MSD assay.
- RIA radioimmunoassay
- MSD bioluminescence interferometry
- the binding is performed, for example, by radioimmunoassay (RIA), biofilm thin layer interferometry (BLI), biolight interferometry, MSD assay or surface plasmon resonance (SPR) or flow cytometry technically measured.
- RIA radioimmunoassay
- BLI biofilm thin layer interferometry
- MSD assay MSD assay
- SPR surface plasmon resonance
- full-length antibody “complete antibody” and “intact antibody” are used interchangeably herein to refer to an antibody that is substantially structurally similar in structure to a native antibody or has a heavy chain comprising an Fc region as defined herein.
- antibody fragment includes a portion of an intact antibody.
- the antibody fragment is an antigen-binding fragment.
- an "antigen-binding fragment” refers to a molecule other than an intact antibody that comprises a portion of the intact antibody and binds the antigen to which the intact antibody binds.
- antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; dAb (domain antibody); linear antibody; single chain antibody (eg, scFv); single domain antibody such as VHH ; a dAb; or a camelid antibody.
- CDR regions are loops in the variable domains of antibodies that are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ( "antigen contact point”).
- the CDRs are mainly responsible for binding to antigenic epitopes.
- the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus.
- the CDRs located within the variable domains of antibody heavy chains are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the variable domains of antibody light chains are referred to as LCDR1, LCDR2 and LCDR3.
- each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and topology of CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S.
- the residues of each CDR are as follows.
- a CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
- a residue position in an antibody variable region refers to the numbering system according to the Kabat ( Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
- the heavy chain variable region CDRs of the antibodies of the present invention are determined according to the following rules:
- HCDR1 was determined according to the AbM rule, and HCDR2 and HCDR3 were determined according to the Kabat rule.
- the light chain variable region CDRs of the antibodies of the present invention are determined according to the following rules:
- LCDR1, LCDR2 and LCDR3 are determined according to Kabat's rule.
- the CDR boundaries of the variable region of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different.
- the scope of said antibodies also covers antibodies whose variable region sequences comprise said specific CDR sequences, but due to the application of different schemes (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined by the present invention.
- Antibodies with different specificities have different binding sites for different antigens
- CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding.
- the minimal binding unit can be a sub-portion of a CDR.
- the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.
- an antibody in the IgG format refers to the IgG format to which the heavy chain constant region of the antibody belongs.
- the heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different.
- an antibody in the IgG4 format means that its heavy chain constant region is derived from IgG4, or an antibody in the IgG1 format means that its heavy chain constant region is derived from IgG1.
- a “humanized” antibody refers to an antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs.
- a humanized antibody will comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the CDRs (eg, CDRs) correspond to those of the non-human antibody, and all Or substantially all of the FRs correspond to those of a human antibody.
- a humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody.
- a "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.
- Human antibody or “fully human antibody” or “fully human antibody” are used interchangeably and refer to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody derived from a human Either human cells are generated or derived from non-human sources using human antibody repertoires or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
- binding means that binding is selective for an antigen or target and can be distinguished from unwanted or non-specific interactions.
- the ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or by bioluminescence interferometry or by thin biofilms Interferometry or MSD assay or Surface Plasmon Resonance (SPR) assay.
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- MSD bioluminescence interferometry
- MSD Surface Plasmon Resonance
- TGF ⁇ RII or "TGF ⁇ receptor II” means having the wild-type human TGF ⁇ receptor type 2 isoform A sequence (eg, the amino acid sequence of NCBI Reference Sequence (RefSeq) Accession No. NP_001020018 or the amino acid sequence of Uniprot ID: P37173) , or a polypeptide having a wild-type human TGF ⁇ receptor type 2 isoform B sequence (e.g., the amino acid sequence of NCBI RefSeq Accession No. NP_003233), or a polypeptide having substantially the same sequence as said wild-type amino acid sequence.
- RefSeq NCBI Reference Sequence Accession No. NP_001020018
- Uniprot ID: P37173 or a polypeptide having a wild-type human TGF ⁇ receptor type 2 isoform B sequence (e.g., the amino acid sequence of NCBI RefSeq Accession No. NP_003233), or a polypeptide having substantially the same sequence as said wild
- TGF[beta]RII can retain at least 75%, 80%, 85%, 90%, 95%, or 99% of the TGF[beta]-binding activity of the wild-type sequence, or equal to or higher than the binding activity of the wild-type sequence.
- the TGF ⁇ RII of the present invention comprises the amino acid sequence shown in SEQ ID NO:45, or has at least 85%, 86%, 87%, 88%, 89% with the amino acid sequence shown in SEQ ID NO:45 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
- Extracellular domain of TGF ⁇ RII or “ECD of TGF ⁇ RII” or “ECD” refers to the extracellular domain of TGF ⁇ RII that can bind TGF- ⁇ and block its activity.
- the ECD is determined according to the Topological domain of Uniprot.
- the ECD of TGF ⁇ RII is generally set forth in SEQ ID NO: 41 and has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
- ECDs can be truncated at the C-terminus to obtain "C-terminally truncated ECDs" without affecting their ability to bind TGF-beta.
