Bifunctional molecules formed by fusing PD1 antibodies with interleukin-2

Abstract

The present invention relates to a bifunctional molecule formed by fusing a PD1 antibody with an interleukin 2, comprising a heterodimer consisting of (1) a first monomer formed by linking interleukin 2 (IL2) to a single immunoglobulin Fc chain and (2) a second monomer formed by linking a Fab / ScFv of an antibody against a T cell surface molecule to the single Fc chain. The bifunctional molecule may also be a homodimer comprising a monomer comprising (1) an interleukin 2 functional block and (2) a monomer of an antibody or a monomer formed by linking a Fab / ScFv of an antibody to a single Fc chain, the antibody being an antibody against a T cell surface molecule. The present invention further relates to the use of the bifunctional molecule in the manufacture of an antitumor drug.

Description

[Technical field]

[0001] The present invention is in the field of biomedical technology and specifically relates to a bifunctional molecule formed by the fusion of PD1 antibody with interleukin-2. [Background technology]

[0002] IL-2 cytokines are potent growth factors for T cells. IL-2 cytokines exert their activity by binding to the IL-2 receptor (IL-2R) on the surface of T cells and NK cells, phosphorylating itself through a JAK / STAT5-dependent pathway, and ultimately inducing the activation and proliferation of the corresponding cells (Non-Patent Document 1). The exact mechanism by which IL-2 exerts its sustained complete response (CR) remains controversial (Non-Patent Document 2). The stimulatory effect of IL-2 has been verified in multiple pathways required for successful generation of adaptive and CTL-mediated antitumor responses (Non-Patent Document 3). Following recognition of antigens in MHC by the T cell receptor (TCR) and binding of the costimulatory molecule CD28 to B7, IL-2 is considered as the third essential signal required for T cell clonal expansion and effector function (Non-Patent Documents 1, 3). Similarly, the function of CD8+ CTLs is also highly dependent on IL-2, and it has been shown that their effectiveness or cytotoxic function is reduced in IL-2-deficient or IL-2R-deficient mice (Non-Patent Documents 4-6). IL-2 increases the trafficking of CTLs to extralymphatic infection or tumor sites (Non-Patent Documents 1, 7). IL-2 is produced by Th1 cells in response to dendritic cell (DC) activation, which activates and proliferates CD8 (Non-Patent Document 3). A unique mechanism of IL-2 antitumor activity may be mediated by activating natural killer (NK) cells (Non-Patent Document 8).

[0003] The efficacy of IL-2 in inducing sustained complete response (CR) and partial response (PR) in patients with clear cell renal cell carcinoma (RCC) (Non-Patent Documents 9-11) was clinically validated in multiple phase II and phase III trials. In contrast, molecular targeted therapy could neither induce CR nor cure cancer. The response rate (RR) of IL-2 therapy (600,000-800,000 IU / kg, q8h x 14 tolerated) reported in multiple phase III trials ranged from 20% to 23.2%, while CR ranged from 7% to 9% (Non-Patent Documents 9-11). In patients who achieved CR, efficacy was sustained in most patients, with median survival exceeding 10 years (Non-Patent Documents 1, 15, 16). Alternative therapies and reduced dose administration of IL-2 were further tested but did not show any improved efficacy (Non-Patent Documents 10-12).

[0004] The tumor microenvironment (TME) usually limits the efficacy of immunotherapy by increasing the production of regulatory T cells (Tregs) and / or decreasing T cell growth factors or their signaling. The main challenge is to provide sufficient cytokines to reactivate cytotoxic T cells (CTLs) or inhibit Tregs. IL-2 is a "T cell growth factor" and a pleiotropic cytokine that is produced upon antigen activation and plays a key role in the immune response. IL-2 is a potent inducer of cytotoxic T cells and NK cells. It is therefore a sought-after treatment in a variety of cancers. However, there are two main obstacles to the use of IL-2 in anticancer immunotherapy. Some T cells, such as Tregs, express a heterotrimeric high-affinity receptor composed of CD25 (IL-2Rα), CD122 (IL-2Rβ) and CD132 (common cytokine receptor γ chain) subunits. In contrast, immature CD8 T cells, CD4 / CD8 memory T cells and NK cells express low affinity dimeric receptors (lacking the CD25 subunit) (Non-Patent Documents 13, 14). When immature CD8 T cells are activated, they upregulate the expression of CD25 (Non-Patent Document 15). Thus, Tregs are better able to compete with effector T cells for the use of IL-2. Currently, IL-2 immunotherapy requires high doses and multiple injections. In addition to selective proliferation of Tregs, high doses of IL-2 can cause vascular leak syndrome, which can lead to increased vascular permeability, hypotension, pulmonary edema, hepatocellular injury and renal failure (Non-Patent Documents 16-18).

