WO2021098834A1 - Psma抗体及其应用 - Google Patents

Psma抗体及其应用 Download PDF

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WO2021098834A1
WO2021098834A1 PCT/CN2020/130511 CN2020130511W WO2021098834A1 WO 2021098834 A1 WO2021098834 A1 WO 2021098834A1 CN 2020130511 W CN2020130511 W CN 2020130511W WO 2021098834 A1 WO2021098834 A1 WO 2021098834A1
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seq
psma
amino acid
acid sequence
scfv
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PCT/CN2020/130511
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French (fr)
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王峰
郑花鸯
张雨菡
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上海一宸医药科技有限公司
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Priority to US17/778,422 priority Critical patent/US20220411530A1/en
Priority to CN202080009706.8A priority patent/CN113677704B/zh
Priority to EP20889117.6A priority patent/EP4063387A4/en
Publication of WO2021098834A1 publication Critical patent/WO2021098834A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention provides an antibody fragment that specifically binds to PSMA, and specifically relates to a single-chain Fv (scfv) that specifically binds to PSMA, a double antibody containing the single-chain Fv, a CAR, and a composition and application thereof.
  • scfv single-chain Fv
  • Prostate cancer is the second leading cause of death in men, second only to lung cancer.
  • current methods such as surgery, radiation therapy, chemotherapy, and androgen deprivation therapy, have limited effects on those advanced cancers. So far, radical surgery is still the main treatment for prostate cancer. Therefore, emerging precision medicine, especially precision medicine based on tumor-targeted antibodies, is expected to improve the outcome of prostate treatment.
  • Prostate-specific membrane antigen is mainly expressed by prostate epithelial cells.
  • PSMA Prostate-specific membrane antigen
  • the expression of PSMA increases (Gregorakis AK et al. (1998) Seminars in Urologic Oncology 16: 2-12; Silver, DA (1997) Clinical Cancer Research 3: 85-515).
  • Low-level expression of PSMA was found in extra-prostate tissues such as the small intestine, salivary glands, duodenal mucosa, proximal renal tubules, and brain (Silver, DA (1997) Clinical Cancer Research 3: 85-515).
  • PSMA is also expressed in the endothelial cells of the capillaries in the peripheral and intratumor regions of some malignant tumors (including renal cell carcinoma and colon cancer), but not in the blood vessels of normal tissues.
  • PSMA is associated with tumor angiogenesis (Silver, D.A. (1997) Clinical Cancer Research 3: 81-85).
  • PSMA is expressed in the endothelial cells of the tumor-associated neovascular system in colon cancer, breast cancer, bladder cancer, pancreatic cancer, kidney cancer, and melanoma (Chang, SS (2004) Curr Opin Investing Drugs 5:611 -5), laying the foundation for the development of targeted precision medicine for prostate cancer.
  • the present invention uses phage display technology to screen and obtain a series of high-affinity antibodies against PSMA.
  • the inventors further found that in the form of these antibody sequences in the form of scFv, the type of amino acid residue at position 42 of the light chain VL has a significant effect on the affinity; further, bispecific antibodies containing a specific type of VL at amino acid position 42 , Its protein yield has been greatly improved.
  • the aPSMA scfv antibody fragment in the present invention can be used to construct bispecific antibodies, chimeric antigen receptors, etc., and can meet the affinity requirements for aPSMA scfv in the field of tumor targeted therapy.
  • VH The heavy chain variable region (VH) with the amino acid sequence shown in SEQ ID NO: 2;
  • VL light chain variable region
  • the antibody fragment that specifically binds to PSMA is a single chain Fv (scfv).
  • the PSMA-targeted scfv has VH with the amino acid sequence shown in SEQ ID NO: 2 and VL with the amino acid sequence shown in SEQ ID NO: 6.
  • the PSMA-targeted scfv has VH with the amino acid sequence shown in SEQ ID NO: 2 and VL with the amino acid sequence shown in SEQ ID NO: 8.
  • the PSMA-targeted scfv has VH with the amino acid sequence shown in SEQ ID NO: 2 and VL with the amino acid sequence shown in SEQ ID NO: 10.
  • the PSMA-targeted scfv has an amino acid sequence as shown in SEQ ID NO:20. In some embodiments, the PSMA-targeted scfv has an amino acid sequence as shown in SEQ ID NO:22. In some embodiments, the PSMA-targeted scfv has an amino acid sequence as shown in SEQ ID NO: 24. In some embodiments, the PSMA-targeted scfv has an amino acid sequence as shown in SEQ ID NO:26. In some embodiments, the PSMA-targeted scfv has an amino acid sequence as shown in SEQ ID NO:28. In some embodiments, the PSMA-targeted scfv has an amino acid sequence as shown in SEQ ID NO:30.