- 1-30 amino acids can be terminally truncated without significantly affecting its activity.
- the C-terminal truncated ECD is an ECD truncated at the terminal by 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5 amino acids, eg at the C-terminal Truncating the ECD of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid (including any range thereof as an endpoint).
- the C-terminal truncated ECD of the invention comprises the amino acid sequence set forth in SEQ ID NO:42, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
- typically 1 or 2 amino acids at the N-terminus can be mutated to a G to increase its flexibility for attachment to other molecules (eg, antibodies, eg, anti-PD-L1 antibodies).
- references herein to "ECD” or “C-terminally truncated ECD” generally also encompass ECDs or C-terminally truncated ECDs that have 1-2 amino acid mutations at the N-terminus for the purpose of constituting a fusion protein.
- therapeutic agent encompasses any substance that is effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (eg, immunosuppressive agents) ).
- cytotoxic agent refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction.
- “Chemotherapeutic agents” include chemical compounds useful in the treatment of diseases of the immune system.
- small molecule drug refers to low molecular weight organic compounds capable of modulating biological processes.
- Small molecule is defined as a molecule with a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD.
- Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As therapeutic agents, small molecules can be more cell permeable, less susceptible to degradation, and less susceptible to eliciting an immune response than macromolecules.
- immunomodulator refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response.
- the immune response can be a humoral response or a cellular response.
- Immunomodulators include immunosuppressants.
- an “immunosuppressant,” “immunosuppressive drug,” or “immunosuppressant” is a therapeutic agent used in immunosuppressive therapy to suppress or prevent the activity of the immune system.
- an effective amount refers to an amount or dose of an antibody or fragment or conjugate or composition or combination of the invention which, after administration to the patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention .
- a “therapeutically effective amount” refers to an amount effective to achieve the desired therapeutic result, at the required dose and for the required period of time.
- a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or conjugate or composition or combination thereof are outweighed by the therapeutically beneficial effects.
- a “therapeutically effective amount” preferably inhibits a measurable parameter (eg, tumor volume) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70% relative to an untreated subject .
- prophylactically effective amount refers to an amount effective to achieve the desired prophylactic result, at the required dose and for the required period of time. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because a prophylactic dose is administered in a subject prior to or at an earlier stage of the disease.
- host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
- Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny screened or selected for the same function or biological activity in the originally transformed cell.
- “Individual” or “subject” includes mammals. Mammals include, but are not limited to, domestic animals (eg, cattle, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, , mice and rats). In some embodiments, the individual or subject is a human.
- an “isolated” antibody is one that has been separated from components of its natural environment.
- the antibody is purified to greater than 95% or 99% purity, such as by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed phase) HPLC) determined.
- electrophoresis eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
- chromatography eg, ion exchange or reversed phase
- isolated nucleic acid encoding a fusion protein molecule or fragment thereof refers to one or more nucleic acid molecules that encode the heavy or light chain (or fragment thereof, eg, heavy or light variable region) of a fusion protein molecule ), including such nucleic acid molecules in a single vector or in separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.
- the sequences are aligned for optimal comparison purposes (e.g., between the first and second amino acid sequences or nucleic acid sequences for optimal alignment. Gaps are introduced in one or both or non-homologous sequences can be discarded for comparison purposes).
- the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
- the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
- Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms.
- the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (at http://www.gcg.com) is used that has been integrated into the GAP program of the GCG software package available), using the Blossum 62 matrix or the PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6, to determine the distance between two amino acid sequences percent identity.
- the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences.
- a particularly preferred set of parameters is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. It is also possible to use the PAM120 weighted remainder table, gap length penalty of 12, gap penalty of 4), using the E. Meyers and W.
- nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
- anti-tumor effect refers to a biological effect that can be exhibited by a variety of means including, but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.
- tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
- cancer and “cancerous” refer to or describe a physiological disorder in mammals that is usually characterized by unregulated cell growth.
- cancers suitable for treatment by the antibodies of the invention include colon cancer.
- tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
- cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
- cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
- pharmaceutical adjuvant refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., with which the active substance is administered.
- composition refers to a composition that is in a form that allows the biological activity of the active ingredients contained therein to be effective and does not contain additional ingredients.
- non-fixed combination means that the active ingredients (eg, (i) a fusion protein of the invention, and (ii) other therapeutic agents) are combined as separate entities simultaneously, without specific time constraints or at the same or different time intervals, Administration to the patient is performed sequentially, wherein such administration provides prophylactically or therapeutically effective levels of the two or more active agents in the patient.
- the fusion proteins of the invention and other therapeutic agents used in pharmaceutical combinations are administered at levels no greater than when they are used alone.
- the term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity.
- the doses and/or time intervals of the two or more active agents are preferably selected so that the combined use of the parts produces a greater effect in the treatment of a disease or condition than either component alone can achieve.
- the ingredients may each be in separate formulations, which may be the same or different.
- combination therapy refers to the administration of two or more therapeutic agents or treatment modalities (eg, radiation therapy or surgery) to treat the diseases described herein.
- administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, eg, in a single capsule having a fixed ratio of active ingredients.
