Anti-MUC17*CD3*CD28 triple specific antibody

Abstract

This invention provides an anti-MUC17*CD3*CD28 trispecific antibody. Specifically, the trispecific antibody of this invention targets the tumor-specific antigen MUC17, and simultaneously targets CD3 and the costimulatory molecule CD28 expressed by T cells. By providing costimulatory signals to T cells, it is expected to stimulate more potent and effective killing activity and effectively overcome the resistance of solid tumors to T cell engagers.

Description

[Technical Field]

[0001] This invention relates to the field of antibodies, and more specifically to an anti-MUC17*CD3*CD28 trispecific antibody. [Background technology]

[0002] According to data from the World Health Organization's (IARC) "2020 World Cancer Report," there were 19.29 million new cancer cases worldwide in 2020, with 4.57 million reported in China alone, accounting for 23.7% of the global total. China's number of new cancer cases far exceeds that of other countries. In 2020, the top 10 most common cancer types in China were lung cancer, breast cancer, stomach cancer, colorectal cancer, liver cancer, esophageal cancer, cervical cancer, thyroid cancer, uterine cancer, and prostate cancer. Lung cancer was the most common cancer, followed by colorectal cancer and stomach cancer, accounting for 12.3% and 10.5% respectively. Therefore, discovering and exploring therapeutic targets related to gastrointestinal tumors is clinically crucial for developing more effective clinical treatments.

[0003] MUC17 is a large O-glycosylated mucin (Mucin) and, along with several other mucins (MUC1, 3, 12, 13, and 16), belongs to the SEA family of transmembrane mucins. Clinical cancer research data have shown that MUC17 is highly expressed in various gastrointestinal tumors (including gastric and colorectal cancer), making it a potential target associated with gastrointestinal cancers.

[0004] Bispecific T-cell Engagers (BiTEs) are bispecific antibodies formed by fusing fragments of two different antibodies. They bind to CD3 molecules on the surface of T cells and antigen molecules on the surface of tumor cells, respectively, inducing T-cell killing of tumor cells, a process that is not restricted by MHC. Currently, this therapy shows good efficacy in hematopoietic malignancies, and blinatumomab, one of the CD3×CD19 BiTEs, has received FDA approval as a treatment for B-cell malignancies. However, due to the physical barriers and immunosuppressive microenvironment of solid tumors, BiTEs have not achieved the expected efficacy in the clinical treatment of many solid tumors.

[0005] Therefore, in this technological field, there is an urgent need to provide novel T-cell engagers that can overcome the shortcomings of BiTEs, such as trispecific T-cell engagers (TriTEs) that can target tumor-specific antigens like MUC17. [Overview of the project]

[0006] The objective of the present invention is to provide an anti-MUC17*CD3*CD28 trispecific antibody.

[0007] The object of the present invention is to provide a triple-specific T cell engager (TriTE) that targets the tumor-specific antigen MUC17, as well as the T cell-expressed CD3 and the co-stimulatory molecule CD28.

[0008] In a first aspect of the present invention, a multispecific T-cell Engager is provided, the multispecific T-cell Engager is The first target domain D1 binds to a target protein selected from the group consisting of CD3, CD28, CD40, and CD137, An optional second target domain D2 that binds to a target protein selected from the group consisting of CD3, CD28, CD40, and CD137, It comprises a third target domain D3 containing one or more MUC17 antigen-binding domains.

[0009] In another preferred embodiment, D1, D2, or D3 are each independently selected from a single-domain antibody (sdAb), Fab, Fab', F(ab')2, TriFab, Fv fragment, fragment-variable (Fv) heterodimer, single-stranded Fv(scFv) fragment, diabody, bispecific T-cell engager (BiTE), or single-domain fragment, and are preferably selected from single-stranded Fv(scFv), Fv fragment, or Fab fragment.

[0010] In another preferred embodiment, the MUC17 antigen-binding domain comprises a third heavy chain variable region and a third light chain variable region, wherein the third heavy chain variable region is H-CDR1 containing the amino acid sequence shown in SEQ ID NO: 3, H-CDR2 containing the amino acid sequence shown in SEQ ID NO: 4, H-CDR3 contains three heavy chain variable regions CDRs with the amino acid sequence shown in SEQ ID NO: 5. The third light chain variable region is, L-CDR1 containing the amino acid sequence shown in SEQ ID NO: 6, L-CDR2 containing the amino acid sequence shown in SEQ ID NO: 7, It contains three light chain variable regions (CDRs) of L-CDR3, including the amino acid sequence shown in SEQ ID NO: 8.

[0011] In another preferred embodiment, the MUC17 antigen-binding domain includes a third heavy chain variable region indicated by SEQ ID NO: 1 or 9, and a third light chain variable region indicated by SEQ ID NO: 2 or 10.

[0012] In another preferred embodiment, the MUC17 antigen-binding domain comprises a third heavy-chain variable region having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence shown in SEQ ID NO: 1 or 9 (where the CDR sequences are invariant or substantially invariant), and a third light-chain variable region having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequence shown in SEQ ID NO: 2 or 10 (where the CDR sequences are invariant or substantially invariant).

[0013] In another preferred embodiment, the second target domain is selected from the group consisting of a CD3 antigen-binding domain, a CD28 antigen-binding domain, a CD40 antigen-binding domain, a CD137 antigen-binding domain, a CD40L sequence, or a CD137L sequence.

[0014] In another preferred embodiment, the third target domain is selected from the group consisting of a CD3 antigen-binding domain, a CD28 antigen-binding domain, a CD40 antigen-binding domain, a CD137 antigen-binding domain, a CD40L sequence, or a CD137L sequence.

[0015] In another preferred embodiment, the multispecific T cell engager also comprises an Fc fragment.

[0016] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4.

[0017] In another preferred embodiment, the Fc fragment is derived from the Fc fragment of IgG1.

[0018] In another preferred embodiment, the Fc fragment derived from the above IgG1 is N297A, L234F / L235E / P331S, Y349C / K370E / K409D / K439E, S354C / D356K / E357K / D399K, S354C / T366W, S354C / T366W / H435R / Y436F, Y349C / T366S / L368A / Y407V, L234F / L235E / P331S / Y349C / K370E / K409D / K439E, L234F / L235E / P331S / S354C / D356K / E357K / D399K, L234F / L235E / P331S / S354C / T366W, L234F / L235E / P331S / Y349C / T366S / L368A / Y407V, N297A / Y349C / K370E / K409D / K439E, N297A / S354C / D356K / E357K / D399K, N297A / S354C / T366W, N297A / Y349C / T366S / L368A / Y407V, and / or S267E;S267E / G236D;S267E / S239D;S267E / L328F It has a mutation selected from the group consisting of the following:

[0019] In another preferred embodiment, the Fc fragment derived from IgG1 is L234A / L235A / G237A, S354C / T366W, S354C / T366W / H435R / Y436F, Y349C / T366S / L368A / Y407V It has a mutation selected from the group consisting of the following:

[0020] In another preferred embodiment, the multispecific T cell engager is a dual / triple specific T cell engager.

[0021] In another preferred embodiment, the multispecific T cell engager is a bispecific T cell engager.

[0022] In another preferred embodiment, the triplicate T cell engager is The first target domain is the CD28 antigen-binding domain, The second target domain is the CD3 antigen-binding domain, It comprises a third target domain containing one or more MUC17 antigen-binding domains.

[0023] In another preferred embodiment, the first target domain, the second target domain, and the third target domain each include one or more antibody-variable regions.

[0024] In another preferred embodiment, the multispecific T cell engager is a triple-specific T cell engager.

[0025] In another preferred embodiment, the multispecific T cell engager is homodimer or heterodimer.

[0026] In another preferred embodiment, the triple-specific T cell engager is a triple-specific antibody.

[0027] In another preferred embodiment, the structure of the triplicate antibody is symmetrical or asymmetrical.

[0028] In another preferred embodiment, the triplicate antibody is a bivalent, trivalent, tetravalent, or polyvalent molecule.

[0029] In another preferred embodiment, the triplicate antibody is a trivalent triplicate antibody having an asymmetric structure.

[0030] In another preferred embodiment, the CD3 antigen-binding domain comprises a second heavy chain variable region and a second light chain variable region, wherein the second heavy chain variable region is H-CDR1 containing the amino acid sequence shown in SEQ ID NO: 17, H-CDR2 containing the amino acid sequence shown in SEQ ID NO: 18, H-CDR3 contains three heavy chain variable regions CDRs with the amino acid sequence shown in SEQ ID NO: 19. The second light chain variable region is, L-CDR1 containing the amino acid sequence shown in SEQ ID NO: 20, L-CDR2 containing the amino acid sequence shown in SEQ ID NO: 21, It contains three light chain variable regions (CDRs) of L-CDR3, including the amino acid sequence shown in SEQ ID NO: 22, or The second heavy chain variable region is, H-CDR1 containing the amino acid sequence shown in SEQ ID NO: 17, H-CDR2 containing the amino acid sequence shown in SEQ ID NO: 18, H-CDR3 contains three heavy chain variable regions CDRs with the amino acid sequence shown in SEQ ID NO: 51. The second light chain variable region is, L-CDR1 containing the amino acid sequence shown in SEQ ID NO: 20, L-CDR2 containing the amino acid sequence shown in SEQ ID NO: 21, It contains three light chain variable regions (CDRs) of L-CDR3, including the amino acid sequence shown in SEQ ID NO: 52.

[0031] In another preferred embodiment, the CD3 antigen-binding domain includes a second heavy chain variable region having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence shown in SEQ ID NO: 15 or 23, and a second light chain variable region having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence shown in SEQ ID NO: 16 or 24.

[0032] In another preferred embodiment, the CD3 antigen-binding domain includes a second heavy chain variable region shown in SEQ ID NO: 15 and a second light chain variable region shown in SEQ ID NO: 16, or includes a second heavy chain variable region shown in SEQ ID NO: 23 and a second light chain variable region shown in SEQ ID NO: 24.

[0033] In another preferred embodiment, the CD28 antigen-binding domain comprises a first heavy chain variable region and a first light chain variable region, wherein the first heavy chain variable region is H-CDR1 containing the amino acid sequence shown in SEQ ID NO: 30, H-CDR2 containing the amino acid sequence shown in SEQ ID NO: 31 or 53, H-CDR3 contains three heavy chain variable regions CDRs, including the amino acid sequence shown in SEQ ID NO: 32. The first light chain variable region is, L-CDR1 containing the amino acid sequence shown in SEQ ID NO: 33, L-CDR2 containing the amino acid sequence shown in SEQ ID NO: 34, It contains three light chain variable regions (CDRs) of L-CDR3, which include the amino acid sequence shown in SEQ ID NO: 35.

[0034] In another preferred embodiment, the CD28 antigen-binding domain includes a first heavy chain variable region having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence shown in SEQ ID NO: 28 or 50, and a first light chain variable region having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequence shown in SEQ ID NO: 29.

[0035] In another preferred embodiment, the CD28 antigen-binding domain includes a first heavy chain variable region indicated by SEQ ID NO: 28 or 50 and a first light chain variable region indicated by SEQ ID NO: 29.

[0036] In another preferred embodiment, the triplicate antibody is a homodimer or a heterodimer.

[0037] In another preferred embodiment, the first target domain and the second target domain exhibit a heterodimer structure.

[0038] In another preferred embodiment, the first target domain includes an anti-CD28 Fv domain, a Fab domain, or an anti-CD28 scFv domain.

[0039] In another preferred embodiment, the second target domain includes an anti-CD3 Fv domain, a Fab domain, or an anti-CD3 scFv domain.

[0040] In another preferred embodiment, the third target domain comprises an anti-MUC17 Fab domain.

[0041] In another preferred embodiment, the triplicate antibody is located from the N-terminus to the C-terminus. (a) First strand: VL1-L1-VH2-L2-VL2-L3-VH1-L4-Fc1, (b) Second chain: VH3-CH1-Fc2, and (c) Third chain: Having the structure shown in VL3-CL, Alternatively, the triplicate antibody may have a structure from the N-terminus to the C-terminus. (a) First strand: VL1-L1-VH1-L2-VL2-L3-VH2-L4-Fc1, (b) Second chain: VH3-CH1-Fc2, and (c) Third chain: Having the structure shown in VL3-CL, Here, VH1 is the first heavy chain variable region, and VL1 is the first light chain variable region. VH2 is the second heavy chain variable region, and VL2 is the second light chain variable region. VH3 is the third heavy chain variable region, and VL3 is the third light chain variable region. CL is the light chain constant region, and CH1 is the CH1 domain. L1, L2, L3, and L4 are, independently, none, coupled, or linker. Fc1 or Fc2 are each independent Fc elements. The hyphen "-" indicates a peptide bond.

[0042] In another preferred embodiment, the linker is a rigid linker or a flexible linker.

[0043] In another preferred embodiment, L1, L2, L3, and L4 are each independently none, or (GS)n, (G3S)n, and (G4S)n (where n is selected from 1 to 6).

[0044] In another preferred embodiment, the trispecific antibody is administered in combination with a checkpoint inhibitor.

[0045] In another preferred embodiment, the triplicate antibody is administered in combination with an anti-PD1 and / or PDL1 antagonist.

[0046] In another preferred embodiment, the light chain steady region is the human Kappa light chain steady region.

