Anti-CD28 humanized single domain antibody

Abstract

The invention provides an anti-CD28 humanized single-domain antibody and a preparation method of the anti-CD28 humanized single-domain antibody. Specifically, the invention discloses a plurality of single-domain antibodies targeting CD28, humanized antibodies thereof, derived proteins, gene sequences for encoding the single-domain antibodies and the humanized antibodies, and expression vectors and expression systems for producing the single-domain antibodies and the humanized antibodies. The single-domain antibody provided by the invention is a CD28 non-agonistic antibody, can block the binding of CD28 and CD80, and can specifically bind to CD28 of human and monkeys. The single-domain antibody provided by the invention has high safety, and provides a research and development basis for development of antibody coupling drugs and multispecific antibody drugs. A bispecific antibody composed of the CD28 single-domain antibody and the targeting tumor antigen antibody can significantly enhance the activity of the CD3 bispecific antibody. The activity of a multi-specific antibody composed of the CD28 single-domain antibody, the targeting tumor antigen antibody and the CD3 antibody is significantly superior to that of the CD3 bispecific antibody.

Description

Anti-CD28 humanized single domain antibody Technical Field

[0001] The present invention belongs to the field of biomedicine technology. Specifically, the present invention relates to an anti-CD28 humanized single-domain antibody. Background Art

[0002] The clinical success of immune checkpoint inhibitors, including anti-PD-1 (e.g., Keytruda, Merck & Co.; Opdivo, Bristol-Myers Squibb), anti-CTLA-4 (e.g., Yervoy, Bristol-Myers Squibb), and anti-PD-L1 (e.g., Tecentriq, Roche), has made cancer immunotherapy an increasingly standard treatment for malignancies, generating significant and durable responses in cancers such as melanoma, non-small cell lung cancer, and renal cell carcinoma. While immune checkpoint inhibitors are now becoming standard of care for many cancer types or the cornerstone of combination therapies, only a subset of patients (<25%) benefit from these therapies. Furthermore, many cold tumors, including prostate cancer, colorectal cancer, pancreatic cancer, sarcomas, and non-triple-negative breast cancer, remain resistant to immune checkpoint inhibitors. Numerous studies have shown that the absence of effector T cells and a suppressive tumor microenvironment in so-called cold tumors are the primary reasons for non-response or suboptimal response in some patients. While existing immunotherapies have demonstrated excellent efficacy in certain tumor types, extending their benefits to a wider range of tumor types and populations remains a daunting task.

[0003] CD28 is a co-stimulatory molecule expressed on the surface of T lymphocytes. Researchers have proposed that anti-CD28 antibodies can be used for therapeutic purposes involving the activation of T cells. CD28 agonistic antibodies can be divided into two categories: (i) CD28 superagonistic antibodies (CD28SA) and (ii) CD28 conventional agonistic antibodies. In the prior art, CD28 superagonistic antibodies and CD28 conventional agonistic antibodies still have many shortcomings. For example, CD28 superagonistic antibodies are prone to cause life-threatening cytokine storms. Bispecific single-chain antibodies constructed using CD28 conventional agonistic antibodies (such as clone 9.3) are also prone to produce "superagonistic" activity.

[0004] Therefore, there is still a need in the art to develop a novel non-agonistic CD28 antibody to avoid the "superagonistic" activity of CD28 antibodies, which is conducive to the development of single-domain antibodies against CD28 and bispecific / multispecific antibodies based on CD28 single-domain antibodies.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide an anti-CD28 single-domain antibody and a humanized antibody thereof.

[0007] In the first aspect of the present invention, a single domain antibody targeting CD28 is provided, wherein the complementarity determining region (CDR) of the VHH chain of the single domain antibody is one or more selected from the group consisting of

[0008] (1) CDR1 shown in SEQ ID NO. 3,

[0009] CDR2 shown in SEQ ID NO. 4, and

[0010] CDR3 shown in SEQ ID NO.5; or

[0011] CDR1 shown in SEQ ID NO.6,

[0012] CDR2 shown in SEQ ID NO. 7, and

[0013] CDR3 shown in SEQ ID NO.5; or

[0014] CDR1 shown in SEQ ID NO.8,

[0015] CDR2 shown in SEQ ID NO. 9, and

[0016] CDR3 shown in SEQ ID NO.5; or

[0017] CDR1 shown in SEQ ID NO.10,

[0018] CDR2 shown in SEQ ID NO. 11, and

[0019] CDR3 shown in SEQ ID NO. 12;

[0020] or,

[0021] (2) CDR1 shown in SEQ ID NO. 15,

[0022] CDR2 shown in SEQ ID NO. 16, and

[0023] CDR3 shown in SEQ ID NO. 17; or

[0024] CDR1 shown in SEQ ID NO.18,

[0025] CDR2 shown in SEQ ID NO. 19, and

[0026] CDR3 shown in SEQ ID NO. 17; or

[0027] CDR1 shown in SEQ ID NO.20,

[0028] CDR2 shown in SEQ ID NO. 21, and

[0029] CDR3 shown in SEQ ID NO. 17; or

[0030] CDR1 shown in SEQ ID NO.22,

[0031] CDR2 shown in SEQ ID NO. 23, and

[0032] CDR3 shown in SEQ ID NO. 24;

[0033] or,

[0034] (3) CDR1 shown in SEQ ID NO. 27,

[0035] CDR2 shown in SEQ ID NO. 28, and

[0036] CDR3 shown in SEQ ID NO. 29, or

[0037] CDR1 shown in SEQ ID NO.30,

[0038] CDR2 shown in SEQ ID NO. 31, and

[0039] CDR3 shown in SEQ ID NO. 29, or

[0040] CDR1 shown in SEQ ID NO.32,

[0041] CDR2 shown in SEQ ID NO. 33, and

[0042] CDR3 shown in SEQ ID NO. 29, or

[0043] CDR1 shown in SEQ ID NO.34,

[0044] CDR2 shown in SEQ ID NO. 35, and

[0045] CDR3 shown in SEQ ID NO.36.

[0046] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain CD28 binding affinity.

[0047] In another preferred embodiment, the CDR region of the single-domain antibody VHH chain comprises an amino acid sequence having a sequence similarity of at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% to any of the above sequences.

[0048] In another preferred embodiment, the amino acid sequence of the CDR region of the single-domain antibody VHH chain comprises one or more amino acid substitutions compared with any of the above sequences, preferably conservative amino acid substitutions.

[0049] In another preferred embodiment, the VHH chain comprises CDR1, CDR2 and CDR3 selected from the following combinations:

[0050] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0051] In another preferred embodiment, the VHH chain of the single-domain antibody further includes a framework region (FR).

[0052] In another preferred embodiment, the framework region FR is of human, mouse, rabbit or camel origin.

[0053] In another preferred embodiment, the framework region FR includes a human FR region, a mouse FR region or a camel FR region.

[0054] In another preferred example, the VHH chain of the single-domain antibody targeting CD28 has an amino acid sequence as shown in SEQ ID NO.: 1, SEQ ID NO. 13, or SEQ ID NO. 25.

[0055] In another preferred example, the VHH chain of the single-domain antibody targeting CD28 has an amino acid sequence as shown in SEQ ID NO.: 2, SEQ ID NO. 14, or SEQ ID NO. 26.

[0056] In a second aspect, the present invention provides an antibody targeting CD28, wherein the antibody comprises one or more VHH chains of the single-domain antibody targeting CD28 according to the first aspect of the present invention.

[0057] In another preferred example, the VHH chain of the single-domain antibody targeting CD28 has an amino acid sequence as shown in SEQ ID NO.: 1, SEQ ID NO. 13, SEQ ID NO. 25, SEQ ID NO.: 2, SEQ ID NO. 14, or SEQ ID NO. 26.

[0058] In another preferred embodiment, the antibody is a monomer, a bivalent antibody, and / or a multivalent antibody.

[0059] In another preferred embodiment, the antibody is an animal-derived antibody, a humanized antibody, a chimeric antibody or a chimeric antigen receptor antibody (CAR).

[0060] In another preferred embodiment, the CDR region of the humanized antibody comprises 1, 2, or 3 amino acid changes.

[0061] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit, or camel.

[0062] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0063] In another preferred embodiment, the antibody is a monoclonal antibody.

[0064] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0065] In another preferred embodiment, 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%.

[0066] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-7, preferably 1-3, and more preferably 1.

[0067] In another preferred embodiment, the at least one amino acid sequence that has been added, deleted, modified and / or substituted has an amino acid sequence with a homology of at least 80%.

[0068] In another preferred embodiment, the derivative sequence after addition, deletion, modification and / or substitution of at least one amino acid has the function of inhibiting the catalytic function of cell surface CD28 or recombinant CD28 protease.

[0069] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0070] In another preferred embodiment, the antibody has one or more characteristics selected from the following group:

[0071] (a) specifically binds to CD28, preferably binds to human and monkey CD28, and does not bind or weakly binds to rat CD28 or mouse CD28;

[0072] (b) blocking the binding of CD28 to CD80 (blocking antibody);

[0073] (c) is a non-agonistic CD28 antibody.

[0074] In another preferred embodiment, the "CD28 non-agonistic antibody" refers to an antibody that blocks the binding of CD28 to CD80 and does not substantially agonize CD28 signal transduction.

[0075] In a third aspect, the present invention provides a multispecific antibody, wherein the multispecific antibody comprises: the single-domain antibody targeting CD28 according to the first aspect of the present invention or the antibody targeting CD28 according to the second aspect of the present invention.

[0076] In another preferred embodiment, the multispecific antibody further comprises a second antigen binding region targeting a target selected from the group consisting of BCMA, CD73, GPC3, HER2, PMSA, 4-1BB, OX40, GLP-1, Trop2, FGL1, LFA-3, 2B4, 5T4, α-4 integrin, α-V integrin, α4β7 integrin, α4β7 integrin, α-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, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD 51. CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD107a, CD117, CD123, CD12 5. CD132(IL-2Rg), CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CEACAM5(CEA), CEACAM6(NC A-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), FSP-1, GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIlb / IIIa receptor, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL -12R, IL-12Rβl, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SL C44A4, Siglecl5, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, DLL3 (Delta-like canonical Notch ligand 3), or a combination thereof.

[0077] In another preferred embodiment, the second antigen binding region is a single domain antibody.

[0078] In another preferred embodiment, the multispecific antibody comprises one or more second antigen binding regions.

[0079] In another preferred embodiment, the multispecific antibody further comprises an antibody Fc segment.

[0080] In another preferred embodiment, the antigen binding region is an antibody or antibody fragment, and the antibody fragment includes: (i) Fab fragment; (ii) F(ab')2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single-chain Fv (scFv) molecule; and (vi) dAb fragment.

[0081] In another preferred embodiment, the multispecific antibody comprises one or more single-domain antibodies targeting CD28 as described in the first aspect of the present invention.

[0082] In another preferred embodiment, the multispecific antibody further comprises a second antigen binding region targeting DLL3 or B7H3.

[0083] In another preferred embodiment, the multispecific antibody is a trispecific antibody, comprising one or more second antigen binding regions.

[0084] In another preferred embodiment, the Fc segment includes a knob-in-hole structure; preferably, it also includes amino acid mutations S354C and Y349C.

[0085] In another preferred embodiment, the Fc segment further comprises amino acid mutations capable of reducing effector function: L234A, L235A and G237A (according to the "EU" numbering of Kabat) amino acid mutations.

[0086] In another preferred embodiment, the multispecific antibody is a bispecific antibody, comprising a first antigen-binding region targeting CD28, and a second antigen-binding region targeting a tumor-associated antigen (TAA).

[0087] In another preferred embodiment, the TAA is selected from the following group: DLL3, B7H3.

[0088] In another preferred embodiment, the first antigen binding region targeting CD28 comprises one or more single domain antibodies targeting CD28 according to the first aspect of the present invention.

[0089] In another preferred embodiment, the second antigen binding region targeting a tumor-associated antigen (TAA) is an antibody or an antibody fragment, and the antibody fragment includes: (i) a Fab fragment; (ii) a F(ab')2 fragment; (iii) a Fd fragment; (iv) a Fv fragment; (v) a single-chain Fv (scFv) molecule; (vi) a dAb fragment; preferably a Fab fragment.

[0090] In another preferred example, the second antigen binding region targeting a tumor-associated antigen (TAA) targets DLL3.

[0091] In another preferred embodiment, the bispecific antibody has a heavy chain and a light chain selected from the following group:

[0092] (1) a first heavy chain as shown in SEQ ID NO.38; a light chain as shown in SEQ ID NO.39; and a second heavy chain as shown in SEQ ID NO.40; or

[0093] (2) a first heavy chain as shown in SEQ ID NO.38; a light chain as shown in SEQ ID NO.39; and a second heavy chain as shown in SEQ ID NO.41.

[0094] In another preferred embodiment, the second antigen binding region targeting a tumor-associated antigen (TAA) targets B7H3.

[0095] In another preferred embodiment, the bispecific antibody has a heavy chain and a light chain selected from the following group:

[0096] The first heavy chain is shown in SEQ ID NO. 42; the light chain is shown in SEQ ID NO. 43; and the second heavy chain is shown in SEQ ID NO. 40.

