[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2022089601A1 - 一种il-2与抗体亚单位构成的双功能融合蛋白 - Google Patents

一种il-2与抗体亚单位构成的双功能融合蛋白 Download PDF

Info

Publication number
WO2022089601A1
WO2022089601A1 PCT/CN2021/127523 CN2021127523W WO2022089601A1 WO 2022089601 A1 WO2022089601 A1 WO 2022089601A1 CN 2021127523 W CN2021127523 W CN 2021127523W WO 2022089601 A1 WO2022089601 A1 WO 2022089601A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
monomer
scfv
mutant
Prior art date
Application number
PCT/CN2021/127523
Other languages
English (en)
French (fr)
Inventor
傅阳心
彭华
曹帅帅
Original Assignee
中国科学院生物物理研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院生物物理研究所 filed Critical 中国科学院生物物理研究所
Publication of WO2022089601A1 publication Critical patent/WO2022089601A1/zh

Links

Images

Classifications

    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • 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
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39566—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against immunoglobulins, e.g. anti-idiotypic antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention belongs to the technical field of biomedicine, and specifically relates to a bifunctional fusion protein composed of IL-2 and antibody subunits.
  • Interleukin 2 also known as T cell growth factor
  • T cell growth factor Interleukin 2
  • IL2 is a four ⁇ -helix glycoprotein with a molecular weight of 15.5Kd[2]. It is mainly secreted by activated CD4 T cells. Other cells such as activated CD8 cells, NK cells, NKT cells and ILCs cells can also produce a small amount of IL2[2]. 3,4]. IL2 binds to receptor cells through autocrine and paracrine pathways to function, and plays an important role in regulating T and NK cells.
  • the receptor of IL2 consists of three subunits: IL2R ⁇ (CD25), IL2R ⁇ (CD122) and IL2R ⁇ c (CD132). There are differences in the affinity between IL2 and the three subunits [5,6]. IL2 binds with low affinity to ⁇ subunit (Kd ⁇ 10-8M), binds with moderate affinity to ⁇ dimer (Kd ⁇ 10-9M), and binds to ⁇ trimer with high affinity (Kd ⁇ 10-11M)[7 ,8].
  • ⁇ receptors are mainly expressed on resting T cells, CD8 T memory cells and NK cells; ⁇ receptors are up-regulated after cell activation, and activated T cells Cells express high-affinity ⁇ receptors. Treg cells continue to highly express CD25 molecules, and Treg cells have high affinity for IL2 molecules [9]. Due to differences in receptor expression, low doses of IL2 preferentially expand Treg cells expressing high-affinity receptors, and high doses of IL2 can effectively activate CD8 + T cells and NK cells [10].
  • IL2 has the highest affinity for the trimeric receptor, and the trimeric receptor is mainly continuously expressed on Treg cells and only temporarily up-regulated on activated effector cells; clinical use of low doses of IL2 will preferentially expand Treg cells, The use of high doses of IL2 to activate effector T cells. High doses of IL2 can lead to more toxic side effects and limit the use of IL2.
  • the molecular weight of IL2 is 15.5KD, and the half-life in serum in vivo is 10-85min. In order to achieve the therapeutic effect, repeated administration is required. PEGylated IL2 increases the overall molecular weight of IL2 and prolongs the half-life of IL2. At the same time, IL2 can be biased to bind to IL2-R ⁇ or IL2-R ⁇ through different PEGylation sites [11].
  • the combination of IL2 and aIL2 antibody complexes can prolong the half-life of IL2.
  • the IL2/aIL2 antibody complexes show different tendencies to bind to receptors CD25 or CD122. sex.
  • the affinity of IL2 to different receptor subunits can also be changed by point mutation engineering of different receptor binding sites on IL2.
  • point mutation engineering of different receptor binding sites on IL2.
  • L80F, R81D, L85V, I86V and I92F the conformation of IL2 is changed, the binding to CD122 is increased, and the downstream signal transduction is independent of CD25 molecule.
  • Mutated IL2 is biased towards the expansion of CD8+ T cells and NK cells, has lower toxic side effects and shows better tumor therapeutic effects in mouse tumor models [12].
  • Double-mutated IL2 to both decrease CD25 subunit binding (F42A) and increase CD122 subunit binding.
  • This double-mutated IL2 molecule has a better therapeutic effect than either the CD25 subunit alone or the CD122 subunit alone with increased affinity [13].
  • Double-mutated IL2 not only has a good therapeutic effect in the MC38 tumor model, but also has a good therapeutic effect in combination with TKI or 7.16.4 antibody in cold tumor models such as TUBO. It shows that while increasing the affinity of CD122 subunit, further reducing the affinity of CD25 subunit can further improve the therapeutic effect of IL2.
  • co-stimulatory or co-inhibitory molecules of the TNF family are also highly expressed on Treg cells and are target molecules that regulate the function of Treg cells.
  • Common molecules include ICOS, GITR, OX40, and 4-1BB.
  • Some co-stimulatory molecules such as GITR and OX40 are highly expressed on intratumoral Treg and also up-regulated on activated CD4 and CD8 T cells.
  • the OX40 molecule is expressed on T cells in the tumor, and it is highly expressed on the Treg in the tumor; systemic administration of activating aOX40 antibody can lead to a decrease in the number and proportion of Treg in the tumor and play an anti-tumor effect [14].
  • aOX40 antibodies no significant decrease in the number of Treg was observed, but an increase in the number of effector T cells was observed [15].
  • the OX40 molecule is co-expressed on effector T cells and Treg cells.
  • the currently used OX40 antibody can play an anti-tumor effect, which can both activate effector cells and delete Treg cells.
  • the anti-tumor effect of OX40 depends on the expansion of intratumoral CD4 T and CD8 T cells, and the tumor will not recur after the mice have immune memory re-challenge after treatment [16].
  • the OX40 antibody OX86 used in mouse experiments can also delete Tregs, and this deletion depends on activating Fc ⁇ R but not on inhibitory Fc ⁇ RIIB.
  • IL2/aOX40 bifunctional molecules as well as CTLA4 and other TNF family co-stimulatory or co-inhibitory molecules ICOS, GITR, 4-1BB, etc. antibodies and IL-2 cytokines constitute fusion protein bifunctional antibodies. It is a new tumor treatment solution with great potential for development.
  • the present invention first relates to a bifunctional fusion protein, the fusion protein is a heterodimer, and is characterized in that:
  • Described heterodimer includes:
  • the first monomer and the second monomer are connected by dimerization of the Fc single chain to form the heterodimer;
  • co-stimulatory or co-inhibitory molecules of CTLA4 or TNF family include but are not limited to: OX40, 4-1BB, ICOS, GITR.
  • the immunoglobulin Fc single chain is a natural immunoglobulin Fc single chain or an immunoglobulin Fc single chain with ADCC effect knocked out through gene mutation;
  • the immunoglobulin Fc single chain is a natural immunoglobulin Fc single chain or an immunoglobulin Fc single chain with ADCC effect knocked out through gene mutation; more preferably, the immunoglobulin Fc single chain is human Fc single chain of IgG.
  • the co-stimulatory or co-inhibitory molecules of the CTLA4 or TNF family are OX40, 4-1BB, and the antibody of the anti-CTLA4 antibody (aCTLA4) or the co-stimulatory or co-inhibitory molecule of the anti-TNF family is anti-OX40 Antibody (aOX40), anti-4-1BB antibody (a4-1BB);
  • the Fab of the antibody is the Fab of a humanized antibody or the Fab of a fully humanized antibody;
  • the ScFv of the antibody is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody;
  • the second monomer is:
  • the antibody monomer comprises one light chain and one heavy chain; preferably, the antibody is a humanized antibody or a fully human antibody.
  • the heterodimer includes:
  • the first monomer comprises sequentially from the N-terminus:
  • connection structure (G4S connection sequence), preferably, described connection sequence is as shown in SEQ ID NO.6;
  • the second monomer comprises:
  • the heterodimer includes:
  • the first monomer is a polypeptide (IL2-Fc) whose sequence is shown in SEQ ID NO.10;
  • Second monomer which is:
  • a second monomer composed of an anti-OX40 antibody VH-CH1-Fc (knob) whose sequence is shown in SEQ ID NO.11 and an anti-OX40 antibody light chain VL-KCL whose sequence is shown in SEQ ID NO.7;
  • the present invention also relates to a bifunctional fusion protein, the fusion protein is a homodimer, and is characterized in that:
  • the monomer of the homodimer is:
  • a molecule of interleukin 2 (IL-2) and a molecule of anti-OX40 antibody Fab are linked in any way to form a monomer, or,
  • IL-2 interleukin 2
  • ScFv single-chain antibody
  • the monomers of the homodimer include sequentially from the N-terminus:
  • connection structure preferably, described connection sequence is as shown in SEQ ID NO.6;
  • the Fab is the Fab of a humanized antibody or the Fab of a fully humanized antibody
  • the ScFv is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody
  • the Fc of the antibody is a fully human wild-type Fc or a No-ADCC mutant Fc.
  • the monomer of described homodimer is:
  • SEQ ID NO. 13 (aOX40-IL2:VL-VH(ScFv)-Fc(wt)-IL2)
  • the present invention also relates to another bifunctional fusion protein, the bifunctional fusion protein is a bifunctional fusion protein composed of an anti-CTLA4 antibody and IL2, and the fusion protein is a heterodimer;
  • Described heterodimer includes:
  • the first monomer comprises sequentially from the N-terminus:
  • connection structure (G4S connection sequence), preferably, described connection sequence is as shown in SEQ ID NO.6;
  • the second monomer comprises:
  • the heterodimer includes:
  • the first monomer is a polypeptide (IL2-Fc) whose sequence is shown in SEQ ID NO.10;
  • Second monomer which is:
  • a second monomer composed of a polypeptide (aCTLA4 VH-CH1-Fc(knob)) whose sequence is shown in SEQ ID NO.24 and an anti-CTLA4 antibody light chain whose sequence is shown in SEQ ID NO.21; or
  • the present invention also relates to a bifunctional fusion protein, the fusion protein is a homodimer, and is characterized in that:
  • the monomer of the homodimer is:
  • a molecule of interleukin 2 (IL-2) and a molecule of anti-CTLA4 antibody Fab are linked in any way to form a monomer, or,
  • IL-2 interleukin 2
  • ScFv single-chain antibody
  • the monomers of the homodimer include sequentially from the N-terminus:
  • connection structure preferably, described connection sequence is as shown in SEQ ID NO.6;
  • the Fab is the Fab of a humanized antibody or the Fab of a fully humanized antibody
  • the ScFv is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody
  • the Fc of the antibody is a fully human wild-type Fc or a No-ADCC mutant Fc.
  • the monomer of described homodimer is:
  • SEQ ID NO.26 (aCTLA4-IL2:VL-VH(ScFv)-Fc(wt)-IL2)
  • the present invention also relates to another bifunctional fusion protein, the bifunctional fusion protein is a bifunctional fusion protein composed of anti-4-1BB antibody and IL2, and the fusion protein is a heterologous or homodimer ;
  • Described heterodimer includes:
  • the first monomer comprises sequentially from the N-terminus:
  • connection structure (G4S connection sequence), preferably, described connection sequence is as shown in SEQ ID NO.6;
  • the second monomer comprises:
  • the heterodimer includes:
  • the first monomer is a polypeptide (IL2-Fc) whose sequence is shown in SEQ ID NO.10;
  • Second monomer which is:
  • composition of the polypeptide (a4-1BB VH-CH1-Fc(knob)) whose sequence is shown in SEQ ID NO.34 and 35 and the light chain of anti-4-1BB antibody whose sequence is shown in SEQ ID NO.28 and 29 the second monomer; or
  • fusion protein is homodimer, it is characterized in that,
  • the monomer of the homodimer is:
  • a molecule of interleukin 2 (IL-2) and a molecule of anti-4-1BB antibody Fab are linked in any way to form a monomer, or,
  • IL-2 interleukin 2
  • ScFv anti-4-1BB single-chain antibody
  • the monomers of the homodimer starting from the N-terminus, sequentially include:
  • connection structure preferably, described connection sequence is as shown in SEQ ID NO.6;
  • the Fab is the Fab of a humanized antibody or the Fab of a fully humanized antibody
  • the ScFv is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody
  • the Fc of the antibody is a fully human wild-type Fc or a No-ADCC mutant Fc.
  • the monomer of described homodimer is: as
  • the present invention also relates to nucleotide sequences encoding said heterodimers and homodimers.
  • the nucleotide sequence encoding the first monomer of the heterodimer is the nucleotide sequence shown in SEQ ID NO.15 (IL2-Fc(hole));
  • the nucleotide sequence encoding the second monomer of the heterodimer is:
  • the nucleotide sequence encoding the homodimer is:
  • SEQ ID NO. 48 (a4-1BB(LOB12.3)-IL2:VL-VH(ScFv)-Fc(wt)-IL2)
  • the present invention also relates to the following application of the bifunctional fusion protein:
  • the immune checkpoint inhibitor is PD-L1 antibody
  • the TKI antagonist is a small molecule TKI antagonist; more preferably, the small molecule TKI antagonist is afatinib or a structural analog thereof.
  • IL2/aOX40-hIgG1 antibody can delete intratumoral Treg cells while retaining the activation of effector cells by IL2, which overcomes the unfavorable factors that can lead to Treg expansion during the use of IL2.
  • IL2-Fc fusion protein can partially control tumor growth.
  • 1A Molecular structure of IL2-Fc fusion protein.
  • the lower dimer is the Fc region dimer of human IgG.
  • the N-terminal of each Fc monomer is coupled to One molecule of wild-type IL-2 molecule; 1B, the inhibitory effect of IL2-Fc fusion protein on tumor growth.
  • aOX40 antibody stimulates T cell activation and exerts anti-tumor function. 2A and aOX40 antibodies can effectively activate CD3 T cells; 2B and aOX40 antibodies can effectively control tumor growth.
  • FIG. 4 Construction of IL2/aOX40-Fc bispecific antibody, 4A, structural pattern diagram of IL2/aOX40-Fc bispecific antibody; 4B, SDS-PAGE analysis of the constructed IL2/aOX40-Fc bispecific antibody; 4C, IL2 /aOX40-Fc heterodimer form, aOX40 is in Fab or scFv form, the antibody contains 1 IL2 molecule; 4D, IL2/aOX40-Fc, aOX40 is in scFv form, connected at the N-terminus of V region or the C-terminus of Fc 1 or 2 IL2 molecules; 4E, IL2/aOX40-Fc homodimer form, aOX40 in Fab form, linked at N- or C-terminus of light chain, N-terminus of heavy chain or C-terminus of Fc 2 1 IL2 molecule; 4F, IL2/aOX40-Fc heterodimer form,
  • IL2/aOX40-Fc antibody has synergistic therapeutic effect, 5A, in vitro CTLL2 proliferation assay; 5B, tumor inhibitory effect of IL2/aOX40 wt Fc fusion protein (MC38 tumor model); 5C, in IL2/aOX40-Fc treatment Group re-challenge test results (MC38 tumor model); tumor inhibitory effect of 5D, IL2/aOX40 wt Fc fusion protein (B16F10 tumor model).
  • FIG. 6 The therapeutic effect of IL2/aOX40-Fc antibody does not depend on NK cells; 6A, One day after NK cell-depleting antibody injection, the proportion of NK cells in the peripheral blood of mice was analyzed by flow cytometry; 6B, The growth of MC38 tumors in different treatment groups curve.
  • FIG. 7 The function of IL2/aOX40 wt Fc depends on CD8 T cells; 7A, One day after injection of 200ug deletion antibody, the deletion efficiency of CD4 T cells and CD8 T cells in peripheral blood was detected by flow cytometry; 7B, The tumor growth curves of different treatment groups.
  • Intratumoral T cells play a key role in the treatment of IL2/aOX40-Fc; 8A, MC38 tumor-bearing mice were intraperitoneally injected with 20ug FTY720, and peripheral blood CD3 T cells were detected 2 days later; 8B, tumor growth curves in different treatment groups.
  • Figure 9 In situ tumor local treatment-induced immune response can control distal tumor growth; 9A, Tumor treatment effect on the right side (administration side) of different treatment groups; 9B, Tumors on the left side (non-treatment side) of different treatment groups treatment effect.
  • CD25 and OX40 are highly expressed on intratumoral Treg cells; 10A. Flow cytometry analysis of the expressions of CD25 and OX40 on intratumoral CD4, CD8 and Treg cells, comparing the levels of CD25 and OX40 on intratumoral, draining lymph node and spleen Treg cells Expression; 10B, MFI statistical analysis of expression levels in different cells.
  • FIG. 11 Deletion of intratumoral Treg by IL2/aOX40-Fc treatment increased the ratio of CD8 T cells to Treg; 11A, flow cytometry of intratumoral Treg cells in different treatment groups; 11B, intratumoral Treg/CD4 T and CD8 T/Treg cells in different treatment groups Treg ratio; 11C, draining lymph node and spleen Treg/CD4 T ratio.
  • aPDL1 antibody can synergize with IL2/aOX40-Fc to improve the anti-tumor effect.
  • IL2/aOX40-Fc can overcome the resistance of afatinib treatment, 14A, 14B, more T cells infiltrated into the tumor during afatinib treatment, but the proportion of Treg did not change; 14C, afatinib Combination therapy with IL2/aOX40-Fc antibody can effectively control tumor growth and recurrence.
  • IL2/aCTLA4 wt Fc fusion protein enhances tumor suppressive effect (MC38 tumor model).
  • pEE12.4-IgG ⁇ -hIgG1 which contains the signal peptide of mouse IgG ⁇ and the Fc sequence of human IgG1, is used for antibody expression.
  • pEE12.4-IgG ⁇ -hIgG1-Fc-hole and pEE12.4-IgG ⁇ -hIgG1-Fc-knob were used to express heterodimeric proteins.
  • Wild-type C57BL/6, BALB/c mice and BALB/c-nude mice were purchased from Weitong Lihua Laboratory Animal Center in Beijing, China. Unless otherwise stated, female mice aged 8-10 weeks were used in all experiments. Mice were raised in a specific pathogen-free (SPF) barrier environment. Animal breeding and experimental operations followed the relevant regulations of the Animal Management Committee of the Institute of Biophysics, Chinese Academy of Sciences.
  • SPF pathogen-free
  • MC38 is a C57 background mouse colorectal cancer cell line
  • MC38-EGFR5 are monoclonal cell lines obtained by infecting and screening lentivirus expressing human-mouse chimeric epidermal growth factor (EGFR) using MC38, respectively.