[0047] In another preferred embodiment, VH1 and VL1 are a first heavy chain variable region and a first light chain variable region contained in the CD28 antigen-binding domain, respectively.

[0048] In another preferred embodiment, VH2 and VL2 are a second heavy chain variable region and a second light chain variable region contained within the CD3 antigen-binding domain, respectively.

[0049] In another preferred embodiment, VH3 and VL3 are a third heavy chain variable region and a third light chain variable region contained within the MUC17 antigen-binding domain, respectively.

[0050] In another preferred embodiment, VH1 comprises HCDR1, HCDR2, and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively, and VL1 comprises LCDR1, LCDR2, and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively, or The VH1 comprises HCDR1, HCDR2, and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 30, SEQ ID NO: 53, and SEQ ID NO: 32, respectively, and the VL1 comprises LCDR1+LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively.

[0051] In another preferred embodiment, the VH2 comprises HCDR1, HCDR2, and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and the VL2 comprises LCDR1, LCDR2, and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively, or The VH2 comprises HCDR1, HCDR2, and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 51, respectively, and the VL2 comprises LCDR1, LCDR2, and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 35, respectively.

[0052] In another preferred embodiment, VH3 comprises HCDR1, HCDR2, and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, and VL3 comprises LCDR1, LCDR2, and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

[0053] In another preferred embodiment, in the triplicate antibody, The amino acid sequence of the first chain is shown in SEQ ID NO. 38 or 42.

[0054] In another preferred embodiment, in the triplicate antibody, The amino acid sequence of the second chain is shown in SEQ ID NO. 40, and / or The amino acid sequence of the third chain is shown in SEQ ID NO. 14.

[0055] In another preferred embodiment, the triplicate antibody further comprises an active fragment and / or derivative of the triplicate antibody, the derivative of the antibody having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence homology with the triplicate antibody of the present invention.

[0056] A second aspect of the present invention provides a polynucleotide encoding a multispecific T cell engager as described in the first aspect of the present invention.

[0057] A third aspect of the present invention provides a vector comprising the polynucleotide described in the second aspect of the present invention.

[0058] In another preferred embodiment, the vector includes plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses and retroviruses, or other vectors.

[0059] In a fourth aspect of the present invention, a host cell containing a genome is provided, comprising the vector described in the third aspect of the present invention or the polynucleotide described in the second aspect of the present invention.

[0060] In another preferred embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell.

[0061] In a fifth aspect of the present invention, (i) A procedure for culturing host cells according to the fourth aspect of the present invention under appropriate conditions to obtain a mixture containing a multispecific T cell engager according to the first aspect of the present invention, The present invention provides a method for preparing a multispecific T cell engager according to a first aspect of the present invention, comprising the step of (ii) purifying and / or separating the mixture obtained in step (i) to obtain a multispecific T cell engager according to a first aspect of the present invention.

[0062] In a sixth aspect of the present invention, (I) A multispecific T cell engager according to a first aspect of the present invention, (II) To provide a pharmaceutical composition containing a pharmaceutically acceptable vector.

[0063] In another preferred embodiment, the pharmaceutical composition further provides other pharmaceutically active reagents.

[0064] In another preferred embodiment, the other pharmaceutically active reagent comprises one or more checkpoint modulators or chemotherapeutic agents.

[0065] In another preferred embodiment, the checkpoint modifier includes, but is not limited to, an anti-PD1 and / or anti-PDL1 antagonist.

[0066] In another preferred embodiment, the pharmaceutical composition is in the form of an injectable preparation.

[0067] In a seventh aspect of the present invention, (a) A multispecific T cell engager according to a first aspect of the present invention, (b) Provide an immune complex comprising a complex portion selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0068] In another preferred embodiment, the complex portion is selected from the group consisting of fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, and nanomagnetic particles.

[0069] An eighth aspect of the present invention provides the use of a multispecific T cell engager according to the first aspect of the present invention, a pharmaceutical composition according to the sixth aspect of the present invention, or an immune complex according to the seventh aspect of the present invention in the preparation of (a) a detection reagent or kit, and / or (b) a drug for the prevention and / or treatment of cancer / tumor.

[0070] In another preferred embodiment, the cancer / tumor is a cancer / tumor associated with MUC17.

[0071] In another preferred embodiment, the cancer / tumor includes solid tumors and hematological malignancies.

[0072] In another preferred embodiment, the cancer / tumor is a solid tumor.

[0073] In another preferred embodiment, the cancer / tumor is selected from the group consisting of stomach cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, gastroesophageal cancer, pancreatic adenocarcinoma, lung cancer, breast cancer, liver cancer, cervical cancer, thyroid cancer, uterine cancer, prostate cancer, or a combination thereof.

[0074] A ninth aspect of the present invention provides a kit comprising a multispecific T cell engager as described in the first aspect of the present invention, a polynucleotide as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, a host cell as described in the fourth aspect of the present invention, a pharmaceutical composition as described in the sixth aspect of the present invention, or an immune complex as described in the seventh aspect of the present invention.

[0075] A tenth aspect of the present invention provides the use of a multispecific T cell engager according to the first aspect of the present invention, a polynucleotide according to the second aspect of the present invention, a vector according to the third aspect of the present invention, a host cell according to the fourth aspect of the present invention, a pharmaceutical composition according to the sixth aspect of the present invention, or an immune complex according to the seventh aspect of the present invention in the preparation of agents for the prevention, treatment and / or detection of cancer / tumors.

[0076] An eleventh aspect of the present invention provides a method for preventing, treating, and / or detecting cancer / tumors by administering a safe and effective amount of the multispecific T cell engager described in the first aspect of the present invention, the pharmaceutical composition thereof, or its immune complex to a subject who requires such a dose.

[0077] It should be understood that each of the above technical features of the present invention and each of the technical features specifically described below (for example, in the examples) can be combined with each other within the scope of the present invention to constitute novel or preferred technical solutions. Due to space limitations, each will not be explained in detail here. [Brief explanation of the drawing]

[0078] [Figure 1A] Figure 1 is a schematic diagram showing the structure of the triply specific antibody of the present invention. Here, Figure 1A is a schematic diagram showing the structure of the CD28-44 / SP34-Dia+44H4-KIH antibody. [Figure 1B] Figure 1 is a schematic diagram showing the structure of the triply specific antibody of the present invention. Here, Figure 1B is a schematic diagram showing the structure of the CD28-44 / SP34-ScFv+44H4-KIH antibody. [Figure 2A]Figure 2A shows the antigen-binding ability of the anti-MUC17*CD3*CD28 trispecific antibody against MUC17 (Figure 2A) as measured by ELISA. [Figure 2B] Figure 2B shows the antigen-binding ability of the anti-MUC17*CD3*CD28 tripspecific antibody against CD3 (Figure 2B) as measured by ELISA. [Figure 2C] Figure 2C shows the antigen-binding ability of the anti-MUC17*CD3*CD28 trispecific antibody against CD28 (Figure 2C) as measured by ELISA. [Figure 3A] Figure 3A shows the binding ability of the anti-MUC17*CD3*CD28 trispecific antibody to MUC17 on the surface of human colon adenocarcinoma cells LS174T (Figure 3A), as measured by flow cytometry. [Figure 3B] Figure 3B shows the binding ability of the anti-MUC17*CD3*CD28 trispecific antibody to human T cell surface CD3 / CD28 (Figure 3B) as measured by flow cytometry. [Figure 4A] Figure 4 shows the killing activity of the anti-MUC17*CD3*CD28 trispecific antibody against target cell LS174T-luciferase. Figure 4A is one of the results from two independent replicated experiments, and the effector cells used in the two experiments were derived from different human individuals. [Figure 4B] Figure 4 shows the killing activity of the anti-MUC17*CD3*CD28 trispecific antibody against target cell LS174T-luciferase, and Figure 4B shows one of the results from two independent replicated experiments, with the effector cells used in the two experiments originating from different human individuals. [Figure 5A] Figure 5 shows the superiority of the anti-MUC17*CD3*CD28 tripspecific antibody compared to the bispecific antibody. Here, the mutant SP34-Hu-IgG1 (A101D) in Figure 5A no longer binds to CD3-Epsilon. [Figure 5B] Figure 5 shows the superiority of the anti-MUC17*CD3*CD28 tripspecific antibody compared to the bispecific antibody. The mutant Anti-CD28-Hu-IgG1(H35A+H96A) in Figure 5B no longer binds to CD28. [Figure 6A] Figure 6 shows the killing activity of the three-specific antibodies and their variants against target cells (Figure 6A). [Figure 6B] Figure 6 shows the IFN-γ secretion stimulating activity of T cells against target cells by the three specific antibodies of the present invention and their variants (Figure 6B). [Figure 7A] Figure 7 shows the superiority of the anti-MUC17*CD3*CD28 trispecific antibody over CODV-IgG, and Figure 7A shows a comparison of the killing activity against target cells. [Figure 7B] Figure 7 shows the superiority of the anti-MUC17*CD3*CD28 trispecific antibody over CODV-IgG, and Figure 7B shows a comparison of their ability to stimulate IFN-γ secretion from CD3-positive T cells. [Figure 8A] Figure 8 shows the in vitro safety evaluation of the anti-MUC17*CD3*CD28 trispecific antibody, demonstrating that the SP34-Hu-IgG1 monoclonal antibody can strongly stimulate IL-2 in PBMCs (Figure 8A). [Figure 8B] Figure 8 shows the in vitro safety evaluation of the anti-MUC17*CD3*CD28 trispecific antibody, demonstrating that it can strongly stimulate IFN-gamma secretion (Figure 8B). [Figure 8C] Figure 8 shows the in vitro safety evaluation of the anti-MUC17*CD3*CD28 trispecific antibody, demonstrating its ability to stimulate PBMC proliferation (Figure 8C). [Figure 9A] Figure 9 shows the HPLC-SEC purity analysis of the anti-MUC17*CD3*CD28 trispecific antibody. Here, Figure 9A shows the HPLC-SEC spectrum of CD28-44 / SP34-Dia+44H4-KIH. [Figure 9B] Figure 9 shows the HPLC-SEC purity analysis of the anti-MUC17*CD3*CD28 trispecific antibody. Here, Figure 9B shows the HPLC-SEC spectrum of CD28-44 / SP34-ScFv+44H4-KIH. [Figure 10A]Figure 10 shows the CE-SDS analysis of the MUC17*CD3*CD28 trispecific antibody. Here, Figure 10A shows the NR-CE-SDS spectrum of CD28-44 / SP34-Dia+44H4-KIH. [Figure 10B] Figure 10 shows the CE-SDS analysis of the MUC17*CD3*CD28 trispecific antibody. Figure 10B shows the R-CE-SDS spectrum of CD28-44 / SP34-Dia+44H4-KIH. [Figure 10C] Figure 10 shows the CE-SDS analysis of the MUC17*CD3*CD28 trispecific antibody. Here, Figure 10C shows the NR-CE-SDS spectrum of CD28-44 / SP34-ScFv+44H4-KIH. [Figure 10D] Figure 10 shows the CE-SDS analysis of the MUC17*CD3*CD28 trispecific antibody. Here, Figure 10D shows the R-CE-SDS spectrum of CD28-44 / SP34-ScFv+44H4-KIH. [Figure 11] Figure 11 shows the tumor-suppressing effect of the anti-MUC17*CD3*CD28 trispecific antibody on animals (including mouse body weight and tumor volume size). [Modes for carrying out the invention]

[0079] As a result of extensive and intensive research, the inventors have unexpectedly developed, for the first time, a multispecific T cell engager targeting MUC17. Preferably, the multispecific T cell engager of the present invention comprises humanized modified MUC17, CD3 and CD28 targeting domains. In addition to binding to CD3, the multispecific T cell engager of the present invention can target CD28, providing a co-stimulatory signal to T cells, thereby stimulating more potent and effective killing activity and effectively overcoming solid tumor resistance to T cell engagers. Based on this, the present invention was completed.

[0080] term Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention pertains. The term "approximately" refers to a value or composition within a tolerance range for a particular value or composition as determined by those skilled in the art, which may depend on the measurement method, measurement value, or composition. In this specification, the terms “contain” or “include” may be open, semi-closed, or closed. In other words, the terms also include the meaning of “essentially consisting of” or “consisting of.”

[0081] MUC17 MUC17 is a large molecular weight O-glycosylated mucin (Mucin) and, along with several other mucins (MUC1, 3, 12, 13, and 16), belongs to the transmembrane mucin family of SEA (Sea urchin sperm protein, Enterokinase, and Agrin). Transmembrane mucins play a crucial role in maintaining mucosal barrier function, not only preventing pathogens from entering the mucosal surface but also being involved in the progression of inflammatory bowel disease. Through analysis of HPA (Human Protein Atlas, genome database) data, the applicant found that MUC17 RNA is mainly expressed in colorectal and gastric-related organs (small intestine, duodenum, rectum, colon, and stomach). Clinical cancer research data have shown that MUC17 is highly expressed in various gastrointestinal tumors (including gastric and colorectal cancer), making MUC17 a potential target associated with gastrointestinal cancers.