[0097] In another preferred embodiment, the multispecific antibody is a trispecific antibody comprising:

[0098] Targeting the first antigen binding region of CD28,

[0099] a second antigen binding region that targets a tumor-associated antigen (TAA); and

[0100] Targets the third antigen-binding region of CD3.

[0101] In another preferred embodiment, the second antigen binding region targeting a tumor-associated antigen (TAA) is an antibody or an antibody fragment, and the antibody fragment includes: (i) a Fab fragment; (ii) a F(ab')2 fragment; (iii) a Fd fragment; (iv) an Fv fragment; (v) a single-chain Fv (scFv) molecule; (vi) a dAb fragment; preferably, an scFv or Fab fragment.

[0102] In another preferred embodiment, the third antigen binding region targeting CD3 is an anti-CD3 bispecific antibody.

[0103] In another preferred example, the second antigen binding region targeting a tumor-associated antigen (TAA) targets DLL3.

[0104] In another preferred embodiment, the second antigen binding region is a scFv targeting DLL3, and the preferred amino acid sequence is shown in SEQ ID NO.44.

[0105] In another preferred embodiment, the trispecific antibody has a heavy chain and a light chain selected from the group consisting of:

[0106] (1) a first heavy chain as shown in SEQ ID NO.44; a second heavy chain as shown in SEQ ID NO.45; and a light chain as shown in SEQ ID NO.46;

[0107] (2) a first heavy chain as shown in SEQ ID NO.44; a second heavy chain as shown in SEQ ID NO.47; and a light chain as shown in SEQ ID NO.46; or

[0108] (3) a first heavy chain as shown in SEQ ID NO.44; a second heavy chain as shown in SEQ ID NO.45; and a light chain as shown in SEQ ID NO.48.

[0109] In a fourth aspect, the present invention provides a recombinant protein having:

[0110] (i) the single domain antibody targeting CD28 as described in the first aspect of the present invention, or the antibody targeting CD28 as described in the second aspect of the present invention; and

[0111] (ii) optionally a polypeptide molecule or fragment having therapeutic function; and / or

[0112] (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

[0113] In another preferred embodiment, the improving the physicochemical properties or drugability of the protein includes prolonging the half-life of the single-domain antibody targeting CD28.

[0114] In another preferred embodiment, the recombinant protein further comprises: (iv) an optional tag sequence for facilitating expression and / or purification.

[0115] In another preferred embodiment, the tag sequence is selected from the following group: 6His tag, GGGS sequence, FLAG tag.

[0116] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0117] In another preferred embodiment, the polypeptide molecules or fragments with therapeutic functions are not limited to: targeting BCMA, CD73, GPC3, HER2, PMSA, 4-1BB, OX40, GLP-1, Trop2, FGL1, LFA-3, 2B4, 5T4, α-4 integrin, α-V integrin, α4β7 integrin, α4β7 integrin, α-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, CD107a, CD117, CD123, CD125, CD132 (IL-2 Rg), CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CEACAM5(CEA), CEACAM6(NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), FSP-1, GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIlb / IIIa receptor, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL -12R, IL-12Rβl, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSG R, RAAG12, RAGE, SLC44A4, Siglecl5, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6 , TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, polypeptide molecules or fragments.

[0118] In another preferred embodiment, the 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, and cytokines.

[0119] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0120] In another preferred embodiment, the fusion protein includes a multispecific antibody or a chimeric antibody.

[0121] In another preferred embodiment, the functional domain for improving the physicochemical properties or drugability of a protein includes an Fc segment and human serum albumin (HSA).

[0122] In another preferred embodiment, the fusion protein has the following elements from the NC end:

[0123] AB;

[0124] Among them, the A component is a single-domain antibody targeting CD28; the B component is the Fc segment or human serum albumin (HSA);

[0125] “-” represents a peptide bond.

[0126] In another preferred example, the VHH chain of the single-domain antibody targeting CD28 has an amino acid sequence as shown in SEQ ID NO.: 1, SEQ ID NO. 13, SEQ ID NO. 25, SEQ ID NO.: 2, SEQ ID NO. 14, or SEQ ID NO. 26.

[0127] In another preferred embodiment, the Fc segment is a human IgG Fc segment.

[0128] In another preferred embodiment, the amino acid sequence of the Fc segment is shown as SEQ ID NO.37.

[0129] In a fifth aspect, the present invention provides a CAR construct, wherein the antigen binding region of the CAR construct is the VHH chain of the single-domain antibody as described in the first aspect of the present invention.

[0130] In the sixth aspect, the present invention provides a recombinant immune cell, wherein the immune cell expresses an exogenous CAR construct as described in the fifth aspect of the present invention.

[0131] In another preferred embodiment, the immune cells are selected from NK cells or T cells.

[0132] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0133] In a seventh aspect, the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0134] (a) an antibody portion, wherein the antibody portion is the single domain antibody targeting CD28 according to the first aspect of the present invention or the antibody targeting CD28 according to the second aspect of the present invention; and

[0135] (b) a conjugated moiety conjugated to the single-domain antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

[0136] In another preferred embodiment, the immunoconjugate is a single domain antibody drug conjugate.

[0137] In another preferred embodiment, the single domain antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0138] In another preferred embodiment, the coupling moiety is a chemical label or a biological label.

[0139] In another preferred embodiment, the chemical label is an isotope, an immunotoxin and / or a chemical drug.

[0140] In another preferred embodiment, the biomarker is biotin, avidin or an enzyme label.

[0141] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0142] In another preferred embodiment, the drug is a cytotoxic drug.

[0143] In another preferred embodiment, the cytotoxic drug is selected from the following group: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0144] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0145] In another preferred embodiment, the toxin is selected from the following group:

[0146] auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis officinalis) inhibitors, glucocorticoids, or a combination thereof.

[0147] In another preferred embodiment, the coupling moiety is a detectable label.

[0148] In another preferred embodiment, the detectable marker comprises a radionuclide, and the radionuclide comprises:

[0149] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0150] (ii) therapeutic isotopes selected from the group consisting of 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-133, Yb-169, Yb-177, or a combination thereof.

[0151] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold single-domain particles / single-domain rods, viral particles, liposomes, single-domain magnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of single-domain particles, etc.

[0152] In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) single-domain antibody targeting CD28 as described in the first aspect of the present invention or an antibody targeting CD28 as described in the second aspect of the present invention.

[0153] In another preferred embodiment, the multivalency means that the amino acid sequence of the immunoconjugate contains multiple repeats of the single domain antibody targeting CD28 as described in the first aspect of the present invention or the antibody targeting CD28 as described in the second aspect of the present invention.

[0154] In another preferred embodiment, the detection is in vivo detection or in vitro detection.

[0155] In another preferred embodiment, the immunoconjugate is used for diagnosing and / or treating tumors expressing CD28 protein.

[0156] In another preferred embodiment, the immunoconjugate has the following molecular formula:

[0157] in:

[0158] nAbs are single-domain antibodies targeting CD28, antibodies targeting CD28, or multispecific antibodies;

[0159] LU is the linker (also called connector);

[0160] D is for medicine;

[0161] Furthermore, the subscript p is a value selected from 1-10.

[0162] In another preferred embodiment, LU is selected from maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC) linkers connected to the antibody portion, and comprises one or more linkers of valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), and polyethylene glycol (PEG).

[0163] In another preferred embodiment, the antibody portion is covalently bound to the linker by reacting with a portion selected from the group consisting of maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC), and the like.

[0164] In another preferred embodiment, D is selected from the following group of compounds having anti-tumor activity:

[0165] (i) Tubulin inhibitors, such as maytansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF);

[0166] (ii) DNA-acting toxins, such as duocarmycin and pyrrolobenzodiazepine (PBD);

[0167] (iii) Topoisomerase inhibitors, such as camptothecin, SN38, exitecan, and Dxd.

[0168] In an eighth aspect, the present invention provides a pharmaceutical composition comprising:

[0169] (i) the single-domain antibody targeting CD28 according to the first aspect of the present invention, the antibody targeting CD28 according to the second aspect of the present invention, the multispecific antibody according to the third aspect of the present invention, the recombinant protein according to the fourth aspect of the present invention, 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;

[0170] (ii) a pharmaceutically acceptable carrier.

[0171] In another preferred embodiment, the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.

[0172] In another preferred embodiment, the pharmaceutical composition further comprises other biologically active substances, such as drugs for treating tumors.

[0173] In another preferred embodiment, 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 spraying and aerosol inhalation.

[0174] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid, solid, or gel.

[0175] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0176] In another preferred embodiment, the pharmaceutical composition is an injection.

[0177] In another preferred embodiment, the pharmaceutical composition further comprises an antibody or antibody fragment targeting CD3.

[0178] In another preferred embodiment, the antibody fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; and (vi) dAb fragments.

[0179] In another preferred embodiment, the antibody is a monomer, a bivalent antibody, and / or a multivalent antibody.

[0180] In another preferred embodiment, the antibody is a bispecific or multispecific antibody.

[0181] In the ninth aspect of the present invention, a use of an active ingredient is provided, wherein the active ingredient is selected from the following group: the single-domain antibody targeting CD28 as described in the first aspect of the present invention, the antibody targeting CD28 as described in the second aspect of the present invention, the multispecific antibody as described in the third aspect of the present invention, the recombinant protein as described in the fourth aspect of the present invention, the recombinant immune cell as described in the sixth aspect of the present invention, the immunoconjugate as described in the seventh aspect of the present invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a detection reagent, a detection plate or a kit; and / or (b) preparing a drug for preventing and / or treating CD28-related diseases.

[0182] In another preferred embodiment, the detection reagent, detection plate or kit is used for:

[0183] (1) detecting CD28 protein in a sample; and / or

[0184] (2) detecting endogenous CD28 protein in tumor cells; and / or

[0185] (3) Detect tumor cells expressing CD28 protein.

[0186] In another preferred embodiment, the detection types include but are not limited to flow cytometry, cell immunofluorescence detection, enzyme-linked immunosorbent assay, immunoblotting detection, etc.

[0187] In another preferred embodiment, the detection reagent, detection plate or kit is used to diagnose CD28-related diseases.

[0188] In another preferred embodiment, the drug is used to treat or prevent tumors with high CD28 expression, tumor migration, or tumor drug resistance.

[0189] In another preferred embodiment, the tumor drug resistance includes: resistance to tumor immunotherapy drugs, resistance to tumor targeted therapy drugs, resistance to conventional tumor chemotherapy, and insensitivity to radiotherapy.

[0190] In another preferred embodiment, the drug is used for a purpose selected from the group consisting of:

[0191] (a) specifically binds to CD28, preferably binds to human and monkey CD28, and does not bind or weakly binds to rat CD28 or mouse CD28;

[0192] (b) Blocking the binding of CD28 to CD80 (blocking antibody).

[0193] (c) Blocks the binding of CD28 to CD80 without stimulating CD28 signaling.

[0194] In another preferred embodiment, the CD28-related disease is selected from the group consisting of cancer, autoimmune disease, metabolism-related disease, or a combination thereof.

[0195] In another preferred embodiment, the CD28-related diseases include: tumor occurrence, growth and / or metastasis.

[0196] In another preferred embodiment, the cancer includes solid tumors and blood cancers.

[0197] In another preferred embodiment, the metabolism-related diseases include: diabetes, diet-induced obesity and adipose inflammation.

[0198] In another preferred embodiment, the cancer is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, colorectal cancer, glioma, melanoma, leukemia, lymphoma, or a combination thereof.

[0199] In another preferred embodiment, 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's syndrome, psoriatic arthropathy, infectious arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis.

[0200] In another preferred embodiment, the metabolism-related diseases include (but are not limited to): diabetes, diet-induced obesity and adipose inflammation.

[0201] In a tenth aspect, the present invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:

[0202] (1) the single-domain antibody targeting CD28 according to the first aspect of the present invention, the antibody targeting CD28 according to the second aspect of the present invention, or the multispecific antibody according to the third aspect of the present invention;

[0203] (2) the recombinant protein according to the fourth aspect of the present invention; or

[0204] (3) The CAR construct as described in the fifth aspect of the present invention.

[0205] In another preferred embodiment, the polynucleotide includes RNA, DNA or cDNA.

[0206] The eleventh aspect of the present invention provides a vector, wherein the vector contains the polynucleotide as described in the tenth aspect of the present invention.

[0207] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0208] The twelfth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the eleventh aspect of the present invention or the polynucleotide described in the tenth aspect of the present invention is integrated into its genome.

[0209] In a thirteenth aspect, the present invention provides a method for detecting CD28 in a sample (including diagnostic or non-diagnostic) in vitro, the method comprising the steps of:

[0210] (1) contacting the sample in vitro with the single domain antibody targeting CD28 described in the first aspect of the present invention, the antibody targeting CD28 described in the second aspect of the present invention, or the immunoconjugate described in the seventh aspect of the present invention;

[0211] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD28 in the sample.

[0212] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0213] A fourteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:

[0214] (a) culturing the host cell according to the twelfth aspect of the present invention under conditions suitable for expression;

[0215] (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is 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, or a recombinant protein as described in the fourth aspect of the present invention.