- administration includes co-administration of the individual active ingredients in multiple or separate containers such as tablets, capsules, powders and liquids. Powders and/or liquids can be reconstituted or diluted to the desired dose prior to administration.
- such administration also includes the sequential use of each type of therapeutic agent at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the drug combination in the treatment of the disorders or conditions described herein.
- treating refers to slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
- prevention includes the inhibition of the occurrence or progression of a disease or disorder or symptoms of a particular disease or disorder.
- subjects with a family history of cancer are candidates for preventive regimens.
- prevention refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.
- vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
- the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
- a "subject/patient/individual sample” refers to a collection of cells or fluids obtained from a patient or subject.
- the source of a tissue or cell sample can be solid tissue like from a fresh, frozen and/or preserved organ or tissue sample or biopsy or biopsy; blood or any blood component; bodily fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid); ), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject.
- Tissue samples may contain compounds that are not naturally mixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
- fusion protein molecules of the invention or fragments thereof bind PD-L1 (eg, human PD-L1 ) with high affinity. In some embodiments, the fusion protein molecules or fragments thereof of the invention bind TGF[beta] (eg, human TGF[beta]) with high affinity.
- PD-L1 eg, human PD-L1
- TGF[beta] eg, human TGF[beta]
- the fusion protein molecules or fragments thereof of the invention bind human PD-L1 with higher affinity than known anti-PD-L1 antibodies. In some embodiments, the fusion protein molecules or fragments thereof of the invention bind human TGF[beta] with higher affinity than known fusion protein molecules. In some embodiments, the affinity of the fusion protein molecule or fragment thereof is determined by biolight interferometry.
- the fusion protein molecules of the invention, or fragments thereof bind human TGF ⁇ 1 with an equilibrium dissociation constant (K D ) that is less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM or 0.5nM.
- K D equilibrium dissociation constant
- the fusion protein molecules of the invention, or fragments thereof bind to human PD-L1 with an equilibrium dissociation constant (K D ) that is less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM.
- K D equilibrium dissociation constant
- the molecules of the invention or fragments thereof are capable of blocking TGF ⁇ 1 signaling while blocking the binding of PD-1 to PD-L1.
- the molecules of the invention or fragments thereof are capable of reversing the inhibition of T cells by TGF[beta]1, and/or activating T cells (eg, increasing the secretion of cytokines such as IFN-[gamma]).
- the molecules of the invention or fragments thereof are more stable than known fusion protein molecules.
- the molecules or fragments thereof of the invention are capable of inhibiting tumor cells, eg, tumor cells expressing PD-1 or PD-L1 or PD-L2, and/or TGF ⁇ .
- the molecules of the invention or fragments thereof can be used to treat cancer.
- the molecules of the present invention or fragments thereof can effectively inhibit tumor growth, and the tumor inhibition rate is greater than or equal to about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%.
- fusion protein molecules of the invention comprise
- the anti-PD-L1 antibody is directly linked to the TGF ⁇ RII molecule at the C-terminus of the heavy chain.
- the anti-PD-L1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-L1 antibody is a complete antibody.
- the anti-PD-L1 antibodies or antigen-binding fragments thereof of the invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3.
- HCDRs complementarity determining regions
- the anti-PD-L1 antibodies or antigen-binding fragments thereof of the invention comprise three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3.
- LCDRs complementarity determining regions
- the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises 3 complementarity determining regions (HCDRs) from the heavy chain variable region and 3 complementarity determining regions (LCDRs) from the light chain variable region ).
- HCDRs complementarity determining regions
- LCDRs complementarity determining regions
- an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH). In some aspects, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL). In some aspects, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3.
- CDRs complementarity determining regions
- the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention further comprises an antibody heavy chain constant region HC. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention further comprises an antibody light chain constant region LC. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention further comprises a heavy chain constant region HC and a light chain constant region LC.
- the heavy chain variable regions of the invention are identical to one another.
- (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 4; or
- amino acid changes comprising 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably The amino acid sequence of amino acid substitutions, more preferably conservative substitutions of amino acids, consists of the amino acid sequence, preferably, the amino acid changes do not occur in the CDR regions.
- the light chain variable regions of the invention are identical to each other.
- amino acid changes (preferably) compared to the amino acid sequence selected from SEQ ID NO: 11
- amino acid sequence of amino acid substitutions more preferably conservative substitutions of amino acids, consists of the amino acid sequence, preferably, the amino acid changes do not occur in the CDR regions.
- the three complementarity determining regions (HCDRs) from the heavy chain variable region of the invention are selected from the three complementarity determining regions contained in the VH as set forth in SEQ ID NO:4 HCDR1, HCDR2 and HCDR3
- the three complementarity determining regions (LCDRs) from the light chain variable region of the invention are selected from the three complementarity determining regions contained in the VL as set forth in SEQ ID NO: 11 LCDR1, LCDR2, and LCDR3.
- HCDRl comprises or consists of the amino acid sequence of SEQ ID NO: 1.
- the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2.
- the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:3.
- LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:8.
- LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:9.
- LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10.
- the antibody heavy chain constant region CH of the invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1.
- the antibody light chain constant region CL of the invention is a lambda or Kappa light chain constant region.
- the antibody heavy chain constant region CH of the present invention is provided.
