WO2024022516A1 - Anticorps à domaine unique humanisé anti-cd28 - Google Patents

Anticorps à domaine unique humanisé anti-cd28 Download PDF

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WO2024022516A1
WO2024022516A1 PCT/CN2023/109962 CN2023109962W WO2024022516A1 WO 2024022516 A1 WO2024022516 A1 WO 2024022516A1 CN 2023109962 W CN2023109962 W CN 2023109962W WO 2024022516 A1 WO2024022516 A1 WO 2024022516A1
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antibody
seq
targeting
single domain
antibodies
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PCT/CN2023/109962
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Chinese (zh)
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刘登念
何雨轩
周晓凤
曹旭
徐树涛
李婉丽
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四川思柏沃生物技术有限公司
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Publication of WO2024022516A1 publication Critical patent/WO2024022516A1/fr

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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2815—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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Definitions

  • the invention belongs to the technical field of biomedicine. Specifically, the present invention relates to an anti-CD28 humanized single domain antibody.
  • immune checkpoint inhibitors including anti-PD-1 (eg, Keytruda, Merck; Opdivo, BMS), anti-CTLA-4 (eg, Yervoy, BMS), and anti-PD-L1 (eg, Tecentriq, Roche), cancer immunotherapy is increasingly becoming the standard of care for malignancies, producing significant and durable responses in types of cancer such as melanoma, non-small cell lung cancer and renal cell carcinoma.
  • Immune checkpoint inhibitors are now becoming the standard of care for many types of cancer or as the basis of combination therapies, however, only a subset of patients ( ⁇ 25%) benefit from such therapies.
  • CD28 is a costimulatory molecule expressed on the surface of T lymphocytes, and anti-CD28 antibodies have been proposed by researchers for therapeutic purposes involving activation of T cells.
  • CD28 agonistic antibodies can be divided into two categories: (i) CD28 super-agonistic antibodies (CD28SA) and (ii) CD28 conventional agonistic antibodies.
  • CD28 super-agonistic antibodies and CD28 conventional agonistic antibodies still have many shortcomings.
  • CD28 super-agonistic antibodies can easily cause life-threatening cytokine storms.
  • CD28 conventional agonistic antibodies (such as clone 9.3) are used to participate in the construction. Bispecific single-chain antibodies are also prone to produce "super-agonistic" activity.
  • CD28 non-agonistic antibody to avoid the "super-agonistic" activity of CD28 antibodies and facilitate the development of CD28 single domain antibodies and bi/multi-specific antibodies based on CD28 single domain antibodies.
  • the purpose of the present invention is to provide an anti-CD28 single domain antibody and its humanized antibody.
  • a single domain antibody targeting CD28 is provided, and the complementarity determining region CDR of the VHH chain of the single domain antibody is one or more selected from the group below:
  • any one of the above amino acid sequences also includes a derivative sequence optionally added, deleted, modified and/or substituted at least one amino acid, and capable of retaining CD28 binding affinity.
  • the CDR region of the single domain antibody VHH chain contains at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% of any of the above sequences. Sequence similarity of amino acid sequences.
  • the amino acid sequence of the CDR region of the VHH chain of the single domain antibody contains one or more amino acid substitutions compared with any of the above sequences, preferably conservative amino acid substitutions.
  • the VHH chain includes CDR1, CDR2 and CDR3 selected from the following combinations:
  • the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • the VHH chain of the single domain antibody further includes a framework region (FR).
  • the framework region FR is of human, mouse, rabbit or camel origin.
  • the framework region FR includes a human-derived FR region, a mouse-derived or camel-derived FR region.
  • the VHH chain of the single domain antibody targeting CD28 has the amino acid sequence shown in SEQ ID NO.: 1, SEQ ID NO. 13, or SEQ ID NO. 25.
  • the VHH chain of the single domain antibody targeting CD28 has the amino acid sequence shown in SEQ ID NO.: 2, SEQ ID NO. 14, or SEQ ID NO. 26.
  • a second aspect of the present invention provides an antibody targeting CD28, said antibody comprising one or more VHH chains of the single domain antibody targeting CD28 as described in the first aspect of the present invention.
  • the VHH chain of the CD28-targeting single domain antibody has the following characteristics: SEQ ID NO.:1, SEQ ID NO.13, SEQ ID NO.25, SEQ ID NO.:2, SEQ ID NO. .14, or the amino acid sequence shown in SEQ ID NO.26.
  • the antibody is a monomer, a bivalent antibody, and/or a multivalent antibody.
  • the antibody is an animal-derived antibody, a humanized antibody, a chimeric antibody or a chimeric antigen receptor antibody (CAR).
  • CAR chimeric antigen receptor antibody
  • the CDR region of the humanized antibody contains 1, 2, or 3 amino acid changes.
  • the animal is a non-human mammal, preferably a rat, sheep, rabbit, or camel.
  • the antibody is a diabody or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • the number of added, deleted, modified and/or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, and more preferably 10%.
  • the number of added, deleted, modified and/or substituted amino acids is 1-7, preferably 1-3, more preferably 1.
  • the at least one amino acid sequence that has been added, deleted, modified and/or substituted has a homology of At least 80% of the amino acid sequence.
  • the derived sequence with addition, deletion, modification and/or substitution of at least one amino acid has the catalytic function of inhibiting cell surface CD28 or recombinant CD28 protease.
  • the antibody is in the form of a drug conjugate.
  • the antibody has one or more properties selected from the following group:
  • CD28 specifically binds to CD28, preferably binds to human and monkey CD28, and does not bind or weakly binds to rat CD28 or mouse CD28;
  • (c) is a CD28 non-agonistic antibody.
  • the "CD28 non-agonistic antibody” means that the antibody blocks the binding of CD28 to CD80 and does not substantially stimulate CD28 signaling.
  • the present invention provides a multispecific antibody.
  • the multispecific antibody includes: the CD28-targeting single domain antibody described in the first aspect of the present invention or the CD28-targeting antibody described in the second aspect of the present invention. Antibodies to CD28.
  • the multispecific antibody further includes a second antigen-binding region targeting a target selected from the following group: BCMA, CD73, GPC3, HER2, PMSA, 4-1BB, OX40, GLP-1, Trop2 , FGL1, LFA-3, 2B4, 5T4, ⁇ -4 integrin, ⁇ -V integrin, ⁇ 4 ⁇ 7 integrin, ⁇ 4 ⁇ 7 integrin, ⁇ -SMA, AGR2, Apelin J receptor, APRIL, B7-H3, B7- H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, carbonic anhydrase 9, CD2, CD3, CD6, CD9, CDlla, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81,
  • the second antigen-binding region is a single domain antibody.
  • the multispecific antibody includes one or more second antigen-binding regions.
  • the multispecific antibody further includes the Fc segment of the antibody.
  • the antigen-binding region is an antibody or an antibody fragment
  • the antibody fragment includes: (i) Fab fragment; (ii) F(ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single chain Fv (scFv) molecule; (vi) dAb fragment.
  • the multispecific antibody includes one or more single domain antibodies targeting CD28 according to the first aspect of the present invention.
  • the multispecific antibody further includes a second antigen-binding region targeting DLL3 or B7H3.
  • the multispecific antibody is a trispecific antibody, including one or more second antigen-binding regions.
  • the Fc segment includes a knob-in-hole structure; preferably, it also includes amino acid mutations of S354C and Y349C.
  • the Fc segment also includes amino acid mutations that can weaken effector function: L234A, L235A and G237A (according to Kabat's "EU” numbering) amino acid mutations.
  • the multispecific antibody is a bispecific antibody, including a first antigen-binding region targeting CD28 and a second antigen-binding region targeting tumor-associated antigen (TAA).
  • TAA tumor-associated antigen
  • the TAA is selected from the following group: DLL3, B7H3.
  • the first CD28-targeting antigen-binding region includes one or more CD28-targeting single domain antibodies according to the first aspect of the present invention.
  • the second antigen-binding region targeting tumor-associated antigen is an antibody or antibody fragment
  • the antibody fragment includes: (i) Fab fragment; (ii) F(ab') 2 Fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single chain Fv (scFv) molecule; (vi) dAb fragment; preferably Fab fragment.
  • the second antigen-binding region targeting tumor-associated antigen (TAA) targets DLL3.
  • the bispecific antibody has a heavy chain and a light chain selected from the following group:
  • the second antigen-binding region targeting tumor-associated antigen (TAA) targets B7H3.
  • the bispecific antibody has a heavy chain and a light chain selected from the following group:
  • the multispecific antibody is a trispecific antibody, including:
  • TAA tumor-associated antigen
  • the second antigen-binding region targeting tumor-associated antigen is an antibody or antibody fragment
  • the antibody fragment includes: (i) Fab fragment; (ii) F(ab') 2 Fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single chain Fv (scFv) molecule; (vi) dAb fragment; preferably scFv or Fab fragment.
  • the third antigen-binding region targeting CD3 is an anti-CD3 bispecific antibody.
  • the second antigen-binding region targeting tumor-associated antigen (TAA) targets DLL3.
  • the second antigen-binding region is a scFv targeting DLL3, and the preferred amino acid sequence is as shown in SEQ ID NO. 44.
  • the trispecific antibody has a heavy chain and a light chain selected from the following group:
  • the present invention provides a recombinant protein, which has:
  • improving the physicochemical properties or druggability of the protein includes extending the half-life of a single domain antibody targeting CD28.
