CN110627906B - anti-PD-L1/4-1 BB bispecific antibody and application thereof - Google Patents

anti-PD-L1/4-1 BB bispecific antibody and application thereof Download PDF

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CN110627906B
CN110627906B CN201910958176.0A CN201910958176A CN110627906B CN 110627906 B CN110627906 B CN 110627906B CN 201910958176 A CN201910958176 A CN 201910958176A CN 110627906 B CN110627906 B CN 110627906B
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antibody
bispecific
bispecific antibody
nanobody
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CN110627906A (en
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万亚坤
朱敏
盖军伟
李光辉
沈晓宁
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Shanghai Luoqi Biomedical Technology Co ltd
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    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Abstract

The invention provides an anti-PD-L1/4-1 BB bispecific antibody and application thereof, in particular to a bispecific antibody which comprises (a) a PD-L1 nano antibody and (b) a 4-1BB nano antibody, a coding sequence for coding the bispecific antibody, a corresponding expression vector, a host cell capable of expressing the bispecific antibody and a production method of the bispecific antibody, wherein the bispecific antibody can effectively target PD-L1 and 4-1BB, and has good biological activity and application prospect.

Description

anti-PD-L1/4-1 BB bispecific antibody and application thereof
Technical Field
The invention relates to the technical field of biomedicine or biopharmaceutical, and more particularly relates to an anti-PD-L1/4-1 BB bispecific antibody and application thereof.
Background
Programmed cell death factor 1 (PD-1) is a member of the CD28 superfamily, as a T cell suppressor receptor, can limit the function of T cell effectors in tumor cells, has an important role in tumor immune escape, blocking the interaction of PD-1 and PD-L can effectively restore the killing function of T cells to tumors, PD-1/PD-L immunotherapy has been a revolution that is currently receiving attention all over the world, leading to cancer treatment, bringing a new promising new class of anticancer immunotherapy to patients, aiming at fully exploiting the human immune system to combat cancer, dying cancer cells by blocking the PD-1/PD-L signaling pathway, has the potential to treat multiple types of tumors, substantially improving the overall survival of patients, however, therapy against PD-1/PD-L is not entirely satisfactory, some patients experience rapid and persistent tumors, but most patients acquire little or no significant effect, in order to increase the response to immune therapy, respond to the development of immune receptor stimulation, a promising strategy, including a strategy of stimulating CD-OX, and stimulating immune receptor activation, 3663, and a promising therapeutic strategy for clinical immune therapy of multiple types of patients, wherein a variety of immune receptor activation, and immune therapy, including GITR, and T cell activation, and T cell.
The first anti-4-1 BB therapeutic agent entering clinical trials, Urelumab (BMS-663513), which is a fully human monoclonal antibody of the IgG4 type, does not block the interaction of 4-1BB with its ligand, has been shown to have encouraging efficacy in the clinic, but the data from the PhaseI and PhaseII phases show that hepatotoxicity appears to be related to the target and dosage, thus preventing its clinical development.
In view of the above, the use of bispecific antibodies limits the activation to tissues expressing the target antigen, thereby reducing systemic toxicity, and at the same time, the tumor-mediated 4-1BB cross linking effect can effectively play an agonistic role, no effective PD-L1/4-1 BB bispecific antibody drug product exists in the market at present, and the combination of PD-L1 nanobody and nanobody targeting 4-1BB may play a better anti-tumor effect, and the technology needs to be further researched.
Disclosure of Invention
The invention aims to provide an anti-PD-L1/4-1 BB bispecific antibody and application thereof.
In a first aspect of the invention, a bispecific antibody is provided, which comprises an anti-PD-L1 nanobody and an anti-4-1 BB nanobody,
wherein, the CDR of the complementarity determining region of the anti-PD-L1 nanobody comprises:
CDR1 shown in SEQ ID NO. 6, CDR2 shown in SEQ ID NO.5 and CDR3 shown in SEQ ID NO. 7.
In another preferred embodiment, the bispecific antibody comprises 2-4 anti-PD-L1 nanobodies, preferably 2 anti-PD-L1 nanobodies, and more preferably the two anti-PD-L1 nanobodies form an anti-PD-L1 nanobody dimer.
In another preferred embodiment, the bispecific antibody comprises 2-4 anti-4-1 BB nanobodies, preferably 2 anti-4-1 BB nanobodies, and more preferably, the two anti-4-1 BB nanobodies form an anti-4-1 BB nanobody dimer.
In another preferred embodiment, the bispecific antibody further comprises an Fc fragment, preferably the Fc fragment comprises a CH2 domain and a CH3 domain.
In another preferred embodiment, the Fc fragment is an IgG4 type Fc fragment.
In another preferred embodiment, the bispecific antibody has a structure represented by formula I from N-terminus to C-terminus:
P-L1-P-L2-Fc-L3-B-L4-B formula I
Wherein the content of the first and second substances,
"-" is a peptide bond;
l1, L2, L3, and L4 are each independently a peptide bond or a linker element;
p is anti-PD-L1 nano antibody,
b is an anti-4-1 BB nanobody, and
fc is the Fc segment of the antibody.
In another preferred embodiment, the anti-PD-L1 nanobody comprises a framework region FR and a complementarity determining region CDR.
In another preferred embodiment, the framework region FR of the anti-PD-L1 nanobody includes:
FR1 shown in SEQ ID NO.1, FR2 shown in SEQ ID NO. 2, FR3 shown in SEQ ID NO. 3, and FR4 shown in SEQ ID NO. 4.
In another preferred example, the amino acid sequence of the anti-PD-L1 nano antibody is shown in SEQ ID No. 8.
In another preferred example, the PD-L1 is human PD-L1.
In another preferred example, the anti-PD-L1 nanobody can block the interaction between PD-1 and PD-L1.
In another preferred embodiment, the anti-4-1 BB nanobody comprises a framework region FR and a complementarity determining region CDR.
In another preferred embodiment, the CDR of the anti-4-1 BB nanobody comprises:
CDR1 shown in SEQ ID No. 14, CDR2 shown in SEQ ID No. 15, and CDR3 shown in SEQ ID No. 16.
In another preferred embodiment, the framework region FR of the anti-4-1 BB nanobody comprises:
FR1 shown in SEQ ID NO. 10, FR2 shown in SEQ ID NO. 11, FR3 shown in SEQ ID NO. 12, and FR4 shown in SEQ ID NO. 13.
In another preferred example, the amino acid sequence of the anti-4-1 BB nanobody is shown in SEQ ID No. 17.
In another preferred embodiment, the 4-1BB is human 4-1 BB.
In another preferred embodiment, the anti-4-1 BB nanobody may block the interaction between 4-1BB and 4-1BB L.
In another preferred embodiment, L1, L3, and L4 are tab members.
In another preferred embodiment, the sequence of the linker element is (4GS) n, wherein n is a positive integer (e.g. 1, 2, 3, 4, 5 or 6), preferably n ═ 4.
In another preferred embodiment, the linker element is as shown in SEQ ID No. 19.
In another preferred embodiment, L2 is peptide bond.
In another preferred embodiment, the Fc segment is shown as SEQ ID NO. 20 at position 273 and 501.
In another preferred embodiment, the amino acid sequence of the bispecific antibody is shown in SEQ ID No. 20.
In another preferred embodiment, the bifunctional antibody has a binding affinity to PD-L1 with a KD value of less than 10E-08M, preferably less than 6E-08M.
In another preferred embodiment, the bifunctional antibody has a binding affinity for 4-1BB with a KD value of less than 5E-08M, preferably less than 3E-08M.
In another preferred embodiment, the bifunctional antibody inhibits the IC of PD-1 and PD-L1 binding50Less than 2ug/ml, preferably less than 1.5ug/ml, more preferably less than 1.2 ug/ml.
In another preferred embodiment, the bifunctional antibody inhibits IC binding of 4-1BB and 4-1BB L50Less than 20ug/ml, preferably less than 15ug/ml, more preferably less than 12 ug/ml.
In a second aspect of the invention, there is provided a bispecific fusion protein which is a dimer formed from two bispecific antibodies according to the first aspect of the invention.
In another preferred embodiment, the bispecific fusion protein comprises 4 anti-PD-L1 nanobodies and 4-1BB nanobodies.
In another preferred embodiment, the bispecific fusion protein forms a dimer through the Fc segment.
In another preferred embodiment, the dimer includes a homodimer and a heterodimer.
In another preferred embodiment, the bispecific fusion protein has a structure represented by formula II from N-terminus to C-terminus:
Figure GDA0002490661480000041
wherein the content of the first and second substances,
"-" is a peptide bond and "|" is a disulfide bond;
l1, L2, L3, and L4 are each independently a peptide bond or a linker element;
p is anti-PD-L1 nano antibody,
b is an anti-4-1 BB nanobody, and
fc is the Fc segment of the antibody.
In a third aspect of the present invention, there is provided an anti-4-1 BB nanobody, wherein the CDR of the complementarity determining region of the anti-4-1 BB nanobody comprises:
CDR1 shown in SEQ ID No. 14, CDR2 shown in SEQ ID No. 15, and CDR3 shown in SEQ ID No. 16.
