WO2022222961A1 - 结合ctla-4的抗体及其用途 - Google Patents

结合ctla-4的抗体及其用途 Download PDF

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WO2022222961A1
WO2022222961A1 PCT/CN2022/087922 CN2022087922W WO2022222961A1 WO 2022222961 A1 WO2022222961 A1 WO 2022222961A1 CN 2022087922 W CN2022087922 W CN 2022087922W WO 2022222961 A1 WO2022222961 A1 WO 2022222961A1
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ctla
heavy chain
chain antibody
single heavy
dose
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PCT/CN2022/087922
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English (en)
French (fr)
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甘馨
项金峰
陶晓璐
姬金凤
陈小祥
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和铂医药(上海)有限责任公司
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Priority to CN202280028639.3A priority Critical patent/CN117203235A/zh
Publication of WO2022222961A1 publication Critical patent/WO2022222961A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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

Definitions

  • the present invention relates to the field of biomedicine, and particularly relates to an antibody that binds CTLA-4 and its use in cancer treatment. More specifically, the present invention relates to methods of depleting regulatory T cells (Treg) and/or increasing the ratio of CD8+ T cells/Treg cells in a subject, methods of treating refractory cancer in a subject, treating cancer Combination therapy of anti-CTLA-4 antibody, method for reducing side effects of CTLA4 antibody in cancer treatment, method for long-term administration of cancer patients or when necessary, administration method of anti-CTLA-4 single heavy chain antibody, unit dose composition, anti-CTLA-4 The use of the single heavy chain antibody in the preparation of medicine, the method for determining the administration dosage of the anti-tumor drug, and the method for determining the administration form of the anti-tumor drug.
  • Cancer immunotherapy uses a patient's own immune system to attack tumor cells. Promoting a robust CD8 T cell-dependent cytotoxic response in the tumor microenvironment is important for generating an effective antitumor immune response.
  • tumors tend to evade immune surveillance by exploiting T-cell suppressive mechanisms.
  • Activated T cells tend to up-regulate the expression of CTLA-4 and compete with CD28 to bind ligands CD80/CD86 (B7-1/B7-2 ) to achieve its negative regulation, preventing the activation and proliferation of T cells.
  • Tumor cells also often suppress immune responses by expressing CTLA-4, thereby achieving "immune escape”. Therefore, CTLA-4 inhibitor drugs developed based on the above theory can promote the activation of immune cells by blocking CTLA-4-related inhibitory signals, and achieve the recovery and enhancement of anti-tumor immune function.
  • CTLA-4 monotherapy and combination therapy have excellent antitumor effects in a variety of tumors.
  • the already approved ipilimumab (Ipilimumab) has achieved good efficacy in the treatment of advanced metastatic melanoma and other tumors.
  • clinical trials from different anti-CTLA-4 drugs have shown that the anti-CTLA-4 high-dose group has nearly doubled the efficacy gain compared with the low-dose group; at the same time, the high-dose single-agent group also showed good clinical results. beneficial.
  • the combination therapy also showed significant efficacy gains: by combining CTLA-4, the combination therapy group showed a 2-3 times ORR improvement compared to the original monotherapy; in some refractory When combined with CTLA-4 in tumor types or tumor types that are not sensitive to single immunotherapy (such as sarcoma, gastric cancer, cervical cancer, etc.), the therapeutic effect has achieved breakthroughs1-23 .
  • the present disclosure relates to anti-CTLA-4 single heavy chain antibodies, eg, fully human single heavy chain antibodies.
  • the anti-CTLA-4 single heavy chain antibody of the present disclosure has lower steric hindrance and high affinity. Tissue permeability and high stability.
  • the inventors have surprisingly found that, compared with traditional antibodies, the anti-CTLA-4 single heavy chain antibody of the present disclosure achieves low in vivo exposure and narrow and controllable adverse reactions; it has a shorter half-life, However, the drug effects (including anti-tumor effect and Treg clearance effect) lasted for a long time.
  • the present disclosure relates to methods of depleting regulatory T cells (Treg) and/or increasing the CD8+ T cell/Treg cell ratio in a subject.
  • the method comprises administering to the subject an anti-CTLA-4 single heavy chain antibody. Such as the steps for fully human single heavy chain antibodies.
  • the subject is a cancer patient.
  • the subject is endometrial cancer, renal non-clear cell carcinoma, renal clear cell carcinoma, non-small cell lung cancer, head and neck cancer, breast cancer, castration-resistant prostate cancer, testicular cancer, urinary Patients with epithelial cancer, liver cancer, esophageal cancer, mesothelioma, melanoma, neuroendocrine tumor.
  • the present disclosure relates to methods of treating refractory cancer in a subject.
  • the method comprises administering to the subject an anti-CTLA-4 single heavy chain antibody.
  • the refractory cancer is unresponsive or resistant to one or more immune checkpoint inhibitors, such as PD-1/PD-L1 axis signaling pathway inhibitors.
  • the cancer is HCC. In other embodiments, the cancer is castration-resistant prostate cancer (CRPC).
  • the present disclosure relates to a method of treating cancer.
  • the method comprises the step of administering to the subject an anti-CTLA-4 single heavy chain antibody and a second therapeutic agent.
  • the second therapeutic agent is selected from one or more immune checkpoint inhibitors, such as PD-1/PD-L1 axis signaling pathway inhibitors.
  • the second therapeutic agent is an anti-PD-1 monoclonal antibody, eg, Perbrolizumab, Nivolumab, or Toripalimab.
  • the second therapeutic agent is targeted to HER-2, HER-3, EGFR, EpCAM, PD-1/PD-L1, CD27, CD28, ICOS, CD40, CD122, OX43, 4- 1BB, GITR, B7-H3, B7-H4, BTLA, LAG-3, CD15, CD52, CA-125, CD34, A2AR, VISTA, TIM-3, KIR, CD30, CD33, CD38, CD20, CD24, CD90, Antibodies or antigen-binding fragments of CA-15-3, CA-19-9, CEA, CD99, CD117, CD31, CD44, CD123, CD133, ABCB5 and CD45, etc.
  • the second therapeutic agent is a chemotherapeutic drug, eg, selected from paclitaxel, cisplatin, carboplatin, gemcitabine, pemetrexed, oxaliplatin, epirubicin, 5-fluorouracil, and the like.
  • chemotherapeutic drug eg, selected from paclitaxel, cisplatin, carboplatin, gemcitabine, pemetrexed, oxaliplatin, epirubicin, 5-fluorouracil, and the like.
  • the second therapeutic agent is administered before the anti-CTLA-4 single heavy chain antibody, concurrently with the anti-CTLA-4 single heavy chain antibody, or after the anti-CTLA-4 single heavy chain antibody.
  • the present disclosure relates to methods of preventing and/or reducing the side effects of CTLA4 antibody therapy for cancer.
  • the method comprises administering to the subject an anti-CTLA-4 single heavy chain antibody.
  • the present disclosure relates to methods of administering anti-CTLA-4 single heavy chain antibodies.
  • the method comprises infusing the anti-CTLA-4 single heavy chain antibody into the patient as an intravenous drip, the anti-CTLA-4 single heavy chain antibody in 0.9% sodium chloride or 5 It is provided in the form of % glucose solution, the concentration of the anti-CTLA-4 single heavy chain antibody in the solution is 0.1 mg/mL-10.0 mg/mL, and the time of the intravenous drip does not exceed 4 hours.
  • the above method comprises administering to the subject an anti-CTLA-4 single heavy chain antibody at a dose of 0.2 mg to 1 mg/kg body weight.
  • the above-described methods include dosing cycles ranging from weekly to every 12 weeks, eg, dosing cycles are weekly, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every Administer once every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, or every 12 weeks.
  • dosing cycles are weekly, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every Administer once every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, or every 12 weeks.
  • the "administration period" described herein refers to the time interval between two adjacent administrations.
  • the anti-CTLA-4 single heavy chain antibody is administered to the subject at a dose of -0.5 mg/kg, 0.35-0.45 mg/kg, 0.45-0.6 mg/kg, 0.45-0.55 mg/kg, or 0.45-0.5 mg/kg.
  • the anti-CTLA-4 single heavy chain antibody is administered to the subject at a dose of 0.3 mg/kg once a week.
  • the anti-CTLA-4 single heavy chain antibody is administered to the subject at a dose of 0.45 mg/kg every 3 weeks.
  • the anti-CTLA-4 single heavy chain antibody is administered to the subject at a dose of 0.6 mg/kg every 3 weeks.
  • the present disclosure relates to methods of achieving long-term or when necessary dosing in cancer patients.
  • the method comprises administering to the subject an anti-CTLA-4 single heavy chain antibody.
  • the long-term administration refers to an administration period of not less than 4 weeks, preferably, not less than 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks, eg, 6 weeks or 12 weeks.
  • the subject's inability to maintain stable disease (SD), partial remission (PR), complete remission (CR) status is indicative of dosing as necessary.
  • SD means that the sum of the maximum diameters of target lesions does not reduce to PR, or does not increase to PD
  • PR means that the sum of the maximum diameters of target lesions decreases by more than or equal to 30% for at least 4 weeks
  • CR means that all target lesions disappear , no new lesions appeared, and the tumor markers were normal for at least 4 weeks
  • PD means that the sum of the maximum diameters of target lesions increased by at least 20%, or new lesions appeared.
  • the subject is unable to maintain an SD, PR or CR status, and the subject is administered an anti-CTLA-4 single heavy chain antibody until the subject recovers the SD, PR or CR status.
  • the anti-CTLA-4 single heavy chain antibody is administered for a duration of 2 to 4 dosing cycles.
  • the anti-CTLA-4 single heavy chain antibody is administered to the subject at a dose of 0.2 mg to 1 mg/kg body weight, preferably 0.3 to 0.6 mg/kg.
  • the present disclosure relates to a unit dose composition.
  • 20-200 mg of anti-CTLA-4 single heavy chain antibody is included. eg 30-200mg, 40-200mg, 50-200mg, 60-200mg, 70-200mg, 80-200mg, 90-200mg, 100-200mg, 60-100mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg , 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 110mg, 120mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg.
  • the present disclosure relates to the use of an anti-CTLA-4 single heavy chain antibody in the preparation of a medicament for the treatment of a CTLA-4-related disease such as cancer in a subject, the medicament being formulated at a dose of 0.2 mg to The 1 mg/kg dose is administered in patient form.
  • the anti-CTLA-4 single heavy chain antibody has a CDR having the amino acid sequence set forth in any of SEQ ID NOs: 1-24.
  • the anti-CTLA-4 single heavy chain antibody has CDR1, 2 and 3, the CDR1 has the amino acid sequence set forth in any of SEQ ID NOs: 1-8; The CDR2 has the amino acid sequence shown in any of SEQ ID NOs: 9-16; the CDR3 has the amino acid sequence shown in any of SEQ ID NOs: 17-24.
  • the anti-CTLA-4 single heavy chain antibody has CDR1 , 2 and 3, said CDR1, CDR2 and CDR3 having SEQ ID NOs: 1, 9 and 17, respectively amino acid sequence shown.
  • the anti-CTLA-4 single heavy chain antibody has CDR1 , 2 and 3, said CDR1, CDR2 and CDR3 having SEQ ID NOs: 2, 10 and 18, respectively amino acid sequence shown.
  • the anti-CTLA-4 single heavy chain antibody has CDR1 , 2 and 3, said CDR1, CDR2 and CDR3 having SEQ ID NOs: 3, 11 and 19, respectively amino acid sequence shown.
  • the anti-CTLA-4 single heavy chain antibody has CDR1, 2 and 3, said CDR1, CDR2 and CDR3 having the SEQ ID NOs: 4, 12 and 20, respectively amino acid sequence shown.
  • the anti-CTLA-4 single heavy chain antibody has CDR1 , 2 and 3, said CDR1, CDR2 and CDR3 having SEQ ID NOs: 5, 13 and 21, respectively amino acid sequence shown.
  • the anti-CTLA-4 single heavy chain antibody has CDR1 , 2 and 3, said CDR1, CDR2 and CDR3 having SEQ ID NOs: 6, 14 and 22, respectively amino acid sequence shown.
  • the anti-CTLA-4 single heavy chain antibody has CDR1 , 2 and 3, said CDR1, CDR2 and CDR3 having SEQ ID NOs: 7, 15 and 23, respectively amino acid sequence shown.
  • the anti-CTLA-4 single heavy chain antibody has CDR1, 2 and 3 having the CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 8, 16 and 24, respectively amino acid sequence shown.
  • the anti-CTLA-4 single heavy chain antibody has a heavy chain variable region having any of SEQ ID NOs: 25-32 amino acid sequence shown.
  • the anti-CTLA-4 single heavy chain antibody has the amino acid sequence set forth in any of SEQ ID NOs: 33-40.
  • the present disclosure relates to a method of administering an anti-CTLA-4 single heavy chain antibody, the anti-CTLA-4 single heavy chain antibody having the amino acid sequence shown in SEQ ID NO:33.
  • the method comprises infusing the anti-CTLA-4 single heavy chain antibody into the patient as an intravenous drip, the anti-CTLA-4 single heavy chain antibody in 0.9% sodium chloride or 5 It is provided in the form of % glucose solution, the concentration of the anti-CTLA-4 single heavy chain antibody in the solution is 0.1 mg/mL-10.0 mg/mL, and the time of the intravenous drip does not exceed 4 hours.
  • the anti-CTLA-4 single heavy chain antibody is infused into the patient at a dose of 0.2 mg to 1 mg/kg, preferably 0.3 to 0.6 mg/kg.
  • the dosing cycle is weekly to once every 12 weeks, in some embodiments, the dosing cycle is weekly, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every Administer once every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, or every 12 weeks.
  • the anti-CTLA-4 single heavy chain antibody is administered at 0.3-0.6 mg/kg, 0.3-0.55 mg/kg, 0.3-0.5 mg/kg, 0.3-0.45 mg/kg, 0.35-0.6 mg /kg, 0.35-0.55mg/kg, 0.35-0.5mg/kg, 0.35-0.45mg/kg, 0.45-0.6mg/kg, 0.45-0.55mg/kg or 0.45-0.5mg/kg into the patient body, Dosing every 3 weeks.
  • the anti-CTLA-4 single heavy chain antibody is infused into the patient at a dose of 0.3 mg/kg administered once a week.
  • the anti-CTLA-4 single heavy chain antibody is infused into the patient at a dose of 0.45 mg/kg administered every 3 weeks.