  • the recombinant protein further includes: (iv) an optional tag sequence to assist expression and/or purification.
  • the tag sequence is selected from the following group: 6His tag, GGGS sequence, and FLAG tag.
  • the recombinant protein is a monomer, dimer, or multimer.
  • the polypeptide molecules or fragments with therapeutic functions include but are not limited to: targeting BCMA, CD73, GPC3, HER2, PMSA, 4-1BB, OX40, GLP-1, Trop2, FGL1, LFA- 3. 2B4, 5T4, ⁇ -4 integrin, ⁇ -V integrin, ⁇ 4 ⁇ 7 integrin, ⁇ 4 ⁇ 7 integrin, ⁇ -SMA, AGR2, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA , C5 complement, C-242, CA9, CA19-9, carbonic anhydrase 9, CD2, CD3, CD6, CD9, CDlla, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L , CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95,
  • polypeptide molecules or fragments with therapeutic functions include but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, cells factor.
  • the recombinant protein includes a fusion protein.
  • the fusion protein includes multispecific antibodies and chimeric antibodies.
  • the functional domain that improves protein physicochemical properties or druggability includes Fc segment and human serum albumin (HSA).
  • the fusion protein has the following elements from the N-C terminus:
  • the A element is a single domain antibody targeting CD28;
  • the B element is the Fc segment or human serum albumin (HSA);
  • the VHH chain of the CD28-targeting single domain antibody has the following characteristics: SEQ ID NO.:1, SEQ ID NO.13, SEQ ID NO.25, SEQ ID NO.:2, SEQ ID NO. .14, or the amino acid sequence shown in SEQ ID NO.26.
  • the Fc segment is a human IgG Fc segment.
  • amino acid sequence of the Fc segment is shown in SEQ ID NO. 37.
  • the fifth aspect of the present invention provides a CAR construct, the antigen-binding region of the CAR construct being the VHH chain of the single domain antibody as described in the first aspect of the present invention.
  • a sixth aspect of the present invention provides a recombinant immune cell expressing an exogenous CAR construct as described in the fifth aspect of the present invention.
  • the immune cells are selected from NK cells or T cells.
  • the immune cells are from humans or non-human mammals (such as mice).
  • the seventh aspect of the present invention provides an immunoconjugate, the immunoconjugate containing:
  • a coupling moiety coupled to the single domain antibody moiety being selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.
  • the immunoconjugate is a single domain antibody drug conjugate.
  • the single domain antibody part and the coupling part are coupled through chemical bonds or linkers.
  • the coupling moiety is a chemical label or a biological label.
  • the chemical label is an isotope, an immunotoxin and/or a chemical drug.
  • the biomarker is biotin, avidin or enzyme label.
  • the coupling moiety is a drug or toxin.
  • the drug is a cytotoxic drug.
  • the cytotoxic drug is selected from the following group: anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolite drugs, chemotherapy Sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.
  • cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecins (camptothecins), docarmycins/duocarmycins, etoposides, maytansines and maytansinoids (such as DM1 and DM4), taxanes ( taxanes), benzodiazepines or benzodiazepine containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines (indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • auristatins camptothecins (camptothecins), docarmycins/duocarmycins, etoposides, maytansines and maytansinoids (such as DM1 and DM4), taxanes (
  • the toxin is selected from the following group:
  • Aristatins e.g., aristatin E, aristatin F, MMAE, and MMAF
  • chlortetracycline maytansetol, ricin, ricin A-chain, combretastatin, docarmicin, poly Lastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, autumn Narcissus, dihydroxyanthracindione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, caprytoxin A chain, ⁇ - Sarcina, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotonin, calicheamicin,
  • the coupling moiety is a detectable label.
  • the detectable label includes a radionuclide
  • the radionuclide includes:
  • Diagnostic isotope the said diagnostic isotope is selected from the following group: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or
  • Therapeutic isotope the therapeutic isotope is selected from the following group: Lu-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-133Yb-169, Yb-177, or combination thereof.
  • the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized X-ray tomography) contrast agents, or can produce detectable Product enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold single domain particles/single domain rods, virus particles, liposomes, single domain magnetic particles, prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin), or any form of single-domain particles, etc.
  • fluorescent or luminescent markers e.g., radioactive markers, MRI (magnetic resonance imaging) or CT (computerized X-ray tomography) contrast agents, or can produce detectable Product enzymes, radionuclides, biotoxins, cytokines (such as IL-2
  • the immunoconjugate contains: a multivalent (such as bivalent) CD28-targeting single domain antibody as described in the first aspect of the present invention or a target as described in the second aspect of the present invention.
  • a multivalent (such as bivalent) CD28-targeting single domain antibody as described in the first aspect of the present invention or a target as described in the second aspect of the present invention.
  • Antibodies to CD28 are described in the first aspect of the present invention or a target as described in the second aspect of the present invention.
  • the multivalent means that the amino acid sequence of the immunoconjugate contains multiple repeats of the CD28-targeting single domain antibody as described in the first aspect of the present invention or the second CD28-targeting single domain antibody of the present invention.
  • the CD28-targeting antibody described in the second aspect is the same or the same.
  • the detection is in vivo detection or in vitro detection.
  • the immunoconjugate is used for diagnosis and/or treatment of tumors expressing CD28 protein.
  • the immunoconjugate has the following molecular formula:
  • nAb is a single domain antibody targeting CD28, an antibody targeting CD28, or a multispecific antibody
  • LU is a connector (also called a linker);
  • LU is selected from maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl) ring Hexane-1-carboxylate (SMCC) linker is attached to the antibody moiety and contains valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine - One or more linkers of glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), and polyethylene glycol (PEG).
  • MC maleimidocaproyl
  • MAL maleimide
  • SCC succinimidyl 4-(N-maleimidomethyl) ring Hexane-1-carboxylate
  • VC valine-citrulline
  • VA valine-alanine
  • GGFG glycine-glycine-phenylalanine
  • PAB p-aminobenz
  • the antibody moiety is covalently bound to the linker by reaction with a moiety selected from the group consisting of: maleimidocaproyl (MC), maleimide (MAL), Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC), etc.
  • MC maleimidocaproyl
  • MAL maleimide
  • SMCC Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • D is selected from the following group of compounds with anti-tumor activity:
  • Tubulin inhibitors such as maytansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF);
  • Topoisomerase inhibitors such as camptothecin, SN38, ixotecan, and Dxd.
  • the present invention provides a pharmaceutical composition, said pharmaceutical composition comprising:
  • a single domain antibody targeting CD28 as described in the first aspect of the present invention, an antibody targeting CD28 as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, as described in the third aspect of the present invention The recombinant protein according to the fourth aspect, the recombinant immune cell according to the sixth aspect of the present invention, or the immunoconjugate according to the seventh aspect of the present invention;
  • the pharmaceutical composition includes single drugs, compound drugs, or synergistic drugs.
  • the pharmaceutical composition also contains other biologically active substances, such as drugs for treating tumors.
  • the administration method of the pharmaceutical composition is selected from the following group: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal spray and mist Inhaled.
  • the dosage form of the pharmaceutical composition is selected from the following group: liquid, solid, or gel.
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition further includes an antibody or antibody fragment targeting CD3.
  • the antibody fragments include: (i) Fab fragment; (ii) F(ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single chain Fv (scFv ) molecule; (vi) dAb fragment.
  • the antibody is a monomer, a bivalent antibody, and/or a multivalent antibody.
  • the antibody is a bispecific or multispecific antibody.
  • a ninth aspect of the present invention provides the use of an active ingredient selected from the group consisting of: a single domain antibody targeting CD28 as described in the first aspect of the present invention, as described in the second aspect of the present invention Antibodies targeting CD28, multispecific antibodies as described in the third aspect of the present invention, recombinant proteins as described in the fourth aspect of the present invention, recombinant immune cells as described in the sixth aspect of the present invention, as described in the seventh aspect of the present invention
  • the immunoconjugate described in the aspect, or a combination thereof, the active ingredient is used for (a) preparing detection reagents, detection plates or kits; and/or (b) preparing drugs for preventing and/or treating CD28-related diseases.
  • the detection reagent, detection plate or kit is used for:
  • the detection type includes but is not limited to flow cytometry detection, cell immunofluorescence detection, enzyme-linked immunosorbent detection, western blot detection, etc.
  • the detection reagent, detection plate or kit is used to diagnose CD28-related diseases.
  • the drug is used to treat or prevent tumors with high CD28 expression, tumor migration, or tumor drug resistance.
  • the tumor resistance includes: resistance to tumor immunotherapy drugs, resistance to tumor targeted therapy drugs, resistance to conventional tumor chemotherapy, and insensitivity to radiotherapy.
  • the drug is used for a purpose selected from the following group:
  • CD28 specifically binds to CD28, preferably binds to human and monkey CD28, and does not bind or weakly binds to rat CD28 or mouse CD28;
  • the CD28-related disease is selected from the group consisting of cancer, autoimmune diseases, metabolism-related diseases, or combinations thereof.
  • the CD28-related diseases include: tumor occurrence, growth and/or metastasis.
  • the cancer includes solid tumors and blood cancers.
  • the metabolism-related diseases include: diabetes, food-borne obesity and fat inflammation.
  • the cancer is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer, glioma, melanoma, leukemia, lymphoma, or a combination thereof.