  • TUBO is a mouse breast cancer cell line in BALB/c background
  • B16F10 is a mouse melanoma cell line in C57 background.
  • DMEM complete medium containing 10% inactivated fetal bovine serum, 2 mmol/l L-glutamine, 0.1 mmol/l non-essential amino acids, 100 U penicillin and 100 ⁇ g/ml streptomycin.
  • TIB-210TM hybridoma cell line ATCC for expression of CD8+ T cell deletion antibody (clone: 2.43).
  • TIB-207TM hybridoma cell line for expression of CD4+ T cell deletion antibody (clone: GK1.5).
  • the PK136 hybridoma cell line was used to produce NK cell deletion antibodies.
  • HB-197TM hybridoma cell line for expression of an antibody that blocks Fc ⁇ RII/III in mice (clone: 2.4G2).
  • FreeStyleTM 293F cell line (Invitrogen) is a suspension cell derived from HEK293 cell line, cultured in SMM293-TII or CD OptiCHOTM medium, and is mainly used for transient transfection and expression of fusion proteins.
  • CTLL-2 cell line is a mouse T cell line used to detect the biological activity of IL-2
  • the above cell lines were cultured in RPMI1640 complete medium (containing 10% inactivated fetal bovine serum, 2mmol/L L-glutamine, 0.1mmol/L non-essential amino acids, 100U penicillin and 100 ⁇ g/ml streptomycin, 100IU/ml recombinant IL2 ) in culture.
  • RPMI1640 complete medium containing 10% inactivated fetal bovine serum, 2mmol/L L-glutamine, 0.1mmol/L non-essential amino acids, 100U penicillin and 100 ⁇ g/ml streptomycin, 100IU/ml recombinant IL2 ) in culture.
  • the human wild-type and mutant IL-2 gene sequences are shown in SEQ ID NO.1 below.
  • the primers used in the experiments were designed by DNAMAN software and synthesized by Invitrogen Company.
  • 5-7.5 ⁇ 10 5 single TUBO cells were suspended in 100 ⁇ l PBS and inoculated subcutaneously on the back of BALB/c mice.
  • mice with tumor regression When re-challenge experiments with the same tumor cells were performed on mice with tumor regression, the number of tumor cells inoculated was 5 times that of the initial tumor modeling, and the inoculation site was subcutaneous on the other side of the back of the mice.
  • the antibody or antibody fusion protein was injected intraperitoneally, and some experiments also used intratumoral administration, and the specific dosage will be described in the specific experiment.
  • the CD4+ T cell-depleted antibody GK1.5, CD8+ T cell-depleted antibody TIB210, NK cell-deleted antibody PK136 and FcRII/III blocking antibody used in the experiment were all derived from the corresponding hybridoma cells, produced and purified by our laboratory. .
  • IL-2 or IL-2 fusion protein treatment 200 ⁇ g of GK1.5 or TIB210 antibody was injected intraperitoneally to delete CD4+ T cells and CD8+ T cells, and then injected every 3 days, and the number of injections was adjusted according to the treatment cycle. Detection of removal efficiency by streaming.
  • PK136 or 1A8 antibody was injected intraperitoneally to delete NK cells or neutrophils, respectively. Streaming detection removal efficiency.
  • FTY720 (purchased from Sigma) is an immunosuppressive agent that reduces the migration of T cells from lymphoid organs out of the peripheral blood circulation.
  • FTY720 blockade was performed at different periods of tumor inoculation in mice to alter the tumor microenvironment. Blocking during tumor treatment in mice: intraperitoneal injection of 20 ⁇ g FTY720 1 day before tumor treatment, followed by intraperitoneal injection of 10 ⁇ g every other day. There are no new T cells that have colonized the tumor tissue. With the help of the FTY720 blockade protocol, the importance of infiltrating lymphocytes in tumor tissue can be investigated.
  • the molecular structures of the IL2/aOX40-Fc bispecific antibodies in Examples 1-5 below are all the molecular structures shown in FIG. 4A , that is, a molecule of IL2-Fc and a molecule of aOX40(Fab)-Fc composed of heterodimers.
  • Example 1 IL2/aOX40-Fc bispecific antibody significantly improved the effect of treatment alone
  • Treatment with IL2-Fc fusion protein can partially control tumors
  • Free free IL2 alone has a short half-life and requires multiple administrations.
  • we designed an IL2-Fc fusion protein see Figure 1A for a schematic diagram of the structure), and verified the function of the fusion protein in the MC38 tumor model.
  • IL2-Fc was able to effectively control tumor growth compared to untreated controls, but IL2-Fc treatment alone failed to completely eliminate tumors when tumors were larger (Fig. 1B).
  • ip intraperitoneal injection
  • Anti-OX40 antibody promotes T cell activation and exerts anti-tumor function
  • OX40 molecule is only expressed on Treg cells and activated T cells It is not expressed on T cells, so the antibody targeting OX40 has higher specificity; and the expression abundance of OX40 on Treg cells and effector cells is different, which also provides a theoretical basis for the same target to produce different effects on different cells.
  • spleen T cell stimulation experiments in vitro. The specific method is as follows: Isolate mouse spleen cells and spread 3 ⁇ 10 5 cells/well in 96-well plates.
  • aCD3 and aCD28 or aCD3 and aOX40 antibodies After stimulation with aCD3 and aCD28 or aCD3 and aOX40 antibodies for 72 h, flow cytometry analysis of the expression level of CD69 on CD3 T cells, aCD3 , aCD28 antibody concentration is 1ug/ml, aOX40 antibody concentration is 2ug/ml.
  • mice We further verified the function of anti-OX40 in anti-tumor experiments in mice.
  • IL2-Fc alone can expand Treg, while aOX40 antibody can delete intratumoral Treg cells.
  • aOX40 antibody can delete intratumoral Treg cells.
  • IL2-Fc and aOX40 antibody can activate effector T cells and delete expanded Treg, it can play a synergistic therapeutic effect.
  • the method was as follows: C57BL6 mice were subcutaneously inoculated with 5 ⁇ 10 5 MC38 tumor cells, and the tumor grew to D14 to start treatment, and the individual treatment groups were intraperitoneally injected (ip) with 6ug IL2 -Fc or 9ug aOX40 antibody, the combined treatment group was injected with both antibodies at the same time; once every three days for a total of three treatments.
  • IL2/aOX40-Fc bispecific antibody has synergistic therapeutic effect
  • Fc monomer is connected to the WT IL2 protein, and the other Fc monomer Linked to the Fab fragment of aOX40 antibody.
  • the constructed fusion protein particles were transfected into 293F cells, and the cell supernatant was collected seven days later. After centrifuging the supernatant, the protein was purified by protein A column, and the purified antibody was identified by protein gel. The molecular composition of the protein was analyzed by SDS-PAGE, and it was proved that the bispecific molecule could fully express the light chain, heavy chain and IL2 molecule of aOX40 antibody simultaneously (Fig. 4B).
  • the structure shown in Figure 4C is a heterodimer, the antibody contains an IL2 molecule, aOX40 (anti-OX40 antibody) is in the form of Fab or scFv, and the Fc dimer is the Fc monomer of human IgG and the Fc of human IgG with the ADCC effector function deleted. Heterodimers formed from monomers.
  • aOX40 (anti-OX40 antibody) is in the form of scFv, one or two IL2 molecules are connected to the N-terminus of the V region or the C-terminus of the Fc, and the Fc dimer is the Fc homologous two of wild-type human IgG.
  • aOX40 (anti-OX40 antibody) is in the form of Fab, and one or two IL2 molecules are connected to the N-terminus or C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain or the C-terminus of the Fc.
  • the dimer is an Fc homodimer of wild-type human IgG, or a heterodimer formed by an Fc monomer of human IgG and an Fc monomer of human IgG with the ADCC effector function deleted.
  • C57BL6 mice were subcutaneously inoculated with 5 ⁇ 10 5 MC38 tumor cells, and treatment was started at D14 after tumor inoculation; 9ug IL2-Fc, 16ug aOX40 antibody, IL2-Fc + aOX40 antibody or IL2 in equimolar numbers of aOX40 antibody were injected intraperitoneally, respectively /aOX40 wt Fc protein.
  • Three treatments were performed on days D14, D17, and D20, and tumor size was measured twice a week; the results showed that the IL2/aOX40-wtFc bispecific antibody was better than aOX40 antibody alone, IL2-Fc, and the combination of both. treatment effect (Figure 5B).
  • mice that had completely cleared tumors were treated with tumor re-challenge for two months.
  • mice that had completely cleared tumors were treated with tumor re-challenge for two months.
  • Five times the dose (2.5 ⁇ 10 6 cells) of MC38 tumor cells were subcutaneously inoculated in the re-challenge. Only tumor cells were inoculated for re-challenge. , after inoculation, each group was no longer given any treatment.
  • Example 2 IL2/aOX40-Fc can activate CD8 T cells
  • IL2/aOX40-Fc bispecific antibody does not depend on NK cells
  • CD25 IL-2 receptor alpha
  • OX40 are mainly expressed on activated effector T cells and NK cells, in order to determine which population of immune cells the treatment of IL2/aOX40-Fc antibody mainly depends on, we performed different cell populations. delete experiment.
  • C57BL6 mice were subcutaneously inoculated with 5 ⁇ 10 5 MC38 tumor cells, and 25ug IL2/aOX40 wt Fc fusion protein was injected intraperitoneally on days 12, 15 and 18.
  • 400ug of NK cell-depleting antibody PK136 was intraperitoneally injected one day before the treatment, once every 4 days, for a total of 3 injections.
  • Figure 6A shows the changes of peripheral blood NK cells 1 day after deletion of NK cells.
  • the IL2/aOX40-Fc antibody still had a therapeutic effect after NK cell deletion, indicating that NK cells were not the main effector cells for the antibody to exert its therapeutic effect (Fig. 6B).
  • IL2/aOX40-Fc antibody The therapeutic effect of IL2/aOX40-Fc antibody depends on CD8 T cells
  • mice were subcutaneously inoculated with 5 ⁇ 10 5 MC38 tumor cells on the back of the 12th day, and were intraperitoneally injected with 25ug IL2/aOX40 wt Fc protein (administered on the 12th, 15th, and 18th days after tumor inoculation, respectively).
  • 25ug IL2/aOX40 wt Fc protein administered on the 12th, 15th, and 18th days after tumor inoculation, respectively.
  • 200ug CD4 T cell-depleting antibody (clone number: GK1.5, prepared in our laboratory)
  • 200ug CD8 T cell-depleting antibody (clone number: TIB210, prepared in our laboratory) or both were injected intraperitoneally. Deleting antibodies were injected three times in total.
  • IL2/aOX40-Fc bispecific antibody depends on intratumoral T cells
  • FTY720 is a clinical drug that can inhibit immune rejection during organ transplantation, and inhibit the migration of T cells from lymphoid organs to peripheral blood [19].
  • IL2/aOX40 wt Fc antibody treatment alone can clear the tumor, and block FTY720 at the same time of treatment.
  • the experimental protocol is as follows:
  • C57BL6 mice were subcutaneously inoculated with 4 ⁇ 10 5 MC38 tumor cells on the back, and 15ug IL2/aOX40-Fc antibody was injected intraperitoneally on days 7, 10 and 13 after tumor inoculation.
  • 15ug IL2/aOX40-Fc antibody was injected intraperitoneally on days 7, 10 and 13 after tumor inoculation.
  • 25ug of FTY720 was injected intraperitoneally on the 6th day after tumor inoculation, and 10ug was injected on the 8th, 10th and 12th days, respectively.
  • C57BL6 mice were inoculated with 2 ⁇ 10 5 MC38 and 4 ⁇ 10 5 MC38 tumor cells on the left and right sides of the back (4 ⁇ 10 5 MC38 tumor cells were inoculated on the right side of the back and 2 ⁇ 10 5 MC38 tumor cells were inoculated on the left side) .
  • the tumor on the right side grew to around 50mm3 and the tumor on the left side started treatment at around 30mm3 :
  • treatment group 1 7.5ug IL2/aOX40 wt Fc antibody was injected into the tumor on the right side (the therapeutic antibody was treated three times on days 7, 10, and 13), and the tumor on the left side was not treated;
  • treatment group 2 7.5ug of IL2/aOX40 wt Fc antibody was injected into the right tumor, and 20ug of TIB210 antibody was injected into the right tumor to delete intratumoral CD8 T cells. Treatment and deletion antibodies were treated three times on days 7, 10, and 13, and left tumors were left untreated.
  • Example 3 IL2/aOX40-Fc can delete intratumoral Treg and increase the ratio of CD8/Treg cells
  • Intratumoral Treg cells highly express CD25 and OX40 molecules
  • mice After inoculation with MC38 tumors, we analyzed the expression of IL2 receptors CD25 and OX40 on T cells in the tumor, draining lymph nodes, and spleen of mice on the 12th day: specific protocol: mice were inoculated with 5 ⁇ 10 5 MC38 tumor cells, and on the 12th day The tumor tissue, spleen and draining lymph node (dLN) of the tumor-bearing mice were collected on the 1st day respectively, and the expressions of CD25 and OX40 on different cells were detected by flow cytometry.
  • specific protocol mice were inoculated with 5 ⁇ 10 5 MC38 tumor cells, and on the 12th day The tumor tissue, spleen and draining lymph node (dLN) of the tumor-bearing mice were collected on the 1st day respectively, and the expressions of CD25 and OX40 on different cells were detected by flow cytometry.
  • IL2/aOX40-Fc antibody deletes intratumoral Treg cells and increases the ratio of CD8/Treg cells
  • IL2/aOX40-Fc antibody might bind more to Treg after intraperitoneal injection.
  • MC38 tumor-bearing mice were treated on the 12th day after tumor inoculation, and 25ug IL2/aOX40-Fc antibody was injected intraperitoneally. On the 15th day, tumor tissue, draining lymph nodes and spleen were collected, and the proportion of Treg cells in the tumor, CD8 T/Treg.
  • IL2/aOX40-Fc can more effectively delete intratumoral Treg cells and increase the ratio of CD8 T/Treg to have the best therapeutic effect (FIG. 11A,B).
  • IL2/aOX40-Fc did not change the Treg/CD4 T ratio in peripheral tumor-draining lymph nodes and spleen, indicating that IL2/aOX40-Fc treatment does not break peripheral immune tolerance and has a better effect.
  • Safety FIG. 11C).
  • IL2/aOX40-Fc antibody has the effect of deleting Treg.
  • the IL2/aOX40-no ADCC Fc antibody was constructed by point mutation of the Fc region. The mutated Fc did not bind to the Fc ⁇ R receptor and lost the deletion function mediated by ADCC and ADCP.
  • C57BL6 mice were inoculated with MC38 tumor cells, and treatment was initiated on day 13 after inoculation. 15ug wt Fc or no ADCC Fc antibody was injected intraperitoneally, once every three days, for a total of three treatments.
  • aPDL1 can synergize with IL2/aOX40-Fc antibody to enhance anti-tumor effect
  • IL2/aOX40-Fc antibody treatment alone had good clearance when tumors in MC38 tumor-bearing mice were below 200 mm 3 .
  • antibody therapy alone can only control the growth of the tumor and cannot completely remove it; in order to better improve the therapeutic effect, we will use a combination of bispecific antibodies and immune checkpoint inhibitory antibodies to observe whether the therapeutic effect can be improved. .
  • aOX40 and aCTLA4 combination group was injected intraperitoneally with 250ug of antibody on day 16 and then injected with 25ug of IL2/aOX40-Fc (aOX40 and aCTLA4 antibodies were administered only once, and 25ug of IL2/aOX40-Fc was administered by intraperitoneal injection three times);
  • the aPDL1 combination treatment group was injected with 25ug IL2/aOX40-Fc and 250ug aPDL1 antibody (days 16, 19, and 22).
  • Afatinib is a second-generation kinase inhibitor that irreversibly inhibits the tyrosine kinase activity of receptors in a covalent manner.
  • the mechanism of action of afatinib was analyzed on the TUBO model, and it was found that the ratio of CD3 T/CD45 lymphocytes in the tumor increased after treatment, indicating that more T cells infiltrated into the tumor during the treatment of chemotherapy drugs (Fig. 14A). ).
  • Treg cells in the tumor inhibit the killing of effector cells on the tumor, which in turn leads to tumor recurrence after treatment.
  • IL2/aOX40-Fc In order to verify whether IL2/aOX40-Fc can antagonize the recurrence after chemotherapeutic drug treatment, we used IL2/aOX40-Fc antibody in combination with chemotherapy drug treatment in tumor-bearing mice.
  • mice were subcutaneously inoculated with 5 ⁇ 10 5 TUBO tumor cells,
  • Treatment plan 1 On the 9th day, 1 mg of Afatinib was given by gavage for treatment, and the tumor tissue was taken for flow analysis 6 days later.
  • Treatment scheme 2 Tumor-bearing mice started treatment on the 14th day after tumor inoculation,
  • the Afatinib treatment group was given 1 mg of Afatinib on the 14th day,
  • the combined treatment group was administered afatinib once by intragastric administration according to the above scheme, and was treated with 25ug IL2/aOX40-Fc intraperitoneal antibody three times.
  • Example 6 IL2/aCTLA4-Fc bispecific antibody significantly improves tumor treatment effect
  • mice were subcutaneously inoculated with 5 ⁇ 10 5 MC38 tumor cells, and treatment was started at D11 after tumor inoculation; 15ug of IL2/aCTLA4 wt Fc protein (the specific A schematic diagram of the structure is shown in Figure 15). Three treatments were performed on D11, D14, and D17, and tumor size was measured twice a week;
  • Example 7 IL2/a4-1BB-Fc bispecific antibody significantly improves tumor treatment effect
  • C57BL/6 mice were subcutaneously inoculated with 5 ⁇ 10 5 MC38 tumor cells, and treatment was started at D10 after tumor inoculation; 10ug 3H3-IL2 and 10ug LOB12.3-IL2 wt Fc fusion protein (IL2/a4-1BB- A schematic diagram of the structure of the Fc fusion protein is shown in Figure 16). Three treatments were performed on the 10th, 16th and 19th days after tumor inoculation, and the tumor size was measured twice a week; the results showed that compared with the control group, the tumor volume of the treatment group with the two fusion proteins was significantly reduced, and the difference was extremely significant.
  • CHIBA K.FTY720 a new class of immunomodulator, inhibits lymphocyte egress from secondary lymphoid tissues and thymus by agonistic activity at sphingosine 1-phosphate receptors[J].Pharmacology&therapeutics,2005,108(3):308-19.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)