[0216] In a fifteenth aspect, the present invention provides a method for treating a CD28-related disease, the method comprising: administering to a subject in need thereof 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, a recombinant protein as described in the fourth aspect of the present invention, a recombinant immune cell as described in the sixth aspect of the present invention, an immunoconjugate as described in the seventh aspect of the present invention, or a pharmaceutical composition as described in the eighth aspect of the present invention, or a combination thereof.

[0217] In another preferred embodiment, the method further comprises: administering other drugs or treatment methods to a subject in need for combined treatment.

[0218] In another preferred embodiment, the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.

[0219] In another preferred embodiment, the anti-tumor immunotherapy drugs include PD-1 and PD-L1 monoclonal antibodies.

[0220] In a sixteenth aspect, the present invention provides an active ingredient combination, wherein the active ingredient combination comprises

[0221] First active ingredient: the single domain antibody targeting CD28 as described in the first aspect of the present invention, the antibody targeting CD28 as described in the second aspect of the present invention, the multispecific antibody as described in the third aspect of the present invention, or the recombinant protein as described in the fourth aspect of the present invention; and

[0222] The second active ingredient is an antibody or antibody fragment targeting CD3; preferably, it is a bispecific antibody or multispecific antibody against CD3.

[0223] In another preferred embodiment, the active ingredient combination comprises: the multispecific antibody as described in the multispecific antibody of the third aspect of the present invention; and an anti-CD3 bispecific antibody.

[0224] In another preferred embodiment, the active ingredient combination is used to treat CD28-related diseases; preferably tumors or cancers, autoimmune diseases, metabolic-related diseases, or a combination thereof.

[0225] In another preferred embodiment, the anti-CD3 bispecific antibody or multispecific antibody is an anti-CD3 bispecific antibody or multispecific antibody that activates T cells specific for tumor-associated antigens.

[0226] In another preferred embodiment, the anti-CD3 bispecific antibody includes but is not limited to: CD20 / CD3 bispecific antibody, DLL3 / CD3 bispecific antibody, anti-CEA / anti-CD3 bispecific antibody, or anti-MCSP / anti-CD3 bispecific antibody.

[0227] In a seventeenth aspect, the present invention provides a medicine kit, comprising:

[0228] (a) a first formulation comprising the multispecific antibody according to the third aspect of the present invention and a pharmaceutically acceptable carrier;

[0229] (b) a second formulation comprising an anti-CD3 bispecific antibody and a pharmaceutically acceptable carrier;

[0230] (c) instructions, which describe a method for treating tumors or cancer by combining the multispecific antibody according to the third aspect of the present invention and the anti-CD3 bispecific antibody.

[0231] In an eighteenth aspect, the present invention provides an in vitro non-therapeutic method for synergistically inhibiting tumor cell growth, comprising the steps of adding the active ingredient combination as described in the sixteenth aspect of the present invention to a tumor cell culture system, thereby synergistically inhibiting tumor cell growth.

[0232] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0233] Figure 1A and Figure 1B show the affinity detection of anti-CD28 recombinant single domain antibody to human CD28 protein.

[0234] Figures 2A and 2B show the affinity detection of anti-CD28 recombinant single domain antibody to CHO-human CD28 cells.

[0235] FIG3 shows the detection of the ability of anti-CD28 recombinant single domain antibody to block the binding of CD28 to CD80.

[0236] FIG4A shows the affinity detection of anti-CD28 recombinant single domain antibody to human peripheral blood mononuclear cells.

[0237] FIG4B shows the affinity detection of anti-CD28 recombinant single domain antibody to monkey peripheral blood mononuclear cells.

[0238] FIG5 shows the anti-CD28 recombinant single domain antibody fluorescence reporter cell functional activity assay.

[0239] FIG6A shows the affinity detection of anti-CD28 recombinant single domain antibody to rat CD28 protein.

[0240] FIG6B shows the affinity detection of the anti-CD28 recombinant single domain antibody to mouse CD28 protein.

[0241] FIG7 shows the affinity detection of anti-CD28 humanized single domain antibody to human CD28 protein.

[0242] FIG8A and FIG8B show the affinity detection of anti-CD28 humanized single domain antibody to Jurkat cells.

[0243] Figure 9A and Figure 9B show the affinity detection of anti-CD28 humanized single domain antibody to human peripheral blood mononuclear cells.

[0244] FIG10 shows the detection of the ability of anti-CD28 humanized single domain antibody to block the binding of CD28 to CD80.

[0245] Figure 11 shows schematic diagrams of exemplary bispecific and trispecific antibody structures, wherein TAA is a tumor-associated antigen.

[0246] Figure 12-1 shows the detection of SHP77 cell activity by combining DLL3 / CD28 bispecific antibody and DLL3 / CD3 bispecific antibody.

[0247] Figure 12-2 shows the detection of SHP77 cell activity using a combination of DLL3 / CD3 / CD28 trispecific antibody and DLL3 / CD3 bispecific antibody.

[0248] Figure 12-3 shows the activity detection of NCI-H716 cells using a combination of B7H3 / CD28 bispecific antibody and DLL3 / CD3 bispecific antibody.

[0249] Figure 13-1 shows the cytotoxicity test of target cell SHP77 by combining DLL3 / CD28 bispecific antibody and DLL3 / CD3 bispecific antibody.

[0250] Figure 13-2 shows the cytotoxicity test of DLL3 / CD28 bispecific antibody and DLL3 / CD3 bispecific antibody combined with non-target cell NCI-H358 cells.

[0251] Figure 14-1 shows that the combination of DLL3 / CD28 bispecific antibody and DLL3 / CD3 bispecific antibody inhibits tumor growth in mice

[0252] Figure 14-1 shows the effects of DLL3 / CD28 bispecific antibody and DLL3 / CD3 bispecific antibody combination on the body weight of tumor-transplanted mice

[0253] Figure 15-1 shows that DLL3 / CD3 / CD28 trispecific antibody inhibits mouse tumor growth

[0254] Figure 15-2 shows the effect of DLL3 / CD3 / CD28 trispecific antibody on the body weight of mice with transplanted tumors DETAILED DESCRIPTION

[0255] After extensive and in-depth research, the inventors unexpectedly developed for the first time a CD28 non-agonistic blocking single-domain antibody and a humanized antibody thereof. The antibody has similar affinity to traditional CD28 non-agonistic antibodies and has no activation effect on T cells under monoclonal antibody conditions, ensuring the safety of the antibody during future use. In particular, the performance of the single-domain antibody of the present invention (e.g., affinity to human peripheral blood mononuclear cells) is further improved after humanization. In addition, the inventors unexpectedly discovered that the CD28 antibody of the present invention, when used in combination with a CD3 bispecific antibody in the form of a bispecific antibody, significantly enhances the activity of the CD3 bispecific antibody. The present invention was completed on this basis.

[0256] the term

[0257] As used herein, the terms "Nanobodies of the invention," "anti-CD28 Nanobodies of the invention," and "CD28 single-domain antibodies of the invention" are used interchangeably and refer to single-domain antibodies that specifically recognize and bind to CD28 (including human CD28), particularly including antibodies that block binding of CD28 to co-stimulatory molecules (e.g., co-stimulatory molecules CD80 and / or CD86) and do not activate CD28 signaling. Particularly preferred are single-domain antibodies whose VHH chain amino acid sequences are shown in SEQ ID NOs.: 1, 2, 13, 14, 25, and 26.

[0258] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while 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 region (VH) at one end, followed by multiple constant regions. 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 the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0259] As used herein, 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. A single-domain antibody (VHH) consisting solely of a single heavy chain variable region is constructed, which is the smallest antigen-binding fragment with complete function. Typically, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting solely of a single heavy chain variable region.

[0260] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0261] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the anti-CD28 protein antibodies or fragments thereof.

[0262] As used herein, the terms "heavy chain variable region" and "V H ” can be used interchangeably.

[0263] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0264] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.

[0265] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0266] In the present invention, the terms "antibody of the present invention," "protein of the present invention," or "polypeptide of the present invention" are used interchangeably to refer to polypeptides that specifically bind to CD28 protein, such as proteins or polypeptides having a heavy chain variable region. These may or may not contain an initial methionine.

[0267] The present invention also provides other proteins or fusion expression products comprising the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) comprising a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0268] Generally, an antibody's antigen-binding properties are described by three specific regions within the heavy chain variable region, known as the variable region (CDR). This region is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0269] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in antigen binding. Thus, the present invention includes molecules having antibody heavy chain variable regions with CDRs that are 90% or more (preferably 95% or more, and most preferably 98% or more) homologous to the CDRs identified herein.

[0270] CD28 and anti-CD28 single domain antibodies

[0271] CD28 is a co-stimulatory molecule expressed on the surface of T lymphocytes. It is a type I transmembrane protein that primarily exists on the cell surface as a homodimer, with an extracellular portion containing an Ig-V-like domain. CD80 (B7.1) and CD86 (B7.2) are the two primary ligands for CD28, and CD28 is primarily activated by CD80 or CD86 expressed on antigen-presenting cells (APCs). Binding of CD28 to CD80 or CD86 provides a co-stimulatory signal that is important for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation via CD28 also provides a potent signal for the production of various interleukins. Following TCR activation, CD28 also enhances cellular signaling, such as pathways controlled by the NFκB transcription factor. CD28 co-signaling is important for efficient T cell activation, including T cell differentiation, proliferation, cytokine release, and cell death.

[0272] Researchers have proposed the use of anti-CD28 antibodies for therapeutic purposes involving T cell activation. CD28 agonistic antibodies can be divided into two categories: (i) CD28 superagonist antibodies (CD28SA) and (ii) CD28 conventional agonist antibodies. CD28 superagonists are CD28-specific monoclonal antibodies that can autonomously activate T cells without significant T cell receptor engagement. CD28SA antibodies have therapeutic effects in various autoimmune, inflammatory, and transplant models. However, a Phase I study of the human CD28SA antibody TGN1412 caused a life-threatening cytokine storm in 2006. TGN1412 (an anti-CD28 superagonist) was administered intravenously to six healthy volunteers at a dose of 0.1 mg / kg. Within two hours, all six patients had a significant inflammatory response (cytokine storm), and within sixteen hours, all patients developed multi-organ failure. In the Phase I study (associated with CRS), the starting dose of 0.1 mg / kg was based on a 500-fold multiple of the "NOAEL" of 50 mg / kg in macaques. Unfortunately, TGN1412 induced a cytokine storm that was not well predicted in preclinical toxicology studies in cynomolgus macaques or by ex vivo human PBMC studies.

[0273] Conventional agonist antibodies to CD28 (such as clone 9.3) mimic the natural ligand of CD28 and can enhance T cell activation only in the presence of a T cell receptor signal (signal 1). Published insights indicate that the binding epitope of the antibody has a significant impact on whether the agonist antibody is a superagonist or a conventional agonist. The superagonist TGN1412 binds to the side motif of CD28, while the conventional agonist molecule 9.3 binds tightly to the ligand binding epitope. Due to the difference in binding epitopes, the ability of superagonist antibodies and conventional agonist antibodies to form linear complexes of CD28 molecules on the surface of T cells is also different. To be precise, TGN1412 is able to effectively form linear arrays of CD28, which probably results in aggregated signaling components sufficient to exceed the threshold of T cell activation. On the other hand, the conventional agonist 9.3 results in a complex whose structure is not linear. Attempts to convert conventional agonist binders based on the 9.3 clone using recombinant bispecific single-chain antibodies against melanoma-associated proteoglycans and CD28 have been previously disclosed. Based on the intrinsic tendency of bispecific single chain antibodies to form multimeric constructs, the reported bispecific single chain antibodies were reported to exert "superagonistic" activity despite the use of the conventional CD28 agonistic binder 9.3.

[0274] It has been found that when a limited amount of anti-CD3 bispecific antibodies (i.e., T cell bispecific antibodies such as PSMA-CD3 and MUC1-CD3) are combined with agonistic anti-CD28 molecules, better T cell activation and anti-tumor killing activity can be achieved. Given the baseline expression of CD28 on T cells in various tumor indications, and the activation of CD28 signaling enhances T cell receptor signaling, it is expected that this combination of CD3 bispecific antibody molecules or immune checkpoint inhibitors with tumor-targeted CD28 molecules can act synergistically to induce strong and long-lasting anti-tumor responses.

[0275] As used herein, "CD28 activity" refers to the activity resulting from or involving the binding of CD80, CD86, and / or another ligand to CD28, and includes, but is not limited to, activation of CD28-mediated cell signaling. CD28 activity also includes inducing T cell proliferation and inducing T cell secretion of cytokines, such as interleukin 2 (IL-2).