  • the PSMA-targeted scfv has an amino acid sequence as shown in SEQ ID NO:32. In some embodiments, the PSMA-targeted scfv has an amino acid sequence as shown in SEQ ID NO:34. In some embodiments, the PSMA-targeted scfv has an amino acid sequence as shown in SEQ ID NO: 36. In some embodiments, the PSMA-targeted scfv has an amino acid sequence as shown in SEQ ID NO:38. In some embodiments, the PSMA-targeted scfv has an amino acid sequence as shown in SEQ ID NO:54. In some embodiments, the PSMA-targeted scfv has an amino acid sequence as shown in SEQ ID NO:56.
  • bispecific antibody comprising:
  • the first antigen-binding domain that specifically binds to PSMA which includes:
  • the first antigen binding domain of the bispecific antibody is a single chain Fv (scfv) that specifically binds to PSMA.
  • the first antigen binding domain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:20.
  • the first antigen binding domain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:22.
  • the first antigen binding domain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 24.
  • the first antigen binding domain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:26.
  • the first antigen binding domain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:28. In some embodiments, the first antigen binding domain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:30. In some embodiments, the first antigen binding domain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:32. In some embodiments, the first antigen binding domain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:34. In some embodiments, the first antigen binding domain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 36. In some embodiments, the first antigen binding domain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:38. In some embodiments, the first antigen binding domain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:54. In some embodiments, the first antigen binding domain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:56.
  • the second antigen binding domain of the bispecific antibody binds to a specific receptor molecule of T cells, wherein the specific receptor molecule of T cells is preferably CD3.
  • the second antigen binding domain that specifically binds to CD3 has a VH shown in SEQ ID NO: 40 and a VL shown in SEQ ID NO: 42.
  • the bispecific antibody has the sequence shown in SEQ ID NO: 44 and SEQ ID NO: 46. In some embodiments, the bispecific antibody has the sequence shown in SEQ ID NO: 58 and SEQ ID NO: 60. In some embodiments, the bispecific antibody has the sequence shown in SEQ ID NO: 62 and SEQ ID NO: 64. In some embodiments, the bispecific antibody has the sequence shown in SEQ ID NO: 58 and SEQ ID NO: 66. In some embodiments, the bispecific antibody has the sequence shown in SEQ ID NO: 68 and SEQ ID NO: 64.
  • One aspect of the present invention provides a chimeric antigen receptor (CAR), wherein the CAR comprises scfv, a transmembrane domain and an intracellular domain that specifically bind to PSMA.
  • CAR chimeric antigen receptor
  • the scfv of the CAR that specifically binds to PSMA has a VH shown in SEQ ID NO: 2 and a VL shown in SEQ ID NO: 6. In some embodiments, the scfv of the CAR that specifically binds to PSMA has a VH shown in SEQ ID NO: 2 and a VL shown in SEQ ID NO: 8. In some embodiments, the scfv of the CAR that specifically binds to PSMA has a VH shown in SEQ ID NO: 2 and a VL shown in SEQ ID NO: 10.
  • the scfv of the CAR that specifically binds to PSMA has an amino acid sequence as shown in SEQ ID NO:22. In some embodiments, the scfv of the CAR that specifically binds to PSMA has an amino acid sequence as shown in SEQ ID NO: 24. In some embodiments, the scfv of the CAR that specifically binds to PSMA has an amino acid sequence as shown in SEQ ID NO: 26. In some embodiments, the scfv of the CAR that specifically binds to PSMA has an amino acid sequence as shown in SEQ ID NO:28. In some embodiments, the scfv of the CAR that specifically binds to PSMA has an amino acid sequence as shown in SEQ ID NO:32.
  • the scfv of the CAR that specifically binds to PSMA has an amino acid sequence as shown in SEQ ID NO:34. In some embodiments, the scfv of the CAR that specifically binds to PSMA has an amino acid sequence as shown in SEQ ID NO: 36. In some embodiments, the scfv of the CAR that specifically binds to PSMA has an amino acid sequence as shown in SEQ ID NO: 38. In some embodiments, the scfv of the CAR that specifically binds to PSMA has an amino acid sequence as shown in SEQ ID NO:54. In some embodiments, the scfv of the CAR that specifically binds to PSMA has an amino acid sequence as shown in SEQ ID NO:56. In some embodiments, the CAR has an amino acid sequence as shown in SEQ ID NO:50.
  • polynucleotide that encodes an antibody fragment, bispecific antibody or CAR that specifically binds to PSMA in any one of the foregoing embodiments.
  • the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 44, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67.
  • a vector which comprises the aforementioned polynucleotide.
  • composition comprising the antibody fragment, bispecific antibody, CAR, polynucleotide, carrier or cell of any one of the foregoing, the composition further comprising a pharmaceutically acceptable carrier.
  • One aspect of the present invention provides a method for treating a subject suffering from a disease related to PSMA expression, which comprises administering to the subject an effective amount of the aforementioned composition.
  • the present invention provides a method for diagnosing a disease associated with PSMA expression in a mammal, the method comprising: using the antibody fragment of any one of claims 1 to 3 in a mammal isolated from the mammal The binding to human PSMA is detected in the tissue sample, and thus the specific binding of the antibody fragment to the human PSMA in the tissue sample is indicative of a disease associated with PSMA expression in the mammal.