[0082] T-cell Engagers Bispecific T-cell Engagers (BiTEs) are bispecific antibodies formed by fusing fragments of two different antibodies. They bind to CD3 molecules on the surface of T cells and antigen molecules on the surface of tumor cells, respectively, thereby inducing T-cell killing of tumor cells. This process is not restricted by MHC. In February 2022, Sanofi developers published a paper in Nature on their research findings regarding trispecific T-cell engagers (TriTEs) (using a CODV-IgG structure) that target HER2 × CD3 × CD28 (Reference: Seung E, Xing Z, Wu L, et al. A trispecific antibody targeting HER2 and T cells inhibits breast cancer growth via CD4 cells[J]. Nature, 2022, 603(7900): 328-334.). This study showed that TriTEs stimulate IL-2 secretion more effectively than BiTEs, activate the NF-κB signaling pathway, and possess more potent killing activity. In immunodeficient NSG mice reconstituted with primary cultured human CD3-positive T cells, TriTEs increased granzyme expression in CD8-positive T cells by 6.8 times, and HER2×CD3×CD28 was able to induce tumor regression even at a low dose of 10 μg / kg. According to this paper, TriTEs targeting HER2×CD3×CD28 have already progressed to Phase 1 clinical development and are expected to be therapeutic agents for HER2-positive solid tumors.

[0083] antibody Typically, "antibodies," also called "immunoglobulins," can be natural or conventional antibodies, consisting of two heavy chains linked to each other by disulfide bonds, with each heavy chain linked to a light chain by disulfide bonds. Two types of light chains exist: λ(l) and κ(k). The heavy chains that determine the functional activity of antibody molecules have five main types (or isotypes): IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. The light chain contains two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a heavy chain variable region (VH) and three constant regions (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant domain (CL) of the light chain and the constant region (CH) of the heavy chain confer important biological properties to antibody chains, such as binding, secretion, placental transfer, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and consists of a variable region of one light chain and one heavy chain. Antibody specificity depends on the structural complementarity of the antibody binding site and the antigen-determining region. The antibody binding site is mainly composed of residues in the highly variable region or complementarity-determining region (CDR). Occasionally, residues in the non-highly variable region or framework region (FR) affect the binding site by influencing the overall structure of the domain. The complementarity-determining region or CDR refers to an amino acid sequence that simultaneously defines binding affinity and the specificity of the native Fv region of the native immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, called CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, and CDR3-H, respectively. Therefore, the typical antibody-antigen binding site consists of six CDRs, which are aggregates of CDRs from each heavy and light chain v region.

[0084] The precise amino acid sequence boundaries of each CDR in the light chain variable region or heavy chain variable region of a given antibody can be determined using one of many well-known antibody CDR assignment systems or combinations thereof, such as Chothia, Kabat, AbM, Contact, the International ImmunoGeneTics database (IMGT), and the EU numbering system.

[0085] It should be understood that the precise amino acid sequence boundaries of the CDR of the present invention may optionally be defined using the different assignment systems described above. Preferably, unless otherwise stated, when the present invention refers to residue positions in the variable region of the antibody (including heavy chain variable region residues and light chain variable region residues), it refers to numbered positions according to the Kabat numbering system.

[0086] In this specification, the term "variable" refers to a difference in the sequence of a portion of the variable region of an antibody, which determines the binding affinity and specificity of different antibodies to specific antigens. However, variability is not uniformly distributed throughout the variable region of an antibody. It is concentrated in three fragments of the light and heavy chain variable regions called complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portion of the variable region is called the framework region (FR). The four FR regions in the native heavy and light chain variable regions basically exhibit a β-sheet structure connected by three CDRs that form a connecting loop, and can sometimes form a partial β-sheet structure. The CDRs of each chain are held in close proximity to each other via the FR region and, together with the CDR of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but exhibits various effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0087] In this specification, the term “framework region” (FR) refers to the amino acid sequence inserted between CDRs, i.e., the relatively conserved portion of the variable regions of the light and heavy chains of immunoglobulins among different immunoglobulins of a single species. The light and heavy chains of immunoglobulins each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Thus, the light chain variable domain can be called (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain can be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, the derivative of the human antibody FR being substantially identical to a naturally occurring human antibody FR, i.e., having 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology. If the amino acid sequence of the CDR is known, a person skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0088] In this specification, the term "human framework region" refers to a framework region that is substantially identical (approximately 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to the framework region of naturally occurring human antibodies.

[0089] In this specification, the terms “fragment,” “derivative,” and “analog” refer to polypeptides that possess substantially the same biological function or activity as the antibodies of the present invention. Polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituents on one or more amino acid residues; or (iii) polypeptides formed by the 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) polypeptides formed by the fusion of a polypeptide sequence with an additional amino acid sequence (e.g., a leader sequence, a secretion sequence, a sequence used to purify the polypeptide, a proprotein sequence, or a fusion protein formed with a 6His tag). As taught herein, these fragments, derivatives, and analogs are known to those skilled in the art.

[0090] In this specification, the terms “antibody fragment,” “antigen-binding fragment,” “target domain,” or “antigen-binding domain” refer to parts of antibodies such as F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fvs(scFv), single-chain antibodies, disulfide-linked Fvs(sdFv), fragments containing VL or VH domains, fragments produced by Fab expression libraries, and anti-idiotype (anti-Id) antibodies. Regardless of their structure, antibody fragments bind to the same antigen recognized by complete antibodies. Examples of target domains in this invention include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv(scFv) fragments, and single-domain fragments.

[0091] The "Fv" fragment is the smallest fragment of an antibody that contains the complete target recognition and binding site. This region consists of a dimer (VH-VL dimer) in which one heavy chain and one light chain variable structural domains are tightly bound non-covalently. In this configuration, three CDRs in each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Typically, six CDRs give the antibody target binding specificity. However, in some cases, a single variable structural domain (or half of the Fv containing only three target-specific CDRs) may have the ability to recognize and bind to the target, although with lower affinity than the entire binding site.

[0092] A "single-stranded Fv" or "scFv" antibody-binding fragment contains the VH and VL domains of the antibody, and these domains are present on a single polypeptide chain. Generally, Fv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows scFv to form a structure favorable for target binding. A "single-domain fragment" consists of a single VH or VL domain that exhibits sufficient affinity for coronavirus RBD. In certain specific embodiments, the single-domain fragment is camelized.

[0093] In this specification, the term “light chain constant region (CL)” includes an amino acid sequence CL derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant κ domain or a constant λ domain.

[0094] In this specification, the term “heavy chain constant region (CH)” includes the amino acid sequence derived from the immunoglobulin heavy chain. Polypeptides containing a heavy chain constant region include at least one of the CH1 domain, hinge region (such as the upper, middle, and / or lower hinge region), CH2 domain, CH3 domain, or variants or fragments thereof. It should be understood that the heavy chain constant region may be modified so that its amino acid sequence differs from that of the native immunoglobulin molecule.

[0095] In this specification, the terms “antigen” or “target antigen” refer to a molecule or part of a molecule to which an antibody or antibody-like binding protein can bind. The term further refers to a molecule or part of a molecule that can be used in an animal to produce an antibody that can bind to an antigen’s epitope. A target antigen may have one or more epitopes. For each target antigen recognized by an antibody or antibody-like binding protein, the antibody-like binding protein may compete with a complete antibody that recognizes the target antigen.

[0096] In this specification, the term “linker” refers to one or more amino acid residues that are inserted into an immunoglobulin domain and provide sufficient mobility for the light-chain and heavy-chain domains to fold into an interchangeable bivariable-region immunoglobulin. Examples of suitable linkers include a single glycine (Gly) or serine (Ser) residue, and the labeling and sequence of amino acid residues in the linker may vary depending on the type of secondary structure element that needs to be achieved in the linker. Preferred linkers may be (GS)n, (G3S)n, or (G4S)n (where n is selected from 1 to 6).

[0097] In this specification, the term “variant” of an antibody, antibody fragment, or antibody domain means the following antibodies, antibody fragments, or antibody domains: (1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the original antibody, antibody fragment, or antibody domain; and (2) specifically binding to the same target that specifically binds to the original antibody, antibody fragment, or antibody structural domain. When sequence homology is expressed in the form of “at least x% homology” or “at least x% homology,” such embodiments should be understood to include any numerical percentage above the lower limit. Furthermore, when an amino acid sequence is present in this application, it should be understood to mean that an additional disclosure or inclusion of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to that amino acid sequence is disclosed or included.

[0098] T cell engager of the present invention In this specification, the term “multispecific T cell engager” refers to a molecule having target domains having at least two distinct binding specificities, of which at least one target domain specifically binds to a T cell surface antigen. In some embodiments, the multispecific inhibitor is a polypeptide comprising a scaffold and two or more immunoglobulin antigen-binding domains targeting different antigens or epitopes. In certain embodiments, the multispecific T cell engager is a bispecific or triplicate antibody.

[0099] In this specification, the terms “bispecific antibody” and “bispecific T cell engager” are used interchangeably and refer to antibodies that specifically bind to two different antigens (or epitopes).

[0100] In this specification, the terms “triply specific antibody of the present invention,” “triply specific T cell engager of the present invention,” and “3Sbody” are used interchangeably and relate to a molecule comprising three target domains having three different binding specificities. Each target domain can specifically bind to a target molecule and inhibits the biological function of the target molecule upon binding. In some embodiments, the triply specific antagonist is a polymer molecule having two or more peptides. In some embodiments, the target domain comprises an antigen-binding domain or CDR of the antibody. In some embodiments, the target domain comprises a ligand or fragment thereof that specifically binds to a target protein.

[0101] The triplicate T cell engager of the present invention is essentially a triplicate antibody, an antibody molecule capable of simultaneously and specifically binding to three antigens. Based on symmetry, triplicate antibodies are classified into symmetrical or asymmetrical molecules. Depending on the number of binding sites, bivalent antibodies are classified into bivalent, trivalent, tetravalent, and polyvalent molecules. The two triplicate antibodies of the present invention are structurally asymmetrical trivalent triplicate antibodies, monovalent to their respective specific targets.

[0102] In this specification, the terms “Fc fragment” or “Fc” refer to the crystalline portion of an antibody that is initially observed but lacks antigen-binding activity, and are therefore named Fc fragments (crystallinity of fragments). Such fragments correspond to a pair of CH2 and CH3 domains and are part of the antibody molecule that interacts with effector molecules and cells. The Fc fragments described herein may be derived from IgG1, IgG2, and IgG4 antibodies. For specific applications, certain IgG subclasses may be preferred. Furthermore, the effector function of an antibody can be enhanced or reduced by introducing one or more mutations into the Fc. Preferably, the Fc segment may contain L234A / L235A / G237A mutations to weaken the interaction between Fc and the Fcγ receptor.

[0103] Furthermore, mutations can be introduced to form a knob-into-hole (KIH) structure. Its main function is to promote the heterodimerization of the two different heavy chains of a bispecific antibody. Its structural feature is that, of the two heavy chains constituting the bispecific antibody, the CH3 region of one heavy chain is mutated to form a protruding "knob" structure, while the CH3 region of the other heavy chain is mutated to form a recessed "hole" structure. Designing a knob-into-hole structure helps in the correct assembly of the two heterologous antibody heavy chains. Specifically, the 366th T in the CH3 domain of the first heavy chain is mutated to W, which has a larger side chain volume, forming a protruding "knob" structure. At the same time, the 366th T in the CH3 domain of the second heavy chain is mutated to S, the 368th L to A, and the 407th Y to V. These three mutations all reduce the volume of the side chains and form a recessed "hole" structure. Furthermore, by mutating the 354th S in the first heavy chain to C and the 349th Y in the second heavy chain to C, a knob-hole structure can be formed between the first and second heavy chains, and a covalent disulfide bond can be added, further stabilizing the knob-hole structure.

[0104] Specifically, for the knob-hole type, a "knob" is formed by introducing the T366W mutation into the first Fc, and a "hole" is formed by introducing the T366S, L368A, and Y407V mutations into the second Fc. For the charge-pair type, ion interactions are stabilized and heterodimerization is promoted by introducing charge residues linked to opposing Fc domains. For example, D356K, E357K, and D399K in the first Fc domain, and K370E, K409D, and K439E mutations in the second Fc domain, or combinations thereof (residue numbers are assigned using the Kabat EU numbering system). Furthermore, cysteine ​​can be introduced to stabilize heterodimer pairing. For example, S234C in the first Fc and Y349C in the second Fc, or Y349C in the first Fc and S344C in the second Fc. By mutating the 435th H in the "hole" chain to R and the 436th Y to F, the binding between the "hole" chain and protein A is eliminated, facilitating the removal of homodimers of the "hole" chain during the purification process.

[0105] The multispecific molecules of the present invention include asymmetric IgG-like antibodies (e.g., triomab / quadroma), knob-into-hole antibodies, cross monoclonal antibodies (Cross MAb), electrostatic matching antibodies, LUZ-Y, chain exchange gene manipulation domains (SEED), Fab exchange antibodies, symmetric IgG-like antibodies, two-in-one antibodies, cross-linked monoclonal antibodies, mAb2, and Cov This can include a variety of structures selected from the group consisting of X-body, dual variable region domain (DVD)-Ig fusion protein, IgG-like bispecific antibody, Ts2Ab, BsAb, scFv / Fc fusion, double (scFv)2-Fabs;F(ab)2 fusion protein, dual-acting or Bis-Fab, Dock-and-Lock (DNL), Fab-Fv, scFv antibody and biantibody (e.g., bispecific T cell engagers (BiTEs)), tandem biantibody (Tandab), DARTs, single-chain biantibody, TCR-like antibody, human serum albumin scFv fusion protein, COMBODIES, and IgG / non-IgG fusion protein.