- amino acid sequence comprising 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared to the amino acid sequence selected from SEQ ID NO: 5
- the amino acid sequence that is altered is or consists of said amino acid sequence.
- the amino acid change occurs in the Fc region.
- the amino acid change is N297A, L234A or L235A.
- the amino acid changes are L234A and L235A.
- the amino acid change in the Fc region attenuates the CDC or ADCC activity of the antibody.
- the antibody light chain constant region CL of the invention is provided.
- (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12; or
- amino acid sequence comprising 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 12
- the amino acid sequence that is altered is or consists of said amino acid sequence.
- the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises: the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 4, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 11.
- the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises:
- HCDR1, HCDR2, HCDR3, respectively as shown in the following amino acid sequences: SEQ ID NOs: 1, 2, and 3, and LCDR1, LCDR2, and LCDR3, respectively, as shown in the following amino acid sequences: SEQ ID NOs: 8, 9, and 10.
- the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises:
- amino acid sequence set forth in SEQ ID NO:4 comprising the amino acid sequence set forth in SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or A VH consisting of the amino acid sequence, and comprising the amino acid sequence shown in SEQ ID NO: 11 and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% therewith or an amino acid sequence of 99% identity or a VL consisting of said amino acid sequence.
- the anti-PD-L1 antibody of the present invention is an antibody in the form of IgGl or an antibody in the form of IgG2 or an antibody in the form of IgG3 or an antibody in the form of IgG4, preferably an antibody in the form of IgGl.
- the anti-PD-L1 antibody is humanized.
- the anti-PD-L1 antibody is a human antibody.
- the anti-PD-L1 antibody is a chimeric antibody.
- the anti-PD-L1 antibodies of the present invention also encompass antibody fragments thereof (eg, antigen-binding fragments), preferably antibody fragments selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single chain antibody (eg scFv), (Fab') 2 , single domain antibodies such as VHHs, dAbs (domain antibodies) or linear antibodies.
- antibody fragments thereof eg, antigen-binding fragments
- Fab' fragments selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single chain antibody (eg scFv), (Fab') 2 , single domain antibodies such as VHHs, dAbs (domain antibodies) or linear antibodies.
- the ECD or C-terminally truncated ECD of the TGF ⁇ RII molecules of the invention is mutated to a G at 1 or 2 amino acids at the N-terminus. In a preferred embodiment, the ECD or C-terminally truncated ECD of the TGF ⁇ RII molecule of the invention is mutated to a G at 1 amino acid at the N-terminus.
- the ECD of the TGF ⁇ RII molecules of the invention is the ECD of the TGF ⁇ RII molecules of the invention.
- amino acid sequence comprising 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 41
- the amino acid sequence that is altered is or consists of said amino acid sequence.
- the C-terminally truncated ECD is one that is truncated at the C-terminus by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids ECD.
- the C-terminally truncated ECD of the TGF ⁇ RII molecules of the invention is a C-terminally truncated ECD of the TGF ⁇ RII molecules of the invention.
- (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 42 or 43; or
- amino acid sequence of amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions of amino acids) of or consisting of said amino acid sequences.
- the fusion protein molecule of the invention comprises
- chain A anti-PD-L1 antibody light chain
- Chain B an anti-PD-L1 antibody heavy chain with the ECD of TGF ⁇ RII or a C-terminally truncated ECD linked at the C-terminus.
- the fusion protein molecule of the invention consists of 2 chains A and 2 chains B.
- the anti-PD-L1 antibody light chain in chain A is as defined above.
- the anti-PD-L1 antibody heavy chain in chain B is as defined above.
- the ECD or C-terminally truncated ECD of TGF ⁇ RII is as defined above.
- chain A of the fusion protein molecule of the invention is:
- chain B of the fusion protein molecule of the invention is a fusion protein molecule of the invention.
- the amino acid changes described herein include amino acid substitutions, insertions or deletions.
- the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
- the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes described in the present invention occur in regions outside the variable region of the heavy chain and/or outside the variable region of the light chain. In some embodiments, the amino acid changes described herein occur in the Fc region of an antibody heavy chain constant region, and in one embodiment, the amino acid changes are N297A, L234A, L235A, or L234A and L235A. . In preferred embodiments, the amino acid changes in the Fc region attenuate the ADCC and/or CDC effects of the antibody.
- substitutions are conservative substitutions.
- Conservative substitutions refer to the substitution of one amino acid by another amino acid within the same class, e.g., substitution of an acidic amino acid by another acidic amino acid, substitution of a basic amino acid by another basic amino acid, or substitution of a neutral amino acid by another neutral amino acid replacement. Exemplary permutations are shown in the following table:
- the substitutions occur in the CDR regions of the antibody.
- the variant obtained has a modification (eg, improvement) in certain biological properties (eg, increased affinity) relative to the parent antibody and/or will have certain biological properties that are substantially retained of the parent antibody.
- exemplary substitutional variants are affinity matured antibodies.
- the antibodies provided herein are altered to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites.
- an antibody contains an Fc region, the carbohydrate attached to it can be varied.