  • Figure 1 shows the aPSMA LH RM and RM2 monoclonal phage ELISA test results, where RM is the first round of panning and RM2 is the second round of panning.
  • Figure 2 shows the results of aPSMA HL monoclonal phage ELISA test.
  • Figure 3A shows the results of ELISA detection of aPSMA scfv antibody with amino acid mutation at position 42 of VL and PSMA-his;
  • Figure 3B shows the flow cytometric detection results of binding of scfv-Fc antibody targeting PSMA (with mutation of amino acid at position 42 of VL) to LnCap cells.
  • Figure 4 shows the results of flow cytometric detection of the binding of bispecific antibodies to Jurkat T cells.
  • Figure 5 shows the results of flow cytometric detection of the binding of bispecific antibodies to LnCap cells.
  • Figure 7 shows the PK curve of the bispecific antibody in mice.
  • Figure 1 shows the affinity detection results of the clones of the mutant library aPSMA LH RM and aPSMA LH RM2. It can be seen from the figure that except for aPSMA LH RM-R13 and aPSMA LH RM2-R15, the affinity of the other clones is much higher.
  • Figure 2 is the affinity detection result of the mutant library aPSMA HL RM, only aPSMA HL RM-R1/R10/R15 has a higher affinity.
  • LnCap cells were fed in RPMI 1640 medium containing 10% FBS and placed in a 5% CO2 incubator. 2*10 5 cells were washed 3 times with pre-cooled PBS. Blocked with 2% FBS diluted in PBS, and incubated with serially diluted antibodies (100nM successively 3 times serially diluted) at 4°C for 1 h. Unbound antibody is washed away by 2% FBS. Mouse anti-human IgG Fc-APC (southern biotech) was incubated for 1 hour at 4 degrees, washed with 2% FBS, and analyzed by flow cytometry.
  • the two chains of the eukaryotic expression vector verified by the above sequencing were transiently co-transfected into FreeStyle HEK293 cells (ThermoFisher): 28ml FreeStyle HEK 293 (3 ⁇ 10 7 cells/ml) was inoculated into a 125ml cell culture flask, and the plasmid was used 1ml Opti- MEM (Invitrogen) was diluted and added to 1 ml of Opti-MEM containing 60 ⁇ l 293Fectin (Invitrogen, Inc), allowed to stand at room temperature for 30 minutes, and the plasmid-293fectin mixture was added to the cell culture medium at 125 rpm, 372, and 5% CO2.
  • the cell culture supernatant was collected 48h and 96h after transfection, and purified by Protein A Resin (Genscript) according to the manufacturer's instructions, and then detected by SDS-PAGE, and the yield was calculated.
  • the results show that the yield of aCD3-aPSMA (15-20mg/L) based on the 42th mutation of VL is significantly higher than the yield of wild-type aCD3-aPSMA (2.5-5mg/L) at the 42nd VL.
  • Jurkat cells were fed in RPMI 1640 medium containing 10% FBS and placed in a 5% CO2 incubator. Wash 2 ⁇ 10 5 cells with pre-cooled PBS for 3 times, after blocking with 2% FBS in PBS, incubate with 4 pairs of double antibodies with concentrations of 100nM, 33.3nM, 11.1nM, 3.7nM, 1.2nM or 0.4nM 1h. The unbound antibody was washed away with PBS containing 2% FBS, and the secondary antibody mouse anti-human IgG Fc-APC (southern biotech) was added to incubate at 4°C for 1 h. After washing three times in PBS containing 2% FBS, flow cytometry analysis was performed. The results are shown in Figure 4, that the bispecific antibody can effectively bind to Jurkat cells.
  • LnCap cells were fed in RPMI 1640 medium containing 10% FBS and placed in a 5% CO2 incubator. 2*10 5 cells were washed 3 times with pre-cooled PBS. Blocked with 2% FBS diluted in PBS, and incubated with fusion protein samples with concentrations of 100nM, 33.3nM, 11.1nM, 3.7nM, 1.2nM or 0.4nM at 4°C for 1h. Unbound antibody is washed away by 2% FBS. Mouse anti-human IgG Fc-APC (southern biotech) was incubated for 1 hour at 4 degrees, washed with 2% FBS, and analyzed by flow cytometry. The results are shown in Figure 5. The bispecific antibody can effectively bind to LnCap cells.
  • LnCap cells 1 ⁇ 10 4 LnCap cells (RPMI medium containing 5% FBS) were mixed with activated PBMCs at a ratio of 1:5, and then incubated with different dilutions of double antibodies at 37°C for 16 hours.
  • the content of LDH (lactate dehydrogenase) in the supernatant was detected with Cytotox-96 nonradioactive cytotoxicity assay kit (Promega).
  • the measured value of the wells containing only target cells treated with the lysate was maximum killing, and the measured value of the wells containing effector cells and target cells treated with PBS (vehicle) was Spontaneous killing.
  • %cytotoxicity (absorbance experimental-absorbance spontaneous average)/(absorbance maximum killing average-absorbance spontaneous average).