[0276] The cell signal transduction of CD28 mediation is included in one or more cellular events induced directly or indirectly in immune cells, and it expresses CD28 on its surface by ligand binding of activation (such as cross-linking) cell surface CD28.In appropriate cases, the signal transduction of CD28 mediation causes the rise of immune cell to immune response.Blocking the signal transduction of CD28 mediation causes the downward regulation of immune cell immune response.Be combined with CD28 to effectively block the signal (for example, by blocking ligand binding) of CD28 mediation without the reagent of self-activation CD28 receptor (for example, by the aggregation of receptor) will effectively block the signal transduction of CD28 mediation.Preferably, reagent specifically blocks the signal transduction of CD28 mediation, i.e. blocks the signal transmitted by CD28, and does not block the signal transmitted by another cell surface molecule such as CTLA4. Preferably, the reagent includes the CD28 single-domain antibody or a humanized antibody thereof of the present invention, which has the characteristics of specifically recognizing CD28 and blocking the ability of CD28 to bind to CD80 ligand, without binding to other immune checkpoint molecules of the CD28 family (such as PDL1, PDL2, CTLA4, B7-1, B7-2, B7H3, B7H4, B7H5, B7H6, B7H7).

[0277] As used herein, the term "substantially non-agonistic" refers to a given agent, such as an anti-CD28 single domain antibody, that substantially inhibits one or more CD28-mediated activities, wherein the term "activation" is as defined herein. Specifically, a "substantially non-agonistic" agent 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 of the activity activated by CD80 and / or CD86 binding to CD28, including zero activation (no activation). As a non-limiting example, the CD28 activity excited by the single domain antibody described herein that does not substantially activate CD28 activity is no more than 5% of the activity obtained by stimulating CD28 activity under otherwise identical assay conditions by anti-CD28 mAb 9.3 (Gibson, et al. (1996) JBC, 271: 7079-7083).

[0278] As used herein, the terms "activate" and "activation" refer to an increase of a given measurable activity relative to a reference by at least 5%, such as at least 10%, 25%, 50%, 75%, or even 100% or more.

[0279] As used herein, the term "inhibit" refers to a decrease of at least 10% in a given measurable activity (e.g., binding activity) relative to a reference. Where 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., complete inhibition or loss of a given activity. Inhibition of CD28 binding to CD80 or CD86 can be measured as described in the working examples herein. As used herein, "inhibit binding" when referring to domain antibodies binding to CD28, or CD80 binding to CD28, or CD86 binding to CD28 refers to a decrease in binding of at least 10% relative to a reference. "Inhibit binding" refers to a decrease in binding of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including 100%.

[0280] As used herein, "Chothia", "Kabat", "IMGT", and "AbM" refer to the determination of complementary binding determining regions (CDRs) under the above-mentioned different assignment systems. The 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, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International Immuno GeneTics database (IMGT), and Chothia definitions based on loop structural positions.

[0281] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0282] Preferably, any one of the above amino acid sequences further comprises a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and is capable of retaining binding affinity to CD28.

[0283] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0284] Preferably, the antibody has the ability to inhibit the catalytic function of cell surface and recombinant CD73 protease, and the antibody can be rapidly internalized into lysosomes by cells.

[0285] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a human-animal chimeric antibody, and preferably a humanized antibody.

[0286] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other antibody derivatives known in the art, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0287] Wherein, the animal is preferably a mammal, such as a mouse or a camel.

[0288] In a preferred embodiment, the present invention discloses a variety of high-specificity and high-affinity Nanobodies targeting CD28, which only include heavy chains, and the heavy chains contain heavy chain variable region (VHH) amino acid sequences listed in Table 11.

[0289] Recombinant protein (or fusion protein)

[0290] In the present invention, a recombinant protein (or fusion protein) containing the CD73 single domain antibody of the present invention is also included. A preferred fusion protein is a multispecific antibody, which 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, Trop2, FGL1, LFA-3, 2B4, 5T4, α-4 integrin, α-V integrin, α4β7 integrin, α4β7 integrin, α-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, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD 51. CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD107a, CD117, CD123, CD12 5. CD132(IL-2Rg), CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CEACAM5(CEA), CEACAM6(NC A-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), FSP-1, GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIlb / IIIa receptor, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL -12R, IL-12Rβl, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PS GR, RAAG12, RAGE, SLC44A4, Siglecl5, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TL R6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, or a combination thereof.

[0291] Preferably, the multispecific antibody comprises one or more second antigen-binding regions, or further comprises a third antigen-binding region.

[0292] In addition, the recombinant protein (or fusion protein) of the present invention, in addition to containing the CD28 single-domain antibody of the present invention, may also include an optional tag sequence that assists in expression and / or purification (e.g., a 6His tag, a GGGS sequence, a FLAG tag); or an optional polypeptide molecule or fragment with therapeutic function; or an optional protein functional domain that assists in physicochemical or pharmaceutical functions (e.g., a molecule that can extend the in vivo half-life of a single-domain antibody, such as HLE or HSA).

[0293] For example, a molecule that increases half-life in an organism may be selected from the group consisting of:

[0294] Proteins from the extracellular matrix; for example, collagen, laminin, integrin, and fibronectin. Collagen is the main protein of the extracellular matrix. There are currently about 15 known types of collagen molecules, which are found in different parts of the body, such as type I collagen (which represents 90% of the collagen in the body) found in bones, skin, tendons, ligaments, corneas, and internal organs, or type II collagen found in cartilage, intervertebral discs, spinal cord, and the vitreous humor of the eye.

[0295] Proteins from the 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;

[0296] Enzymes and inhibitors such as plasminogen, lysozyme, cystatin C, alpha-1-antitrypsin, and pancreatic trypsin inhibitor. Plasminogen is the inactive precursor of the trypsin-like serine protease plasmin. It is normally found circulating through the bloodstream. When plasminogen becomes activated and converts to plasmin, it unfolds its powerful enzymatically active domain and dissolves the fibrinogen fibers that entangle blood cells in a blood clot. This is called fibrinolysis;

[0297] Immune system proteins such as IgE, IgG, and IgM.

[0298] 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 the antibodies.

[0299] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0300] The antibodies of the present invention refer to polypeptides that have CD28 protein binding activity and include the above-mentioned CDR regions. The term also includes variant forms of polypeptides that have the same function as the antibodies of the present invention and include the above-mentioned CDR regions. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not change the function of the protein. For another example, addition of one or more amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0301] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0302] The present invention also provides other polypeptides, such as fusion proteins comprising single-domain antibodies or fragments thereof. In addition to substantially full-length polypeptides, the present invention also encompasses fragments of the single-domain antibodies of the invention. Typically, the fragment comprises 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.

[0303] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0304] Table A

[0305] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0306] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0307] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0308] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0309] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0310] Antibody preparation

[0311] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form a single-chain antibody.

[0312] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0313] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0314] Currently, DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

[0315] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0316] 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 and HEK-293 cells.

[0317] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0318] The resulting monoclonal antibodies can be characterized using conventional methods. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, using the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0319] The antibodies of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated 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 renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0320] Immunoconjugates

[0321] The present invention also provides an immunoconjugate (ADC) based on the antibody of the present invention, preferably a single-domain antibody-drug conjugate (NDC).

[0322] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0323] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0324] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly linked to antibodies through linkers.

[0325] Single-domain antibodies can be conjugated to drugs to form antibody-drug conjugates (NDCs). Typically, NDCs contain a linker positioned between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers typically readily degrade in the intracellular environment, for example, at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. Peptide linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0326] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0327] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0328] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0329] In the present invention, drug-linkers can be used to form NDCs in a single step. In other embodiments, bifunctional linker compounds can be used to form NDCs in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug to form an NDC.

[0330] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached 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 alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, when a complementary pair of reactive functional groups is selected for selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0331] The present invention also provides a method for preparing NDC, which may further comprise: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (NDC).

[0332] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0333] In some embodiments, the structure of the immunoconjugate, preferably a single domain antibody drug conjugate (NDC), is shown in the following molecular formula:

[0334] in:

[0335] nAb is a single domain antibody targeting CD28, an antibody targeting CD28, or a multispecific antibody.

[0336] LU is the linker / connector;

[0337] D is for medicine;

[0338] And the subscript p is a value selected from 1 to 10.

[0339] application

[0340] The present invention also provides uses of the antibodies of the present invention, such as for preparing diagnostic agents or medicaments for preventing and / or treating CD28-related diseases, including tumor development, growth, and / or metastasis, tumor resistance-related diseases, inflammation, and metabolic diseases.

[0341] The uses of the antibodies, NDCs, or CAR-Ts of the present invention include (but are not limited to):

[0342] (i) Diagnosis, prevention and / or treatment of tumor occurrence, growth and / or metastasis, especially tumors with high CD28 expression. Such 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, colorectal 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, malignant glioma, more preferably triple-negative breast cancer and / or non-small cell lung cancer.

[0343] (ii) Diagnosis, prevention and / or treatment of autoimmune diseases, including but not limited to systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, and multiple sclerosis.

[0344] (iii) diagnosing, preventing and / or treating inflammation, including but not limited to rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Reiter's syndrome, psoriatic arthropathy, infectious arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis.

[0345] (iv) diagnosing, preventing and / or treating metabolic diseases, including but not limited to diabetes, diet-induced obesity and adipose inflammation.

[0346] Pharmaceutical composition

[0347] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its NDC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0348] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0349] The pharmaceutical composition of the present invention can be used to directly bind to CD28 protein molecules, and thus can be used to prevent and treat diseases such as tumors. In addition, other therapeutic agents can also be used simultaneously.

[0350] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0351] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0352] For NDC, since the single domain antibody-drug conjugate provided by the present invention can target a specific cell population and bind to a specific cell surface protein (antigen), thereby releasing the drug into the cell in an active form through endocytosis of the conjugate or drug penetration, 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 (e.g., a human) in a therapeutically effective amount through an appropriate route. The subject in need of treatment can be a patient who is at risk or suspected of having a disease related to the activity or expression of a specific antigen. Such patients can be identified through routine physical examinations.

[0353] When treating with the single domain antibody-drug conjugates of the present invention, delivery can be performed by conventional methods in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or vector can be delivered locally by direct injection or by using an infusion pump.

[0354] The main advantages of the present invention include

[0355] (1) The single-domain antibody of the present invention has similar affinity to traditional CD28 super-agonist antibodies, has no activation effect on T cells when present alone (non-agonist antibody), and has good safety.

[0356] (2) The single-domain antibody of the present invention has a molecular weight of only 12.5 kDa and has better tissue penetration ability than traditional CD28 antibodies.

[0357] (3) The single-domain antibody of the present invention can be constructed into bispecific / multispecific antibodies with other traditional tumor-targeting antibodies or single-domain antibodies, greatly enhancing the diversity of future drug development and the potential for combined drug use.

[0358] (4) The bispecific or multispecific antibodies constructed from the single-domain antibodies of the present invention significantly enhance the activity of CD3 bispecific antibodies. In vivo animal experiments suggest that the above combination can significantly inhibit tumor growth.

[0359] (5) The bispecific antibody or multispecific antibody constructed from the single-domain antibody of the present invention has good stability.

[0360] Abbreviations

[0361] CDR: complementarity determining region

[0362] VHH: single heavy chain variable region

[0363] Kabat: Immunoglobulin alignment and numbering system proposed by Elvin A. Kabat

[0364] IMGT: numbering system based on the International Immunogenetics Information System initiated by Lefranc et al.

[0365] EC50: Half-maximal effect concentration, the concentration that can cause 50% of the maximum effect

[0366] ELISA: enzyme-linked immunosorbent assay

[0367] PCR: polymerase chain reaction

[0368] HRP: horseradish peroxidase

[0369] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0370] Example 1: Preparation of anti-CD28 single domain antibody

[0371] 1.1 Material preparation:

[0372] In order to prepare the antigen protein required for immunization and screening evaluation, the extracellular domain of human CD28 antigen (Asn19-Pro152, Uniport ID: P10747) was constructed into fusion proteins 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), respectively. The proteins were transiently transfected into 293EBNA cells, and after 7 days of expression, the target proteins in the supernatant were purified by Protein A.

[0373] To meet the screening and evaluation requirements during the discovery of anti-CD28 single-domain antibodies, CHO-S cells were modified and the CD28-overexpressing cell line, CHO-S-CD28, was constructed via lentiviral infection. The full-length amino acid sequence of the human CD28 protein (Uniprot ID: P10747) was codon-optimized by Universal Genetics. The optimized nucleotide sequence was then synthesized into the pLVX-Puromycin vector. The synthesized human CD28-expressing lentiviral plasmid was then synthesized using standard viral packaging procedures. The viral supernatant was then added to CHO-S cells for infection by centrifugation. After pressure treatment with puromycin, the CHO-S-hCD28 stable cell line was generated. Expression in the stable cell line was confirmed by flow cytometry.

[0374] The anti-CD28 humanized single-domain control antibody C1 used in the present invention has a sequence derived from Mab (UNII: LCT264LTYE); the control antibody amino acid sequence was synthesized by Universal Genetics into PTT5-hFc, expressed by transient transfection of CHO-E cells, and obtained by ProA purification 7 days later.