Abstract

本发明提供了IL-2与抗体亚单位构成的双功能融合蛋白及其在制备抗肿瘤药物中的应用，所述双功能融合蛋白包括异源二聚体或同源二聚体。

Description

一种IL-2与抗体亚单位构成的双功能融合蛋白 技术领域
本发明属于生物医药技术领域，具体的，涉及一种IL-2与抗体亚单位构成的双功能融合蛋白。
背景技术
白细胞介素2(IL2)又称为T细胞生长因子，发现于1976年，在体外实验中刺激T细胞克隆扩增[1]。IL2为四个α螺旋的糖蛋白，分子量为15.5Kd[2]，主要由活化的CD4 T细胞分泌，其它细胞如活化的CD8细胞、NK细胞、NKT细胞以及ILCs细胞也能产生少量的IL2[3,4]。IL2以自分泌和旁分泌途径结合于受体细胞上发挥功能，对T、NK细胞具有重要的调节作用。
IL2的受体由IL2Rα(CD25)，IL2Rβ(CD122)和IL2Rγc(CD132)三个亚基构成，IL2与三种亚基之间的亲和力存在差异[5,6]。IL2与α亚基之间低亲和力结合(Kd～10-8M)，与βγ二聚体中等亲和力结合(Kd～10-9M)，与αβγ三聚体高亲和力结合(Kd～10-11M)[7,8]。
不同亲和力受体在不同细胞上的表达水平存在差异：βγ受体主要表达在静息状态的T细胞、CD8 T记忆细胞和NK细胞上；α受体在细胞活化后表达上调，活化后的T细胞表达高亲和力的αβγ受体。Treg细胞持续高表达CD25分子，Treg细胞对IL2分子具有很高的亲和力[9]。由于受体表达的差异性，低剂量的IL2优先扩增表达高亲和力受体的Treg细胞，高剂量的IL2才能有效活化CD8 +T细胞和NK细胞[10]。
IL2与三聚体受体的亲和力最高，而三聚体受体主要在Treg细胞上持续表达而仅仅在活化的效应细胞上短暂上调表达；临床上使用低剂量的IL2会优先扩增Treg细胞，而使用高剂量的IL2才能活化效应T细胞。高剂量的IL2会导致更大的毒副作用而限制了IL2的使用。
为了克服这一问题，尝试通过不同的手段对IL2分子进行改造，使其降低对Treg的结合而提高对效应T细胞和NK细胞的结合来增强其在肿瘤治疗中的效果，同时避免高剂量使用而导致的毒性。目前对IL2的改造有以下几种方法：
(1)PEG化的IL2
IL2的分子量为15.5KD，在体内血清中的半衰期为10-85min，为了达到治疗效果需要多次重复给药。PEG化的IL2提高了IL2的总体分子量延长了IL2的半衰期，同时通过PEG化修饰位点的不同可以使IL2偏向于结合IL2-Rα或者IL2-Rβ[11]。
(2)IL2/aIL2抗体复合物
IL2与aIL2抗体复合物的结合一方面可以延长IL2的半衰期，另一方面由于aIL2抗体可以与IL2分子不同部位的结合，使IL2/aIL2抗体复合物表现出与受体CD25或者CD122结合的不同倾向性。
(3)IL2突变改造
通过对IL2上面不同受体结合位点的点突变改造，也可以改变IL2与不同受体亚基的亲和力。通过对IL2分子L80F、R81D、L85V、I86V和I92F五个碱基的点突变，使IL2发生构象改变，增加与CD122的结合同时使下游信号的转导不依赖于CD25分子。突变的IL2偏向于扩增CD8+T细胞和NK细胞，具有更低的毒副作用并且在小鼠的肿瘤模型中表现出更好的肿瘤治疗效果[12]。
在我们实验室之前的改造中，我们对IL2同时进行了降低CD25亚基结合(F42A)和增加CD122亚基结合的突变。这种双突变的IL2分子比单独的CD25亚基亲和力下降或者单独的CD122亚基亲和力上升的突变都具有更好的治疗效果[13]。双突变的IL2不仅在MC38肿瘤模型中具有很好的治疗效果；而在TUBO等冷肿瘤模型中，与TKI或者7.16.4抗体的联合治疗也具有很好的治疗效果。说明在增加CD122亚基亲和力的同时进一步降低CD25亚基的亲和力可以更进一步提高IL2的治疗效果。
上述针对IL2的种种改造都是通过降低IL2对CD25分子的结合降低或者对CD122亚基的结合增加。 虽然这一改造的策略相比于野生型的IL2分子的治疗效果有提升，但是仍不能完全消除瘤内Treg细胞对治疗效果的抑制作用。 如何进一步比较和分析瘤内Treg细胞和效应T细胞上CD25以及CD122受体表达的 差异性来进一步提高IL2治疗效果仍是一个关键的问题。
TNF家族的共刺激或共抑制性分子也在Treg细胞上高表达，是调节Treg细胞功能的靶点分子，常见的分子有ICOS、GITR、OX40、4-1BB等。
一些共刺激分子如GITR和OX40分子在瘤内Treg上高表达，在活化的CD4和CD8 T细胞上也上调表达。OX40分子表达于瘤内的T细胞上，而且在瘤内的Treg上高表达；全身给予激活性aOX40抗体后可以导致瘤内Treg数目和比例的下降而发挥抗肿瘤的作用[14]。在aOX40抗体的临床试验中未观察到明显的Treg数目下降，但是能观察到效应T细胞的数目上升[15]。
OX40分子共同表达于效应T细胞和Treg细胞上，目前使用的OX40抗体发挥抗肿瘤作用可以既发挥活化效应细胞又发挥删除Treg细胞作用。OX40发挥抗肿瘤作用依赖于瘤内CD4 T和CD8 T细胞的扩增，而且治疗后小鼠具有免疫记忆re-challenge后肿瘤不会再复发[16]。另一方面，小鼠实验中使用的OX40抗体OX86也可以发挥删除Treg的功能，这种删除作用依赖于活化性的FcγR而不依赖于抑制性的FcγRIIB。而且将OX86的rIgG1Fc分别换成小鼠的IgG2a和IgG2An297A(不与FcR结合)后，发现IgG2a的Fc具有更好的肿瘤控制能力，说明在此肿瘤模型上OX40抗体功能的发挥主要依赖于删除Treg的功能；而且删除作用主要发生在Treg细胞，而非Treg的CD4 T细胞以及CD8 T细胞的数目和绝对数并未下降[17]。在使用OX40靶点进行治疗时，我们可以利用Treg和效应T细胞上OX40表达丰度的不同，对不同T细胞群发挥不同作用。除了OX40抗体单独使用外，OX40分子还可以同化疗、放疗以及其它细胞因子联合使用，都具有协同作用而发挥更好的抗肿瘤作用[18]。
因此，IL2/aOX40双功能分子，以及CTLA4和其他TNF家族的共刺激或共抑制性分子ICOS、GITR、4-1BB等的抗体与IL-2细胞因子构成的融合蛋白双功能抗体，在临床上是非常具有开发潜力的肿瘤治疗新方案。
发明内容
本发明首先涉及一种双功能融合蛋白，所述的融合蛋白为异源二聚体，其特征在于，
所述的异源二聚体包括：
(1)白介素2(IL-2)与免疫球蛋白Fc单链连接而成的异源二聚体第一单体；
(2)抗CTLA4分子或TNF家族的共刺激或共抑制性分子的抗体的Fab或ScFv与免疫球蛋白Fc单链连接而成的异源二聚体第二单体；
所述的第一单体与第二单体通过Fc单链的二聚化连接，构成所述的异源二聚体；
所述的CTLA4或TNF家族的共刺激或共抑制性分子包括但不限于：OX40、4-1BB、ICOS、GITR。
所述的免疫球蛋白Fc单链为天然免疫球蛋白Fc单链或通过基因突变敲除ADCC效应的免疫球蛋白Fc单链；
优选的，所述的免疫球蛋白Fc单链为天然免疫球蛋白Fc单链或通过基因突变敲除ADCC效应的免疫球蛋白Fc单链；更优选的，所述的免疫球蛋白Fc单链为人IgG的Fc单链。
所述的CTLA4或TNF家族的共刺激或共抑制性分子为OX40、4-1BB，所述的抗CTLA4的抗体(aCTLA4)或抗TNF家族的共刺激或共抑制性分子的抗体为抗OX40的抗体(aOX40)、抗4-1BB的抗体(a4-1BB)；
所述的第二单体中，
所述的抗体的Fab为人源化抗体的Fab或全人化抗体的Fab；
所述的抗体的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；
更优选的，所述的第二单体为：
抗CTLA4或抗TNF家族的共刺激或共抑制性分子的抗体的单体，所述抗体的单体包含一条轻链和一条重链；优选的，所述抗体为人源化抗体或全人抗体。
更优选的，所述的异源二聚体包括：
(1)第一单体，所述的第一单体自N端开始依次包含：
1)序列如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；
2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；
3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；
(2)第二单体，所述的第二单体包含：
1)序列如SEQ ID NO.7所示的抗OX40抗体轻链VL-KCL和序列如SEQ ID NO.8所示的抗OX40抗体重链VH&CH1组成的抗OX40抗体Fab区；
或：2)序列如SEQ ID NO.9所示的抗OX40单链抗体(ScFv)：
和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；
更优选的，所述的异源二聚体包括：
第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；
第二单体：其为：
(1)序列如SEQ ID NO.11所示的抗OX40抗体VH-CH1-Fc(knob)和序列如SEQ ID NO.7所示的抗OX40抗体轻链VL-KCL组成的第二单体；
或(2)序列如SEQ ID NO.12所示的多肽(aOX40 ScFv-Fc(knob))。
本发明还涉及一种双功能融合蛋白，所述的融合蛋白为同源二聚体，其特征在于，
所述的同源二聚体的单体为：
一分子白介素2(IL-2)与一分子抗OX40抗体Fab通过任意方式连接构成的单体，或，
一分子白介素2(IL-2)与一分子抗OX40单链抗体(ScFv)通过任意方式连接构成的单体。
优选的，所述的同源二聚体的单体自N端开始依次包含：
(1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；
(2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；
(3)抗OX40抗体的Fab或ScFv；所述的Fab为人源化抗体的Fab或全人化抗体的Fab，所述的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；
(4)抗体的Fc；所述的抗体Fc为全人野生型Fc或No-ADCC突变型Fc。
更优选的，所述的同源二聚体的单体为：
(1)SEQ ID NO.13(aOX40-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
(2)SEQ ID NO.14(IL2-aOX40：IL2-VL-VH(ScFv)-Fc(wt))所示序列。
本发明还涉及另一种双功能融合蛋白，所述的双功能融合蛋白为抗CTLA4抗体与IL2的组合成的双功能融合蛋白，所述的融合蛋白为异源二聚体；
所述的异源二聚体包括：
(1)第一单体，所述的第一单体自N端开始依次包含：
1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；
2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；
3)如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；
(2)第二单体，所述的第二单体包含：
1)序列如SEQ ID NO.21所示的抗CTLA4抗体轻链VL-KCL与序列如SEQ ID NO.22所示的抗CTLA4抗体重链VH&CH1组成的抗体Fab区；
或：2)序列如SEQ ID NO.23所示的抗CTLA4单链抗体(ScFv)：
和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；
更优选的，所述的异源二聚体包括：
第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；
第二单体：其为：
(1)序列如SEQ ID NO.24所示的多肽(aCTLA4 VH-CH1-Fc(knob))和序列如SEQ ID NO.21所示的抗CTLA4抗体轻链组成的第二单体；或
(2)序列如SEQ ID NO.25所示的多肽(a CTLA4 ScFv-Fc(knob))。
本发明还涉及一种双功能融合蛋白，所述的融合蛋白为同源二聚体，其特征在于，
所述的同源二聚体的单体为：
一分子白介素2(IL-2)与一分子抗CTLA4抗体Fab通过任意方式连接构成的单体，或，
一分子白介素2(IL-2)与一分子抗CTLA4单链抗体(ScFv)通过任意方式连接构成的单体。
优选的，所述的同源二聚体的单体自N端开始依次包含：
(1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；
(2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；
(3)抗CTLA4抗体的Fab或ScFv；所述的Fab为人源化抗体的Fab或全人化抗体的Fab，所述的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；
(4)抗体的Fc；所述的抗体Fc为全人野生型Fc或No-ADCC突变型Fc。
更优选的，所述的同源二聚体的单体为：
(1)SEQ ID NO.26(aCTLA4-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
(2)SEQ ID NO.27(IL2-a CTLA4：IL2-VL-VH(ScFv)-Fc(wt))所示序列。
本发明还涉及另一种双功能融合蛋白，所述的双功能融合蛋白为抗4-1BB抗体与IL2的组合成的双功能融合蛋白，所述的融合蛋白为异源或同源二聚体；
所述的异源二聚体包括：
