WO2023236954A1 - Pd-1变体及其用途 - Google Patents

Pd-1变体及其用途 Download PDF

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WO2023236954A1
WO2023236954A1 PCT/CN2023/098627 CN2023098627W WO2023236954A1 WO 2023236954 A1 WO2023236954 A1 WO 2023236954A1 CN 2023098627 W CN2023098627 W CN 2023098627W WO 2023236954 A1 WO2023236954 A1 WO 2023236954A1
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variant
seq
amino acid
cells
fusion protein
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PCT/CN2023/098627
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French (fr)
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谷为岳
高斌
梅小伟
李雪龙
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北京卡替医疗技术有限公司
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Publication of WO2023236954A1 publication Critical patent/WO2023236954A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of immunotherapy. Specifically, the present invention provides PD-1 variants.
  • the invention also provides nucleic acid molecules encoding the PD-1 variants, fusion proteins or compositions comprising the PD-1 variants, and uses of the PD-1 variants.
  • Programmed death protein 1 is a protein mainly expressed on the surface of activated lymphocytes.
  • PD-1 is an immune checkpoint protein with immune braking ability.
  • inhibiting PD-1 can relieve the suppression of the immune system and attack tumors, making PD-1 a popular immune target in recent years.
  • a variety of antibodies against PD-1 have been approved and marketed.
  • PD-1 binds to its ligand PD-L1, producing an immunosuppressive signal.
  • Tumor cells express PD-L1 and thereby achieve immune evasion through the PD-1/PD-L1 pathway.
  • Studies have been conducted to prevent the interaction between PD-1 and PD-L1 by developing PD-1 analogues that can bind to PD-L1, thereby inhibiting PD-1.
  • these PD-1 analogs with binding specificity to PD-L1 can also be used to recognize and bind to tumor cells.
  • PD-1 variant sequences are described in Stanford University's PCT patent publication WO2016/023001. These variants have increased affinity for its ligand PD-L1 relative to human wild-type PD-1 and lack the transmembrane region. However, the variants in this application all have multiple amino acid differences relative to the wild type. The application did not study the affinity of PD-1 variants for anti-PD-1 antibodies.
  • PD-1 variants sometimes involves combination with anti-PD-1 antibodies.
  • anti-PD-1 antibodies are able to recognize and bind to the PD-1 variant, the two treatment pathways could interfere with each other, reducing the efficacy of both.
  • the inventors of the present invention constructed a PD-1 mutant plasmid library and screened out a series of PD-1 variants through flow sorting, which not only retain high affinity to its ligand PD-L1, At the same time, it does not bind to various commercially available anti-PD-1 monoclonal antibodies, thereby completing the present invention.
  • the present invention provides PD-1 variants that have a different amino acid sequence from a wild-type human PD-1 polypeptide and that bind PD-1 with high affinity, e.g., higher than the affinity of a wild-type human PD-1 polypeptide. -L1, while essentially not binding to multiple anti-PD-1 monoclonal antibodies.
  • the invention provides a fusion protein comprising the PD-1 variant of the first aspect.
  • the fusion protein may be a transmembrane fusion protein with the PD-1 variant as the extracellular segment.
  • the fusion protein is a fusion protein comprising immunoglobulin Fc.
  • the present invention provides a nucleic acid molecule encoding the PD-1 variant of the first aspect or the fusion protein of the second aspect.
  • the present invention provides an expression vector comprising the nucleic acid molecule of the third aspect.
  • the present invention provides a host cell comprising the nucleic acid molecule of the third aspect or the expression vector of the fourth aspect.
  • the present invention provides a pharmaceutical composition comprising the PD-1 variant of the first aspect or the fusion protein of the second aspect.
  • the present invention provides the use of the PD-1 variant of the first aspect or the fusion protein of the second aspect in the preparation of medicines.
  • the medicament is used in cancer treatment.
  • the present invention provides a pharmaceutical combination comprising (1) the PD-1 variant of the first aspect or the fusion protein of the second aspect, and (2) an anti-PD-1 antibody.
  • the plasmid diagram in Figure 1 shows the structure of the plasmid vector used for constructing a PD-1 mutant library in Example 1.
  • Figure 2 shows a schematic diagram of sorting cells that bind to PD-L1 but not anti-PD-1 antibodies by flow cytometry in Example 3.
  • Figure 3 is the SDS-PAGE result of expressing wild-type PD-1 or PD-1 variant and rabbit IgG Fc fusion protein in Example 5.
  • Lane 1 is wild-type PD-1, lanes 2-7 are PD-1 variants shown in SEQ ID Nos: 2-7, and lane 8 is molecular weight markers.
  • Figure 4 shows the blocking effect of the PD-1 variant Fc fusion proteins tested in Example 7 on T cell IL-2 release.
  • Figure 5 shows that the PD-1-CD28 enhanced receptor (108-CD28) armed T cells constructed using PD-1 variants in Example 8 can be stimulated by target cells expressing PD-L1 and secrete the cytokine IL-2.
  • Figure 6 shows the PD-1-CD28 enhanced receptor constructed from PD-1 variants in Example 9 (108-CD28) can enhance T cells to kill corresponding target cells.
  • the terms “about” or “approximately” mean within an acceptable error range for a particular value, and such error ranges can be determined by one of ordinary skill in the art. In some cases, the acceptable error range depends in part on how the value is measured or determined, ie, the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, in accordance with practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with regard to biological systems or processes, the term may mean within one order of magnitude of the value, preferably within 5 times the value, more preferably within 2 times the value. Where specific values are described in this application and in the claims, it should be assumed that the term “about” means within an acceptable error range for the specific value, unless otherwise stated.
  • nucleotide generally refers to a base-sugar-phosphate combination.
  • the nucleotides may comprise synthetic nucleotides.
  • the nucleotides may include synthetic nucleotide analogs.
  • Nucleotides may be monomeric units of nucleic acid sequences such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • nucleotide may include ribonucleoside triphosphate adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP); and deoxyribonucleoside triphosphates, such as dATP , dCTP, dITP, dUTP, dGTP, dTTP; or derivatives thereof.
  • Such derivatives may include, for example, [ ⁇ S]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to the nucleic acid molecules containing them.
  • nucleotide as used herein may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • ddNTPs dideoxyribonucleoside triphosphates
  • Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.
  • the nucleotides may be unlabeled or may be detectably labeled by well-known techniques. Labeling can also be done with quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels.
  • polynucleotide refers to a polymeric form of nucleotides of any length, whether in the form of a single-stranded, double-stranded, or multi-stranded deoxyribonucleic acid nucleotide or ribose Nucleotides or their analogs.
  • Polynucleotides may be exogenous or endogenous to the cell. Polynucleotides can exist in a cell-free environment.
  • the polynucleotide can be a gene or a fragment thereof.
  • the polynucleotide can be DNA.
  • the polynucleotide can be RNA.
  • Polynucleotides can have any three-dimensional structure and can perform any known or unknown function.
  • Polynucleotides may contain one or more analogs (eg, altered backbones, sugars, or nucleobases).
  • analogs include: 5-bromouracil, peptide nucleic acids, xenogeneic nucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threonucleosides, dideoxynucleotides, cordycepin, 7- Deazo-GTP, fluorophores (such as rhodamine or sugar-linked fluorescein), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7- Guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, braidin and wyosine.
  • analogs include: 5-bromour
  • Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci/locus defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA ( tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors , isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides (including cell-free DNA (cfDNA) and cell-free RNA (cfRNA)), nucleic acid probes and primers.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • expression refers to one or more processes by which a polynucleotide is transcribed from a DNA template (eg, into mRNA or other RNA transcripts) and/or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein.
  • peptide refers to a polymer of at least two amino acid residues linked by one or more peptide bonds. This term does not imply a specific length of the polymer and is not intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis or is naturally occurring.
  • the term applies to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, polymers can be interrupted by non-amino acids.
  • the term includes amino acid chains of any length, including full-length proteins, as well as proteins with or without secondary and/or tertiary structure (eg, domains).
  • amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation and any other manipulation, such as conjugation to a labeling component.
  • amino acid generally refers to natural and unnatural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and unnatural amino acids that have been chemically modified to include groups or chemical moieties that are not naturally present on the amino acid. Amino acid analogs may refer to amino Acid derivatives.
  • amino acid includes both D-amino acids and L-amino acids.
  • variants or fragments refers to a substance that is different from the wild type, e.g., by amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity), and/or function.
  • Peptide-related peptides may contain one or more amino acid changes (eg, mutations, insertions and deletions), truncations, modifications, or combinations thereof compared to the wild-type polypeptide.
  • fusion protein refers to a protein produced by joining two or more originally independent genes or fragments thereof. Fusion proteins may contain one or more non-natural amino acid sequences. Fusion proteins can be chimeric proteins. Fusion proteins can contain peptide affinity tags.
  • the term "antigen" refers to a molecule or fragment thereof capable of being bound by a selective binding agent.
  • the antigen can be a ligand that can be bound by a selective binding agent, such as a receptor.
  • an antigen may be an antigenic molecule that can be bound by a selective binding agent, such as an immune protein (e.g., an antibody).
  • An antigen may also refer to a molecule that can be used in an animal to generate antibodies capable of binding to the antigen or fragments of it.
  • antibody refers to a protein-binding molecule with immunoglobulin-like functions.
  • the term antibody includes antibodies (eg, monoclonal and polyclonal antibodies), as well as derivatives, variants and fragments thereof.
  • Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (ie, IgA, IgG, IgM, IgD, and IgE) and subclasses (eg, IgGl, IgG2, etc.).
  • Ig immunoglobulins
  • a derivative, variant or fragment of an antibody may refer to a functional derivative, variant or fragment that retains the binding specificity (eg, completely and/or partially) of the corresponding antibody.
  • Antigen-binding fragments include Fab, Fab', F(ab') 2 , variable fragments (Fv), single-chain variable fragments (scFv), small antibodies, diabodies, and single domain antibodies ("sdAb” or “nanobodies” ” or “ camelids ”).
  • the term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered, or chemically coupled. Examples of antibodies that have been optimized include affinity matured antibodies. Examples of antibodies that have been engineered include Fc-optimized antibodies (eg, antibodies optimized in fragment crystallizable regions) and multispecific antibodies (eg, bispecific antibodies).
  • “Sintilimab” or “Sintilimab” is a PD-1 monoclonal antibody injection jointly developed by Innovent Biologics and Eli Lilly and Company.
  • Nivolumab or “Nivolumab” is an anti-PD-1 monoclonal antibody jointly developed by Ono Pharmaceuticals and Medarex (the latter has been acquired by Bristol-Myers Squibb). Nivolumab is sold under the trade name Opdivo, and is also known as the "O drug” for this reason. Opdivo is the first approved and most representative anti-PD-1 monoclonal antibody in the world.
  • Tilelizumab or “Tilelizumab” is an anti-PD-1 antibody developed by BeiGene.
  • Camrelizumab or “Camrelizumab” is an anti-PD-1 antibody developed by Jiangsu Hengrui.
  • Pembrolizumab or “Pembrolizumab” is an anti-PD-1 antibody developed by Merck. Pembrolizumab is sold under the trade name Keytruda, and is therefore also known as the "K drug.”
  • Toripalimab or “Toripalimab” is an anti-PD-1 antibody developed by Junshi Biologics and is also the first domestically developed anti-PD-1 antibody approved in China.
  • subject refers to a vertebrate, preferably a mammal, such as a human.
  • Mammals include, but are not limited to, rats, apes, humans, farm animals, sporting animals, and pets.
  • treatment refers to a method for obtaining beneficial or desired results including, but not limited to, therapeutic benefits and/or prophylactic benefits.
  • Therapeutic benefit means any treatment-related improvement or effect on one or more diseases, conditions, or symptoms being treated.
  • the composition may be administered to a subject at risk of developing a particular disease, disorder, or symptom, or to a subject who reports one or more physiological symptoms of the disease, even though the disease, disorder, or symptom may not have manifested yet.
  • an effective amount refers to an amount of an agent (eg, a variant, fusion protein of the invention, or a composition containing the same) sufficient to produce the desired activity upon administration to a subject in need thereof.
  • therapeutically effective amount may refer to an amount of an agent sufficient to delay the manifestation of the condition to be treated, to arrest the progression of the condition to be treated, to alleviate or alleviate at least one symptom of the condition to be treated.
  • “Synergy” means that when two or more drugs are administered together, they produce an additive or synergistic effect, preferably a synergistic effect, that is, the effect when administered together is better than the sum of the effects of each drug when administered alone.
  • the human PD-1 sequence shown in SEQ ID NO: 1 is used as a basis.
  • the amino acids at positions 1-20 are the signal peptide
  • the amino acids at positions 21-170 are the extracellular region of PD-1
  • the amino acids at positions 171-191 are The amino acids are the transmembrane region
  • the amino acids at positions 192-288 are the intracellular region.
  • the extracellular region is a sequence of 150 amino acids from proline at position 21 to valine at position 170.
  • the transmembrane region is a sequence of 21 amino acids from valine at position 171 to isoleucine at position 191.
  • the position of the mutated amino acid in SEQ ID NO: 1 is recorded.
  • the amino acid type corresponding to the amino acid position in SEQ ID NO:1 is used as the amino acid before mutation and is placed before the position number (on the left), and the amino acid type present in the mutated PD-1 variant is Amino acids are placed after the position number (on the right).
  • the same position can be mutated into different amino acids, separate multiple optional amino acids with "/".
  • A129S mutation refers to the mutation of alanine to serine at position 129 of SEQ ID NO:1.
  • A129S/H means that alanine at position 129 can be mutated into serine or histidine.
  • the "PD-1 variant" of the present invention refers to a polypeptide sequence that has one or more amino acid differences compared with the amino acid sequence of wild-type human PD-1 shown in SEQ ID NO: 1.
  • the PD-1 variant of the invention is a variant of a truncated fragment of human PD-1 wild type, for example, a variant of a truncated fragment that does not contain an intracellular domain, or does not contain an intracellular domain. Variants of structural domains and transmembrane domains.
  • the PD-1 variant of the present invention has one or more of the following characteristics:
  • PD-1 variant does not bind to an anti-PD-1 antibody
  • PD-1 variant may mean that the PD-1 variant binds to a specific anti-PD-1 antibody with a lower affinity than the instrument used detection limit, or higher than the detection limit of the instrument used when expressing affinity as a K D value.
  • affinity for example, when determining affinity using the BIACORE T200 instrument, the measurable K D value should be in the range of 10 -12 M to 10 -3 M.
  • the instrument cannot detect the binding curve, that is, the PD-1 variant referred to in this article does not bind to the anti-PD-1 antibody.
  • the PD-1 variant of the present invention contains mutations at one or more amino acid positions selected from the two regions E84 to G90 and A129 to Q133 relative to the wild-type PD-1 polypeptide.
  • at least one mutation is selected from the region consisting of E84 to G90, and at least one mutation is selected from the region consisting of A129 to Q133.
  • the PD-1 variant does not include the region from M70 to K78 relative to the wild-type PD-1 polypeptide. Contains any mutations and has the same amino acid residues at corresponding positions as the wild-type PD-1 polypeptide shown in SEQ ID NO: 1.
  • the PD-1 variant of the invention has one or more amino acid mutations selected from the group consisting of: E84W, E84F; D85L, D85M; R86F; S87N, S87C, S87R, S87F, S87I; Q88L, Q88F, Q88N, Q88T; P89C, P89V, P89R; G90T, G90S; A129S, A129G, A129Q, A129Y; P130T; K1 31P; A132V, A132G; Q133W.
  • the PD-1 variant of the present invention includes the mutation combination contained in the variant having the amino acid sequence shown in any one of SEQ ID NOs: 2-26. Specifically, these mutation combinations are:
  • the PD-1 variant of the invention comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2-26, or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 2-26 sequence composition.
  • the PD-1 variant of the invention is a variant of the extracellular domain of wild-type human PD-1 as set forth in SEQ ID NO: 1, which includes SEQ ID NOs: 2-26 The amino acid sequence shown in any one of SEQ ID NOs: 2-26, or consisting of the amino acid sequence shown in any one of SEQ ID NOs: 2-26; and does not contain the intracellular domain of wild-type PD-1, or does not contain wild-type PD- The intracellular domain and transmembrane domain of 1.