  • the autoimmune diseases include (but are not limited to): systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis, ankylosing Spondylitis, asthma, atherosclerosis, colitis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Reiter syndrome, psoriatic arthropathy, infectious arthritis, tuberculous arthritis, viruses Arthritis, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis.
  • the metabolism-related diseases include (but are not limited to): diabetes, food-borne obesity and fat inflammation.
  • the present invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:
  • the polynucleotide includes RNA, DNA or cDNA.
  • the eleventh aspect of the present invention provides a vector, said vector containing the polynucleotide according to the tenth aspect of the present invention.
  • the vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • a twelfth aspect of the present invention provides a host cell containing the vector of the eleventh aspect of the present invention or the polynucleotide of the tenth aspect of the present invention integrated into the genome.
  • a thirteenth aspect of the present invention provides a method for in vitro detection of CD28 in samples (including diagnostic or non-diagnostic), the method comprising the steps:
  • the sample is mixed with the CD28-targeting single domain antibody described in the first aspect of the present invention, the CD28-targeted antibody described in the second aspect of the present invention, or the seventh aspect of the present invention.
  • Immunoconjugate exposure ;
  • the detection includes diagnostic or non-diagnostic.
  • a fourteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:
  • a fifteenth aspect of the present invention provides a method for treating CD28-related diseases, the method comprising: administering to a subject in need a CD28-targeting single domain antibody as described in the first aspect of the present invention, or as described in the second aspect of the present invention.
  • the method further includes: administering other drugs or treatment methods to the subject in need for combined treatment.
  • the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.
  • the anti-tumor immunotherapy drugs include PD-1 and PD-L1 monoclonal antibodies.
  • a sixteenth aspect of the present invention provides a combination of active ingredients.
  • the combination of active ingredients includes
  • First active ingredient a single domain antibody targeting CD28 as described in the first aspect of the present invention, an antibody targeting CD28 as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, such as The recombinant protein according to the fourth aspect of the present invention.
  • the second active ingredient an antibody or antibody fragment targeting CD3; preferably an anti-CD3 bispecific antibody or multispecific antibody.
  • the active ingredient combination includes: a multispecific antibody as described in the multispecific antibody of the third aspect of the present invention; and an anti-CD3 bispecific antibody.
  • the combination of active ingredients is used to treat CD28-related diseases; preferably tumors or cancers, autoimmune diseases, metabolism-related diseases, or combinations thereof.
  • the anti-CD3 bispecific antibody or multispecific antibody is a T cell-activated anti-CD3 bispecific antibody or multispecific antibody that is specific for a tumor-associated antigen.
  • the anti-CD3 bispecific antibodies include but are not limited to: CD20/CD3 bispecific antibodies, DLL3/CD3 bispecific antibodies, anti-CEA/anti-CD3 bispecific antibodies, or anti-MCSP /anti-CD3 bispecific antibody.
  • the present invention provides a medicine box, which includes:
  • the eighteenth aspect of the present invention provides an in vitro non-therapeutic method for synergistically inhibiting the growth of tumor cells, including the steps of: adding a combination of active ingredients as described in the sixteenth aspect of the present invention to a tumor cell culture system, thereby synergistically inhibiting Tumor cells grow.
  • Figure 1A and Figure 1B show the affinity detection of anti-CD28 recombinant single domain antibody and human CD28 protein.
  • Figure 2A and Figure 2B show the affinity detection of anti-CD28 recombinant single domain antibody with CHO-human CD28 cells.
  • Figure 3 shows the detection of the ability of anti-CD28 recombinant single domain antibodies to block the binding of CD28 to CD80.
  • Figure 4A shows the affinity detection of anti-CD28 recombinant single domain antibody and human peripheral blood mononuclear cells.
  • Figure 4B shows the affinity detection of anti-CD28 recombinant single domain antibody and monkey peripheral blood mononuclear cells.
  • Figure 5 shows anti-CD28 recombinant single domain antibody fluorescent reporter cell functional activity assay.
  • Figure 6A shows the affinity detection of anti-CD28 recombinant single domain antibody and rat CD28 protein.
  • Figure 6B shows the affinity detection of anti-CD28 recombinant single domain antibody and mouse CD28 protein.
  • Figure 7 shows the affinity detection of anti-CD28 humanized single domain antibody and human CD28 protein.
  • FIGS 8A and 8B show the affinity detection of anti-CD28 humanized single domain antibodies with Jurkat cells.
  • Figure 9A and Figure 9B show the affinity detection of anti-CD28 humanized single domain antibody and human peripheral blood mononuclear cells.
  • Figure 10 shows the detection of the ability of anti-CD28 humanized single domain antibodies to block the binding of CD28 to CD80.
  • FIG 11 shows a schematic diagram of example bispecific and trispecific antibody structures.
  • TAA is a tumor-associated antigen.
  • Figure 12-1 shows the combined use of DLL3/CD28 bispecific antibody and DLL3/CD3 bispecific antibody to detect SHP77 cell activity.
  • Figure 12-2 shows the combined use of DLL3/CD3/CD28 trispecific antibody and DLL3/CD3 bispecific antibody to detect SHP77 cell activity.
  • Figure 12-3 shows the combined use of B7H3/CD28 bispecific antibody and DLL3/CD3 bispecific antibody to detect NCI-H716 cell activity.
  • Figure 13-1 shows the detection of target cell SHP77 cell killing activity by the combination of DLL3/CD28 bispecific antibody and DLL3/CD3 bispecific antibody.
  • Figure 13-2 shows the detection of non-target cell NCI-H358 cell killing activity using DLL3/CD28 bispecific antibody and DLL3/CD3 bispecific antibody.
  • Figure 14-1 shows that the combination of DLL3/CD28 bispecific antibody and DLL3/CD3 bispecific antibody inhibits tumor growth in mice.
  • Figure 14-1 shows the effect of the combination of DLL3/CD28 bispecific antibody and DLL3/CD3 bispecific antibody on the body weight of mice with transplanted tumors.
  • Figure 15-1 shows that the DLL3/CD3/CD28 trispecific antibody inhibits tumor growth in mice.
  • Figure 15-2 shows the effect of DLL3/CD3/CD28 trispecific antibodies on the body weight of mice with transplanted tumors.
  • the inventor accidentally developed a CD28 non-agonistic blocking single domain antibody and its humanized antibody for the first time.
  • the antibody has similar affinity to traditional CD28 non-agonistic antibodies. , has no activating effect on T cells under the condition of monoclonal antibody, ensuring the safety of this antibody in future use.
  • the performance for example, affinity to human peripheral blood mononuclear cells
  • the single domain antibody of the present invention is further improved after humanization.
  • the inventor unexpectedly discovered that the CD28 antibody of the present invention in the form of a bispecific antibody combined with a CD3 bispecific antibody can significantly enhance the activity of the CD3 bispecific antibody. On this basis, the present invention was completed.
  • Single domain antibodies include antibodies that block the binding of CD28 to costimulatory molecules (eg, costimulatory molecules CD80 and/or CD86) and do not activate signaling by CD28.
  • costimulatory molecules eg, costimulatory molecules CD80 and/or CD86
  • Particularly preferred are single domain antibodies whose amino acid sequences of the VHH chain are shown in SEQ ID NO.: 1, 2, 13, 14, 25, and 26.
  • antibody or "immunoglobulin” is a heterotetrameric protein of approximately 150,000 daltons with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains.
  • VH variable domain
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. .
  • Special amino acid residues form the interface between the variable regions of the light and heavy chains.
  • single domain antibody VHH
  • nanobody the terms “single domain antibody (VHH)” and “nanobody” have the same meaning and refer to the variable region of the heavy chain of a monoclonal antibody, constructing a single domain consisting of only one heavy chain variable region Antibody (VHH), which is the smallest fully functional antigen-binding fragment.
  • VHH single domain antibody
  • CH1 light chain and heavy chain constant region 1
  • the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.
  • variable means that certain portions of the variable regions of an antibody differ in sequence and contribute to the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments in the variable regions of the light and heavy chains called complementarity determining regions (CDRs) or hypervariable regions. The more conserved part of the variable region is called the framework region (FR).
  • CDRs complementarity determining regions
  • FR framework region
  • the variable regions of natural heavy and light chains each contain four FR regions, which are generally in a ⁇ -sheet configuration and are connected by three CDRs forming a connecting loop. In some cases, a partial ⁇ -sheet structure can be formed.
  • the CDRs in each chain are held closely together by the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pp. 647-669 (1991)). Constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules formed by combining with the antibodies of the present invention or fragments thereof of conjugates.
  • the present invention also includes cell surface markers or antigens that bind to the anti-CD28 protein antibody or fragment thereof.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.
  • antibody of the invention protein of the invention
  • polypeptide of the invention are used interchangeably, and all refer to polypeptides that specifically bind to CD28 protein, such as proteins or polypeptides with heavy chain variable regions. . They may or may not contain starting methionine.
  • the invention also provides other proteins or fusion expression products with the antibodies of the invention.
  • the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to the heavy chain of the antibody of the invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are separated into four framework regions (FRs), and the amino acid sequences of the four FRs. Relatively conservative and not directly involved in binding reactions. These CDRs form a cyclic structure, and the ⁇ -sheets formed by the FRs between them are close to each other in spatial structure.