  • aCD3-aPSMA can effectively recruit T cells in PBMC and exert the specific killing effect of T cells on the target cell LnCap.
  • the bispecific antibody was intraperitoneally injected (IP) C57 female mice (3 mice/group, dose 10mg/kg), and the whole was collected 30min, 1h, 2h, 4h, 10h, 24h, 3d, 5d, 7d, 14d after injection. blood. After centrifugation, plasma was collected and stored at -80°C for later use. The detection of the content of bispecific antibodies in plasma was carried out with reference to Example 2.2. The results are shown in the figure. The half-life of the bispecific antibody in mice is about 5 days.

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Abstract

涉及特异性结合PSMA的抗体片段，尤其是单链Fv(scfv)，包含所述scFv的双特异性抗体和嵌合抗原受体(CAR)及其制备与应用。

Description

PSMA抗体及其应用 技术领域
本发明提供了特异性结合PSMA的抗体片段，具体涉及特异性结合PSMA的单链Fv(scfv)、包含所述单链Fv的双抗、CAR及其组合物和应用。
背景技术
前列腺癌是导致男性死亡的第二大主要病因，仅次于肺癌。然而，目前的方法，例如手术、放射疗法、化学疗法和雄激素剥夺疗法，对那些晚期癌症具有有限的效果。迄今为止，根治性手术仍是前列腺癌的主要治疗方法。因此，新兴的精准医学，特别是基于肿瘤靶向抗体的精确医学，有望改善前列腺治疗的结果。
前列腺特异性膜抗原(PSMA)主要由前列腺上皮细胞表达。在前列腺癌中，特别是在低分化的、转移的和激素难以治疗的癌中，PSMA的表达增加(Gregorakis AK等(1998)Seminars in Urologic Oncology 16：2-12；Silver，DA(1997)Clinical Cancer Research 3：85-515)。在前列腺外组织例知小肠、唾液腺、十二指肠粘膜、近端肾小管和脑中发现低水平PSMA表达(Silver，DA(1997)Clinical Cancer Research 3：85-515)。在一些恶性肿瘤(包括肾细胞癌和结肠癌)的肿瘤外周和肿瘤内区域中的毛细血管的内皮细胞中也表达PSMA，但是在正常组织的血管中不表达。另外，据报道PSMA与肿瘤血管生成相关(Silver，D.A.(1997)Clinical Cancer Research 3：81-85)。最近，已经证明PSMA在结肠癌、乳腺癌、膀胱癌、胰腺癌、肾癌和黑素瘤中的肿瘤相关新血管系统的内皮细胞中表达(Chang，S.S.(2004)Curr Opin Investig Drugs 5：611-5)，为前列腺癌靶向精准医学的发展奠定了基础。
发明内容
本发明利用噬菌体展示技术筛选获得一系列针对PSMA的高亲和力抗体。发明人进一步发现在这些抗体序列呈现为scFv的形式中，轻链VL的第42位氨基酸残基种类对亲和力有显著的影响；进一步的，含有特定类型第42位氨基酸的VL的双特异性抗体，其蛋白产率有大幅度提高。利用本发明中的aPSMA scfv抗体片段可用于构建双特异性抗体、嵌合抗原受体等，可满足肿瘤靶向治疗领域内对aPSMA scfv亲和力的要求。
本发明的一个方面，提供了一种特异性结合PSMA的抗体片段，其包括：
A)具有如SEQ ID NO:2所示氨基酸序列的重链可变区(VH)；
B)选自具有如下氨基酸序列的轻链可变区(VL)：SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14或SEQ ID NO:52。
在一些实施方案中，所述特异性结合PSMA的抗体片段为单链Fv(scfv)。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:2所示氨基酸序列的VH和SEQ ID NO:6所示氨基酸序列的VL。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:2所示氨基酸序列的VH和SEQ ID NO:8所示氨基酸序列的VL。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:2所示氨基酸序列的VH和SEQ ID NO:10所示氨基酸序列的VL。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:2所示氨基酸序列的VH和SEQ ID NO:12所示氨基酸序列的VL。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:2所示氨基酸序列的VH和SEQ ID NO:14所示氨基酸序列的VL。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:2所示氨基酸序列的VH和SEQ ID NO:52所示氨基酸序列的VL。