- the Fc region effector function is eliminated by mutating the glycosylation site, eg, N297A, or L234A and L235A mutations, thereby reducing ADCC activity or CDC activity.
- one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants, to alter one or more functional properties of the antibody, such as serum half-life, serum half-life, Complement fixation, complement dependent cytotoxicity, Fc receptor binding and/or antibody dependent cytotoxicity.
- Fc region variants can include human Fc region sequences (eg, human IgGl, IgG2, IgG3, or IgG4 Fc regions) comprising amino acid changes (eg, substitutions) at one or more amino acid positions.
- the antibodies described herein introduce changes in the Fc region to attenuate the ADCC activity or CDC activity of the antibody.
- the present invention provides nucleic acids encoding any of the above fusion protein molecules.
- a vector comprising the nucleic acid is provided.
- the vector is an expression vector such as pcDNA3.1.
- a host cell comprising the nucleic acid or the vector is provided.
- the host cell is eukaryotic.
- the host cell is selected from yeast cells, mammalian cells (eg, CHO cells (eg, CHO-S) or 293 cells (eg, 293F or HEK293 cells)), or other cells suitable for the production of antibodies or fragments thereof.
- the host cell is prokaryotic.
- the nucleic acid of the present invention comprises a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 7, or encoding the amino acid sequence shown in SEQ ID NO: 7 with at least 85%, 90%, 91%, 92%, 93%, A nucleic acid having an amino acid sequence of 94%, 95%, 96%, 97%, 98% or 99% identity.
- one or more vectors are provided comprising the nucleic acid.
- the vector is an expression vector, such as a eukaryotic expression vector.
- Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
- YACs yeast artificial chromosomes
- the vector is pV120 or pCHO1.0.
- a host cell comprising the vector.
- Suitable host cells for cloning or expressing the vector encoding the fusion protein include the prokaryotic or eukaryotic cells described herein.
- the host cell is eukaryotic.
- the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of fusion proteins or fragments thereof.
- eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for the encoded vector.
- fungal and yeast strains whose glycosylation pathways have been "humanized” result in fusion proteins that produce antibodies with partially or fully human glycosylation patterns.
- Host cells suitable for expression of fusion proteins of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
- mammalian cell lines engineered for growth in suspension can be used.
- useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; the human embryonic kidney line (HEK293, 293F or 293T cells) and the like.
- Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NSO, and Sp2/0. Suitable mammalian host cell lines for the production of fusion proteins are known in the art.
- the present invention provides a method of making a fusion protein molecule of the invention or a fragment thereof (preferably an antigen-binding fragment), wherein the method comprises under conditions suitable for expression of a nucleic acid encoding a fusion protein molecule of the invention or a fragment thereof
- the host cell is cultured and optionally the fusion protein or fragment thereof is isolated.
- the method further comprises recovering the fusion protein molecule or fragment thereof of the invention from the host cell.
- a method of making a fusion protein molecule of the present invention comprises, under conditions suitable for expression of the fusion protein, culturing a fusion protein containing chain A and chain B (optionally in one) encoding the fusion protein.
- a nucleic acid in a vector) or a host cell comprising an expression vector of the nucleic acid, as provided above, and optionally the fusion protein is recovered from the host cell (or host cell culture medium).
- the nucleic acid encoding the strand of the fusion protein is isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
- Such nucleic acids are readily isolated and sequenced using routine procedures.
- Fusion protein molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
- the actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.
- the purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
- the fusion proteins provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
- the fusion proteins of the invention are tested for their PD-L1 and/or TGF[beta] binding activity, eg, by known methods such as bio-optical interference and the like.
- the invention also provides assays for identifying biologically active fusion proteins.
- Biological activities can include, for example, binding PD-L1 or TGF ⁇ (eg, binding human PD-L1 or TGF ⁇ ), blocking TGF ⁇ 1 signaling, blocking PD-1 binding to PD-L1, reversing TGF ⁇ 1 inhibition of T cells, and /or activation of T cells (eg, increased secretion of cytokines such as IFN- ⁇ ), inhibition of tumor cells, etc.
- antibodies of the invention are tested for such biological activity.
- Cells for use in any of the above in vitro assays include cell lines that either naturally express PD-L1 or TGF ⁇ or are engineered to express or overexpress PD-L1 or TGF ⁇ . Such cells also include cell lines transfected with DNA encoding PD-L1 or TGF ⁇ that express PD-L1 or TGF ⁇ and do not normally express PD-L1 or TGF ⁇ .
- the present invention provides compositions comprising any of the fusion protein molecules described herein or fragments thereof, preferably the compositions are pharmaceutical compositions.
- the composition further comprises pharmaceutical excipients.
- a composition eg, a pharmaceutical composition, comprises a fusion protein molecule of the invention, or a fragment thereof, in combination with one or more other therapeutic agents.
- compositions comprising the fusion protein molecules described herein or fragments thereof, or compositions comprising polynucleotides encoding the fusion protein molecules or fragments thereof described herein (including pharmaceutical composition or pharmaceutical preparation).
- compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
- compositions of the present invention may be in a variety of forms.
- forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), powders or suspensions, liposomes, and suppositories.
- liquid solutions eg, injectable solutions and infusible solutions
- powders or suspensions e.g., liposomes, and suppositories.