  • the anti-CTLA-4 single heavy chain antibody is infused into the patient at a dose of 0.6 mg/kg administered every 3 weeks.
  • a unit dose of the composition comprises 60-100 mg of an anti-CTLA-4 single heavy chain antibody having an amino acid sequence as described above, eg selected from SEQ ID NO. : the amino acid sequence represented by any one of 33-40.
  • the "unit dose composition" mentioned in this application refers to a dosage composition suitable for one-time administration, that is, the unit dosage composition contains the amount of active ingredient suitable for one-time administration.
  • the unit dose composition according to the embodiment of the present invention has high safety and good efficacy for one-time administration.
  • a unit dose of the composition comprises 80 mg of anti-CTLA-4 single heavy chain antibody.
  • the present disclosure relates to the use of an anti-CTLA-4 single heavy chain antibody in the preparation of a medicament, the anti-CTLA-4 single heavy chain antibody having the amino acid sequence described above, such as selected from SEQ ID NO: 33 ⁇ 40.
  • the medicament is formulated to be administered once every 3 weeks at a dose of 0.3 mg/kg to 0.6 mg/kg; preferably, the medicament is formulated to be administered at a dose of 0.3 to 0.45 mg/kg
  • the dosage is in the form of a once every 3 week dose; preferably, the medicament is formulated for a once every 3 week dose at a dose of 0.45 mg/kg.
  • the present disclosure relates to a "Tick-Tock pendulum motion" type antitumor drug dosage determination method.
  • the method includes: 1) performing a dose-escalation experiment in a wide range of tumor types with the drug to be tested according to a predetermined dose gradient, so as to obtain the efficacy data and toxicity data of the drug to be tested at the predetermined dose; 2. ) Based on the efficacy data and toxicity data of the drug to be tested at a predetermined dose, determine whether the drug to be tested is subjected to a dose expansion experiment in a specific tumor species at the predetermined dose; 3) Repeat step 2) to determine whether the drug to be tested is in a specific tumor type. Whether to conduct dose-expansion experiments in specific tumor species at higher predetermined doses.
  • the two parts of the dose escalation experiment and the dose expansion experiment can be performed simultaneously: that is, during the dose escalation process, once a dose group with reliable safety and a signal of efficacy appears, the dose is performed in a specific tumor type.
  • the method includes "dose ramping" and "dose expansion” parts; the “dose ramping” part is: according to the i3+3 dose ramping algorithm (FIG. 1, FIG. 2), according to each The dose-limiting toxicity of each dose level and the comprehensive toxicity performance are combined with the algorithm to make the decision of "climbing", “staying” or “de-escalating” to determine the dose group that obtains the "efficacy signal”.
  • the "dose expansion” step is: to evaluate the PD/PK characteristics and curative effect status presented in each dose group, and after regular scientific review and discussion to confirm whether the dose "has a good benefit/risk ratio", it can be used. Dose escalation in specific tumor types starts at a certain dose.
  • dose escalation is continued to continue exploring the safety margin of the drug, further confirming the next dose with a favorable benefit/risk ratio; if the "benefit/risk ratio" of the higher dose group is still.
  • the anti-tumor drug is an anti-CTLA-4 single heavy chain antibody.
  • the CDRs, heavy chain variable region, and heavy chain of the anti-CTLA-4 single heavy chain antibody have the amino acid sequences shown in SEQ ID NOs: 1-40.
  • the present disclosure relates to a method of determining the administration form of an antineoplastic drug.
  • the method comprises: administering a drug to be tested to a test model, and performing tests at predetermined doses, times of administration and periods of administration, so as to obtain a dominant form of administration of the drug to be tested, wherein , the SD, PR, or CR of the test model is in steady state and is an indication of the predominant form of dosing.
  • it further comprises administering the second therapeutic agent to a test model, and performing tests at predetermined doses, times of administration, and periods of administration to obtain a dominant form of administration of the second therapeutic agent, wherein the test The SD, PR or CR of the model remains in a steady state and is an indication of the predominant form of administration of the second therapeutic agent.
  • the SD, PR or CR of the test model cannot be maintained in a steady state, and the drug to be tested is re-administered to the test model in order to bring the SD, PR or CR of the test model back into a steady state.
  • a specific tumor type may have a specific drug dose and interval pattern and number of cycles; and if necessary, further exploration will be conducted on the "required" (PRN) dosing mode, that is, pre-set At the end of the fixed dosing duration (such as 4 dosing cycles), if the result is SD or PR, CR, it will be maintained with a second drug (such as PD-1), and periodic tumor assessments will be performed during subsequent treatment, if When the disease progresses or the progression trend is likely, temporarily add the anti-tumor drug to be tested, such as anti-CTLA-4 single heavy chain antibody for several dosing cycles (such as 2-4 dosing cycles), as a "consolidation" treatment The purpose is to repeat the above logic until the patient has stable disease again or remission
  • the CDRs, heavy chain variable region, and heavy chain of the anti-CTLA-4 single heavy chain antibody have the amino acid sequences shown in SEQ ID NOs: 1-40.
  • the present disclosure relates to anti-CTLA-4 single heavy chain antibodies for use in depleting regulatory T cells (Treg) and/or increasing the CD8+ T cell/Treg cell ratio in a subject.
  • the present disclosure relates to anti-CTLA-4 single heavy chain antibodies for the treatment of refractory cancer in a subject.
  • the present disclosure relates to an anti-CTLA-4 single heavy chain antibody and a second therapeutic agent for the treatment of cancer.
  • the present disclosure relates to anti-CTLA-4 single heavy chain antibodies for use in reducing the side effects of CTLA4 antibodies in the treatment of cancer.
  • the present disclosure relates to anti-CTLA-4 single heavy chain antibodies for use in achieving long-term or when necessary dosing in cancer patients.
  • Figure 1 is a schematic diagram of "dose ramping" and “dose expansion” experiments according to embodiments of the present invention
  • Fig. 2 is an i3+3 dose ramping algorithm presentation diagram according to an embodiment of the present invention
  • FIG. 3 is a graph showing the results of killing Treg cells by anti-CTLA-4 single heavy chain antibodies according to an embodiment of the present invention.
  • ISO isotype control
  • FIG. 4 is a graph showing the results of killing T reg cells in CD45 + cells in tumor samples by anti-CTLA-4 single heavy chain antibodies according to an embodiment of the present invention.
  • G1 hIgG1, 10 mg/kg
  • G2 ipilimumab Mock, 10 mg/kg
  • G3 hIgG1HCAb, 5.4 mg/kg
  • G4 HBM4003, 5.4 mg/kg
  • G5 HBM4003, 1.5 mg/kg;
  • Figure 5 is a graph of mean serum concentration-time profiles of anti-CTLA-4 single heavy chain antibodies following intravenous dose administration of 1 mg/kg and 5 mg/kg according to an embodiment of the present invention
  • Figure 6 is a graph of mean serum concentration-time profiles of anti-CTLA-4 single heavy chain antibodies following single 1, 3 and 10 mg/kg intravenous dose administration in cynomolgus monkeys according to embodiments of the present invention
  • Figure 7 is the pharmacokinetic results of anti-CTLA-4 single heavy chain antibody in patients according to an embodiment of the present invention.
  • a and B serum anti-CTLA-4 single heavy chain antibody concentration-time curves of C1D1-C1D7 after the first dose of 0.3mg/kg and 0.45mg/kg and 0.6mg/kg after the first dose of C1D1-C1D21, A Plots on a logarithmic scale, B plots on a linear scale.
  • C and D Serum anti-CTLA-4 single heavy chain antibody concentration-time curves of C1D22-C1D28 after multiple administration of 0.3 mg/kg, C is the logarithmic scale plot, D is the linear scale plot.
  • Antibody antibody, Ab
  • Immunoglobulin immunoglobulin, Ig
  • Heavy chain heavy chain, HC
  • light chain light chain, LC
  • Heavy chain variable region heavy chain variable domain, VH;
  • Heavy chain constant region heavy chain constant domain, CH;
  • Light chain variable region light chain variable domain, VL;
  • Light chain constant region light chain constant domain, CL;
  • Complementarity determining region complementarity determining region, CDR;
  • Fab fragment antigen binding fragment, Fab
  • Fc region fragment crystallizable region, Fc;
  • Monoclonal antibody monoclonal antibody, mAb
  • Single heavy chain antibody An antibody that lacks the light chain.
  • antibody referred to in this disclosure is a single heavy chain antibody.
  • Antibodies also include murine antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies of other origins. Antibodies may contain additional alterations such as unnatural amino acids, Fc effector function mutations and glycosylation site mutations.
  • Antibodies also include post-translationally modified antibodies, fusion proteins comprising antigenic determinants of the antibody, and immunoglobulin molecules comprising any other modifications to the antigen recognition site, so long as these antibodies exhibit the desired biological activity.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain at least one antigen-binding domain.
  • variable regions refer to the paired light and heavy chain domain portions that are directly involved in antibody and antigen binding.
  • Each VH and VL region consists of three hypervariable or complementarity determining regions (CDRs) and four framework regions (FRs) arranged from N-terminal to C-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 , FR4.
  • CDR refers to the complementarity determining regions within antibody variable sequences. For each variable region, there are three CDRs in each variable region of the heavy and light chains, which are referred to as CDR1, CDR2, and CDR3. The exact boundaries of these CDRs are defined differently from system to system.
  • the system described by Kabat et al (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides unambiguous residue numbering for antibody variable regions system, but also provides residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs.
  • Each complementarity determining region may comprise amino acid residues from the "complementarity determining regions" defined by Kabat. Chothia et al. (Chothia & Lesk, J. Mol. Biol, 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain sub-portions within the Kabat CDRs adopt nearly identical peptide backbone conformations, although in There is diversity at the amino acid sequence level. These subsections are referred to as L1, L2 and L3 or H1, H2 and H3, respectively, where “L” and “H” represent light and heavy chain regions, respectively.
  • Chothia CDRs regions may be referred to as Chothia CDRs , which has overlapping boundaries with the Kabat CDRs.
  • CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with the Kabat CDRs.
  • the methods used herein can utilize CDRs defined according to any of these systems, although it is preferred to implement
  • the protocol uses the CDRs defined by Kabat or Chothia. This application uses the Kabat system to define CDR sequences.
  • the amino acid modifications do not alter the CDR sequences of the antibody, ie, the amino acid modifications are made in the framework regions (FRs) of the variable regions.
  • the one or several amino acid modifications refer to 1-10 amino acid modifications or 1-5 amino acid modifications, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modification.
  • the amino acid modification is selected from substitutions, deletions, additions and/or insertions of amino acid residues. In some embodiments, the amino acid modification is an amino acid substitution, eg, a conservative substitution.
  • the antibody has the following VH:
  • VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 25, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;
  • a VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 26, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;
  • a VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 27, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;
  • VH comprising an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 28;
  • a VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;
  • VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 30, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;
  • a VH comprising an amino acid sequence having at least 80% sequence identity, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 31;
  • a VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 32, eg, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • sequence identity As understood by those skilled in the art, the relatedness between two amino acid sequences or between two nucleotide sequences can be described by the parameter "sequence identity".
  • the percent sequence identity between two sequences can be determined, for example, by using a mathematical algorithm.
  • Non-limiting examples of such mathematical algorithms include the algorithm of Myers and Miller (1988) CABIOS 4: 11-17, the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482, Needleman and Wunsch ( 1970) Homology Alignment Algorithm of J. Mol. Biol. 48:443-453, Method for Searching for Homology of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
  • Sequence comparisons for determining sequence identity can be performed by using programs based on such mathematical algorithms.
  • the program can be appropriately executed by a computer. Examples of such programs include, but are not limited to, the CLUSTAL of the PC/Gene program, the ALIGN program (Version 2.0), and the GAP, BESTFIT, BLAST, FASTA, and TFASTA of the Wisconsin Genetics Package. Alignment using these programs can be performed, for example, by using initial parameters.
  • the antibody may have the amino acid sequence set forth in any one of SEQ ID NOs: 33-40.
  • the present disclosure relates to methods of depleting regulatory T cells (Treg) and/or increasing the ratio of CD8+ T cells/Treg cells in a subject, methods of treating refractory cancer, methods of treating cancer, and methods of reducing the side effects of CTLA4 antibody therapy for cancer method.
  • Treg regulatory T cells
  • treating refers to therapeutic treatment wherein the purpose is to reverse, alleviate, ameliorate, inhibit, slow or stop the progression or severity of a condition associated with a disease or disorder.
  • the term “treating” includes reducing or alleviating at least one side effect or symptom of a disease or disorder.
  • a treatment is generally “effective” if one or more symptoms or clinical markers are reduced.
  • a treatment is “effective” if the progression of the disease is reduced or stopped, that is, “treatment” includes not only the improvement of symptoms, but also the cessation of the progression or worsening of symptoms expected in the absence of treatment, Slow down at least.
  • Beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction of disease severity, stabilization (ie, not worsening) of disease state, delay or slowdown of disease progression, improvement or remission of disease state, and remission (whether partial or not) all), whether detectable or undetectable.
  • the terms “subject”, “patient” and “individual” are used interchangeably herein and refer to animals, such as humans.
  • the term subject also includes “non-human mammals” such as, for example, rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates.
  • the subject is a human subject.
  • the disease is cancer.
  • cancers include, but are not limited to: basal cell carcinoma, cholangiocarcinoma; bladder cancer; bone cancer; breast cancer; peritoneal cancer; cervical cancer; cholangiocarcinoma; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer ; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer; glioblastoma; liver cancer; squamous cell carcinoma of the lung); lymphomas, including Hodgkin lymphoma and non-Hodgkin lymphoma; melanoma; myeloma; neuroendocrine tumors (eg, neuroblastoma); oral cancer; ovarian cancer; pancreatic cancer ; Prostate cancer; Retinoblastoma; Rhabdomyosarcoma; Rectal cancer; Respiratory system cancer; Salivary gland cancer; Sarcoma; Skin cancer; S
  • the treatment-refractory cancer is primary liver cancer (HCC) or castration-resistant prostate cancer (CRPC).
  • HCC primary liver cancer
  • CRPC castration-resistant prostate cancer
  • the method further comprises the step of administering one or more additional therapies.
  • the therapy is selected from chemotherapy, radiation therapy, immunotherapy, and surgical therapy.
  • the immunotherapy is selected from the group consisting of therapies directed against immune checkpoint molecules, CAR-T cell therapy, and CAR-NK cell therapy.