[0106] Encoding nucleic acids and expression vectors The present invention further provides a polynucleotide molecule encoding the antibody or a fragment thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. Examples of DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence of the antibody of the present invention, or it may be a degenerate mutant. In this specification, “degenerate mutant” refers to a nucleic acid sequence that encodes the same amino acid sequence as the polypeptide of the present invention but has a different coding region sequence.

[0107] The polynucleotides encoding mature polypeptides of the present invention include a coding sequence encoding only mature polypeptides, a coding sequence of mature polypeptides and various additional coding sequences, a coding sequence of mature polypeptides (and any additional coding sequences) and non-coding sequences.

[0108] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide containing the polypeptide encoding, additional coding, and / or non-coding sequences.

[0109] The present invention also relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% homology between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, “stringent conditions” means (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2 × SSC, 0.1% SDS, 60°C; (2) addition of a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol, 42°C; and (3) hybridization occurs only when the homology between the two sequences is at least 90%, preferably at least 95%. Furthermore, polypeptides encoded by hybridizable polynucleotides have the same biological function and activity as the mature polypeptides shown in SEQ ID NO. 4 and SEQ ID NO. 9.

[0110] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. In particular, when the fragment length is short, it is also possible to synthesize the sequence artificially. In many cases, very long fragments can be obtained by synthesizing several smaller fragments and ligating them. Furthermore, a fusion protein can also be formed by fusing the heavy chain coding sequence with an expression tag (e.g., 6His).

[0111] Once the desired sequence is obtained, it can be acquired in large quantities by recombinant DNA. This is typically done by cloning it into a vector, transplanting it into cells, and then isolating the desired sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) related to this invention include biomolecules that exist in isolated forms.

[0112] Currently, DNA sequences encoding the protein (or its fragment or derivative thereof) of the present invention are readily available through chemical synthesis. These DNA sequences can be introduced into various existing DNA molecules (or vectors, etc.) or cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0113] The present invention also relates to vectors comprising a suitable DNA sequence and a suitable promoter or regulatory sequence as described above. These vectors can be used to transform suitable host cells so that they can express proteins.

[0114] The host cell may 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. Typical examples include bacterial cells of Escherichia coli, Streptomyces species, and Salmonella typhimurice; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0115] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, transformation-receptor cells capable of absorbing DNA can be collected after the exponential growth phase and treated with the CaCl2 method; the procedure for using this method is well known in the art. Another method involves using MgCl2. Transformation can also be performed by electroporation if necessary. When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, microinjection, electroporation, and conventional mechanical methods such as liposome packaging can be selected.

[0116] The resulting transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cells used, the culture medium can be selected from a variety of conventional media. Culture is carried out under conditions suitable for host cell proliferation. After the host cells have proliferated to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction), and the cells are cultured for a further period.

[0117] The recombinant polypeptides in the above method may be expressed intracellularly or at the cell membrane, or secreted extracellularly. If necessary, the recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical, and other properties. These methods are known to those skilled in the art. Examples of these methods include, but are not limited to, conventional regeneration processes, treatment with protein precipitants (salting-out), centrifugation, osmotic bacterial disruption, superprocessing, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations thereof.

[0118] The antibodies of the present invention can be used alone, or they can be conjugated or coupled with detection markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modification sites, or any combination thereof.

[0119] Detectable markers for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0120] Potentially coupling agents include, but are not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, cytokines, and hormones.

[0121] composition The present invention also provides compositions. In preferred embodiments, the composition is a drug-pharmaceutical composition comprising the antibody or its activated fragment or a fusion protein, and a pharmaceutically acceptable vector. Typically, these substances may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous vector medium, in which case the pH may typically be about 5 to 8, preferably about 6 to 8, and the pH may vary depending on the properties of the substances being formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including, but not limited to, oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

[0122] The pharmaceutical composition of the present invention can be used for the treatment of cancer / tumors, particularly solid tumors, especially solid tumors with high expression of MUC17.

[0123] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, particularly preferably 0.1-80 wt%) of the monoclonal antibody (or its complex) described in the present invention and a pharmaceutically acceptable vector or excipient. Such vectors include, but are not limited to, sodium chloride, buffer, glucose, water, glycerin, ethanol, and combinations thereof. The formulation needs to be adapted to the dosage form. The pharmaceutical composition of the present invention can be prepared, for example, by conventional methods from physiological saline or an aqueous solution containing glucose and other excipients to be made into an injectable preparation. Pharmaceutical compositions such as injectable preparations and solutions should preferably be prepared under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example, about 1 μg / kg body weight to about 10 mg / kg body weight per day. Furthermore, the pharmaceutical composition of the present invention can also be used in combination with other therapeutic agents.

[0124] When using a drug composition, a safe and effective amount of immune complexes should be administered to the mammal, where a safe and effective amount is typically at least about 10 μg / kg body weight, and in most cases about 8 mg / kg body weight or less, preferably about 10 μg / kg body weight to about 1 mg / kg body weight. Of course, the specific dosage should take into account factors such as the mode of administration and the patient's health condition, all of which are within the scope of a skilled physician's expertise.

[0125] application The present invention relates to a method for preventing, treating and / or detecting cancer / tumors or autoimmune diseases. The method comprises administering an effective amount of the multispecific T-cell engager or triplicate antibody of the present invention to a subject in need. In another embodiment, the method for preventing, treating and / or detecting cancer / tumors comprises administering an effective amount of one or more expression vectors expressing the triplicate antibody of the present invention to a subject in need. In another preferred embodiment, the cancer / tumor is a MUC17-associated cancer / tumor characterized by a disease / condition in which the number / percentage / activity of cells expressing MUC17 is increased compared to the number / percentage / activity of cells expressing MUC17 in the absence of the disease / condition. The cancer / tumor includes solid tumors and hematological malignancies. Preferably, it is selected from the group consisting of gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, gastroesophageal cancer, pancreatic adenocarcinoma, lung cancer, breast cancer, liver cancer, cervical cancer, thyroid cancer, uterine cancer, prostate cancer, or a combination thereof.

[0126] The main advantages of this invention are as follows: (1) The triplicate antibody of the present invention simultaneously targets the tumor-specific antigen MUC17, CD3 expressed by T cells, and the costimulatory molecule CD28, stimulating a more potent and effective killing activity against target cells, and has good in vitro safety. (2) The triplicate antibody of the present invention is highly pure and has good physicochemical properties, and can avoid problems such as aggregation, degradation fragments, and incomplete assembly that tend to occur in the production and storage of antibody molecules, thus contributing to industrial production.

[0127] The present invention will be described in more detail below, in accordance with specific embodiments. It should be understood that this is used solely to illustrate the present invention and not to limit its scope. In the following embodiments, experimental methods where specific conditions are not indicated generally follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or manufacturer-recommended conditions. Unless otherwise specified, percentages and quantities are on a weight basis.

[0128] Example 1. Origin and sequence of the anti-MUC17 monoclonal antibody According to CN202111500224.5, the amino acid sequence of the heavy chain variable region of the anti-MUC17 mouse monoclonal antibody 44H4 is as follows (SEQ ID NO:1). EVQLQQSGAELVKPGASVKLSFTASGFNIKDTYIHWVKQRPEQGLEWIGRIDPANGYTKYGPRFQGKATITADTASNAAYLQLSSLTSEDTAVYYCARNYGTSYPNAMDYWGQGTSVTVSS The amino acid sequence of the light chain variable region of the anti-MUC17 mouse monoclonal antibody 44H4 is as follows (SEQ ID NO:2). DIVLTQSPASLAVSLGQRATISCRASESVETYGNSFMHWYQQKPGQPPKLLIYRASSLESGIPARFSGSGSRTDFTLTITPVEADDVATYFCQQSNEDPYTFGGGTKLEIK The amino acid sequences of the heavy chain variable region and light chain variable region of the mouse monoclonal antibody 44H4 were analyzed, and the complementarity determining region (CDR) and framework region of the heavy chain and light chain variable regions were determined according to Kabat numbering rules. The amino acid sequences of the 44H4 heavy chain variable region CDR are as follows: H-CDR1:DTYIH (SEQ ID NO:3), H-CDR2:RIDPANGYTKYGPRFQG (SEQ ID NO:4), and H-CDR3:NYGTSYPNAMDY (SEQ ID NO:5). The amino acid sequences of the light chain variable regions (CDRs) are L-CDR1:RASESVETYGNSFMH (SEQ ID NO:6), L-CDR2:RASSLES (SEQ ID NO:7), and L-CDR3:QQSNEDPYT (SEQ ID NO:8), respectively.

[0129] The humanization process of mouse 44H4 is as follows: The homology between the heavy chain variable region of mouse monoclonal antibody 44H4 and the germline sequence of the heavy chain variable region of human antibody was compared. IGHV1-46*01 was selected as a model for humanization. Three heavy chain CDRs from mouse 44H4 were transplanted into IGHV1-46*01, and the corresponding CDRs in IGHV1-46*01 were replaced. A fourth framework region was added after H-CDR3 to obtain the amino acid sequence of the CDR-transplanted heavy chain variable region. Similarly, the homology between the light chain variable region of mouse 44H4 and the germline sequence of the light chain variable region of human antibody was compared. IGKV7-3*01 was selected as a model for humanization. Three light chain CDRs from mouse 44H4 were transplanted into the framework region of IGKV7-3*01, and the corresponding CDRs in IGKV7-3*01 were replaced. A fourth framework region was added after L-CDR3 to obtain the amino acid sequence of the CDR-transplanted light chain variable region. Based on the CDR transplantation variable region, reverse mutations were performed at several sites in the framework region (reverse mutations involve changing several amino acid residues in the human framework region to amino acid residues at the same positions in the mouse framework region; the sites of reverse mutations are usually crucial for maintaining the antibody structure and / or affinity). Kabat numbering was performed on the amino acid sequences during the reverse mutation process, and the locations of the sites were indicated by the Kabat numbers.

[0130] Preferably, in the heavy chain variable region after CDR transplantation, the 27th Y was reversed to mouse F, the 28th T was changed to N, the 29th F to I, the 30th T to K, the 69th M to I, and the 71st R to A. In the light chain variable region after CDR transplantation, the 81st N was changed to D to remove the N glycosylation site of the light chain. The heavy chain and light chain variable region having the above mutations were defined as the 44H4 humanized heavy chain variable region (SEQ ID NO: 9) and the light chain variable region (SEQ ID NO: 10), respectively.

[0131] The DNA encoding the humanized heavy chain and light chain variable regions was synthesized by Shanghai Bioengineering Co., Ltd. The synthesized humanized heavy chain variable region was ligated to the human IgG1 heavy chain constant region (SEQ ID NO: 11) using genetic engineering techniques to obtain a full-length humanized heavy chain, which was named 44H4-Hu-HC (SEQ ID NO: 12). The humanized light chain variable region was ligated to the human Kappa light chain constant region (SEQ ID NO: 13) to obtain a full-length humanized light chain, which was named 44H4-Hu-LC (SEQ ID NO: 14).

[0132] The coding genes for 44H4-Hu-HC and 44H4-Hu-LC were cloned into pcDNA3.4 expression vectors, respectively. To express antibodies, the heavy and light chain expression vectors were co-introduced into HEK293F cells using PEI. After 5 days, the FreeStyle® 293-F cell culture supernatant was collected, and the antibody was purified by Protein A affinity chromatography. The resulting antibody was named 44H4-Hu-IgG1.

[0133] Example 2. Origin and sequence of the anti-CD3 monoclonal antibody The amino acid sequences of the heavy chain and light chain variable regions of the mouse anti-human CD3 monoclonal antibody (abbreviated as SP34) are derived from SEQ ID NO: 2 and 4 in US8236308B2, respectively, and correspond to SEQ ID NO: 15 and 16 of the present invention, respectively. Amino acid sequence of the SP34 heavy chain variable region (SEQ ID NO: 15): EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA Amino acid sequence of the SP34 light chain variable region (SEQ ID NO: 16): QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL The amino acid sequences of the heavy and light chain variable regions of SP34 were analyzed, and the complementarity determination regions (CDRs) and framework regions of the SP34 heavy and light chains were determined according to Kabat numbering rules. The amino acid sequences of the three CDRs in the SP34 heavy chain are H-CDR1:TYAMN (SEQ ID NO:17), H-CDR2:RIRSKYNNYATYYADSVKD (SEQ ID NO:18), and H-CDR3:HGNFGNSYVSWFAY (SEQ ID NO:19), respectively. The amino acid sequences of the three CDRs in the light chain were L-CDR1:RSSTGAVTTSNYAN (SEQ ID NO:20), L-CDR2:GTNKRAP (SEQ ID NO:21), and L-CDR3:ALWYSNLWV (SEQ ID NO:22), respectively.