In a fourth aspect of the invention, there is provided a polynucleotide encoding a protein selected from the group consisting of: the bispecific antibody of the first aspect of the invention, the bispecific fusion protein of the second aspect of the invention, or the anti-4-1 BB nanobody of the third aspect of the invention.
In another preferred embodiment, the polynucleotide encodes the bispecific antibody of the first aspect of the invention and the polynucleotide has a nucleotide sequence as shown in SEQ ID No. 20.
In another preferred example, the polynucleotide encodes an anti-PD-L1 nanobody, and the polynucleotide has a nucleotide sequence as shown in SEQ ID No. 9.
In another preferred embodiment, the polynucleotide encodes the anti-4-1 BB nanobody of the third aspect of the present invention, and the polynucleotide has a nucleotide sequence as shown in SEQ ID No. 18.
In another preferred embodiment, the polynucleotide comprises DNA or RNA.
In a fifth aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the fourth aspect of the invention.
In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
In a sixth aspect of the invention, there is provided a host cell comprising an expression vector according to the fifth aspect of the invention, or having a polynucleotide according to the fourth aspect of the invention integrated into its genome;
alternatively, the host cell expresses a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention, or an anti-4-1 BB nanobody according to the third aspect of the invention.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of: escherichia coli, yeast cells, mammalian cells.
In a seventh aspect of the invention, there is provided a method of producing a bispecific antibody or nanobody, comprising the steps of:
(a) culturing the host cell of the sixth aspect of the invention under suitable conditions, thereby obtaining a culture comprising the bispecific antibody or nanobody; and
(b) purifying and/or isolating the culture obtained in step (a) to obtain said bispecific or nanobody.
In another preferred example, the purification can be performed by protein a affinity column purification and separation to obtain the target antibody.
In another preferred embodiment, the purity of the purified and separated target antibody is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, and preferably 100%.
In an eighth aspect of the invention, there is provided an immunoconjugate comprising:
(a) a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention, and/or an anti-4-1 BB nanobody according to the third aspect of the invention; and
(b) a conjugating moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein or V L P, or a combination thereof.
In another preferred embodiment, the radionuclide includes:
(i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, L u-177, Re-188, or combinations thereof, and/or
(ii) A therapeutic isotope selected from the group consisting of L u-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133 Yb-169, Yb-177, or a combination thereof.
In another preferred embodiment, the coupling moiety is a drug or toxin.
In another preferred embodiment, the drug is a cytotoxic drug.
In another preferred embodiment, the cytotoxic agent is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folate antagonist, an anti-metabolite drug, a chemotherapeutic sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycins/duocarmycins (duocarmycins), etoposides (etoposides), maytansinoids (maytansinoids) and maytansinoids (e.g., DM1 and DM4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indoloprazoids (indobenzodiazepines) and benzodiazepines (oxyphenodiazepines)), or combinations thereof.
In another preferred embodiment, the toxin is selected from the group consisting of:
auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, maytansinoid, ricin a-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide (tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthrax dione, actinomycin, diphtheria toxin, Pseudomonas Exotoxin (PE) A, PE40, abrin a-chain, anemonin a-chain, α -sarcina, gelonin, mitogellin (mitogellin), restrictocin (rettricin), phenomycin, enomycin, curcin (curcin), crotin, calicheamicin, soapworthia officinalis (safilin), glucocorticoid, or a combination thereof.
In another preferred embodiment, the conjugated moiety is a detectable label.
In another preferred embodiment, the conjugate is selected from the group consisting of a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine (e.g., I L-2, etc.), an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle/nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPH L)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle.
In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g. bivalent) bispecific fusion protein according to the second aspect of the invention and/or an anti-4-1 BB nanobody according to the third aspect of the invention.
In another preferred embodiment, the multivalent refers to the bispecific fusion protein according to the second aspect of the invention and/or the anti-4-1 BB nanobody according to the third aspect of the invention comprising multiple repeats in the amino acid sequence of the immunoconjugate.
In a ninth aspect of the invention, there is provided use of a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention, or a bispecific antibody according to the first aspect of the invention, for the manufacture of (a) an agent for the detection of a PD-L1 and/or 4-1BB molecule, (b) a medicament for the treatment of a tumor.
In another preferred embodiment, the conjugated moiety of the immunoconjugate is a diagnostic isotope.
In another preferred embodiment, the agent is one or more agents selected from the group consisting of: isotope tracer, contrast agent, flow detection reagent, cell immunofluorescence detection reagent, nano magnetic particles and imaging agent.
In another preferred example, the agent for detecting PD-L1 and/or 4-1BB molecules is a contrast agent for detecting PD-L1 and/or 4-1BB molecules (in vivo).
In another preferred embodiment, the assay is an in vivo assay or an in vitro assay.
In another preferred embodiment, the detection comprises flow detection and cell immunofluorescence detection.
In another preferred embodiment, the medicament is for blocking the interaction of PD-1 and PD-L1, while blocking the interaction of 4-1BB and 4-1BB L.
In a tenth aspect of the present invention, there is provided a pharmaceutical composition comprising: (i) a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention, an anti-4-1 BB nanobody according to the third aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention; and (ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the conjugation moiety of the immunoconjugate is a drug, toxin, and/or therapeutic isotope.
In another preferred embodiment, the pharmaceutical composition further comprises other drugs for treating tumors, such as cytotoxic drugs.
In another preferred embodiment, the other drug for treating tumor comprises paclitaxel, doxorubicin, cyclophosphamide, axitinib, lenvatinib, or pembrolizumab.
In another preferred embodiment, the pharmaceutical composition is used to block the interaction of PD-1 and PD-L1, while blocking the interaction of 4-1BB and 4-1BB L.
In another preferred embodiment, the pharmaceutical composition is used for blocking PD-1/PD-L1 signal pathway.
In another preferred embodiment, the pharmaceutical composition is used for treating tumors expressing PD-L1 protein (i.e., PD-L1 positive).
In another preferred embodiment, the pharmaceutical composition is in the form of injection.
In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating tumors.
In another preferred embodiment, the tumor is selected from the group consisting of: colorectal cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestinal cancer, bone cancer, prostate cancer, cervical cancer, lymph cancer, adrenal tumor, or bladder tumor.
In an eleventh aspect of the invention, there is provided the use of one or more of the bispecific antibody of the first aspect of the invention, the bispecific fusion protein of the second aspect of the invention, or the anti-4-1 BB nanobody of the third aspect of the invention, selected from (i) for the detection of human PD-L1 molecule and/or 4-1BB molecule, (ii) for flow detection, (iii) for immunofluorescence detection, (iv) for the treatment of tumors, (v) for tumor diagnosis, (vi) for blocking the interaction of PD-1 and PD-L1, and (vii) for blocking the interaction of 4-1BB and 4-1BB L.
In another preferred embodiment, the tumor is a tumor expressing PD-L1 protein (i.e., PD-L1 positive).
In another preferred embodiment, the use is non-diagnostic and non-therapeutic.
In the twelfth aspect of the invention, the invention also provides an antibody, wherein the antibody comprises the anti-PD-L1 nanobody and the anti-4-1 BB nanobody of the third aspect of the invention.
In another preferred embodiment, the antibody is an antibody against PD-L1 protein and/or 4-1BB protein.
In a thirteenth aspect of the present invention, there is provided a recombinant protein having: (i) a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention, an anti-4-1 BB nanobody according to the third aspect of the invention; and (ii) optionally a tag sequence to facilitate expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag, an HA tag and an Fc tag.
In another preferred embodiment, the recombinant protein specifically binds to PD-L1 protein and/or 4-1BB protein.
In a fourteenth aspect of the invention, there is provided use of a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention, an anti-4-1 BB nanobody according to the third aspect of the invention, or an immunoconjugate according to the eighth aspect of the invention for the preparation of a medicament, a reagent, a detection plate or a kit for detecting PD-L1 protein and/or 4-1BB protein in a sample, wherein the medicament is for the treatment or prevention of a tumor expressing PD-L1 protein (i.e. PD-L1 positive) or a tumor expressing 4-1BB L.
In a fifteenth aspect of the invention, there is provided a method for detecting PD-L1 protein and/or 4-1BB protein in a sample, the method comprising the steps of (1) contacting the sample with a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention, or an anti-4-1 BB nanobody according to the third aspect of the invention, and (2) detecting the formation of an antigen-antibody complex, wherein the formation of a complex indicates the presence of PD-L1 protein in the sample.
In a sixteenth aspect of the invention, there is provided a method of treating a disease, the method comprising administering to a subject in need thereof a bispecific antibody according to the first aspect of the invention, a bispecific fusion protein according to the second aspect of the invention, an anti-4-1 BB nanobody according to the third aspect of the invention or an immunoconjugate according to the eighth aspect of the invention.
In another preferred embodiment, the subject comprises a mammal, such as a human.
In a seventeenth aspect of the invention, there is provided a PD-L1 protein and/or 4-1BB protein detection reagent, wherein the detection reagent comprises the immunoconjugate of the eighth aspect of the invention and a detectably acceptable carrier.
In another preferred embodiment, the conjugated moiety of the immunoconjugate is a diagnostic isotope.