在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:20所示的氨基酸序列。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:22所示的氨基酸序列。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:24所示的氨基酸序列。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:26所示的氨基酸序列。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:28所示的氨基酸序列。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:30所示的氨基酸序列。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:32所示的氨基酸序列。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:34所示的氨基酸序列。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:36所示的氨基酸序列。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:38所示的氨基酸序列。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:54所示的氨基酸序列。在一些实施方案中，所述靶向PSMA的scfv具有如SEQ ID NO:56所示的氨基酸序列。
本发明的一个方面，提供了一种双特异性抗体，所述双特异性抗体包括：
1)特异性结合PSMA的第一抗原结合结构域，其包括：
A)具有如SEQ ID NO:2所示的氨基酸序列的重链可变区(VH)；和
B)具有选自如下氨基酸序列的轻链可变区(VL)：SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14或SEQ ID NO:52，和
2)第二抗原结合结构域。
在一些实施方案中，所述双特异性抗体的第一抗原结合结构域为特异性结合PSMA的单链Fv(scfv)。在一些实施方案中，所述双特异性抗体的第一抗原结合结构域具有如SEQ ID NO:20所示的氨基酸序列。在一些实施方案中，所述双特异性抗体的第一抗原结合结构域具有如SEQ ID NO:22所示的氨基酸序列。在一些实施方案中，所述双特异性抗体的第一抗原结合结构域具有如SEQ ID NO:24所示的氨基酸序列。在一些实施方案中，所述双特异性抗体的第一抗原结合结构域具有如SEQ ID NO:26所示的氨基酸序列。在一些实施方案中，所述双特异性抗体的第一抗原结合结构域具有如SEQ ID NO:28所示的氨基酸序列。在一些实施方案中，所述双特异性抗体的第一抗原结合结构域具有如SEQ ID NO:30所示的氨基酸序列。在一些实施方案中，所述双特异性抗体的第一抗原结合结构域具有如SEQ ID NO:32所示的氨基酸序列。在一些实施方案中，所述双特异性抗体的第一抗原结合结构域具有如SEQ ID NO:34所示的氨基酸序列。在一些实施方案中，所述双特异性抗体的第一抗原结合结构域具有如SEQ ID NO:36所示的氨基酸序列。在一些实施方案中，所述双特异性抗体的第一抗原结合结构域具有如SEQ ID NO:38所示的氨基酸序列。在一些实施方案中，所述双特异性抗体的第一抗原结合结构域具有如SEQ ID NO:54所示的氨基酸序列。在一些实施方案中，所述双特异性抗体的第一抗原结合结构域具有如SEQ ID NO:56所示的氨基酸序列。
在一些实施方案中，所述双特异性抗体的第二抗原结合结构域结合至T细胞的特异性受体分子，其中所述T细胞的特异性受体分子优选为CD3。在一些实施方案中，所述特异性结合CD3的第二抗原结合结构域具有如SEQ ID NO:40所示的VH和SEQ ID NO:42所示的VL。
在一些实施方案中，所述双特异性抗体具有如SEQ ID NO:44和SEQ ID NO:46所示的序列。在一些实施方案中，所述双特异性抗体具有如SEQ ID NO:58和SEQ ID NO:60所示的序列。在一些实施方案中，所述双特异性抗体具有如SEQ ID NO:62和SEQ ID NO:64所示的序列。在一些实施方案中，所述双特异性抗体具有如SEQ ID NO:58和SEQ ID NO:66所示的序列。在一些实施方案中，所述双特异性抗体具有如SEQ ID NO:68和SEQ ID NO:64所示的序列。
本发明的一个方面，提供了一种嵌合抗原受体(CAR)，其中所述CAR包含特异性结合PSMA的scfv、跨膜结构域和胞内结构域。
在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:2所示的VH和SEQ ID NO:6所示的VL。在一些实施方案中，所述CAR的特异性结合PSMA的scfv 具有如SEQ ID NO:2所示的VH和SEQ ID NO:8所示的VL。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:2所示的VH和SEQ ID NO:10所示的VL。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:2所示的VH和SEQ ID NO:12所示的VL。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:2所示的VH和SEQ ID NO:14所示的VL。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:2所示的VH和SEQ ID NO:52所示的VL。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:20所示的氨基酸序列。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:22所示的氨基酸序列。