  • the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody. You can determine which amino acids constitute the FR or CDR region by comparing the amino acid sequences of antibodies of the same type.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least part of them is involved in binding the antigen. Accordingly, the present invention includes those molecules having antibody heavy chain variable regions with CDRs as long as the CDRs are more than 90% (preferably more than 95%, optimally more than 98%) homologous to the CDRs identified herein sex.
  • CD28 is a costimulatory molecule expressed on the surface of T lymphocytes.
  • CD28 is a type I transmembrane protein. The protein mainly exists on the cell surface in the form of homodimers, and the extracellular part contains an Ig-V-like domain.
  • CD80 (B7.1) protein and CD86 (B7.2) protein are the two main ligands of CD28.
  • CD28 is mainly activated by CD80 or CD86 expressed on antigen-presenting cells (APC). Binding of CD28 to CD80 or CD86 provides a costimulatory signal important for T cell activation and survival.
  • T cell stimulation through CD28 also provides a potent signal for the production of various interleukins.
  • CD28 also enhances cellular signaling following TCR activation, such as pathways controlled by the NF ⁇ B transcription factor.
  • CD28 co-signaling is important for efficient T cell activation such as T cell differentiation, proliferation, cytokine release and cell death.
  • CD28 agonistic antibodies can be divided into two categories: (i) CD28 super-agonistic antibodies (CD28SA) and (ii) CD28 conventional agonistic antibodies.
  • CD28 superagonists are CD28-specific monoclonal antibodies that can autonomously activate T cells without obvious T cell receptor involvement.
  • CD28SA antibodies have therapeutic efficacy in multiple autoimmune, inflammatory and transplant models.
  • TGN1412 anti-CD28 superagonist
  • TGN1412 anti-CD28 superagonist
  • CD28 conventional agonistic antibodies (such as clone 9.3) mimic the CD28 natural ligand and enhance T cell activation only in the presence of a T cell receptor signal (Signal 1).
  • a T cell receptor signal (Signal 1).
  • the superagonist TGN1412 binds to the side motifs of CD28, whereas the conventional agonist molecule 9.3 binds tightly to the ligand-binding epitope. Due to different binding epitopes, super-agonistic antibodies and conventional agonistic antibodies also have different abilities to form linear complexes of CD28 molecules on the surface of T cells.
  • TGN1412 is able to efficiently form linear arrays of CD28, which presumably results in aggregated signaling components sufficient to exceed the T cell activation threshold.
  • the conventional agonist 9.3 resulted in a complex whose structure was not linear.
  • Attempts to convert conventional agonistic binders based on the 9.3 clone using recombinant bispecific single chain antibodies against melanoma-associated proteoglycans and CD28 have been previously published. Based on the inherent tendency of bispecific single chain antibodies to form multimeric constructs, the reported bispecific single chain antibodies were reported to exert "super-agonistic" activity despite the use of the conventional CD28 agonistic binder 9.3.
  • CD28 activity is activity involving, or resulting from, CD80, CD86, and/or another ligand binding to CD28, and includes, but is not limited to, activation of CD28-mediated cellular signaling.
  • CD28 activity also includes inducing T cell proliferation and inducing T cells to secrete cytokines, such as interleukin 2 (IL-2).
  • IL-2 interleukin 2
  • CD28-mediated cell signaling includes one or more cellular events induced directly or indirectly in immune cells through ligand binding that activates (eg, cross-links) cell surface CD28 to express CD28 on their surface. Under appropriate circumstances, CD28-mediated signaling leads to the upregulation of immune responses by immune cells. Blocking CD28-mediated signaling leads to downregulation of immune cell immune responses. Agents that bind to CD28 to effectively block CD28-mediated signaling (e.g., by blocking ligand binding) without themselves activating the CD28 receptor (e.g., by aggregation of the receptor) will effectively block CD28-mediated Signaling.
  • the agent specifically blocks CD28-mediated signaling, ie, blocks the signal transmitted by CD28 without blocking the signal transmitted by another cell surface molecule, such as CTLA4.
  • the reagent includes the CD28 single domain antibody of the present invention or its humanized antibody, which has the ability to specifically recognize CD28 and block CD28 and CD80 ligands without binding to other immune checkpoint molecules of the same family of CD28 ( For example, the characteristics of PDL1, PDL2, CTLA4, B7-1, B7-2, B7H3, B7H4, B7H5, B7H6, B7H7).
  • the term "substantially not agonistic” means that a given agent, e.g., an anti-CD28 single domain antibody, has substantially one or more CD28-mediated activities, wherein the term “activating” is as defined herein.
  • an agent that is “substantially non-agonistic” means that the agent does not activate more than 20% of the activity activated by CD80 and/or CD86 binding to CD28, and in one aspect, the agent does not activate more than about 10% , 8%, 5%, 3% or 2% or less activity activated by CD80 and/or CD86 binding to CD28, including zero activation (no activation).
  • single domain antibodies described herein that do not substantially activate CD28 activity stimulate no more CD28 activity than that produced by anti-CD28 mAb 9.3 (Gibson, et al. (1996) JBC, 271:7079-7083). 5% of the activity obtained by agonizing CD28 activity under otherwise identical assay conditions.
  • activation and “activation” refer to an increase in a given measurable activity of at least 5% relative to a reference, such as at least 10%, 25%, 50%, 75%, or even 100% or more.
  • the term “inhibition” refers to a reduction of a given measurable activity (eg, binding activity) by at least 10% relative to a reference. When inhibition is desired, such inhibition is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, up to and including 100%, i.e., of a given activity Complete inhibition or loss. Inhibition of CD28 binding to CD80 or CD86 can be measured as described in the working examples herein. As used herein, “inhibiting binding” when referring to a domain antibody binding to CD28, or CD80 binding to CD28, or CD86 binding to CD28, means that binding is reduced by at least 10% relative to the reference. “Inhibiting binding” means reducing binding by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, up to and including 100%.
  • Chothia refers to the complementary binding determining region CDRs determined under the different assignment systems described above.
  • assignment systems include, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins” ”, Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International Immuno GeneTics database (IMGT), and Chothia definition based on loop structure position.
  • any one of the above amino acid sequences also includes a derivative sequence optionally added, deleted, modified and/or substituted at least one amino acid, and capable of retaining the binding affinity to CD28.
  • the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, and more preferably at least 90%. %, optimally at least 95% of the amino acid sequence.
  • the antibody has the function of inhibiting cell surface and recombinant CD73 protease catalysis, and the antibody can be quickly endocytosed by cells and enter lysosomes.
  • the antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, human-animal chimeric antibodies, preferably humanized antibodies.
  • the antibody derivatives of the present invention can be single-chain antibodies and/or antibody fragments, such as: Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as IgA, IgD, IgE , IgG and IgM antibodies or any one or more of other subtypes of antibodies.
  • the animal is preferably a mammal, such as a mouse or a camel.
  • the present invention discloses a variety of highly specific and high-affinity Nanobodies targeting CD28, which only include heavy chains containing heavy chain variable region (VHH) amino acid sequences, as shown in Table 11 listed.
  • VHH heavy chain variable region
  • Recombinant protein or fusion protein
  • the present invention also includes recombinant proteins (or fusion proteins) containing the CD73 single domain antibody of the present invention.
  • a preferred blend The fusion protein is a multispecific antibody that further includes a second antigen-binding region targeting a target selected from the group consisting of: BCMA, CD73, GPC3, HER2, PMSA, 4-1BB, OX40, GLP- 1.
  • the multispecific antibody includes one or more second antigen-binding regions, or further includes a third antigen-binding region.
  • the recombinant protein (or fusion protein) of the present invention in addition to containing the CD28 single domain antibody of the present invention, also includes optional tag sequences to assist expression and/or purification (such as 6His tag, GGGS sequence, FLAG tag); or Including optional polypeptide molecules or fragments with therapeutic functions; or optional protein functional domains that assist in physicochemical or pharmaceutical medicine (for example, molecules that can extend the half-life of single domain antibodies in vivo, such as HLE and HSA).
  • tag sequences to assist expression and/or purification such as 6His tag, GGGS sequence, FLAG tag
  • optional polypeptide molecules or fragments with therapeutic functions or optional protein functional domains that assist in physicochemical or pharmaceutical medicine (for example, molecules that can extend the half-life of single domain antibodies in vivo, such as HLE and HSA).
  • molecules that extend half-life in organisms may be selected from the following group:
  • Proteins from the extracellular matrix such as collagen, laminin, integrins, and fibronectin.
  • Collagen is the main protein of the extracellular matrix.
  • type I collagen (representing 90% of the collagen in the body) found in bones, skin, tendons, ligaments, cornea, internal organs white), or type II collagen found in cartilage, intervertebral discs, notochord, and vitreous fluid of the eye;
  • Proteins from blood including plasma proteins such as fibrin, alpha-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen B, serum amyloid A, heptagolbin, actin arrestin, ubiquitin, uteroglobulin, and beta-2 microglobulin;
  • Enzymes and inhibitors such as plasminogen, lysozyme, cystatin C, alpha-1-antitrypsin, and pancreatic trypsin inhibitors.
  • Plasminogen is the inactive precursor of the trypsin-like serine protease plasmin. It is usually found circulating through the bloodstream. When plasminogen becomes activated and converted to plasmin, it opens the folds of the powerful enzymatically active domain and dissolves the fibrin fibrils that entangle blood cells in the blood clot. This is called fibrinolysis;
  • Immune system proteins such as IgE, IgG and IgM.