In another preferred embodiment, the detectably acceptable carrier is a non-toxic, inert, aqueous carrier medium.
In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of: isotope tracer, contrast agent, flow detection reagent, cell immunofluorescence detection reagent, nano magnetic particles and imaging agent.
In another preferred embodiment, the detection reagent is used for in vivo detection.
In another preferred embodiment, the dosage form of the detection reagent is liquid or powder (such as water solution, injection, freeze-dried powder, tablet, buccal agent and aerosol).
In the eighteenth aspect of the invention, a kit for detecting PD-L1 protein and/or 4-1BB protein is provided, and the kit contains the immunoconjugate according to the eighth aspect of the invention or the detection reagent according to the seventeenth aspect of the invention, and instructions.
In another preferred embodiment, the instructions describe that the kit is used for non-invasively detecting the expression of PD-L1 and/or 4-1BB in a test subject.
In another preferred embodiment, the kit is used for detecting tumors expressing PD-L1 protein (i.e., PD-L1 positive).
In a nineteenth aspect of the invention, there is provided a use of the immunoconjugate of the eighth aspect of the invention for the preparation of a contrast agent for the in vivo detection of PD-L1 protein and/or 4-1BB protein.
In another preferred embodiment, the detection is used for the diagnosis or prognosis of cancer.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the results of the epitope difference of antigen recognition between 4-1BB nanobody and marketed antibody. The results indicated that the binding site for Urelumab to 4-1BB was N42, while the binding sites for Utomillumab to 4-1BB were M101 and I132. None of the three sites is the binding site of the 4-1BB nanobody of the present invention and 4-1BB, so that the epitope recognized by the nanobody of the present invention is considered to be different from those of the two control antibodies.
FIG. 2 shows the inhibitory effect of 4-1BB nanobody on mouse colon cancer MC 38. Wherein, the tumor inhibition rate TGI of the contrast antibody Utomillumab is 68.3 percent, and the tumor inhibition rate TGI of the candidate 4-1BB nano antibody is 75.8 percent.
FIG. 3A shows a schematic structural diagram of a PD-L1/4-1 BB bispecific antibody (i.e., an octave diabody).
Figure 3B shows a schematic of the structure of hexavalent bis-antibody a.
Figure 3C shows a schematic of the structure of the hexavalent bis-antibody B.
Fig. 3D shows a schematic of the structure of a tetravalent diabody.
FIG. 4 shows the SDS-PAGE detection of PD-L1/4-1 BB bispecific antibody the results show that the double antibody was more than about 85% pure by one step of affinity purification.
The results show that the binding activity of the octave diabody (EC50 ═ 6.43nM)4-1BB moiety is superior to that of the hexavalent diabody A (EC50 ═ 20.1nM), the hexavalent diabody B (EC50 ═ 19.2nM) and the tetravalent diabody (EC50 ═ 30.1nM) at the cellular level, and that the binding activity of the octave diabody is significantly superior to that of the hexavalent and tetravalent diabodies.
FIG. 6 shows the blocking activity of different structures of PD-L1/4-1 BB bispecific antibodies against 293T/4-1BB cells and 4-1BB L. the results show that the blocking activity of the 4-1BB moiety is superior to that of hexavalent diabody A (IC 50: 99.7nM), hexavalent diabody B (IC 50: 105nM) and tetravalent diabody (IC 50: 126nM) at the cellular level, and that the blocking activity of the octahedral diabody cell level is significantly superior to that of both hexavalent and tetravalent diabodies.
FIG. 7A shows the detection results when 4-1BB antigen and IgG1 protein were coated, and the results indicate that the anti-PD-L1/4-1 BB bispecific nanobody can bind to 4-1BB and PD-L1 simultaneously.
FIG. 7B shows the detection results when PD-L1 antigen and IgG1 were coated, and the results indicate that the anti-PD-L1/4-1 BB bispecific nanobody can bind to PD-L1 and 4-1BB simultaneously.
FIG. 8 shows the affinity of Fortebio detection anti-PD-L1/4-1 BB bispecific nanobody for PD-L1, the affinity is 5.87E-08M.
FIG. 9 shows the affinity of Fortebio detection anti-PD-L1/4-1 BB bispecific nanobody for 4-1BB, which is 2.24E-08M.
FIG. 10 shows the anti-PD-L1/4-1 BB bispecific nanobody, PD-L1 nanobody and the analysis of PD-L1/PD-1 blocking activity at the Tecnriq cell level, wherein, the IC of the anti-PD-L1/4-1 BB bispecific antibody50IC of 1.194ug/ml, PD-L1 nano antibody50IC 0.6437ug/ml, Tecntriq50It was 1.136 ug/ml.
FIG. 11 shows the analysis of the cell-level 4-1BB/4-1BB L blocking activity of anti-PD-L1/4-1 BB bispecific nanobody, 4-1BB nanobody and Utomilumab, wherein the IC of anti-PD-L1/4-1 BB bispecific antibody50IC of 4-1BB nanobody at 11.70ug/ml50IC at 4.640ug/ml, Utomillumab503.069 ug/ml.
FIG. 12 shows the results of the detection of the biological activity of an anti-PD-L1/4-1 BB bispecific antibody using the PD-1/PD-L1 Reporter Assay system, wherein the EC of the anti-PD-L1/4-1 BB bispecific antibody50Is 0.3021ug/ml, EC of PD-L1 nano antibody500.3232ug/ml, EC of Tecntriq500.6198 ug/ml.
FIGS. 13A and 13B show in vitro activation experiments of anti-PD-L1/4-1 BB bispecific nanobody, PD-L1 nanobody and 4-1BB nanobody against PBMC cells in Donor 1 and Donor 2, respectively, wherein the bispecific antibody can effectively activate T cells, and the activation effect is significantly stronger than that of the individual PD-L1 nanobody and 4-1BB nanobody.
14A, 14B, 14C and 14D show that anti-PD-L1/4-1 BB bispecific nanobody-mediated killing of T cells on tumor cells is PD-L1 dependent, wherein the candidate double antibodies shown in 14A and 14B can significantly kill PD-L1 over-expressed A375 cells against T cells of different individual sources, and the killing effect is significantly better than that of 4-1BB nanobody alone and PD-L1 nanobody alone, and the results of 14C and 14D show that the IFNr amount generated by the candidate double anti-stimulated T cell/A375 cell over-expressing PD-L1 combination is significantly higher than that of 4-1BB nanobody alone and PD-L1 nanobody alone.
Detailed Description
The inventor has extensively and deeply studied and unexpectedly obtained an anti-PD-L1/4-1 BB bispecific antibody which comprises an anti-PD-L1 nanobody and an anti-4-1 BB nanobody, experiments show that the bispecific antibody has better binding activity to PD-L1 and 4-1BB molecules, can block the interaction between PD-1 and PD-L1 and the interaction between 4-1BB and 4-1BB L, and has good anti-tumor activity, and the invention is completed on the basis.
Applicants have found that an 8-valent fusion protein of the invention has significantly superior binding activity compared to a 4-valent fusion protein comprising 2 anti-PD-L nanobodies and 2 anti-4-1 BB nanobodies, or a 6-valent fusion protein comprising 2 anti-PD-L nanobodies and 4 anti-4-1 BB nanobodies, or 4 anti-PD-L nanobodies and 2 anti-4-1 BB nanobodies.
In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.
As used herein, the terms "bispecific antibody of the invention", "diabody of the invention", "anti-PD-L1/4-1 BB bispecific antibody" have the same meaning, all referring to bispecific antibodies that specifically recognize and bind to PD-L1 and 4-1BB the invention also provides PD-L nanobody and 4-1BB nanobody.
As used herein, the term "antibody" or "immunoglobulin" is an isotetraglycan protein of about 150000 daltons having the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains (H), each light chain being linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different.two types of light chains, λ (l) and κ (k), five major heavy chain species (or isotypes) exist which determine the functional activity of the antibody molecule, IgM, IgD, IgG, IgA and IgE, each chain comprising different sequence domains.A light chain comprises two domains or regions, a light chain variable domain (V L) and a constant domain (C L) comprises four domains, a heavy chain variable region (VH) and three constant regions (CH1, CH 632 and CH3, collectively referred to as CH) light chain (V463) and a constant region (VH) and three constant region (CDR L) which are derived from the natural heavy chain variable domain binding sites of the Fc-Constant Domain (CDR) and which are derived from the amino acid binding sites of the Fc-heavy chain receptor binding of the Fc region (CDR-V) which are defined by the amino acid binding sites of the amino acid sequence of the Fc-V binding of the Fc region of the Fc-Fc region which is complementary heavy chain binding of the Fc region (CDR-Fc region).
As used herein, the terms "nanobody VHH", "nanobody" have the same meaning, referring to the variable region of a cloned antibody heavy chain, constructing a nanobody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Nanobodies (VHHs) consisting of only one heavy chain variable region are typically constructed by first obtaining an antibody that naturally lacks light and heavy chain constant region 1(CH1) and then cloning the variable region of the antibody heavy chain.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, in a substantially-folded configuration, connected by three CDRs that form a connecting loop, and in some cases may form a partially-folded structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.