  • the PD-1 variant can be allowed to be used in other position phase
  • the PD-1 variant contains one or several conservative amino acid substitutions, such as 1-20 conservative amino acid substitutions, preferably 1-15 conservative amino acid substitutions, more preferably 1-10 conservative amino acid substitutions, and even more preferably 1 -5 conservative amino acid substitutions.
  • the PD-1 variants of the invention comprise an amino acid identical to any one of SEQ ID NOs: 2-26, in the presence or absence of specific mutations and/or combinations of mutations as defined above.
  • the sequence has an extracellular region of at least 85% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, such as at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • Methods for determining sequence identity between two amino acid sequences are well known to those skilled in the art. For example, amino acid sequences can be aligned by NCBI's BLAST program, using the BLOSUM62 scoring matrix.
  • extracellular region and “extracellular domain” are used interchangeably and have the same meaning, and are generally used to describe PD-1 variants derived from the extracellular structure of wild-type human PD-1 area.
  • the PD-1 variant of the present invention can only be a polypeptide fragment corresponding to all or part of the extracellular region of wild-type PD-1, that is, an "extracellular region variant".
  • the PD-1 variant of the present invention can be regarded as a variant polypeptide in which the transmembrane region and intracellular region of wild-type PD-1 are removed and further includes the mutations as described above.
  • the PD-1 variant may not contain any amino acids corresponding to the intracellular and transmembrane regions of wild-type PD-1.
  • the PD-1 variant may comprise an extracellular signal peptide in wild-type PD-1, such as the signal peptide shown in amino acids 1 to 20 of SEQ ID NO: 1.
  • the exemplary PD-1 variants of the present invention are all polypeptides with a length of 155 amino acids, which correspond to amino acids 1-155 of wild-type human PD-1 as shown in SEQ ID NO: 1.
  • the PD-1 variant of the present invention can only or must have a length of 155 amino acids, nor does it mean that it cannot contain a transmembrane region or an intracellular region.
  • you wish to take advantage of the binding properties of the PD-1 variant of the present invention and use it to prepare a fusion protein, especially a transmembrane protein you can still include more amino acids corresponding to the PD-1 polypeptide, for example, the wild type The amino acids of the transmembrane region of PD-1 are incorporated into the transmembrane region of the fusion protein.
  • the PD-1 variants of the present invention Due to its unique binding properties, the PD-1 variants of the present invention, especially the PD-1 extracellular region variants, have broad application prospects.
  • the binding properties of the PD-1 variant can be utilized as part of a fusion protein, such as an extracellular binding domain, for targeting PD-L1 and/or PD-L2, such as targeted expression of PD- L1 and/or PD-L2 cells.
  • PD-1 variants can be constructed as antibody-like molecules.
  • the PD-1 variant can be directly constructed into a fusion protein with the Fc region of an immunoglobulin to form a functional polypeptide that specifically binds PD-L1 similar to an anti-PD-L1 antibody.
  • the Fc region is preferably an Fc region of a mammalian, such as a human immunoglobulin, such as an Fc region of human IgG1.
  • the fusion protein comprising the PD-1 variant is a chimeric stimulatory molecule, which may also be referred to as an enhancer receptor, a switch molecule, or a switch receptor.
  • the chimeric stimulatory molecule includes an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain is a PD-1 variant of the present invention, especially a PD-1 extracellular region variant, It is capable of binding PD-L1 and/or PD-L2, and the intracellular domain is the intracellular domain of costimulatory molecules that mediate activation of immune cells such as T cells.
  • the costimulatory molecule can be selected from interleukin 2 receptor (IL-2R), interleukin 12 receptor (IL-12R), CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB/CD137, ICOS, lymphocyte function-associated antigen 1 (LFA-1), LIGHT, NKG2C or OX40.
  • the costimulatory molecule is selected from OX40, 4-1BB, CD28, ICOS, CD27.
  • the costimulatory molecule is CD28.
  • the transmembrane domain of the fusion protein can be from wild-type PD-1 or a variant thereof, for example, it can be the transmembrane domain itself of wild-type PD-1 or a variant thereof, or a fragment of the transmembrane domain.
  • the transmembrane domain is from wild-type PD-1, for example, comprising amino acids 171-191 of the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof, or consisting of the amino acid sequence set forth in SEQ ID NO: 1 The composition of amino acids 171-191 of the amino acid sequence or its fragment is shown.
  • the transmembrane domain comprises at least 70% homology, at least 80% homology, or 90% homology to amino acids 171-191 of the amino acid sequence set forth in SEQ ID NO: 1 source sequence.
  • the transmembrane domain of the fusion protein may also be derived from other sources, such as from a costimulatory molecule.
  • Immune cells can be modified so that they express the chimeric stimulatory molecules. When immune cells containing the chimeric stimulatory molecule combine with PD-L1, immune cell activation signals will be generated in the immune cells, instead of immune cell inactivation signals usually brought about by PD1/PD-L1 binding. . In this way, the immunosuppressive signal is converted into an activating signal, thereby activating the function of immune cells.
  • the PD-1 variants of the present invention can be prepared by molecular biology methods.
  • the PD-1 variant or fusion polypeptide of the invention can be prepared by recombinant techniques. Nucleotide fragments, such as DNA or RNA, encoding PD-1 variants or fusion polypeptides can be synthesized in vitro, It is then constructed into a suitable expression vector and expressed in a suitable host cell. For example, sequences to be expressed can be delivered to host cells via viral vectors such as lentiviral vectors. The desired polypeptide is obtained by harvesting and optionally purifying the protein expressed by the host cell.
  • Methods for purifying polypeptides are known in the art, for example, by chromatography techniques such as column chromatography, HPLC, affinity chromatography, size exclusion chromatography, and the like. Proteins can also be purified using magnetic beads.
  • PD-1 variants or fusion polypeptides can be prepared through a cell-free polypeptide synthesis system.
  • Equipment useful for performing cell-free polypeptide synthesis is known in the art.
  • the PD-1 variants of the invention have a variety of potential uses due to their unique binding properties.
  • the PD-1 variant of the present invention retains the ability to bind to ligands PD-L1 and PD-L2, and therefore can be used to replace the functions of wild-type human PD-1 or its extracellular domain in a variety of applications, especially They are applications that utilize the binding function of PD-1, PD-L1, and PD-L2. Since the PD-1 variant of the present invention has improved binding ability to ligands, it may even produce better effects than wild-type PD-1.
  • the chimeric stimulatory molecule comprising the PD-1 variant of the invention when expressed in immune cells, can function to convert inhibitory PD-1/PD-L1 signals into stimulatory signals, by This enhances the function of immune cells such as T cells. Therefore, it can be used in cell therapy using immune cells to modify immune cells for use as therapy.
  • Such therapeutic immune cells can be the subject's own immune cells, or they can come from a donor that is half identical to the subject.
  • the immune cells may be obtained from the peripheral blood of the subject, for example, by sorting peripheral blood mononuclear cells.
  • the immune cells may be derived from tumor tissue, for example, they may be tumor-infiltrating lymphocytes (TIL).
  • T cells expressing the chimeric stimulatory molecule when they come into contact with tumor cells expressing PD-L1, they will increase the secretion of interleukin-2 (IL-2) by the T cells, which in turn can interact with the T cells. Receptor binding further stimulates T cells.
  • IL-2 interleukin-2
  • an antibody analog comprising a PD-1 variant of the invention has a similar effect to a PD-1 monoclonal antibody.
  • the fusion protein formed by the PD-1 variant of the present invention and Fc can block the PD-1/PD-L1 signaling pathway, thereby inhibiting the release of IL-2. Therefore, the Fc fusion protein of PD-1 mutants can play the same role of lifting the "immune brake" as PD-1 or PD-L1 monoclonal antibodies.
  • the PD-1 variant of the present invention does not bind to a variety of commercially available PD-1 monoclonal antibodies, which allows it to be used in combination with these PD-1 monoclonal antibodies without affecting their respective efficacy, providing the possibility of combined use. This is using This cannot be achieved with wild-type human PD-1 or its extracellular region.
  • the PD-1 variant of the present invention also retains the ability to bind to a specific PD-1 monoclonal antibody, allowing it to use the monoclonal antibody for purification and other operations.
  • the PD-1 variant of the present invention or the fusion protein containing the variant is particularly suitable for treating cancer.
  • Cancer in the context of the present invention includes malignant tumors occurring in different organs, tissues and cell types, including solid tumors and hematological cancers.
  • the present invention at least also includes the following items:
  • a PD-1 variant characterized by:
  • the PD-1 variant is capable of binding to the human PD-L1 polypeptide shown in SEQ ID NO: 28;
  • the PD-1 variant does not bind to one or more anti-PD-1 antibodies selected from the following group: sintilimab, nivolumab, camrelizumab Camrelizumab, Pemrolizumab and Toripalimab.
  • the PD-1 variant according to item 1 wherein compared with the wild-type human PD-1 polypeptide shown in SEQ ID NO: 1, the PD-1 variant has one or more of the following amino acid positions: There are mutations in all places: E84, D85, R86, S87, Q88, P89, G90, A129, P130, K131, A132 and Q133. The amino acid positions are numbered with the amino acid sequence shown in SEQ ID NO:1.
  • the PD-1 variant according to item 1 or 2 wherein compared with the wild-type human PD-1 polypeptide shown in SEQ ID NO: 1, the PD-1 variant has one or more options.
  • the PD-1 variant has one or more selected from the group consisting of: Group of amino acid mutations: E84W, E84F; D85L, D85M; R86F; S87N, S87C, S87R, S87F, S87I; Q88L, Q88F, Q88N, Q88T; P89C, P89V, P89R; G90T, G90S; A129S, A129G, A129Q, A12 9Y ;P130T; K131P; A132V, A132G; Q133W.
  • Group of amino acid mutations E84W, E84F; D85L, D85M; R86F; S87N, S87C, S87R, S87F, S87I; Q88L, Q88F, Q88N, Q88T; P89C, P89V, P89R; G90T, G90S; A129S, A129G, A129Q, A12 9Y ;P130T; K131P; A132V, A132
  • the PD-1 variant according to any one of items 2 to 4, wherein it is identical to SEQ ID NO:1 Compared with the wild-type human PD-1 polypeptide shown, the PD-1 variant has mutations at one or more amino acid positions selected from E84, D85, R86, S87, Q88, P89, G90, and at one or more amino acid positions selected from the group consisting of A129, P130, K131, A132 and Q133 have mutations at one or more amino acid positions.
  • the PD-1 variant according to any one of items 1 to 5, wherein compared with the wild-type human PD-1 polypeptide shown in SEQ ID NO: 1, the PD-1 variant comprises or only Comprising an amino acid mutation combination selected from any one of the following (1) to (25):
  • the PD-1 variant according to item 6 wherein compared with the wild-type human PD-1 polypeptide shown in SEQ ID NO: 1, the PD-1 variant contains or only contains as (1) to Amino acid mutation combinations of any group in (11).
  • the PD-1 variant according to item 7 wherein compared with the wild-type human PD-1 polypeptide shown in SEQ ID NO: 1, the PD-1 variant contains or only contains as (1) to Amino acid mutation combinations of any group in (6).
  • PD-1 variant according to any one of items 1 to 8, wherein the PD-1 variant does not contain an intracellular region, or does not contain a transmembrane region and an intracellular region.
  • the PD-1 variant according to item 10 wherein the variant comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 12 or an amino acid sequence having at least 85% sequence identity thereto, Or consist of an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 12 or an amino acid sequence having at least 85% sequence identity thereto.
  • the PD-1 variant according to item 11 wherein the variant comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 7 or an amino acid sequence having at least 85% sequence identity thereto, Or consist of an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 7 or an amino acid sequence having at least 85% sequence identity thereto.
  • the PD-1 variant according to any one of items 1 to 13, having a signal peptide as shown in amino acids 1 to 20 in SEQ ID NO: 1.
  • PD-1 variant according to any one of items 1 to 17, wherein the PD-1 variant binds to the PD-L2 with a higher affinity compared to wild-type human PD-1 polypeptide. Peptide binding.
  • a fusion protein comprising the PD-1 variant according to any one of items 1 to 23.
  • the fusion protein according to item 24 further comprising an immunoglobulin Fc fragment.
  • fusion protein according to item 27 wherein the costimulatory molecule is selected from the group consisting of: OX40, 4-1BB, CD28, ICOS, CD27.
  • nucleic acid molecule according to item 30 which comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 56-61, or a nucleotide sequence as shown in any one of SEQ ID NOs: 56-61 Nucleotide sequence A nucleotide sequence having at least 85% homology.
  • An expression vector comprising the isolated nucleic acid molecule of any one of items 30 to 32.
  • a host cell comprising the isolated nucleic acid molecule according to any one of items 30-32, or the expression vector according to item 33.
  • a pharmaceutical composition comprising the PD-1 variant according to any one of items 1 to 23, and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising the fusion protein according to any one of items 24 to 29, and a pharmaceutically acceptable carrier.
  • a pharmaceutical combination comprising (1) a PD-1 variant according to any one of items 1 to 23 or a fusion protein according to any one of items 24 to 29, and (2) an anti-PD-1 antibody .
  • anti-PD-1 antibody selected from the group consisting of: Sintilimab, Nivolumab, Camrelizumab , Pemrolizumab and Toripalimab.
  • the cell according to item 42 which is an immune cell.
  • the cell of item 43, wherein the immune cell is derived from peripheral blood mononuclear cells or is a tumor-infiltrating lymphocyte before modification.
  • This example describes the construction process of PD-1 mutant library.
  • SEQ ID NO: 1 three regions in SEQ ID NO: 1 were randomly mutated.
  • the first region is amino acid No. 70-78
  • the second region is amino acid No. 84-90
  • the third region is amino acid No. 84-90.
  • six sets of random mutation primers were designed. Each pair of primers can randomly mutate the corresponding codons of 5 consecutive amino acids belonging to the above-mentioned region.
  • the six sets of primers respectively include random mutations of amino acids 70-74 (7074-For/Rev), amino acids 74-78 (7478-For/Rev), and amino acids 84-88 (8488- For/Rev), the codons for amino acids 86-90 (8690-For/Rev), amino acids 127-131 (12731-For/Rev), and amino acids 129-133 (12933-For/Rev).
  • the codons corresponding to these amino acids were included in the upstream primers, and these codons were designed as 5 consecutive NNK Codon, where N represents any base among A, T, C, and G, and K represents any base among G or T.
  • P1-For and P1-Rev are used together with six other pairs of mutation primers to amplify the non-mutated region and mutated region, and are used to amplify the complete PD-1 DNA sequence through overlapping PCR.
  • primers used are 7074-For+P1-Rev and P1-For+7074-Rev respectively.
  • Amplification conditions were: 98°C for 10 seconds, 58°C for 5 seconds, and 72°C for 5 seconds, a total of 30 cycles.
  • the PCR product of the full-length sequence and the lentiviral vector pLV2-PD1-CD28 were digested using Sal I and Xba I restriction enzymes respectively (the plasmid diagram is shown in Figure 1; the complete sequence of the plasmid is shown in SEQ ID NO: 63 shown, a total of 8417bp, of which 1368-1877 is the PD1 extracellular region, 1878-1940 is the PD1 transmembrane region, and 1941-2063 is the CD28 intracellular region).
  • T4 DNA Ligase ligates the digested full-length sequence containing random mutations to the vector to prepare a PD-1 random mutation plasmid library.
  • Each ligation product was used to transform E. coli Trans1-T1 strain (Beijing Quanshijin Biotechnology, Cat. No. CD501-03). Specifically, the ligation product was added to E. coli Trans1-T1 chemically transformed competent cells thawed on ice and left on ice for 30 minutes. Then incubate at 42°C for 45 seconds, then place on ice for 2 minutes, add LB medium, and incubate on a shaker at 37°C for 45 minutes. After culturing, the bacterial solution was spread on an LB agar plate containing ampicillin and cultured at 37°C overnight. After cultivation, the total number of library clones was counted and library plasmids were extracted.
  • This example describes using the PD-1 random mutation library plasmid constructed in Example 1 to transfect 293T cells in order to construct a 293T cell line expressing PD-1 mutants.