[0005] Two fundamentally important strategies can be used to improve the use of IL-2 in immunotherapy: (1) how to maintain activity in tumor tissue while limiting systemic side effects, and (2) how to selectively activate effector T cells while limiting stimulation of Tregs. For the first problem, many research groups hope to reduce the toxicity of IL-2 by using antibody-based IL-2 delivery (Non-Patent Documents 19-24). For the second problem, some researchers have constructed IL-2 mutants that selectively reduce binding to CD25 and improved the proliferation of non-Treg populations (Non-Patent Documents 25-27). Christopher Garcia's team constructed the IL-2 superkine (also called super 2), which eliminated the functional requirement of IL-2 for CD25 expression and increased the binding affinity for IL-2Rβ. Compared to IL-2, the IL-2 superkine induced hyperproliferation of cytotoxic T cells, improved antitumor responses in vivo, and reduced systemic side effects by proportionally reducing the proliferation of T regulatory cells (Non-Patent Document 26). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Malek TR, and Bayer AL. Tolerance, not immunity, crucially depends on IL-2. Nature reviews Immunology. 2004;4(9):665-74. [Non-Patent Document 2] [ PubMed ] Rosenberg SA, Mule JJ, Spiess PJ, Reichert CM, Schwarz SL. Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2. The Journal of Experimental Medicine. 1985;161(5):1169-88.

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[0007] Whether the approach of targeted IL-2 can improve efficacy is controversial. A recent study showed that F8-IL-2, an immunocytokine composed of F8 antibody fused to human IL-2, has a strong and improved inhibition of lymphoma progression compared to targeted IL-2 (Non-Patent Document 21). Another recent study suggested that antigen specificity may not be important for the efficacy and biodistribution of immunocytokines (Non-Patent Documents 28, 29). They found that immunocytokines with unrelated specificity and / or inactive mutant Fc domains behave similarly to tumor-specific IL-2, so that immunocytokine antigen specificity and Fcγ receptor interaction do not seem to be necessary for therapeutic efficacy or biodistribution pattern. They speculated that the biodistribution of IL-2 is mainly related to innate immune cells expressing IL-2R. This difference is thought to be related to the tumor model, the targeting ability of the antibody, and the affinity between IL-2 and IL-2 receptor. Therefore, the targeting effect needs to be evaluated. [Means for solving the problem]

[0008] Therefore, because tumor-infiltrating lymphocytes (TILs) express more T cell surface antigen PD1 than other cells, we designed a bifunctional molecule (IL-2 linked to an antibody against the T cell surface antigen PD1) to target TILs. To reduce IL-2 binding to Tregs, we selected an IL-2 mutant (abIL2) that had significantly reduced binding to IL-2α and IL-2β. We conjugated abIL2 to an antibody against the T cell surface antigen PD1 (anti-PD1-abIL2) to increase its affinity for CD8+ T cells in tumors. Anti-PD1-abIL2 showed improved intratumoral T cell binding and strong antitumor effects. By using such bifunctional molecules, PDL1 treatment resistance can also be overcome.

[0009] Therefore, the anti-PD1-abIL2 bifunctional molecule is a novel and promising tumor treatment in the clinic.

[0010] The present invention relates primarily to bifunctional molecules that are heterodimers, The heterodimer is (1) a first monomer of the heterodimer formed by linking interleukin 2 (IL2) to a single Fc chain of an immunoglobulin; (2) a second monomer of the heterodimer formed by linking a Fab or ScFv of an antibody against a T cell surface molecule to a single Fc chain of an immunoglobulin; the first monomer and the second monomer are linked by dimerization of the single Fc chain to form the heterodimer; the T cell surface molecule includes, but is not limited to, PD1, TIM-3, LAG-3, OX40, 4-1BB, ICOS, and GITR; The single Fc chain of the immunoglobulin is a natural single Fc chain of an immunoglobulin or a single Fc chain of an immunoglobulin whose ADCC effect has been knocked out by genetic mutation; Preferably, the single immunoglobulin Fc chain is a natural single immunoglobulin Fc chain or a single immunoglobulin Fc chain whose ADCC effect has been knocked out by genetic mutation; More preferably, the single immunoglobulin Fc chain is a single human IgG Fc chain.

[0011] the T cell surface molecule is PD1, and the antibody against the T cell surface molecule is an anti-PD1 antibody (aPD1); In the second monomer, the Fab of the antibody is a Fab of a humanized antibody or a Fab of a fully human antibody, the ScFv of the antibody is a humanized antibody ScFv or a fully human antibody ScFv, More preferably, the second monomer is A monomer of an antibody against a T cell surface molecule, comprising a light chain and a heavy chain, Preferably, the antibody is a humanized or fully human antibody.