[0375] 1.2 Alpaca immunization and phage library construction

[0376] To induce a humoral immune response against human CD28 in camelids, a human CD28 antigen protein fused to Llama Fc (Sichuan Sibowo Biotechnology Co., Ltd.) was mixed with an equal volume of Freund's adjuvant. Healthy adult alpacas were subcutaneously injected with 0.2-0.5 mg of the antigen. Serum was collected after multiple immunizations, and the antigen titer was determined by ELISA. 50 ml of blood samples were collected 6-7 days after the last two injections. Peripheral blood mononuclear cells (PBMCs) were isolated and purified by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Total RNA was extracted from PBMCs using Trizol reagent, and reverse transcribed into cDNA using oligo-dT and random primers using the SuperScript III First-Strand Synthesis SuperMix System (Invitrogen). A complete VHH repertoire was amplified using VHH gene-specific primers, and the PCR product was ligated into a phage vector.

[0377] 1.3 Phage library panning

[0378] To select single-domain antibodies that effectively bind to CD28, multiple rounds of protein panning were performed. Human CD28 antigen was directly coated onto an ELISA plate and incubated at 4°C overnight. After coating, the plate was washed once with PBST, and 300 μl of 2% BSA blocking buffer was added to each well and blocked at 37°C for 2 hours. The phage library was resuspended in 1 ml of 2% BSA, incubated at 37°C for 30 minutes, added to the ELISA-labeled wells, and incubated at 37°C for 60 minutes. The supernatant was discarded. The plate was washed six times with 0.1% PBST, and 800 μl of Gly-HCl eluate was added and incubated at 37°C for 10 minutes. The eluate was transferred to a 1.5 ml sterile centrifuge tube and quickly neutralized with 120 μl of Tris-HCl, typically at room temperature for 30 minutes. The phage library obtained in the first round of panning was amplified using TG1 cells and then enriched in the second round. The eluted library from the second round was serially diluted, and 10 μl of the diluted phage was added to 90 μl of TG1 culture medium and incubated at 37°C for 30 minutes. 100 μl of each aliquot was spread onto a 2×YT plate (containing Amp and 2% Glu) and incubated at 37°C overnight.

[0379] 1.4 ELISA identification of anti-CD28 single domain antibody positive clones

[0380] From each plate containing the second-round panning eluate, single colonies were randomly selected using a sterile pipette tip and inoculated into 400 μL of 2×YT-A medium. The cells were cultured at 37°C with shaking at 220 rpm for 4 hours. To this, 200 μL of the above culture was added with M13K07 phage at a cell:phage ratio of 1:20. The cells were allowed to stand at 37°C for 15 minutes and then incubated with shaking at 220 rpm for 45 minutes. An additional 200 μL of 2×YT-AK was added and the cells were cultured overnight at 30°C with vigorous shaking. The next day, the cells were centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected for single-clone ELISA analysis. Human CD28 protein was diluted to 1 μg / mL in CBS buffer and added to a 96-well microtiter plate. The plates were incubated at 4°C overnight. After blocking and washing, 50 μL of the E. coli supernatant was transferred to the coated plate. 50 μL of 2% BSA was added to each well and the cells were incubated at 37°C for 2 hours. The plate was then washed three times with PBST buffer, followed by the addition of HRP-conjugated anti-M13 antibody (100 μL / well) and incubation at 37°C for 1 hour. The plate was then washed five times with PBST and developed with TMB colorimetric solution (100 μL / well) at 37°C for 7 minutes. The reaction was terminated with 2M H₂SO₄ (50 μL / well), and the optical density was measured at 450 nm. A threshold of 3 times the reading in negative wells was used as the screening threshold to identify clones that bind to the CD28 protein and proceed to subsequent characterization.

[0381] 1.5 FACS identification of anti-CD28 single domain antibody positive clones

[0382] In order to evaluate whether the positive clones that bind to CD28 recombinant protein identified by ELISA bind to CD28 protein on the cell membrane surface, we used flow cytometry to analyze the supernatant of the clones. The general process includes: CHO-S-hCD28 cells in the logarithmic growth phase are centrifuged at 500g for 3 minutes to collect the cells, washed twice with PBS, resuspended in 1% BSA, and transferred to a 96-well pointed bottom plate, 50ul per well; phage supernatant identified as positive clones by ELISA is added to the cell wells, 50ul per well, and incubated at 4 degrees for 50 minutes; cells are washed twice with PBS, centrifuged at 500g for 3 minutes, and cells are resuspended in 1% BSA solution (containing biotinylated M13 antibody, Sino Biological #11973-MM05T-B), 100ul per well, incubated at 4 degrees for 40 minutes, and PBS is added. Cells were washed twice, centrifuged at 500g for 3 minutes, and resuspended in 1% BSA solution (containing FITC-SA, Biolegend #405202). 100 μl of solution was added to each well (containing 1 μl of fluorescent secondary antibody) and incubated at 4°C for 30 minutes. Cells were then washed three times with PBS, centrifuged at 500g for 3 minutes, and resuspended in 400 μl of PBS. Fluorescence signal was measured by flow cytometry. The results in Table 1 indicate that, with the exception of clone A04#16, which did not bind to CHO-S-hCD28 cells, the supernatants of the remaining 15 clones all showed varying degrees of binding activity to CHO-S-hCD28 cells.

[0383] Table 1. Anti-CD28 single domain antibody binding analysis of CHO-S-hCD28 cells

[0384] Example 2: Evaluation of anti-CD28 single domain antibodies

[0385] 2.1 Expression of anti-CD28 recombinant single-domain antibody

[0386] The anti-CD28 positive monoclonal clone obtained by flow cytometry identification in Example 1.5 was cloned into the PTT5-hFc vector by PCR recombination. After the plasmid construction was completed, CHO-E cells were transiently transfected. 293-EBNA cells in good growth state and in the logarithmic phase were adjusted to a density of 2.5×10 6The cells were then cultured in a CO2 shaker for 10 minutes. Twenty-four hours after transfection, 1 / 5 volume of OPM-293CD05 medium (Shanghai Aopuma Biotech Co., Ltd.) was added, supplemented with an anti-aggregating agent at a final concentration of 0.5%. Forty-eight hours after transfection, 4% volume of OPM-293ProFeed (Shanghai Aopuma Biotech Co., Ltd.) was added, and feed was added every other day. Cell status was monitored daily. After 7 days of culture, the cell culture supernatant was harvested by centrifugation. The antibody was purified by affinity chromatography (Protein A) using Mab Select SuRe (GE Biosystems).

[0387] 2.2 Quality testing of anti-CD28 recombinant single domain antibody

[0388] An anti-CD28 recombinant single-domain antibody was obtained as described in Example 2.1, and its purity was determined by SEC. The purity of the preferred recombinant anti-CD28 single-domain antibody of the present invention was determined using a Tosoh size exclusion chromatography column (TSKgel G3000SWXL) on an Agilent HPLC instrument with a mobile phase of 100 mM Na₂HPO₄, 100 mM NaCl, pH 7.0, and 5% IPA. Instrument parameters were set to: sample chamber temperature: 8°C, column temperature: 30°C, flow rate: 0.5 ml / min, and detection wavelength: 280 nm. The sample to be tested was diluted with ddH₂O as the mobile phase to a final concentration of 1 mg / ml, and 20 μl of the sample was injected for purity analysis. The specific results are shown in Table 2. The purities of A04#1, A04#11, A04#12, and A04#14 anti-CD28 recombinant single-domain antibodies were all lower than 90%, while the purities of the remaining A04#2, A04#3, A04, A04#5, A04#6, A04#7, A04#8, A04#9, A04#13, and A04#15 anti-CD28 recombinant single-domain antibodies were all higher than 97%, and they could be used for further evaluation.

[0389] Table 2. SEC purity analysis of anti-CD28 recombinant single domain antibody

[0390] 2.3 ELISA affinity determination of anti-CD28 recombinant single domain antibody and human CD28 protein

[0391] In order to accurately quantify the binding activity of anti-CD28 recombinant single-domain antibodies with CD28 recombinant protein, we used ELISA to quantitatively detect all antibodies. First, human CD28-LIamaFc protein was diluted with CBS coating solution to a final concentration of 1 μg / ml, 100 ng / well, and coated overnight at 4°C; the experiment was continued on the next day, and 300 μl PBST was used for washing once; 100 μl 2% BSA was added to each well and blocked at 37°C for 2 hours; the CD28 recombinant antibody to be tested was diluted with 2% BSA, and the starting concentration of the anti-CD28 recombinant single domain antibody was 26.6 nM, 3-fold gradient, 12 concentration points, duplicate wells, mixed and placed at 37°C for incubation for 2 hours; the blocking solution was discarded, and 300 μl PBST was used for washing 3 times; 100 μl of HRP-Goat-Anti-human IgG (jackson immunoresearch) diluted in 2% BSA was added to the corresponding wells and incubated at 37°C for 1 hour; 300 μl PBST was used for washing 5 times; 100 μl TMB (Biopanda) was added to the corresponding wells for color development; 50 μl 2N The reaction mixture was terminated with H₂SO₄ and read on a microplate reader at 450 nm. GraphPad Prism software was used to perform nonparametric fitting of the absorbance values ​​at 450 nm corresponding to different antibody concentrations. The results are shown in Figures 1A and 1B. The specific affinities are shown in Table 3. A04#2, A04#3, A04#5, A04#6, A04#09, and A04#13 exhibited high affinities for human CD28-LlamaFc, below 1 nM.

[0392] Table 3. Affinity of anti-CD28 recombinant single domain antibodies and human CD28-LlamaFc

[0393] 2.4 Affinity determination of anti-CD28 recombinant single-domain antibody and CHO-S-hCD28 cells

[0394] In order to detect the affinity of anti-CD28 recombinant antibodies to the target protein CD28 on the cell surface, flow cytometry was used to perform affinity detection on the antibodies to be tested. First, CHO-S-hCD28 cells were collected by centrifugation, washed twice with PBS, and resuspended in 8X10 6 / ml density, 50ul / well, recombinantly expressed CD28 antibody was diluted with 1% BSA, starting at 100ug / ml, 3-fold gradient, added to the cell wells, 50ul per well, mixed, and incubated at 4°C for 50min; washed twice with PBS, and the cells were finally resuspended with 1% BSA solution (containing PE anti-human IgG), 100ul per well (containing 1ul fluorescent secondary antibody), mixed, reacted at 4°C in the dark for 0.5 hour, washed twice with PBS, and the fluorescence signal value and positive rate were detected by flow cytometry. The fluorescence signal values ​​corresponding to different antibody concentrations were curve fitted using GraphPad Prism software. The results are shown in Figures 2A and 2B . The specific affinities are shown in Table 4 . A04#2, A04#3, A04#6, and A04#13 had high affinities for CHO-S-hCD28 cells, below 5 nM, while A04#4, A04#5, A04#7, A04#8, and A04#15 had affinities for CHO-S-hCD28 cells between 10 and 50 nM, and A04#9 bound weakly to CHO-S-hCD28 cells.

[0395] Table 4. Affinity of anti-CD28 recombinant single domain antibodies and CHO-S-hCD28

[0396] 2.5 Determination of ligand blocking activity of anti-CD28 recombinant single-domain antibody

[0397] CD28 has two main ligands in the human body, namely the high-affinity ligand CD80 and the low-affinity ligand CD86. The two ligands bind to the same position on CD28, so we used a competitive ELISA method to study the ligand blocking activity of anti-CD28 recombinant single-domain antibodies. CD80-mFc (ACROBiosystems) protein was diluted to 1ug / ml with CBS coating buffer, and 100ul was added to each well of a 96-well microplate. Incubate at 4℃ overnight; drain the coating solution and wash once with PBST washing buffer; drain the microplate and add 100ul to each well. 2% BSA (PBS) blocking solution, blocked in a 37°C incubator for 2 hours; CD28-hFc-biotin was diluted to 2ug / ml with BSA diluent, and this was used as the diluent for diluting the anti-CD28 recombinant antibody, starting from 1uM, diluted 3-fold, and 100ul per well was added to each well of a 96-well plate; the liquid in the wells was drained and the cells were washed three times with PBST washing buffer; HRP-conjugated SA secondary antibody (Proteintech) was diluted 1:10000 with 2% BSA, and 100ul per well was added to each well of a 96-well plate; the liquid in the wells was drained and the cells were washed five times with PBST washing buffer; 100ul of color development solution was added for several minutes, and then 50ul of 2N HCl was added to terminate the reaction; the OD450nm reading was read on a microplate reader, and the absorbance values ​​at 450nm of different concentrations were curve fitted using GraphPad Prism software.

[0398] The results are shown in Figure 3 and Table 5. Among them, A04#2 and A04#4 had the strongest ability to block the binding of CD28 and CD80, which was less than 10nM, while A04#3, A04#5, A04#6, A04#7, A04#8, A04#13, and A04#15 had the ability to block CD28 and CD80 between 10-30nM, and A04#9 had the weakest ability to block CD28 and CD80.