  • 293T cells were cultured as follows. Cells of the human embryonic kidney 293T cell line with less than 20 passages were taken out of the liquid nitrogen and quickly placed in a 37°C water bath until completely melted. The thawed cells were added to 10 ml of D10 complete medium (DMEM containing 10% fetal calf serum) medium. After mixing, centrifuge at 400 g for 5 min and discard the medium. Resuspend with appropriate amount of culture medium and add to T25 or T75 cell culture flask for culture. The starting density of cells is approximately 2x10 5 /ml. The cells were cultured at 37°C in an incubator with 5% carbon dioxide for 72 h, and then the medium was aspirated.
  • D10 complete medium DMEM containing 10% fetal calf serum
  • the constructed variant plasmid library containing PD-1 random mutations was transformed into 293T cells through PEI (Poly(ethylenimine), polyethylenimine) transfection method.
  • PEI Poly(ethylenimine), polyethylenimine
  • Each library plasmid and PEI were mixed at a mass ratio of 1:3 and transiently transfected into 293T cells. 293T cells containing randomly mutated PD-1 variant library plasmids were thus obtained.
  • Example 3 Flow cytometry screening of cells that bind to PD-L1 but not to anti-PD-1 antibodies
  • This example describes the process of sorting a population of cells with the desired binding capacity by flow cytometry.
  • cells were digested with trypsin-EDTA solution. Centrifuge at 400g for 5 minutes, aspirate the culture medium and resuspend in phosphate buffer saline. Take a portion of the cell suspension and incubate it with PD-L1-Fc (R&D Systems, 156-B7-100) and Dylight650-labeled goat anti-human IgG Fc secondary antibody (Abcam, ab97006) for 30 minutes at 4°C.
  • PD-L1-Fc R&D Systems, 156-B7-100
  • Dylight650-labeled goat anti-human IgG Fc secondary antibody Abcam, ab97006
  • biotin-labeled anti-PD-1 antibodies including sintilimab, nivolumab, tislelizumab, camrelizumab, pembrolizumab, and Ripalimab (one of the following) and PE-labeled streptavidin (BD Bioscience, 554061).
  • Cell populations that bind to PD-L1 and do not bind to anti-PD-1 antibodies are analyzed and collected using a flow cytometry sorter. Sorting was first performed by flow cytometry using pemrolizumab, see Figure 2. Specifically, cells in the red box area in the flow cytometry diagram in Figure 2 were sorted. These cells were then subjected to binding experiments with five other antibodies, and cell populations that did not bind to four or five antibodies were selected. As a result of the preliminary screening, a total of 5000 cells were collected.
  • Example 4 Isolation and identification of plasmids in cells obtained by sorting
  • This example describes the isolation and identification process of the PD-1 variant plasmid contained in the cells obtained in Example 3.
  • Example 3 Take 5000 cells that were sorted and collected by flow cytometry in Example 3, and perform plasmid extraction using a plasmid mini-extraction kit (Quanjin Biotechnology, EM101-02).
  • the extracted plasmid was used to transform E. coli Trans1-T1 strain. After culturing, single clones of E. coli were picked and sequenced with EF-1 ⁇ oligonucleotide primers to obtain the nucleotide sequence of the PD-1 variant contained in the clone, and based on this, the corresponding amino acid sequence was obtained. Of the 5000 cells used, approximately 300 clones were obtained and sequenced. Among the clones sequenced, a total of about 60 clones (about 20%) were sequenced as irrelevant sequences, and about 30 clones (about 10%) contained successful PD-1 mutations but contained stop codons. The mutations were successful and There are about 210 clones that can express effectively (about 70%).
  • SEQ ID NO: 1 is wild-type PD-1
  • SEQ ID NOs: 2-26 are the sequences of representative PD-1 variants obtained.
  • the mutation sites of the variant of the present invention are concentrated at 12 sites: E84, D85, R86, S87, Q88, P89, G90, A129, P130, K131, A132 and Q133. Comparing with the three regions that were randomly mutated when originally designed (see Example 1), it can be found that these mutations are concentrated in the last two regions, that is, the second region from E84 to G90, and the second region from S127 to Q133. The third area (more specifically A129 to Q133). Among the representative variants, none contained amino acid mutations in the region from M70 to K78, nor mutations at S127 and L128.
  • Plasmids from these clones were extracted and used to transiently transfect 293T cells respectively. The transfection method was as described in Example 2.
  • the cells were digested 48 hours after transfection, and some cells were incubated with human IgG1-derived Fc fusion-expressed PD-L1 (R&D Biosystems) and Dylight650-labeled goat anti-human IgG Fc secondary antibody (Abcam) for 30 minutes at 4°C. Then centrifuge at 400g for 5 minutes, resuspend and wash the cells in PBS phosphate buffer. Repeat centrifugation and washing once.
  • human IgG1-derived Fc fusion-expressed PD-L1 R&D Biosystems
  • Dylight650-labeled goat anti-human IgG Fc secondary antibody Abcam
  • the wild-type PD-1 used is a fragment of wild-type PD-1 with the same length and corresponding amino acid position as the variant PD-1, and its sequence corresponds to the amino acid sequence of wild-type human PD-1 as shown in SEQ ID NO:1 1 to 155th.
  • wild-type human PD-1 was able to bind to PD-L1 and PD-L2, and was also able to bind to all six tested anti-PD-1 antibodies.
  • all 25 representative PD-1 variants of the present invention can bind to PD-L1; most variants can also bind to PD-L2 (except PD1_49#_93 and 56#-2AA). All variants can bind to tilelizumab and do not bind to at least three or more anti-PD1 monoclonal antibodies.
  • the human T cell isolation, viral infection, and culture processes were performed as follows. Take 1 ⁇ 10 8 peripheral blood mononuclear cells (PBMC) frozen in liquid nitrogen. After rapid thawing in a 37°C water bath, add 5 times the volume of pre-warmed T cell culture medium (X-VIVO15+0.5% HSA+300U/ml IL-2) and mix well. Then centrifuge at 400 g for 5 min, and discard the supernatant. Resuspend the cells in 20 ml of the T cell culture medium and place them in a T175 culture flask for culture in an incubator at 37°C and 5% carbon dioxide.
  • PBMC peripheral blood mononuclear cells
  • the preparation process of lentivirus is as follows. Add 0.75 mL of DMEM culture medium (Biological Industries, 06-1055-57-1A) and plasmid solution (VSVG 2.5 ⁇ g + dR8.91 7.5 ⁇ g + PLV2-PD1-CD28 10 ⁇ g, the total amount is 20 ⁇ g) in tube 1. Add 0.75 mL of DMEM culture medium and 60 ⁇ l of polyethylenimine (PEI) solution to tube 2. Mix the solutions in tube 1 and tube 2 and let stand for 30 minutes, then add them to the T75 culture flask growing 293T cells. Collect the virus supernatant after 24 hours, centrifuge at 100,000g for 2 hours, and discard the supernatant. Resuspend the virus in 500ul X-VIVO15 medium and use it to infect T cells.
  • DMEM culture medium Biological Industries, 06-1055-57-1A
  • plasmid solution
  • T cells cultured for 7-14 days were taken and incubated with PD-L1 (R&D Biosystems) fused with human IgG1-derived Fc and Dylight650-labeled goat anti-human IgG Fc secondary antibody (Abcam).
  • PD-L2 fused with human IgG1-derived Fc
  • Dylight650-labeled goat anti-human IgG Fc secondary antibody Abcam, ab97006
  • washed with phosphate buffer saline cells twice washed with phosphate buffer saline cells twice.
  • some cells were also taken and incubated with biotin-labeled anti-PD-1 antibody and PE-labeled streptavidin.
  • PD-1 variants (PD-1 variants of SEQ ID NOs: 2-7) were fused with rabbit IgG Fc, expressed and purified, and wild-type PD-1 (SEQ ID NO: 1) was used as a control. Specific steps are as follows.
  • PCR was used to amplify nucleotide sequences corresponding to the wild type (SEQ ID NO: 1) and 6 variants (variants with amino acid sequences SEQ ID NOs: 2-7) shown in Table 2 (sequences such as SEQ ID NOs:55-61 shown).
  • the primers are P32-For and P32-Rev (SEQ ID NO: 43 and 44) as shown below, using the pLV2-PD1-CD28 vector containing PD-1 wild type and variants constructed in Example 1 as a template.
  • the amplification reagents, system, and amplification conditions are the same as the PCR conditions in Example 1.
  • the PCR product and the protein expression vector pcDNA3.4-RFc (pcDNA3.4 vector (Thermo) containing the rabbit IgGc (RFc) gene) were digested with Nhe I and BamHI restriction enzymes respectively.
  • HEK293 cells (Zhuhai Kairui) were cultured to a density of 1 ⁇ 10 6 cells/ml at 37°C, 5% CO 2 , and 120 rpm.
  • the constructed pcDNA3.4-PD1-RFc vector was transfected into the HEK293 cells using 5 mL/L transfection reagent TA-293 (Zhuhai Kerry), and the transfection plasmid concentration was 1.5 mg/L.
  • the cells were cultured at 37°C, 8% CO 2 and 120 rpm for 7 days. train After the culture was completed, the culture was centrifuged at 4000 rpm and the supernatant was collected.
  • the protein was purified with Protein A beads, eluted with 500uL 0.1M Gly-HCL eluent at pH 2.6-3.0, and the eluate was collected to obtain the purified fusion protein.
  • FIG. 3 shows electrophoresis under non-reducing conditions
  • Figure 3B shows electrophoresis under reducing conditions.
  • the expected molecular weight of the fusion protein is approximately 43KDa. Under reducing conditions, band sizes are closer to expected. Under non-reducing conditions, the fusion protein formed a dimer and was modified such as glycosylation, so the molecular weight was higher than 86KDa.
  • bands in lanes 3, 4, and 6 there are obvious multiple bands in lanes 3, 4, and 6, and the bands have higher molecular weights, which may indicate that the bands with higher molecular weights have different forms of aggregation, such as the production of multimers, or There are varying degrees and types of protein modifications.
  • Wild-type PD-1 and representative PD-1 variants of the invention were determined by Biacore T200 with PD-L1, PD-L2 and the 6 species described in Example 3 Affinity of PD-1 monoclonal antibodies.
  • variant 1, variant 3 and variant 4 The binding affinity of variant 1, variant 3 and variant 4 to PD-L2 is lower than that of wild-type PD-1.
  • Variant 1 SEQ ID NO: 2
  • variant 6 SEQ ID NO: 7 are comparable to wild-type PD-1 in terms of binding affinity to the anti-PD-1 antibody tilelizumab.
  • PD1-CD28-expressing T cells When PD1-CD28-expressing T cells (PD1-CD28-T) are co-cultured with PD-L1-positive tumor cells (J82-PD-L1), the T cells release IL-2.
  • anti-PD-1 antibodies anti-PD-1 The antibody binds to PD-1, thereby blocking T cells from binding to tumor cells through PD-1, thereby inhibiting the release of IL-2.
  • the PD-1 mutant Fc fusion protein when the PD-1 mutant Fc fusion protein is added, it binds to PD-L1 on the target cells, preventing PD-L1 from binding to PD-1 on the T cell membrane. It also has similar properties to anti-PD-1 antibodies. Blocks the release of IL-2.
  • the pLV2-PD1-CD28 plasmid (PD-1 is human wild-type PD-1) described in Example 1 was prepared into a lentiviral vector as described in Example 4 and used to infect human T cells to obtain PD1-CD28-T cell.
  • the T cell culture method was as described in Example 4.
  • the pLV2-PDL1 plasmid (PD-L1 is human wild-type PD-L1) was prepared into a lentiviral vector as described in Example 4 and used to infect human bladder cancer cell lines (purchased from the Chinese Academy of Medical Sciences, Cat. No. 1101HUM-PUMC000346 ) to obtain J82-PDL1 cell line.
  • PD-1 variant Fc fusion protein blocks IL-2 secretion experiment
  • Table 5 shows the measured values of the concentration of IL-2 secreted by T cells after adding different concentrations of antibodies or fusion proteins.
  • Figure 5 is a trend chart of the data in Table 5.
  • the data in Table 5 shows that there is no significant change in IL-2 secretion when the concentration of tislelizumab is lower than 6.4nM. When the concentration is ⁇ 6.4nM, IL-2 secretion is significantly inhibited; 56#-16 fusion As the protein concentration increases, IL-2 secretion shows a downward trend, but IL-2 secretion It is not completely inhibited; as the concentration of 56#-108 fusion protein increases, IL-2 secretion shows a downward trend.
  • This example tested the effect of the enhanced receptor based on the PD1 variant of the present invention on T cell function through an IL-2 secretion experiment.
  • PD1-CD28-expressing T cells When PD1-CD28-expressing T cells (PD1-CD28-T) are co-cultured with PD-L1-positive tumor cells, the T cells release the cytokine IL-2 through the interaction of PD-1 and PD-L1. Therefore, by detecting the IL-2 content in the co-cultured culture medium using enzyme-linked immunosorbent assay (ELISA), the effect of PD1-CD28 in promoting IL-2 secretion can be determined.
  • ELISA enzyme-linked immunosorbent assay
  • ELISA method Human IL-2 detection kit (ELISA method) (Daktronix, Cat. No. 1110203) and perform ELISA according to the following procedure.
  • Microplates were previously coated with anti-human IL-2 capture antibody.
  • SA-HRP horseradish peroxidase-labeled streptavidin
  • SA-HRP horseradish peroxidase-labeled streptavidin
  • the meter reads the optical absorption value (OD) at 450nm. Within a certain range, the concentration of human IL-2 is proportional to the OD value.
  • IL-2 standard was used for ELISA detection and a standard curve was obtained. The concentration of IL-2 in each co-culture was calculated using this standard curve.
  • the target cell's PD-L1 is knocked out, whether it is a control T cell or a T cell armed with a PD-1 variant enhancing receptor (108T, that is, 108-CD28-T)
  • a PD-1 variant enhancing receptor 108T, that is, 108-CD28-T
  • 108-CD28 a PD-1 variant enhancing receptor
  • This example relates to cell killing experiments.
  • T cells By causing T cells to express the PD1-C28 enhanced receptor containing the PD-1 variant of the present invention, it was confirmed that the PD-1 variant enhanced receptor can enhance the killing ability of T cells expressing tumor cells with PD-L1.
  • Tumor cells express HLA-peptide complex antigens on their surfaces, and T cells express T cell receptors (TCRs) that target this antigen.
  • TCRs T cell receptors
  • the tumor cells were J82 cells (J82Eso1PDL1) expressing Eso1 antigen and PD-L1.
  • J82Eso1PDL1 tumor cell line lentiviral vectors expressing human PD-L1 and ESO1 genes were prepared according to the method in Example 4, and were named pLV2-PDL1 and pLV2-ESO1 respectively.
  • the pLV2-PDL1 lentivirus was used to infect the human bladder cancer cell line (purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences, Cat. No. 1101HUM-PUMC000346) to obtain J82-PDL1 cells expressing human PD-L1. .
  • the J82-PDL1 cells were then infected with the pLV2-ESO1 lentivirus prepared as described above. After the ESO1 antigen of this cell is expressed and processed intracellularly, it is presented extracellularly in the form of ESO1peptide and forms an antigen complex with the HLA*A0201 protein expressed on the surface of J82 cells. This complex can be accepted by specific T cells. Recognized by T cell receptor (TCR).
  • TCR T cell receptor
  • T cells Three different T cells were prepared, including: (1) T cells expressing TCR targeting HLA-Eso1 peptide (TCR-T); (2) expressing PD1 variants (56#-108 mutant, i.e., variants 6) T cells with enhanced receptors composed of CD28 and TCR targeting HLA-Eso1 peptide (108-TCRT); and (3) T cells expressing GFP green fluorescent protein (GFP-T) as a control.
  • TCR-T T cells expressing TCR targeting HLA-Eso1 peptide
  • PD1 variants 56#-108 mutant, i.e., variants 6
  • T cells with enhanced receptors composed of CD28 and TCR targeting HLA-Eso1 peptide 108-TCRT
  • GFP-T GFP green fluorescent protein
  • PD1 + T cells were first prepared. Frozen whole blood cells from healthy donors were quickly resuscitated in a water bath at 37°C, and added to 5 times the volume of preheated T cell culture medium containing 0.5% human serum albumin (Xingir, Shuanglu Pharmaceutical Industry) X-VIVO15 (Lonza; BE02-053Q). After mixing evenly, centrifuge at 400g for 5 minutes and remove the supernatant. Resuspend in PBS-0.5% HSA to a density of 10 8 total cells/ml. Add biotin-labeled anti-PD1 antibody Biotin-anti-PD1 (Biolegend, Cat. No.