[0012] More preferably, the heterodimer is: (1) a first monomer comprising, in order from the N-terminus, the following: 1) a wild-type IL-2 protein having the sequence shown in SEQ ID NO:1 or a variant thereof comprising any one or any combination of the following mutations: R38L, F42A, D20K, R38A, F42K and K43E; 2) a required linker structure (G4S linker sequence) preferably having the sequence shown in SEQ ID NO:6, and 3) IgG Fc single chain having the sequence shown in SEQ ID NO:2, or No-ADCC mutant IgG Fc having the sequence shown in SEQ ID NO:3, or knob mutant Fc having the sequence shown in SEQ ID NO:4, or hole mutant Fc having the sequence shown in SEQ ID NO:5. (2) a second monomer comprising: 1) an anti-PD1 antibody Fab region consisting of an anti-PD1 antibody light chain VL-KCL having the sequence shown in SEQ ID NO: 7 and an anti-PD1 antibody heavy chain VH&CH1 having the sequence shown in SEQ ID NO: 8; or 2) an anti-PD1 single chain antibody (ScFv) having the sequence shown in SEQ ID NO: 9; and 3) IgG Fc single chain having the sequence shown in SEQ ID NO:2, or No-ADCC mutant IgG Fc having the sequence shown in SEQ ID NO:3, or knob mutant Fc having the sequence shown in SEQ ID NO:4, or hole mutant Fc having the sequence shown in SEQ ID NO:5. Including, More preferably, the heterodimer is a first monomer (abIL2-Fc) which is a polypeptide having the sequence shown in SEQ ID NO: 10; (1) a second monomer consisting of an anti-PD1 antibody VH-CH1-Fc(knob) having the sequence shown in SEQ ID NO: 11 and an anti-PD1 antibody light chain VL-KCL having the sequence shown in SEQ ID NO: 7; or (2) Polypeptide having the sequence shown in SEQ ID NO: 12 (aPD1ScFv-Fc(knob)) and a second monomer which is

[0013] The present invention also relates to bifunctional molecules which are homodimers, The monomers of the homodimer are a monomer formed by linking a molecule of interleukin 2 (IL2) to a molecule of an anti-PD1 antibody Fab by any means; or A monomer formed by linking a molecule of interleukin 2 (IL2) to a molecule of anti-PD1 single chain antibody (ScFv) by any means. It is.

[0014] Preferably, the monomers of the homodimer are, in order from the N-terminus: (1) a wild-type IL-2 protein having the sequence set forth in SEQ ID NO:1, or a mutant thereof comprising any one or any combination of the following mutations: R38L, F42A, D20K, R38A, F42K, and K43E; (2) an essential linker structure (G4S linker sequence) preferably having the sequence shown in SEQ ID NO:6; (3) an anti-PD1 antibody Fab or ScFv, wherein the Fab is a humanized antibody Fab or a fully human antibody Fab, and the ScFv is a humanized antibody ScFv or a fully human antibody ScFv; (4) an antibody Fc that is a fully human wild-type Fc or a No-ADCC mutant Fc; Includes.

[0015] More preferably, said monomers of said homodimer have the sequence shown below: (1) SEQ ID NO: 18 (aPD1-abIL2: VL-VH(ScFv)-Fc-abIL2); (2) SEQ ID NO: 19 (abIL2-aPD1: abIL2-VL-VH(ScFv)-Fc).

[0016] The present invention also relates to another bifunctional molecule, which is a bifunctional molecule comprising an anti-PD1 antibody (K) and abIL2, said bifunctional molecule being a heterodimer; The heterodimer is (1) a first monomer comprising, in order from the N-terminus, the following: 1) a wild-type IL-2 protein having the sequence shown in SEQ ID NO:1 or a variant thereof comprising any one or any combination of the following mutations: R38L, F42A, D20K, R38A, F42K and K43E; 2) a required linker structure (G4S linker sequence) preferably having the sequence shown in SEQ ID NO:6, and 3) IgG Fc single chain having the sequence shown in SEQ ID NO:2, or No-ADCC mutant IgG Fc having the sequence shown in SEQ ID NO:3, or knob mutant Fc having the sequence shown in SEQ ID NO:4, or hole mutant Fc having the sequence shown in SEQ ID NO:5. (2) a second monomer comprising: 1) an antibody Fab region consisting of an anti-PD1 antibody (K) light chain VL-KCL having the sequence shown in SEQ ID NO: 13 and an anti-PD1 antibody (K) heavy chain VH&CH1 having the sequence shown in SEQ ID NO: 14; or 2) an anti-PD1 single chain antibody (K) (ScFv) having the sequence shown in SEQ ID NO: 15; and 3) IgG Fc single chain having the sequence shown in SEQ ID NO:2, or No-ADCC mutant IgG Fc having the sequence shown in SEQ ID NO:3, or knob mutant Fc having the sequence shown in SEQ ID NO:4, or hole mutant Fc having the sequence shown in SEQ ID NO:5. Including, More preferably, the heterodimer is a first monomer which is a polypeptide having the sequence shown in SEQ ID NO: 10 (abIL2-Fc); (1) a second monomer consisting of a polypeptide having the sequence set forth in SEQ ID NO: 16 (aPD1(K)VH-CH1-Fc(knob)) and an anti-PD1 antibody (K) light chain having the sequence set forth in SEQ ID NO: 13; or (2) A polypeptide having the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 22 (aPD1(K)ScFv-Fc(knob)) and a second monomer which is

[0017] The present invention also relates to bifunctional molecules which are homodimers, The monomers of the homodimer are A monomer formed by linking a molecule of interleukin 2 (IL2) to a molecule of anti-PD1 antibody (K) Fab by any means; or It is a monomer formed by linking a molecule of interleukin 2 (IL2) to a molecule of anti-PD1 single chain antibody (K) (ScFv) by any means.