  • the immune checkpoint molecule can be selected from PD-1, PD-L1, PD-L2, CTLA4, OX40, LAG3, TIM3, TIGIT, and CD103.
  • the chemotherapy is selected from a combination chemotherapy regimen comprising epirubicin, oxaliplatin, and 5-fluorouracil.
  • This example relates to a phase I, open-label, international multicenter study evaluating the safety, tolerability, pharmacokinetics, and antitumor activity of anti-CTLA-4 single heavy chain antibodies in subjects with advanced solid tumors. The trial is divided into 2 parts: “dose escalation” and “dose escalation”.
  • the first part is the dose escalation phase, according to the i3+3 dose escalation algorithm ( Figure 1, Figure 2), according to the dose-limiting toxicity (DLT) of each dose level and the comprehensive toxicity performance (including delayed toxicity) Combining the algorithm with the decision to "climb", “stay” or “de-escalate”, there is still the possibility of trying again, unless the toxic conditions exhibited by the cohort directly trigger DU (going to the previous dose level and not trying the same again). dose).
  • DLT dose-limiting toxicity
  • this trial evaluates PD/PK characteristics and efficacy status (imaging changes of tumors, tumor marker changes, etc.)
  • Dose expansion (Tock stage) in specific tumor types can be started from a certain dose after the discussion confirms whether the dose "has a good benefit/risk ratio".
  • dose escalation continues to explore the safety margin of the drug, further confirming the next dose with "stretch” potential (with a good benefit/risk ratio) (Tick phase). If the "benefit/risk ratio" of the higher dose group is still confirmed by the above-mentioned clinician-based SRC as acceptable and with expansion potential, expansion in specific tumor types (Tock stage) will also be carried out in the same manner as described above.
  • the “benefit/risk ratio” evaluation is completed while the safety confirmation is completed through dose escalation in a wide range of solid tumors. Once confirmed, it is immediately pushed to the expansion part. POC validation is carried out in the "efficacy validation model" (specific tumor type with potential); at the same time, the trial will continue to enter the next "pendulum cycle”.
  • Tumors involved include: endometrial cancer, renal non-clear cell carcinoma, renal clear cell carcinoma, non-small cell lung cancer, head and neck cancer, breast cancer, castration-resistant prostate cancer, testicular cancer, urothelial cancer, liver cancer, Esophageal cancer, mesothelioma, penile cancer, colorectal cancer, etc.
  • CTLA-4 In terms of safety, compared with other CTLA-4 drugs reported adverse events involving multiple organs (the most common top 5 are rash, diarrhea or colitis, hepatitis, hypophysitis, pneumonia, etc.), anti-CTLA-4 The adverse reactions of single heavy chain antibodies were concentrated in the digestive tract, mainly diarrhea (grade 2 or 3) or colitis (grade 1 or 2).
  • the toxicity spectrum of anti-CTLA-4 single heavy chain antibodies is narrower/concentrated than that of drugs with the same target, and the existing data suggest that the gastrointestinal symptoms and pathological findings after exposure to anti-CTLA-4 single heavy chain antibodies are compared with drugs of the same target
  • the reports of G3 diarrhea are simple watery diarrhea, and some patients are accompanied by G1-2 colitis without mucus, pus and blood and no abdominal pain, but most of the pathological pathologies are mild under the microscope, which is consistent with the preclinical findings. similar.
  • Colonoscopy and pathological biopsy prompts except for 1 patient (occurring after 12 intraperitoneal injections) with typical ulcerative colitis manifestations, all other patients showed atypical manifestations (the report suggested that "enteritis cannot be excluded, it may be related") or biopsy Mild inflammatory cell activity under the microscope, and the diarrhea symptoms of the above-mentioned patients can be quickly relieved after drug intervention.
  • the comprehensive diagnosis of related digestive tract can be obtained by clinical use of anti-diarrheal drugs and conventional doses of hormones (equivalent to 0.5mg- 1 mg/kg prednisone) was well controlled and eventually relieved.
  • Table 1 below shows a summary of adverse events in the 0.3 mg/kg QW and 0.6 mg/kg Q3W dose groups.
  • Human T reg cell scavenging activity of anti-CTLA-4 single heavy chain antibodies was evaluated in an in vitro ADCC killing assay.
  • human T reg cells were differentiated from undifferentiated CD4 T cells in vitro, then labeled with the marker calcein AM, and incubated with antibody and primary human PBMC for several hours.
  • Numerical detection of calcein AM in the supernatant revealed that the anti-CTLA-4 single heavy chain antibody (PR218) had potent T reg cell killing activity compared to the ipilimumab mimic (PR149) ( Figure 3).
  • the primary endpoint was to determine changes in the proportions of different immune cell populations in tumor, blood, and spleen in tumor-bearing mice following administration.
  • One-way repeated measures analysis of variance (ANOVA) was used to analyze differences in cell populations between different groups.
  • Bonferroni's multiple comparison method was used for comparison with the vehicle group.
  • Tregs in CD4 + T cells were observed in the anti- CTLA -4 single heavy chain antibody dosed groups at 5.4 mg/kg or 1.5 mg/kg The ratio decreased, whereas no change was observed with the ipilimumab mimetic.
  • neither anti-CTLA-4 single heavy chain antibody nor ipilimumab mimics were observed to significantly alter the proportion of T reg in CD4 + cells.
  • the purpose of this study was to determine the PK profile of an anti-CTLA-4 single heavy chain antibody (PR218) following intravenous administration in female C57BL/6 mice.
  • Blood serum samples were collected at the following time points: pre-dose, 0.083 hr, 1, 2, 4, 7, 10 and 14 days.
  • the concentration of anti-CTLA-4 single heavy chain antibody in serum of C57BL/6 mice was determined by ELISA method.
  • the PR218PK parameters in serum (t 1/2 , C max , total clearance [CL], volume of distribution at steady state [Vss], mean residence time [MRT] and area under the concentration-time curve [ AUC]).
  • the non-compartmental analysis method was used to estimate the parameters ( Professional 6.4, Pharsight, Mountain View, USA). Any values below quantifiable levels were excluded from the PK parameter calculations using WinNonlin.
  • Figure 5 shows mean serum concentration-time profiles of anti-CTLA-4 single heavy chain antibodies following 1 mg/kg and 5 mg/kg intravenous doses. Serum concentrations of anti-CTLA-4 single-heavy-chain antibodies increased with increasing dose and were biphasic over time.
  • AUC INF , CL Z , MRT last and V ss were 2.22 ⁇ 0.0281 days, 10.4 ⁇ 0.758 ⁇ g/mL, 16.1 ⁇ 1.48 days* ⁇ g/mL, 16.2 ⁇ 1.52 days* ⁇ g/mL, 62.1 ⁇ 58.0mL, respectively /kg/day, 2.05 ⁇ 0.228 days and 134 ⁇ 12.1 mL/kg (Table 3).
  • t 1/2 , C max , AUC last , AUC INF , CL Z , MRT last and V ss were 1.94, respectively ⁇ 0.718 days, 36.6 ⁇ 15.9 ⁇ g/mL, 73.3 ⁇ 15.3 days* ⁇ g/mL, 74.0 ⁇ 15.9 days* ⁇ g/mL, 69.8 ⁇ 15.4mL/kg/day, 2.41 ⁇ 0.144 days and 176 ⁇ 40.5mL/kg ( table 3).
  • the terminal elimination half-life and CLz remained constant over the 1 mg/kg-5 mg/kg dose range, with mean values of approximately 2.1 days and 66.0 mL/kg/day, respectively.
  • the total exposure measured with AUCINF as a parameter showed a dose-proportional increase in the dose range of 1 mg/kg-5 mg/kg.
  • the data show that the PK of anti-CTLA-4 single heavy chain antibody in mice is linear over the intravenous dose range of 1 mg/kg-5 mg/kg.
  • the PK of anti-CTLA-4 single heavy chain antibody was evaluated in cynomolgus monkeys following single intravenous bolus administration of anti-CTLA-4 single heavy chain antibody at doses of 1, 3 and 10 mg/kg. A total of 18 cynomolgus monkeys (9 males and 9 females) were randomly assigned into 3 groups and tested at each dose level.
  • Plasma samples for PK assessment were collected at the following time points: pre-dose, 0.033, 0.5, 2, 4, 8, 24, 48, 72, 96, 120, 144, 312, 480, 648, and 816 hr post-dose ( Day 35).
  • Serum concentrations of anti-CTLA-4 single heavy chain antibodies were determined using a validated ELISA method.
  • Anti-CTLA-4 single heavy chain antibody PK parameters in serum (t1 /2 , Cmax at C0 , CL, Vss , AUC) were determined from the mean concentration-time data. A non-compartmental analysis was used to estimate the parameters.
  • ADA anti-drug antibodies
  • Figure 6 shows mean serum concentration-time profiles of anti-CTLA-4 single heavy chain antibodies following single 1, 3 and 10 mg/kg intravenous doses in cynomolgus monkeys.
  • t 1/2 and CL remained constant between the 1, 3 and 10 mg/kg dose groups.
  • Co and total exposure as measured by AUC INF showed a dose-proportional increase.
  • the data show that the PK of anti-CTLA-4 single heavy chain antibody in cynomolgus monkeys is linear over the 1 mg/kg-10 mg/kg intravenous dose range.
  • each bottle contains 4.0 mL of liquid medicine, containing 80 mg of anti-CTLA-4 single heavy chain antibody (with the amino acid sequence shown in SEQ ID NO: 33), administered on the same day, dosage Store at 2-8°C by body weight and use the original packaging to store the drug product upright.
  • the dispensing method is: extract the required dose of anti-CTLA-4 single heavy chain antibody from the bottle, add 0.9% sodium chloride injection or 5% glucose injection infusion bag to dilute, the dilution concentration range is 0.12mg/mL ⁇ 10.0 mg/mL.
  • Intravenous infusion of the diluent is required for more than 90 minutes, but should not exceed 4 hours, that is, the infusion time is controlled within 90 minutes to 4 hours. If the anti-CTLA-4 single heavy chain antibody injection is prepared into a 50 mL diluent, the infusion rate should be controlled to be 0.21 mL/min to 0.56 mL/min; if the anti-CTLA-4 single heavy chain antibody injection is prepared into a 100 mL diluent, The infusion rate should be controlled to be 0.42mL/min to 1.1mL/min.
  • an intravenous drip device containing a 0.2-micron in-line filter, sterile, non-pyrogenic, low-protein tuberculosis filter should be used.
  • use 20 mL of 0.9% sodium chloride injection or 5 % Dextrose injection flushes the tubing of the intravenous drip device.
  • Some cancer patients were given an infusion of anti-CTLA-4 single heavy chain antibody injections and were divided into three cohorts: the first cohort was dosed at 0.3 mg/kg once a week; the second cohort was dosed The design was 0.6 mg/kg administered every three weeks; the third cohort of patients was designed to be dosed at 0.45 mg/kg every three weeks.
  • Another group of cancer patients were infused with ipilimumab, which was divided into 2 cohorts: the first cohort was designed to be dosed at 0.3 mg/kg every three weeks; the second cohort was given 0.3 mg/kg.
  • the drug design is 3 mg/kg administered once every three weeks.
  • Peripheral blood was collected from patients (Australia Patients) before administration and at different time points after one cycle of administration to detect pharmacokinetic (PK) data and serum anti-CTLA-4 single heavy chain antibody concentrations and anti-drug antibodies ( ADA).
  • PK pharmacokinetic
  • ADA serum anti-CTLA-4 single heavy chain antibody concentrations and anti-drug antibodies
  • ECL electrochemiluminescence method
  • Anti-drug antibodies were analyzed using a fully validated bridging electrochemiluminescence (ECL) method, using a multi-tiered analytical approach that first performed a screening test on all samples, followed by a confirmatory test for the specificity of suspected antibody-positive samples, A titer test is performed on samples that have been determined to be positive for antibodies.
  • ECL electrochemiluminescence
  • the screening sensitivity of this analytical method was 34.0 ng/mL. The drug was well tolerated at the collected sample drug concentration levels.
  • PK parameters were estimated using non-compartmental analysis methods.
  • Serum anti-CTLA-4 single heavy chain antibody concentration-time profiles at various dose levels of anti-CTLA-4 single heavy chain antibody tested in subjects with advanced solid tumors are shown in Figure 7, and PK parameters are summarized in Table 5.
  • the half-life is short (about 2-4 days), so the accumulation after multiple doses is small; the half-life compared with ipilimumab is 14.7 days;
  • the exposure (AUC) was close to the predicted value, much lower than the exposure of ipilimumab at the same dose;
  • the AUC increases with the dose and is basically proportional to the dose
  • each bottle contains 4.0 mL of liquid medicine, containing 80 mg of anti-CTLA-4 single heavy chain antibody (with the amino acid sequence shown in SEQ ID NO: 33), administered on the same day, dosage Store at 2-8°C by body weight and use the original packaging to store the drug product upright.
  • the dispensing method is: extract the required dose of anti-CTLA-4 single heavy chain antibody from the bottle, add 0.9% sodium chloride injection or 5% glucose injection infusion bag to dilute, the dilution concentration range is 0.12mg/mL ⁇ 10.0 mg/mL.
  • Intravenous infusion of the diluent is required for more than 90 minutes, but should not exceed 4 hours, that is, the infusion time is controlled within 90 minutes to 4 hours. If the anti-CTLA-4 single heavy chain antibody injection is prepared into a 50 mL diluent, the infusion rate should be controlled to be 0.21 mL/min to 0.56 mL/min; if the anti-CTLA-4 single heavy chain antibody injection is prepared into a 100 mL diluent, The infusion rate should be controlled to be 0.42mL/min to 1.1mL/min.
  • an intravenous drip device containing a 0.2-micron in-line filter, sterile, non-pyrogenic, low-protein tuberculosis filter should be used.
  • use 20 mL of 0.9% sodium chloride injection or 5 % Dextrose injection flushes the tubing of the intravenous drip device.
  • Tumor patients were infused with anti-CTLA-4 single heavy chain antibody injections. A total of 2 anti-tumor evaluations were performed in tumor patients. In each anti-tumor evaluation, they were administered in three cohorts: the first cohort patient dosing design was 0.3 mg/kg once weekly; 0.6 mg/kg once every three weeks for patients in the second cohort; and 0.45 mg/kg once every three weeks for patients in the third cohort.