  • the present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of the antibodies of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative 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 substituent groups in one or more amino acid residues, or (iii) a mature polypeptide combined with another compound (such as a compound that extends the half-life of the polypeptide, e.g.
  • polyethylene glycol or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a protein sequence, or with Fusion protein formed by 6His tag).
  • additional amino acid sequence such as a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a protein sequence, or with Fusion protein formed by 6His tag.
  • the antibody of the present invention refers to a polypeptide that has CD28 protein-binding activity and includes the above-mentioned CDR region.
  • the term also includes variant forms of polypeptides containing the above-described CDR regions that have the same function as the antibodies of the invention. These variant forms include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids with similar or similar properties generally do not alter the function of the protein. As another example, adding one or more amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA that can hybridize with the DNA encoding the antibody of the invention under high or low stringency conditions.
  • the invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof.
  • the invention also encompasses fragments of the single domain antibodies of the invention.
  • the fragment has at least about 50 contiguous amino acids of an 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.
  • “conservative variants of the antibody of the present invention” means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, optimally at most 3 Amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotides of the invention may be in DNA form or RNA form.
  • Forms of DNA include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • Polynucleotides encoding mature polypeptides of the present invention include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optional additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide may include polynucleotides encoding such polypeptides, or may also include polynucleotides that also include additional coding and/or non-coding sequences.
  • the invention also relates to polynucleotides that hybridize to the sequences described above and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides of the invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90%, more It is best when hybridization occurs only when the ratio is above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombinant or artificial synthesis.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained using conventional techniques, such as PCR amplification or genome library screening.
  • the coding sequences of the light and heavy chains can also be fused together to form single-chain antibodies.
  • recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.
  • artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.
  • the DNA sequence encoding the antibody (or fragment thereof, or derivative thereof) of the present invention can be obtained entirely through chemical synthesis.
  • the DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the invention through chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences as described above and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.
  • the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other techniques in the art.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other techniques in the art.
  • the antibody of the present invention can be purified by conventional separation and purification means well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the antibody of the present invention can be expressed within the cell, on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, infiltration sterilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the invention also provides an immunoconjugate (ADC) based on the antibody of the invention, preferably a single domain antibody drug conjugate (nanobody-drug conjugate, NDC).
  • ADC immunoconjugate
  • NDC single domain antibody drug conjugate
  • the antibody-conjugated drug includes the antibody and an effector molecule, and the antibody is coupled to the effector molecule, and preferably is chemically coupled.
  • the effector molecule is preferably a drug with therapeutic activity.
  • the effector molecule may be one or more of toxic proteins, chemotherapeutic drugs, small molecule drugs or radionuclides.
  • the antibody of the present invention and the effector molecule can be coupled through a coupling agent.
  • the coupling agent may be any one or more of non-selective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds.
  • the non-selective coupling agent refers to a compound that allows the effector molecule and the antibody to form a covalent bond, such as glutaraldehyde, etc.
  • the coupling agent utilizing carboxyl groups may be any one or more of aconitic anhydride coupling agents (such as aconitic anhydride) and acyl hydrazone coupling agents (the coupling site is an acyl hydrazone).
  • antibodies are used to connect to a variety of functional groups, including imaging reagents (such as chromophores and fluorescent groups), diagnostic reagents (such as MRI contrast agents and radioisotopes) , stabilizers (such as ethylene glycol polymers) and therapeutic agents.
  • imaging reagents such as chromophores and fluorescent groups
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers such as ethylene glycol polymers
  • therapeutic agents such as ethylene glycol polymers
  • Antibodies can be coupled to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg drugs, detection reagents, stabilizers
  • the functional agent can be linked to the antibody directly or indirectly through a linker.
  • Single-domain antibodies can be conjugated with drugs to form antibody-drug conjugates (NDCs).
  • NDC antibody-drug conjugates
  • the NDC contains a linker between the drug and the antibody.
  • Linkers can be degradable or non-degradable linkers.
  • Degradable linkers are typically susceptible to degradation in the intracellular environment, such as at the target site, allowing the drug to be released from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases, such as lysosomal or endosomal proteases, or sugar linkers, such as those that can be degraded by glucuronides. Enzymatic degradation of glucuronide-containing linkers.
  • Peptidyl linkers may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
  • Other suitable degradable linkers include, for example, pH-sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers).
  • Nondegradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by proteases.
  • the linker Before being connected to the antibody, the linker has an active reactive group that can react with certain amino acid residues, and the connection is achieved through the active reactive group.
  • Thiol-specific reactive groups are preferred and include, for example, maleimides, halogenated amides (e.g., iodine, bromo, or chlorinated); halogenated esters (e.g., iodine, bromo, or chlorinated). ); Halogenated methyl ketones (e.g. iodine, bromo or chlorinated), benzyl halides (e.g.
  • Linkers may include, for example, maleimides linked to the antibody via thiosuccinimide.
  • the drug can be any cytotoxic, cytostatic, or immunosuppressive drug.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can form a bond with the linker.
  • the drug is directly attached to the linker, the drug has reactive groups before being attached to the antibody.
  • Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors , Catharanthus roseus alkaloids, etc.
  • particularly useful classes of cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecins (camptothecins), docarmycins/duocarmycins, etoposides, maytansines and maytansinoids (such as DM1 and DM4), taxanes ( taxanes), benzodiazepines or benzodiazepine containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines (indolinobenzodiazepines) and oxazolidinobenzodiazepines (oxazolidinobenzodiazepines) and vinca alkaloids.
  • auristatins camptothecins (camptothecins), docarmycins/duocarmycins, etoposides
  • maytansines and maytansinoids such as DM1 and DM4
  • taxanes taxanes
  • benzodiazepines or benzodiazepine containing drugs
  • drug-linkers can be used to form NDCs in one simple step.
  • bifunctional linker compounds can be used to form NDCs in a two- or multi-step process. For example, a cysteine residue reacts with the reactive part of the linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug, forming an NDC.
  • functional groups on the linker are selected to facilitate specific reaction with appropriate reactive groups on the drug moiety.
  • azide-based moieties can be used to specifically interact with drug moieties.
  • Reactive alkynyl groups react.
  • the drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides), isocyanates and isothiocyanates (suitable for reaction with amines) and activated esters, such as N-hydroxysuccinimide ester (suitable for reactions with amines and alcohols).
  • ketones and aldehydes suitable for reaction with hydrazides and alkoxyamines
  • phosphines suitable for reaction with azides
  • isocyanates and isothiocyanates suitable for reaction with amines
  • activated esters such as N-hydroxysuccinimide ester (suitable for reactions with amines and alcohols).
  • the present invention also provides a method for preparing NDC, which may further include: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (NDC).
  • NDC antibody conjugate
  • methods of the invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate.
  • the methods of the present invention further comprise: conjugating the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.
  • the structure of the immunoconjugate preferably a single domain antibody drug conjugate NDC, is as follows:
  • nAb is the above-mentioned single domain antibody targeting CD28, an antibody targeting CD28 or a multispecific antibody,
  • LU is the joint/linker
  • the present invention also provides the use of the antibody of the present invention, for example, for the preparation of diagnostic preparations or the preparation of medicaments for the prevention and/or treatment of CD28-related diseases.
  • the CD28-related diseases include tumor occurrence, growth and/or metastasis, tumor drug resistance-related diseases, inflammation, metabolism-related diseases, etc.
  • the uses of the antibodies, NDC or CAR-T of the present invention include (but are not limited to):
  • the tumors include (but are not limited to): breast cancer (such as triple negative breast cancer), lung cancer (such as non-small cell lung cancer), pancreatic cancer, malignant glioma, gastric cancer, liver cancer, esophageal cancer, kidney cancer, tuberculosis Rectal cancer, bladder cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, leukemia, bone marrow cancer, angiosarcoma, etc.; especially triple-negative breast cancer, non-small cell lung cancer, pancreatic cancer, and malignant glioma, More preferred are triple negative breast cancer and/or non-small cell lung cancer.
  • autoimmune diseases include (but are not limited to): systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, and multiple sclerosis.
  • the inflammation includes (but is not limited to): rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Reiter syndrome, psoriatic arthritis, infectious arthritis, tuberculous arthritis, disease Toxic arthritis, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis.
  • the metabolism-related diseases include (but are not limited to): diabetes, food-borne obesity, and adipose inflammation.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its NDC or the corresponding CAR-T cell, and a pharmaceutically acceptable carrier.
  • these materials may be formulated in a nontoxic, inert, and pharmaceutically acceptable aqueous carrier medium.
  • the formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to) intratumoral, intraperitoneal, intravenous, or topical administration.
  • the antibody of the present invention can also be expressed in cells from a nucleotide sequence and used for cell therapy.
  • the antibody can be used for chimeric antigen receptor T cell immunotherapy (CAR-T).
  • the pharmaceutical composition of the present invention can be directly used to bind CD28 protein molecules, and thus can be used to prevent and treat tumors and other diseases.
  • other therapeutic agents may be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and pharmaceutical acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day. Additionally, the polypeptides of the invention may be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 mg per kilogram of body weight, Preferably the dose is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.
  • the single domain antibody-drug conjugate provided by the present invention can target a specific cell population and bind to specific cell surface proteins (antigens), thereby making the drug active through endocytosis of the conjugate or drug penetration. Therefore, the single domain antibody-drug conjugate of the present invention can be used to treat target diseases.