[0018] Preferably, the monomers of the homodimer are, in order from the N-terminus: (1) a wild-type IL-2 protein having the sequence set forth in SEQ ID NO:1, or a mutant thereof comprising any one or any combination of the following mutations: R38L, F42A, D20K, R38A, F42K, and K43E; (2) an essential linker structure (G4S linker sequence) preferably having the sequence shown in SEQ ID NO:6; (3) a Fab or ScFv of an anti-PD1 antibody (K), wherein the Fab is a Fab of a humanized antibody or a Fab of a fully human antibody, and the ScFv is a ScFv of a humanized antibody or a ScFv of a fully human antibody; (4) an antibody Fc that is a fully human wild-type Fc or a No-ADCC mutant Fc; Includes.

[0019] More preferably, said monomers of said homodimer have the sequences shown below: (1) SEQ ID NO: 20 (aPD1(K)-abIL2: VL-VH(ScFv)-Fc-abIL2), (2) SEQ ID NO: 21 (IL2-a PD1(K):IL2-VL-VH(ScFv)-Fc)

[0020] The present invention also provides (1) In the manufacture of antitumor drugs, (2) In the manufacture of an antitumor drug for use in combination with an immune checkpoint inhibitor, (3) In the manufacture of antitumor drugs that overcome resistance to immune checkpoint inhibitors, (4) in the manufacture of an antitumor drug for use in combination with T cell adoptive transfer, or (5) in the manufacture of antitumor drugs that overcome non-responsiveness to T cell adoptive transfer; The present invention relates to the use of said bifunctional molecules.

[0021] Preferably, the immune checkpoint inhibitor is an anti-PDL1 antibody.

[0022] Preferably, the T cells are anti-tumor T cells, more preferably, the T cells are anti-tumor CAR T cells or structural analogs thereof. Effect of the Invention

[0023] The beneficial effects of the invention are as follows: (1) The present invention provides a solution to address the existing bottlenecks in the clinical application of IL-2. Specifically, the abIL2 / aPD1-hIgG1 antibody overcomes the adverse effects of Treg proliferation caused by the use of IL2 by reducing the binding of IL2 to intratumoral Tregs while retaining the activation of IL2 on effector cells. These results provide a new idea for the clinical use of IL2. (2) Overcoming resistance to immune checkpoint blockade therapy and non-response to T cell adoptive transfer is of great importance. [Brief description of the drawings]

[0024] [Figure 1] Figure 1 shows that anti-PD1-abIL2 has low in vitro activity. [Diagram 2] FIG. 2 shows that anti-PD1-abIL2 can effectively control the growth of A20 tumors. [Diagram 3] FIG. 3 shows that anti-PDL1-abIL2 cannot effectively control the growth of A20 tumors. [Figure 4] Figure 4 shows that the anti-tumor effect of anti-PD1-abIL2 is significantly better than that of anti-PDL1-abIL2. [Diagram 5] FIG. 5 shows that anti-PD1-abIL2 can effectively control the growth of MC38 tumors. [Figure 6] Figure 6 shows that the anti-tumor effect of anti-PD1-abIL2 is significantly better than that of anti-PD1-IL2 in A20 tumors. [Figure 7] Figure 7 shows that the anti-tumor effect of anti-PD1-abIL2 is significantly better than that of anti-PD1-IL2 in MC38 tumors. [Figure 8] FIG. 8 shows that the anti-tumor effect of anti-PD1-abIL2 is independent of CD4 T cells. [Figure 9] Figure 9 shows that the anti-tumor effect of anti-PD1-abIL2 is independent of NK cells. [Figure 10] FIG. 10 shows that the anti-tumor effect of anti-PD1-abIL2 is dependent on CD8 T cells. [Figure 11] FIG. 11 shows that anti-hPD1-abIL2 can better control human tumors. [Figure 12] FIG. 12 shows that anti-PD1-abIL2 can act synergistically with T cell engraftment in tumor control. [Figure 13] FIG. 13 shows that anti-PD1-abIL2 can act synergistically with anti-PDL1 in tumor control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Experimental materials

[0026] 1. Strains and Plasmids

[0027] Bacterial strains: Top10 E. coli and DH5α E. coli competent cells (Beijing TransGen Biotech)

[0028] Plasmids: pEE6.4-IgGκ-hIgG1, which contains the signal peptide of mouse IgGκ and the Fc sequence of human IgG1, was used for antibody expression. pEE6.4-IgGκ-hlgG1-Fc-hole and pEE6.4-IgGκ-hlgG1-Fc-knob were used for expression of heterodimeric proteins. pEE6.4-PD1 VH-CH1-Fc-knob and pEE6.4-PD1 VL-CL were used to express the antibody portion of the heterodimeric protein, and pEE6.4-abIL2-Fc-hole was used to express the abIL2 portion of the heterodimeric protein.

[0029] 2. Experimental Animals

[0030] Wild-type C57BL / 6 mice, BALB / c mice and BALB / c-Rag mice were purchased from Beijing Vital River Institute Animal Center. Unless otherwise specified, 8-10 week-old female mice were used in all experiments. Mice were housed in a specific pathogen-free (SPF) barrier environment. Animal care and experimental procedures were performed in accordance with the relevant regulations of the Animal Care Committee of the Institute of Biophysics, Chinese Academy of Sciences.