  • Cancer patient 1 was given an intravenous infusion of anti-CTLA-4 single heavy chain antibody, the patient information is shown in Table 7 below, the patient had previously received surgery and radiation therapy, and had previously taken lenvatinib, sorafenib, and SHR1701-001 ( 2020-Sep-08, Hengrui PD-1; PD), the reason for discontinuation is unknown, and he has previously received PD-1/PD-L1 immunotherapy.
  • each bottle contains 4.0 mL of liquid medicine, containing 80 mg of anti-CTLA-4 single heavy chain antibody (sequence shown in SEQ ID NO: 33), administered on the same day , the dosage is calculated according to body weight, stored at 2-8 °C, and the original packaging is used to store the medicine upright.
  • the dispensing method is: extract the required dose of anti-CTLA-4 single heavy chain antibody from the bottle, add 0.9% sodium chloride injection or 5% glucose injection infusion bag to dilute, the dilution concentration range is 0.12mg/mL ⁇ 10.0 mg/mL.
  • Intravenous infusion of the diluent is required for more than 90 minutes, but should not exceed 4 hours, that is, the infusion time is controlled within 90 minutes to 4 hours. If the anti-CTLA-4 single heavy chain antibody injection is prepared into a 50 mL diluent, the infusion rate should be controlled to be 0.21 mL/min to 0.56 mL/min; if the anti-CTLA-4 single heavy chain antibody injection is prepared into a 100 mL diluent, The infusion rate should be controlled to be 0.42mL/min to 1.1mL/min.
  • an intravenous drip device containing a 0.2-micron in-line filter, sterile, non-pyrogenic, low-protein tuberculosis filter should be used.
  • use 20 mL of 0.9% sodium chloride injection or 5 % Dextrose injection flushes the tubing of the intravenous drip device.
  • Patient 1 was administered a dose of 0.45 mg/kg, once every three weeks, three times.
  • Table 8 Patient 1 Lesion Indicators
  • each bottle contains 4.0 mL of liquid medicine, containing 80 mg of anti-CTLA-4 single heavy chain antibody, administered on the same day.
  • the dispensing method is: extract the required dose of anti-CTLA-4 single heavy chain antibody from the bottle, add 0.9% sodium chloride injection or 5% glucose injection infusion bag to dilute, the dilution concentration range is 0.12mg/mL ⁇ 10.0 mg/mL.
  • Intravenous infusion of the diluent is required for more than 90 minutes, but should not exceed 4 hours, that is, the infusion time is controlled within 90 minutes to 4 hours. If the anti-CTLA-4 single heavy chain antibody injection is prepared into a 50 mL diluent, the infusion rate should be controlled to be 0.21 mL/min to 0.56 mL/min; if the anti-CTLA-4 single heavy chain antibody injection is prepared into a 100 mL diluent, The infusion rate should be controlled to be 0.42mL/min to 1.1mL/min.
  • an intravenous drip device containing a 0.2-micron in-line filter, sterile, non-pyrogenic, low-protein tuberculosis filter should be used.
  • use 20 mL of 0.9% sodium chloride injection or 5 % Dextrose injection flushes the tubing of the intravenous drip device.
  • Patient 2 was administered a dose of 0.6 mg/kg, once every three weeks, three times.
  • the overall clinical manifestations of anti-CTLA-4 single heavy chain antibodies include: patients tolerated anti-CTLA-4 single heavy chain antibodies well, and the adverse events occurred in line with the same target.
  • the toxicity profile is expected to be more concentrated in the digestive tract system than similar target drugs, and less likely to occur in the skin, liver, kidney, lung and other organs, and no serious digestive system-specific pathological changes and related clinical risks (gastrointestinal perforation) have been observed. risk, severe intestinal mucosal damage, etc.).
  • the anti-CTLA-4 single heavy chain antibody has a short half-life (about 2-4 days, relative to the half-life of ipilimumab, which is 14.7 days), so the accumulation after multiple doses is small.
  • the exposure (AUC) was close to the predicted value and much lower than that of ipilimumab at the same dose. AUC increased with increasing dose.
  • long-lasting PD marker changes (CD8 T cell/Treg ratio, ICOS+ T cells and Ki67) were still observed in patients with shorter half-life, lower drug accumulation and exposure. + T cells increase) and significant antitumor effect.

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Abstract

提供在受试者中清除调节性T细胞(Treg)和/或增加CD8+T细胞/Treg细胞比例的方法、在受试者中治疗难治性癌症的方法、治疗癌症的组合疗法、减少CTLA4抗体治疗癌症副作用的方法、实现癌症患者长周期给药或者必要时给药的方法、抗CTLA-4单重链抗体的给药方法、单位剂量组合物、抗CTLA-4单重链抗体在制备药物中的用途、确定抗肿瘤药物的给药剂量的方法以及确定抗肿瘤药物的给药形式的方法。

Description

结合CTLA-4的抗体及其用途
本国际专利申请要求于2021年4月21日提交的中国专利申请号CN202110429291.6和2022年4月14日提交的中国专利申请号202210392059.4的权益，以上申请的全部内容通过引用并入本文。
技术领域
本发明涉及生物医药领域，并具体涉及结合CTLA-4的抗体及其在癌症治疗中的用途。更具体地，本发明涉及在受试者中清除调节性T细胞(Treg)和/或增加CD8+T细胞/Treg细胞比例的方法、在受试者中治疗难治性癌症的方法、治疗癌症的组合疗法、减少CTLA4抗体治疗癌症副作用的方法、实现癌症患者长周期给药或者必要时给药的方法、抗CTLA-4单重链抗体的给药方法、单位剂量组合物、抗CTLA-4单重链抗体在制备药物中的用途、确定抗肿瘤药物的给药剂量的方法以及确定抗肿瘤药物的给药形式的方法。
发明背景
癌症免疫疗法是癌症治疗中的最新突破，它利用患者自身的免疫系统攻击肿瘤细胞。在肿瘤微环境中促进强大的CD8T细胞依赖性细胞毒性应答对于产生有效的抗肿瘤免疫应答非常重要。然而，肿瘤倾向于通过利用T细胞抑制机制来逃避免疫监视。
T细胞的活化需要双重正信号的刺激实现，即第一信号和第二信号。第一信号通过抗原识别过程，即TCR与MHC分子-抗原肽复合物的特异性结合实现。第二信号激活依靠协同共刺激分子实现，其中T细胞表面的CD28分子与抗原呈递细胞表面相应配体CD80/CD86(B7-1/B7-2)结合，从而最终启动免疫应答。CTLA-4是负向调控T细胞活化/增殖的重要免疫检查点，活化的T细胞往往会通过上调表达CTLA-4，通过与CD28竞争性结合配体CD80/CD86(B7-1/B7-2)实现其负向调控，阻止T细胞的活化和增殖。肿瘤细胞往往也会通过表达CTLA-4抑制免疫应答，从而实现“免疫逃逸”。因此，基于上述理论开发的CTLA-4抑制剂药物，通过阻断CTLA-4相关的抑制信号促进免疫细胞的活化，实现抗肿瘤免疫功能的恢复和增强。
CTLA-4单药以及联合用药已经在多种肿瘤中具有优异的抗肿瘤效应。例如，已经获批的伊匹单抗(Ipilimumab)在治疗晚期转移性黑色素瘤等肿瘤上取得了较好的疗效。此外，来自不同抗CTLA-4药物的临床试验均显示出抗CTLA-4高剂量组相较于低剂量组有接近翻倍的疗效增益；同时，高剂量单药组也表现出不错的临床获益。在多个类型的晚期实体瘤中，联合用药亦展示出显著的疗效增益：通过联合使用CTLA-4，联合治疗组相较于原单药治疗展现出2-3倍的ORR提升；在部分难治性瘤种或对单免疫治疗不敏感的瘤种中(如肉瘤，胃癌，宫颈癌等)联合CTLA-4后，疗效获得突破 1-23。
尽管疗效增益显著，但目前的抗CTLA-4单抗和联合治疗均受到其毒性特征限制了疗效进一步上探的潜力。目前受限于毒性天花板，该靶点的临床应用模式普遍采用低剂量的非维持治疗。然而，对于HCC等瘤种目前已有数据显示，疗效与CTLA-4剂量显著正相关，即使是目前已获批的用药形式(伊匹单抗4周期+纳武单抗维持)仍存在多数患者不易耐受导致停药的现实问题，由此突显出开发改善该靶点药物安全性的下一代产品的必要性和巨大价值。因此，下一代抗体进一步挖掘将是通过疗效增强和安全性改善潜在带来更高的临床获益。
发明内容
在一方面，本公开涉及抗CTLA-4单重链抗体，例如全人源单重链抗体。相比于具有经典抗体结构(即包含两条重链和两条轻链的抗体结构)的传统抗体，本公开的抗CTLA-4单重链抗体具有更低的空间位阻和高亲和力，高组织渗透性能和高稳定性等特点。本发明人令人惊讶的发现，相比于传统抗体，本公开的抗CTLA-4单重链抗体实现了低体内暴露量，以及窄范围且可控的不良反应；其具有较短的半衰期，但药效(包括抗肿瘤效应和Treg清除效应)延续时间长。
在一方面，本公开涉及在受试者中清除调节性T细胞(Treg)和/或增加CD8+T细胞/Treg细胞比例的方法。根据本发明的实施方案，所述方法包括给予受试者施用抗CTLA-4单重链抗体。例如全人源单重链抗体的步骤。在一些实施方案中，所述受试者是癌症患者。
在一些实施方案中，所述受试者为子宫内膜癌，肾非透明细胞癌，肾透明细胞癌，非小细胞肺癌，头颈癌，乳腺癌，去势抵抗性前列腺癌，睾丸癌，尿路上皮癌，肝癌，食管癌，间皮瘤，黑色素瘤，神经内分泌瘤患者。
在一方面，本公开涉及在受试者中治疗难治性癌症的方法。根据本发明的实施方案，所述方法包括给予受试者施用抗CTLA-4单重链抗体。在一些实施方案中，所述难治性癌症对一种或多种免疫检查点抑制剂例如PD-1/PD-L1轴信号通路抑制剂不响应或耐受。在一些实施方案中，所述癌症为HCC。在另一些实施方案中，所述癌症为去势抵抗性前列腺癌(CRPC)。