  • the above-mentioned antibody-drug conjugate can be administered to a subject in a therapeutically effective amount through a suitable route. (such as people).
  • a subject in need of treatment may be a patient at risk for, or suspected of having, a condition related to the activity or expression of a particular antigen. Such patients can be identified through routine physical examination.
  • delivery can be performed by methods routine in the art. For example, it can be introduced into cells through the use of liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres.
  • the nucleic acid or vector may be delivered locally by direct injection or by use of an infusion pump.
  • the single domain antibody of the present invention has similar affinity to traditional CD28 super-agonistic antibodies, has no activating effect on T cells when alone (non-agonistic antibody), and has good safety.
  • the single domain antibody of the present invention has a molecular weight of only 12.5kDa and has better tissue penetration ability than traditional CD28 antibodies.
  • the single-domain antibody of the present invention can be constructed with other traditional tumors-targeting antibodies or single-domain antibodies to form bi/multi-specific antibodies, which greatly enhances the diversity of future drug development and the potential for combined drugs.
  • the bispecific antibody or multispecific antibody constructed from the single domain antibody of the present invention can significantly enhance the activity of the CD3 bispecific antibody. In vivo animal experiments suggest that the above combination can significantly inhibit tumor growth.
  • the bispecific antibody or multispecific antibody constructed from the single domain antibody of the present invention has good stability.
  • VHH single heavy chain variable region
  • IMGT Numbering system based on the International Immunogenetic Information System initiated by Lefranc et al.
  • EC50 half maximum effect concentration, that is, the concentration that can cause 50% of the maximum effect
  • the extracellular domain of human CD28 antigen (Asn19-Pro152, Uniport ID: P10747) was combined with the Fc region of camelid IgG (Glu 1-Ser 243, AAX73259.1). and the Fc region of human IgG1 (Pro100-Lys330, Uniport ID: P01857) were constructed into a fusion protein and transiently transfected into 293EBNA cells. After 7 days of expression, the target protein in the supernatant was purified by Protein A.
  • CHO-S cells were modified and the CD28 overexpressing cell line CHO-S-CD28 was constructed through lentiviral infection.
  • the full-length amino acid sequence of human CD28 protein (Uniprot ID: P10747) was submitted to General Genomics Company for nucleotide codon optimization, and the optimized nucleotide sequence was synthesized into the pLVX-Puromycin vector.
  • After obtaining the synthetic lentiviral plasmid expressing human CD28 prepare the virus supernatant according to the conventional virus packaging process. The virus supernatant is added to CHO-S cells and centrifuged for infection. After puromycin is pressurized, the stable CHO-S-hCD28 is obtained. Cell lines, stable cell line expression were identified and confirmed by flow cytometry.
  • the anti-CD28 humanized single domain control antibody C1 used in the present invention has a sequence derived from Mab (UNII:LCT264LTYE); the amino acid sequence of the control antibody was synthesized into PTT5-hFc by General Gene Company, and expressed by transient transfection into CHO-E cells. ProA was purified after 7 days.
  • human CD28 antigen protein fused with Llama Fc (Sichuan Sibowo Biotechnology Co., Ltd.) was mixed with an equal volume of Freund's adjuvant, and adult healthy alpacas were selected.
  • the antigen was injected subcutaneously at a dose of 0.2-0.5 mg.
  • the alpaca serum was collected and the antigen immune titer was determined by ELISA.
  • Peripheral blood mononuclear cells (PBMC) were isolated and purified by Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation.
  • protein panning method was used to conduct multiple rounds of enrichment.
  • the human CD28 antigen was directly coated on the enzyme plate and left at 4°C overnight. After coating is completed, wash the enzyme plate once with PBST, add 300ul of 2% BSA blocking solution to each well, and block at 37°C for 2 hours. Resuspend the phage library in 1 ml of 2% BSA, incubate at 37°C for 30 minutes, add it to the enzyme-labeled well, incubate at 37°C for 60 minutes, and discard the supernatant.
  • Human CD28 protein was diluted to 1ug/ml with CBS buffer and added to a 96-well enzyme plate, and left at 4°C overnight. After blocking and washing, 50 ul of the E. coli supernatant was transferred to the coated well plate, and 50 ul of 2% BSA was added to each well, and incubated at 37°C for 2 hours. Then wash the well plate three times with PBST buffer, then add HRP-labeled anti-M13 antibody, 100 ⁇ L/well, and incubate at 37°C for 1 hour.
  • the general process includes: 500 g of CHO-S-hCD28 cells in the logarithmic growth phase, centrifuge for 3 minutes to collect the cells, wash twice with PBS, resuspend with 1% BSA, transfer the cells to a 96-well tip bottom plate, 50ul per well ; Add the phage supernatants identified as positive clones by ELISA to the cell wells, 50ul per well, and incubate at 4 degrees for 50 minutes; wash the cells twice with PBS, 500g, centrifuge for 3 minutes, and use 1% BSA solution (containing biotinylation) to M13 antibody, Yiqiao Shenzhou #11973-MM05T-B) was resuspended, 100ul per well, incubated at 4 degrees for 40 minutes,
  • the fluorescence signal value was detected by flow cytometry. According to the analysis results in Table 1, except for clone A04#16, which did not bind to CHO-S-hCD28 cells, the supernatants of the remaining 15 clones had varying degrees of binding activity to CHO-S-hCD28 cells.
  • Anti-CD28 recombinant single domain antibody was obtained through Example 2.1, and its purity was detected by SEC.
  • the purity of the preferred recombinant anti-CD28 single domain antibody of the present invention is measured on Agilent HPLC equipment using Tosoh's size exclusion chromatography column (TSKgel G3000SWXL).
  • the mobile phase is 100mM Na 2 HPO 4 , 100mM NaCl, pH 7.0. 5% IPA;.
  • the instrument parameters are set as sample chamber temperature: 8°C, column temperature: 30°C, flow rate: 0.5ml/min, and detection wavelength: 280nm.
  • the sample to be tested was diluted with mobile phase ddH 2 O to a final concentration of 1 mg/ml, and 20 ul was injected for purity analysis.
  • the specific results are shown in Table 2.
  • the purity of A04#1, A04#11, A04#12, and A04#14 anti-CD28 recombinant single domain antibodies are all lower than 90%, and the purity of the remaining A04#2, A04#3, A04, and A04#5 , A04#6, A04#7, A04#8, A04#9, A04#13, and A04#15 anti-CD28 recombinant single domain antibodies have purity higher than 97% and can be used for further evaluation.
  • the starting concentration of the anti-CD28 recombinant single domain antibody is 26.6nM, 3-fold gradient, 12 concentration points, double wells, mix and incubate at 37°C. 2 hours; discard the blocking solution, wash 3 times with 300 ⁇ l PBST; add 100 ⁇ l of HRP-Goat-Anti-human IgG (jackson immunoresearch) diluted with 2% BSA into the corresponding well, and incubate at 37°C for 1 hour; wash 5 times with 300 ⁇ l PBST; Add 100 ⁇ l TMB (Biopanda) to the corresponding well to develop color; add 50 ⁇ l 2N H 2 SO 4 to terminate, and read with a microplate reader at 450 nm.
  • the fluorescence signal values corresponding to different concentrations of antibodies were curve-fitted using GraphPad Prism software. The results are shown in Figure 2A and Figure 2B.
  • the specific affinities are shown in Table 4, among which A04#2, A04#3, A04#6, A04#13 and The affinity of CHO-S-hCD28 cells is higher, lower than 5nM, while the affinity of A04#4, A04#5, A04#7, A04#8, and A04#15 to CHO-S-hCD28 cells is between 10-50nM.
  • A04#9 has weak binding to CHO-S-hCD28 cells.
  • CD28 mainly has two ligands in the human body, namely the high-affinity ligand CD80 and the low-affinity ligand CD86.
  • the two ligands bind to CD28 at the same position, so we used a competition ELISA method to resist CD28 recombinant single domain antibodies. Ligand blocking activity was studied.
  • PBS BSA
  • Dilution solution the antibody started from 1uM, diluted 3 times, 100ul per well was added to each well of the 96-well plate; the liquid in the well was drained, and washed three times with washing buffer PBST; HRP-coupled SA secondary antibody (Proteintech ) was diluted with 2% BSA at a dilution ratio of 1:10000, and 100ul per well was added to each well of the 96-well plate.
  • A04#2 and A04#4 have the strongest ability to block the binding of CD28 and CD80, which is lower than 10nM, while A04#3, A04#5, A04#6, A04#7, and A04#8 , A04#13, and A04#15 have the ability to block CD28 and CD80 between 10-30nM, while A04#9 has the weakest ability to block CD28 and CD80.
  • A04#2 and A04#6 have the strongest affinity with human PBMC cells, which is lower than 30 nM, while A04#3 and A04#5 have the strongest affinity with human PBMC cells at 40 nM. -100nM, A04#3 has the weakest affinity; A04#2, A04#3, A04#5 and A04#6 all have strong affinity to monkey PBMC.
  • the first clinical trial of the CD28 antibody TGN1412 resulted in severe systemic inflammatory reactions in all volunteers, and four of them developed multiple organ failure.
  • the side effects came from the rapid and large release of cytokines induced by TGN1412, so the T of the CD28 antibody It is very important to study the properties of cell activation.