(1)第一单体，所述的第一单体自N端开始依次包含：
1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；
2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；
3)如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；
(2)第二单体，所述的第二单体包含：
1)序列如SEQ ID NO.28、29所示的抗4-1BB抗体轻链与序列如SEQ ID NO.30、31所示的抗4-1BB抗体重链VH&CH1组成的抗体Fab区；
或：2)序列如SEQ ID NO.32、33所示的抗4-1BB单链抗体(ScFv)：
和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；
更优选的，所述的异源二聚体包括：
第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；
第二单体：其为：
(1)序列如SEQ ID NO.34、35所示的多肽(a4-1BB VH-CH1-Fc(knob))和序列如SEQ ID NO.28、29所示的抗4-1BB抗体轻链组成的第二单体；或
(2)序列如SEQ ID NO.36、37所示的多肽(a4-1BB ScFv-Fc(knob))。
所述的融合蛋白为同源二聚体，其特征在于，
所述的同源二聚体的单体为：
一分子白介素2(IL-2)与一分子抗4-1BB抗体Fab通过任意方式连接构成的单体，或，
一分子白介素2(IL-2)与一分子抗4-1BB单链抗体(ScFv)通过任意方式连接构成的单体。
所述的同源二聚体的单体自N端开始依次包含：
(1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；
(2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；
(3)抗4-1BB抗体的Fab或ScFv；所述的Fab为人源化抗体的Fab或全人化抗体的Fab，所述的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；
(4)抗体的Fc；所述的抗体Fc为全人野生型Fc或No-ADCC突变型Fc。
更优选的，所述的同源二聚体的单体为：如
SEQ ID NO.38(a4-1BB(LOB12.3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
SEQ ID NO.39(a4-1BB(3H3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
SEQ ID NO.40(IL2-a4-1BB(LOB12.3)：IL2-VL-VH(ScFv)-Fc(wt))、
SEQ ID NO.41(IL2-a4-1BB(3H3)：IL2-VL-VH(ScFv)-Fc(wt))所示序列。
本领域技术人员同样也应当明确：
使用如：
SEQ ID NO.52-55所示的氨基酸序列编码的抗人OX40抗体的功能结构
SEQ ID NO.56-57所示的氨基酸序列编码的抗人CTLA4抗体的功能结构
SEQ ID NO.58-61所示的氨基酸序列编码的抗人4-1BB抗体的功能结构
替换上述各个双功能融合蛋白的相似功能嵌段，同样可以达到本发明所述的技术效果。
本发明还涉及编码所述异源二聚体和同源二聚体的核苷酸序列。
优选的，
编码所述异源二聚体第一单体的核苷酸序列为SEQ ID NO.15(IL2-Fc(hole))所示的核苷酸序列；
编码所述异源二聚体第二单体的核苷酸序列为：
SEQ ID NO.16(aOX40：VL-KCL)、
SEQ ID NO.17(aOX40：VH-CH1-Fc(knob))、
SEQ ID NO.18(aOX40 ScFv-Fc(knob))、
SEQ ID NO.42(a4-1BB(LOB12.3)：VL-KCL)、
SEQ ID NO.43(a4-1BB(3H3)：VL-KCL)、
SEQ ID NO.44(a4-1BB(LOB12.3)：VH-CH1-Fc(knob))、
SEQ ID NO.45(a4-1BB(3H3)：VH-CH1-Fc(knob))、
SEQ ID NO.46(a4-1BB(LOB12.3)：ScFv-Fc(knob))、
SEQ ID NO.47(a4-1BB(3H3)：ScFv-Fc(knob))所示的核苷酸序列；
编码所述同源二聚体的核苷酸序列为：
SEQ ID NO.19(aOX40-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
SEQ ID NO.20(IL2-aOX40：IL2-VL-VH(ScFv)-Fc(wt))、
SEQ ID NO.48(a4-1BB(LOB12.3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
SEQ ID NO.49(a4-1BB(3H3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
SEQ ID NO.50(IL2-a4-1BB(LOB12.3)：IL2-VL-VH(ScFv)-Fc(wt))、
SEQ ID NO.51(IL2-a4-1BB(3H3)：IL2-VL-VH(ScFv)-Fc(wt))所示的核苷酸序列。
本发明还涉及所述的双功能融合蛋白的如下应用：
(1)制备抗肿瘤药物；
(2)制备与免疫检查点抑制剂联用的抗肿瘤药物；
(3)制备克服免疫检查点抑制剂耐受的抗肿瘤药物；
(4)制备与TKI拮抗剂联用的抗肿瘤药物；
(5)制备克服TKI拮抗剂耐受的抗肿瘤药物。
优选的，所述的免疫检查点抑制剂为PD-L1抗体；
优选的，所述的TKI拮抗剂为小分子TKI拮抗剂；更优选的，所述的小分子TKI拮抗剂为阿法替尼或其结构类似物。
本发明的有益效果在于：
(1)提出了IL2临床应用的瓶颈和解决方案，IL2/aOX40-hIgG1抗体在保留IL2对效应细胞活化的同时删除瘤内Treg细胞，克服了IL2使用过程中会导致Treg扩增的不利因素，这些结果为临床使用IL2提供了新思路；
(2)对克服免疫检查点阻断疗法耐受和TKI化疗药使用后产生的抗性具有重要意义。
附图说明
图1、IL2-Fc融合蛋白能部分控制肿瘤的生长，1A、IL2-Fc融合蛋白的分子结构，下部的二聚体为人IgG的Fc区二聚体，每个Fc单体N端偶联了一分子的野生型IL-2分子；1B、IL2-Fc融合蛋白对肿瘤生长的抑制效果。
图2、aOX40抗体刺激T细胞活化并发挥抗肿瘤功能，2A、aOX40抗体能够有效活化CD3 T细胞；2B、aOX40抗体能有效控制肿瘤生长。
图3、IL2-Fc和aOX40联合治疗未见明显的协同效果
图4、构建IL2/aOX40-Fc双特异性抗体，4A、IL2/aOX40-Fc双特异性抗体结构模式图；4B、SDS-PAGE分析构建的IL2/aOX40-Fc双特异性抗体；4C、IL2/aOX40-Fc异源二聚体形式，aOX40为Fab或scFv形式，抗体包含1个IL2分子；4D、IL2/aOX40-Fc，aOX40为scFv形式，在V区的N端或Fc的C端连接1个或2个IL2分子；4E、IL2/aOX40-Fc同源二聚体形式，aOX40为Fab形式，在轻链的N端或C端、在重链的N端或Fc的C端连接2个IL2分子；4F、IL2/aOX40-Fc异源二聚体形式，aOX40为Fab形式，在轻链的N端或C端、在重链的N端或Fc的C端连接1个IL2分子。
图5、IL2/aOX40-Fc抗体具有协同治疗的效果，5A、体外CTLL2增殖实验；5B、IL2/aOX40 wt Fc融合蛋白的肿瘤抑制效果(MC38肿瘤模型)；5C、在IL2/aOX40-Fc治疗组re-challenge试验结果(MC38肿瘤模型)；5D、IL2/aOX40 wt Fc融合蛋白的肿瘤抑制效果(B16F10肿瘤模型)。
图6、IL2/aOX40-Fc抗体治疗效果不依赖于NK细胞；6A、NK细胞删除性抗体注射后一天，流式分析小鼠外周血中NK细胞的比例；6B、不同治疗组MC38肿瘤的生长曲线。
图7、IL2/aOX40 wt Fc功能依赖于CD8 T细胞；7A、注射200ug删除性抗体一天后，流式检测外周血中CD4 T细胞和CD8 T细胞删除效率；7B、不同治疗组肿瘤生长曲线。
图8、瘤内T细胞对IL2/aOX40-Fc的治疗起关键作用；8A、MC38荷瘤小鼠腹腔注射20ug FTY720，2天后检测外周血CD3 T细胞；8B、不同治疗组肿瘤生长曲线。
图9、原位肿瘤局部治疗诱导的免疫应答可以控制远端肿瘤生长；9A、不同治疗组右侧(给药侧)的肿瘤治疗效果；9B、不同治疗组左侧(非治疗侧)的肿瘤治疗效果。
图10、CD25和OX40在瘤内Treg细胞上高表达；10A、流式分析肿瘤内CD4、CD8和Treg细胞上CD25、OX40的表达，比较瘤内、引流淋巴结和脾脏Treg细胞上CD25、OX40的表达；10B、不同细胞表达水平的MFI统计分析。
图11、IL2/aOX40-Fc治疗删除瘤内Treg提高CD8 T细胞与Treg的比例；11A、不同治疗组瘤内Treg细胞流式图；11B、不同治疗组瘤内Treg/CD4 T和CD8 T/Treg比例；11C、引流淋巴结和脾脏Treg/CD4 T比例。
图12、抗体功能的发挥部分依赖于Fc与FcγR的结合。
图13、aPDL1抗体能够协同IL2/aOX40-Fc提高抗肿瘤效果。
图14、IL2/aOX40-Fc能够克服Afatinib治疗的抗性，14A、14B、阿法替尼治疗过程中有更多的T细胞浸润到瘤内，但是Treg的比例没有改变；14C、阿法替尼和IL2/aOX40-Fc抗体联合治疗能够有效控制肿瘤的生长和复发。
图15、IL2/aCTLA4 wt Fc融合蛋白增强肿瘤抑制效果(MC38肿瘤模型)。
图16、IL2/a4-1BB-wt Fc双特异性抗体显著改善肿瘤治疗效果。
具体实施方式
实验材料
1、菌种和质粒
菌种：Top10 E.coli、DH5αE.coli感受态细胞(北京全式金生物技术有限公司)
质粒：
pEE12.4-IgGκ-hIgG1，包含有小鼠IgGκ的信号肽和人IgG1的Fc序列，用于表达抗体。
pEE12.4-IgGκ-hIgG1-Fc-hole和pEE12.4-IgGκ-hIgG1-Fc-knob用于表达异二聚体蛋白。
001-OX40 VH-CH1-Fc-knob、002-OX40 VL-CL用于表达异二聚体蛋白的抗体部分；pEE12.4-Wt IL-2-Fc-hole、用于表达异二聚体蛋白的IL-2部分。
2、实验动物
野生型C57BL/6、BALB/c小鼠和BALB/c-nude小鼠购于中国北京维通利华实验动物心。除特殊说明外，所有实验使用的均为8-10周龄的雌性小鼠。小鼠均在无特定病原微生物(specific pathogen-free，SPF)的屏障环境中饲养。动物的饲养和实验操作遵从中国科学院生物物理研究所动物管理委员会的相关规定。
3、细胞系
MC38为C57背景小鼠结直肠癌细胞系，
MC38-EGFR5是分别利用MC38感染表达人鼠嵌合的表皮生长因子(EGFR)的慢病毒并筛选得出的单克隆细胞系，
TUBO为BALB/c背景的小鼠乳腺癌细胞系，B16F10为C57背景小鼠黑色素瘤细胞系。
上述这些细胞系均在DMEM完全培养基(含10％灭活胎牛血清，2mmol/l L-谷氨酰胺，0.1mmol/l非必需氨基酸，100U青霉素和100μg/ml链霉素)中培养。
TIB-210TM杂交瘤细胞系(ATCC)，用于表达CD8+T细胞的删除抗体(clone:2.43)。
TIB-207TM杂交瘤细胞系(ATCC)，用于表达CD4+T细胞的删除抗体(clone:GK1.5)。
PK136杂交瘤细胞系用于生产NK细胞的删除抗体。
HB-197TM杂交瘤细胞系(ATCC)，用于表达阻断小鼠的FcγRII/III的抗体(clone:2.4G2)。
FreeStyleTM 293F细胞系(Invitrogen)为悬浮细胞，源自于HEK293细胞株，培养在SMM293-TII或者CD OptiCHOTM培养基中，主要用于瞬时转染表达融合蛋白。
CTLL-2细胞系为小鼠T细胞系，用于检测IL-2的生物活性
上述细胞系在RPMI1640完全培养基(含10％灭活胎牛血清，2mmol/L L-谷氨酰胺，0.1mmol/L非必需氨基酸，100U青霉素和100μg/ml链霉素，100IU/ml重组IL2)中培养。
基因及引物的设计与合成
人的野生型及突变型的IL-2基因序列如下SEQ ID NO.1所示。实验中所用引物均通过DNAMAN软件设计并由Invitrogen公司合成。
小鼠肿瘤接种及治疗
(1)肿瘤接种及测量：
肿瘤模型的建立，
5-7.5×10 5个MC38、MC38-EGFR5单细胞悬于100μl PBS中，皮下接种于C57BL/6小鼠背部；
5-7.5×10 5个TUBO单细胞悬于100μl PBS中,接种于BALB/c小鼠背部皮下。
对肿瘤消退的小鼠进行同一种肿瘤细胞的re-challenge实验时，肿瘤细胞的接种数量为初始肿瘤造模时候的5倍，接种部位为小鼠背部另一侧皮下。每周监测两次肿瘤大小，使用游标卡尺测量肿瘤长径(a)、短径(b)和高(c)，小鼠肿瘤体积＝a×b×c/2。
(2)治疗：
抗体或者抗体融合蛋白采用腹腔注射方式，部分实验也采用肿瘤内给药的方式，具体给药剂量将在具体实验中叙述。
单克隆抗体制备(小鼠腹水法)
实验中用到的CD4+T细胞删除型抗体GK1.5、CD8+T细胞删除型抗体TIB210、NK细胞删除抗体PK136以及FcRII/III封闭抗体均来自相应的杂交瘤细胞，由本实验室生产、纯化。