在一方面，本公开涉及一种治疗癌症的方法。根据本发明的实施方案，所述方法包括对所述受试者施用抗CTLA-4单重链抗体和第二治疗剂的步骤。在一些实施方案中，所述第二治疗剂选自一种或多种免疫检查点抑制剂，例如PD-1/PD-L1轴信号通路抑制剂。
在一些实施方案中，所述第二治疗剂是抗PD-1单克隆抗体，例如派姆单抗(Perbrolizumab)、纳武单抗(Nivolumab)或特瑞普利(Toripalimab)。
在一些实施方式中，所述第二治疗剂是靶向于HER-2、HER-3、EGFR、EpCAM、PD-1/PD-L1、CD27、CD28、ICOS、CD40、CD122、OX43、4-1BB、GITR、B7-H3、B7-H4、BTLA、LAG-3、CD15、CD52、CA-125、CD34、A2AR、VISTA、TIM-3、KIR、CD30、CD33、CD38、CD20、CD24、CD90、CA-15-3、CA-19-9、CEA、CD99、CD117、CD31、CD44、CD123、CD133、ABCB5和CD45等的抗体或抗原结合片段。
在一些实施方案中，所述第二治疗剂为化疗药物，例如选自紫杉醇，顺铂，卡铂，吉西他滨，培美曲塞，奥沙利铂、表柔比星、5-氟尿嘧啶等。
在一些实施方案中，所述第二治疗剂在抗CTLA-4单重链抗体之前，与抗CTLA-4单重链抗体同时，或在抗CTLA-4单重链抗体之后施用。
在一方面，本公开涉及预防和/或减少CTLA4抗体治疗癌症副作用的方法。根据本发明的实施方案，所述方法包括给予所述受试者施用抗CTLA-4单重链抗体。
在一方面，本公开涉及抗CTLA-4单重链抗体的给药方法。根据本发明的实施方案，所述方法包括将所述抗CTLA-4单重链抗体以静脉滴注的形式输入患者体内，所述抗CTLA-4单重链抗体以0.9％氯化钠或5％葡萄糖溶液的形式提供，所述抗CTLA-4单重链抗体在所述溶液中的浓度为0.1mg/mL-10.0mg/mL，所述静脉滴注的时间不超过4小时。
在一些实施方案中，上述方法包括给予受试者以0.2mg～1mg/kg体重的剂量施 用抗CTLA-4单重链抗体。
在一些实施方案中，上述方法包括的给药周期为每周～每12周给药一次，例如，给药周期为每周、每2周、每3周、每4周、每5周、每6周、每7周、每8周、每9周、每10周、每11周或每12周给药一次。需要说明的是，本文中所述的“给药周期”是指相邻两次给药间隔的时间。
在一些实施方案中，以0.3-0.6mg/kg、0.3～0.55mg/kg、0.3～0.5mg/kg、0.3～0.45mg/kg、0.35～0.6mg/kg、0.35～0.55mg/kg、0.35～0.5mg/kg、0.35～0.45mg/kg、0.45～0.6mg/kg、0.45～0.55mg/kg或0.45～0.5mg/kg的剂量向受试者施用抗CTLA-4单重链抗体。
在一些实施方案中，以0.3-0.6mg/kg、0.3～0.55mg/kg、0.3～0.5mg/kg、0.3～0.45mg/kg、0.35～0.6mg/kg、0.35～0.55mg/kg、0.35～0.5mg/kg、0.35～0.45mg/kg、0.45～0.6mg/kg、0.45～0.55mg/kg或0.45～0.5mg/kg每3周一次给予受试者施用抗CTLA-4单重链抗体。
在一些实施方案中，以0.3mg/kg的剂量每周一次给予受试者施用抗CTLA-4单重链抗体。
在一些实施方案中，以0.45mg/kg的剂量每3周一次给予受试者施用抗CTLA-4单重链抗体。
在一些实施方案中，以0.6mg/kg的剂量每3周一次给予受试者施用抗CTLA-4单重链抗体。
在一方面，本公开涉及实现癌症患者长周期给药或者必要时给药的方法。根据本发明的实施方案，所述方法包括给予所述受试者施用抗CTLA-4单重链抗体。
在一些实施方案中，所述长周期给药是指给药周期不小于4周，优选地，不小于6周、7周、8周、9周、10周、11周或12周，例如，6周或12周。
在一些实施方案中，所述受试者的不能维持疾病稳定(SD)、部分缓解(PR)、完全缓解(CR)状态，是必要时给药的指示。其中，SD是指靶病灶最大径之和缩小未达PR，或增大未达PD；PR是指靶病灶最大径之和减少大于等于30％，至少维持4周；CR是指所有靶病灶消失，无新病灶出现，且肿瘤标志物正常，至少维持4周；PD是指靶病灶最大径之和至少增加20％，或出现新病灶。
在一些实施方案中，所述受试者的不能维持SD，PR或CR状态，给予所述受试者施用抗CTLA-4单重链抗体直到受试者恢复SD，PR或CR状态。
在一些实施方案中，所述施用抗CTLA-4单重链抗体的持续时间为2～4个给药周期。
在一些实施方案中，向受试者以0.2mg～1mg/kg体重的剂量施用所述抗CTLA-4单重链抗体，优选为0.3-0.6mg/kg。
在一方面，本公开涉及一种单位剂量组合物。根据本发明的实施方案，包含20-200mg的抗CTLA-4单重链抗体。例如30-200mg，40-200mg，50-200mg，60-200mg，70-200mg，80-200mg，90-200mg，100-200mg，60-100mg，20mg，25mg，30mg，35mg，40mg，45mg，50mg，55mg，60mg，65mg，70mg，75mg，80mg，85mg，90mg，95mg，100mg，110mg，120mg，130mg，140mg，150mg，160mg，170mg，180mg，190mg，200mg。
在一方面，本公开涉及抗CTLA-4单重链抗体在制备药物中的用途，所述药物用于治疗受试者中的CTLA-4相关疾病例如癌症，所述药物配制成以0.2mg～1mg/kg剂量施用病人的形式。
在上述方法，单位剂量组合物或用途的一些实施方案中，所述抗CTLA-4单重链抗体具有CDR，所述CDR具有SEQ ID NO:1～24任一所示的氨基酸序列。
在上述方法，单位剂量组合物或用途的一些实施方案中，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1具有SEQ ID NO:1～8任一所示的氨基酸序列；所述CDR2具有SEQ ID NO:9～16任一所示的氨基酸序列；所述CDR3具有SEQ ID NO:17～24任一所示的氨基酸序列。
在上述方法，单位剂量组合物或用途的一些实施方案中，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:1，9和17所示的氨基酸序列。
在上述方法，单位剂量组合物或用途的一些实施方案中，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:2，10和18所示的氨基酸序列。
在上述方法，单位剂量组合物或用途的一些实施方案中，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:3，11和19所示的氨基酸序列。
在上述方法，单位剂量组合物或用途的一些实施方案中，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:4，12和 20所示的氨基酸序列。
在上述方法，单位剂量组合物或用途的一些实施方案中，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:5，13和21所示的氨基酸序列。
在上述方法，单位剂量组合物或用途的一些实施方案中，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:6，14和22所示的氨基酸序列。
在上述方法，单位剂量组合物或用途的一些实施方案中，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:7，15和23所示的氨基酸序列。
在上述方法，单位剂量组合物或用途的一些实施方案中，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:8，16和24所示的氨基酸序列。
在上述方法，单位剂量组合物或用途的一些实施方案中，所述抗CTLA-4单重链抗体具有重链可变区，所述重链可变区具有SEQ ID NO:25～32任一所示的氨基酸序列。
在上述方法，单位剂量组合物或用途的一些实施方案中，所述抗CTLA-4单重链抗体具有SEQ ID NO:33～40任一所示的氨基酸序列。
在一方面，本公开涉及一种抗CTLA-4单重链抗体的给药方法，所述抗CTLA-4单重链抗体具有SEQ ID NO:33所示的氨基酸序列。根据本发明的实施方案，所述方法包括将所述抗CTLA-4单重链抗体以静脉滴注的形式输入患者体内，所述抗CTLA-4单重链抗体以0.9％氯化钠或5％葡萄糖溶液的形式提供，所述抗CTLA-4单重链抗体在所述溶液中的浓度为0.1mg/mL-10.0mg/mL，所述静脉滴注的时间不超过4小时。
在一些实施方案中，将所述抗CTLA-4单重链抗体以0.2mg～1mg/kg的剂量输入患者体内，优选以0.3～0.6mg/kg的剂量输入患者体内。在一些实施方案中，给药周期为每周～每12周给药一次，在一些实施方案中，给药周期为每周、每2周、每3周、每4周、每5周、每6周、每7周、每8周、每9周、每10周、每11周或每12周给药一次。
在一些实施方案中，将所述抗CTLA-4单重链抗体以0.3～0.6mg/kg、 0.3～0.55mg/kg、0.3～0.5mg/kg、0.3～0.45mg/kg、0.35～0.6mg/kg、0.35～0.55mg/kg、0.35～0.5mg/kg、0.35～0.45mg/kg、0.45～0.6mg/kg、0.45～0.55mg/kg或0.45～0.5mg/kg的剂量输入患者体内，每3周给药一次。
在一些实施方案中，将所述抗CTLA-4单重链抗体以0.3mg/kg的剂量输入患者体内，每周给药1次。
在一些实施方案中，将所述抗CTLA-4单重链抗体以0.45mg/kg的剂量输入患者体内，每3周给药一次。
在一些实施方案中，将所述抗CTLA-4单重链抗体以0.6mg/kg的剂量输入患者体内，每3周给药一次。
在一方面，本公开涉及一种单位剂量组合物。根据本发明的实施方案，单位剂量组合物包含60-100mg的抗CTLA-4单重链抗体，所述抗CTLA-4单重链抗体具有上文中所述的氨基酸序列，例如选自SEQ ID NO:33～40任一所示的氨基酸序列。需要说明的是，本申请所述的“单位剂量组合物”是指适于一次性给药的剂量份组合物，即，所述单位剂量组合物包含适于一次性给药的活性成分的量。根据本发明实施方案的单位剂量组合物一次性给药安全性高、疗效好。在一些实施方案中，单位剂量组合物包含80mg的抗CTLA-4单重链抗体。
在一方面，本公开涉及抗CTLA-4单重链抗体在制备药物中的用途，所述抗CTLA-4单重链抗体具有上文中所述的氨基酸序列，例如选自SEQ ID NO:33～40任一所示的氨基酸序列，所述药物用于治疗CTLA-4相关疾病例如癌症，所述药物配制成以0.2mg～1mg/kg剂量施用病人的形式。
在一些实施方案中，将所述药物配制成以0.3mg/kg～0.6mg/kg的剂量每3周一次剂量施用的形式；优选地，将所述药物配置成以0.3～0.45mg/kg的剂量每3周一次剂量施用的形式；优选地，将所述药物配置成以0.45mg/kg的剂量每3周一次剂量施用的形式。
在一方面，本公开涉及一种“Tick-Tock钟摆运动”式抗肿瘤药物的给药剂量确定方法。根据本发明的实施方案，所述方法包括：1)将待测定药物按照预定剂量梯度在广泛瘤种中进行剂量爬坡实验，以便获得待测定药物在预定剂量下的疗效数据以及毒性数据；2)基于待测定药物在预定剂量下的疗效数据以及毒性数据，确定待测定药物在所述预定剂量下是否在特定瘤种中进行剂量扩展实验；3)重复步骤2)，以便确定待测定药物在更高预定剂量下是否在特定瘤种中进行剂 量扩展实验。
在一些实施方案中，剂量爬坡实验和剂量扩展实验两部分可以同时进行：即在剂量爬坡过程中，一旦出现安全性可靠且具有疗效信号发生的剂量组，即进行该剂量在特定瘤种中的剂量扩展，与此同时继续进行更高剂量在广泛实体瘤中的爬坡，一旦确定下一个“可扩展剂量”继续依照上述方法进行扩展，最终将在“扩展”部分中同一瘤种中获得不同的剂量水平数据，以及同一剂量水平在特定瘤种的数据，最终选定“各瘤种最适宜剂量”进入后续二期和三期(PH2/3)开发。
在一些实施方案中，所述方法包括“剂量爬坡”和“剂量扩展”部分；所述“剂量爬坡”部分为：按照i3+3剂量爬坡算法(图1、图2)，根据每个剂量水平的剂量限制性毒性以及综合毒性表现结合算法进行“爬升”、“停留”或者“降级”的决定，确定获得“疗效信号”的剂量组。所述“剂量扩展”步骤为：就各剂量组中呈现出的PD/PK特征，疗效状况进行评估，定期经科学审评讨论确认该剂量“是否具有良好的获益/风险比”后，可以从某一剂量即开始在特定的瘤种中进行剂量扩展。在一些实施方案中，继续进行剂量爬坡以继续探索药物的安全性边际，进一步确认下一个具有良好的获益/风险比的剂量；若更高剂量组的“获益/风险比”仍被上述科学审评确认为可接受和具备扩展潜力，同样会按照上述方式在特定的瘤种中进行扩展。
在一些实施方案中，所述抗肿瘤药物为抗CTLA-4单重链抗体。
在一些实施方案中，所述抗CTLA-4单重链抗体的CDRs、重链可变区、重链具有SEQ ID NO:1～40所示的氨基酸序列。
在一方面，本公开涉及一种确定抗肿瘤药物的给药形式的方法。根据本发明的实施方案，所述方法包括：将待测药物施于测试模型，在预定给药剂量、给药次数和给药周期的进行试验，以便获得待测药物的优势给药形式，其中，测试模型的SD，PR或CR处于稳定状态，是优势给药形式的指示。
在一些实施方案中，进一步包括将第二治疗剂施于测试模型，在预定给药剂量、给药次数和给药周期的进行试验，以便获得第二治疗剂的优势给药形式，其中，测试模型的SD，PR或CR依然处于稳定状态，是第二治疗剂的优势给药形式的指示。
在一些实施方案中，测试模型的SD，PR或CR不能维持稳定状态，将待测药物再次施于测试模型，以便使得测试模型的SD，PR或CR重新处于稳定状态。
在一些实施方案中，除预先设定的待测抗肿瘤药物的给药持续时间和给药剂量或给药周期外，允许增加格外的给药周期，和增加新的队列以探索新的给药间隔。该探索实验可能实现的结局有：可能特定的瘤种具备有特定的药物剂量和间隔模式和周期数；并且必要时，尚会进一步探索“必要时”(PRN)给药的模式，即预先设定的给药持续时间结束(如4个给药周期)，如结果为SD或PR,CR，则以第二药物(如PD-1)进行维持，在后续治疗过程中进行定期肿瘤评估，如果出现疾病进展或者进展趋势可能时，临时加用待测的抗肿瘤药物，如抗CTLA-4单重链抗体若干给药周期(如2-4个给药周期)，用以作为“巩固”治疗目的，直至患者再次出现疾病稳定或者缓解后停用重复上述逻辑，或者出现疾病进展从而放弃本药物方案。
在一些实施方案中，所述抗CTLA-4单重链抗体的CDRs、重链可变区、重链具有SEQ ID NO:1～40所示的氨基酸序列。
在一方面，本公开涉及抗CTLA-4单重链抗体，用于在受试者中清除调节性T细胞(Treg)和/或增加CD8+T细胞/Treg细胞比例。