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:24所示的氨基酸序列。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:26所示的氨基酸序列。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:28所示的氨基酸序列。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:32所示的氨基酸序列。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:34所示的氨基酸序列。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:36所示的氨基酸序列。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:38所示的氨基酸序列。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:54所示的氨基酸序列。在一些实施方案中，所述CAR的特异性结合PSMA的scfv具有如SEQ ID NO:56所示的氨基酸序列。在一些实施方案中，所述CAR具有如SEQ ID NO:50所示的氨基酸序列。
本发明的一个方面，提供了一种多核苷酸，所述多核苷酸编码前述实施方案中任一项特异性结合PSMA的抗体片段、双特异性抗体或CAR。在一些实施方案中，所述多核苷酸包含选自下组的核苷酸序列：SEQ ID NO:5、7、9、11、13、19、21、23、25、27、29、31、33、35、37、39、41、43、44、45、47、49、51、53、55、57、59、61、63、65、67。
本发明的一个方面，提供了一种载体，其包含前述的多核苷酸。
本发明的一个方面，提供了一种细胞，其包含前述的载体。
本发明的一个方面，提供了包含前述任一项的抗体片段、双特异性抗体、CAR、多核苷酸、载体或细胞的组合物，所述组合物还包括药学可接受的载体。
本发明的一个方面，提供了治疗患有与PSMA表达相关的疾病的受试者的方法，其包括对所述受试者施用有效量的前述的组合物。
在一个方面，本发明提供了诊断哺乳动物中与PSMA表达相关的疾病的方法，所述方法包括：使用如权利要求1-3中任一项所述的抗体片段在从所述哺乳动物分离的组织样品中检测与人PSMA的结合，由此所述抗体片段对所述组织样品中人PSMA的特异性结合指示所述哺乳动物中与PSMA表达相关的疾病。
附图说明
构成本申请的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定，在附图中：
图1为aPSMA LH RM和RM2单克隆噬菌体ELISA检测结果，其中RM为第一轮淘选、RM2为第二轮淘选。
图2为aPSMA HL单克隆噬菌体ELISA检测结果。
图3A为VL第42位氨基酸突变的aPSMA scfv抗体与PSMA-his ELISA检测结果；图3B为靶向PSMA的scfv-Fc抗体(其中VL第42位氨基酸突变)与LnCap细胞结合流式检测结果。
图4为双特异性抗体与JurkatT细胞结合流式检测结果。
图5为双特异性抗体与LnCap细胞结合流式检测结果。
图6为双特异性抗体召集PBMC对LnCap细胞的细胞毒作用。
图7为双特性抗体在小鼠体内的PK曲线。
实施例
需要说明的是，在不冲突的情况下，本申请中的实施例仅为举例说明，不旨在对本发明造成任何方式上的限制。
实施例
实施例1 噬菌体文库筛选
合成aPSMA HL(SEQ ID NO:37)和aPSMA LH(SEQ ID NO:39)，将其分别克隆进phagemid载体中，采用GeneMorph II random mutagenesis(Agilent)进行随机突变后，转化XL1-blue感受态细胞，待OD600＝0.6-0.8时，用辅助性噬菌体M13K07感染，12小时后收集噬菌体并测定噬菌体滴度(库容)。
微孔板包被抗原Fc-PSMA并使用蛋白BSA封闭，300ng/孔，对前述两个噬菌体文库进行2-3轮淘选并进行亲和力检测，从淘选的板子上分别随机挑选单克隆送去测序，并对测 序结果进行分析。将有序列富集和在CDR区有突变的单克隆进行了噬菌体包装和亲和力检测。检测结果如图1所示。
图1是突变库aPSMA LH RM和aPSMA LH RM2的单克隆的亲和力检测结果，从图中可以看出，除了aPSMA LH RM-R13和aPSMA LH RM2-R15外，其余单克隆的亲和力都远远高出原序列；图2是突变库aPSMA HL RM的单克隆的亲和力检测结果，仅aPSMA HL RM-R1/R10/R15的亲和力较高。
对上述亲和力较高的序列分析后发现，在突变库aPSMA LH RM、aPSMA LH RM2和aPSMA HL RM中，亲和力较高的单克隆都在VL的第42位氨基酸发生了突变。
表1 序列表
Figure PCTCN2020130511-appb-000001
Figure PCTCN2020130511-appb-000002
实施例2 靶向PSMA的抗体与细胞的亲和力检测
2.1靶向PSMA的scfv-Fc抗体克隆及表达
将aPSMA HL(SEQ ID NO:15)、aPSMA LH(SEQ ID NO:17)、aPSMA HL(L42)(SEQ ID NO:19)、aPSMA HL(H42)(SEQ ID NO:21)、aPSMA HL(T42)(SEQ ID NO:23)、aPSMA HL(S42)(SEQ ID NO:25)、aPSMA HL(Q42)(SEQ ID NO:27)及aPSMA LH(Q42)(SEQ ID NO:29)通过传统酶切连接方法分别克隆至pFuse-hIgG1-Fc2的N端，测序验证。