As used herein, the term "framework region" (FR) refers to amino acid sequences inserted between CDRs, i.e. those portions of the light and heavy chain variable regions of an immunoglobulin which are relatively conserved between different immunoglobulins in a single species, the light and heavy chains of an immunoglobulin each have four FRs, designated FR 1-L, FR 2-L, FR 3-L0, FR 4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively, the light chain variable domain may thus be referred to as (FR 1-L) - (CDR 1-L) - (FR 2-L) - (CDR 2-L) - (FR 3-L) - (CDR 3-L) - (FR 4-L) and the heavy chain variable domain may thus be referred to as (FR1-H) - (CDR1-H) - (FR2-H) - (CDR2-H) - (FR 39 3-H) - (CDR3-H) - (FR 4-48356) preferably, the human antibody derivatives of the invention are substantially identical, i.e.g. human antibody derivatives of the same, human antibody sequences as FR 95%, or derivatives thereof.
Knowing the amino acid sequences of the CDRs, one skilled in the art can readily determine the framework regions FR 1-L, FR 2-L, FR 3-L, FR 4-L and/or FR1-H, FR2-H, FR3-H, FR 4-H.
As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework regions of a naturally occurring human antibody.
The affinity of the diabodies of the invention may be assessed or determined by KD values (dissociation constants) (or other means of determination), for example, by biofilm interference techniques (Bio-layer interference B L I) as determined by measurement using a FortebioRed96 instrument.
As used herein, the term "linker" refers to an insertion into an immunoglobulin domain that provides sufficient mobility for the domains of the light and heavy chains to fold into one or more amino acid residues that exchange the dual variable region immunoglobulin.
Immunoconjugates and fusion expression products include conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules conjugated to the antibodies or fragments thereof of the present invention, as will be appreciated by those skilled in the art, as well as cell surface markers or antigens conjugated to the PD-L1/4-1 BB bispecific antibody or fragment thereof.
As used herein, the terms "heavy chain variable region" and "VH"may be used interchangeably.
As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".
In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR 3.
In a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and heavy chain constant region.
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and refer to a polypeptide that specifically binds to PD-L1 and/or 4-1BB protein, e.g., a protein or polypeptide having a heavy chain variable region.
The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.
In general, the antigen binding properties of an antibody can be described by 3 specific regions located in the variable region of the heavy chain, called the variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction, the CDRs forming a loop structure, β folds formed by the FRs between them being spatially close to each other, the CDRs on the heavy chain and the CDRs on the corresponding light chain constituting the antigen binding site of the antibody.
The variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding to antigen. Thus, the invention includes those molecules having an antibody heavy chain variable region with CDRs whose homology to the CDRs identified herein is greater than 90% (preferably greater than 95%, most preferably greater than 98%).
The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.
The term also includes variants of the polypeptides comprising the CDR regions described above having the same function as the antibody of the invention, including, but not limited to, deletion, insertion and/or substitution of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or more (typically up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus.
Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
The invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full-length polypeptides, fragments of the nanobodies of the invention are also encompassed by the present invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.
In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.
TABLE 1
Figure GDA0002490661480000151
Figure GDA0002490661480000161
The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.
The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.
The present invention also relates to polynucleotides which hybridize to the above-described sequences and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences, and the present invention particularly relates to polynucleotides which hybridize to the polynucleotides of the present invention under stringent conditions, where "stringent conditions" refer to (1) hybridization and elution at lower ionic strength and higher temperatures, e.g., 0.2 × SSC, 0.1% SDS,60 ℃, or (2) hybridization with denaturing agents, e.g., 50% (v/v) formamide, 0.1% bovine serum/0.1% Ficoll, 42 ℃, etc., or (3) hybridization only if the identity between the two sequences is at least 90% or more, and more preferably 95% or more, and the polypeptides encoded by the hybridizable polynucleotides have the same biological function and activity as the mature polypeptides.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.
At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.
Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl2Methods, the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptides in the above methods can be expressed intracellularly, or on the cell membrane, or secreted extracellularly, if desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties, which are well known to those skilled in the art.
The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that can be conjugated or conjugated to the antibodies of the invention include, but are not limited to, 1. radionuclides, 2. biotoxicants, 3. cytokines such as I L-2, etc., 4. gold nanoparticles/nanorods, 5. viral particles, 6. liposomes, 7. nanomagnets, 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPH L)), 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.
Bispecific antibodies
The invention provides an anti-PD-L1/4-1 BB bispecific antibody, which comprises an anti-PD-L1 nano antibody and an anti-4-1 BB nano antibody.
In a preferred embodiment, the bispecific antibody comprises two anti-PD-L1 nanobodies and two anti-4-1 BB nanobodies, and the bispecific antibody can form a dimer, i.e., a bispecific fusion protein comprising 4 anti-PD-L1 nanobodies and 4 anti-4-1 BB nanobodies, as shown in fig. 3A.
In fact, based on the anti-4-1 BB nanobody and the anti-PD-L1 nanobody of the present invention, the applicant constructed a plurality of structures of bispecific antibodies in the construction process of bispecific antibodies, including 1 anti-PD-L1 nanobody and 1 anti-4-1 BB nanobody, or including 1 anti-PD-L1 nanobody and 2 anti-4-1 BB nanobodies, or including 2 anti-PD-L1 nanobodies and 1 anti-4-1 BB nanobody, so that the bispecific antibodies of the structure can also form a dimeric fusion protein, the structures are shown in FIGS. 3B, 3C and 3D, and the experimental results show that the fusion protein shown in FIG. 3A has significantly better binding activity than the fusion protein shown in FIGS. 3B, 3C and 3D.
Pharmaceutical composition
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above antibody or an active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
The pharmaceutical composition of the present invention can be used directly to bind to PD-L1 and/or 4-1BB protein molecules and thus can be used to treat tumors.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the nanobody (or its conjugate) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
Labeled nanobodies
In a preferred embodiment of the invention, the nanobody carries a detectable label. More preferably, the marker is selected from the group consisting of: isotopes, colloidal gold labels, coloured labels or fluorescent labels.
In a preferred embodiment of the present invention, the anti-PD-1 nanobody, the anti-4-1 BB nanobody, and the PD-L1/4-1 BB bispecific antibody may be labeled with colloidal gold to obtain a colloidal gold-labeled nanobody.
The PD-L1/4-1 BB bispecific antibody has good specificity and high titer.
Detection method
The present invention also relates to a method for detecting PD-L1 and/or 4-1BB protein, which method comprises the steps of obtaining a cell and/or tissue sample, solubilizing the sample in a medium, and detecting the level of PD-L1 and/or 4-1BB protein in the solubilized sample.
The sample used in the detection method of the present invention is not particularly limited, and a typical example is a cell-containing sample present in a cell preservation solution.
Reagent kit
The present invention also provides a kit comprising an antibody (or fragment thereof) or assay plate of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.
The invention also provides a detection kit for detecting the level of PD-L1 and/or 4-1BB, which comprises an antibody for identifying the PD-L1 and/or 4-1BB protein, a lysis medium for dissolving a sample, general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates and the like.
Applications of
As described above, the nanobody of the present invention has wide biological and clinical application values, and its applications relate to many fields such as diagnosis and treatment of diseases related to PD-L1 and/or 4-1BB, research in basic medicine, biological research, etc. one preferred application is in clinical diagnosis and targeted therapy against PD-L1 and/or 4-1BB, such as tumor therapy.
The main advantages of the invention include:
(a) the 4-1BB nano antibody has different antigen recognition epitopes with the products on the market, namely Urelumab and Utomilumab;
(b) the bispecific antibody of the invention can simultaneously block the binding of PD-1/PD-L1 and the binding of 4-1BB/4-1BB L;
(c) the bispecific antibody of the invention has significant anti-tumor activity.
(d) The killing effect of the T cell mediated by the antibody on the tumor cell has PD-L1 dependency, and hepatotoxicity can be avoided in the future.
The present invention is further illustrated below with reference to specific examples, which are intended to illustrate the invention only and not to limit the scope of the invention the experimental procedures, for which specific conditions are not indicated in the following examples, are generally according to conventional conditions, such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: Cold Spring Harbor L laboratory Press,1989), or according to manufacturer's recommendations.
Example 1 screening and humanization of PD-L1 Nanobodies
Construction of the PD-L1 antibody library:
(1) the method comprises the steps of (1) mixing 1mg of hPD-L1 (ECD) -Fc antigen with Freund's adjuvant in an equal volume, immunizing one Xinjiang bactrian camel once a week for 7 times, and stimulating B cells to express antigen-specific nano antibodies, (2) extracting L m camel peripheral blood lymphocytes and extracting total RNA after 7 times of immunization, (3) synthesizing cDNA and amplifying VHH by nested PCR, (4) digesting 20 mu g of pMECS phage display vectors and 10 mu g of VHH by using restriction enzymes PstI and NotI and connecting the two fragments, (5) transforming the connecting products into electrotransformation competent cells TG1 to construct a PD-L1 nano antibody library.