在一方面，本公开涉及抗CTLA-4单重链抗体，用于在受试者中治疗难治性癌症。
在一方面，本公开涉及抗CTLA-4单重链抗体和第二治疗剂，用于治疗癌症。
在一方面，本公开涉及抗CTLA-4单重链抗体，用于减少CTLA4抗体治疗癌症时副作用。
在一方面，本公开涉及抗CTLA-4单重链抗体，用于实现癌症患者长周期给药或者必要时给药。
上述技术方案的附加技术特征和技术效果与前面所述内容相同，在此不再赘述。
附图说明
图1是根据本发明实施方案的“剂量爬坡”和“剂量扩展”实验的示意图；
图2是根据本发明实施方案的i3+3剂量爬坡算法展示图；
图3是根据本发明实施方案的抗CTLA-4单重链抗体杀伤Treg细胞的结果图；ISO：同种型对照；
图4是根据本发明实施方案的抗CTLA-4单重链抗体杀伤肿瘤样本中CD45 + 细胞中的T reg细胞的结果图；其中，G1：hIgG1，10mg/kg；G2：伊匹木单抗模拟物，10mg/kg；G3：hIgG1HCAb，5.4mg/kg；G4：HBM4003，5.4mg/kg；G5：HBM4003，1.5mg/kg；
图5是根据本发明实施方案的1mg/kg和5mg/kg静脉剂量给药后抗CTLA-4单重链抗体的平均血清浓度-时间曲线图；
图6是根据本发明实施方案的食蟹猴中单次1、3和10mg/kg静脉剂量给药后抗CTLA-4单重链抗体的平均血清浓度-时间曲线图；
图7是根据本发明实施方案的抗CTLA-4单重链抗体在患者体内的药代动力学结果；
其中，A和B:0.3mg/kg首次给药后C1D1～C1D7和0.45mg/kg及0.6mg/kg首次给药后C1D1～C1D21的血清抗CTLA-4单重链抗体浓度-时间曲线，A为对数尺度作图，B为线性尺度作图。C和D:0.3mg/kg多次给药后C1D22～C1D28的血清抗CTLA-4单重链抗体浓度-时间曲线，C为对数尺度作图，D为线性尺度作图。
发明详述
除非本文另有定义，与本发明结合使用的科学和技术术语及其缩略语应具有本发明所属领域的普通技术人员通常理解的含义。以下列举了本文中使用的部分术语和缩略语。
抗体：antibody，Ab；免疫球蛋白：immunoglobulin，Ig；
重链：heavy chain，HC；轻链：light chain，LC；
重链可变区：heavy chain variable domain，VH；
重链恒定区：heavy chain constant domain，CH；
轻链可变区：light chain variable domain，VL；
轻链恒定区：light chain constant domain，CL；
互补决定区：complementarity determining region，CDR；
Fab片段：antigen binding fragment，Fab；
Fc区：fragment crystallizable region，Fc；
单克隆抗体：monoclonal antibody，mAb；
单重链抗体：缺失轻链的抗体。
本公开所提及的术语“抗体”为单重链抗体。抗体还包括鼠源抗体、人源化抗 体、嵌合抗体、人抗体以及其它来源的抗体。抗体可以含有另外的改变，如非天然氨基酸，Fc效应功能突变和糖基化位点突变。抗体还包括翻译后修饰的抗体、包含抗体的抗原决定簇的融合蛋白，以及包含对抗原识别位点的任何其它修饰的免疫球蛋白分子，只要这些抗体展现出所期望的生物活性。换句话说，抗体包括免疫球蛋白分子和免疫球蛋白分子的免疫活性片段，即至少含有一个抗原结合结构域的分子。
如本文所用，“可变区”(重链可变区VH和轻链可变区VL)表示成对的轻链和重链结构域部分，其直接参与抗体和抗原的结合。每个VH和VL区由以下顺序从N末端到C末端排列的三个高变区或互补决定区(CDR)和四个框架区(FR)组成：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
如本文所用，术语“CDR”是指抗体可变序列内的互补决定区。对于每个可变区，在重链和轻链的每个可变区中有三个CDR，其称为CDR1、CDR2和CDR3。这些CDR的确切边界根据不同的系统而不同定义。Kabat等人(Kabat et al,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987)和(1991))描述的系统不仅提供了适用于抗体可变区的明确的残基编号系统，而且还提供了限定三个CDR的残基边界。这些CDR可以称为Kabat CDR。每个互补决定区可以包含由Kabat定义的“互补决定区”的氨基酸残基。Chothia等人(Chothia&Lesk,J.Mol.Biol,196:901-917(1987)和Chothia et al.,Nature 342:877-883(1989))发现，Kabat CDR内的某些子部分采用几乎相同的肽骨架构象，尽管在氨基酸序列水平上具有多样性。这些子部分分别称为L1、L2和L3或H1、H2和H3，其中“L”和“H”分别表示轻链和重链区。这些区域可以称为Chothia CDR，其具有与Kabat CDR重叠的边界。还有其它CDR边界定义可以不严格遵循上述系统之一，但是仍将与Kabat CDR重叠。本文使用的方法可以利用根据任何这些系统定义的CDR，尽管优选实施方案使用Kabat或Chothia定义的CDR。本申请使用Kabat系统来定义CDR序列。
在一些实施方案中，所述氨基酸修饰不改变抗体的CDR序列，即在可变区的框架区(FR)中进行所述氨基酸修饰。
在一些实施方案中，所述一个或几个氨基酸修饰是指1-10个氨基酸修饰或1-5个氨基酸修饰，例如1、2、3、4、5、6、7、8、9或10个氨基酸修饰。
在一些实施方案中，所述氨基酸修饰选自氨基酸残基的取代、缺失、添加和 /或插入。在一些实施方案中，所述氨基酸修饰是氨基酸取代，例如保守取代。
在本公开的单重链抗体的一些实施方案中，所述抗体具有以下VH：
a.包含与SEQ ID NO:25的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH；
b.包含与SEQ ID NO:26的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH；
c.包含与SEQ ID NO:27的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH；
d.包含与SEQ ID NO:28的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH；
e.包含与SEQ ID NO:29的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH；
f.包含与SEQ ID NO:30的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH；
g.包含与SEQ ID NO:31的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH；
h.包含与SEQ ID NO:32的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH。
如本领域技术人员所理解的，两个氨基酸序列之间或两个核苷酸序列之间的相关性可以通过参数“序列同一性”描述。可以通过例如使用数学算法测定两种序列间的序列同一性的百分比。此类数学算法的非限制性实例包括Myers和Miller(1988)CABIOS 4:11-17的算法、Smith等(1981)Adv.Appl.Math.2:482的局部同源性算法、Needleman和Wunsch(1970)J.Mol.Biol.48:443-453的同源性比对算法、Pearson和Lipman(1988)Proc.Natl.Acad.Sci.85:2444-2448的用于搜索同源性的方法、和Karlin和Altschul(1990)Proc.Natl.Acad.Sci.USA 87:2264的算法的修改形式，记载于Karlin和Altschul(1993)Proc.Natl.Acad.Sci.USA 90:5873-5877的算法。通过使用基于此类数学算法的程序，可以实施用于测定序列同一性的序列比较(即比对)。程序可以由计算机适当执行。此类程序的实例包括但不限于PC/Gene程序的CLUSTAL、ALIGN程序(Version 2.0)、和Wisconsin遗传学软件包的GAP、BESTFIT、BLAST、FASTA、和TFASTA。可以例如通过使用初始参数 实施使用这些程序的比对。
在本公开的抗CTLA-4单重链抗体的一些实施方案中，所述抗体可以具有如SEQ ID NO:33～40任一所示的氨基酸序列。
本公开涉及受试者中清除调节性T细胞(Treg)和/或增加CD8+T细胞/Treg细胞比例的方法、治疗难治性癌症的方法、疗癌症的方法、减少CTLA4抗体治疗癌症副作用的方法。
如本文所用，术语“治疗”是指治疗性处理，其中目的是反转、减轻、改善、抑制、减缓或停止与疾病或病症相关的状况的进展或严重性。术语“治疗”包括减少或减轻疾病或病症的至少一种副作用或症状。如果减少一种或多种症状或临床标志物，则治疗通常是“有效的”。或者，如果疾病的进展减少或停止，则治疗是“有效的”，也就是说，“治疗”不仅包括症状的改善，而且还包括在缺乏治疗的情况下预期的症状的进展或恶化的停止，至少减慢。有益或期望的临床结果包括但不限于减轻一种或多种症状、减少疾病程度，稳定(即不恶化)疾病状态、延迟或减缓疾病进展、改善或缓解疾病状态、和缓解(不管是部分还是全部)，无论是可检测的还是检测不到的。
如本文所用，术语“受试者”、“患者”和“个体”在本文中可互换使用，并且是指动物，例如人类。术语受试者还包括“非人哺乳动物”，例如如大鼠、小鼠、兔、绵羊、猫、狗、牛、猪和非人灵长类动物。在优选的实施方案中，所述受试者是人类受试者。
在上述方法的一些实施方案中，所述疾病是癌症。癌症的具体实例包括但不限于：基底细胞癌、胆管癌；膀胱癌；骨癌；乳腺癌；腹膜癌；宫颈癌；胆管癌；绒毛膜癌；结肠和直肠癌；结缔组织癌；消化系统癌症；子宫内膜癌；食道癌；眼癌；头颈癌；胃癌；胶质母细胞瘤；肝癌；肾癌；喉癌；白血病；肺癌(例如，小细胞肺癌、非小细胞肺癌、肺腺癌和肺鳞状细胞癌)；淋巴瘤，包括霍奇金淋巴瘤和非霍奇金淋巴瘤；黑色素瘤；骨髓瘤；神经内分泌瘤(例如，神经母细胞瘤)；口腔癌；卵巢癌；胰腺癌；前列腺癌；视网膜母细胞瘤；横纹肌肉瘤；直肠癌；呼吸系统癌；唾液腺癌；肉瘤；皮肤癌；鳞状细胞癌；睾丸癌；甲状腺癌；子宫或子宫内膜癌；泌尿系统癌症；B细胞淋巴瘤；慢性淋巴细胞性白血病(CLL)；急性成淋巴细胞性白血病(ALL)；毛细胞白血病；慢性成髓细胞性白血病等。在优选的实施方案中，所述癌症选自、肝癌、黑色素瘤、非小细胞肺癌、晚期神经 内分泌瘤(NEN)。
在上述方法的一些实施方案中，所述治疗难治性癌症为原发性肝癌(HCC)或去势抵抗性前列腺癌(CRPC)。
在上述方法的一些实施方案中，所述方法还包括施用一种或多种另外的疗法的步骤。例如，在一些实施方案中，所述疗法选自化学疗法、放射疗法、免疫疗法和手术疗法。
在一些实施方案中，所述免疫疗法选自针对免疫检查点分子的疗法、CAR-T细胞疗法和CAR-NK细胞疗法。例如，所述免疫检查点分子可以选自PD-1、PD-L1、PD-L2、CTLA4、OX40、LAG3、TIM3、TIGIT和CD103。
在一些实施方案中，所述化学疗法选自包括表柔比星、奥沙利铂和5-氟尿嘧啶的联合化疗方案。
实施例
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
以下实验，以具有SEQ ID NO:33所示氨基酸序列的抗CTLA-4单重链抗体为例，探索了抗CTLA-4单重链抗体的给药剂量、方式、Treg杀伤效应、药代动力学活性和抗肿瘤效果。
实施例1.抗CTLA-4单重链抗体的剂量爬坡和剂量扩展
本实施例涉及评估抗CTLA-4单重链抗体在晚期实体瘤受试者中的安全性、耐受性、药代动力学和抗肿瘤活性的I期、开放标签、国际多中心研究。本试验分为2个部分：“剂量爬坡”和“剂量扩展”。第1部分为剂量爬坡阶段，按照i3+3剂量爬坡算法(图1，图2)，根据每个剂量水平的剂量限制性毒性(DLT)以及综合毒性表现(包含迟发性毒性反应)结合算法进行“爬升”、“停留”或者“降级”的决定，与此同时尚有再次尝试的可能，除非该队列所表现的毒性条件直接触发DU(将至前一剂量水平并不再尝试该剂量)。
该爬坡方法除给与临床试验爬坡更大灵活性的同时，简单直观的视觉图表亦易于操作，并且其底层数学原理使得该方法相较于以往传统爬坡方式，优化了“错误估计”的风险，从而保证了各剂量组队列的安全性评估更为准确和公平。
与此同时，试验队列在第一部分随着剂量的爬升，预计将会在某一剂量组开始出现疗效信号。考虑到免疫药物的效应特点，往往会存在多个“临床可用剂量”的情况。已有研究提示同一药物对于不同类型肿瘤的“最优目标剂量”并不一致(参见例如伊匹单抗已获批瘤种及用药方法)，并且最大限制剂量(MTD)并非必要探及。
因此，除安全性评估外，本试验就各剂量组中呈现出的PD/PK特征，疗效状况(肿瘤的影像学改变，肿瘤标记物变化等)进行评估，定期经由临床医生为主的科学审评讨论确认该剂量“是否具有良好的获益/风险比”后，可以从某一剂量即开始在特定的瘤种中进行剂量扩展(Tock阶段)。与此同时，仍然继续进行剂量爬坡以继续探索本药物的安全性边际，进一步确认下一个具备“拓展”潜力(具有良好的获益/风险比)的剂量(Tick阶段)。若更高剂量组的“获益/风险比”仍被上述临床医生为主组成的SRC确认为可接受和具备扩展潜力，同样会按照上述方式在特定的瘤种中进行扩展(Tock阶段)。由此，类似钟摆运动(Tick-Tock)，不断通过广泛实体瘤中的剂量爬坡完成安全性确认的同时完成“获益/风险比”评价，一经确认即立即推送至扩展部分，在选定的“疗效验证模型”(具备潜力的特定瘤种)中进行POC验证；与此同时，试验将继续进入下一个“钟摆周期”。
此方法相较于以往“找到MTD后进入下一步”的理念，充分考虑了免疫药物量效多样化(不同瘤种的“最优剂量水平”不同，“起效剂量”亦可能不同)的特点，瘤种差异等因素，保证爬坡伦理安全性的同时，加速了进入剂量扩展的速度。同时，由于“钟摆模式”这一设定，将使得剂量扩展阶段的各瘤种会具有不同的剂量水平下的数据，这样有利于更全面的理解和分析“同一剂量下不同瘤种”和“同一瘤种不同剂量下”的药代药效特点以及临床获益差异。此外，考虑到可能存在的“特定瘤种的敏感起效剂量不同”假设，保留了扩展阶段队列设置的灵活性，允许根据在上述“钟摆模式”任何剂量阶段中发现的新信号增加额外的探索队列，从而增加该试验设计针对抗CTLA-4单重链抗体所具备的潜在应用提供足够的灵活性和兼容性。
已经完成了2个队列，合计19位患者中的剂量爬坡试验，其中0.3mg/kg，每周 一次给药：7人；0.6mg/kg，每三周一次给药：6人；0.45mg/kg，每三周一次给药：6人。所涉及的肿瘤包含：子宫内膜癌，肾非透明细胞癌，肾透明细胞癌，非小细胞肺癌，头颈癌，乳腺癌，去势抵抗性前列腺癌，睾丸癌，尿路上皮癌，肝癌，食管癌，间皮瘤，阴茎癌，结直肠癌等。