[0399] Table 5. Anti-CD28 recombinant single domain antibodies block the binding of CD28 to CD80

[0400] 2.6 Affinity determination of anti-CD28 recombinant single-domain antibody and human monkey PBMC (peripheral blood cells)

[0401] To test the affinity of the anti-CD28 recombinant antibody for the target protein CD28 on the surface of human natural cells, as well as its cross-reactivity and affinity with the target protein CD28 on the surface of monkey natural cells, human peripheral blood mononuclear cells (hPBMC) were obtained by Ficoll separation from peripheral blood collected from healthy volunteers, and monkey peripheral blood mononuclear cells (cynoPBMC) were obtained by Ficoll separation from peripheral blood collected from 3-5 year old cynomolgus macaques. PBMC were collected by centrifugation, washed twice with PBS, and resuspended in 1% BSA buffer. Human TruStain FcX (Biolegend) was added to the hPBMC cell suspension and incubated at room temperature for 20 minutes. After incubation, the cell suspension was added to a 96-well conical bottom plate, 50 μl per well. Recombinant CD28 antibody was diluted in 1% BSA, starting at 100 μg / ml, in a 5-fold gradient. 50 μl was added to the cell wells, mixed, and incubated at 4°C for 50 min. The cells were washed twice with PBS. Finally, the cells were resuspended in 1% BSA solution (containing PE anti-human IgG), 100 μl per well (containing 1 μl of fluorescent secondary antibody), mixed, and reacted at 4°C in the dark for 0.5 h. The cells were washed twice with PBS. Fluorescence signal and positive rate were measured by flow cytometry. Fluorescence signal values ​​corresponding to different concentrations were curve fitted using GraphPad Prism software.

[0402] The results are shown in Figures 4A and 4B and Table 6. A04#2 and A04#6 have the strongest affinity for human PBMC cells, below 30 nM, while A04#3 and A04#5 have the strongest affinity for human PBMC cells, between 40-100 nM, and A04#3 has the weakest affinity; A04#2, A04#3, A04#5 and A04#6 all have strong affinity for monkey PBMC.

[0403] Table 6. Affinity of anti-CD28 recombinant single domain antibodies to human and monkey PBMC

[0404] 2.7 In vitro functional assay of anti-CD28 recombinant single-domain antibody

[0405] The first clinical trial of the CD28 antibody TGN1412 caused severe systemic inflammatory reactions in all volunteers, and 4 of them developed multiple organ failure. This side effect comes from the rapid and large-scale release of cytokines induced by TGN1412. Therefore, it is very important to study the T cell activation properties of CD28 antibodies. The CD28 recombinant antibody to be tested (OKT3 antibody as a control) was diluted to 200nM with 1640+10% FBS, and then diluted 3 times to 12 concentration points. Then 50ul of each sample was added to a 96-well white plate (Corning). The same volume of culture medium was added to the negative control wells, and all wells were set up in duplicate. Jurkat-NFAT-Luciferase cells (Sichuan Sibowo Biotechnology Co., Ltd.) were diluted to 1*10 with 1640+10% FBS. 6 / ml, 50 μl of cell suspension was added to the sample well and blank well, and incubated at 37°C for 6 hours. After 6 hours, 20 μl of One-Glu (Promega) was added to each well. Fluorescence signals were read on a microplate reader, and curve fitting of the fluorescence signals corresponding to different concentrations was performed using GraphPad Prism software.

[0406] The results are shown in Figure 5, indicating that A04#2, A04#3, A04#5 and A04#6 were unable to activate T cells.

[0407] 2.8 Determination of cross-reactivity of anti-CD28 recombinant single-domain antibody with rat and mouse CD28 proteins

[0408] In order to explore whether the anti-CD28 recombinant antibody of the present invention has cross-reactivity with rat and mouse CD28 proteins, we used ELISA to study this issue. First, rat CD28 and mouse CD28 proteins were diluted with CBS coating solution to a final concentration of 1 μg / ml, 100 μL / well, and incubated at 4°C overnight; the experiment was continued the next day, and the cells were washed once with 300 μl PBST; 100 μl 2% BSA was added and blocked at 37°C for 2 hours; the CD28 antibody to be tested was diluted with 2% BSA, starting at 5 ug / ml, with a 3-fold gradient and 12 concentration points, in duplicate wells, mixed and incubated at 37°C for 2 hours; the blocking solution was discarded and the cells were washed three times with 300 μl PBST; 100 μl of HRP-Goat-Anti-human IgG (Jackson Immunoresearch) diluted with 2% BSA was added to the corresponding wells and incubated at 37°C for 1 hour; 300 μl PBST was washed five times; 100 μl TMB was added to the corresponding wells for color development; 50 μl 2N H2SO4 was added to terminate the reaction and the plate was read at 450 nm on a microplate reader. The 450 nm signal values ​​at different concentrations were fitted nonparametrically using GraphPad Prism software, and the results are shown in Figures 6A and 6B .

[0409] The results showed that A04#2, A04#3, A04#5 and A04#6 had no significant binding to rat CD28 or mouse CD28.

[0410] Example 3: Evaluation of anti-CD28 humanized single domain antibodies

[0411] 3.1 Sequencing of anti-CD28 recombinant single-domain antibody

[0412] The clones that specifically bound to CD28 protein evaluated by recombinant expression were sequenced to obtain the corresponding anti-CD28 single-domain antibody nucleotide sequences, which were then translated into the corresponding amino acid sequences. See the sequence listing for A04#2 (Seq. ID NO: 1), A04#3 (Seq. ID NO: 13), and A04#6 (Seq. ID NO: 25).

[0413] 3.2 Humanization of anti-CD28 single-domain antibody

[0414] Camel-derived single-domain antibodies (ADCs), A04#2, A04#3, and A04#6, were humanized using a CDR-grafted antibody humanization method. Briefly, humanization involves the following steps: The amino acid sequences of the ADCs were compared with those of human germline antibodies to identify sequences with high homology and superior physicochemical properties as human germline framework sequences; HLA-DR affinity was analyzed to select human germline framework sequences with low affinity; and the CDRs of the ADCs were then transplanted onto the selected heavy chain framework sequences.

[0415] Specifically, the CDR regions of camelid antibodies A04#2, A04#3, and A04#6 were transplanted onto the FR frameworks of their corresponding humanized templates. The humanized template for A04#2 was based on the human germline gene sequence IGHV3-66*01; the humanized templates for A04#3 and A04#6 were based on the human germline gene sequence IGHV3-23*01.

[0416] Furthermore, computer simulations and molecular docking were used to analyze the variable region and its surrounding framework amino acid sequences to examine their spatial binding patterns. By calculating electrostatic forces, van der Waals forces, hydrophilicity, and entropy, key amino acids within the camel antibody amino acid sequence that interact with CD28 and maintain the spatial framework were identified. These camelid amino acids were then retained in the transplanted antibody. Specifically, a series of back mutations were performed on the amino acid residues in the FR region of the humanized template to ensure that the humanized antibody retained the antigen-binding ability of the camel antibody as much as possible.

[0417] Using the above method, one humanized antibody, A04#2-Hz, was constructed based on the CDRs of camelid single-domain antibody A04#2. Another humanized antibody, A04#3-Hz, was constructed based on the CDRs of camelid single-domain antibody A04#3. Another humanized antibody, A04#6-hz, was constructed based on the CDRs of camelid single-domain antibody A04#6.

[0418] The amino acid sequences of the variable and constant regions of the above humanized antibodies are shown in Table 7.

[0419] Table 7. Anti-human CD28 humanized single domain antibody variable region and CDR amino acid sequences

[0420] 3.3 Recombinant Expression of Humanized Anti-CD28 Single Domain Antibody

[0421] The amino acid sequence of the humanized anti-CD28 single-domain antibody was synthesized by universal gene synthesis into the PPT5 expression vector, and all synthesized plasmids were confirmed by sequencing. The correctly sequenced humanized antibody expression plasmid was transiently transfected into 293EBNA cells in the manner of Example 2.1 for recombinant expression of the antibody. The 293EBNA cell culture expressed for 7 days was first centrifuged at a low speed of 1000g for 15 minutes to separate the supernatant and the cell pellet; then centrifuged at a high speed of 8000g for 20 minutes to obtain a clarified liquid. The antibody was purified by affinity chromatography (Protein A), and all the purified antibodies were ultrafiltered and replaced into PBS buffer and maintained at -80C.

[0422] 3.4 Dynamic affinity determination of anti-CD28 humanized recombinant single-domain antibody and human CD28 protein

[0423] The dynamic affinity of the humanized CD28 single domain antibody for antigen binding was determined using a method similar to that of Example 2.4. The results in Table 8 indicate that the affinity of the humanized single domain antibody was not significantly affected compared to that before humanization, and the affinity level was in the nM range.

[0424] Table 8. Dynamic affinity of humanized CD28 single domain antibodies binding to CD28 protein

[0425] 3.5 ELISA affinity determination of anti-CD28 humanized recombinant single domain antibody and human CD28 protein

[0426] To analyze the target protein binding activity of the recombinantly expressed anti-CD28 humanized single-domain antibody, hCD28-His protein (Sinobiological) and hCD28-Llama Fc (Chengdu Sibowo Biotechnology Co., Ltd.) were first added to a 96-well ELISA plate (Thermofisher) at 100 ng / well and coated overnight at 4°C. The next day, the solution in the wells was discarded and washed once with wash buffer. PBS solution containing 2% BSA was added at 100 μl / well and blocked at 37°C for 2 h. The recombinantly expressed anti-CD28 humanized single-domain antibody was diluted 3-fold at 11 concentration points starting at 66.66 nM at 100 μl / well. The ELISA plate was incubated at 37°C for 2 h and washed three times with wash buffer. HRP-conjugated Goat Anti-Human IgG (Jackson Immunoresearch solution (100 μl / well) was added and incubated at 37°C for 1 hour. TMB solution (100 μl / well) was added and allowed to react at room temperature for approximately 5 minutes. Stop solution (50 μl / well) was added and the plate was placed in a microplate reader for OD450 absorbance readings. Fluorescence signal values ​​at different concentrations were curve fitted using GraphPad Prism 6 software.

[0427] As shown in Figure 7 , the affinities of A04#2-Hz, A04#3-Hz, and A04#6-Hz were 0.43 nM, 0.43 nM, and 0.22 nM, respectively.

[0428] 3.6 Affinity determination of anti-CD28 humanized recombinant single-domain antibody and Jurkat cells

[0429] Jurkat cells are a known cell line that naturally expresses the CD28 protein. Flow cytometry was used to analyze the affinity of the antibody for Jurkat cells, following the procedures in Example 2.3. Curve fitting of the mean fluorescence signal values ​​at different concentrations was performed using GraphPad Prism 6 software. As shown in Figure 8A, the affinity of A04#2-Hz (0.46 nM) was significantly higher than that of A04#3-Hz (13.31 nM) and A04#6-Hz (31.89). As shown in Figure 8B, the affinity of A04#2-Hz (0.61 nM) was significantly higher than that of the control antibody (9.93 nM).

[0430] 3.7 Affinity determination of anti-CD28 humanized recombinant single-domain antibody and human peripheral blood mononuclear cells

[0431] Human peripheral blood mononuclear cells were isolated according to the method in Example 2.6, and the binding of anti-CD28 to the human CD28 target protein expressed on human peripheral blood mononuclear cells was determined by flow cytometry. PBMCs were centrifuged, washed twice with PBS, and resuspended in 1% BSA buffer. Human TruStain FcX (Biolegend) was added to the PBMC suspension and incubated at room temperature for 20 minutes. After incubation, the cell suspension was added to a 96-well conical bottom plate at 50 μl per well. Recombinantly expressed CD28 antibody was diluted in 1% BSA, starting at 100 nM and progressing through a 3-fold gradient. 50 μl was added to the cells, mixed, and incubated at 4°C for 50 minutes. After washing twice with PBS, the cells were finally resuspended in 1% BSA solution (containing PE anti-human IgG), 100 μl per well (containing 1 μl of fluorescent secondary antibody), mixed, and incubated at 4°C in the dark for 0.5 hour. The cells were washed twice with PBS, and the fluorescence signal and positive rate were determined by flow cytometry. The average fluorescence signal values ​​corresponding to different antibody concentrations were curve fitted using GraphPad Prism software. The results are shown in Figure 9A . The affinity (EC50 value) of A04#2-Hz (1.35 nM) was much higher than that of A04#3-Hz (6.87 nM) and A04#6-Hz (15.90).

[0432] For PBMC from another volunteer, the results are shown in Figure 9B , the affinity of A04#2-Hz is much higher than that of the control antibody C1, and the affinity of A04#2-Hz is about 3.5 times higher than that of the control antibody C1 (calculated according to EC50 value).

[0433] 3.8 Determination of CD80 ligand blocking activity of anti-CD28 humanized recombinant antibody

[0434] Dilute CD80-mFc (ligand) protein to 1ug / ml with CBS coating buffer, add 100ul per well to each well of a 96-well plate and incubate at 4°C overnight; drain the coating solution and wash once with wash buffer (PBST); drain the ELISA plate and add 100ul of 2% BSA (PBS) blocking solution to each well and block in a 37°C incubator for 2h; dilute CD28-hFc-biotin to 2ug / ml with BSA diluent and use this as the diluent for anti-CD28 recombinant antibody, starting from 1uM and diluted 3-fold, and add 100ul per well to each well of a 96-well plate; drain the liquid in the well and wash three times with wash buffer (PBST); dilute SA secondary antibody with 2% BSA (PBS) at a dilution ratio of 1:10000 and add 100ul per well to each well of a 96-well plate; drain the liquid in the well and wash five times with wash buffer (PBST); add 100ul of color development solution for several minutes, then add 50ul of 2N The reaction was terminated with HCl; the OD at 450 nm was read on a microplate reader, and the absorbance values ​​at 450 nm corresponding to different antibody concentrations were curve fitted using GraphPad Prism software.