小鼠体内细胞删除
(1)CD4+T细胞、CD8+T细胞的删除：
IL-2或IL-2融合蛋白治疗前一天分别腹腔注射200μg GK1.5或者TIB210抗体用来删除CD4+T细胞、CD8+T细胞，之后每隔3天注射一次，依据治疗周期调整注射次数。通过流式检测删除效率。
(2)NK细胞以及中性粒细胞的删除：
IL-2或IL-2融合蛋白治疗前一天分别腹腔注射400μg PK136或者1A8抗体删除NK细胞或者中性粒细胞。流式检测删除效率。
T细胞迁出阻断
FTY720(购自Sigma公司)是一种免疫抑制剂，可以减少T细胞从淋巴器官迁出外周血液循环。在本发明中，小鼠肿瘤接种的不同时期进行FTY720阻断以改变肿瘤微环境。在对小鼠肿瘤治疗过程中进行阻断：肿瘤治疗前1天腹腔注射20μg FTY720，之后每隔一天进行腹腔注射10μg，阻断时间依据治疗周期而定，这样会造成在肿瘤治疗的过程中，没有新迁入肿瘤组织的T细胞。借助FTY720阻断方案，可以研究肿瘤组织中浸润的淋巴细胞的重要性。
如无特别说明，以下实施例1-5中的IL2/aOX40-Fc双特异性抗体的分子结构都为图4A所示的分子结构，即一分子IL2-Fc和一分子aOX40(Fab)-Fc组成的异源二聚体。
实施例1、IL2/aOX40-Fc双特异性抗体比单独治疗效果显著改善
1、IL2-Fc融合蛋白的治疗能部分控制肿瘤
单独的游离free IL2半衰期短且需要多次给药，为了延长IL2的半衰期我们设计了IL2-Fc融合蛋白(结构示意图见图1A)，并在MC38肿瘤模型中验证融合蛋白的功能。与未治疗的对照组相比IL2-Fc能够有效控制肿瘤的生长，但是在肿瘤较大时单独的IL2-Fc治疗未能完全清除肿瘤(图1B)。C57BL/6小鼠皮下接种5 ×10 5MC38肿瘤细胞，荷瘤小鼠在D12天开始分组治疗(n＝5/组)：腹腔注射(ip)9ug IL2-Fc融合蛋白，每三天治疗一次，共治疗三次。
结果显示，单独给予IL2-Fc融合蛋白治疗并不能持续抑制肿瘤的生长。
2、anti-OX40抗体促进T细胞活化并发挥抗肿瘤功能
单独IL2-Fc融合蛋白的治疗只能部分控制肿瘤的生长，为了进一步提高治疗的效果我们采取联合治疗的策略。OX40分子只在Treg细胞和活化的T细胞上表达而在
Figure PCTCN2021127523-appb-000001
T细胞上不表达，这样靶向OX40的抗体具有较高的特异性；而且OX40在Treg细胞和效应细胞上的表达丰度不同，也为相同靶点对不同细胞产生不同效应提供了理论基础。为了验证OX40信号通路能否活化T细胞并发挥抗肿瘤作用，我们进行了脾脏
Figure PCTCN2021127523-appb-000002
T细胞体外刺激实验。具体方法为：分离小鼠的脾脏细胞并按3×10 5细胞/孔铺96孔板，分别用aCD3和aCD28或者aCD3和aOX40抗体刺激72h后流式分析CD3 T细胞上CD69的表达水平，aCD3、aCD28抗体浓度为1ug/ml，aOX40抗体浓度为2ug/ml。
结果显示，相比较aCD3和aCD28对T细胞的共刺激效应，aOX40抗体和aCD3联合使用发挥了和aCD28相类似的刺激作用，能够有效活化CD3 T细胞(图2A)。
我们进一步在小鼠的抗肿瘤实验中验证anti-OX40的功能，具体方法为：野生型C57BL/6小鼠皮下接种5×10 5MC38肿瘤细胞，肿瘤生长到100-150mm 3开始分组治疗(n＝5/组)：腹腔注射(ip)16ug aOX40抗体，三天治疗一次共治疗三次。
结果显示，与未治疗组相比，腹腔注射(ip)16ug aOX40抗体能有效控制肿瘤生长(图2B)。
3、IL2-Fc融合蛋白和aOX40联合治疗并未明显提高肿瘤的治疗效果
IL2-Fc单独使用会扩增Treg，而aOX40抗体能够删除瘤内Treg细胞，为了验证IL2-Fc和aOX40抗体联合使用是否能够在活化效应T细胞同时删除扩增的Treg，发挥协同治疗的效果。我们在MC38荷瘤小鼠上同时给予两种蛋白治疗，方法为：C57BL6小鼠皮下接种5×10 5MC38肿瘤细胞，肿瘤生长到D14开始治疗，单独的治疗组分别腹腔注射(ip)6ug IL2-Fc或者9ug aOX40抗体，联合治疗组同时腹腔注射两种抗体；每三天治疗一次共治疗三次。
结果显示，与单独的aOX40抗体或者IL2-Fc治疗相比，联合治疗并未能进一步提高IL2-Fc的治疗效果(图3)。
4、IL2/aOX40-Fc双特异性抗体具有协同治疗的效果
4.1、双特异性抗体的构建
由上述实验结果分析发现，简单的混合性联合治疗并不具有明显的协同治疗效果，甚至没有叠加效果，提示我们两种抗体单独结合并不能同时发挥出既活化效应细胞又删除Treg的效果。据此，为了降低IL2结合、扩增Treg细胞，我们构建了两种结构一致的双特异性抗体(IL2/aOX40 wt Fc和IL2/aOX40 no ADCC Fc)，每个抗体分子仅含有一个IL2分子(融合蛋白结构见图4A)，该分子中，Fc二聚体为人IgG的Fc二聚体或删除ADCC效应功能的Fc二聚体，一条Fc单体与WT型IL2蛋白连接，另一条Fc单体与aOX40抗体的Fab段连接。构建完成的融合蛋白质粒转染293F细胞，七天后收细胞上清。离心上清后用protein A柱子纯化蛋白，纯化后的抗体经蛋白胶鉴定。经SDS-PAGE分析蛋白分子组成，证明双特异性分子能够同时完好表达aOX40抗体的轻链、重链和IL2分子(图4B)。
我们同时设计和构建了包含IL2和aOX40不同形式的抗体：
图4C示意的结构为异源二聚体，抗体包含一个IL2分子，aOX40(抗OX40抗体)为Fab或scFv形式，Fc二聚体为人IgG的Fc单体和删除ADCC效应功能的人IgG的Fc单体形成的异源二聚体。
图4D示意的结构中，aOX40(抗OX40抗体)为scFv形式，在V区的N端或Fc的C端连接一个或两个IL2分子，Fc二聚体为野生型人IgG的Fc同源二聚体，或人IgG的Fc单体和删除ADCC效应功能的人IgG的Fc单体形成的异源二聚体；
图4 E-F示意的结构中，aOX40(抗OX40抗体)为Fab形式，在抗体轻链的N端或C端、在抗体重链的N端或Fc的C端连接一个或两个IL2分子，Fc二聚体为野生型人IgG的Fc同源二聚体，或人IgG的Fc单体和删除ADCC效应功能的人IgG的Fc单体形成的异源二聚体。
4.2、验证双特异性抗体的功能，
(1)体外CTLL2增殖实验：
在CTLL2细胞培养基中加入不同浓度的IL2-Fc、IL2/aOX40 wt Fc和IL2/aOX40 no ADCC Fc蛋白(图4A所示结构)，72h后通过CCK8试剂盒检测CTLL2细胞在不同蛋白浓度下的增值水平，结果显示IL2/aOX40-Fc抗体和IL2-Fc分子一样能够有效的扩增CTLL2细胞，说明构建的抗体分子保留了IL2活性(图5A)。
(2)体内小鼠荷瘤实验：
在C57BL6小鼠皮下接种5×10 5MC38肿瘤细胞，在接种肿瘤后D14开始治疗；分别通过腹腔注射9ug IL2-Fc、16ug aOX40抗体、与aOX40抗体等摩尔数的IL2-Fc+aOX40抗体或IL2/aOX40 wt Fc蛋白。在D14、D17、D20天共治疗三次，每周两次测量肿瘤大小；结果显示，与单独的aOX40抗体、IL2-Fc以及两者混合治疗相比，IL2/aOX40-wtFc双特异抗体具有更好的治疗效果(图5B)。
除了MC38模型外，在B16F10荷瘤小鼠模型上也进行同样的实验，在C57BL6小鼠皮下接种5×10 5B16F10肿瘤细胞，在接种肿瘤后D8开始治疗；通过腹腔注射15ug IL2/aOX40 wt Fc蛋白；在D8、D11和D14天共治疗三次，结果显示，IL2/aOX40 wt Fc也能发挥显著的肿瘤控制作用(图5D)。
进一步的，在IL2/aOX40-Fc治疗组，肿瘤清除后两个月进行肿瘤re-challenge实验。在IL2/aOX40 wt Fc组，治疗完全清除肿瘤的小鼠两个月后进行肿瘤re-challenge试验，皮下接种五倍剂量的(2.5×10 6个)MC38肿瘤细胞，re-challenge只接种肿瘤细胞，接种后各组均不再给药治疗。
结果显示，与对照组相比，再次接种五倍剂量的MC38细胞并不会引起肿瘤的生长，说明双特异性抗体治疗诱导小鼠产生记忆性免疫细胞(图5C)。
实施例2、IL2/aOX40-Fc能够激活CD8 T细胞
1、IL2/aOX40-Fc双特异抗体治疗效果不依赖于NK细胞
由于CD25(IL-2受体α)和OX40主要表达在活化的效应T细胞和NK细胞上，为了确定IL2/aOX40-Fc抗体的治疗主要依赖于哪一群免疫细胞，我们分别进行了不同细胞群的删除实验。
C57BL6小鼠皮下接种5×10 5MC38肿瘤细胞，在第12、15、18天腹腔注射25ug IL2/aOX40 wt Fc融合蛋白。在治疗开始前一天腹腔注射400ug NK细胞删除性抗体PK136(本实验室制备)，每4天一次，共注射3次。
图6A显示在删除NK细胞1天后，外周血NK细胞的变化情况。在小鼠肿瘤实验中，删除NK细胞后IL2/aOX40-Fc抗体仍然具有治疗效果，说明NK细胞不是抗体发挥治疗效果的主要效应细胞(图6B)。
2、IL2/aOX40-Fc抗体治疗效果依赖于CD8 T细胞
我们进一步验证T细胞在抗体治疗过程中发挥的作用。
C57小鼠背部皮下接种5×10 5MC38肿瘤细胞后第12天开始治疗，腹腔注射25ug IL2/aOX40 wt Fc蛋白(肿瘤接种后分别在第12、15、18天给药)。在治疗的同时腹腔注射200ug CD4 T细胞删除性抗体(克隆号：GK1.5，本实验室制备)、200ug CD8 T细胞删除性抗体(克隆号：TIB210，本实验室制备)或者同时注射两种删除性抗体，共注射三次。
CD4 T和CD8 T细胞的删除实验表明，删除CD4 T细胞后抗体治疗效果无显著下降，但在删除CD8 T细胞后，治疗效果有明显的降低；同时删除CD4 T和CD8 T细胞后，抗体治疗效果也完全消失。说明IL2/aOX40-Fc抗体的治疗效果依赖于T细胞，而且主要是CD8 T细胞(图7B)。
3、IL2/aOX40-Fc双特异型抗体的功能依赖于瘤内的T细胞
为了进一步检测治疗过程中是瘤内浸润的T细胞发挥主要作用还是外周迁移的T细胞发挥主要作用，我们进行了FTY720阻断实验。FTY720是临床上可以抑制器官移植过程中免疫排斥反应的一种药物，抑制T细胞由淋巴器官向外周血的迁移[19]。
单独的IL2/aOX40 wt Fc抗体治疗可以清除肿瘤，在治疗的同时进行FTY720的阻断，实验方案如下：
C57BL6小鼠背部皮下接种4×10 5MC38肿瘤细胞，在肿瘤接种后第7、10和13天，腹腔注射15ug IL2/aOX40-Fc抗体。在FTY720处理组，在肿瘤接种后第6天腹腔注射25ug FTY720，并继续在第8，10和12天分别注射10ug。
结果显示，对外周淋巴细胞向肿瘤迁移的抑制并不影响双特异抗体治疗效果(图8B)，表明IL2/aOX40-Fc抗体发挥抗肿瘤作用主要依赖于浸润到瘤内的效应T细胞。
4、IL2/aOX40-Fc的治疗对远端转移型肿瘤的保护作用
为了验证原位的肿瘤治疗产生的免疫保护是否对其它转移位点的肿瘤也具有抑制作用，我们进行了小鼠双肿瘤模型实验。我们同时在小鼠皮下左右两侧分别接种肿瘤细胞并只在其中一侧进行原位治疗，观察远端的肿瘤是否也能得到控制。实验方案如下：
C57BL6小鼠在背部的左右两侧分别接种2×10 5MC38和4×10 5MC38肿瘤细胞(在背部右侧接种4×10 5MC38肿瘤细胞，在左侧接种2×10 5MC38肿瘤细胞)。在第七天右侧的肿瘤长到50mm 3左右，左侧的肿瘤在30mm 3左右开始治疗：
治疗组1，在右侧肿瘤的瘤内注射7.5ug IL2/aOX40 wt Fc抗体(治疗抗体在第7、10、13天共治疗三次)，左侧肿瘤不治疗；
治疗组2，在右侧肿瘤的瘤内注射7.5ug IL2/aOX40 wt Fc抗体，同时在右侧肿瘤瘤内注射20ug TIB210抗体，删除瘤内CD8 T细胞。治疗和删除抗体在第7、10、13天共治疗三次，左侧肿瘤不治疗。
结果如图9所示，
(1)仅对右侧肿瘤进行瘤内注射的原位治疗，不仅能够清除原位的肿瘤而且能够控制对侧远端未治疗的肿瘤。
(2)同时为了验证远端肿瘤的控制是否依赖于治疗侧原位肿瘤的CD8 T细胞，在治疗的同时我们删除原位肿瘤的CD8 T细胞。在删除CD8 T细胞后，原位和远端肿瘤治疗效果都消失了，说明原位肿瘤内的CD8 T细胞对控制远端肿瘤发挥重要的作用。
实施例3、IL2/aOX40-Fc能够删除瘤内Treg并提高CD8/Treg细胞比例
1、瘤内Treg细胞高表达CD25和OX40分子
为了进一步研究IL2/aOX40-Fc抗体的抗肿瘤机制，我们分析了荷瘤小鼠T细胞上CD25和OX40分子的表达水平以分析IL2/aOX40-Fc抗体主要结合到哪一群细胞上发挥功能。在接种MC38肿瘤后，第12天我们分别分析小鼠肿瘤内、引流淋巴结和脾脏T细胞上IL2受体CD25和OX40的表达：具体方案：小鼠接种5×10 5MC38肿瘤细胞，在第12天分别取荷瘤小鼠的肿瘤组织、脾脏和引流淋巴结(dLN)，流式检测不同细胞上CD25和OX40表达。
发现在荷瘤小鼠中，CD25和OX40均在Treg细胞上高表达，而且瘤内Treg比外周引流淋巴结和脾脏Treg表达更高水平CD25和OX40。非Treg的CD4 T和CD8 T的表达水平比Treg细胞低，瘤内的CD8 T细胞的CD25和OX40表达水平最低(图10A、B)。
2、IL2/aOX40-Fc抗体删除瘤内Treg细胞，提高CD8/Treg细胞比例