[0134] The humanization process for mouse SP34 is as follows: The homology between the heavy chain variable region of the mouse monoclonal antibody SP34 and the germline sequence of the heavy chain variable region of the human antibody was compared, and IGHV3-23*04 was selected as the humanization model. Three heavy chain CDRs from mouse SP34 were transplanted into IGHV3-23*04, and the corresponding CDRs in IGHV3-23*04 were replaced. A fourth framework region was added after H-CDR3, and the amino acid sequence of the CDR-transplanted heavy chain variable region was obtained. Similarly, the homology between the germline sequences of the light chain variable region of SP34 and the heavy chain variable region of human antibodies was compared. IGLV7-46*01 was selected as the light chain CDR transplantation model. Three light chain CDRs from mouse SP34 were transplanted into IGLV7-46*01, and the corresponding CDRs in IGLV7-46*0 were replaced. A fourth framework region was added after L-CDR3, and the amino acid sequence of the CDR-transplanted light chain variable region was obtained. Based on the CDR-transplanted variable region, reverse mutations were performed at several sites in the framework region. Kabat numbering was performed on the amino acid sequences during the reverse mutation process, and the site locations are indicated by the Kabat numbers.

[0135] Preferably, in the CDR transplanted heavy chain variable region, the 73rd N was reversed to D and the 94th K to R. In the CDR transplanted light chain variable region, the 36th F was reversed to V, the 46th T to G, the 49th Y to G, the 57th W to G, and the 58th T to V. In addition, the 2nd A in the CDR transplanted heavy chain variable region was reversed to T, and the 96th G (located in H-CDR3) was reversed to D. In addition, the 24th R in the CDR transplanted light chain variable region was reversed to G, the 33rd A to P, and the 94th N (located in L-CDR3) was reversed to D. These modifications can further improve the performance of the SP34 humanized antibody, such as affinity and stability. The heavy chain and light chain variable regions having the above mutations are defined as the SP34 humanized heavy chain variable region (SEQ ID NO: 23) and the light chain variable region (SEQ ID NO: 24), respectively.

[0136] The DNA encoding the humanized heavy chain and light chain variable regions was synthesized by Shanghai Bioengineering Co., Ltd. The synthesized humanized heavy chain variable region was ligated to the human IgG1 heavy chain constant region (SEQ ID NO: 11) using genetic engineering techniques to obtain a full-length humanized heavy chain, which was named SP34-Hu-HC (SEQ ID NO: 25). The humanized light chain variable region was ligated to the human Lambda light chain constant region (SEQ ID NO: 26) to obtain a full-length humanized light chain, which was named SP34-Hu-LC (SEQ ID NO: 27).

[0137] The coding genes for SP34-Hu-HC and SP34-Hu-LC were cloned into pcDNA3.4 expression vectors, respectively. To express antibodies, the heavy and light chain expression vectors were co-introduced into FreeStyle® 293-F cells using PEI. After 5 days, the FreeStyle® 293-F cell culture supernatant was collected, and the antibody was purified by Protein A affinity chromatography. The resulting antibody was named SP34-Hu-IgG1.

[0138] Example 3. Origin and sequence of the anti-CD28 monoclonal antibody The amino acid sequences of the heavy chain and light chain variable regions of the anti-human CD28 monoclonal antibody (abbreviated as Anti-CD28) are derived from SEQ ID NO: 46 and 48 in US20060286104A1, respectively, and correspond to SEQ ID NO: 28 and 29 of the present invention, respectively. Amino acid sequence of the Anti-CD28 heavy chain variable region (SEQ ID NO: 28): QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSS Amino acid sequence of the Anti-CD28 light chain variable region (SEQ ID NO: 29): DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIK The amino acid sequences of the heavy and light chain variable regions of Anti-CD28 were analyzed, and the complementarity-determining regions (CDRs) and framework regions of the Anti-CD28 heavy and light chains were determined according to Kabat rules. The amino acid sequences of the three CDRs of the Anti-CD28 heavy chain were H-CDR1:SYYIH (SEQ ID NO:30), H-CDR2:CIYPGNVNTNYNEKFKD (SEQ ID NO:31), and H-CDR3:SHYGLDWNFDV (SEQ ID NO:32), respectively. The amino acid sequences of the three CDRs in the light chain are L-CDR1:HASQNIYVWLN (SEQ ID NO:33), L-CDR2:KASNLHT (SEQ ID NO:34), and L-CDR3:QQGQTYPYT (SEQ ID NO:35), respectively.

[0139] To improve the degree of humanization of the anti-CD28 monoclonal antibody, the amino acid sequence of its heavy chain variable region was modified. The process was as follows: The homology between the germline sequences of the anti-CD28 heavy chain variable region and the heavy chain variable region of the human antibody was compared, and IGHV1-46*01 was selected as the reference model. Specific amino acid residues in the framework region of the anti-CD28 heavy chain variable region were mutated to amino acid residues at the same positions in the framework region of IGHV1-46*01. Kabat numbering was performed on the amino acid sequences during the mutation, and the site locations were indicated by the Kabat numbers.

[0140] Preferably, the amino acid sequence of the Anti-CD28 heavy chain variable region was mutated, with the 48th I being changed to M, the 67th A being changed to V, and the 91st F being changed to Y. In addition, to avoid the formation of covalent disulfide bonds between antibody molecules during the production process, the first amino acid residue C of the Anti-CD28 heavy chain variable region H-CDR2 was mutated to S.

[0141] The amino acid sequence of the modified Anti-CD28 heavy chain variable region is as follows: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGSIYPGNVNTNYNEKFKDRVTLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGQGTTVTVSS(SEQ ID NO. 50) The DNA encoding the modified Anti-CD28 heavy chain variable region and the unmodified Anti-CD28 light chain variable region was synthesized by Shanghai Bioengineering Co., Ltd. The synthesized humanized heavy chain variable region was ligated to the human IgG1 heavy chain constant region (SEQ ID NO: 11) by genetic engineering to obtain a full-length humanized heavy chain, which was named Anti-CD28-Hu-HC (SEQ ID NO: 36). The Anti-CD28 light chain variable region was ligated to the human Kappa light chain constant region (SEQ ID NO: 13) to obtain a full-length humanized light chain, which was named Anti-CD28-Hu-LC (SEQ ID NO: 37).

[0142] The coding genes for Anti-CD28-Hu-HC and Anti-CD28-Hu-LC were cloned into pcDNA3.4 expression vectors, respectively. To express the antibodies, the heavy and light chain expression vectors were co-introduced into FreeStyle® 293-F cells using PEI. After 5 days, the FreeStyle® 293-F cell culture supernatant was collected, and the antibody was purified by Protein A affinity chromatography. The resulting antibody was named Anti-CD28-Hu-IgG1.

[0143] Example 4. Construction and preparation of an anti-MUC17*CD3*CD28 trispecific antibody. The initial structure consists of three polypeptide chains with different sequences, encoded by three genes, and its design and construction method are as follows.

[0144] The method for constructing the first polypeptide chain or gene is as follows: Using genetic engineering techniques, the light chain variable region of Anti-CD28-Hu-IgG1 (including the Q100C mutation) was linked to the heavy chain variable region of SP34-Hu-IgG1 with a short artificial linker (the linker used here is G4S), and the ends were linked to the light chain variable region of SP34-Hu-IgG1 with a long artificial linker (the linker used here is four tandem G4S), the ends were linked to the heavy chain variable region of Anti-CD28-Hu-IgG1 (including the G44C mutation) with a short artificial linker (the linker used here is G4S), and the ends were linked to the hinge region and Fc segment of human IgG1 (the Fc segment represents the CH2 and CH3 domains of the human IgG1 heavy chain constant region) with a long artificial linker (the linker used here is three tandem G4S).

[0145] The Fc segment contains L234A / L235A / G237A mutations to weaken the interaction between Fc and the Fcγ receptor (Reference: Liu R, Oldham RJ, Teal E, et al. Fc-engineering for modulated effector functions - improving antibodies for cancer treatment [J]. Antibodies, 2020, 9(4): 64.), and S354C / T366W to form heterodimers with other heavy chain polypeptides having a Knob-Into-Hole (KIH) structure, as described below (Reference: Ha JH, Kim JE, Kim Y S. Immunoglobulin Fc heterodimer platform technology: from design to applications in therapeutic antibodies and proteins [J]. Frontiers in Immunology, 2016, 7: 394.).

[0146] The first polypeptide chain or gene was named CD28-44-SP34-Dia-IgG1-Knob-HC (amino acid sequence SEQ ID NO:38, nucleotide sequence SEQ ID NO:39).

[0147] The method for constructing the second polypeptide chain or gene is as follows: The heavy chain variable region of 44H4-Hu-IgG1 is linked to the human IgG1 heavy chain constant region (including the CH1, CH2, and CH3 domains), which contains multiple mutations. Here, the Fc segment contains the L234A / L235A / G237A mutation to weaken the interaction between Fc and the Fcγ receptor, and the Y349C / T366S / L368A / Y407V mutation to form a heterodimer with the other heavy chain polypeptides mentioned above, which has a Knob-Into-Hole (KIH) structure. The second polypeptide chain or gene was named 44H4-IgG1-Hole-HC (amino acid sequence SEQ ID NO: 40, nucleotide sequence SEQ ID NO: 41).

[0148] The third polypeptide chain or gene is the light chain of 44H4-Hu-IgG1, and therefore was not modified or altered. The coding genes for CD28-44-SP34-Dia-IgG1-Knob-HC, 44H4-IgG1-Hole-HC, and 44H4-Hu-LC were cloned into pcDNA3.4 expression vectors, and these expression vectors were co-introduced into FreeStyle® 293-F cells using PEI to express antibodies. After 5 days, the FreeStyle® 293-F cell culture supernatant was collected, and the antibody was purified by Protein A affinity chromatography. The resulting antibody was named CD28-44 / SP34-Dia+44H4-KIH.

[0149] Figure 1A shows a schematic diagram of the structure of the triply specific antibody. Here, VL1 and VH1 are the light and heavy chain variable regions of the anti-CD28 antibody, respectively; VH2 and VL2 are the heavy and light chain variable regions of the anti-CD3 antibody, respectively; and VH3 and VL3 are the heavy and light chain variable regions of the anti-MUC17 antibody, respectively. Straight and curved arrows indicate artificially designed engagers and their directions, and bidirectional straight arrows indicate the position of artificially designed disulfide bonds. In Figure 1A, VL1, VH2, VL2, and VH1 are sequentially linked via engagers of different lengths, and finally fold and join to form a Diabody structure (for the structure of the Diabody, see Wu C. Diabodies: molecular engineering and therapeutic applications[J]. Drug News Perspect, 2009, 22(8): 453.).

[0150] The second structure consists of three polypeptide chains with different sequences, encoded by three genes, and its design and construction method are as follows.

[0151] The method for constructing the first polypeptide chain or gene is as follows: Using genetic engineering techniques, the light chain variable region of Anti-CD28-Hu-IgG1 (including the Q100C mutation) was linked to the heavy chain variable region of Anti-CD28-Hu-IgG1 (including the G44C mutation) with a long artificial linker (the linker used here is four tandem G4S). The ends were then linked to the light chain variable region of SP34-Hu-IgG1 with a short artificial linker (the linker used here is two tandem G3S). The ends were then linked to the heavy chain variable region of SP34-Hu-IgG1 with a long artificial linker (the linker used here is four tandem G3S). The ends were then linked to the hinge region and Fc segment of human IgG1 (the Fc segment represents the CH2 and CH3 domains of the human IgG1 heavy chain constant region) with a short artificial linker (the linker used here is two tandem GGGS). Here, the Fc segment includes the L234A / L235A / G237A mutation to weaken the interaction between Fc and the Fcγ receptor, and the S354C / T366W mutation to form a heterodimer with other heavy chain polypeptides described later, which have a Knob-Into-Hole (KIH) structure. The first polypeptide chain or gene was named CD28-44-SP34-2ScFv-IgG1-Knob-HC (amino acid sequence SEQ ID NO: 42, nucleotide sequence SEQ ID NO: 43).

[0152] The second polypeptide chain or gene is 44H4-IgG1-Hole-HC.

[0153] The third polypeptide chain or gene is 44H4-Hu-LC. The coding genes for CD28-44-SP34-2ScFv-IgG1-Knob-HC, 44H4-IgG1-Hole-HC, and 44H4-Hu-LC were cloned into pcDNA3.4 expression vectors, and these expression vectors were co-introduced into FreeStyle® 293-F cells using PEI to express antibodies. After 5 days, the FreeStyle® 293-F cell culture supernatant was collected, and the antibody was purified by Protein A affinity chromatography. The resulting antibody was named CD28-44 / SP34-ScFv+44H4-KIH.

[0154] Figure 1B shows a schematic diagram of the structure of the triply specific antibody. Here, VL1 and VH1 are the light and heavy chain variable regions of the anti-CD28 antibody, respectively; VL2 and VH2 are the light and heavy chain variable regions of the anti-CD3 antibody, respectively; and VH3 and VL3 are the heavy and light chain variable regions of the anti-MUC17 antibody, respectively. Curved arrows indicate artificially designed engagers and their directions, and bidirectional straight arrows indicate the positions of artificially designed disulfide bonds.

[0155] In Figure 1B, VL1, VH1, VL2, and VH2 are sequentially linked via engagers of different lengths and eventually fold to form two tandem ScFv structures (for the structure of ScFv, see Ahmad ZA, Yeap SK, Ali AM, et al. scFv antibody: principles and clinical application [J]. Clinical and developmental immunology, 2012, 2012:980250).