[0031] 3. Cell lines

[0032] MC38 cell line, a colon cancer cell line derived from C57 mice, was cultured in 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 μg / mL streptomycin).

[0033] The A20 cell line, a B cell lymphoma cell line derived from BALB / c mice, was cultured in RPMI1640 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 μg / mL streptomycin).

[0034] The FreeStyle™ 293F cell line (Invitrogen), a suspension cell derived from the HEK293 cell line, was cultured in SMM293-TII medium or CD OptiCHOTM medium and was primarily used for transient transfection to express bifunctional molecules.

[0035] The biological activity of IL2 was detected using the CTLL-2 cell line, a murine T cell line, cultured in RPMI1640 complete medium (containing 10% inactivated fetal bovine serum, 2 mmol / L L-glutamine, 0.1 mmol / L non-essential amino acids, 100 U penicillin, 100 μg / mL streptomycin, and 100 IU / mL recombinant IL2).

[0036] Design and synthesis of genes and primers

[0037] The human wild-type IL2 gene sequence is shown in SEQ ID NO: 1. Primers used in the experiments were designed by DNAMAN software and synthesized by Invitrogen.

[0038] The proteins used in the examples below are all heterodimers, specifically as follows:

[0039] 1. aPD1-abIL2 consists of the first and second monomers described below. (1) A polypeptide formed by fusing an Fc fragment to IL2 having the sequence shown in SEQ ID NO:10 and containing the mutations R38L, F42A, D20K, R38A, F42K and K43E. (2) A second monomer of aPD1 having the sequence shown in SEQ ID NO:22

[0040] 2. aPDL1-abIL2 consists of the first and second monomers described below. (1) A polypeptide formed by fusing an Fc fragment to IL2 having the sequence shown in SEQ ID NO:10 and containing the mutations R38L, F42A, D20K, R38A, F42K and K43E. (2) A second monomer of aPDL1 having the sequence shown in SEQ ID NO: 23

[0041] 3. ahPD1-abIL2 consists of the first and second monomers shown below. (1) A polypeptide formed by fusing an Fc fragment to IL2 having the sequence shown in SEQ ID NO:10 and containing the mutations R38L, F42A, D20K, R38A, F42K and K43E. (2) A second monomer of aPD1 having the sequence shown in SEQ ID NO:17

[0042] 4. The aPDL1 antibody is the commercialized atezolizumab, which consists of the first and second monomers described below. First monomer: A polypeptide formed by fusing an Fc fragment to IL2 containing the mutations R38L, F42A, D20K, R38A, F42K and K43E, wherein when including the murine mode, said polypeptide has the sequence shown in SEQ ID NO: 10 (abIL2-Fc). Second monomer: scFv-Fc of PD1 antibody, including the mouse mode, said polypeptide has the sequence shown in SEQ ID NO: 10 (abIL2-Fc).

[0043] Tumor inoculation and treatment in mice

[0044] (1) Tumor inoculation and measurement

[0045] Establishment of tumor models

[0046] 5×10 5 -7.5×10 5 The MC38 and MC38-EGFR5 single cells were suspended in 100 μL of PBS and then inoculated subcutaneously into the backs of C57BL / 6 mice.

[0047] 2×10 6 A20 single cells were suspended in 100 μL of PBS and then inoculated subcutaneously into the backs of BALB / c mice.

[0048] When the same tumor cell rechallenge experiment was 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 opposite side of the mouse's back. The tumor size was measured twice a week using a caliper to measure the long axis (a), short axis (b) and height (c) of the tumor. The tumor volume of the mouse = a × b × c / 2.

[0049] (2)Treatment

[0050] Antibodies or bifunctional molecules were injected intraperitoneally. In some experiments, intratumoral administration was also performed. Specific doses are described in the specific experiments.

[0051] In vivo cell deletion in mice

[0052] (1) Depletion of CD4+ T cells and CD8+ T cells

[0053] 200μg of GK1.5 antibody or TIB210 antibody was intraperitoneally injected the day before IL2 therapy or IL2 bifunctional molecule therapy to deplete 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. The depletion efficiency was detected by flow cytometry.

[0054] (2) Depletion of NK cells

[0055] 20 μL of NK cell depleting antibody was injected intraperitoneally the day before IL2 or IL2 bifunctional molecule therapy to deplete NK cells. Depletion efficiency was detected by flow cytometry. EXAMPLES

[0056] Example 1 aPD1-abIL2 has a low ability to activate the receptor, thereby avoiding peripheral side effects.

[0057] To reduce the toxicity of IL2, the binding ability of IL2 to IL2Rα and IL2Rβ was reduced. This design was then verified by an in vitro CTLL2 proliferation experiment. CTLL2 cells were added with different concentrations of IL-2 or aPD1-abIL2 and cultured for 72 hours, and the proliferation levels at different concentrations of IL2 or aPD1-abIL2 were detected by CCK8 kit. The results showed that aPD1-abIL2 had a low ability to proliferate CTLL2 cells (Figure 1), indicating that the constructed antibody reduced IL2 activity.