  • the lentiviral vector pLV2-TCR containing the TCR coding sequence was prepared as described in Example 4.
  • the TCR is a T cell receptor that specifically recognizes the HLA*A0201/ESO1peptide complex, and the coding sequence is shown in SEQ ID NO: 41.
  • the lentivirus was then used to infect the above-mentioned PD1 + T cells according to the method described in Example 4 to obtain TCR-T cells.
  • a lentiviral vector containing PD1 variant enhanced receptor PD1-CD28 (referred to as 108-CD28) and TCR coding sequences was prepared according to the method described in Example 4.
  • the lentivirus was then used to infect the above-mentioned PD1 + T cells, and the infection method was as described in Example 4 to obtain 108-TCRT cells.
  • the gene sequence of 108-TCR is shown in SEQ ID NO:42.
  • the transmembrane region in the enhanced receptor is that derived from PD1.
  • Killing experiments were performed using real-time label-free dynamic cell analysis (RTCA) to detect the survival of target cells.
  • RTCA real-time label-free dynamic cell analysis
  • J82Eso1PDL1 target cells were first placed in a 96-well culture plate and cultured (100ul per well, target cell concentration 8x10 4 /ml).
  • TCR-T cells or TCR-T cells modified with enhanced receptors were added to the corresponding wells and observed. And record the growth of target cells, and the results are shown in Figure 6.
  • the cell index on the ordinate reflects the adhesion of cells. The higher the value, the more cells are attached, which means there are more living cells and good growth.
  • the abscissa is time, expressed in hours.
  • TCR-T cells expressing enhanced receptors exhibited stronger inhibition of tumor cells than TCR-T alone.
  • J82Eso1PDL1 co-cultured with GFP-T a control cell without TCR, had some non-specific killing effect, it still maintained growth (blue curve in Figure 6); while 82Eso1PDL1 co-cultured with Eso1-TCR-T showed significant growth. is inhibited (green curve).
  • the number of target cells co-cultured with TCR-T (108-TCRT) expressing the enhanced receptor of the present invention decreased significantly, indicating that the target cells were killed (red curve in Figure 6), indicating that the TCR-T expressing the enhanced receptor The killing effect is stronger.

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Abstract

本发明涉及PD-1变体及其用途。具体而言，本发明的PD-1变体能够以高亲和力结合PD-L1，但与一种或多种抗PD-1单克隆抗体不结合，优选与至少两种抗PD-1单克隆抗体不结合。本发明还涉及包含所述PD-1变体的融合蛋白。本发明还涉及所述PD-1变体和融合蛋白的编码核酸分子以及用途。

Description

PD-1变体及其用途 技术领域
本发明涉及免疫治疗领域。具体而言，本发明提供PD-1变体。本发明还提供编码所述PD-1变体的核酸分子，包含所述PD-1变体的融合蛋白或组合物，以及所述PD-1变体的用途。
背景技术
程序性死亡蛋白1，即PD-1，是主要表达于活化淋巴细胞表面的一种蛋白。PD-1是一种免疫检查点蛋白，具有免疫刹车的能力。相应地，抑制PD-1能够解除针对免疫系统的抑制来攻击肿瘤，使得PD-1成为近年来热门的免疫靶标。多种针对PD-1的抗体已经获批并上市。
PD-1与其配体PD-L1结合，产生免疫抑制信号。肿瘤细胞通过表达PD-L1，从而借助PD-1/PD-L1通路来实现免疫逃逸。已有研究通过开发能够结合PD-L1的PD-1类似物来阻止PD-1和PD-L1的相互作用，从而实现对PD-1的抑制。另外，也可以利用这些与PD-L1具有结合特异性的PD-1类似物来识别并结合肿瘤细胞。
例如，斯坦福大学的PCT专利公开WO2016/023001中描述了多种PD-1变体序列。这些变体相对于人的野生型PD-1与其配体PD-L1具有提高的亲和力，并且缺少跨膜区。然而，该申请中的变体相对于野生型均具有多个氨基酸的差异。该申请并没有研究PD-1变体与抗PD-1抗体的亲和力。
在将PD-1变体用于治疗用途时，有时会涉及到与抗PD-1抗体的联用。在这样的情况下，如果抗PD-1抗体能够识别并结合PD-1变体，那么这两种治疗途径会彼此影响，降低二者的功效。
因此，本领域仍然需要改进的PD-1多肽变体以适用于更广泛的应用场景。
发明内容
为了解决上述问题，本发明的发明人构建了PD-1突变质粒文库，并通过流式分选，筛选出一系列PD-1变体，它们既保留了与其配体PD-L1的高亲和力，同时不与多种市售的抗PD-1单克隆抗体结合，由此完成了本发明。
因此，第一方面，本发明提供PD-1变体，其与野生型人PD-1多肽具有不同的氨基酸序列，并且以高亲和力，例如高于野生型人PD-1多肽的亲和力，结合PD-L1，同时基本上不结合多种抗PD-1单克隆抗体。
第二方面，本发明提供包含第一方面的PD-1变体的融合蛋白。在一个实施方案中，所述融合蛋白可以是以所述PD-1变体为胞外段的跨膜融合蛋白。在一个实施方案中，所述融合蛋白是包含免疫球蛋白Fc的融合蛋白。
第三方面，本发明提供编码第一方面的PD-1变体或第二方面的融合蛋白的核酸分子。
第四方面，本发明提供包含第三方面的所述核酸分子的表达载体。
第五方面，本发明提供包含第三方面所述核酸分子或第四方面的表达载体的宿主细胞。
第六方面，本发明提供包含第一方面的PD-1变体或第二方面的融合蛋白的药物组合物。
第七方面，本发明提供第一方面的PD-1变体或第二方面的融合蛋白在制备药物中的用途。在具体的实施方案中，所述药物用于癌症治疗。
第八方面，本发明提供一种药物组合，其包含(1)第一方面的PD-1变体或第二方面的融合蛋白，和(2)抗PD-1抗体。
附图说明
图1的质粒图显示了实施例1中用于构建PD-1突变体文库所使用的质粒载体的结构。
图2显示了实施例3中通过流式细胞术分选与PD-L1结合但不与抗PD-1抗体结合的细胞的示意图。
图3为实施例5中表达野生型PD-1或PD-1变体与兔IgG Fc融合蛋白的SDS-PAGE结果。(A)非还原条件；(B)还原条件。泳道1为野生型PD-1，泳道2-7为SEQ ID Nos:2-7所示的PD-1变体，泳道8为分子量标志物。
图4显示了实施例7中测试的PD-1变体Fc融合蛋白对T细胞IL-2释放的阻断作用。
图5显示了实施例8中用PD-1变体构建的PD-1-CD28增强受体(108-CD28)武装T细胞，可被表达PD-L1靶细胞刺激，分泌细胞因子IL-2。
图6显示了实施例9中PD-1变体构建的PD-1-CD28增强受体 (108-CD28)可增强T细胞杀伤相应靶细胞。
发明详述
定义
除非另有说明，否则本文所公开的一些方法的实践采用免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA的常规技术，它们在本领域的技术范围之内。参见例如Sambrook和Green,《分子克隆实验指南》(Molecular Cloning:A Laboratory Manual),第4版(2012)。
术语“约”或“大约”意指在特定值的可接受误差范围内，并且所述误差范围是本领域普通技术人员能够确定的。在一些情况中，所述可接受的误差范围部分取决于如何测量或确定所述值，即测量系统的局限性。例如，根据本领域的实践，“约”可以意指在1个或超过1个标准偏差内。可替代地，“约”可以意指给定值的至多20％、至多10％、至多5％或至多1％的范围。可替代地，特别是关于生物系统或过程，所述术语可以意指在值的一个数量级内、优选地在值的5倍内、更优选地在值的2倍内。在本申请和权利要求书中描述特定值的情况下，除非另有说明，否则应当假设术语“约”意指在所述特定值的可接受误差范围内。
如本文所用，术语“核苷酸”通常是指碱基-糖-磷酸组合。核苷酸可以包含合成核苷酸。核苷酸可以包含合成核苷酸类似物。核苷酸可以是核酸序列(例如脱氧核糖核酸(DNA)和核糖核酸(RNA))的单体单元。术语核苷酸可以包括核糖核苷三磷酸腺苷三磷酸(ATP)、尿苷三磷酸(UTP)、胞嘧啶三磷酸(CTP)、鸟苷三磷酸(GTP)；以及脱氧核糖核苷三磷酸，如dATP、dCTP、dITP、dUTP、dGTP、dTTP；或其衍生物。此类衍生物可以包括例如[αS]dATP、7-脱氮-dGTP和7-脱氮-dATP，以及对含有它们的核酸分子赋予核酸酶抗性的核苷酸衍生物。如本文所用的术语核苷酸可以是指双脱氧核糖核苷三磷酸(ddNTP)及其衍生物。双脱氧核糖核苷三磷酸的说明性例子可以包括但不限于ddATP、ddCTP、ddGTP、ddITP和ddTTP。核苷酸可以是未标记的，或者可以通过熟知的技术可检测地标记。标记也可以用量子点进行。可检测的标记可以包括例如放射性同位素、荧光标记、化学发光标记、生物发光标记和酶标记。
术语“多核苷酸”、“寡核苷酸”和“核酸”可互换使用，是指任何长度的核苷酸的聚合形式，无论是呈单链、双链或多链形式的脱氧核糖核苷酸或核糖 核苷酸或其类似物。多核苷酸对于细胞而言可以是外源的或内源的。多核苷酸可以存在于无细胞环境中。多核苷酸可以是基因或其片段。多核苷酸可以是DNA。多核苷酸可以是RNA。多核苷酸可以具有任何三维结构，并且可以执行任何已知或未知的功能。多核苷酸可以包含一种或多种类似物(例如，改变的骨架、糖或核碱基)。类似物的一些非限制性例子包括：5-溴尿嘧啶、肽核酸、异种核酸、吗啉代、锁核酸、乙二醇核酸、苏阿糖核酸、双脱氧核苷酸、虫草素、7-脱氮-GTP、荧光团(例如罗丹明或与糖连接的荧光素)、含硫醇的核苷酸、生物素连接的核苷酸、荧光碱基类似物、CpG岛、甲基-7-鸟苷、甲基化核苷酸、肌苷、硫代尿苷、假尿苷、二氢尿苷、辫苷和怀俄苷。多核苷酸的非限制性例子包括基因或基因片段的编码或非编码区、由连锁分析定义的基因座(loci/locus)、外显子、内含子、信使RNA(mRNA)、转移RNA(tRNA)、核糖体RNA(rRNA)、短干扰RNA(siRNA)、短发夹RNA(shRNA)、微小RNA(miRNA)、核酶、cDNA、重组多核苷酸、支链多核苷酸、质粒、载体、任何序列的分离DNA、任何序列的分离RNA、无细胞多核苷酸(包括无细胞DNA(cfDNA)和无细胞RNA(cfRNA))、核酸探针和引物。核苷酸的序列可以被非核苷酸组分中断。
术语“表达”是指多核苷酸从DNA模板转录(如转录成mRNA或其他RNA转录物)的一个或多个过程和/或转录的mRNA随后翻译成肽、多肽或蛋白质的过程。
术语“肽”、“多肽”和“蛋白质”在本文中可互换使用，是指通过一个或多个肽键连接的至少两个氨基酸残基的聚合物。这一术语并不意味着聚合物的特定长度，也不旨在暗示或区分肽是使用重组技术、化学或酶促合成产生的还是天然存在的。所述术语适用于天然存在的氨基酸聚合物以及包含至少一种经修饰的氨基酸的氨基酸聚合物。在一些情况下，聚合物可以被非氨基酸中断。所述术语包括任何长度的氨基酸链，包括全长蛋白质，以及具有或不具有二级和/或三级结构(例如，结构域)的蛋白质。所述术语还涵盖已经例如通过二硫键形成、糖基化、脂化、乙酰化、磷酸化、氧化和任何其他操作(如与标记组分缀合)被修饰的氨基酸聚合物。如本文所用，术语“氨基酸”通常是指天然和非天然氨基酸，包括但不限于经修饰的氨基酸和氨基酸类似物。经修饰的氨基酸可以包括天然氨基酸和非天然氨基酸，它们已经被化学修饰以包括非天然存在于氨基酸上的基团或化学部分。氨基酸类似物可以是指氨基 酸衍生物。术语“氨基酸”包括D-氨基酸和L-氨基酸两者。
当涉及多肽时，术语“变体”或“片段”是指例如通过氨基酸序列、结构(例如，二级和/或三级)、活性(例如，酶促活性)和/或功能而与野生型多肽相关的多肽。与野生型多肽相比，多肽的变体和片段可以包含一种或多种氨基酸变化(例如，突变、插入和缺失)、截短、修饰或其组合。
术语“融合蛋白”指通过连接两个或更多个原本相互独立的基因或其片段而产生的蛋白质。融合蛋白可以包含一个或多个非天然的氨基酸序列。融合蛋白可以是嵌合蛋白。融合蛋白可以包含肽亲和标签。
如本文所用，术语“抗原”是指能够被选择性结合剂结合的分子或其片段。例如，抗原可以是可以被选择性结合剂(如受体)结合的配体。又如，抗原可以是可以被选择性结合剂(如免疫蛋白(例如，抗体)结合的抗原分子。抗原也可以是指能够被用于动物中以产生能够与所述抗原结合的抗体的分子或其片段。
如本文所用，术语“抗体”是指具有免疫球蛋白样功能的蛋白质结合分子。术语抗体包括抗体(例如，单克隆和多克隆抗体)，以及其衍生物、变体和片段。抗体包括但不限于不同类别(即IgA、IgG、IgM、IgD和IgE)和亚类(如IgG1、IgG2等)的免疫球蛋白(Ig)。抗体的衍生物、变体或片段可以是指保留对应抗体的结合特异性(例如，完全和/或部分)的功能性衍生物、变体或片段。抗原结合片段包括Fab、Fab′、F(ab′)2、可变片段(Fv)、单链可变片段(scFv)、小抗体、双抗体和单结构域抗体(“sdAb”或“纳米抗体”或“骆驼抗体(camelids)”)。术语抗体包括已经被优化、被工程化或被化学偶联的抗体和抗体的抗原结合片段。已经被优化的抗体的例子包括亲和力成熟的抗体。已经被工程化的抗体的例子包括Fc优化的抗体(例如，在片段可结晶区域中优化的抗体)和多特异性抗体(例如，双特异性抗体)。
“Sintilimab”或“信迪利单抗”是由信达生物和礼来制药联合开发的PD-1单抗注射剂。