Enrichment of PD-L1 antibody positive clones:
(1) dissolving in 100mM NaHCO 310 μ g of hPD-L1 (ECD) -Fc antigen (10 μ g of Fc inNaHCO) in pH8.23As a control), coupling on NUNC enzyme label plate, standing overnight at 4 ℃, (2) adding 100 mu L0.1% BSA the next day, blocking for 2h at room temperature, (3) adding 100 mu L phage (2 × 10) after 2h11CFU immune camel nano antibody phage display geneA library) is acted for 1 hour at room temperature, (4) the phage which is specifically combined with PD-L1 is washed for 5 times by 0.05 percent PBS + Tween-20 to wash off the non-specific phage, (5) the phage which is specifically combined with PD-L is dissociated by 100mM triethanolamine and infected into Escherichia coli TG1 cells which grow in logarithmic phase, the cells are cultured for 1 hour at 37 ℃, the phage is generated and purified for the next round of screening, and the enriched phage clone strain is obtained by repeating 3 rounds of the same screening process.
Screening of single positive clones for the PD-L1 antibody:
(1) from the phage-containing cell culture dishes selected as described above, 200 single colonies were selected and inoculated into TB medium containing 100. mu.g/m L ampicillin (2.3 g KH in 1L TB medium)2PO4，12.52g K2HPO412g of peptone, 24g of yeast extract and 4m of L glycerol), adding IPTG (1 mM final concentration) after growth to a logarithmic phase, and culturing at 28 ℃ overnight, (2) obtaining crude antibodies by using an osmosis method, transferring the antibodies to an E L ISA plate coated with antigen, and standing at room temperature for 1h, (3) washing unbound antibodies by using PBST, adding mouse anti-HA antibodies, purchased from Beijing kang, century Biotechnology Co., Ltd, and standing at room temperature for 1h, (4) washing unbound antibodies by using PBST, adding goat anti-mouse alkaline phosphatase labeled antibodies, and standing at room temperature for 1h, (5) washing unbound antibodies by using PBST, adding an alkaline phosphatase developing solution on an E L ISA instrument, reading an absorption value at a wavelength of 405nm, and (6) judging the obtained product to be a positive clone well when the OD value of the sample well is more than 3 times larger than the OD value of a control well (Ratio +/-3), and carrying out sequencing identification and comparing and analyzing the CDR sequence of the nano antibodies.
Preliminary identification of the blocking function of the nano antibody:
(1) the method for preparing hPD-1(ECD) -Biotin protein and biotinylation of the protein refers to a Biotin reagent specification, (2) instantly transferring a PD-L1 gene to HEK293F cells to enable the cell surface to express the PD-L1 protein, (3) preparing PD-L1 nano antibody TG1 strain crude lysis solution, the preparation method is the same as that of Zhu Min et al, Nanoscale Res L et, 2014Sep26, (9) (1) 528, (4) taking 1 × 10 from each sample6HEK293F cells transiently transfected with PD-L1 were resuspended in 0.5% BSA-PBS buffer, and 100. mu. L of the crude extract was added while setting negativeControl (hIgG1) and positive control (Tecntriq), 5. mu.g of hPD-1(ECD) -Fc-Biotin was added per well and incubated at 4 ℃ for 20 min; (5) and (3) washing the cells for 2 times by PBS, adding SA-PE of eBioscience, incubating for 20min at 4 ℃, washing the cells for 2 times by PBS, and detecting by a flow cytometer (BD FACS Calibur), wherein the obtained 1 strain of nano antibody has an obvious blocking effect.
Carrying out humanized transformation:
(1) the method comprises the steps of firstly, taking a PD-L1 nano antibody sequence shown in SEQ ID NO.19 as a template, searching a homologous structure in a structure database, taking a structure in which Evalue is 0.0 and the sequence identity is more than or equal to 70 percent, (2) carrying out structure comparison on the structures, carrying out multi-template homologous modeling based on the PD-L1 nano antibody sequence shown in SEQ ID NO.19 according to the resolution of crystal structures and a constructed evolutionary tree, selecting a structure with the lowest molpdf according to the high-low ordering of a scoring function, (3) calculating the solvent accessibility of the residues by using a ProtSA server according to the optimal structure of the constructed structure, wherein the ratio of the accessible areas of the folded state of the residues relative to the unfolded state of the solvent is a criterion, and taking more than 40 percent of the residues as the optimal residues exposed outside the solvent, (4) carrying out sequence comparison on the constructed structure of the modal and DP-47, and replacing the corresponding residues exposed to the solvent, and finally determining a humanized PD-L1 nano antibody, wherein the amino acid sequence shown in SEQ ID NO.8 and the amino acid sequence region shown in a coding sequence table 1 are amino acid region shown in a CDR 1.
Example 2: screening and humanization of 4-1BB nanobody
By referring to the method of example 1, library construction, enrichment and screening of positive clones, preliminary identification of blocking function, and humanization modification of 4-1BB nanobody were carried out to successfully obtain a humanized 4-1BB nanobody encoded by the amino acid sequence shown in SEQ ID NO.17, and the amino acid sequences of each CDR region and FR region are shown in Table 1.
TABLE 1
Figure GDA0002490661480000221
Example 3: antigen recognition epitope analysis of 4-1BB nano antibody
(1) The method comprises the steps of coating an enzyme label plate with 100u L0.2 ug/M L of human 4-1BB antigen protein and three mutant proteins (N42R, M101R and I132R), incubating at 37 ℃ for 2h, (2) washing with PBST for 5 times, adding 1% BSA (bovine serum albumin) of 300u L into each hole after drying, sealing at room temperature for 2h, (3) washing with PBST for 5 times, adding 2ug/M L4-1 BB nano antibody and control antibodies (Urelumab and Utomillumab) respectively after drying, incubating at room temperature for 1h, (4) washing with PBST for 5 times, adding 100u L diluted goat anti-human IgG antibody (1:100000 dilution) after drying, incubating at room temperature for 1h, (5) washing with PBST for 5 times, adding 100u L B liquid respectively after drying, standing at room temperature for 10min, and reading at the wavelength of 450nm in a dark place.
The results are shown in FIG. 1, where the site where Urelumab binds 4-1BB is N42, and the site where Utomilumab binds 4-1BB is M101 and I132. the results are consistent with the results reported in the Structure of the 4-1BB/4-1BB L complex recognition and functional properties of utilliumand ureluab, published in the Nature communications journal, by S.Michael Chin, Christopher R.Kimberlin, Zygy Roe-Zurz et al, all three sites are not the sites where the 4-1BB nanobody of the present invention binds 4-1BB, and thus the epitope recognized by the nanobody of the present invention can be considered different from the two control antibodies.
Example 4: anti-tumor activity of 4-1BB nano antibody
(1) Culturing mouse colon cancer MC38 cells at 37 deg.C and 5% CO2(2) Dulbecco's Modified Eagle's Medium containing 10% inactivated fetal bovine serum (PBS-resuspended MC38 colon cancer cells at 1 × 10%5One/0.1 m L concentration, 0.1m L/volume inoculated subcutaneously on the right side of B-h4-1BB humanized mice (3) when the mean tumor volume reached about 100-150mm3In the process, mice with appropriate individual tumor volume are selected into groups, animals are randomly distributed into each experimental group according to the tumor volume, 7 animals in each group are divided into groups, administration is started on the same day, and administration is performed once every 3 days for 6 times. (4) After the last administration, the body weight and tumor growth status of the experimental animals were observed for 1 week, and during the observation period, the tumor volume and the animal body weight were measured twice per week, and the measured values were recorded.
The results are shown in FIG. 2, the antibodies of both experimental groups have good tumor inhibition effect, and the 4-1BB nano antibody is more excellent. The tumor inhibition rate TGI of the control antibody Utomillumab was 68.3%, and the tumor inhibition rate TGI of the candidate 4-1BB nanobody was 75.8%.
Example 5 anti-PD-L1/4-1 BB bispecific antibody design
The PD-L1 nanobody obtained in example 1 and example 2 and the 4-1BB nanobody are connected in series and are connected by a linker (SEQ ID NO.19) to form the following structure:
NbPD-L1-4(GGGGS)-NbPD-L1-Fc-4(GGGGS)-Nb4-1BB-4(GGGGS)-Nb4-1BB
the schematic structure is shown in fig. 3A.
The amino acid sequence is shown as follows, wherein the underlined part is the linker sequence, and the italic part is the Fc sequence:
QVQLQESGGGLVQPGGSLRLSCTASGYNLSPSCMGWFRQAPGKGLEGVAFTDADGSTRYADSVKARFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADFFSYCSVVFRASARDKYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCTASGYNLSPSCMGWFRQAPGKGLEGVAFTDADGSTRYADSVKARFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADFFSYCSVVFRASARDKYRGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCAASGYTYSSNCMGWFRQAPGKGLEGVAVICTGGGSPSYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADLLRAGTPLSSYEFNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLSCAASGYTYSSNCMGWFRQAPGKGLEGVAVICTGGGSPSYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADLLRAGTPLSSYEFNYWGQGTLVTVSS
example 6 anti-PD-L1/4-1 BB bispecific antibody expression purification
The plasmid containing the double-antibody encoding gene in example 5 is transiently transferred into HEK293F cells for expression and purification of the antibody, and the specific method is as follows:
(1) preparing a large amount of plasmids by using an MN plasmid large-extraction kit, and filtering and sterilizing in a superclean workbench for later use; (2) HEK293F cells were cultured to 2.0×106M L, (3) mixing the plasmid and PEI in the proportion of 1:3 evenly to the transfection medium F17(Gibco), standing for 20min, then adding into HEK293F cells, at 37 ℃, 6% CO2Culturing for 6 days in a shaking incubator, (4) centrifuging to obtain a supernatant, combining the supernatant with proteinA A beads for 1h at room temperature, (5) washing off foreign proteins and other impurities by using a phosphate buffer solution with pH7.0, and then eluting a target antibody protein by using 0.1M of Glycine with pH3.0, and (6) ultrafiltering the eluted antibody into a 1 × PBS solution, and sampling for SDS-PAGE analysis.