[0435] The results are shown in Figure 10. The blocking ability of CD28 and CD80 ligands of A04#2-Hz (14.58 nM) and A04#6-Hz (13.61 nM) was much higher than that of A04#3-Hz (42.48 nM) and the control antibody (57.70 nM).

[0436] 3.9 Binding Specificity of Anti-CD28 Humanized Recombinant Antibody

[0437] Each protein in Table 10 was diluted to 1ug / ml with CBS coating buffer, and 100ul was added to each well of a 96-well plate and incubated at 4°C overnight. The coating solution was drained and the plate was washed once with PBST. The plate was drained and 100ul of 2% BSA (PBS) blocking solution was added to each well and the plate was blocked in a 37°C incubator for 2h. The anti-CD28 recombinant antibody dilution was diluted with BSA diluent to antibody concentrations of 10ug / ml and 2ug / ml, respectively, and 100ul was added to each well of a 96-well plate. The well liquid was drained and the plate was washed three times with PBST. The specific Goat anti-human IgG (SOUTHERNBIOTECH, Catalog No. 2049-05) secondary antibody was diluted 1:8000 in 2% BSA (PBS), and 100 μl was added to each well of a 96-well plate. The wells were drained and washed five times with PBST. 100 μl of developer solution was added for several minutes, followed by 50 μl of 2N HCl to terminate the reaction. OD readings were taken at 450 nm using a microplate reader. The results are shown in Table 10. The results demonstrate that the anti-CD28 humanized single-domain antibody of the present invention has excellent specificity, specifically recognizing CD28 without binding to other immune checkpoint molecules in the CD28 family.

[0438] Table 9. List of specific detection antigens for humanized single-domain antibodies against CD28

[0439] Table 10. Specificity test results of CD28 humanized single domain antibody

[0440] Example 4.1 Production of multispecific antibodies of the present invention

[0441] A total of three bispecific antibody molecules in a 1+1 format were designed (DLL3 / CD28, DLL3 / CD3, and B7H3 / CD28);

[0442] Two bispecific antibody molecules in a 1+2 format (DLL3 / CD28 / CD28) were designed.

[0443] Two trispecific antibody molecules (DLL3 / CD3 / CD28 Trispecific-1 and DLL3 / CD3 / CD28 Trispecific-2) were designed in a 1+1+1 format. Trispecific-1 was linked to the VH of the anti-CD3 Fab, and Trispecific-2 was linked to the VL of the anti-CD3 Fab.

[0444] The Fc "knob-in-hole" technique was used to address heavy chain mispairing in bispecific antibodies. S354C and Y349C were introduced into both heavy chains to stabilize proper heavy chain pairing. The Fc region was an IgG1 heavy chain constant region, and the amino acid mutations L234A, L235A, and G237A (according to Kabat's "EU" numbering) were introduced to reduce effector function. A schematic diagram of an example bispecific antibody structure is shown in Figure 11.

[0445] According to Table 12, anti-human DLL3 (Rovalpituzumab, KEGG #10683) and B7H3 (WO2022040482, SEQ ID: 1008) sequences were combined with A04#2-Hz to construct two 1+1 bispecific antibodies, DLL3 / CD28 and B7H3 / CD28, respectively, and one 1+2 bispecific antibody, DLL3 / CD28 / CD28. Anti-human DLL3 (Tarlatamab, KEGG #10683) and CD3 (Teclistamab, KEGG #D12177) were combined to construct a DLL3 / CD3 1+1 bispecific antibody. 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.

[0446] The above-described multispecific antibody sequences were synthesized into the pTT5 vector and then transiently expressed in 293F cells. After 7 days of expression, the resulting cell fermentation broth was filtered, clarified, and captured using a Protein A column (Hitrap Mabselect Sure, GE 11-0034-95). Antibody concentration was determined using the A280 method, and purity was determined using SEC-HPLC. The ten multispecific antibodies obtained were used in the following examples.

[0447] Table 11. List of example multispecific antibodies

[0448] 4.2 ELISA affinity detection of the multispecific antibody of the present invention

[0449] Using a method similar to that in Example 3.4, the single-end affinity of the bispecific antibodies of the present invention was tested. The CD28 end was coated with hCD28-ECD-lamFc, the DLL3 end was coated with hDLL3-ECD-his, and the B7H3 end was coated with hB7H3-ECD-his. The antibody concentration started at 10 μg / ml, and a three-fold dilution gradient was used with 11 concentration points. The calculated affinity EC50 values ​​are shown in Table 12.

[0450] Table 12. Example multispecific antibody protein ELISA affinities

[0451] 4.3 Activation experiment of Jurkat-NFAT-Luc cells by the bispecific antibody of the present invention

[0452] 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, activating the NFAT-Luc downstream signaling pathway through DLL3-dependent CD3 cross-linking. The DLL3 / CD28 dual antibody simultaneously binds to DLL3 on the surface of small cell lung cancer cells and CD28 on the surface of primary T cells, activating the NFAT-Luc downstream signaling pathway through DLL3-dependent CD28 cross-linking. This study used a luciferase reporter gene assay to assess the luciferase expression of Jurkat-NFAT-Luc cells co-cultured with DLL3-positive cells for 6 hours after the addition of the example antibodies. The study also evaluated the differences in the synergistic effects of the DLL3 / CD28 and DLL3 / CD28 / CD28 dual antibodies on the DLL3 / CD3 dual antibody.

[0453] Specific experimental steps:

[0454] In experiments conducted as described in the above assay, as shown in Figure 12-1, the exemplary DLL3 / CD3 bispecific antibody was able to activate NFAT signaling with an EC50 value of 0.192 nM. The CD28 bispecific antibody significantly enhanced the activation of NFAT signaling by the CD3 bispecific antibody, demonstrating enhanced activity compared to the DLL3 / CD3 bispecific antibody. The monovalent CD28 bispecific antibody significantly outperformed the bivalent CD28 bispecific antibody, with EC50 values ​​of 0.040 nM and 0.086 nM, respectively.

[0455] 4.4 Activation experiment of Jurkat-NFAT-Luc cells by the trispecific antibody of the present invention

[0456] 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. Through DLL3-dependent cross-linking of CD3 and CD28, it activates the NFAT-Luc downstream signaling pathway. The present invention utilizes a luciferase reporter gene assay to detect the activation ability of Jurkat-NFAT-Luc cells co-cultured with DLL3-positive cells. Luciferase expression after six hours of incubation with the example antibody is used to assess the antibody's activation ability. The activity of the DLL3 / CD3 / CD28 trispecific antibody is also compared with that of the DLL3 / CD3 bispecific antibody.

[0457] The specific experimental methods are described in Example 4.3. The results are shown in Figure 12-2. The example DLL3 / CD3 bispecific antibody was able to activate NFAT signaling with an EC50 value of 0.122 nM. The activity of DLL3 / CD3 / CD28 Trispecific-1 was 1-fold more potent than that of DLL3 / CD3 / CD28 Trispecific-2, with EC50 values ​​of 0.043 nM and 0.080 nM, respectively.

[0458] Therefore, both EC50 and maximum signal value of the two structural types of trispecific antibodies described in the present invention are significantly improved compared with bispecific antibodies.

[0459] 4.5 Dynamic affinity detection of the DLL3 / CD28 bispecific antibody of the present invention

[0460] After biotinylation of the DLL3 / CD28 dual antibody, the dynamic affinity of the DLL3 and CD28 ends was measured using Fortebio. The experimental steps are as follows: 1. Sensor preparation: Remove the SA sensor and pre-wet it with PBST (pH 7.4) for 10 minutes. 2. Sample dilution: Dilute the antibodies to be immobilized to 5 μg / ml. For the antigens hDLL3-ECD-His and hCD28-ECD-lamaFc, start at 500 nM and dilute 2-fold to five concentration points, including a zero concentration point. 3. Set up the program, insert the sensor plate and sample plate, start the program, and regenerate the sensor with 20 mM glycine solution (pH 1.7). 4. Analyze the data using Octet analysis software and export the results graph.

[0461] The results of dynamic affinity calculation are shown in Table 13. The affinity of the DLL3 end is much higher than that of the CD28 end, with KD values ​​of 5.90E-10M and 1.09E-08M, respectively.

[0462] Table 13. Dynamic affinity of DLL3 / CD28 bispecific antibodies

[0463] 4.5 Activation experiment of Jurkat-NFAT-Luc cells by the bispecific antibody of the present invention

[0464] A method similar to that of Example 4.3 was used. Since NCI-H716 cells (human colorectal adenocarcinoma cells) highly express B7H3 and DLL3 proteins on their membrane surface, NCI-H716 cells were used as target cells to evaluate the synergistic effect of the B7H3 / CD28 dual antibody and the DLL3 / CD3 dual antibody.

[0465] In experiments performed as described in the above assay, the exemplary CD28 bispecific antibody was able to increase the activation of NFAT signaling by the CD3 bispecific antibody. The results are as follows:

[0466] As shown in Figure 12-3, the B7H3 / CD28 bispecific antibody can enhance the activity of DLL3 / CD3, especially the maximum signal value of the DLL3 / CD3 activation signal is increased by nearly 60%.

[0467] The above data fully demonstrate that the CD28 of the present invention in the form of a bispecific antibody has a significant synergistic effect on the activity of the CD3 bispecific antibody, and this synergistic effect can be reflected in different target combinations of the same tumor.

[0468] 4.6 In vitro tumor cell killing experiment of the bispecific antibody of the present invention

[0469] The DLL3 / CD3 dual antibody simultaneously binds to DLL3 on the surface of small cell lung cancer cells and CD3 on primary T cells. Through DLL3-dependent CD3 cross-linking, it provides the primary T cell activation signal, activating T cells. The DLL3 / CD28 dual antibody simultaneously 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 secondary T cell activation signal, enhancing T cell activity. Ultimately, it synergizes with DLL3 / CD3 to mediate T cell cytotoxicity against DLL3-positive tumor cells. In this example, the Cell titer Glu assay was used to assess the cytotoxicity of human T cells against DLL3-positive tumor cells by co-culturing human PBMCs with DLL3-positive cells. Tumor cell viability was measured 48 hours after the addition of the example antibodies.

[0470] The specific detection methods are as follows:

[0471] DLL3-positive SHP77 cells (human small cell lung cancer purchased from Nanjing Kebai Biotechnology) were used as target cells, and NCI-H358 cells (human non-small cell lung cancer obtained from Sichuan Xiapaison Biotechnology Co., Ltd.) were used as non-target cells. After trypsinization, the cells were centrifuged at 400g for 5 minutes and resuspended in 1640 medium containing 10% FBS. The cell density was adjusted to 1×10^5 cells / ml. 100 μl of target cells were plated per well in a 96-well clear-bottom culture plate and incubated overnight at 37°C. PBMCs were removed from a liquid nitrogen tank, rapidly thawed at 37°C, and added dropwise to preheated 1640 medium containing 10% FBS to obtain a 10 ml mixture. The mixture was then centrifuged at 100g for 5 minutes and resuspended in 10 ml of 1640 medium containing 10% FBS to adjust the cell density of the human PBMCs to 1×10^6 cells / ml for use as effector cells. At a target-to-effect ratio of 1:5, 50 μl of human PBMC cell suspension was added to the target cell wells. 50 μl of serially diluted test antibodies was added to each well. The cells were placed in a CO2 incubator and incubated at 37°C for 48 hours. After 48 hours, the supernatant was carefully removed, and 200 μl of PBS was added to each well to rinse the cells. After removing the PBS, 100 μl of Cell titer Glu (Novozymes, DD1101-02) detection reagent was added to each well. The cells were shaken at room temperature for 3 minutes and read using a microplate reader (SpectraMax i3X).

[0472] In experiments conducted according to the above assay, as shown in Figures 13-1 and 13-2 , the DLL3 / CD3 bispecific antibody alone exhibited some cytotoxicity against target cells (SHP-77), with an EC50 of 0.541 nM. The DLL3 / CD28 bispecific antibody, when used alone, showed no cytotoxicity against target cells (SHP-77). However, when the two bispecific antibodies were used in combination, the DLL3 / CD28 bispecific antibody significantly enhanced the cytotoxicity of the DLL3 / CD3 bispecific antibody against target cells (SHP-77), with an EC50 of 0.0264 nM.

[0473] For non-target cells (NCI-H358, DLL3-negative), when the DLL3 / CD3 bispecific antibody was used alone or in combination with the DLL3 / CD28 bispecific antibody, there was only slight cell killing at the highest concentration point, indicating that the bispecific antibody of the present invention exhibited good specificity and target-dependent activity.