由于CD25和OX40高表达在瘤内的Treg细胞上，我们推测IL2/aOX40-Fc抗体腹腔注射后可能更多的结合到Treg上。在MC38荷瘤小鼠在接种肿瘤后第12天开始治疗，腹腔注射25ug IL2/aOX40-Fc抗体，在第15天取肿瘤组织、引流淋巴结和脾脏，流式分析瘤内Treg细胞的比例、CD8 T/Treg。通过流式实验分析，我们发现相比于IL2-Fc和aOX40的单独治疗，IL2/aOX40-Fc能够更有效的删除瘤内的Treg细胞，并提高CD8 T/Treg比例而具有最好的治疗效果(图11A，B)。相比较于对瘤内Treg的删除，IL2/aOX40-Fc并不改变外周肿瘤引流淋巴结和脾脏中Treg/CD4 T比例，说明IL2/aOX40-Fc治疗不会打破外周的免疫耐受具有更好的安全性(图11C)。
3、IL2/aOX40-Fc抗体治疗效果依赖于Fc与FcγR的结合
通过对MC38荷瘤小鼠瘤内淋巴细胞的分析，我们发现IL2/aOX40-Fc抗体具有删除Treg的作用，为了验证Treg删除是否是IL2/aOX40-Fc抗体发挥抗肿瘤作用的主要机制，我们通过对Fc区域点突变构建了IL2/aOX40-no ADCC Fc抗体，突变后的Fc不与FcγR受体结合而失去ADCC、ADCP介导的删除功能。使用MC38肿瘤模型，C57BL6小鼠接种MC38肿瘤细胞，接种后第13天开始治疗。腹腔注射15ug wt Fc或者no ADCC Fc抗体，每三天治疗一次，共治疗三次。
结果显示，接种肿瘤后，对比wt Fc与no ADCC Fc的治疗效果，发现突变的Fc仅部分降低了IL2/aOX40抗体的治疗效果(图12)。IL2/aOX40-no ADCC Fc抗体治疗组中，抗体仍然保留部分抗肿瘤作用，说明IL2/aOX40-Fc抗体功能的发挥不仅仅依赖对Treg细胞进行删除，还可以通过与效应T细胞的结合发挥治疗作用。
实施例4、IL2/aOX40-Fc联合PDL1抗体治疗
1、aPDL1能够协同IL2/aOX40-Fc抗体增强抗肿瘤效果
在MC38荷瘤小鼠肿瘤在200mm 3以下时，单独的IL2/aOX40-Fc抗体治疗具有很好的清除作用。但是当肿瘤更大时，单独的抗体治疗只能控制肿瘤生长而不能完全清除；为了更好的提高治疗效果，我们将双特异性抗体和免疫检查点抑制性抗体联合使用观察能否提高治疗效果。
具体试验方案：
方案一：C57BL6小鼠皮下接种MC38细胞并在第16天开始治疗，
(1)单独的IL2/aOX40-Fc治疗组第16、19和22天腹腔注射25ug IL2/aOX40-Fc；
(2)aOX40和aCTLA4联合组第16天腹腔注射250ug抗体两天后注射25ug IL2/aOX40-Fc(aOX40和aCTLA4抗体仅给药一次，25ug IL2/aOX40-Fc腹腔注射给药三次)；
(3)aPDL1联合治疗组在注射25ug IL2/aOX40-Fc同时注射250ug aPDL1抗体(16、19、22天)。
方案二：MC38荷瘤小鼠，第12、15和18天腹腔注射200ug aPDL1抗体，第14、17和20天腹腔注射15ug wt Fc或者no ADCC Fc的IL2/aOX40抗体。
结果显示：
(1)在IL2/aOX40-Fc与aCTLA4或aOX40联合治疗的过程中，发现这两类抗体的联合使用并不能进一步提高IL2/aOX40-Fc的治疗效果(图13A)。
(2)在和aPDL1的联合治疗过程中发现，两者的联合使用具有协同作用效果(图13A)。
(3)为了进一步分析aPDL1与IL2/aOX40-Fc抗体的作用机制，我们分别使用wt Fc或者no ADCC Fc的IL2/aOX40-Fc抗体与aPDL1联合使用，发现更好的联合治疗效果依赖于wt Fc(图13B)。
实施例5、IL2/aOX40-Fc双功能抗体拮抗TKI治疗后的肿瘤复发
临床上针对EGFR突变肿瘤患者的治疗方案主要是使用靶向受体的抗体或者针对下游信号通路的激酶抑制剂。TKI对于ErBB基因家族相关的肿瘤具有很好的治疗效果，阿法替尼(Afatinib)是一种第二代激酶抑制剂，以共价结合的方式不可逆抑制受体的酪氨酸激酶活性。在TUBO模型上分析了阿法替尼的作用机制，发现治疗后瘤内CD3 T/CD45淋巴细胞的比例上升，说明在化疗药的治疗过程中有更多的T细胞浸润到瘤内(图14A)。单独的Afatinib治疗虽然增加了T细胞向肿瘤的浸润，但不能有效降低瘤内Treg细胞的比例(图14B)。临床上单独的TKI治疗难以完全清除肿瘤，我们推测瘤内的Treg细胞抑制了效应细胞对肿瘤的杀伤，进而导致治疗后的肿瘤复发。
为了验证IL2/aOX40-Fc是否能够拮抗化疗药治疗后的复发，我们在给荷瘤小鼠化疗药治疗的同时联合使用IL2/aOX40-Fc抗体。
具体实验方案：
BALB/c小鼠皮下接种5×10 5TUBO肿瘤细胞，
治疗方案1：在第9天给予1mg Afatinib灌胃进行治疗，6天后取肿瘤组织进行流式分析。
治疗方案2：荷瘤小鼠在肿瘤接种后第14天开始治疗，
(1)Afatinib治疗组在第14天灌胃1mg Afatinib，
(2)IL2/aOX40-Fc治疗组则在第14、17和20天腹腔注射25ug抗体，
(3)联合治疗组按照上述方案Afatinib灌胃一次，25ug IL2/aOX40-Fc腹腔抗体治疗三次。
结果显示，治疗方案1中的单独的阿法替尼治疗组，肿瘤在得到部分控制后很快复发；而在治疗方案2中，和IL2/aOX40-Fc抗体联合治疗组，可以有效的控制肿瘤的生长，部分小鼠的肿瘤被完全清除(图14C)。
实施例6、IL2/aCTLA4-Fc双特异性抗体显著改善肿瘤治疗效果
体内小鼠荷瘤实验：在C57BL6小鼠皮下接种5×10 5MC38肿瘤细胞，在接种肿瘤后D11开始治疗；分别通过腹腔注射15ug IL2/aCTLA4 wt Fc蛋白(该IL2/aCTLA4 wt Fc蛋白的具体结构示意图如图15所示)。在D11、D14、D17天共治疗三次，每周两次测量肿瘤大小；
结果显示，IL2/aCTLA4-Fc双特异抗体有效控制肿瘤(图15)。
实施例7、IL2/a4-1BB-Fc双特异性抗体显著改善肿瘤治疗效果
在C57BL/6小鼠皮下接种5×10 5MC38肿瘤细胞，在接种肿瘤后D10开始治疗；分别通过腹腔注射10ug 3H3-IL2和10ug LOB12.3-IL2 wt Fc融合蛋白(IL2/a4-1BB-Fc融合蛋白的结构示意图如图16所示)。在肿瘤接种后的第10、16和19天治疗三次，每周两次测量肿瘤大小；结果显示，与对照组相比，2种融合蛋白的治疗组肿瘤体积明显缩小，差异极显著。
实验证明IL2/a4-1BB-Fc融合蛋白具有良好的肿瘤治疗效果(图16)。
最后需要说明的是，以上实施例仅用作帮助本领域技术人员理解本发明的实质，不用于限制本发明的保护范围。
[参考文献]
[1]MORGAN D A,RUSCETTI F W,GALLO R.Selective in vitro growth of T lymphocytes from normal human bone marrows[J].Science,1976,193(4257):1007-8.
[2]ROBB R J,SMITH K A.Heterogeneity of human T-cell growth factor(s)due to variable glycosylation[J].Molecular immunology,1981,18(12):1087-94.
[3]TANIGUCHI T,MINAMI Y.The IL-2IL-2 receptor system:a current overview[J].Cell,1993,73(1):5-8.
[4]MALEK T R.The biology of interleukin-2[J].Annu Rev Immunol,2008,26(453-79.
[5]LEONARD W J,DEPPER J M,CRABTREE G R,et al.Molecular cloning and expression of cDNAs for the human interleukin-2 receptor[J].Nature,1984,311(5987):626-31.
[6]NIKAIDO T,SHIMIZU A,ISHIDA N,et al.Molecular cloning of cDNA encoding human interleukin-2 receptor[J].Nature,1984,311(5987):631-5.
[7]LIAO W,LIN J-X,LEONARD W J.Interleukin-2 at the crossroads of effector responses,tolerance,and immunotherapy[J].Immunity,2013,38(1):13-25.
[8]FLYNN M J,HARTLEY J A.The emerging role of anti‐CD 25 directed therapies as both immune modulators and targeted agents in cancer[J].British journal of haematology,2017,179(1):20-35.
[9]FONTENOT J D,RASMUSSEN J P,GAVIN M A,et al.A function for interleukin 2 in Foxp3-expressing regulatory T cells[J].Nature immunology,2005,6(11):1142-51.
[10]SIEGEL J P,SHARON M,SMITH P L,et al.The IL-2 receptor beta chain(p70):role in mediating signals for LAK,NK,and proliferative activities[J].Science,1987,238(4823):75-8.
[11]CHARYCH D,KHALILI S,DIXIT V,et al.Modeling the receptor pharmacology,pharmacokinetics,and pharmacodynamics of NKTR-214,a kinetically-controlled interleukin-2(IL2)receptor agonist for cancer immunotherapy[J].PloS one,2017,12(7):e0179431.
[12]LEVIN A M,BATES D L,RING A M,et al.Exploiting a natural conformational switch to engineer an interleukin-2‘superkine’[J].Nature,2012,484(7395):529-33.
[13]SUN Z,REN Z,YANG K,et al.A next-generation tumor-targeting IL-2 preferentially promotes tumor-infiltrating CD8+T-cell response and effective tumor control[J].Nature communications,2019,10(1):1-12.
[14]MARABELLE A,KOHRT H,SAGIV-BARFI I,et al.Depleting tumor-specific Tregs at a single site eradicates disseminated tumors[J].The Journal of clinical investigation,2013,123(6):2447-63.
[15]CURTI B D,KOVACSOVICS-BANKOWSKI M,MORRIS N,et al.OX40 is a potent immune-stimulating target in late-stage cancer patients[J].Cancer research,2013,73(24):7189-98.
[16]SONG A,SONG J,TANG X,et al.Cooperation between CD4 and CD8 T cells for anti‐tumor activity is enhanced by OX40 signals[J].European journal of immunology,2007,37(5):1224-32.
[17]BULLIARD Y,JOLICOEUR R,ZHANG J,et al.OX40 engagement depletes intratumoral Tregs via activating FcγRs,leading to antitumor efficacy[J].Immunology and cell biology,2014,92(6):475-80.
[18]LINCH S N,MCNAMARA M J,REDMOND W L.OX40 agonists and combination immunotherapy:putting the pedal to the metal[J].Frontiers in oncology,2015,5(34).
[19]CHIBA K.FTY720,a new class of immunomodulator,inhibits lymphocyte egress from secondary lymphoid tissues and thymus by agonistic activity at sphingosine 1-phosphate receptors[J].Pharmacology&therapeutics,2005,108(3):308-19.