“Nivolumab”或“纳武单抗”是由小野制药和Medarex公司(后者已被百时美施贵宝收购)联合开发的抗PD-1单抗。纳武单抗以商品名Opdivo销售，也因此被称为“O药”。Opdivo是世界上最早获批，也是最具代表性的抗PD-1单抗。
“Tilelizumab”或“替雷利珠单抗”是百济神州开发的抗PD-1抗体。
“Camrelizumab”或“卡瑞利珠单抗”是江苏恒瑞开发的抗PD-1抗体。
“Pemrolizumab”或“帕博利珠单抗”是默沙东开发的抗PD-1抗体。帕博利珠单抗以Keytruda为商品名销售，也因此被称为“K药”。
“Toripalimab”或“特瑞普利单抗”是君实生物开发的抗PD-1抗体，也是中国首个获批的本国研发的抗PD-1抗体。
术语“受试者”、“个体”和“患者”在本文中可互换使用，是指脊椎动物，优选地哺乳动物，如人。哺乳动物包括但不限于鼠类、猿猴、人类、农场动物、运动动物和宠物。
如本文所用，术语“治疗”是指用于获得有益或所需的结果的方法，所述有益或所需的结果包括但不限于治疗益处和/或预防益处。治疗益处意指对治疗中的一种或多种疾病、病症或症状的任何治疗相关的改进或作用。对于预防益处，可以将组合物给予有发生特定疾病、病症或症状的风险的受试者，或给予报告疾病的一种或多种生理症状(即使疾病、病症或症状可能尚未表现)的受试者。
术语“有效量”或“治疗有效量”是指足以在给予有需要的受试者后产生所需的活性的作用剂(例如本发明的变体、融合蛋白或包含它们的组合物)的量。在本公开文本的上下文中，术语“治疗有效量”可以指足以延迟待治疗病症的表现、阻止待治疗病症的进展、缓解或减轻待治疗病症的至少一种症状的作用剂的量。
“协同作用”指两种或多种药物共同施用时，产生了加合或增效作用，优选增效作用，即共同施用时的效果优于各种药物单独施用时的效果之和。
突变的命名规则
在本发明的上下文中描述PD-1变体中所含的突变时，如果没有特殊说明，则均以如SEQ ID NO:1所示的人PD-1序列作为基础。在SEQ ID NO:1所示的人PD-1的氨基酸序列中，第1-20位的氨基酸为信号肽，第21-170位的氨基酸为PD-1胞外区，第171-191位的氨基酸为跨膜区，第192-288位的氨基酸为胞内区。根据该编号体系，在如SEQ ID NO:1所示的人PD-1中，胞外区是从第21位的脯氨酸到第170位的缬氨酸组成的共150个氨基酸的序列，且跨膜区是从第171位的缬氨酸到第191位的异亮氨酸组成的共21个氨基酸的序列。
在本发明中描述氨基酸突变位置时，以被突变的氨基酸在SEQ ID NO:1中的位置来记录。在描述氨基酸突变的类型时，将SEQ ID NO:1中对应氨基酸位置的氨基酸类型作为突变前的氨基酸，放在位置号之前(左侧)，而将突变后的PD-1变体中存在的氨基酸放在位置号之后(右侧)。当同一位置可以突变为不同的氨基酸时，以“/”隔开多种可选的氨基酸。
基于上述规则，以A129S突变为例，其指代参考SEQ ID NO:1在第129位的丙氨酸突变为丝氨酸。另外，A129S/H则指代第129位的丙氨酸可以突变为丝氨酸或组氨酸。
PD-1变体
本发明的“PD-1变体”指与SEQ ID NO:1中所示的野生型人PD-1的氨基酸序列相比，具有一个或多个氨基酸差异的多肽序列。在具体的实施方案中，本发明的PD-1变体是人PD-1野生型的截短片段的变体，例如不含胞内结构域的截短片段的变体，或不含胞内结构域和跨膜结构域的变体。
本发明的PD-1变体相对于野生型人PD-1而言，具有如下一种或多种特征：
(1)改进的与PD-L1配体的亲和力，
(2)改进的与PD-L2配体的亲和力，
(3)与两种或更多种市售抗PD-1抗体不结合。
在本发明的上下文中，在本发明的上下文中，“PD-1变体不与抗PD-1抗体结合”可以指PD-1变体与特定抗PD-1抗体的结合亲和力低于所用仪器的检测限，或在以KD值表示亲和力时高于所用仪器的检测限。例如，在使用BIACORE T200仪器测定亲和力时，可以测得的KD值应在10-12M至10-3M的范围。当KD值高于10-3M，该仪器检测不到结合曲线，也就是本文中所指的PD-1变体不与抗PD-1抗体结合。
由于本发明的PD-1变体最重要的性质是结合亲和力的改变，因此其突变主要集中在胞外段。具体而言，本发明的PD-1变体相对于野生型PD-1多肽而言，在选自E84至G90以及A129至Q133这两个区域内的一个或多个氨基酸位置处包含突变。优选地，至少有一个突变选自E84至G90组成的区域，且至少有一个突变选自A129至Q133组成的区域。优选地，所述PD-1变体相对于野生型PD-1多肽而言，在M70至K78组成的区域内不包 含任何突变，且具有与如SEQ ID NO:1所示的野生型PD-1多肽在对应位置相同的氨基酸残基。
在优选的实施方案中，本发明的PD-1变体与SEQ ID NO:1所示的野生型人PD-1多肽相比，具有一种或多种选自下组的氨基酸突变：E84W、E84F；D85L、D85M；R86F；S87N、S87C、S87R、S87F、S87I；Q88L、Q88F、Q88N、Q88T；P89C、P89V、P89R；G90T、G90S；A129S、A129G、A129Q、A129Y；P130T；K131P；A132V、A132G；Q133W。
本发明的PD-1变体包含具有SEQ ID NOs:2-26中任一所示的氨基酸序列的变体中所含的突变组合。具体来说，这些突变组合为：
(1)A129S、P130T、K131P、A132V和Q133W；
(2)E84F、D85L、R86F、S87N、Q88L、A129S、P130T、K131P、A132V和Q133W；
(3)E84W、D85M、R86F、S87C、Q88F、A129S、P130T、K131P、A132V和Q133W；
(4)R86F、S87R、Q88N、P89C、G90T、A129S、P130T、K131P、A132V和Q133W；
(5)R86F、S87F、Q88T、P89V、G90S、A129S、P130T、K131P、A132V和Q133W；
(6)S87I、Q88N、P89R、G90S、A129S、P130T、K131P、A132V和Q133W；
(7)E84F、D85L、R86F、S87N、Q88L、P130T、K131P和Q133W；
(8)R86F、S87R、Q88N、P89C、G90T、A129G、P130T、K131P和Q133W；
(9)R86F、S87R、Q88N、P89C、G90T、P130T、K131P、A132G和Q133W；
(10)R86F、S87R、Q88N、P89C、G90T、A129Q、P130T、K131P和Q133W；或
(11)R86F、S87F、Q88T、P89V、G90S、A129Y、P130T、K131P和Q133W；
(12)A129H、P130F、K131R、A132F和Q133N；
(13)A129S、P130E、K131P、A132I和Q133V；
(14)S87L、Q88C、P89V、G90F、A129H、P130F、K131R、A132F和Q133N；
(15)R86S、S87F、Q88E、P89V、G90R、A129H、P130F、K131R、A132F和Q133N；
(16)R86L、S87Y、Q88L、P89K、G90Y、A129S、P130E、K131P、A132I和Q133V；
(17)R86F、S87R、Q88N、P89C、G90T、A129W、P130T、K131P、A132M和Q133W；
(18)R86F、S87F、Q88T、P89V、G90S、A129H、P130T、K131P、A132F和Q133W；
(19)R86F、S87F、Q88T、P89V、G90S、A129G、P130T、K131P、A132F和Q133W；
(20)R86F、S87F、Q88T、P89V、G90S、A129I、P130T、K131P、A132G和Q133W；
(21)S87I、Q88N、P89R、G90S、A129T、P130T、K131P、A132F和Q133W；
(22)S87I、Q88N、P89R、G90S、A129V、P130T、K131P和Q133W；
(23)S87I、Q88N、P89R、G90S、P130T、K131P和Q133W；
(24)R86F、S87R、Q88N、P89C、G90T、P130T、K131P和Q133W；
(25)R86F、S87F、Q88T、P89V、G90S、P130T、K131P和Q133W。
在优选的实施方案中，本发明的PD-1变体包含如SEQ ID NOs:2-26中任一所示的氨基酸序列，或者由如SEQ ID NOs:2-26中任一所示的氨基酸序列组成。在优选的实施方案中，本发明的PD-1变体是如SEQ ID NO:1所示的野生型人PD-1的胞外结构域的变体，其包含如SEQ ID NOs:2-26中任一所示的氨基酸序列，或者由如SEQ ID NOs:2-26中任一所示的氨基酸序列组成；并且不含野生型PD-1的胞内结构域，或不含野生型PD-1的胞内结构域和跨膜结构域。
在如上限定的具体突变和/或突变组合存在或不存在的基础上，应该理解可以允许在所述PD-1变体的一个或几个氨基酸位置包含保守氨基酸取代，而包含这些保守氨基酸取代的PD-1变体仍然保留本发明期望的活性和结合特异性。保守氨基酸取代的替代规则是本领域技术人员已知的。例如，在满足包含如上(1)至(25)中任一氨基酸组合，且在M70至K78组成的区域内具有与野生型相同的氨基酸的前提下，可以允许所述PD-1变体在其他位置相 对于野生型人PD-1而言包含一个或几个保守氨基酸取代，如1-20个保守氨基酸取代，优选1-15个保守氨基酸取代，更优选1-10个保守氨基酸取代，还更优选1-5个保守氨基酸取代。
在另一个实施方案中，在如上限定的具体突变和/或突变组合存在或不存在的基础上，本发明的PD-1变体包含与SEQ ID NO:2-26中任一所示的氨基酸序列具有至少85％序列同一性的胞外区，优选至少90％序列同一性，更优选至少95％序列同一性，例如至少96％，至少97％，至少98％，至少99％序列同一性。两个氨基酸序列之间序列同一性的确定方法是本领域技术人员熟知的。例如，可以通过NCBI的BLAST程序来比对氨基酸序列，其中使用BLOSUM62评分矩阵。
在本发明的上下文中，“胞外区”和“胞外结构域”可以互换使用并具有相同含义，其通常用于描述PD-1变体源自野生型人PD-1的胞外结构域。在以上任何一种突变方式中，本发明的PD-1变体都可以只是对应于野生型PD-1的全部或部分胞外区的多肽片段，即“胞外区变体”。换言之，在这种情况下，可以将本发明的PD-1变体视作是将野生型PD-1的跨膜区和胞内区去除，并进一步包含如上所述的突变的变体多肽。例如，所述PD-1变体可以不含任何对应于野生型PD-1的胞内区和跨膜区的氨基酸。例如，所述PD-1变体可以包含野生型PD-1中位于胞外的信号肽，例如SEQ ID NO:1的氨基酸1至20所示的信号肽。例如，本发明的示例性PD-1变体均为长度为155个氨基酸的多肽，其对应于如SEQ ID NO:1所示的野生型人PD-1的第1-155位氨基酸。然而，需要理解的是，这并不意味着本发明的PD-1变体只能或必需具有155个氨基酸的长度，也不意味着其不能含有跨膜区、胞内区。例如，在希望利用本发明的PD-1变体的结合特性，并利用它来制备融合蛋白特别是跨膜蛋白时，仍然可以包括更多的对应于PD-1多肽的氨基酸，例如将野生型PD-1的跨膜区的氨基酸纳入作为融合蛋白的跨膜区。
包含PD-1变体的融合蛋白
由于具有独特的结合特性，本发明的PD-1变体，特别是PD-1胞外区变体具有广泛的应用前景。例如，可以利用所述PD-1变体的结合特性，将其作为融合蛋白的一部分，如胞外结合结构域，用于靶向PD-L1和/或PD-L2，如靶向表达PD-L1和/或PD-L2的细胞。
在一个实施方案中，可以将PD-1变体构建成抗体样分子。例如，可以将所述PD-1变体直接与免疫球蛋白的Fc区构建成融合蛋白，形成类似于抗PD-L1抗体的特异性结合PD-L1的功能性多肽。所述Fc区优选哺乳动物例如人的免疫球蛋白的Fc区，例如人IgG1的Fc区。
在另一个实施方案中，包含所述PD-1变体的融合蛋白是嵌合刺激分子，其也可成称作增强受体、转换分子(switch molecule)或转换受体。所述嵌合刺激分子包含胞外结构域、跨膜结构域和胞内结构域，其中所述胞外结构域为本发明的PD-1变体，特别是PD-1胞外区变体，其能够结合PD-L1和/或PD-L2，并且胞内结构域是介导免疫细胞如T细胞激活的共刺激分子的胞内结构域。所述共刺激分子可以选自白细胞介素2受体(IL-2R)、白细胞介素12受体(IL-12R)、CD2、CD3、CD4、CD7、CD8、CD27、CD28、CD30、CD40、4-1BB/CD137、ICOS、淋巴细胞功能相关抗原1(LFA-1)、LIGHT、NKG2C或OX40。在优选的实施方案中，所述共刺激分子选自OX40、4-1BB、CD28、ICOS、CD27。在特别优选的实施方案中，所述共刺激分子为CD28。
所述融合蛋白的跨膜结构域可以来自野生型PD-1或其变体，例如可以是野生型PD-1或其变体的跨膜结构域本身，或者是所述跨膜结构域的片段。在一个实施方案中，所述跨膜结构域来自野生型PD-1，例如包含如SEQ ID NO:1所示氨基酸序列的第171-191位氨基酸或其片段，或由SEQ ID NO:1所示氨基酸序列的第171-191位氨基酸或其片段组成。在一个实施方案中，所述跨膜结构域包含与如SEQ ID NO:1所示氨基酸序列的第171-191位氨基酸具有至少70％同源性，至少80％同源性，或90％同源性的序列。
所述融合蛋白的跨膜结构域也可以来自其他来源例如来自共刺激分子的跨膜结构域。可以修饰免疫细胞以使其表达所述嵌合刺激分子。包含所述嵌合刺激分子的免疫细胞在与PD-L1的结合时，在所述免疫细胞中会产生免疫细胞激活信号，而非通常由PD1/PD-L1结合带来的免疫细胞失活信号。如此一来，将免疫抑制信号转换为激活信号，从而激活免疫细胞的功能。
制备方法
本发明的PD-1变体可以通过分子生物学方法制备。
例如，可以通过重组技术来制备本发明的PD-1变体或融合多肽。可以在体外合成编码PD-1变体或融合多肽的核苷酸片段，例如DNA或RNA， 然后将其构建到合适的表达载体中，并在合适的宿主细胞中进行表达。例如，可以通过病毒载体如慢病毒载体将需要表达的序列递送到宿主细胞中。通过收获宿主细胞表达的蛋白并任选地进行纯化，由此获得希望的多肽。
纯化多肽的方法是本领域已知的，例如可以通过层析技术，例如柱层析、HPLC、亲和层析、大小排阻层析等。也可以通过磁珠对蛋白进行纯化。
例如，可以通过无细胞的多肽合成体系来制备PD-1变体或融合多肽。可用于进行无细胞多肽合成的设备是本领域已知的。
用途
如上所述，本发明的PD-1变体由于其独特的结合特性，具有多种潜在的用途。
本发明的PD-1变体保留了与配体PD-L1、PD-L2的结合能力，因此可以用于在多种应用中替代野生型人PD-1或其胞外结构域的功能，特别是那些利用PD-1与PD-L1、PD-L2的结合功能的应用。由于本发明的PD-1变体在与配体的结合能力上有所改进，甚至会产生比野生型PD-1更好的效果。
在一个实施方案中，包含本发明的PD-1变体的嵌合刺激分子在表达于免疫细胞时，可以起到将抑制性PD-1/PD-L1信号转变为刺激性信号的作用，由此增强免疫细胞如T细胞的功能。因此，其可以在使用免疫细胞的细胞治疗中用于修饰用作治疗的免疫细胞。这样的治疗性免疫细胞可以是受试者自体的免疫细胞，也可以来自与受试者半相合的供者。所述免疫细胞可以来自受试者的外周血，例如通过分选外周血单个核细胞获得。所述免疫细胞可以来自肿瘤组织，例如可以是肿瘤浸润淋巴细胞(TIL)。另外，表达所述嵌合刺激分子的T细胞在与表达PD-L1的肿瘤细胞接触时，会增加T细胞的白介素-2(IL-2)分泌，白介素-2反过来可以与T细胞上的受体结合，进一步刺激T细胞。
在一个实施方案中，包含本发明的PD-1变体的抗体类似物具有与PD-1单抗类似的作用。例如，本发明的PD-1变体与Fc形成的融合蛋白可以阻断PD-1/PD-L1信号通路，从而抑制IL-2的释放。因此，PD-1突变体的Fc融合蛋白可以起到与PD-1或PD-L1单抗相同的解除“免疫刹车”的作用。