在安全性方面，相较于其他CTLA-4类药物报道的涉及多器官的不良事件(最常见前5种分别为皮疹，腹泻或结肠炎，肝炎，垂体炎，肺炎等)，抗CTLA-4单重链抗体的不良反应集中在消化道，主要是腹泻(2级或3级)或结肠炎(1级或2级)。抗CTLA-4单重链抗体的毒性谱相较同靶点药物窄/集中，且现有数据提示抗CTLA-4单重链抗体暴露后发生的消化道症状和病理发现相较同靶点药物的报道呈现明显的“分离”现象：G3腹泻为单纯性水样泻，部分患者伴随无粘液脓血便且无腹痛的G1-2结肠炎，但镜下病理大多轻微，该现象与临床前发现相类似。
肠镜及病理活检提示：除1例患者(腹腔注射12剂后发生)出现典型的溃疡性结肠炎表现外，其他患者均提示不典型表现(报告提示“肠炎不能除外，可能有关”)或活检镜下轻度炎症细胞活动，且上述患者腹泻症状可在药物干预后快速缓解，相关消化道综合诊断(腹泻/肠炎)均可通过临床使用抗腹泻药物及常规用量的激素(等量0.5mg-1mg/kg泼尼松)得到良好控制并最终缓解。
除消化道相关不良事件外，仅在0.3mg/kg，每周一次给药剂量组报道3例皮疹(≤2级)，而在其他剂量组均未提示皮肤相关不良事件。未观察到明确的免疫相关肝炎，垂体炎，肺炎，肾炎发生。3级治疗相关不良反应(TRAE)均为腹泻，无4级或以上TRAE发生。
0.45mg/kg Q3W剂量组受试者有6人，腹泻0人，结肠炎0人，疲劳0人，皮疹0人，瘙痒1人，整体耐受性良好。
以下表1显示了0.3mg/kg QW和0.6mg/kg Q3W剂量组中的不良事件汇总。
表1.各个剂量组中的不良事件结果汇总
Figure PCTCN2022087922-appb-000001
Figure PCTCN2022087922-appb-000002
实施例2.抗CTLA-4单重链抗体的体外和体内Treg杀伤效应
在体外ADCC杀伤试验中评价了抗CTLA-4单重链抗体的人T reg细胞清除活性。在该实验中，人T reg细胞在体外从未分化的CD4T细胞分化得到，然后用标记钙黄绿素AM标记，之后与抗体和原代人PBMC共孵育数个小时。对上清液中钙黄绿素AM的数值检测发现，抗CTLA-4单重链抗体(PR218)相较伊匹木单抗模拟物(PR149)具有强效的T reg细胞杀伤活性(图3)。相反在原代人总T细胞中无明显的由抗CTLA-4单重链抗体介导的细胞杀伤。
为评估疗效模型中肿瘤浸润细胞群的改变，在小鼠模型中进行了另一项抗CTLA-4单重链抗体研究性试验。对每一只敲入人CTLA-4基因的C57BL/6小鼠皮下接种MC38肿瘤细胞(1×10 6)，肿瘤接种后第10天的平均肿瘤大小为373mm 3。将动物随机分组，接受人IgG1 10mg/kg(G1)、伊匹木单抗模拟物10mg/kg(G2)、hIgG1HCAb(PR271)5.4mg/kg(G3)和抗CTLA-4单重链抗体5.4mg/kg(G4)和1.5mg/kg(G5)给药。在第3天给予小鼠第二剂，24小时后实施安乐死，收集肿瘤、脾脏和血液样本进行FACS分析。
主要终点是确定给药后携带有肿瘤的小鼠中肿瘤、血液和脾脏中不同免疫细胞群比例的变化。采用单向重复测量方差分析(ANOVA)方法分析不同组之间的细胞群差异。采用Bonferroni's多重比较方法与溶媒组进行比较。
如图4和表2中的结果所示，在肿瘤样本中，于5.4mg/kg或1.5mg/kg剂量抗CTLA-4单重链抗体给药组中观察到CD4 +T细胞中的T reg比例降低，而在使用伊匹木单抗模拟物时未观察到变化。在脾脏和血液样本中，使用抗CTLA-4单重链抗体或伊匹木单抗模拟物时均未观察到CD4 +细胞中的T reg比例有明显改变。
表2.T细胞和NK细胞的比例
Figure PCTCN2022087922-appb-000003
实施例3.抗CTLA-4单重链抗体的药代动力学结果
雌性C57BL/6小鼠单次静脉给药后的抗CTLA-4单重链抗体药代动力学
本研究的目的是确定雌性C57BL/6小鼠静脉给药后抗CTLA-4单重链抗体(PR218)的PK特征。6只SPF级C57BL/6小鼠(雌性)分别给予1和5mg/kg的抗CTLA-4单重链抗体(N＝3)。在以下时间点采集血液血清样本：给药前、0.083hr、第1、2、4、7、10和14天。通过ELISA方法测定C57BL/6小鼠血清中的抗CTLA- 4单重链抗体浓度。根据平均浓度-时间数据确定血清中的PR218PK参数(t 1/2、C max、总清除率[CL]、稳态分布容积[Vss]、平均停留时间[MRT]和浓度-时间曲线下面积[AUC])。采取非房室分析方法估计参数(
Figure PCTCN2022087922-appb-000004
Professional 6.4,Pharsight,Mountain View,USA)。在使用WinNonlin进行的PK参数计算中，排除任何低于可计量水平的数值。
图5中显示了1mg/kg和5mg/kg静脉剂量给药后抗CTLA-4单重链抗体的平均血清浓度-时间曲线图。抗CTLA-4单重链抗体的血清浓度随着剂量的增高而增高，随着时间的推移，呈双相特征。
在雌性C57BL/6小鼠中，1mg/kg抗CTLA-4单重链抗体静脉给药后，t 1/2、C max、时间0至末次可计量浓度间的浓度-时间曲线下面积(AUC last)、AUC INF、CL Z、MRT last和V ss分别是2.22±0.0281天、10.4±0.758μg/mL、16.1±1.48天*μg/mL、16.2±1.52天*μg/mL、62.1±58.0mL/kg/天、2.05±0.228天和134±12.1mL/kg(表3)。
在雌性C57BL/6小鼠中，5mg/kg抗CTLA-4单重链抗体静脉给药后，t 1/2、C max、AUC last、AUC INF、CL Z、MRT last和V ss分别是1.94±0.718天、36.6±15.9μg/mL、73.3±15.3天*μg/mL、74.0±15.9天*μg/mL、69.8±15.4mL/kg/天、2.41±0.144天和176±40.5mL/kg(表3)。
在1mg/kg-5mg/kg剂量范围内，终末清除半衰期和CLz保持恒定，平均值分别约为2.1天和66.0mL/kg/天。以AUCINF为参数测得的总暴露量显示，在1mg/kg-5mg/kg剂量范围内，与剂量成比例增高。数据表明，1mg/kg-5mg/kg的静脉剂量范围内，抗CTLA-4单重链抗体在小鼠中的PK呈线性。
表3.雌性C57BL/6小鼠中抗CTLA-4单重链抗体的药代动力学参数
Figure PCTCN2022087922-appb-000005
Figure PCTCN2022087922-appb-000006
食蟹猴单次静脉推注给药后抗CTLA-4单重链抗体的药代动力学研究
按照1、3和10mg/kg剂量单次静脉推注给予抗CTLA-4单重链抗体后，在食蟹猴中评价了抗CTLA-4单重链抗体的PK。将总计18只食蟹猴(9只雄性和9只雌性)随机分配至3组中，在每一剂量水平下测试。
在以下时间点采集用于PK评估的血液样本：给药前、给药后0.033、0.5、2、4、8、24、48、72、96、120、144、312、480、648和816hr(第35天)。采用经过验证的ELISA方法测定抗CTLA-4单重链抗体的血清浓度。根据平均浓度-时间数据确定血清中的抗CTLA-4单重链抗体PK参数(t 1/2、C 0时的C max、CL、V ss、AUC)。采取非房室分析估计参数。
为检测抗CTLA-4单重链抗体给药后生成的抗药抗体(ADA)，在以下时间点采集血液血清样本：给药前、给药后312hr(第14天)、480hr(第21天)、816hr(第35天)。采用经过验证的点化学发光方法检测ADA浓度。
给药前，所有动物均未显示任何可测得的ADA。在给药后第14天，多数动物显示ADA阴性，但1mg/kg组3只雌性动物中的1只、3mg/kg组3只雌性动物中的2只和10mg/kg组所有3只雄性和3只雌性动物中的1只例外，显示ADA阳性，但滴度较低。第21天，1mg/kg组3只雌性动物中的1只、3mg/kg组3只雄性中的2只和3只雌性动物中的2只和10mg/kg组所有3只雄性和3只雌性动物中的1只显示ADA阳性，但滴度较低。
第35天时，ADA阳性率增高，分别有1mg/kg组3只雄性中的1只和3只雌性中的2只、3mg/kg组3只雄性中的2只和所有雌性和10mg/kg组所有3只雄性和3只雌性动物中的2只显示ADA阳性。
图6显示了食蟹猴中单次1、3和10mg/kg静脉剂量给药后抗CTLA-4单重链抗体的平均血清浓度-时间曲线图。
食蟹猴中静脉给予1、3和10mg/kg剂量的抗CTLA-4单重链抗体后，初始时间点C 0达到的C max(首次注射后立即测量的浓度)分别为25.2±2.33μg/mL、82.9±12.6μg/mL和244±22.9μg/mL；t1/2分别是3.02±0.490天、2.77±0.482天和2.55±0.412天；AUC last分别是26.2±3.74μg*天/mL、75.9±4.48μg*天/mL和231±23.0μg*天/mL；AUC INF分别是26.6±3.71μg*天/mL、76.8±4.69μg*天/mL和233±23.2 μg*天/mL；CL分别是38.4±6.05mL/天/kg、39.2±2.52mL/天/kg和43.3±4.38mL/天/kg；Vd分别是112±14.3mL/kg、109±8.67mL/kg和118±19.3mL/kg(表4)。
1、3和10mg/kg剂量组间，t 1/2和CL保持恒定。在1mg/kg-10mg/kg剂量范围内，C 0和以AUC INF为参数测量的总暴露量显示与剂量成比例增高。数据表明，在1mg/kg-10mg/kg静脉剂量范围内，抗CTLA-4单重链抗体在食蟹猴中的PK呈线性。
表4.雄性和雌性食蟹猴中抗CTLA-4单重链抗体的药代动力学参数
Figure PCTCN2022087922-appb-000007
实施例4.抗CTLA-4单重链抗体在患者体内的药代动力学结果
药物信息
抗CTLA-4单重链抗体注射剂，每瓶含有4.0mL药液，含80mg的抗CTLA-4单重链抗体(具有SEQ ID NO:33所示的氨基酸序列)，当日给药，给药量按体重计算，保存于2-8℃下，使用原始包装直立储存药品。
配药方式为：从瓶中抽取所需剂量的抗CTLA-4单重链抗体，加入0.9％氯化钠注射液或5％葡萄糖注射液输液袋中稀释，稀释浓度范围为0.12mg/mL～10.0mg/mL。
要求静脉滴注稀释液90分钟以上，但不应超过4小时，即输注时间控制在90分钟～4小时内。如果抗CTLA-4单重链抗体注射剂配制成50mL的稀释液，应控制滴注速率为0.21mL/min～0.56mL/min；如果抗CTLA-4单重链抗体注射剂配制成 100mL的稀释液，应控制滴注速率为0.42mL/min～1.1mL/min。静脉滴注时，需采用含有0.2微米的在线滤膜、无菌、无致热源、低蛋白结核的过滤器的静脉滴注器，静脉输注后，使用20mL 0.9％氯化钠注射液或5％葡萄糖注射液冲静脉滴注器管道。
给药信息
给予一部分癌症患者输注抗CTLA-4单重链抗体注射剂，分为三队列给药：第一个队列患者给药设计是0.3mg/kg，每周一次给药；第二个队列患者给药设计是0.6mg/kg，每三周一次给药；第三个队列患者给药设计是0.45mg/kg，每三周一次给药。
另一部分癌症患者输注伊匹木单抗(Ipilimumab)，分为2个队列给药：第一个队列患者给药设计是0.3mg/kg，每三周一次给药；第二个队列患者给药设计是3mg/kg，每三周一次给药。
数据采集
在患者(Australia Patients)给药前以及给药一个循环后不同时间点采集外周血，以检测药代动力学(PK)数据及血清中的抗CTLA-4单重链抗体浓度以及抗药抗体(ADA)。
采用经全面验证的电化学发光方法(ECL)检测人血清中抗CTLA-4单重链抗体的浓度。分析方法的定量下限(LLOQ)为20.5ng/mL，定量上限为5000.0ng/mL，超出定量上限的样品可进行最高400倍稀释。样品在室温、冷藏及冷冻条件下均有很好的稳定性。
采用经全面验证的桥接电化学发光方法(ECL)分析抗药抗体(ADA)，采用多层级的分析方法，即首先对所有样品进行筛选试验，其次对疑似抗体阳性样品的特异性进行确证试验，对已确定抗体阳性的样品进行滴度试验。该分析方法筛选分析灵敏度为34.0ng/mL。在采集的样品药物浓度水平下药物耐受良好。
采取非房室分析方法估计PK参数。
在晚期实体瘤受试者中测试的各剂量水平抗CTLA-4单重链抗体的血清抗CTLA-4单重链抗体浓度-时间曲线见图7，PK参数总结见表5。
表5 受试者中抗CTLA-4单重链抗体的药代动力学参数
Figure PCTCN2022087922-appb-000008
在晚期实体瘤受试者中观察到的PK特性与临床前预测的类似：
半衰期短(大约2～4天)，因而多次给药后积蓄小；对比伊匹木单抗的半衰期为14.7天；
暴露量(AUC)与预测值接近，远低于伊匹木单抗同剂量下的暴露量；
AUC随剂量增加而增加，与剂量基本呈比例；
另外，数据显示抗CTLA-4单重链抗体免疫原性低：13位患者中只有一人在一个访视时检测到ADA阳性，滴度为2。
实施例5.抗CTLA-4单重链抗体的抗肿瘤活性
药物信息
抗CTLA-4单重链抗体注射剂，每瓶含有4.0mL药液，含80mg的抗CTLA-4单重链抗体(具有SEQ ID NO:33所示的氨基酸序列)，当日给药，给药量按体重计算，保存于2-8℃下，使用原始包装直立储存药品。
配药方式为：从瓶中抽取所需剂量的抗CTLA-4单重链抗体，加入0.9％氯化钠注射液或5％葡萄糖注射液输液袋中稀释，稀释浓度范围为0.12mg/mL～10.0mg/mL。
要求静脉滴注稀释液90分钟以上，但不应超过4小时，即输注时间控制在90分钟～4小时内。如果抗CTLA-4单重链抗体注射剂配制成50mL的稀释液，应控制滴注速率为0.21mL/min～0.56mL/min；如果抗CTLA-4单重链抗体注射剂配制成100mL的稀释液，应控制滴注速率为0.42mL/min～1.1mL/min。静脉滴注时，需采用含有0.2微米的在线滤膜、无菌、无致热源、低蛋白结核的过滤器的静脉滴注 器，静脉输注后，使用20mL 0.9％氯化钠注射液或5％葡萄糖注射液冲静脉滴注器管道。
抗肿瘤评估
给肿瘤患者输注抗CTLA-4单重链抗体注射剂，在肿瘤患者中共进行了2次抗肿瘤评估，每次抗肿瘤评估中，分为三队列给药：第一个队列患者给药设计是0.3mg/kg，每周一次给药；第二个队列患者是0.6mg/kg，每三周一次给药；第三个队列患者是0.45mg/kg，每三周一次给药。
临床疗效方面，11例患者完成了第1次肿瘤评估，其中9例维持疾病稳定(SD)。HCC患者中观察到肿瘤较基线缩小22％，AFP下降至正常水平。CRPC患者实现“PSA缓解”，PSA从基线210μg/L下降超过50％至91μg/L，维持至今(近8月)，最近一次复查显示PSA仍维持在78μg/L，患者感觉良好。EC和PRCC两位患者观察到6-14％肿瘤退缩。7例患者完成了第2次肿瘤评估，2例维持疾病稳定(SD)，1例疾病缓解(PR，肿瘤较基线下降约50％，AFP维持正常至今)，1例RCC患者观察到部分靶病灶显著退缩。抗CTLA-4单重链抗体的治疗结果总结于表6中。