The results are shown in FIG. 4, where the antibody purity was about 85% or higher. Subpackaging the rest proteins, and storing in a refrigerator at-80 deg.C.
Example 7 analysis of the cellular level binding and blocking Activity of PD-L1/4-1 BB bispecific antibody 4-1BB parts of different structures
The PD-L1/4-1 BB bispecific antibody with different structures has the binding activity to 293F/4-1BB cells by the following specific method:
(1) each sample was taken 3 × 105293T/4-1 BB-transfected cells were individually transferred to PBS buffer, diluted anti-PD-L1/4-1 BB bispecific antibodies (prepared in example 6) of different structures (antibody concentrations: 241nM, 120nM, 60.2nM, 30 nM, 15.1nM, 7.53nM, 3.77nM, 1.88nM, 0.94nM, 0.47nM, 0.24nM, 0.12nM) were added, each sample was incubated at 100u L for 20min at 4 ℃, (2) the cells were washed with PBS for 2 times, Goat 4 ℃ anti Human Fc-FITC of Abcam was added, incubated for 20min at 4 ℃, washed with PBS for 2 times and then detected with a flow cytometer (BD FACS Calibur) using graphpadprism 6 software for data processing.
As a result, as shown in fig. 5, the cell-level binding activity of the octavalent diabody (EC50 ═ 6.43nM)4-1BB moiety was superior to that of the hexavalent diabody a (EC50 ═ 20.1nM), the hexavalent diabody B (EC50 ═ 19.2nM) and the tetravalent diabody (EC50 ═ 30.1 nM).
The PD-L1/4-1 BB bispecific antibody with different structures blocks the binding activity of 293T/4-1BB cells and 4-1BB L by the following specific method:
(1) each sample was taken 3 × 105293T/4-1 BB-transfected cells were placed in PBS buffer, and diluted anti-PD-L1/4-1 with different structures was addedBB bispecific antibody (prepared in example 6) (antibody concentrations: 1651nM, 826nM, 413nM, 206nM, 103nM, 51.7nM, 25.8nM, 12.9nM, 6.45nM, 3.23nM, 1.61nM, 0.81nM), 100u L per sample, 0.6ug/ml of h4-1BB L (ECD) -Fc-Biotin (ECD) added simultaneously to all samples, incubation at 4 ℃ for 20min, (2) PBS washing of 2 cells, addition of SA-PE from eBioscience, incubation at 4 ℃ for 20min, washing of 2 cells with PBS, detection with flow cytometer (BD FACS Calibur), data processing using graphpad prism 6 software.
As a result, as shown in fig. 6, the blocking activity at the cellular level of the 4-1BB moiety of the octavalent diabody (IC50 ═ 59.5nM) was superior to that of the hexavalent diabody a (IC50 ═ 99.7nM), the hexavalent diabody B (IC50 ═ 105nM) and the tetravalent diabody (IC50 ═ 126 nM).
Example 8 identification of Activity of anti-PD-L1/4-1 BB bispecific antibodies to simultaneously bind Dual targets
The double antibodies prepared in example 6 were subjected to activity identification by the following method
(1) With NaHCO respectively3PD-L1-Fc or 4-1BB-Fc100ul which is diluted by the solution is coated and stays overnight at 4 ℃, (2) PBST is washed for 5 times, (3) 300ul of 1% BSA in each hole is sealed for 2 hours at 37 ℃, (4) PBST is washed for 5 times, (5) 100ul of diluted primary antibody in each hole is incubated for 1 hour at 37 ℃, (6) PBST is washed for 5 times, (7) 100ul of 4-1BB-biotin or PD-L1-biotin sample in each hole is incubated for 1 hour at 37 ℃, (8) PBST is washed for 5 times, (9) 100ul of SA-HRP (1:5000PBS) is added and incubated for 1 hour at 37 ℃, (10) PBST is washed for 5 times, (11) 100ul of TMB developing solution is added in each hole, the reaction is carried out at room temperature and is carried out in a dark place for 5-7 minutes, (12) 50ul of 2M H is added2SO4The reaction was stopped and absorbance was read at 450 nm.
Results as shown in FIGS. 7A and 7B, FIG. 7A is the results of the assay when 4-1BB-Fc was coated, and FIG. 7B is the results of the assay when PD-L1-Fc was coated, indicating that the anti-PD-L1/4-1 BB bispecific antibody of the present invention can bind to 4-1BB and PD-L1 simultaneously.
Example 9: double antibody affinity assay
The binding kinetics of the diabody prepared in example 6 to the recombinant human PD-L1 was determined by a biofilm interference technique (Bio-layer interference B L I) and a Fortebio Red96 instrument, and the specific method is as follows:
the diabody prepared in example 6 was diluted to 4ug/m L with PBST buffer, PD-L1 antigen (expressed by HEK293F, prepared in the same manner as in example 1, with Fc-tagged Protein removed after cleavage with TEV enzyme) was diluted with PBST buffer in 1.5-fold gradient with six concentration gradients (200nM, 133.3nM, 88.9nM, 59.3nM, 39.5nM, 26.3nM), set the instrument operating conditions at 30 ℃ and Shake speed 1000rpm, antibody capture using probes coated with Protein A (Fortebio Part No:18-5010), capture time 180s, antigen bound to the gradient dilution, binding time 70s, dissociation time 120s, 10mM glycine (PH1.7) was regenerated 3 times, analysis was performed using Fortebio Analysis9.0 version 5s each time, 1:1 binding model Glnal model was performed in1 mode, and the Kd dissociation rate (Kois) and dissociation rate (Kois) constant were calculated.
The results are shown in FIG. 8, where the affinity of the diabody for PD-L1 is 5.87E-08M.
Similarly, the binding kinetics of the diabody prepared in example 6 to recombinant human 4-1BB was determined by biofilm layer interference technique (Bio-layer interference B L I) and FortebioRed96 instrument, as follows:
the diabody prepared in example 6 was diluted to 5ug/m L with PBST buffer, 4-1BB antigen (expressed by HEK293F, prepared in a manner similar to the preparation of PD-L1 antigen in example 1, with Fc-tagged Protein removed after cleavage by TEV enzyme) was diluted with PBST buffer 2-fold gradient in six concentration gradients (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM), the instrument was operated at 30 ℃ under Shake 1000rpm, antibody capture with a probe coated with Protein A (Fortebio Part No:18-5010), capture time 60s, antigen bound to the gradient dilution, binding time 70s, dissociation time 120s, 10mM glycine (PH1.7) was regenerated 2 times, KD was analyzed at 5s each time using Fortebio analysis version 9.0, 1:1 binding model was fitted, binding rate (Glal mode) was calculated, dissociation rate (Kdis), dissociation rate (Kobis) was calculated, and dissociation rate (Kobis) was calculated.
The results are shown in FIG. 9, where the affinity of the diabody for 4-1BB is 2.24E-08M.
Example 10 FACS detection of the Activity of the Biantibodies of the invention to block the binding of PD-1/PD-L1 and 4-1BB/4-1BB L
The activity of the anti-PD-L1/4-1 BB bispecific antibody of the invention for blocking the binding of PD-1/PD-L1 is detected by the following specific method:
(1) each sample was taken 3 × 105A375/PD-L1 cells were stably transfected into PBS buffer, and diluted anti-PD-L1/4-1 BB bispecific antibody (prepared in example 6), PD-L1 nanobody (prepared in example 1) and positive control antibody (Tecntriq) (antibody concentrations: 20ug/ml, 10ug/ml, 5ug/ml, 2.5ug/ml, 1.25ug/ml, 0.625ug/ml, 0.3125ug/ml, 0.1563u/ml, 0.0781ug/ml, 0.0391ug/ml, 0.0196ug/ml, 0.0098ug/ml) were added to each sample at 100u L, all samples were incubated with 50ug/ml hPD-1(ECD) -BD-Biotin at 4 ℃ for 20min, and 2) cells were washed, SA-PD-L incubated with PBS, and cells were washed with FACS 2 min, and the data were measured using a flow cytometry (FACS) instrument after 2 min (PBS buffer).
The results are shown in FIG. 10, IC for candidate double antibody501.194ug/ml, and positive control antibody (Tecntriq, IC)501.136ug/ml), the candidate double antibody has PD-L1/PD-1 blocking activity equivalent to the performance of the candidate double antibody, and the candidate double antibody has PD-L1 nano antibody (IC)500.6437ug/ml), the diabody of the present invention retained better PD-L1/PD-1 blocking activity.