此外，本发明的PD-1变体不与多种市售PD-1单抗结合，这使其可以和这些PD-1单抗联用而不影响各自的效力，为联用提供了可能。这是使用 野生型人PD-1或其胞外区时无法实现的。同时，本发明的PD-1变体也保留了与特定PD-1单抗结合的能力，使其能够利用该单抗进行纯化等操作。
基于上述用途，本发明的PD-1变体或包含该变体的融合蛋白特别适合于治疗癌症。“癌症”在本发明的上下文中包括发生在不同器官、组织和细胞类型中的恶性肿瘤，包括实体肿瘤和血液癌症。
本发明至少还包括如下各项：
1.一种PD-1变体，其特征在于：
(1)所述PD-1变体能够与如SEQ ID NO:28所示的人PD-L1多肽结合；并且
(2)所述PD-1变体不与一种或多种选自下组的抗PD-1抗体结合：信迪利单抗(Sintilimab)、纳武单抗(Nivolumab)、卡瑞利珠单抗(Camrelizumab)、帕博利珠单抗(Pemrolizumab)和特瑞普利单抗(Toripalimab)。
2.根据项1所述的PD-1变体，其中与SEQ ID NO:1所示的野生型人PD-1多肽相比，所述PD-1变体在如下氨基酸位置中的一个或多个处具有突变：E84、D85、R86、S87、Q88、P89、G90、A129、P130、K131、A132和Q133，所述氨基酸位置以SEQ ID NO:1所示的氨基酸序列进行编号。
3.根据项1或2所述的PD-1变体，其中与SEQ ID NO:1所示的野生型人PD-1多肽相比，所述PD-1变体具有一种或多种选自下组的氨基酸突变：E84W、E84F；D85L、D85M；R86F；S87N、S87C、S87R、S87F、S87I、S87L、S87Y；Q88L、Q88F、Q88N、Q88T、Q88C、Q88E；P89C、P89V、P89R、P89K；G90T、G90S、G90F、G90R、G90Y；A129S、A129G、A129Q、A129Y、A129H、A129W、A129V、A129T、A129I；P130T、P130F、P130E；K131P、K131R；A132V、A132G、A132F、A132I、A132M；Q133W、Q133N、Q133V。
4.根据项3所述的PD-1变体，其中与SEQ ID NO:1所示的野生型人PD-1多肽相比，所述PD-1变体具有一种或多种选自下组的氨基酸突变：E84W、E84F；D85L、D85M；R86F；S87N、S87C、S87R、S87F、S87I；Q88L、Q88F、Q88N、Q88T；P89C、P89V、P89R；G90T、G90S；A129S、A129G、A129Q、A129Y；P130T；K131P；A132V、A132G；Q133W。
5.根据项2至4中任一项所述的PD-1变体，其中与SEQ ID NO:1所 示的野生型人PD-1多肽相比，所述PD-1变体在选自E84、D85、R86、S87、Q88、P89、G90的一个或多个氨基酸位置处具有突变，并且在选自A129、P130、K131、A132和Q133的一个或多个氨基酸位置处具有突变。
6.根据项1至5中任一项所述的PD-1变体，其中与SEQ ID NO:1所示的野生型人PD-1多肽相比，所述PD-1变体包含或仅包含选自如下(1)至(25)中任一组的氨基酸突变组合：
(1)A129S、P130T、K131P、A132V和Q133W；
(2)E84F、D85L、R86F、S87N、Q88L、A129S、P130T、K131P、A132V和Q133W；
(3)E84W、D85M、R86F、S87C、Q88F、A129S、P130T、K131P、A132V和Q133W；
(4)R86F、S87R、Q88N、P89C、G90T、A129S、P130T、K131P、A132V和Q133W；
(5)R86F、S87F、Q88T、P89V、G90S、A129S、P130T、K131P、A132V和Q133W；
(6)S87I、Q88N、P89R、G90S、A129S、P130T、K131P、A132V和Q133W；
(7)E84F、D85L、R86F、S87N、Q88L、P130T、K131P和Q133W；
(8)R86F、S87R、Q88N、P89C、G90T、A129G、P130T、K131P和Q133W；
(9)R86F、S87R、Q88N、P89C、G90T、P130T、K131P、A132G和Q133W；
(10)R86F、S87R、Q88N、P89C、G90T、A129Q、P130T、K131P和Q133W；或
(11)R86F、S87F、Q88T、P89V、G90S、A129Y、P130T、K131P和Q133W；
(12)A129H、P130F、K131R、A132F和Q133N；
(13)A129S、P130E、K131P、A132I和Q133V；
(14)S87L、Q88C、P89V、G90F、A129H、P130F、K131R、A132F和Q133N；
(15)R86S、S87F、Q88E、P89V、G90R、A129H、P130F、K131R、A132F和Q133N；
(16)R86L、S87Y、Q88L、P89K、G90Y、A129S、P130E、K131P、A132I 和Q133V；
(17)R86F、S87R、Q88N、P89C、G90T、A129W、P130T、K131P、A132M和Q133W；
(18)R86F、S87F、Q88T、P89V、G90S、A129H、P130T、K131P、A132F和Q133W；
(19)R86F、S87F、Q88T、P89V、G90S、A129G、P130T、K131P、A132F和Q133W；
(20)R86F、S87F、Q88T、P89V、G90S、A129I、P130T、K131P、A132G和Q133W；
(21)S87I、Q88N、P89R、G90S、A129T、P130T、K131P、A132F和Q133W；
(22)S87I、Q88N、P89R、G90S、A129V、P130T、K131P和Q133W；
(23)S87I、Q88N、P89R、G90S、P130T、K131P和Q133W；
(24)R86F、S87R、Q88N、P89C、G90T、P130T、K131P和Q133W；
(25)R86F、S87F、Q88T、P89V、G90S、P130T、K131P和Q133W。
7.根据项6所述的PD-1变体，其中与SEQ ID NO:1所示的野生型人PD-1多肽相比，所述PD-1变体包含或仅包含如(1)至(11)中任一组的氨基酸突变组合。
8.根据项7所述的PD-1变体，其中与SEQ ID NO:1所示的野生型人PD-1多肽相比，所述PD-1变体包含或仅包含如(1)至(6)中任一组的氨基酸突变组合。
9.根据项1至8中任一项所述的PD-1变体，其中所述PD-1变体不含胞内区，或不包含跨膜区和胞内区。
10.根据项1至9中任一项所述的PD-1变体，其中所述变体包含如SEQ ID NOs:2至26中任一所示的氨基酸序列或与之具有至少85％序列同一性的氨基酸序列，或由如SEQ ID NOs:2至26中任一所示的氨基酸序列或与之具有至少85％序列同一性的氨基酸序列组成。
11.根据项10所述的PD-1变体，其中所述变体包含如SEQ ID NOs:2至12中任一所示的氨基酸序列或与之具有至少85％序列同一性的氨基酸序列，或由如SEQ ID NOs:2至12中任一所示的氨基酸序列或与之具有至少85％序列同一性的氨基酸序列组成。
12.根据项11所述的PD-1变体，其中所述变体包含如SEQ ID NOs:2至7中任一所示的氨基酸序列或与之具有至少85％序列同一性的氨基酸序列，或由如SEQ ID NOs:2至7中任一所示的氨基酸序列或与之具有至少85％序列同一性的氨基酸序列组成。
13.根据项1至12中任一项所述的PD-1变体，其中所述PD-1变体在如下位置与SEQ ID NO:1所示的野生型人PD-1多肽具有相同的氨基酸：M70、S71、P72、S73、N74、Q75、T76、D77和K78。
14.根据项1至13中任一项所述的PD-1变体，所述PD-1变体具有如SEQ ID NO:1中第1至20位氨基酸所示的信号肽。
15.根据项1至14中任一项所述的PD-1变体，其中与野生型人PD-1多肽相比，所述PD-1变体以更高的亲和力与所述PD-L1多肽结合。
16.根据项1至15中任一项所述的PD-1变体，其中所述PD-1变体以低于10-7M的KD值与所述PD-L1多肽结合。
17.根据项1至16中任一项所述的PD-1变体，其中所述PD-1变体以低于10-6M的KD值与人PD-L2多肽结合。
18.根据项1至17中任一项所述的PD-1变体，其中与野生型人PD-1多肽相比，所述PD-1变体以更高的亲和力与所述PD-L2多肽结合。
19.根据项1至18中任一项所述的PD-1变体，其中所述PD-1变体与至少两种，优选至少三种，选自下组的抗PD-1抗体不结合：信迪利单抗(Sintilimab)、纳武单抗(Nivolumab)、卡瑞利珠单抗(Camrelizumab)、帕博利珠单抗(Pemrolizumab)和特瑞普利单抗(Toripalimab)。
20.根据项19所述的PD-1变体，其中所述PD-1变体与如下抗PD-1抗体不结合：信迪利单抗(Sintilimab)、纳武单抗(Nivolumab)和帕博利珠单抗(Pemrolizumab)。
21.根据项20所述的PD-1变体，其中所述PD-1变体与信迪利单抗(Sintilimab)、纳武单抗(Nivolumab)、卡瑞利珠单抗(Camrelizumab)、帕博利珠单抗(Pemrolizumab)和特瑞普利单抗(Toripalimab)中的每一个均不结合。
22.根据项1至21中任一项所述的PD-1变体，其中所述PD-1变体与替雷利珠单抗(Tilelizumab)结合。
23.根据项22所述的PD-1变体，其中所述PD-1变体与替雷利珠单抗(Tilelizumab)的结合亲和力以低于10-9M的KD值表示。
24.一种融合蛋白，其包含根据项1至23中任一项所述的PD-1变体。
25.根据项24所述的融合蛋白，其进一步包含免疫球蛋白Fc片段。
26.根据项25所述的融合蛋白，其中所述免疫球蛋白Fc片段来源于哺乳动物，优选人。
27.根据项24所述的融合蛋白，其包含所述PD-1变体作为胞外结构域，所述PD-1变体不包含胞内区，并进一步包含源自共刺激分子的胞内信号传递结构域。
28.根据项27所述的融合蛋白，所述共刺激分子选自下组：OX40、4-1BB、CD28、ICOS、CD27。
29.根据项27或28所述的融合蛋白，其进一步包含PD-1的跨膜区。
30.一种分离的核酸分子，其编码如项1至23中任一项所述的PD-1变体。
31.根据项30所述的核酸分子，其包含如SEQ ID NOs:56-61中任一项所示的核苷酸序列，或与如SEQ ID NOs:56-61中任一项所示的核苷酸序列具有至少85％同源性的核苷酸序列。
32.一种分离的核酸分子，其编码如项24至29中任一项所述的融合多肽。
33.一种表达载体，其包含如项30至32中任一项所述的分离的核酸分子。
34.一种宿主细胞，其包含如项30-32中任一项所述的分离的核酸分子，或如项33所述的表达载体。
35.一种药物组合物，其包含根据项1至23中任一项所述的PD-1变体，和药学上可接受的载体。
36.一种药物组合物，其包含根据项24至29中任一项所述的融合蛋白，和药学上可接受的载体。
37.根据项1至23中任一项的PD-1变体或根据项24至29中任一项的融合蛋白在制备用于治疗癌症的药物中的用途。
38.一种药物组合，其包含(1)根据项1至23中任一项的PD-1变体或根据项24至29中任一项的融合蛋白，和(2)抗PD-1抗体。
39.根据项38所述的药物组合，其中所述抗PD-1变体不与所述抗PD-1抗体结合。
40.根据项39所述的药物组合，其中所述抗PD-1抗体选自下组：信迪利单抗(Sintilimab)、纳武单抗(Nivolumab)、卡瑞利珠单抗(Camrelizumab)、帕博利珠单抗(Pemrolizumab)和特瑞普利单抗(Toripalimab)。
41.根据项38至40中任一项所述的药物组合，其用于治疗癌症。
42.一种细胞，其经修饰而表达如项24至29所述的融合蛋白。
43.项42所述的细胞，其为免疫细胞。
44.项43所述的细胞，其中所述免疫细胞在经修饰之前源自外周血单个核细胞或为肿瘤浸润淋巴细胞。
45.项44所述的细胞，所述免疫细胞是T细胞。
46.项42至45中任一项的细胞，其经修饰后用于治疗癌症。
实施例
为了更全面地理解和应用本发明，下文将参考实施例和附图详细描述本发明，所述实施例仅是意图举例说明本发明，而不是意图限制本发明的范围。本发明的范围由后附的项具体限定。
实施例1.PD-1突变体文库构建
本实施例描述PD-1突变体文库的构建过程。
概括而言，对PD-1(SEQ ID NO:1)中参与了PD-1和PD-L1，或PD-1和PD-L2的相互作用的位点进行随机突变，因为这些区域通常也是抗体结合表位所在的区域。由此构建了突变体质粒文库，并转入293T细胞中用于后续分析。
具体而言，对SEQ ID NO:1中的三个区域进行了随机突变，第一个区域为第70-78号氨基酸，第二个区域为第84-90号氨基酸，第三个区域为第127-133号氨基酸。为了实现随机突变，设计了六组随机突变引物，每对引物能够随机突变属于上述区域的5个连续氨基酸的相应密码子。具体来说，6组引物分别包含用于随机突变第70-74号氨基酸(7074-For/Rev)、第74-78号氨基酸(7478-For/Rev)、第84-88号氨基酸(8488-For/Rev)、第86-90号氨基酸(8690-For/Rev)、第127-131号氨基酸(12731-For/Rev)、第129-133号氨基酸的密码子(12933-For/Rev)。在用于随机突变的六组引物中，在上游引物中包含对应于这些氨基酸的密码子，并且将这些密码子设计为5个连续的NNK 密码子，其中N代表A、T、C、G中任意一种碱基，K代表G或T中的任意一种碱基。具体的六组引物序列(SEQ ID NOs:27-40)如下表1所示。还设计了另外两条引物P1-For和P1-Rev(SEQ ID NOs:35和36)，其序列也列于表1。P1-For和P1-Rev与其它六对突变引物配合用于扩增非突变区和突变区，并通过重叠PCR扩增完整PD-1的DNA序列使用。
表1.用于随机突变的引物设计
使用表1中的7组引物对，以PrimeStar DNA聚合酶(Takara，R045B)进行聚合酶链式反应，分别扩增包含PD-1突变区域和不包含PD-1突变区域的片段。以突变第70-74号氨基酸为例，使用的引物分别为7074-For+P1-Rev和P1-For+7074-Rev。扩增条件为：98℃10秒、58℃5秒、72℃5秒，共30个循环。
接下来进行重叠PCR。以突变第70-74号氨基酸为例，以上述两个PCR扩增的产物的等摩尔比混合物为模板，使用P1-For和P1-Rev引物扩增包含第70-74号氨基酸随机突变的PD-1全长序列。扩增条件为：98℃10秒、58℃5秒、72℃5秒，共30个循环。使用Sal I和Xba I限制性内切酶分别消化所述全长序列的PCR产物和慢病毒载体pLV2-PD1-CD28(质粒图如图1所示；质粒的完整序列如SEQ ID NO:63所示，共8417bp，其中1368-1877为PD1胞外区，1878-1940为PD1跨膜区，1941-2063为CD28胞内区)。使用T4 DNA 连接酶将经过消化的包含随机突变的全长序列和载体连接，由此制备PD-1随机突变质粒文库。
使用各个连接产物转化大肠杆菌Trans1-T1菌株(北京全式金生物，货号CD501-03)。具体而言，将连接产物添加至冰上解冻的大肠杆菌Trans1-T1化学转化感受态细胞中，并在冰上放置30分钟。然后于42℃孵育45秒，之后在冰上放置2分钟，并加入LB培养基，在37℃摇床上培养45分钟。培养之后，将菌液涂布于含有氨苄青霉素的LB琼脂平板，并在37℃培养过夜。培养之后，计算文库克隆总数并提取文库质粒。
实施例2.使用PD-1随机突变文库质粒瞬时转染哺乳动物293T细胞
本实施例描述用实施例1中构建的PD-1随机突变文库质粒来转染293T细胞，以便构建表达PD-1突变体的293T细胞系。