  • the CD28 recombinant antibody to be tested (OKT3 antibody as a control) was diluted to 200nM with 1640+10% FBS, starting with 3-fold gradient dilution and 12 concentration points, and then 50ul of each sample was added to a 96-well white plate (Corning) , add the same volume of culture medium to the negative control wells, and set all duplicate holes.
  • Jurkat-NFAT-Luciferase cells (Sichuan Sibowo Biotechnology Co., Ltd.) were diluted to 1*10 6 /ml with 1640+10% FBS, and 50ul of cell suspension was added to the sample wells and blank wells, and cultured at 37 degrees for 6 hours. . After 6 hours, 20ul One-Glu (Promega) was added respectively, and the fluorescence signal value was read by a microplate reader. The fluorescence signal values corresponding to different concentrations were curve-fitted using GraphPad Prism software.
  • rat CD28 and mouse CD28 proteins were diluted with CBS coating solution to a final concentration of 1 ⁇ g/ml, 100 ⁇ L/well, overnight at 4°C; continue the test the next day, wash once with 300 ⁇ l PBST; add 100 ⁇ l 2% BSA, 37 Block at °C for 2 hours; dilute the CD28 antibody to be tested with 2% BSA, starting at 5ug/ml, 3-fold gradient, 12 concentration points, double wells, mix and incubate at 37°C for 2 hours; discard the blocking solution, wash 3 times with 300 ⁇ l PBST; add 100 ⁇ l of 2% BSA diluted HRP-Goat-Anti-human IgG (jackson immunoresearch) to the corresponding wells, and incubate at 37°C for 1 hour; wash 5 times with 300 ⁇ l
  • the clones that specifically bind to the CD28 protein evaluated through recombinant expression are sent for sequencing to obtain the corresponding anti-CD28 single domain antibody nucleotide sequence, and then translated into the corresponding amino acid sequence, A04#2 (Seq.ID NO: 1), A04 #3(Seq.ID NO:13) and A04#6(Seq.ID NO:25) are detailed in the sequence list.
  • the CDR transplanted antibody humanization method was used to humanize camel-derived single domain antibodies, A04#2, A04#3 and A04#6.
  • humanization transformation involves the following steps: Compare the amino acid sequence of camel-derived single domain antibodies with the amino acid sequences of human germline antibodies to find sequences with high homology and better physical and chemical properties as human embryonic lines. Framework sequence; analyze and examine HLA-DR affinity, select human embryonic line framework sequences with low affinity; then transplant the CDRs of camel-derived antibodies to the selected heavy chain framework sequences.
  • the humanized template for A04#2 is the human germline gene sequence IGHV3-66*01; the humanized template for A04#3 and A04#6 is the human germline gene sequence IGHV3-23*01.
  • a humanized antibody was constructed based on the CDR of camel-derived single domain antibody A04#2, named A04#2-Hz. Based on the CDR of camel-derived single domain antibody A04#3, a total of 1 humanized antibody was constructed, named A04#3-Hz; based on the CDR of camel-derived single domain antibody A04#6, a total of 1 humanized antibody was constructed The humanized antibody strain was named A04#6-hz.
  • the amino acid sequence of the humanized anti-CD28 single domain antibody was synthesized into the PPT5 expression vector by universal genes, and all synthesized plasmids were sequenced and confirmed.
  • the correctly sequenced humanized antibody expression plasmid was transiently transfected into 293EBNA cells according to the method in Example 2.1 for recombinant expression of the antibody.
  • For 7-day-old 293EBNA cell culture first centrifuge at 1000g for 15 minutes at low speed to separate the supernatant and cell pellet; then centrifuge at 8000g for 20 minutes at high speed to obtain a clear feed liquid.
  • Antibodies were purified by affinity chromatography (Protein A). All purified antibodies were ultrafiltrated and replaced with PBS buffer and maintained at -80C.
  • the antigen-binding dynamic affinity of the humanized CD28 single domain antibody was measured using a method similar to that of Example 2.4.
  • the results in Table 8 show that the affinity of the single-domain antibody after humanization is not significantly affected compared with that before humanization, and the affinity level is at the nM level.
  • hCD28-His protein (Sinobiological) and hCD28-Llama Fc (Chengdu Sinobiological Biotechnology Co., Ltd.) were first added at 100ng/well.
  • the affinities of A04#2-Hz, A04#3-Hz and A04#6-Hz are 0.43nM, 0.43nM and 0.22nM respectively.
  • Jurkat cells are a known cell line that naturally expresses CD28 protein.
  • Human peripheral blood mononuclear cells were separated according to the method of Example 2.6, and flow cytometry was used to determine the binding of anti-CD28 to the human CD28 target protein expressed on human peripheral blood mononuclear cells. Centrifuge the PBMC, wash twice with PBS, and resuspend in 1% BSA buffer. Add Human TruStain FcX (Biolegend) to the PBMC cell suspension and incubate at room temperature for 20 minutes. After the incubation is completed, the cell suspension is added to a 96-well pointed bottom plate, 50ul per well.
  • Trispecific-1 is connected to the VH in the anti-CD3 Fab;
  • Trispecific-2 is connected to the VL in the anti-CD3 Fab.
  • Fc knock-in-hole
  • S354C and Y349C are introduced into the two heavy chains respectively to stabilize the correct pairing of the heavy chains.
  • Fc is the heavy chain of IgG1. Chain constant region, while introducing L234A, L235A and G237A (according to Kabat's "EU” numbering) amino acid mutations that weaken the effector function.
  • Figure 11 A schematic diagram of the structure of an example bispecific antibody is shown in Figure 11.
  • Anti-human DLL3 (Tarlatamab, KEGG#10683) was combined with CD3 (Teclistamab, KEGG#D12177) and A04#2hz to construct two 1+1+1 trispecific antibodies DLL3/CD28/CD28.
  • the above example multi-specific antibody sequences were synthesized into the pTT5 vector, and then transiently expressed in 293F cells. After 7 days of expression, the cell fermentation broth was filtered and clarified, and processed using a Protein A column (Hitrap Mabselect Sure, GE 11-0034-95). capture. After detecting the antibody concentration using the A280 method, SEC-HPLC was used for purity identification.
  • the above 10 multispecific antibodies obtained were used in the following examples.
  • the single-end affinity of the bispecific antibody of the present invention was detected respectively, in which the CD28 end was coated with hCD28-ECD-lamFc for detection, the DLL3 end was coated with hDLL3-ECD-his, and the B7H3 end was coated with hB7H3 -ecd-his.
  • the antibody concentration started at 10ug/ml, with a 3-fold dilution gradient and 11 concentration points.
  • the affinity EC50 calculation results are shown in Table 12.
  • the DLL3/CD3 dual antibody simultaneously binds to DLL3 on the surface of small cell lung cancer cells and CD3 on the surface of Jurkat-NFAT-Luc cells.
  • the DLL3/CD28 dual antibody simultaneously binds to DLL3 and CD3 on the surface of small cell lung cancer cells.
  • CD28 binding on the surface of primary T cells activates the NFAT-Luc downstream signaling pathway through DLL3-dependent CD28 cross-linking.
  • the luciferase reporter gene method was used to detect the co-culture conditions of Jurkat-NFAT-Luc cells and DLL3-positive cells, and the expression of luciferase after adding the sample antibody for 6 hours of culture was used to reflect the activation ability of the antibody. Evaluate the difference between DLL3/CD28 and DLL3/CD28/CD28 on the synergistic effects of DLL3/CD3 dual antibodies.
  • the example DLL3/CD3 bispecific antibody was able to activate NFAT signaling with an EC50 value of 0.192 nM.
  • the CD28 bispecific antibody can significantly increase the activation of NFAT signal by the CD3 biantibody, and the activity of the DLL3/CD3 bispecific antibody is enhanced.
  • the monovalent CD28 bispecific antibody is significantly better than the bivalent CD28 bispecific antibody.
  • the EC50 values of the two are 0.040nM and 0.086 respectively. nM.
  • the DLL3/CD3/CD28 trispecific antibody simultaneously binds to DLL3 on the surface of small cell lung cancer cells and CD3 and CD28 on the surface of Jurkat-NFAT-Luc cells, and activates the NFAT-Luc downstream signaling pathway through DLL3-dependent cross-linking of CD3 and CD28.
  • the present invention uses the luciferase reporter gene method to detect the co-culture conditions of Jurkat-NFAT-Luc cells and DLL3-positive cells, and reflects the strength of the antibody activation ability by detecting the expression of luciferase after adding an example antibody and culturing for 6 hours. Compare the activity differences between DLL3/CD3/CD28 trispecific antibody and DLL3/CD3 bispecific antibody.
  • Example 4.3 For specific experimental methods, refer to Example 4.3. The results are shown in Figure 12-2.
  • the example DLL3/CD3 bispecific antibody can activate NFAT signaling with an EC50 value of 0.122nM.
  • the activity of DLL3/CD3/CD28 Trispecific-1 is 1 times stronger than that of DLL3/CD3/CD28 Trispecific-2, and their EC50 values are 0.043nM and 0.080nM respectively.
  • both the EC50 and the maximum signal value of the two structural types of trispecific antibodies described in the present invention are significantly improved compared to bispecific antibodies.
  • the dynamic affinity calculation results are shown in Table 13.
  • the affinity of DLL3 end is much higher than the affinity of CD28 end, and the KD values are 5.90E-10M and 1.09E-08M respectively.