- liposomes e.g., liposomes, and suppositories.
- suppositories e.g., suppositories.
- the preferred form depends on the intended mode of administration and therapeutic use.
- compositions comprising the antibodies described herein can be prepared by admixing an antibody of the invention of the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.
- compositions or formulations of the present invention may also contain more than one active ingredient required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other.
- active ingredients such as chemotherapeutic agents, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs or immunomodulatory agents, and the like.
- the active ingredients are suitably combined in amounts effective for the intended use.
- the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising a fusion protein molecule or fragment thereof of the present invention, and one or more other therapeutic agents (eg, chemotherapeutic agents, cytokines, cytotoxic agents) , other antibodies, small molecule drugs or immunomodulators, etc.).
- therapeutic agents eg, chemotherapeutic agents, cytokines, cytotoxic agents
- other antibodies small molecule drugs or immunomodulators, etc.
- Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably the kit is in the form of a pharmaceutical dosage unit. Dosage units can thus be provided according to the dosing regimen or the interval between drug administrations.
- kit of parts of the present invention contains in the same package:
- a first container containing a pharmaceutical composition comprising a fusion protein molecule described herein or a fragment thereof;
- One aspect of the present invention provides a method of preventing or treating a tumor (eg, cancer) in a subject, comprising administering to the subject an effective amount of a fusion protein molecule or fragment thereof, pharmaceutical composition, pharmaceutical combination or kit of the present invention .
- a tumor eg, cancer
- the tumor (eg, cancer) patient has (eg, elevated levels, eg, nucleic acid or protein levels) PD-L1 or PD-L2 or PD-1.
- the tumor (eg, cancer) patient has (eg, elevated levels, eg, nucleic acid or protein levels) TGF[beta].
- the tumor (eg, cancer) patient has (eg, elevated levels, eg, nucleic acid or protein levels) TGF ⁇ and PD-L1 (or PD-1 or PD-L2).
- the tumor eg, cancer
- the tumor includes solid and hematological tumors and metastatic lesions.
- examples of solid tumors include malignant tumors. Cancer can be in early, intermediate or advanced stages or metastatic.
- the tumor therapy will benefit from inhibition of PD-L1 or PD-L2 or PD-1, and/or TGF ⁇ at the nucleic acid or protein level.
- the fusion protein molecules or fragments thereof of the present invention are capable of inhibiting tumor cell proliferation, such as tumor cells expressing TGF ⁇ and/or PD-L1 (or PD-1 or PD-L2).
- the tumor is tumor immune escape.
- the tumor is cancer, such as colon cancer.
- the subject can be a mammal, eg, a primate, preferably a higher primate, eg, a human (eg, an individual having or at risk of having a disease described herein).
- the subject has or is at risk of having a disease described herein (eg, cancer).
- the subject receives or has received other treatments, such as chemotherapy treatment and/or radiation therapy.
- the subject has previously received or is receiving immunotherapy.
- the present invention provides the use of a fusion protein molecule or fragment thereof or a pharmaceutical composition or pharmaceutical combination or kit in the manufacture or manufacture of a medicament for the use described herein, eg for prophylaxis or treatment herein The related disease or condition mentioned.
- a fusion protein molecule or fragment thereof or pharmaceutical composition or pharmaceutical combination or kit of the invention delays the onset of the disorder and/or symptoms associated with the disorder.
- the fusion protein molecules or fragments thereof or pharmaceutical compositions of the invention can also be administered in combination with one or more other therapies, eg, therapeutic modalities and/or other therapeutic agents, for the uses described herein, eg For the prevention and/or treatment of the related diseases or conditions mentioned herein.
- therapies eg, therapeutic modalities and/or other therapeutic agents, for the uses described herein, eg For the prevention and/or treatment of the related diseases or conditions mentioned herein.
- the treatment modality includes surgery; radiation therapy, localized or focused radiation, and the like.
- the therapeutic agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs, or immunomodulatory agents.
- immunomodulatory agents include immunosuppressive or anti-inflammatory agents.
- combination therapy encompasses combined administration (eg, two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case the additional therapeutic agents and/or agents may be administered
- Administration of the fusion protein molecules or fragments thereof of the invention occurs prior to, concurrently with, and/or subsequent to.
- the route of administration of the pharmaceutical composition is according to known methods, eg, orally, by intravenous injection, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional Routes; via sustained release systems or via implanted devices.
- the compositions can be administered by bolus injection or by continuous infusion or by implanted devices.
- the bifunctional antibody fusion protein of the present invention is composed of 1) anti-PD-L1 antibody and 2) ECD of TGF ⁇ receptor 2 (TGF ⁇ RII).
- the anti-PD-L1 antibody part adopts the published antibody sequence (WO2019129136A1, HZ3266-IgG1N297A molecule).
- the N-terminus of TGF ⁇ RII (Uniprot ID: P37173) is prone to breakage (WO 2018/205985 A1).
- WO 2018/205985 A1 In order to analyze the possible reasons for the breakage, we fitted the conformations of 10 models in the NMR structure of TGF ⁇ RII.