[0156] In this example, mutations were introduced into the Fc segment of the human IgG1 heavy chain constant region to construct a heavy chain constant region with a "knob" structure and a heavy chain constant region with a "hole" structure, respectively. By mutating the 435th H to R and the 436th Y to F in the "hole" chain, the binding effect of the "hole" chain to Protein A was eliminated, and the homodimer of the "hole" chain could be removed in the purification procedure (Reference: Smith E, Olson K, Haber L, et al. A novel, native-format bispecific antibody triggering T-cell killing of B-cells is robustly active in mouse tumor models and cynomolgus monkeys[J]. Scientific Reports, 2016, 5(1): 17943-17943.). The aforementioned "knob" heavy chain constant region and the improved "hole" heavy chain constant region Fc segment were named IgG1-Knob-HC (SEQ ID NO:44) and IgG1-Hole-HC (SEQ ID NO:45), respectively.

[0157] Example 5. Antigen-binding ability of anti-MUC17*CD3*CD28 trispecific antibody. The method for expressing and purifying the recombinant protein is as follows. The human MUC17 amino acid sequence was obtained from Uniprot (Entry: Q685J3), and amino acid sequences from position 4131 to 4390 were selected. A signal peptide was introduced at the N-terminus, and one engager (G4S) and six histidine residues were introduced at the C-terminus. The coding gene for the above recombinant amino acid sequence was obtained by gene synthesis, and the coding gene for the recombinant protein was cloned into a pcDNA3.4 (Thermo Fisher Scientific) expression vector. The expression vector was introduced into FreeStyle® 293-F cells (Thermo Fisher Scientific) using PEI to express the recombinant protein. After an appropriate time, the cell culture supernatant was collected and filtered, and the recombinant protein was purified from the supernatant by nickel affinity chromatography. The obtained recombinant protein was named MUC17-His (SEQ ID: 46). The human CD28 amino acid sequence was obtained from Uniprot (Entry: P10747), and amino acid sequences from position 9 to 152 were selected. A signal peptide was introduced at the N-terminus, and one engager (G4S) and six histidine residues were introduced at the C-terminus. The coding gene for the above recombinant amino acid sequence was obtained by gene synthesis, and the recombinant protein was expressed and purified using the same method as described above. The resulting recombinant protein was named CD28-His (SEQ ID: 47).

[0158] In this example, the binding affinity of an anti-MUC17*CD3*CD28 tripspecific antibody to MUC17, CD3, and CD28 was measured using ELISA (Enzyme-Linked Immunosorbent Assay). The method is as follows. Recombinant proteins MUC17-His, CD3-Epsilon (ACROBiosystems, catalog number: CDE-H5223), and CD28-His were coated at 10 ng / well in a microplate (96 wells), and the microplate was blocked with PBST containing 1% bovine serum albumin (PBST represents a phosphate buffer containing 0.05% Tween-20). The antibody to be measured was gradient diluted and transferred to the microplate coated with the recombinant proteins. The plate was incubated at room temperature for 30 minutes and then washed. Appropriately diluted HRP (Horseradish Peroxidase)-labeled goat anti-human antibody (Fc specific, Sigma, catalog number: SAB3701283) was added, and the plate was incubated at room temperature for 30 minutes. 100 μL of a colorimetric solution using TMB (3,3′,5,5′-Tetramethylbenzidine) as a substrate was added to each well, and the mixture was incubated at room temperature for 1–5 minutes. The reaction was stopped by adding 50 μL of reaction stop solution (2M H2SO4). The OD450 was read using a Microplate Reader (model SpectraMax 190), plotted, and data analysis was performed using GraphPad Prism7. 50 The concentration for 50% of the maximal effect was calculated. [Table 1]

[0159] Table 1 summarizes the calculation results for each graph in Figure 2A / B / C. As shown in Figure 2A / B / C and Table 1, 44H4-Hu-IgG1, SP34-Hu-IgG1, and Anti-CD28-Hu-IgG1 can effectively bind to MUC17-His, CD3-Epsilon, and CD28-His, respectively, and EC 50 The concentrations were 0.06507 nM, 0.05533 nM, and 0.1166 nM, respectively. CD28-44 / SP34-Dia+44H4-KIH can effectively bind to MUC17-His, CD3-Epsilon, and CD28-His, and EC50 These values ​​were 0.06246 nM, 0.9199 nM, and 0.8207 nM, respectively. CD28-44 / SP34-ScFv+44H4-KIH can also be effectively coupled to MUC17-His, CD3-Epsilon, and CD28-His, EC 50 These values ​​were 0.1732 nM, 3.363 nM, and 1.711 nM, respectively. Both CD28-44 / SP34-Dia+44H4-KIH and CD28-44 / SP34-ScFv+44H4-KIH were shown to be triply specific antibodies, effectively binding to MUC17-His, CD3-Epsilon, and CD28-His.

[0160] Example 6. Binding ability of anti-MUC17*CD3*CD28 tripspecific antibody to cell surface antigens. The binding ability of anti-MUC17*CD3*CD28 trispecific antibodies to MUC17 on the surface of human colon adenocarcinoma cells LS 174T (ATCC, catalog number: CL-188) and to CD3 / CD28 on the surface of human T cells was measured by flow cytometry. The specific procedure is as follows: LS 174T cells or fresh human peripheral blood mononuclear cells (abbreviated as PBMCs, manufactured by Shanghai Aoneng Biotechnology Co., Ltd., catalog number: FPB002-C-200) were inoculated into a 96-well round-bottom microplate (300,000 cells per well). After centrifugation at 300g for 5 minutes, the supernatant was aspirated and removed using a multichannel pipette. 200 μL of phosphate buffer (PBS) containing 1% bovine serum albumin (BSA) (referred to as PBS + 1% BSA) was added to each well to resuspend the cell aggregate, and the plate was centrifuged at 300g for 5 minutes, with the supernatant aspirated and removed. Gradient-diluted antibodies were added, the cell aggregate was resuspended, and the plate was incubated at room temperature for approximately 30 minutes. Centrifuged at 300g for 5 minutes, the supernatant was aspirated and removed, and the cells in each well were washed twice with 200 μL of PBS. An appropriate amount of R-phycoerythrin-labeled goat anti-human IgG (Jackson ImmunoResearch, catalog number: 109-115-098), diluted 1:1000 in PBS + 1% BSA, was added to each well, the cell aggregate was resuspended, and incubated at room temperature for approximately 30 minutes. For PBMCs, APC Mouse Anti-Human CD4 (BD Biosciences, catalog number: 555349) was added to label CD4-positive T cells. Centrifuged at 300g for 5 minutes, the supernatant was aspirated and removed, and the cells in each well were washed twice with 200 μL of PBS. Fix Buffer I (BD Biosciences, catalog number: 557870) was added to each well to fix the cells, and incubated at room temperature for approximately 5 minutes. Centrifuged at 300g for 5 minutes, the supernatant was aspirated and removed, the cells in each well were washed twice with 200 μL of PBS, and finally the cells were resuspended in 200 μL of PBS. The fluorescence intensity of PE channels was detected using a flow cytometer, the CytoFLEX Cytometer System (Beckman Coulter). For PBMCs, the PE fluorescence intensity of CD4-positive T cells was measured. The data was processed using flow cytometer software, and the mean fluorescence intensity (MFI) of the PE channels for each sample was exported. The data was analyzed and plotted using GraphPad Prism 7, and EC (European Clinical Laboratory) analysis was performed. 50 The result was calculated.

[0161] Here, the anti-MUC17*CD3 bispecific antibody AMG199, developed by Amgen, was introduced as a control antibody. The amino acid sequence of AMG199 was derived from SEQ ID NO: 171 of WO2022060901A1. AMG199 was prepared using the expression and purification methods described in the above example. [Table 2] Table 2 summarizes the calculation results for each graph in Figure 3A / B. "Top" indicates the high-frequency range of the fitting curve, representing the theoretical maximum signal intensity of antibody binding.

[0162] As shown in Figure 3A and Table 2, 44H4-Hu-IgG1, CD28-44 / SP34-Dia+44H4-KIH, CD28-44 / SP34-ScFv+44H4-KIH, and AMG199 can all effectively bind to LS 174T cells, and EC 50 These values ​​were 0.652 nM, 2.265 nM, 1.844 nM, and 0.4247 nM, respectively, with their top values ​​being 3745, 8072, 7494, and 4068.

[0163] The results above indicate that 44H4-Hu-IgG1 and AMG199 have equivalent binding abilities to LS174T cells, and that CD28-44 / SP34-Dia+44H4-KIH and CD28-44 / SP34-ScFv+44H4-KIH also have equivalent binding abilities to LS174T cells. At high concentrations, the top binding levels of CD28-44 / SP34-Dia+44H4-KIH and CD28-44 / SP34-ScFv+44H4-KIH were significantly higher than those of 44H4-Hu-IgG1 and AMG199, indicating that the triply specific antibodies of the present invention have superior binding ability to cell surface antigens compared to the latter two.

[0164] As shown in Figure 3B and Table 2, SP34-Hu-IgG1, Anti-CD28-Hu-IgG1, CD28-44 / SP34-Dia+44H4-KIH, CD28-44 / SP34-ScFv+44H4-KIH, and AMG199 can all effectively bind to CD4-positive T cells, and EC 50 These values ​​were 0.6691nM, 2.38nM, 4.175nM, 2.802nM, and 2.714nM, respectively, with their top values ​​being 39823, 21894, 31073, 26579, and 12929, respectively.

[0165] The results above indicate that the monoclonal antibody SP34-Hu-IgG1 had the highest binding ability to CD4 T cells, while Anti-CD28-Hu-IgG1, CD28-44 / SP34-Dia+44H4-KIH, and CD28-44 / SP34-ScFv+44H4-KIH had similar binding abilities to CD4 T cells, and AMG199 had the lowest binding ability.

[0166] Example 7. Killing activity of anti-MUC17*CD3*CD28 trispecific antibody against target cells. The target cells were prepared as follows. The Luciferase gene sequence was derived from GenBank (MK484108.1), and after the coding gene was synthesized by Bioengineering (Shanghai) Co., Ltd., the Luciferase gene was cloned into the pLVX-puro lentiviral vector (Clontech, catalog number: 632164). The resulting expression vector was named pLVX-Luciferase-puro. The lentiviral packaging plasmid and pLVX-Luciferase-puro were co-introduced into HEK293 cells to prepare a recombinant lentivirus into which the Luciferase gene was introduced. The cell culture supernatant containing the recombinant lentivirus was collected and used to infect LS 174T cells. After 3 days, the cells were screened with puromycin under pressure to obtain cell clones into which the target gene had been introduced. The final cell line that stably expressed Luciferase was named LS174T-Luciferase.

[0167] CD3-positive T cells (manufactured by Shanghai Aoneng Biotechnology Co., Ltd., catalog number: FPB009-1-C-100) were used as effector cells. Through the mediation of the anti-MUC17*CD3*CD28 trispecific antibody, the effector cells can effectively activate and kill the tumor target cell LS174T-Luciferase established above. By measuring the Luciferase intensity, the killing effect of the effector cells, or the degree of target cell killing, can be reflected, and the functional activity of the trispecific antibody can be calculated.

[0168] LS174T-Luciferase and CD3-positive T cells were mixed in a 1:10 ratio, centrifuged, and the supernatant was discarded. The cell aggregate was then resuspended in Advanced RPMI 1640 complete medium (containing 10% FBS, 1% Penicillin Streptomycin, 1% GlutaMAX, and 1‰ 2-Mercaptoethanol), and 10,000 LS174T-Luciferase cells were seeded per well in a flat-bottomed 96-well cell culture plate. Various antibodies were then added in gradient dilutions. Furthermore, target cells were completely killed by adding 0.01% Triton X-100 to the borrowed wells, and several other wells were supplemented with antibody-free complete medium. After 72 hours, luciferase intensity was measured using the Bio-Glo® Luciferase Assay System (Promega, catalog number: G7940). The average luciferase intensity of a few wells containing Triton X-100 was used as the minimum background value, and the average luciferase intensity of a few wells without antibody was used as the maximum threshold for cell proliferation. The formula for calculating antibody cytotoxicity is: Cytotoxicity = (Maximum threshold - Luciferase) / (Maximum threshold - Minimum background value) * 100% [Table 3A] [Table 3B]

[0169] Figures 4A and 4B show the results of two independent repeated experiments respectively. The effector cells used in the two experiments are from different human individuals. Tables 3A / B summarize the calculation results of each group in Figures 4A / B respectively. Top shows the high frequency of the fitting curve and represents the theoretical maximum intensity of cytotoxicity.

[0170] The results of Figure 4A and Table 3A show that the monoclonal antibodies 44H4-Hu-IgG1 and Anti-CD28-Hu-IgG1 have no activity to kill the target cells LS174T-Luciferase, and SP34-Hu-IgG1 has relatively weak killing activity (EC 50 was 432.4 pM and Top was 37.76%). CD28-44 / SP34-Dia+44H4-KIH, CD28-44 / SP34-ScFv+44H4-KIH and AMG199 can all effectively kill LS174T-Luciferase cells, and EC 50 was 8.418 pM, 25.71 pM and 13.63 pM respectively. Their Tops were 97.11%, 85.14% and 83.04% respectively. The killing degree of CD28-44 / SP34-Dia+44H4-KIH against target cells was the highest (97.11%). As a result of comprehensive evaluation, the order of the high killing activities of the three antibodies against target cells is as follows: CD28-44 / SP34-Dia+44H4-KIH > AMG199 > CD28-44 / SP34-ScFv+44H4-KIH.