  • NCI-H716 was used as the target cell to evaluate the synergy of B7H3/CD28 dual antibodies and DLL3/CD3 dual antibodies. effect.
  • the exemplary CD28 bispecific antibody was able to increase CD3 biantibody-activated NFAT signaling. The result is as follows:
  • the B7H3/CD28 bispecific antibody can enhance DLL3/CD3 activity, especially the maximum signal value of DLL3/CD3 activation signal is increased by nearly 60%.
  • DLL3/CD3 dual antibodies simultaneously bind to DLL3 on the surface of small cell lung cancer cells and CD3 on the surface of primary T cells. Through DLL3-dependent CD3 cross-linking, they provide the first signal for T cell activation and activate T cells. DLL3/CD28 dual antibodies At the same time, it binds to DLL3 on the surface of small cell lung cancer cells and CD28 on the surface of primary T cells. Through DLL3-dependent CD28 cross-linking, it provides the second signal of T cell activation, enhances T cell activity, and finally cooperates with DLL3/CD3 to mediate T Cell killing of DLL3-positive tumor cells.
  • This example uses the Cell titer Glu method to detect the co-culture conditions of human PBMC cells and DLL3-positive cells, and adds the example antibody for 48 hours to detect the tumor cell viability, thereby evaluating the killing ability of human T cells against DLL3-positive tumor cells.
  • the specific detection methods are as follows:
  • DLL3-positive cells SHP77 human small cell lung cancer purchased from Nanjing Kebai Biotech
  • NCI-H358 human non-small cell lung cancer purchased from Sichuan Sharpson Biotechnology Co., Ltd.
  • PBMC Take out the PBMC from the liquid nitrogen tank and quickly melt it at 37°C, then add it dropwise into the preheated 1640 culture medium containing 10% FBS to obtain 10 ml of mixed solution. Centrifuge at 100 g for 5 minutes, resuspend in 10 ml of 1640 culture medium containing 10% FBS, and adjust The cell density of human PBMC is 1 ⁇ 10 ⁇ 6 cells/ml as effector cells. Add 50 ⁇ l of human PBMC cell suspension to the target cell wells at a target-effectiveness ratio of 1:5, and add 50 ⁇ l of serially diluted antibody to be tested into each well. Place in a carbon dioxide incubator and incubate at 37°C for a total of 48 hours.
  • NCG mice Five-week-old female NCG mice (18-20 g) were purchased from Chengdu Yaokang Biotechnology Co., Ltd. The grade is SPF. Mice were acclimated and quarantined for 7 days upon arrival before the study began. Mice were intravenously injected with PBMC cells and tumor cells, and each NCG mouse was subcutaneously inoculated with SHP77 cells and human T cells (5*10 ⁇ 6 cells each) on the right scapula of each NCG mouse. The inoculation volume is 0.2mL (50 ⁇ L SHP77+50 ⁇ L PBMC cells+100 ⁇ L Matrigel). When the average tumor volume of the mice reached 150 mm3, they were divided into groups (4 mice in each group) for administration.
  • DLL3/CD3 dual antibody dose 1mg/kg Dosing once a week, 2 times in total, DLL3/CD3 dual antibody dose 1mg/kg, DLL3/CD28 dual antibody dose 10mg/kg, combination group DLL3/CD3 dose 1mg/kg and DLL3/CD28 dose 10mg/kg .
  • the tumor volume and body weight of the mice were monitored twice a week.
  • TGI% On the 22nd day after inoculation, calculate the relative tumor inhibition rate (TGI%), calculate the tumor volume, and draw the tumor growth curve.
  • TGI% On the 22nd day after inoculation, calculate the relative tumor inhibition rate (TGI%), calculate the tumor volume, and draw the tumor growth curve.
  • TGI% On the 22nd day after inoculation, calculate the relative tumor inhibition rate (TGI%), calculate the tumor volume, and draw the tumor growth curve.
  • V The calculation formula of tumor volume (V) is:
  • V 1/2 ⁇ a ⁇ b 2where a and b represent the long diameter and short diameter of the tumor respectively.
  • TGI% (1-T/C) ⁇ 100 (T and C are the relative tumor volumes (RTV) of the treatment group and the control group at a specific time, respectively).
  • the tumor growth curve is shown in Figure 14-1.
  • the DLL3-CD28 antibody can significantly enhance the DLL3-CD3 antibody to inhibit the growth of SHP77 cells.
  • the tumor size was counted on the 22nd day, and the tumor inhibition rate was calculated.
  • the tumor inhibition rate of DLL3/CD28+DLL3/CD28 in the combination group was 100% (p ⁇ 0.01), and all tumors disappeared at 18 days.
  • no significant weight loss was found in the administered mouse groups ( Figure 14-2), indicating that the drug safety in all groups was good.
  • the anti-tumor activity of the trispecific antibody DLL3/CD3/CD28 Trispecific-1 of the present invention was tested using a protocol similar to Example 4.7. It was administered once a week at a dose of 0.5 mg/kg for a total of 2 administrations.
  • the tumor growth curve is shown in Figure 15-1.
  • the tumor size was counted on the 22nd day, and the tumor inhibition rate was calculated.
  • the tumor inhibition rate of the example antibody DLL3/CD28/CD3 was 100% (p ⁇ 0.01).
  • no significant weight loss was found in the administered mouse group ( Figure 15-2).
  • the T m value of DLL3-CD28 in PBS buffer is approximately 63.7°C, indicating that the DLL3-CD28 double antibody has good thermal stability.
  • the SEC detection method is as described above.
  • the CE-SDS detection method is as follows: use ultrapure water to dilute the sample to 4mg/ml. Take 25uL in a centrifuge tube, add 75uL premix (SDS Sample buffer + iodoacetamide), mix well, and centrifuge at 10000g for 1 minute. Process at 70°C for 5 minutes. After taking it out, let it cool at room temperature, centrifuge at 10000g for 1 minute, mix well and transfer it to the injection bottle.
  • Capillary tube 30.2cm*50um (bare tube), effective length: 20cm
  • Detection wavelength setting 220nm; collection frequency: 4Hz.
  • the experimental results are shown in Table 14.
  • the DLL3/CD28 double antibody has good accelerated stability, and its purity and affinity are well maintained under accelerated conditions.
  • the present invention discloses a variety of high specificity and high affinity Nanobodies targeting CD28, which only include heavy chains containing heavy chain variable region (VHH) amino acid sequences, as listed in the following table.
  • VHH heavy chain variable region

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Abstract

La présente invention concerne un anticorps à domaine unique humanisé anti-CD28. Plus particulièrement, l'invention concerne une pluralité d'anticorps à domaine unique ciblant CD28 et des anticorps humanisés de ceux-ci, des protéines dérivées, des séquences de gènes codant pour ceux-ci, des vecteurs d'expression et des systèmes d'expression pour leur production. L'anticorps à domaine unique selon la présente invention est un anticorps non agoniste de CD28, peut bloquer la liaison de CD28 à CD80, et peut se lier de manière spécifique à CD28 humain et de singe. L'anticorps à domaine unique selon la présente invention a une sécurité élevée et fournit une base de recherche et de développement pour le développement de conjugués anticorps-médicament et de médicaments anticorps multispécifiques. Un anticorps bispécifique formé par l'anticorps à domaine unique CD28 selon la présente invention et un anticorps ciblant un antigène tumoral peuvent améliorer de manière remarquable l'activité d'un anticorps bispécifique CD3, et un anticorps multispécifique formé par l'anticorps à domaine unique CD28 selon la présente invention, l'anticorps ciblant un antigène tumoral, et l'anticorps CD3 ayant une activité remarquablement supérieure à celle de l'anticorps bispécifique CD3.
PCT/CN2023/109962 2022-07-28 2023-07-28 Anticorps à domaine unique humanisé anti-cd28 WO2024022516A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1563092A (zh) * 2004-04-01 2005-01-12 北京安波特基因工程技术有限公司 基因工程重组抗cea抗cd3抗cd28单链三特异抗体
CN108264560A (zh) * 2016-12-30 2018-07-10 上海欣百诺生物科技有限公司 一种结合cd3和cd28的双功能分子及其应用
WO2021155071A1 (fr) * 2020-01-29 2021-08-05 Inhibrx, Inc. Anticorps cd28 à domaine unique et constructions multivalentes et multispécifiques de ceux-ci

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
CN1563092A (zh) * 2004-04-01 2005-01-12 北京安波特基因工程技术有限公司 基因工程重组抗cea抗cd3抗cd28单链三特异抗体
CN108264560A (zh) * 2016-12-30 2018-07-10 上海欣百诺生物科技有限公司 一种结合cd3和cd28的双功能分子及其应用
WO2021155071A1 (fr) * 2020-01-29 2021-08-05 Inhibrx, Inc. Anticorps cd28 à domaine unique et constructions multivalentes et multispécifiques de ceux-ci

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CHENG JULONG, WANG XIANGBIN, LIU JING, GU YING: "The Study of a Reshaping Anti-CD28 Heavy-chain Variable Domain(VH) Antibody with CDR Mutations", GAOJISHU TONGXUN - HIGH TECHNOLOGY LETTERS, BEIJING, CN, no. 04, 28 April 2001 (2001-04-28), CN , pages 11 - 15, XP009552309, ISSN: 1002-0470 *

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