将构建好的真核表达载体的分别瞬时转染FreeStyle HEK293细胞(ThermoFisher)：将28ml FreeStyle HEK 293(3×10 7细胞/ml)接种至125ml细胞培养瓶，质粒用1ml Opti-MEM(Invitrogen)稀释后加至1ml含60μl 293Fectin(Invitrogen,Inc)的Opti-MEM中，室温静置30min，将质粒-293fectin mixture加至细胞培养液中125rpm，372，5％CO2培养。分别于转染后48h和96h收集细胞培养上清，并根据制造商的说明书，使用Protein A Resin(Genscript)纯化后经SDS-PAGE检测。
2.2靶向PSMA的scfv-Fc抗体与PSMA-His的亲和力检测
包被PSMA-His(Acro)(100ng/孔)于96孔板，4℃孵育过夜；含2％脱脂奶粉的PBST(0.5％Tween-20in PBS)室温封闭1小时，分别加入梯度稀释的scfv-Fc抗体室温孵育2h，含2％脱脂奶粉的PBST洗4-5次后，加入anti-human Fc-HRP二抗室温孵育1h，含2％脱脂奶粉的PBST洗4-5次后，采用TMB显色试剂(BioLegend,Cat.421101)显色后于650nm(未 终止)或450nm(终止)处读数。Prizm Graphpad软件用specific binding model对数据进行非线性回归。
结果如图3A所示，VL野生型的aPSMA LH-Fc或aPSMA LH-Fc抗体与PSMA-his的亲和力弱，VL第42位氨基酸突变的aPMSA HL(Q42)-Fc或aPSMA LH(Q42)-Fc抗体与PSMA-his的亲和力大大增强。
2.3靶向PSMA的scfv-Fc抗体与LnCap细胞的亲和力检测
LnCap细胞饲养于含10％FBS的RPMI 1640的培养基，置于5％CO2的培养箱。2*10 5个细胞用预冷的PBS洗3次。PBS稀释的2％FBS封闭，与梯度稀释的抗体(100nM依次3倍梯度稀释)4℃共孵育1h。未结合的抗体由2％FBS洗去。小鼠抗人IgG Fc-APC(southern biotech)4度孵育1h，2％FBS洗涤，流式细胞计数分析。结果如图3所示，VL第42位发生氨基酸突变的aPSMA单链scfv融合到Fc后对LnCap表面的PSMA均有较高的亲和力，比野生型VL的亲和力提高190-220倍。
实施例3 靶向PSMA和CD3的双特异性抗体
3.1靶向PSMA和CD3的双特异性抗体构建和表达
采用标准分子生物学方法构建含有序列表1所示的不同aCD3-aPMSA双抗的chain-1和chain-2，测序验证。
将上述测序验证的真核表达载体的两条链瞬时共转染FreeStyle HEK293细胞(ThermoFisher)：将28ml FreeStyle HEK 293(3×10 7细胞/ml)接种至125ml细胞培养瓶，质粒用1ml Opti-MEM(Invitrogen)稀释后加至1ml含60μl 293Fectin(Invitrogen,Inc)的Opti-MEM中，室温静置30min，将质粒-293fectin mixture加至细胞培养液中125rpm，372，5％CO2培养。分别于转染后48h和96h收集细胞培养上清，并根据制造商的说明书，使用Protein A Resin(Genscript)纯化后经SDS-PAGE检测，并计算产率。结果显示：基于VL第42位突变的aCD3-aPSMA的产率(15-20mg/L)比VL第42位野生型的aCD3-aPSMA的产率(2.5-5mg/L)有明显提升。
3.2靶向PSMA和CD3的双特异性抗体体外活性检测
3.2.1与Jurkat(CD3+)细胞结合的流式细胞术分析
Jurkat细胞饲养于含10％FBS的RPMI 1640的培养基，置于5％CO2的培养箱。2х10 5个细胞用预冷的PBS洗3次，经含2％FBS的PBS封闭后，与浓度分别为100nM,33.3nM,11.1nM,3.7nM,1.2nM or 0.4nM的双抗于4双孵育1h。用含2％FBS的PBS洗去未结合的抗 体，加二抗小鼠抗人IgG Fc-APC(southern biotech)4℃孵育1h，含2％FBS的PBS洗三次后，流式细胞计数分析。结果如图4所示，双特异性抗体能有效结合Jurkat细胞。
3.2.2与LnCap(PSMA+)细胞结合的流式细胞术分析
LnCap细胞饲养于含10％FBS的RPMI 1640的培养基，置于5％CO2的培养箱。2*10 5个细胞用预冷的PBS洗3次。PBS稀释的2％FBS封闭，与浓度分别为100nM,33.3nM,11.1nM,3.7nM,1.2nM or 0.4nM的融合蛋白样品4度共孵育1h。未结合的抗体由2％FBS洗去。小鼠抗人IgG Fc-APC(southern biotech)4度孵育1h，2％FBS洗涤，流式细胞计数分析。结果如图5所示，双特异性抗体能有效结合LnCap细胞。
表2 双特异性抗体与Jurkat和LnCap结合的EC 50
EC 50(nM) aCD3-aPSMA aCD3 aPSMA
Jurkat 0.51 0.51 NA
LnCap 0.05 NA 0.01
3.2.3依赖PBMC的细胞毒性试验
采用常规Ficoll-Hypaque梯度离心法从新鲜健康供体血液中分离纯化出单个核细胞(PBMC)。洗涤分离后的PBMC，在含10％(vol/vol)FBS的RPMI培养基中孵育1h，去除贴壁细胞。用CD3和CD28活化PBMC后，加IL-2培养10天。