[0171] The results of Figure 4B and Table 3B show that the monoclonal antibodies 44H4-Hu-IgG1 and Anti-CD28-Hu-IgG1 have no activity to kill the target cells LS174T-Luciferase, and SP34-Hu-IgG1 has relatively weak killing activity (EC 50It was shown that the concentration was 253.3 pM and the top concentration was 49.58%. CD28-44 / SP34-Dia+44H4-KIH, CD28-44 / SP34-ScFv+44H4-KIH, and AMG199 can all effectively kill LS174T-Luciferase cells, and EC 50 The concentrations were 9.284 pM, 20.59 pM, and 10.48 pM, respectively, with top percentages of 93.55%, 85.72%, and 83.91%. CD28-44 / SP34-Dia+44H4-KIH showed the highest degree of target cell killing (93.55%).

[0172] Based on an overall evaluation, the order of the three antibodies' killing activity against target cells, from highest to lowest, is as follows: CD28-44 / SP34-Dia+44H4-KIH > AMG199 > CD28-44 / SP34-ScFv+44H4-KIH.

[0173] Example 8. Evaluation of the superiority of an anti-MUC17*CD3*CD28 tripspecific antibody over a bispecific antibody. Site-directed mutations were introduced into the major CDR sites of the monoclonal antibodies SP34-Hu-IgG1 and Anti-CD28-Hu-IgG1, respectively, to create variants of each monoclonal antibody. By evaluating their binding activity using the ELISA method described in Example 5, monoclonal antibody variants that no longer bound to their respective targets were obtained. Figure 5A shows that the variant SP34-Hu-IgG1(A101D), created by introducing the A101D mutation into CDR3 of the SP34-Hu-IgG1 heavy chain, no longer bound to CD3-Epsilon. Figure 5B shows that the variant Anti-CD28-Hu-IgG1(H35A+H96A), created by introducing the H35A mutation into CDR1 and the H96A mutation into CDR3 of the Anti-CD28-Hu-IgG1 heavy chain, no longer bound to CD28.

[0174] CD28-44 / SP34-Dia+44H4-KIH and CD28-44 / SP34-ScFv+44H4-KIH contain VH and VL of SP34-Hu-IgG1. By introducing the A101D mutation into VH and regenerating the mutation, the resulting antibodies were named CD28-44 / SP34-Dia+44H4-KIH(-CD3) and CD28-44 / SP34-ScFv+44H4-KIH(-CD3), respectively. These two mutated antibodies lose the ability to bind to CD3-Epsilon and are theoretically bispecific antibodies capable of binding to MUC17 and CD28.

[0175] CD28-44 / SP34-Dia+44H4-KIH and CD28-44 / SP34-ScFv+44H4-KIH contain VH and VL of Anti-CD28-Hu-IgG1. By introducing H35A and H96A mutations into VH and regenerating the mutations, the resulting antibodies were named CD28-44 / SP34-Dia+44H4-KIH(-CD28) and CD28-44 / SP34-ScFv+44H4-KIH(-CD28), respectively. These two mutated antibodies lose the ability to bind to CD28 and are theoretically bispecific antibodies capable of binding to MUC17 and CD3.

[0176] The killing activity of the triplicate antibody and its variants against target cells was measured using the method described in Example 7. Table 4 summarizes the calculation results for each graph in Figure 6A.

[0177] The results in Figure 6A and Table 4 show that CD28-44 / SP34-Dia+44H4-KIH and CD28-44 / SP34-ScFv+44H4-KIH can still effectively kill target cells, and EC 50The concentrations were 6.6 pM and 12.99 pM, respectively, with Top concentrations of 97.51% and 96.41%, respectively. CD28-44 / SP34-Dia+44H4-KIH(-CD3) and CD28-44 / SP34-ScFv+44H4-KIH(-CD3) did not show any killing activity against target cells. The EC2 of CD28-44 / SP34-Dia+44H4-KIH(-CD28) and CD28-44 / SP34-ScFv+44H4-KIH(-CD28) showed killing activity against target cells. 50 The activity levels were 117.8 pM and 87.59 pM, respectively, and the top activity levels were 98.15% and 92.51%, respectively, indicating that the activity of the mutant was significantly reduced compared to the parental triplicate antibody. [Table 4]

[0178] In the aforementioned cell toxication test, the effector cells were CD3-positive T cells, and the target cells were LS174T-Luciferase. Through the intervention of a trispecific antibody or its variant, T cells recognize and are activated by a specific antigen expressed on the surface of the target cells. Activated T cells secrete several cytokines, which can further activate the T cells and enhance their toxic activity. To further verify the superiority of the trispecific antibody compared to a bispecific antibody, the INF-gamma concentration in the supernatant of the cell toxication reaction system was also measured in this example. The method for measuring the INF-gamma concentration in the supernatant is as follows.

[0179] Anti-human IFN-gamma antibody (BD Biosciences, catalog number: 551221) was coated at 0.3 μg / well in an ELISA microplate, and the microplate was blocked with PBST containing 1% bovine serum albumin (PBST represents a phosphate buffer containing 0.05% Tween-20). An IFN-gamma standard (BD Biosciences, catalog number: 554617) was prepared in PBST containing 1% BSA. The supernatant of the cell killing reaction system was diluted fourfold with PBST containing 1% BSA. The IFN-gamma standard, gradient diluted at 100 μL / well, and the four-fold diluted supernatant were transferred to the microplate coated with the anti-human IFN-gamma antibody and incubated at room temperature for 1 hour. After washing the plate three times with PBST, biotinylated anti-human IFN-gamma antibody (BD Biosciences, catalog number: 554550) was added to each well. Immediately before use, the solution was diluted 1000-fold with PBST containing 1% BSA and incubated at room temperature for 0.5 hours. After washing the plate three times with PBST, 100 μL of Streptavidin HRP (BD Biosciences, catalog number: 554066) was added to each well. Immediately before use, the solution was diluted 1000-fold with PBST containing 1% BSA and incubated at room temperature for 0.5 hours. After washing the plate three times with PBST, 100 μL of a colorimetric solution using TMB (3,3′,5,5′-Tetramethylbenzidine) as a substrate was added to each well and incubated at room temperature for 1-5 minutes. The reaction was stopped by adding 50 μL of stop solution (2M H2SO4). The OD450 was read using a Microplate Reader (model SpectraMax 190), and the IFN-gamma concentration in each well was calculated using a calibration curve. The data was plotted and analyzed using GraphPad Prism7. 50 The result was calculated.

[0180] The results in Figure 6B show that CD28-44 / SP34-Dia+44H4-KIH and CD28-44 / SP34-ScFv+44H4-KIH effectively stimulate CD3-positive T cells and promote IFN-gamma secretion, and EC 50At concentrations of 16.65 pM and 165.8 pM, respectively, the antibodies were able to secrete up to 10 ng / mL of IFN-gamma at high concentrations. CD28-44 / SP34-Dia+44H4-KIH(-CD3) and CD28-44 / SP34-ScFv+44H4-KIH(-CD3) did not show activity to promote IFN-gamma secretion. Compared to the parental triplicate antibody, the activity of CD28-44 / SP34-Dia+44H4-KIH(-CD28) and CD28-44 / SP34-ScFv+44H4-KIH(-CD28) to stimulate T cells to secrete IFN-gamma was very weak.

[0181] The results of this example demonstrate a correlation and consistency between the target cell-killing activity of CD28-44 / SP34-Dia+44H4-KIH and CD28-44 / SP34-ScFv+44H4-KIH and their activity in stimulating T cells to secrete IFN-gamma. CD28 is a classic costimulatory molecule expressed on the surface of T cells. The triplicate antibody of the present invention can bind to both CD3 and CD28 simultaneously, thus providing a dual signal for T cell activation and exhibiting more effective T cell killing activity than conventional bispecific antibodies.

[0182] Example 9. Evaluation of the superiority of an anti-MUC17*CD3*CD28 trispecific antibody compared to CODV-IgG. CODV-Ig is Sanofi's multispecific antibody platform (Reference: Steinmetz A, Vallee F, Beil C, et al. CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications[C] / / MAbs. Taylor & Francis, 2016, 8(5): 867-878.). Sanofi used this platform to design a trispecific antibody targeting HER2*CD3*CD28 (Reference: Seung E, Xing Z, Wu L, et al. A trispecific antibody targeting HER2 and T cells inhibits breast cancer growth via CD4 cells[J]. Nature, 2022, 603(7900): 328-334.).

[0183] To demonstrate the superiority of the anti-MUC17*CD3*CD28 trispecific antibody of the present invention compared to CODV-IgG, this example prepared an anti-MUC17*CD3*CD28 trispecific antibody having a CODV-IgG structure and a related anti-CD3 / CD28 antibody variable region sequence. The preparation process is as follows. The amino acid sequence of the heavy chain variable region of the CODV-IgG-related anti-CD3 / CD28 antibody was derived from SEQ ID:64 of US20200140552A1. This sequence was spliced ​​with the heavy chain constant region sequence of the polypeptide CD28-44-SP34-Dia-IgG1-Knob-HC, and the coding gene encoding this recombinant sequence was obtained by genetic engineering. This polypeptide and its coding gene were named CD28-CD3-CODV-IgG1-Knob-HC (SEQ ID:48). The amino acid sequence of the light chain of the CODV-IgG-related anti-CD3 / CD28 antibody was derived from SEQ ID:61 of US20200140552A1, and the polypeptide and its coding gene were named CD3-CD28-CODV-LC (SEQ ID:49).

[0184] The coding genes for CD28-CD3-CODV-IgG1-Knob-HC and CD3-CD28-CODV-LC were cloned into pcDNA3.4 expression vectors, respectively. These vectors were then combined with the 44H4-IgG1-Hole-HC and 44H4-Hu-LC expression vectors from the above example in a predetermined ratio. The four expression vectors were then co-introduced into FreeStyle® 293-F cells using PEI to induce antibody expression. After 5 days, the FreeStyle® 293-F cell culture supernatant was collected, and the antibody was purified by Protein A affinity chromatography. The resulting antibody was named 44H4-CD3-CD28-CODV.

[0185] In this example, the activity of the present invention and the trispecific antibody with the CODV-IgG structure were investigated and compared, and the killing activity of these trispecific antibodies and IFN-gamma secretion in the cellular reaction system were measured using the method described in the above example.

[0186] The results in Figure 7A show that CD28-44 / SP34-Dia+44H4-KIH and CD28-44 / SP34-ScFv+44H4-KIH can still effectively kill target cells, and EC 50 The concentrations were 6.6 pM and 12.99 pM, respectively. EC2 of the 44H4-CD3-CD28-CODV killing activity against target cells. 50 The concentration was 173.2 pM. Compared to the two triplicate antibodies of the present invention, the activity of 44H4-CD3-CD28-CODV was significantly weaker.

[0187] The results in Figure 7B show that CD28-44 / SP34-Dia+44H4-KIH and CD28-44 / SP34-ScFv+44H4-KIH effectively stimulate CD3-positive T cells and promote IFN-gamma secretion, and EC 50The concentrations are 16.65 pM and 165.8 pM, respectively, and the antibody can secrete up to 10 ng / mL of IFN-gamma at high concentrations. Compared to the two triplicate antibodies of the present invention, the activity of stimulating 44H4-CD3-CD28-CODV T cells to secrete IFN-gamma is very weak.

[0188] Example 10. In vitro safety evaluation of an anti-MUC17*CD3*CD28 trispecific antibody. PBMC (manufactured by Shanghai Aoneng Biotechnology Co., Ltd., catalog number: FPB002-C-200) was washed twice with PBS, and the cell aggregate was resuspended in Advanced RPMI 1640 complete medium (containing 10% FBS, 1% Penicillin Streptomycin, 1% GlutaMAX, 1‰ 2-Mercaptoethanol). After counting and adjusting the cell density, the cells were inoculated at 150 μL / well into a 96-well round-bottom microplate. In another 96-well microplate, the antibody was gradient diluted with medium, and 50 μL of the gradient-diluted antibody was added to the cell culture plate. The 96-well cell culture plate with the added antibody was incubated in a CO2 cell incubator at 37°C for 2 days. An appropriate amount of cell culture supernatant was taken, and the secretion levels of IL-2 and IFN-gamma were measured.

[0189] The method for measuring IL-2 secretion was almost the same as the IFN-gamma measurement method in the above example, and the double antibody sandwich method was also used. The relevant reagents were anti-human IL-2 antibody (Catalog No.: 9133977), IL-2 standard (Catalog No.: 9220085), and biotinylated anti-human IL-2 antibody (Catalog No.: 9120557), purchased from BD Biosciences. After discarding the excess supernatant in the cell culture plate, the cell proliferation status was measured using the Cell-Titer-Glo Luminescent Cell Viability Assay Kit (Promega, Catalog No.: G7572).