- the N-terminus of TGF ⁇ RII is structurally different in 10 conformations, indicating that its conformation is very active. Therefore, we speculate that if the flexible GGGGS linking peptide is used to connect it to the antibody, the activity will be further increased, which may be the reason for the breakage.
- the inventors of the present application directly linked the PD-L1 antibody domain TGF ⁇ RII ECD (TGF ⁇ RII extracellular domain) for the first time without using an additional linker.
- the schematic diagram of the obtained fusion protein is shown in FIG. 1B .
- the PTR2 fusion protein is constructed from the HZ3266-IgG1 N297A molecule disclosed in WO2019129136A1, which is directly linked to the ECD region of TGF ⁇ RII at the C-terminus of the heavy chain (shown in SEQ ID NO: 43), wherein the N297A amino acid mutation is replaced by L234A and L235A mutations.
- the M7824 fusion protein is disclosed by WO2015118175A2 and is named Anti-PDL1/TGF ⁇ Trap in this patent application, wherein between Anti-PDL1 and TGF ⁇ RII ECD is linked with GGGGSGGGGSGGGGGSGGGGSG;
- the SHR1701 fusion protein is disclosed by WO2018205985A1 and named fusion protein 9 in this patent application, wherein the anti-PD-L1 and TGF ⁇ RII ECD are linked by GGGGSGGGGSGGGGSGGGGSG.
- the three exemplary fusion proteins described above were expressed and purified in Expi293 cells.
- the vector pcDNA3.1 (Invitrogen) was used.
- the complete heavy chain and complete light chain of the fusion protein were cloned into separate pcDNA3.1 vectors to obtain plasmid DNA.
- the pcDNA3.1 vector with the complete heavy chain of the fusion protein molecule and the pcDNA3.1 vector with the complete light chain were co-transfected into Expi293 cells by chemical transfection.
- the chemical transfection reagent used was PEI (purchased from Polysciences), and the cultured Expi293 was transiently transfected according to the protocol provided by the manufacturer. The specific steps are as follows:
- Biofilm thin-layer interferometry was used to determine the equilibrium dissociation constant (KD) of the antibody of the present invention binding to human PD-L1 and TGF ⁇ .
- KD equilibrium dissociation constant
- the BLI method affinity determination was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270-8).
- the instrument setting parameters are as follows: running steps: Baseline, Antibody Loading ⁇ 1nm, Baseline, Antigen Association and Dissociation; the running time of each step depends on the speed of sample binding and dissociation, the rotation speed is 1000rpm, and the temperature is 30°C. KD values were analyzed using ForteBio Octet analysis software.
- the ForteBio affinity assay was performed again as described above, except that the target recombinant human TGF ⁇ 1 protein was replaced with recombinant human PD-L1.
- Example 3 The fusion protein molecule of the present invention blocks the experiment of SMAD2 reporter cell signal
- the molecular PTR2 of the present invention together with two control molecules M7824 and SHR1701, were detected to block the effect of the SMAD2 reporter cell on the signal generated under the stimulation of TGF ⁇ .
- the detailed experimental process is as follows:
- the 4T1 cell line (ATCC: CRL-2539) was infected with the SMAD2 luciferase reporter system (Qiagen) in the form of lentivirus, and the 4T1-SMAD2 reporter cell line was obtained by screening.
- SMAD2 luciferase reporter system Qiagen
- 50,000 cells were plated in each well, and then recombinant human TGF ⁇ 1 (R&D Systems) was added to each well to a final concentration of 2ng/ml.
- the fusion protein molecules to be tested prepared in Example 1 were added to the cells at a final concentration of up to 80 nM, diluted 4 times, with a total of 9 gradient points, and incubated overnight in a cell incubator.
- Example 4 The fusion protein of the present invention blocks the experiment that Jurkat PD-1 reporter cells are inhibited by PD-L1
- the molecule PTR2 of the present invention can efficiently block the combination of PD-L1 and PD-1, and restore the signal intensity of reporter cells overexpressing PD-1.
- the efficiency of PTR2 and SHR1701 in blocking the binding of PD-L1 to PD-1 was slightly higher than that of M7824, which is consistent with the difference in the affinity of PD-L1 at the protein level.
- Example 5 The fusion protein molecule of the present invention reverses the proliferation of CD4+ T cells
- the experimental steps are as follows:
- AIM-V medium to dilute the fusion protein molecule PTR2 and control molecules M7824 and SHR1701 prepared in Example 1 (starting at 100nM, 3-fold dilution, 50ul/well), and then add TGF at a final concentration of 20ng/ml - ⁇ protein (R&D, 7754-BH), 50ul/well, incubate at 37°C for 30 minutes after mixing.
- Example 6 Activation experiment of human T cells by the molecules of the present invention
- MLR mixed lymphocyte reaction
- the fusion protein molecule PTR2 prepared in Example 1 was diluted with AIM-V medium, the control molecule M7824 or SHR1701 and the anti-PD-L1 antibody (WO2019129136A1, HZ3266-IgG1N297A molecule, prepared according to the method in this document), the highest The concentration is 400nM, 4-fold serial dilution;
- the results are shown in Figure 5.