Claims (18)

  1. 一种双功能融合蛋白，所述的融合蛋白为异源二聚体，其特征在于，
    所述的异源二聚体包括：
    (1)白介素2(IL-2)与免疫球蛋白Fc单链连接而成的第一单体；
    (2)抗CTLA4或TNF家族的共刺激或共抑制性分子的抗体的Fab或ScFv与免疫球蛋白Fc单链连接而成的第二单体；
    所述的第一单体与第二单体通过Fc单链的二聚化连接，构成所述的异源二聚体；
    所述的CTLA4或TNF家族的共刺激或共抑制性分子包括但不限于：OX40、ICOS、GITR、4-1BB。
  2. 根据权利要求1所述的双功能融合蛋白，其特征在于，所述的免疫球蛋白Fc单链为天然免疫球蛋白Fc单链或通过基因突变敲除ADCC效应的免疫球蛋白Fc单链；优选的，所述的免疫球蛋白Fc单链为人IgG的Fc单链。
  3. 根据权利要求1任一所述的双功能融合蛋白，其特征在于，
    所述的第二单体中，
    所述的抗体的Fab为人源化抗体的Fab或全人化抗体的Fab；
    所述的抗体的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；
    优选的，所述的第二单体为：
    抗CTLA4或TNF家族的共刺激或共抑制性分子的抗体的单体，所述抗体的单体包含一条轻链和一条重链；
    更优选的，所述的第二单体为：
    人源化抗CTLA4或抗TNF家族的共刺激或共抑制性分子的抗体的单体或全人化抗CTLA4或抗TNF家族的共刺激或共抑制性分子的抗体抗体的单体，所述抗体的单体包含一条轻链和一条重链。
  4. 根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，
    所述的CTLA4或TNF家族的共刺激或共抑制性分子为OX40、4-1BB，
    所述的抗CTLA4的抗体(aCTLA4)或抗TNF家族的共刺激或共抑制性分子的抗体为抗OX40的抗体(aOX40)、或抗4-1BB的抗体(a4-1BB)。
  5. 根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，
    所述的异源二聚体包括：
    (1)第一单体，所述的第一单体自N端开始依次包含：
    1)序列如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；
    2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；
    3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；
    (2)第二单体，所述的第二单体包含：
    1)序列如SEQ ID NO.7所示的抗OX40抗体轻链VL-KCL和序列如SEQ ID NO.8所示的抗OX40抗体重链VH&CH1组成的抗OX40抗体Fab区；
    或：2)序列如SEQ ID NO.9所示的抗OX40单链抗体(ScFv)：
    和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc。
  6. 根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，
    所述的异源二聚体包括：
    第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；
    第二单体：其为：
    (1)序列如SEQ ID NO.11所示的抗OX40抗体VH-CH1-Fc(knob)和序列如SEQ ID NO.7所示的抗OX40抗体轻链VL-KCL组成；
    或(2)序列如SEQ ID NO.12所示的多肽(aOX40 ScFv-Fc(knob))。。
  7. 根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，
    所述的异源二聚体包括：
    (1)第一单体，所述的第一单体自N端开始依次包含：
    1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；
    2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；
    3)如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；
    (2)第二单体，所述的第二单体包含：
    1)序列如SEQ ID NO.21所示的抗CTLA4抗体轻链与序列如SEQ ID NO.22所示的抗CTLA4抗体重链VH&CH1组成的抗体Fab区；
    或：2)序列如SEQ ID NO.23所示的抗CTLA4单链抗体(ScFv)：
    和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc。
  8. 根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，
    所述的异源二聚体包括：
    第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；
    第二单体：其为：
    (1)序列如SEQ ID NO.24所示的多肽(aCTLA4 VH-CH1-Fc(knob))和序列如SEQ ID NO.21所示的抗CTLA4抗体轻链可变区组成；或
    (2)序列如SEQ ID NO.25所示的多肽(a CTLA4 ScFv-Fc(knob))。
  9. 根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，
    所述的异源二聚体包括：
    (1)第一单体，所述的第一单体自N端开始依次包含：
    1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；
    2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；
    3)如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；
    (2)第二单体，所述的第二单体包含：
    1)序列如SEQ ID NO.28、29所示的抗4-1BB抗体轻链与序列如SEQ ID NO.30、31所示的抗4-1BB 抗体重链VH&CH1组成的抗体Fab区；
    或：2)序列如SEQ ID NO.32、33所示的抗4-1BB单链抗体(ScFv)：
    和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc。
  10. 根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，
    所述的异源二聚体包括：
    第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；
    第二单体：其为：
    (1)序列如SEQ ID NO.34、35所示的多肽(a4-1BB VH-CH1-Fc(knob))和序列如SEQ ID NO.28、29所示的抗4-1BB抗体轻链组成；或
    (2)序列如SEQ ID NO.36、37所示的多肽(a4-1BB ScFv-Fc(knob))。
  11. 一种双功能融合蛋白，所述的融合蛋白为同源二聚体，其特征在于，
    所述的同源二聚体的单体为：
    一分子白介素2(IL-2)与一分子抗OX40的抗体(aOX40)、抗CTLA4的抗体(aCTLA4)或抗4-1BB的抗体(a4-1BB)的Fab通过任意方式连接构成的单体，或，
    一分子白介素2(IL-2)与一分子抗OX40的抗体(aOX40)、抗CTLA4的抗体(aCTLA4)或抗4-1BB的抗体(a4-1BB)的(ScFv)通过任意方式连接构成的单体。
  12. 根据权利要求11所述的双功能融合蛋白，其特征在于，
    所述的同源二聚体的单体自N端开始依次包含：
    (1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；
    (2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.4、SEQ ID NO.5所示；
    (3)抗OX40的抗体(aOX40)的Fab/ScFv、抗CTLA4的抗体(aCTLA4)的Fab/ScFv或抗4-1BB的抗体(a4-1BB)的Fab/ScFv；所述的Fab为人源化抗体的Fab或全人化抗体的Fab，所述的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；
    (4)抗体的Fc；所述的抗体Fc为全人野生型Fc或No-ADCC突变型Fc。
  13. 编码所述权利要求1-12任一所述的双功能融合蛋白的核苷酸序列。
  14. 根据权利要求13所述的核苷酸序列，其特征在于，
    编码所述异源二聚体第一单体的核苷酸序列为SEQ ID NO.11所示的核苷酸序列；
    编码所述异源二聚体第二单体的核苷酸序列为：
    SEQ ID NO.16(aOX40：VL-KCL)、
    SEQ ID NO.17(aOX40：VH-CH1-Fc(knob))、
    SEQ ID NO.18(aOX40 ScFv-Fc(knob))、
    SEQ ID NO.42(a4-1BB(LOB12.3)：VL-KCL)、
    SEQ ID NO.43(a4-1BB(3H3)：VL-KCL)、
    SEQ ID NO.44(a4-1BB(LOB12.3)：VH-CH1-Fc(knob))、
    SEQ ID NO.45(a4-1BB(3H3)：VH-CH1-Fc(knob))、
    SEQ ID NO.46(a4-1BB(LOB12.3)：ScFv-Fc(knob))、
    SEQ ID NO.47(a4-1BB(3H3)：ScFv-Fc(knob))所示的核苷酸序列；
    编码所述同源二聚体的核苷酸序列为：
    SEQ ID NO.19(aOX40-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
    SEQ ID NO.20(IL2-aOX40：IL2-VL-VH(ScFv)-Fc(wt))、
    SEQ ID NO.48(a4-1BB(LOB12.3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
    SEQ ID NO.49(a4-1BB(3H3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、
    SEQ ID NO.50(IL2-a4-1BB(LOB12.3)：IL2-VL-VH(ScFv)-Fc(wt))、
    SEQ ID NO.51(IL2-a4-1BB(3H3)：IL2-VL-VH(ScFv)-Fc(wt))所示的核苷酸序列。
  15. 权利要求1-12任一所述的双功能融合蛋白的如下应用：
    (1)制备抗肿瘤药物；
    (2)制备与免疫检查点抑制剂联用的抗肿瘤药物；
    (3)制备克服免疫检查点抑制剂耐受的抗肿瘤药物；
    (4)制备与TKI拮抗剂联用的抗肿瘤药物；
    (5)制备克服TKI拮抗剂耐受的抗肿瘤药物。
  16. 根据权利要求15所述的应用，其特征在于，所述的免疫检查点抑制剂为抗PD-L1的拮抗剂，优选的，所述的抗PD-L1的拮抗剂为抗PD-L1的抗体。
  17. 根据权利要求15所述的应用，其特征在于，所述的TKI拮抗剂为小分子TKI拮抗剂；优选的，所述的小分子TKI拮抗剂为阿法替尼或其结构类似物。
  18. 包含权利要求1-12任一所述的双功能融合蛋白的药物或药物组合物。
PCT/CN2021/127523 2020-10-30 2021-10-29 一种il-2与抗体亚单位构成的双功能融合蛋白 WO2022089601A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011189127.4A CN114437228B (zh) 2020-10-30 2020-10-30 一种il-2与抗体亚单位构成的双功能融合蛋白
CN202011189127.4 2020-10-30