将293T细胞按照如下方法培养。将低于20代次的人胚肾293T细胞系细胞从液氮中取出，并迅速置于37℃水浴锅中至完全融化。将融化后的细胞加入到10ml D10完全培养基(含10％胎牛血清的DMEM)培养基中。在混匀之后，以400g离心5min并弃去培养基。以适量培养基重悬，并加入至T25或T75细胞培养瓶进行培养。细胞起始密度约为2x105/ml。将细胞置于37℃，在5％二氧化碳的培养箱中培养72h，之后吸弃培养基。以磷酸盐缓冲液清洗贴壁细胞，并吸弃缓冲液。以0.5％胰酶-EDTA溶液静置消化细胞3-5min，至大部分细胞从培养瓶底部脱落。加入2mL D10完全培养基并混合。以400g离心5分钟，吸弃培养基并以1mL D10完全培养基重悬。取适量细胞进行流式反应或传代培养。
将构建的包含PD-1随机突变的变体质粒文库通过PEI(Poly(ethylenimine)，聚乙烯亚胺)转染法转入293T细胞中。将各文库质粒与PEI以质量比1:3混合，瞬时转染293T细胞。由此获得了包含随机突变的PD-1变体文库质粒的293T细胞。
实施例3.流式细胞术筛选与PD-L1结合但不与抗PD-1抗体结合的细胞
本实施例描述了通过流式细胞术分选具有期望的结合能力的细胞群体的过程。
瞬时转染48小时后，以胰酶-EDTA溶液消化细胞。以400g离心5分钟，吸弃培养基并以磷酸盐缓冲液重悬。取一部分细胞悬液与PD-L1-Fc(R&D Systems,156-B7-100)、Dylight650标记的山羊抗人IgG Fc二抗(Abcam,ab97006)在4℃孵育30分钟。再取一部分细胞悬液与生物素标记的抗PD-1抗体(包括信迪利单抗、纳武单抗、替雷利珠单抗、卡瑞利珠单抗、帕博利珠单抗、特瑞普利单抗中的一种)、PE标记的链霉亲和素(BD Bioscience，554061)孵育。
以流式细胞分选仪分析并收集与PD-L1结合且与抗PD-1抗体不结合的细胞群体。首先使用帕博利珠单抗(pemrolizumab)通过流式细胞术进行了分选，参见图2。具体而言，分选出如图2流式细胞图中红色方框区域的细胞。再将这些细胞分别与其它五种抗体进行结合实验，选择与4种或5种抗体不结合的细胞群体。作为初步筛选的结果，总共收集了5000个细胞。
实施例4.分离和鉴定通过分选获得的细胞中的质粒
本实施例描述了对实施例3中获得的细胞中所含PD-1变体质粒的分离和鉴定过程。
测序
取实施例3中通过流式细胞术分选并收集的5000个细胞，以质粒小量提取试剂盒(全式金生物，EM101-02)进行质粒提取。
按照与实施例1中相同的方法，用提取的质粒转化大肠杆菌Trans1-T1菌株。培养之后，挑取单克隆大肠杆菌，并以EF-1α寡核苷酸引物进行测序，以获得克隆中所含PD-1变体的核苷酸序列，并基于此得出对应的氨基酸序列。在使用的5000个细胞中，获得并测序了约300个克隆。在测序的克隆中，共有约60个克隆(约占20％)测序结果为无关序列，约30个克隆(约占10％)中所含PD-1突变成功但是包含终止密码子，突变成功且能有效表达的克隆约为210个(约占70％)。
在表2中示出了筛选出的PD-1变体相对于野生型PD-1的突变氨基酸位置和氨基酸类型(未示出所有测序克隆的序列)。在表2中，SEQ ID NO:1为野生型PD-1，而SEQ ID NOs:2-26为获得的代表性PD-1变体的序列。

从表2中可知，本发明的变体的突变位点集中在E84、D85、R86、S87、Q88、P89、G90、A129、P130、K131、A132和Q133这12个位点上。与最初设计时(参见实施例1)进行随机突变的三个区域进行比对，可以发现这些突变都集中在后两个区域，即从E84至G90的第二个区域，和从S127至Q133的第三个区域(更具体为A129至Q133)。在各个代表性变体中，无一在M70至K78的区域内包含氨基酸突变，也没有在S127和L128发生突变。
基于293T细胞的流式细胞分析验证各种突变体与PD-L1、PD-L2及各 种抗体的结合能力
基于测序结果，选择如上所述在预期引入突变的三个区域内的相应位点突变成功且未突变为终止密码子的克隆。提取这些克隆的质粒，并分别用于瞬时转染293T细胞。转染方法如实施例2中所述。
转染48小时后消化细胞，并取部分细胞与人IgG1来源的Fc融合表达的PD-L1(R&D Biosystems)和Dylight650标记的山羊抗人IgG Fc二抗(Abcam)在4℃孵育30分钟。然后在400g离心5分钟，并以PBS磷酸盐缓冲液重悬、洗涤细胞。重复离心和洗涤一次。同时取部分细胞与人IgG1来源的Fc融合表达的PD-L2(R&D Biosystems,PD2-H5251)和Dylight650标记的山羊抗人IgG Fc二抗(Abcam,ab97006)孵育，然后以磷酸盐缓冲液洗涤细胞两次。同时，还取部分细胞与生物素标记的各个抗PD-1抗体和PE标记的链霉亲和素孵育。
以流式细胞术分析各个变体以及与野生型PD-1与PD-L1、PD-L2和抗PD-1抗体的结合情况，并将结果示于表2(原始数据图未显示)。表2中的“+”代表可以结合，“-”代表不能结合(即低于检出值)。使用的野生型PD-1是长度与变体PD-1相同且氨基酸位置对应的野生型PD-1的片段，其序列对应于如SEQ ID NO:1所示的野生型人PD-1氨基酸序列的第1至155位。
在210个PD-1的目标序列成功突变且不包含终止密码子的克隆中，无法与PD-L1结合的约为60个(约占20％)，能够结合PD-L1但结合4-6种测试抗体的约为120个(约占40％)，而同时满足能够结合PD-L1且仅和1-3种测试抗体结合的克隆仅有约30个(约占10％)。最终确定了具有如表2所示的25种序列的变体。
如表2所示，基于流式细胞术分析，野生型的人PD-1能够与PD-L1和PD-L2结合，并且也能够与全部6种检测的抗PD-1抗体结合。与之相比，本发明的25种代表性PD-1变体均能与PD-L1结合；大多数变体同时也能够结合PD-L2(PD1_49#_93和56#-2AA除外)。全部变体均可与Tilelizumab结合，且至少与三种以上的抗PD1单抗不结合。值得注意的是，仅第三个区域中包含突变的变体(PD1_56#，PD1_49#和PD1_110#)保留了对三种抗体(包括Tilelizumab)的结合能力，而其它变体在第二和第三这两个区域中都包含突变并且仅保留了对一种抗体(即Tilelizumab)的结合能力。
基于人T细胞的流式细胞分析验证各种突变体与PD-L1、PD-L2及各种 抗体的结合能力
虽然上文在293T表达了各种突变体并测试了各种突变体与PD-L1、PD-L2及各种抗体的结合能力，鉴于各种突变体主要通过T细胞实现其效应功能，还将各种突变体通过慢病毒表达于人T细胞，用流式细胞术对筛选出的变体与PD-L1、PD-L2及各种抗体的结合能力进行了验证，并将结果示于表3(原始数据图未显示)。
人T细胞分离、病毒感染与培养过程如下进行。取1×108个液氮冻存的外周血单个核细胞(PBMC)。在37℃水浴中快速解冻之后，加入到5倍体积的、预热的T细胞培养基(X-VIVO15+0.5％HSA+300U/ml IL-2)中，并混匀。然后以400g，5min离心，并弃去上清。以20ml所述T细胞培养基重悬细胞，并置于T175培养瓶中在37℃，5％二氧化碳的培养箱中培养。
1天后，将3倍细胞数量的CD3/CD28磁珠(Thermo)加入到细胞悬液中，并继续培养。
1天后，向T细胞培养物中加入MOI＝3数量的慢病毒，并继续培养。慢病毒的制备过程如下。在管1中加0.75mL的DMEM培养基(Biological Industries,06-1055-57-1A)和质粒溶液(VSVG 2.5μg+dR8.91 7.5μg+PLV2-PD1-CD28 10μg，总量为20μg)。在管2中加0.75mL的DMEM培养基和聚乙烯亚胺(PEI)溶液60μl。将管1与管2溶液混合后静置30分钟，之后加入到生长293T细胞的T75培养瓶中。24小时后收集病毒上清，以100000g离心2小时，弃上清。以500ul X-VIVO15培养基重悬病毒，用于感染T细胞。
5天后，将细胞与磁珠复合物吹打均匀，尽量使磁珠与细胞分离，然后将离心管置于磁力架上进行吸附。1分钟后，将细胞悬液转移至新的离心管，并重复上述吹打和吸附步骤3次。细胞以T细胞培养基重悬后继续培养。
取培养7-14天的T细胞，与人IgG1来源的Fc融合表达的PD-L1(R&D Biosystems)、Dylight650标记的山羊抗人IgG Fc二抗(Abcam)孵育。同时取部分细胞与人IgG1来源的Fc融合表达的PD-L2(R&D Biosystems,PD2-H5251-100ug)和Dylight650标记的山羊抗人IgG Fc二抗(Abcam,ab97006)孵育，以磷酸盐缓冲液洗涤细胞两次。同时，还取部分细胞与生物素标记的抗PD-1抗体、PE标记的链霉亲和素孵育。
以流式细胞术分析与PD-L1和抗PD-1抗体的结合情况，并将结果示于表3。表3中的“+”代表可以结合，“-”代表不能结合(即低于检出值)。发现在结合特性上的结果与在293T细胞中获得的完全一致。
实施例5.制备PD-1变体-兔IgGFc融合蛋白
将部分PD-1变体(SEQ ID NOs:2-7的PD-1变体)与兔IgG Fc进行融合表达和纯化，并以野生型PD-1(SEQ ID NO:1)作为对照。具体步骤如下。
使用PCR扩增对应于表2中所示的野生型(SEQ ID NO:1)和6种变体(氨基酸序列为SEQ ID NOs:2-7的变体)的核苷酸序列(序列如SEQ ID NOs:55-61所示)。引物为如下所示的P32-For和P32-Rev(SEQ ID NO:43和44)，使用实施例1中构建的含有PD-1野生型和变体的pLV2-PD1-CD28载体作为模板。扩增试剂、体系、扩增条件同实施例1中的PCR条件。
P32-For:TCAGTAGCTAGCGGTACCGCCGCCACCatgcagatcccacagg
P32-Rev:GTTGAGGATCCgtgggctgtgggcacttctg
以Nhe I和BamH I限制性内切酶分别消化PCR产物和蛋白表达载体pcDNA3.4-RFc(包含兔IgGFc(RFc)基因的pcDNA3.4载体(Thermo))中。使用T4DNA连接酶将经过消化的PD-1编码序列和载体连接，由此制备pcDNA3.4-PD1-RFc表达载体，用于转染细胞。
在转染之前，将HEK293细胞(珠海凯瑞)在37℃、5％CO2、120rpm条件下培养至1×106个细胞/ml的密度。将构建的pcDNA3.4-PD1-RFc载体使用5mL/L的转染试剂TA-293(珠海凯瑞)转染到所述HEK293细胞中，转染质粒浓度为1.5mg/L。转染后在37℃、8％CO2、120rpm条件下培养7天。培 养结束后在4000rpm离心培养物，收集上清液。用Protein A珠纯化蛋白，用500uL pH2.6-3.0的0.1M Gly-HCL洗脱液洗脱，并收集洗脱液以获得纯化的融合蛋白。
对纯化的融合蛋白在还原和非还原条件下进行了SDS-PAGE。蛋白质电泳图如图3所示。在图3中，泳道1-7分别为包含野生型PD-1和PD-1变体1-6的融合蛋白，泳道8为蛋白分子量标准。图3A为非还原条件下电泳，图3B为还原条件下电泳。融合蛋白的预期分子量约为43KDa。在还原条件下，条带大小与预期较为接近。在非还原条件下，融合蛋白形成了二聚体，并有糖基化等修饰，因此分子量高于86KDa。在非还原条件下，第3、4、6泳道有明显的多条带，且条带分子量较高，这可能表明分子量更高的条带有不同形式的聚合，如多聚体的产生，或者存在不同程度、不同类型的蛋白质修饰。
实施例6.通过BiacoreT200测定亲和力
通过Biacore T200测定了野生型PD-1以及本发明的代表性PD-1变体(SEQ ID NOs:2-7的变体)与PD-L1、PD-L2以及实施例3中所述6种PD-1单抗的亲和力。
亲和力统计结果见表4A-B所示。在表4A中以“不结合”表示测定结果低于Biacore T200仪器检测下限，即表示亲和力的KD值高于10-3M。
表4A.PD-1突变体与PD-L1、PD-L2和6种PD-1单抗的亲和力测定
表4B.表4A的原始数据

从表4的结果可知，Biacore实验结果验证了前述实施例中通过流式细胞术确定的各种结合特性，并提供了具体的结合亲和力数值。全部6种变体与PD-L1和PD-L2以及Tilelizumab均具有较高的结合亲和力，KD值均小于10-6。全部6种变体均具有至少比野生型PD-1低两个数量级的KD值，意味着显著更高的与PD-L1的结合亲和力。变体2(SEQ ID NO:3)、变体5(SEQ ID NO:6)和变体6(SEQ ID NO:7)还具有比野生型PD-1高的PD-L2结合亲和力。变体1、变体3和变体4与PD-L2的结合亲和力则低于野生型PD-1。在与抗PD-1抗体Tilelizumab的结合亲和力方面，变体1(SEQ ID NO:2)和变体6(SEQ ID NO:7)与野生型PD-1相当。
实施例7.108-CD28 Fc阻断细胞因子分泌的功能实验
将PD1-CD28表达T细胞(PD1-CD28-T)与PD-L1阳性肿瘤细胞(J82-PD-L1)共培养时，T细胞会释放IL-2。在加入抗PD-1抗体时，抗PD-1 抗体会结合PD-1，从而阻断T细胞通过PD-1与肿瘤细胞结合，进而抑制IL-2的释放。同样，加入PD-1突变体Fc融合蛋白时，其与靶细胞上的PD-L1结合，使PD-L1不能和T细胞膜上的PD-1结合，也具有和抗-PD-1抗体类似的阻断IL-2释放的效果。
PD1-CD28-T细胞制备过程
将实施例1中所述的pLV2-PD1-CD28质粒(PD-1为人野生型PD-1)如实施例4所述制备成慢病毒载体并用于感染人T细胞，以获得PD1-CD28-T细胞。T细胞培养方法如实施例4所述。
J82-PDL1细胞制备过程
将pLV2-PDL1质粒(PD-L1为人野生型PD-L1)如实施例4所述制备成慢病毒载体并用于感染人膀胱癌细胞系(购自中国医学科学院，货号1101HUM-PUMC000346)，以获得J82-PDL1细胞系。
PD-1变体Fc融合蛋白阻断IL-2分泌实验
1.用0.05％胰酶消化、清洗并重悬J82-PDL1细胞，方法如实施例2中所述，密度调整为1×105/mL。取PD1-CD28-T细胞，清洗、重悬，如实施例4所述，调整密度为1×106/mL，添加6ng/mL的PMA(佛波醇12-十四酸酯13-乙酸酯，Sigma，货号P8139-1MG)。
2.向96孔板中分别加入100ul J82-PDL1细胞和100ul PD1-CD28-T细胞，并分别加入终浓度为0.8、1.6、3.2、6.4、12.8、25.6、51.2nM的Anti-IgG4-RFc(阴性对照，不与PD-1或PD-L1结合)、替雷利珠单抗(阳性对照)、实施例5中制备的2种PD1-RFc(变体56#-16、56#-108)融合蛋白，37℃培养16小时。
3.取96孔板，500g×3min离心，取50μL上清用IL-2ELISA试剂盒(达科为，货号1110203)检测IL-2浓度。
表5为不同浓度抗体或融合蛋白加入后，T细胞分泌IL-2浓度的测定值，图5为表5数据的趋势图。表5中的数据说明，替雷利珠单抗在浓度低于6.4nM情况下，IL-2分泌无明显变化，当浓度≥6.4nM时，IL-2分泌被显著抑制；56#-16融合蛋白随着浓度提高，IL-2分泌呈下降趋势，但IL-2分泌 没有被完全抑制；56#-108融合蛋白随着浓度提高，IL-2分泌呈下降趋势，趋势较56#-16融合蛋白组明显，不如替雷利珠单抗组明显，但在浓度≥25.6nM时，IL-2分泌抑制效果较替雷利珠单抗组强。该结果说明PD-1突变体融合蛋白具有与PD-1单抗类似的阻断PD-1/PD-L1信号传递的作用。
表5
实施例8.细胞因子分泌实验
本实施例通过IL-2分泌实验测试了基于本发明PD1变体的增强受体对T细胞功能的作用。
将表达PD1-CD28的T细胞(PD1-CD28-T)与PD-L1阳性肿瘤细胞共培养时，T细胞会通过PD-1与PD-L1的相互作用而释放细胞因子IL-2。因此，通过酶联免疫吸附法(ELISA)检测共培养的培养液中IL-2的含量，可以判定PD1-CD28促进IL-2分泌的效果。