The activity of the anti-PD-L1/4-1 BB bispecific antibody of the invention for blocking the binding of 4-1BB/4-1BB L is detected by the following specific method:
(1) each sample was taken 3 × 105293T/4-1 BB-transfected cells were cultured in PBS buffer, diluted anti-PD-L1/4-1 BB bispecific antibody (prepared in example 6), 4-1BB nanobody (prepared in example 2) and positive control antibody (Utomillumab) (antibody concentrations: 160ug/ml, 80ug/ml, 40ug/ml, 20ug/ml, 10ug/ml, 5ug/ml, 2.5ug/ml, 1.25u/ml, 0.625ug/ml, 0.3125ug/ml, 0.1563ug/ml, 0.0781ug/ml) were added to each sample of 100u L, all samples were incubated with 0.6ug/ml of h4-1BB L (ECD) -Fc-Biotin at 4 ℃ for 20min, 2) washed cells, PBS-bioscience added, PE-1 BB-cell incubation at 4 ℃ for 20min, and then treated with PBS buffer and PBS buffer, and the data were measured using a flow cytometry (PBS buffer) FACS-2 BD-wash.
The results are shown in FIG. 11IC with double-antibody candidate5011.70ug/ml, and 4-1BB nanobody (IC)504.64ug/ml) retained better 4-1BB/4-1BB L blocking activity.
Example 11 luciferase reporter Gene System for detecting the Activity of the PD-L1 antibody in bispecific antibodies
The PD-L1/PD-L1 reporter gene detection system (purchased from Nanjing Kingsley Biotech Co., Ltd.) can be used in the processes of batch release of biological samples, stability detection, process development, etc., for evaluating the biological activity of PD-L antibody drugs, the PD-L antibody effectively blocks the mutual regulation of the surface PD-L and the luciferase gene expression of target cells (GS-C5/PD-L) L, when the added PD-L antibody effectively blocks the mutual regulation of the luciferase gene expression of the NFase gene in cells, the AT-L reporter gene is reported.
Using this principle, (1) 4000 GS-C2/PD-L1 per well were inoculated into a 96-well plate for overnight culture, (2) bispecific antibody (prepared in example 6), PD-L1 nanobody (prepared in example 1), and positive control antibody (Tecntriq) were diluted in a gradient (66.67nM, 22.22nM, 7.41nM, 2.47nM, 0.82nM, 0.27nM, 0.09nM, 0.03nM, 0.01nM), respectively, and the diluted antibodies were mixed with 1X10 nM, respectively6Mixing GS-J2/PD-L1 cells, adding into target cells at 37 deg.C with 5% CO2Culturing for 6 hr, (3) adding L uciferase detecting substrate, reacting at room temperature for 5 min, and reading with enzyme labeling instrument to obtain standard value (R L U)Sample to be tested-RLUBlank control)/(RLUEC100 of Tecentriq–RLUBlank control) 100 processing.
The results are shown in FIG. 12, EC of the positive control antibody Tecntriq50Is 0.6198ug/ml of the solution,EC of PD-L1 nano-antibody50EC for bispecific antibody at 0.3232ug/ml50The results showed that the biological activity of the bispecific antibody of the invention was similar to that of the PD-L1 nanobody, better than that of the control antibody Tecentriq, 0.3021 ug/ml.
Example 12: effect of the Biantibody of the invention on T cell activation
The detection method comprises the following steps:
(1) 50ul of PBMC cells, 1E5/well, were added to the corresponding 96-well cell culture plates;
(2) 100ul of antibody (final concentration: 25nM, 5nM, 1nM, 0.2nM, 0.04nM, 0.008nM, 0nM) and 50ul of SEB (final concentration: 0.1ug/ml) were added to the corresponding wells, respectively; (3)37 ℃ and 5% CO2Culturing for 72h, and (4) detecting the content of I L-2 in the supernatant by using a BDI L-2E L ISA kit.
As shown in fig. 13A and 13B, the candidate double antibodies showed significant activation on T cells in both Donor 1 (fig. 13A) and Donor 2 (fig. 13B), and the activation was significantly stronger than PD-L1 nanobody and 4-1BB nanobody alone.
Example 13 Dual antibody mediated killing of tumor cells by T cells of the invention is PD-L1 dependent
(1) Recovering PBMC cells of different donors, and separating CD3+ T cells from the PBMC by using a CD3 kit, (2) co-incubating antibodies to be detected and cell cells, namely adding 1E 5T cells and 3E 4A 375 or A375/PD-L1 cells into each well, adding 50u L antibody mixed liquor or CD3 antibody diluent, uniformly mixing, placing in an incubator, and incubating for 72 hours, (3) taking supernatant after incubation, and respectively detecting the contents of lactate dehydrogenase and IFN-r by using E L ISA, wherein the detection method refers to BD L otNO.55142 kit specifications and Promega L NO. ot.G1780 kit specifications.
Results as shown in fig. 14A and 14B, the killing effect of candidate dual-anti-mediated T cells on PD-L1-overexpressed a375 is significantly better than that of 4-1BB nanobody alone and PD-L1 nanobody alone, whereas the killing effect of candidate dual-anti-mediated T cells on native a375 cells is not significantly different compared to 4-1BB nanobody alone and PD-L1 nanobody alone, and the results in fig. 14C and 14D indicate that the IFNr generated by the candidate dual-anti-stimulated T cell/PD-L1-overexpressed a375 cell combination is significantly higher than that of 4-1BB nanobody alone and PD-L1 nanobody alone, and the IFNr generated by the candidate dual-anti-stimulated T cell/native a375 cell combination is not significantly different from that of 4-1BB nanobody alone and PD-L1 nanobody alone.
The above examples show that the anti-PD-L1/4-1 BB bispecific antibody of the present invention can be expressed in HEK293F cells, can be further purified by affinity chromatography, and the purity after only one-step purification can reach 85%. the obtained bispecific antibody can bind to 4-1BB positive BxPC3 cells, and simultaneously block the interaction between 4-1BB and 4-1BB L on the cell surface, and in addition, the double antibody can simultaneously block the interaction between PD-L and PD-1 on the cell surface.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Shanghai Luoqi biomedical technology, Inc
<120> anti-PD-L1/4-1 BB bispecific antibody and use thereof
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Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
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caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcaccg ccagcggcta caacctgagc cccagctgca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccttc accgacgccg acggcagcac caggtacgcc 180
gacagcgtga aggccaggtt caccatcagc agggacaaca gcaagaacac cctgtacctg 240
cagatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc cgacttcttc 300
agctactgca gcgtggtgtt cagggccagc gccagggaca agtacagggg ccagggcacc 360
ctggtgaccg tgagcagc 378
<210>10
<211>25
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>10
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210>11
<211>15
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>11
Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala Val
1 510 15
<210>12
<211>38
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>12
Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210>13
<211>11
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>13
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210>14
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>14
Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly
1 5 10
<210>15
<211>8
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>15
Ile Cys Thr Gly Gly Gly Ser Pro
1 5
<210>16
<211>18
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>16
Ala Ala Asp Leu Leu Arg Ala Gly Thr Pro Leu Ser Ser Tyr Glu Phe
1 5 10 15
Asn Tyr
<210>17
<211>125
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>17
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Val Ile Cys Thr Gly Gly Gly Ser Pro Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Leu Leu Arg Ala Gly Thr Pro Leu Ser Ser Tyr Glu Phe
100 105 110
Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210>18
<211>375
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta cacctacagc agcaactgca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccgtg atctgcaccg gcggcggcag ccccagctac 180
gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa caccctgtac 240
ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc cgccgacctg 300
ctgagggccg gcacccccct gagcagctac gagttcaact actggggcca gggcaccctg 360
gtgaccgtga gcagc 375
<210>19
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
ggggsggggs ggggsggggs 20
<210>20
<211>791
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>20
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Asn Leu Ser Pro Ser
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Phe Thr Asp Ala Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys
50 55 60
Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Phe Phe Ser Tyr Cys Ser Val Val Phe Arg Ala Ser Ala Arg
100 105 110
Asp Lys Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro
145 150 155 160
Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Asn Leu Ser
165 170 175
Pro Ser Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu
180 185 190
Gly Val Ala Phe Thr Asp Ala Asp Gly Ser Thr Arg Tyr Ala Asp Ser
195 200 205
Val Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
210 215 220
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
225 230 235 240
Cys Ala Ala Asp Phe Phe Ser Tyr Cys Ser Val Val Phe Arg Ala Ser
245 250 255
Ala Arg Asp Lys Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
260 265 270
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