Publications (1)

Publication Number Publication Date
WO2022089601A1 true WO2022089601A1 (zh) 2022-05-05

Family

ID=81357118

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/127523 WO2022089601A1 (zh) 2020-10-30 2021-10-29 一种il-2与抗体亚单位构成的双功能融合蛋白

Country Status (2)

Country Link
CN (1) CN114437228B (zh)
WO (1) WO2022089601A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11896648B2 (en) 2020-10-22 2024-02-13 Gilead Sciences, Inc. Interleukin-2 variant proteins fused to human IgG4 Fc and uses thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116554344A (zh) * 2022-01-30 2023-08-08 中国科学院生物物理研究所 一种pd1抗体与白介素2融合的双功能分子

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102199218A (zh) * 2011-04-29 2011-09-28 中国人民解放军军事医学科学院基础医学研究所 一种抗Her2抗体-白细胞介素2融合蛋白及其用途
CN102372780A (zh) * 2010-08-23 2012-03-14 上海市计划生育科学研究所 抗人绒毛膜促性腺激素抗体-白介素2融合蛋白的制备及其应用
WO2013130102A2 (en) * 2012-03-02 2013-09-06 Providence Health & Services - Oregon Dual ox40 agonist/il-2 cancer therapy methods
CN103304669A (zh) * 2012-03-12 2013-09-18 复旦大学 人源化肿瘤免疫细胞因子tntil2及其制备方法和用途
WO2016180715A1 (en) * 2015-05-08 2016-11-17 Philogen S.P.A. Il2 and tnf immunoconjugates
CN108503714A (zh) * 2018-04-10 2018-09-07 浙江科途医学科技有限公司 一种人白介素2和抗人上皮细胞黏附分子单链抗体融合蛋白及其应用
CN110088134A (zh) * 2016-08-02 2019-08-02 阿杜罗生物科技控股有限公司 针对人ctla-4的抗体
CN110256583A (zh) * 2019-07-11 2019-09-20 中国科学院生物物理研究所 一种il-2突变体与抗体的融合蛋白及其应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2006343459A1 (en) * 2005-12-16 2007-11-22 Genentech, Inc. Anti-OX40L antibodies and methods using same
CN102174111B (zh) * 2011-01-25 2013-01-09 江苏省弗泰生物科技有限公司 人白介素2-Fc融合蛋白及其用途
SG10201604160WA (en) * 2011-02-10 2016-07-28 Roche Glycart Ag Mutant Interleukin-2 Polypeptides
CA3026477A1 (en) * 2016-06-20 2017-12-28 Kymab Limited Anti-pd-l1 antibodies
CR20190227A (es) * 2016-10-14 2019-08-29 Xencor Inc Proteínas de fusión heterodiméricas biespecíficas que contienen proteínas de fusión fc il-15/il-15ra y fragmentos de anticuerpo pd-1
CN112646031B (zh) * 2019-10-10 2021-12-07 天境生物科技(上海)有限公司 抗4-1bb纳米抗体及其用途

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102372780A (zh) * 2010-08-23 2012-03-14 上海市计划生育科学研究所 抗人绒毛膜促性腺激素抗体-白介素2融合蛋白的制备及其应用
CN102199218A (zh) * 2011-04-29 2011-09-28 中国人民解放军军事医学科学院基础医学研究所 一种抗Her2抗体-白细胞介素2融合蛋白及其用途
WO2013130102A2 (en) * 2012-03-02 2013-09-06 Providence Health & Services - Oregon Dual ox40 agonist/il-2 cancer therapy methods
CN103304669A (zh) * 2012-03-12 2013-09-18 复旦大学 人源化肿瘤免疫细胞因子tntil2及其制备方法和用途
WO2016180715A1 (en) * 2015-05-08 2016-11-17 Philogen S.P.A. Il2 and tnf immunoconjugates
CN110088134A (zh) * 2016-08-02 2019-08-02 阿杜罗生物科技控股有限公司 针对人ctla-4的抗体
CN108503714A (zh) * 2018-04-10 2018-09-07 浙江科途医学科技有限公司 一种人白介素2和抗人上皮细胞黏附分子单链抗体融合蛋白及其应用
CN110256583A (zh) * 2019-07-11 2019-09-20 中国科学院生物物理研究所 一种il-2突变体与抗体的融合蛋白及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WILLIAM L. REDMOND, TODD TRIPLETT, KEVIN FLOYD, ANDREW D. WEINBERG: "Dual Anti-OX40/IL-2 Therapy Augments Tumor Immunotherapy via IL-2R-Mediated Regulation of OX40 Expression", PLOS ONE, vol. 7, no. 4, pages e34467, XP055214862, DOI: 10.1371/journal.pone.0034467 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11896648B2 (en) 2020-10-22 2024-02-13 Gilead Sciences, Inc. Interleukin-2 variant proteins fused to human IgG4 Fc and uses thereof

Also Published As

Publication number Publication date
CN114437228A (zh) 2022-05-06
CN114437228B (zh) 2024-02-06

Similar Documents

Publication Publication Date Title
US20220002383A1 (en) Multimeric il-15 soluble fusion molecules and methods of making and using same
US20220193197A1 (en) Synergistic tumor treatment with il-2 and integrin-binding-fc fusion protein
EP3013859B1 (en) Bispecific molecules capable of specifically binding to both ctla-4 and cd40
KR102609197B1 (ko) 인터류킨 15 단백질 복합체 및 그의 용도
RU2689717C2 (ru) Гетеродимерный белок il-15 и его применения
JP2021511069A (ja) 抗体Ｆｃ領域を主鎖として用いた融合タンパク質二量体及びその使用
WO2019201161A1 (zh) 一种ifn与抗pd-l1抗体的融合蛋白及其应用
AU2018202982A1 (en) Fusion immunomodulatory proteins and methods for making same
CN110256583B (zh) 一种il-2突变体与抗体的融合蛋白及其应用
US11926654B2 (en) Fusion proteins composed of an interleukin-2 mutein and type I interferon
US10603358B2 (en) Combination tumor treatment with an integrin-binding-Fc fusion protein and immune stimulator
CN108026534B (zh) 嵌合抗原受体和其中表达有嵌合抗原受体的t细胞
CA3120564A1 (en) Plasmid constructs for treating cancer and methods of use
US20230149509A1 (en) Il15/il15r alpha heterodimeric fc-fusion proteins for the treatment of cancer
WO2022089601A1 (zh) 一种il-2与抗体亚单位构成的双功能融合蛋白
CN110396133A (zh) 一种以白介素12为活性成分的融合蛋白型药物前体
WO2023143308A1 (zh) 一种pd1抗体与白介素2融合的双功能分子
CN118027212A (zh) Il-15与t细胞共刺激分子抗体构成的双功能融合蛋白
CN118974101A (zh) 一种pd1抗体与白介素2融合的双功能分子

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21885327

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21885327

Country of ref document: EP

Kind code of ref document: A1