[0474] 4.7 In vivo tumor activity experiments of the bispecific antibodies of the present invention

[0475] To test the in vivo pharmacodynamics of the bispecific antibody of the present invention in mice bearing SHP77 tumor cells, immunodeficient mice were reconstituted with a human immune system and inoculated with human tumor cells to test the anti-tumor activity of the bispecific antibody of the present invention. The specific procedures are as follows:

[0476] Five-week-old female NCG mice (18-20 g) were purchased from Chengdu Yaokang Biotechnology Co., Ltd. They were SPF-grade. Mice were acclimated and quarantined for 7 days upon arrival before the study began. PBMCs and tumor cells were injected intravenously into the mice. Each NCG mouse was subcutaneously inoculated with a mixture of SHP77 cells and human T cells (5 x 10 cells each) at the right shoulder blade. 6 Each mouse was inoculated with 0.2 mL of 50 μL SHP77 medium + 50 μL PBMC cells + 100 μL Matrigel. When the average tumor volume reached 150 mm³, mice were divided into groups (4 mice per group) and dosed. Dosing was performed once weekly for a total of two doses, with a DLL3 / CD3 dual antibody dose of 1 mg / kg and a DLL3 / CD28 dual antibody dose of 10 mg / kg. The combination group received 1 mg / kg of DLL3 / CD3 and 10 mg / kg of DLL3 / CD28. Tumor volume and body weight were monitored twice weekly.

[0477] On the 22nd day after inoculation, the relative tumor inhibition rate (TGI%) was calculated, the tumor volume was calculated, and the tumor growth curve was drawn. The calculation formula of tumor volume (V) was:

[0478] V=1 / 2×a×b 2 Where a and b represent the long diameter and short diameter of the tumor, respectively.

[0479] Relative tumor growth rate (T / C) (%) = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the control group; RTV = Vt / V0, V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment);

[0480] Relative tumor inhibition rate 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).

[0481] The tumor growth curve is shown in Figure 14-1. The DLL3-CD28 antibody can significantly enhance the growth inhibition of SHP77 cells by the DLL3-CD3 antibody. The tumor size was counted on day 22, and the tumor inhibition rate was calculated.

[0482] Compared to the PBS group, the combination group (DLL3 / CD28 + DLL3 / CD28) achieved a 100% tumor inhibition rate (p<0.01), with complete tumor regression by day 18. In contrast, the TGIs for DLL3 / CD3 and DLL3 / CD28 alone were only 36.3% (p=0.564) and -10.4% (p=1), respectively, with no tumor regression observed in either group. Furthermore, no significant weight loss was observed in any of the treated mice (Figure 14-2), demonstrating the good safety profile of all drug groups.

[0483] 4.8 In vivo tumor activity experiments of the trispecific antibodies of the present invention

[0484] The anti-tumor activity of the trispecific antibody DLL3 / CD3 / CD28 Trispecific-1 of the present invention was tested using a protocol similar to that of Example 4.7, with administration once a week at a dose of 0.5 mg / kg for a total of 2 doses.

[0485] The tumor growth curve is shown in Figure 15-1. Tumor size was measured on day 22, and the tumor inhibition rate was calculated. Compared to the saline group, the tumor inhibition rate of the exemplary antibody DLL3 / CD28 / CD3 was 100% (p<0.01). Furthermore, no significant weight loss was observed in the treated mice (Figure 15-2).

[0486] 4.7 Thermal stability test of the bispecific antibody of the present invention

[0487] Adopt DSF to measure the humanized antibody Tm value, to react the thermal stability of antibody.Use real-time fluorescence quantitative PCR (QuantStudio 7 Flex, Thermo Fisher Scientific) to carry out DSF determination.In brief, 19 μ L concentration is the antibody solution of 1 mg / ml and 1 μ L 62.5 X SYPRO Orange solution (Invitrogen) is mixed and added in 96-well plates (Biosystems).The plate is heated from 25 ° C to 95 ° C at a rate of 2 ° C / min, and the fluorescence data generated are collected.Calculate the negative derivative of fluorescence change relative to different temperatures, and the maximum value is defined as the melting temperature Tm.Data acquisition and T h calculation are automatically performed by operating software (QuantStudio Real-Time PCR PCR Software v1.3).

[0488] The T m value of DLL3-CD28 in PBS buffer was approximately 63.7°C, indicating that the DLL3-CD28 bispecific antibody has good thermal stability.

[0489] 4.8 Accelerated Stability Test of the Bispecific Antibody of the Present Invention

[0490] The DLL3-CD28 bispecific antibody was ultrafiltered into PBS buffer at an antibody concentration of 11.36 mg / ml, sterilized by filtration, and stored at 37°C for 0, 14, and 21 days, followed by eight freeze-thaw cycles at -80°C. Protein purity was determined by SEC-HPLC, non-reduced CE-SDS, and reduced CE-SDS. Charge variants were determined by iCIEF. Activity changes were measured by ELISA.

[0491] The SEC detection method is as described above. The CE-SDS detection method is as follows: Dilute the sample to 4 mg / mL with ultrapure water. Transfer 25 μL of the sample to a centrifuge tube, add 75 μL of the premix (SDS sample buffer + iodoacetamide), mix thoroughly, and centrifuge at 10,000 g for 1 minute. Incubate at 70°C for 5 minutes. Remove the sample, cool to room temperature, centrifuge at 10,000 g for 1 minute, mix thoroughly, and transfer to a sample vial.

[0492] The SCIEX PA800plus capillary electrophoresis instrument was used for analysis, and the instrument parameters were set as follows:

[0493] Capillary: 30.2cm*50um (bare tube), effective length: 20cm

[0494] Capillary column temperature: 25°C, sample tray temperature: 15°C

[0495] Detection wavelength setting: 220 nm; acquisition frequency: 4 Hz.

[0496] The ELISA method is described in Example 2.

[0497] The experimental results are shown in Table 14. The DLL3 / CD28 bispecific antibody has good accelerated stability, and its purity and affinity are well maintained under accelerated conditions.

[0498] Table 14 Accelerated stability quality testing of DLL3 / CD28 bispecific antibody

[0499] The present invention discloses a variety of high-specificity and high-affinity nanobodies targeting CD28, which only include heavy chains, and the heavy chains contain heavy chain variable region (VHH) amino acid sequences listed in the following table.

[0500] Table 15. Antibody sequences of the present invention

[0501] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A single-domain antibody targeting CD28, characterized in that: The complementarity determining region (CDR) of the VHH chain of the single-domain antibody is one or more selected from the following groups: (1) CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 4, and CDR3 shown in SEQ ID NO.5; or CDR1 shown in SEQ ID NO.6, CDR2 shown in SEQ ID NO. 7, and CDR3 shown in SEQ ID NO.5; or CDR1 shown in SEQ ID NO.8, CDR2 shown in SEQ ID NO. 9, and CDR3 shown in SEQ ID NO.5; or CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO. 11, and CDR3 shown in SEQ ID NO. 12; or, (2) CDR1 shown in SEQ ID NO. 15, CDR2 shown in SEQ ID NO. 16, and CDR3 shown in SEQ ID NO. 17; or CDR1 shown in SEQ ID NO.18, CDR2 shown in SEQ ID NO. 19, and CDR3 shown in SEQ ID NO. 17; or CDR1 shown in SEQ ID NO.20, CDR2 shown in SEQ ID NO. 21, and CDR3 shown in SEQ ID NO. 17; or CDR1 shown in SEQ ID NO.22, CDR2 shown in SEQ ID NO. 23, and CDR3 shown in SEQ ID NO. 24; or, (3) CDR1 shown in SEQ ID NO. 27, CDR2 shown in SEQ ID NO. 28, and CDR3 shown in SEQ ID NO. 29, or CDR1 shown in SEQ ID NO.30, CDR2 shown in SEQ ID NO. 31, and CDR3 shown in SEQ ID NO. 29, or CDR1 shown in SEQ ID NO.32, CDR2 shown in SEQ ID NO. 33, and CDR3 shown in SEQ ID NO. 29, or CDR1 shown in SEQ ID NO.34, CDR2 shown in SEQ ID NO. 35, and CDR3 shown in SEQ ID NO. 36; Any of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain CD28 binding affinity.

2. The single domain antibody according to claim 1, wherein The VHH chain of the single-domain antibody further comprises a framework region (FR).

3. An antibody targeting CD28, characterized in that The antibody comprises one or more VHH chains of the CD28-targeting single-domain antibody according to claim 2.

4. A multispecific antibody, characterized in that The multispecific antibody comprises: the single-domain antibody targeting CD28 according to claim 1 or the antibody targeting CD28 according to claim 3.

5. The multispecific antibody according to claim 4, wherein The multispecific antibody is a bispecific antibody comprising a first antigen-binding region targeting CD28 and a second antigen-binding region targeting a tumor-associated antigen (TAA).

6. The multispecific antibody according to claim 4, wherein The multispecific antibody is a trispecific antibody comprising: Targeting the first antigen binding region of CD28, a second antigen binding region that targets a tumor-associated antigen (TAA); and Targets the third antigen-binding region of CD3.

7. A recombinant protein, characterized in that The recombinant protein has: (i) the CD28-targeting single domain antibody according to claim 1 or 2, or the CD28-targeting antibody according to claim 3; and (ii) optionally a polypeptide molecule or fragment having therapeutic function; and / or (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

8. A CAR construct, characterized in that The antigen binding region of the CAR construct is the VHH chain of the single-domain antibody according to claim 1.

9. A recombinant immune cell, characterized in that The immune cells express the exogenous CAR construct as described in claim 8.

10. An immunoconjugate, characterized in that The immunoconjugate contains: (a) an antibody portion, wherein the antibody portion is the CD28-targeting single domain antibody according to claim 1 or 2 or the CD28-targeting antibody according to claim 3; and (b) a coupling moiety coupled to the single domain antibody portion, wherein the coupling moiety is selected from the group consisting of a detectable marker, a drug drugs, toxins, cytokines, enzymes, or combinations thereof.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (i) the CD28-targeting single-domain antibody according to claim 1 or 2, the CD28-targeting antibody according to claim 3, the multispecific antibody according to any one of claims 4 to 6, the recombinant protein according to claim 7, the recombinant immune cell according to claim 9, or the immunoconjugate according to claim 10; (ii) a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, wherein The pharmaceutical composition further comprises an antibody or antibody fragment targeting CD3; preferably, an anti-CD3 bispecific antibody or multispecific antibody.

13. A use of an active ingredient, wherein the active ingredient is selected from the group consisting of the single-domain antibody targeting CD28 according to claim 1 or 2, the antibody targeting CD28 according to claim 3, the multispecific antibody according to any one of claims 4 to 6, the recombinant protein according to claim 7, the recombinant immune cell according to claim 9, the immunoconjugate according to claim 10, or a combination thereof, wherein: The active ingredient is used for (a) preparing a detection reagent, a detection plate or a test kit; and / or (b) preparing a drug for preventing and / or treating CD28-related diseases.

14. A polynucleotide, characterized in that The polynucleotide encodes a polypeptide selected from the group consisting of: (1) The single-domain antibody targeting CD28 according to claim 1 or 2, the antibody targeting CD28 according to claim 3, or the multispecific antibody according to any one of claims 4 to 6; (2) the recombinant protein according to claim 7; or (3) The CAR construct as described in claim 8.

15. A carrier, characterized in that The vector contains the polynucleotide according to claim 14.

16. A host cell, characterized in that The host cell contains the vector according to claim 15 or the polynucleotide according to claim 14 is integrated into its genome.

17. A method for detecting CD28 in a sample in vitro (including diagnostic or non-diagnostic), characterized in that: The method comprises the steps of: (1) contacting the sample in vitro with the CD28-targeting single domain antibody of claim 1 or 2, the CD28-targeting antibody of claim 3, or the immunoconjugate of claim 10; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD28 in the sample.

18. A method for preparing a recombinant polypeptide, characterized in that: The method comprises: (a) culturing the host cell according to claim 13 under conditions suitable for expression; (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is a single-domain antibody targeting CD28 according to claim 1 or 2, an antibody targeting CD28 according to claim 3, a multispecific antibody according to any one of claims 4 to 6, or a recombinant protein according to claim 7.

19. An active ingredient combination, characterized in that The combination of active ingredients includes First active ingredient: the single domain antibody targeting CD28 according to claim 1 or 2, the antibody targeting CD28 according to claim 3, the multispecific antibody according to any one of claims 4 to 6, or the recombinant protein according to claim 7; and Second active ingredient: an antibody or antibody fragment targeting CD3; preferably a bispecific antibody or multispecific antibody against CD3 Antibodies.

20. A medicine box, characterized in that: The medicine box comprises: (a) a first formulation comprising the multispecific antibody of claim 5 and a pharmaceutically acceptable carrier; (b) a second formulation comprising an anti-CD3 bispecific antibody and a pharmaceutically acceptable carrier; (c) instructions describing a method for treating tumors or cancer by combining the multispecific antibody of claim 5 and an anti-CD3 bispecific antibody.

21. A non-therapeutic in vitro method for synergistically inhibiting tumor cell growth, characterized in that: The method comprises the steps of: adding the active ingredient combination as claimed in claim 19 to a tumor cell culture system, thereby synergistically inhibiting the growth of tumor cells.

Images