如图5所示，在96孔板中，将过表达人PD-L1的Malme-3M肿瘤细胞(3M-PDL1-OE)和敲除了人PD-L1的Malme-3M肿瘤细胞(3M-PDL1-KO)作为靶细胞，并分别以表达PD1变体(56#-108突变体)和CD28构成的增强受体的T细胞(图5中显示为108T，也可称为108-CD28-T)和无基因修饰的T细胞(T)作为效应细胞，按照靶细胞:108T＝10:1的比例，即靶细胞1×105/孔， 108T细胞1×104/孔，在添加3ng/ml PMA(佛波酯)的培养基中共培养。将过夜培养的上清作为待检测样品，与检测标准品一起进行IL-2ELISA分析。
使用人IL-2检测试剂盒(ELISA法)(达科为，货号1110203)，按照如下流程进行ELISA。预先用抗人IL-2捕获抗体包被微孔板。将标准品或待检样品和检测抗体(生物素标记的抗人IL-2抗体)同时加入微孔板，然后加入辣根过氧化物酶标记的链霉亲和素(SA-HRP)，其特异性结合免疫复合物中的生物素。加入酶底物四甲基联苯胺(TMB)，出现蓝色，颜色的深浅与标准品或样本中IL-2浓度相关，显色5-10分钟后加入终止液终止反应，通过多功能酶标仪读取450nm处光吸收值(OD)。在一定范围内人IL-2浓度与OD值成正比，使用IL-2标准品进行ELISA检测并得到了标准曲线。使用该标准曲线计算出了每种共培养物中的IL-2浓度。
如图5所示，在靶细胞的PD-L1被敲除的情况下，无论是对照T细胞还是武装有PD-1变体增强受体的T细胞(108T，即108-CD28-T)，因没有第二信号刺激源，均没有IL-2分泌(红色柱图)；而与高表达PD-L1靶细胞共培养时，108T增强受体武装的T细胞在受到刺激时释放了大量IL-2，表明PD1变体108与CD28的融合蛋白(108-CD28)具有传递刺激T细胞的信号的功能。
实施例9.肿瘤细胞杀伤实验
本实施例涉及细胞杀伤实验。通过使T细胞表达包含本发明的PD-1变体的PD1-C28增强受体，证实了该PD-1变体增强受体可增强T细胞PD-L1表达肿瘤细胞的杀伤能力。
肿瘤细胞表面会表达HLA-肽复合抗原，而T细胞会表达靶向该抗原的T细胞受体(TCR)。将这样的肿瘤细胞和与之对应的T细胞进行共同孵育，T细胞上的TCR与肿瘤细胞上的复合抗原的接触就会介导一系列T细胞的杀伤反应，将肿瘤细胞裂解。
在本实验中，肿瘤细胞为表达Eso1抗原和PD-L1的J82细胞(J82Eso1PDL1)。为了制备J82Eso1PDL1肿瘤细胞系，按照实施例4中的方法分别制备了表达人PD-L1和ESO1基因的慢病毒载体，分别命名为pLV2-PDL1和pLV2-ESO1。如实施例4中所述，使用所述pLV2-PDL1慢病毒感染人膀胱癌细胞系(购自中国医学科学院基础医学研究所，货号1101HUM-PUMC000346)，获得表达人PD-L1的J82-PDL1细胞。然后， 再用如上所述制备的pLV2-ESO1慢病毒感染所述J82-PDL1细胞。该细胞的ESO1抗原在胞内表达并被加工后，以ESO1peptide的形式递呈到胞外并与J82细胞表面表达的HLA*A0201蛋白形成抗原复合物，该复合物能被特异性的T细胞受体(T cell receptor,TCR)所识别。
制备了三种不同的T细胞，包括：(1)表达靶向HLA-Eso1肽的TCR的T细胞(TCR-T)；(2)表达PD1变体(56#-108突变体，即变体6)和CD28构成的增强受体以及靶向HLA-Eso1肽的TCR的T细胞(108-TCRT)；和(3)作为对照的表达GFP绿色荧光蛋白的T细胞(GFP-T)。
首先制备了PD1+T细胞。取冻存的健康捐献者的全血细胞于37℃快速水浴复苏，加入到5倍体积预热的T细胞培养基中，所述培养基为包含0.5％人血清白蛋白(欣吉尔，双鹭药业)的X-VIVO15(Lonza；BE02-053Q)。混合均匀后，以400g离心5min，去除上清。以PBS-0.5％HSA重悬，使密度为108总细胞/ml。加入生物素标记的抗PD1抗体Biotin-anti-PD1(Biolegend,货号329934)，在4℃孵育20分钟，以PBS-0.5％HSA清洗两次。加入抗生物素微珠(Anti-Biotin Microbeads)(美天旎，货号130-090-485)，混匀，并在4℃孵育20分钟。以PBS-0.5％HSA清洗两次。使用LS分选柱(美天旎，货号130-042-401)分选PD1+的T细胞。将获得的PD1+T细胞参照实施例4所述的方式培养。
为了制备TCR-T细胞，如实施例4中所述制备包含TCR编码序列的慢病毒载体pLV2-TCR。所述TCR为特异性识别HLA*A0201/ESO1peptide复合物的T细胞受体，编码序列如SEQ ID NO:41所示。然后按照实施例4所述的方法使用所述慢病毒感染上述PD1+T细胞，以获得TCR-T细胞。
为了制备表达增强受体的T细胞，按照实施例4所述的方法制备包含PD1变体增强受体PD1-CD28(简称108-CD28)和TCR编码序列的慢病毒载体。再使用该慢病毒感染上述PD1+T细胞，感染方式如实施例4所述，获得108-TCRT细胞。108-TCR的基因序列如SEQ ID NO:42所示。增强受体中的跨膜区为源自PD1的跨膜区。
杀伤实验利用实时无标记动态细胞分析技术(RTCA)进行，以检测靶细胞的存活。具体而言，先将J82Eso1PDL1靶细胞置于培养板的96孔板中培养(每孔100ul，靶细胞浓度8x104/ml)。在18小时的时间点，将TCR-T细胞或经增强受体修饰的TCR-T细胞(108-TCR-T)分别加入到对应的孔中，观察 并记录靶细胞的生长情况，结果示于图6。
在图6中，纵坐标的细胞指数反映的是细胞的贴壁情况，数值越高表明贴壁的细胞越多，也意味着活细胞多且生长情况良好。横坐标为时间，并以小时表示。
从图6中可见，表达增强受体的TCR-T细胞均比单独的TCR-T展现出对肿瘤细胞更强的抑制。与不带TCR的对照细胞GFP-T共培养的J82Eso1PDL1虽然有些非特异杀伤作用，但仍保持生长状态(图6蓝色曲线)；而和Eso1-TCR-T共培养的82Eso1PDL1，则可见生长明显受到抑制(绿色曲线)。而与表达本发明的增强受体的TCR-T(108-TCRT)共培养的靶细胞数量显著下降，说明靶细胞被杀灭(图6红色曲线)，表明表达增强受体的TCR-T的杀伤效果更强。
关键序列信息
SEQ ID NO:1(PD1-wt)
SEQ ID NO:2(Mut1/56#)
SEQ ID NO:3(Mut2/56#-2)
SEQ ID NO:4(Mut3/56#-3)
SEQ ID NO:5(Mut4/56#-15)
SEQ ID NO:6(Mut5/56#-16)

SEQ ID NO: 7 (Mut6/56#-108)
SEQ ID NO: 8 (Mut7/56#-2-129A132A)
SEQ ID NO: 9 (Mut8/56#-15-38)
SEQ ID NO: 10 (Mut9/56#-15-61)
SEQ ID NO: 11 (Mut10/56#-15-69)
SEQ ID NO: 12 (Mut11/56#-16-68)
SEQ ID NO: 13 PD1_49#
SEQ ID NO: 14 PD1_110#
SEQ ID NO: 15 PD1_49#_93
SEQ ID NO: 16 PD1_49#_260
SEQ ID NO: 17 PD1_110#_48
SEQ ID NO: 18 56#-15-7
SEQ ID NO: 19 56#-16-12
SEQ ID NO: 20 56#-16-13
SEQ ID NO: 21 56#-16-50
SEQ ID NO: 22 56#-108-51
SEQ ID NO: 23 56#-108-60
SEQ ID NO: 24 56#-108AA
SEQ ID NO: 25 56#-15AA
SEQ ID NO: 26 56#-16AA

Claims (20)

  1. 一种PD-1变体，其特征在于：
    (1)所述PD-1变体能够与如SEQ ID NO:28所示的人PD-L1多肽结合；并且
    (2)所述PD-1变体不与一种或多种选自下组的抗PD-1抗体结合：信迪利单抗(Sintilimab)、纳武单抗(Nivolumab)、卡瑞利珠单抗(Camrelizumab)、帕博利珠单抗(Pemrolizumab)和特瑞普利单抗(Toripalimab)。
  2. 根据权利要求1所述的PD-1变体，其中与SEQ ID NO:1所示的野生型人PD-1多肽相比，所述PD-1变体在如下氨基酸位置中的一个或多个处具有突变：E84、D85、R86、S87、Q88、P89、G90、A129、P130、K131、A132和Q133，所述氨基酸位置以SEQ ID NO:1所示的氨基酸序列进行编号。
  3. 根据权利要求1或2所述的PD-1变体，其中与SEQ ID NO:1所示的野生型人PD-1多肽相比，所述PD-1变体具有一种或多种选自下组的氨基酸突变：E84W、E84F；D85L、D85M；R86F；S87N、S87C、S87R、S87F、S87I、S87L、S87Y；Q88L、Q88F、Q88N、Q88T、Q88C、Q88E；P89C、P89V、P89R、P89K；G90T、G90S、G90F、G90R、G90Y；A129S、A129G、A129Q、A129Y、A129H、A129W、A129V、A129T、A129I；P130T、P130F、P130E；K131P、K131R；A132V、A132G、A132F、A132I、A132M；Q133W、Q133N、Q133V。
  4. 根据权利要求2或3所述的PD-1变体，其中与SEQ ID NO:1所示的野生型人PD-1多肽相比，所述PD-1变体在选自E84、D85、R86、S87、Q88、P89、G90的一个或多个氨基酸位置处具有突变，并且在选自A129、P130、K131、A132和Q133的一个或多个氨基酸位置处具有突变。
  5. 根据权利要求1至4中任一项所述的PD-1变体，其中与SEQ ID NO:1所示的野生型人PD-1多肽相比，所述PD-1变体包含或仅包含选自如下(1)至(25)中任一组的氨基酸突变组合：
    (1)A129S、P130T、K131P、A132V和Q133W；
    (2)E84F、D85L、R86F、S87N、Q88L、A129S、P130T、K131P、A132V和Q133W；
    (3)E84W、D85M、R86F、S87C、Q88F、A129S、P130T、K131P、A132V和Q133W；
    (4)R86F、S87R、Q88N、P89C、G90T、A129S、P130T、K131P、A132V和Q133W；
    (5)R86F、S87F、Q88T、P89V、G90S、A129S、P130T、K131P、A132V和Q133W；
    (6)S87I、Q88N、P89R、G90S、A129S、P130T、K131P、A132V和Q133W；
    (7)E84F、D85L、R86F、S87N、Q88L、P130T、K131P和Q133W；
    (8)R86F、S87R、Q88N、P89C、G90T、A129G、P130T、K131P和Q133W；
    (9)R86F、S87R、Q88N、P89C、G90T、P130T、K131P、A132G和Q133W；
    (10)R86F、S87R、Q88N、P89C、G90T、A129Q、P130T、K131P和Q133W；或
    (11)R86F、S87F、Q88T、P89V、G90S、A129Y、P130T、K131P和Q133W；
    (12)A129H、P130F、K131R、A132F和Q133N；
    (13)A129S、P130E、K131P、A132I和Q133V；
    (14)S87L、Q88C、P89V、G90F、A129H、P130F、K131R、A132F和Q133N；
    (15)R86S、S87F、Q88E、P89V、G90R、A129H、P130F、K131R、A132F和Q133N；
    (16)R86L、S87Y、Q88L、P89K、G90Y、A129S、P130E、K131P、A132I和Q133V；
    (17)R86F、S87R、Q88N、P89C、G90T、A129W、P130T、K131P、A132M和Q133W；
    (18)R86F、S87F、Q88T、P89V、G90S、A129H、P130T、K131P、A132F和Q133W；
    (19)R86F、S87F、Q88T、P89V、G90S、A129G、P130T、K131P、A132F和Q133W；
    (20)R86F、S87F、Q88T、P89V、G90S、A129I、P130T、K131P、A132G和Q133W；
    (21)S87I、Q88N、P89R、G90S、A129T、P130T、K131P、A132F和Q133W；
    (22)S87I、Q88N、P89R、G90S、A129V、P130T、K131P和Q133W；
    (23)S87I、Q88N、P89R、G90S、P130T、K131P和Q133W；
    (24)R86F、S87R、Q88N、P89C、G90T、P130T、K131P和Q133W；
    (25)R86F、S87F、Q88T、P89V、G90S、P130T、K131P和Q133W。
  6. 根据权利要求1至5中任一项所述的PD-1变体，其中所述PD-1变体不含胞内区，或不包含跨膜区和胞内区。
  7. 根据权利要求1至6中任一项所述的PD-1变体，其中所述变体包含如SEQ ID NOs:2至26中任一所示的氨基酸序列或与之具有至少85％序列同一性的氨基酸序列，或由如SEQ ID NOs:2至26中任一所示的氨基酸序列或与之具有至少85％序列同一性的氨基酸序列组成。
  8. 根据权利要求1至7中任一项所述的PD-1变体，其中所述PD-1变体与至少两种，优选至少三种，选自下组的抗PD-1抗体不结合：信迪利单抗(Sintilimab)、纳武单抗(Nivolumab)、卡瑞利珠单抗(Camrelizumab)、帕博利珠单抗(Pemrolizumab)和特瑞普利单抗(Toripalimab)。
  9. 一种融合蛋白，其包含根据权利要求1至8中任一项所述的PD-1变体。
  10. 根据权利要求9所述的融合蛋白，其进一步包含免疫球蛋白Fc片段，优选所述免疫球蛋白Fc片段来源于哺乳动物，优选人。
  11. 根据权利要求9所述的融合蛋白，其包含所述PD-1变体作为胞外结构域，所述PD-1变体不包含胞内区，并进一步包含源自共刺激分子的胞内信号传递结构域，并且所述融合蛋白任选地包含PD-1的跨膜区。
  12. 一种分离的核酸分子，其编码如权利要求1至8中任一项所述的PD-1变体或根据权利要求9至11中任一项所述的融合蛋白。
  13. 根据权利要求12所述的核酸分子，其包含如SEQ ID NOs:56-61中任一项所示的核苷酸序列，或与如SEQ ID NOs:56-61中任一项所示的核苷酸序列具有至少85％同源性的核苷酸序列。
  14. 一种表达载体，其包含如权利要求12或13所述的分离的核酸分子。
  15. 一种宿主细胞，其包含如权利要求12或13所述的分离的核酸分子，或如权利要求14所述的表达载体。
  16. 一种药物组合物，其包含(1)根据权利要求1至8中任一项所述的PD-1变体或根据权利要求9至11中任一项所述的融合蛋白，和(2)药学上可接受的载体。
  17. 根据权利要求1至8中任一项的PD-1变体或根据权利要求9至11中任一项的融合蛋白在制备用于治疗癌症的药物中的用途。
  18. 一种药物组合，其包含(1)根据权利要求1至8中任一项的PD-1变体或根据权利要求9至11中任一项的融合蛋白，和(2)抗PD-1抗体；其中所述抗PD-1变体不与所述抗PD-1抗体结合。
  19. 一种细胞，其经修饰而表达如权利要求9至11中任一项所述的融合蛋白。
  20. 权利要求9所述的细胞，其中所述细胞是免疫细胞，并且在经修饰之前源自外周血单个核细胞或为肿瘤浸润淋巴细胞。
PCT/CN2023/098627 2022-06-06 2023-06-06 Pd-1变体及其用途 WO2023236954A1 (zh)

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