275 280 285
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
290 295 300
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
305 310 315 320
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
325 330 335
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
340 345 350
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
355 360 365
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
370 375 380
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
385 390 395 400
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
405 410 415
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
420 425 430
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
435 440 445
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
450 455 460
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
465 470 475 480
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
485 490 495
Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
500 505 510
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser
515 520 525
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
530 535 540
Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Phe Arg Gln
545 550 555 560
Ala Pro Gly Lys Gly Leu Glu Gly Val Ala Val Ile Cys Thr Gly Gly
565 570 575
Gly Ser Pro Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
580 585 590
Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
595 600 605
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Leu Leu Arg Ala
610 615 620
Gly Thr Pro Leu Ser Ser Tyr Glu Phe Asn Tyr Trp Gly Gln Gly Thr
625 630 635 640
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
645 650 655
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu
660 665 670
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
675 680 685
Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Phe Arg
690 695 700
Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala Val Ile Cys Thr Gly
705 710 715 720
Gly Gly Ser Pro Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
725 730 735
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
740 745 750
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Leu Leu Arg
755 760 765
Ala Gly Thr Pro Leu Ser Ser Tyr Glu Phe Asn Tyr Trp Gly Gln Gly
770 775 780
Thr Leu Val Thr Val Ser Ser
785 790
<210>21
<211>2373
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
caggtgcagc tgcaggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60
tcctgcaccg cctccggcta caacctgtcc ccctcctgca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccttc accgacgccg acggctccac caggtacgcc 180
gactccgtga aggccaggtt caccatctcc agggacaact ccaagaacac cctgtacctg 240
cagatgaact ccctgagggc cgaggacacc gccgtgtact actgcgccgc cgacttcttc 300
tcctactgct ccgtggtgtt cagggcctcc gccagggaca agtacagggg ccagggcacc 360
ctggtgaccg tgtcctccgg cggcggcggc tccggcggcg gcggctccgg cggcggcggc 420
tccggcggcg gcggctccca ggtgcagctg caggagtccg gcggcggcct ggtgcagccc 480
ggcggctccc tgaggctgtc ctgcaccgcc tccggctaca acctgtcccc ctcctgcatg 540
ggctggttca ggcaggcccc cggcaagggc ctggagggcg tggccttcac cgacgccgac 600
ggctccacca ggtacgccga ctccgtgaag gccaggttca ccatctccag ggacaactcc 660
aagaacaccc tgtacctgca gatgaactcc ctgagggccg aggacaccgc cgtgtactac 720
tgcgccgccg acttcttctc ctactgctcc gtggtgttca gggcctccgc cagggacaag 780
tacaggggcc agggcaccct ggtgaccgtg tcctccgagt ccaagtacgg ccccccctgc 840
cccccctgcc ccgcccccga gttcctgggc ggcccctccg tgttcctgtt cccccccaag 900
cccaaggaca ccctgatgat ctccaggacc cccgaggtga cctgcgtggt ggtggacgtg 960
tcccaggagg accccgaggt gcagttcaac tggtacgtgg acggcgtgga ggtgcacaac 1020
gccaagacca agcccaggga ggagcagttc aactccacct acagggtggt gtccgtgctg 1080
accgtgctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt gtccaacaag 1140
ggcctgccct cctccatcga gaagaccatc tccaaggcca agggccagcc cagggagccc 1200
caggtgtaca ccctgccccc ctcccaggag gagatgacca agaaccaggt gtccctgacc 1260
tgcctggtga agggcttcta cccctccgac atcgccgtgg agtgggagtc caacggccag 1320
cccgagaaca actacaagac cacccccccc gtgctggact ccgacggctc cttcttcctg 1380
tactccaggc tgaccgtgga caagtccagg tggcaggagg gcaacgtgtt ctcctgctcc 1440
gtgatgcacg aggccctgca caaccactac acccagaagt ccctgtccct gtccctgggc 1500
aagggcggcg gcggctccgg cggcggcggc tccggcggcg gcggctccgg cggcggcggc 1560
tcccaggtgc agctgcagga gtccggcggc ggcctggtgc agcccggcgg ctccctgagg 1620
ctgtcctgcg ccgcctccgg ctacacctac tcctccaact gcatgggctg gttcaggcag 1680
gcccccggca agggcctgga gggcgtggcc gtgatctgca ccggcggcgg ctccccctcc 1740
tacgccgact ccgtgaaggg caggttcacc atctccaggg acaacgccaa gaacaccctg 1800
tacctgcaga tgaactccct gagggccgag gacaccgccg tgtactactg cgccgccgac 1860
ctgctgaggg ccggcacccc cctgtcctcc tacgagttca actactgggg ccagggcacc 1920
ctggtgaccg tgtcctccgg cggcggcggc tccggcggcg gcggctccgg cggcggcggc 1980
tccggcggcg gcggctccca ggtgcagctg caggagtccg gcggcggcct ggtgcagccc 2040
ggcggctccc tgaggctgtc ctgcgccgcc tccggctaca cctactcctc caactgcatg 2100
ggctggttca ggcaggcccc cggcaagggc ctggagggcg tggccgtgat ctgcaccggc 2160
ggcggctccc cctcctacgc cgactccgtg aagggcaggt tcaccatctc cagggacaac 2220
gccaagaaca ccctgtacct gcagatgaac tccctgaggg ccgaggacac cgccgtgtac 2280
tactgcgccg ccgacctgct gagggccggc acccccctgt cctcctacga gttcaactac 2340
tggggccagg gcaccctggt gaccgtgtcc tcc 2373

Claims (14)

1. A bispecific antibody is characterized in that the bispecific antibody comprises an anti-PD-L1 nanobody and an anti-4-1 BB nanobody,
wherein the CDR of the complementarity determining region of the anti-4-1 BB nanobody comprises:
CDR1 shown in SEQ ID No. 14, CDR2 shown in SEQ ID No. 15, and CDR3 shown in SEQ ID No. 16;
and, the bispecific antibody has a structure represented by formula I from N-terminus to C-terminus:
P-L1-P-L2-Fc-L3-B-L4-B formula I
Wherein the content of the first and second substances,
"-" is a peptide bond;
l1, L2, L3, and L4 are each independently a no or a linker element;
p is anti-PD-L1 nano antibody,
b is an anti-4-1 BB nanobody, and
fc is the Fc segment of the antibody.
2. The bispecific antibody of claim 1, wherein the complementarity determining region CDRs of the anti-PD-L1 nanobody comprise:
CDR1 shown in SEQ ID NO. 6, CDR2 shown in SEQ ID NO.5 and CDR3 shown in SEQ ID NO. 7.
3. The bispecific antibody of claim 1, wherein the sequence of the linker element is (4GS) n, wherein n is 1, 2, 3, 4, 5 or 6.
4. A bispecific fusion protein characterized in that said bispecific fusion protein is a dimer formed by two bispecific antibodies of claim 1.
5. A polynucleotide encoding the bispecific antibody of claim 1 or the bispecific fusion protein of claim 4.
6. An expression vector comprising the polynucleotide of claim 5.
7. A host cell comprising the expression vector of claim 6, or having the polynucleotide of claim 5 integrated into its genome;
alternatively, the host cell expresses the bispecific antibody of claim 1.
8. A method of making a bispecific antibody comprising the steps of:
(a) culturing the host cell of claim 7 under suitable conditions, thereby obtaining a culture comprising the bispecific antibody; and
(b) purifying and/or isolating the culture obtained in step (a) to obtain said bispecific antibody.
9. An immunoconjugate, comprising:
(a) the bispecific antibody of claim 1 or the bispecific fusion protein of claim 4; and
(b) a coupling moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a nanomagnetic particle, a viral coat protein or V L P, or a combination thereof.
10. The immunoconjugate of claim 9, wherein the detectable label comprises a radionuclide, a gold nanoparticle/nanorod.
11. The bispecific antibody of claim 1 or the bispecific fusion protein of claim 4, for the preparation of (a) a reagent or a kit for the detection of PD-L1 and/or 4-1BB molecules, and (b) a medicament for the treatment of tumors.
12. A pharmaceutical composition, comprising: (i) the bispecific antibody of claim 1, the bispecific fusion protein of claim 4, or the immunoconjugate of claim 9; and (ii) a pharmaceutically acceptable carrier.
13. A recombinant protein, said recombinant protein having: (i) the bispecific antibody of claim 1, the bispecific fusion protein of claim 4; and (ii) optionally a tag sequence to facilitate expression and/or purification.
14. A PD-L1 protein and/or 4-1BB protein detection reagent, wherein the detection reagent comprises (i) the bispecific antibody of claim 1, the bispecific fusion protein of claim 4, or the immunoconjugate of claim 9, and (ii) a detectably acceptable carrier.
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CN116003601A (en) * 2019-12-05 2023-04-25 启愈生物技术(上海)有限公司 anti-PD-L1 nano antibody and application thereof
US20230203167A1 (en) * 2020-03-31 2023-06-29 Biotheus Inc. Anti-pd-l1 and pd-l2 antibody and derivatives and use thereof
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WO2022002038A1 (en) * 2020-06-30 2022-01-06 和铂医药(上海)有限责任公司 Multispecific binding protein of immune cell engager, preparation therefor and application thereof
CN114106190A (en) * 2020-08-31 2022-03-01 普米斯生物技术(珠海)有限公司 anti-VEGF/PD-L1 bispecific antibody and application thereof
CN114195900B (en) * 2020-09-17 2024-02-23 普米斯生物技术(珠海)有限公司 Anti-4-1 BB/PD-L1 bispecific antibody and application thereof
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CN114437228B (en) * 2020-10-30 2024-02-06 中国科学院生物物理研究所 Double-function fusion protein composed of IL-2 and antibody subunit
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