[0058] Example 2 The aPD1-abIL2 bispecific antibody showed better anti-tumor activity.

[0059] 1. The aPD1-abIL2 bispecific antibody showed significantly improved therapeutic effects compared with the aPD1 antibody alone.

[0060] 2 × 10 6 A20 tumor cells were subcutaneously inoculated, and 10 μg of aPD1 antibody protein or 20 μg of aPD1-abIL2 antibody protein were intraperitoneally injected on the 14th day (D14) after tumor inoculation. Tumor size was measured twice a week. The results showed that the aPD1-abIL2 bispecific antibody had a better therapeutic effect than the aPD1 antibody alone (Figure 2).

[0061] 2. The aPDL1-abIL2 bispecific antibody failed to significantly improve the therapeutic effect.

[0062] 2 × 10 6 A20 tumor cells were subcutaneously inoculated, and 10 μg of aPDL1 antibody protein or 20 μg of aPDL1-abIL2 antibody protein were intraperitoneally injected on the 14th day (D14) after tumor inoculation. Tumor size was measured twice a week. The results showed that the aPD1-abIL2 bispecific antibody had no better therapeutic effect compared with the aPD1 antibody alone (Figure 3).

[0063] 3. The aPD1-abIL2 bispecific antibody showed significantly improved therapeutic effects compared with the aPDL1-abIL2 bispecific antibody.

[0064] 2 × 10 6 A20 tumor cells were inoculated subcutaneously, and 20 μg of aPD1-abIL2 antibody protein or 20 μg of aPDL1-abIL2 antibody protein were injected intraperitoneally on the 14th day (D14) after tumor inoculation. Tumor size was measured twice a week. The results showed that the aPD1-abIL2 bispecific antibody had a better therapeutic effect than the aPDL1-abIL2 antibody alone (Figure 4).

[0065] 4. The aPD1-abIL2 bispecific antibody significantly improved the therapeutic effect in MC38 colon cancer tumors compared with the aPD1 antibody alone.

[0066] 5 × 10 5 MC38 tumor cells were subcutaneously inoculated, and 10 μg of aPD1 antibody protein or 20 μg of aPD1-abIL2 antibody protein were intraperitoneally injected on day 18 (D18) after tumor inoculation. Tumor size was measured twice a week. The results showed that the aPD1-abIL2 bispecific antibody had a better therapeutic effect than the aPD1 antibody alone (Figure 5).

[0067] 5. The aPD1-abIL2 bispecific antibody showed significantly improved therapeutic effects compared with the aPDL1-abIL2 bispecific antibody.

[0068] 2 × 10 6 A20 tumor cells were subcutaneously inoculated, and 20 μg of aPD1-IL2 antibody protein or 20 μg of aPDL1-abIL2 antibody protein were intraperitoneally injected on day 14 (D14) after tumor inoculation. Tumor size was measured twice a week. The results showed that the aPD1-abIL2 bispecific antibody had a better therapeutic effect than the aPDL1-IL2 antibody alone (Figure 6).

[0069] 5 × 10 5 MC38 tumor cells were subcutaneously inoculated, and 20 μg of aPD1-IL2 antibody protein or 20 μg of aPD1-abIL2 antibody protein were intraperitoneally injected on day 18 (D18) after tumor inoculation. Tumor size was measured twice a week. The results showed that the aPD1-abIL2 bispecific antibody had a better therapeutic effect than the aPD1-IL2 antibody alone (Figure 7).

[0070] Example 3 aPD1-abIL2 could activate CD8 T cells.

[0071] 1. The therapeutic effect of the aPD1-abIL2 bispecific antibody was independent of NK cells.

[0072] Because CD25 (IL2 receptor α) and PD1 were mainly expressed on activated effector T cells and NK cells, depletion experiments of different cell populations were performed separately to determine the population of immune cells on which aPD1-abIL2 antibody treatment primarily depends.

[0073] 2×10 6 A20 tumor cells were subcutaneously inoculated into BALB / c mice, and 20 μg of aPD1-abIL2 bifunctional molecule was injected intraperitoneally on day 17, and 20 μL of NK cell-depleting antibody was injected intraperitoneally every 4 days for a total of three times on the day before treatment.

[0074] In the above experiment, the aPD1-abIL2 antibody still had a therapeutic effect even after NK cell depletion, indicating that NK cells are not the main effector cells for the therapeutic effect of the antibody (Figure 8).

[0075] 2. The therapeutic effect of the aPD1-abIL2 antibody was dependent on CD8 T cells.

[0076] The role of T cells in antibody therapy was further examined.

[0077] 2×10 6A20 tumor cells were subcutaneously inoculated into BALB / c mice, and 17 days after tumor inoculation, 20 μg of aPD1-abIL2 protein was injected intraperitoneally together with 200 μg of CD4 T cell depleting antibody (clone number: GK1.5, prepared in our laboratory), 200 μg of CD8 T cell depleting antibody (clone number: TIB210, prepared in our laboratory), or both, for a total of three injections.