- the experimental results show that after adding the molecule PTR2 of the present invention and the control molecules M7824 and SHR1701 to the mixed lymphocyte reaction experimental system, the activation of T cells is improved compared with adding anti-PD-L1 monoclonal antibody alone.
- SHR1701 has a weaker effect on T cell activation, while PTR2 and M7824 can significantly increase the degree of T cell activation.
- Example 7 The efficacy of the fusion molecule of the present invention in the MC38 tumor-bearing human PD-L1 knock-in mouse model
- Human PD-L1 knock-in C57 mice female mice, 4-6 weeks old, Shanghai Southern Model Center
- 10 6 MC38-hPDL1 mice Murine colon cancer cells (Nanjing Galaxy).
- hIgG Equitech-Bio, Cat. No.: 161206-0656
- PTR2, M7824 and SHR1701 prepared in Example 1, or 2 mg/kg were injected intraperitoneally, respectively.
- PTR2 prepared in Example 1.
- Anti-PD-L1 antibody (WO2019129136A1, HZ3266-IgG1N297A molecule, prepared according to the method in the literature) was intraperitoneally administered in equimolar number at 4.11 mg/kg.
- Example 8 In vitro activity detection of the fusion protein molecule of the present invention before and after being placed at 40°C for 14 days
- the ExpiCHO-S TM cell line expressing the fusion protein molecule was generated using the ExpiFectamine TM CHO Transfection Kit (Gibco) according to the manufacturer's instructions.
- OptiPRO TM -SFM medium Gibco
- plasmid DNA 80 ⁇ g/100ml
- the molecule PTR2 of the present invention does not have an additional linker peptide added between anti-PD-L1 and TGF ⁇ RII to increase the stability of the molecule.
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Abstract
L'invention concerne une nouvelle protéine de fusion comprenant un anticorps PD-L1 et un récepteur de TGFβ, un acide nucléique codant pour la protéine de fusion ou un fragment de celle-ci, une cellule hôte la comprenant, et leur utilisation pertinente. De plus, la présente invention concerne l'utilisation thérapeutique desdites protéines de fusion ou d'un fragment de celles-ci.
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105658672A (zh) * | 2013-08-22 | 2016-06-08 | 阿塞勒隆制药公司 | TGF-β受体II型变体及其用途 |
WO2018064190A1 (fr) * | 2016-09-27 | 2018-04-05 | Epicentrx, Inc. | Protéines de fusion immunomodulatrices |
WO2018205985A1 (fr) * | 2017-05-12 | 2018-11-15 | 江苏恒瑞医药股份有限公司 | PROTÉINE DE FUSION CONTENANT UN RÉCEPTEUR DE TGF-β ET UTILISATIONS MÉDICALES ASSOCIÉES |
CN109970857A (zh) * | 2017-12-27 | 2019-07-05 | 信达生物制药(苏州)有限公司 | 抗pd-l1抗体及其用途 |
WO2019241625A1 (fr) * | 2018-06-15 | 2019-12-19 | Acceleron Pharma Inc. | Protéines de fusion bi- et tri-fonctionnelles et utilisations associées |
-
2021
- 2021-11-16 WO PCT/CN2021/130930 patent/WO2022105751A1/fr active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105658672A (zh) * | 2013-08-22 | 2016-06-08 | 阿塞勒隆制药公司 | TGF-β受体II型变体及其用途 |
WO2018064190A1 (fr) * | 2016-09-27 | 2018-04-05 | Epicentrx, Inc. | Protéines de fusion immunomodulatrices |
WO2018205985A1 (fr) * | 2017-05-12 | 2018-11-15 | 江苏恒瑞医药股份有限公司 | PROTÉINE DE FUSION CONTENANT UN RÉCEPTEUR DE TGF-β ET UTILISATIONS MÉDICALES ASSOCIÉES |
CN109970857A (zh) * | 2017-12-27 | 2019-07-05 | 信达生物制药(苏州)有限公司 | 抗pd-l1抗体及其用途 |
WO2019241625A1 (fr) * | 2018-06-15 | 2019-12-19 | Acceleron Pharma Inc. | Protéines de fusion bi- et tri-fonctionnelles et utilisations associées |
Non-Patent Citations (2)
Title |
---|
TAN YI, QIN ZHONG-QIANG, ZHOU WAN-FEI, LI HONG-JUN, LIU MING-ZHU, XU WEN-QING, LI ZHENG-HONG, LI YONG-HAI: "Preparation of fusion protein of PD-L1 single-chain antibody and verification of its antitumor activity", ZHONGGUO MIANYIXUE ZAZHI = CHINESE JOURNAL OF IMMUNOLOGY, JILIN SHENG YIXUE QIKANSHE, CN, vol. 35, no. 2, 31 December 2019 (2019-12-31), CN , pages 202 - 206, XP055931301, ISSN: 1000-484X, DOI: 10.3969/j.issn.1000-484X.2019.02.015 * |
WANG, X. H. ET AL.: "Expression, purification, and evaluation of in vivo anti-fibrotic activity for soluble truncated TGF-β receptor II as a cleavable His-SUMO fusion protein", WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, vol. 34, 24 November 2018 (2018-11-24), XP036656890, DOI: 10.1007/s11274-018-2565-x * |
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