表6.抗CTLA-4单重链抗体的抗肿瘤效应
Figure PCTCN2022087922-appb-000009
Figure PCTCN2022087922-appb-000010
实施例6.抗CTLA-4单重链抗体在典型患者中的治疗结果
患者1
给予癌症患者1静脉输注抗CTLA-4单重链抗体，患者信息如下表7所示，患者先前接受过外科手术和放射治疗，先前服用伦伐替尼、索拉非尼和SHR1701-001(2020-Sep-08,恒瑞PD-1；PD)，停药原因不明，并且先前接受过PD-1/PD-L1免疫治疗。
表7.患者1的信息
Figure PCTCN2022087922-appb-000011
给予患者1输注抗CTLA-4单重链抗体注射剂，其中每瓶含有4.0mL药液，含80mg的抗CTLA-4单重链抗体(序列如SEQ ID NO:33所示)，当日给药，给药量按体重计算，保存于2-8℃下，使用原始包装直立储存药品。
配药方式为：从瓶中抽取所需剂量的抗CTLA-4单重链抗体，加入0.9％氯化 钠注射液或5％葡萄糖注射液输液袋中稀释，稀释浓度范围为0.12mg/mL～10.0mg/mL。
要求静脉滴注稀释液90分钟以上，但不应超过4小时，即输注时间控制在90分钟～4小时内。如果抗CTLA-4单重链抗体注射剂配制成50mL的稀释液，应控制滴注速率为0.21mL/min～0.56mL/min；如果抗CTLA-4单重链抗体注射剂配制成100mL的稀释液，应控制滴注速率为0.42mL/min～1.1mL/min。静脉滴注时，需采用含有0.2微米的在线滤膜、无菌、无致热源、低蛋白结核的过滤器的静脉滴注器，静脉输注后，使用20mL 0.9％氯化钠注射液或5％葡萄糖注射液冲静脉滴注器管道。
患者1的给药剂量0.45mg/kg，每三周一次给药，给药三次。
患者输注抗CTLA-4单重链抗体后的指标性状如表8-9所示。
表8：患者1病变指标
Figure PCTCN2022087922-appb-000012
表9：患者1其它指标
Figure PCTCN2022087922-appb-000013
由表8和表9可以看出，患者1输注抗CTLA-4单重链抗体后，肿瘤明显减小，肿瘤基线变化超50％，肿瘤生物标志物明显减少，肝功明显恢复。
患者2
给予癌症患者2静脉输注抗CTLA-4单重链抗体，患者信息如下表10所示，患者先前接受过前列腺切除术(2001年2月15日)和未接受放射治疗，先前进行过Docetaxel、Cabazitaxel、Cosudex、Zoladex化疗，没有接受过PD-1/PD-L1免疫治疗，现在服用过地高辛(Digoxin)；利伐沙班(rivaroxaban)；维拉帕米(verapamil)；钙盐；维生素D；地诺单抗(denosumab)；二噻嗪(dithiazide)药物。
表10.患者2的信息
Figure PCTCN2022087922-appb-000014
给予患者2输注的抗CTLA-4单重链抗体注射剂，每瓶含有4.0mL药液，含80mg的抗CTLA-4单重链抗体，当日给药，给药量按体重计算，保存于2-8℃下，使用原始包装直立储存药品。
配药方式为：从瓶中抽取所需剂量的抗CTLA-4单重链抗体，加入0.9％氯化钠注射液或5％葡萄糖注射液输液袋中稀释，稀释浓度范围为0.12mg/mL～10.0mg/mL。
要求静脉滴注稀释液90分钟以上，但不应超过4小时，即输注时间控制在90 分钟～4小时内。如果抗CTLA-4单重链抗体注射剂配制成50mL的稀释液，应控制滴注速率为0.21mL/min～0.56mL/min；如果抗CTLA-4单重链抗体注射剂配制成100mL的稀释液，应控制滴注速率为0.42mL/min～1.1mL/min。静脉滴注时，需采用含有0.2微米的在线滤膜、无菌、无致热源、低蛋白结核的过滤器的静脉滴注器，静脉输注后，使用20mL 0.9％氯化钠注射液或5％葡萄糖注射液冲静脉滴注器管道。
患者2的给药剂量0.6mg/kg，每三周一次给药，给药三次。
患者抗CTLA-4单重链抗体给药后的指标性状如表11-12所示。
表11.患者2病变指标
Figure PCTCN2022087922-appb-000015
表12.患者2其它指标
Figure PCTCN2022087922-appb-000016
由表11和表12可以看出，患者2输注抗CTLA-4单重链抗体后，肿瘤明显减小，肿瘤基线变化超50％，肿瘤生物标志物明显减少。
综上所述，结合临床数据及观察，抗CTLA-4单重链抗体的总体临床表现包括：患者对于抗CTLA-4单重链抗体的耐受性良好，所发生的不良事件符合同靶点毒性预期，较同类靶点药物毒性谱更集中于消化道系统，而较少发生于皮肤肝肾肺等器官，且未观察到严重的消化系统特异性病理改变及其相关临床风险(消化道穿孔风险，严重的肠黏膜损伤等)。包含起始剂量在内的不同剂量组，均观察到了明确的抗肿瘤效应，且在部分难治性瘤种中(如多线治疗失败后的冷肿瘤“CRPC”；多线治疗失败后的末线HCC；)观测到符合现行指南所规定的“显著临床获益”(“疾病缓解”；“肿瘤标记物PSA缓解”等)并持续至今。
抗CTLA-4单重链抗体的半衰期短(约2-4天，相对于伊匹单抗的半衰期为14.7天),因而多次给药后积蓄小。暴露量(AUC)与预测值接近，远低于伊匹木单抗同剂量下的暴露量。AUC随剂量增加而增加。出人意料的是，在较短的半衰期、较低的药物积蓄和暴露量的情况下，在患者中仍然观察到长时间维持的PD标志物变化(CD8 T细胞/Treg比例、ICOS+T细胞和Ki67+T细胞增加)和显著的抗肿瘤效应。此发现支持抗CTLA-4单重链抗体的Q3W给药，同时表明抗CTLA-4单重链抗体在短半衰期减小药物暴露的同时仍然可以维持可靠的长时间药效，具备进一步开发长周期给药或者必要时给药的临床应用可能性。
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Figure PCTCN2022087922-appb-000017
Figure PCTCN2022087922-appb-000018
Figure PCTCN2022087922-appb-000019
Figure PCTCN2022087922-appb-000020
Figure PCTCN2022087922-appb-000021
Figure PCTCN2022087922-appb-000022
Figure PCTCN2022087922-appb-000023
Figure PCTCN2022087922-appb-000024

Claims (23)

  1. 一种在受试者中清除调节性T细胞(Treg)和/或增加CD8+T细胞/Treg细胞比例的方法，其特征在于，给予受试者施用抗CTLA-4单重链抗体。
  2. 根据权利要求1所述的方法，其特征在于，所述受试者为癌症患者；
    任选地，所述受试者为子宫内膜癌、肾非透明细胞癌、肾透明细胞癌、非小细胞肺癌、头颈癌、乳腺癌、去势抵抗性前列腺癌、睾丸癌、尿路上皮癌、肝癌、食管癌、间皮瘤、黑色素瘤或神经内分泌瘤患者。
  3. 一种在受试者中治疗难治性癌症的方法，其特征在于，给予受试者施用抗CTLA-4单重链抗体。
  4. 根据权利要求3所述的方法，其特征在于，所述难治性癌症对一种或多种免疫检查点抑制剂例如PD-1/PD-L1轴信号通路抑制剂不响应或耐受。
  5. 根据权利要求3或4所述的方法，其特征在于，所述治疗难治性癌症为原发性肝癌(HCC)或去势抵抗性前列腺癌(CRPC)。
  6. 一种治疗癌症的方法，其特征在于，包括对所述受试者施用抗CTLA-4单重链抗体和任选地第二治疗剂的步骤。
  7. 根据权利要求6所述的方法，其特征在于，所述第二治疗剂选自一种或多种免疫检查点抑制剂例如PD-1/PD-L1轴信号通路抑制剂；
    任选地，所述第二治疗剂抗PD-1单克隆抗体，例如派姆单抗(Perbrolizumab)、纳武单抗(Nivolumab)、或特瑞普利(Toripalimab)。
  8. 根据权利要求6所述的方法，其特征在于，所述第二治疗剂为化疗药物，例如紫杉醇或卡铂。
  9. 一种预防和/或减少CTLA4抗体治疗癌症时副作用的方法，其特征在于，向所述受试者施用抗CTLA-4单重链抗体。
  10. 一种抗CTLA-4单重链抗体的给药方法，其特征在于，包括将所述抗CTLA-4单重链抗体以0.1mg/mL-10.0mg/mL的浓度通过静脉滴注的形式输入患者体内；优选地，所述抗CTLA-4单重链抗体以0.9％氯化钠或5％葡萄糖溶液的形式提供；优选地，所述静脉滴注的时间不超过4小时。
  11. 根据权利要求1～10任一项所述的方法，其特征在于，给予受试者以0.2mg～1mg/kg体重的剂量施用抗CTLA-4单重链抗体；
    任选地，给药周期为每周～每12周给药一次，任选地，给药周期为每周、每2周、每3周、每4周、每5周、每6周、每7周、每8周、每9周、每10周、每11周或每12周给药一次；
    优选地，以0.3-0.6mg/kg的剂量向受试者施用抗CTLA-4单重链抗体；
    优选地，以0.3mg/kg-0.6mg/kg的剂量每3周一次给予受试者施用抗CTLA-4单抗重链抗体；
    优选地，以0.3mg/kg-0.45mg/kg的剂量每3周一次给予受试者施用抗CTLA-4单抗重链抗体；
    优选地，以0.3mg/kg的剂量每周一次给予受试者施用抗CTLA-4单重链抗体；
    优选地，以0.45mg/kg的剂量每3周一次给予受试者施用抗CTLA-4单重链抗体；
    优选地，以0.6mg/kg的剂量每3周一次给予受试者施用抗CTLA-4单重链抗体。
  12. 一种实现癌症患者长周期给药或者必要时给药的方法，其特征在于，向所述受试者施用抗CTLA-4单重链抗体。
  13. 根据权利要求12所述的方法，其特征在于，所述长周期给药是指给药周期不小于4周，优选地，不小于6周、7周、8周、9周、10周、11周或12周，例如，6周或12周。
  14. 根据权利要求12所述的方法，其特征在于，所述受试者的不能维持疾病稳定(SD)、部分缓解(PR)或完全缓解(CR)状态，是必要时给药的指示；
    任选地，所述受试者不能维持SD，PR或CR状态，给予所述受试者施用抗CTLA-4单重链抗体直到受试者恢复SD，PR或CR状态；
    任选地，所述施用抗CTLA-4单重链抗体的持续时间为2～4个给药周期；
    任选地，向受试者以0.2mg～1mg/kg体重的剂量施用所述抗CTLA-4单重链抗体，优选为0.3-0.6mg/kg。
  15. 一种单位剂量组合物，其特征在于，包含20-200mg的抗CTLA-4单重链抗体。
  16. 根据权利要求15所述的单位剂量组合物，其特征在于，包含60-100mg的所述抗CTLA-4单重链抗体，优选地，包含80mg的所述抗CTLA-4单重链抗体。
  17. 抗CTLA-4单重链抗体在制备药物中的用途，所述药物用于治疗受试者 中的CTLA-4相关疾病例如癌症，所述药物配制成以0.2mg～1mg/kg剂量施用的形式。
  18. 根据权利要求17所述的用途，所述药物配制成以0.3mg/kg-0.6mg/kg的剂量每3周一次剂量施用的形式；
    优选地，0.45mg/kg-0.6mg/kg的剂量每3周一次给予受试者施用抗CTLA-4单抗重链抗体；
    优选地，所述药物配置成以0.45mg/kg的剂量每3周一次剂量施用的形式。
  19. 根据权利要求1～18任一项所述的方法、单位剂量组合物或用途，其特征在于，所述抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1具有SEQ ID NO:1～8任一所示的氨基酸序列；所述CDR2具有SEQ ID NO:9～16任一所示的氨基酸序列；所述CDR3具有SEQ ID NO:17～24任一所示的氨基酸序列；
    任选地，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:1，9和17所示的氨基酸序列；
    任选地，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:2，10和18所示的氨基酸序列；
    任选地，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:3，11和19所示的氨基酸序列；
    任选地，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:4，12和20所示的氨基酸序列；
    任选地，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:5，13和21所示的氨基酸序列；
    任选地，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:6，14和22所示的氨基酸序列；
    任选地，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:7，15和23所示的氨基酸序列；
    任选地，抗CTLA-4单重链抗体具有CDR1，2和3，所述CDR1，CDR2和CDR3分别具有SEQ ID NO:8，16和24所示的氨基酸序列。
  20. 根据权利要求19所述的方法、单位剂量组合物或用途，其特征在于，所述抗CTLA-4单重链抗体具有重链可变区，所述重链可变区具有SEQ ID NO:25～32任一所示的氨基酸序列。
  21. 根据权利要求19所述的方法、单位剂量组合物或用途，其特征在于，所述抗CTLA-4单重链抗体具有SEQ ID NO:33～40任一所示的氨基酸序列。
  22. 一种“钟摆运动”式抗肿瘤药物的给药剂量确定方法，其特征在于，
    1)将待测定药物按照预定剂量梯度在广泛瘤种中进行剂量爬坡实验，以便获得待测定药物在预定剂量下的疗效数据以及毒性数据；
    2)基于待测定药物在预定剂量下的疗效数据以及毒性数据，确定待测定药物在所述预定剂量下是否在特定瘤种中进行剂量扩展实验；
    3)重复步骤2)，以便确定待测定药物在更高预定剂量下是否在特定瘤种中进行剂量扩展实验。
  23. 一种确定抗肿瘤药物的给药形式的方法，其特征在于，包括：
    将待测药物施于测试模型，在预定给药剂量、给药次数和给药周期的进行试验，以便获得待测药物的优势给药形式，其中，测试模型的SD，PR或CR处于稳定状态，是优势给药形式的指示；
    优选地，进一步包括将第二治疗剂施于测试模型，在预定给药剂量、给药次数和给药周期的进行试验，以便获得第二治疗剂的优势给药形式，
    其中，测试模型的SD，PR或CR依然处于稳定状态，是第二治疗剂的优势给药形式的指示；
    任选地，测试模型的SD，PR或CR不能维持稳定状态，将待测药物再次施于测试模型，以便使得测试模型的SD，PR或CR重新处于稳定状态。
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