[0078] The depletion experiments of CD4 T cells and CD8 T cells showed that the therapeutic effect of antibody therapy was not significantly decreased after depletion of CD4 T cells, but was significantly decreased after depletion of CD8 T cells, and was completely abolished after depletion of both CD4 T cells and CD8 T cells. These results showed that the therapeutic effect of aPD1-abIL2 antibody was T cell-dependent, mainly CD8 T cells-dependent (Figures 9 and 10).

[0079] Example 4 The aPD1-abIL2 bispecific antibody showed better anti-tumor activity in humanized mice.

[0080] 2 × 10 in humanized mice 6 A549 tumor cells were subcutaneously inoculated, and 10 μg of ahPD1 antibody protein + 10 μg of abIL2 protein, or 20 μg of ahPD1-abIL2 antibody protein were intraperitoneally injected on the 9th day (D9) after tumor inoculation. Tumor size was measured twice a week. The results showed that the ahPD1-abIL2 bispecific antibody had a better therapeutic effect compared with the combined antibody (Figure 11).

[0081] Example 5 aPD1-abIL2 was able to overcome non-response to T cell adoptive transfer therapy.

[0082] 2 × 10 6 A20-HA tumor cells were inoculated subcutaneously at 2 x 10 6of OTI cells were adoptively transferred and 20 μg of aPD1-abIL2 was injected intraperitoneally. The tumor size was measured twice a week. The results showed that the aPD1-abIL2 bispecific antibody had a better therapeutic effect compared with T cell adoptive transfer therapy alone (Figure 12).

[0083] Example 6 aPD1-abIL2 could overcome non-response to PDL1 antibody therapy.

[0084] The tumor in the A20-bearing mouse was 150 mm 3 In the following cases, aPD1-abIL2 antibody therapy alone showed good tumor elimination effects. However, when the tumor was larger, antibody therapy alone could not completely eliminate the tumor by controlling tumor growth alone. In order to improve the therapeutic effect, we investigated whether the therapeutic effect could be improved by combining bispecific antibodies and immune checkpoint inhibitor antibodies.

[0085] Specific protocol: BALB / c mice were injected with 2 × 10 6 A20 tumor cells were subcutaneously inoculated, and 50 μg of aPDL1 antibody protein or / and 20 μg of aPDL1-abIL2 antibody protein were intraperitoneally injected on the 20th day (D20) after tumor inoculation. Tumor size was measured twice a week. The results showed that the combination therapy had a better therapeutic effect compared with aPDL1 antibody or aPD1-abIL2 bispecific antibody alone (Figure 13).

[0086] Finally, it should be noted that the above examples are only used to assist those skilled in the art to understand the essence of the present invention, and are not intended to limit the protection scope of the present invention.

Claims

1. A heterodimer, The heterodimer is (1) a first monomer formed by linking interleukin 2 (IL2) to a single Fc chain of an immunoglobulin; (2) a second monomer formed by linking a Fab or ScFv of an antibody against a T cell surface molecule to a single Fc chain of an immunoglobulin; the first monomer and the second monomer are linked by dimerization of the single Fc chain to form the heterodimer; The T cell surface molecule includes, but is not limited to, PD1, TIM-3, LAG-3, OX40, 4-1BB, ICOS, and GITR; Bifunctional molecules.

2. The single immunoglobulin Fc chain is a natural single immunoglobulin Fc chain or a single immunoglobulin Fc chain whose ADCC effect has been knocked out by genetic mutation; Preferably, the single Fc chain of the immunoglobulin is a single human IgG Fc chain. The bifunctional molecule of claim 1 .

3. In the second monomer, the Fab of the antibody is a Fab of a humanized antibody or a Fab of a fully human antibody; the ScFv of the antibody is a ScFv of a humanized antibody or a ScFv of a fully human antibody, Preferably, the second monomer is a monomer of an antibody against a T-cell surface molecule, comprising a light chain and a heavy chain; More preferably, the antibody is a humanized antibody against a T cell surface molecule or a fully human antibody against a T cell surface molecule, comprising a light chain and a heavy chain. The bifunctional molecule of claim 1 .

4. the T cell surface molecule is PD1; The antibody against the T cell surface molecule is an anti-PD1 antibody (aPD1); The bifunctional molecule according to any one of claims 1 to 3.

5. The heterodimer is (1) a first monomer comprising, in order from the N-terminus, the following: 1) a wild-type IL-2 protein having the sequence set forth in SEQ ID NO:1, or a mutant thereof comprising any one or any combination of the following mutations: R38L, F42A, D20K, R38A, F42K and K43E; 2) a required linker structure (G4S linker sequence), preferably having the sequence shown in SEQ ID NO:6; and 3) a single chain IgG Fc having the sequence shown in SEQ ID NO:2, or an ADCC knockout mutant IgG Fc having the sequence shown in SEQ ID NO:3, or a knob mutant Fc having the sequence shown in SEQ ID NO:4, or a hole mutant Fc having the sequence shown in SEQ ID NO:5 (2) a second monomer comprising: 1) an anti-PD1 antibody Fab region consisting of an anti-PD1 antibody light chain VL-KCL having the sequence shown in SEQ ID NO: 7 and an anti-PD1 antibody heavy chain VH&CH1 having the sequence shown in SEQ ID NO: 8; or An anti-PD1 single chain antibody (ScFv) having the sequence shown in SEQ ID NO:9, and 2) a single chain IgG Fc having the sequence shown in SEQ ID NO:2, or an ADCC knockout mutant IgG Fc having the sequence shown in SEQ ID NO:3, or a knob mutant Fc having the sequence shown in SEQ ID NO:4, or a hole mutant Fc having the sequence shown in SEQ ID NO:5 The bifunctional molecule according to any one of claims 1 to 4, comprising:

6. The heterodimer is a first monomer which is a polypeptide having the sequence shown in SEQ ID NO: 10 (abIL2-Fc); (1) An anti-PD1 antibody Fab construct consisting of an anti-PD1 antibody light chain VL-KCL having the sequence shown in SEQ ID NO: 7 and an anti-PD1 antibody VH-CH1-Fc (knob) having the sequence shown in SEQ ID NO: 11; or (2) Polypeptide having the sequence shown in SEQ ID NO: 12 (aPD1ScFv-Fc (knob)) and a second monomer, The bifunctional molecule according to any one of claims 1 to 5, comprising:

7. The second monomer is 1) an antibody Fab region consisting of an anti-PD1 antibody (K) light chain having the sequence shown in SEQ ID NO: 13 and an anti-PD1 antibody (K) heavy chain VH&CH1 having the sequence shown in SEQ ID NO: 14; or 2) an anti-PD1 single chain antibody (K) (ScFv) having the sequence shown in SEQ ID NO: 15; and 3) A single chain IgG Fc having the sequence shown in SEQ ID NO:2, or a No-ADCC mutant IgG Fc having the sequence shown in SEQ ID NO:3, or a knob mutant Fc having the sequence shown in SEQ ID NO:4, or a hole mutant Fc having the sequence shown in SEQ ID NO:

5. The bifunctional molecule of claim 5 , comprising:

8. The second monomer is (1) A Fab construct of an anti-PD1 antibody consisting of a polypeptide having the sequence set forth in SEQ ID NO: 16 (aPD1(K)VH-CH1-Fc(knob)) and the variable region of the anti-PD1 antibody light chain having the sequence set forth in SEQ ID NO: 13; or (2) A polypeptide having the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 22 (aPD1(K)ScFv-Fc(knob)) The bifunctional molecule of claim 6 ,

9. A homodimer, The monomers of the homodimer are a monomer formed by linking a molecule of interleukin 2 (IL2) to a molecule of an anti-PD1 antibody Fab by any means; or A monomer formed by linking a molecule of interleukin 2 (IL2) to a molecule of anti-PD1 single chain antibody (ScFv) by any means. A bifunctional molecule.

10. The monomers of the homodimer are, in order from the N-terminus, (1) a wild-type IL-2 having the sequence set forth in SEQ ID NO:1, or a mutant thereof comprising any one or any combination of the following mutations: R38L, F42A, D20K, R38A, F42K, and K43E; (2) an essential linker structure (G4S linker sequence) preferably having the sequence shown in SEQ ID NO:6; (3) Fab / ScFv of an anti-PD1 antibody (aPD1), in which Fab is a Fab of a humanized antibody or a Fab of a fully human antibody, and ScFv is a ScFv of a humanized antibody or a ScFv of a fully human antibody; (4) An antibody Fc that is a fully human wild-type Fc or a No-ADCC mutant Fc; The bifunctional molecule of claim 9 comprising:

11. The monomer of the homodimer is (1) A polypeptide having the sequence shown in SEQ ID NO: 18 (aPD1-abIL2: aPD1(ScFv)-Fc-abIL2), or (2) a polypeptide having the sequence shown in SEQ ID NO: 19 (abIL2-aPD1:abIL2-aPD1(ScFv)-Fc), or (3) A polypeptide having the sequence shown in SEQ ID NO: 20 (aPD1(K)-abIL2: aPD1(K)(ScFv)-Fc-abIL2), or (4) Polypeptide having the sequence shown in SEQ ID NO: 21 (IL2-aPD1(K):IL2-aPD1(K)(ScFv)-Fc) 10. The bifunctional molecule of claim 9 ,

12. (1) In the manufacture of an antitumor drug, (2) In the manufacture of an antitumor drug for use in combination with an immune checkpoint inhibitor, (3) In the manufacture of an antitumor drug that overcomes resistance to immune checkpoint inhibitors, (4) in the manufacture of an antitumor medicament for use in combination with T cell adoptive transfer, or (5) In the manufacture of an antitumor drug that overcomes non-responsiveness to adoptive T cell transfer, Use of a bifunctional molecule according to any one of claims 1 to 11.

13. the immune checkpoint inhibitor is an anti-PDL1 antagonist; Preferably, the anti-PDL1 antagonist is an anti-PDL1 antibody.

13. The use according to claim 12.

14. the T cells are anti-tumor T cells; More preferably, the T cells are anti-tumor CAR T cells or structural analogs thereof.

13. The use according to claim 12.

15. A medicament or pharmaceutical composition comprising a bifunctional molecule according to any one of claims 1 to 12.

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