1х10 4个LnCap细胞(含5％FBS的RPMI培养基)与活化后的PBMCs按1:5的比例混合后，与不同稀释度的双抗于37℃孵育16h。上清中LDH(乳酸脱氢酶)的含量用Cytotox-96 nonradioactive cytotoxicity assay kit(Promega)检测。用裂解液处理的仅含靶细胞的孔测得的值为maximum killing，用PBS(vehicle)处理的含效应细胞、靶细胞的孔测得的值为Spontaneous killing。根据产品说明书进行孵育显色，BMG LABTECH CLARIOstar酶标仪(Bio-Gene Technology Ltd.)读取490nm处的吸光度。计算细胞毒百分比：％cytotoxicity＝(absorbance experimental-absorbance spontaneous average)/(absorbance maximum killing average-absorbance spontaneous average)。
结果如图6所示，与对照aPSMA相比，aCD3-aPSMA能有效召集PBMC中的T细胞，发挥T细胞对靶细胞LnCap的特异性杀伤作用。
表3 双特异性抗体召集PBMC对靶细胞LnCap的杀伤
  aCD3-aPSMA aCD3 aPSMA
LDH Killing EC50(pM) 184.4 712.3 NA
LDH max killing(pM) 79.79 42.76 NA
3.3靶向PSMA和CD3的双特异性抗体PK研究
将双特异性抗体腹腔注射(I.P.)C57雌性小鼠(3只/组，剂量10mg/kg)，于注射后30min、1h、2h、4h、10h、24h、3d、5d、7d、14d采集全血。离心后取血浆，-80℃保存备用。血浆中双特异性抗体的含量检测参照实施例2.2进行。结果如图所示，双特异性抗体在小鼠体内的半衰期5天左右。

Claims (20)

  1. 一种特异性结合人PSMA的抗体片段，其包括：
    A)具有如SEQ ID NO:2所示的氨基酸序列的重链可变区(VH)；和
    B)具有选自如下氨基酸序列的轻链可变区(VL)：SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14或SEQ ID NO:52。
  2. 根据权利要求1所述的抗体片段，其为单链Fv(scfv)。
  3. 根据权利要求2所述的抗体片段，其中所述scfv具有如SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:54或SEQ ID NO:56所示的氨基酸序列。
  4. 一种双特异性抗体，所述双特异性抗体包括：
    1)特异性结合PSMA的第一抗原结合结构域，其包括：
    A)具有如SEQ ID NO:2所示的氨基酸序列的重链可变区(VH)；和
    B)具有选自如下氨基酸序列的轻链可变区(VL)：SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14或SEQ ID NO:52，和
    2)第二抗原结合结构域。
  5. 根据权利要求4所述的双特异性抗体，其中所述第一抗原结合域是scfv，优选如权利要求3中所述的scfv。
  6. 根据权利要求4所述的双特异性抗体，其中所述第二抗原结合结构域结合T细胞的特异性受体分子，其中所述T细胞的特异性受体分子优选为CD3。
  7. 根据权利要求6所述的双特异性抗体，其中所述特异性结合CD3的第二抗原结合结构域具有如SEQ ID NO:40所示的VH和SEQ ID NO:42所示的VL。
  8. 根据权利要求4-7任一项所述的双特异性抗体包括可结合CD3和PMSA的两条多肽链组成的组：SEQ ID NO:44和SEQ ID NO:46；SEQ ID NO:58和SEQ ID NO:60；SEQ ID NO:62和SEQ ID NO:64；SEQ ID NO:58和SEQ ID NO:66；SEQ ID NO:68和SEQ ID NO:64。
  9. 一种嵌合抗原受体(CAR)，其中所述CAR包含特异性结合PSMA的scfv、跨膜结构域和胞内结构域，其中所述scfv包含：
    A)具有如SEQ ID NO:2所示的氨基酸序列的重链可变区(VH)；和
    B)选自具有如下氨基酸序列的轻链可变区(VL)：SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14或SEQ ID NO:52。
  10. 根据权利要求9所述的CAR，其具有如SEQ ID NO:50所示的氨基酸序列。
  11. 一种多核苷酸，其编码根据权利要求1-3中任一项所述的抗体片段、或根据权利要求4-8中任一项所述的双特异性抗体、或根据权利要求9-10中任一项所述的CAR。
  12. 根据权利要求11所述的多核苷酸，其具有SEQ ID NO:19、21、23、25、27、29、31、33、35、37、43、45、49、51、53、55、57、59、61、63、65或67所示的核苷酸序列。
  13. 一种载体，其包含根据权利要求11或12所述的多核苷酸。
  14. 一种细胞，其包含根据权利要求11-12任一项所述的多核苷酸或根据权利要求13所述的载体。
  15. 根据权利要求14所述的细胞，其中该细胞是T细胞。
  16. 组合物，其包含根据权利要求1-3中任一项所述的抗体片段、根据权利要求4-8中任一项所述的双特异性抗体、根据权利要求9-10中任一项所述的CAR、根据权利要求11-12中任一项所述的多核苷酸、根据权利要求13所述的载体，或根据权利要求14-15任一项所述的细胞。
  17. 权利要求16的组合物，其还包括药学可接受的载体。
  18. 权利要求16或17的组合物，其用于嵌合抗原受体修饰T细胞(CAR-T)治疗。
  19. 治疗患有与PSMA表达相关的疾病的受试者的方法，其包括对所述受试者施用有效量的根据权利要求16-18任一项所述的组合物。
  20. 诊断哺乳动物中与PSMA表达相关的疾病的方法，所述方法包括：使用如权利要求1-3中任一项所述的抗体片段在从所述哺乳动物分离的组织样品中检测与人PSMA的结合，由此所述抗体片段对所述组织样品中人PSMA的特异性结合指示所述哺乳动物中与PSMA表达相关的疾病。
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