[0190] The test results showed that the SP34-Hu-IgG1 monoclonal antibody was effective against IL-2 in PBMCs (Figure 8A, EC 50(It was 7.596 pM), IFN-gamma (Figure 8B, EC) 50 (The level was 24.49 pM) This strongly stimulated secretion and also stimulated PBMC proliferation (Figure 8C, EC) 50 The concentration was 0.4244 pM. The above in vitro test results showed that the two types of triplicate antibodies of the present invention did not nonspecifically activate PBMC cells within a reasonable concentration range.

[0191] Example 11. Evaluation of the physicochemical properties of the anti-MUC17*CD3*CD28 trispecific antibody. Antibodies are high-molecular-weight proteins with highly complex secondary and tertiary structures. Antibodies exhibit heterogeneous biochemical and biophysical properties due to changes such as post-translational modifications, aggregation, and degradation. Analysis of bispecific antibodies using separation techniques often reveals variants, aggregates, and degradation fragments, their presence potentially compromising safety and efficacy. Aggregates, degradation fragments, and incompletely assembled molecules are prone to occurring during antibody production and storage.

[0192] This invention uses high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) to measure the content of the aforementioned impurities in a sample. Since the molecular weight of aggregates is larger than that of monomers, the retention time of the corresponding peak is shorter. On the other hand, since the molecular weight of decomposed fragments or incompletely assembled molecules is smaller than that of monomers, the retention time of the corresponding peak is longer. The chromatograph used for HPLC-SEC was a Dionex Ultimate 3000, and the mobile phase was prepared as follows: an appropriate amount of 20 mM sodium dihydrogen phosphate mother liquor was taken, and the pH was adjusted to 6.8 ± 0.1 with 20 mM disodium hydrogen phosphate. Injection volume: 20 μg, column: TSK G3000SWXL, specifications: 7.8 × 300 mm 5 μm, flow rate: 0.5 mL / min, elution time: 30 min, column temperature: 25 °C, sample chamber temperature: 10 °C, detection wavelength: 214 nm.

[0193] Figure 9A shows the HPLC-SEC spectrum of CD28-44 / SP34-Dia+44H4-KIH, where the main peak at a retention time of 16.263 minutes accounts for 98.8%. Figure 9B shows the HPLC-SEC spectrum of CD28-44 / SP34-ScFv+44H4-KIH, where the main peak at a retention time of 15.997 minutes accounts for 98.2%. The main peaks of the two trispecific antibodies show similar proportions, indicating equivalent SEC purity.

[0194] In this invention, the content of degraded fragments or incompletely assembled molecules in a sample was analyzed using CE-SDS (Capillary Electrophoresis-Sodium Dodecyl Sulfate). CE is divided into two types: non-reducing and reduced. Samples used for the former do not require the use of the reducing agent DTT to break the intramolecular disulfide bonds during denaturation, while samples used for the latter require the use of the reducing agent DTT to break the intramolecular disulfide bonds during denaturation. Non-reducing and reduced CE-SDS are denoted as NR-CE-SDS and R-CE-SDS, respectively. The capillary electrophoresis apparatus used was a ProteomeLab™ PA800 plus (Beckman Coulter) equipped with a UV 214 nm detector. The capillary model was a Bare Fused-Silica Capillary, with specifications of 30.7 cm × 50 μm and an effective length of 20.5 cm. Other related reagents were purchased from Beckman Coulter. The main instrument parameters are as follows. Capillary and sample chamber temperature: 20±2℃, separation voltage: 15 kV.

[0195] Figures 10A and 10B show the NR-CE-SDS and R-CE-SDS spectra of CD28-44 / SP34-Dia+44H4-KIH, respectively, while Figures 10C and 10D show the NR-CE-SDS and R-CE-SDS spectra of CD28-44 / SP34-ScFv+44H4-KIH, respectively. The main NR-CE-SDS peak of CD28-44 / SP34-Dia+44H4-KIH accounted for 97.5%, while the main NR-CE-SDS peak of CD28-44 / SP34-ScFv+44H4-KIH accounted for 98.1%. The main peaks 3, 8, and 11 of the R-CE-SDS for CD28-44 / SP34-Dia+44H4-KIH (corresponding to the three polypeptide chains of the triplicate antibody, in order of increasing molecular weight) accounted for 15.5%, 32.5%, and 50.7%, respectively, with a total peak area of ​​98.7%. The main peaks 2, 6, and 10 of the R-CE-SDS for CD28-44 / SP34-ScFv+44H4-KIH (corresponding to the three polypeptide chains of the triplicate antibody, in order of increasing molecular weight) accounted for 15.6%, 32.2%, and 50.8%, respectively, with a total peak area of ​​98.6%. The results above indicate that the SEC purity of the two types of triplicate antibodies of the present invention is 98% or higher, and the purity measured by the CE method is close to or exceeds 98%, demonstrating that the antibodies are highly pure and possess good physical and chemical properties.

[0196] Example 12: Evaluation of the tumor-suppressing effect of an anti-MUC17*CD3*CD28 trispecific antibody in animals. The human immune system was reconstituted in NOG (Vittong Lihua) mice using human peripheral blood mononuclear cells (hPBMCs), and a modified human colon cancer LS174T-MUC17 subcutaneous transplant tumor model was established in these mice. The in vivo tumor suppressive effect of a MUC17*CD3*CD28 trispecific antibody was evaluated. The target cell preparation method is as follows: The MUC17 gene was cloned into a pLVX-puro lentiviral vector (Clontech, catalog number: 632164), and the resulting expression vector was named Muc17-plvx. The lentiviral packaging plasmid and Muc17-plvx were co-introduced into HEK293 cells, and the cell culture supernatant containing the recombinant lentivirus was collected and used to infect LS 174T cells. After 3 days, the cells were screened with puromycin under pressure to obtain cell clones into which the target gene had been introduced. The final cell line that stably expressed MUC17 was named LS174T-MUC17. The specific procedure is as follows: Ten days before tumor cell inoculation, the purchased PBMCs (Peripheral blood mononuclear cells, manufactured by Aoneng Bio-Corp, product code FPB004F-C) are resuscitated in vitro, the PBMC cells are resuspended in PBS, and the concentration of the PBMC suspension is set to 2.5 × 10⁻⁶. 7 The concentration was adjusted to / mL. 200 μL of PBMC cell suspension was administered to each mouse by tail vein injection, i.e., 5 × 10⁶ cells per mouse. 6 PBMC cells were inoculated. After 10 days, modified human colon cancer LS174T-MUC17 cells cultured in vitro were harvested, and the cell suspension concentration was increased to 4 × 10⁶. 7 The cell suspension was adjusted to 1 / mL and mixed with the matrix gel in a 1:1 ratio. Under sterile conditions, 100 μL of the cell suspension was subcutaneously inoculated into the right thoracic cavity of NOG mice. The inoculated tumor cells formed a solid tumor subcutaneously, with a volume of approximately 50-100 mm². 3The patients were randomly divided into groups according to tumor volume. A blank control group, which received only saline injections, was used as the control group. The other groups received either 0.5 mg / kg of the positive control antibody AMG199 or 0.5 mg / kg of MUC17*CD3*CD28. Subsequently, the drugs were administered twice a week for a total of five times according to the above regimen, and tumor volume was measured twice a week. Finally, the tumor growth curves measured over time for each group are shown in Figure 11. As a result, the anti-MUC17*CD3*CD28 trispecific antibody showed a relatively higher tumor-suppressing effect compared to AMG199.

[0197] All documents relating to the present invention are cited herein by reference, so that each document may be cited independently. Furthermore, after reading the foregoing, those skilled in the art may make various variations and modifications to the present invention, and it should be understood that equivalent forms of these variations are included within the scope of the claims of the present invention.

Claims

1. The first target domain D1 binds to a target protein selected from the group consisting of CD3, CD28, CD40, and CD137, An optional second target domain D2 that binds to a target protein selected from the group consisting of CD3, CD28, CD40, and CD137, A multispecific T-cell engager characterized by comprising a third target domain D3 containing one or more MUC17 antigen-binding domains.

2. The multispecific T cell engager according to claim 1, wherein D1, D2, or D3 are each independently selected from a single-domain antibody (sdAb), Fab, Fab', F(ab')2, TriFab, Fv fragment, fragment-variable (Fv) heterodimer, single-stranded Fv(scFv) fragment, biantibody (diabody), bispecific T cell engager (BiTE), or single-domain fragment, and are preferably selected from single-stranded Fv(scFv), Fv fragment, or Fab fragment.

3. The MUC17 antigen-binding domain comprises a third heavy chain variable region and a third light chain variable region, wherein the third heavy chain variable region is H-CDR1 containing the amino acid sequence shown in SEQ ID NO: 3, H-CDR2 containing the amino acid sequence shown in SEQ ID NO: 4, Three heavy chain variable regions including H-CDR3 containing the amino acid sequence shown in SEQ ID NO: 5, The third light chain variable region is, L-CDR1 containing the amino acid sequence shown in SEQ ID NO: 6, L-CDR2 containing the amino acid sequence shown in SEQ ID NO: 7, The multispecific T cell engager according to claim 1, characterized by comprising three light chain variable regions including L-CDR3 containing the amino acid sequence shown in SEQ ID NO:

8.

4. The multispecific T cell engager according to claim 1, further comprising an Fc fragment, preferably an Fc fragment derived from IgG1 or IgG4.

5. The Fc fragment derived from the aforementioned IgG1 is N297A; L234F / L235E / P331S; Y349C / K370E / K409D / K439E; S354C / D356K / E357K / D399K; S354C / T366W; S354C / T366W / H435R / Y436F; Y349C / T366S / L368A / Y407V; L234F / L235E / P331S / Y349C / K370E / K409D / K439E; L234F / L235E / P331S / S354C / D356K / E357K / D399K; L234F / L235E / P331S / S354C / T366W; L234F / L235E / P331S / Y349C / T366S / L368A / Y407V; N297A / Y349C / K370E / K409D / K439E; N297A / S354C / D356K / E357K / D399K; N297A / S354C / T366W; N297A / Y349C / T366S / L368A / Y407V; and / or S267E; S267E / G236D; S267E / S239D; S267E / L328F The multispecific T cell engager according to claim 1, characterized by having a mutation selected from the group consisting of the following.

6. The multispecific T cell engager is a triplicate antibody, and the triplicate antibody is located from the N-terminus to the C-terminus. (a) First chain: VL 1 -L1-VH 2 -L2-VL 2 -L3-VH 1 -L4-Fc1 (b) Second strand: VH 3 -CH1-Fc2, and (c) Third strand: VL 3 -Having the structure shown in CL, Alternatively, the triplicate antibody may have a structure from the N-terminus to the C-terminus. (a) First chain: VL 1 - L1 - VH 1 - L2 - VL 2 - L3 - VH 2 - L4 - Fc1 (b) Second strand: VH 3 -CH1-Fc2, and (c) Third strand: VL 3 -Having the structure shown in CL, Here, VH 1 This is the first heavy chain variable region, VL 1 This is the first light chain variable region, VH 2 This is the second heavy chain variable region, VL 2 This is the second light chain variable region, VH 3 This is the third heavy chain variable region, VL 3 This is the third light chain variable region, CL is the light chain constant region, and CH1 is the CH1 domain. L1, L2, L3, and L4 are, independently, none, coupled, or linker. Fc1 or Fc2 are each independent Fc elements. The multispecific T cell engager according to claim 1, characterized in that "-" is a triply specific antibody exhibiting a peptide bond.

7. A polynucleotide characterized by encoding a multispecific T cell engager according to any one of claims 1 to 6.

8. A vector characterized by comprising the polynucleotide described in claim 7.

9. A host cell characterized in that the vector described in claim 8 or the polynucleotide described in claim 7 is incorporated into the host cell's genome.

10. (i) A procedure for culturing the host cells described in claim 9 under appropriate conditions to obtain a mixture containing the multispecific T cell engager described in any one of claims 1 to 6, A method for preparing a multispecific T cell engager according to any one of claims 1 to 6, comprising the step of (ii) purifying and / or separating the mixture obtained in step (i) to obtain a multispecific T cell engager according to any one of claims 1 to 6.

11. (I) A multispecific T cell engager according to any one of claims 1 to 6, (II) A pharmaceutical composition characterized by comprising a pharmaceutically acceptable vector.

12. (a) A multispecific T cell engager according to any one of claims 1 to 6, (b) An immune complex comprising a complex portion selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

13. (a) Use of a multispecific T cell engager according to any one of claims 1 to 6, the pharmaceutical composition according to claim 11, or the immune complex according to claim 12 in the preparation of a detection reagent or kit and / or an agent for the prevention and / or treatment of cancer / tumor.

14. The use according to claim 13, characterized in that the cancer / tumor is MUC17-related cancer / tumor.

15. The use according to claim 13, characterized in that the cancer / tumor includes solid tumors and hematological malignancies.

16. The use according to claim 13, characterized in that the cancer / tumor is selected from the group consisting of stomach cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, gastroesophageal cancer, pancreatic adenocarcinoma, lung cancer, breast cancer, liver cancer, cervical cancer, thyroid cancer, uterine cancer, prostate cancer, or a combination thereof.

17. A kit comprising a multispecific T cell engager according to any one of claims 1 to 6, a polynucleotide according to claim 7, a vector according to claim 8, a host cell according to claim 9, a pharmaceutical composition according to claim 11, or an immune complex according to claim 12.

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